Summer Research Program

The Summer Research Training Program helps veterinary students explore research-focused career paths.

…the work I contributed to a project even landed my name as a co-author on a paper that will soon be published… Research is definitely something I want to keep in mind as I pursue a residency and a future in academia.

Teddy Hristova, Class of 2018

The Summer Research Training Program at Illinois is one of many similar programs nationally and internationally and is funded in part by the National Institutes of Health. Students receive a stipend for participation in the program.

During the 10-week program, students are expected to:

  • Work on a research project in the mentor’s laboratory for 40 hours per week
  • Complete training modules to learn about laboratory safety and the use of animals and human subjects in research
  • Complete training in the ethical conduct of scientific research
  • Attend a seminar series covering topics from research compliance to career opportunities and planning. Careers in academia, industry, and government are discussed
  • Participate in a scientific writing series;  prepare an abstract, a poster presentation and a short manuscript
  • Participate in a field trip to pharmaceutical companies;  network with veterinary students in the summer research program at Purdue University
  • Write a research abstract and submit it to the National Veterinary Scholars Symposium
  • Present a research poster at the National Veterinary Scholars Symposium
    If you have questions regarding the application process, please send an email to the Program Directors ( prior to submitting an application.


  • The program will begin on Monday, May 23, and end on Friday, July 29, 2022.
  • The National Veterinary scholars Symposium will be held at the University of Minnesota during the first week of August.
  • Students will receive a stipend of $5,382.50 for participation in the program.
  • Complete the online application form by January, 24, 2022.


Eligibility Requirements

  • Enrollment in the first or second year of veterinary school at the time of application.
  • Career interests that would benefit from research training. (Veterinary students from other universities are encouraged to apply).

Submitting Your Application

Selection Process

  • All applications will be reviewed by the Program Directors.
  • The student-mentor matching process will begin immediately after the application deadline and continue until all places in the program are filled.

Student-Mentor Matching

  • Students will be matched with a mentor based on the student’s interests and mentor’s availability. Applicants may learn about potential mentors under “Meet our Mentors” (below).
  • Students will be notified of a potential match in the program and will meet with the mentor to discuss possible research projects. Following that meeting, the student and mentor will provide feedback to the Program Directors to indicate whether they have agreed to work together.


View the list of institutions participating in the Boehringer Ingelheim Veterinary Scholars Program.

See the summer opportunities available through Morris Animal Foundation.


A list of program mentors is provided below. Mentors may be added to accommodate student research interests.

Department of Animal Sciences

Department of Comparative Biosciences

Department of Chemistry

Department of Entomology

Department of Evolution, Ecology, and Behavior

Department of Food Science and Human Nutrition

Department of Kinesiology and Community Health

Department of Microbiology

Department of Molecular and Integrative Physiology

Department of Pathobiology

Department of Psychology

Department of Veterinary Clinical Medicine


Is participation in the program a full-time commitment?

Yes, the program is a full-time commitment. You are expected to work 40 hours per week during the program. Accepting a traineeship in the Summer Research Training Program means that you agree not to participate in other programs or expect vacation time during the 10-week program.

What will my hours and schedule be?

Hours and scheduling will vary depending on your project (for example, lab work vs. field work) and are determined by your mentor.  By accepting a traineeship in the Summer Research Training Program, you agree not to participate in other programs or expect vacation time during the 10-week program.

Can I hold another job as long as it does not interfere with the Summer Research Training Program?

Many students use the summer to earn as much money as they can to finance their education and living expenses. It is common for students who are participating in the Summer Research Training Program to hold a part-time job, either on or off campus. These students work in the evenings or on weekends, when they are not committed to their research project. Because of the way that the Summer Research Training Program pays its stipends, students who hold an hourly job on the university campus cannot work more than 6 hours per week at the other campus job. Failure to disclose the campus job will delay processing of paychecks.

Is my summer stipend taxable?

Funds may be considered taxable income to the recipient, but are not reported as taxable income by the University on Forms W-2 or 1099-MISC. Taxability of the stipend depends on personal circumstances, so it is advisable to consult with a tax professional for advice on your own situation.

Should I pre-arrange a project with a mentor before applying to the program?

No, you should not assume that pre-arrangements with a potential mentor will lead to acceptance in the program. It is important that the application process provides equal opportunity for each student. Mentors and students are matched based on information provided in the program application.

Can I participate in Summer Research Training Program activities even if I do not have a program traineeship?

Yes. Program activities are open to all veterinary students. Contact the Program Directors to request a schedule of activities.

Read the abstracts for projects in the 2021 Summer Research Training Program

2021 Research Project Abstracts

Cytochrome P450 reaction phenotyping of hydroxyitraconazole metabolism in canine liver

Jennifer Applebaum, Zhong Li, and Jennifer M. Reinhart
Department of Veterinary Clinical Medicine, College of Veterinary Medicine (Applebaum, Reinhart), and the Roy J. Carver Biotechnology Center (Li), University of Illinois at Urbana-Champaign, Urbana, IL

Itraconazole (ITZ) is a valuable systemic antifungal agent and, although it is a safer long-term treatment option, abundant evidence has established adverse effects, particularly hepatotoxicity, in canines. Alterations in ITZ metabolic pathways result in the production of toxic metabolites, which are thought to be linked to hepatotoxicity. In humans and rodents, ITZ is metabolized by the hepatic cytochrome P450 (CYP) 3A4 enzyme to hydroxyitraconazole (OH-ITZ) and then to keto-ITZ. Our laboratory has recently established that CYP3A12, a canine ortholog to CYP3A4, is responsible for converting ITZ to OH-ITZ, but the CYP responsible for converting OH-ITZ to keto-ITZ in canines is unknown. This study aims to identify the CYP(s) responsible for metabolizing OH-ITZ in canines in vitro and we hypothesize that this enzyme is CYP3A12. Initial experiments in canine hepatic microsomes (MICs), which contain aggregated CYPs, established that oxidation of OH-ITZ to keto-ITZ optimally occurred under the following conditions: 1 mg/mL MIC, 1 mM NADPH, and a 30-minute incubation period, with keto-ITZ generation detected using HPLC-MS. Michaelis-Menten reaction kinetics were established in MICs using OH-ITZ (0-40 µM) and yielded the following parameter values: Vmax = 11.06 pmol keto-ITZ/min/mg MIC, km = 148.6 mM. Final assays will be performed using Bactosomes, which contain recombinant isolated canine CYPs, to identify the specific CYP(s) responsible for OH-ITZ metabolism. This study will establish a better understanding of ITZ metabolic pathways, which will contribute to the knowledge of ITZ-associated hepatotoxicity. Such advances may permit development of new treatment and preventative strategies in canine patients.

Research Grant: Partially supported by the Companion Animal Memorial Fund, University of Illinois College of Veterinary Medicine

Student Support: Office of the Director, NIH, T35 OD011145

Variation in tick species abundance between two collection methods at three central Illinois study sites

Alexandra K. Doran, Heather Kopsco, Nohra Mateus-Pinilla, and Rebecca L. Smith
Department of Pathobiology, College of Veterinary Medicine (Doran, Kopsco, Mateus-Pinilla), Illinois Natural History Survey-Prairie Research Institute (Mateus-Pinilla), University of Illinois at Urbana-Champaign, Urbana, IL

As global climate change continues to facilitate the spread of ticks and tick-borne illnesses, it is more important than ever to understand the habitat preferences of these tiny yet dangerous arachnids. There are currently at least 15 species of ticks known to inhabit Illinois. These species spread many serious illnesses, including Lyme disease, to humans and domestic animals. Habitat preferences of these tick species are well-documented, but previously collected data suggest a surprising lack of ticks at three central Illinois study sites with habitats that appear conducive to ticks. The purpose of this study is to determine if collecting ticks via small mammal trapping will yield different relative tick species abundances compared to using a drag cloth in these sites. The study will also provide the opportunity to examine the prevalence of Babesia microti, Borrelia burgdorferi, Anaplasma phagocytophylum, and Ehrlichia spp. in small mammals to determine if the number and species of ticks found on a particular host can be used to predict disease status. We will trap field mice using live Sherman traps baited with peanut butter. Traps will be placed at sites with prior approval from site managers. Animals will be restrained within a clear plastic tube that limits movement during sample collection, then released. Ear punches and blood samples will be collected for parasite identification. Ectoparasites will be removed and placed in 70% ethanol for subsequent identification. Understanding why seemingly ideal locations do not have the expected abundance of ticks may yield clues about how the spread of ticks may be mitigated.

Research Grant: CDC Cooperative Agreement U01 CK000505; DOD TB180052

Student Support: Office of the Director, NIH, T35 OD00011145

The effects of perinatal exposure to phthalates on hippocampal cell death and cognitive flexibility in juvenile rats

Hope Fenton, Ellen Sellinger, and Janice Juraska
College of Veterinary Medicine (Fenton), Neuroscience Program (Sellinger, Juraska), and Department of Psychology (Juraska), University of Illinois at Urbana-Champaign, Urbana, IL

Phthalates are a group of chemicals used as plasticizers, most prominently in polyvinyl chloride plastics, food packaging and processing, and to infuse fragrance in personal care products. Early exposure to phthalates decreases neurons and synapses in the medial prefrontal cortex (mPFC), resulting in decreased cognitive flexibility. This effect on the developing brain seems to be due, in part, to increased levels of apoptosis during neonatal and postnatal periods. However, the effects of phthalates have not been explored in other brain areas, including the hippocampus. Here, we examine how perinatal exposure to a human-equivalent level and mixture of phthalates affects cell death in the dorsal and ventral hippocampi, as well as cognitive function. Pregnant rats were dosed with phthalates daily, beginning at gestational day 2 through postpartum day 25. Pups were euthanized at one of the following postnatal days: P0, P2, P5, and P10, and hippocampal sections were stained using TUNEL to mark for cell death. One group of littermates was tested in the Morris water maze at P30, while a second group was tested at P90. We hypothesize that early exposure to phthalates will result in an increase in hippocampal cell death, as seen in the mPFC, causing a decrease in cognitive flexibility, especially spatial ability in the water maze. The information obtained in this study will contribute to a better understanding of the developmental and lasting effects that phthalates have on different brain regions.

Research Grant: NIH P01 ES002948-Project3

Student Support: HF was supported by NIH T35 OD011145; ES was supported by NIH T32 ES007326

Micromanaging resistance: miRNA expression and chemosensitivity in testicular germ cell tumors

Kelly Kries, Ratnakar Singh, and Michael J. Spinella
Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL

Testicular germ cell tumors (TGCTs) are the most common type of cancer in young men and their prevalence has increased over the past 40 years. Although 70% of metastatic TGCT patients can be cured with chemotherapy, the tumors that become refractory to treatment are largely fatal. After failing to find clear genetic drivers of chemotherapy sensitivity and resistance in TGCTs, research focus has shifted to explore the role of epigenetics in maintaining sensitivity to cisplatin, azacytidine, and similar drugs. Epigenetic mechanisms such as DNA methylation, histone modifications, and miRNAs modify gene expression and protein function. We hypothesize that the response of TGCTs to cisplatin and azacytidine is regulated in part by alterations in miRNA expression. To assess this relationship, we used qPCR to compare the relative expression of ten candidate miRNAs between parental and matching, isogenic drug-resistant TGCT cells. The miRNAs were chosen based on reports that suggested a role in TGCT biology. Preliminary data demonstrated that the hsa-miR-302/367 cluster was upregulated in cisplatin-resistant TGCT cells compared to parental cells. This cluster is known to act as an oncogene in other tumor types by increasing proliferation and survival through the MAPK/ERK pathway and increasing levels of survivin, an apoptosis inhibitor. Future experiments will use RNA-seq to assess miRNA populations in these TGCT cell lines in an unbiased, genome-wide manner. Understanding the epigenetic landscape of drug sensitivity and resistance in TGCT cells is the first step in developing novel therapeutic strategies to treat resistant TGCTs and possibly other cancers that are not curable in the metastatic setting.

Research Grant: NIH, 5R01CA211875-02

Student Support: Office of the Director, NIH, T35 OD011145

Cryptosporidium phosphoenolpyruvate carboxylase as a molecular target for novel potent antiparasitic drugs

Rachael Lahar, Shahbaz M. Khan, and William H. Witola
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Cryptosporidium parvum is a zoonotic parasite that causes a diarrheal syndrome in neonatal and immunocompromised animals and humans worldwide. Currently, there is no fully effective drug nor vaccine against C. parvum. Moreover, parasite oocysts contaminate the environment and are resistant to most chemical disinfectants. Thus, it is urgent to identify strategies for developing efficacious drugs against C. parvum. The C. parvum genome lacks the tricarboxylic acid cycle and oxidative phosphorylation steps, suggesting that the parasite depends solely on glycolysis for metabolic energy. C. parvum glycolytic enzymes differ from mammalian orthologs, making them ideal targets for anti-cryptosporidial drug development. In previous work, our laboratory identified inhibitors for C. parvum glycolytic enzymes lactate dehydrogenase and pyruvate kinase that prevented parasite growth and disease in mice. The focus of the present study was another glycolytic enzyme, C. parvum phosphoenolpyruvate carboxylase (CpPEPc), which contributes to synthesis of pyruvate that is utilized for generation of metabolic energy. We will develop and validate an in vitro coupled-enzymatic assay involving the sequential catalytic activities of CpPEPc and C. parvum malate dehydrogenase, leading to the synthesis of pyruvate. The assay will be used to screen compound libraries for inhibitors of CpPEPc that will then be analyzed for anti-cryptosporidial activity both in vitro and in vivo. Ultimately, our goal is to unveil validated lead compounds for developing new effective drugs for treating C. parvum infection.

Research Grant: Bill and Melinda Gates Foundation (completed), USDA (pending)

Student Support: Office of the Director, NIH, T35 OD011145

Molecular components of oocyst formation in Cryptosporidium parvum

Ixzacil Marquez, Derek Pinto, Josh A. Lain, Maria G. Nava, and Sumiti Vinayak
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Cryptosporidium parvum is a protozoan parasite that causes watery diarrhea and mortality in agricultural animals and young children following ingestion of oocysts. The thick wall of the C. parvum oocyst allows it to survive harsh environments and resist chemical disinfectants. The wall consists of an outer glycocalyx, rigid lipid bilayer, and an inner layer. The glycocalyx is made of glycoproteins while the innermost layer is composed of Cryptosporidium oocyst wall proteins (COWPs). Electron microscopy and mass spectrometry showed that sporozoites within the oocyst tether to the inner layer and that these tethers are composed of polysaccharides. However, the molecular machinery that synthesizes these polysaccharides has not been characterized. We identified two genes in the C. parvum genome that encode for enzymes required for the synthesis of these polysaccharides and hypothesize they provide structural stability to the oocyst wall for productive transmission of infection. To understand the localization of these proteins and determine their essentiality for parasite survival, we utilized CRISPR/Cas9 genome editing and the C. parvum immunocompromised mouse infection model. We generated targeted Cas9/guide RNA constructs and linear repair DNA templates consisting of a luciferase reporter and a drug selection marker to select for resistant transgenic parasites in the animal model. Experiments are focused on creating these transgenic parasite strains and determining localization as well as essentiality of these enzymes for viable oocyst formation and transmission. Understanding oocyst structure provides unique targets for the development of anti-cryptosporidial drugs.

Research Grant: University of Illinois College of Veterinary Medicine

Student Support: Office of the Director, NIH, T35 OD011145

Immunohistochemical evaluation of the modular vascularity in the inferior colliculus of the mouse

Josefina Nordenstahl, Nathiya Vaithiyalingam Chandra Sekaran, and Daniel A. Llano
College of Veterinary Medicine (Nordenstahl), Beckman Institute for Advanced Science and Technology (Vaithiyalingam Chandra Sekaran, Llano), Department of Molecular and Integrative Physiology (Llano), University of Illinois at Urbana-Champaign, Urbana, IL

The inferior colliculus (IC), located within the midbrain, serves as a major auditory integration center in the brain. Our laboratory has contributed to the mounting evidence that suggests the IC plays a key role in multisensory processing. Thus, understanding IC microvascular structure is necessary to investigate multisensory integration disorders. In previous studies, we found a pattern of clearly defined modular regions that develop in the lateral cortex of the inferior colliculus (LCIC) during early development and remain distinguishable across the mouse lifespan. The goal of the current study is to further our physiological understanding of the LCIC modular regions by characterizing their microvascular density. We hypothesized that a higher microvascular density is present inside the modular regions in comparison to the surrounding matrix as they are concentrated hubs for neurochemical activity. Experiments were conducted on glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) knock-in mice in which LCIC modular regions were apparent due to their concentrated GAD67-GFP expression. Antibodies against endothelial markers were used to visualize blood vessels in brain sections. Overlapping images of GAD67-GFP and endothelial markers were captured microscopically and analyzed. Early results suggested that microvascular density was uniform across the LCIC. Greater understanding of brain microvasculature and IC structure will contribute to knowledge about multisensory processing, a critical process in both humans and animals.

Research Grant: R01 DC013073

Student Support: Office of the Director, NIH, T35 OD011145

The impact of periarticular osteophytes of the distal tarsus on race performance of Standardbred horses

Kara N. Scolman and Annette M. McCoy
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Periarticular osteophytes are bony outgrowths adjacent to a joint space that occur in response to various stressors on the joint. While they are commonly recognized as a radiographic hallmark of degenerative joint disease (DJD), periarticular osteophytes of the distal tarsus have also been reported in non-lame yearling horses on routine pre-sale radiographs. Distal tarsal periarticular osteophytes in yearling Quarter Horses have been associated with poorer performance, but effects in Standardbreds (SB) have not been investigated. The purpose of this study was to identify the radiographic prevalence of periarticular osteophytes of the distal tarsus in non-lame yearling SB horses as well as to evaluate the potential impact on race performance. We hypothesized that larger osteophytes would have a significantly negative impact on race performance. Of 416 SB yearlings from a single farm, 113 had distal tarsal periarticular osteophytes. Osteophytes were measured on the radiographs and categorized based on size. To assess the clinical impact of these findings, racing records of horses with and without osteophytes were obtained from the U.S. Trotting Association. Collected data included lineage, gait (pace vs. trot), number of races started, number of races won, earnings per start, lifetime earnings, and fastest qualifying time. Regression analysis revealed no association between the presence of periarticular osteophytes and performance parameters. Within the affected group, size of the osteophyte was not associated with performance parameters. These data highlight breed differences in the clinical relevance of osteophytes and suggest that osteophytes are of little concern in SB yearlings.

Research Grant: Morris Animal Foundation (DEQ16-311)

Student Support: Office of the Director, NIH, T35 OD011145

Bone’s black hole: Comparing the osteolytic mechanisms of hemangiosarcoma of the bone with multiple myeloma 

Corrine Thomas, Bahaa Fadl-Alla, and Timothy M. Fan
College of Veterinary Medicine (Thomas, Fadl-Alla, Fan) and Cancer Center at Illinois (Fan), University of Illinois at Urbana-Champaign, Urbana, IL

Multiple myeloma (MM) is a well-studied, aggressive cancer of plasma cells (a type of white blood cell) that can proliferate in the bone marrow of humans. In canines, hemangiosarcoma (HSA) of the bone presents similarly to MM, being predominantly osteolytic in nature. HSA has a high mortality rate and is a common, invasive cancer of endothelial cells (a type of cell that lines blood vessels) that can also proliferate in the bone. Clinically, both bone cancers compromise the structural integrity of the affected bone resulting in similar signs of spiral bone fractures and/or osteolytic lesions seen on radiography. Given their similar osteolytic presentation, canine HSA of the bone may adopt a MM-like mechanism of action. This study uses literature on the mechanism of MM in humans to focus analysis of canine HSA of the bone on specific features of the mechanism. Receptor Activator of Nuclear Factor-κB-Ligand (RANKL) activates osteoclasts, which are cells that break down bone, while Osteoprotegerin (OPG) inhibits RANKL to create a balance of bone remodeling in healthy bones. In severe cases of MM, the RANKL/OPG ratio is increased to favor bone resorption through increased osteoclast activation. The RANKL/OPG ratio for canine HSA cells was analyzed, using total RNA extraction followed by RT-PCR, and calculated. In MM, interleukin (IL)-1beta, IL-3, and IL-6 are responsible for osteoclast activation while IL-7 is responsible for osteoblast inhibition, and together these effects favor osteolysis. These interleukins were analyzed using sandwich ELISA tests to determine their expression from canine HSA cells. This information may lead to new treatments and/or earlier methods of detection of canine HSA of the bone.

Research Grant: Internal funding

Student Support: Office of the Director, NIH, T35 OD011145

2020 Research Project Abstracts

Is there a bias toward consideration of adaptationist explanations for behavioral phenomena?

Elisabeth A. Bacon, Coltan G. Parker, and Justin S. Rhodes

College of Veterinary Medicine (Bacon), Beckman Institute for Advanced Science and Technology (Parker, Rhodes), and Department of Psychology (Rhodes), University of Illinois at Urbana-Champaign, Urbana, IL

Researchers often seek to understand biological traits in terms of adaptive functioning, assuming that a trait is selected for a specific function that increases fitness. However, over the years, several critics have pointed out potential issues with this assumption. Namely, Gould and Lewontin in their 1979 seminal paper entitled “The Spandrels of San Marco and the Panglossian Paradigm: A Critique of the Adaptationist Programme” demonstrated that alternative evolutionary explanations to adaptation often exist for the occurrence of a trait. They went on to call out followers of the “Adaptationist Programme” for failing to consider such alternative explanations for phenomena. In this study, we examined ten prominent behavioral research journals to determine if there is a bias toward consideration of adaptationist explanations over alternative explanations for the occurrence of animal behaviors and if the extent of such a possible bias has changed since 1979. Starting with the year 1980, articles every fifth year were examined manually for both adaptationist and alternative keywords, as well as for relevant context. Preliminary results suggest there is a bias in the behavioral sciences toward explaining traits as evolutionary adaptations even when equally or more plausible alternative evolutionary explanations could have been offered. We speculate that this bias may contribute to prejudiced beliefs and misleading conclusions about the origins of behavioral traits, slowing progress in uncovering the true nature of nature.

Research Grant: None

Student Support: Office of the Director, NIH, T35 OD011145

Epigenetic control of tumorigenicity of testicular germ cell tumors 

Hannah L. Baldwin, Emmanuel Bikorimana, Ratnakar Singh, Zeeshan Fazal, Clifford Yerby, Michael J. Spinella

Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Testicular germ cell tumors (TGCTs) are aggressive yet can be extremely sensitive to cisplatin-based chemotherapy. Alternative therapies are needed for tumors that become refractory to cisplatin with the DNA methyltransferase inhibitor 5-aza deoxycytidine (5-Aza) providing one possibility. Two series of cell models were generated to better understand mechanisms of chemotherapy sensitivity and resistance: one was resistant to cisplatin and the other to 5-Aza. Interestingly, cisplatin resistance was associated with increased sensitivity to 5-Aza and 5-Aza resistance was associated with increased sensitivity to cisplatin. Preliminary data demonstrated reciprocal epigenetic alterations and a substantial decrease in tumorigenicity in cisplatin-resistant cells. The goal of the current study was to determine the tumorigenicity status of 5-Aza-resistant cells and to investigate reciprocal changes in gene expression that may account for tumorigenicity alterations. Transcriptomics revealed tumorigenicity genes (ZEB2, ID2, CCND1, CAPN2, COL12A1, and MMP2) upregulated in 5-Aza-resistant cells and downregulated in cisplatin-resistant cells. Inversely, tumor suppressor genes (PTMA, MEG3, ZNF208, KLF17, LRRC4B, and ARL10) were upregulated in cisplatin-resistant cells and downregulated in 5-Aza-resistant cells. Tumorigenicity assays were initiated using 5-Aza-resistant cells which are expected to exhibit increased tumorigenicity compared to cisplatin-resistant cells. This study provides insight into the mechanism of TGCT cell sensitivity to 5-Aza and cisplatin and suggests that epigenetics may reciprocally control cisplatin sensitivity and tumorigenicity of TGCTs.

Research Grant: NIH, 5R01CA211875-02

Student Support: Office of the Director, NIH, T35 OD011145

Development of a method for reducing the incidence of rotavirus transmission between farrowing groups

Sarah R. Botkin, Benjamin W. Blair, Suzanna M. Storms, James F. Lowe

College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Porcine rotavirus is a pathogen of neonatal pigs that is increasing in frequency in commercial swine breeding herds. Porcine rotavirus is spread from carrier sows that shed the virus in feces. When piglets ingest the feces, the resulting small intestine enteritis and diarrhea cause reduced growth rate and high economic losses. The most-concerning source of rotavirus is an infected farrowing group. Without adequate cleaning, rotavirus remaining in the farrowing crate is spread to the next group of pigs. Conventional control methods of vaccination and high-pressure washing with disinfection have been ineffective against this environmentally stable pathogen. We hypothesize that enhanced hygiene with surfactant, detergent, and/or physical removal (power washing) will reduce viral loads on flooring surfaces in swine farrowing facilities and reduce the incidence of virus transmission to new sows and piglets. Clean rubber mats were infected with a single dose of modified live porcine rotavirus vaccine mixed with 5 grams of feces and 5 ml of water spread over a 4-square-inch sample area. Mats were subjected to various cleaning methods involving combinations of pressure washing, surfactant, and disinfectant. Samples were collected from the mats using sterile cotton swabs, as well as a toothbrush to simulate pigs foraging and rooting. Cell culture will be used to quantify recovery of infectious virus from the mats. Results from this model system will be tested in a commercial swine operation experiencing clinical disease. Reduction of the transmission of rotavirus between groups of farrowing sows would provide a meaningful improvement to current swine production methods.

Research Grant: FFAR AAVMC Fellowship 100579

Student Support: FFAR AAVMC Fellowship 100579

The role of glycoprotein C in horizontal transmission of turkey alphaherpesvirus

Valeria Campos, Widaliz Vega-Rodriguez, Keith W. Jarosinski

Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Members of the Alphaherpesvirinae subfamily of the Herpesviridae infect mammalian, avian and reptilian species, and continuously cause problems affecting humans and food animals. Previous research showed that the alphaherpesvirus-conserved glycoprotein C (gC) is essential for horizontal transmission of Marek’s disease alphaherpesvirus (MDV) in chickens. MDV causes a highly contagious infection in chickens that clinically presents with neoplasms and neurological signs. We hypothesized that gC homologs of other avian alphaherpesviruses are also necessary for horizontal transmission. MDV is controlled in the poultry industry by vaccination with modified-live vaccines (MLV) including homologous turkey alphaherpesvirus (HVT). Preliminary data in our laboratory showed that HVT does not spread from chicken to chicken during in vivo studies. This study aims to determine whether HVT gC is expressed during replication in cell culture and whether HVT gC replaced with MDV gC will facilitate horizontal transmission. To test these hypotheses, two viruses were generated using the 2-step Red recombination method: one with an HA epitope in frame at the N-terminus of the HVT gC protein to facilitate its identification, and another in which MDV gC will be inserted into the previously generated HVTΔgC to replace HVT gC. Viruses were characterized in cell culture for replication and then subsequently used in chicken-to-chicken transmission studies. The information provided in this study will allow us to understand more about the HVT vaccine strain commonly used in chickens and its future implications in gene modification research.

Research Grant: USDA-NIFA-AFRI 2019-67015-29262

Student Support: Office of the Director, NIH, T35 OD011145

Pharmacokinetics of Stokes compounding pharmacy extended-release theophylline formulation

Gabriela A. R. de Oliveira, Zhong Li, Jennifer M. Reinhart

Department of Veterinary Clinical Medicine, College of Veterinary Medicine (de Oliveira, Reinhart), Roy J. Carver Biotechnology Center (Li), University of Illinois at Urbana-Champaign, Urbana, IL

Theophylline is used in veterinary medicine as a bronchodilator to treat canine bronchitis. However, immediate-release formulas require administration four times daily, leading to lower owner compliance and poor management of chronic disease. Additionally, only one commercially available extended-release formulation has been tested in dogs and showed poor bioavailability. These shortcomings led compounding pharmacies to create new formulations for which the pharmacokinetics need to be established. This study will use eight healthy dogs to establish the pharmacokinetics of oral extended-release theophylline compounded by Stokes Pharmacy. The design will be a randomized, two-way crossover with a 7-day washout period between intravenous aminophylline (IVA, 8.6 mg/kg theophylline equivalent), to determine absolute bioavailability, and oral Stokes theophylline formula (STF, 10 mg/kg) administration. Plasma theophylline will be quantified by liquid chromatography and tandem mass spectrometry. Non-compartmental pharmacokinetics analysis will be performed for both IVA and STF. The accumulation ratio and the superposition methods will be used to estimate whether steady state plasma concentrations fall within the therapeutic range established for adult humans (10 – 20 mg/mL), as there are currently no studies determining the canine therapeutic range. We predict that the STF will have similar bioavailability to previously established theophylline products and produce adequate plasma concentrations and durations to be used at a twice-daily regimen. Validating STF will provide another product alternative for treating canine bronchitis, while improving client compliance and disease management.

Research Grant: Stokes Pharmacy

Student Support: Office of the Director, NIH, T35 OD011145

Validating respiratory pathogen qPCR for use with feces as a non-invasive diagnostic tool for great apes

Jennifer D. Despotovich, Nadia Ahmed, Karen Terio

Zoological Pathology Program, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Human respiratory pathogens pose a serious threat to great ape populations, often causing high morbidities and occasional mortalities. Because research and tourism result in frequent contact between human and apes, quick and safe pathogen identification are essential for species health. Many respiratory pathogens are coughed up, swallowed, and passed in the feces, which are a non-invasive sample that is easy to collect for testing. Although previous studies screening for respiratory pathogens in wild great ape populations have used real-time PCR assays developed for humans, to our knowledge, these assays have not been validated for use with feces. To validate these assays, two different fecal DNA/RNA extraction methods were compared for identification of the respiratory pathogens Streptococcus pneumoniae and human Metapneumovirus (hMPV) in chimpanzee and gorilla feces. Varying concentrations of pathogens or their nucleic acids were added to fecal samples from clinically normal apes to evaluate assay performance compared to a standard curve. Results showed an effect of extraction method on target amplification and assay efficiency, demonstrating the need for assay validation. Newly validated assays will be used to test fecal samples collected from free-ranging chimpanzees in Gombe National Park, Tanzania during a respiratory disease outbreak of unknown etiology.

Research Grant: Zoological Pathology Program

Student Support: Office of the Director, NIH, T35 OD011145

Unmanned aerial vehicles (UAVs) for counting wildlife: a review of their efficacy compared to other methods

Annalisa M. Fama, Rachel G. Miller, Brian F. Allan

College of Veterinary Medicine (Fama) and School of Integrative Biology (Miller, Allan), University of Illinois at Urbana-Champaign, Urbana, IL

Population counts of free-roaming wildlife are essential for scientists to monitor dynamics of a species and provide information for improved management and conservation practices. Unmanned Aerial Vehicles (UAVs) are a novel technology for scientists to conduct research on free-roaming wildlife, including to evaluate population density, monitor health and behavior, collect remote images, and identify threats to their environments. The purpose of this literature review was to evaluate the existing literature that compares the efficacy of UAVs compared to other methods for counting free-roaming wildlife. We hypothesized that UAVs are more efficient and welfare-conscious for evaluating free-roaming wildlife compared to traditional methods. Eighty relevant publications were retrieved from Thomson Reuters Web of Science that utilized UAVs to evaluate free-roaming wildlife: 9 literature reviews, 43 studies that evaluated terrestrial wildlife, 22 studies that evaluated marine animals, 7 studies that evaluated aquatic animals, and 2 studies that evaluated livestock. Publications that discussed behavior offered important insights to the welfare implications of UAVs. Published studies from 2006-2020 indicated researchers are using UAVs to study wildlife with increased frequency. Preliminary analyses indicated that UAVs have a shorter flight distance than manned flights, and offer overall improved accuracy, increased safety, and reduced costs, compared to traditional wildlife counting methods. Important future frontiers in the use of this technology include standardization of methodology and development of artificial intelligence technologies for processing large volumes of wildlife imagery.

Research Grant: None

Student Support: Office of the Director, NIH, T35 OD011145

The genetics of paternal behavior in unique stickleback fish populations 

Emily Grzeda, Colby Behrens, Alison M. Bell

College of Veterinary Medicine (Grzeda), Department of Evolution, Ecology, and Behavior (Behrens, Bell), University of Illinois at Urbana-Champaign, Urbana, IL

Parental care is crucial for offspring survival but can vary widely across populations of the same species. Stickleback fish (Gasterosteus aculeatus) provide a unique opportunity for study in this area. Stickleback parental care is male-exclusive: males build a nest, court a female, fertilize her eggs, then chase her away. However, different populations (called commons and whites) exhibit highly divergent parenting strategies. Commons care for eggs with frequent nest fanning and poking until they hatch, but whites immediately disperse eggs into surrounding algae before building a new nest. Behavior of pure common or white fish was compared to common/white F2 progeny. Assessing F2 survival, willingness to mate, and presentation of characterizable behaviors was a major goal of this study as they had never been produced. Nesting behaviors post-fertilization were recorded in 15-minute intervals twice daily for 4 days, at which time patterns were clearly identifiable. Parenting data was designated as time 0 (immediately post-fertilization) and long-term parenting (days 0.5-4). Data analysis, including principal component analysis, was performed using R. F2s exhibited high survival rates and readily mated with females. There was extensive variation in paternal behavior among F2 males in nesting time, fanning time, fanning bouts, and long-term nest attendance. Most F2 males provided care instead of dispersing eggs. These data provide a foundation for the long-term goal of this project, which is to use quantitative trait locus (QTL) mapping to identify genes that underlie differences in paternal care.

Research Grant: NSF IOS 1645170, NIH 2R01GM082937-06A1, Arnold A. Beckman Award #20069 from the University of Illinois Campus Research Board

Student Support: Office of the Director, NIH, T35 OD011145

Does misoprostol reduce inflammatory cytokine release caused by circulating endotoxin in equine patients?

Samantha Hammack, Kara Lascola, Shune Kimura, Annette M. McCoy

Department of Veterinary Clinical Medicine, University of Illinois Urbana-Champaign, Urbana, IL (Hammack, McCoy); Department of Clinical Sciences, Auburn University, Auburn, AL (Lascola, Kimura)

Inflammation mediated by pro-inflammatory cytokines can have a profound negative effect on diseased equine patients. These cytokines are produced in response to circulating endotoxin and can lead to a severe inflammatory state known as Systemic Inflammatory Response Syndrome (SIRS) which can lead to further systemic, possibly fatal disease. NSAIDs are a common choice to treat inflammation but are often contraindicated. Thus, it is vital to identify other pharmaceuticals that can combat increased cytokine production. Misoprostol, a prostaglandin E1 analog, has anti-inflammatory effects in multiple species and may provide an alternative to conventional therapies. In vitro, misoprostol decreases the release of inflammatory cytokines by peripheral blood equine leukocytes. The aim of this study is to determine the effect of misoprostol on inflammatory cytokine release in vivo. Using a crossover design, six healthy horses were given a saline control or oral misoprostol and then challenged with lipopolysaccharide (LPS). Blood was then collected serially over 24 hours. Neutrophilia in all horses confirmed the expected inflammatory response to the LPS challenge and was not different between groups. RNA was extracted from frozen peripheral blood leukocyte pellets. Inflammatory cytokine production (TNFα, IL-1β, IL-10, and IL-6) will be measured using quantitative PCR and results compared between the saline control and misoprostol treatment samples. This study will hopefully provide clinicians with evidence to support this novel therapy for equine patients with endotoxemia and systemic inflammation.

Research Grant: American Quarter Horse Foundation, Auburn University Animal Health and Disease Research Program

Student Support: USDA Multistate Research Funds

Genetic analysis of swine and human influenza A virus subtype H3N2 from 2014-2019 in the United States

Dayna Kinkade, Suzanna Storms, Benjamin Blair, James F. Lowe

College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Influenza A virus (IAV) surveillance is a vital aspect of both swine and human health industries because new IAV strains can arise by inter-species transmission, antigenic mutations (drift) and genetic reassortment. These characteristics create a genetically variable virus which can result in a pandemic with significant public health and economic implications. Previous research has shown that the H3N2 subtype has a high potential for cross-species transmission, yet little is known about IAV transmission between humans and swine. H3N2 sequences of human and swine isolates from July 2014 to July 2019 were collected from NCBI’s Influenza Virus Database. All RNA segment sequences from these strains were then aligned. The best-fitting models were selected using a Bayes factor with marginal likelihoods implemented in Beast. The resulting trees were then summarized in a maximum clade credibility tree after performing a 10% burn-in using TreeAnnotator. FigTree was then used to differentiate the human and swine strains. Swine strains found in human clades were considered human-to-swine spillover while human strains in swine clades were considered swine-to-human spillover. Results showed that the current influenza strains were more adapted to move from human hosts to swine hosts which was contrary to expectations from previous studies. Continued genetic surveillance and analysis of IAV is crucial to ensure the health and safety of swine and human populations.

Research Grant: Dr. Kenneth T. and Betty Wright Endowment, Lutz Family Endowment-IFAMS Award

Student Support: Foundation for Food and Agriculture Research

Characterizing the erythrocyte sedimentation rate in free-ranging Blanding’s turtles (Emydoidea blandingii)

Kayla LaDez, Samantha Bradley, Gary Glowacki, Laura Adamovicz, Matthew Allender

Wildlife Epidemiology Laboratory, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL (LaDez, Bradley, Adamovicz, Allender); Lake County Forest Preserve, Libertyville, IL (Glowacki)

Erythrocyte sedimentation rate (ESR) is an indicator of inflammation across taxa but most commonly is measured in mammals. Our group is assessing the utility of ESR in evaluating the health of select chelonian species. The focus of this study is the Blanding’s turtle (Emydoidea blandingii), which is endangered in Illinois. As an environmental indicator species, the health of the Blanding’s turtle supports general ecosystem wellness which is vital for other free-ranging wildlife already being threatened by human impact. We hypothesized that ESR is elevated in adult Blanding’s turtles compared to juveniles and in turtles with clinical signs of injury or disease. ESR will be measured in two free-ranging Blanding’s turtle populations in Lake County, Illinois. ESR measurement would be a valuable addition to traditional chelonian hematological tests because it requires little blood, equipment, or expertise. Characterization of ESR will advance the knowledge and conservation of Blanding’s and other semi-aquatic and aquatic turtles, by increasing the number of validated methods to assess health.

Research Grant: Preservation Foundation of the Lake County Forest Preserve District and the Wildlife Epidemiology Lab

Student Support: Office of the Director, NIH, T35 OD011145

Ticks and tick-borne diseases: assessing knowledge, attitudes, and practices of medical professionals in southern Illinois 

Emma N. Sandstrom, Rebecca L. Smith

Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

The prevention and control of tick-borne diseases in human populations is dependent on the expertise of the medical professionals who identify and treat them. Since their harmful effects were first discovered, tick-borne diseases have been an important part of epidemiological education for medical professionals. However, medical professionals need to keep pace with rapidly changing information about tick-borne diseases and their spread. The warming climate and altered migratory patterns of tick-carrying animals have distributed tick species and associated diseases to regions where they previously did not exist. Information on these spreading populations is available through a variety of health campaigns and educational programs, but the efficacy of communicating this information to medical professionals warrants analysis. To explore this topic, the Illinois tick survey team created an anonymous survey to assess the general tick knowledge of medical professionals, as well as the attitudes and practices that inform their patient differentials and treatments. Emergency and general practice physicians, registered nurses, and nurse practitioners in southern Illinois were invited to participate in the study, due to their diverse clientele and the rapidly changing tick-borne disease situation in this area. The results obtained from this survey will determine how well the information is being distributed across southern Illinois and highlight which aspects of tick and tick-borne disease education should be further emphasized in future campaigns. In time, this more-targeted education will hopefully reduce the rising incidence of tick-borne diseases in the southern Illinois populace.

Research Grant: Cooperative Agreement with Centers for Diseases Control and Prevention, U01 CK000505

Student Support: Office of the Director, NIH, T35 OD011145

Biomechanical risk factors of pacing and trotting on the incidence of osteochondrosis in Standardbred foals

Marissa L. Zerkowski, Annette M. McCoy

Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Osteochondrosis (OC) is a common developmental orthopedic disease that occurs when endochondral ossification is delayed. Both genetic and environmental risk factors contribute to OC. It was previously reported that pacers and trotters are susceptible to developing OC lesions at different locations in the tarsocrural joint (hock). This observation may be due to the biomechanical differences between pacing and trotting that could affect which OC lesions heal and which become permanent. The goal of this study was to quantify activity in Standardbred foals in their typical farm environment and correlate these data with the development and locations of OC lesions. We hypothesized that the amount of time foals spend pacing or trotting influences the distribution of OC lesions. Standardbred foals (n=137) on three farms were prospectively followed from 2-12 months of age. Radiographs were taken of their hocks every two months from 2-12 months of age to look for OC lesions. Foals were also video recorded in their normal paddock/pasture turnout for at least two consecutive hours per week. Based on the video recordings, activity was assessed in 30-second time intervals and categorized as nursing/eating, standing quietly, lying down, walking, trotting/pacing, and running. Foals spent ~82% of their time standing or eating with <18% of their time in active movement. Less than 1% of their time was spent pacing or trotting. Permanent OC lesions developed in 26% of foals; the majority of these were at the distal intermediate ridge of the tibia. Lesion distribution did not differ between pacers and trotters. More work is needed to elucidate the potential role of biomechanics on OC lesions.

Research Grant: Morris Animal Foundation D16EQ-311

Student Support: Office of the Director, NIH, T35 OD011145

2019 Research Project Abstracts

Analysis of SorCS1 haplotypes associated with tame and aggressive behavior in selectively bred red foxes

Kylie Ayers, Jennifer Johnson, and Anna Kukekova

College of Veterinary Medicine (Ayers), Department of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences (Johnson, Kukekova), University of Illinois at Urbana-Champaign, Urbana, IL

Since 1959, the Russian farm-fox experiment has used the red fox (Vulpes vulpes) as a model organism for the study of domestication. Through years of selective breeding, the experiment has created lineages of tame and aggressive foxes as well as maintained a strain of conventional farm foxes that were not under behavioral selection. Recently, genetic mapping and whole genome sequencing analyses identified the SorCS1 gene as a candidate gene associated with tame behavior in the fox. SorCS1 (sortilin related VPS10 domain containing receptor 1) encodes the main trafficking protein for AMPA glutamate receptors and neurexins, suggesting a role for synaptic plasticity in fox domestication. Subsequent analysis identified three common SorCS1 haplotypes in tame and aggressive populations with one haplotype strongly associated with tame behavior. Samples used for this analysis were approximately ten years old. The goal of the current study was to evaluate SorCS1 haplotypes in samples collected during 2018, which represent ten additional generations of selection beyond the initial work. Results showed that the frequency of the previously described tame or aggressive SorCS1 haplotypes remained constant in the tame and aggressive populations suggesting that these haplotypes were not strongly selected in recent years. Although most haplotypes present in the conventional population resembled those found in the aggressive population, a novel haplotype was identified. Results from this study will be used to develop breeding plans to further investigate the role of SorCS1 in fox behavior.

Research Grant: NIH, GM120782

Student Support: Office of the Director, NIH, T35 OD011145

The effect of formalin and paraffin storage time on RNAscope in situ hybridization signal amplification

Megan E. Colburn, Martha A. Delaney, and Karen Terio

Zoological Pathology Program, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

RNAscope (ACD Diagnostics) in situ hybridization (ISH) is a relatively new technique for detection of RNA targets and for studying gene expression in formalin-fixed, paraffin-embedded (FFPE) tissue samples. RNAscope protocols suggest that prolonged FFPE tissue storage and formalin fixation times may limit the ability to detect RNA in archived tissues. Yet, the technology has been successfully used in retrospective studies where tissue fixation time in formalin and block storage time were unknown and/or prolonged. To better understand the limits of storage and fixation time on the ability to detect RNA targets in archival FFPE tissues, we will compare RNAscope staining in FFPE tissues (n=15) known to be infected with Canine Distemper Virus (CDV) that have been stored for 6 months to 18 years at room temperature. Tissues were obtained from raccoons immunohistochemistry-positive for CDV and were fixed in formalin for up to 10 days. To determine the effect of prolonged exposure to formalin, staining of housekeeping gene mRNA expression will be compared in tissues fixed in formalin for 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, and 28 days prior to embedding in paraffin. RNA target signal amplification will be scored using a semi-quantitative method described in the RNAscope Assay user manual. Results obtained from this study will determine the utility of RNAscope ISH for archival tissues with prolonged storage and/or fixation time and will guide protocol development for the use of RNAscope technology in future veterinary retrospective studies

Research Grant: Zoological Pathology Program

Student Support: Office of the Director, NIH, T35 OD011145

Effect of nerve growth factor-β in pregnancy outcomes in dairy cows

Shelby Crump, Andrew Hubner, Phillip Peixoto, Wilson Meireles Coelho Junior, and Fabio Soares de Lima

Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Early pregnancy loss due to luteal insufficiency in high-producing dairy cows compromises the sustainability of the dairy industry. Nerve Growth Factor-Beta (NGF) is a natural seminal plasma protein that has been shown to improve luteinizing hormone release, luteal development, and signs of maternal recognition of pregnancy in our previous studies. We are currently operating a large field study to test the hypothesis that NGF improves luteal function, which in turn translates into reduced pregnancy loss and improved fertility in dairy cows. The study includes 600 lactating Holstein dairy cows that will be blocked by parity. Cows within each block will be randomly allocated to either receive 1 ml of phosphate buffer solution (PBS) containing 300 µg of purified NGF intramuscularly (NGF, n = 300) or 1 ml of PBS (Control, n = 300). Cows will be synchronized for the first service and the treatment will be performed at day of artificial insemination. A subset of 120 cows will have blood samples collected and receive ultrasonography examination at day of insemination, and then 7, 14, and 19 days later to evaluate progesterone concentration and corpus luteum development. Blood samples collected on day 19 post insemination will also be used to extract RNA and measure expression of interferon stimulated genes (ISG 15, MX1, MX2, and RTP4) to assess markers for maternal recognition of pregnancy. Pregnancy outcomes will be evaluated at days 37 and 65 post insemination. The development of a novel tool to mitigate the negative impact of subfertility would improve milk production efficiency, environmental stewardship, and the global food supply.

Research Grant: USDA Multistate Research Funds

Student Support: Foundation for Food and Agriculture Research

Salmonella spp. prevalence and antibiotic resistance in free-ranging Eastern box turtles

Greta Doden, Carol Maddox, and Matthew C. Allender

Wildlife Epidemiology Lab (Doden, Allender), Department of Pathobiology (Maddox), College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Salmonellosis is an important zoonotic infection and exposure to pet reptiles has been implicated in several human outbreaks. Although several studies report a low prevalence of this pathogen in free-ranging chelonians, they may serve as a reservoir for human-pathogenic strains. In May-July 2019, free-ranging Eastern box turtles (Terrapene carolina carolina) from three populations in Illinois (rural) and Tennessee (urban) were collected through canine and visual search. We hypothesized that the prevalence of Salmonellae in free-ranging Eastern box turtles is less than 5%. Cloacal swab samples were collected from each turtle, selectively enriched with tetrathionate broth, then plated on selective and differential media to isolate Salmonella spp. Genus was confirmed via MALDI-TOF and antibiotic sensitivities were performed. Of the 96 turtles sampled, three (3.1%; 95% CI: 0.6-8.9%) were detected with Salmonella spp. One isolate was serogroup PolyA and was resistant to penicillin, ampicillin, and doxycycline. The other two isolates were serogroup C1 and were resistant to amikacin, erythromycin, and gentamicin. All confirmed Salmonellae will be sent to the National Veterinary Services Laboratory for serotyping. The serogroup PolyA contains multiple pathogens associated with foodborne outbreaks in humans, including serotypes Typhimurium and Enteritidis. Serogroup C1 contains the highly pathogenic serotype Choleraesuis. Documenting the prevalence of pathogenic Salmonella serotypes in animal indicators like the Eastern box turtle will further our understanding of the spread of these serotypes between humans, animal agriculture, and the environment.

Research Grant: Wildlife Epidemiology Lab

Student Support: Office of the Director, NIH, T35 OD011145

Effects of a CSF1R inhibitor on microglia and their interactions with glioblastoma

Victoria Fisher, Katiria Soto-Diaz, Jan Lumibao, H. Rex Gaskins, and Andrew J. Steelman

College of Veterinary Medicine (Fisher), Department of Animal Sciences (Soto-Diaz, Gaskins, Steelman), Divison of Nutritional Sciences (Lumibao, Gaskins), Carl R. Woese Institute for Genomic Biology (Gaskins), Cancer Center at Illinois (Gaskins), University of Illinois at Urbana-Champaign, Urbana, IL

Glioblastoma (GBM, WHO grade IV glioma) is the most common, malignant, and aggressive form of primary brain tumor in adults, accounting for approximately 50% of diagnosed gliomas each year. The transformed cells in GBM are star shaped glial cells of the central nervous system called astrocytes. Under normal physiologic conditions, astrocytes are responsible for providing energy for the neurons they support, maintaining the blood brain barrier, and modifying the transmission of an action potential across a neuronal synapse by maintaining extracellular ion concentrations and removing excess neurotransmitter. After transformation, astrocytes with activated oncogenes also gain the ability to assemble and control cells of the central nervous immune system, namely tumor associated microglia and macrophages (TAM). Intercellular cross talk between these two cell groups can be directly extrapolated to the clinical symptoms associated with tumor progression. Of these, the most concerning is the peritumoral edema and hemorrhage resulting from the proinflammatory state created by activated TAM. A theoretical remedy for this has been to diminish TAM proliferation and activation using a colony stimulating factor receptor (CSF1R) inhibitor to compete with the factor released by glioblastoma cells to target the TAM. In vivo and in vitro studies are being conducted to examine the extent to which CSF1R inhibition suppresses tumor progression. If significant, the results of this study would provide further support for the progression of an approved CSF1R inhibitor to enter clinical trials and help people with glioblastoma.

Research Grant: NMSS 1-550124-538031-191100

Student Support: Office of the Director, NIH, T35 OD011145

A non-invasive measurement of prostate tumor hypoxia: systemic utilization of hypoxia probe 1 (HyP-1)

Katherine K. Havighorst, Catherine C. Applegate, Hailey J. Knox, Jefferson Chan, and John W. Erdman Jr.

College of Veterinary Medicine (Havighorst), Division of Nutritional Sciences (Applegate, Erdman), Beckman Institute for Advanced Science and Technology (Knox, Chan, Erdman), Department of Chemistry (Knox, Chan), Department of Food Science and Human Nutrition (Erdman), University of Illinois at Urbana-Champaign, Urbana, IL

Hypoxia is a pathological hallmark of many cancers, and increased hypoxia has been associated with increased tumor aggressiveness and treatment resistance in prostate cancer. The highest standard hypoxia measurements are acquired through an invasive procedure that places oxygen sensitive electrodes directly into the tissue of interest. While less invasive, fluorescent probes cannot measure hypoxia in tissue deeper than 1mm. Hypoxia probe 1 (HyP-1) was developed to address the limitations of available hypoxia detection methods and successfully measures hypoxia when locally injected into tumor tissue. The aim of this study was to determine if HyP-1 is taken up by prostate tumors following systemic injection and whether probe reactivity is indicative of a low oxygen tumor environment. Transgenic mice (TRAMP; n=4) were injected with varying doses of HyP-1 retro-orbitally, and probe uptake and activity were measured via IVIS fluorescent imaging. Average HyP-1 activity was determined within a standardized region of interest (ROI) and normalized to tumor volume to assess changes over time. Tumor volume was assessed prior to IVIS imaging via VisualSonics 3D ultrasound measurement. Results confirmed intratumoral HyP-1 uptake and showed that as tumor volume increased, ratiometric fluorescence of HyP-1 within the ROI increased, suggesting increasing hypoxia of the tumor tissue. Results of HyP-1 reactivity will be compared against intratumoral protein expression of the low oxygen responsive transcription factor, hypoxia inducible factor-1α (HIF-1α). Taken together, these data will assess the feasibility of a systemic injection of HyP-1 as a reliable, non-invasive marker for tumor hypoxia.

Research Grant: USDA NIFA ILLU-971-334; NIH T32EB019944; NIH R37EB002641

Student Support: Office of the Director, NIH, T35 OD011145

Evaluation of subcutaneous estradiol benzoate capsule implantation to sterilize male hamsters

Monika A. Liszka, Po-Ching Lin, Shah Tauseef Bashir, Radwa Barakat, Sherry Qi Zhou, and CheMyong J. Ko

Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

There are approximately 1.1 million pet hamsters in the U.S. Many hamster owners report unwanted, accidental litters creating a need for effective sterilization methods. Supraphysiologic levels of estradiol interfere with germ cell development in males. This study tests the hypothesis that sustained high levels of estradiol in neonatal hamsters inhibit male gonadal development. A silastic capsule containing 3 mg/kg estradiol benzoate (EB) or sesame oil (vehicle control) was implanted subcutaneously at postnatal day (PND) 21 in male hamsters (n=5). Blood estradiol levels were monitored on the day of capsule implantation and weekly afterward. Gonadal development was assessed by measuring serum levels of sex steroids and performing gonadal histology. These measurements will be repeated until the hamsters reach the age of sexual maturity when reproductive function will also be evaluated. Initial data from morphological examination of PND30 animals showed reduced testes size in the EB capsule group compared to the control. Inhibition of gonadal development is expected to induce sterility. EB-treated animals are expected to show no or significantly decreased sperm production and lower serum testosterone levels than the controls. EB-treated animals are expected to show low interest in mating and are not expected to produce pups. Development of an easily implantable, effective sterilization method for hamsters would provide an attractive alternative to costly and invasive surgical methods. The implantable capsule is expected to be adapted for use in other species, too.

Research Grant: INSIGNA TTA

Student Support: Office of the Director, NIH, T35 OD011145

Suppression of superoxide sensitivity in Salmonella Typhimurium

Caraline Nack, Rouhallah Ramezanifard, and James Slauch

College of Veterinary Medicine (Nack) and Department of Microbiology (Ramezanifard, Slauch), University of Illinois at Urbana-Champaign, Urbana, IL

Salmonella Typhimurium is a gram-negative bacterium that survives the harsh conditions within macrophages during systemic infection. In this environment, Salmonella is exposed to reactive oxygen species such as superoxide and hydrogen peroxide. A previous genetic screen demonstrated that the ytfLMN locus is involved in resistance to oxidative stress in the macrophage. Mutations in these genes confer sensitivity to high concentrations of hydrogen peroxide in vitro. However, the mechanism of this sensitivity is unknown. To further characterize this phenotype, mutations that suppress the sensitivity to hydrogen peroxide in the ytfL mutant were sought. Exponentially growing ytfL mutants and wild-type cells were exposed to 1.5 mM, 1.75 mM, and 2 mM hydrogen peroxide for 3-4 hours. Dilutions of cells were plated, and the resulting colonies counted. Although results were highly variable, the ytfL mutant was generally several orders of magnitude more sensitive to hydrogen peroxide relative to the wild-type. Occasionally, a ytfL-derived culture showed resistance to hydrogen peroxide, presumably due to suppressor mutations. Seven putative suppressor strains were isolated; at least two of them exhibited increased resistance compared to their ytfL parent. Suppressor mutations will be identified by sequencing chromosomal DNA from these strains. The identity of these genes will provide a greater understanding of molecular mechanisms of Salmonella sensitivity to reactive oxygen species.

Research Grant: NIH R01 AI123381

Student Support: Office of the Director, NIH, T35 OD011145

Effects of oral tributyrin and g-cyclodextrin supplementation on swine ascending colon microbiota composition

Claudia Perkowski, Marcia H. Monaco, Mei Wang, and Sharon M. Donovan

College of Veterinary Medicine (Perkowski), Department of Food Science and Human Nutrition (Monaco, Wang, Donovan), University of Illinois at Urbana-Champaign, Urbana, IL

Some gastrointestinal bacteria produce butyrate, a short chain fatty acid (SCFA) recognized for its anti-inflammatory effects. Despite its ability to reduce inflammation, oral butyrate supplementation poses a challenge to administer due to its noxious odor and taste. Encapsulating butyrate as tributyrin (TB) with g-cyclodextrin (g-CD) not only improves palatability, but g-CD can also function as a prebiotic. The aim of this study was to assess the effect of two concentrations of TB/g-CD or g-CD on seven species of bacteria in the ascending colon (AC) of piglets. Piglets (2-day-old; n=40) were randomized into 5 treatment groups: control formula (CON); formula+4.15 g/L TB/g-CD (CDTBL); formula+8.3 g/L TB/g-CD (CDTBH); formula+3.69 g/L g-CD (CDL); formula+7.39 g/L g-CD (CDH). On Day 21 AC contents were collected and extracted for DNA. The abundance (Log10 number of 16s rRNA gene copies/gram of content) of total bacteria, Prevotella, Roseburia, Faecalibacterium prausnitzii, Clostridium perfringens, Lactobacillus, and Bifidobacterium were quantified via Quant Studio 6 and 7 Real-time qPCR using SYBR Green assays. Data were analyzed by one-way ANOVA via the MIXED procedure with Tukey-Kramer adjustments for diet effects. Data followed a normal distribution and no significant changes were seen among the bacterial populations in response to the treatments. Supplementation with TB and g-CD was well tolerated by the piglets, but did not affect the abundance of the specific bacterial taxa assessed. Ongoing investigations using next-generation sequencing of the V3-V4 region of the 16S rDNA gene will determine whether differences in diversity or the abundance of other bacterial taxa were changed in response to TB and g-CD.

Research Grant: NIFA, USDA 2017-67017-26519

Student Support: Office of the Director, NIH, T35 OD011145

Impact of in vitro expansion on enrichment of osteoprogenitors from equine periosteum

Daniel Shinsako and Matt Stewart

Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Mesenchymal stem cells (MSC) exist in low numbers in most body tissues. In vitro expansion is routinely used to enrich samples for MSCs, based on their capacity for prolonged replication. Recently, we found that in vivo osteogenic activity of periosteal osteoprogenitor cells (POC) in foals is far greater than in adult horses. However, this difference is lost after in vitro expansion. This study assessed the impact of in vitro expansion on the osteogenic phenotype of POCs and tested the hypothesis that in vitro expansion of isolated periosteal cells enriches for POCs. Periosteum was collected from five foals and five adult horses. A sample was frozen in liquid nitrogen and the remainder was digested in collagenase for 4 hours. Isolated periosteal cells were seeded in DMEM/10% FBS, expanded to near confluence and passaged twice. Samples were collected of the original periosteum, after initial isolation and at each passage for RNA isolation. Changes in Runx2 and Osterix (OSX) levels, both mandatory for the osteogenesis, were assessed by RT-qPCR. Both Runx 2 (3-fold) and OSX (6-fold) levels were higher in foal periosteum than in adult tissue. During in vitro expansion, Runx2 levels only modestly changed in both foal and adult cultures. In contrast, OSX expression fell significantly and dramatically in both age groups. These results emphatically disprove our hypothesis. In vitro expansion does not enrich for active POCs. Outer zone fibroblastic periosteal cells might dominate during in vitro proliferation, preventing POC enrichment. Alternatively, in vitro conditions might lack critical factors required for OSX expression in these cells. Further work is required to clarify this issue.

Research Grant: USDA Hatch Funds

Student Support: Office of the Director, NIH, T35 OD011145

What the FOXA2?!—protein degradation causes mucus hypersecretion in canine and feline fungal pneumonia

Aaron Sieve, Woosuk Choi, Alina Xiaoyu Yang, Miranda Vieson, Som Nanjappa, Carol W. Maddox, and Gee W. Lau

Department of Pathobiology (Sieve, Choi, Yang, Nanjappa, Maddox, Lau), Veterinary Diagnostic Laboratory (Vieson, Maddox), and Department of Veterinary Clinical Medicine (Vieson), College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Pulmonary mycoses are more difficult to treat than bacterial pneumonia and cause similar detrimental effects to the patient’s lungs. Microbial infections modulate lung immune response, inducing goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion in the airways. Mucus overproduction clogs small airways and reduces pulmonary function by decreasing oxygen exchange in the lung, leading to respiratory distress. Forkhead box protein A2 (FOXA2) is a transcription factor that regulates mucus homeostasis in the airways. Previous studies in bacteria-infected human and canine lungs showed that FOXA2 depletion in airway epithelial cells causes mucus hypersecretion. However, there are no published studies investigating whether pulmonary mycosis modulates the expression of FOXA2 resulting in excessive mucus in either canine or feline lungs. In this study, we used a combination of immunohistochemical staining of clinical canine and feline fungal pneumonia cases from the University of Illinois Veterinary Diagnostic Lab and molecular analysis of Blastomyces dermatidis-infected immortalized canine bronchoalveolar cancer cell line (BACA) to study the relationships between fungal infection, FOXA2, and mucin expression. The results indicated that fungal-infected canine and feline lung downregulates FOXA2 expression, resulting in overexpression of MUC5AC and MUC5B mucins. Inhibition of FOXA2 was mediated through the activation of the Dectin-ROS-EGFR-AKT/ERK1/2 pathway. Further understanding the role of FOXA2 in mucus hypersecretion may lead to novel therapeutics to aid treatment of fungal pulmonary infections for human and veterinary patients.

Research Grant: University of Illinois College of Veterinary Medicine Companion Animal Research Grant Program (James R. Harkness Fund), R01 AI080710-06A1, and R01 HL142626-01A1

Student Support: Office of the Director, NIH, T35 OD011145

2018 Research Project Abstracts

Differentiation of early and late lytic gene expression using a Marek’s disease alphaherpesvirus model
Michael R. Brooks, Yung-Tien Tien, Keith W. Jarosinski
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

The Herpesviridae family of viruses establishes a latent infection in hosts that can last for life, reactivating to produce infectious virions. Little is known about the genes involved in latency and reactivation. We are addressing these questions using Marek’s disease virus (MDV), an alphaherpesvirus that affects poultry. Lymphocytes are targeted for latent infection and can be transformed to tumors. We constructed a recombinant MDV (v2001) that expresses monomeric red fluorescent protein (mRFP) inserted in frame with the early lytic RLORF4 gene (RLORF4mRFP) and enhanced green fluorescent protein (eGFP) inserted in frame with the late lytic UL47 gene (UL47eGFP). We hypothesize that viruses in the early lytic phase express only RLORF4mRFP and viruses in the late lytic phase express both RLORF4mRFP and UL47eGFP. Our goal is to develop transformed cell lines in which we can separate cells at early and late phases of lytic replication following reactivation. This approach will allow us to identify viral genes important for reactivation. Seven-day-old chickens (n=11) were infected with v2001. At 7, 14, and 21 days post-infection, the spleen, bursa of Fabricius, and thymus were collected from infected chickens and used to prepare cell suspensions of infected lymphocytes. Flow cytometry was used to detect viral antigens and host surface markers. B and T lymphocytes could be differentiated with this technique, but infection could not be verified. We are also developing ex vivo cell lines from tumors induced by v2001. The long-term goal of this project is to identify proteins important for reactivating herpesviruses from latently infected lymphocytes that could be used in the development of drug or antibody targets.

Research Grant: Agriculture and Food Research Initiative Competitive Grant 2013-67015-26787, USDA National Institute of Food and Agriculture
Student Support: Office of the Director, NIH, T35 OD011145

Adverse azole antifungal effects: a retrospective study of fluconazole, itraconazole, ketoconazole
Cora Kubra Catak, Jennifer M. Reinhart
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Fluconazole, itraconazole, and ketoconazole are the most-common medications prescribed for canine fungal diseases, such as blastomycosis, aspergillosis, and dermatophytosis. These medications may have adverse effects with long-term usage. The most-common, serious adverse effect is hepatotoxicosis, usually characterized by an increase in alanine transaminase activity (ALT). The goal of this study is to use retrospective analysis of University of Illinois Veterinary Teaching Hospital records to identify factors that predispose dogs to azole-induced hepatotoxicity. Records were included for dogs i) at least 6 months of age receiving systemic fluconazole, itraconazole, or ketoconazole, ii) with at least 3 months of therapy, and iii) monitoring with serial biochemistry profiles. Exclusion criteria included i) systemic illness other than fungal infection, ii) medications known to increase liver enzyme activity, iii) steroid use at the initiation of antifungal therapy, and iv). steroid use for > 14 days at or after antifungal therapy initiation. Hepatotoxicity was defined as an ALT increase above the reference range. Medical records will be separated into groups according to whether or not the patient developed hepatotoxicity. Possible predisposing factors will be compared between groups. Fisher’s exact test or Chi Square test will be used for non-continuous data, such as type of drug. Depending on normality, a Student’s t test or Mann-Whitney U test will be used for continuous data including drug dose and patient age. Identifying predisposing factors for azole-induced hepatotoxicity will help clinicians choose antifungal medications for individual patients with the goal of reducing the incidence of adverse drug reactions.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Can IS711 be used as a surrogate for MLST genotyping of Brucella ceti in wild cetaceans?
Allison Dianis, Jeffrey Curtiss, Kathleen Colegrove, Marine Mammal Stranding Network, Karen Terio
Zoological Pathology Program, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL (Dianis, Curtiss, Colegrove, Terio) and Marine Mammal Health and Stranding Response Program, National Marine Fisheries Service, Silver Spring, MD (Marine Mammal Stranding Network)

Brucella ceti, identified in dolphins, whales, and porpoises beginning in the 1990s, has 3 main sequence types (ST): ST23, ST26, and ST27, which vary by species and geographic region. B. ceti produces a range of pathologic lesions and can induce abortions, negatively affecting already-declining wild cetacean populations. Additionally, as the human-cetacean interface increases, so does the need for a rapid genotyping assay to determine the zoonotic risk to exposed individuals, as at least ST27 has been identified as zoonotic. Although the ST of B. ceti isolates can be differentiated using the nine-gene multilocus sequence typing (9-MLST), the method is cumbersome for diagnostic testing. An insertion sequence called IS711 can be used to differentiate between STs of many Brucella species, but it is unknown if it can differentiate among B. ceti STs. Here, we investigate the use of IS711 genotyping for differentiation of ST23, ST26, and ST27 in B. ceti isolates. We hypothesize that IS711 genotype will be predictive of 9-MLST genotype, allowing for the identification of B. ceti type by a single PCR. Both the IS711 and 9-MLST methods were used to genotype B. ceti isolates (n=56) that were collected from cetacean tissues on necropsy. 9-MLST genotyping identified the isolates as ST23 (n=3), ST26 (n=25) and ST27 (n=28). Preliminary comparisons indicate that the IS711 genotyping may provide a more-rapid method for distinguishing among B. ceti STs of pathogenic importance.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: University of Illinois College of Veterinary Medicine

Variation in cytosine arabinoside protocols based on dosage and route of administration in canine patients
Mary Kate E. Feldner, Kari D. Foss, Devon W. Hague, David J. Schaeffer
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Cytosine arabinoside (CA) is one of many chemotherapeutics used as an immunosuppressant or anti-neoplastic agent to treat a variety of diseases in veterinary medicine. It is particularly useful in the treatment of diseases of the central nervous system due to its ability to cross the blood brain barrier. However, given its side effects (myelosuppression, GI disturbance, hepatotoxicity), it is critical to monitor patients that receive CA. The goal of this study is to collect and compare data about CA treatment protocols in veterinary medicine. Delegates of the American College of Veterinary Internal Medicine were offered a 26-question survey via email about the administration, monitoring, and side effects in dogs being treated with CA. The University of Illinois Web Services Toolbox was used to collect and collate responses. Survey data were tabulated for each response and for selected cross-tabulations. Compiled survey data were compared statistically to similar data extracted from records of dogs treated with CA at the University of Illinois Veterinary Teaching Hospital. Results of this study will provide insight into best practices for use of CA in veterinary medicine.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Essentiality and functional analysis of Calcium Dependent Protein Kinase 1 (CDPK1) in Cryptosporidium parvum
Brina Gartlan, Sumit Rathore, Savannah Rose, Rachel Han, Sumiti Vinayak
Department of Pathobiology, College of Veterinary Medicine (Gartlan, Rathore, Vinayak) and School of Molecular and Cellular Biology (Rose, Han), University of Illinois at Urbana-Champaign, Urbana, IL

Cryptosporidium parvum, a protozoan parasite, is a leading cause of diarrheal disease in young children and agricultural animals. There is no effective drug to treat cryptosporidiosis, which makes C. parvum highly relevant to veterinary medicine and research. The development of novel therapeutics against C. parvum has been challenging since there is limited understanding of the parasite biology. With new molecular genetic tools now available for C. parvum, it is possible to dissect gene function. One of the leading drug targets in C. parvum is the Calcium Dependent Protein Kinase-1 (CDPK1). The ortholog of CpCDPK1 in other protozoan parasites is essential for parasite growth. However, there is no information available regarding the essentiality of CDPK1 for C. parvum. To determine essentiality of CDPK1, we used the genome-editing CRISPR/Cas9 tool and immunocompromised mouse model system to create a transgenic CDPK1 knockout strain. To create this strain, we attempted to delete the cdpk1 gene and replace it with a sensitive luciferase reporter fused to a drug-resistance marker. Fecal luminescence measurements were performed to monitor the emergence of drug-resistant parasites. When compared to our positive control, there was no rise in luciferase readings for the CDPK1 knockout strain, indicating that the CDPK1 was essential for C. parvum. We are repeating our experiments to confirm our results. We also created another transgenic strain to append an epitope tag to the cdpk1 gene. Efforts are underway to purify these tagged transgenic oocysts and determine the localization of CDPK1 in the parasite. Discovery and validation of new drug targets will aid in development of effective treatments against cryptosporidiosis.

Research Grant: Bill and Melinda Gates Foundation, Grand Challenges Exploration award number OPP1171934 & University of Illinois College of Veterinary Medicine
Student Support: Office of the Director, NIH, T35 OD011145

Effect of dose rate on expression of immunologic mediators in irradiated tumor cells
Jordan Hampel, Bahaa Fadl-Alla, Kim A. Selting
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Radiation therapy can elicit a systemic immune response against tumor cells and improve the effectiveness of cancer treatment. Extended radiation treatment times at low dose rates can allow for repair of DNA damage and we hypothesized that this could impact the interaction between the cancer cell and the immune system. The goal of this study was to compare the effect of different radiation dose rates on two cell lines representing tumor types that have been treated with immunotherapy in dogs: osteosarcoma (OSA) and melanoma. Cells (400,000/well in 6-well plates) were irradiated with 8 Gy using a Co-60 at 25 cGy/min or a Linear Accelerator (Linac) at 20, 400, or 1000 cGy/min, then harvested at 2 h and 24 h. RT-PCR was used to quantify changes in gene expression of immunologic endpoints including MHC-I, Fas-L, PD-L1, and HMGB-1. Comparisons to untreated control at 24 h post treatment showed the greatest differences. Low-dose-rate treatment had similar results of gene downregulation, except for MHC-I where Co-60 showed upregulation. Inhibition of PD-L1 is a popular strategy for immunotherapy, however, our results revealed no consistent pattern in its expression and thus an insensitivity to dose-rate. Unexpectedly, HMGB-1 (marker of DNA damage) appeared to be downregulated at the 24-hour time point. The most remarkable finding was Fas-L, with a 75-fold increase in the OSA cell line at the highest dose rate and over a 3-fold increase in the melanoma cell line at the standard dose rate (400 cGy/min). Expression of genes related to the response of canine melanoma and OSA cells to the immune system can vary with dose rate of radiation therapy. These findings could impact the use of radioimmunotherapy in cancer treatment.

Grant support: College of Veterinary Medicine
Student support: Office of the Director, NIH, T35 OD011145

Hypoglycemia in dogs after ingestion of metformin: a retrospective evaluation (2001 – 2017)
Jessica Kerr, Charlotte Means
College of Veterinary Medicine, University of Illinois at Urbana-Champaign (Kerr) and American Society for the Prevention of Cruelty to Animals Animal Poison Control Center (Means), Urbana, IL

Metformin is a biguanide oral antihyperglycemic agent used in the treatment of type 2 diabetes. Metformin intoxication can occur in pet dogs that ingest the prescription drug that is intended for human use. Most cases will resolve with fluid therapy and the prescription of gastrointestinal (GI) protectants. The Animal Poison Control Center (APCC) is a 24-hour consultation service that provides support to poisoned animals through advising both animal owners and licensed veterinarians across North America and collecting case information to be stored in the AnTox database. In this study, the AnTox database was searched for calls regarding metformin monointoxications in dogs to specifically search for common characteristics in previous cases that led to the development of hypoglycemia. Between the years 2001 and 2017, the APCC received 1,833 calls regarding metformin monointoxication and 4,281 regarding multiple-point intoxications with metformin. Twenty-four dogs ingested only metformin and developed hypoglycemia. Doses ranged from 29.76 mg/kg to 859.80 mg/kg, with a mean of 259.02 mg/kg. The other 1,809 dogs developed signs of GI upset, lactic acidosis, or no clinical signs. In these cases, the average metformin dose was 202.67 mg/kg, with a range of 1 to 3,000 mg/kg. Dogs that developed metformin-induced hypoglycemia were generally young, smaller breeds (0.2 – 2 years of age). There did not appear to be a link between metformin dosage and the development of hypoglycemia. Identifying the variables associated with metformin-induced hypoglycemia would improve monitoring of cases. This information is important because severe hypoglycemia can lead to a worse prognosis and more lethal sequelae, such as seizure and death.

Research Grant: None
Student Support: University of Illinois College of Veterinary Medicine

Predicting West Nile: a spatial analysis of human activity and environmental factors in Cook County, Illinois
Bennett Lamczyk, Patrick Irwin, Karki Surendra, John Uelmen, Marilyn Ruiz O’Hara
Department of Pathobiology (Lamczyk, Surendra, Uelmen, O’Hara), College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL; Northwest Mosquito Abatement District (Irwin), Wheeling, IL

In 2002, northeast Illinois experienced a large outbreak of West Nile virus (WNV) with higher-than-average human caseloads. WNV continues to be a significant human infectious disease in the area. The WNV transmission cycle is dependent on a mosquito vector, Culex sp., which feeds on birds preferentially over mammals, and is most active during the dusk hours. Using multisource data, a model was developed to predict human cases within 1000-meter hexagon sites in the Cook and DuPage counties of northeastern Illinois. This model ran its predictions from the years 2005 to 2016 while creating residual values to describe prediction accuracy against recorded human cases. In this study, 30 hexagon sites within the Northwest Mosquito Abatement District of Cook County, with varying residual values, were selected for human observation and interview. These data were collected to determine differences in outdoor activity between sites during the hours from 5 pm to 9 pm which may account for differences between predicted and observed human WNV illness. Mosquito infection rate, catch basin location, open water bodies, and percent forest data will be evaluated, as well, to further describe differences between sites. Results from this study may allow researchers to further increase accuracy of the prediction model and improve public health in the region. With over 46,086 cases and 2,017 deaths from WNV in the U.S. since its introduction in 1999, there is an urgent need for improved preventative practices and tools.

Research Grant: Centers for Disease Control and Prevention
Student Support: Office of the Director, NIH, T35 OD011145

A retrospective study of prognostic indicators for survival in eastern grey squirrels (Sciurus carolinensis)
Ivana H. Levy, Matthew C. Allender, Stephanie Principati, Julia Whittington, Sarah Reich, Krista A. Keller
Wildlife Epidemiology Laboratory (Levy, Allender, Keller), Wildlife Medical Clinic (Whittington, Reich), Department of Veterinary Clinical Medicine (Allender, Principati, Whittington, Reich, Keller), College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

The eastern grey squirrel (EGS), Sciurus carolinensis, is a tree squirrel native to the eastern United States. This species commonly presents to wildlife medical clinics for a variety of human-related injuries including road traffic and pet predation. The purpose of this study was to determine prognostic indicators for survival in young or orphaned EGS. Retrospective data were collected from January 1, 2012 to June 12, 2018 for all EGS weighing less than 300 g that presented to the Wildlife Medical Clinic at the University of Illinois Veterinary Teaching Hospital. Information regarding each animal’s predefined weight class, month of the year the animal presented, overall health status (sick or healthy), presence of diarrhea, necessity for assisted feeding, and presence of respiratory, integumentary, and neurologic signs was collected. Outcome factors (survivor or non-survivor) were modeled using a logistic regression model and all single factors were considered individually. A total of 872 EGS were included in this study. Factors that significantly predicted a non-survivor status included EGS that presented to the clinic in any type of diseased state (OR 4.8, p < 0.0001), specifically those with respiratory signs (OR 1.54, p < 0.0001), or neurologic signs (OR 2.85, p < 0.001). The month the animal presented was also significant because EGS that presented during the second quarter were more likely to be non-survivors (OR 1.54, p = 0.019). These findings can be used by wildlife clinicians to identify EGS with negative prognostic indicators, thereby informing treatment decisions. Further research will reveal how these negative indicators are associated with EGS non-survivor status.

Research Grant: None
Student Support: University of Illinois College of Veterinary Medicine

Comparison of axillary and inguinal temperature to rectal temperature in healthy guinea pigs (Cavia porcellus)
Ivana H. Levy, Matthew C. Allender, Caitlin M. Hemby, Krista A. Keller
Wildlife Epidemiology Laboratory (Levy, Allender, Hemby, Keller), Department of Veterinary Clinical Medicine (Allender, Keller), College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Core body temperature is an essential health parameter. Temperature aberrations can indicate certain disorders, influence clinical management decisions, and serve as a prognostic indicator for patient recovery. Historically, rectal temperature measurements have been utilized in companion animals, however there is growing interest in less-invasive methods including auricular and axillary measurements to determine core body temperature. In this study, triplicate temperature measurements (axillary (AT), inguinal (IT), and rectal (RT)) were performed in 19 healthy guinea pigs (7 male, 12 female). Paired sample t-tests were utilized to evaluate differences in the mean body temperature measurements between each body site. There was no agreement in body temperature measurements between the body sites. The IT (36.3 C – 39.7 C) was significantly lower than both AT (36.8 C – 39.9 C; t = -3.401, p = 0.003) and RT (38.2 C – 40 C; t = -5.068, p < 0.0001), and AT was significantly lower than RT (t = -3.485, p = 0.003). These results indicate that AT, IT, and RT cannot be used interchangeably to represent core body temperature. However, these data provide a basis for clinicians to infer RT from AT and IT (RT = IT + 0.83 C, RT = AT + 0.44 C). Additional investigation is planned to assess these temperature relationships in animals suffering from disease.

Research Grant: None
Student Support: University of Illinois College of Veterinary Medicine

The effects of acute exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate on hormone levels in mice
Lily Lewis, Catheryne Chiang, Liying Gao, Jodi A. Flaws
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL

Di(2-ethylhexyl) phthalate (DEHP) is a synthetic chemical used ubiquitously in the environment in a variety of plastics, medical equipment, and children’s toys. Individuals can absorb DEHP via dermal contact, inhalation, and ingestion. Previous studies show that DEHP affects fertility and causes endocrine disruption in both males and females, however, most of these studies focused on males. This information led to a shift to replace DEHP with other plasticizers. One such replacement is diisononyl phthalate (DiNP). Unfortunately, little is known about the reproductive effects of DiNP. Thus, the purpose of this study was to fill the knowledge gaps of how DEHP and DiNP affect female reproduction, and to test the hypothesis that acute exposure to DEHP or DiNP has negative, long-term effects on female reproductive health. Adult female CD-1 mice were orally dosed with either vehicle control (corn oil), DEHP (20 or 200 µg/kg/day, or 20 or 200 mg/kg/day), or DiNP (20 or 100 µg/kg/day, or 20 or 200 mg/kg/day) for 10 days. Mice were euthanized three months post-dosing and blood was collected to measure sex steroid hormone levels via enzyme-linked immunosorbent assays. Treatment with 100 µg/kg DiNP significantly decreased progesterone and testosterone levels compared to controls (n=5-6; p≤0.05). DiNP also moderately decreased progesterone (20 µg/kg, p=0.082) and increased estradiol (200 mg/kg, p=0.083) levels compared to controls. DEHP exposure did not significantly affect hormone levels compared to controls at any time. In conclusion, these data show that DEHP does not alter hormone levels significantly and that DiNP, a common replacement for DEHP, alters hormone levels in mice three months after exposure has stopped.

Research Grant: NIH R56 ES025147
Student Support: Office of the Director, NIH, T35 OD011145

Genomics of methicillin-resistant Staphylococcus pseudintermedius
Lyan Padilla Velez, Tarannum A. Khan, Jason B. Pieper, Carol W. Maddox, Md Tauqeer Alam
Department of Pathobiology (Padilla Velez, Khan, Maddox, Alam) and Department of Veterinary Clinical Medicine (Pieper, Alam), College of Veterinary Medicine, University of Illinois at Urbana-Champaign

The gram-positive bacterium, Staphylococcus pseudintermedius, is a major cause of canine skin and soft tissue infections (SSTIs), pyoderma, ear infections, otitis externa and urinary tract infections (UTIs). Because methicillin and multidrug-resistant S. pseudintermedius cases are becoming more frequent, it is important to understand the evolution and mechanism of acquired antibiotic resistance. This project utilizes whole-genome sequencing to identify genetic changes associated with antibacterial resistance. S. pseudintermedius genome sequences from public databases will be analyzed using a pipeline established in our laboratory. The multilocus sequence type (MLST), phenotype profile, antibiotic resistant genes, and virulence genes of each strain will be identified. We expect that an isolate exhibiting elevated minimum inhibitory concentration (MIC) against an antibiotic will have a corresponding mutation or gene associated with phenotypic resistance. The strains with high MICs to multiple antibiotics are expected to bear a greater number of resistance mutations. In addition to known mutations, we expect to discover some novel mutations that may be associated with high MIC and multidrug resistance. Information from this study will provide insight for better management and control of the pathogen, and future development of treatment options.

Research Grant: College of Veterinary Medicine
Student Support: Office of the Director, NIH, T35 OD011145

Ticking all the boxes: bureaucracy, research, and reality in reports of tick-borne diseases
Melanie Repella, Lee Ann Lyons, William Brown, Marilyn O’Hara Ruiz
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

To address the increasing public-health concern about tick-borne diseases it is necessary to know where people are exposed to ticks. This study addresses the efficiency of the disease-reporting process and comparisons between five states: Illinois, Iowa, Michigan, Minnesota, and Wisconsin. Data were collected from discussions with public-health officials and by assessing each state’s reporting documents. Currently, each state uses a different methodology to report tick-borne diseases, with particular differences in how geographic exposure location and travel data are collected. One important variation between states was the number of follow-up reports to gather additional information about the illness from either the patient or healthcare provider. This value also varied by disease. Another discrepancy between states was who does the follow-up, varying from public health officials to a team of student employees. Additionally, some states follow up cases with the provider and others follow up with the patient. This process results in varied detail regarding information where the tick may have been acquired. Data for Wisconsin from 2007 to 2016 revealed that at least half of the reported cases of Lyme disease every year had no information about the location the tick was encountered. Overall, these disparate results reveal a need for improved surveillance methods of tick-borne diseases in the Midwest. Increased funding and manpower, a universal form for each disease, and an equal emphasis on all tick-borne diseases would improve information collected in the future and provide increased information about tick exposure. Understanding where people are exposed can allow for improved public health knowledge and control efforts.

Research Grant: Centers for Disease Control (Midwest Center for Vector-Borne Diseases)
Student Support: Office of the Director, NIH, T35 OD011145

K9 officer down: lifesaving field training for working-dog handlers
Danielle Schneider, Ashley Mitek, David Schaeffer, Maureen McMichael
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

With the increase in K9 officers, and state legislatures proposing to exempt responders providing emergency care to working animals from the Veterinary Practice Act, veterinary educators need to be ready to train first responders. Veterinarians, emergency medical service paraprofessionals, medical doctors, and law-enforcement officers evaluated the most common emergency conditions that affect working dogs in the field and created a hands-on training program for K9 handlers. The training program focused on recognition and care of common medical conditions in working dogs including gastric dilatation and volvulus, heat stroke, blunt force trauma-pneumothorax, CPR, vital signs and oxygen supplementation, fracture stabilization, wound care, opioid overdose, toxicity, dental trauma/care, and ophthalmic injury. Thirty-six K9 handlers from the Illinois State Police and local municipalities completed a pre-training survey that was followed by hands-on training. Before training, approximately 70% of handlers were least comfortable with gastric dilatation and volvulus, administering apomorphine, recognizing signs of pneumothorax, and knowing how to place a chest seal. A similarly worded post-training survey indicated that 100% of respondents were somewhat or very comfortable recognizing clinical signs and providing care for the covered conditions. This study demonstrates that K9 handlers benefit from hands-on education in providing emergency care. This study is a template for training all first responders in states that allow, or propose to allow, emergency care for K9 officers. Veterinarians should initiate collaboration with K9 handlers and provide the medical information needed to save lives.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Phytocannabinoid metabolism by canine cytochrome P450s
Ashley Sommerkamp, Austin Weigle, Aditi Das
Department of Comparative Biosciences (Sommerkamp, Das), Department of Chemistry (Weigle), University of Illinois at Urbana-Champaign, Urbana, IL

Phytocannabinoids (pCBs) are natural compounds found in Cannabis plants (marijuana). Recent popularity of pCB use in human and veterinary medicine, combined with an insufficient understanding of pCB interactions on animal homeostasis, warrants investigation of pCB metabolism and pharmacokinetics. Cytochrome P450s (CYPs) are a class of enzymes involved in phase 1 drug metabolism playing a significant role in pCB biotransformation. In this study, metabolism of selected pCBs by canine CYPs 3A12 and 3A26 enzymes was explored and compared to metabolite formation by the human ortholog CYP3A4. We focused on two medically relevant pCBs found in Cannabis: ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD). These enzymes may produce similar metabolites due to conserved substrate and protein structures. To analyze human metabolism, we recombinantly expressed and purified CYP3A4 and its redox partner, cytochrome P450 reductase (CPR) in E. coli. Due to differences in expression between CYPs 3A12 and 3A26 in canine liver and peripheral tissues, native canine liver microsomes (containing both enzymes) and CYP3A12-specific bactosomes were obtained. Experiments will involve incubation of rCYP3A4, canine liver microsomes and bactosomal CYP3A12, with ∆9-THC and CBD. Resulting metabolites will be extracted prior to high performance liquid chromatography-mass spectrometry (HPLC-MS), so that fragmentation analysis can enable structure identification. Since dogs are commonly used in drug trials and pCBs are becoming increasingly popular in veterinary medicine, the comparison and discovery of human-versus-canine-derived pCB metabolites will create a foundation for future studies supporting Cannabis-based therapy.

Research Grant: American Heart Association Scientist Development Grant 15SDG25760064 (A.D.), National Institutes of Health (NIH) Grant R01 GM1155884 (A.D.), and R03 DA042365 (A.D.)
Student Support: Office of the Director, NIH, T35 OD011145

Effects of Nerve Growth Factor-β on ovulatory cascade factors from follicular fluid of pre-ovulatory follicles
Stephanie Stella, Jamie Stewart, Fabio Lima
Department of Veterinary Clinical Medicine (Stella, Stewart, Lima), and Department of Comparative Biosciences (Stewart, Lima), College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Nerve growth factor-β (NGF) is a seminal plasma protein that induces ovulation in camelids. Although NGF is not responsible for ovulation in the bovine species, previous results revealed that it enhances luteal development and markers of maternal recognition of pregnancy. There is, therefore, a critical need to understand what components of the ovulatory cascade are affected by NGF. We hypothesized that administration of NGF to Holstein heifers stimulates ovulatory cascade factors that can help understand the mechanism of action of NGF on pre-ovulatory follicles. Virgin Holstein heifers (n = 24) had their estrous cycle synchronized using a 5-day CIDR Synch program. On day 8 of the cycle, heifers with pre-ovulatory follicle (> 12 mm) and no corpus luteum noted by ultrasonography were deemed eligible for the study. Eligible heifers (n = 17) were randomly assigned to one of two treatments: CON (n = 7), intramuscular injection of 12 mL phosphate buffer solution (PBS, pH=7.4), or NGF (n = 10), intramuscular injection of 296 μg of NGF-β diluted in 12 mL of PBS. Follicular fluid samples were obtained from pre-ovulatory dominant follicles 8 hours after treatment via follicular aspiration, added to TRIzol (Invitrogen, Carlsbad, CA) and flash-frozen in liquid nitrogen. Samples were evaluated for gene expression of estrogen receptor 1 (ESR1) and Prostaglandin E Synthase (PGES) using quantitative real-time PCR. Data were analyzed with the GLIMMIX procedure of SAS. While no differences were revealed for ESR1 (P = 0.99) and PGES (P = 0.89), additional genes associated with the ovulatory cascade will be analyzed in the near future to provide a further understanding of the mechanism of action of NGF.

Donor Funding: Lima Reproductive Biology Research Funds
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Increasing the diagnostic utility of canine cerebrospinal fluid

Megan M. Stuart, Devon W. Hague, Kari D. Foss, Peter M. Yau, Anne M. Barger, David J. Schaeffer
Department of Veterinary Clinical Medicine (Stuart, Hague, Foss, Barger, Schaeffer), Roy J. Carver Biotechnology Center (Yau), University of Illinois at Urbana-Champaign, Urbana, IL

Cerebrospinal fluid (CSF) is used to characterize neurologic disease. Increased CSF protein concentration (PC) can be due to changes in permeability of the blood brain barrier secondary to a variety of infectious, inflammatory, compressive, or neoplastic processes. While this change indicates pathology, it is not specific for etiology. We are conducting several lines of investigation to increase the diagnostic utility of CSF. One aim evaluates the relationship between specific disease processes and CSF-PC. Medical records for dogs that underwent CSF analysis at the University of Illinois Veterinary Teaching Hospital (UI-VTH) were examined retrospectively. Information gathered included the CSF-PC (mg/dl), site of collection (cisternal or lumbar), and diagnosis (presumptive or confirmed). Data analysis will evaluate the relationship between specific disease processes, CSF-PC, and site of collection. Prospectively, CSF was collected from UI-VTH clinical patients. CSF-PC measurements made with a urine dipstick will be compared to data from a Beckman Coulter AU Analyzer to determine whether the dipstick can provide quick, reliable readings of PC. Liquid chromatography/mass spectroscopy (LC/MS) will identify proteins in canine CSF. Discriminant analysis will identify proteins that are associated with specific disease processes. This combination of approaches may expand the diagnostic information that can be derived from canine CSF and aid in neurodiagnostics and treatment of canine patients.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Effect of cocoa flavanols on endothelial nitric oxide synthase (eNOS) expression in brains of aged mice
Allison M. Tomasino, Anastassia Sorokina, Justin S. Rhodes, Catarina Rendeiro
Department of Psychology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL (Tomasino, Sorokina, Rhodes) and School of Sport, Exercise & Rehabilitation Sciences, University of Birmingham, Birmingham, UK (Rendeiro)

Cocoa flavanols improve peripheral vascular function in humans, both acutely and chronically, through increases in the bioavailability of nitric oxide (NO) in circulation. Recent work in aging populations suggests that such beneficial effects on the vasculature might extend into the central nervous system (CNS). Our preliminary work showed that feeding a high-cocoa-flavanol diet in aged mice starting at approximately 12 months old for 6 months prevented age-associated declines in blood perfusion throughout the brain, compared to a control (low-cocoa-flavanol) group. Our current study aimed to determine the mechanism for these beneficial effects. We hypothesized that improved blood perfusion in the high-cocoa-flavanol group was due to higher levels of endothelial nitric oxide synthase (eNOS), the enzyme responsible for NO production. Brains from the original study (n=5 cocoa and n=5 control) were sectioned in the coronal plane at 40 microns. A 1-in-6 series (with 240 microns separating adjacent sections) was stained using an anti-eNOS primary antibody with Diaminobenzidine (DAB) as the chromogen. Area fraction covered by eNOS-positive labeling was quantified using stereological methods in the granular and molecular layer of the dentate gyrus and CA1 regions of the hippocampus using Image J software. Results showed similar levels of eNOS expression between control and cocoa treated mice for all 3 brain regions. Taken together, results suggest that increased eNOS expression is an unlikely mechanism for increased blood perfusion in the high-cocoa-flavanol group.

Research Grant: Abbott Nutrition (Center for Nutrition, Learning and Memory at the University of Illinois)
Student Support: Office of the Director, NIH, T35 OD011145

The effect of simvastatin on CD44 shedding in canine osteosarcoma
Christine K. Tran Hoang, Timothy M. Fan
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Osteosarcoma (OS) is the most common primary bone tumor in dogs. Despite multimodality therapy with surgery and chemotherapy, 90% of dogs develop distant metastases and die from disease progression. Thus, there is a need to identify novel therapeutics that slow metastatic progression and increase survival time. Simvastatin belongs to a drug class called statins and is used primarily for controlling hypercholesterolemia in people. Some studies suggest that statins also exert anticancer effects. These effects may be mediated through shedding of CD44, a transmembrane receptor that normally mediates cell adhesion by interacting with hyaluronan and also contributes to tumor progression and metastasis. Simvastatin stimulates CD44 shedding in human cancers and decreases cell migration, but this effect has not yet been evaluated in veterinary oncology. We hypothesize that simvastatin stimulates CD44 shedding in canine immortalized OS cells. Following simvastatin treatment for 48 hours at concentrations of 100 nM, 1 uM, and 10 uM, flow cytometry was used to assess CD44 expression on the canine OS cells. Exposure to simvastatin decreased median fluorescent intensity of detectable CD44 in treated cells compared to untreated controls. ELISA will be used to quantify the amount of CD44 that is shed, and a transwell assay will be used to assess the effect of simvastatin on cell migration. In addition, an outcome-linked tissue microarray will be used to evaluate the relationship between basal CD44 expression and patient clinical outcomes. Demonstrating that simvastatin induces CD44 shedding would make it a promising candidate for future in vivo studies.

Research Grant: Internal Research Funds
Student Support: Office of the Director, NIH, T35 OD011145

Cloning and expression of feline calicivirus P domain protein
Jack Tribble, Leyi Wang
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Feline Calicivirus (FCV), a single-stranded positive sense RNA virus of the family Caliciviridae, is a highly infectious and genetically diverse virus.  FCV mainly causes respiratory disease in cats. Outbreaks of the related virulent systemic FCV, which causes disease of greater severity, occur throughout the world. Our recent sequencing data indicated that currently circulating FCV strains showed only 80% nucleotide identity to those deposited into GenBank. The high percentage of nucleotide disparity will possibly contribute to false negatives in conventional molecular diagnosis, and vaccination failure in cats. The viral P protein forms a protruding domain of the FCV capsid and is responsible for immune recognition and host receptor interaction. The goal of this project is to clone and express P domain protein of FCV. We produced two different constructs: one with the hinge region that connects the S and P domain of the capsid, and one without the hinge region. The constructs were expressed via the pGEX-4T-1 vector in E. coli strain BL21. Recombinant proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant protein will be purified using chromatographic methods. This work provides the foundation for FCV diagnosis and perhaps disease prevention through vaccination.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Boehringer Ingelheim Veterinary Scholars Program

2017 Research Project Abstracts

A preliminary validation of molecular tools for quantifying biomarkers of respiratory pathology
Amanda S Aker, Mohamed M. Zeineldin, Kayla H. Wade, Elsbeth D. Grimmer, James F. Lowe, Brian M Aldridge
Integrated Food Animal Medicine Systems, Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Respiratory disease is a significant cause of morbidity and economic loss in the cattle industry. Quantifying the nature and severity of respiratory pathology in affected animals is useful in determining prognosis and in selecting optimal therapeutics. The objective of this study was to validate the use of quantitative real-time PCR and Western Blot analysis in measuring gene expression and protein concentration of candidate biomarkers of lung pathology in growing cattle. Bronchoalveolar lavage (BAL) samples were taken from healthy and diseased feed lot cattle and lung tissue samples were taken from healthy slaughterhouse animals immediately postmortem. Protein and RNA were extracted using conventional techniques from the BAL and lung tissue samples respectively. Protein concentrations were evaluated and Western Blot analysis was performed on the BAL samples with the highest protein concentration samples using antibodies against Surfactant protein A, MMP2, and MUC1. Quantitative real time PCR was performed using primers designed for the genes that encode these proteins. Preliminary data suggests that all three proteins are produced in both the healthy and diseased lung. Additional studies will quantify the amount of each protein in the healthy versus diseased lung. It is anticipated that these assays can be used in the diagnosis and treatment of respiratory pathology.

Research Grant: National Institute of Food and Agriculture, U.S. Department of Agriculture
Student Support: University of Illinois College of Veterinary Medicine

Measurement of IL-1 beta in chelonian plasma by ELISA using anti-human IL-1 beta antibodies
Michelle I. Beermann, Matthew C. Allender, Jeremy M. Rayl
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Interleukin-1 beta (IL-1 beta) is a cytokine produced by monocytes and macrophages that contributes to the acute phase response of inflammation. In mammals, IL-1 beta concentrations in the blood are elevated during inflammatory disease states. In comparison, cytokine activity is poorly understood in reptiles and reagents specific for detection of reptile cytokines have not been developed. The enzyme-linked immunosorbent assay (ELISA) uses antibodies to detect and quantify proteins and is used frequently for cytokine measurements. The purpose of this study was to determine if a commercial ELISA kit containing anti-human IL-1 beta antibodies could be used to measure IL-1 beta in chelonian plasma. Plasma from red-eared sliders (Trachemys scripta elegans) that were experimentally infected with ranavirus (frog virus 3) was used as a positive control for cross-reactivity with human antibodies. Plasma from both infected turtles and uninfected turtles had measurable concentrations of IL-1 beta. The presence of IL-1 beta in uninfected turtles may be due to stress associated with shipment of the turtles or the presence of other inflammatory processes. Plasma from wild Eastern box turtles (Terrapene carolina carolina) was also tested for IL-1 beta by ELISA. A better understanding of the activity of cytokines in reptiles can provide insight into the manifestation and course of inflammation in these species. The ability to measure cytokines by ELISA could serve as a valuable tool for wildlife epidemiology by providing a practical and quantitative means of disease surveillance.

Research Grant: Wildlife Epidemiology Laboratory
Student Support: University of Illinois College of Veterinary Medicine

Development of a fecal microbiota transplant preparation protocol for dogs
Tony Bieser, Patrick Barko, David Williams, Maureen McMichael
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Fecal microbiota transplants (FMT) are highly effective in humans for treating recurrent Clostridium difficile, but have mixed efficacy for other gut dysbiosis conditions. Oxygenation during donor sample processing, and its negative impact on anaerobic bacteria, is a likely cause for this observation. Many processing methods for donor stool samples have been reported, but little is known about their effect on bacterial viability. As there are no established protocols for FMT in veterinary medicine, our goal was to analyze the impact of sample processing on fecal bacteria and create a clinically useful FMT processing protocol. We hypothesize that oxygen-exposed FMT samples will have decreased viability of anaerobic bacteria, and that the addition of L-cysteine, a reducing agent, to fecal homogenate solutions will mitigate the deleterious effect of oxygen exposure. Fecal samples from six healthy dogs were collected and homogenized in normal saline with 10% glycerol or 0.1% L-cysteine in normal saline with 10% glycerol. An aliquot of the donor homogenate solution was sparged with air to mimic sample oxygenation during homogenization. An aliquot was also treated with propidium monoazide (PMA) to assess bacterial viability. PMA, a membrane-impermeable DNA binding agent, modifies DNA from dead bacterial cells allowing for sequencing of living cell DNA only. Additional aliquots of each buffer were subjected to several freeze-thaw cycles before the addition of PMA. Sample processing is underway and, when completed, genomic DNA will undergo purification and Illumina sequencing of 16S rDNA. Overall, a novel FMT protocol for veterinarians would enhance the treatment options available for animals with intestinal dysbiosis.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Effect of intracoelomic dexmedetomidine-alfaxalone on righting reflex in garter snakes (Thamnophis sirtalis)
Kelly Chen, Stephanie Keating, Danielle Strahl-Heldreth, Lynelle Graham, Stuart Clark-Price
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Reptiles often require anesthesia for clinical procedures, but little is known about their response to common anesthetic agents. While alfaxalone has been evaluated in snakes, the effects of alfaxalone combined with sedatives such as dexmedetomidine are currently unknown. A randomized, controlled study was used to evaluate the effects of intracoelomic dexmedetomidine-alfaxalone in 8 common garter snakes (Thamnophis sirtalis). Loss and return of righting reflex (LRR and RRR) were examined following the administration of alfaxalone (30 mg/kg) combined with either 0.05 or 0.1 mg/kg of dexmedetomidine (DEX0.05 and DEX0.1) administered through a single intracoelomic injection. If LRR occurred, heart and respiratory rates were monitored until RRR. Loss of righting reflex occurred in 63% (5/8) and 38% (3/8) of snakes following administration of DEX0.05 and DEX0.1, respectively. For DEX0.05, the onset of LRR was variable with a range of 1 to 20 minutes, and duration of 62-124 minutes. Following DEX0.1, the onset of LRR ranged from 3 to 15 minutes, and duration ranged from 15-174 minutes. Following LRR, heart rate ranged between 20-40 beats per minute and respiratory rate ranged between 0-11 breaths per minute for both treatments at recorded time points. The co-administration of dexmedetomidine and alfaxalone in snakes resulted in marked variability in the presence and duration of LRR; however, fewer snakes demonstrated LRR at DEX0.1 compared to DEX0.05. A second phase of the study will evaluate dexmedetomidine and alfaxalone administered at separate injection sites to determine if the variability is due to pharmacokinetic or pharmacodynamic interactions.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Effects of intrauterine growth restriction on peripheral inflammatory mediators in a neonatal piglet model
Kyle Christensen, Megan Caputo, Rodney Johnson
Division of Nutritional Sciences, Department of Animal Sciences, College of Agricultural, Consumer, and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL

Infants born small for gestational age (SGA) due to intrauterine growth restriction (IUGR) are more likely to exhibit abnormalities in brain development and greater susceptibility to infection than their average for gestational age (AGA) counterparts. The purpose of this study was to determine if there was a difference in the level of expression of various inflammation mediating proteins between SGA and AGA infants using a piglet model. We hypothesized that the peripheral tissues of SGA piglets would contain lower inflammation-associated gene expression levels, and lower circulating plasma IL-6 levels, than their AGA counterparts. SGA and AGA piglets were weaned at postnatal day (PD) 2, and at PD 14 injected intraperitoneally with E. coli lipopolysaccharide (LPS) or sterile saline. Blood was drawn pre- and 4 hours post injection, and ELISAs were performed to quantify plasma IL-6 levels. Four hours after injection, piglets were sacrificed and thymus, liver, and spleen was collected. RNA was isolated for cDNA synthesis and qPCR was performed for gene expression. We found no association between SGA status and plasma IL-6 levels. The thymus samples of SGA piglets showed decreased expression of the genes TMPO and RUNX1. Spleen and liver samples had a main effect of LPS injection with varying expression of several inflammation mediating genes. Only SGA piglet liver had increased levels of pro-inflammatory CCL2 gene after LPS injection. Possible implications of this work include further research targeting the thymus of SGA infants to better determine how hematopoietic proteins like TMPO and RUNX1 contribute to immunodeficiency, in order that effective therapeutics or preventatives can be developed in the future. 

Research Grant: NIH HD069899
Student Support: Office of the Director, NIH, T35 OD011145

Circadian disruption impacts impulsivity and attention: examining neurochemical mechanisms in Long-Evans rats
Bridget M. Clancy, Rekha C. Balachandran, Megan A. Sieg, Katherine M. Hatcher, Megan M. Mahoney, Paul A. Eubig
Department of Comparative Biosciences, College of Veterinary Medicine (Clancy, Balachandran, Mahoney, Eubig), Neuroscience Program (Sieg, Hatcher, Mahoney, Eubig), University of Illinois at Urbana-Champaign, Urbana, IL

Circadian rhythms are 24-hour cycles that control physiological processes, and their disruption can have negative effects on health. Two forms of circadian disruption in people are working overnight shifts and exposure to artificial light at night. The purpose of this study is to examine the effects of circadian disruption on attention and impulsivity in adult Long-Evans rats using the five-choice serial reaction time task (5-CSRTT). The three circadian conditions were testing in dark phase (control), testing in dark phase with pulse of light (modeling artificial light-at-night), and testing in light phase (modeling shift work). Our results showed that both models of circadian disruption caused decreased attention and greater impulsivity than the control, with the light-at-night group exhibiting greatest impulsivity. This indicates that artificial light-at-night may be more harmful to cognitive functions than overnight shift work. Considering the neurochemical basis for the effects, dopamine modulates impulsive behavior while acetylcholine (ACh) modulates both attention and circadian rhythms. Therefore, we hypothesized that impulsivity resulting from circadian disruption results from an interaction between ACh and dopamine. To explore this, we tested the effects of a nicotinic acetylcholine receptor agonist (nicotine) and a dopamine-1 (D1) receptor antagonist (SCH 23390) on attention and impulsivity. Nicotine increased impulsivity while SCH decreased impulsivity. When administered together, SCH attenuated the effect of nicotine on impulsivity. These results suggest that D1 receptors are involved in impulsivity and that cholinergic signaling interacts with the dopamine system to influence impulsive behavior.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Office of the Director, NIH, T35 OD011145

Evaluating the effect of Helicobacter pylori VacA toxin on parietal cell function
Kalinda Clark, Steven Blanke, Robin Holland
Department of Pathobiology (Clark, Holland) and Department of Microbiology (Blanke, Holland), University of Illinois at Urbana-Champaign, Urbana, IL

Helicobacter pylori (Hp) is a human-specific gastric pathogen, infecting 50% of the world’s population and increasing the risk of gastric cancer. Hp infection results in peripheral immune cell infiltration, inflammation, and parietal cell proliferation. The vacuolating cytotoxin (VacA), named for its ability to induce vacuole production in human cells, has an unclear role in these processes. Parietal cells are dynamic, gastric-acid-secreting cells that exist in a resting or active state. The resting state sequesters apical membrane proton pumps into the cytosol as tubulovesicles. We hypothesize that VacA targets tubulovesicles to create a microenvironment for Hp colonization by maintaining inactive parietal cells. Parietal cells were isolated and cultured from mouse gastric mucosa, then challenged with VacA in a resting or histamine-stimulated active state. The apical and basolateral pH and ion concentrations were measured by metabolomics, and cell morphology and vacuolation visualized by TEM and light microscopy. We predict that VacA-challenged apical media will have an increased pH and concentration of anions, and minimal morphologic changes with co-histamine stimulation compared to controls. The parietal cell tubulovesicles are anticipated to be the observed VacA-mediated vacuoles. Because Hp infection induces a prolonged ineffective parietal cell state coupled with proliferation, we propose that VacA targets parietal cells to gather nutrients and create a microenvironment for Hp colonization. Understanding the role of VacA in Hp infection addresses a fundamental knowledge gap in the study of Hp infection, and provides new insight into host-microbe interactions and the importance of secreted microbial effectors.

Research Grant: NIH R01 AI045928, NIH R21 AI117497
Student Support: Office of the Director, NIH, T35 OD011145

The use of SP-10 protein as an acrosome marker in stallion seminiferous tubules
Anamaria Cruz, Derek Sullivan, Igor F. Canisso, Prabhakara P. Reddi
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Optimal breeding stallion fertility is multifactorial; understanding the reasons for infertility would require employment of proper tests to evaluate the progression of spermatogenesis. Previous studies have shown the intra-acrosomal antigen SP-10 as a useful marker for round spermatids and spermatozoa in mice and men. The goal of this study was to evaluate the usefulness of the SP-10 antibody in the staging of the stallion seminiferous epithelium using immunohistochemistry. Adult stallion testis cross sections were treated with an in-house guinea pig polyclonal antibody raised against the mouse SP-10 antigen at a concentration of 1:750. The Zeiss Axiovert 200M Widefield microscope, the Axiocam 506 Color camera, and ZenPro software were used to capture images. We have found the SP-10 antibody labeling to be highly specific for the acrosome and were able to follow the progression of acrosome development. Historically, staging of cross sections of the seminiferous tubules was done using the morphology of the acrosome as the criterion. In mice, for example, any given cross section of the tubule represents one of the 12 stages of the cycle. Because the SP-10 antibody clearly outlined the acrosome in round spermatids of the stallion, we were able to discern the stage represented in each cross section. In general, we observed more than one stage in a given cross section. Based on observations of 100 cross sections each from three different stallions, 66% had more than one stage represented. In this regard, the stallion exhibited similarities to the human seminiferous epithelium wherein multiple stages are found in a given cross section. These studies provide a reference for staging of the horse seminiferous epithelium.

Research Grant: NIH HD36239
Student Support: Office of the Director, NIH, T35 OD011145

Itchy eyes no more: the effects of Zaditor on the healthy canine eye
Aislinn A. Klein, Tatiane Villar, Flor D. Y. Chacaltana-Claros, Kathryn M. Fleming, Bianca C. Martins
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

The precorneal tear film consists of multiple components that contribute to vision as well as ocular health and nutrition. Previous research demonstrated that the tear film of humans can be negatively affected by certain topical and oral medications; however, such phenomenon has not been determined in dogs. Zaditor is an over-the-counter antihistamine drop frequently prescribed to dogs that have irritated eyes due to allergies. At this time, it is unknown if Zaditor has a detrimental effect on the canine tear film. The purpose of this study is to determine if Zaditor alters the quantity or quality of tears produced in healthy dogs when administered twice daily. We hypothesize that the use of Zaditor will not influence the tear quantity and quality. Healthy dogs were enrolled, and ocular examination and tear production and quality tests performed, including Schirmer tear test (STT), tear osmolality, tear ferning, and meibometry. All parameters were evaluated at baseline, on days 7 and 14 of treatment, and on day 30 of the study (2-week washout period). To date, baseline values from 25 dogs were obtained. Analysis of variance was used to verify any differences between right (OD) and left (OS) eyes. Mean STT ± SD (mm/min) were 22.3 ± 2.9 (OD) and 21.8 ± 3.9 (OS). Mean osmolarity ± SD (mOsm/L) were 305.4 ± 35.3 (OD) and 304.2 ± 33.0 (OS). Mean tear ferning grades ± SD were 2.1 ± 0.8 (OD) and 2.0 ± 0.8 (OS). Mean meibometry ± SD (MU) were 115.5 ± 56.7 (OD) and 173.7 ± 139.3 (OS). No significant differences between eyes were observed at baseline day for any test (p > 0.05). The results of this work will contribute to better understanding the safety of this topical drug in dogs.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Making a list and checking it twice – the effects of implementing a surgical checklist in veterinary medicine
Briana McConnell, Laura Selmic, Daniel Duffy, Tisha Harper, Heidi Phillips
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Following development by the World Health Organization, surgical checklists have become an adopted standard in many human hospitals, and studies have shown that they drastically reduce intra-operative and post-operative complications. Despite the proven benefits in human medicine, there has yet to be comprehensive evaluation in veterinary medicine. The aim of this study was to evaluate the impact of adopting a surgical safety checklist in a veterinary medical academic center. We hypothesized that implementing a surgical checklist would decrease the number of perioperative errors and post-operative complications. To begin this study, we looked retrospectively at the orthopedic, neurology, oncology, and soft tissue surgeries that took place in our hospital between March 2017 and May 2017. Of the 301 surgeries that occurred during that time, 21.6% had intra-operative complications and 36.2% had post-operative complications. Next, we observed three weeks of surgeries to observe any perioperative complications prospectively. During this time, a sample checklist was drafted and review by surgeons, anesthesia personnel, and technicians so that a final customized file could be constructed. This final checklist was then implemented for three weeks, during which time surgeries were also observed and perioperative error rates were determined. The number and type of surgical complications associated with the pre-checklist surgeries will be compared to those that used the checklist. We will then extend the study over another three-month period to collect more data and compare results to the retrospective data collected earlier. Data from this study will assist clinicians in deciding whether to implement use of a surgical checklist.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

How does reproduction affect the personality of female threespined sticklebacks (Gasterosteus aculeatus)?
Laura Molinet, Chloe Monestier, Alison Bell
Department of Animal Biology, School of Integrative Biology, University of Illinois Urbana-Champaign, Urbana, IL

Evidence has shown that the personality of an animal impacts its life history traits and fitness, but we know little about how life history events like reproduction affect personality. Reproduction is one of the most important events for any individual to experience. The aim of this project is to investigate how personality changes after reproduction in female threespined sticklebacks (Gasterosteus aculeatus). We hypothesize that females will be bolder and more risk-prone post reproduction, because the life history theory predicts a trade-off between current and future reproduction. We tested the personality of females before and after reproduction (control: did not reproduce) using three personality assays. The first assay assessed the females’ activity level in a new environment, the second assessed how females reacted to conspecifics, and the final assay was a trade-off between resource acquisition (i.e. food) and risk of predation (i.e. wooden bird). All three personality assays were performed on the same experimental and control fish for three consecutive days prior to reproduction. After the final  personality assay on day 3, a water sample was obtained for a hormonal assay to evaluate stress and sex hormone levels. Once the “before” assays were complete, the gravid experimental females were placed in a tank with a male for courtship. The females and their controls were then evaluated again using the same assays. The data from the “before” and “after” assays will be compiled and compared to  determine if the experience of reproduction changes the personality of females. The results should  show that life history could impact personality and provide more evidence about the proximate mechanism for personality change.

Research Grant: NIH PHS 1 R01 GM082937 to AMB
Student Support: Office of the Director, NIH, T35 OD011145

Do the biomechanical differences between the trot and pace affect tarsal osteochondrosis lesions in Standardbred horses?
Amy L. Narotsky, Christine T. Lopp, Annette M. McCoy
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Osteochondrosis (OC) is a developmental orthopedic disease that occurs when normal endochondral ossification is disturbed. Standardbred horses are predisposed to OC, particularly in their tarsocrural joint (hock), and it can become a problem when young horses begin race training, as they may show signs of lameness or joint effusion. Both genetic and environmental factors (such as diet and exercise) are known to play a role in the risk of developing osteochondrosis. Previous research has indicated that Standardbred pacers and trotters are prone to developing OC lesions at different locations within the hock. The reason for this difference is unclear but the differing biomechanics of the trot (a two-beat diagonal gait) and pace (a two-beat lateral gait) could play a role. We hypothesize that the amount of time young Standardbreds spend pacing or trotting in the field correlates with the location and severity of the tarsal OC lesions that develop. Our goal is to quantify the activity of these foals in the field and correlate these findings with differences in development and progression of tarsal OC lesions. Horses in the study will be followed from 2 to 12 months of age and observed for a minimum of 2 hours per week with observations made every 30 seconds. In addition, 4 standard views of hock radiographs will be taken and evaluated for OC lesions every two months. By investigating the role of gait biomechanics as a risk factor for OC, we will have a more complete picture of the etiology of this disease and hopefully be able to improve the health of the Standardbred horse.

Research Grant: Morris Animal Foundation D16EQ-311
Student Support: Morris Animal Foundation D16EQ-311

Static posturography as a novel diagnostic tool in dogs with suspected thoracolumbar spinal cord disease
Aaron M. Paushter, Kari D. Foss, Devon W. Hague, Tisha A. Harper, Jacob Sosnoff
Department of Veterinary Clinical Medicine (Paushter, Foss, Hague, Harper) and Department of Kinesiology and Community Health (Sosnoff), University of Illinois at Urbana-Champaign, Urbana, IL

Dogs with spinal cord disease (SCD) are evaluated through the application of subjective scoring systems during serial neurologic examinations. Post-treatment outcome is measured subjectively through the direct observation of an animal’s gait, and may overestimate or underestimate a patient’s recovery. Static posturography provides an objective, quantitative alternative to subjective gait analysis through measurement of characteristics of an animal’s balance in quiet stance. The purpose of this study was to evaluate the utility of static posturography as an objective diagnostic adjunct and prognostic indicator in dogs with suspected thoracolumbar SCD. Control dogs demonstrated a normal neurologic examination and had no known history of neurologic or musculoskeletal disease. Affected dogs presented with clinical signs of thoracolumbar SCD, including pain, weakness and incoordination. All dogs were placed in quiet stance on a Tekscan WalkwayTM force and pressure measurement system. Measurements related to each dog’s balance were taken during five trials, including aggregate distance traveled by the dog’s center of force (COF), maximal cranial/caudal and medial/lateral movement of COF, and weight distribution by limb. The authors hypothesize that these values will be significantly larger in dogs with thoracolumbar SCD. This study has important implications for the development of static posturography as a novel method of evaluating recovery and efficacy of treatment in dogs with SCD.

Research Grant: None
Student Support: Boehringer Ingelheim Veterinary Scholars Program

Integrating prediction of parturition and calcium supplementation for prevention of subclinical hypocalcemia
Karli N. Robinson, Jair C. Ferreira, Ziyao Zhou, Jamie L. Stewart, Ameer A. Megahed, Peter D. Constable, Fabio S. Lima
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Accurately predicting parturition has the potential to allow for strategic prevention of metabolic disorders such as subclinical hypocalcemia, which affects between 25% and 54% of lactating dairy cows in the U.S. Adequate cytosolic ionized calcium concentration (ICC) is vital for the regulation of neurotransmission and muscle contraction. Cows diagnosed with hypocalcemia can develop problems such as displaced abomasum, uterine prolapse, and retained placenta due to the low ICC suppressing smooth muscle contraction. Moocall is a tail-mounted sensor that uses an algorithm to predict when cows are likely to calve by measuring tail movement patterns triggered by labor contractions. This device sends 2 text messages for prediction of parturition when tail movements reach a certain level of intensity that generally suggests that parturition will occur within 1-2 hours. We hypothesize that the first message Moocall sends predicting parturition can be used as a time point for strategic prevention of hypocalcemia in dairy cows. Devices will be placed on multiparous Holstein dairy cows. Excel random function will be used to assign cows to treatments (calcium supplemented and control). Calcium-supplemented cows will receive an oral bolus containing 43 g of calcium (Bovikalc) at the time of the first message predicting parturition followed by two boluses at 0 and 24 h after calving. Control cows will not receive calcium supplementation, but will receive a sham bolus administration. By evaluating the accuracy of Moocall and the effects that administering an oral calcium bolus prior to calving has on cows, the welfare of the cow can be improved by regulation of calcium homeostasis at calving and associated health disorders.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: University of Illinois College of Veterinary Medicine

The effect of anal fin color on male mating depth preference in Bluefin Killifish (Lucania goodei)
Jessica Schult, Chia-Hao Chang, Rebecca Fuller
Department of Animal Biology, School of Integrative Biology, University of Illinois at Urbana-Champaign, Urbana, IL

Bluefin killifish (Lucania goodei) populations provide the opportunity to study the population biology of color polymorphism. In most natural systems, one color morph in a species with color polymorphism will go to fixation due to natural selection or genetic drift. However, in L. goodei populations found in springs, males display both red and yellow anal fin colors. This polymorphism is controlled by a single locus, and each naturally occurring population has both color morphs. One hypothesis for the maintenance of polymorphisms within populations is that there are different microenvironments where each color morph has a higher level of fitness. The aim of this study was to determine the effect that male L. goodei anal fin color has on preferential mating depth in order to determine if mating depth could be a driving factor in the presence of color polymorphism in this species. Red is more vibrant in clear water at shallower depths, so males with red anal fins should display higher preference for mating in shallow water when compared with yellow-finned males. One male and five female L. goodei were placed in a tank set up with spawning mops at three different depths. The mops were removed twice daily for five days and the eggs were removed from the mops and counted. The eggs were placed in a solution of methylene blue to determine whether they had been fertilized. Preliminary data showed that the eggs were most often laid and fertilized at the top of the tank regardless of the coloration of the male. These results indicate that depth is unlikely a major factor in maintaining polymorphism within L. goodei populations. Alternative hypotheses to explain maintenance of color polymorphism are being tested.

Research Grant: None
Student Support: Office of the Director, NIH, T35 OD011145

Loss of USP18 disrupts brown adipose tissue and causes multi-organ auto-inflammation
Nicole Sidebotham, Genesis Romo, Emmanuel Bikorimana, Michael J. Spinella, Sarah J. Freemantle
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Ubiquitin-Specific Protease 18 (USP18) is a dual-function protein that removes interferon stimulated gene 15 (ISG15) protein modifications and inhibits the activity of the interferon receptor. Necropsy analysis of USP18-null mice revealed immune infiltration of multiple tissues including brown adipose, pancreas, salivary glands and skin. The mice exhibit phenotypes similar to those in autoimmune diseases such as systemic lupus erythematosus and Sjogren’s Syndrome. USP18-null mice showed elevated liver glycogen levels, which are associated with liver damage and stress. Gene expression analysis of peripheral blood lymphocytes (PBL), liver, brown adipose tissue (BAT) and white adipose tissue (WAT) were performed using samples derived from USP18-null, heterozygous, and wildtype mice. Interferon signature genes were upregulated in PBL of USP18-null mice and this effect was amplified in BAT where IFIT1, IFR7, OAS1, OASL and ISG15 were each increased over 100-fold. Immunohistochemical analysis demonstrated that both infiltrating immune cells and the brown adipose cells had upregulated ISG15. USP18-null mice cannot maintain body temperature in a cold-tolerance test, suggesting a defect in BAT function. We hypothesize that USP18 reduces responsiveness to type 1 interferon signaling in BAT to maintain its normal thermogenic activity. In the absence of USP18, BAT function is disrupted through amplified interferon signaling causing dysregulation of lipid metabolism, cold intolerance and potentially autoimmunity. These studies demonstrate the usefulness of the USP18-null mouse for studying signaling pathways maintaining normal BAT function and the link between dysregulated interferon signaling and autoimmune disease.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Office of the Director, NIH, T35 OD011145

Rapid identification of Mannheimia haemolytica tetracycline resistance in Illinois cattle
Whitnie Smartt, Sara Lanka, Carol Maddox
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Bovine Respiratory Disease (BRD) is a major health concern in North American feedlot cattle contributing to major economic losses. Mannheimia haemolytica is a bacterium considered to be the predominant pathogen associated with BRD. Antibiotics, specifically tetracyclines, have often been used as a feed additive for prevention and treatment of bacterial infections common in feedlot cattle. Because the increased use of antibiotic treatment can select for antibiotic resistance, a method for rapidly identifying resistance can aid in drug selection and treatment success. This study aims to evaluate MALDI-TOF mass spectrometry as a method for rapid identification of M. haemolytica tetracycline resistance in Illinois cattle. M. haemolytica isolates were collected from tissues taken from cattle with respiratory disease submitted for necropsy to the University of Illinois Veterinary Diagnostic Laboratory. Isolates were subjected to MALDI-TOF, an evolving diagnostic methodology that can provide rapid identification of microbial isolates, as well as information about antimicrobial resistance profiles. Traditional PCR methods were used to detect the tetH genotype commonly associated with tetracycline resistance in M. haemolytica. Mass spectrometry peaks from tetracycline-resistant and susceptible isolates will be analyzed to detect the presence of unique protein peaks that might identify resistant isolates. Data from these analyses will be combined with information from the PCR assays, as well as clinical and demographic information. A method for rapid detection of tetracycline resistant M. haemolytica isolates would improve treatment outcomes for cattle with respiratory disease.

Research Grant: Donor Funds
Student Support: Office of the Director, NIH, T35 OD011145

Does proximity to railroads increase arsenic-related health risks?
Nicole Staton, Maya P. Scott, Marilyn O’Hara Ruiz
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Arsenic-contaminated soil and water pose serious health concerns for both humans and animals.  Exposure to arsenic can occur through ingestion of contaminated water or through food grown in arsenic-containing soil, through aerosolized particles or through dermal contact.  Railroads have historically transported arsenical-based pesticides and housed arsenic-containing cattle-dipping vats. Moreover, railroad rails are coated in arsenic. We hypothesize that the risk of arsenic exposure increases with proximity to railroads.  Escambia and Santa Rosa counties in Florida provided test cases for the characterization of arsenic contamination.  These counties were chosen because of past arsenic use on cotton production, the transportation of cattle, and the presence of arsenic-containing superfund sites.  A previous study of 89 soil samples from these counties provided data for a spatial statistical analysis of arsenic levels. An additional 25 soil samples and 10 water samples were collected in summer 2017. Samples were collected near railroads and elsewhere in the counties for comparison. Data from this work will help confirm the locally variable presence of arsenic and better equip scientists and public health officials to raise public awareness and prioritize remediation of arsenic-contaminated soil and water.

Research Grant: Donor Funds
Student Support: Office of the Director, NIH, T35 OD011145

Effects of in utero exposure to DEHP and high-fat diet on uterine development in mice
Suzanna M. Storms, Arpita Bhurke, Indrani Bagchi
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Di(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical found in many consumer products. DEHP exposure is related to endocrine and metabolic diseases in humans, and food ingestion is the main source of DEHP exposure. Studies have suggested that DEHP and adipose signaling may act through similar metabolic pathways, specifically PPARg. It is well-documented that obesity increases the risk of developing endometrial carcinomas, but no link has yet been found to suggest DEHP involvement. In this study, we investigated the effects of prenatal exposure to DEHP and high-fat diet on the development of the uterus. Pregnant dams were orally dosed with either a corn oil control or 20 µg/kg/BW DEHP, and fed either a control diet of a balanced maintenance chow or a “western” high-fat diet with 45% of calories derived from fat. Diets were fed from day 0.5 of pregnancy, and continued until parturition. Uterine tissues from post-natal day (PND) 8 and PND 21 pups were collected from all treatment groups. Preliminary results indicated in utero exposure to a high-fat diet resulted in accelerated adenogenesis, uterine gland development, at PND 8. By PND 21 the pups exposed to high-fat diets or DEHP in utero showed increased proliferation of the uterine luminal epithelium. These results suggest that in utero exposure to DEHP or a hig-fat diet may lead to uterine epithelial hyperplasia later in life. Uterine epithelial hyperplasia is associated with implantation failure in women, as well as being a premalignant lesion of endometrial carcinoma.

Research Grant: EPA RD-83543401
Student Support: Office of the Director, NIH, T35 OD011145

Proteinuria in dogs with atopic dermatitis: a retrospective analysis
MaryKate Tully, Jason B. Pieper
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Atopic dermatitis is an inflammatory skin condition in dogs, resulting in intense pruritus, self-trauma, and papules, amongst a variety of other cutaneous lesions. These animals are most commonly treated with immunomodulators, such as cyclosporine or oclacitinib, or systemic anti-inflammatories such as glucocorticoids. Given the inflammatory nature of atopic dermatitis, proteinuria is expected to occur in some atopic dogs. In this retrospective study, the records of 77 client-owned dogs with atopic dermatitis were analyzed for evidence of proteinuria. Animals with proteinuria before or at the time of diagnosis of atopy were further studied to determine whether the proteinuria persisted after treatment with oclacitinib, cyclosporine, or glucocorticoids. It was hypothesized that systemic treatment of atopic dermatitis would decrease or eliminate proteinuria in these dogs. Preliminary results of this on-going analysis indicated that most dogs exhibiting proteinuria prior to, or at the time of diagnosis with atopic dermatitis, did not experience lessening or resolution of proteinuria following treatment of atopy. This study revealed that proteinuria is present in a small number of dogs and can be attributed to atopic dermatitis. In addition, proteinuria is unlikely to resolve, even after the atopic dermatitis becomes well-controlled with treatment.

Research Grant: Gift Funds
Student Support: Boehringer Ingelheim Veterinary Scholars Program

In vivo gene knockdown approach for validation of gene function in Cryptosporidium parvum
Tori Worthen, Xuejin Zhang, Chi Yong Kim, William H. Witola
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Cryptosporidium parvum is a parasitic protozoan of medical and veterinary significance worldwide. Infections with C. parvum occur through ingestion of contaminated water or food, and cause a diarrheal syndrome called Cryptosporidiosis, that may be life-threatening in immunosuppressed individuals. Currently, there is only one approved drug, nitazoxanide, used for the treatment of Cryptosporidium infections but it is ineffective in malnourished children and immunosuppressed individuals. In this study, we endeavored to develop an in vivo assay for interrogating gene function throughout the life cycle of C. parvum using phosphorodiamidate morpholino oligomers. By targeting the C. parvum lactate dehydrogenase (LDH) gene, we achieved over 90% knockdown of its expression during C. parvum growth and development in IFN-gamma knockout mice. To analyze the effect of LDH knockdown, we performed real-time PCR to quantify the amount of C. parvum oocysts shed by infected mice treated with LDH-target morpholino compared to those treated with control (off-target) morpholino. We then used the oocysts shed by the two groups of mice to infect HCT-8 cells to assess the effect of LDH knockdown on viability. We found that LDH knockdown significantly decreased the growth and development of C. parvum, and that the oocysts shed contained daughter parasites with significantly reduced infectivity. These studies have developed an antisense gene knockdown approach for studying gene function in Cryptosporidium, and validated LDH as an essential gene for growth, development and viability of C. parvum. This technology will be useful in functionally characterizing and validating potential molecular drug targets in Cryptosporidium.

Research Support: Bill & Melinda Gates Foundation
Student Support: Boehringer Ingelheim Veterinary Scholars Program

2016 Research Project Abstracts

Biomechanical risk factors in the development of osteochondrosis in Standardbred pacers and trotters
Samantha R. Carter, Christine T. Lopp, Annette M. McCoy
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Osteochondrosis (OC) is a developmental orthopedic disease in which the process of endochondral ossification is disrupted. Although the etiology of OC is not clearly understood, there are genetic and environmental risk factors (i.e. diet, exercise) that can increase the likelihood of developing the disease. In previous work, it was found that pacers and trotters had a predilection to develop hock OC lesions at different locations. Trotters were more likely to develop medial malleolus lesions, whereas pacers developed distal intermediate ridge of the tibia lesions. The reason behind these differences is unknown, but biomechanical differences between the pace and the trot may play a role. We hypothesize that the amount of time young foals spend pacing or trotting in the field correlates with the subsequent lesions that develop, and with their severity. The aim of this study is to quantify spontaneous activity of Standardbred pacer and trotter foals and correlate these findings with differences in development and progression of OC lesions. Horses will be followed prospectively from 2 to 12 months of age. Field observations will be made weekly, and hock radiographs taken every two months. Observations of activity (sleeping, nursing, walking, trotting/pacing, cantering/galloping) will be recorded at 30 second time intervals for a minimum of 2 hours per foal per week, focusing on the duration of time spent at either the pace or trot. At each radiographic time point, 4 standard views of hock radiographs will be taken and evaluated for the presence or absence of OC lesions. We expect the lesions to be dynamic (i.e. capable of forming or healing) until 8 months of age and our work to contribute to knowledge of OC pathophysiology.

Research Grant: Morris Animal Foundation D16EQ-311 and USDA Hatch Fund
Student Support: Merial Veterinary Scholars Program

Do male sticklebacks use visual or olfactory cues to assess the history of a potential mate?
Marion Dellinger, Jennifer Hellmann, Alison M. Bell
Department of Animal Biology, School of Integrative Biology, University of Illinois at Urbana-Champaign, Urbana, IL

Differential allocation occurs when individuals alter their reproductive investment based on their mate’s traits. Previous studies have shown that male threespined sticklebacks (Gasterosteus aculeatus) alter their behavior toward a potential mate depending on her previous experience with predators: when a male encountered a female who had been exposed to predators while yolking her eggs, he showed fewer courtship displays as well as less care to her offspring relative to an encounter with an unexposed female. Although both chemical and visual cues mediate conspecific interactions and mate choice in this species, we do not know the mechanism males use to detect previous predator-exposure in mates. To test the extent to which those cues aid in male detection of female predation exposure, we compared male courtship behavior across four treatments in a 2×2 repeated measures design manipulating a potential mate’s previous experience (predator-exposed or not) as well as the presence or absence of olfactory cues from predator-exposed females. We confined females to a transparent box providing visual cues only, while olfactory cues were added via water from the tanks of predator-exposed females (experimental) or predator-naïve ones (control). We predicted that if males use olfactory cues to assess female predation exposure, males exposed to experimental water would behave differently compared to males exposed to control water, regardless of female predation status. If males use visual cues, however, only the female status should alter male courtship. Determining the relative importance of visual versus chemical cues will help us better understand the mechanisms by which males adjust their behavior based on female phenotypes.

Research Grant: NSF 1121980, NIH GM082937
Student Support: Merial Veterinary Scholars Program

Make food great again: effect of protein source and amount on whole-body metabolism and weight gain
Danielle Engel, Kristy Du, Justin Rhodes
College of Veterinary Medicine (Engel), Department of Animal Sciences (Du) and Department of Psychology (Rhodes), University of Illinois at Urbana-Champaign, Urbana, IL

Recent studies have shown that over 66% of the U.S. adult population is overweight or obese. Approximately 30% of dogs and cats are overweight, indicating that obesity is a significant problem in the pet population as well. Protein is an important element of the daily diet. Previous studies indicated that certain types of protein, such as egg white (EW) and wheat gluten (WG), have satiating and anti-satiating effects on rats, respectively. The goal of this study was to determine if animal and plant-based protein sources affect metabolism, leading to weight loss or slower weight gain in rats over a 30-day period. Higher metabolic rates and slower weight gain were expected in WG treatment groups due to a predicted increase in energy expenditure based on amino acid composition. After an overnight fast, adult Sprague-Dawley rats were provided 30 minutes of access to a meal of manipulated protein source and amount. These meals were either 20% or 35% WG or EW and the amount was calculated to be 10% of daily food consumption. Food intake and body weight were recorded daily. EchoMRI was used to measure body composition on days 1, 15, and 30. Columbus Lab Animal Monitoring System (CLAMS) cages were used to analyze respiratory exchange ratio (RER) and heat produced during the first and last 3 days of the study. No significant difference was found between treatment groups with regard to weight gain and metabolism. RER data suggested that as rats adapted to their new diet regimen, protein became a larger source of fuel. Although the results did not support the overall hypothesis, RER data suggest that long-term effects of high protein diets on protein digestion merit further study.

Research Grant: National Institute of Diabetes and Digestive and Kidney Diseases, NIH, DK082609
Student Support: Office of the Director, NIH, T35 OD011145

Phthalates and diet: effects on the developing cortex
Anthony Franceschelli, Daniel Kougias, Carly Drzewiecki, Steven Rhoads, Janice Juraska
College of Veterinary Medicine (Franceschelli), Neuroscience Program (Kougias, Drzewiecki, Juraska), Department of Psychology (Rhoads, Juraska), and Children’s Environmental Health and Disease Prevention Research Center, University of Illinois at Urbana-Champaign, Urbana, IL

Phthalates, a group of endocrine disruptors used widely in consumer products, disrupt thyroid function and are known to have estrogenic and anti-androgenic effects.  Moreover, high-fat diets interfere with cognition in both rodents and human children.  Phthalates and high-fat diets increase oxidative stress and inflammation, which negatively affect nervous system function and plasticity, including that of the cerebral cortex.  Because infants and young children are exposed to higher levels of these chemicals than adults, this study tested the hypothesis that pre- and post-natal exposure to a phthalate mixture combined with a maternal high-fat diet will result in long-term alterations in cortical development and in cognitive behavior accompanied by indices on oxidative stress and inflammation as well as epigenetic alterations. Pregnant hooded Long-Evans rats were administered one of two doses of a phthalate mixture (based on the profile of urinary phthalate metabolites in pregnant women in Champaign-Urbana, Illinois) or vehicle and placed on either a high-fat or control diet. Slices of the medial prefrontal cortex of offspring were mounted and stained for anatomical examination. Intra- and extra-dimensional shifts were examined as a test of cognition. We hypothesize that the combination of exposure to endocrine disruptors and a high-fat diet during development will be especially damaging for cognitive behavior and the neural regions of the brain that are important for this behavior.  The results of this study will contribute to a better understanding of the effects of our abundant food and consumer products on cognition and the development of the nervous system.

Research Grant: Children’s Environmental Health and Disease Prevention Research Center, NIEHS, P01 ES002848 and USEPA, 83543401
Student Support: Office of the Director, NIH, T35 OD011145

Connectional modularity of the mouse inferior colliculus
Teodora Hristova, Alexandria Lesicko, Daniel Llano
College of Veterinary Medicine (Hristova), Neuroscience Program (Lesicko, Llano) and Department of Molecular and Integrative Physiology (Llano), University of Illinois at Urbana-Champaign, Urbana, IL

Auditory and somatosensory structures in the brain send information to the lateral cortex of the inferior colliculus, a structure that is believed to be important in multisensory integration. Previous studies have shown that this nucleus contains a network of modules, or compartmental zones which stain densely for certain neurochemical markers, such as glutamic acid decarboxylase-67 (GAD-67). Though the function of these modules remains unknown, the termination patterns of sensory inputs to the lateral cortex appear structurally similar to the distribution of neurochemical modules. The goal of the present study is to determine whether the auditory and somatosensory inputs to the lateral cortex project into the modules, interdigitate with the modules, or show no relationship with these neurochemical structures. The auditory and somatosensory projections to the lateral cortex were labeled with the anterograde tracer, biotinylated dextran amine (BDA) and GAD-67 immunohistochemistry was done to expose the modular network in the lateral cortex of the mouse brain. GAD-labeled modules and BDA-labeled input terminals within the inferior colliculus were visualized using confocal microscopy, then images were imported in Neurolucida®, a software program in which axonal reconstructions were performed. Analysis revealed that somatosensory inputs from the somatosensory cortex and dorsal column nuclei project into the modules, while auditory inputs from the auditory cortex and central nucleus of the inferior colliculus interdigitate with the modules.  These results suggest that the auditory and somatosensory inputs to the mouse lateral cortex form isolated processing pathways.

Research Grant: NIH R01 DC013073 and NSF IGERT Fellowship 0903622
Student Support: Office of the Director, NIH, T35 OD011145

Validation of putative chromosome 14 risk alleles for osteochondrosis in Standardbred horses
Eric J. Ledesma, Megan Darragh, Annette M. McCoy
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Osteochondrosis (OC) is a developmental orthopedic disease characterized by focal failure of endochondral ossification. It is believed that vascular failure leading to ischemic chondronecrosis results in OC, although the etiology of this pathology is unknown. Both genetic and environmental factors are believed to play a role in the risk of developing OC. Previously, two regions of equine chromosome 14 were validated for association with risk of hock OC in Standardbreds. We hypothesize that specific variants within these regions have a moderate effect on hock OC risk. In this study, specific single nucleotide polymorphisms (SNPs) located within these regions were assessed for association with OC in an independently sampled Standardbred population. This population will be prospectively assessed radiographically for OC development, between 2 and 12 months of age, with a definitive OC diagnosis made at >8 months. Primers surrounding 14 SNPs of interest were designed using the assembly Equus caballus 2.0.  Subsequently, NEBcutter v2.0 was used to design restriction enzymes for genotyping via restriction fragment length polymorphism (RFLP). SNPs for which RFLP could not be designed were genotyped by Sanger sequencing. Simultaneously, candidate genes in these regions were Sanger sequenced to discover novel SNPs. These will be tested by similar methods in the experimental population. Our long-term goal is to develop a diagnostic genetic panel to identify at-risk animals, which would allow clinicians and owners/trainers to make management changes to decrease environmental risk factors and overall risk of developing OC. The diagnostic genetic panel would also allow more-informed breeding decisions within high-risk pedigrees.

Research Grant: Morris Animal Foundation D16EQ-311 and USDA Hatch Funds
Student Support: Office of the Director, NIH, T35 OD011145

Morphology of peritoneal endometriotic lesions using an in-vivo mouse model of retrograde menstruation
Victoria McLean, Quanxi Li, Romana Nowak
Department of Animal Sciences (McLean, Nowak) and Department of Comparative Biosciences (Li), University of Illinois at Urbana-Champaign, Urbana, IL

Endometriosis is a painful, chronic disease in which endometrial tissue grows outside of the uterus in women of childbearing age.  The condition commonly causes pelvic pain, dysmenorrhea, and dyspareunia, while also resulting in infertility in 30-50% of cases.  The theory of retrograde menstruation is the most commonly accepted mechanism of endometriosis, stating that blood and endometrial fragments flow backward through the oviduct and exit through the ovarian bursa into the peritoneal cavity.  The purpose of this preliminary experiment is to investigate the morphology of peritoneal lesions using a mouse model of endometriosis that mimics the process of retrograde menstruation found in humans.  Endometrial tissues from female mice primed with pregnant mare serum gonadotropin (PMSG) were transplanted into the peritoneal cavities of ovariectomized, syngeneic mice treated with estradiol (E2) at regular intervals.  Immunohistochemistry (IHC) was performed on lesion sections using antibodies against epithelial marker claudin-4, proliferation marker Ki67, endothelial marker CD31, smooth muscle cell marker ASMA, and mesothelial marker calretinin.  Previous studies reveal an abundance of proliferating stromal cells, myofibroblasts, and mature blood vessels within these sections, which this study also confirms, but it is unknown what processes are occurring in the mesothelial cells that are in contact with the endometrial cells.  If the results confirm the disappearance of mesothelial cells, further studies will determine whether these cells are undergoing a mesothelial-to-mesenchymal-transition (MMT), causing them to lose their adhesive properties and secrete factors that may aid in the establishment of endometriotic lesions.

Research Grant: NIH 1R21 ES026388
Student Support: Office of the Director, NIH, T35 OD011145

Licorice root: a phytoestrogen supplement with neuroprotective effects on cognition
Caitlin E. Ondera, Payel Kundu, William G. Helferich, Donna L. Korol, Ikhlas A. Khan, and Susan L. Schantz
Dept of Comp Biosci (Ondera, Schantz), Neurosci Prog (Kundu, Schantz), Beckman Inst (Schantz), and Dept of Food Sci & Human Nut (Helferich), University of Illinois Urbana-Champaign, Urbana, IL; Dept of Biol (Korol), Syracuse University, Syracuse, NY; Natl Ctr for Natural Prod Res (Khan), University of Mississippi, University, MS

Licorice root (Glycyrrhiza glabra) is a dietary supplement that is widely used for the treatment of menopausal symptoms in women. It contains the phytoestrogens liquiritigenin and isoliquiritigenin that exhibit estrogenic activity. Phytoestrogens are a promising alternative to estrogen replacement therapy, which increases the risk of cancer, stroke, and cardiovascular disease, but their efficacy and safety are not well understood. Estradiol administration to ovariectomized rats improves performance on hippocampal-dependent tasks but causes deficits on prefrontal and striatum-dependent tasks. Conversely, our previous research showed isoliquiritigenin improves performance on a hippocampal task and has no effect on a prefrontal task. This study focused on the effects of licorice root on the striatum to determine if it causes deficits similar to estradiol. Licorice root powder, licorice root extract, and isoliquiritigenin were administered orally to ovariectomized Long Evans rats. Estradiol positive and negative control groups were also included. After 3 weeks of exposure, rats were subjected to a striatum-dependent object recognition task. Rats were placed in a testing chamber and allowed to habituate to two stimulus objects. The familiar objects were then replaced with two novel objects. Object exploration time was recorded, and a pattern separation index was calculated between habituation and test trials. Rats that recognize the second pair of objects as novel have a greater pattern separation index, indicating better performance. This work may lead to the identification of a dietary supplement for treatment of menopausal symptoms that confers neuroprotective effects without the negative effects of other estrogens.

Research Grant: NIH, 5 P50 AT006268-06
Student Support: Office of the Director, NIH, T35 OD011145

The molecular basis of mate preference: the retinal mosaic of the Bluefin Kilifish (Lucania goodei)
Zachary Osterholz, Alexa Sadier, Rebecca Fuller
Department of Animal Biology (Osterholz, Fuller) and Department of Cell and Developmental Biology (Sadier), University of Illinois at Urbana-Champaign, Urbana, IL

Discerning links between physiology and behavior is essential to a better understanding of the evolutionary forces acting to determine how an organism responds to particular stimuli. The visual system of L. goodei is an effective model for understanding how evolution regulates the physiology of sensory systems. L. goodei is a freshwater fish that occurs in a range of lighting environments from tea-stained swamps (low transmission of UV/blue light) to clear springs (high transmission of UV/blue light). Selection appears to favor blue anal fins in tea-stained habitats while males with red anal fins are more abundant in clear springs. The reigning hypothesis is that visual contrast against the background water column is essential for mate recognition. These differences in male abundance also correlate with variation in cone cell abundance and opsin expression, which may be related to differences in mating preference. This study sought to further investigate the molecular basis of the differences underlying variation in mate preference. Ten fish from a swamp population and ten from a spring population were collected. Their retinae were dissected and the retinal pigment epithelium bleached. Retinae were then stained for opsins, which are light-sensitive proteins containing visually active pigments that receive particular wavelengths of light ranging from red to UV. By simultaneously staining six distinct opsins using multiplexed in-situ hybridization, a “mosaic” of the spatial distribution of opsins was created. The mosaics of clear and swamp populations were compared to determine if population-level molecular dissimilarities resulting from variable selection underlay the behavioral differences in these populations.

Research Grant: University of Illinois School of Integrative Biology
Student Support: Office of the Director, NIH, T35 OD011145

Venturing into the unknown: characterization of the aerobic cultivable bacteria in wounds of wildlife species
Courtney Pike, Lois Hoyer, Carol Maddox, Matthew Allender, Julia Whittington
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Traumatic injuries, including blunt-force trauma and bites, are common in wildlife species. A lack of data detailing the microbiota of wildlife wounds, as well as unknown history and limited resources, hamper treatment of these injuries. The goal of this work was to characterize the aerobic cultivable bacteria from wounded animals brought to a wildlife hospital during summer 2016. We hypothesized that the bacterial populations in wounds and on the peripheral skin would be similar. Our secondary hypothesis was that wounds caused by contaminated sources would contain additional bacteria not found on the skin. Upon admission, open wounds and the peripheral skin of wildlife species were individually sampled with a double-tipped swab, taking care to avoid cross-contamination. One swab from each site was used to plate the sample on blood, MacConkey, and Columbia Naladixic Acid agar, then incubated for 16 hours. The second swab was used for Gram staining. Pure cultures were derived for further identification. Isolates were analyzed using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry, which is a newly emerging, cost-effective, and time-saving method in diagnostic microbiology. Genus-level identifications were accurately achieved and deemed clinically sufficient for this study. Preliminary data suggest that MALDI-TOF is an attractive and inexpensive option for the wildlife veterinarian. Understanding the normal bacterial populations on the skin of wildlife species provides insight regarding bacteria that may be present in a wound and facilitates judicious antimicrobial use.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Office of the Director, NIH, T35 OD011145

Anti-Toxoplasma efficacy of green algae extracts
Jonathan L. Powers, William H. Witola
Department of Pathobiology, College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL

Toxoplasma gondii is an obligate, intracellular, zoonotic, parasitic protozoa that infects about one third of the world population. There are no drugs nor effective vaccines to control T. gondii in livestock. In humans, current drugs used against T. gondii are limited by hypersensitivity and toxicity, and are only effective against the tachyzoite stage of T. gondii. Treatment is necessary in immunocompromised patients and infants infected congenitally who may experience severe symptoms such as eye damage, neurological pathologies, and difficulty breathing. The main goal of this study is to evaluate the efficacy of green algae extracts in killing T. gondii proliferative-tachyzoite stages in vitro. Algae has been shown to possess antibacterial and antifungal properties, however, no studies have been performed to test whether it has any anti-protozoal effects. We obtained a pure culture of green algae, Chlorophyceae, and an isolate of pond green algae. The samples were dried and treated with either methanol or hexane. The methanol and hexane extracts were dried by evaporation and reconstituted in DMSO. Confluent human foreskin fibroblasts were infected with T. gondii tachyzoites expressing YFP. Varying concentrations of the algae extracts were added and the cultures incubated for 48 h. Parasite growth was quantified by fluorescent microscopy. We found that both the methanol and hexane extracts from the pure culture and pond algae had concentration-dependent anti-Toxoplasma effect at low microgram concentrations. We will derive anti-Toxoplasma IC50 values and determine the cytotoxic IC50 values in HFF cells. Possible implications of this work may include a new treatment for a T. gondii infection in humans and animals.

Research Grant: USDA Agriculture and Food Research Initiative
Student Support: Office of the Director, NIH, T35 OD011145

Evaluating body condition score with body weight on serum drug levels of phenobarbital and potassium bromide
Chelsea F. Randall, Devon W. Hague
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Epilepsy is one of the most common neurological diseases affecting companion animals and seizures are managed through the use of anticonvulsants. Anticonvulsants require regular therapeutic drug monitoring and medication adjustment by the use of a first-order kinetics equation; however it is not always successful. The aim of this retrospective project was to assess whether a patient’s body condition score (BCS) affects the serum level of phenobarbital and potassium bromide and determine if inclusion of this factor would result in an improved approach for medication adjustments. Data were collected from a retrospective review of canine epilepsy patients at University of Illinois Veterinary Teaching Hospital from January 1, 2000 to June 1, 2016. Records that reported a phenobarbital and/or potassium bromide dosage (mg/kg), serum level, and BCS were included in the study. The data set currently includes 113 dogs on phenobarbital, 29 dogs on potassium bromide, and 27 dogs on both therapies. Once the data set is complete, statistical analysis will assess whether BCS affects serum drug levels. A novel drug adjustment approach using BCS could transform epilepsy treatment and increase the patient’s quality of life.

Research Grant: None
Student Support: Merial Veterinary Scholars Program

Demonstrating C. botulinum neurotoxin heavy chain cytosolic localization via modified Kirby-Bauer assays
Abby Reising, Mengfei Ho, Jamie Perry, Melissa Pires-Alves, Ema Khan, Brenda A. Wilson
College of Veterinary Medicine (Reising) and Department of Microbiology, School of Molecular and Cellular Biology (Ho, Perry, Pires-Alves, Khan, Wilson), University of Illinois at Urbana-Champaign, Urbana, IL

Clostridium botulinum produces the most potent toxins known to man, which inhibit acetylcholine release from neuronal cells causing flaccid paralysis.  The long half-life of toxins in cells paired with the lack of an available antitoxin that can act once the protein has entered the cell makes exposure untreatable and lethal after the critical prophylactic window.  This notion and the possibility of toxin use in bioterrorism makes it crucial to find a novel antitoxin.  This study harnesses the binding domain (BD) and the translocation domain (TD) of the heavy chain (HC) of botulism neurotoxin serotype A (BoNT/A) to bind to neuronal cells, transfer from vesicles into the cytosol, and deliver therapeutic machinery necessary to prevent further neurotransmitter inhibition.  The heavy chain was linked to a prototype cargo, beta-lactamase (Bla), to exemplify a means by which the cargo was successfully delivered to the cytosol with enzymatic activity intact.  To test for this delivery, modified Kirby-Bauer assays were developed.  The positive control protein of Bla-LD-BD that utilized a modified translocation domain (LD) and negative control protein of Bla-BD that lacks a TD or LD have been expressed, purified, and assessed for their activity via Kirby-Bauer assays.  Absence of a zone of clearing around an ampicillin-treated disk, indicating the localization of beta-lactamase in the cytosol and the maintenance of its ability to cleave the beta-lactam ring of ampicillin, reflects successful delivery of the Bla cargo.  If efficacious, the HC of BoNT/A or the modified LD-BD could be used not only as a vehicle for delivery of antitoxin cargo, but also as a means to deliver other new therapeutics to neuronal cells.

Research Grant: National Institute of Allergy and Infectious Diseases, NIH, R33 AI101504
Student Support: Office of the Director, NIH, T35 OD011145

2015 Research Project Abstracts

Can exogenous insulin improve sperm motility in cooled-stored stallion semen?
Shelby M. Adcock, Robyn Ellerbrock, Lindsey Rothrock, Vitoria Muller, Igor F. Canisso
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL (Adcock, Ellerbrock, Rothrock, Canisso) Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil (Muller)

Unlike many domesticated large animal species, stallions are selected based on athletic and phenotypic characteristics rather than fertility. Stallion semen is often extended, cooled, and shipped to the location of the mare prior to breeding, and sperm motility inevitably decreases as semen ages despite the addition of semen extender. There is active interest in identifying additives to extend the life and improve motility of sperm, to increase the likelihood of successfully impregnating mares. Recent studies in human males showed that insulin may play a role in sperm metabolism and motility. We hypothesized that addition of insulin to stallion semen would have a positive effect on sperm viability and motility, compared to an insulin-negative control. To test this hypothesis, semen was collected from eight stallions using the Missouri artificial vagina for a total of 24 separate ejaculates. Semen extender was used to dilute each ejaculate to a final volume of 50 ml and a concentration of 25 million sperm/ml. The extended ejaculate was divided into three separate 15 ml aliquots. One was designated as the control, 0.25 IU insulin/ml was added to the second and 1.0 IU insulin/ml was added to the third. Aliquots were further divided into three smaller 5-ml aliquots and stored in a passive cooling device for subsequent examination of motility. A computer-aided sperm analysis machine was used for the testing. Motilities were analyzed at 0, 24, 48, and 72 hours post-collection. Completion of this analysis will indicate whether addition of exogenous insulin aids in preservation of semen quality. Positive results would indicate utility of the method for improving mare breeding and production of new foals for the horse industry.

Research Grant: University of Illinois Department of Veterinary Clinical Medicine
Student Support: Merial Veterinary Scholars Program

Evaluating efficacy of hygiene plans in reducing prevalence of M. avium ssp. paratuberculosis in beef cattle
Hilary Antosh, Sara W. McReynolds, and Rebecca Smith
College of Veterinary Medicine, University of Illinois, Urbana, IL (Antosh, Smith), North Dakota Department of Agriculture (McReynolds)

Johne’s Disease (Paratuberculosis) is a chronic, contagious enteric infection that primarily affects cattle and small ruminants. It is caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). Young animals are believed to be most susceptible. Because infected animals typically do not begin shedding the bacterium or showing clinical signs for several years, identifying them is difficult. Current recommendations include improved hygiene practices to minimize disease, particularly in calving areas. The purpose of this study is to determine the longterm effect of improved hygiene interventions on the prevalence of MAP in individual herds. Surveys conducted at multiple beef cattle farms in North Dakota over the course of seven years were gathered and analyzed. Scores that reflected hygiene standards amongst different age groups were tracked over the years and contrasted with the prevalence of MAP in the herd. Preliminary data suggest efficacy of the hygiene interventions because there was a marked decrease in MAP prevalence 2-3 years after the interventions were implemented. Demonstrating that the hygiene interventions are effective would confirm the rationale for their use, and encourage more herd owners to adopt these practices.

Research Grant: Office of Research Infrastructure Programs, NIH Grant Number 8K01OD01968-04
Student Support: Office of the Director, NIH, T35 OD011145

Immersion anesthetics in freshwater stingrays: Comparing the effects of MS-222 and alfaxalone on Potamotrygon
Maryssa Beckman, Tricia Spooner, Allyn C. Meyer, Michelle A. Waltenburg, and Mark Mitchell
College of Veterinary Medicine, University of Illinois, Urbana, IL

Freshwater stingrays (Potamotrygon spp.) are becoming increasingly popular additions to home and institutional aquariums. Consequently, veterinarians may be asked to treat them when medical issues arise. The purpose of this study was to evaluate two different types of immersion anesthetics for freshwater stingrays and develop anesthetic protocols to safely accommodate them for examinations, diagnostic testing, and surgery. Eight juvenile stingrays were used for this crossover study. The average body weight for these animals was 61.2 g (39.5 – 103.6 g). Two different commonly available anesthetics were used: tricaine methanesulfonate (MS-222) and alfaxalone. Stingrays were randomly assigned to two groups. Group 1 received MS-222 first, followed by alfaxalone, while group 2 received alfaxalone first, followed by MS-222. A 2-week wash-out period was used between trials. Baseline heart rate, respiration rate, and response to noxious stimuli were assessed prior to induction and every 3 minutes until the stingrays were recovered. Blood samples for blood gases were taken before induction (baseline), when a surgical level anesthesia was reached, and after full recovery. Average induction times were 2 minutes for MS-222 and 2.5 minutes for alfaxalone. Both anesthetics successfully anesthetized the stingrays to a level deep enough for surgical anesthesia, with full recoveries being achieved, on average, of 12 minutes for MS-222 and 24 minutes for alfaxalone. By comparing blood gas and electrolyte values, induction rates, response to noxious stimuli, and recovery times, we were able to determine that both anesthetics could be used safely in these animals.

Research Grant: None
Student Support: Merial Veterinary Scholars Program

Promotion of malignant osteolysis via secretion of bone resorptive cytokines in canine osteosarcoma
Matthew R. Berry, Holly C. Pondenis, and Timothy M. Fan
College of Veterinary Medicine, University of Illinois, Urbana, IL

Interleukins (IL-1β, IL-6, IL-8, and IL-11) and tumor necrosis factor (TNFα) are inflammatory cytokines that contribute to pathologic bone resorption. Cytokines typically promote bone resorption through RANKL-dependent pathways to enhance osteoclastogenesis. Canine osteosarcoma (OS) is a common bone tumor characterized by focal bone destruction. We hypothesize canine OS cells will produce and secrete IL-1β, IL-6, IL-8, IL-11, and TNFα, and will consequently enhance osteoclastogenesis and bone resorption. The expression and secretion of cytokines were investigated by western blot, immunohistochemistry, and ELISA using four immortalized canine OS cell lines and normal canine osteoblasts. The ability of cytokines to promote osteoclastogenesis is being evaluated by counting osteoclast numbers derived from canine peripheral blood mononuclear cells stimulated with M-CSF, RANKL, and ± cytokine. Our data indicate canine OS cells express IL-1β, IL-6, IL-8, IL-11, and TNFα, however only IL-6 and IL-8 are basally secreted. Furthermore, OS cells secrete markedly greater concentrations of IL-6 and IL-8 compared to normal osteoblasts. The synergies of OS-derived cytokines with RANKL for promoting osteoclastogenesis are being studied. In vivo studies will be conducted to describe the involvement of IL-6 and IL-8 in dogs with OS through the measurement of circulating cytokine levels and localized tumor expression studies. This study suggests that canine OS cells can secrete IL-6 and IL-8, which could promote unregulated bone resorption within the primary tumor microenvironment. As such, small molecule inhibitors of IL-6 and IL-8 may serve as treatment options for slowing tumor growth and alleviating malignant bone pain in dogs with OS.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Merial Veterinary Scholars Program

Effect of combinative treatment with metformin and dichloroacetate in feline carcinoma cell line
Zachary Cowden, Holly Pondenis, and Jacqueline Wypij
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Existing drugs with putative anti-neoplastic activity may be repurposed and brought to clinical manifestation more rapidly than novel drug compounds. Metformin, an anti-diabetic agent that reduces cancer risk in people, shows promise in feline cancer. In cats and people, metformin therapy can result in the development of hyperlactatemia and potential subsequent acidosis. Dichloroacetate (DCA) is one clinical option for treating human hyperlactatemia. Both metformin and DCA have been shown to promote apoptosis in human carcinoma in vitro and DCA may also attenuate metformin-induced lactate production with potential additive cytotoxicity. We hypothesize that metformin and DCA have cytotoxic and metabolic effects in feline carcinomas. Two feline cell lines (CAT-MT mammary carcinoma and SCCF1 oral squamous cell carcinoma) were treated with DCA and metformin at various concentrations. Cell viability/cytotoxicity was monitored with a tetrazolium-based assay, lactate dehydrogenase assay, colony formation assay, and Trypan blue exclusion. Western blotting was used to assess protein expression of total and phosphorylated pyruvate dehydrogenase E1α (PDE1α/p-PDE1α), an enzyme involved in lactate production. Effect on lactate metabolism was assessed via supernatant lactate quantification and pH. Preliminary results demonstrate that 1) metformin and DCA decrease cell viability in a dose-dependent manner, 2) feline carcinomas express PDE1α/p-PDE1α in vitro with modulation by drug therapy, and 3) DCA ameliorates metformin’s decrease in supernatant pH. This study demonstrates anti-neoplastic activity of DCA and metformin in feline cell lines and may lay the groundwork for clinical metformin/DCA combination therapy of feline carcinoma.

Research Grant: J. Wypij, Donor gift funds
Student Support: Merial Veterinary Scholars Program

Effect of pre-plating on tendon-derived progenitor cell enrichment
Anna Cymerman, Sushmitha Durgam and Matthew Stewart
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, University of Illinois, Urbana IL

Interest in cell-based therapy for tendon injuries in horses has grown vastly in the past decade. Accepting the great promise stem cell (SC) therapy holds for improving tissue repair in many contexts, identifying and enriching the small SC numbers in primary samples for ex vivo expansion is important for clinical applications. Several methods have been developed to select stem/progenitor cells from tissue and body fluids. One of them, the pre-plate technique was originally developed for muscle-derived SC isolation and is based on the differential attachment of non-stem (rapid) and SC (slow) to cell culture plastic surfaces. The objective of this study was to determine whether pre-plating enriches for tendon-derived stem cells (TDSC). Immuno-phenotypic characteristics (fluorescent activated cell sorting-FACS) and tri-lineage differentiation capacities of cells obtained at early, mid and late stages of pre-plating were assessed. Donor-matched bone marrow-mesenchymal stem cells (BM-MSCs) were used as control. There were no differences in immuno-phenotype characteristics among the different pre-plates of TDSC and BM-MSCs. FACS analysis showed that more than 90% of cells from each pre-plate were CD29+, CD44+, CD90+ and CD45-. In addition, phenotypic assays confirmed similar adipogenic, chondrogenic and osteogenic differentiation in all TDSC pre-plates. RT-PCR analyses for lineage-specific gene expression were similar among the pre-plates and about 3-4 fold less than BM-MSCs. These data show that pre-plating does not enrich for tendon-derived progenitors. Rather, un-segregated tendon digests are adequate for purification of stem cells via monolayer expansion.

Research Grant: Animal Health and Disease Research Funds
Student Support: Office of the Director, NIH, T35 OD011145

Glowing guts? Exploring the use of fluorescent Candida albicans in a piglet model of oro-GI tract colonization
Price A. Dickson, Soon-Hwan Oh, David A. Coleman and Lois L. Hoyer
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Candida albicans (C.a.) is a commensal fungus of humans. Disruption of normal microbiota or immune function can lead to candidiasis. In its disseminated form, the disease can be life-threatening. The patient’s oro-gastrointestinal tract (oro-GIT) often serves as the source of C.a. in these cases. Reducing a patient’s fungal burden may prevent disseminated disease. The mouse is commonly used for studying C.a. colonization, even though antibacterial pre-treatment is needed for C.a. to colonize the murine oro-GIT. In previous work, we developed a piglet C.a. colonization model that is established simply by feeding C.a. cells to the animals. Quantification of fungal burden is labor-intensive with this model, however. It requires many workers to harvest and homogenize tissues, plate serial dilutions of homogenates, and count the resulting colonies. It is difficult to achieve consistency in methods among individual workers. We hypothesize that use of fluorescent C.a. will improve the piglet model because tissue sections can be viewed microscopically, morphology of fungal cells documented, and fungal burden quantified using image analysis software. A C.a. strain that produces green fluorescent protein and another that produces a near-infrared fluorescent protein were evaluated. In vitro analyses assessed growth and morphology of the strains, and stability of fluorescence following treatment with common tissue fixatives. In vivo experiments investigated piglet colonization, and provided tissue specimens for developing visualization and quantification protocols. A relevant animal model of C.a. colonization will facilitate development of methods to reduce fungal burden in patients at-risk for disseminated candidiasis.

Research Grant: University of Illinois College of Veterinary Medicine
Student Support: Office of the Director, NIH, T35 OD011145

Isoliquiritigenin reduces antral follicle growth and estradiol levels in the mouse ovary
Jacqueline L. Eisner, Liying Gao, Marni E. Gonnering, and Jodi A. Flaws
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Isoliquiritigenin (ISL) is a phytoestrogen that can be extracted from licorice root. ISL has been used as an anti-inflammatory, antimicrobial, and chemotherapeutic agent, but little is known about the effects of this phytoestrogen on the ovary. Thus, the purpose of this study was to test the hypothesis that exposure to ISL affects ovarian antral follicle growth and estradiol levels. To test this hypothesis, antral follicles (260-400 µM) were mechanically isolated from the ovaries of CD-1 mice, and cultured with vehicle control (dimethylsufoxide) or varying doses of ISL (600 nM, 6 µM, 36 µM, and 100 µM) for 48 or 96 hours. The sizes of the follicles were measured every 24 hours. At the end of the cultures, media were collected and subjected to measurements of estradiol using enzyme-linked immunosorbent assays. Exposure to the lower doses of ISL (600 nM, 6-36 µM) did not significantly affect follicle growth at any time point. However, the highest dose of ISL (100 µM) significantly reduced follicle growth at 72 hours (DMSO: 121.72 ± 6.48; ISL 100 µM: 103.10 ± 1.69 % change, n=3, p≤0.05) and 96 hours (DMSO: 145.75 ± 7.41; ISL 100 µM: 101.67 ± 1.58 % change, n=3, p≤0.05) compared to control. Further, the low doses of ISL (600 nM, 6-36 µM) did not significantly impact estradiol levels compared to the control. However, the highest dose of ISL (100 µM) significantly inhibited estradiol levels compared to controls (DMSO: 1996.28 ± 666.31; ISL 100 µM: 134.81 ± 15.06 pg/mL, n=3, p≤0.05). Collectively, these data indicate that ISL at 100 µM, but not at 600 nM-36 µM, reduces antral follicle growth and estradiol production. These data suggest that high doses, but not low doses, of ISL may be ovarian toxicants.

Research Grant: National Institute of Environmental Health Sciences, NIH, R03 ES023972
Student Support: Office of the Director, NIH, T35 OD011145

Not all soy is digested “equol”: equol’s adverse effects on ovarian follicular growth and estradiol production
Marni E. Gonnering, Liying Gao, Jacqueline L. Eisner, and Jodi A. Flaws
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL

Equol is a non-steroidal isoflavone derived from the soy phytoestrogen daidzein by gastrointestinal microbiota. Equol is commonly used as a dietary supplement and as a pharmaceutical agent for hormone imbalances. This lab previously demonstrated that equol exposure for 96 hr inhibits growth and estradiol production in intact antral follicles in vitro. However, it is unknown whether acute exposure to equol has similar effects. We hypothesize that acute equol exposure causes decreased follicular growth, resulting from lower estradiol production. To test this hypothesis, antral follicles from CD-1 mice were mechanically isolated and cultured in vehicle control (dimethylsulfoxide; DMSO) or equol (600 nM, 6 µM, 36 µM, 100 µM) for 48 or 96 hr. Follicle diameters were measured every 24 hr. Upon culture completion, media were collected to measure estradiol levels by enzyme-linked immunosorbent assays. The results indicate that at 48 hr, equol (100µM) did not significantly affect follicular growth, but it significantly increased the production of estradiol compared to the vehicle control (DMSO: 118±34.4 pg/mL; 100 µM equol: 369.1±35.6 pg/mL; n=3, p=0.001). At 72-96 hr, equol significantly decreased follicular growth (72 hr DMSO: 138.5±2.6 µm; 72 hr 100 µM equol: 111.9±5.1 µm; n=3; p=0.015; 96 hr DMSO: 156.1±4 µm; 96 hr 100 µM equol: 121.5±3.6 µm; n=3; p=0.002). At 96 hr, equol (100 µM) also significantly decreased estradiol production compared to the vehicle control (DMSO: 6203.9±1050.6 pg/mL; 100 µM equol: 1803.5±265.7 pg/mL; n=3; p=0.033). These results indicate that short-term exposure to equol (24-48 hr) does not inhibit follicle growth, but longer-term exposure (72-96 hr) inhibits follicle growth and estradiol production.

Research Grant:  Office of the Director, NIH, T35 OD011145
Student Support: National Institute of Environmental Health Sciences, NIH, R03 ES023972

The effects of metritis on dairy herd reproduction, production and profitability
Julie Gunter and Fabio Lima
College of Veterinary Medicine, University of Illinois, Urbana, IL

Metritis is a postpartum disease that negatively affects the health and well-being of cows and the sustainability of dairy farms. To date, there have not been controlled studies thoroughly evaluating the economic impact of metritis. We hypothesized that an economic analysis taking into account differences in antibiotic costs, mean time to pregnancy, differences in cure rate between treatments, and decrease in milk production would reveal the least costly approach to treat metritis. To test this hypothesis, cows diagnosed with metritis were blocked by parity and within each block allocated randomly to receive either ampicillin (N=259), or ceftiofur (N=269). Additionally, a no metritis control group matching parity and day in milk was enrolled (N=268) to be used as baseline for comparison. Mean time to pregnancy was analyzed using PROC PHREG and LIFETEST on SAS 9.4, while milk production and cull rate were evaluated using PROC GLIMMIX. While there was no significant difference between cull rates, the mean time to pregnancy was 12 days longer for cows with metritis, and those treated with ampicillin produced nearly 700 pounds of milk more than those treated with ceftiofur. We will account for these differences in a subsequent economic analysis using market prices from the time period in which the study was conducted. Results from this analysis will provide practical guidelines for the management of metritis in dairy herds.

Research Support: University of Illinois College of Veterinary Medicine
Student Support: Merial Veterinary Scholars Program

Does mother know best? Factors that influence habitat attractiveness for Culex pipiens oviposition
Brit’nee Haskins, Allison Gardner, Brian Allan, and Ephantus Muturi
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL (Haskins)
Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL (Gardner, Allan)
Illinois Natural History Survey, Champaign, IL (Muturi)

Management of the mosquito Culex pipiens is crucial to public health because it is the main vector for West Nile Virus (WNV) in the Midwest, a common concern not only for humans, but also for populations of birds and animals. The main predictor for risk of WNV infection is local abundance of vectors; the abundance of these vectors is determined by the attractiveness and quality of larval habitats. Leaf detritus present in these larval habitats is an important factor in creating an ideal environment. A previous study found that leaf infusions of an exotic invasive plant, Lonicera maackii (Amur honeysuckle), and a native plant, Rubus allegheniensis (blackberry), attracted the most female mosquitoes to lay eggs. While honeysuckle provided a high-quality habitat for larval development, blackberry leaves acted as an ecological trap that prevented larvae maturation. This study aims to determine the mechanism that makes these habitats attractive for mosquito oviposition: the microbial community formed by the leaves, or chemical aspects of the leaves. We hypothesized that oviposition behavior is determined largely by the microbial community of the leaf infusions. Binary choice assays were conducted in which field-collected gravid females were put in cages with two cups where each cup contained one of the following treatments: whole leaf infusion of Amur honeysuckle or blackberry leaves, microbes, leaf extract, or deionized water. Egg rafts were counted after 3 days. Findings from this study may allow for effective management of Cx. pipiens and WNV infection incidence. Results from this study may also indicate whether mosquito-attracting leaves can be used to lure mosquitos into certain habitats for insecticide application.

Research Grant: U.S. Environmental Protection Agency Science to Achieve Results (STAR) Fellowship
Student Support: Office of the Director, NIH, T35 OD011145

Does prenatal exposure to di-ethylhexyl phthalate affect spleen cytokine levels in mouse pups and their dams?
Molly K. Houle, Sulalita Chaki, Sidonie N. Lavergne
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL

Di-(ethylhexyl) phthalate (DEHP) is a plasticizer found in numerous products including food containers, toys, and medical equipment. Unfortunately, DEHP can leach out of plastics and bio- accumulate in fats after absorption. This capability represents a significant health issue because phthalates, including DEHP, are associated with endocrine toxicity and neurotoxicity, as well as immunotoxicity. For instance, DEHP acts as an adjuvant that can disrupt normal and pathological immune pathways. Further, the immune system in young animals appears more sensitive to immunotoxicants, especially with in utero exposure. There is also evidence that immune markers and functions can be sex-specific. Yet, very little is known about DEHP developmental immunotoxicity and its potential gender dimorphism. Thus, this project aimed to investigate cytokine levels in the offspring of CD-1 mice that were exposed to DEHP orally throughout pregnancy (0, 20 or 200 ug/kg/day, 500 or 750 mg/kg/day). At various time points (pups at Post- Natal Day 8, PND21, and PND60; dams at PND21), spleens were collected to measure levels of selected cytokines (Th2 markers: IL-4, IL-5, IL-13; Th1 markers: IL-12p70 and IFNγ) using a Luminex assay. Our initial data in the dams suggests an increased inflammatory response towards Th2 > Th1 when exposed to DEHP at 750 mg/kg/day during pregnancy, even three weeks after discontinuation of the toxin. This Th2 imbalance was not observed in pups’ spleens. Interestingly, IL-12 levels increased between PND21 and PND60, suggesting a mounting Th1 response in these juveniles. Further work is underway to analyze any sex differences in our selected markers.

Research Grant: University of Illinois Campus Research Board (RB13207)
Student Support: Department of Comparative Biosciences; NIH/USEPA P01 (ES022848-2, RD835434010)

Risky business: Factors that contribute to the presence of human cases of West Nile virus in the Chicago area
Cassie J. Lothery, Surendra Karki, William M. Brown, and Marilyn O. Ruiz
College of Veterinary Medicine, University of Illinois, Urbana, IL

West Nile virus (WNV) is a mosquito-borne pathogen transmitted to humans via Culex mosquitoes. Transmission is via a dynamic mosquito-bird-mosquito transmission cycle that sometimes spills over to humans, horses, and other mammals. In humans, fewer than 1% of WNV infections develop into a neuro-invasive disease, but 10% of those cases are fatal, and WNV is an ongoing public health concern in the United States. Prevention efforts have focused on active surveillance and reduction of the vector mosquito population, monitoring of reported human cases, and promoting preventive practices, including mosquito repellants and avoiding outdoor activities during peak mosquito activity. Previous studies have identified weather conditions, landscape features and several demographic features as the risk factors for human WNV, but this varies spatially. The human illness reported to public health authorities is a primary indicator of risk in an area, but passive surveillance systems can lead to an incomplete picture of risk and be biased toward specific neighborhoods. Using historical data of human cases and mosquito infection rates in northeastern Illinois during two years of high prevalence of human cases (2005 and 2012) this analysis determined spatial associations between human WNV cases and WNV mosquito infection rate, while also accounting for socio-economic, weather related, and land use factors. The resultant model was then used to identify areas of spatial mismatch between human illness and mosquito infection and to determine the factors that are related to this incongruity. Also reported are the preliminary results of the use of canine surveillance to supplement the human case data as a measure of potential exposure to WNV.

Research Grant: NSF Ecology of Infectious Disease Grant 0429124, Mariangela Segre Student Support Research Grant 2015, Department of Pathobiology, University of Illinois, Urbana, IL
Student Support: Office of the Director, NIH, T35 OD011145

Time to return of righting reflex in Bufo marinus anesthetized with isoflurane, sevoflurane or desflurane
Kaleigh Morrison and Stuart Clark-Price
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL

Volatile anesthetics are commonly used in mammals for general anesthesia. Isoflurane, sevoflurane, and desflurane induce similar states of anesthesia, however they have different solubilities in various tissues resulting in different recovery times. Potency of inhalant anesthetics in non-mammals is expressed as minimum anesthetic concentration (MAC), the concentration of anesthetic required to immobilize 50% of the subjects exposed. Loss of righting reflex (LRR) is commonly used to demonstrate anesthetic effects in non-mammals. It is generally recognized that 1.5 X MAC is necessary to produce an anesthetic plane in 100% of subjects. We tested the hypothesis that there would be a difference in time to return of righting reflex in toads anesthetized with isoflurane, sevoflurane, or desflruane. In a random, cross-over design, 10 toads were anesthetized with each of the anesthetic agents at 1.5 X MACLRR. Time to return of righting reflex was determined from discontinuation of exposure to an anesthetic to when a toad spontaneously returned to sternal position from dorsal recumbency. Median (range) recovery times were 175 min (123-211) for isoflurane, 192 min (116-383) for sevoflurane, and 74 min (52-220) for desflurane. Toads in the desflurane group recovered 2.5 times faster than isoflurane or sevoflurane toads. These findings suggest that the use of desflurane may be a more appropriate choice of anesthetic for amphibian species when a shorter recovery period is desirable.

Research Grant: Clark-Price Anesthesia Laboratory External Unrestricted Gift Funds
Student Support: Merial Veterinary Scholars Program

Battling feline oral squamous cell carcinoma with deoxynyboquinone compounds in combination with radiation
Malgorzata K. Pajak1, 2, Elizabeth I. Parkinson1, Alycen Lundberg2, Holly Pondenis2, Paul J.
Hergenrother1, Timothy Fan2
1University of Illinois at Urbana-Champaign, Department of Chemistry, Roger Adams Laboratory, Urbana, Illinois
2University of Illinois at Urbana-Champaign, Veterinary Medicine Basic Sciences Building, College of Veterinary Medicine, Urbana, Illinois

Oral squamous cell carcinoma (SCC) is the most common malignant tumor in cats. It responds poorly to available therapies including surgery, chemotherapy, and radiation therapy. Novel therapeutics which are effective against this aggressive cancer would be extremely valuable for the treatment of these animals. Additionally, cats are excellent natural models for human head and neck squamous cell carcinoma and treatments that benefit them may also be effective for humans. NQO1, a 2-electron reductase commonly overexpressed in solid tumors, is typically responsible for the detoxification of quinones. However, reduction of certain quinones such as deoxynyboquinone (DNQ) by NQO1 leads to an unstable hydroquinone that generates reactive oxygen species (ROS) resulting in selective cancer cell death. We show here that two out of three feline SCC cell lines were sensitive to treatment with DNQ and IB-DNQ (isobutyl DNQ). Western blot confirmed that the sensitive cell lines overexpressed NQO1 while the insensitive cell line did not. Additionally, studies using NQO1 inhibitors confirmed the dependence of compound activity on NQO1. The resistant cell line was exposed to ionizing radiation in order to induce NQO1 expression and increase its sensitivity to DNQ. Together these results showed that feline SCC was sensitive to the deoxynyboquinone class of compounds in cell culture and new treatments involving IB-DNQ or DNQ may be effective either alone or in combination with other therapeutic strategies.

Research Grant: University of Illinois Department of Chemistry
Student Support: Office of the Director, NIH, T35 OD011145

Development of a PEDV transmission model for live haul transport at swine lairage facilities
Alyssa Storino, Ryan McCann, Dennis French and James Lowe
Integrated Food Animal Medicine Systems, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL

Porcine Epidemic Diarrhea Virus (PEDV) is an alpha coronavirus that is found globally. It was introduced into the United States in 2013, causing high neonatal mortality. Previous studies showed that live animal transport to harvest plants is a key potential route of transmission during an outbreak. We developed a model to replicate the process of unloading pigs into lairage. The model allowed us to test the effects of several environmental and management variables on PEDV transmission and survival. The trailer was modeled using an aluminum sheet; a plastic tub filled with a mix of manure and shavings was used to simulate the unloading dock. A foot contact event was mimicked using a clean plastic boot to step on the model dock and directly onto the model trailer. Variables such as conditions on the dock (temperature, UV light, scraping, PEDV concentration) and in the trailer (temperature, humidity) were tested. PEDV was detected pre- and post-contamination using real-time polymerase chain reaction (RT-PCR). In trial 1 we investigated the impact of trailer temperature on PEDV persistence over 1 hour. Trial 2 focused on dock conditions including scraping, temperature, UV light intensity and time post-contamination on PEDV contamination in the trailer at 1 hour post contact. Under the conditions of the study, transmission of PEDV was highly efficient as PEDV RT-PCR CT values were similar on the dock, at time 0 and at 1 hour post contact on the trailer. The results of trial 2 are pending. This model confirms that lairage is a significant risk for the dissemination of PEDV between swine production sites.

Research Grant: National Pork Board Grant #14-266
Student Support: Merial Veterinary Scholars Program

The effect of low-dose aspirin on the primary feline urinary thromboxane metabolite
Jason Tarricone, Maureen McMichael, Brittney Heggem-Perry, Stephanie A. Smith
College of Veterinary Medicine, University of Illinois, Urbana, IL

Thrombus formation is a common, life-threatening sequela to heart failure in cats and is, in part, driven by the arachadonic acid (AA) pathway, where inflammation results in the production of eicosanoids. Thromboxane A2 (TXA2), the leading eicosanoid participating in thrombus formation, undergoes rapid, non-enzymatic conversion to thromboxane B2 (TXB2) in vivo making it difficult to quantify. TXB2 is enzymatically metabolized to 11-dehydro-thromboxane B2 (11-dehyrdroTXB2) and 2,3-dinorTXB2. There is a species-specific preference for one metabolite over the other. In humans and cats, 11-dehydroTXB2 is the primary metabolite whereas in dogs 2,3-dinorTXB2 is the major metabolite. Urinary concentration of the primary metabolite is representative of platelet activity in humans and dogs. Aspirin is a readily available NSAID known to irreversibly block thromboxane A2 formation in platelets via the cyclooxygenase pathway. Low-dose (1mg/kg q24h) aspirin is administered as a thromboprophylaxis in humans. The advantage of the dose is that it minimizes systemic side effects. The effectiveness of the 1 mg/kg dose is unknown in cats. Eight male neutered purpose-bred cats were administered approximately 1 mg/kg of aspirin every 24 hours for 2 days, and after a 7 day washout period they were given 1 mg/kg q24h for 5 days. Urine was collected at time 0, 48h, 72h, 120h post administration. Creatinine corrected urinary 11-dehydroTXB2 concentrations were measured by a commercially available ELISA. Decrease in the concentration of urinary 11-dehyrdroTXB2 with the low-dose aspirin makes this a convenient test for aspirin effectiveness. If identified, this relationship could be important in changing the management of cats with heart failure.

Research Grant: Morris Animal Foundation
Student Support: Merial Veterinary Scholars Program

The effects of perinatal propylthiouracil exposure on novel object and location recognition in Long-Evans rats
Catherine A. Townes, Megan L. Sieg, Rekha C. Balachandran, Mary E. Gilbert, Paul A. Eubig
Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL (Townes, Sieg, Balachandran, Eubig)
U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Toxicity Assessment Division, Research Triangle Park, NC (Gilbert)

Developmental exposure of rats to higher doses of the anti-thyroid drug, propylthiouracil (PTU), results in persistent neurodevelopmental effects. PTU inhibits enzymes required for the synthesis of thyroid hormone (TH), and is transplacentally and lactationally transmitted to offspring. Therefore, perinatal PTU exposure is a potentially useful model for examining the effects and mechanisms of environmental contaminants that perturb developmental thyroid homeostasis. However, research using more subtle PTU exposures could yield a more accurate model of human developmental hypothyroidism. In this study, we compared cognitive performance of offspring of both sexes from Long-Evans rat dams exposed to PTU prenatally, postnatally, or during both stages, with offspring of unexposed dams. Recognition and spatial memory were evaluated using two well-established tasks: novel-object recognition (NOR) and novel-location recognition (NLR). Over 15 days, rats were placed in an arena with 2 identical objects and removed 3 minutes later. Then one object was either exchanged with a different object (NOR) or moved (NLR), and rats were placed back into the arena after 1, 4, or 24 hours. We measured time spent exploring and number of visits to the novel versus familiar object or location as indices of recognition. We hypothesize that rats exposed to PTU will show recognition and spatial memory deficits, and that these deficits may depend upon age of PTU exposure. Experimental results are pending. These results could identify sensitive periods for the behavioral effects of developmental hypothyroidism, and could improve our understanding of the role of thyroid hormone in neurodevelopment. Does not reflect EPA policy.

Research grant: National Institute of Environmental Health Sciences, NIH, K08 ES017045
Student support: Office of the Director, NIH, T35 OD011145

Characterization of immunological gene expression in the intestine of healthy calves
Kayla H Wade, Elena Bichi, Brian M Aldridge
College of Veterinary Medicine, University of Illinois, Urbana, IL

Gastrointestinal (GI) disease is a cause of morbidity and economic loss in young livestock. Understanding the pathophysiological significance of each element of the gut health ‘triad’ (immune system, microbiome, epithelium) will be useful in designing strategies to improve GI health. In this study, we characterized mucosal inflammatory gene expression (IGE) profiles at different biogeographical sites of the healthy calf intestine during the first 3 weeks of life. Twelve calves from second parity cows on a single commercial dairy were removed from their dams upon delivery and fed clean, high quality colostrum. Calves were fed milk replacer and health-monitored daily. A subset of calves (n=3) were euthanized at days 1, 3, 7, and 21, and mucosal tissue samples were taken from the duodenum, jejunum, ileum, and colon. Quantitative PCR analysis was performed to quantify the expression of interleukin 10 (IL10), toll-like receptors (TLR) 2, 4, and 10, and tumor necrosis factor-α (TNF-α) genes. There was a correspondingly low level of IGE at all intestinal locations and time points. The ileal mucosa showed a significantly higher pattern of IGE than all other sites (p< 0.05) for IL10 and TNF-α. TLR 4 had a three-fold higher expression than all other genes. As expected, the IGE profile of this calf group was subdued. There appears to be higher inflammatory activation in ileal mucosa, possibly due to the relative abundance of Peyer’s patches. In future studies, we will compare the IGE profiles with the intestinal microbiota and epithelial function of sick and healthy calves. Gaining an understanding of the immune system and microbial population could lead to management solutions for GI disease and reduce the use of antimicrobials.

Research Grant – National Institute of Food and Agriculture, U.S. Department of Agriculture, award number 1002450
Student Support – Merial Veterinary Scholars Program

The effect of FOXA2 inactivation on mucus overproduction in canine respiratory infections
Michelle Waltenburg, Woosuk Choi, Brendan McKiernan, Richard Fredrickson, Elizabeth Driskell, Carol Maddox and Gee W. Lau
College of Veterinary Medicine, University of Illinois, Urbana, IL

Dogs naturally develop several pulmonary diseases similar to humans. Older dogs, especially of small breeds, develop infectious canine tracheobronchitis (kennel cough), chronic bronchitis (CB), and chronic obstructive pulmonary disease (COPD) with excessive mucus due to chronic exposure to environmental pollutants or infectious agents.  Two previous clinical studies involving 766 canine lower respiratory tract infection cases showed that major pathogens included those on the ESKAPE list: Pseudomonas aeruginosaStaphylococcusE. coli + other Enterobacteriaceae and Enterococcus. FOXA2 is a key transcriptional regulator that maintains airway mucus at healthy levels. FOXA2 is inactivated in human airways with respiratory infection, resulting in excessive mucus. Previously, our laboratory demonstrated that pyocyanin (PCN), a virulence factor of P. aeruginosa, causes goblet cell hyperplasia and metaplasia and mucus hypersecretion by repressing the expression of FOXA2. However, the role of FOXA2 regulation in canine respiratory diseases remains unexplored. In this study, we retrospectively examined cases of canine respiratory infection received by the University of Illinois Veterinary Diagnostic Laboratory. We also used immortalized dog bronchial epithelial BACA cell lines to examine the mechanism of FOXA2 inactivation by PCN, in a time and concentration-dependent manner. Finally, we examined bronchial sections from dogs with CB and COPD by immunohistochemistry to determine whether microbial pathogens found in these airways inactivate FOXA2. The results of these studies will reveal the contribution of FOXA2 inactivation by microbial pathogens to excessive mucus in canine respiratory diseases.

Research Support: American Lung Association DeSouza Research Award (DS-192835-N) and NIH (HL090699) to Gee W. Lau
Student Support: Office of the Director, NIH, T35 OD011145

2014 Research Project Abstracts

Outbreak Modeling of an Amphibian and Reptilian Emerging Infectious Disease
A.C. Barthel, R.C. Lacy, and M.C. Allender
College of Veterinary Medicine, University of Illinois, Urbana, Illinois

Ranavirus has caused mass mortality events in wildlife populations of reptiles and amphibians worldwide, and is proposed as a significant threat to biodiversity. It is a World Organization for Animal Health (OIE) reportable disease known to infect fish, amphibians, and reptiles with the potential for interclass transmission via skin-skin contact, ingestion of diseased tissue, or through contact with contaminated water. Local epizootics have led to outbreak mortality rates as high as 90-100%; hence, Ranavirus can be catastrophic if introduced to a local community of ectotherms. In particular, free-ranging Eastern box turtle populations (Terrapene carolina carolina) have demonstrated increased susceptibility to Ranavirus with mass mortality events. Utilizing data from a long-term surveillance study of a closed population of T. carolina carolina in Tennessee, population viability analysis (PVA) will be completed. PVAs bring together ecology and statistics to provide information on population health, stability, and extinction risk under various conditions. The Tennessee focus population has undergone thorough mark-recapture collection and monitoring for the past eight years, resulting in population estimates of 1655 individuals. Outbreak scenarios will be modeled in the presence of a Ranavirus outbreak by meta-modeling utilizing both the software Vortex for individual-based demographic modeling and the software Outbreak for epidemiological modeling. Model estimates will be used to influence management decisions that aim to minimize the impact of this disease on biodiversity.

Student support: Merial Veterinary Scholars Program
Research support: Wildlife Epidemiology Laboratory, University of Illinois

West Nile Virus Risk Perceptions and Preventative Behavior
Claire Behnke and Marilyn O’Hara Ruiz
College of Veterinary Medicine, University of Illinois, Urbana, IL

Public health efforts to reduce mosquito-borne disease originated with the work of Dr. Ronald Ross in the late 1880s, who recognized the role of mosquitoes in transmission of malaria. Through his work and that of others who followed, epidemiologists developed models to show that the control of malaria could be achieved through mosquito control. The evidence from these experiences led to the creation of mosquito-control programs across the United States in the early 1900s. By 2002, with the introduction of West Nile Virus (WNV) into Illinois, local mosquito-control programs were challenged to mitigate this novel disease, and both organizational and epidemiological impediments became apparent. In DuPage County, Illinois, as many as 45 different entities carry out mosquito control at an institutional level. For many decades prior to WNV in the region, many of these mosquito-control programs focused on control of nuisance mosquitoes, rather than disease vectors. The objective of this project was to evaluate risk of WNV, control strategies and public health responses to vector mosquitoes in DuPage County, in the Chicago metropolitan region. The suburban environment of DuPage County provides ideal breeding habitats for Culex species mosquitoes, which are the main vector for WNV. Between 2002 and 2013, there were 231 human WNV cases in the County. The different entities governing mosquito control in the County differ in the level of government at which they are organized and by characteristics of the land that they cover. By comparing data from interviews with personnel involved in governing mosquito control, and quantitative mosquito infection and human infection numbers within each entity, this project examines surveillance methods and resulting perceptions of WNV risk in thesedifferent entities in order to understand mosquito control decisions and their effect on WNV mitigation across the County.

Student support: Office of the Director, NIH, T35 OD011145
Research support: Wheaton Mosquito Abatement District

Effects of Isoflurane on the Hematology of Wild Raptors Following Serial Anesthetic Episodes
Kyra Berg, Kelly Wiggen, Julia K. Whittington, and Mark A. Mitchell
College of Veterinary Medicine, University of Illinois, Urbana, IL

Isoflurane is an inhalant anesthetic agent commonly used in veterinary medicine. Volatile inhalant anesthetic agents, like isoflurane, have significant effects on multiple biological parameters in anesthetized patients. Hematological effects include changes in total cell counts and circulating cell distribution. Clinical management of free-living raptors is complicated by the need to minimize stress and to provide secure restraint to avoid potential injury to both patient and handler. Inhalant anesthesia is often implemented in these patients to facilitate medical and surgical treatments. A previous study found no significant difference in hematological parameters between manually restrained owls and anesthetized owls. The purpose of the current study was to evaluate the hematological effects of isoflurane anesthesia in owls during serial administration. Healthy native North American owls that were presented to the University of Illinois Wildlife Medical Clinic were anesthetized for 15 minutes on 3 consecutive days. Blood was collected at times 0 and 15 minutes. Blood was collected within 3 minutes of handling to prevent any confounding factors caused by stress-induced catecholamine release. A final sample was collected 24 hours after the last anesthetic episode to detect effects on the leukogram that can be delayed 24 hours or longer. Preliminary results demonstrated an overall leukopenia characterized by heterophilia and lymphopenia. This result is similar to the hematological changes that are often attributed to a stress response in birds. It is undetermined how the leukopenia affects immune function in these patients. In all cases to date, the white blood cell count did not return to pre-anesthetic levels within 24 hours. Our results suggest that owls requiring serial administration of isoflurane anesthesia may be at increased risk for immune compromise and opportunistic infections.

Student support: University of Illinois Wildlife Medical Clinic
Research support: National Wildlife Rehabilitators Association

Effects of Thiostrepton on the FOXM1 Transcriptional Pathway in Canine Lymphoma
S. Goldschmidt, H. Pondenis, and J. Wypij
College of Veterinary Medicine, University of Illinois, Urbana, IL

Forkhead box M1 (FOXM1) is a transcription factor regulating cell cycle progression, angiogenesis, and apoptosis. Increased FOXM1 is observed in human lymphoma/leukemia. Thiostrepton, an antibiotic used in commercial veterinary topicals, is a putative FOXM1 inhibitor. We hypothesize that canine lymphoma cells express FOXM1 and that thiostrepton has anti-cancer activity via FOXM1 inhibition. Four different canine lymphoma cell lines (17-71, GL-1, CL-1, and OSW) were treated with thiostrepton (0-100μM). Expression of FOXM1 and FOXM1 pathway proteins cyclin B, survivin, and Cdc25 were assessed by Western blotting. Cell viability was monitored using the tetrazolium compound MTS and by Trypan Blue exclusion. Preliminary results demonstrated that FOXM1 and downstream pathway proteins were expressed in canine lymphoma cell lines and that thiostrepton decreased cell viability in the 17-71 cell line in a dose-dependent manner. Follow-up experiments will use flow cytometry to assess cell cycle progression, with an expected arrest at G2/M. Vascular endothelial growth factor (VEGF) will be detected by enzyme-linked immunosorbent assay (ELISA). We predict that thiostrepton would decrease VEGF secretion in vivo, with a concomitant decrease in angiogenesis. Therefore, the FOXM1 pathway may be a viable target in canine lymphoma and thiostrepton may have anti-cancer activity via FOXM1 inhibition. These results warrant further evaluation of the potential to treat cutaneous lymphoma with topical drugs containing thiostrepton.

Source of student support: J. Wypij, Donor gift funds
Source of research support: J. Wypij, Donor gift funds

Evaluation of Impulsivity, Attention, and Coordination in High-Active Mice: a Model for ADHD
Gabrielle Hofmann, Petra Majdak, and Justin Rhodes
College of Veterinary Medicine and Department of Psychology, University of Illinois, Urbana, IL

Attention deficit-hyperactivity disorder (ADHD) is a common developmental disorder characterized by increased levels of hyperactivity, impulsivity and inattention. Though ADHD is highly heritable, the specific risk factors and genes involved largely remain undefined, in part due to lack of animal models. One emerging neurobiological correlate of ADHD is decreased cerebellar volume. From an initial population of eight diverse mouse strains, a High-Active line was selectively bred for high locomotor activity. The purpose of these experiments was to evaluate the High-Active line as a potential model for ADHD. Previous studies indicated that low doses of amphetamine (the active ingredient in Adderall, a common ADHD medication) decrease the activity of High-Active mice while paradoxically increasing the activity of randomly bred Control mice, encouraging further examination of the High-Active line as an ADHD model. We evaluated attention with the Y-maze paradigm, in which intact attentional capabilities prompt spontaneous alternation behavior. High-Active mice tend to exhibit lower percentages of spontaneous alternations during the first 20 arm entries than Control mice. We indirectly assessed cerebellar function (coordination) via performance on the accelerating rotarod. High-Active mice performed significantly worse than Controls, with lower average and maximum latency to fall. We examined impulsivity by quantifying nose-pokes during the Go/No-go task. We administered amphetamine or saline to High-Active and Control mice to determine whether amphetamines ameliorate the impulsivity inherent in High-Active mice, as they do for many humans with ADHD. We expect that High-Active mice will display more inappropriate nose pokes than controls, but that this trend will be less significant in High-Active mice treated with amphetamine. Use of this animal model will advance our understanding of the risk factors and genes involved in ADHD.

Student Funding: Office of the Director, National Institutes of Health, T35 OD011145
Research Funding: National Institutes of Health, R01 MH083807 and R01 DA027487

Effects of Water Management and Quality on the Ecology of Vector Mosquitoes
Matt Holland, Andrew Mackay, and Brian Allan
College of Veterinary Medicine and Department of Entomology, University of Illinois, Urbana, IL

In urban environments, stormwater management infrastructure provides an abundance of aquatic habitats that are utilized by Culex restuans and Culex pipiens, the primary vectors of West Nile Virus in Illinois. Habitats enriched with decomposing plant detritus can greatly affect the potential to produce these mosquitoes. The overall objective of this study was to examine how two common plant substrates, turfgrass and cattail, influence the potential of aquatic habitats to be colonized by, and to support the juvenile development of, vector mosquitoes. The study tested two hypotheses: 1) oviposition site selection is influenced by the type and amount of plant substrate enrichment in aquatic habitats, and 2) oviposition site selection by Cx. restuans is positively associated with the capacity of these habitats to support juvenile development. We first performed a field assay to assess oviposition response. At five study sites in Urbana, IL, oviposition of Cx. restuans and Cx. pipiens in ovitraps representing seven (three turfgrass, four cattail) discrete densities were randomly assigned to sheltered locations at least 10 m apart along the margins of woody vegetation. We measured Cx. restuans egg quantity of each ovitrap each day for three weeks. In a parallel laboratory experiment, we evaluated how the potential of these type of decaying plant infusions (turfgrass vs. cattail) and concentration of plant substrate present in the aquatic environment influences juvenile development of Cx. restuans and their effects on adult Cx. pipiens mosquito fitness. Preliminary data from these experiments support the hypotheses. Preventing juvenile mosquito development is considered one of the most effective strategies for reducing adult mosquito abundance and public health risk due to mosquito-borne disease. Municipalities may use results from our experiment in their urban mosquito control programs.

Student Support: Office of the Director, NIH, T35 OD011145
Research Support: Illinois Water Resources Center, U.S. Geological Survey

Analysis of Putative Serum Biomarkers in Canine Multicentric Lymphoma
K.A. Jacobs, H. Pondenis, and J. Wypij
College of Veterinary Medicine, University of Illinois, Urbana, IL

Canine multicentric lymphoma is one of the most common types of canine cancer. Current methods to predict survival time, guide treatment choices, and determine remission status are based on clinical staging tests that are limited in predicting outcome. Thus, there is a need for alternative methods to differentiate patient status in a clinical setting. Molecular markers including serum levels of interleukin 10 (IL-10), IP-10, LR-11 (SorLA) and IL-2R are biomarkers in human lymphoma/leukemia. We hypothesize that these proteins will also serve as biomarkers for canine lymphoma. Our experimental plan involves using Western blots to evaluate basal protein expression in four canine lymphoma cell lines: CL-1, GL-1, 17-71 and OSW. Enzyme-linked immunosorbent assays (ELISA) will be used to compare abundance of the four putative biomarkers in serum samples from dogs diagnosed with varying stages of lymphoma, compared to normal dogs. This work may identify a differential, measurable biomarker in canine serum that will aid disease identification and prognosis.

Student support: Merial Veterinary Scholars Program
Research support: Donor Gift Funds

Contamination Rate of a Battery Operated Drill During Orthopedic Surgery
J. Karnia1, T.A.M. Harper1, J.J. Biskup2, and K.S. Aulakh1
College of Veterinary Medicine, 1University of Illinois, Urbana, IL and 2University of Tennessee, Knoxville, TN

Background: Surgical site infections (SSIs) are a major concern for surgeons as they can result in increased duration of hospital stay, increased patient morbidity and increased overall cost to the client. There are different potential sources of contamination leading to SSIs, including surgical instruments.
Methods: In this clinical study, samples for aerobic bacterial culture were collected as follows: sterile saline (to moisten swabs prior to sampling); drill handle (battery casing) prior to draping the patient, battery, drill handle after battery placement and drill handle after completion of surgery.
Preliminary Results: 55 samples were collected from 11 cases. The mean age of the patients was 3.5 years with 3 males and 8 females. The most common diagnosis was cranial cruciate ligament rupture (45.5%), and the most common procedure was tibial plateau leveling osteotomy (36.4%). Six out of eleven (54.5%) cases had bacterial growth at some point during the procedure, and 10/55 (18.2%) samples were positive for bacteria. Staphyloccus spp., Bacillus spp., and Corynebacterium spp. were recovered, with Staphylococcus spp. being the most common (60.0%). Three out of eleven (27.3%) cases had bacterial growth on the drill handle upon completion of surgery, although only two of these cases had a previous positive culture. Three out of eleven (27.3%) cases had positive cultures from the battery, and only one case had a positive culture at the end of surgery. Susceptibility results for bacteria identified are pending.
Conclusions: Bacterial contamination of the drill handle in this study was 27.3%. Loading of non-sterile batteries may not be a cause of contamination of the drill during orthopedic surgery.

Research support: College of Veterinary Medicine

Synergistic Enhancement of Anticancer Activity with DNQ and glu4j
A. Lee, B. Parkinson, H.Y. Lee, and P.J. Hergenrother
College of Veterinary Medicine and Department of Chemistry, University of Illinois, Urbana, IL

Drug combination therapies are often utilized in the clinic for anticancer regimens to exploit drug synergy. Synergy occurs when drug combinations are more efficacious than the sum of their individual components. Deoxynyboquinone (DNQ) is a NQO1 substrate that is reduced in cancer cells to a hydroquinone that undergoes spontaneous reduction-oxidation cycling producing toxic reactive oxygen species that cause cell death. Glu4j is a lactate dehydrogenase inhibitor that kills cancer cells by depriving them of ATP. Because both NQO1 and lactate dehydrogenase are overexpressed in cancer cells, DNQ and glu4j have potential to serve as highly notable anticancer compounds due to their selectivity. Previous in vitro experiments demonstrated significant synergy between DNQ and glu4j in non-small cell lung cancer cell line A549. Current work discovered a similar synergy between these compounds when tested using a breast cancer cell line. These results demonstrate a powerful synergistic effect between DNQ and glu4j across multiple types of cancer. In vivo efficacy studies will be conducted to determine the synergistic effects of DNQ and glu4j in vivo, using murine models xenografted with A549.

Student support: Office of the Director, NIH, T35 OD011145
Research support: University of Illinois

Exploring TrkA Signaling in Canine Osteosarcoma Cell Proliferation and Survival
Alison R. Masyr, Holly C. Pondenis, and Timothy M. Fan
College of Veterinary Medicine, University of Illinois, Urbana, IL

Tropomyosin-related kinase A (TrkA) and its ligand, nerve growth factor (NGF) are involved in neuronal growth, differentiation, and maintenance. Additionally, TrkA and NGF are expressed in other cell types (e.g. developing osteoblasts). Autocrine and paracrine signaling of the TrkA/NGF axis has an antiapoptotic effect in these bone forming cells, leading to tumorigenesis. AZ-23, a highly selective inhibitor of TrkA, is novel in its oral bioavailability. This study sought to determine if AZ-23 hinders proliferation and antiapoptotic pathways in canine osteosarcoma (OSA) cells. All methodologies were performed in vitro. The presence of TrkA in canine OSA cell lines was verified through western blotting. The effect of AZ-23 on cell proliferation was investigated using commercially available assays. The effect of AZ-23 on TrkA phosphorylation was evaluated through western blotting. Across five cell lines, one human OSA and four canine OSA, all expressed TrkA as well as the other two members of its receptor family, TrkB and TrkC. AZ-23 did not have a significant inhibitory effect on cell proliferation in any of these lines. To determine the effect of AZ-23 on TrkA phosphorylation, preliminary experiments were conducted to first establish that the presence of NGF leads to TrkA activation. OSA cells will be stimulated with human recombinant NGF and lysed for use in a western blot. Once the receptor is phosphorylated, AZ-23 will be added with NGF. Cells will be collected and evaluated in the same manner. It is anticipated that the addition of AZ-23 will reduce phosphorylation, perhaps dose-dependently. The results of this study continue to explore the implications of a novel and potentially powerful inhibitor of the TrkA/NGF axis in canine OSA cells. AZ-23 did not inhibit the unregulated proliferation for which cancer cells have gained notoriety. However, AZ-23 may inhibit TrkA phosphorylation and potentially impede the downstream antiapoptotic effects of the receptor.

Student Support: Office of the Director, NIH, T35 OD011145
Research Support: College of Veterinary Medicine

The Effects of Prenatal Di(2-ethylhexyl) Phthalate Exposure on MHC II Expression in the Mouse Spleen
Kelly M Patchett and Sidonie N Lavergne
College of Veterinary Medicine, University of Illinois, Urbana, IL

Background: Phthalates are widely used industrial chemicals that are found ubiquitously, including vinyl floors, plastic kitchenware, food packaging, cosmetics, shoes, toys, and medical devices. They are not chemically bound to the products in which they are found, and thus leach into food, water, and air. Di(2-ethylhexyl) phthalate (DEHP), the most commonly used phthalate, causes developmental and reproductive toxicity. DEHP also appears to affect the immune system, but little is known about this toxic effect.

Objective: The goal of this study was to evaluate the effect of prenatal exposure to DEHP on the Major Histocompatibility Complex class II (MHC II) expression in mouse spleens. MHC II was used as a marker of inflammatory status and innate immune activation.
Methods: DEHP doses were administered orally to the dams throughout pregnancy as follows: 20 μg/kg/day; 200 μg/kg/day; 200 mg/kg/day; 500 mg/kg/day; 750 mg/kg/day; or corn oil (vehicle). The pups were humanely euthanized at post natal day (PND) 1, 8, 21, and 60, and dams at PND 21. Spleens from both male and female mice of each PND group were collected, immediately frozen in liquid nitrogen, and stored at -80°C. Western immunoblotting will be performed using a mouse anti-rat MHC II monoclonal antibody that cross-reacts with the mouse protein (AbDSerotec).
Results: Our results will indicate whether prenatal exposure to DEHP affects neonatal and prepuberty MHC II levels in mouse spleens. Because the study included both males and females, we will also be able to determine whether this effect is sexually dimorphic.
Conclusion: To fully determine the immune effects of DEHP, future studies should explore a variety of inflammatory markers, including cytokines. These markers should be measured in different immune organs, like the thymus or blood, and in other species, possibly the rat or human.

Student support: Merial Veterinary Scholars Program
Research support: University of Illinois Campus Research Board and NIH/USEPA grant NIEHS (ES022848-01) – USEPA (RD 835434010)

What’s in a Personality? The Relationship between Individual Steroid Profiles, Learning and Behavior in Three-spined Stickleback Fish
Laura Pereira, Miles Bensky, Ryan Paitz, and Alison Bell
College of Veterinary Medicine and Department of Animal Biology, University of Illinois, Urbana, IL

Many animals including mammals, fish and birds exhibit consistent individual variation in behavior. In some species, individuals exhibit consistent individual differences in how they respond to changes in the environment, known as coping styles. Some individuals (proactive) are relatively exploratory, aggressive and routinized relative to other individuals (reactive), which tend to pay more attention to environmental cues and are more responsive to changes in the environment. It is thought that differences in coping styles reflect differences in the stress response system. Here, we evaluate evidence for the proactive-reactive axis in three-spined stickleback fish (Gasterosteus aculeatus), a species well-known for its consistent individual variation in behavior. We repeatedly measured the steroid production, exploratory behavior, antipredator behavior and performance in a learning assay on individual fish. We observed the time it took each individual to emerge from a shelter and counted the number of unique areas they visited in a pool, both before and after an attack by a model predator. The learning assay involved training each fish a color discrimination task, and then conducting reversal learning trials once the initial learning criterion was met. Preliminary analyses of the data show that individuals that emerged faster from a refuge were more exploratory of a novel environment both before and after a model predator was present. If stickleback behavior is consistent with the proactive-reactive axis, we predict that highly exploratory individuals will perform relatively well on the initial discrimination task, but then struggle with the reversal learning. We also predict that proactive individuals will have high levels of androgens and relatively low levels of glucocorticoids, while reactive individuals will have cortisol and relatively low androgens. This research may show a relationship between steroid production, learning and behavior.

Publication: Testing the predictions of coping styles theory in threespined sticklebacks
Student support: Office of the Director, NIH, T35 OD011145
Research support: NIH R01 GM082937 and NSF IOS 1121980

Host Factors Contributing to Restriction of Canine Influenza Virus Infection in Canine Respiratory Tissue Explants
A.E. Pohly and E.A. Driskell
College of Veterinary Medicine, University of Illinois, Urbana, IL

Influenza virus is widely studied because of its high impact in endemic populations and broad range of host species. Canine influenza virus has made a recent evolutionary jump from equine influenza virus, providing a model for the study of viral mutation and transmission. In a previous study, we observed that canine influenza virus replicated poorly in tracheal explant tissues compared to nasal and bronchial explant tissues. In the current study, we examined the effects of local mucus production on efficiency of viral infection in different canine respiratory tissues. Because mucus glycoproteins are similar to those on the cell surface receptor of the virus, mucus binds the virus and prevents its attachment to the host. Therefore, we hypothesized that virus would not replicate as well in areas that had a greater amount of mucus production. Using PAS/Alcian Blue staining, we compared the ratio of surface area of mucus (µm2) to length of basement membrane within the respiratory epithelium of nasal, tracheal, and bronchial tissues 1 hour, 24 hours, and 48 hours post infection with canine influenza virus isolate A/Ca/CO/6723-14/08. Our results showed that 3 of 5 dogs had higher mucus levels in their bronchial tissue compared to nasal tissue suggesting other factors play a role in influenza virus’s infection of the host. We will explore induction of the innate immune response to viral infection using ELISA Canine Immunoassays, quantifying the production of IFN-γ, IL-6, and IL-8 in the inoculated canine respiratory explants 1 hour, 24 hours, and 48 hours post infection. Correlation between differences in cytokine levels and a robust or poor viral replication in the tissue may allow for development of diagnostics or treatments for canine influenza virus infections.

Student support: Merial Veterinary Scholars Program
Research Support: Department of Pathobiology

Sex Differences in Attention Measured in Rats by the 5-Choice Serial Reaction Time Task
Audrey Robertson, Rekha Balachandran, and Paul Eubig
College of Veterinary Medicine, University of Illinois, Urbana, IL

The ability to pay attention is a vital aspect of cognitive functioning, but impaired attention is a key deficit in neurobehavioral disorders such as attention deficit hyperactivity disorder (ADHD). Sex differences affect the manifestation of attention-related disorders such as ADHD. While ADHD is more common in males, males exhibit a more impulsive phenotype as opposed to a more inattentive phenotype that females exhibit. The 5-Choice Serial Reaction Time Task (5-CSRTT) is a well-established behavioral model for measuring different aspects of attention. Baseline performance of this task requires rats to identify a light cue in one of five ports and respond with a nose-poke in the correct port within a given time. This study (6 Long-Evans rats of each sex) served as a pilot in which this task was implemented in our laboratory and sex differences in performance were examined. Duration of cue stimulus, inter-trial interval, and the presence of a distractor were individually manipulated throughout the study to increase the difficulty of the task and the type of attention to be measured. The parameters evaluated in this study included percent accuracy, percent of correct and incorrect responses, percent premature responses, and the number of perseverative responses. We hypothesized that there would be sex differences in performance across these parameters. With baseline testing conditions, preliminary analyses suggested that males performed better than females with greater percent accuracy and greater percent correct responses. Females had greater percent of omitted responses, meaning they failed to respond within a given time after the cue stimulus, and a greater percent incorrect. Females had a greater latency to respond both correctly and incorrectly. This information about sex differences in 5-CSRTT performance is important for planned studies to investigate influences of environmental factors on attention.

Student support: Office of the Director, NIH, T35 OD011145
Research support: National Institute of Environmental Health Sciences, NIH, K08 ES017045

Characterizing the Role of Blanding’s Turtle in the Epidemiology of Leptospira spp.
Kelly E Rockwell, Dan Thompson, Carol Maddox, and Mark A Mitchell
College of Veterinary Medicine, University of Illinois, Urbana, IL and DuPage County Forest Preserve, Glen Ellyn, IL

Leptospira spp. is a bacterial pathogen that is ubiquitous in nature and considered a re-emerging zoonotic disease. Previous research found that Blanding’s turtles experimentally infected with Leptospira interrogans serovars became leptospiremic and shed leptospires in their urine, suggesting that they could play an important role in the transmission of leptospires in an aquatic ecosystem. The purpose of this study was to determine whether Blanding’s turtles could become infected with an actively shed Leptospira spp. under natural conditions. The hypotheses tested were that: 1) Blanding’s turtles would actively shed leptospires in their urine and 2) that infected turtles would show no clinical signs of disease. A cross-sectional study was performed using a population of Blanding’s turtles previously found to be exposed to Leptospira spp. within the Forest Preserve District of DuPage County, IL. Thirty-four turtles were sampled for the study. Each turtle was given a physical exam to assess its general health status. Blood samples were collected from the jugular vein or subcarapacial vein for complete blood counts, chemistry profiles, and serologic testing. Free catch urine produced during the examination was collected for polymerase chain reaction (PCR) testing. All 34 of the turtles were seropositive for Leptospira spp. and 24 (70.6%, 95% Confidence interval: 55.1-85.9) urine samples were PCR positive. All of the turtles in this study appeared clinically healthy and showed no apparent signs of infection or disease. The results of this study suggest that the majority of Blanding’s turtles shed Leptospira spp., making them a significant reservoir for this important pathogen.

Student support: Morris Animal Foundation
Research support: Fluker Farms, Port Allen, LA

Impact of Phthalates on Emotional Behavior in Mice
S. Scholz, J.S. Kim, and M.M. Mahoney
College of Veterinary Medicine, University of Illinois, Urbana, IL

Phthalates are ubiquitous synthetic chemicals with a wide array of applications. Commonly referred to as plasticizers, phthalates are present in oral drugs, insect repellants, food storage containers, and baby and pet toys. Phthalates are part of a larger group of endocrine disrupting chemicals that can mimic or block endogenous hormones. Phthalates cause deficits in reproductive and developmental processes in both humans and animals. However, very little is known about the effect of phthalates on behavior. Gonadal hormones modulate emotional behaviors, specifically anxiety and depression. Thus, we hypothesize that phthalate mimicry or antagonism of gonadal hormones will affect these behavioral states. In experiment one, adult male and female gonadally intact C57BL/6 (n=7/group) were dosed with oil (control) or the model phthalate at two doses. Di-ethylhexyl phthalate (DEHP) was administered orally as a low dose of 250 µg/kg or a high dose of 250 mg/kg daily. Tests to assess anxiety or depression were administered beginning on day 13. Four different tests were used: forced swim test (day 13), elevated plus maze (day 15), burrowing (day 16), and open field test (day 17). Mice were then gonadectomized and after recovery, DEHP treatment and tests were repeated as in experiment one. Preliminary data analysis suggested that intact females and males treated with a low dose of DEHP had more anxiety (burrowing behavior) when compared to other groups. Data analysis is ongoing for the other assessments of emotional behavior. These data will be among the first to describe the effects of phthalates on anxiety and depression, and will have broad significance for laboratory animals, pets, and humans.

Student support: Merial Veterinary Scholars Program
Research support: College of Veterinary Medicine

Pre-Natal Bisphenol A Decreases Expression of Follicle Growth Regulators in the Rat Ovary
Mallory Sczygelski, Liying Gao, and Jodi A. Flaws
College of Veterinary Medicine, University of Illinois, Urbana, IL

Bisphenol A (BPA) is a chemical found in polycarbonate plastics and epoxy resins commonly used in the linings of beverage and food product containers. BPA can be released from these products and consumed by humans and pets. Previous studies have shown BPA exposure during adult life inhibits follicle growth and induces atresia in the rodent ovary. However, little is known about the effects of in utero BPA exposure on the ovaries of the offspring (F1 generation). This study was designed to test the hypothesis that pre-natal exposure to BPA decreases expression of key regulators of ovarian function in the offspring. This study also examined the response of the ovary to BPA when dams are on a high-fat diet because pregnant females are often simultaneously exposed to BPA and a high-fat diet. Pregnant Sprague-Dawley dams were placed on a control or high-fat diet and were orally dosed with either vehicle (tocopherol-stripped corn oil) or BPA (50 µg/kg/day body weight) from gestational day 6 until post-natal day (PND) 21. Ovaries were then collected from the pups. RNA was extracted, transcribed to cDNA, and subjected to quantitative PCR for measurement of expression of genes that control ovarian function. The genes analyzed are known to regulate cell growth (cyclin D2 and cyclin-dependent kinase 4), cell death (B-cell lymphoma 2 and bcl2-associated X protein), or steroidogenesis (cyp450scc, cyp450 17α, cyp450 aromatase, and steroid acute regulatory protein). Results indicate that in pregnant dams on a control diet, BPA significantly decreased mRNA expression levels of cyclin D2 (n=4; p=0.017) in the ovaries of the F1 offspring, but did not affect expression of the other genes. In contrast, BPA did not affect the expression of any of the selected genes in pregnant dams on a high-fat diet. These data suggest that BPA may inhibit factors that

Student Support: Office of the Director, NIH, T35 OD011145
Research Support: National Institute of Environmental Health Sciences, NIH, R01 ES019178

Effects of Arachidonic Acid and Docosahexaenoic Acid on Spermiogenesis in Mice
Richard Sheng, Timothy Abbott, and Manabu Nakamura
College of Veterinary Medicine and Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL

Arachidonic acid (20:4n6; AA) and Docosahexaenoic acid (20:6n3;DHA) are highly unsaturated fatty acids (HUFAs) found at high levels in the testes of mammals. These fatty acids are synthesized from the essential fatty acids linoleic acid (LA) and alpha-linolenic acid (ALA) respectively. The gene FADS2 encodes the protein Delta-6 desaturase (D6D), which catalyzes the rate-limiting step in the synthesis of the AA and DHA. AA and DHA play a variety of physiological roles including the inflammation response, vision, and brain function. Furthermore, deficiencies in AA and DHA lead to abnormalities in spermatogenesis specifically the final stages of the process—spermiogenesis. This study used FADS2 knockout mice to determine the level of AA or DHA supplementation required to restore normal spermiogenesis. Wild type mice were fed a diet sufficient in LA and ALA with no supplementation of AA or DHA. FADS2 knockout mice (-/-) were fed the same diet, but were also supplemented with varying concentrations of either AA or DHA: none, 0.1, or 0.2% (grams of fat/ grams of food). Sections of the seminiferous tubules and cauda epididymis were collected and analyzed histologically to check for abnormalities in sperm. Results showed that there was abnormal release of spermatids in the other knockout treatment groups. However, feeding 0.2% DHA to knockout mice fully restored normal spermiogenesis as observed in both the seminiferous tubules and the cauda epididymis. In order to study the process in more detail, immunofluorescence for adhesion proteins between Sertoli cells and spermatids will be conducted. Results from this study demonstrated that HUFAs play a critical role in spermatogenesis, and that diet can be a determining factor for proper sperm production.

Student Support: Office of the Director, NIH, T35 OD011145
Research Support: USDA NIFA 1LLU-971-354

Interaction of Helicobacter pylori Vacuolating Cytotoxin with the Host Cell Inflammasome
Matthew R Shockey, Robin Holland, and Steven Blanke
College of Veterinary Medicine and Department of Microbiology, University of Illinois, Urbana, IL>

Helicobacter pylori is a human gastric bacterium that can cause serious disease, including peptic ulcers and gastric adenocarcinomas. Nearly 50% of the world’s population is infected with H. pylori, which underscores its capacity to remodel the gastric environment, establishing a chronic infection that can persist over an individual’s lifetime. An important H. pylori virulence factor is VacA, a pore-forming vacuolating cytotoxin that binds and alters gastric mucosal epithelial cell functions. To be effective, VacA must be taken into the intracellular environment, where the toxin modulates membrane permeability, altering mitochondrial function and membrane trafficking within the endolysosomal system. Typically, pathogen-mediated damage to host cell membranes is sensed by intracellular inflammasomes, which are large, multiprotein complexes responsible for the activation of the protein caspase-1 in the initial, cellular inflammatory pathways, specifically those involved in host defense against pathogens. Inflammasome activation requires two signals: one that primes the complex and another that activates caspase-1 cleavage activity. Caspase-1 cleaves the pro- forms of interleukin-1β and interleukin-18, which function in immunity against bacteria and viruses. We evaluated the relationship between VacA-mediated alterations in intracellular membrane integrity and the capacity of intoxicated cells to respond through inflammasome activation. Preliminary studies showed that VacA alone (e.g. in the absence of H. pylori infection), induced gastric damage in a murine model. Gastric tissues collected 24 h after exposure exhibited some blunting of gastric pits and infiltration of leukocytes. Immunohistochemistry will be performed to determine the presence of VacA and expression of IL-1β in tissue, indicating possible VacA induction of inflammasomes. Understanding VacA effects in vivo and requirements for inflammasome activation may reveal ways to combat H. pylori infection.

Student Support: Office of the Director, NIH, T35 OD011145
Research Support: University of Illinois Department of Microbiology

Prognostic Parameters in Equine Head Trauma Patients
N.D. Wettstein, D.W. Hague, K.M. Lascola, and S.M. Reed
College of Veterinary Medicine, University of Illinois, Urbana, IL and Rood & Riddle Equine Hospital, Lexington, KY

Background: While the Modified Glascow Coma Scale (MGCS) and hyperglycemia have been successfully used to establish a meaningful prognosis in humans and dogs with head trauma, little research has been done in horses to assess its prognostic value.
Objective: To correlate the MGCS, Small Animal Coma Scale (SACS), blood glucose concentrations and heart rate at admission to an outcome prognosis in equine patients with head trauma.
Animals: 100 equine patients, 1 day to 27 years of age
Methods: In this retrospective study design, records spanning 1999-2014 from the University of Illinois Veterinary Teaching Hospital (n=56) and Rood and Riddle Equine Hospital (n=34) were selected by the following search terms: “head trauma”, “injury of the head”, “open head wound”, “fracture of bone of head” and “closed wound of head”. Information evaluated included: signalment, final diagnosis, medications administered, blood glucose concentration within 24 hours of admission, heart rate, temperature and outcome. Each case was given a MGCS, a SACS and a modified MGCS (mMGCS) based on information present in the records.
Results: We anticipate the mMGCS to be more accurate at predicting patient outcome, as compared to the MGCS and the SACS. Additionally, we expect to find that hyperglycemia and tachycardia in adult horses correlates with a poor outcome.
Conclusions and clinical relevance: An altered version of the MGCS and SACS that primarily used pupillary light reflex as an indicator of brain stem reflexes and a modified motor scale to accommodate a wider range of motor dysfunction present in horses was useful in determining the prognosis of equine patients. Hyperglycemia and heart rate can also be used as indicators of poor outcome. The mMGCS, hyperglycemia, and heart rate parameters may be applied together in clinical practice in addition to possible prospective studies to further establish these guidelines for patient evaluation.

Student support: Merial Veterinary Scholars Program

Hemoparasites and Hematology of Dickcissels (Spiza americana), a Grassland Bird Breeding in Central Illinois
Steven Zachar, Todd M. Jones, and Jeffrey D. Brawn
College of Veterinary Medicine and Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL

Blood parasites impact a number of mammalian, reptilian, and avian species. Previous studies have shown that pathogenic effects from blood parasites may result in decreased fitness and survival of infected wild birds. Although such studies highlight how hematologic references may be useful in monitoring individual and population health, such parameters have been established for only a limited number of avian species. We seek to establish hematologic parameters for the Dickcissel (Spiza americana), a migratory songbird commonly found breeding in the grassland habitats of the Midwestern United States. We will use polymerase chain reaction (PCR)-based methods to detect Trypanosoma spp, Haemoproteus spp, Plasmodium spp, and filaroid nematodes, and investigate differences in hematocrit, plasma protein levels, estimated white blood cell count, and white blood cell differentials of 34 adult and juvenile Dickcissels captured in central Illinois. We anticipate that individuals infected with blood parasites will display lower hematocrits and elevated lymphocytes, eosinophils, and basophils in comparison with unaffected individuals. Results of this study will be incorporated into a larger body of research investigating the breeding ecology and survival of adult and juvenile Dickcissels.

Student support: College of Veterinary Medicine
Research support: Department of Natural Resources and Environmental Sciences and College of Veterinary Medicine

2013 Research Project Abstracts

Di(2-Ethylhexyl) Phthalate and its Metabolite Mono(2-Ethylhexyl) Phthalate Inhibit Steroidogenesis
Katherine E. Brannick, Wei Wang, Patrick R. Hannon, and Jodi A. Flaws
Department of Comparative Biosciences, University of Illinois, Urbana, IL

Phthalate esters are a group of synthetic plasticizers that impart flexibility to polyvinylchloride plastics and are widely used in food packaging, toys, cosmetics, and medical devices. Di(2-ethylhexyl) phthalate (DEHP) is the most commonly used plasticizer, and mono(2-ethylhexyl) phthalate (MEHP) is its active metabolite. Phthalates are known endocrine disrupting chemicals and have been shown to inhibit estradiol synthesis in previous studies, however, the mechanism of this inhibition is unknown. Estradiol synthesis requires production of progesterone, which is converted to androstenedione, then testosterone, and lastly, estradiol. Thus, the goal of this study was to identify where DEHP and MEHP disrupt estradiol production in the steroidogenic pathway. Antral follicles were isolated from CD-1 mice and exposed to vehicle, DEHP (1-100 μg/ml) or MEHP (0.1-100 μg/ml) for 96 h. After culture, media were subjected to measurements of hormone levels. The results indicate that DEHP did not affect progesterone levels compared to controls. In contrast, the highest dose of MEHP increased progesterone compared to controls (n = 5-7; p ≤ 0.05). Both DEHP and MEHP decreased androstenedione and testosterone levels compared to controls (p ≤ 0.05). Further, high DEHP doses and all doses of MEHP decreased estradiol levels compared to controls (p ≤ 0.05). These results indicate that both DEHP and MEHP inhibit estradiol levels by targeting upstream hormones, primarily androstenedione and testosterone. Further, the results suggest that follicles are more sensitive to MEHP-induced inhibition of steroidogenesis than to DEHP-induced inhibition of steroidogenesis. Understanding the mechanism by which phthalates inhibit steroidogenesis will aid in the development of methods to prevent and treat adverse effects of phthalates.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145
Research Support: National Institute of Environmental Health Sciences, NIH R01 ES019178

The Effect of Zoledronate on Chemokine Receptor Expression in Canine Osteosarcoma Cells
Mark Byrum, Bahaa A. Fadl-Alla, and Timothy M. Fan
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Canine osteosarcoma (OS) is the most common primary bone tumor affecting dogs of large and giant breeds. OS causes aggressive localized bone destruction and metastases to distant organs, particularly the lung parenchyma. Although localized disease can be effectively managed with surgery, effective therapies for inhibiting metastatic progression remain in their infancy. Standardized palliative pain therapy for canine OS incorporates the use of zoledronate, a potent third-generation aminobisphosphonate that inhibits osteoclast activities, in combination with radiation therapy. Effective pain relief has been achieved with this specific protocol, suggesting adequate localized OS control. Interestingly, dogs treated with zoledronate and radiation subsequently die from distant metastases involving atypical anatomic sites. One potential explanation for the observed altered metastatic pattern could be zoledronate’s capacity to modulate surface chemokine receptor expression, which regulate cell trafficking patterns during metastatic progression. We hypothesized that biologically achievable concentrations of zoledronate (1 uM and 5 uM) have the capacity to perturb expression of various chemokine receptors (CCR6, CCR7, CCR9, CCR10, CXCR4, and CXCR7) in canine OS cells. RNA and protein will be extracted from cultured Abrams canine OS cells. Quantitative PCR will be used to measure expression of the chemokine receptor genes; Western blotting will be used to detect the presence of chemokine receptor proteins. Results will indicate whether zoledronate enhances chemokine receptor expression, leading to abnormal metastatic lesions in canine OS.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145

Molecule PAC-1 Activates Apoptotic Enzyme Caspase-3 to Induce Osteosarcoma Cancer Stem Cell Death
Yael E. Farah and Timothy M. Fan
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Osteosarcoma cancer stem cells are resistant to apoptosis, presumably due to their ability to maintain the apoptotic enzyme caspase-3 in its inactive form, procaspase-3. A small molecule, PAC-1, has been found to directly activate procaspase-3 in a manner proportional to procaspase-3 concentrations. PAC-1 thus induces apoptosis in procaspase-3-heavy cancer cells. In this study, Abrams osteosarcoma cancer stem cells were isolated via sphere culture assay to determine procaspase-3 concentrations and to be probed with PAC-1. The effectiveness of Abrams stem cell isolation was assessed by showing equivalent expression of embryonic stem cell-associated genes Oct4 and Nanog via qualitative PCR relative to adherent Abrams cells. Western blotting followed by densitometry testing showed that procaspase-3 levels increased five-fold in the spheres relative to adherent cells. Accordingly, the procaspase-3-heavy osteosarcoma cancer stem cell spheres should fall subject to increased apoptosis when probed with PAC-1 and therefore reduce the survival of this highly metastatic and therapy-resistant disease.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145

The Influence of Environmental Conditions on the Abundance of a West Nile Virus Vector
Mackenzie Kelling and Marilyn O’Hara
Department of Pathobiology, University of Illinois, Urbana, IL

West Nile Virus results in significant morbidity and mortality of people, horses and birds. Epidemiologists have a need to understand Culex mosquito distribution, specifically Culex pipiens Linnaeus (Diptera: Culicidae) and Culex restuans Theobald, because of their ability to transmit West Nile Virus. Elevated numbers of infected vector mosquitoes increases the risk of illness, and mosquito abundance varies spatially and temporally based on variation in environmental conditions. Culex mosquitoes prefer to oviposit in stagnant water, making catch basins an established ovipositing site in the Chicago, Illinois area. Alternative sites for mosquito breeding include stagnant natural water sources, household containers, and discarded tires. We tested the hypothesis that adult Culex mosquito abundance is higher where there are more larval sites and that this relationship will vary depending on weather. We compared adult mosquito abundance recorded from 2006 to 2012 and the location of catch basins, natural standing water, and residential larval sites in a West Nile focus area in south Cook County, Illinois. The methods used included a geographical mapping of catch basins and identification of other larval habitat. Between June and August 2013, catch basins, houses, and natural water sources were sampled for larval presence. The number and species of larvae was recorded for each water source tested. A spatial statistical analysis was conducted to determine the relationship between available water sources, mosquito larvae abundance, and adult abundance. Results indicate that the abundance of mosquitoes varies from year to year, while the structure of the urban environment remains relatively constant.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145

Shedding Some Light on Phthalate Activity in Circadian Rhythms
Molly E. King, Patrick R. Hannon, Sara Roysten, Jodi A. Flaws, and Megan M. Mahoney
Department of Comparative Biosciences, University of Illinois, Urbana, IL

Phthalates are ubiquitous plasticizers that act as endocrine disrupters, causing significant behavioral and health changes in rodents, domestic animals and humans. Daily biological rhythms regulate all physiological functions from body temperature to hormone production, but the effect of phthalates on rhythms has never been explored. To examine this issue we dosed mice with two model phthalates, Diethylhexyl phthalate (DEHP) or Di-n-butyl phthalate (DnBP) and analyzed behavioral rhythms and clock-controlled protein expression found in the master oscillator, the suprachiasmatic nucleus (SCN). Albumin D site-binding protein (DaBP) and peroxisome proliferator-activated receptor alpha (PPARα) are two rhythmically expressed genes that play a role in daily behavioral rhythms and are involved in metabolism of phthalates. In the first experiment adult male CD-1 mice were gonadectomized and testosterone capsules were inserted to regulate hormone levels. The mice were then orally dosed with DEHP (750mg/kg/day) or oil daily for 30 days. In the second experiment, gonadectomized adult male and female CD-1 mice were orally dosed with DnBP (0.1 mg/kg/day) for 30 days. For both experiments the timing, amount and distribution of wheel activity was quantified. In a third experiment immunocytochemistry was used to assess numbers of cells expressing DaBP and PPARα in the SCN of mice dosed with DEHP for 10 days. We predict that phthalate-treated mice will show an increase in overall activity as well as changes in cell numbers of rhythmic genes in the SCN. The results of this study will be the first to determine the impact of daily exposure to phthalates on the functioning of the fundamental circadian timekeeping system.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145
Research Support: University of Illinois Campus Research Board

Mitigation of Negative Effects of Corticosteroids on Biosynthetic Cartilage Matrix Production by TGF-β1 or BMP-2
Melody Martychenko, Sushmitha Durgam, and Matthew Stewart
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Intra-articular corticosteroid (CS) injections are commonly used to treat equine lameness. In addition to their potent anti-inflammatory effects, CSs also suppress articular chondrocyte (AC) biosynthetic activities. We have shown that CSs significantly down-regulate expression of several BMP and TGF-β ligands by ACs. We hypothesize that co-administration of TGF-β1 or BMP-2 to chondrocytes will mitigate the suppressive effects of CSs on collagen type II (Coll II). ACs were treated with 10-5 M methylprednisolone acetate (MPA) alone or with BMP-2 (100 ng/mL) or TGF-β1 (10 ng/mL) for 5 days. Expression of collagen type II mRNAs was assessed by quantitative PCR. Collagen secretion was measured by ELISA. One-way ANOVA was used to analyze the outcomes. Administration of TGF-β or BMP-2 alone variably increased Coll II. However, neither growth factor was able to increase synthesis of these cartilage matrix components in the presence of MPA. Exogenous TGF-β1 or BMP-2 administration did not mitigate the suppressive effects of MPA on AC matrix biosynthesis. This finding suggests that other elements in the TGF-β/BMP signaling pathway are also affected by CSs, or that factors directly required for AC matrix synthesis are negatively impacted by CS administration.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145

Effect of Zoledronate on Osteogenesis of Equine Bone Marrow-Derived Mesenchymal Stems Cells
Michelle Piccione, Sushmitha Durgam, and Matthew Stewart
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Zoledronate (ZOL) is a bisphosphonate (BP) used to prevent osteoporosis and pathological fracture. BPs localize to mineralized matrix and, consequently, target bone-resorptive osteoclasts. Osteoprogenitor cells (OPCs) are critical for skeletal homeostasis. We hypothesize that ZOL will inhibit OPC aggregation and subsequent osteogenesis. Bone marrow-derived OPCs were collected from three healthy horses. Primary cells were seeded at 5×103 cells/cm2 and passaged twice. After P2, cells were transferred to osteogenic medium +/- zoledronate [0.1, 0.5 and 1.0 µM]. OPC aggregation was monitored by direct counting. After 7 and 10 days, OPC phenotype was assessed by staining for mineralized matrix (Alizarin red), alkaline phosphatase (ALP) activity and qPCR of osteogenic gene expression. Data were analyzed by ANOVA and Bonferroni’s post hoc test. Over the first 7 days, ZOL did not affect cell number (DNA). However, at day 10, there was a clear reduction in OPC numbers in 1.0 µM ZOL cultures, reflected by a 50% drop in [DNA]. ZOL significantly reduced OPC aggregation in osteogenic cultures. Osteogenic medium induced ALP activity in OPCs. ZOL did not affect bulk ALP activity. The preliminary results of this study show that ZOL inhibits OPC aggregation and multicellular nodule formation but does not substantively affect ALP induction. Further phenotypic analyses will be required to establish the extent of ZOL’s effect on OPC osteogenesis.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145
Research Support: USDA Animal Health and Disease Program

Cytokine Expression in the Presence of Lactoferrin and Probiotic Supplementation in a Systemic Staphylococcus aureus Piglet Model
Kathryn L. St. John1, Elizabeth A. Reznikov2, and Sharon M. Donovan2,3
1College of Veterinary Medicine, 2Division of Nutritional Sciences and 3Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL

Sepsis due to Staphylococcus species is a significant problem in preterm neonates, particularly those that are formula-fed rather than breastfed. The aim of this study was to examine the effect of supplementing infant formula with the probiotic Bifidobacterium longum subsp infantis (B. infantis) and/or bovine lactoferrin (bLf) on cytokine expression in a piglet model of systemic Staphylococcus aureus (S. aureus) infection. Piglets are an especially good model for the human infant, due to similarities in gastrointestinal physiology, immune response and anatomy. The hypothesis was that the combination of B. infantis and bLf would promote a synergistic effect on the immune system, allowing the piglets to mount an appropriate immune response during the course of infection. It was also hypothesized bLf would promote B. infantis colonization in the intestine thereby increasing the beneficial effects of B. infantis on the immune system. Female, colostrum-deprived newborn piglets (n = 15) were individually housed and randomized to receive formula supplemented with one of the four treatments: bovine whey (4 g/L) (control), whey (4 g/L) + B. infantis (109 CFU/mL), bLf (4 g/L), or combined bLf (4 g/L) + B. infantis (109 CFU/mL). Piglets were infected with S. aureus (105 CFU/mL at 1 mL/kg body weight) on day 7 and tissues were collected at euthanasia on day 12. Cytokine expression (IFN-γ, IL-6, and IL-10) was analyzed in spleen, lung, and liver tissues using quantitative real-time polymerase chain reaction. The aim of this research is to reduce neonatal morbidity due to systemic S. aureus infections. Formula supplemented with B infantis and bLf has the potential to improve the neonatal immune response to systemic infections.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145
Research support: National Institute of Child Health and Human Development, National Institutes of Heath, R01 HD061929

Analysis of Streptococcus equi M-Protein: Comparisons Between Wild-Type, covS Mutant and Pinnacle IN Strains
Mara Varvil, Saraswathi Lanka, and Carol Maddox
Department of Pathobiology and Veterinary Diagnostic Laboratory, University of Illinois, Urbana, IL

Streptococcus equi subsp. equi (S. equi) is the causative agent of strangles, a highly contagious disease of horses that causes inflammation and swelling of the nasopharyngeal lymph nodes. Vaccination against strangles is recommended for young horses. The commercially available modified live vaccine, Pinnacle IN, causes clinical symptoms of strangles in 20-25% of weanlings. Pinnacle IN has a covS single nucleotide mutation that may contribute to its attenuation, but this site has the ability to revert causing adverse vaccine reactions. CovS is a sensor of virulence and produces a signaling protein when it detects there are appropriate conditions for host infection. CovS then signals covR, which produces a protein that can up- or down-regulate several virulence genes, including SeM that encodes M protein. A covS deletion mutant was created to remove the gene, thereby eliminating the chance for reversion and producing a strangles vaccine. Testing of the deletion mutation in horses demonstrated partial protection of the animals from a S. equi challenge. We hypothesize that reduced expression of surface immunogens in the deletion strain, such as M protein, reduce the efficacy of the vaccine. Our work will compare the DNA sequences of SeM in the various strains, use quantitative PCR to assess alterations in SeM expression, and use Western blotting to examine differences in abundance of M protein in the various isolates. These data will indicate whether there is a connection between covS deletion, M protein abundance, and the efficacy of the putative new vaccine strain.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145

The Influence of Urban Stormwater Management Practices on Mosquito Blood Feeding Behavior and the Potential for West Nile Virus Enzootic Amplification
Arsema Weldu, Andrew Mackay, Page Fredericks, and Brian Allan
Department of Entomology, University of Illinois, Urbana, IL

West Nile Virus (WNV) transmission intensity and exposure risk are not homogeneous across an urban landscape. Some of this variability in transmission intensity can be attributed to differences in host utilization patterns of the primary mosquito vectors, which determine the proportion of vector-host interactions that result in virus transmission. This project is designed to determine whether the species of birds that mosquitoes (Culex sp.) feed upon differs between detention basins and the surrounding residential land use, as well as the influence of the presence of invasive plants or communal bird roosts in these basins. We extracted host DNA from mosquito blood meals using the QIAamp DNA Micro Kit. Vertebrate host DNA was amplified using avian- and mammalian-specific primer pairs. These PCR products were purified, sequenced, and then analyzed using the National Center for Biotechnology Information database. This study will help identify habitat factors that facilitate the amplification of WNV in suburban environments by identifying how stormwater detention basins and their flora influence which bird species are fed on. This information could assist in the development of surveillance and control protocols for monitoring and reducing risk of WNV exposure for humans, domestic animals, and wildlife.

Student Support: Office of the Director, National Institutes of Health, T35 OD011145

2012 Research Project Abstracts

Evaluation of the Effect of Transforming Growth Factor Bets (TGFB) on Malignant Osteoblasts
Jessica Corrie, Holly Pondenis, Timothy M. Fan
Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL

Transforming Growth Factor Beta (TGFB) plays an important role in malignant osteoblast progression and the development of osteosarcomas (OS). Malignant osteoblasts produce and secrete TGFB and its associated receptors, TGFBRI and TGFBRII, for signaling purposes. TGBF also causes downstream effects like phosphorylation of Smad 2/3 and subsequent activation of target genes via intracellular signaling. The role of TGFB as a protumorigenic cytokine is complex and variable, as evidenced by its mitogenic, angiogenic, and immunosuppressive effects as well as its ability to act in an autocrine or paracrine fashion. Clonally-derived OS cell lines of variant metastatic potential derived from 3 different species (human, murine, and canine) will be utilized to assess differences in TGFB expression. OS production and secretion of TGFB will be demonstrated by Western Blot and ELISA. OS expression of TGFBRI and TGFBRII will be demonstrated by Western Blot and IHC-P TGFB induction of P-Smad 2/3 will be demonstrated by Western Blot. Western Blot and ELISA for TGFB suggest the presence of a feedback loop between OS and TGFB in all 3 species. Western Blot and IHC-P suggest expression of both TGFBRI and TGFBRII by malignant osteoblasts in all 3 species. TGFB, TGFBRI and TGFBRII are present in OS cell lines from various species. We expect to demonstrate the mitogenic, angiogenic , and immunosuppressive effects of TGFB in future studies. Ultimately, we hope to demonstrate that blocking TGFB signaling can have beneficial biologic effects including enhanced survival time and decreased number/size of pulmonary metastases.

Research supported by NIH T35 OD011145

Equine Strangles: Evqluation in Weanlings of Two Novel Modified Live Vaccine Candidates Against Streptococcus equi
Elise M. Hall, Gabriela Calzada-Nova, Kara Lascola, Stephanie Freese, Carol W. Maddox
College of Veterinary Medicine, University of Illinois, Urbana, IL

Streptococcus equi, is the causative agent of “strangles,” a common bacterial disease of horses. A novel mutant of S. equi, C3-19, has delections in genes encoding control of virulences (covS), superoxide dismutase (sodA), and strep Zooepidemicus-like protein (szp). We hypothesized that C3-19 would induce humoral immunity in weanling horses and safely prevent S. equi infection. Four weanlings (5-11 mo.) received live intranasal C3-19 vaccine. Antibody levels following immune response were evaluated using direct ELISA and Western Blots. C3-19 vaccination was protective against S. Equi. There were no significant fevers following vaccination (p>0.14). Daily lymph node and nasal discharge scores did not exceed 2 on a scale of 0 to 3. Mild increases in lymphadenopathy and nasal discharge were observed following vaccination but no abscessed lymph nodes or purulent nasal discharges were noted. Nasal cultures demonstrated vaccine clearance within 3 days post inoculation. At necropsy, all weanlings were culture negative for the S. equi challenge and vaccine strain. Serum ELSIA titers against C3-19 demonstrated a rise in antibody titer levels following vaccination for 2 weanlings: levels went from undetectable at day 0 to 17-33% of positive control. Titer levels of 2 weanlings showed a decrease in titer, but remained signaficant at 26-55% of positive control. M-Protein titers performed by Equine Diagnostic Solutions declined during the trial. This study may allow development of a safer vaccine against S. equi, crucial to minimizing economic losses in the equine industry. The testing of another strain, #34, which has a sole gene deletion of covS, is ongoing.

Research supported by NIH T35 OD011145
Research supported by USDA-HATCH funds

Does Female Predator Exposure Affect Male Courtship in Three-Spined Stickleback Fish?
Sagan R. Leasure, Sally Feng, Katie McGhee, Alison Bell
College of Veterinary Medicine and the Department of Animal Biology, University of Illinois, Urbana, IL

The behavior of an individual often depends on the characteristics of their audience. For example, courtship behavior might vary with the traits of the individuals involved. In three-spined stickleback fish (Gasterosteus aculeatus), males might be particularly choosy in female mate choice since males provide costly offspring care. Previous studies have shown that maternal predator exposure increases cortisol levels in eggs and negatively affects offspring anti-predator behavior and survival. Thus, males might be expected to adjust their courtship behavior based on the previous predator exposure of the female. The aim of this study was to determine whether males change their courtship behavior based on the predator-exposure experience of the female. On one hand, males might prefer to court unexposed over predator-exposed females because high cortisol levels in the eggs induced by predator exposure result in lower-quality offspring. Alternatively, males might be equally motivated to court both types of females but predator-exposed females might be less receptive to courtship behavior requiring males to compensate by courting them more intensely. To examine these hypotheses, we used a within-subjects design where males interacted both with a predator-exposed and unexposed female in random order. Data was recorded on male courtship behavior for five minutes. Our study will determine whether males can assess the predation exposure or stress level of potential mates and whether they use this information in their courtship decisions. It will also provide insight into how ecologically relevant maternal stressors, such as predation risk, might affect male mating behavior and subsequent reproductive success.

Research supported by NIH T35 OD011145
Research supported by NSF IOS 1121980

Modifications of Airway Mucin Glycoproteins by Pseudomonas aeruginosa Alginate and Flagella
Rebecca Powell, Jayme Jeffries, Gee Lau
Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL

Alginate and flagella are two of many virulence factors produced by Pseudomonas aeruginosa during chronic lung infections, as commonly seen in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients. Alginate characterizes the mucoid phenotype of P. aeruginosa that is most commonly isolated from patients with exacerbated infections. Flagella, usually associated with motility, have been shown in P. aeruginosa to function as an adhesin to mucins found in human lungs. P. aeruginosa infection exacerbations have been linked with structural changes in the glycosylation of mucin glycoproteins that make up the thick mucus obstructing the airways of these diseases. In order to determine if alginate and flagella contribute to these changes, mice were treated intratracheally with 140 ug/treatment of alginate or intranasally with 2 ug/treatment of flagella for 1, 3 and 7 days. Lungs from these mice were examined under a High Iron Diamine/Alcian Blue stain as well as a Periodic Acid-Schiff (PAS) stain. The PAS stain showed evidence of goblet cell hyperplasia around large airways in both the alginate and flagella samples. Additionally, evidence of sialylated mucins was seen in these same locations. Goblet cell hyperplasia was seen at a higher frequency in the flagella sections compared to the alginate. This supports the claim that alginate and flagella induce goblet cell hyperplasia and changes in mucin glycosylation. Knowing which virulence factors of P. aeruginosa contribute to mucin modifications in CF and COPD airways can lead to development of better treatments.

Research supported by NIH T35 OD011145, R01 HL090699
Research supported by American Lung Association DeSouza Research Award

The Effects of Dopaminergic Drugs on Impulsive Behavior in Long Evans Rats Exposed to Polychlorinated Biphenyls and Polybrominated Diphenyl Ethers
Lindsay Awsumb, Paul Eubig, Susan Schantz
Department of Comparative Biosciences, College of Veterinary Medicine University of Illinois, Urbana, IL

Impulsivity, a component of neurobehavioral disorders including attention deficit/ hyperactivity disorder (ADHD), is associated with dysfunctional dopaminergic activity in the brain. Differential reinforcement of low rates of responding (DRL), a behavioral test of impulsive action, requires that a subject waits for a predetermined amount of time before pressing a lever to earn a food reward. If a subject prematurely presses the lever, the clock resets with no reward. In this study, Long-Evans rats were dosed perinatally with environmentally relevant mixtures of either polychlorinated biphenyls or polybrominated diphenyl ethers, toxicants that affect dopaminergic signaling in the brain. The offspring were trained on the DRL task and then were treated with the dopaminergic agents amphetamine, flupenthixol, or both in order to allow assessment of the role of perturbed dopaminergic signaling in mediating the effects of toxicant exposure. We hypothesized that toxicant exposure would increase impulsive action manifested as increased lever pressing and decreased food rewards earned. Furthermore, we hypothesized that toxicant exposed subjects, as compared to controls, would be relatively more impulsive after flupenthixol and less impulsive after amphetamine. Preliminary results did not reveal effects of toxicant exposure on DRL performance but the drug challenges changed behavior. Amphetamine increased impulsive behavior as demonstrated by increased lever pressing and less food rewards earned. Flupenthixol resulted in decreased lever pressing and less food rewards earned, suggesting that flupenthixol caused subjects to wait for relatively longer intervals between lever presses, thus failing to maximize the number of rewards earned.

Research Supported by NIH T35 OD011145, K08 ES017045 and R01 ES015687

Invasion of the Black-Legged Tick in Illinois
Cari Rasmussen, Natalie Pawlikowski, Daniel Girzadas, Brian Allan
College of Veterinary Medicine, School of Integrative Biology, and the Department of Entomology, University of Illinois, Urbana, IL

The black-legged tick, Ixodes scapularis, is the main vector of Lyme disease in the United States and is becoming established in woodland areas in Illinois, initially in the northwest part of the state and moving south and southeast. While the purported mechanism of establishment is long-distance movements by wildlife hosts, little is known about the pattern of establishment in novel habitats in relation to landscape features. Predictions from the Theory of Island Biogeography would suggest that migrating hosts are more likely to encounter large habitat fragments than small, and more connected than isolated, while dispersing through an otherwise inhospitable matrix habitat (e.g., row-crop agriculture). The pattern of this invasion was studied and it was predicted ticks would establish in large sites first, while smaller sites farther from these large foci would exhibit the lowest tick densities, contradicting the pattern seen in long-term established areas. Ticks were collected in 4 counties in each of 3 regions of Illinois: northwest, north central, and east central. In each county, 5 sites were sampled: a large site, 2 “medium” sites and 2 “small” sites and 1 of each near and 1 of each far from the large site. There was no significant relationship between invasion history and the abundance of I. scapularis nymphs (P = 0.983). However, across the entire invasion gradient, there was a significant effect of site type on nymph abundance (P = 0.039). Large sites supported significantly more ticks than Small, Far (P = 0.042), and there was a marginally significant trend that Large sites supported more ticks than Medium, Far (P = 0.066). Understanding the establishment patterns of I. scapularis during an invasion are vital for implementing preventative measures not only for inhibiting the establishment of I. scapularis, but stopping the spread of Lyme disease into new regions.

Research supported by NIH, T35 OD011145

Microscopic and Ultrastructural Lung Injury Following Intravenous Bolus LPS Administration to Healthy Adult Horses
Stefanie Reed, Pamela Wilkins, Kara Lascola
College of Veterinary Medicine University of Illinois, Urbana, IL

Endotoxemia is a serious complication in several diseases of horses that exacerbates disease severity and may result in death. Endotoxin (LPS) is released from the cell membrane of Gram-negative bacteria and can induce a pro-inflammatory systemic reaction. Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) may result. Additionally LPS administration causes significant pulmonary hypertension, known to damage the pulmonary vasculature, creating ‘vascular leak syndrome’. This study investigates the severity of secondary lung injury induced in horses approximately 3 hours after intravenous bolus administration of 100 ng/kg LPS (Escherichia coli O55:B5) to healthy adult horses as part of a larger study evaluating pulmonary changes associated with LPS administration. Percutaneous lung biopsies from a randomly chosen lung field were collected from 8 healthy horses (BASELINE) after sedation with xylazine hydrochloride (0.2-0.3 mg/kg IV) for analysis by histopathology, micro-computed tomography and transmission electron microscopy. Following two weeks of recovery, horses underwent LPS or saline administration via a jugular catheter. LPS resulted in pulmonary hypertension as anticipated. The maximum increase in mean pulmonary artery pressure [128% (+/- 54%)] was noted in the horses at about28.5 (+/- 2.4) minutes after start of the LPS infusion. This was transient for most of the horses and, the pressures had begun to return towards baseline values for several horses near time zero. At this time tissue sample analysis is still ongoing. A better understanding of clinical symptoms and internal tissue damage of infected horses is essential to creating more efficient treatment and management protocols.

Research supported by Merial Veterinary Scholars Program

Influence of wetland flora and avian fauna density and diversity on vector-host interactions and WNV transmission risk
Sarah Reich, Andrew Mackay, Tyler Hedlund, Christine Roy, Brian Allan
Department of Entomology, University of Illinois, Urbana, IL

Urban and suburban environments are known to be favorable for the transmission of West Nile Virus (WNV) by providing both an abundance of competent avian hosts and an advantageous aquatic habitat for mosquito vector development (stormwater drains and other forms of standing water). However, within these environments, differences in vegetative and host density and diversity can greatly affect not only WNV transmission but also disease occurrence. In this study, we compared the diversity and density of plants, birds, and mosquitoes in wetland and corresponding residential environments to determine if the differences in habitat and avian abundance had an effect on the mosquito population. In each of our three study cities (Bloomington, Decatur, and Springfield) we chose wetland sites that were within 200 m of urbanized areas and a corresponding residential site within a 500 m radius of the wetland. Mosquitoes were collected via light and gravid traps at each site over 3 consecutive days each month (collections will continue May through September). Species and blood meal status will be determined for all female mosquitoes collected. Reproductive status and fecundity of females will be assessed and host DNA will be extracted from blood fed females for identification. A subsample of females collected from gravid traps will also be tested for the presence of WNV. Avian surveys (roosting and point count) will also be completed at least twice at every site in July to determine bird diversity and density and to assess roosting behavior. Results from this project will aid in assessing how wetlands influence the risk of WNV transmission in urbanized areas in Illinois.

Research supported by NIH T35 OD011145 and the School of Integrative Biology

Does Osteosarcoma-Derived-IL-8 Participate in Tumor Microenvironment Bone Resorption?
Jean-Benoît Tanis, Holly Pondenis, Timothy M. Fan
Department of Veterinary Clinical Medicine, College of Veterinary Medicine University of Illinois, Urbana, IL

Interleukin-8 (IL-8) is a cytokine which participates in many inflammatory pathologies, and has been recently incriminated as a pro-tumorigenic cytokine. Indeed, breast carcinoma cells that have metastasized to skeletal sites secrete IL-8 which leads to the overexpression of Receptor Activator of NF-kB Ligand (RANKL) by osteoblasts, thereby which promoting osteoclastogenesis and malignant bone resorption. Although metastatic breast carcinoma cells can directly influence malignant osteolysis through IL-8 secretion, whether this resorptive mechanism is conserved across various skeletal pathologies, such as osteosarcoma (OS), remains undetermined. The purposes of this investigation are 1) to assess the production and secretion of IL-8 by canine OS cells; 2) to assess the presence of IL-8 receptors (CXCR1 and CXCR2) on canine OS cells; and 3) to characterize if an autocrine or paracrine loop exists between IL-8 secretion and RANKL/OPG expression in canine OS cells. IL-8, RANKL, OPG, CXCR1 and CXCR2 expressions were assessed by RT-PCR and Western blot in 6 canine OS cell lines. Secreted IL-8 will be measured by ELISA. IL-8 was expressed at varying levels among the 6 OS cell lines (high, intermediate, and low); however, IL-8 expression did not directly correlate with RANKL/OPG ratios. Preliminary data support the expression of CXCR1 and CXCR2 by OS cells; supporting the possible existence of an autocrine or paracrine feedback loop. IL-8 might directly participate in OS-associated bone resorption and understanding osteoclastogenesis induction though IL-8 secretion by OS cells might lead to new treatment options for attenuating malignant osteolysis in dogs with OS.

Research supported by Merial Veterinary Scholars Program

Bisphenol A Exposure Inhibits Follicular Growth and May Induce Oxidative Stress
Krista A. Turey, Jackye Peretz, Jodi A. Flaws
Department of Comparative Biosciences, College of Veterinary Medicine University of Illinois, Urbana, IL

Bisphenol A (BPA) is used commonly as a monomer for polycarbonate products. Previous studies have shown that BPA advances puberty, inhibits follicular growth, and inhibits steroidogenesis by ovarian follicles. However, the mechanism by which BPA causes these effects is unknown. BPA may cause ovarian toxicity by inducing oxidative stress. Thus this study was designed to identify changes in levels of three antioxidant enzymes: superoxide dismutase (SOD1), catalase (CAT), and glutathione peroxidase (GPX) after BPA exposure. We hypothesized that BPA reduces levels of follicular antioxidant enzyme expression, creating oxidative stress. Antral follicles were isolated from post-natal day 31 CD1 mice and cultured with either vehicle (dimethylsulfoxide, DMSO) or BPA (1 ug/ml, 10 ug/ml, or 100 ug/ml) in essential alpha minimum media. Every 24 hours, growth was measured on perpendicular axes. At selected times (6, 12, 18, 96 hours), follicles were collected and subjected to quantitative polymerase chain reaction for measurement of SOD1, CAT, and GPX. The results indicate that BPA 10 ug/ml significantly inhibits follicular growth at 48-72 hours compared to DMSO controls (DMSO at 72 hours = 33% growth +/- 5.52%, BPA 10 ug/ml at 72 hours = 12.40% growth +/- 5.52%; n=43, p < 0.05). Further, BPA 100 ug/ml significantly inhibits follicular growth at 24-96 hours compared to DMSO controls (DMSO at 96 hours = 42.43% growth +/- 10.79%, BPA 100 ug/ml at 96 hours = 1.22% growth +/- 10.79%; n=38, p < 0.05). Collectively, these data suggest that BPA may cause oxidative stress.

Research supported by NIH T35 OD011145, NIH R21 ES12061

Formation of Drug-Protein Adducts by Dog Keratinocytes Exposed to Sulfamethoxazole
Olga I. Vinogradova, Dayna R. Mierke, Benjamin Lucas, Abigail Asangba, Sidonie N. Lavergne
Department of Comparative Biosciences, College of Veterinary Medicine University of Illinois, Urbana, IL

Drug hypersensitivity (HS) reactions are common in both dogs and humans and frequently target the skin. The drugs themselves are too small to trigger an immune response, but one theory postulates that covalent drug-protein adducts can be formed and induce an immune response in allergic patients. Sulfamethoxazole (SMX)-protein adducts have been detected in vivo and in vitro in humans, rats, and dogs. They have been identified in immune cells, in the blood, and in the liver. In humans, they have also been detected in skin cells, but this has not been evaluated yet in dog cells. We therefore hypothesize that dog keratinocytes will produce drug-protein adducts after treatment with SMX in vitro. Immortalized dog keratinocytes (CPEK cell line) were grown in vitro and treated with SMX at five different doses (2000 uM, 500 uM, 100 uM, 20 uM and 2 uM) for 24 hours. An ELISA assay was then used to determine the relative amount of SMX-protein adducts present in each condition, using a rabbit antiserum raised against a highly immunogenic SMX keyhole limpet hemocyanin (KLH) conjugate (Panigen Inc, IL, USA) and an alkaline phosphatase anti-rabbit IgG (AbdSerotec). We predict a dose-dependent increase in adduct formation. If found in vivo, such SMX-keratinocyte adducts could be the first step towards an SMX-specific immune response targeting the skin. Further work is underway with beta-lactam protein adducts in CPEK cells.

Research supported by the Merial Veterinary Scholar’s Program
Research supported by the College of Veterinary Medicine Companion Animal Fund

Determining Tissue Factor Pathway Inhibitor Release Induced by Unfractionated and Low-Molecular Weight Heparins in the Dog
Stacy Whitton, Maureen McMichael, Stephanie A. Smith
College of Veterinary Medicine and the Department of Biochemistry, University of Illinois, Urbana, IL

Tissue Factor Pathway Inhibitor (TFPI) is a physiologic anticoagulant responsible for the feedback inhibition of coagulation by binding Factor Xa (FXa) and Factor VIIa-Tissue Factor complex (FVIIa-TF). Heparin therapy, commonly used for prevention of thrombosis, has been shown to induce a 2-4 fold increase in plasma TFPI concentration in humans, due to release from the endothelial surface. Neither the normal physiologic levels of plasma TFPI nor the change induced by heparins have been described in the dog. Fifteen healthy dogs were randomly assigned to receive unfractionated heparin (UFH, n=5), enoxaparin (Enox, n=5), or dalteparin (Dal, n=5). Blood samples were collected at 0, 1, 3, 5, 7, and 9 hours after subcutaneous administration. Plasma was frozen for later analysis. A functional microplate chromogenic assay (Actichrome) performed poorly with canine plasma likely due to lack of ability of canine TFPI to inhibit the human derived enzymes. Dot blots with recombinant human TFPI and canine pooled normal plasma (PNP), using MAb antibodies against TFPI Kunitz 1, or Kunitz 2 indicated cross-reactivity with canine samples. Western blot conditions were then optimized for canine plasma. These conditions will be used to quantitatively evaluate plasma TFPI concentration in the stored samples. The determination of the relative effectiveness of different heparins in inducement of TFPI release may allow for more effective heparin therapy in canine patients.

Research supported by Merial Veterinary Scholars Program and University of Illinois Start-up Funds

The effect of an endocrine disrupter on sexually dimorphic neuropeptide expression in the brain of adult female intact mice
Kelly E. Wiggen, Megan M. Mahoney
Department of Comparative Biosciences, College of Veterinary Medicine University of Illinois, Urbana, IL

Di-2-ethylhexyl phthalate (DEPH) is a plasticizer used in polyvinyl chloride (PVC) plastics that has been shown to suppress ovarian hormone production and cause anovulatory cycles in rodents; however, the effects of DEHP on the expression of steroid-dependent expression of neuropeptides is unknown. Adult intact female C57/B6 mice were treated daily orally for at 30-32 days with DEPH (corn oil vehicle, 20 ug/kg/day, 200 ug/kg/day, 20 mg/kg/day, 200 mg/kg/day). Estrous cyclicity was determined daily via vaginal cytology and brains were collected from animals on the day of diestrus. Using immunocytochemistry, the number of cell bodies and density of fibers of tyrosine hydroxylase (TH) and kisspeptin (KISS) were identified in several areas of the brain, including the dorsomedial hypothalamic nucleus (TH, KISS), anteroventral paraventricular nucleus (TH, KISS), zona incerta (TH), periventricular nucleus (TH), and arcuate nucleus (TH, KISS). These are both peptides for which expression is influenced by circulating steroid hormones. We predict that the administration of DEHP will result in a decrease in the number of cell bodies and fiber density throughout the brain for both peptides, with a greater decrease in cell body number and fiber density as the dosage of DEHP increases. Because humans and other animals are regularly exposed to DEHP through plastics, determining DEHP’s estrogenic effect on the brain is crucial for understanding how oral exposure may impact neural growth and development.

Research supported by the Merial Veterinary Scholars Program
Research supported by the Department of Comparative Biosciences, College of Veterinary Medicine University of Illinois

Aerobic Exercise Accelerates Extinction of Cocaine-Induced Condition Placed Preference in Nestin-Thymidine Kinase Transgenic Mice
Anne M. Wyer, Martina L. Mustroph, Chessa N. Kilby, Justin S. Rhodes
Department of Psychology, University of Illinois, Urbana, IL

C57BL/6J mice readily acquire conditioned place preference (CPP) for cocaine. Wheel running after conditioning can facilitate extinction of CPP, possibly due to increased numbers of new hippocampal neurons from the exercise. Nestin-thymidine kinase transgenic mice (NTK) have a C57BL/6J background with a thymidine kinase (TK) gene inserted downstream of the Nestin promoter. When NTK mice are fed ganciclovir, TK phosphorylates the chemical, exclusively disrupting cell division in the Nestin-expressing neural progenitor cells, thereby selectively reducing neurogenesis. The purpose of this study was to establish whether the NTK mice fed normal chow exhibit accelerated extinction of CPP from running as did C57BL/6J. If so, NTK mice can be used to test the extent to which new running-generated neurons are required for the accelerated extinction. Mice were habituated to the CPP apparatus and then evaluated for a baseline preference of two textures. Subsequently, mice were conditioned to associate cocaine on one texture and saline on the other. After conditioning, half the mice were placed in sedentary cages and half in cages with running wheels interfaced to pedometers to record rotations daily for 28 days. All mice received bromodeoxyuridine (BrdU) injections during the first 10 days of runner or sedentary treatment to label dividing cells. After 28 days, mice will undergo place preference testing without cocaine followed by reinstatement testing with cocaine injections. Three days after testing, mice will be transcardially perfused and hippocampal neurogenesis measured in the extracted brain. These results will set the foundation for future experiments in showing the different facets of learning, memory and neurogenesis.

Research supported by NIH T35 OD011145 and R01 DA027487

The Effects of Experimentally Induced Acute Endotoxemia on Pulmonary TNFalpha and Lung Inflammation in Healthy Adult Horses
Kaitlin M. Young, Kara M. Lascola, Gabriela Calzada-Nova, Pamela A. Wilkins, Carol W. Maddox
Departments of Veterinary Clinical Medicine and Pathobiology, College of Veterinary Medicine University of Illinois, Urbana, IL

Endotoxemia is a major cause of morbidity and mortality in critically ill horses. The contribution of a specific endotoxin, lipopolysaccharide (LPS), to acute lung injury has been described in several species. Although many systemic effects of LPS have been characterized, little is known regarding the contribution of LPS to acute lung injury in the horse. The objectives of this study were to determine whether systemic administration of LPS is associated with increased TNFalpha concentration in bronchoalveolar lavage fluid (BALF) and sera and to characterize changes in inflammatory cell distribution in BALF and lung tissue of healthy adult horses. Baseline BAL, percutaneous lung biopsy, TNFalpha, and white blood cell (WBC) counts were collected from 6 healthy adult horses. Following a 2-week recovery period, LPS from Escherichia coli O55:B5 (100 ng/kg IV) was administered to each horse. Serum TNFalpha, and WBC counts were determined prior to LPS administration and at 0, 0.5, 1, 1.5, 2, 4, 6, and 24 hours post-LPS infusion. BAL and percutaneous lung biopsy samples were collected at 3 hours post-infusion and were submitted for cytologic and histopathologic analysis. Serum and BAL TNFalpha concentrations were determined using a sandwich ELISA. A Student’s T-Test and Repeated Measure ANOVA were used to determine differences compared to baseline and over time with significance defined as P < 0.05. WBC counts decreased (P=0.000) and serum TNFalpha increased (P=0.000) compared to baseline, with the maximum change occurring at 1 hour post-infusion. No change in BALF cytology was observed. Analysis of BALF TNFalpha concentration and lung histology are pending. This research will expand our understanding of endotoxin-mediated acute lung injury in the horse and will contribute towards improved characterization of systemic effects of LPS. Ultimately this may aid in the management of critically ill adult and neonatal equine patients.

Research supported by NIH T35 OD011145

Loss and return of righting reflex in American Green Tree Frogs (Hylidae cinerea) after topical application of compounded sevoflurane or isoflurane jelly
Stephanie Zec, Stuart Clark-Price, Jordyn Boesch, Mark Mitchell
Department of Veterinary Clinical Medicine, College of Veterinary Medicine University of Illinois, Urbana, IL

Amphibians respire through pulmonary and cutaneous routes, allowing for topical anesthetic delivery. Anesthesia in frogs can be estimated by loss and return of righting reflex. Topical isoflurane or sevoflurane jellies can be used to anesthetize anurans. Sevoflurane is less tissue-soluble than isoflurane resulting in shorter induction and recovery times in mammals. It is unknown if tissue solubility affects topical anuran anesthesia. Loss and return of righting reflex was determined in 8 Hylidae cinerea weighing 6.5 ± 1.7 g in a randomized study. Frogs were placed into a container with 2 mL of isoflurane or sevoflurane jelly spread on the floor. Containers were intermittently inverted until loss of righting reflex. Frogs were then removed, rinsed clean of jelly and placed in new containers in dorsal recumbency. Frogs were observed until return of righting reflex. Four frogs initially tested with isoflurane developed skin lesions. Two died and were submitted for necropsy, histopathology and bacteriology; results pending. Use of isoflurane jelly was discontinued and only sevoflurane jelly was tested on remaining frogs. Data was analyzed after log transformation with t-tests. Data is reported as mean ± SD. Frogs receiving isoflurane jelly lost righting reflex at 90 ± 20 sec and returned righting reflex at 2659 ± 135 sec. Frogs receiving sevoflurane jelly lost righting reflex at 112 ± 33 sec and returned righting reflex at 1085 ± 1204 sec and no skin lesions were observed. There was no significant difference between loss of righting reflex times (p = 0.3); however there was a significant difference in return of righting reflex times (p = 0.04), isoflurane recovery was 4.5 times longer than sevoflurane. Topically applied sevoflurane jelly appears to be a satisfactory anesthetic agent compared to isoflurane which may cause fatal skin lesions in frogs. Options for providing anesthesia should be investigated and identified in order to advance care for this diverse species.

Research supported by Merial Veterinary Scholars Program

2011 Research Project Abstracts

Ecological Determinants of Lyme Disease Risk
Sarah E. Andermann1 and Brian F. Allan2
College of Veterinary Medicine, Department of Entomology, University of Illinois

Ecological determinants of disease risk play a critical role in the understanding and prevention of Lyme disease, a vector-borne zoonosis caused by the bacterium Borellia burgdorferi, and transmitted to humans by the bite of an infected black-legged tick, Ixodes scapularis. Due to human activity, the Midwestern United States has experienced fragmentation of what were once contiguous habitats of deciduous forest and tall-grass prairie. The white-tailed deer, Odocoileus virginianus, which has adapted extremely well to habitats fragmented by human activity, also serves as a critical host organism for the black-legged tick. The purpose of this study was to determine the types of habitats colonized first by I. scapularis and B. burgdorferi in east-central Illinois, a region located at the front wave in the expanding distribution of Lyme disease. Due to the movements of hosts such as O. virginianus, we hypothesized that I. scapularis will colonize large forest fragments (>40 ha) first, later progressing to smaller fragments (1-2 ha) nearby. Tick density and prevalence of infection with the bacterium B. burgdorferi were sampled from 18 forest fragments extending over 3 counties in east-central Illinois. Of these fragments, 6 were large and 12 were small, with half of the small sites being near (4 km) from the large sites. A higher density of infected ticks associated with large forest fragments at the invasion front may indicate that the encroachment and establishment of I. scapularis and B. burgdorferi are predictable processes associated with host movements through fragmented landscapes. Eventually, this pattern may be used to forecast areas that will be at high risk for the establishment of Lyme disease and targeted preventative measures may be utilized.

Student Support: Merial Veterinary Scholars Program
Research Support: The USDA North Central Integrated Pest Management Center and the EPA Pesticide Registration Improvement Renewal Act Partnership Grant

The Effects of Bisphenol A on Steroidogenesis in the Rat Ovary
Elizabeth Beesley, Wei Wang, and Jodi A. Flaws
Department of Comparative Biosciences, University of Illinois, Urbana, IL

Bisphenol A (BPA) is an endocrine disrupting compound frequently used in polycarbonate plastics and epoxy resins. BPA has been declared toxic for infants in many countries including China and Canada. In the United States, BPA has been banned in products that are intended for infant use in both California and the city of Chicago. The decision to reduce the amount of BPA used in infant products arises from new research demonstrating adverse effects of this chemical on endocrine parameters, specifically those in reproductive systems. Previous studies indicate that BPA exposure inhibits steroid hormone production. The goal of this study was to expand previous studies by determining if BPA affects steroid hormone production by altering the expression of genes encoding the steroidogenic enzymes, steroidogenic acute regulatory protein (Star) and aromatase (Cyp19a1), in rat ovaries. Our hypothesis is that BPA exposure decreases the expression of these steroidogenic genes, decreasing the amount of testosterone and therefore estradiol produced by the ovaries. To test this hypothesis, pregnant adult rats were dosed orally with vehicle or BPA (2.5 – 300,000 µg/kg) from gestation day 11.5 until birth. After delivery, the pups were dosed with vehicle or BPA until ovaries were collected on postnatal day 21. Once the ovaries from the pups were harvested, the mRNA levels were determined by quantitative real-time PCR, specifically investigating changes in gene expression of Star and Cyp19a1. Preliminary results indicate that middle concentrations of BPA affect the amount of Star mRNA found in the rat ovaries. We are currently determining the effect of BPA on the expression of the other steroidogenic gene. These results indicate that BPA may affect the expression of key enzymes required for normal steroidogenesis in rats. The main concern about BPA is that exposure will cause infertility by targeting the ovary.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: National Institute of Environmental Health Sciences, NIH, R01 ES019178

Intra- and Inter-Day Variability in Plasma [tCO2] in Horses
B.E. Bergstrom, J.H. Foreman, C.R. Foreman, and A.M. Barger
College of Veterinary Medicine, University of Illinois, Urbana, IL

Sodium bicarbonate and other alkalinizing solutions (“milkshakes”) have been administered to horses to buffer the lactic acid accumulated during high velocity exercise. Governing jurisdictions measure plasma [tCO2] as a proxy for plasma [bicarbonate] to determine if horses have been administered exogenous bicarbonate illegally. Our hypothesis was that plasma [tCO2] varies measurably over a multi-day monitoring interval. Our objective was to determine the intra- and inter-day variability of plasma [tCO2] and other plasma strong ion variables. Eight sedentary horses fed only alfalfa hay (5.3 kg twice daily at 7 AM and 3 PM after sampling) were sampled 3 times daily (7 AM, 11 AM, and 3 PM) for 5 days. Anaerobically-obtained heparinized jugular venous samples were analyzed immediately for plasma [tCO2], [Na+], [K+], and [Cl-], on an Olympus AU680 chemistry analyzer which was internally calibrated by the manufacturer’s recommendations. Mean ± s.e. was determined for each sampling interval with results compared by RMANOVA and other tests where appropriate. P

Student Support: Merial Veterinary Scholars Program
Research Support: Fédération Equestre Internationale and Maria Caleel Fund for Equine Sports Medicine Research

Microfilarial Nematodes and West Nile Virus Co-Infection in an Urban Setting
Amanda Dolinski1, Bethany Krebs2, Gabe Hamer3, Jeff Brawn2
1College of Veterinary Medicine and 2Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL; 3Department of Microbiology and Molecular Biology, Michigan State University, East Lansing, MI

West Nile Virus (WNV) is an emerging infectious disease posing increasing risks to wildlife populations, domestic animals, and humans. Microfilarial nematodes are thought to potentially increase the risk of WNV transmission by two mechanisms. First, by penetrating the mid-gut barrier of the mosquito, microfilarial nematodes may decrease the time it takes for the mosquito to be able to transmit the virus. This would enable the virus to be spread through a population at a much quicker rate. Second, infection with nematodes may suppress the host’s immune system making the host more susceptible to falling ill from WNV. If filarial nematodes play a role in WNV transmission, then we expect the prevalence of microfilaria to be high in important avian hosts. In order to determine the role of microfilarial nematodes in WNV transmission, it is important to understand how common microfilarial nematode infection is in wild bird populations. Five species of important avian hosts for WNV located in the Chicago suburbs were tested for microfilaria and West Nile Virus: the American Robin (Turdus migratorius), House Finch (Carpodacus mexicanus), House Sparrow (Passer domesticus), Mourning Dove (Zenaida macroura), and Northern Cardinal (Cardinalis cardinalis). Catching the birds was accomplished by mist-netting in the morning and late at night. Blood was collected by jugular venipuncture, and approximately 40 uL of each sample was centrifuged in a capillary tube and examined by light microscope for microfilaria. The remainder of each blood sample was saved for West Nile Virus testing. Birds positive for microfilaria were euthanized and necropsied to recover the adult filaroid nematodes for species identification. Collecting the parasites and correct identification of the nematodes is crucial to understanding the relationship between WNV and microfilaria.

Student Support: University of Illinois College of Veterinary Medicine
Research Support: National Science Foundation

Of Mice and Pigs: Optimization of Animal Models of Candida albicans Colonization and Disease
Cassandra Kerwin1, Sandra Manfra2, Michael Miller3, Jennifer Hoeflinger3, Dimitri Kashtanov3, Soon-Hwan Oh1, David A. Coleman4, and Lois L. Hoyer1
1Department of Pathobiology, 2Department of Veterinary Clinical Medicine, 3Department of Food Science and Human Nutrition, and 4Veterinary Diagnostic Laboratory, University of Illinois, Urbana, IL

Animal models of human disease are useful to study the interaction between microbes and host. C. albicans is a commensal fungus that can cause disease when the normal flora or immune system of the host is compromised. Oral candidiasis is one of the hallmarks associated with development of AIDS, and also affects people who wear dentures. Disseminated candidiasis occurs as the result of immunosuppression associated with chemotherapy or organ transplantation, for example, with the gastrointestinal tract believed to serve as the source of C. albicans. This work focused on optimization of two animal models to study C. albicans, one in its commensal state and the other in the context of oral disease. Because pigs are used as a model system to study the human gastrointestinal tract, we explored the possibility of stably colonizing pigs with C. albicans. We identified a source of pigs that were C. albicans-free. Piglets were farrowed normally and left with the sow for three days. On the fourth day, piglets were transferred to an artificial rearing environment and fed milk replacer. Following oral inoculation with C. albicans, pigs were monitored daily to detect C. albicans using swabs of the oral cavity and rectum, as well as the environment. Pigs were necropsied to assess the relative abundance of C. albicans throughout the gastrointestinal tract. Results showed that pigs could be stably colonized with C. albicans and the fungus recovered readily. The mouse model of oral candidiasis involved immunosuppression of mice with cortisone acetate and subsequent oral inoculation with C. albicans. Variables optimized included inoculum size and length of infection. The model was used to assess the effect of gene deletions on C. albicans pathogenesis. Both models will be used in future studies of C. albicans colonization and pathogenesis to better understand the interaction of the fungus with its host.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: National Institute for Dental and Craniofacial Research, NIH, DE14158; University of Illinois Campus Research Board

A Retrospective Evaluation of Long-Term Survival in Dogs Diagnosed with SARDS
MacDonell JL, Labelle AL, Hamor RE
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL.

Sudden acquired retinal degeneration syndrome (SARDS) is an acquired syndrome causing spontaneous and irreversible blindness in dogs. The goal of this retrospective study was to determine incidence of mortality and cause of death in dogs diagnosed with SARDS at the University of Illinois Veterinary Teaching Hospital. Medical records were reviewed and data regarding age at SARDS diagnosis, age at time of death, and other systemic illness was collected from the medical record and from both the referring veterinarian and owner via telephone. A total of 51 dogs were included in the study, with mixed breed dogs and Dachshunds representing the most common breeds. Dogs were diagnosed with SARDS at an average age of 8.09 ± 3.20 years. A total of 28/51 (54.9%) dogs were deceased and 23/51 (45.1%) dogs survived. The average age of the surviving dogs was 9.07 ± 3.10 years. The average age at time of death for non-survivors was 11.42 ± 2.89 years. The median interval since diagnosis of SARDS in surviving dogs was 1.38 years (IQR 1.10-4.48). The mean interval since diagnosis of SARDS in non-surviving dogs was 1.72 ± 1.58 years. Of the 28 non-survivors, 7 dogs died spontaneously and 21 were euthanized. The reason for euthanasia was reported for 15/21 euthanized dogs. The most common reasons for euthanasia were cardiac failure, renal failure, and musculoskeletal disease. Mortality rate for dogs diagnosed with SARDS was high, and cause of death often resulted from systemic disease rather than owner-perceived decrease in quality of life from SARDS-associated vision loss. Furthering the understanding of SARDS and its effects on the canine species is fundamental for the development of future treatment and prevention.

Student Support: University of Illinois College of Veterinary Medicine
Research Support: University of Illinois College of Veterinary Medicine

A Comparative Study of Osteogenesis in Equine Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue and Synovium
Brittany Naumann, Antonella Andretti, Yu-Wen Chen, and Matthew Stewart
College Of Veterinary Medicine, University of Illinois, Urbana, IL

Mesenchymal stem cells (MSC) are present in many tissues and are capable of multilineage differentiation, including osteogenesis. Osteogenesis is critical for embryonic skeletogenesis and for the repair of orthopedic injuries. The goal of this study was to determine the relative osteogenic potency of equine MSCs isolated from adipose tissue, synovium and bone marrow. Equine MSCs were isolated from bone marrow [BM], adipose tissue [AD] and synovium [SYN], expanded in monolayer for two passages, then transferred to standard osteogenic medium. At Day 7 and 14, the phenotypic status of the cells was evaluated by measuring alkaline phosphatase (ALP) activity, and staining for mineralized matrix (Alizarin red and Von Kossa). Robust osteoblastic differentiation occurred in cells derived from BM and SYN. Staining of the mineralized matrix of BM MSCs demonstrated a much higher uptake of both Alizarin red (AR) and Von Kossa (VK) stains in treated cells as well as a 4-fold increase in osteogenic nodule formation. SYN cells showed a 2-fold increase in nodule formation between osteogenic cells and control cells. In contrast, there were minimal differences between the control and treated AD cells. There was no nodule formation in either AD group. Osteogenic medium increased ALP activity in both BM and SYN MSCs in comparison to the controls. However, AD MSCs did not show any difference in ALP activity between treated and control groups. These findings suggest that both BM and SYN MSCs are capable of osteogenesis, although BM MSCs exhibit more robust differentiation. In contrast, equine AD cells were poorly responsive to osteogenic stimulation. On the basis of these data, BM MSCs should be preferred for clinical applications involving bone repair.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: National Institute for Arthritis Musculoskeletal and Skin Diseases and The Grayson Jockey Club Research Foundation.

Evaluating Impulsive Behavior: Effects of Dopaminergic Drugs on Rat Performance of a Delay Discounting Task
Terese E. Noe1, Paul A. Eubig1, and Susan L. Schantz1,2
1Department of Comparative Biosciences and 2Neuroscience Program, University of Illinois, Urbana, IL

Studies have suggested that dopaminergic activity may play a direct role in impulsive behavior, which is a component of neuropsychological disorders such as attention deficit/hyperactivity disorder (ADHD). One aspect of impulsive decision making manifests as intolerance for reinforcement delay, which can be assessed using a delay discounting task that evaluates delay tolerance. Based on a lever-press, normal individuals choose between a smaller but immediate food reward (the more impulsive choice) and a larger, delayed reward, with preference for the larger reward decreasing over longer delays. Decreased dopamine is associated with greater discounting, or higher impulsivity, in this task. The goal of this pilot study was to determine appropriate dosages of dopaminergic drugs that will be used in subsequent studies observing the behavior and performance of rats developmentally exposed to toxicants that may perturb dopamine signaling in the brain. Six male and six female toxicant-naïve Long-Evans rats were trained on the delay discounting task. Their performance in the same task after systemic administration of α-flupenthixol, d-amphetamine, and both drugs combined was then evaluated. Flupenthixol is a dopamine antagonist, while amphetamine increases synaptic concentrations of dopamine. Relative to baseline levels of delay tolerance, impulsivity was found to increase after α-flupenthixol injection, but administration of d-amphetamine did not produce a significant change in performance. The combined effect of these drugs will be determined as the drug trials are completed. These preliminary results indicate that, in normal Long-Evans rats, performance of the delay discounting task may not be sensitive to alterations in synaptic dopamine concentrations, although dopamine does influence performance of the task. These data help us further understand the relationship between dopamine and impulsivity, and may provide further insight into the mechanisms of neurological disorders that affect impulsive behavior.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: NIEHS, K08 ES017045 and NIEHS, R01 ES015687

Inhibitory Effect of Curcumin on Uterine Leiomyoma Cells Obtained from Aged Laying Hens
Alyssa Ortega, Malavika Adur, Olga Vinogradova and Romana Nowak
Department of Animal Sciences, University of Illinois, Urbana, IL

Uterine leiomyomas (fibroids) are noncancerous tumors that form within the myometrial wall of the uterus. The incidence of these benign tumors is 77% in reproductive-aged women in the United States, with the most common symptom being abnormal uterine bleeding. In serious cases, infertility or miscarriages also occur. The specific pathogenesis of human uterine leiomyomas has not been determined. Several types of animals have been suggested as in vivo models to study uterine leiomyomas. Laying hens have been validated as an appropriate model for uterine leiomyoma studies because hens develop smooth muscle leiomyomas on the wall of the oviduct with age and hormonal cycling patterns similar to humans. Previous studies have shown the polyphenol curcumin has an inhibitory effect on uterine leiomyoma cell proliferation, but optimal dose ranges for treatment were not determined. In the present study, we used three chicken leiomyoma cell lines (2P, 302P and 624P) established from leiomyomas harvested from three different birds. We determined the doubling time for each cell line, which was 0.66, 0.89, and 1.24 days, respectively. We carried out dose-response curves using concentrations of 20 µM, 50 µM, 100 µM, and 250 µM curcumin on each cell line, with corresponding concentrations of vehicle (DMSO) to determine whether it had any effect on cells. Using titrated-thymidine assays, we assessed the effects that the varying concentrations of curcumin had on DNA synthesis as a measure of cell proliferation at 24-, 48- and 72-hour time points. Preliminary results suggested that curcumin doses ranging from 100-500 µM had the greatest inhibitory effect on chicken leiomyoma cell lines. It is possible that the optimal dose for a particular cell line depends on doubling time. The results from our study showed that chicken leiomyoma cells can be used to screen compounds and to identify effective therapies for uterine leiomyomas.

Student Support: University of Illinois College of Veterinary Medicine
Research Support: NIH R01 HD046227

The Effectiveness of PAC-1 on Peripheral and Central Compartment Lymphomas
Nicole Pach1, Holly Pondenis1, Paul J. Hergenrother2, and Timothy M. Fan1
1Department of Veterinary Clinical Medicine and 2Department of Chemistry, University of Illinois, Urbana, IL

Apoptosis evasion is a hallmark of cancer, and studies suggest that proapoptotic compounds maybe promising anticancer agents. The induction of apoptosis can occur extrinsically or intrinsically, and both pathways lead to the activation of protein degrading enzymes called caspases. Procaspase-3 is an inactive zymogen that is cleaved to caspase-3, the key executioner caspase. In 2006, a small molecule named Procaspase Activating Compound 1 (PAC-1) was discovered, which activates procaspase-3 through zinc chelation, and induces apoptosis in cancer cells. Many cancer cells, including lymphomas, express procaspase-3, and should be susceptible to apoptosis induction. Uniquely, PAC-1 rapidly penetrates the blood brain barrier, however, the mechanisms for how PAC-1 may enter the brain remain uncharacterized. Given its CNS penetrant characteristics, the purposes of this study were to demonstrate the effectiveness of PAC-1 as an anticancer treatment for peripheral and central compartment lymphomas and to further characterize potential mechanisms for how PAC-1 permeates the CNS. Murine lymphoma cells (EL-4) were exposed to different concentrations of PAC-1 at various time points and the activation of procaspase-3 was quantified using flow cytometry, Western blotting, and a colorimetric assay. The in vivo mechanism of PAC-1 action was studied in mice intraperitoneally inoculated with EL-4 cells, and subsequently treated with PAC-1 for varying times. The ability of PAC-1 to traverse vascular endothelial cell barriers was studied using a commercially available vascular permeability assay. In vitro, PAC-1 activated procaspase-3 in EL-4 cells at biologically achievable concentrations (25-50 uM) and exposure durations (4-8 hours), based upon prior pharmacokinetic and toxicity studies. Significantly, PAC-1 administration to EL-4-bearing mice also activated procaspase-3 in vivo. Finally, PAC-1 did not increase vascular permeability, suggesting that its rapid CNS penetrating effects are unlikely dependent upon direct vascular permeation. These results suggest that PAC-1 maybe a promising anti-lymphoma agent and warrants further investigation.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: NIH, R01 CA120439

Competitive Revision-Small Molecule Activators of Procaspases as Anti-Cancer Agents: The Effect of Acute Exercise on the Central Immune and Behavior Responses of LPS-Challenged Mice
EB Parsons1, SA Martin2 and JA Woods2
1 College of Veterinary Medicine and 2 Integrative Immunology and Behavior Program, Department of Kinesiology and Community Health, University of Illinois, Urbana, IL

Upon pathogen recognition in the periphery, the innate immune system produces pro-inflammatory cytokines which signal the brain to induce sickness behavior, characterized by weight loss, anorexia, fever, and lethargy. Under normal conditions, sickness is an adaptive response aimed at conserving energy while the host clears the infection. However, under certain pathological conditions (e.g. aging, cancer, autoimmune disorders), sickness behavior often progresses toward a depressive behavior phenotype, characterized by helplessness, anhedonia, fatigue, and hyperalgesia. While both long-term moderate exercise training and acute exhaustive exercise have been shown to decrease pro-inflammatory cytokine levels in the periphery (serum & tissues) following innate immune activation with E. coli-derived lipopolysaccharide (LPS), it is unclear if this translates to reduced brain inflammation and attenuated depressive behavior. The aim of this study was to determine if acute exercise attenuates LPS-induced sickness behavior and depressive-like behavior in adult CD-1 mice. We hypothesized that acute treadmill exercise would decrease peripheral inflammation, which would translate to reduced brain inflammation and behavioral abnormalities. Male CD-1 mice (n=40) were randomized into exhaustive treadmill running and sedentary groups (10/group). After an 80-minute acute exercise or sedentary session, mice were injected intraperitoneally with either LPS at a dose of 0.33 mg/kg or saline. Bodyweight, food intake, and novel cage motor activity were assessed at 3 and 8 hours post-injection. Depressive-like behavior was assessed by the tail suspension test at 9 hours post-injection. If the mechanism of acute exercise’s effects on immune function can be clarified, it may have implications for treating the chronic low-grade inflammatory status associated with aging, autoimmune disease, and cancer, which could decrease the likelihood of depressive episodes subsequent to disease onset.

Student Support: Merial Veterinary Scholars Program
Research Support: National Institutes of Health, R01 AG029573

Effect of Voluntary Exercise on Hippocampal Microglia, Neurogenesis, and Learning in Aged Mice
Lindsey N. Peterson1, Rachel A. Kohman2, and Justin S. Rhodes2
1College of Veterinary Medicine and 2Department of Psychology, University of Illinois, Urbana, IL

Microglia, the brain’s primary immune cells, undergo age-related changes that may be responsible, in part, for the cognitive decline seen in older adults. Exercise has been shown to combat cognitive decline and improve brain health by enhancing learning and stimulating neurogenesis in the hippocampus. It was the goal of this study to elucidate what effect exercise has on microglia proliferation and phenotype, as microglia can take on a classic inflammatory phenotype or an alternative neuroprotective phenotype. Additionally, we assessed the potential correlation between changes in microglia, neurogenesis, and learning. In order to do this, aged and young mice were housed with or without access to a running wheel for eight weeks. Mice received daily injections of bromodeoxyuridine (BrdU) for either the first or last 10 days of the study to label cell division. After seven weeks of running, mice underwent auditory and contextual fear conditioning to test learning. Brains were collected, sectioned, and labeled for BrdU, NeuN, Iba-1, and/or IGF-1 using immunohistochemistry to estimate neurogenesis, microglia proliferation, and microglia phenotype. If exercise has an effect on microglia that is correlated with a decrease in neuroinflammation and an increase in neurogenesis and learning, then exercise may be beneficial as a therapy for disorders such as Alzheimer’s disease and may be used to slow cognitive decline.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: National Institutes of Health, MH083807 and DA027487

Oranges Might Contain the Key for Your Memory Formation
Elizabeth Rivera-Cruz1, Lori Newman2,3, Paul Gold2,3,4
1School of Molecular and Cellular Biology, 2Department of Psychology, 3Neuroscience Program, 4Departments of Psychiatry, Integrative and Molecular Biology, Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana and Champaign, Champaign, IL

Ascorbic acid, the reduced form of Vitamin C, is highly concentrated in the central nervous system (CNS), and is able to inhibit glucose consumption and stimulate lactate uptake in neuronal cells. Astrocytes in the CNS produce lactate, which will be preferably used as an energy source by neurons during memory formation. Therefore, it is hypothesized that ascorbic acid functions as a mediator between lactate and glucose during neuronal activity. In this experiment, a biosensor probe able to detect lactate, ascorbic acid or glucose, was inserted in the hippocampus of young Sprague Dawley male rats to determine the concentration of the mentioned molecules during training on hippocampus-dependent place or striatum-dependent response learning tasks. The hippocampus-dependent place learning task was performed by placing a rat in a maze in which a reward will always be in the same location of a cue-rich room. On the other hand, the striatum-dependent response learning task was performed in a maze in which the rat would learn to obtain a reward every time it turns to the same direction in a cue-poor room. It is hypothesized that the levels of ascorbic acid and lactate will be higher during place learning. Future work will consist of inserting a biosensor with the same probes into the striatum to determine the levels of glucose, lactate and ascorbic acid during both hippocampus-dependent place and striatum-dependent response tasks. It is expected that the levels of lactate and ascorbic acid in the striatum will be higher during the response task. These findings suggest a specific role for astrocytes during memory formation in controlling ascorbic acid and lactate release. Such results point to novel targets for treatment of memory loss.

Student Support: University of Illinois College of Veterinary Medicine
Postdoctoral Support: NIH Training Grant HD007333
Research Support: NIH grants AG07648 and DA024129, and an award from the Alzheimer’s Association.

Effects of Sedation on Standard Equine Coagulation Parameters
Brandi Saas1, Stephanie Smith2, Georgean Zyrkowski1, and Kara Lascola1
1College of Veterinary Medicine and 2College of Medicine, University of Illinois, Urbana, IL

Acepromazine and xylazine are commonly administered to critically ill equine patients to reduce anxiety, provide analgesia and enable safe evaluation of the animal. These medications cause a decrease in hematocrit and may impact platelet function. This study evaluates whether administration of acepromazine or xylazine impacts thromboelastometry (TEM) or other coagulation assays in healthy horses. Using a random crossover design 7 adult horses were administered a single dose of xylazine (1.1 mg/kg IV) or acepromazine (0.1 mg/kg IV) with a 3-week washout period between treatments. Hematocrit, total protein, platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT), TEM and thrombin antithrombin complexes (TATc) were measured at baseline, 30 minutes and two hours for both medications, and at 6 hours and 12 hours for acepromazine. At 30 minutes post xylazine administration (n=3) hematocrit decreased by 15.7%. Changes in TEM included an 8.0% decrease in clot formation time (CFT) and a 5.3% increase in alpha angle. At 30 minutes, 2 hours and 6 hours post acepromazine administration (n=3), hematocrit decreased by 18.0%, 19.7%, and 14.3%, respectively. Changes in TEM (n=2) included a 9.0% decrease in CFT and a 3.0% increase in alpha angle at 30 minutes. As expected, hematocrit decreased after the administration of acepromazine and xylazine. Changes in TEM parameters at 30 minutes suggested hypercoagulability. Evaluation of additional animals is required to determine statistical significance. Measurement of PT, aPTT and TATc will help determine whether changes in TEM represent true alterations in coagulation or are due to decreases in hematocrit. Coagulation abnormalities

Student Support: Merial Veterinary Scholars Program
Research Support: Lascola Equine Medicine Laboratory

Prevalence and Spatial Distribution of Frog Virus 3 in Wild Populations of Northern Cricket Frog (Acris crepitans) in Western Illinois
Joanna Sekowska and Matthew Allender
College of Veterinary Medicine, University of Illinois, Urbana, IL

Ranavirus is a lethal and highly contagious disease responsible for mass mortality events in wild amphibian populations. Due to the drastic decline of amphibian species witnessed in the last three decades, determining susceptible locations and populations is a critical key to slowing down the present extinction rates. This study focuses on determining the prevalence and spatial distribution of Frog Virus 3 (FV3), a species of ranavirus, in western Illinois ponds. The northern cricket frog was chosen due to its unexplained decline from being one of the most abundant frogs in Illinois. Using a sterile technique, each wild-caught frog was physically examined and swabbed for virus particles in the oral cavity and cloaca. DNA was extracted and will be examined using real-time PCR to determine if and how many individuals test positive for this deadly pathogen. A positive result does not prove the individual was infected, but indicates the presence of the virus in the pond. Knowing if and where FV3 exists in Illinois is extremely useful in aiding efforts to save frog populations.

Student Support: Merial Veterinary Scholars Program
Research Support: Allender Research Lab

Is Dermatophyte Morphology Only Skin-Deep? Identification of Veterinary Dermatophyte Isolates Using Molecular Methods
Ursula Sieklucki, Soon-Hwan Oh, and Lois L. Hoyer
College of Veterinary Medicine, University of Illinois, Urbana, IL

Dermatophytes are pathogenic fungi that can invade keratinized tissue (skin, hair and nails) of humans and animals. The familiar name for the resulting infection is ringworm. The most common dermatophytes that cause ringworm in animals are Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. Identification of dermatophytes traditionally has relied upon the appearance of the colony on an agar plate and microscopic visualization of fungal structures. For example, an isolate can be placed into the genus Microsporum, rather than into the genus Trichophyton, based upon relative differences in the abundance of microconidia and macroconidia, whereas the species M. canis and M. gypseum can be differentiated from each other based on the shape of the macroconidia. The use of molecular methods to identify dermatophyte isolates is becoming more developed, demonstrating that previous taxonomic groupings at the species level are actually comprised of more than one dermatophyte. For example, T. mentagrophytes is now recognized as a species complex, that includes several distinguishable species. This study applies molecular identification methods to a collection of dermatophyte isolates from veterinary clinical cases. We hypothesize that molecular identification methods will reveal greater taxonomic variation in the collection of isolates than recognized based on morphological identifications. Genomic DNA from each dermatophyte isolate will be amplified by polymerase chain reaction (PCR) using primers specific to the internal transcribed spacer (ITS) region of the nuclear-encoded ribosomal RNA genes (rDNA), as well as primers specific for the 28S rRNA-encoding gene. DNA sequences will be compared to those in public databases to aid in identification of the dermatophyte isolate. Clinical case records that are available for many of the isolates will be used to evaluate potential associations between specific dermatophyte species and animal hosts. Information from this analysis will further knowledge of the epidemiology of dermatophytosis in animal species.

Student Support: National Center for Research Resources, NIH, T35 RR020292

Brain-Behavior Changes in Female Three-Spined Sticklebacks (Gasterosteus aculeatus) Using Immediate Early Gene Expression as an Indicator of Brain Activation
Amanda Smith1, Molly Kent2, Travis Weinsheim2, and Alison Bell2
1College of Veterinary Medicine and 2Department of Animal Biology, University of Illinois, Urbana, IL

The three-spined stickleback (Gasterosteus aculeatus) is a model organism for studying behavior. The goal of the study is to determine which regions of the female brain are activated in response to courtship from a male. Females were placed in a flask and then introduced into a male’s tank for ten minutes. We compared immediate early gene expression in the brains of females placed in a flask and exposed to a courting male, to females that were placed in a flask (but not exposed to a courting male), and to females that were sampled directly from their holding tank. To detect brain activation, we measured the expression of an immediate early gene, Egr-1, in the brain using whole mount in situ hybridization. Briefly, brains were excised and placed into 4% paraformaldehyde. After 24 hours, the dura mater and remaining debris were cleaned from the brains and the brains were placed in 100% methanol. Brains were incubated in a probe specific for stickleback Egr-1. Next, the brains were treated with an antibody specific for the created probe and visualized through a chemical reaction using a FITC fluorescent marker. Finally, brains were incubated in DAPI, a neuronal cell marker, to allow all brains to be compared across treatments regardless of activation. This study will enable us to further understand the neuronal mechanisms that mediate the dramatic behavioral shifts seen during the breeding cycle in this male uniparental species.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: NIH, R01 GM082937

Topically Applied Sevoflurane Jelly: an Alternative Method of Anesthesia for Bufo marinus
Sabrina Stone, Stuart Clark-Price, Jordyn Boesch, and Mark Mitchell
College of Veterinary Medicine, University of Illinois, Urbana, IL, USA

Amphibians have a unique respiratory system that allows gas exchange by two routes: pulmonary and cutaneous. Studies have shown that anurans can be anesthetized by topical application of isoflurane. Sevoflurane, an anesthetic similar to isoflurane, has not been tested for topical application. We developed and tested a topically applied sevoflurane jelly-mixture on Bufo marinus to determine anesthesia induction and recovery times. Eight toads were used for the study. Sevoflurane jelly was made by mixing 3 parts of sevoflurane liquid, 1.5 parts of distilled water, and 3.5 parts of aqueous jelly. The anesthetic mixture was applied to the dorsum of toads at 0.025 ml/g of body weight. Toads were placed in an airtight, custom-built anesthetic chamber that allowed manipulation of the toads and determination of sevoflurane vapor. Toads were turned to dorsal recumbency every 30 seconds until righting ability was lost. During the anesthetic induction phase, gular movement slowed and eventually became absent. After loss of righting ability, anesthetic mixture was rinsed off and toads were placed back in dorsal recumbency. Toads were observed until return of righting ability and normal gular movements. Anesthetic chamber air was measured for sevoflurane vapor throughout. Results from a single toad trial showed an induction time of eight minutes and recovery in 52 minutes. Anesthetic chamber sevoflurane reached a maximum concentration of 0.9% during induction and was 0% during recovery. Topical application of sevoflurane jelly mixture may be a convenient method of anesthesia for anurans, particularly in field settings, eliminating the need for euthanasia.

Student Support: Merial Veterinary Scholars Program
Research Support: Clark-Price Anesthesiology Laboratory

Detection and Differentiation of Canine Lymphoma Using Quantitative PCR
Zachary Westrick, Kuldeep Singh, Sheila Patterson, Therese Eggett, and Carol Maddox
Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Illinois, Urbana, IL

Lymphoma is the most common cancer found in dogs. It arises from a clonal expansion of either B cells or T cells and rarely NK cells. The diagnosis of lymphosarcoma is frequently corroborated by B- and T-cell immunohistochemistry; however, this technique is somewhat subjective and takes time. Quantitative Polymerase Chain Reaction (qPCR) provides the opportunity for a rapid and definitive diagnosis. Using qPCR allows one to differentiate between B- and T-cell lymphomas more objectively, which is important for treatment and prognosis. The common mechanism in the pathogenesis of lymphosarcoma is generally a monoclonal expansion of lymphocytes. Single or double amplicons obtained using qPCR suggest lymphoma, contrary to multiple amplicons that appear in healthy lymphoid tissue. Template DNA was isolated from both formalin-fixed tissues embedded in paraffin and fresh tissue samples. Tissue samples from both positive and negative lymphosarcoma cases were used for optimization of a qPCR protocol. Primers specific to the VDJ region of B- and T-cell receptors were used in each assay. Differences in these joining regions account for different products formed from the qPCR reaction. Melting curve analysis was used to determine whether positive samples had unique amplicon numbers and sizes compared to negative samples. Gel electrophoresis was used to isolate the reaction products for cloning and DNA sequence analysis. Sequence results were compared to gene databases to ensure the correct gene segments were amplified. Having this molecular diagnostic tool will help to specifically treat lymphomas in a timelier manner.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: College of Veterinary Medicine, University of Illinois

Effect of Lactoferrin on Piglet Gastrointestinal and Immune Development
Amanda K. Wolff1, Sarah S. Comstock3, Elizabeth A. Reznikov2, Marcia Monaco3, and Sharon M. Donovan2,3
College of Veterinary Medicine1, Division of Nutritional Sciences2 and Department of Food Science and Human Nutrition3, University of Illinois, Urbana, IL

Human breast milk protects the infant by providing maternal antibodies that infant formula does not contain. Lactoferrin (LF), an antimicrobial breast milk-protein, is known to strengthen immunity. By adding LF to formula, we can study its effects compared to infant formula alone. Since neonatal pig gastrointestinal physiology is similar to infants, piglets make an excellent model to study gastrointestinal and immune development. This study used naturally farrowed piglets to study LF’s effect on piglet gastrointestinal and immune development. Piglets were colostrum-deprived and therefore lacked maternal antibody transfer, resulting in immunocompromised animals. Piglets were randomly divided into three groups (n=10-13 per time point): formula without added LF (0.36 g LF/L), formula with 1.02 g LF/L, and formula with 3.62 g LF/L. The piglets were euthanized on day 7 or day 14 and serum samples from the heart, tissue from the middle of the jejunum, and contents of the ascending colon were collected. We hypothesized that LF would increase IgG and IgA antibody concentrations as well as modulate gastrointestinal genes related to LF, proliferation and innate immunity. Total serum IgG concentration was measured by ELISA as was total ascending colon IgA concentration. Intestinal gene expression was measured using jejunal tissue extracts and quantitative real-time PCR. The results of this study will help us to better understand the effect of lactoferrin on human infant gastrointestinal and immune development.

Student Support: National Center for Research Resources, NIH, T35 RR020292
Research Support: Pfizer Nutrition, Collegeville, PA

2010 Research Project Abstracts

Inhibition of NFĸB by the MC159 Protein in A375 Human Melanoma Cells
Alice Bugman, Crystal Randall, and Joanna Shisler
College of Veterinary Medicine and Department of Microbiology, University of Illinois, Urbana, IL

Oncolytic viruses are viruses that selectively replicate in cancer cells and provide a promising alternative therapeutic treatment for proliferative cancers. Myxoma virus (MYXV), a poxvirus that solely infects rabbits, has shown great potential as an oncolytic virus in humans. Some cancers, such as melanoma, have increased cellular proliferation as a result of constitutive activation of Nuclear Factor-Kappa B (NFĸB), a nuclear transcription factor involved in proinflammatory responses. Inhibition of NFĸB induces cell death in A375 cells, a human melanoma cell line. Poxviruses produce immunomodulatory proteins capable of inhibiting NFĸB activation. We are interested in the MC159 protein, a known viral inhibitor of NFĸB activation produced by the molluscum contagiosum virus (MCV). Our goal is to characterize the effects of MC159 protein expression on NFĸB activation and proliferation of A375 cells. We found that MC159 inhibits NFĸB activation in A375 cells through electromobility shift assays (EMSA) and luciferase assays. The ability of MC159 to induce cell death in A375 cells is still being analyzed. MC159 has potential as both a solitary therapeutic or as part of an oncolytic viral system such as MYXV. This development could lead to further advances in the field of virotherapeutic treatments for numerous cancers affecting both humans and animals.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Institute of Allergy and Infectious Diseases, NIH, R01 AI055530

Effects of Lighting Environment on Expression on Opsins, the Alteration of Ellipsosomes, and Genes Linked to Retinal Damage in the Bluefin Killifis
Kristin M. Claricoates and Rebecca C. Fuller
College of Veterinary Medicine and Department of Animal Biology, University of Illinois, Urbana, IL

Bluefin killifish live in various lighting habitats ranging from tannin-stained swamps to clear springs. Spring and swamp populations vary in the relative abundance of various cone cells, as well as in the expression of the opsins that determine the spectral absorbance. This project analyzes the effects of light on the expression of opsins (8 genes) to determine whether opsin expression is plastic and how quickly these changes occur. This project also examines the expression of 3 genes associated with ellipsosomes – a light filter at the base of cone cells, and 2 genes associated with retinal damage due to high light. Fish from three different natural Florida habitats (clear, tannin-stained or mixed water) were placed into a tank of either clear or tannin-stained water. Fish were euthanized at 7 different time points from day 0 to 28 and eyes saved for RNA extraction. We hypothesize that (1) spring fish in clear water will express high levels of UV and violet opsins and high levels of genes associated with ellipsosomes, (2) swamp fish in high light will show high expression of genes associated with retinal damage, and (3) fish from mixed water conditions will show rapid changes in gene expression between treatments. Primer sets for real-time reverse transcription polymerase chain reaction were designed from bluefin killifish gene sequences. This research will indicate the extent to which gene expression is readily plastic with respect to lighting environment and the extent to which this differs among populations. This work will also indicate whether the bluefin killifish can be used as a model organism for studying adaptation to stressful high light conditions.

Student support: Abbott Laboratories
Research support: National Science Foundation and the University of Illinois

Minimally Invasive Diagnostic Evaluation of Meniscal Disease in Dogs with Cranial Cruciate Ligament Rupture
Dayle Dillon, Wanda Gordon-Evans, Dominique Griffon, Kim Knap, and Carrie Bubb
College of Veterinary Medicine, University of Illinois, Urbana, IL

Meniscal disease is common in dogs presenting with cranial cruciate ligament (CCL) injuries. The most definitive diagnostic tests for detecting meniscal disease are MRI and arthroscopy. However, both procedures are expensive, and can be stressful for the animal. We evaluated common, minimally invasive examination findings to see which factor(s) would be most accurate for diagnosing meniscal disease. Eighty dogs presenting with partial or complete CCL tears were evaluated preoperatively for factors that may increase the risk or aid in the diagnosis of meniscal disease. All dogs were evaluated arthroscopically to evaluate the menisci and cruciate ligaments. Our results showed that body condition score, weight, pain in the stifle upon extension, thigh circumference, cranial-drawer, thrust, duration of injury, and partial tears of the CCL had no significance in detecting or increasing the risk of meniscal disease. A positive meniscal “click” increased the likelihood that dogs with CCL damage would also have meniscal disease by a factor of 11.3 (confidence intervals: 2.2, 58.4). Dogs with pain upon flexion of the stifle were 4.3 times more likely to have meniscal disease and dogs with a complete CCL tear were 9.6 times more likely to have meniscal disease. The diagnostic accuracy using all three risk factors was 77% with 81% sensitivity and 60% specificity. Combining diagnostic factors including meniscal “click”, severity of CCL tear, and pain upon flexion of the stifle provide a novel approach to diagnosing meniscal disease.

Student support: Merial Veterinary Scholars Program
Research support: Morris Animal Foundation

Correlation Between Body Size and Intestinal Size, Nutrient Processing Capacity, and Rate of Gain in Growing Piglets
Sarah Francik, Heather Mangian, and Kelly A. Tappenden
College of Veterinary Medicine and Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL

It is believed that intestinal size in growing pigs decreases as body size increases. The implications of this relationship are that nutrient processing capacity and hence growth also decreases as pigs gain more weight. We hypothesize the nutrient processing capacity and feed efficiency decrease as body size increases in growing piglets. The objective of this study is to assess the validity of a correlation between intestinal size and function and body size of growing barrows. Growing barrows (n=72) at 9 weeks of age will be divided into three replicates (n=24) with the pigs divided into two time points. Half of the pigs from each replicate will be fed a corn soybean meal diet for 7 days and the other half will be fed the same diet for 14 days. The pigs will be weighed at 0, 7, and 14 days, if applicable, to track growth, rate of gain and feed efficiency. At the end of a given time point pigs will be euthanized and intestinal samples will be collected for analysis of structure and function. The intestine will be divided into four segments: the duodenum, jejunum, ileum and colon. Each segment will be weighed and measured to assess size. Ussing chambers will be used to analyze mucosal ion and nutrient transport in the segments. The data will give further insight into the correlation between body size, intestinal size, and nutrient processing capacity which could lead to more efficient diet formulation in growing pigs.

Student support: National Center for Research Resources, NIH, T35 RR020292

Arachidonic Acid Specific Deficiency Results in Inflammation at the Ileocecal Junction
Caitlyn Getty, Manuel Roqueta-Rivera, Timothy Abbott, Kevin Hadley, and Manabu Nakamura
College of Veterinary Medicine, Department of Food Science andHuman Nutrition, and Division of Nutritional Sciences, University of Illinois, Urbana, IL; Martek Biosciences, Columbia, MD

Arachidonic acid (20:4n6, AA) and docosahexaenoic acid (22:6n3, DHA) are important in gastrointestinal cell function, but the roles of each are not fully elucidated. AA and DHA are metabolites of the dietary essential polyunsaturated linoleic acid(18:2n6) and α-linolenic acid(18:3n3), and the rate-limiting enzyme is delta-6-desaturase(D6D). In order to study the gut requirement for AA, D6D knockout (-/-) mice were used as the experimental model of specific AA deficiency and wild-type (+/+) mice were used as a control. Three age groups were utilized: 1 month (+/+), 3 months (+/+, -/-), and 4 months (+/+, -/-); (n=3-4) for each group. Mice were fed a diet containing 0.1% DHA and no arachidonic acid. At each time point, fatty acid and histological analyses (3 and 4 months) were performed on the gut tissue. Tissue AA levels decreased in the (-/-) mice over the course of the study and were 92.1% depleted at 3 months and 96.6% at 4 months in the (-/-) mice compared to the controls. Histological analysis showed normal epithelium in the (+/+) mice and compromised epithelium at the ileocecal junction in the 3 and 4 month (-/-) mice. Increased lymphocyte infiltration and decreased goblet cell number were seen in all (-/-) mice before erosions or ulcers appeared. Erosions were seen in two 3-month mice, and more severe erosions or ulcers were seen in three 4-month mice. In conclusion, the study has shown essentiality of AA in gastrointestinal function; in particular, AA may play a critical role in goblet cell function.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: Martek Biosciences

Meaningful Assessment of Behavior in the Study of Laying Hen Preference
Jennifer Carr, Angela Green, Patricia Paulausky, and Tatiana Sales
College of Veterinary Medicine and Department of Agricultural and Biological Engineering, University of Illinois, Urbana, IL

Laying hens (Gallus gallus) provide an ideal subject for preference research, as their welfare in agricultural environments is a subject of ongoing moral and economic debate. In such preference studies, conclusions are often based on the pretext that a hen’s choices will reflect the pressures of its primal–or ethological–needs. The addition of behavioral analysis to these studies can provide further insight into the birds’ environmental or resource preferences. For example, the correlation of defecation with a presumably negative environment (such as one high in ammonia) offers a logical explanation for lack of aversion. To investigate the relationship between preference and innate behavior in the hen, a video analysis was performed concurrently with a preference study on passageways. Hens were trained to use three types of doors to move between two cages, one containing food and the other water. They were then allowed to choose between the three doors. Their choices were recorded via sensors in the connecting passages, and their behavior was monitored by video. The aim of this experiment is to explore the degree to which the hens’ choices are reflected in their general behavior. For behavioral analysis to be considered a meaningful tool in preference research, it should provide evidence of the birds’ experiential perceptions of the environment presented to them, and how these perceptions inform their choices. The results of this study will enhance the value of preference studies in the design of housing systems that reduce stress and optimize welfare for laying hens.

Student support: Merial Veterinary Scholars Program
Research support: Multistate project NE-1042: Poultry Production Systems: Optimization of Production and Welfare Using Physiological, Behavioral, and Physical Assessments and University of Illinois Urbana-Champaign

Spatial Analysis of Human Behavior and its Relation to Exposure to West Nile Virus in Suburban Chicago
Alicia M. Hines and Marilyn O. Ruiz
College of Veterinary Medicine, University of Illinois, Urbana, IL

West Nile Virus (WNV) poses a significant public health threat, but the risk of infection for humans varies by location. Spatial analysis is useful to identify infection patterns in mosquitoes and hosts, but few studies have examined how to best measure the locations of people when assessing risk of infection. In this study, we examined a region south of Chicago, Illinois, where we selected 40 sites from a random stratified sampling of three environments (natural, residential, and other) and monitored human distribution and activity during evening mosquito feeding hours. Sex, age group (child, adult, senior) and activity were recorded for each observed human and additional subsets of humans were interviewed about their knowledge of WNV, use of mosquito repellent, place of residence, and frequency of visits to the interview area. To determine how human behaviors affect WNV exposure risk, the demographic and behavioral data were analyzed in conjunction with mosquito WNV infection rates. Estimates of human location from survey data, U.S. Census data, and house locations were also used to compare estimated exposure risk given different data. Greater risk was expected in natural areas near residential spaces due to increased human use of the areas during mosquito feeding hours and a high abundance of mosquito hosts in which WNV is amplified. Pairing the knowledge of how the location of specific human behaviors influence WNV exposure with the ecology and epidemiology of the virus will aid in developing more accurate predictive models and improve protocols for controlling potential outbreaks.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Science Foundation grant number 0840403

The Affect of TNF-α on Meiotic Maturation and Blastocyst Development
Jennifer Ida, Y. Yuan, E. Silva, and R.L. Krisher
College of Veterinary Medicine and Department of Animal Sciences, University of Illinois, Urbana, IL

Female infertility has been linked to elevated Tumor Necrosis Factor alpha (TNFα) in multiple tissues. TNFα is a cytokine released in response to inflammation and could potentially have an effect on gametes in vivo. Previous work in our laboratory has demonstrated elevated expression of TNFα in oocytes with reduced competence. The objective of this study was to determine the effects of TNFα on the meiotic maturation and developmental potential of immature porcine oocytes. Oocyte treatments were; 1) control with no TNFα, 2) 0.1 ng/mL TNFα, 3) 1 ng/mL TNFα and 4) 100 ug/mL anti-TNFα antibody. Porcine oocytes were aspirated from abattoir derived sow ovaries, and selected to assure good, homogeneous quality. Oocytes were placed into PPM maturation medium and matured in vitro for 42-44 hours. Then, a sample of oocytes was assessed for meiotic stage. The remaining oocytes were fertilized (mTBM medium) and cultured (NCSU23 medium) in vitro (IVF/C) to assess cleavage and development to the blastocyst stage. Preliminary data demonstrate there is no difference (p>0.05) in embryo cleavage or development after exposure of oocytes to TNFα during maturation, despite previous reports that TNFα reduced blastocyst formation. Compared to control oocytes, anti-TNFα tended to reduce development to the blastocyst stage after IVF/C, suggesting that some level of TNFα is necessary to support oocyte quality. Overall, these results suggest, however, that TNFα is not a critical factor during oocyte maturation in supporting oocyte development following fertilization. These results enhance our understanding of oocyte competence and factors affecting infertility.

Student support: National Center for Research Resources, NIH, T35 RR020292

The Use of Anti-Inflammatory Therapy as Adjunctive Treatment in Dogs with Blastomycosi
Melissa J. Munks, Julie K. Byron, Sarah K. Dowling, Nick Szigetvari, Marcella D. Ridgway, and Mark Mitchell
College of Veterinary Medicine, University of Illinois, Urbana, IL

Medical records were reviewed for dogs diagnosed with blastomycosis at the University of Illinois Veterinary Teaching Hospital between 1992 and 2007 and dogs with a presenting PaO2 of ≤ 80 mmHg and clinical or radiographic signs of respiratory blastomycosis were included. All dogs were treated with either itraconazole, fluconazole, amphoteracin B, or a combination of these. Group 1 (G1) dogs were treated with NSAIDs and Group 2 (G2) dogs were treated with anti-inflammatory doses of steroids as adjunctive therapy for blastomycosis treatment. The following comparisons were made: number of days of oxygen supplementation, number of days in hospital, survival to discharge, and long term survival of the infection. G1 consisted of 31 dogs and G2 consisted of 37 dogs. The two groups were found to be similar in weight, age, and sex distribution. There was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and survival of the infection. There does not appear to be a difference in the efficacy of NSAIDs and anti-inflammatory steroids in dogs with severe pulmonary blastomycosis. Further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ARDs and additional respiratory complications.

Student support: Merial Veterinary Scholars Program

Fecal Endocrine Monitoring in Snow Leopards (Uncia Uncia)
Jamie L. Reichert and Jason R. Herrick
College of Veterinary Medicine, University of Illinois, Urbana, IL

Little is known about the complex endocrine interactions controlling ovarian function and conception in the endangered snow leopard. The goal of this work is to utilize an enzyme immunoassay (EIA) to analyze fecal concentrations of estrogen and progesterone metabolites to characterize ovarian activity throughout the estrous cycle, non-pregnant luteal phase (pseudopregnancy), and gestation in female snow leopards. Measuring fecal steroid metabolites to assess endocrine function is extremely useful in non-domestic species because it eliminates the need for anesthesia or restraint during sample collection, and greatly reduces stress on the animals. Fecal steroid metabolite measurements provide an accurate average concentration estimate because the hormone metabolites are excreted over a period of hours, compared to measurements from serum samples that represent a single point in time. For this study, fecal samples were acquired from 7 female snow leopards maintained at 5 different zoos accredited by the Association of Zoos and Aquariums. Zoo staff collected 3-4 fecal samples per week during routine daily cleaning beginning in January 2010 and continued until the end of the breeding season (May or June 2010) or gestation. The samples were lyophilized and a standard mass prepared for analysis (0.250 +/- 0.05g). Steroids will be extracted from these samples and EIAs will be performed. The results of this study can be used to promote the success of snow leopard captive breeding programs by identifying the best time for mating and allowing early pregnancy detection and monitoring.

Student support: Morris Animal Foundation
Research support: Department of Veterinary Biosciences

Effects of Population Density and Fecundity on Levels of Polyandry in the Least Killifish, Heterandria formosa
Melissa Rohrs, Matthew Schrader, and Rebecca Fuller
College of Veterinary Medicine and Department of Animal Biology, University of Illinois, Urbana, IL

The genetic mating system of a species will determine the strength of sexual selection, which can in turn influence the evolution of morphological and behavioral traits involved in mating. In species with post-fertilization parental care the genetic mating system will also determine the intensity of conflicts between parents and offspring over parentally supplied resources. These conflicts are expected to influence the evolution of traits involved in parent-offspring interactions (e.g. begging) as well as life-history traits, reproductive structures, and epigenetic phenomena such as genomic imprinting. In this study, we used microsatellites to estimate the level of multiple mating in two populations of the placental fish, Heterandria formosa, a species whose mating system involves polyandry and superfetation. We then combined these data with previously collected data from four other populations to determine if there is a consistent relationship between population density and levels of multiple mating in H. formosa and if a relationship exists between female reproductive success (i.e. fecundity) and mating success (i.e. the number of mates) in this species.

Student support: Merial Veterinary Scholars Program
Research support: National Science Foundation

Dendritic Morphology of Hippocampal CA1 Pyramidal Cell Neurons in the Newborn Domestic Pig (Sus scrofa domesticus)
Renee E. Seager, Matthew S. Conrad, Ryan N. Dilger, and Rodney W. Johnson
College of Veterinary Medicine, Department of Animal Sciences, and Division of Nutritional Sciences, University of Illinois, Urbana, IL

The domestic pig is emerging as a neurodevelopmental model. This study involves characterizing normal development of the hippocampus. The hippocampus is important in learning and memory, and is involved in many neurological and mental health disorders. The gross morphology of porcine hippocampal formation closely resembles that of the human, however there are no studies characterizing the complexity of pyramidal cell neurons within the hippocampus proper. In this study, dendritic spine density and apical and basal dendritic tree complexity of pyramidal neurons in the CA1 region were quantified using Golgi-Cox stained hippocampal tissues from 3 male and 3 female pigs at birth. The basal dendritic complexity including tree length, number of nodes, and spine density were not significantly different between the sexes. In both sexes, the number of intersections off of the basal tree decreased as the distance away from the soma increased. Female pigs had a significantly larger soma volume (P < 0.001), while the numbers of apical intersections were similar throughout the length of the dendritic tree, and the complexity was similar between the genders at this age. Thus, at birth, CA1 pyramidal cell complexity is very similar between males and females with significant differences only evident in the volume of the soma. Future studies will analyze dendritic complexity over time to fully characterize the normal pyramidal cell development in the pig. This work will allow comparisons of pyramidal cell complexity and development with humans in order to further evaluate use of the pig as a neurodevelopmental model.

Student support: National Center for Research Resources, NIH, T35 RR020292

Mono-OH Methoxychlor (mono-OH) Inhibits Antral Follicle Growth through Oxidative Pathways
Julia Seitz, Zelieann Craig, and Jodi A. Flaws
College of Veterinary Medicine, University of Illinois, Urbana, IL

Female mammals are born with ovaries containing a finite number of follicles. Once these follicles mature from the primordial to the antral stage, they become capable of ovulation and synthesize the hormones that regulate and maintain normal estrous cycles. The organochlorine pesticide methoxychlor (MXC) has been shown to cause atresia of antral follicles through oxidative pathways. Although it was banned in the U.S. in 2002, it remains a concern because of its persistence in soil and insolubility in water. Cytochrome P450 enzymes metabolize MXC to two derivative compounds, mono-OH methoxychlor (mono-OH) and bis-OH methoxychlor, which are thought to be more toxic than MXC, though little is known of their effect on ovarian follicles. Thus, we tested the hypothesis that mono-OH inhibits growth of antral follicles via oxidative pathways. To test this hypothesis, antral follicles were mechanically isolated from CD-1 mice at 32-35 days of age. Follicles were placed in culture and treated with vehicle (dimethylsulfoxide [DMSO]) or a selected concentration of mono-OH (0.1, 1.0, 10.0 µg/ml). Every 24 hours follicle growth was measured in perpendicular axes for 96 hours. At the end of the culture, follicles were collected and subjected to RNA isolation and quantitative real time polymerase chain reaction for Cu/Zn superoxide dismutase (SOD1), catalase (CAT), and glutathione peroxidase (GPX). Results indicated that mono-OH significantly inhibited growth of antral follicles. Further, they showed that mono-OH may alter the expression of some anti-oxidant genes. Collectively, these data suggest that mono-OH may inhibit follicle growth via an oxidative pathway.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: NIH, R01 ES012893 and R01 ES019178

A Retrospective Study of Seasonal and Circadian Presentations of Dogs with Congestive Heart Failure
Gary Steinberg Jr., Julie K. Byron, and Megan M. Mahoney
College of Veterinary Medicine, University of Illinois, Urbana, IL

Humans have circadian variations in cardiovascular events such as myocardial infarction and congestive heart failure (CHF), and other diseases such as asthma caused by bronchoconstriction. Previous studies of CHF in cats have shown daily (circadian) and seasonal variations in disease presentation. This work tested the hypothesis that there is a daily or seasonal rhythm in the presentation of CHF in dogs. Medical records from the University of Illinois Veterinary Teaching Hospital were searched from 1997 to 2009 for acute cardiac problems (CHF). The effect of admission time, weekday, month, and season were analyzed separately using Chi-square tests. The results identified 163 presentations of 156 dogs with a clinical diagnosis of CHF. CHF presentations were largely confined to Monday and Tuesday (56%) and between 9:00-12:00 (56%). CHF was more common during September, October, and November (38%). Information from this study may assist veterinarians in the development of a preventative medicine plan, aid in educating clients about the timing of clinical signs, and enable veterinary hospitals to anticipate CHF cases during certain times of the day and year.

Student support: Abbott Laboratories

The Effect of Mate Choice on Immediate Early Genes in the Stickleback Brain
Sarah Turek, Molly Kent, Lauren Hostert, and Alison Bell
College of Veterinary Medicine and Department of Animal Biology, University of Illinois, Urbana, IL

Stickleback fish are widely used to study the evolution of behavior. During mating, females show a preference for certain males by exhibiting a “heads-up” posture. This “heads-up” posture is exemplified when a female lifts up the front half of her body facing a male that she has “chosen”. Three treatments were used to determine what areas of the stickleback brain were active during courtship: 1) females demonstrating heads up to a male, 2) females not interested in males (no heads up) and 3) a control group with no exposure to a male. After each treatment was completed, the females were placed into an isolation tank for ninety minutes before they were perfused and brains were dissected out then isolated and sectioned. Immunohistochemistry was used to detect proteins encoded by immediate early genes, specifically analyzing the optic tectum, hypothalamus and telencephalon. We hypothesized that changes would be visible in all three regions, but that changes in the telencephalon would be visible between females that adopted the “heads-up” posture and those that did not. Results from this study will provide new information about brain activity that accompanies animal courtship behavior and may be important for studies of human behavior because the human brain has analogous structures.

Student support: Merial Veterinary Scholars Program

Exercise-Induced Neurogenesis May Enhance Associative Fear Memory Formation in the Hippocampus
Christine E. Venghaus, Rachel A. Kohman, and Justin S. Rhodes
College of Veterinary Medicine and Department of Psychology, University of Illinois, Urbana, IL

Exercise enhances performance in a number of cognitive tasks, in part, through facilitating acquisition of new memories. However, whether exercise improves memory recall after a delay has yet to be investigated. Pro-cognitive effects of exercise may be related to increased production of new neurons within the hippocampus. The objective was to evaluate whether exercise training improves performance on a fear conditioning task when the time delay between training and testing is increased and whether number of new hippocampal neurons are associated with the performance differences. Four-month-old male C57BL/6J mice were housed in cages with or without running wheels. During the initial ten days, mice received BrdU injections (50 mg/kg) to label dividing cells. After 30 days of wheel access, mice were trained in a contextual fear conditioning task. Half of the mice were placed into a novel context and received seven footshocks at 1 min intervals. The remaining mice were placed into the context but no shocks were delivered (no-shock controls). Freezing to the context was assessed on day 1, day 7, and day 21 post-training. Mice were euthanized by transcardial perfusion 90 min after testing. Results show that exercise improved performance when testing occurred 1 day after training. No differences were observed between the exercised and sedentary mice 7 and 21 days after training. Furthermore, exercise is expected to increase the survival of new neurons in the hippocampus. These data will further our understanding of how exercise improves cognitive function and the potential contribution of new neurons to memory retrieval.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Institutes of Health, MH083807 and DA027487

Health Survey of the Free-Ranging Eastern Box Turtle (Terrapene carolina carolina)
Brittany Way and Matthew C. Allender
College of Veterinary Medicine, University of Illinois, Urbana, IL

Turtle populations across the world are declining, the Eastern box turtle included. They are the victims of increased vehicular traffic, urban sprawl, and increased disease prevalence. For this reason it is important for us to study the health status of these populations to determine the effects these factors may have on them. During the summer of 2010, physical examinations, blood, feces, and ectoparasites were collected from Eastern Box Turtles (Terrapene carolina carolina) in Oak Ridge, Tennessee. The box turtles were collected through incidental encounters and canine searches. A total of 131 turtles were collected. Physical examinations were performed on 100 turtles. The most common physical exam findings were healthy animals (n=85), but rare cases of nasal discharge and conjunctivitis were encountered (n=5). Blood samples were analyzed for a complete blood count (n=125) and plasma biochemistries (n=100). The parameters measured included packed cell volume, white blood cell count, total solids, white cell differential, asparatate aminotransferase, bile acids, creatinine kinase, uric acid, glucose, phosphorus, calcium, total protein, albumin, globulin, potassium, and sodium. Reference ranges were established for all parameters. Fecal examinations were performed on four turtles and no parasites were present. Ectoparasites were observed on four of the turtles. The purpose of these samples is to serve as baseline health parameters for ongoing health assessments of this population and future investigations into similar turtle populations. With these parameters established, populations with a higher susceptibility to diseases and other stressors can be identified and the proper action to protect these reptiles can be taken.

Student support: Morris Animal Foundation

Assessment of Microbial Communities and Comparison of Culture-Based vs. PCR-Based Detection of Campylobacter or Salmonella from Laying Hen Facilities
Kathleen Whelan and Angela Kent
College of Veterinary Medicine and Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL

Management of laying-hen facilities directly influences the health and production of the flock. Efficient and accurate detection of pathogenic bacteria is necessary for assessing the health status of these animals. Molecular methods based on amplification of specific genes using polymerase chain reaction (PCR) allow quick screening of environmental samples with high throughput technique. The objectives of this research were (1) determine the most informative sample types (e.g., hen feces) to test for bacterial community differences between hen facilities, (2) assess which microbial populations are responsible for the differences between the communities within the samples that test positive and negative for Campylobacter or Salmonella samples, and (3) design PCR-based detection of Campylobacter and Salmonella in environmental samples collected from hen facilities under different management regimes. Surface and environmental samples (dust, feces, water) were collected in 2009 from hen facilities under different management strategies: caged-belt, caged-high rise, and caged-free. DNA was extracted from environmental samples and DNA fingerprint analysis was used to assess bacterial community composition. Culture-based techniques were used to determine presence/absence of Campylobacter or Salmonella in a subset of samples. From this subset of samples, PCR-based methods were used to screen the samples for Campylobacter or Salmonella. Fecal and surface samples (besides drinkers) were similar among different management schemes. PCR-based methods were compared to culture-based methods. If detection without enrichment from environmental samples is possible, then molecular-based screening of Campylobacter or Salmonella in hen facilities could provide another option for screening the health status of laying-hens and the microbial ecology of their housing facilities.

Student support: Merial Veterinary Scholars Program

Pseudosubstrate Inhibition of Botulinum Neurotoxin A Through Modification of the SNAP-25 Cleavage Site
Abigail Wolf, Jasmine Lee, Julianna Lui, Mengfei Ho, and Brenda A. Wilson
College of Veterinary Medicine, School of Molecular and Cellular Biology, Department of Microbiology, and Institute for Genomic Biology, University of Illinois, Urbana, IL

Botulinum neurotoxin A (BoNT/A), produced by the bacterium Clostridium botulinum, is the most potent bacterial toxin known to man. BoNT/A causes flaccid paralysis that can last for weeks to months by cleaving the SNAP-25 protein and inhibiting the release of acetylcholine from peripheral motor neurons. We hypothesize that modifying the cleavage site of the SNAP-25 protein will inhibit the catalytic activity of the BoNT/A light chain while still allowing the protein to be recognized as a substrate, thereby serving as a pseudosubstrate. Residues 141-206 of SNAP-25 (SNAP-25 peptide) are required for recognition by BoNT/A, with the toxin cleaving between Q 197 and R198. We generated a series of DNA constructs that replaced the normal site of cleavage, ANQR, with 2-4 His residues followed by 1 or 2 Arg residues, which are thought to be necessary for recognition by the toxin’s catalytic site. The DNA constructs were created using PCR mutagenesis of the gene encoding the SNAP-25 protein, and the SNAP-25 peptide was cloned into an E. coli expression vector for heterologous production of the mutant proteins. The resulting proteins will be purified by affinity and anion exchange chromatography and compared to the wild-type SNAP-25 peptide to determine their inhibitory activities using FRET-based assay analysis. Proteins that exhibit increased inhibitory activity compared to the wild-type peptide may be useful in post-exposure therapeutics for BoNT/A intoxication.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Institute of Allergy and Infectious Diseases, NIH, U01 AI075502

The Role of Lactoferrin in Immunomodulation in Porcine Model of Human Infants
Krystal M. Woo, Sarah S. Comstock, Elizabeth A. Reznikov, and Sharon M. Donovan
College of Veterinary Medicine, and Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL

Diet plays a critical role in the development of the newborn’s immune system. Because they are omnivores that display similar intestinal physiology and function to the human infant, neonatal piglets are an outstanding model to study the impact of infant nutrition on development. Dendritic cells (DC) are an immune cell type that extend into the intestinal lumen, interact with intestinal tract contents and react to dietary proteins. One of these proteins is lactoferrin (Lf), an iron-binding milk glycoprotein with antimicrobial function, which may interact with DC to produce immunomodulatory effects. In addition, Lf is also released from neutrophils and apoptotic cells to prevent further migration of granulocytes to sites of inflammation. We hypothesized the addition of Lf to the diet of colostrum-deprived, formula-fed piglets would alter the phenotype and function of intestinal DC and decrease neutrophil numbers. Neonatal piglets (n=36) were randomized into 3 treatment groups: 1) Control (sow-reared); 2) FORM (colostrum-deprived/formula-fed); 3) Lf (colostrum-deprived/formula-fed+3.75 g/L bovine Lf). Tissue samples (duodenum, jejunum, ileum, ascending and descending colon) were collected on day 7 and 14. Tissue sections were analyzed via immunoflourescence, using antibodies previously demonstrated to identify porcine DC and neutrophils. The results of this study will improve our understanding of the influence of Lf on intestinal immune development in neonates.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: Pfizer Nutrition, Collegeville, PA

2009 Research Project Abstracts

Ontogeny of Uterine Gland Development in the Neonatal Dog
Daniel C. Borsdorf, Gail C. Ekman, and Paul S. Cooke
Department of Veterinary Biosciences, University of Illinois, Urbana, IL

Ovine models suggest that administration of progesterone during neonatal life can permanently inhibit uterine gland development and cause infertility in the adult animal. Although extensive information is available on uterine gland development in other species, postnatal uterine development and uterine gland ontogeny in the dog has not been studied. The goal of this study was to evaluate neonatal canine uterine development and uterine gland ontogeny to identify the most efficacious window for neonatal/juvenile administration of progesterone as a contraceptive method in the dog. Neonatal canine uteri at 7, 14, 28, 42, and 56 days of age postnatal (n = 3 for all ages except 6 weeks, where n = 2) were evaluated by morphometric methods and Ki-67 immunohistochemistry (IHC) to evaluate proliferation in both epithelial and stromal compartments. Initiation of uterine gland development began around day 7, with epithelial cells beginning to bud off from the uterine luminal epithelium. Both luminal epithelial and stromal cells were proliferating rapidly, with a Ki-67 labeling index (LI) of 36.8% ± 5.8% and 19.2% ± 4.9%, respectively at day 7. At 14 days, glands were clearly identifiable, and proliferation of luminal, glandular and stromal cells remained active (LI= 30.3% ± 4.8%, 45.4% ± 3.7% and 14.4% ± 1.4%, respectively). By day 28, glands had invaded further into the stroma, although proliferative activity had decreased in all cell compartments (LI= 9.5% ± 0.6%, 16.0% ± 1.5% and 2.2% ± 0.5% in luminal, glandular and stromal cells respectively). By days 42 to 56, uterine gland development was more extensive than at day 28, although the amount of gland development was substantially less than seen in the dog at proestrous and estrous, indicating that substantial gland development takes place during the adult estrous cycle. Luminal, glandular, and stromal LI was minimal by day 42 (LI= 1.9% ± 1.0%, 2.7% ± .3%, and 0.2% ± 0.1% respectively) indicating that the uterus is nearly mitotitcally quiescent by this age. In summary, luminal and stromal proliferation are maximal at day 7 and decrease to minimal levels by day 56. The onset of uterine gland ontogeny occurs at day 7, thus the optimal time frame of a progesterone induced-sterilization protocol would be from day 7 to day 42.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: University of Illinois Billie A. Field Endowment, National Institute on Aging, NIH, R01 AG24387.

A Combinatorial Genomic and Histopathology Approach to Studying the Effect of Surfactant Protein A on Fungal Growth and Pathogenesis
Caroline Cua, Yonghua Hao, Brent Walling, and Gee W. Lau
Department of Pathobiology, University of Illinois, Urbana, IL

Inhalation of fungal spores is the means of contracting several fungal diseases of importance in humans and animals. Thousands of fungal spores are deposited onto the surfactant-lining layer of pulmonary alveoli during the course of daily life. The Surfactant Protein A (SP-A) is a calcium-dependent innate immunity protein that enhances the clearance of microbial pathogens from the lung by opsonization and membrane permeabilization. Little is known about SP-A/fungal interactions. A yeast deletion library was used to identify fungal factors that interact with SP-A. A library consisting of approximately 5,000 yeast deletion mutants was screened for their susceptibility to aggregation and killing by SP-A. Pools of yeast mutants were grown and replicated in 96-well microtiter plates and exposed to 50 microgram/ml SP-A in aggregation buffer. Yeast aggregation was scored by visual observation using an inverted light microscope. Aggregated yeast mutants were LIVE/DEAD stained to determine the extent of killing by SP-A. The homologs of these fungal targets will be identified by bioinformatics using genome sequences of pulmonary fungal pathogens. Immunohistochemistry was also performed to determine the relationship between SP-A and fungal infection. Paraffin-embedded lungs of birds and companion animals with known fungal infections were sectioned and stained for SP-A using a rabbit polyclonal antibody to determine SP-A levels. We hypothesize that fungal infection will decrease SP-A levels in the affected lungs and that deletion of fungal targets in pathogenic fungi will attenuate their virulence in the lungs.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Heart, Lung and Blood Institute, NIH, R01 HL090699

Phenotypic Evaluation of a Candida albicans pir1/pir1 Mutant and Analysis of PIR1 Allelic Variability in a Diverse Collection of Candida albicans Isolates
Simuel Hampton, Xiaomin Zhao, Soon-Hwan Oh, and Lois L. Hoyer
Department of Pathobiology, University of Illinois, Urbana, IL

The opportunistic fungus Candida albicans causes various forms of disease including disseminated disease, most commonly associated with immunocompromised patients, and superficial, mucosal disease that can affect immunocompromised or normally healthy individuals. C. albicans PIR1 encodes a protein associated with beta-1,3-glucan in the C. albicans cell wall. Published studies suggested that Pir1 is required for proper cell wall architecture and that PIR1 is an essential gene. Our work suggested that Pir1 might contribute to adhesive interactions between C. albicans and mammalian cells. Contrary to literature predictions, we constructed a viable pir1/pir1 mutant strain. The phenotype of the strain was compared to wild-type and reintegrant control strains for growth rate, cellular morphology, germ tube formation, sensitivity to compounds that interfere with polymerization of cell wall components, and adhesion to cultured vascular endothelial cells and freshly collected buccal epithelial cells. PIR1 allelic variation was also studied. Many genes encoding C. albicans cell wall proteins have considerable allelic variability, often arising from regions of repeated sequences. Based on differences between the PIR1 alleles from strain SC5314, we developed a PCR assay and assessed allelic variability in collections of diverse C. albicans isolates from humans and wildlife. Strains passaged for many generations in vitro or in vivo were used to assess stability of the repeated sequences within PIR1 alleles. The C. albicans cell wall provides fungal cell integrity and is a structure not present in mammalian host cells. As such, the cell wall is an important antifungal drug target and its study is significant.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Institute of Dental and Craniofacial Research, NIH, R01 DE14158

Genetic Characterization of Epizootic Hemorrhagic Disease Virus Using the Capsid (VP2) and Core (VP3) Protein Genes
Andrew Hennenfent, Yijun Du, and Dongwan Yoo
Department of Pathobiology, University of Illinois, Urbana, IL

Epizootic hemorrhagic disease virus (EHDV) is an insect-transmitted Orbivirus infecting ruminants and white-tailed deer. EHDV outbreaks occurred in 2007 in U.S. white-tailed deer populations, including those in Illinois. The purpose of this study was to determine the genotype of an EHDV isolate from the 2007 outbreak. EHDV contains 10 segments of double-stranded RNA that code for seven viral proteins and three non-structural proteins. Of these proteins, the major neutralizing antigen protein VP2 and the major structural protein VP3 were selected for genotyping. EHDV was grown in HeLa cells and the viral genomic RNA was purified. RT-PCR was used to reverse transcribe and amplify gene segments from VP2 and VP3. DNA sequences of the gene segments were compared to those from EHDV isolates available in GenBank. Comparison of VP2 gene segments representing positions 1318-1644 (327bp), 1716-2075 (360bp), and 2384-2873 (490bp) showed 97%, 95%, and 96% amino acid sequence identity, respectively, with EHDV sequences from an serotype-2 isolate from Alberta, Canada. Comparison of VP3 gene segment representing position 1654-2670 (1017bp) with an Alberta isolate showed 100% identity. These preliminary data suggest the 2007 outbreaks in the U.S. were most likely attributed to the EHDV serotype-2 strain common in North America. Completion of the VP2 and VP3 sequences will allow a more comprehensive analysis. Given the limited availability of EHDV sequences, information from this work will aid in identifying the source of future EHDV outbreaks.

Student support: National Center for Research Resources, NIH, T35 RR020292

Regulation of Aromatase Expression in Mouse Uterus
Kalyn Herzog, Amrita Das, and Indrani Bagchi Department of Veterinary Biosciences, University of Illinois, Urbana, IL

Implantation is initiated when the embryo attaches to the uterine luminal epithelium during early pregnancy, triggering the transformation of uterine stromal cells to decidual cells in a process tightly coordinated by estrogen (E) and progesterone (P). Recent studies have shown that the intra-uterine biosynthesis of local E via induction of P450 aromatase is critical to sustain pregnancy in addition to the ovarian source of P. Plausible candidates involved in regulating aromatase expression in the decidual uterus include members of the monomeric orphan nuclear receptor family, which are key aromatase transcription regulators in other E-producing tissues. Liver-receptor homolog-1 (LRH-1) and steroidogenic factor 1 (SF1) were the two major factors studied with an overall aim to identify the specific molecule expressed during decidualization. Uterine stromal cells from pregnant mice were isolated during days 3 -7 of pregnancy. RNA was isolated and subjected to cDNA preparation. Expression patterns of LRH-1 and SF1 were monitored via q-PCR analysis, and protein expression during pregnancy was identified via immunolocalization using an antibody against the specific protein. Expression of decidualization markers like alkaline phosphatase, prolactin-related peptide, and aromatase was also analyzed. A significant induction of LRH-1 in the isolated stromal cell population was observed, however, the expression of SF1 was not markedly induced. LRH-1 may be the transcription factor of interest that up-regulates aromatase expression in uterine stromal cells during implantation and decidualization. Future experiments should determine expression profiles in ovariectomized virgin mice to understand if the regulation of aromatase is specific to differentiating stromal cells.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: NICHD Center for Research in Reproduction and Infertility, NIH, U54 HD055787<

Determining the Dosing Regimen of PAC-1-II-9 for Treatment of Murine Lymphoma
Jared A. Holtgrave1, Quinn P. Peterson2, and Paul J. Hergenrother2 1College of Veterinary Medicine and 2
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL

Caspase-3 is a critical executioner enzyme in apoptosis that exists intracellularly as the proenzyme procaspase-3. In cancer cells, abnormal expression of proteins in apoptotic pathways prevents the activation of procaspase-3 to caspase-3, thus preventing apoptosis. Since procaspase-3 is often upregulated in cancer, it represents a viable and cancer-specific target for chemotherapeutics. Recently, the small molecule PAC-1 was shown to activate procaspase-3 to caspase-3, thereby inducing apoptosis in vitro. Studies in mice showed that PAC-1 inhibits tumor growth, but causes neurotoxicity. A sulfonamide derivative of PAC-1 (PAC-1-II-9) was synthesized which induces apoptosis in vitro without producing neurotoxicity in vivo at predicted therapeutic concentrations. Prior to testing PAC-1-II-9 in treatment of murine lymphoma a proper dosing regimen must be established. To determine the importance of drug concentration versus exposure time for cytotoxicity, IC50s of PAC-1-II-9 were completed using a sulfhorodamine B assay for times ranging from 1 to 72 hours and compared to those of PAC-1, doxorubicin, and paclitaxel. Results showed that PAC-1-II-9 was dependent upon both exposure time and concentration for cytotoxicity. The pharmacokinetics of 150 mg/kg subcutaneous injections of PAC-1-II-9 maintained a therapeutic concentration for two hours. Blood samples were taken at time points between 10 minutes and 1 day post-injection and analyzed via liquid chromatography-mass spectrometry. Determining the dependence of PAC-1-II-9 on exposure time and concentration, and determining its pharmacokinetics in vivo are important precursors to testing the efficacy of PAC-1-II-9 against cancer in vivo.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: University of Illinois Department of Chemistry

The Effects of the Antifibrotic Drugs Halofuginone and Trichostatin A on Leiomyoma Smooth Muscle Cells of Aged Laying Hens
Alyssa M. Kritzman1, Sergio A. Machado2, and Romana A. Nowak2
1College of Veterinary Medicine and 2Department of Animal Sciences, University of Illinois, Urbana, IL

Uterine leiomyomas (fibroids) are the leading cause of hysterectomies for women in the United States. These benign tumors occur in women during their reproductive years and are characterized by increased smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) production. We recently observed that aged laying hens, Gallus domesticus, spontaneously develop fibroid-like polyps on their oviducts similar to human uterine leiomyomas. We used primary SMC cultures derived from hen fibroids as a potential in vitro model to test novel, alternative therapeutic compounds as treatments for human uterine fibroids. Two antifibrotic drugs, Halofuginone and Trichostatin A, were tested on hen leiomyoma cells for their effects on cell proliferation, apoptosis, and production of the ECM proteins collagen type I and type III. We hypothesized that the anti-fibrotic drugs Halofuginone and Trichostatin A inhibit cell proliferation and collagen production while increasing apoptosis in chicken leiomyoma cells. Time-course and dose-response studies assessed effects of both drugs on proliferation using thymidine uptake assays and manual cell counting. Changes in collagen type I, type III, and TGF-beta 1 production were analyzed using real-time PCR, while effects on apoptosis were assessed by immunoblotting using an anti-poly (ADP-ribose) Polymerase-1 (PARP) antibody. Initial results showed that both Halofuginone and Trichostatin A inhibited proliferation of chicken leiomyoma cells. We anticipate that both Halofuginone and Trichostatin A significantly inhibit growth of chicken leiomyoma SMCs, reinforcing the concept of using aged laying hens as a model species for further research on uterine leiomyomas in women.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: Department of Animal Sciences and National Institute of Child Health and Human Development, NIH, RO1 HD04227

The Effect of Female Presence on Risk-Taking Behavior of Male Sticklebacks
Angela Nguyen and Alison Bell
Department of Animal Biology, University of Illinois, Urbana, IL

Reproduction is often associated with increased predation risk, considering that the attributes used to attract females may also heighten conspicuousness to predators. Additionally, males will often engage in riskier behavior in the presence of a female and her cues. Using Three-Spine Sticklebacks, we investigated whether males behave more boldly toward a predator in the presence of a female compared to her absence. Because males might engage in risk-taking behavior in the presence of conspecifics due to risk dilution with increased density, we tested the focal male in the presence of male to ensure that changes in boldness correlated specifically to female presence. Males were placed in individual tanks containing materials used for constructing nests, the completion of which indicated his availability for testing. Once ready, a predator model was placed in the tank of the focal male, while a jar, empty or containing a stimulus fish, was placed in an adjacent tank. An opaque screen allowed the focal male and stimulus fish to interact with each other, but prevented the stimulus fish from seeing the predator. We predict that in the presence of a female, males will react more boldly to the predator by increasing the number of risk-taking behaviors. Increases in risky behavior can inform males of the potential threat of predation, indicate high fitness to the female, or be a spillover effect from courtship activity. Further research can determine the purpose and benefit, if any, of female induced increased risk-taking behavior in stickleback males.

Jonathan Stack1, Joshua Turner2, Michael Tencati2, and Richard Tapping2
1College of Veterinary Medicine and 2Department of Microbiology, University of Illinois, Urbana, IL

We have identified two strains of mice, BALB/cJ and 129S1/SvImJ, that are resistant to Yersinia pestis, the pathogen responsible for plague; however, the mechanism of resistance has not yet been elucidated. In comparison to susceptible C57BL/6J mice, the resistant substrains have markedly decreased bacterial burdens in the spleen early after infection. Nevertheless, spleens from all infected mouse strains exhibit high numbers of infiltrating neutrophils which are known to play a general role in controlling bacterial growth in the early stages of infection. To assess their potential contribution to resistance, peritoneal neutrophils from different strains of mice were co-incubated for varying times with Y. pestis D27 and samples were plated to determine bacterial growth. Here we report that neutrophils from resistant mice exhibited an enhanced capacity to control the growth of Y. pestis when it is cultured at 23oC but not when cultured at 37oC. Two important virulence factors, an antiphagocytic capsule and a Type III Secretion System, are induced by this change in temperature as Y. pestis adapts from the flea vector to the mammalian host following a flea bite. Two Y. pestis mutants, each lacking one of these virulence factors, are currently being utilized to investigate their role in bacterial evasion of neutrophils from resistant mice. Initial results support the idea that neutrophils play an important role in controlling Y. pestis growth in the early stages of infection.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Institute of Allergy and Infectious Disease, NIH, R01 AI056148

Alterations in Hepatic mRNA Expression of CMO-I/CMO-II in Mice Fed Carotenoid-Containing Diets
James Weidner1, Nikki Ford2, and John Erdman2
1College of Veterinary Medicine and 2Division of Nutritional Sciences, University of Illinois, Urbana, IL

Epidemiological studies suggest that increased intake of tomato products and higher blood levels of the cartenoid, lycopene, reduce the risk of development or progression of certain cancers. 15,15’carotenoid monooxygenase (CMO-I) and 9’,10’-carotenoid monooxygenase (CMO-II) are the principal cleavage enzymes for carotenoids. CMO-I is recognized as the primary enzyme responsible for converting provitamin A carotenoids to vitamin A. Theoretically, CMO-II eccentrically cleaves lycopene into apo-10’-lycopenal and other lycopenoids. Previous research has shown alterations in the bioaccumulation of carotenoids in CMO-I and CMO-II knockout (KO) mice when fed either a lycopene or a beta-carotene containing diet, suggesting alteration in the expression of CMO-I/CMO-II. To measure alteration in expression, we fed either a lycopene diet (100 mg lyc/kg diet in beadlet form), a placebo beadlet diet, a 10% tomato power diet, or an AIN-93G based diet to CMO-I KO, CMO-II KO, and wild-type (WT) mice for 30 days. Because carotenoids are stored and found in high concentrations in the liver, and the liver is the primary storage site for beta-carotene conversion to vitamin A, we collected liver tissue at sacrifice. Real-time PCR was used to measure the relative abundance of CMO-I and CMO-II mRNA. Preliminary data suggest that CMO-I is down-regulated in CMO-II KO and WT mice fed lycopene and CMO-II is down-regulated in WT mice fed lycopene. These changes are important because carotenoid cleavage enzymes are essential for the production of bioactive carotenoid metabolites that can combat chronic diseases.

Student support: National Center for Research Resources, NIH, T35 RR020292
Research support: National Cancer Institute, NIH, R01 CA125384

Microbial Ecology and Environmental Quality in Egg-Laying Facilities
Caroline Chu1, Glogerley Sales2, Angela Green2, and Angela Kent1
1Department of Natural Resources and Environmental Sciences and 2Department of Agricultural and Biological Engineering, University of Illinois, Urbana, IL

Laying-hen housing currently exists in production agriculture as caged or cage-free systems. While both categories are commonly found in practice, there still remains inadequate information regarding the comparisons and consequences of alternative housing schemes. Issues of particular importance are those that affect food safety and human health as poultry products may become rapidly contaminated by environmental bacteria and pathogens. We hypothesize that management strategy differences impact the diversity of microorganisms within different housing operations and their associated environmental variables. Samples of air, litter, water, surfaces, eggs, and feces were collected at different types of laying-hen facilities in Illinois and Indiana. Environmental variables such as temperature, relative humidity, carbon dioxide, and atmospheric ammonia levels were measured simultaneously. We then performed DNA extraction followed by automated ribosomal intergenic space analysis (ARISA) to identify the microbial community composition of each sample. Detection of zoonotic pathogens Salmonella and Campylobacter within the laying-hen environment was completed via selective enrichment of fecal, egg, and dust samples. Characterization of microbial populations in the laying-hen environment and identification of the environmental factors that influence community structure may provide key insight into the control and prevention of the contamination of poultry meat and eggs.<

Student support: Merck-Merial Veterinary Scholars Program
Research support: Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, Project No. ILLU 875-374

Evaluating the Potential for the Als2 Cell-Surface Glycoprotein to Function in Candida albicans Cell Growth and Division
Nicki Dahm, Soon-Hwan Oh, Xiaomin Zhao, and Lois L. Hoyer
Department of Pathobiology, University of Illinois, Urbana, IL

The fungus Candida albicans causes disseminated and mucosal disease. Risk factors for disseminated candidiasis are often associated with clinical settings. Oropharyngeal candidiasis is one form of mucosal disease and is strongly associated with progression from HIV infection to AIDS. The C. albicans genome encodes several gene families, some of which are associated with pathogenesis. Proteins in the Als (agglutinin-like sequence) family are most commonly considered as adhesins. Recently, a different role for Als1 was described. Deletion of ALS1 from the C. albicans genome results in cells that are smaller than those of a wild-type control strain; reintegration of ALS1 restores the wild-type phenotype. The purpose of this work is to expand on observations regarding Als1 and cell size and to determine if Als2 also plays a role in cell growth and division. ALS1 was overexpressed in two C. albicans backgrounds. We hypothesized that Als1 production at levels above those in the wild-type strain would result in cells larger than wild-type. Less is known about Als2 than other Als proteins because the ALS2 open reading frame and ALS2 locus have proven difficult to manipulate. ALS2 experiments involved developing standard growth conditions in a rich and a minimal growth medium, comparing growth characteristics of the mutant and control strains, and measuring ALS2 transcription over the course of culture growth. These studies provide new insights into the function of proteins within a gene family, and contribute to a greater understanding of the biology of an important fungal pathogen.

Student support: Merck-Merial Veterinary Scholars Program
Research support: National Institute of Dental and Craniofacial Research, NIH, R01 DE14158

Development of an Assay for the Detection and Quantification of Cephapirin and Ceftiofur in Goat Milk
Jessica Haydel1, Tushara Chakkath1, Wes Hoffmann1, Edgar Garrett2, and Levent Dirikolu1
1Department of Veterinary Biosciences and 2Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Cephapirin and ceftiofur are both cephalosporin antibiotics that inhibit bacterial cell wall synthesis. Cephapirin, a first generation cephalosporin, is predominantly active against gram positive organisms, while the third generation ceftiofur has a broader spectrum covering both gram positive and gram negative bacteria. These antibiotics are commonly used on and off label in dairy animals for the treatment of mastitis. The FDA has set a maximum residue limit of 20 ng/ml for cephapirin and 100 ng/ml for ceftiofur in milk. In the present study, we developed and validated an LC-MS method for the detection and quantification of these two antibiotics in goat milk. The simple extraction procedure involved centrifugation of the samples for defatting, precipitation of the proteins with acetonitrile and passage of the samples through Amicon Ultra 4 filters (Millipore Corporation, Temecula, CA). Following extraction, 30 µl of the filtrate was injected into the LC-MS system. The extraction efficiency ranged from 54-72% for cephapirin and 40-51% for ceftiofur. The within and between assay accuracies for cephapirin were 80-92% and 91-101%, and for ceftiofur were 96-106% and 88-95%, respectively. The precision of the method (% CV) for both compounds was equal to or less than 15%. This validated method can be used to determine the pharmacokinetics of cephapirin and ceftiofur following intramammary infusions in goats, and also to accurately determine their withdrawal times in milk samples.

Student support: Merck-Merial Veterinary Scholars Program
Research support: USDA Hatch Funds and Section 1433 Animal Health and Disease Research Funds

The Effect of the Spatial Organization of Vegetation on the Productivity of Culex Mosquito Larvae in Catch Basins in an Area of High West Nile Virus Infection
Dana Johnson1, Kate Varela1, Gabe Hamer2, Edward Walker3, Zach Allison4, Kelly DeBane1, Joanna Ganning5 and Marilyn Ruiz5
1University of Illinois at Urbana-Champaign College of Veterinary Medicine, 2University of Wisconsin at Madison Department of Pathobiological Sciences, 3Michigan State University Department of Microbiology 4University of Illinois at Urbana-Champaign Department of Natural Resources and Environmental Sciences, 5University of Illinois at Urbana-Champaign Department of Pathobiology

Selected residential areas southwest of Chicago have historically been hotspots for St. Louis Encephalitis and West Nile Virus, both mosquito-borne diseases. Culex species mosquitoes are the primary transmitters of West Nile Virus and are known to use the stagnant water found in the sumps of stormwater catch basins to deposit their eggs. Previous studies have found that some catch basins are significantly more productive than others, but analyses across multiple dimensions of the basins and their neighborhoods have not clarified a causal mechanism. We hypothesized that the type and spatial organization of vegetation surrounding catch basins may influence the productivity of Culex within basins. To test these hypotheses, we collected data for mosquito both productivity and vegetation. We measured mosquito productivity by sampling weekly for number of larvae and pupae present in sixty catch basins that have had high levels of Culex larvae in previous study years. For vegetation data, we surveyed the species and number of trees with a height greater than three meters within the study areas. Additionally, the number, type, height and area of shrubs and plants less than three meters in height were surveyed in regions within a 75-foot radius of each basin. Water depth and quality were also monitored within the basins in an effort to assess the effect of the surrounding vegetation on the microhabitat within the basins. The mosquito productivity and vegetation data will be analyzed and the results will add to the current understanding of mosquito-borne disease management.

Student support: Merck-Merial Veterinary Scholars Program
Research support: National Science Foundation Award Number 0840403 and University of Illinois AESIS Program

Auditory Function in Rats Exposed to a PCB and PBDE Mixture During Development
Ruth M. McAlonan,1 Emily Poon,2 Brian E. Powers,2 and Susan L. Schantz2,3
1College of Veterinary Medicine, 2Neuroscience Program, and 3Department of Veterinary Biosciences, University of Illinois, Urbana, IL

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are chemically similar substances that are present in the environment and bioaccumulate in the food chain. PCBs were a component of dielectric fluids in transformers and capacitors. These compounds are no longer manufactured since their toxicity is known; however, they are very stable and still present in the food chain. PBDEs are currently used as flame retardants in many household products. Their toxicity is not as well-studied, but they have a similar chemical structure to PCBs. Previous studies concluded that rats developmentally exposed to an environmentally relevant PCB mixture have hearing deficits. We hypothesized that due to the similarity in structure to PCBs, PBDE exposure during gestation and lactation will also cause hearing deficits, and that a combination of PCBs and PBDEs will result in an additive effect on auditory function. In this study, we examined auditory function in rats exposed to PCBs and/or PBDEs during gestation and lactation. Dams were exposed to 3 mg/kg/day or 6 mg/kg/day of PCBs, 5.7 mg/kg/day or 11.4 mg/kg/day of PBDEs, or a combination of 3 mg/kg/day PCBs + 5.7 mg/kg/day PBDEs or 6 mg/kg/day PCBs + 11.4 mg/kg/day PBDEs orally. Auditory function was assessed when the pups reached adulthood using two methods: auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs), which evaluate the integrity of the outer hair cells of the cochlea. These data will provide important information on the ability of two widespread environmental contaminants to cause hearing loss following developmental exposure.

Student support: Merck-Merial Veterinary Scholars Program
Research support: National Institute of Environmental Health Sciences, R01 ES015687

A Potential Novel Cancer Treatment: Evaluation of Myxoma Virus Oncolysis in Feline Cancer Cells
Tiffany Moldenhauer and Amy MacNeill Department of Pathobiology, University of Illinois, Urbana, IL

Although there is a high incidence of cancer in dogs and cats, treatment options for canine and feline cancer patients lag behind treatments available for humans. We investigated the effectiveness of myxoma virus-induced oncolysis in two feline cancer cell cultures as a potential cancer treatment. Single- and multi-step growth curves quantified myxoma virus replication and spread in tumor cells. Flow cytometry assays assessed the extent of virus-induced cell death. The level of phosphorylated Akt was detected by immunoblot to determine if Akt activation (which inhibits apoptosis) was necessary for myxoma virus infection. Each cancer cell line studied demonstrated differences in susceptibility to viral infection and cellular lysis. Feline metastatic carcinoma cells (Stude) showed significant cytopathic effects 24 h post infection (hpi), whereas, at 72 hpi there was minimal cell lysis in feline squamous cell carcinoma cells (SCCF1). At 24 hpi, peak virus titers were reached, but Stude had a three-fold higher virus load compared to SCCF1. Compared to fully permissive rabbit kidney cells, feline cancer cell cultures produced far fewer infectious virus particles. Even though there was production of viral proteins (shown by viral expression of red fluorescent protein) there was little viral spread in feline cancer cells. We will collect additional feline tumor cells to assess whether feline sarcoma cells are more permissive to infection than carcinoma cells. As companion animals live longer, the prevalence of cancer is increasing. Studies investigating novel cancer treatments are essential for advancing the standard of care in cancer Patients.

Student support: Merck-Merial Veterinary Scholars Program
Research support: The Winn Feline Foundation

Prevalence of Leptospira in Wildlife and Watersheds: a Measure of Ecosystem Health
Kourtney Cone1, Nohra Mateus-Pinilla1,2, and Carol Maddox1,3
1College of Veterinary Medicine, 2Illinois Natural History Survey, and 3Department of Pathobiology, University of Illinois, Urbana, IL

Leptospirosis is a waterborne bacterial disease. Wildlife and domestic animals can serve as reservoirs for multiple pathogenic serovars. Urban sprawl has increased wildlife interactions with humans and domestic animals. Therefore, assessments of the prevalence and incidence of leptospirosis in wildlife and leptospires in watersheds can be used as an initial evaluation of pathogen distribution and potential risk of disease transmission to humans, wildlife, and domestic animals. Serum samples from raccoons, opossums, and feral cats from a local natural area were analyzed using a Microscopic Agglutination Test (MAT) for the presence of antibodies against 7 pathogenic Leptospira serovars (Leptospira interrogans serovars Autumnalis, Bratislava, Canicola, Icterohaemorrhagiae, Pomona; Leptospira kirschneri serovar Grippotyphosa; Leptospira borgpetersenii serovar Hardjo). MAT titers greater than 1:25 were considered positive; titers higher than 1:800 suggested an active or recent infection. Titers were not detected in serum from feral cats (prevalence = 0%, n = 9). Of 51 opossums, 29 (57%) were positive, 3 with evidence of active or recent infection. Forty-four raccoons (45.4% of n = 99) had positive titers of which 13 were 1:800 or greater. Watersheds most likely used by the sampled animals were collected and analyzed by real-time PCR using primers that recognize the Leptospira genus. Nine of 17 water samples were Leptospira-positive. When analyzed by chi-square test, the watersheds were not significantly related to the MAT titers.

Student support: Morris Animal Foundation
Research support: Illinois Department of Natural Resources, Fish and Wildlife Service, Robert Allerton Park, University of Illinois: Illinois Natural History Survey and Cooperative Extension

The Effects of Detomidine Sedation During Pulmonary Function Testing in Horses
Ashley Mitek and Kara Lascola
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Objective—To evaluate the bronchodilatory effects of detomidine sedation on airway responsiveness in horses undergoing pulmonary function testing.
Animals—6 adult horses (1 gelding, 1 stallion, 4 mares).
Procedures—Airway responsiveness using flowmetric plethysmography and histamine bronchoprovocation will be determined without sedation and after administration of low dose (LD = 0.005 mg/kg) and high dose (HD = 0.01 mg/kg) detomidine with a 72 hour minimum washout between tests. PC35 (provocative concentration of histamine needed to increase flow by 35%) will be used as a measure of airway responsiveness.
Results—Six horses completed testing without sedation and with LD detomidine. Nonsedated horses moved more frequently during testing and variation in response to sedation was noted. Baseline flow values were higher in nonsedated horses. PC35 with and without sedation did not differ for three horses. PC35 significantly decreased in two horses (8.94 mg/ml vs 2 mg/ml; 16.28 mg/ml vs 5.54 mg/ml) and increased in one horse (3.18 mg/ml vs 27.67 mg/ml) after sedation.
Conclusions and Clinical Relevance—Increased PC35 values after sedation suggests a bronchodilatory effect of detomidine. One horse demonstrated an increase in PC35 after sedation, however decreased PC35 in two horses suggests factors other than bronchodilation may influence test outcome after sedation. Many of the observed differences may be attributable to increased movement and agitation in nonsedated horses, or individual differences in response to sedation. Further evaluation of the potential bronchodilatory effects of detomidine sedation on airway responsiveness is needed before its use can be recommended in a clinical setting.

Student support: Morris Animal Foundation

Investigating Phospholipase D2 Expressions in Canine Osteosarcoma
Zachary Levi Neumann, Holly Pondenis, Jackie M. Wypij, Laura D. Garrett, and Timothy M. Fan
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Introduction: In dogs with osteosarcoma (OS), a limited number of poor prognostic factors have been consistently identified including elevations in serum bone alkaline phosphatase (bALP). Despite its accepted value for prognostication, it remains poorly understood why elevated bALP is associated with more aggressive OS biology. Under physiologic conditions, the release of bALP into circulation is regulated through the enzyme phospholipase D2 (PLD2), which promotes and regulates cancer cell motility, cytoskeletal rearrangement, proliferation, and survival. As such, elevated bALP in dogs with OS may be a surrogate biomarker of increased PLD2 activity originating from a sub-population of metastatic malignant osteoblasts.
Methods: Clonally-derived OS cell lines of variant biologic aggressiveness derived from 3 different species (human, canine, and murine) will be utilized to assess differences in PLD2 expressions. Gene transcription of PLD2 will be assessed with real-time polymerase chain reaction. Protein translation of PLD2 will be determined by western blot analysis using the human U937 cell lysate as a positive control.
Results: For gene transcription, PLD2 expressions were not statistically different between clonally-derived variants of known differing metastatic capacity (non-metastatic versus highly-metastatic). For western blot analysis, PLD2 protein is identified for all cell lineages regardless of metastatic capacity.
Conclusions: PLD2 is expressed by OS cell lines from various species. No obvious differences in gene expression and protein translation of PLD2 were identified. However, differences in enzymatic activity may be present between clonally-derived variants requires further investigation and may be the underlying reason for why bALP concentrations are elevated in dogs with more aggressive OS.

Student support: Morris Animal Foundation

Effect of Tendon-Derived Progenitor Cells on Extracellular Matrix Production and Collagen Fiber Alignment in a Collagenase-Induced Model of Tendinitis in Horses
DN Avery, AA Stewart, SS Durgam, MK Coleman, and DM Sivaguru
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Tendon injuries are a common cause of lameness in performance horses. Stem cell-based treatments for tendon injuries show promise, but are not widely used or universally successful. Furthermore, no studies thus far have evaluated the effects of cell injections on fibre alignment or extracellular matrix production, which is responsible for tissue mechanical strength. We hypothesize that autogenous tendon-derived progenitor cells promote tendon healing with improved tissue architecture and fibre alignment in a collagenase-induced tendinitis model in horses. Collagenase-induced tendinitis was created in the superficial digital flexor (SDF) tendons of the forelimbs of eight clinically normal horses. Subsequently, a randomly chosen forelimb was injected with 10 million tendon-derived cells, and saline was injected into the contralateral forelimb serving as a control. The horses were euthanized 12 weeks post treatment, and the SDF tendons of both forelimbs and a normal hind limb were harvested for histologic evaluation, which included picro-sirius red staining for fibre alignment and quantitative estimation of collagen and toluidine blue staining for the estimation of proteoglycan using image analysis software. The results obtained from this study will help determine effects of cell-based therapies for the clinical treatment of tendinitis. In addition, these results could revolutionize tendinitis treatments by promising a healthier, stronger tendon and quicker healing of the injured tendon.

Student support: Pfizer, Inc.
Research support: American Quarter Horse Foundation

Temporal Evaluation of Collagen Fibre Alignment, Extracellular Matrix Production in a Collagenase Model of Tendinitis in Horses
MK Coleman, AA Stewart, SS Durga, DN Avery, and DM Sivaguru
Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Tendinitis is a common cause of breakdown injuries in equine athletes and accounts for up to 30% of all racing injuries. The purpose of this project is to evaluate the temporal and spatial effects of tendon-derived progenitor cells on tendon healing in a collagenase model of tendinitis. Our hypothesis is that the progenitor cell-treated tendon will have an improved fibre alignment and increasing extracellular matrix production over time with reference to normal tendon. Autogenous tendon-derived progenitor cells were isolated from the lateral digital extensor tendon using a differential adherence technique. Collagenase-induced tendonitis was created in the superficial digital flexor tendon by injecting 2000 units of collagenase at 3, 4, 6, and 8 weeks prior to euthanasia. Two weeks post injury, all 8 horses were injected with 10 million tendon-derived progenitor cells into two sites of maximal injury. The opposite normal control tendon and the collagenase injured tendon were collected for analysis 1, 2, 4, and 6 weeks (n=2) after treatment with progenitor cells. Tendons harvested were subjected for histologic evaluation of collagen, proteoglycan content and collagen fibre alignment. The aim of this study is to determine the progression of tendon-derived progenitor cells in the healing process of tendinitis. Results of this study are important to assess the viability of cells over time and its ability to fuse to native tenocytes, and also its effect on the healing process to evaluate the usefulness of cell-based therapy for clinical treatment of tendinitis.

Student support: Pfizer, Inc.
Research support: American Quarter Horse Foundation

Mono-OH Methoxychlor (Mono-OH) and Steroidogenesis in the Mouse Antral Follicle
T Leslie, KP Hatfield, RK Gupta, and JA Flaws
Department of Veterinary Biosciences, University of Illinois, Urbana, IL

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by causing ovarian atrophy, decreasing antral follicle numbers and increasing follicular atresia. MXC is readily metabolized in the body, and previous studies have shown the metabolite mono-OH to be more ovotoxic than the parent compound. MXC exposure at 10-100µg/mL decreases the production of estradiol, a steroid hormone that is essential for normal ovarian function. However, the effects of mono-OH on estradiol levels were unknown. Thus, this work tested the hypothesis that mono-OH exposure decreases production of sex steroid hormones in the estradiol biosynthesis pathway. To test this hypothesis, antral follicles were isolated from 39-day-old CD-1 cycling adult mouse ovaries and cultured in supplemented _-minimum essential media. Follicles were either untreated, exposed to dimethylsulfoxide (DMSO; vehicle), or exposed to mono-OH MXC (0.1-10µg/mL) for 96 hrs. After culture, the media was collected and estradiol levels were measured using enzyme-linked immunosorbent assays. The results indicate that mono-OH significantly decreases estradiol levels at the 10µg/mL dose when compared to DMSO treated control groups (control = 3009.72 ± 744.99 ng/mL; mono-OH 0.1µg/mL = 1679.66 ± 461.99 ng/mL; mono-OH 1µg/mL = 1752.72 ± 532.41 ng/mL; mono-OH 10µg/mL = 45.89 ± 33.83 ng/mL; n = 11; p ≤ 0.05). Collectively, these data suggest that mono-OH exposure significantly decreases the production of the steroid hormone estradiol by mouse antral follicles. This reduction in levels of steroid hormones could lead to mono-OH MXC induced ovotoxicity.

Student support: Department of Veterinary Biosciences
Research support: National Institute of Environmental Health Sciences, NIH, RO1 ES012893

Biological Characteristics of Catch Basins Affect Mosquito Larvae Productivity, the Influence of Vector Habitat on the Potential for West Nile Virus Transmission.
Kate Varela1, Dana Johnson1, Gabe Hamer2, Edward Walker3, Zach Allison4, Kelly DeBane1, Joanna Ganning5 and Marilyn Ruiz5
1University of Illinois at Urbana-Champaign College of Veterinary Medicine, 2University of Wisconsin at Madison Department of Pathobiological Sciences, 3Michigan State University Department of Microbiology, 4University of Illinois at Urbana-Champaign Department of Natural Resources and Environmental Sciences, 5University of Illinois at Urbana-Champaign Department of Pathobiology

Catch basins in suburban areas provide an ideal habitat for Culex pipiens and Culex restuans mosquitoes, potential vectors of West Nile virus (WNV), to deposit their eggs. Basins vary in larvae productivity, but the reasons for these differences are not clear. The goal of this study was to determine the characteristics of productive catch basins in an area southwest of Chicago, a hot spot for WNV. We hypothesized that catch basins with higher levels of organic nutrients, and less variation in water level and temperature, would be more productive. Sensors were placed in 60 catch basins in the study area to record changes in temperature, light level, and water level. Basin water pH was measured and the water was tested for organic enrichment through ammonia, phosphate, and nitrate concentrations. Larvae and pupae were collected from the catch basins on a weekly basis, quantified by basin, and identified to the species level. The number of larvae and pupae were compared between basins and within the same basins over time and were compared with basin characteristics. We used SPSS for statistical analysis and ESRI ArcGIS to process and map spatial data. The results of this study provide insight into biological characteristics that increase the abundance of Culex mosquitoes. This information will provide guidance for more efficient, effective and targeted mosquito control and, therefore, potentially decrease disease transmission.

Student support: Department of Pathobiology
Research support: National Science Foundation Award Number 0840403 and University of Illinois AESIS Program