CZR Veterinary Student Summer Training at University of Illinois

University of Illinois at Urbana-Champaign

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Summer Research Training Program for Veterinary Students

Past Student Projects

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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.

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 STUDENT PROJECTS

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 5x103 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 STUDENT PROJECTS

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 STUDENT PROJECTS

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 (<1 km) and half being far (>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<0.05 was considered significant. Results documented mild variation in mean plasma [tCO2] (range 28.9-31.6 mmol/L). Individual horses' plasma [tCO2] ranged over 6 units (n=1: 26-32 mmol/L) and 5 units (n=3: 29-34 [2 horses] and 28-33 mmol/L [1 horse]). When daily samples were pooled, there was a significant time-of-day effect, with 7 AM values (31.0±0.2) higher than 11 AM (30.1±0.2, P=0.005) and 3 PM (29.8±0.2, P<0.001). Similarly, measured Strong Ion Difference (SIDm: [Na+]+[K+] – [Cl-]) was higher at 7 AM (40.1±0.2) than at 11 AM (38.7±0.2, P<0.001) and 3 PM (38.9±0.2, P<0.001). Mean plasma [tCO2] and SIDm differed mildly but significantly by time-of-day. There was considerable intra- and inter-individual variability in plasma [tCO2]. Governing jurisdictions must acknowledge that mean [tCO2] data are not predictive of individual variability and that some horses may naturally test higher or over wider ranges than the population mean.

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 STUDENT PROJECTS

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 STUDENT PROJECTS

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


2008 STUDENT PROJECTS

Gastrointestinal Biota of Wild Birds and Associated Cattle in Illinois Habitats
Brininger, Craig; Burdorf, Kristin; Herrmann, John A.; Myint, Maung S.; Wheeler, Emily;  Johnson, Yvette J.
College of Veterinary Medicine, University of Illinois, Urbana, IL

There has been a growing concern about the role of wildlife in the transmission of pathogens to domestic livestock.  Outbreaks of clinical salmonellosis in livestock and poultry operations have been associated with carriage by resident wild birds, leading to an assumption that wildlife are the reservoir for such infections. A total of 146 samples were collected which included 30 wild bird and 30 environmental samples collected from each of the University of Illinois beef/dairy farm, poultry farm, and a remote site away from domestic animals.  The samples were then tested for Salmonella spp. and Enterococcus spp. using standard culture methods.  The positive samples were tested for antibiotic sensitivity and submitted to NVSL for serotyping.


The Fate of Tendon-Derived Progenitor Cells in Equine Tendonitis Model
Coleman, Kelli; Durgam, SS; Stewart, AA.
Department of Veterinary Clinical Medicine, University of Illinois

Tendon and ligament injuries are a common cause of lameness in horses, and often result in poor athletic potential after a prolonged period of rest, regardless of treatment.  Stem cell-based treatments for tendon injuries show promise, but are not widely used or universally successful. Our hypothesis is that tendon-derived progenitor cells will survive over time and integrate into native tendon during healing in a collagenase model of tendonitis.  Autogenous tendon-derived progenitor cells were isolated from the lateral digital extensor tendon of 8 horses using a differential adherence technique. Collagenase-induced tendonitis was created in the superficial digital flexor tendon of the hind limb by injecting 2000 units of collagenase at 3, 4, 6, and 8 weeks prior to euthanasia (n=2). Two weeks post injury, all 8 horses were injected with 10 million DiI labeled tendon-derived progenitor cells into the site of maximal injury. The opposite normal control tendon and the collagenase injured tendon were collected for analysis 1, 2, 4, and 6 weeks after treatment with DiI labeled progenitor cells. DNA content (ug/mL) was quantified using a fluorometric assay. Treated tendons were cryosectioned and fluorescence microscopy was used to evaluate the persistence and distribution of DiI labeled cells relative to the native tendon cells. Statistical significance was determined for the DNA content using a two way ANOVA. Histologic evaluation was reported descriptively.  The progenitor cells were viable in high numbers during the first week.  By the second week the progenitor cells began to distribute longitudinally from the site of injection and decline in number.  There were still a small number of progenitor cells localized in regions of tendon repair at 6 weeks. Total DNA content in the progenitor cell-treated tendon was significantly (p=0.041) higher (3.2 fold) than in the control tendon at the 1 week time points. There was no significant difference between the treated and control tendon at the 2, 4, and 6 week time points.  In the progenitor cell-treated tendons there was a significant (p<0.001) increase (2.8-9.5 fold) in DNA content 1 week post injection compared to the treated tendons at later time points.  The decline in DiI labeled cells was due to cell turnover and migration from the site.  Further work needs to be done to determine the effects during this time that progenitor cells have on tendon healing.


Mosquito Production and Catch Basins in West Nile Virus “Hot Spots” of Suburban Chicago
DeBaene, Kelly1; Messina, Jane1; Brown, William1; Hamer, Gabriel2; Walker, Edward2; Ruiz, Marilyn O.1
1Department of Pathobiology, University of Illinois, 2Department of Microbiology & Molecular Genetics, Michigan State University

The complexity of the WNV transmission cycle has been a major  focal point of past research. Birds are the primary hosts of the  virus while mosquitoes act as vectors of the disease due to their  necessity to feed on blood. Two species, Culex pipiens and Culex  restuans, have been identified as chief vectors in the virus’s  transmission in our Chicago suburban study areas. Because both  mosquito species require stagnant water for oviposition and the  subsequent life cycle, storm water catch basins are ideal sites for  female mosquitoes to lay their eggs. The study areas were chosen  based on past high human infection rates of WNV and positive  tests from mosquito pools within the region. By examining the  local ecology and catch basin characteristics alongside larvae  numbers, we hope to determine specifically what type of habitat  in which immature mosquitoes are able to thrive. Using ESRI  ArcGIS software and spatial analysis, we are able to visualize and  analyze mosquito production in catch basin sites both spatially  and temporally.


Epidemiology of Fresh Produce Availability in Champaign County, IL
Delcore, Keely; Brown, William; Lee, Soojae; and Ruiz, Marilyn,
University of Illinois at Urbana-Champaign, Department of Pathobiology, Urbana, IL

Access to grocery stores is geographically unequal. Specifically, lower socioeconomic status (SES) neighborhoods have fewer grocery stores with quality products and there are typically more convenience stores than grocery stores. This disparity can lead to difficulties in lower SES residents meeting nutritional needs and contributes to increased disease incidence due to nutritional deficiencies.  This study gives insight into whether a less urban county follows the same pattern as prior urban studies.  We measured the availability of fresh fruits and vegetables in Champaign County, IL using a survey instrument from the Nutrition Environment Measures Survey in Stores (NEMS-S). We mapped the geographic location of 43 stores and markets that sold fresh produce and graded 10 fruits and 10 vegetables according to availability and quality. Access to fresh produce at the level of a census block group was measured based on geographic proximity. The availability and access data was then compared with several factors. First, we used US census SES data to provide a general picture of the county at the block group level. Second, we mapped the residential locations and nutritional status of clients in the Women Infants & Children (WIC) program and summarized weight levels, low levels of hemoglobin, high levels of lead, incidence of gestational diabetes and breastfeeding rates in block groups. We then tested the hypothesis that poor SES and poor nutrition levels in neighborhoods was related to low quality and variety of, and poor access to fresh produce. The main impact of the study is that it will provide empirically-based information for the health district to make plans to improve availability of fresh fruits and vegetables to areas in need of nutritional improvements.  Results show that although stores with fresh produce are typically more accessible to lower SES block groups, more fruit variety and quality and vegetable variety are available to higher SES block groups. 


Effects of Logging on Cryptosporidium sp. and Giardia sp. Infection of Wild Apes in Congo
Deutsch, Joseph1; Kuhlenschmidt, Mark1;Salzer, Johanna1; Morgan, David3; Sanz, Crickette3; Gillespie, Thomas1,2
1Department of Pathobiology, University of Illinois, 2Department of Anthropology, University of Illinois, 3Goualogo Triangle Chimpanzee Project, Nouabale-Ndoki National Park, Congo

Recent studies have indicated zoonotic disease prevalence in wild primates is correlated with changes in ecosystems due to habitat destruction and anthropogenic change. Among pathogens transmitted, Cryptosporidium sp. and Giardia sp. prevalence have been shown to increase in wild African apes with increased habitat destruction and exposure to man and livestock. Cryptosporidium and Giardia are protozoan parasites inhabiting mammalian gastrointestinal tracts with zoonotic potential to cause disease and death. These two parasites prove to be a significant global public health concern and despite their discovery quite some time ago, little has been done to understand how ecological alterations influence transmission of these organisms in wild primates. This project identifies and quantifies infections in chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla) fecal samples (n=103) from the Democratic Republic of Congo (Nouabale-Ndoki National Park / Kabo Logging Concession).  We then examine the influences of habitat change on prevalence of Giardia sp. and Cryptosporidium sp. between a paired track of pristine, undisturbed forest and a neighboring active logging concession, both without significant human presence (<1 person/km2). Our results indicate the effects of logging do not reveal differences in Giardia sp. and Cryptosporidium sp. prevalence rates between these ecosystems. Low prevalence of Giardia sp. (4 of 57 Chimpanzees, 7%; 1 of 46 Gorillas, 2%,) and no Cryptosporidum sp. were present in these populations. These areas, affected by anthropogenic disturbance without significant human presence thereafter, allow for a baseline measurement of the natural prevalence rates of Giardia sp. and Cryptosporidium sp.


Epidemiology of Serovar Specific Leptospira Exposure and Infections
Eisenbart, Valerie; Jung, Namjung; Patterson, Sheila; Ruiz, Marilyn; Maddox, Carol.Veterinary Diagnostic Laboratory, University of Illinois

This project aimed to identify genes of Leptospira that could be of use for clinical diagnosis and epidemiological surveillance.  I chose to examine four genes encoding outer membrane proteins (LigB, LipL21, LipL32 and OmpL1) and one O-antigen polymerase gene (wzy). I used polymerase chain reaction (PCR) to amplify homologous regions among Leptospira interrogans serovar pomona, Leptospira kirscherni serovar grippotyphosa, Leptospira interrogans serovar canicola, and Leptospira interrogans serovar icterohaemorrhagiae.  The amplified genes were then examined for single nucleotide polymorphisms (SNPs) by S1 nuclease digestion (Surveyor kit) of hybridized amplicons.  Data suggests that SNPs exist among the Leptospira serovars tested. These SNPs could be used to determine the serovar identity of Leptospira in clinical and environmental samples, leading to an earlier serovar specific diagnosis than is currently available.  Serovar specific information could also lead to improved vaccine efficacy, since the currently available vaccines only include certain Leptospira serovars.


Ecology of Orthopoxviruses in Uganda
Falendysz, Elizabeth 1, Johanna Salzer2 Innocent Rwego3, Derek Meyer2, Michelle Madonia2, Zach Braden5, Joanna, Shisler4, Inger Damon5, Thomas R. Gillespie2
1 U. of Wisconsin, School of Veterinary Medicine; 2 U. of Illinois, College of Veterinary Medicine; 3 Makerere University, Uganda, Dept. of Zoology;  4 U. of Illinois, School of Medicine;  5 Center for Disease Control, Atlanta, GA.

Poxviruses are a group of DNA viruses that can infect a variety of species. One genus of this family is the Orthopoxvirus genus, and many of these viruses are zoonotic, with the notable exception of smallpox. Since the eradication of smallpox and subsequent cessation of vaccination campaigns, the incidence of orthopoxvirus infection in humans has been increasing. Thus, poxviruses are a serious concern for public health officials. However, the distribution and reservoir species of many of these pathogens remains unclear. To investigate the distribution of orthopoxviruses in East Africa, a survey of rodents was undertaken in western Uganda. This region was of interest because monkeypox is known to occur in humans in the neighboring country of the Democratic Republic of Congo (DRC), although no human cases of this disease have been reported in Uganda. 71 wild rodents were trapped in and around Kibale National Park, near the Rwenzori Mountains, which form the border with DRC. Blood was collected and serum these rodents was assayed for the presence of antibodies reactive to one orthopoxvirus, vaccinia virus, using ELISA. Sera from 11 rodents displayed reactivity to vaccinia antigen. These included 5 dormice (Graphiurus murinus), and 6 rats (Rattus sp.). This is the first report of potential orthopoxvirus infection in rodents of East Africa.


Evaluating the health status of Blanding´s Turtles (Emydoidea blandingii) in northern Illinois.
Heggem, Brittany; 1 Mark A. Mitchell, DVM, MS, PhD,1 Dana Johnson, BS,2 Dan Thompson, BS2
1University of Illinois, College of Veterinary Medicine, Department of Veterinary Clinical Medicine, 2Forest Preserve District of DuPage County, Department of Natural Resources

Blanding’s turtles (Emydoidea blandingii)  are semi-aquatic chelonians that are threatened or endangered throughout much of the their range.  The primary reason for their perilous status is due to habitat loss and fragmentation.  Few studies have been conducted to assess how alterations in the Blanding turtle’s landscape have affected the general health of these animals. With rapidly declining populations, it is important that biologists and veterinarians take note of a population’s overall heath status to develop long term management plans for this species.  The purpose of this study was to collect biologic data from a population of Blanding’s turtles from northern Illinois to characterize their health status. A total of 32 turtles, ranging from juvenile to adult, were collected from DuPage and Will Counties in Illinois.  Each female collected was assessed for reproductive status by radiography.  Blood was taken from the jugular vein in the adult turtles and from the subcarapacial sinus of the juveniles.  Whole blood was analyzed for packed cell volume, total solids, complete blood counts and the presence of hematologic parasites.  A complete biochemistry profile was also performed on each animal. A rectal swab was collected from each animal to determine if it was Salmonella positive. A number of differences between the blood results were noted by gender, age and location. Additional analysis is on-going at this time. This information will be used in developing long-term management plans for these turtles.


Evaluation of the Canine Brief Pain Inventory Questionnaire by its Correlation with Measured Ground Reaction Forces as an Outcome Measure in Orthopedic Patients
Hsia, Gary; Wanda Gordon-Evans, Kimberly Knap, Department of Veterinary Clinical Medicine, University of Illinois

Clinicians often use owner evaluated pain forms to help assess and manage chronic pain in their orthopedic patients. However, these owner evaluation forms can be quite subjective because each patient’s response to pain is different and there is no universal sign for pain. The Canine Brief Pain Inventory Questionnaire (CBPI) is the owner’s assessment of the severity of pain and how the pain interferes with a patient’s normal function. The purpose of the study is to correlate a subjective CBPI questionnaire to an objective gait analysis. We hypothesize that there is an inverse correlation between the pain scores and peak vertical force exerted by orthopedic patients. 22 client-owned dogs of various age, sex, and breed that presented to the University of Illinois Rehabilitation Center for hip pain were included in the study. Each client was given a CBPI questionnaire and dogs were measured for ground reaction forces before treatment. Each subsequent return visit warranted another set of questionnaires and measurements. Data of peak vertical force (PVF) and vertical impulse (VI) of front and hind limbs were correlated with the numerical scores from the CBPI questionnaire. Results showed there was no correlation between single limb PVF or VI with any of the CBPI questions or its average. However, there was a significant correlation (p=0.05) between PVF of back to front limb symmetry ratios in 5 of the 10 questions and the average CBPI scores. Also, between the 1st and 2nd visits, the correlation between the change in PVF and change in average CBPI scores approaches significant. Overall, the poor correlation between the CBPI questionnaire and measured ground reaction forces maybe due to owners incorrectly evaluating non-gait aspects of pain. Also the CBPI questionnaire may not be a good survey when using measured ground reaction forces as an outcome measure.


Investigation of the Oncolytic Nature of Myxoma virus in Canine Tumor Cell Lines
Hynes, Stacy; Somrak,  Amy J.; MacNeill, Amy L. Department of Pathobiology,  University of Illinois

Virotherapy holds promise as a potential treatment for several cancers by using the ability of specific viruses to infect and kill tumor cells while leaving healthy surrounding tissue unaffected. One potential oncolytic virus is Myxoma virus (MYX), a rabbit-specific poxvirus that causes the lethal systemic disease, myomatosis in European rabbits. MYX is oncolytic in human gliomas in vivo and human pancreatic adenocarcinoma cells in vitro. This study examined the infectability of canine cancer cells with MYX that was genetically modified to express the fluorochrome Tomato Red (MYX Red). This virus behaves like wild type MYX in cell culture and is fully virulent in rabbits.  Primary canine tumor cells were extracted from eight tumors obtained post-operatively (Babs- mixed mammary tumor; Casper- intestinal stromal tumor; Cinna- perianal adenocarcinoma; Duke- perianal adenoma; Holly and Sweetie- mast cell tumors; Morris- soft tissue sarcoma; Newbie- hemangioma). Confluent cells from a canine perianal adenocarcinoma (Cinna) were infected with MYX Red at multiplicities of infection (MOI) 0.01, 0.1, 1, and 5 plaque forming units/cell. Observations of cytotoxic effects of MYX Red were taken at 8, 24, and 48 h time points. Rabbit-kidney epithelial cells are fully permissive to MYX Red, and functioned as a positive control. The number of fluorescent cells observed corresponded to the MOI. Slight cytopathic effects were apparent in this initial assay indicating that Cinna was susceptible to MYX Red infection although viral replication and spread were not apparent. Further studies will evaluate the effectiveness of MYX Red replication in Cinna using multi-step growth curves. Additionally, the other seven cell lines will be inoculated with MYX Red at various MOIs to determine if they are permissive to infection and/ or replication. Eventually, we hope to effectively treat canine cancer patients with oncolytic forms of MYX.


Molecular Epidemiology of Cross-species Giardia Transmission in Western Uganda
Johnston, Amanda R.; Gillespie, Thomas R.; Rwego, Innocent B.; Tranby McLachlan, Traci L.; Goldberg,Tony L. Department of Pathobiology, University of Illinois

Forest fragmentation increases ecological overlap among wildlife, humans, and domestic animals, which may result in increased transmission of zoonotic pathogens, such as Giardia spp. Molecular epidemiological methods were used to infer patterns of relatedness among Giardia from different host species near Kibale National Park, western Uganda.  Results suggest that multiple Giardia assemblages are co-circulating in western Uganda, and that transmission among humans, wild primates, and livestock occurs frequently.


Demographics, Resources, and Health Indicators of Pastoralists in Rajasthan, India
Joshi, Vaishali; Yvette Johnson, College of Veterinary Medicine, University of Illinois

This project aims to uncover any public health concerns related to the culture and traditions of pastoralists in Rajasthan, India.  Pastoralists are people who engage in animal husbandry and breeding of domestic animals, and many live a semi-nomadic to nomadic life.  According to several sources, the agriculturalist sect of Rajasthan contributes to about 16-17% of its economy through sale of milk products, wool, and animals.  Additionally, pastoralists contribute to the genetic resources by breeding animals that are able to live in dry, often drought-prone conditions.  With current climate changes occurring world-wide, the knowledge passed down by pastoralists is key in producing animals that will be able to survive such changes. A questionnaire was administered to pastoralists in Rajasthan, India, describing their demographics, general health assessment of the people and their livestock, access to medical and veterinary care and application of traditional and western methods for the treatment and control of disease in the human and animal populations.  Investigators visited homes of pastoralists.  Sample sites were chosen at random, but interviews depended upon the willingness of individuals to sit for the interviews.  The interviews generally took forty-five minutes to complete, and individuals were informed that they were free to answer all, some, or none of the questions.  The study aims to collect information that may potentially provide support to the efforts of organizations and government entities seeking to provide technical or resource support to pastoralist communities in India. Results have shown that the communities generally have limited access to agricultural resources and health care due to financial limitations.  Additionally, many participants expressed disappointment in the government for not providing more opportunities to the pastoralists for improved animal health and nutrition.  Outcomes of this research are to identify human and animal ailments that may be prevented by improved sanitation and treatment protocols for this population.  In addition, the information obtained about indigenous techniques for maintaining human and animal health may provide new areas of research in pharmaceuticals.


Transmission of Methicillin-Resistant Staphylococcus sp. between Pets and Their Owners
King, Leanne;  Johnson, Yvette; Herrmann, John; and Myint, Maung, Department of Veterinary Clinical Medicine, University of Illinois

Methicillin-resistant staphylococcal infections have been identified as a growing health concern for both humans and animals.  According to the CDC, human data indicates that in 1974 methicillin-resistant Staphylococcus aureus (MRSA) accounted for 2% of all staphylococcal infections in the United States; in 1995 it increased to 22%; in 2004 it was 63%.  Coinciding with the increase of MRSA in human staphylococci infections has also been an increase of methicillin-resistant Staphylococcus species (MRS) in household pets.    The objective of this study is to determine the risk factors for transmission of MRS between pets and their owners. The study design was a cross-sectional survey of household pets (dogs and/or cats) and their owners which are students, faculty and staff at the University of Illinois, College of Veterinary Medicine.  Nasal swabs were collected from both the human participants and their pets from the anterior nares.   Each participant also filled out a questionnaire to help determine risk factors such as previous antibiotic exposure, hospitalization, and number/type of pets.  The samples were cultured for bacterial isolation, identification and biochemical confirmation of the Staphylococcus .   A total of 27 people, 19 dogs and 23 cats participated in the study.   The prevalence of MRS in these samples was 18.5%, 16% and 9%, respectively. MRSA was isolated in 4 out the 5 people that tested MRS positive. Difficulty breathing and coughing was found to be the only statistically significant risk factor. This study is important because risk factor data are necessary in order to better understand the dynamics of MRS transmission between owners and their pets.  Owners with infections need to consider that their animal may become a carrier of that agent and a potential source of re-infection if they are not screened and perhaps treated even in the absence of clinical illness.


Human-Wildlife Interactions and Monkeypox Transmission in the Kabarole District, Uganda
Madonia, Michelle MPH*, Innocent Rwego BVM MSc**, Charles Kyalisiima***, Edith Mbabazi***, Mary Reynolds‡, Tom Gillespie PhD MS*‡‡
*Department of Pathobiology, University of Illinois, Champaign, IL; **Department of Zoology, Makerere University, Kampala, Uganda; ***Makerere University Biological Field Station, Kyanyawara, Uganda; Poxvirus and Rabies Branch, CDC; ‡‡ Department of Anthropology, University of Illinois.

Monkeypox virus (MPXV) is primarily a zoonosis that is potentially devastating to both animals and humans. Although it is endemic in Central and Western Africa, no cases of human monkeypox have been reported in East Africa. One explanation for the lack of reported monkeypox cases in this region of Uganda is that there are differences in animal-human interactions.  In this study, we have used a survey instrument to assess monkeypox-like symptoms and animal interactions for residents of six villages surrounding Kibale National Park.  Although we discovered that 6.8% (n=22) of subjects fit one of our case definitions for monkeypox, further consultation with our collaborators at the CDC has led us to conclude that monkeypox was not present in the study subjects.  These data suggest that the manner in which these individuals are interacting with the animals in question does not facilitate monkeypox transmission.


Development of a Feline Thyroglobulin Immunoassay
Reinhart, Jennifer M. and Duncan C. Ferguson, Department of Veterinary Biosciences, University of Illinois, Urbana, IL

In human thyroid disease associated with thyroid gland enlargement, serum concentrations of thyroglobulin (Tg), the major protein in the thyroid, increase in proportion to the size of the gland, and have been used to track the efficacy of treatment regimens.  If this is true for cats, an assay for feline Tg may allow earlier detection and treatment of feline hyperthyroidism, a very important disease in small animal practice.  fTg was isolated from the thyroid glands of eight cats by homogenization and centrifugation, and then purified using size exclusion chromatography yielding ~13 mg fTg (23% yield).  The resulting fractions were concentrated and will be assessed for purity on an SDS-PAGE and using pronase digestion with measurement of thyroxine(T4) release.  Using fTg preparations prepared previously, fTg crossreactivity in two commercially available hTg assays was evaluated.   The hTg immunoassay kits selected for evaluation were: ALPCO EIA (ALPCO Diagnostics, Salem, NH) and Immulite (Siemens, Los Angeles, CA).  The ALPCO EIA showed less than 1% crossreactivity.  All standards read at or below the analytical sensitivity of the Immulite assay.  Because these assays appear species-specific, a novel enzyme-linked immunosorbent assay (ELISA) was developed using two rabbit polyclonal antibodies previously raised against fTg (R870 and R871). Purified fTg was tested in a sandwich immunoassay with a biotinylated secondary antibody and detection with streptavidin-horseradish peroxidase conjugate followed by a colorimetric substrate. The pairing of antibody R871 with itself was found to give the highest colorimetric signal and the best sensitivity. Age-controlled normal ranges for this assay will be established using sera from euthyroid cats of various ages.  Furthermore, sera from cats before and after radioiodide treatment will be used to test the clinical value of this assay.


Assessing Genetic Changes in Candida albicans Isolates from a Murine Candidiasis Model
Sachen, Dusty, Xiaomin Zhao and Lois L. Hoyer, Department of Pathobiology, University of Illinois, Urbana, IL

The pathogenic fungus Candida albicans is diploid, with an incomplete sexual cycle. Following serial transfer of the fungus in culture, C. albicans genome changes are obvious at a gross level, such as by Southern blotting. Repeated sequences, mostly intergenic, are involved in some of these microevolutionary changes (Pujol et al. 1999. Microbiology 145:2635-2646). Baker’s yeast (Saccharomyces cerevisiae, non-pathogenic) has been used as a model for studying C. albicans. In both fungi, genes that encode cell-surface adhesins often have tandemly repeated sequences in the coding region. In S. cerevisiae, recombination between the intragenic tandem repeats results in quantitative alterations in adhesion phenotype at a high frequency (approximately 1 x 10e-5; Verstrepen et al. 2005. Nat Genet 37:986-990). This mechanism of creating adhesive diversity was proposed to apply to C. albicans although tandem repeat copy number in genes encoding cell-surface adhesins is stable over 3000 generations in culture (Zhao et al., 2007. Fungal Genet Biol 44:1298-1309). To date, the stability of tandem repeats within C. albicans cell wall-encoding genes has not been assessed in vivo. The goal of this work is to determine whether the tandem repeats in three cell wall-protein-encoding genes (ALS3, ALS5 and ALS6) change as the fungus is passaged through the murine disseminated model of candidiasis. Two strains were utilized: SC5314 (originally a bloodstream isolate) and 1-234 (an oral isolate from a healthy human). Each strain was inoculated into the murine lateral tail vein and, after 24 h, C. albicans was recovered from the kidneys. Twenty-four colonies from each mouse were assessed for changes in the tandem repeat copy number of each gene. One of these colonies was used to inoculate the subsequent mouse. After the completion of eight passages the data collected suggest that the tandem repeat copy number in ALS3, ALS5 and ALS6 is highly stable in vivo.


Evaluation of Myxoma virus as an Agent Oncolytic to Canine Primary Tumor Cells
Somrak, Amy; Hynes, Stacy; MacNeill, Amy L., Department of Pathobiology, University of Illinois

Myxoma virus (MYX) is a poxvirus that is pathogenic only to rabbits, and has been shown to productively infect and be oncolytic in vitro to human glioma cells and canine osteosarcoma and hemangiosarcoma primary tumor cells. The main goal of the project is to isolate additional canine primary tumor cells and to study the effects of their infection by MYX; eight cell cultures were isolated and propagated. Further characterization of each tumor cell type is ongoing. The virus used is MYXΔSERP2::lacZ, a genetically modified form of MYX that causes apoptosis in rabbit kidney epithelial cells (RK-13s). This virus is expected to infect the same types of canine primary tumor cells as MYX but be more oncolytic. RK-13s were used as positive controls because they are fully permissive to the MYX virus. Confluent cells grown as explants of canine primary tumor cells (Cinna: perianal adenocarcinoma) were infected with MYXΔSERP2::lacZ at multiplicities of infection (moi) of 0.01, 0.1, 1 and 5 plaque-forming units/cell to evaluate cytopathic effect (CPE). CPE increased as moi increased, suggesting that these canine tumor cells are susceptible to infection by the virus. Further studies will evaluate additional canine primary tumor types for CPE upon infection with MYXΔSERP2::lacZ, including explants of perianal adenoma, hemangioma, mast cell tumor, mixed mammary tumor, intestinal stromal tumor, and soft tissue sarcoma. Cinna primary tumor cells will be used to evaluate cell-to-cell spread of the virus and to quantitate the rate of viral replication in the tumor cells. The ultimate goal of this work is to determine the feasibility of this virus as a cancer therapy.


Infectious Disease Survey of Feral Mice at Brookfield Zoo
Son, Ji-son, Jennifer N. Langan, and Thomas Meehan
College of Veterinary Medicine, University of Illinois

House mice (Mus musculus domesticus) and white-footed mice (Peromyscus leucopus) are two very common feral rodent species in zoological parks worldwide. Since these mice are known to carry a variety of diseases that can potentially infect and cause illness in humans and other animals, a pilot surveillance program was initiated as a part of the preventative medicine program at Brookfield Zoo. Infectious diseases caused by a select group of viral agents (Encephalomyocarditis virus (EMCV), Hantavirus, and Lymphocytic Choriomeningitis virus (LCMV)) and by bacterial agents (Samonella sp., and Leptospiria sp.) were screened for in this study.  Mice were trapped in live traps and euthanized with carbon dioxide gas. Blood was collected via intercardiac puncture. Serum was separated, stored at -70°C, and sent for out for analysis. Mice were necropsied and samples of liver, kidney and spleen were collected and stored for potential later use at -70°C.  The remaining tissues were stored in 10% formalin and were routinely processed for histopathological examination.  Fecal samples from the colon were collected for Samonella sp. culture.  Samples were sent to several diagnostic labs for testing. All samples tested were negative for the diseases. Because, however, there were reports of diseases in neighboring places, the samples size was small, and there could be a diagnostic error, it is important to aware that the possibilities of presence of diseases still exist. 


Evaluating Environmental and Climatic Influences on Nesting Patterns in Leatherback Sea Turtles  (Dermochelys coriacea) in St. Kitts, West Indies
Watson, Megan,1 Mitchell Mark,1 Stewart Kimberly,1,2 Krecek Tammi2.
1University of Illinois, Urbana, IL USA, 2Ross University, St. Kitts, West Indies

Sea turtles are endangered throughout their range. To protect  these animals, research is needed to develop conservation  programs that cover the turtles during all of their life stages,  including foraging, breeding, nesting, and hatching. The purpose of this study was to focus on issues related to nesting by determining if environmental and climatic factors influence nesting patterns for female leatherback sea  turtles. A variety of climatic and environmental samples were collected during the nesting season from May-July 2008. Data was collected during nesting and non-nesting periods for comparison. Sea turtles were found to be significantly more likely to nest during the later lunar phases and when cloud cover was low over the natal beaches. Differences in the conductivity of the sand at nesting sites between the surface layers and nesting layers of sand were also noted. These findings suggest that the climatic and environment factors do influence nesting patterns in this species. This information should be considered when developing conservation plans for this species.


Pharmacokinetics of Toltrazuril Sulfone (Ponazuril®) in Cattle
Yohn, Rachel; E.F. Garrett; D. Ferguson; L. Dirikolu
University of Illinois, College of Veterinary Medicine

Toltrazuril sulfone is a triazine-based anti-protozoal agent with highly specific actions against the apicomplexan group of organisms.  Toltrazuril sulfone (TS) may have clinical applications in the treatment of Neospora caninum and other protozoal infections in cattle. In order to evaluate absorption, distribution and elimination characteristics of toltrazuril sulfone in cattle, a sensitive validated quantitative high-pressure liquid chromatography (HPLC) method for toltrazuril sulfone in bovine biological fluids was developed.  After a single oral doseof toltrazuril sulfone was administered the plasma samples from 6 cows showed good plasma concentrations of toltrazuril sulfone which peaked at 4,821 ng/ml +/- 916 ng/ml (SD) at 48 hours post-administration.  Plasma concentrations declined to 1,950 ng/ml +/- 184 ng/ml at 192 hours after administration with an average plasma elimination half-life of about 58 hours.  Following oral administration of toltrazuril sulfone, the observed peak plasma concentrations were in relatively close agreement ranging from lowest concentration of 3,925 ng/ml to highest of 6,285 ng/ml with the mean peak plasma concentration being 4,821 ng/ml. Toltrazuril sulfone is relatively well absorbed after oral administration in cattle.  These results are consistent with and support the reported clinical efficacy of toltrazuril sulfone in the treatment of experimentally induced clinical cases of N. caninum and other protozoal-mediated bovine diseases. 


2007 STUDENT PROJECTS

Potential Monkeypox Reservoir in Uganda
Elizabeth Falendysz1, Anna Czekala2, Joanna Shisler3, Zach Braden4, Darin Carroll4, Yu Li4, Kevin Karem4, Christy Hudson4, Inger Damon4, Thomas R. Gillespie5,6
1 University of Wisconsin School of Veterinary Medicine, Madison, WI 53706
2 University of Illinois College of Veterinary Medicine, Urbana, IL 61802
3 University of Illinois College of Medicine, Department of Microbiology, Urbana, IL 61802
4 Centers for Disease Control, Division of Viral and Rickettsial Disease, Atlanta, GA 30333
5 University of Illinois Department of Anthropology, Urbana, IL 61802
6 University of Illinois College of Veterinary Medicine, Department of Pathobiology, Urbana, IL 61802

Monkeypox virus (MPXV), a member of the Orthopoxvirus family, is an emerging zoonotic pathogen which can cause serious smallpox-like infection in people. Records of infected humans in West and Central Africa indicate that monkeypox infection is associated with a 10% mortality rate. Since the global eradication of smallpox in 1977, monkeypox virus has been considered the most problematic orthopoxvirus in regards to human health. Unfortunately, very little is known about the virus or its natural host, although it is thought to spread via direct contact with infected animals. There have been no reports of human monkeypox infection in East Africa to date. To determine if this is due to lack of reporting, lack of viral transfer, or to a lack of a natural monkeypox reservoir in East Africa, 61 rodents were trapped and sampled in and around Kibale National Park, in Uganda.  Previous research in this area demonstrated the presence of antibodies recognizing monkeypox virus in one individual of the rodent species Graphiurus murinus (dormouse).  To ascertain whether the dormouse may be a natural reservoir for the monkeypox virus in East Africa, our approach was to target the collection of this species of rodent. To this end, a live-trapping method was used to collect blood and tail tissues from small rodents in Western Uganda.  In addition to 54 other animals, 7 dormice were trapped and anesthetized. Serum and tissue samples were collected from each individual and shipped to the CDC in Atlanta, GA.  Initially, the sera were screened for antibodies to Orthopoxviruses, using an ELISA-based assay. Additionally, DNA extracted from skin samples containing suspicious lesions was tested for orthopox genomic material using real time PCR. Although no clear positive results were obtained from the serology assays, orthopox genomic material was identified in 4 rodents. Lesions from 2 dormice contained viral DNA which is thought to be specific to MPXV. This work is the first to identify MPXV in Uganda and indicates that MPXV may represent a previously unknown public health risk in this region.


Effects of Exposure to the Herbicides Atrazine and s-Metolachlor on Ribeiroia ondatatrae Infections of Bullfrog (Rana catesbeiana) Tadpoles
Elzerman, AL*; Beasley, VR; Levengood, JM
University of Illinois at Urbana-Champaign

Amphibian populations around the world are in precipitous decline.  Multiple hypotheses exist concerning causes of the declines.  One factor that may be involved in amphibian declines is trematode infections.  One species of trematode, Ribeiroia ondatrae, has been linked with malformations and high death losses in field and laboratory studies, yet it is unclear why numbers of infections and malformations are increasing.  The co-occurrence of high infection rates with high numbers of deformed frogs in water bodies altered by human activities suggests that deteriorating environmental conditions are partially to blame.  Two ubiquitous agricultural herbicides, atrazine and s-metolachlor, impair immune function in amphibians.  The mechanisms and extent to which herbicides interact with parasite infection and clearance rates remains to be explored.  We are examining the effects of herbicide exposure on the immune response of the tadpoles to the parasite and the numbers of encysted cercaria.  Bullfrog tadpoles (Rana catesbeiana) hatched from field-collected eggs are being exposed to environmentally relevant concentrations of atrazine and s-metolachlor individually, as well as to a combination of the herbicides together, from hatching until the early limb-bud stage.  Each tadpole will then be exposed to R. ondatrae cercariae.  At the end of the study, tadpoles will be removed, anesthetized, and subjected to euthanasia.  Blood smears will be utilized to count total white blood cell numbers in relation to red blood cell numbers and differential white blood cell counts, including eosinophils and lymphocytes.  Tissues will be fixed in 10% neutral buffered formalin and routinely processed for histopathology including staining with hematoxylin and eosin.  Tissue sections will be evaluated with a light microscope for evidence of metacercariae as well as hyperemia, edema and cell infiltrates as indicators of immune reactions to the parasites.


Effects of forest fragmentation and domestic livestock interaction on the transmission of the water borne microbes Giardia spp. and Cryptosporidium spp. between non-human primates and livestock in Kibale National Park, Uganda
Annie J. Lo1, Innocent Rwego2, Thomas R. Gillespie1, Angela Kent3, Tony L. Goldberg
1 College of Veterinary Medicine, University of Illinois at Urbana-Champaign
2 Makerere University, Kampala, Uganda
3 Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign

The recent emergence of many important infectious pathogens and the increased encroachment of humans into wildlife habitats makes understanding the effects of anthropogenic environmental changes on disease dynamics imperative.
Kibale National Park, Uganda itself is well protected, but outside of the park boundaries exist a series of forest fragments that represent what has been left after agricultural clearing. In particular, these are areas of open wells, streams, and standing water bodies in the ranges of primates that domestic livestock also utilize.

I am investigating two important pathogens that are shared between wild non-human primates and livestock: Cryptosporidium spp. and Giardia spp.  This study focuses on primates and livestock living in fragmented and undisturbed forest habitats in Kibale National Park. These pathogens can cause severe diarrhea and fatal systemic infections. We are testing the hypothesis that open water sources are important reservoirs of infection and inter-species transmission between non-human primates and domestic livestock.  In Kibale, hydrological processes are such that surface water flows through both forest fragments and livestock watering sites.  I have already sampled water sources upstream of, within, and downstream of these forest fragments, as well as in undisturbed forest sites, to examine whether the location and type of the water source affects the presence and concentration of Cryptosporidium spp.and Giardia spp. within it.  Genotyping the parasites in water, primates, and livestock will help indicate whether these water sources are important for interspecies transmission.
I hypothesize that these pathogens are more prevalent in water sources in the fragments than in undisturbed forest habitats, and are particularly high in water sources used by both primates and livestock. This study will help to determine how forest fragmentation affects primate health and will help to improve conservation strategies for primates and forest ecosystems, as well as domestic livestock and human health.


Parasitic Infections of Non-human primates in Fragmented and Undisturbed Forests in Western Uganda
Martha Low*1, Derek Meyer1, Trisha Campbel1, Jed Nicholas Panganiban1, Johanna Salzer1, Tony L. Goldberg1, Innocent B. Rwego2, and Thomas R. Gillespie1
1College of Veterinary Medicine, University of Illinois at Urbana-Champaign
2 Makerere University, Kampala, Uganda

In June 2005, 115 fecal samples were collected from wild primates in Uganda. These samples were analyzed for intestinal parasites using fecal floatation and sedimentation techniques. Primates sampled included red colobus (Pilocolobus tephrosceles), red-tailed guenons (Cercopithecus ascanius), and black and white colobus (Colobus guereza).  Primates were sampled from an undisturbed forest in Kibale National Park and from three highly disturbed forest fragments outside the park.  Fecal samples from livestock in the same areas were also collected and analyzed to determine the parasites present.

The final analysis of the data will involve determining patterns of parasitism in non human primates living in fragmented forests versus undisturbed forests. It will also determine any correlation between livestock and non human primate parasite infections.

To date, more than half of the non-human primate samples and livestock samples have been completed.


Soda as a virulence factor in Streptococcus equi and Streptococcus zooepidemicus infections
*Lisa Lukas and Dr. Carol Maddox
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Streptococcus equi is the etiologic agent of strangles in horses while Streptococcus zooepidemicus is an opportunistic pathogen that can cause disease in many mammals, including humans. The ability to survive in phagocytic immune cells is an important virulence factor of these Lancefield group C Streptococcus. One gene that has been recognized as contributing to macrophage survival by the Streptococcacea Family is sodA. The sodA gene encodes superoxide dismutase, which catalyzes the dismutation of superoxide anion radical to molecular oxygen and hydrogen peroxides. sodA is found ubiquitously in oxygen metabolizing organisms and is thought to protect cells from the cytotoxic superoxide anions. We hypothesize that Sod may contribute to virulence by playing an important role in the survival in the phagocytic cells of the host. To test this hypothesis, deletions of the sodA gene are being constructed in both S. equi and S. zooepidemicus. The effects of the deletion on intracellular survival will then quantified using a J774 murine macrophage cell line assay. Zebrafish will then serve as models to compare mutants for changes in streptococcal infection pathology. The mutant strains, constructed for these studies, could  potentially be applied to vaccines to prevent not just strangles, but respiratory diseases in other hosts as well.


The virulence role of Pseudomonas aeruginosa pyocyanin in chronic suppurative otitis media
Elisabeth F. Peters, YongHua Hao, and Gee W. Lau*
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Pseudomonas aeruginosa is the most common and important causative pathogen of chronic suppurative otitis media (CSOM).  It produces various tricyclic phenazine toxins, with pyocyanin being among the most redox-active and best-studied.  Because pyocyanin is toxic to human cells, and is secreted in high quantities during chronic infections in humans diseases, we hypothesized that pyocyanin is a major virulence factor that contributes to the pathogenesis of CSOM.  Our hypothesis is supported by clinical studies showing that significant concentrations of pyocyanin capable of killing human cells can be recovered from the middle ear secretions of CSOM patients. In this study, we discovered that pyocyanin is synthesized by P. aeruginosa during stationary phase of growth. In addition, mutant strains (phzM, phzS, mvfR and phzB1) with disruption in genes required for pyocyanin biosynthesis retained growth rates similar to their respective wild-type strains, suggesting that pyocyanin biosynthesis genes are dispensable for growth.  We also found pyocyanin was capable of inhibiting the growth of other bacterial and fungal pathogens that cause otitis media or colonize the middle ear cavity. These microorganisms include Streptococcus pneumoniae, Escherichia coli, Candida albicans, and Aspergillus nidulans.   In addition, when exposed to pyocyanin, epithelial cells accumulate oxygen radicals (ROS) in a time and pyocyanin-concentration dependent manner. The increase in intracellular ROS induced a corresponding increased in the activity of catalase and superoxide dismutase, suggesting that the epithelial cells were trying to detoxify the ROS. During in vivo studies, direct instillation of pyocyanin into the middle ear cavity of chinchillas was found to induce the secretion of proinflammatory cytokine IL-8. More importantly, comparative virulence studies demonstrated that pyocyanin-deficient mutants phzM and phzS were attenuated in their ability to colonize the middle ear cavity of chinchillas when compared to their parental wild-type PA01. Cytokine analysis suggests that phzM and phzS mutants were also less able to induce the secretion of IL-1 cytokine (a marker for CSOM) than their wild-type strain. In summary, for the first time, we demonstrated that pyocyanin produced by P. aeruginosa plays an important role in P. aeruginosa-mediated middle ear infection.


Identification of Clostridial Isolates Responsible for Suspected Botulism Outbreak in Horses
Jennifer M. Reinhart*1, Dusty S. Sachen*1, Melissa Pires-Alves2, Mengfei Ho2, and Brenda A. Wilson2,3
1College of Veterinary Medicine, University of Illinois at Urbana-Champaign
2Dept. of Microbiology, University of Illinois at Urbana-Champaign
3Institute for Genomic Biology, University of Illinois at Urbana-Champaign

In June 2006, an Amish farm in Illinois lost a total of seventeen horses to illness. These horses exhibited botulism-like signs including flaccid paralysis. Bacterial isolates from necropsy, fecal and environmental samples submitted to the University of Illinois Veterinary Diagnostic Laboratory (VDL) were identified as Clostridium sp., but the VDL was unable to conclusively determine the presence of a toxin gene. The purpose of this project is to more specifically identify the strain(s) of Clostridium present in these samples and to isolate and identify the responsible toxin gene. Near full-length 16S ribosomal DNA segments (~1.4 kb) were PCR amplified with universal bacteria primers, then cloned and sequenced. These sequences were compared against the NCBI database using the BLASTN algorithm. C. sporogenes/botulinum, C. novyi and C. bifermentans 16S gene sequences have been identified with 99% similarity from the different samples.

Botulism is caused by botulism neurotoxins (BoNTs), which inhibit the release of acetylcholine at the neuromuscular junction, resulting in flaccid paralysis. There are seven known BoNT serotypes, A through G. Novel PCR primers were designed to target the heavy and the light chain of each of these seven serotypes as well as the related tetanus neurotoxin (TeTx). Multiple primer pairs yielded PCR amplicons for the different samples, which were then cloned and sequenced. None of these sequences confirmed the presence of a BoNT or TeTx gene in our samples. Primers targeting the C. novyi alpha, beta and gamma toxins were also designed, since C. novyi was identified by the 16S ribotyping. Primers for the beta and gamma toxins yielded amplicons for the C. novyi sample and sequencing confirmed the presence of these genes.  However, we cannot conclude that these toxins are responsible for the clinical signs observed in the horses.  Furthermore, because no BoNT genes were found, we cannot conclude that the C. sporogenes/botulinum samples are responsible.


Gastrointestinal Salmonella diversity in Galapagos marine iguanas
Emily Wheeler 1, 2, Isaac K. O. Cann 1, and Roderick I. Mackie 1
1. Department of Animal Science, University of Illinois at Urbana-Champaign
2. College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Salmonella bacteria are common residents of reptilian gastrointestinal tracts and are of focal concern in veterinary medicine due to the established risk of pet reptile-associated illness in humans. While most studies of Salmonella in reptiles have focused on such epidemiological concerns, few studies have addressed the role of Salmonella in the physiology and gastrointestinal function of reptiles. Initial anaerobic cultivation of marine iguana fecal samples suggests that Salmonella may play an important role in colonic function in these herbivorous reptiles. While mammals present with the more “classical” sulfate reducing bacteria, such as Desulfovibrio, which perform functions like lactate fermentation and sulfate reduction in the colon, our findings suggest that these are not the predominant cultivable organisms of this type in marine iguanas. In order to explore the functional significance of Salmonella in the marine iguana, we are surveying frozen marine iguana fecal samples to evaluate Salmonella microdiversity using a cultivation based strategy followed by serotyping and molecular fingerprinting of isolates using REP-PCR. From these data, we will select isolates of interest for further genotypic and phenotypic studies in order to better understand the functional ecology of Salmonella in wild reptiles.


Molecular Phylogenetic Analysis of Candida albicans Isolates from Humans and Non-Migratory Wildlife in Central Illinois
Lauren Wrobel1*, Julia K. Whittington2, Claude Pujol3, Soon-Hwan Oh1, Michael A. Pfaller4, Daniel J. Diekema4,5, David R. Soll3, Lois L. Hoyer1
Departments of Pathobiology1 and Veterinary Clinical Medicine2, University of Illinois, Urbana, IL 61802; Departments of Biological Sciences3, Pathology4 and Internal Medicine5, University of Iowa, Iowa City, IA 52242

Candida albicans is a commensal of mucosal surfaces in humans and animals. Under conditions where normal host defenses are compromised, C. albicans can cause disease. Among the different molecular methods used to study the epidemiology of C. albicans strains in human populations, fingerprinting with the complex probe Ca3 has shown that this species is subdivided into a few discrete major genetic clades. While strains from different clades can be found side by side in the same geographical locale, their distribution varies between geographical areas (Soll and Pujol, 2003; FEMS Immunol Med Mycol 39:1-7). This finding was unexpected in light of the high frequency of human travel that should homogenize the worldwide distribution of C. albicans clades. These observations argue in favor of a local reservoir of C. albicans strains that maintain the association between certain groups of C. albicans isolates and a specific geographic area. The goal of this work is to test whether local non-migratory wildlife serves as a reservoir for human C. albicans isolates. To test this hypothesis, we collected oral and anal/cloacal swabs from non-migratory wildlife species immediately upon their admission to the Wildlife Medical Clinic at the University of Illinois College of Veterinary Medicine. A geographically matched set of C. albicans oral isolates was collected from normally healthy human volunteers who attended the annual College of Veterinary Medicine Open House. Molecular phylogenetic analysis will be completed using the well-established Ca3 fingerprinting method and the more recently developed approach of Multilocus Sequence Typing (Bougnoux et al., 2003; J Clin Microbiol 41:5265-5266). The antifungal susceptibilities of the C. albicans isolates will also be determined. Data analysis will indicate the genetic relatedness between the human and animal isolates and also the intrinsic antifungal susceptibilities of the various strains.


2006 STUDENT PROJECTS

A blinded evaluation of the diagnostic accuracy of canine lymphomas using immunohistochemistry and the criteria of the WHO histological classification
Marcia C. Chien*, Victor E Valli
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Lymphoma is the most commonly diagnosed and treated malignant tumor that afflicts dogs of all ages. In animals and humans, there are 30 subtypes of B and T-cell lymphomas which differ markedly in their normal biology, untreated survival and response to therapy.  A generic diagnosis of “lymphosarcoma” is often made in animals based on morphology and increasingly with phenotypic identification of cell lineage based on a widespread acceptance that B-cell lymphomas had better survival upon treatment than T-cell lymphomas.  It is now shown that high and low grade lymphomas are present in both B and T-cell types and that tumor grade is more significant than cell lineage.  This finding indicates a specific diagnosis of lymphoma subtype is essential to properly titer treatment to tumor characteristics and to provide more accurate prognostication to the owner.  A specific diagnosis will assist oncologists to better treat canine lymphoma, plus allow cooperative oncology trials to compare the efficacies of new treatments on a single subtype of lymphoma rather than on tumors grouped by stage of disease or cell lineage.

To assist veterinary pathologists to provide this level of diagnostic specificity, an international study is underway to determine the accuracy and reproducibility of veterinary pathologists in applying a revised system of lymphoma classification. A pilot study of 50 cases, representative of the spectrum of lymphoma subtypes and selected from those intended for the major diagnostic trial, was first conducted to evaluate the accuracy and reproducibility of diagnosis of canine lymphomas by veterinary pathologists using the revised WHO classification system.  In this pilot study there was a high mean accuracy (>90%) in the application of the revised WHO human lymphoma classification scheme for the diagnosis of canine lymphoma.   Since pathology consultations frequently require the production of further microscopic slides to be sent to additional reviewers for interpretation, an added comparison of diagnostic accuracy of lymphoma by routine examination of histological preparations with that of scanned images of the same cases has been initiated.  


Detection of Salmonella in retail raw meat at grocery stores in Champaign County, Illinois
Jared Cohen, Yvette Johnson, Maung Myint, Lee Ann Lyons,
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

The United States Department of Agriculture assesses pathogen presence in raw meat samples at processing plants, but there is a lack of testing meat after further handling at retail outlets.  Sampling was conducted to estimate the prevalence of Salmonella in raw beef, chicken, turkey, pork, fish, and shellfish at retail stores in Champaign County, Illinois.  This estimation provides information for the evaluation of hazardous control points to manage pathogen related food-borne disease in raw meat at retail grocery outlets.  A total of 240 samples between beef, chicken, turkey, pork, fish and shellfish samples were randomly collected from fourteen retail grocery stores in Champaign County, Illinois from May through July 2006.  Store location, brand of meat, packaging location, and ground or non-ground meat status was evaluated for contamination risk.  The samples were tested for Salmonella by culture method.  The overall presence of Salmonella in raw meat samples from retail stores in Champaign County, Illinois was 6.3%.  Of the poultry (chicken and turkey) samples, 22.2% tested positive for Salmonella presence, 71.4% of which were ground samples.  Beef tested positive for Salmonella in 1.6% of the samples.  Salmonella was not isolated from pork, fish, or shellfish samples.  Ground meat products were 5 times more likely than non-ground meat products to have Salmonella contamination with a (95% C.I. 0.0583 - 0.6139).  Non-store brand meat products were 28 times more likely to have Salmonella contamination than store brand meat (95% C.I. 6.1572 - 130.2715).    Stores outside of Champaign-Urbana were 4 times more likely to have Salmonella positive meat samples than Champaign-Urbana stores (95% C.I. 1.2506 – 13.1118).  According to personnel from the Champaign County Public Health District responsible for the inspection o retail grocery stores in Champaign County, this increase in risk may be due to historical differences in the frequency of inspection at stores outside of the Champaign-Urbana city limits. 


Risk factors for ovarian adenocarcinomas in three populations of chickens
Aya Iwai1, Yvette Johnson1, Laura Kohrt1, Maung San Myint1, Janice Bahr2
1 College of Veterinary Medicine, University of Illinois at Urbana-Champaign
2 Department of Animal Sciences, University of Illinois at Urbana-Champaign

Several studies have reported a high prevalence of ovarian tumors in chickens.  Limited work has been done however, to identify risk factors for ovarian cancer in hens.  Previous studies have failed to identify ovarian tumors in SPF chickens reared in controlled laboratory facilities.   The objective of this study is to identify risk factors for ovarian tumors in three populations of chickens:  commercial layers from the UIUC poultry flock that are 165 weeks of age; SPF chickens reared in laboratory conditions that are 165 weeks of age; and aged hens obtained from live bird markets in the Chicago-area.  A total of 30 birds from each population were used for the study.  Data from SPF birds was obtained from previously conducted research.  Thirty UIUC flock birds that were scheduled for culling from the flock were selected for sampling.  A convenience sample of 30 live bird market hens were selected for sampling.  Blood samples were collected and the birds were euthanized and necropsied.  Serology, bacteriology, and histopathology were conducted on all the birds.  The hypotheses were:  1) Vaccinated birds are at increased risk for ovarian tumors; 2) Auto-immune disease (manifested by thyroiditis and nephritis) is significantly associated with ovarian tumors.  Results:  Ovarian tumors were identified in a significantly greater proportion of the UIUC poultry flock birds than in the SPF birds and the live bird market birds.  The results of the serology and bacteriology are still pending.  These preliminary results indicate that ovarian cancer is not evenly distributed across different populations of hens.  There may be genetic, infectious, or management factors that are placing some populations of birds at increased risk for ovarian cancer.  Because previous research has determined that chickens are a good model for human ovarian adenocarcinoma, these results may translate into additional early diagnostic tools, treatment, and prevention options for women.


Inhibition of microneme secretion in cryptosporidium parvum by unsaturated long chain fatty acids
Shannon Karbs*, Theresa Kuhlenschmidt, Joann Schmidt, and Mark Kuhlenschmidt
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Cryptosporidium parvum is a protozoal parasite which infects and causes disease in young calves and many other vertebrate hosts, including humans.  At this time, there is no effective treatment for cryptosporidiosis.  Considerable work has been done recently on the mechanisms of the attachment and invasion of the cryptosporidial sporozoite to the host enterocyte.  Sporozoites attach to host cells and secretory organelles called micronemes begin releasing proteins thought to aid in invasion.  Previous work conducted in this lab has discovered that certain long chain unsaturated fatty acids inhibit the attachment and invasion of the sporozoite possibly by blocking microneme secretion.  A certain fatty acid, linolenic acid, appears to have a very strong inhibitory effect on the secretion of at least one microneme protein, GP900.  By using this fatty acid and newly excysted sporozoites, we examined the effects of fatty acid concentration, exposure time and temperature on GP900 secretion using a western blot technique.  We hope to show that higher concentrations of fatty acid at a determined temperature will effectively inhibit microneme secretion.  This may help further our understanding of the mechanism involved in fatty acid-mediated inhibition.


Antibiotic resistance in humans, primates, and livestock in Kibale National Park, Uganda
Mary H. Lee, Elizabeth E. Estoff, Emily Wheeler, Thomas R. Gillespie, and Tony L. Goldberg
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Many infectious zoonotic agents are transmissible between non-human primates and humans.  However, relatively little is known about prevalence, incidence, or risk of transmission of zoonotic diseases in primates, or about how anthropogenic changes to primate habitats affect rates and patterns of disease transmission.  We surveyed humans, primates, and livestock living in and around the forests of Kibale National Park in Uganda for the presence of antibiotic resistance in Escherichia coli and Salmonella sp. bacteria.  We recovered 624 E. coli isolates from humans, non-human primates, and domestic animals, between June 2004 and June 2006.  We found resistance to at least one antibiotic in 67.4% of isolates (84.7% of individuals) from humans.  We found resistance to multiple antibiotics in 25% of isolates (25% of individuals) from livestock, 6.7% of isolates (10% of individuals) from red-tailed monkeys (Cercopithecus ascanius), 2.5% of isolates (3.2% of individuals) from chimpanzees (Pan troglodytes schweinfurthii), 1.3% of isolates (2.3% of individuals) from black and white colobus (Colobus guereza), and 0% of isolates (0% of individuals) from red colobus (Pilocolobus tephrosceles).   The red-tailed monkeys and black and white colobus with multi-resistant E. coli all lived in forest fragments associated with nearby human villages, whereas none of the monkeys living in undisturbed forest harbored resistant E. coli.  This suggests that close association with humans may be the source of antibiotic resistant pathogens in these primates.  We recovered a single Salmonella isolate from a human living near the park.  This Salmonella isolate was resistant to ampicillin, chloramphenicol, doxycycline, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline.  This antibiotic resistance pattern was found at 2.8% prevalence in human E. coli isolates collected from the Kibale region, indicating there may have been an exchange of genes encoding multi-resistance between bacterial species.  We conclude that high contact rates between humans and non-human primates enhance the transmission of multi-antibiotic resistant bacteria or resistance-conferring genes from humans to wild primates.  We are currently conducting studies to determine the genetic basis of antibiotic resistance in humans, primates and livestock of Kibale, as well as the impact of the spread of antibiotic resistant pathogens on the health status of humans, wildlife, and domestic animals in the region.


The effects of bacterial TLR ligands on TLR expression of articular cartilage explants and chondrocyte aggregates
Anne Love*, Evelyn Caporali, Trina Kuykendall, Matthew Stewart
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Toll-like receptors (TLRs) recognize invading microorganisms and express inflammatory cytokines, interleukins, chemokines, and other effectors that stimulate the host immune response.  To understand the role of TLR4 and TLR2 in bacterial septic arthritis the effects of several bacterial ligands on extracellular matrix turnover of articular chondrocytes were explored.  TLR4’s main ligand is lipopolysaccharide (LPS) and TLR2 recognizes lipoteichoic acid (LTA), peptidoglycan (PGN), and heat killed Staphylococcus aureus (HKSA). Equine articular cartilage explants and chondrocyte aggregates were treated with these bacterial ligands and the expression of the TLRs was determined in several different ways.  Glycosaminoglycans (GAG) are released in response to the activated aggrecanolysis, thus DMMB assays were used to determine the GAG content of the cultures.  Real-time PCR was used to explore gene expression for aggrecanases (MMPs and ADAMTS), interleukins (IL-1, IL-6), and cytokines (TNFα).  Aggrecan western blots assessed aggrecan degradation, a result of TLR activation.  Inhibition of the TLR, IL-1, and TNFα signaling pathway will be explored to determine the ability to suppress aggrecanolysis and GAG release in response to TLR ligands.  This will help determine if these inhibitory agents could protect articular cartilage against matrix degradation in septic arthritis.


SodA as a virulence factor in Streptococcus equi and Streptococcus zooepidemicus infections
Lisa Lukas, Carol Maddox
College of Veterinary Medicine, University of Illinois Urbana-Champaign

Streptococcus equi is the etiologic agent of strangles in horses while Streptococcus zooepidemicus is an opportunistic pathogen that can cause disease in many mammals, including humans. The ability to survive in phagocytic immune cells is an important virulence factor of Lancefield group C Streptococcus. One gene that has been recognized as contributing to macrophage survival by the Streptococcacea Family is sodA. The sodA gene encodes superoxide dismutase, which catalyzes the dismutation of superoxide anion radical to molecular oxygen and hydrogen peroxides. sodA is found ubiquitously in oxygen metabolizing organisms and is thought to protect cells from the cytotoxic superoxide anions. We hypothesize that Sod may contribute to virulence by playing an important role in the survival in the phagocytic cells of the host. To test this hypothesis, sequences of sodA amplicons of several clinical isolates of S. equi were screened for mismatches using S1 nuclease and found to have no differences in their respective sodA genes. The next step was to introduce several base pair mutations into sodA. The effects of the mutation on intracellular survival will then quantified using a J774 murine macrophage cell line assay. Zebrafish will then serve as models to compare mutants for changes in streptococcal infection pathology. The results of this study will be used to construct more effective vaccines to Streptococcus.


Prevalence of Escherichia coli in retail beef, chicken, pork, turkey, fish, and shellfish from Champaign County, Illinois
Lee Ann Lyons, Yvette Johnson, Maung Myint, Jared Cohen
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Escherichia coli is one of the most common causes of food borne disease in the United States.  The CDC has estimated that food borne diseases cause around 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year.    The objectives of this study were to estimate the prevalence of E. coli of raw retail meats (beef, chicken, turkey, pork, fish, and shellfish) within the Champaign County, IL area, and to evaluate the risk factors for increased contamination in retail meat products.  A total of 240 raw retail meat samples were collected from 14 random stores in Champaign County from May through July of 2006.  Store location, condition of sample (ground vs. non-ground), brand of sample, and type of packaging (pre- packaging vs. store packaging) data was collected for risk analysis.  These samples were then tested for E. coli by standard culturing methods.  Overall prevalence of E. coli is 30% with a prevalence of 36.7% in beef, 41.2% in chicken, 33.3% in pork, 48.3% in turkey, 2.9% in fish, and 4.3% in shellfish.  There was no statistically significant difference of E. coli prevalence between samples from Champaign-Urbana and samples from areas in the county outside Champaign- Urbana.  Ground meat products were three times more likely to be contaminated with E. coli when compared to non-ground meat products (95% CI= 1.76 to 5.54).  Non-store brand products were 2.5 times than store brand products (95% CI= 1.31 to 4.57) and pre-package products were 4 times than store packaged products (95% CI= 1.78 to 7.78) to be contaminated with E. coli.  Based on these results it can be inferred that fish and shellfish have a lower prevalence of E. coli contamination when compared to beef, chicken, pork, and turkey.  It can also be inferred that ground, non-store brand, and pre-packaged meats have a higher risk associated with E. coli contamination compared to non-ground, store brand, and store packaged meats.  Determining the prevalence of E. coli in meats at the retail level can be a better indicator of the public health risk associated with meat consumption when compared to the prevalence found at the processing plant. 


Seroprevalence of monkeypox virus in small mammals in Western Uganda
Abigail Mathewson1, Kevin Karem2, Zachary Braden2, Inger Damon2, Joanna Shisler3, Innocent Rwego2, Thomas Gillespie1
1College of Veterinary Medicine, University of Illinois at Urbana-Champaign
2Centers for Disease Control Poxvirus Group
3Department of Microbiology, University of Illinois at Urbana-Champaign

In this study we were interested in the seroprevalence of the Monkeypox virus in extreme western Uganda along its border with The Democratic Republic of Congo (DRC).  The Congo Basin countries and West Africa have had endemic cases of human Monkeypox while Uganda has not.


Our epidemiological study of leptospirosis in Champaign County canines
Jacqueline Scapa, Marilyn Ruiz, NamJung Jung, and Carol Maddox
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Leptospirosis is a bacterial disease caused by any one of shypothesis was that the virus is not currently present in Uganda for there to be transmission to humans and that the lack of cases does not reflect the cultural differences between the people of The DRC and Uganda, but rather geographical ones. To find the seroprevalence, we trapped small mammals in villages and National Parks in western Uganda, collected serum samples and analyzed them with ELISA assays at the Centers for Disease Control and Prevention (CDC) in Atlanta.  One of the samples collected has been shown to have been exposed to an orthopox virus in preliminary laboratory tests.

Several samples tested showed slight positive results but nothing significant above the baseline, except for Sample 15 from a Graphiurus murinas specimen collected at the Kanyawara field station in Kibale National Park.  This is not at what we had expected to see.  By our line of thinking, if we were to find any evidence of Monkeypox or any other Orthopox virus, it should have been in the sites trapped that shared the border with The DRC, not on the eastern side of the Rwenzori Mountains but on the western side.  This sample initially did not seem impressive, but the excessive reaction that it had to the cellular lysate used in the assays warranted further investigation using purified virus in order to completely eliminate any possibility of clouding results by background noise within the ELISA test.  With the last assay, there was undeniable evidence that this animal had had previous exposure to either Monkeypox or another Orthopox virus.


Optimization of terminal restriction fragment length polymorphism (T-RFLP) for assessment of vaginal ecosystem diversity
Corrin McCann*, Noriko Nakamura, Mengfei Ho, Angel Rivera, Claudia Reich, H. Rex Gaskins, Lois L. Hoyer, Steven Blanke, James M. Slauch, Gary J. Olsen, Brenda A. Wilson
Institute for Genomic Biology, Host-Microbe System, University of Illinois at Urbana-Champaign

Multiple genera and species of microbial populations colonize areas of the body such as the vagina. Little is currently known about the diversity of vaginal ecosystems and changes in populations of microbiota in disease states. Our research hopes to gain information as to the normal microbiota of the vagina and what role it plays in vaginal health and disease.The objective of this study is to optimize the conditions for using the analysis method of terminal restriction fragment length polymorphisms (T-RFLP) for the assessment of microbial community profiles based  on the 16S rRNA genes. To accomplish this we examined primer sets (27fbac & 1492r, 27f & 926r), DNA template concentration for PCR amplification, purified PCR product DNA concentration, restriction enzymes (Hae III, Hha I, Msp I), and restriction digestion conditions for optimal resolution of bacteria. Seven pure cultures were used to optimize the protocol conditions (Clostridium bifermentans, Clostridium paraputrificum, Enterococcus faecium, Lactobacillus jensenii, Gardnerella vaginalis, Pseudomonas aeruginosa and Staphylococcus aureus) as well as a mixture of these pure cultures. Human vaginal samples were also used to help calibrate DNA template concentration for PCR since human DNA is more likely to contain things like PCR inhibitors then bacterial isolates.

Once optimization of the protocol for T-RFLP analysis is complete we hope to use it to examine the diversity of bacteria in the vagina of human non-human primates. With a more complete understanding of the role that normal microbial populations play in vaginal health we hope to identify factors that may predispose certain females to acquiring vaginal infections or other STDs.


Enhancing the quality and reliability of diagnostic immunocytochemistry
Elisabeth Peters*, Victor E. Valli
College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Diagnostic immunocytochemistry is frequently conducted on routine submissions from schools of veterinary medicine as well as local practitioners.  Cytology is practical because it offers valuable management information for many types of lesions at very reasonable cost, but it requires skilled diagnostic interpretation.  A recurring problem with this service is that freshly made blood and cytological smears with heavy cellular spreads tend to detach from the slides in the staining process.  For our study, tissues were collected from animals that were electively euthanized.  The tissues were selected from sites where inflammatory, hyperplastic, and neoplastic lesions commonly occur such as the lymph nodes, spleen, bone marrow, skin, thyroid, liver, pancreas, and adrenal gland.  Dispersed cells were collected into a transport media and cytologic preparations of these tissues were used in the study along with specimens submitted to the veterinary teaching hospital.  Two methods of fixation were used prior to staining.  These include drying the slide for a day followed by immersion in acetone for 5 minutes, or immediate fixation in 10% formalin for 30 seconds.  Formalin fixation gives better cell preservation and adhesion, but prevents the diagnostic antibodies form penetrating the cell membranes and requires an antigen retrieval process.  This retrieval is achieved by boiling the slide preparations for 5 minutes in a pressurized chamber containing citrate buffer at pH6.  We have established base protocols for CD3, CD20, CD79, cytokeratin, and vimentin.  Other antibodies in advanced development include CD4, CD8a, CD8b, CD172, FIP, FeLV, thyroglobulin, myeloperoxidase, chromogranin, and synaptophysin.  The results of this study will permit those using immunocytochemistry to access a wider array of reagents and expand their capabilities for making a specific diagnosis.

An epidixteen species and 2,000 serovars of the spirochete Leptospira.  This study is designed to examine the correlation between cases of Leptospirosis in the canine population of Champaign County and its presence ponds or other bodies of water near to those cases.  Suburban retention ponds in the housing developments of Champaign County, for example, may serve as exposure sites for pets and pet owners who frequent the area on walks or during other recreational activities.  A highly detailed digital map of water bodies and GIS software  permitted the selection of water sites based on their proximity to cases of canine Leptospirosis.  Real-time Polymerase Chain Reaction was used to determine the presence of Leptospira in every water sample.  Given the water sampled thus far, there is evidence that the ponds, contaminated by wildlife with endemic Leptospira infections, may serve as the reservoirs responsible for the infection in canines.  Investigation is currently underway to determine if these ponds contain pathogenic Leptospira serovars.


Characterization of nanotube toxicity in primary and established cell lines
Edwina D. Witkowski1, Sharon H. Meachum1, Michael S. Strano2, Thomas E. Eurell1
1 Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign
2 Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign

Nanotechnology is yielding new classes of materials that can provide innovative engineering solutions to traditionally difficult questions, especially in the area of medical research.  Recently, however, there has been an increase of concern over the safety of nanotubes. Studies have shown that nanotubes have the ability to accumulate inside cells at very high concentrations to the point where they can become toxic.  The goal of my study is to characterize the morphologic effects of nanotubes on two cell types: rabbit corneal epithelial cells (RCE; primary culture) and 3T3 mouse fibroblasts (3T3; established line) and to compare their response using an in vitro cytotoxicity assay.  A standardized technique was established for the culture of each cell type and for nanotube delivery.  At 21 hours after exposure, nanotube induced cell toxicity was assessed by a fluorescence-based live/dead assay.  These experiments suggest differences in the interactions of carbon nanotubes between primary and established cell lines and caution when interpreting fluorescent-based live/dead assay results.


2005 STUDENT PROJECTS

Investigation for Biomarkers in Fish Infected with Atypical Mycobacteria
Ainsworth, Ryan J.; Eurell, Thomas E.; Van Bonn, Bill
Center for Zoonoses Research and Department of Veterinary Biosciences, University of Illinois, Urbana, IL

The purpose of this study was to search for diagnostic biomarkers in fish infected with bacteria from the genus Mycobacterium. All fish were obtained from the Shedd Aquarium in Chicago, Illinois and selected by the Shedd personnel as part of their routine health maintenance program. Euthanasia and necropsy of all fish used in this study was conducted by Shedd personnel following guidelines approved by the American Veterinary Medical Association. Two of the nine fish evaluated in this study were found to be positive for Mycobacteria using PCR amplification of a portion of the 16s rRNA gene isolated from a liver and spleen digestion. The Ziehl-Neelsen (Z-N) acid fast stain revealed Mycobacteria in spleen of one of the PCR –positive fish (a T-bar Convict Cichlid; Cichlasoman sajica) but not in any tissue samples from the other PCR-positive fish (a black spot barb; Barbus filamentosus). Proteomic analysis of blood plasma and selected tissue samples revealed reproducible, species-specific protein profiles from all fish evaluated in this study; however, a definitive biomarker for mycobacterium was not determined. As a first step to a refined approach of this study we will plan to use Laser capture microscopy in an attempt to identify the proteomic profile of a laser-captured section of normal liver from a PCR-positive fish compared to the proteomic profile of the isolated granuloma containing acid fast bacteria. This should allow a better understanding of what the bacteria/granuloma contribute to the overall proteomic profile of the liver from PCR-positive fish and a new insight into potential biomarkers of mycobacterial infections.


Immuno Expression of Potential Cell Cycle Regulatory Factors in the Developing Pig Testis
Wanda Averhart, Julia Baldrighi*, Tameka Phillips*, Kay Carnes*, Rex Hess*, Sherrie Clark#
Center for Zoonoses Research, *Department of Veterinary Biosciences, #Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Today’s pork production is dependent on reproductive efficiency. Improvements in the production capability of the animals (number of oocytes ovulated or sperm produced) involved is invaluable.  Researchers have examined methods to improve oocyte production, but have not focused on the concentration of sperm from a single boar used for artificial insemination (AI).  Artificial Insemination allows numerous females to be bred to a single boar, making the total number of sperm per ejaculation the main factor in AI efficiency.  An increase in the number of Sertoli cells, leads to an increase in the number of sperm produced.  Therefore, by understanding the factors that control the growth and differentiation of Sertoli cells, the amount of sperm per ejaculation in a boar can be increased.

The purpose of this research is to begin a careful, systematic analysis of cell cycle regulators expressed in the Sertoli cell during testicular development of the pig.  By using pigs of different ages, we will establish a baseline of what regulatory factors are present at different time points in the developing Sertoli cell.  We will test the hypothesis that alterations in the expression of different factors regulating the cell cycle of Sertoli cells, will lead to the growth or reduction of cells and that the concentration of these factors during periods of growth is the main control of Sertoli cell proliferation.

Immunohistochemistry was performed using the following antibodies to examine the factors controlling Sertoli cell proliferation: GATA-4 (transcription factor specific for developing and adult pig Sertoli cells), Ki67 (a nuclear protein present in all phases of the cell cycle, except G0), Cyclin-dependent kinase inhibitor p27(Kip1), Steroid receptors AR (androgen receptor), LH2 (estrogen receptor).  Immunostaining using p27(Kip1) revealed no positive staining in any of the days tested as there is cell division during all of these time points.  Protein expression for Ki67 stained mildly after day 25 suggesting that Sertoli cells became more active at this stage of development.  The AR weakly stained and GATA-4 stained intensely at all time points.  The data for LH2 was inconclusive and needs to be performed again. 


Localizing the activity domain of Pasteurella multocida toxin
Miranda Bertram#, Leila Aminova*, Brenda A. Wilson*
Center for Zoonoses Research, *Department of Microbiology, University of Illinois, Urbana, IL #College of Veterinary Medicine, Kansas State University, Manhattan, KS

Pasteurella multocida is the causative agent of several respiratory diseases in mammals, including atrophic rhinitis in swine, as well as dermonecrosis and bacteremia in humans exposed to infected animals.  The bacterium produces a toxin, P. multocida toxin (PMT), which is a potent mitogen for various cell types.  PMT is a 146-kDa, 1285-amino acid protein that shows little similarity to other proteins.  It is known to act through the Gq family of G proteins, however there is some debate about the location of the functional domains of the toxin.  One study showed the activity domain in the N terminus, but more recent data puts the activity domain in the C terminus.  To test the hypothesis that the activity domain is in the C terminus, I cloned five vectors that were truncations of the C terminus or of the whole toxin and one vector that coded for only the N terminus.  All constructs included green fluorescent protein (GFP) in the N terminus of the vector.  I tested the activity of these vectors in 293T cells by transfection and dual luciferase assay using serum response element (SRE) as the promoter for the luciferase gene.  None of my truncations showed luciferase activity significantly above the negative control, but the vectors coding for the whole toxin and for the entire C terminus did show significant activity.  I used a fluorescent microscope to visualize the localization of the toxin or toxin part in the cells.  The whole toxin and the C terminal truncations were localized to the cytosol, while the end 302bp and the N terminus vectors did not show any localization within the cell.  The C terminus of the toxin is sufficient to cause localization and SRE activation associated with the toxin, and the beginning of the C terminus to residue 983 is sufficient to cause localization, indicating that the activity domain is in the C terminus.  However, the end 302bp of PMT does not cause localization or SRE activation, nor does the beginning of the C terminus cause SRE activation.  It appears that both ends of the C terminus are necessary to produce cellular effects, at least those mediated by signaling pathways that activate SRE.


Spatial Clustering of 2002 Equine West Nile Virus Cases in East-central Illinois
Katherine Brix-Rutherford and Dr. Marilyn Ruiz
Center for Zoonoses Research and Department of Pathobiology, University of Illinois, Urbana, IL

The current understanding of factors affecting West Nile virus (WNV) spatial clustering of equine cases is insufficient to predict areas of high disease rates. Further knowledge of these factors could decrease the cost of WNV to the equine industry by allowing maximized protection and control measures focused on specific risk factors in specific locations.  This study was conducted to explore equine case spatial clustering and related factors in east-central Illinois in 2002 and to determine horse owners’ attitudes towards WNV risk, vaccination, and vector abatement in the same area.  Individual level equine case data in the counties of Champaign, Piatt, Moultrie, Douglas, Shelby, and Coles were analyzed for space and time relationships and associations with land cover data, human case data, and Amish residence locations using ClusterSeer and SatScan.  A paper and online survey of horse owners’ opinions and husbandry practices covering 2002 to 2005 was conducted in July 2005.

Significant spatial clustering (P<0.05) of WNV equine cases was detected on the Moultrie-Douglas border (a region that includes an Amish settlement) and in southeastern Shelby County.  One significant space-time cluster was identified but was not associated with either of the significant spatial clusters.  Visual assessment of land cover data in the Amish settlement region revealed a possible negative relationship between case location and rural grassland areas (pasture).  No direct relationships were able to be confirmed with human case data.  Horse owner perception of WNV risk peaked in 2003, the year following the highest year of equine cases in Illinois, and a level of unawareness of local equine WNV incidence in 2002 was noted.  The factors affecting spatial clustering of equine WNV cases in east-central Illinois are still unclear, but the potential negative association with percentage of land in pasture is perhaps a reflection of the habitat requirements of birds and mosquitoes necessary for virus amplification and transmission and equine exposure to infected mosquitoes.  The disease incidence may also have been influenced by low or delayed WNV awareness of horse owners in the area.  Further statistical analyses are required to confirm these relationships.


Questing behavior and ambush height of the lone star tick, Amblyomma americanum, and the American dog tick, Dermacentor variabilis, in relation to diurnal environmental variation
Dan Cartwright
Center for Zoonoses Research and Atlantic Veterinary College, University of Prince Edward Island, Canada

The questing behavior of the lone star tick, Amblyomma americanum, and the American dog tick, Dermacentor variabilis, were investigated.  Air temperature and relative humidity were measured concurrently with observations of lone star tick and American dog tick questing heights and activity on wooden dowels and boxes situated in Wolf Creek State Park, Illinois.  Dowels and boxes of varying heights were used to simulate natural surfaces used by questing ticks.  Measurements and observations were conducted 4 times daily, for a total of 11 days in June and July, 2005.  Dragging for ticks in the surrounding area was conducted once a day, on each day of the study, to sample the available tick populations.  Statistical analysis demonstrated that there were no significant relationships between either temperature or humidity and the questing height of either tick species at any life stage.  Also, no correlation between tick abundance and box/dowel height was observed. On average, both adults and nymphal ticks climbed to the top of their respective dowel or box, regardless of dowel or box height.  Significantly more adult ticks quested on boxes compared to nymphs though compared to adults, significantly more nymphs were captured on drag.  Also, significantly fewer adult dog ticks were captured on drags compared to the number observed on boxes.  These results indicate that a greater proportion of the adult tick population was using the box surfaces for questing, in comparison to the proportion of the nymphal tick population that was using these surfaces.  Also, these results suggest that by sampling brush and tree trunks which represent a different layer or stratum for ticks to quest, a different subset of the lone star tick and American dog tick populations may be collected, compared to dragging.  Within our study parameters, these results suggest that due to the toughness and resistance to desiccation of adult A. americanum, and D. variabilis, temperature and humidity are not good indicators of questing activity.


Pathophysiology of chytridiomycosis in amphibians
Mary H. Lee, Gary Iwamoto, and Val. R. Beasley
Center for Zoonoses Research and Department of Veterinary Biosciences, University of Illinois, Urbana, IL

One of the major contributors to global amphibian declines and extinctions is the emerging infectious disease, chytridiomycosis.  Chytridiomycosis is caused by the fungus Batrachochytrium dendrobatidis.  It causes very high mortality (90-100%) in some species of amphibians, and is responsible for catastrophic die-offs of entire frog communities and extinction of species.  Over 94 species of amphibians from 15 families from Australia, New Zealand, South America, North America, Central America, Europe, and Africa have been found infected with B. dendrobatidis.

Chytrid fungi infect the keratinized skin of amphibians, resulting in thickening, erosion, or sloughing of the skin.  Infected individuals typically die within 2-3 days after the onset of clinical signs.  The mechanisms by which chytridiomycosis becomes fatal to frogs is unknown. The thin, well-vascularized skin of frogs is critically important as a respiratory organ and a direct pathway for taking up water to maintain hydration. 

We hypothesize the epidermal changes caused by chytridiomycosis seriously impair water, electrolyte, pH, and blood gas balance in infected amphibians.  We infected four species of frogs and toads with Batrachochytrium dendrobatidis, andmeasured O2 consumption in infected and non-infected individuals.  We attempted to measure blood gasses, pH, and electrolytes, and we will measure complete blood counts (CBC) and total proteins (TP) from infected individuals and compare to normal values.  One group showed increased oxygen consumption after infection.  The infected individuals have not developed signs of the disease and we are continuing to monitor the animals.  We predict a decline in oxygen consumption as the disease progresses to a more serious stage.  Knowing the mechanisms of this disease will give insight on why certain species are highly susceptible, how to treat the disease, and how to prevent the spread to other vulnerable populations.


Plasmodium gallinaceum occysts and sporozoites compared in vitro and in vivo
Catherine Wenkel, April Paulman, Milton McAllister
Center for Zoonoses Research and Department of Pathobiology, University of Illinois, Urbana, IL

The McAllister laboratory at the University of Illinois, College of Veterinary Medicine, is working on a modified live vaccine for Plasmodium falciprium malaria in humans. The model is avian malaria in chickens caused by Plasmodium gallinaceum, using the mosquito vector Aedes aegypti. The lab is growing in vitro oocysts containing Plasmodium sporozoites that will be used to induce protective immunity.

The research presented investigates the development of the in vitro oocysts and sporozoites compared with the in vivo oocysts and sporozoites in the Aedes aegypti mosquito. While conclusive electron microscopy results are pending, the initial results indicate that the development of in vitro oocysts is stalling 2-3 days before complete  maturation. Whether or not they contain infective sporozoites is an important question for further investigation.


Morphologic, Phenotypic, and Quantitative Cellular Characterization of Feline Small Intestinal Immune Cells
Cara E. Williams*, M. Elena Gorman**, Victor E. Valli***
Center for Zoonoses Research, University of Illinois, Urbana, IL
*Junior in Animal Sciences - PreVetMed U of Illinois     **DVM, MS – candidate, Resident, Clinical Pathology Department of Pathobiology CVM U of Illinois     ***DVM, PhD, Professor of Pathobiology CVM U of Illinois

Inflammatory Bowel Disease (IBD) of the domestic cat is a commonly encountered disease that is not well understood. Treatments are often ineffective, and there is little knowledge of the range of normal intestinal composition against which IBD can be measured.  The goal of this project is to determine the range of reactive inflammatory cells present in the small intestine of cats that are outwardly normal. B-cell reactions in the intestinal lamina propria compartment (LPC) and T-cell reactions in the intra-epithelial compartment (IEC) were analyzed by histological, cytological, and flow cytometric studies. Analyses were carried out on the entire small intestinal tract of 11 euthanized cats derived from a local shelter.  Results reveal that the levels and phenotypes of the lymphocytes of conventionally-reared cats vary widely and have more variability than those of laboratory-reared cats.  Overall, essentially all the lymphocytes in the IEC are T-cells, the majority of which are of CD8 subtype.  The LPC, however, contains a mixture of B and T-cells with ratios ranging from 1:8 to 1:37 B to T.


The Role of Toll-like Receptor Signaling in Septic Arthritis
Angela C. Yates and Matthew C. Stewart
Center for Zoonoses Research and Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL

Septic arthritis in equines is potentially lethal if left untreated.  The term “septic arthritis” refers to the condition where bacteria enter the synovial space in a joint and cause inflammation, (1,2).  This condition is common in neonates but does occur in adult equines.  In foals, septic arthritis usually is a result of impaired passive immunity resulting from a partial or complete failure of the transfer of immunoglobulins.  Therefore, umbilical infection, pneumonia, or diarrhea can result in joint sepsis (3, 4, 5, 6). In adults, septic arthritis is typically caused by trauma to the joint; either accidental, or as a consequence of arthrocentesis or intra-articular surgery (1, 3, 7, 8, 9).  

The innate immune system is the primary means to fight sepsis and includes the Toll-like receptors, a family of mammalian homologues to the Drosophila gene Toll.  Their role in the pathogenesis of sepsis is not completely understood, but Toll-like receptor signaling induces several genes involved in infection, such as proinflammatory cytokines and chemokines (10).  Here, we explore the role of Toll-like receptor 2 and Toll-like receptor 4 in equine neonatal sepsis.  Normal cartilage was compared against septic cartilage in two foals using quantitative real-time PCR. While both toll-like receptor 2 and 4 are constitutively expressed in normal cartilage, there is a significant upregulation in septic cartilage.  We next exposed both equine and human normal chondrocytes to a variety of bacterial agents such as lipopolysaccaride (LPS) and peptidoglycan, and then measured the glycosaminoglycan content, as well as Toll-like receptor expression of the chondrocytes.  We were able to induce an upregulation in the mRNA of the Toll-like receptors successfully using this in vitro model.

While we have demonstrated that exposure to LPS and peptidoglycan causes a significant change in the cartilage, more experiments must be performed to determine the relative importance of Toll-like receptors in the inflammatory process of septic arthritis.

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  2. Stover SM.  Infectious arthritis and tenosynovitis in the horse.  In:  Dyke, TM (ed.), Proceedings of the 12th Annual Meeting of the Australian Equine Veterinary Association.  Current Issues in Equine Practice, (1990) 167-173.
  3. Martens RJ and Auer JA.  Hematogenous septic arthritis and osteomyelitis in the foal.  Proc. Am. Assoc. Equine Pract. (1980) 26, 47-63.
  4. Morris PG.  The clinical management of septic arthritis in the horse.  Comp. Cont. Educ. Pract. Vet. Suppl. (1980) 2, 207-219.
  5. Firth EC. Infectious arthritis in foals.  In:  White NA and Moore JN (eds.), Current Practice of Equine Surgery (1990), 577-580.  W.B. Saunders, Philadelphia.
  6. Stoneham SJ.  Septic arthritis in the foal:  practical considerations on diagnosis and treatment.  Equine Vet Ed. (1997) 9, 25-29.
  7. Gustafson SB, McIlwraith CW, and Jones RL.  Comparison of the effect of polysulfated glycosaminoglycan, corticosteroids, and sodium hyaluronate in the potentiation of a subinfective dose of Staphylococcus aureus in the midcarpal joint of horses. Am. J. Vet. Res. (1989) 50, 2014-2018.
  8. Lapointe, JM, Laverty S, and Lavoie LP.  Septic arthritis in 15 standardbred racehorses after intra-articular injection.  Equine Vet. J. (1992) 24, 430-436.
  9. Brusie RW, Sullins KE, White NA, Coffin PCII, Parker PA, Anver MR, and Rosenberger JL.  Evaluation of sodium hyaluronate therapy in induced septic arthritis in the horse. Equine Vet. J. supplement (1992) 11, 18-23.
  10. Roy M-F.  Sepsis in adults and foals.  Vet Clin Equine (2004); 20:  41-61

2004 STUDENT PROJECTS

Influence of season and temperature on questing of immature Ixodes scapularis
Anthony Cappa, Uriel Kitron, and Roberto Cortinas
Center for Zoonoses Research and Department of Veterinary Pathobiology, University of Illinois College of Veterinary Medicine

The influence of season and temperature on questing of immature Ixodes scapularis was examined at Natural Lands Conservation Area, a site in north central Illinois, from June-July 2004.  Seasonal variation in both larva and nymph questing numbers was observed.  Larva numbers displayed a bimodal trend with a smaller peak occurring in early June and a much larger peak occurring in late July to early August, whereas nymph questing numbers peaked in late May to early June and gradually decreased during the rest of the season.  Trends of increasing larval questing numbers in relation to increasing meteorological conditions, specifically surface temperature and surface to soil temperature gradients, were also observed during our study.  Due to the short length of the study, it is difficult to discern seasonal trends in tick numbers from effects due to weather conditions.  A study over several seasons may help differentiate these two effects.


Common pond invertebrates consume Ribeiroia ondatra cercariae
Kim Marie Labak and Anna Schotthoefer
Center for Zoonoses Research and Department of Veterinary Pathobiology, University of Illinois College of Veterinary Medicine.

Since the mid-1990s, amphibian populations have been declining at an unnatural rate. Coinciding with this decline is an increase in the frequency of frog limb deformities, with a 50% or higher deformity rate occurring in many populations. Although these deformities may be attributed to factors such as UV radiation and toxicity, lab research and field studies have pointed to infection of tadpoles by trematode cercariae, especially of the species Ribeiroia ondatrae, as their most likely cause.

This study explored the roles other pond organisms may play in helping or hindering cercariae infection of tadpoles. Several pond species were tested for cercariophagic activity; organisms were placed in wells of multi-well culture plates with discreet numbers of cercariae.  The numbers of swimming cercariae were recorded over time and compared with numbers in control wells that contained no predators. Of the species tested, hydra, damselfly larvae and copepods consumed significant amounts of cercariae in feeding trials. Hydra were also observed paralyzing cercariae on contact, whether or not they consumed the cercariae, greatly decreasing the numbers of swimming cercariae in the wells. Preference studies, which tested the cercariophagic activity of hydra and damselflies in the presence of other foods, indicated some change in the feeding efficiency of predators on cercariae.

These results indicate a potentially significant ecological relationship between common pond invertebrates, R. ondotrae cercariae, and other food organisms that these cercariophages may consume, such as protozoa, algae and micro-crustaceans. Changes in cercariophagic invertebrate populations, or the activity of these populations, in natural pond environments may affect the transmission potential of these parasites.  The results of this study suggest a need for further investigation into the ecological role of pond invertebrates on transmission of R. ondatrae to tadpoles and their effects on limb deformity rates in amphibian populations.  Moreover, these results suggest similar cercariophagic activity may effect the transmission of cercariae-borne infections in humans and livestock, such as schistosomiasis and fascioliasis.


Vectors for the antigenic determination of bordetella dermonecrotic toxin
Caroline Merrill and Brenda Wilson
Center for Zoonoses Research, University of Illinois College of Veterinary Medicine, Department of Microbiology, College of Life Sciences

Bordetella bronchiseptica, a gram-negative coccobaccilli, is an animal pathogen mainly effecting animals kept in close quarters under stressful conditions. Dermonecrotic toxin (DNT) is one of several virulence factors of B. bronchiseptica. Of particular interest is the contribution of DNT to the swine disease atrophic rhinitis, which results in the loss of tubular cancellous bone of the nasal turbinates and fibrotic lung lesions.

The goal of this ongoing study is to identify the antigenic determinants of DNT, using a novel epitope-mapping strategy. The results are to be used in the development of a diagnostic kit to provide rapid confirmation of DNT infection in suspected animals. A plasmid vector was designed and successfully constructed to express fragments of the DNT protein. DNT fragments were created through PCR and inserted into the plasmid vector. Constructs have been created that cover DNT amino acid residues 1-263 and 649-1464. These are currently being tested for correct protein expression. These constructs are to be screened with DNT antibody developed through an scFv phagemid library, to visualize where binding occurs within DNT. Constructs containing DNT epitopes (7-10 aa) will then be created. It will be possible to use these epitope constructs for in vitro protein translation in an E. coli cell-free extract. This can then be used as antigen in a diagnostic kit against the serum of infected animals to detect pathogen exposure.


Serum protein changes and colostral characteristics in dairy cows housed in different photoperiod conditions during the dry period
Stephanie Nelson and Dawn Morin
Center for Zoonoses Research and Department of Veterinary Clinical Medicine, University of Illinois College of Veterinary Medicine

Serum was collected from 81 multiparous Holstein cows at dry-off and at calving along with colostrum from the first milking to study influences on colostral IgG concentration.  Cows were housed in one of four photoperiod conditions: ambient day length, long day length (16 hours per day of light), or short day length (8 hours per day of light) for the entire dry period or for only the last 21 days.  Serum and colostrum were analyzed for IgG concentration and serum protein components were measured.  Volume of colostrum and the time interval from calving until milking were the two variables most highly correlated with a drop in colostral IgG concentration.  Non-IgG globulin, not IgG itself, was found to be the major component in the serum to decrease in concentration during the dry period.  No effects of photoperiod were observed. 


Seroprevalence survey and risk factor analysis of canine Lyme Borreliosis in West Central Illinois
Paula Roney (Mentors, Drs. Kitron and Cortinas)
Center for Zoonoses Research, University of Illinois College of Veterinary Medicine

A seroprevalence survey to detect the presence of Borrelia burgdorferi antibody in healthy canine pets was conducted within counties nearby the Illinois River in West Central Illinois.  317 serum samples were obtained by local veterinarians from 19 different clinics, along with a questionnaire for each dog which included information regarding signalment, medical history, vaccination status, home address, travel history, tick control product usage, tick exposure history, primary function and habitat of the dog.  All submitted serum samples were screened with a commercially available canine Borrelia IgG ELISA kit, and then selected ELISA-positive samples were confirmed by Western blot.  Residential addresses of the dogs were mapped using a geographic information system (GIS) and seroprevalence rates were determined by county.  Each variable from the questionnaire was analyzed individually to establish significance of association with seropositivity.  Seropositivity was positively associated with a history of tick exposure.  Canine seroprevalence studies are an effective tool for estimating the environmental levels of B. burgdorferi, which can help to determine the risk of Lyme disease to the human population in a given area.


Clinical and clinicopathological effects Of Cryptosporidial infection in dairy calves
Sarah E. Vos, Peter D. Constable, and Mark S. Kuhlenschmidt
Center for Zoonoses Research, Departments of Pathobiology and Veterinary Clinical Medicine, University of Illinois College of Veterinary Medicine Urbana, IL

The long-term objective of this study is to characterize the effect of Cryptosporidium parvum infection on clinical parameters in neonatal calves, including the systemic and fecal clinicopathologic values.  Before the impact of cryptosporidial infection on bovine diarrheal disease can be accurately assessed and appropriate treatment strategies developed, it is necessary to characterize the association between fecal oocyst numbers and clinical signs in calves with experimentally induced infections.  The results of the initial pilot study reported here focused on the effect of C. parvum on the following fecal clinicopathologic values measured throughout the time course of infection: oocyst shedding, fecal consistency, volume, mass, pH, fat, protein, carbohydrate, and abomasal emptying rate.  Oocyst shedding is found to follow a cyclic pattern, as seen with many parasitic infections.  Fecal consistency increases (becomes more diarrheic) early in the infection and returns to normal, as the intestine recovers.  Volume and mass of fecal material increases as fecal consistency increases, due to increased water content in the feces.  Fecal pH remains steady throughout infection.  Fecal fat decreases with infection, while fecal protein and carbohydrate concentrations follow a cyclic pattern similar in nature to oocyst shedding.  Abomasal emptying rates decrease with C. parvum infection but gradually return to normal within one week of the onset of diarrhea.  Future studies will focus on serum, blood, and urine clinicopathologic parameters.


Emily Wheeler*, Tom Gillespie*, Colin Chapman**, and Tony Goldberg*

*Department of Veterinary Pathobiology and Center for Zoonoses Research, University of Illinois College of Veterinary Medicine,  ** Department of Anthropology, McGill University, Montreal

Habitat alteration is believed to modify disease transmission dynamics among wild primates, humans and domestic livestock. This is a concern both for human public health and for primate conservation.  This study investigated patterns of disease transmission in Kibale National Park, a mid-altitude rainforest in western Uganda, by examining levels of antibiotic resistance in the common gastrointestinal bacterium Eschericia coli (E. coli).  Bacterial isolates were collected from fecal samples of four species of non-human primates (black-and-white colobus, red colobus, red-tailed guenon and chimpanzee) from forest areas that have experienced different types and degrees of anthropogenic disturbance.  Fecal samples were also collected from populations of humans living near each type of forest.  Bacteria were isolated from freshly deposited feces on on MacConkey agar.  Isolates were then transported to the United States where they were confirmed as E. coli using indole and citrate metabolism assays. Confirmed E. coli isolates were tested for antibiotic susceptibility using the disk diffusion method. Ten antibiotics commonly used in humans and livestock in Uganda were tested (ampicillin, chloramphenicol, ciprofloxin, doxycycline, gentamicin, neomycin, oxytetracycline, streptomycin, tetracycline).  A third-generation veterinary cephalosporin (ceftiofur) was included for comparison, since it is not used in the study region. Zones of inhibition were measured and resistance status assigned using NCCLS (National Committee for Clinical Laboratory Standards) guidelines. The proportion of human isolates clinically resistant (ranging from 2.3 – 58.1% across antibiotics) greatly exceeded that in the wild primate samples (ranging from 0 – 10.3%). Average susceptibility to all 10 antimicrobials included in the study (measured as the mean zone of inhibition across antibiotics) was reduced in colobus monkeys living in more disturbed areas relative to less disturbed areas (Tukey’s post-hoc test, p =0.007) Susceptibility patterns were similar for humans and chimpanzees, with isolates from an ecotourism site having reduced susceptibility relative to isolates from a relatively undisturbed deep-forest site (Tukey’s post-hoc test, p = 0.0016 for humans and p =0.0018 for chimpanzees).  The results of this study suggest that the dissemination of antimicrobial resistance from humans to primates is increased by habitat fragmentation, and that such effects are sufficient to produce parallel patterns of antimicrobial resistance in closely associated human and primate populations.


Spatio-temporal analysis and spatial analysis of Equine West Nile virus cases in Illinois in 2002 and comparison with land cover data
Amy Jo Wolf and Marilyn Ruiz
Center for Zoonoses Research and Department of Veterinary Pathobiology, University of Illinois College of Veterinary Medicine

West Nile Virus (WNV) is a flavivirus historically found in Africa, West Asia and the Middle East (www.cdc.gov/ncidod/dvbid/westnile/qa/overview.htm) with outbreaks also documented in Europe, South Africa and Israel.  In 1999, WNV was detected in New York City and by 2001 the virus had reached Illinois.  The subsequent outbreaks involved humans, equids and both mammalian and avian wildlife. Approximately 1200 equids tested positive for the virus in 2002 (www.idph.state.il.us).  Developing a reliable means of identifying equine outbreak locations and the expected severity of outbreaks before disease occurs could potentially decrease loss of life, both human and non-human, from the virus.  The purpose of the 2004 study was to describe the spatial and temporal pattern of equine WNV in Illinois in 2002 at the individual case level and to determine if vegetation data can be used as a predictor for equine WNV incidence.  It appears that 2002 equine WNV cases were documented at several foci early in the season located in the southwestern, northwestern and northeastern areas of the state.  Subsequent cases seem to overlap these early foci.  Cases documented throughout the season appear to spread outwards from and then recede towards the original foci.  Late season cases appear to be randomly distributed.  The spatio-temporal analysis of 2002 cases revealed a strong correlation between where cases occurred and the time of onset when the date of onset was used.  No significant correlation was seen between space and time when week of onset was used in the analysis.  One reason for the differences between day and week tests is that disease progression occurred quickly throughout the state and therefore weekly documentation of onset dates would group many new cases together, thus losing the ability to detect smaller changes having occurred during that week.  When 2002 cases were compared to land cover data, it appears that most cases occur outside forested areas.  This observation is supported by data from a previous study, completed in 2003, where percentage Upland was identified as being negatively correlated with disease incidence at the county level.  The conclusions reached may be skewed by the usage of the owner’s address for the location of disease occurrence.  Stable address would provide a more accurate depiction of disease dynamics, as many owners do not house their horses at their home.  However, this information is not available from any governmental or private entity at this time.  Future studies should focus on the actual location of WNV cases.  In order to do so, a statewide or national system must be instituted to collect the appropriate information needed for future epidemiological studies of disease outbreaks. 


2003 STUDENT PROJECTS

A Quantitative Real-Time PCR Assay for the Detection of Pathogenic Leptospira spp
Luke Borst – Mentor:  Dr. Carol Maddox

Leptospirosis continues to be an important zoonosis of worldwide concern. Primarily caused by serovars of Leptospira interrogans, leptospirosis, if left untreated or misdiagnosed, can progress to hepatic or renal failure.  Leptospirosis is transmitted via contact with water contaminated with the urine of carrier animals that continually shed the organism.  Although leptospirosis typically exhibits a low mortality and morbitity in this country, current research suggests that there may be a possibility for a re-emergence of the disease.4  For example, an increasing incidence in leptospirosis cases resulting from infections by novel serovars of Leptospira interrogans other than historically significant serovars canicola and icterohemorrhagiae has been observed.2  Further, the encroachment of suburban developments and retention ponds into wildlife habitats could presumably create an environment predisposed to the spread of leptospires.  A rapid, sensitive method for the detection of pathogenic leptospires in urine, tissue and environmental samples would be valuable in obtaining accurate diagnoses and aid in tracking emerging trends.   To this end, we adapted a TaqManâ (quantative) real-time polymerase chain reaction (PCR) assay for use in the Smart Cycler, by Cephied.  The assay is based on the rrs gene (16S rRNA) alignments published in GenBank and has a detection limit of 25 cells and does not cross-react with common urinary pathogens or non-pathogenic leptospires.   Sample inhibition was of no consequence in kidney specimens; however, a slight inhibition was seen with urine specimens resulting in a detection limit of 45 cells.  A blinded test performed on 22 randomly spiked negative urine specimens, subsequently subjected to PCR, yielded a sensitivity and specificity of 93% and 100% respectively.


Diurnal patterns of activity of Ixodes scapularis, the tick vector of Lyme disease
Tony Cappa, Mentor: Dr. Uriel Kitron

As part of a long-term ongoing study of the epidemiology of Lyme disease and the ecology of its tick vector in Illinois and surrounding states, tick bionomics were studied in one infested site in central Illinois. Transmission of Lyme disease is the result of encounters between questing ticks and susceptible vertebrates, thus examining diurnal patterns of tick activity as a function of time of day, temperature, and relative humidity may help determine times of high and low encounter probability.

Ixodes scapularis larvae were collected during a three day period from 14 July to 17 July 2003 using drag-sampling techniques at Natural Land State Park in Putnam County, IL. Three individual grids were established at the park and were dragged during four separate times of the day.  Relationships between I. scapularis larval activity and temperature, relative humidity, and time of day were examined.  The number of larvae on individual drags ranged from 0-14 with most drags containing between zero and three larvae.  Of the three grids, grid one had significantly (P<0.05) more larvae per drag. This difference may be related to vegetation, slope or other land cover features. There was no significant relationship found between the numbers of larvae collected and temperature, relative humidity, and time of day.

Based on extensive collections in the past, we predicted that tick activity would be lowest during the hottest part of the day, due to increased water loss and susceptibility to desiccation. We did not find such a relationship in our study. Because our collection dates were cooler than normal, we will repeat our study under more extreme conditions to verify the lack of association of time of day and tick activity.


Functional complementation in yeast by a vacuolar H+-pyrophosphatase from Trypanosoma cruzi
Rebecca Dieter (from laboratory work with Dr. Roberto Docampo)

Trypanosomatids contain acidic calcium storage organelles called acidocalcisomes.  Within the acidocalcisome is a vacuolar H+-pyrophosphatase enzyme (V-H+ PPase).  Mammals lack a similar enzyme, creating a potential drug target for treating Chagas disease, which is caused by Trypanosoma cruzi.  To characterize this V-H+ PPase in T. cruzi (TcPPase), a mutant strain of yeast, only capable of growth on galactose-containing media, was transformed with TcPPase.  Heterologous expression of the enzyme allowed the transformed yeast to regain the ability to grow in glucose media at different pHs.  Under immunofluorescence microscopy, the heterologous protein localized in the yeast cell plasma membrane and in areas of the cytoplasm.  Membrane fractions were isolated from TcPPase transformed yeast and the presence of the protein was confirmed, in certain fractions, by Western blot with a monoclonal antibody against TcPPase.  Measurements of AMDP-sensitive pyrophosphatase activity in the membrane fractions demonstrated the release of free pyrophosphate by the enzyme.  The functional complementation of TcPPase in mutant yeast has allowed more information to be gained about TcPPase that will contribute to the process of targeting this protein for chemotherapy.


Surveillance of Trypanosoma cruzi and other parasites in sylvatic mammals in northern Argentina
Rebecca Dieter (From fieldwork with Dr. Uriel Kitron)

From July 19-August 4, I participated in field research work on the ecology of Chagas´ disease (NIH-funded collaboration between UIUC and University of Bueno Aires).  The study area is located in the northern province of Santiago del Estero, Argentina.  The purpose of the field work was to conduct a survey of sylvatic mammals.  Animals were live-trapped or caught by local hunters and then processed in a field lab. Processing included sedation, extraction of blood and xenodiagnosis for testing of infection with T. cruzi, hair sample, collection of feces (when available) and a rectal swab for gut parasites, and collection of ectoparasites.  Blood samples will be used for microscopic examination, PCR, and serological tests for T. cruzi.  Following recovery, animals were released near the capture location. A total of 181 mammals were processed, including rodents, armadillos, marmosets, opossums, skunks, cuis, and foxes. 

As part if this comprehensive study of Chagas disease and vector ecology, I also participated in collection of triatomine bugs using light traps, as well as manual flushing out with insecticide of bugs from peridomestic structures.  After collection I was trained to process the bugs, which involves determining species, sex, growth stage, weight, length, and nutritional status. 

Some details of the analysis of data of ectoparasites from armadillos, specifically patterns of flea (Malacopsylla grossiventris) infestation of the mataco bola (Tolypeutes matacus) are reported.  It was found that fleas are not randomly distributed on individual hosts and that fleas are more common and more aggregated on male matacos than on females. 


Effect of abomasal pH on the potential susceptibility of dairy calves to infection by Mycobacterium avium subsp. paratuberculosis
Stacy Furgang – Mentors:  Drs. Peter Constable and Carol Maddox

Mycobacterium avium subsp. paratuberculosis infection causes Johne’s disease, which is a chronic, progressive enteritis of ruminants.  Johne’s disease is a widespread and economically important disease of dairy cattle, with losses occurring through premature culling, reduced milk production, and body weight losses.  In addition, there is a possible association between M. paratuberculosis infection and Crohn’s disease in humans. Cattle appear to be at greatest risk of M. paratuberculosis infection during the first four months of life, with the greatest risk occurring in the first month.  The reason for this age-dependent resistance is not known, but we hypothesize that it is due, in part, to diet and age-dependent changes in abomasal pH that influence survival of M. paratuberculosis during abomasal passage. The objectives of this study were therefore: (1) to determine the effect of a 1 h exposure to a pH of 1.0 to 6.0 on the survival of M. paratuberculosis in vitro, and (2) to characterize the change in abomasal luminal pH of Holstein bull calves during the first 8 weeks of life.  We obtained two M. paratuberculosis isolates from bovine fecal samples submitted to the University of Illinois Veterinary Diagnostic Laboratory. Both isolates were exposed to a broth containing Mycobactin J at different pH (6.0, 5.0, 4.0, 3.0, 2.0. and 1.0) for 1 h at 37° C to simulate abomasal passage. Log dilutions of the broth were then inoculated onto Herrold’s slant tubes and incubated at 37° C for 6 weeks to determine the effect of pH on survival of M. paratuberculosis.We also measured the change in abomasal pH of two Holstein bull calves during weeks 2 through 8 of life by introducing a flexible glass pH electrode through an abomasal cannula into the lumen. This provided a continuous measurement of abomasal pH over a 24 h period for each week.  Calves were fed an all milk protein milk replacer (12% body weight per day divided into two feedings 12 h apart) and ad libitum calf starter ration during the study period, but were weaned once they were consuming more than 1 lb of concentrate per day (between weeks 6 and 7 of life).  The feeding regimen was representative of the US dairy industry.  The in vitro survival of the first isolate was not affected by pH, whereas survival of the second isolate was linearly dependent on pH, with a 50% kill occurring after a 1 h exposure to a pH between 3.0 and 4.0.  Mean 24 h pH for the 2 calves was 3.31, 3.30, 3.51, 3.29, 3.02, 1.88, and 1.91 for weeks 2 through 8 of life, with a marked decrease in mean abomasal pH occurring after weaning from milk replacer between weeks 6 and 7 of life.  Abomasal pH ranged widely from 0.7 to 6.6 when calves suckled milk replacer, but was much more constant (range from 0.5 to 3.1) after weaning.  Because the survival of one of our isolates was pH-dependent, and because we observed a marked effect of diet on abomasal pH, we conclude that the age-dependent resistance to M. paratuberculosis infection in cattle may be due, in part, to diet-induced changes in abomasal pH.


William Love - Mentor:  Dr.Tony Goldberg

Largemouth Bass Virus (LMBV; family Iridoviridae), first identified in 1995, has since been found throughout the United States, where it has been associated with large die-offs of largemouth bass (Micropterus salmoides).  Some suspected modes of transmission include water (transported by boats), or stocking of fish between lakes.  The purpose of this study was to determine the relative efficiency of transmission of LMBV between 1) fish that were allowed direct contact to infected fish, and 2) fish in the same water source, but that did not have direct contact with infected fish.  We placed 10 juvenile largemouth bass in 8 tanks, 4 of which were divided with a double fenestrated barrier, and injected half of the fish in each tank with cultured LMBV.  The viscera of the fish were removed post-mortem and viral load was quantified using real-time quantitative PCR.  Survival was lower in experimental injected (donor) fish than in uninjected (recipient) fish, but did not differ between divided and undivided tanks.  Injected fish were found to have reduced average body condition scores compared to recipient fish.  We found a statistically significant, although small, difference between the viral load of recipient fish in divided tanks and recipient fish in undivided tanks, which indicates that transmission by direct contact was marginally more efficient than transmission through water alone.  Transport of water between lakes and streams may be as important for contrALS (agglutinin-like sequence) genes encode cell-surface glycoproteins that are involved in adhesion of the fungus to the host. The family consists of eight genes that encode proteins with a similar three-domain structure. Each protein has an N-terminal domain that is believed to funolling the spread of LMBV in the United States as limiting the movement of fish between bodies of water.


Marie Sienkewicz -  Mentor:  Dr. Randy Singer

Objective: To investigate the short-term dynamics of antibiotic resistance genes following antibiotic treatment. The AmpC-like ß-lactamase gene family, cmy-2, in enteric Escherichia coli of dairy cattle treated with a third-generation cephalosporin (ceftiofur).
Design: Cohort Study
Animals: 10 dairy cows
Procedures: Five dairy cows treated with ceftiofur (2.2 mg/kg IM, once daily, for five days) were matched to an untreated cohort of five cows in the same herd. Fecal samples were collected prior to (days –1 and 0), during (days 2 and 4) and following (days 5-11, 14, 18, 25 and 32) treatment. Enteric E. coli counts (cfu/g) were estimated for Days –1 through 14. Three random isolated colonies, plus any additional colonies needed so that all preliminary antibiotic susceptibility profiles were represented, were selected for further investigation. Selected isolates (N=228 from Treatment Group; 250 from Control Group) were then identified biochemically as E. coli. A cmy multiplex PCR protocol was used to screen for the presence of the cmy-2 gene family.
Results: The mean log-transformed fecal E. coli count (log cfu/g) of the treatment cohort dropped to levels significantly lower than that of the control group on Days 2, 4, and 5 and returned to pretreatment levels by Day 7 of the study (72 hours following the last antibiotic injection). The presence of cmy-2 was not detected in either cohort except on Days 4-5 of the study, when it was identified in four of the five treatment cows.
Conclusions: Treatment with ceftiofur resulted in a significant drop in the gram-negative enteric bacteria population, allowing for the detection of cmy-2-bearing E. coli. With the removal of ceftiofur as a selection factor, E. coli counts returned to pretreatment levels and cmy-2 frequency returned to a population frequency not detected by our sampling methods.


Candida albicans ALS Gene Family Dynamics
Jason Smith – Mentor:  Dr. Lois Hoyer

Candida albicans ction in adherence, followed by a central tandem repeat domain and a C-terminal domain. These last two domains are heavily glycosylated and likely serve to bring the N-terminal binding domain into contact with host surfaces. Two different studies were conducted to examine the dynamics of the C. albicans ALS gene family.  In the first, we determined whether C. albicans proteolytic activity was responsible for the changing profile of Als cell wall proteins that occurs during the morphological transition from yeast to hyphal forms.  In the second study, we evaluated the stability of the central tandem repeat domain of ALS genes in C. albicans cells serially passaged for 3000 generations.

Previous work showed that as C. albicans mother yeast produced germ tubes, the yeast cells lost Als proteins from their cell wall.  The mechanism responsible for the release was not determined, but could involve proteolytic activity.  One attractive possibility is that the mechanism involves proteins encoded by the SAP (secreted aspartyl proteinase) family that is known to contribute to C. albicans pathogenesis. To investigate this potential, pepstatin A was added at concentrations known to inhibit Sap activity. No difference was observed between the treated and control cultures suggesting that Sap proteins are not involved in the release of Als molecules from the cell surface. Addition of a protease inhibitor cocktail was also evaluated to test the potential for the involvement of different types of proteases. In the presence of this reagent, C. albicans failed to form germ tubes. Although it is suspected that EDTA inhibited germ tube formation, individual components of this cocktail would need to be tested individually to determine which component had this effect on C. albicans morphology

In the second study, we examined the stability of a repeated region of DNA within the ALS gene coding sequence. The central tandem repeat domain of a given ALS gene varies between strains and often between alleles within the same strain. Studies of other C. albicans repeated DNA sequences have shown that such structures undergo recombination that can be observed on Southern blots in as few as several hundred generations. In addition, parallels have been drawn between the ALS family and genes encoding Saccharomyces cerevisiae cell-surface flocculins (FLO). Since the FLO genes have a sub-telomeric localization (23), repeats within their coding regions are relatively unstable. To examine the stability of the central tandem repeat domain of ALS genes, we analyzed a set of four isolates, each grown for 3000 generations. Southern blots of BglII-digested DNA were hybridized with probes specific for each of the various ALS genes. For each of the four isolates, we observed no differences in hybridization pattern between the zero and 3000 generation strains suggesting that none of the ALS repeat regions recombined within this time frame. From these data, we conclude that the ALS tandem repeat sequences are relatively stable compared to other repeated DNA sequences that have been studied, including those in the FLO genes of S. cerevisiae.


Spatial Analysis of Equine West Nile Virus Case Rates By County in Illinois in 2002 and Comparison With Land Cover Data
Amy Jo Wolf – Mentor:  Dr. Marilyn Ruiz

West Nile Virus (WNV) is a flavivirus historically found in Africa, West Asia and the Middle East (www.cdc.gov/ncidod/dvbid/westnile/qa/overview.htm) with outbreaks also documented in Europe, South Africa and Israel.  In 1999, WNV was detected in New York City and by 2001 the virus had reached Illinois.  The subsequent outbreaks involved humans, equids and both mammalian and avian wildlife. Approximately 1200 equids tested positive for the virus in 2002 (www.idph.state.il.us).  Developing a reliable means of identifying equine outbreak locations and the expected severity of outbreaks before disease occurs could potentially decrease loss of life, both human and non-human, from the virus.  The purpose of the study was to both describe the spatial pattern of equine WNV and to determine if vegetation data can be used as a predictor for equine WNV incidence.  Areas of intensive agriculture have higher rates of equine WNV, while upland areas, that is, areas of closed-canopy deciduous forest, and areas of grasses and other agriculture have lower rates of equine WNV.  Although the correlations seen were statistically significant, only the regression coefficient for uplands was found to be significant in a linear regression analysis.  The negative correlations may be due to the fact that the habitat in open grassland and forests are not ideal for vector breeding and survival.  One future hypothesis to be tested is to consider whether the horses in areas with more intensive agriculture have relatively less pasture land, thus putting them in closer contact with barns and other peridomestic structures where the mosquito vectors may find more places to breed.  Further research will need to be completed in these areas to elucidate the processes that are responsible for the results seen.

1  Adamus C., Buggin-Daubie M., Izembart A., et al. 1997. Chronic hepatitis associated with leptospiral infection in vaccintatied beagles.  J Comp Pathol 117:331-328
2  Bolin, C.A. 2000. Leptospirosis, p.185-200. In Corrie Brown and Carole Bolin (ed.), Emerging Diseases of Animals. ASM Press, Washington, DC.