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Veterinary Student Summer Research Training Program at the University of Illinois

Past Student Projects

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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 hypothesis 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 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 sixteen 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

The bacterium 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.

 

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

Patterns of antibiotic resistance in E. coli isolated from Ugandan primates
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 controlling 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.

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

Candida albicans ALS (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 function 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.

 

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.

 

 

 

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