Research Projects

Ongoing and Past Research Projects

Pathoanatomical features of the equine pancreas and the correlation to hyperinsulinemia in horses

Researcher: Grace Bronneberg, Purdue University
Mentor: Francois-Rene Bertin

Equine hyperinsulinemia, or the increased production of insulin, results when the cells within the liver and peripheral tissues produce an ineffective response to insulin, which causes the pancreas to increase insulin and glucagon and decrease somatostatin production to maintain blood sugar levels. Previous clinical evaluation has demonstrated the cellular composition of the equine pancreas in relation to hyperinsulinemia. However, differences in cellular composition between the lobes of the pancreas have not been evaluated as there is a lack of understanding on the gross anatomy, especially regarding the number of lobes, of the equine pancreas. Here we show an experimental approach to analyze our hypothesis of a uniform morphology of three lobes of similar size, shape and mass, and a degree of significant variability of cellular composition between the lobes of the pancreas. We used a sample size of 5 horses of differing backgrounds and harvested pancreatic tissues using a right lateral approach. We documented the gross morphology and fixed the tissues in formalin to be sectioned into slides for later evaluation through immunohistochemistry. Our preliminary results indicate a uniform morphology across our samples. In collecting our samples, we found the pancreas in close association to the right dorsal colon, dorsolateral to the base of the cecum, and dorsal to the right kidney. Additionally, we found three lobes, each of which were within a 5cm difference in length, between each sample. The left and right lobes were found to be the largest, and of equal size, compared to the middle lobe which was 10 cm shorter. Our results have future implications for understanding the cellular development of hyperinsulinemia.

Reliability of Smartphone-Based Two-Dimensional Kinematic Gait Analysis in Healthy Dogs

Researcher: Jaeguk Byun, Purdue University
Mentor: Sun Young Kim

Kinematic analysis is widely used to describe and quantify joint motion in both humans and animals. In quadrupeds, such as dogs, sagittal plane motions, including flexion and extension, are particularly important, and two-dimensional (2D) kinematic analysis is commonly used in gait analysis of dogs. However, traditional 2D kinematic systems require a dedicated video camera, software, and a computer workstation, which makes it difficult for clinicians to use in daily patient evaluations. Recent advances in technology have enabled the measurement of joint angles using a smartphone-based app. Although measuring joint angles using a mobile app is attractive due to its convenience, it has not been validated in quadrupeds. This study aimed to determine the reliability of joint angle measurements obtained using a mobile app. Five reflective markers were placed on anatomical landmarks to measure the hip, stifle, and tarsal joint angles in three healthy dogs. Five valid gaits per dog were recorded using a smartphone camera (4K at 60 fps) as the dogs trotted at a self-selected pace along a 12-meter walkway. Joint angles were measured at six time points within one stride cycle. Intra-rater and inter-rater reliability were evaluated using the two-way intraclass correlation coefficient (ICC). The results demonstrated excellent intra- and inter-rater reliability (ICC ≥ 0.91) for all joints during the swing phase. Reliability was generally good (ICC ≥ 0.80) during the mid-stance and toe-off phases, except for the stifle and tarsal joints, which had slightly lower ICC values. These findings suggest that mobile apps can reliably measure joint angles in dogs and may serve as a practical tool for clinicians in everyday patient assessments.

Is shorter better? Detection of caregiver burden in a canine oncology setting using a single-item measure

Researcher: Fionna Freeman, Purdue University
Mentor: Christopher M. Fulkerson

Owners of pets with cancer experience caregiver burden. Elevated burden is associated
with negative psychosocial outcomes, and early detection may facilitate interventions like
referral to a veterinary social worker. The Zarit Burden Interview (ZBI) adapted for animals is
used to measure burden, but it is a time-intensive tool. This work explores if elevated
burden can be detected using a single-item – “Overall, how burdened do you feel in caring
for your pet?”
Psychometric properties of the single-item screener were examined in relation to the full
ZBI in 315 owners during an initial appointment at a veterinary oncology clinic for a dog with
a confirmed cancer diagnosis. Measures of caregiver burden were completed and
established cutoffs for elevated burden were used. Records were reviewed for the pet's
health status.
Correlation of the single item with ZBI was r = 0.57. One-month test-retest reliability in a
sample subset of owners reporting a dog with stable health was r = 0.39. Receiver
operating characteristic analyses showed area-under-the-curve was 0.81, differentiating
burden groups, (P=.000). Maximized classification accuracy was reached at ≥0.5, with
sensitivity of 0.75 and specificity of 0.83. Elevated caregiver burden was observed in 21.1%
of the sample using the ZBI and 28.7% of cases with the single-item screener.
While the single-item was correlated with the ZBI, test-retest reliability was not acceptable
and over-identified caregiver burden in the sample. If used alone, follow-up of positive
results with the full ZBI is indicated. Further research is needed to optimize a reliable
abbreviated measure to identify burden in owners of pets with cancer.

Innovative delivery of oncolytic adenoviruses expressing IL-27 using adipose-derived mesenchymal stromal cells for prostate cancer treatment

Researcher: Lucie Ganachaud, Purdue University
Mentor: Marxa L. Figueiredo

In the search for less invasive cancer treatments, oncolytic viruses have shown promising therapeutic potential. In fact, these viruses can selectively replicate in cancer cells, modulating their replication, and can alternate tumor microenvironment. Prostate cancer remains a leading cause of cancer-related mortality, necessitating innovative, targeted therapies. Our team previously demonstrated that an oncolytic adenovirus armed with interleukin 27 (oAd-IL27) carried by adipose-derived mesenchymal stromal cells (ASCs) showed anti-tumor effects in multi-cell culture models of prostate cancer. While the in vivo experiment demonstrated prolonged survival potential after treatment, no significant tumor reduction was observed, likely due to insufficient ASCs recruitment at the tumor sites. We hypothesize that enhancing ASC migratory behavior through biological modification may improve oAd-IL27 accumulation at tumor sites. We have begun to characterize the potential for ASCs as oAd-IL27 systemic carriers by performing wound healing assays, which assess cell motility in vitro. Next, we plan to quantify and assess using an IncuCyte whether our biologically modified ASCs show improved migration behavior. Furthermore, transwell migration assays involving prostate cancer cells and oAd-IL27-loaded ASCs will be performed to recapitulate the tumor microenvironment, before final in vivo evaluation. We anticipate that the findings from this study may bring new insights into the field of oncolytic virotherapy by proposing an innovative cell-based oncolytic virus delivery system. Following continuous development of this new system, our long-term goal is to ultimately provide a therapeutic that can be more effective for treating patients with metastatic prostate cancer.

Leukocyte comparison of barbering and dermatitis conditions in laboratory mice

Researcher: Emily Godollei-Jacob, Purdue University
Mentor: Amanda Darbyshire

Idiopathic ulcerative dermatitis in laboratory rodents is a significant source of unplanned euthanasia and confounding variability in laboratory studies. Current literature suggests genetic, environmental, and behavioral elements in this condition. The relationship between ulcerative dermatitis and other behavioral conditions, such as barbering, remains unclear. Given the behavioral roles of barbering and dermatitis, it was hypothesized that the leukocytic parameters of dermatitis and barbering individuals are similar in their deviations from healthy individuals. Context to any physiological relationship between these conditions could provide more appropriate treatment and management methods to improve laboratory animal welfare. In turn, improved rodent welfare leads to more reliable data and research overall. Blood samples were collected primarily via submandibular or submental route from control animals and individuals actively experiencing barbering or dermatitis, with rare instances of postmortem cardiac puncture collection for severe dermatitis cases. Differential leukocyte counts were performed and analyzed for variance via ANOVA tests. Characterized leukocyte counts failed to find a main effect of barbering or dermatitis conditions. This suggests a lack of definitive leukocyte characterization of either condition and no strong immunological link between the two conditions. Neutrophil counts trended towards significance, and may have become apparent with a larger sample size. Further investigations into these findings with larger sample sizes should be performed to more fully encompass variation across strains, sex, and age. Additionally, further exploration into leukocyte characterization of different levels of conditional severity may provide valuable insight into any possible physiological basis for these conditions.

The Effect of ADAMTS10-Mutation on IOP-Associated Optic Neuropathy in Canine Glaucoma

Researcher: Haley Harmeson, Purdue University
Mentor: Shin Ae Park

Glaucoma is one of the leading causes of irreversible blindness in both humans and
canines. Increased intraocular pressure (IOP) and its negative impact, such as apoptosis and
degeneration on retinal ganglion cells (RGCs) and the optic nerve, is a primary risk factor for
glaucoma. In comparison to dogs with other forms of glaucoma, clinical observations suggest
that Beagle dogs that display ADAMTS10 mutations may exhibit more favorable visual outcomes
despite elevated IOP. However, it remains unclear whether this clinical impression reflects an
intrinsic effect of the ADAMTS10 mutation, the gradual elevation of IOP typically seen in this
form of glaucoma, or other unknown factors. From previous studies, our group developed a
canine ocular hypertensive glaucoma model with controlled IOP elevations, allowing consistent
application of the same magnitude and duration of IOP elevation. In this model, the target IOP
range of 36-45 mmHg can best resemble the human glaucomatous disease mechanism. Using
this preliminary model, we induced controlled ocular hypertension to compare its effects on
glaucoma progression between ADAMTS10-mutant (n=1) and non-mutant (n=1) dogs. The
progression of glaucoma was evaluated based on optical coherence tomography (OCT),
electroretinography (ERG), fundus photography (Retcam), and maze vision testing.
Histopathology of the optic nerves and the eyes will be performed at the end of the study.
Preliminary in vivo results include (1) ADAMTS10-mutant dog showed dramatic fundus
photography changes including altered tapetal reflectivity during IOP elevation. (2) The
ADAMTS10-mutant dog showed decreased ERG values and decreased visual performance in
maze vision testing. (3) In OCT, subjectively, the ADAMTS10-mutant dog appeared to have
more retinal thinning. Unlike the clinical impression, when the same magnitude and duration of
IOP elevation was induced, ADAMTS10-mutant dog might be more susceptible to IOP-induced
optic neuropathy.

Azithromycin Drug Resistance in Acanthamoeba

Researcher: Lucas Hendershot, Purdue University
Mentor: Christopher Rice

Acanthamoeba, a genus of pathogenic free-living amoeba, are capable of inflicting
significant diseases in humans and animals alike. Species belonging to Acanthamoeba are
responsible for causing granulomatous amoebic encephalitis (GAE), Acanthamoeba
keratitis, skin and systemic diseases. Currently, the best treatment for these infections
involves a chemical combinational therapy made up of non-specific anti-biotics, antifungals,
and anti-parasitics. However, recent studies have suggested Azithromycin, a
macrolide antibiotic, to be a frontline therapeutic against diseases caused by
Acanthamoeba and other free-living amoeba. Azithromycin has been proven to work by
binding to the 50S subunit, specifically the 23S rRNA subunit in bacterial ribosomes. Upon
binding, azithromycin reduces protein synthesis and prevents cellular growth.
Unfortunately, this mechanism has only been validated in bacteria and not in amoeba
species. This study aims to develop azithromycin resistant Acanthamoeba mutants to
potentially yield the phenotypical mechanism of action or resistance pathway(s).
Developing resistant mutants capable of reaching a 100-fold shift in its IC50 towards a
compound is the first step in discovering a mechanical mode of action (MMOA) within
Acanthamoeba. Mutants were developed using a step-wise approach and now we have
mutants growing in 60x-80x concentrations of the original azithromycin IC50. We will use
specific target sequencing and potentially whole genome sequencing of mutants to identify
azithromycin’s MMOA. If successful, this study would be the first to utilize drug resistant
Acanthamoeba for studying molecular modes of action. Understanding the MMOA of
Acanthamoeba is vital to developing enhanced therapeutic drugs that can e􀆯ectively
mitigate the diseases caused by this organism.

Creating a Xenografted Human Vascularized Chimeric Brain Model

Researcher: Oliver Johnson, Purdue University
Mentor: Ranjie Xu

Cerebral blood vessels are essential for transporting nutrients throughout the brain and regulating the entry of materials into the brain through the Blood-Brain Barrier (BBB). Dysfunction of the brain's vascular system is linked to conditions such as stroke, vascular dementia, and Alzheimer’s disease. Growing evidence shows significant differences between human and mouse vascular cells. For example, human vascular cells express higher levels of Alzheimer 's-associated genes. Therefore, it is urgent to develop human vascular models to better understand how the vascular system affects brain health and disease. An in vivo vascular model, in which human blood vessels are perfused and functionally integrated into the host's brain, has not yet been developed. To create these models, we will inject human Pluripotent Stem Cell (hPSC)-derived vascular progenitor cells into mice and allow the cells to grow and differentiate within the mouse brains. We will examine human vasculature maturation in host brains by harvesting, sectioning, and immunohistochemically staining the brains at different times post-cell injection. The staining will target markers for the main cell types needed for functional blood vessels and the Blood-Brain Barrier: endothelial cells, smooth muscle cells, pericytes, and astrocytes. Developing this chimeric vascularized brain model will provide a more physiologically relevant system for studying neurovascular contributions in healthy and diseased conditions, such as Alzheimer’s disease and Vascular Dementia.

The Effect of Trazodone on Equine Lameness Examinations

Researcher: Will Kendall, Purdue University
Mentor: Michelle L. Tucker

Lameness is one of the most common presenting complaints in equine practice, and
performing complete examinations is crucial for diagnosis. Patient non-compliance is a
frequent roadblock and also presents safety issues to the veterinarian. Trazodone is
known to have a sedative effect in horses, but is not expected to reduce pain and may
facilitate lameness exams in horses. We hypothesize that trazodone given to a horse at
both a high (10 mg/kg) and low (5 mg/kg) dose will not significantly affect subjective or
objective measurements of lameness, and will not create excessive sedation or ataxia
during an exam. For this study, 18 Purdue teaching horses are weighed and receive a
high dose of trazodone, low dose of trazodone, or a placebo on three separate days. A
straight line jogging evaluation is completed prior to drug administration, and then again
at 30, 60, 120, and 240 minutes post administration. For each evaluation, lameness is
graded by a blinded board-certified surgeon, and objective lameness data (Equinosis) is
also collected. Additionally, video recordings of the joggings are taken and will be
randomized and later reviewed by two separate blinded observers to grade lameness.
Horses are actively monitored for adverse effects of trazodone. Once all of the data is
collected, statistical analysis will compare the lameness grades and objective lameness
data between the placebo and two different trazodone doses for each horse. Final
results of the study are pending, but one horse experienced severe ataxia, preventing
data collection at two time points. Should trazodone not significantly affect lameness,
then it can potentially increase the safety and efficacy of lameness examinations with
uncooperative horses.

Immunolocalization of a Rhoptry Protein, SnROP21, in the Merozoite and Schizont

Researcher: NaLaya Lee, Fort Valley State University
Mentor: Sriveny Dangoudoubiyam

Sarcocystis neurona, a protozoan responsible for a debilitating neurological disease called equine protozoal myeloencephalitis, undergoes asexual replication in the central nervous system of the horse. This replication involves two developmental stages: extracellular merozoites and intracellular schizonts. Interestingly, these stages in S. neurona lack a specialized apical secretory organelle called rhoptries, but several rhoptry proteins (ROPs), including SnROP21, were identified in the transcriptome and proteome of S. neurona. In related protozoans, rhoptry proteins typically localize to and are discharged by rhoptries to participate in host-parasite interactions. This prompted questions about rhoptry protein localization and their functional roles in S. neurona in the absence of rhoptry organelles. In this study, we have used a transgenic S. neurona line expressing a C-terminal hemagglutinin (HA)-tagged SnROP21 (SnROP21-HA) to perform immunolocalization assays. Bovine fibroblasts seeded on cover glasses were infected with S. neurona merozoites that transformed into schizonts upon invasion. Cover glasses with intracellular schizonts at four different timepoints over a 72-h period of development were fixed and processed by probing with anti-HA antibodies to localize SnROP21. In extracellular merozoites, SnROP21 was localized predominantly to the apical pole. In the schizonts, SnROP21 showed temporal expression, with it being undetectable at the 48 h timepoint but showing granular cytoplasmic distribution at other timepoints. This dynamic expression of SnROP21 suggests a unique role that might be temporally regulated. Further studies on SnROP21 will enhance our understanding of its role in S. neurona biology and host-parasite interaction.

CLCN1 variant predicted to be responsible for congenital myotonia in a domestic shorthair cat

Researcher: Sarah Nelson, Liberty University
Mentor: Kari Ekenstedt

Congenital myotonia is a neuromuscular disease that causes the muscles to remain tense after
a voluntary contraction for some time after the initial event. This is typically a result of
dysfunction in chloride channels in the skeletal muscle membranes; these channels help
maintain a stable charge across the membrane to prevent spontaneous action potentials.
Variants in the CLCN1 gene (encoding a chloride channel protein) are associated with congenital
myotonia in a myriad of species, including humans, dogs, and the well-known fainting goats.
Recently, a domestic shorthair cat presented with clinical signs that align with congenital
myotonia; the cause of this cat’s disease was unknown but speculated to be genetic. Therefore,
DNA from this cat was subjected to whole genome sequencing, and the data examined for
causative variants private to this cat, compared to >100 other feline genomes (all assumed to
be unaffected by congenital myotonia). A plausible causal variant was identified in this cat’s
CLCN1 gene, specifically a homozygous missense variant (ChrA2:157,195,615 c.979G>T,
p.Val327Phe). In silico testing was explored to predict the effect of this amino acid change,
which likely impairs the ion channel gate function of the protein. Given that this cat was found
as a stray, it can only be speculated that this homozygous variant resulted from inbreeding,
which is not uncommon in feral cat populations. This work adds to our knowledge of congenital
myotonia in cats and across all mammals, which will allow for the continued exploration of new
diagnostic and therapeutic measures in the future.

A Novel TCIRG1 deletion causes autosomal recessive osteopetrosis in a domestic cat

Researcher: Grace Palmer, Purdue University
Mentor: Kari Ekenstedt

Osteopetrosis is a rare genetic disorder in which bones grow overly dense and become brittle, making them more likely to break. This condition results from defects in osteoclasts, which normally help break down old bone to make way for new, healthy bone. When osteoclasts fail to function properly, excessive bone accumulates. In humans, severe forms of osteopetrosis often begin in infancy and can be life-threatening. About half of these early onset cases are caused by mutations in a gene called TCIRG1. Recently, a 3-year-old random-bred cat presented to Purdue University’s Small Animal Hospital with unusual clinical signs, including abnormally dense bones, a fractured patella, and issues with its teeth and eyes. Together, these clinical signs suggested a diagnosis of osteopetrosis. Because the disease is genetic in humans, we carried out whole genome sequencing of the cat’s DNA, comparing it to 115 genomes from unrelated cats. Our standard variant elimination pipeline ultimately identified an exonic, homozygous, in-frame 24bp deletion in TCIRG1 (XM_023240141.2:c.645_668del) which is predicted to eliminate eight amino acids from the protein structure (XP_023095909.1:p.Ala173_Ala180del). We genotyped 59 additional unrelated cats to confirm the absence of this variant from the healthy cat population. The variant was not found in any of these cats, supporting our prediction that this is a novel mutation. To our knowledge, this is the first time a TCIRG1 mutation has been associated with osteopetrosis in a cat; the deletion’s homozygous presence suggests a close inbreeding event, which is not uncommon in random-breeding cat populations.

Mechanical Testing of Equine Laminae Samples

Researcher: Emily E. Rastovski, Purdue University
Mentor: Timothy B. Lescun

Equine laminitis is a condition that causes extreme pain, suffering, and can result in euthanasia of
horses on humane grounds. Failure of the laminar junction, which connects the hoof wall to the
distal phalanx, is the mechanism responsible for clinical manifestation of laminitis. However,
there is limited data published comparing healthy and laminitic mechanical strength. Samples of
laminae collected from euthanized horses can be mechanically tested in vitro using a materials
testing system that measures maximum load supported by the laminae. Furthermore, the effects
of storage conditions on the strength of lamina samples are unknown. This study includes two
aims: 1) to determine if different storage conditions affect mechanical testing results and 2) to
determine differences in load-bearing properties between healthy and laminitic tissue. We
hypothesized that 1) there would be no difference in mechanical testing results among
refrigerated, incubated, or frozen tissue and 2) laminar strength would be lower in laminitic
tissue compared to healthy tissue. Here, we first examined the differences in laminar tensile
strength over time in differing storage conditions. Then, differences in laminar strength between
healthy and laminitic tissue were investigated. In the first phase of this study, hoof samples were
collected after euthanasia and divided between refrigerator, incubator, and freezer storage (all
within 2 hours post-mortem). Refrigerated and incubated samples were randomly chosen to be
tested at n=8 time points over 72 hours following time of death. Freezer samples were thawed at
room temperature for 6 hours, then tested at n=8 time points over the next 72 hours post-thaw.
Mechanical testing involved incising samples to fit within a jig, which was placed onto the
materials testing apparatus. Once testing began, the machine pulled the sample apart vertically at
a rate of about 1mm/second until it reached a set displacement endpoint. Measurements of
maximum force sustained by laminae over the testing period were recorded. Results from this
study will provide a foundation for future research investigating laminitis in horses.

Descriptive analysis of highly pathogenic avian influenza outbreak in U.S. from 2022-present

Researcher: Nasir Roberts, Purdue University
Mentor: Wendy Beauvais

The current outbreak of highly pathogenic avian influenza (HPAI) virus in the U.S. is a result of transmission from wild to domestic fowl. Since early 2022, every U.S. state has detected HPAI-infected birds, and 35 states have reported cases in commercial flocks. Currently, efforts to create models that can predict how HPAI will spread are underway, however these models require analysis of the distribution of HPAI cases for parameterization. Our objective in this project was to statistically analyze the spatial-temporal distribution and seasonality of HPAI cases in the US from 2022-present. We analyzed outbreak data from the U.S. Department of Agriculture (USDA) to create maps and plots that illustrate the data using “R” statistical software. Our results show a higher number of detections in fall and spring, as well as increased occurrence of cases in the northern U.S. These findings are consistent with migration patterns, as birds migrate during the spring and fall months. This presents migratory patterns as a potential predictor for the spread of HPAI.

Investigating IL-17A-Driven Tertiary Lymphoid Structure Cytokine Expression in Canine Bladder Cancer Cell Lines

Researcher: Brayden Spurlock, Wabash College
Mentor: Meaghan M. Broman

Tertiary lymphoid structures (TLS) are immune aggregates associated with improved prognosis in various human cancers, including invasive transitional cell carcinoma (InvTCC) of the bladder. TLS formation is orchestrated by chemokines such as CXCL13 and CCL21, typically induced by pro-inflammatory cytokines, including interleukin-17A (IL-17A), produced by stromal fibroblasts and immune cells. In human and murine models, IL-17A signaling promotes TLS development and enhances anti-tumor immune responses. However, the extent to which this pathway functions in canine tumors remains unclear. Here, we investigated IL-17A-induced expression of TLS-associated chemokines and angiogenic factors in canine UC cells. TLS-associated gene expression was explored in singe cell RNA sequencing (scRNA-Seq) data previously generated from canine UC patient specimens. Cell lines were treated in vitro with recombinant IL-17A, and quantitative PCR was used to assess expression of IL-17ra, CXCL12, CXCL13, CCL19, CCL21, and VEGF. IL-17ra expression was low across cell lines. Cytokine expression varied among cell lines but was not significantly increased by IL-17A treatment. These findings suggest IL-17A alone is insufficient to induce TLS-associated chemokine expression in canine tumor cells under monoculture conditions. These results suggest stromal or immune-derived signals may be necessary for full pathway activation, and may indicate species-specific or context-dependent differences in IL-17 signaling. Our study reveals a knowledge gap in canine cancer immunobiology, supporting the future use of co-culture models incorporating stromal and immune cells to more accurately model TLS-inducing pathways in canine InvTCC anti-tumor responses.

Validation and Temporal Analysis of Differentially Expressed MicroRNAs in Horse Serum Following Intravenous Lipopolysaccharide Administration

Researcher: McKinley Underhill, Purdue University
Mentor: Andrea Pires dos Santos

Sepsis remains one of the leading causes of mortality in horses and presents significant diagnostic challenges, particularly in its early stages. MicroRNAs (miRNAs), small, non-coding RNA molecules that regulate gene expression, offer a promising avenue for early detection of sepsis before clinical signs become evident. However, limited research exists on the role of miRNAs in equine sepsis. Our preliminary analysis identified several miRNAs that were significantly upregulated or downregulated in response to the LPS challenge. Blood plasma samples were collected from horses before and after LPS administration, and digital PCR was used to quantify changes in specific miRNA expression. The aim is to identify miRNAs that are differentially expressed in horses during experimentally induced sepsis via lipopolysaccharide (LPS) infusion. These findings suggest that certain miRNAs may serve as potential biomarkers for the early detection of sepsis in horses. Further research is needed to validate these candidates in clinical cases. Ultimately, understanding miRNA dynamics in sepsis could contribute to faster and more accurate diagnostics, as well as improved outcomes, for equine patients.

Antibiogram development for Clostridium perfringens and in-house anaerobic AST verification

Researcher: Caitlin H. Wager, Purdue University
Mentor: Rachel C. Soltys

Anaerobic bacterial infections often present significant diagnostic and therapeutic challenges in veterinary medicine due to rising antimicrobial resistance. Accurate antibiotic susceptibility testing (AST) is crucial for effective therapy in the clinical setting. However, comprehensive anaerobic antibiotic susceptibility testing is currently unavailable at the Indiana Animal Disease Diagnostic Laboratory (ADDL), limiting diagnostic capabilities and antibiotic stewardship efforts. Therefore, this study aims to perform in-house verification of an anaerobic antibiotic susceptibility testing method at the Indiana ADDL, as well as develop a local antibiogram for Clostridium perfringens, a common and important pathogen in various animal species. For in-house verification of anaerobic AST, a 15-replicate plan per CLSI recommendations was performed using ATCC control strains (Bacteroides fragilis, Bacteroides thetaiotaomicron, and Clostridioides difficile). Briefly, eTest strips for benzylpenicillin, clindamycin, and metronidazole were used as an alternative to agar dilution, and Minimum Inhibitory Concentration (MIC) values were determined after 48 hours of anaerobic incubation. This verified methodology was then applied to banked Clostridium perfringens clinical isolates to generate a comprehensive local antibiogram, reflecting real-world resistance patterns within our region. These efforts will enhance ADDL's diagnostic services, offering clinicians informed antibiotic choices for improved patient outcomes and reduced treatment failures. The C. perfringens antibiogram will serve as a vital tool for tracking resistance trends, guiding antibiotic stewardship efforts and benefiting both animal and public health.

Affect scores in Veterans with posttraumatic stress disorder (PTSD) in an equine-assisted learning program

Researcher: Elisabeth Wolf, Purdue University
Mentor: Leanne Nieforth

About 23 percent of post-9/11 U.S. military veterans have service-related posttraumatic stress disorder (PTSD) (Fulton et al. 2015). Though traditional treatment options exist, complementary interventions are becoming popular. One such intervention is equine-assisted interventions (EAI), which include both equine-assisted psychotherapy (EAP) and equine-assisted learning (EAL). EAP utilizes a licensed mental health professional, whereas EAL is focused on learning horsemanship with an educator or coach. To date, most research focuses on EAP in veterans, but due to availability of resources, EAL is often more commonly used in practice. Few studies have examined the impact of EAL on veteran psychosocial wellbeing. This study explores positive and negative affect in veterans (N=29) involved in an 8-week EAL program. Each week, participants completed the International Positive and Negative Affect Schedule – Short Form (I-PANAS-SF), a validated survey that measures positive and negative affect. Data was analyzed using a linear mixed effects model with an AR(1) structure to account for repeated measures. Results showed a significant effect of week on both positive (p = 0.0218), and negative (p < 0.0001) affect scores. Findings suggest that EAL may promote positive changes to both positive and negative affect in veterans with PTSD. Future studies should continue to explore the psychosocial outcomes of EAL for veterans with PTSD as it may be a promising alternative intervention for veterans who are not interested in a more traditional mental health intervention.

Investigating the association between preterm birth and the development of vocal folds in lamb larynges

Researcher: Gaibrielle Bressler, Purdue
Mentor: Abigail Cox

The development of laryngeal vocal folds (VFs) plays a crucial role in voice production.
Voice abnormalities in humans often stem from premature birth, yet few studies have
explored how prematurity affects laryngeal health and voice outcomes. This study aims
to characterize the preterm lamb larynx for future translational studies, specifically
pertaining to preterm infants and school-age children speech pathology. Microscopic
examination focused on 3 lamb larynges: term, preterm, and extreme preterm.
Hematoxylin and eosin (H&E) stains evaluated VF lamina propria (LP) and epithelia
width, while alcian blue (AB) stains assessed glycosaminoglycan and matrix protein
levels. All developmental stages exhibited typical connective tissue components in the
LP. In preterm and extreme preterm VFs, fibroblasts, collagen, elastin, and vessels were
evenly distributed throughout the LP, contrasting with term VFs showing greater spatial
differences, with higher fibroblast and vessel densities in superficial versus deeper LP
layers. AB staining revealed that the deeper LP layers in term VFs had more
alcianophilic glycosaminoglycans than superficial layers, whereas preterm and extreme
preterm VFs showed more uniform glycosaminoglycan distribution. These findings
indicate significant LP reorganization in fetal lamb VFs during later gestational stages.
Future studies should further investigate these developmental changes to understand
their implications for human premature birth and associated voice pathology.

Investigating serum growth differentiation factor-8 concentrations in cats with early chronic kidney disease

Researcher: Kerrigan Fleming, Purdue
Mentor: William Whitehouse

Growth differentiation factor-8 (GDF-8), also known as myostatin, is a negative regulator of muscle growth. In people, GDF-8 is increased with chronic kidney disease (CKD). The objective of this study is to evaluate if the concentrations of circulating GDF-8 in cats with early CKD are increased compared to healthy cats. Associations of GDF-8 concentrations with age, sex, body weight, body condition score (BCS), muscle condition score (MCS), creatinine, blood urea nitrogen (BUN), symmetric dimethylarginine (SDMA), phosphorus, and urine specific gravity (USG) were also examined. Serum concentrations of GDF-8 in healthy (n=10), International Renal Interest Society (IRIS) stage 1 CKD (n=5), and IRIS stage 2 CKD (n=10) cats were quantified using a commercially available multispecies sandwich Enzyme-Linked Immunosorbent Assay (GDF-8/Myostatin; DGDF80; R&D Systems, Inc., Minneapolis, MN). GDF-8 was not different amongst healthy cats (2137 ±740 pg/mL) and cats with IRIS stage 1 (1785 ±530 pg/mL) and IRIS stage 2 (1961 ±638 pg/mL; P = 0.608) CKD. GDF-8 was negatively correlated to MCS (r = -0.517, P = 0.049), but no association was found between GDF-8 and the other selected renal parameters. However, age was significantly higher in IRIS stage 2 CKD cats compared to the healthy cats (P = 0.036), and GDF-8 was negatively correlated with age (r = -0.429, P = 0.032). In conclusion, GDF-8 could be a marker for muscle mass. Further evaluation of the functional role of GDF-8 with age and CKD in cats is warranted.

Characterization of Biomechanical Properties of Canine Sclera and Lamina Cribrosa Tissue Using Atomic Force Microscopy

Researcher: Lisa Hoard, Purdue
Mentor: Shin Ae Park

Glaucoma is a group of progressive optic neuropathies characterized by progressive degeneration of retinal ganglion cells (RGC) with no known cure. The initial insult responsible for the characteristic axonopathy is thought to occur within the lamina cribrosa (LC) region of the optic nerve head. As intraocular pressures rise, the LC is stretched and displaced, along with the other components of the corneoscleral shell, leading to narrowing of perforations containing RGC axons. While the LC is subject to variations in mechanical stimuli, it is a responsive and reactive structure. This project aims to characterize and analyze the biomechanical properties of the sclera and LC in canine eyes using atomic force microscopy (AFM). AFM uses a cantilever to scan across the surface of the sample and obtain various biomechanical measurements, such as force-displacement curves, by analyzing the deflection of the cantilever when the tip comes in contact with the sample. It is hypothesized that in advanced stages of glaucoma the sclera and LC will exhibit a significantly larger elastic modulus compared to normal control eyes. Thus, characterizing the biomechanical properties of normal canine eyes will serve as a control for future comparison with glaucomatous eyes. Overall analysis of biomechanical properties of tissues at the nanoscale from animals affected with naturally occurring glaucoma in life provides more insight into the adaptability of this fibrous shell in response to disease. This helps researchers identify new targets of intervention to manage or prevent advanced stages of glaucoma. By doing so there is potential for long-term preservation of vision in animals predisposed to glaucoma or diagnosed with early glaucoma. The aim of this study optimizes AFM methods to establish normal AFM data for the canine sclera and LC. Thus, this study establishes control data for future studies utilizing AFM to characterize and analyze biomechanical properties in canine eyes with varying types and stages of glaucoma.

MicroRNA differential expression analysis in placental tissue from cases of miscarriage in Brazil

Researcher: Kyra Holt, Purdue
Mentor: Andrea Pires dos Santos

Ureaplasma parvum is a commensal bacterium of the genitourinary tract that when in disbalance, it has been identified as a possible cause of miscarriage in women. MicroRNAs are known for regulating gene expression and their dysregulation have been associated with miscarriage as well. We hypothesized that miRNAs are differentially expressed in the placenta from women with normal delivery versus subjects who suffered an abortion, despite the presence of Ureaplasma. Placental samples from normal delivery and miscarriage cases in Northeastern Brazil previously placed into subgroups based on Ureaplasma parvum infection were submitted to DNA and RNA extraction and qPCR for microRNA differential expression analysis of miR-23a, miR-494-3p, and miR-146a-5p. This is a continuation of a project that had performed microRNA analysis of different markers. miR-23a was overexpressed in patients with normal delivery, despite the presence of Ureaplasma, when compared to patients that had a miscarriage in addition to Ureaplasma infection. MicroRNA expression differences between these groups could be used as prognostic markers for pregnancy in women. Testing in larger cohorts and serum samples are necessary and can contribute to a better understanding of the miscarriage process.

Comparison of debridement of the canine antebrachiocarpal joint by arthrotomy and arthroscopy

Researcher: Madelynn Luebcke, Purdue
Mentor: Sarah Malek

Pancarpal arthrodesis in dogs involves surgical removal of all articular cartilage to the level of subchondral bone. Arthrotomy is the surgical approach used for cartilage debridement that can increase postoperative complication rates due to damage to adjacent soft tissues and blood supply. The objective of this study was to evaluate the feasibility and efficiency of a minimally invasive approach to antebrachiocarpal (AC) joint debridement compared to arthrotomy. Nine pairs of cadaveric canine forelimbs were randomly assigned to one of three groups of six limbs to undergo antebrachiocarpal joint debridement via arthrotomy or arthroscopy using either a rigid arthroscope or a flexible needle arthroscope. Next, the joints were opened and photographed pre- and post-staining with India ink. Percentage of debrided surface area (%DSA) on pre-stained images and completeness of debridement (%C) on post-stained images were measured. Measurements were made for the articular surfaces of the bones contributing to the AC joint (i.e., radius (R), ulna (U), radiocarpal (RC) and ulnarcarpal (UC) bones). Analysis using a linear mixed model for repeated measures found that neither %DSA nor %C significantly differed between groups (P=0.0622 and P=0.5737, respectively). However, both %DSA and %C did significantly differ amongst bone surfaces (P<0.0001). While the %DSA did not significantly differ between the R and RC, the U and UC differed from both R, RC and from each other (P<0.0001). The %C of U was significantly lower than the other three bones (P<0.001). These findings demonstrate that arthroscopic debridement of the AC joint is feasible with similar efficacy to the traditional arthrotomy technique in achieving cartilage debridement.

Antifungal activity of antimicrobial peptide LL-37 on multidrug-resistant fungal pathogen Candida auris

Researcher: Casey Scarnati, Purdue
Mentor: Shankar Thangamani

Candida auris is a relatively recent emerging fungal pathogen. This pathogen is notable in its multidrug resistance to common antifungal cleaners and medication including fluconazole, amphotericin B, and echinocandins as well as its ability to grow biofilm on surfaces and equipment within medical facilities. The peptide LL-37 is a human peptide with antimicrobial activity against Gram-positive and Gram-negative bacteria. The goal of our research was to test the antifungal properties of LL-37 on the growth of colonies of both the wild-type yeast form (0387) and filamentous form (Δalm1) of Candida auris. Colonies of wild-type and mutated filamentous Candida auris were inoculated in YPD broth overnight. They were then cleaned and diluted with 1x PBS until an acceptable absorbance (0.32-0.35 for 0387 and 1.6-2 for Δalm1) was found on a spectrometer reading at a wavelength of 600 nm. The resulting final solution was combined with LL-37 at various concentrations, diluted further by a factor of ten seven times and grown via spot plating assay onto YPD agar plates. These plates incubated for 24 hours afterward. The colony growth following incubation of the wild-type samples was reduced with increasing concentrations of LL-37 while the filamentous Candida auris samples are expected to be unaffected. Concentrations tested (10-200 µg/mL) showed varying levels of growth restriction with the minimum inhibitory concentration found to be near 25-50 µg/mL. In conclusion, the antimicrobial peptide LL-37 has antifungal properties against certain forms of Candida auris. These results show the viability of LL-37 as a potential tool for the removal of Candida auris growth within patients and on medical surfaces.

Does tunnel handling hinder identification of clinical conditions in mice?

Researcher: Jordan Toney, Purdue
Mentor: Amanda Darbyshire

Refined handling techniques such as tunnel handling and cupping, are becoming increasingly popular as they improve the animal welfare of laboratory mice. Tunnel handling has been shown to reduce anxiety in behavioral tests, increase an animal’s willingness to interact with handlers even after brief periods of restraint, and improve physiological parameters like glucose tolerance, blood glucose, and corticosterone levels. It has also been shown to reduce data variability which has the potential to reduce the number of animals needed for a particular study, aiding in the replication of experiments. There is limited research regarding how tunnel handling affects the ability of technicians to identify clinical conditions of mice during routine cage changes. The purpose of this study is to assess how different types of tunnels affect skin lesion identification during cage changes of mice. 50 C57BL6/N female mice housed 5 per cage will have an artificial skin lesion. These lesions will be created under anesthesia using hair removal (hair plucking and shaving) and sharpie markers at various locations (dorsal neck, ventral neck, front limb, inguinal region, base of tail, or none). Six technicians will be instructed to change all 10 cages using the tunnel that is provided (clear, red, or opaque) or tail handling (control). During the cage change, the technician observes each mouse in the tunnel and documents any lesions noticed. The number of correct skin lesions identified for each type of tunnel and traditional tail handling will be compared. Our hypothesis is that the clear tunnels will allow for similar lesion identification as tail handling, which will aid in implementation of refined handling techniques.

Investigating viral associations in ruminant sinonasal tumors

Researcher: Caitlin Wager, Purdue
Mentor: Viju Pillai

Enzootic nasal adenocarcinoma of sheep is a contagious neoplasm of the ethmoid turbinate mucosa caused by the betaretrovirus Enzootic Nasal Tumor Virus-1 (ENTV-1). Clinically, affected sheep present with respiratory distress, anorexia, weight loss, and unilateral mucopurulent nasal discharge secondary to the tumor in the nasal cavity. This disease poses considerable economic and animal health challenges globally, as it impacts sheep productivity and welfare. Diagnosis primarily relies on histopathological identification of the adenocarcinoma, with molecular confirmation seldom utilized. Recently, the jaagsiekte sheep retrovirus (JSRV), another betaretrovirus known to cause lung tumors in sheep, was identified in a nasal adenocarcinoma case that tested negative for ENTV-1. Given the infrequent use of molecular confirmation in enzootic nasal adenocarcinoma diagnosis and the detection of JSRV in an ENTV-1 negative case, this retrospective study aims to assess the presence of ENTV-1 or JSRV in histologically diagnosed nasal adenocarcinoma cases. DNA was extracted from archived formalin-fixed paraffin-embedded (FFPE) specimens of histologically confirmed nasal adenocarcinomas. A polymerase chain reaction (PCR) was performed using primer pairs specific to ENTV-1 or JSRV to determine the viral presence. Understanding viral etiology is crucial for accurate diagnosis, effective management, and control of the disease. This study seeks to elucidate the viral associations in nasal adenocarcinomas and enhance diagnostic protocols by incorporating molecular techniques.

Analysis of Fluid Dynamics of the Equine Upper Airway Using 3D Printed Models

Researcher: Audrey Wood, Purdue
Mentor: Michelle Tucker

This project investigates the fluid mechanics of the equine upper airway via the creation of 3D printed anatomical replicas of the trachea, larynx, and nasal passages. These models allow us to assess the performance of these structures in a state of health and provide insight into the dynamic airflow patterns within the equine airway.

The objective of this study is to create separate 3D printed models of each portion of the airway (trachea, larynx, and nasal passages) using 3D Slicer v. 5.6.2. These models will be created in such a fashion that they can be analyzed separately or fit together seamlessly to form the entirety of the airway for more specific applications. The fluid dynamics of air flowing through these structures will be evaluated using computational fluid dynamics (CFD) simulations under physiological conditions.

Results from this study may be used to inform treatment protocols for diseases of the upper airway; this is especially applicable to performance horses affected by both symptomatic and subclinical cases of laryngeal neuropathy. This study offers an approach to improving therapeutic procedures in equine respiratory health.

Can injectable antibodies give failure of passive transfer dairy calves a new lease on life?

Researcher: Emma Zaicow, Purdue
Mentor: Andrew Hubner

Bovine placentation prevents the mixing of maternal and fetal blood, leaving neonatal calves completely dependent on colostrum for immune transfer. Failure of passive transfer (FPT) is defined as a serum protein concentration of <5.5g/dL and can predispose a calf to premature morbidity, decreased production, and increased susceptibility to disease. Several management practices exist to prevent FPT in dairy calves, but our laboratory seeks to determine if an injectable antibody solution (Bovi-Sera, Colorado Serum Company; Denver, CO) can improve outcomes for calves with FPT. In an ongoing study across three locations, 408 FPT Holstein calves were randomly assigned to receive 60mL of Bovi-Sera administered subcutaneously at enrollment, which occurred within 7 days of life, or to remain as untreated controls. Prior to treatment and 1-week post-enrollment, serum samples were collected then plated on IgG radial immunodiffusion plates. The diameter of immunodiffusion was measured after 24 hours of incubation at room temperature and converted to mg/dL of IgG. compared between treated and untreated FPT calves. For calves with enrollment IgG concentrations less than 1200 mg/dL, treatment tended to increase circulating IgG one week after enrollment (Control: 841 mg/dL, Treatment: 922 mg/dL; P = 0.09) and significantly decreased farm-diagnosed cases of diarrhea (Control: 32.0%, Treatment: 8.7%; P = 0.04). However, for calves with IgG less than 1200 mg/dL, treatment did not affect average daily gain through 21 days of life (Control: 0.29 kg/day, Treatment: 0.31 kg/day; P = 0.65). Though not labeled specifically for the treatment of FPT, these findings suggest that Bovi-Sera may prevent neonatal diarrhea in Holstein calves.

bTBI-on-a-Chip: Synaptic Loss Following Blast Induced Traumatic Brain Injury or Biochemical Injury in Vitro

Researcher: Maddison Claybrooke, Franklin College
Mentor: Riyi Shi

Annually, up to 74 million people worldwide suffer traumatic brain injuries (TBIs), many of which go untreated. In particular, TBIs sustained as the result of a pressure wave due to an explosive, called blast TBIs (bTBI), are less likely to be adequately treated due to the initial injury often being mild and difficult to diagnose. TBI is classified into primary and secondary injury. Primary injury is the initial injury resulting from tissue damage and is irreversible, while secondary injury is damage due to the immediately ensuing biochemical cascades that spread from the primary injury. These biochemical cascades include inflammation and enhanced presence of reactive species, such as acrolein, which, if left untreated, can persist for years after the TBI is sustained and damage uninjured cells. These spreading biochemical injuries to the brain can result in an increased chance in developing conditions like epilepsy, neurodegeneration, and psychological disorders. One possible factor linking TBI and long-term pathologies is synaptic loss due to damaged neurons and axons. Synapse loss has already been linked to neurodegeneration, but the mechanisms relating synaptic loss, TBI, and secondary injury pathology remain unclear. While animal models have been helpful in understanding the pathological effects of both primary and secondary injury, the cellular and subcellular mechanism underlying the findings in animal studies remain unclear due to the high number of cofounding variables in animal models. To fill this gap in current knowledge, we developed a platform to deliver clinically relevant blast injuries to primary murine cortical networks in vitro, called bTBI-on-a-Chip, which allows direct study of the cellular and subcellular changes that arise from injury. Using this system, we delivered two types of injury to cultures and studied the resulting changes in synapse density. First, primary blast injuries were delivered to neuronal networks. Second, given that acrolein is a key component of secondary injury, we added a clinically relevant concentration of acrolein to uninjured cultures in order to determine if acrolein alone could cause similar synaptic loss as primary and secondary injury. After each type of treatment, we measured the change in number of synaptic terminals 24 hours post treatment using fluorescent imaging. There were significantly fewer pre and post synaptic terminals on each neuron after both bTBI and acrolein treatment. Together, these findings demonstrate that acrolein alone can cause synapse loss and could therefore be crucial in synaptic changes in secondary injury. Furthermore, our results confirm that even mild blast injury disrupts synaptic numbers in single cells. These findings may help elucidate the relationship between injury, synapse loss, neuron rewiring, and brain pathologies.

Identification of a Novel Candidate Variant in CLPX for Spinocerebellar Ataxia in a Mixed Breed Dog

Researcher: Justin Kim, Vanderbilt
Mentor: Kari Ekenstedt

Spinocerebellar ataxia (SCA) is a progressive neurodegenerative disorder primarily affecting the cerebellum, resulting in the loss of motor control and voluntary muscle coordination. While prevalent in Jack and Parson Russell Terriers due to autosomal recessive mutations in electrolyte channel coding genes such as CAPN1, KCNJ10, SCN8A, SLC12A6, and SPTBN2, an atypical case of SCA was recently documented in a mixed breed dog. Health records and necropsy findings identified paraparesis, SCA, anemia, and retinal degeneration in this individual. Because SCA is an inherited condition, whole-genome sequence (WGS) was generated for the affected dog. The known canine mutations above were not present in the mixed-breed dog’s genome. Principal Component Analysis of genomic data was utilized to confirm the breed identity. The dog’s WGS was then screened for private variants compared to >700 unaffected dogs. This revealed a homozygous 4-base-pair frameshift mutation in CLPX, a gene that encodes for caseinolytic mitochondrial matrix peptidase chaperone subunit X involved in mitochondrial protein degradation, as a novel candidate gene for SCA in any species. In-silico tools predict a frameshift and a premature stop codon within 591 amino acids, truncating 6.64% of the protein. Our study is the first to explore the association of a CLPX mutation with SCA. This connection is potentially significant for human health due to the high evolutionary conservation of CLPX across species.

Investigating suspected mosquitos for the presence of Cache Valley Virus in Indiana

Researcher: Rubyleane Linton, Prairie View A & M
Mentor: Rebecca Wilkes

Cache Valley Virus (CVV) is a rare but dangerous orthobunyavirus that causes abortions and
congenital disabilities in small ruminants, such as goats and sheep. The virus is zoonotic, having
implications for human health as well. Mosquitoes are considered the primary vector of CVV. It
is an emerging pathogen in the U.S and recent serologic testing of aborted small ruminants
suggests this virus is present in Indiana.
This study aimed to identify CVV in Indiana by testing mammal-biting mosquito species
collected from different parts of the state. A previously published real-time reverse transcriptase
PCR was verified for use in the lab and utilized to detect target sequences of CVV within nucleic
acids extracted from mosquito pools collected in 2023-2024. The mosquito species that were
analyzed include Aedes albopictus, Aedes canadensis, Aedes j.japonicus, Aedes vexans,
Anopheles punctipennis, Anopheles quadrimaculatus, Coquillettidia perturbans, and Culex
pipiens (mixed).
While the study is ongoing, no CVV was detected in the 165/648 pools of mosquitoes tested so
far. Detection of positive pools would confirm the presence of CVV in the state. Additionally,
testing various species of mosquitoes from different parts of the state would provide information
regarding the location of the virus and the species of mosquitoes responsible. Traps that collect
these mosquitoes mimic preferred hosts and environments to attract particular species. Infected
mosquito species knowledge is essential in determining which traps will be the most effective in
Indiana to increase the possibility of detecting this virus in the mosquito vector.

Identification of mast cells within the tumor immune microenvironment of canine urothelial carcinoma

Researcher: Ally Schimpf, Purdue
Mentor: Meaghan Broman

There are over 80,000 reported cases of human urothelial carcinoma (UC) each year within the United States which contributes to about 17,000 deaths. Human UC research has shown that the tumor immune microenvironment can contain a variable number of mast cells whose presence may be a prognostic indicator for the response to treatment. Certain breeds of dogs such as Scottish and West Highland White terriers have a high incidence of UC which share many histological, morphological and treatment response similarities with human UC. However, little is known about the number and role of mast cells in canine UC. The goal of this project is to classify mast cell involvement within the tumor immune microenvironment of canine UC while determining mast cell association with prognosis for possible subsequent immunotherapy studies. Canine UC samples obtained from patients of the Purdue University Veterinary Hospital were evaluated by histology and immunohistochemistry. Visiopharm AI assisted technology was utilized for mast cell count, location, and structural distances. The recruitment of mast cells into the tumor microenvironment is governed in part by chemokines secreted by tumor cells. Five different canine UC cell lines were propagated in vitro and evaluated by PCR for chemokine expression. This project will establish foundational data validating the use of canine cancer patients as an invaluable model for human cancer immunological research leading to future immunotherapy studies benefiting both people and veterinary patients.