Research Projects

Amyloid deposits have been detected in the majority of feline diabetic patients. These deposits originate from islet amyloid polypeptide (IAPP or amylin). Amylin is a normal satiety hormone that is produced and co-secreted with insulin by beta-cells, which are the most common cell type in the islets of Langerhans in the pancreas. However, amylin misfolding leads to the development of amyloid deposits, which have been associated with beta-cell death during the progression of diabetes. IAPP aggregation can be inhibited by several molecular entities such as silibinin and resveratrol. However, these agents have poor bioavailability and cause a variety of pharmacological effects. Currently, there is no commercially available drug treatment to stop or prevent pancreatic amyloidosis in diabetes mellitus. The goal of this project is to identify inhibitors of feline IAPP (fIAPP) fibril formation, and to demonstrate that the aggregation of fIAPP can be modulated by IAPP-interactive compounds in vitro. Three series of urea-based compounds were developed for this purpose, and their ability to reduce the formation of fibrils from IAPP was assessed in vitro using biophysical methods such as Thioflavin T (ThT) fluorescence assays, dynamic light scattering, and transmission electron microscopy (TEM). Six potent inhibitors of IAPP fibril formation were identified. This study has the potential to point toward new therapeutic strategies for type 2 diabetes in cats.

Many adults struggle with over consumption and binge-like drinking of alcoholic beverages. Almost always these beverages are not pure alcohol but have other taste and flavor components. In preclinical research alcohol is often studied in a pure form in order to avoid confounding effects. However, flavor may be a significant risk factor leading to increased binge-like drinking and alcohol intake. In the present study, we first determined a preferred concentration of flavor (cherry Kool-Aid) to be consumed with 20% alcohol. Mice(8 females, 8 males/group) underwent two bottle choice (2BC) drinking sessions with pure water in one bottle and alcohol flavored with 7 concentrations(0-5%) of Kool-Aid in the other bottle. We tested concentrations for two 24-hour sessions each and measured the amount of alcohol consumed and preference to water. A concentration of .1% was determined to be the preferred concentration based on preference and intake. We next compared the .1% flavored alcohol to regular alcohol in two 2-hour 2BC sessions to determine if flavored alcohol was preferred over regular alcohol. Both male and female mice showed greater intake of flavored alcohol compared to regular alcohol. A second cohort of male and female mice are currently undergoing several weeks of binge-like drinking either consuming .1% flavored alcohol or regular alcohol using a drinking in the dark model. These mice are being tested for binge-like drinking behavior for 6 total weeks to see if flavored alcohol promotes a higher level of binge-like drinking and blood alcohol levels. 

As many as 1 in 5 military veterans are diagnosed with posttraumatic stress disorder (PTSD). Service dogs have recently emerged as a complementary intervention, but additional scientific evidence supporting their use is needed. In this study, we examined veterans with PTSD involved in a service dog program, gathering feedback from them and their spouses to (1) understand their day-to-day experiences, (2) understand the influence of service dog partnership, and (3) assess whether the response rate was associated with demographic characteristics. This study used ecological momentary assessment to obtain real-time information from n=170 veteran and n=87 spouse participants longitudinally (baseline and 3 months later), comparing veterans with a service dog to veterans receiving usual care alone. Open-ended feedback prompts occurred four times daily through a smartphone app and were analyzed using a mixed-methods approach. Four themes were identified through qualitative content analysis: life events, technical issues, health, and sleep. Compared to veterans on the waitlist, veterans with service dogs mentioned having less overall anxiety, more resilience, and a more positive outlook. Service dog partnership did not affect spouse feedback. On average, feedback response rates were 8% for spouses and 11% for veterans. Time of day and demographics were not found to be significantly associated with response frequency. While the quantity of feedback received was relatively low, veteran and spouse responses were rich in detail and showed a meaningful influence of the service dog on day-to-day well-being. Based on these results, service dogs may be a useful adjunctive intervention for military-connected PTSD.

Spinal cord injury (SCI) is marked by primary injury (physical impact) and a secondary injury (chemical injury) that amplifies the damage and functional deficits triggered by the primary trauma. An important hallmark of secondary injury is oxidative stress. Acrolein, a key player in oxidative stress, is a toxic aldehyde that is elevated significantly following SCI. Acrolein increases reactive oxygen species (ROS) and lipid peroxidation, thus furthering the damage. Acrolein is of special importance because it has a longer half-life than known ROS and inhibits important endogenous antioxidative stress enzymes. Previous research has identified aldehyde dehydrogenase 2 (ALDH2) as an important antioxidative enzyme. ALDH2 metabolizes acrolein to suppress oxidative stress, but can also be inhibited by acrolein, especially during acrolein overload. Over 600 million people worldwide exhibit an inactive form of isoenzyme ALDH2 (ALDH2*2) that is linked to several diseases, such as Alzheimer's, Parkinson's Disease, and alcohol flushing response. The overall objective of this study was to assess the role and the potential therapeutic value of ALDH2 and the neuroprotective effect of Alda-1, an ALDH2- selective agonist, in SCI, using a transgenic mouse model with ALDH2*2. There were two central hypotheses for this study: 1) transgenic mice would exhibit a higher concentration of acrolein compared to wild-type following SCI, and 2) treatment with Alda-1 would amplify ALDH2 function in both wild-type and transgenic mice, reducing acrolein concentration in the spinal cord. Findings from this study further illustrated ALDH2 as a target for attenuating secondary injury of SCI and introduced Alda-1 as a potential treatment for SCI.

The integrity of the laryngeal vocal folds is important in the creation of the voice. They are a known target organ for estradiol, and their function is negatively affected by systemic dehydration. Estradiol in humans has been shown to increase capillary permeability by vasodilation, modulate vasopressin release, and respond to changes in blood osmolarity. The objective of this study was to determine if the presence of estradiol influences vocal fold histology during systemic dehydration. Twelve female Sprague Dawley rats were divided into dehydrated (n=6) and euhydrated groups (n=6). Each hydration group had intact (n=3) and ovariectomized (n=3) rats to simulate the loss of sex hormones. Rats were ovariectomized 14 days prior to the start of the study to allow hormone levels to stabilize. Serum estradiol levels and packed cell volume (PCV) were determined at the start of the study. During the 5-day baseline period, all rats were given ad lib food and water; the rats were weighed, and water intake was measured daily. Following the baseline period, a 5-day dehydration period in which the dehydrated group received 4 ml water/100 g of baseline body weight daily and euhydrated continued to receive ad lib water. At the end of the dehydration period all rats were euthanized, blood was collected to measure PCV and estradiol levels, and the larynx was dissected and placed in 10% neutral buffered formalin for histological processing. Larynges were sectioned at 4um in the coronal plane and stained with hematoxylin and eosin for routine morphological assessment. Immunohistochemical labeling of laryngeal estrogen receptors will be quantified using digital software.

Avian Cholera, caused by the bacteria Pasteurella multocida, is a respiratory and septicemic disease of domestic and wild avian species. Disease control is challenged by a lack of effective vaccines, identifying reservoirs of the disease agent, and predicting drivers of outbreaks. We analyzed publicly available outbreak reports to explore the spatio-temporal distribution of avian cholera outbreaks in the US.[1] Reports of sightings of 24 species of birds from each county in the US between January 2002 and May 2022 were extracted from eBird. Daily precipitation, temperature, and humidity data for each county were also extracted. Exploratory mapping and logistic regression were conducted to determine the association between temperature, precipitation, and the distribution of avian cholera within the US. Over the study period, there were 263 reported cases of suspected or confirmed avian cholera events with a median number of 241 birds affected per event. Snow geese (Anser caerulescens) and American Coots (Fulica americana) were the most common avian species present during outbreaks and appeared in 144 and 131 events, respectively. We hypothesize that avian cholera is associated with higher temperatures. The visualization of these spatial and temporal trends in avian cholera could be used to target surveillance in regions and during times where vulnerable species are most at risk.

[1] Retrieved May, 31, 2022, from the Wildlife Health Information Sharing Partnership event reporting system on-line database, https://whispers.usgs.gov

Mild equine asthma is a common disease in racehorses developed in response to particulate matter (PM) exposure and is associated with poor performance.  PM smaller than or equal to 2.5 µm (PM2.5) is one size of dust defined as a health hazard by the EPA. Feeding hay and bedding horses on straw are associated with higher levels of PM exposure in the breathing-zone, in comparison to pelleted feed and wood shavings, respectively. However, particulate exposure varies significantly between horses, despite similar management practices. We hypothesize that within different racetracks, PM2.5 concentrations in the breathing-zone vary based on management practices and horse activity while stalled. . Therefore, real-time particulate monitors and video cameras were placed on Thoroughbred racehorses' halters to quantify breathing-zone PM exposure associated with an activity. Horses were stalled under normal management conditions and monitored with a custom-made personal PM monitor (Plantower PMS 7003) and activity was recorded (Hawkeye Firefly Q6) over 20-30 minutes. Video and PM data were uploaded to the EVADE software (NIOSH) to quantify dust exposure during various activities. We expect that this approach will identify activities and management practices associated with higher PM exposure and provide new solutions to lessen the burden of mild equine asthma.

Zebrafish (Danio rerio) are useful in scientific research due to their close genetic similarity to the human genome and fast reproductive lifecycle. Their increased use in scientific research calls for improved methods of monitoring their health, as current methods involve multiple types of testing including submission of whole fish to identify various pathogens. This study aims to create a novel sampling technique by exposing nitrocellulose filters to sump water over the course of twelve weeks. The filter was compared against other known testing methods of swabbing biofilm from the sump and passing sump water through a vacuum filter.  It was hypothesized that the nitrocellulose filter would identify more pathogens over time, reducing the need for multiple testing methods. Weekly PCR testing was conducted to detect Mycobacterium chelonae, zebrafish picornavirus, Myxidium streisingeri, Mycobacterium fortuitium, and Pseudoloma neurophilia. Nitrocellulose filters were the most consistent in identifying pathogens every week as their sensitivity to identify pathogens increased over time. The vacuum filter was also a consistent and sensitive method, but to a lesser degree than the nitrocellulose filters over time. Sump tank swab samples were the least sensitive in pathogen detection as its positive pathogen identification results were inconsistent. In preliminary results, none of the methods have been able to detect P. neurophilia. This suggests that the nitrocellulose filters may be a useful method of monitoring the health of laboratory zebrafish colonies for most of the agents tested.

Candida auris, an emerging multi-drug resistant fungal pathogen poses a serious threat and causes invasive infection in humans. Majority of C. auris isolates exhibit resistance to the currently FDA-approved antifungal drugs. Thus, there is an urgent need to understand the pathogenesis to develop novel therapeutics to combat this pathogenic yeast. C. auris primarily colonizes in the skin leading to systemic invasive infections. Therefore, understanding the factors regulating C. auris colonization in the skin is critical to gain insights into the pathogenesis of this fungal pathogen. In this project, beta defensin-3 (HBD) which is one of the major antimicrobial peptides expressed in the human skin was tested against C. auris. Antifungal activity of HBD against different clades of C. auris isolates was tested using physiologically relevant conditions using in vitro assays and the results will be discussed. Future research efforts to focus on understanding the antifungal activity of HBD using in vivo mouse studies will open the door to modulate host antimicrobial peptides to prevent and treat this fungal pathogen in humans.

Introduction: Atherosclerosis is characterized by calcification in the walls of arteries that can lead to constriction and ultimately blockage of the whole vessel. Since the most common clinical sign for atherosclerosis in birds is sudden death and the diagnosis is usually determined post-mortem^{[1, 2]}, further diagnostic imaging is needed to improve early diagnosis. High-resolution magnetic resonance imaging (MRI) is a promising modality for assessment of arterial atherosclerotic plaques. This pilot study aims to investigate MRI as a potential technique to diagnose and quantify atherosclerosis in birds.  

Methodology: A total of 5 cadaver of birds with unknown causes of death from different species were used. These birds were collected by the community practice service at Purdue University College of Veterinary Medicine. The cadavers were scanned with a 7T MRI system (Bruker Biospec). The T₂ turbo rare scan was performed without fat suppression with the voxel volume of 0.5*0.5*0.5mm². This implies that the field of view was 50*50mm² and the slice thickness was also 50mm. For the bigger birds like owls and macaws, for ease of scanning, the outer gradient was used, and two separate scans were done to look at the heart and the aorta. Whereas in smaller birds like doves and parakeets, the inner gradient of the MRI was used and only one scan was done to observe the whole area at once. There were no gaps between slices for precision. Morphologic assessment of atherosclerotic plaques (area measurements of calcification) will be performed at the end of the study using ImageJ® software. The birds, after scanning were sent for a necropsy to confirm the presence of atherosclerotic lesions. 

Observations and Results: Preliminary evaluation demonstrates that MRI is feasible and reproducible in cadavers of birds. Given the wide range of body size among the enrolled birds (30g-500g), we plan to enroll a larger sample of subspecies of birds. This will also give us a better representative assessment of atherosclerosis in different sizes of birds before scanning alive animals. By the end of the study, we hope to be able to quantify atherosclerotic lesions in birds which would help to assess the severity of the disease. 

References:

1. Bavelaar, F. J., & Beynen, A. C. (2004). Atherosclerosis in parrots. A review. The veterinary quarterly, 26(2), 50–60. https://doi.org/10.1080/01652176.2004.9695168
2. Bertozzi, G., Cafarelli, F. P., Ferrara, M., Di Fazio, N., Guglielmi, G., Cipolloni, L., Manetti, F., La Russa, R., & Fineschi, V. (2022). Sudden Cardiac Death and Ex-Situ Post-Mortem Cardiac Magnetic Resonance Imaging: A Morphological Study Based on Diagnostic Correlation Methodology. Diagnostics (Basel, Switzerland), 12(1), 218. https://doi.org/10.3390/diagnostics12010218
3. Michaud, K., Genet, P., Sabatasso, S., & Grabherr, S. (2019). Postmortem imaging as a complementary tool for the investigation of cardiac death. Forensic sciences research, 4(3), 211–222. https://doi.org/10.1080/20961790.2019.1630944

This study investigated the effect of the covid pandemic on the relationship between owners and their pets and the behavior issues of the pet cats and dogs. Longitudinal surveys were conducted with participants who were 18 years and older and recruited via Amazon’s crowdsourcing platforms between June 2020 and December 2021. Participants were asked pet-related questions including perceived closeness of pet-owner relationship, behavior issues, and interactions during four different phases of the pandemic: pre-pandemic, lockdown, reopening, and recovering. Pet-owner relationship was quantified using the validated instruments, including the Cat-Owner Relationship Scale (CORS), the Monash Dog Owner Relationship Scale (MDORS), and the Inclusion of Other in the Self (IOS) diagrams, all with a higher score indicating a closer relationship. Both CORS and MDORS have human-animal interaction (HAI), Emotion, and Cost subscales. A total of 1,761 dog owners, 1,849 cat owners, and 657 non-pet owners completed one to six surveys during the study period. The results showed an increase in all pet-owner relationship measures starting from the lockdown phase and maintained the levels in the following phases. The most common behavior issues in cats and dogs are signs of aggression, fear of noise, and excess licking. This study suggests that the initial shock of the pandemic forced pet owners to adapt to the extended periods of staying at home, leading to increased interactions and commitment to both pet cats and dogs, and ultimately positively affecting pet-owner relationships. This study also supports that pet behavior changes during the pandemic resulting from their owners spending more time at home.

Aluminum-containing adjuvants have been widely used for over 90 years in many human and veterinary vaccines to enhance immune response. Today, the most commonly used aluminum adjuvants are aluminum hydroxide (AH) and aluminum phosphate (AP). While often collectively referred to as “alum”, these adjuvants possess different physical and chemical properties. In spite of these differences, most of the current literature on aluminum adjuvants reports only on AH. We are investigating if there are different mechanisms of action between AH and AP. To do this, human THP-1 cells, differentiated with phorbol 12-myristate-13-acetate (PMA), were used as a model system for antigen-presenting cells. The secretion of IL-1α, IL-1β and IL-18 was measured by enzyme-linked immunosorbent assay (ELISA). Priming of the cells with lipopolysaccharide (LPS) followed by treatment with the aluminum adjuvants markedly increased the secretion of IL-1α and IL-1β and had less effect on IL-18.  AP induced greater secretion of  IL-1α, IL-1β, and IL-18 than AH. Incubation of LPS-primed THP-1 cells with inhibitors of different signaling pathways revealed differences in the regulation of IL-1α and IL-1β secretion, and differences between AH and AP.  This indicates that these adjuvants may activate different signaling pathways in antigen-presenting cells. Pointing out the differences between these two aluminum adjuvants may allow for better manipulation of immune response, as well as aid in making informed decisions when choosing an adjuvant for vaccine formulation.

Laboratory mice will interact with intracage shelters when these are provided. However, even though mice spend a significant amount of time in their shelters, our pilot studies suggest that markers associated with chronic stress are elevated. Chronic stress in laboratory rodents is a welfare issue that can alter behavior and physiology, introducing uncontrolled variables and reducing comparability to human disease. In this study, mice were given access to clear, red, yellow, or blue intracage shelters. The control group was not provided an intracage shelter. Once the mice acclimated to the shelters, anxiety was measured using the open field test. A blood sample was also collected to compare serum corticosterone (ELISA) and complete blood count (neutrophil:lymphocyte ratio) between groups. Behavioral and physiological assessments were combined to determine how the provision of an intracage shelter and its color impact well-being. Comparing the no-shelter group to the groups that have shelters will assess the impact of intracage shelter presence on stress whereas comparing the groups with intracage shelters by color will assess the impact of light intensity on stress. As the mice will be acclimating to the shelters until late July, there is no data to report yet. Cage-side observations have shown more mice using the intracage shelters as the acclimation period progresses, with a current average of 30% of mice using the shelters when observed. Therefore, we are hopeful this project may help guide the recommendations for the use of intracage shelters for the enrichment of laboratory rodents.

Osteocytes are the most abundant cells in the bone, comprising more than 90% of the cells within the mineralized bone matrix. Far from being a passive cell, osteocytes are indispensable for bone homeostasis and normal skeletal function. Osteocytes are hypothesized to be sensitive to mechanical loading and produce signals that alter bone formation by osteoblasts, but the mechanisms are poorly understood. The goal of this study is to characterize the osteocyte lacunar-canalicular networks between different species of vertebrates and how these networks may affect bone adaptation to mechanical loading in these different taxa. Tibiae (or tibiotarsi, in the case of birds) were harvested from different vertebrate species including birds (guinea fowl, chukar, emu, ostrich), mammals (rat, mouse, opossum), and reptiles (monitor lizard, iguana). A total of 15 tibiae were studied. Harvested bones were fixed in 10% neutral buffered formalin and embedded in epoxy for structural support. Transverse sections (~700µm) originating near the midshaft of the bones were collected using a diamond blade saw and hand-ground to a thickness of approximately 100µm. Finally, samples were stained using Alexa 488 for imaging by confocal microscopy. A Matlab program developed in our laboratory was used to characterize the lacunae (geometry, orientation) and the dendritic canalicular processes from each osteocyte in the posterior region of the bone. We expect that this comparative research approach will provide novel insights into the role of lacunar-canalicular networks of osteocytes in bone remodeling.

The current gold standard for detecting tick-borne pathogens is quantitative PCR (qPCR). However, this requires individual testing for each pathogen and performing many tests to obtain an accurate diagnosis. The purpose of this study was to develop a targeted next-generation sequencing (NGS) assay for tick-borne pathogens and to perform a feasibility study and initial validation of the protocol. Test feasibility and analytical specificity of the assay were evaluated with a set of validated positive clinical samples from dogs provided by the Vector Borne Disease Diagnostic Lab, North Carolina State University (VBDDL). Diagnostic sensitivity and specificity were evaluated with nucleic acid samples extracted from a set of known positive and negative clinical samples provided by Texas A&M and VBDDL, based on qPCR testing. For each known sample the pathogen target regions were amplified via PCR, DNA library was prepared with the Ion AmpliSeqä Library Kit Plus, loaded onto a chip using the Ion Chefä, and sequenced with the Ion Torrent S5ä. The data were assembled using SPAdes and mapped to a reference file prepared with the sequences from the targeted regions of the tick-borne disease pathogens. Geneiousä software was used to process the raw sequence data and the BLAST analysis was performed to confirm the results. The primer sets used for amplification were determined to be specific for the intended targets, based on sequence analysis of the amplified products. Cohen’s kappa was calculated to be 0.53, which indicates moderate agreement between the qPCR assay and the targeted NGS assay. The positive percent agreement was 92%, and the missed qPCR positives were due to failure to detect pathogens in samples with high Ct values. The negative percent agreement was 59%, due to detection of organisms by targeted NGS that were missed by qPCR. Additional testing will be done to evaluate the NGS analytical sensitivity.

Stereotactic Radiation Therapy (SRT) has risen in prevalence for the treatment with curative intent of canine nasal tumors because it can deliver higher radiation doses to more concentrated areas in less fractions compared to previous forms of radiation. Preliminary studies utilizing SRT technique have reported moderate toxicities. Rare severe cases associated, were reported with single dose fraction or tumor involvement to the hard palate or the skin. Treatment margins surrounding tumor volumes (TV) are variable between studies. The purpose of this study was to analyze Purdue’s experience utilizing tight TV margins and taking 1 day of rest between fractions. The medical records for 11 dogs receiving SRT for nasal tumors between 2014-2019 were reviewed.  Any dogs that received prior radiation therapy (RT) were excluded. Follow-up information was collected, if available, from hospital records, primary veterinarians, and owners. The median survival time (MST) was 627 days.  Nine of 11 (81%) dogs presented mild acute toxicities. Two dogs were euthanized before data was collected for late toxicities. Of the 9 dogs remaining, 5 (55%) had moderate late toxicities. Toxicities may have been under reported.  A linear accelerator was used to deliver 3 fractions with intensity modulated radiation therapy (IMRT) at a dose of 8 Gy every other day, for a cumulative dose of 24 Gy. Modified Adam’s staging for canine nasal tumors was used to classify tumor progression. PTV margins averaged 3 mm, except in 2 cases. Organs at risk (OAR) that received higher radiation doses appeared to have a higher risk for side effects.  Conclusions are yet to be determined.

Baylisascaris procyonis infection is an emerging zoonosis in North America, Europe, and Asia. Adult B. procyonis are found in the small intestine of raccoons and parasitic eggs are shed in their feces. Accidental ingestion of the eggs can result in larva migrans in humans and over 150 different species of mammals and birds. Migrating B. procyonis larvae travel through somatic tissues (visceral larva migrans), the brain (neural larva migrans – NLM), and eyes (ocular larva migrans – OLM), inflicting serious damage. While several cases occur as covert disease, infection can lead to irreversible vision loss, encephalitis, and death. Currently, there is a lack of understanding of B. procyonis larval virulence factors that play a role in the pathogenesis of this parasite. This study aims to characterize the immunoreactive clones that have been identified via screening of the larval cDNA expression library. Sequencing and bioinformatic analyses were performed on the previously identified and isolated cDNA clones, and their putative identities were established. A few select clones were over-expressed in a bacterial expression system and the recombinant proteins were purified using metal-affinity chromatography to further establish their sero-reactivity and to generate antisera. Experiments are ongoing to establish their differential expression between various B. procyonis life cycle stages to identify a larva-specific antigen. Overall, this study is part of a larger project aimed at discovery of B. procyonis larval effector molecules that play a role in host-pathogen interaction during the migratory phase of the parasite in the abnormal human host.

In 2001, the ASPCA Animal Poison Control Center (APCC) reported that a review of calls revealed cases of acute renal failure in dogs that ingested grapes or raisins. These cases were rare, and while very few incidences of grape or raisin ingestion result in acute renal failure, there were sufficient confirmed cases to warrant a warning letter to the veterinary community in JAVMA. Grapes and raisins were analyzed for known renal toxins, but none were identified. In 2021, the ASPCA APCC reported two cases in which dogs developed acute renal failure following ingestion of large amounts of tartaric acid (cream of tartar). Tartaric acid can be present in some grapes and raisins in widely varying amounts, and in some instances is not present at all. The current study aims to evaluate if canine kidneys can be adversely affected by tartaric acid. Our hypothesis is that MDCK cells, a canine kidney cell line, will be adversely affected by exposure to tartaric acid in vitro. MDCK cells were plated in 96 well culture plates and grown to near confluency. The cells were dosed with PBS (vehicle), tartaric acid (100 or 10mM), valproic acid (positive control: 100 or 10mM), or malic acid (negative control: 100 or 10mM) and incubated at 37^°C. At 24hrs, the cells in each well were examined microscopically, and, in each well, an LDH cytotoxicity assay and an MTT cell viability assay were performed. Preliminary results indicate that MDCK, canine kidney cells, are sensitive to the toxic effects of tartaric acid. We conclude that tartaric acid, if present in sufficient amounts in grapes/raisins, or cream of tartar, may contribute to acute renal failure in dogs following its ingestion.

Interventions to combat the opioid crisis are generally focused on opioid use disorder alone and not polysubstance use disorders. However, in the real world, multiple drugs are often used in combination or to self-medicate withdrawal symptoms. The purpose of this study was to design a preclinical model of self-medication with oxycodone during withdrawal from alcohol dependence to further characterize the behavioral and neural effects of polysubstance use disorder. A total of 64 mice were split into two groups, Oxycodone+Chronic Intermittent Ethanol Vapor Exposure (O+CIE; 16 male, 16 female) or Oxycodone alone (OA; 16 male, 16 female). The O+CIE mice first got four weeks of CIE with a rest week in between each CIE week. The mice from all groups received an intravenous catheter to self-administer oxycodone for 5 days. The O+CIE mice had another week of CIE followed by self-administration of oxycodone for 4 days in all mice before behavioral tests were performed. Brains were collected after the last IV self-administration. Male mice of the O+CIE group self-administered more oxycodone during the last 3 days of self-administration compared to the OA group, but there were no differences between groups in the female mice. This suggests a potential sex-dependent effect of withdrawal from alcohol dependence on oxycodone self-administration, but female mice self-administered high levels of oxycodone which may limit the ability to detect if withdrawal from alcohol dependence promotes increased oxycodone intake (ceiling effect). We are currently processing brain tissue from the mice to assess brain-wide neural activity associated with withdrawal from oxycodone and alcohol to determine if there are major sex differences.

Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Destabilization of the medial meniscus (DMM) in mice to model post-traumatic osteoarthritis was first documented in 2004 and now is the most common surgical model used to induce post-traumatic osteoarthritis in mice. Despite its frequent use, difficulties in comparing results across studies have been acknowledged. The goal of this study was to better understand sources of variability by surveying investigators and research groups around the world on their laboratory animal characteristics, management, and surgical techniques used in the DMM procedure. Following Institutional Review Board approval, a comprehensive Qualtrics survey was developed, tested, and distributed to first and last authors of manuscripts published and curated in PubMed that used the DMM technique. Wide variability was reported between research groups in. mouse husbandry, surgical, and post-operative factors, even when research groups cited the use of the originally published DMM model. Understanding and recording these sources of variability, together with improved reporting experimental methods, could allow improvements to be made in repeatability, reproducibility, and enhance the ability to compare across studies. Ultimately these improvements could lead to improved understanding of the pathogenesis of post-traumatic osteoarthritis.

College of Veterinary Medicine, Purdue University (Rishika Virdee), Gluck Equine Research Center, University of Kentucky (Jamie Norris), Department of Comparative Pathobiology, CVM, Purdue University (Sriveny Dangoudoubiyam)

The canine roundworm, Toxocara canis is a well-known agent of zoonotic infection in human hosts, which oftentimes causes visceral and ocular larva migrans (VLM and OLM), as well as neurotoxocarosis. This is a neglected tropical disease prevalent in many countries worldwide; impoverished areas of North America can also be affected. Millions of people in the United States are exposed to T. canis, while larva migrans is reported especially in young children and senior citizens. The parasite is only infectious to humans during the L3 larval stage, and never matures to adulthood in human tissues. Proteins secreted by T. canis larvae facilitate the parasite’s migration through host tissues and help evade immune capture. The aim of this study was to analyze the secretome of the parasite’s L3 stage using bioinformatics tools. The presence of a signal sequence identified 1147 secretory proteins in the L3 stage. These were categorized to predict their functions during the migratory phase. Many belonged to various protein families involved in functions such as transmembrane signaling, carbohydrate/lipid-binding, neuropeptide signaling pathways, and endopeptidase inhibition. Another 313 proteins were hypothetical proteins of unknown function, most likely shared between T. canis and other closely related nematode species. Thirteen secretory proteins were categorized as proteases, which potentially play a role in parasite-host interactions such as host connective tissue and mucus plug degradation, parasite molting, pro-inflammatory response, immune evasion etc. These proteases could also be potential candidates for vaccine development or anthelmintic drug discovery.

GE Miller^*1, J Scaria², SK Narayanan¹, and D Pillai^1,3

¹ Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA

²Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD 57007

³ Indiana Animal Disease Diagnostic Laboratory (ADDL), Purdue University, West Lafayette, IN 47907, USA

Non-typhoidal Salmonella spp. (NTS) are responsible for enteric infections in humans and animals. Considering the fact that reptiles carry and shed NTS through feces, the potential for exotic pets to harbor antimicrobial resistant NTS, a one health concern and needs to be addressed. To fight against the rise in antimicrobial resistance (AMR) and predict outbreaks using virulence matches in serovars from the environment and clinical isolates, we aim to characterize NTS isolates to determine the presence of indicators of virulence and confer AMR using genomic data.

We sequenced the whole genomes of 9 NTS isolates from snake fecal samples submitted at Animal Disease Diagnostic Lab Purdue. Comparison of genome data for AMR and virulence gene determinants were performed among the 9 NTS isolates. Antimicrobial susceptibility testing to 17 different antibiotics were evaluated phenotypically according to the Clinical Laboratory Standards (CLSI) procedures. 

The genetic analysis indicated the presence of several virulence factors such as adherence genes, outer membrane proteins and secretion systems that are of clinical importance. The minimum inhibitory concentration report showed all 9 samples had identical AMR profile with resistance to aminoglycosides and first-generation cephalosporin. Genetic analysis revealed AMR genes conferring resistance to aminoglycoside antibiotics. 

This data provides insightful public health implications to mitigate outbreaks; gain better understanding of host-pathogen interaction and benefits microbial source tracking and prophylactic planning approaches for clinicians in the future.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Hemangiosarcoma (HS) makes up 5-7% of malignant tumors in dogs and has a poor prognosis due to metastatic disease. HS originates from pluripotent endothelial cells and relies on angiogenesis for growth. Initial slow growth allows time for invasion of surrounding tissues and hematogenous dissemination. Visceral HS of the spleen or right auricle is more common than non-visceral HS, which develops in the skin or muscle and has a slightly better prognosis. Treatment of visceral HS with surgery results in an average survival time of 1-3 months, and with chemotherapy, survival time can be up to 6 months. However, reliable factors to help predict survival have been difficult to elicit. In this study, electronic medical records from cases of splenic HS in dogs presenting to a veterinary teaching hospital from 2010-2020 were analyzed to determine if signalment data, CBC, and serum chemistry values had a significant relationship to overall survival time and therefore could be utilized as potential prognostic markers. Twenty-three cases of splenic HS met inclusion criteria and were divided into three groups: G1, <90 days survival; G2, 90-180 days survival, and G3, >180 days survival. Kruskal-Wallis was used for group comparisons. As expected, presence of the tumor in multiple organs (multicentric) and gross metastatic disease were both significantly different between groups (p<0.5). In addition, serum phosphorus levels were decreased in G2 compared to G1 and G3. This retrospective analysis supports the presence of metastasis as a useful prognostic indicator in canine HS. Other potential prognostic markers of HS in dogs are being analyzed, including IHC and microRNA-based markers using the archived biopsy samples from these cases.

College of Veterinary Medicine (Ogburn), Department of Comparative Pathobiology (Weng), College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Early detection through surveillance is critical for control and prevention of outbreaks caused by disease or foodborne illness. Veterinary electronic medical records (EMR), if strategically analyzed, could aid in animal disease surveillance. However, EMR data are often in an unstructured free-text format. The project objective was to develop a signal-detection algorithm that integrated medical knowledge for disease surveillance using EMR. We used 2017 to 2019 EMR of Purdue University Small Animal Hospital for this research. We searched for historical pet food recalls and disease outbreaks in companion animals during study period to compile a list of pathogens, chemicals and corresponding clinical signs. We investigated clinical signs and used an established vocabulary library to define search terms. We then used NVivo to convert the chief complaint data to quantitative measures using the search terms. The results were analyzed using the investigated algorithm and rules for signal detection. A total of 60,226 non-duplicate records were analyzed. Among the 11 defined search terms, search term pertaining to vomiting resulted in most consistent signals. Among 31 detected time periods, 55% were classified as plausible aberrations and 45% as plausible false negatives. Review of medical records found 11% errors from NVivo query with most due to irrelevance to the project objective. Development of a disease surveillance application involving free-text EMR is still a work in progress, however the potential it presents as an effective and robust tool should be recognized, and thus explored further.

Veterinary Clinical Sciences (Hess, Taylor), Basic Medical Sciences (Anderson), College of Veterinary Medicine; Bindley Bioscience Center (Cooper), Purdue University, West Lafayette, IN; Veterinary Clinical Medicine (Reinhart), College of Veterinary Medicine, University of Illinois, Urbana-Champaign, IL

Sepsis is a major cause of morbidity and mortality in neonatal foals and adult horses. Effective treatment of sepsis requires that the excessive inflammatory response, known as Systemic Inflammatory Response Syndrome (SIRS), is interrupted. Thiamine is a vital co-factor in many metabolic processes, including those that are critical in mitigating inflammation. Previous studies in humans have shown that metabolic resuscitation, a term used to reference the use of thiamine hydrochloride (TH), ascorbic acid, and hydrocortisone, decreases inflammation and increases survival of septic patients. The benefits of TH in treating diseases in horses are unknown. Before efficacy studies of TH alone or TH in combination with ascorbic acid and hydrocortisone can be performed in horses, pharmacokinetic (PK) analysis is necessary. We hypothesized that intravenous (IV) TH at increasing doses results in corresponding increases in plasma TH concentrations without causing adverse effects. A PK analysis of 3 doses of TH was performed and any adverse effects were documented. A randomized cross-over study included 9 healthy adult horses treated with IV TH at 5, 10, and 20 mg/kg. For each treatment, blood was collected immediately prior to drug administration (T0) and 5, 10, 15, 20, 30, 45, 60, 90 minutes and 2, 4, 6, 8, 10, 12, 24, and 48 hours after drug administration. A physical examination was completed at T0, 6, 12, 24 and 48 hours. No clinical signs of adverse effects were observed. Results of high-performance liquid chromatography with mass spectrometry to quantify plasma TH concentrations are pending. Understanding the PK profile of TH in horses is the first step in evaluating the potential benefits of exogenous TH in equine patients.

Department of Comparative Pathobiology and Department of Animal Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana.

Subjective scoring methods such as Qualitative Behavioral Assessment (QBA) may inform welfare assessments, but observer bias may influence ratings. This study investigated: 1) whether QBA could be reliably used to score the emotional states of dogs from commercial breeding kennels (CBKs) and 2) the effects of knowledge about the dogs’ sourcing and empathy for animals on veterinary students’ interpretations of dogs’ emotions. Five observers experienced in dog behavior analysis and trained to use QBA terms scored 25 videos of dogs to assess inter- and intra-rater reliability. There was moderate to good agreement (0.40-0.80) across all terms and raters. Using an online survey, students at two Midwestern Colleges of Veterinary Medicine (n=71) used the same 20 terms to score dogs from 8 videos and completed an Animal Empathy Scale (AES).  Students were randomly assigned to informed/uninformed groups relative to being told that the dogs were from CBKs. The level of agreement on each QBA term was lower for students than for experienced observers (< 0.40 for 3/20 terms). Principal component analysis (PCA), used to reduce the QBA variables, extracted 4 components (PCs) explaining 69.2% of the variance. Regression models were used to investigate the effects of treatment group and AES on the 4 PC scores. Being informed about the source of dogs did not impact QBA scoring, but higher AES was associated with scoring dogs as being more sociable/explorative (PC1). Overall, QBA training and experience in dog behavior analysis improved scoring reliability. As in previous studies, empathy towards animals impacted scoring of animal emotions.  The findings suggest that QBA scoring should be used cautiously to avoid bias in assessing welfare.

Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana.

Mild equine asthma (i.e. inflammatory airway disease) affects up to 80% of racehorses worldwide. Respirable dust exposure while eating hay is a major contributor to airway inflammation. Irritants in the dust like beta-glucan and endotoxin play a role in activating the inflammatory response. We hypothesized that horses eating hay will have higher exposure to respirable dust, beta-glucan, and endotoxin than horses consuming steamed hay or haylage. Respirable dust exposure was measured gravimetrically using PVC filters in the breathing zone of 28 Thoroughbred racehorses eating dry hay, steamed hay, or haylage. Concentration of beta-glucan and endotoxin were measured using kinetic chromogenic limulus amebocyte lysate technique (LAL). A generalized mixed model was constructed to examine the effect of the forage on dust exposure, LPS concentration, and beta-glucan concentration. Adjusted P-value <0.05 was considered significant. Horse’s eating hay had significantly higher respirable dust exposure (0.077±0.042 mg/m³) compared to those eating steamed hay (0.05±0.008 mg/m³, P=0.008) or haylage (0.058±0.024 mg/m³, P=0.028). Beta-glucan (n=26) and endotoxin (n=10) exposures did not differ between forage groups. Steamed hay or haylage reduces dust exposure to equine athletes. Further experimentation should be performed to confirm if reduced dust exposure can reduce the prevalence of equine asthma.

Many of Indiana’s freshwater mussels are considered endangered by federal and/or state authorities. Population die-offs are becoming increasingly prevalent, often with no known inciting cause. The aim of this study was to establish detailed plans and procedures to be used for rapid sample collection, diagnosis, and subsequent management during die-off events. Three species were included in this project: the native Fat Mucket (Lampsilis siliquoidea) and Plain Pocketbook (Lampsilis cardium), and the non-native Asian Clam (Corbicula fluminea). Over the course of this study, 20 individuals from each of the three species were sampled from each of three sites along the Wildcat Creek tributary. Once collected, the specimens were sent to appropriate laboratories for evaluation of hemolymph analytes, tissue glycogen and stable isotope levels, presence and relative abundance of microbes, tissue contaminants such as mercury and/or copper, and antimicrobial resistance patterns among identified bacteria. Using the sample collection methods established in this study we were able to collect and sample mollusks in a real-world setting.  The results of this work will prove useful as baseline data in the event of mussel die-off events in the Wildcat Creek tributary.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Lung cancer is the leading cause of cancer-related deaths worldwide (1.76 million deaths in 2018) and the leading cause of brain metastatic disease among all primary cancers.  As tumor cells colonize the neuroparenchyma, they breach the tightest and most effective vascular barrier in the body, the blood-brain barrier (BBB).  The functional components of the BBB include endothelial cells, basement membranes, pericytes and astrocytes.  Herein, we hypothesized dynamic transformation of the BBB to the blood-tumor barrier (BTB) would be correlated with paracellular permeability.  An experimental model of lung cancer brain metastases was developed using twelve 6-week-old athymic nude mice.  Mice were injected with 250,000 brain-seeking cells, which colonized the brain for 4-6 weeks.  Animals were injected with 3kd Texas Red dextran at the time of euthanasia, and brains were harvested, cryosectioned and prepared for immunofluorescence analysis.  Metastatic tumors were characterized as highly or poorly permeable based on the diffusion of Texas Red dextran within and around the tumor parenchyma.  Brain metastases were roughly spherical and measured between 50-500 µm in diameter, irrespective of their permeability status.  The most striking BTB pathology was identified in tight junctions and associated adapter proteins.  Highly permeable tumors exhibited haphazard expression of claudin-5 and a loss of zona occludens-1 adapter protein compared to metastases with low paracellular permeability.   Understanding the cellular and molecular alterations occurring within the blood-brain barrier during brain metastatic disease (from NSCLC) is imperative to the identification of novel therapeutic targets and improved methods of drug delivery.

Post-traumatic stress disorder (PTSD) is a psychological illness that affects about 3.5% of adults in the United States, commonly military veterans. Service dogs (SD) have been found to help clinically alleviate PTSD symptoms when used as complementary therapy. One intra-group comparison study of veteran PTSD symptoms found similarities in the self-reports of both veterans and their spouses. The experiences of those close to veterans with PTSD, such as their spouses, may have effects on treatment efficacy and quality of life. However, minimal research exists on the effects of SD intervention on spouses or the spousal relationship. This qualitative analysis aims to assess self-reported experiences of veterans and spouses through intra-couple comparison. Survey data was gathered from participants of K9s for Warriors, a national provider of service dogs for veterans with PTSD. Participants included both SD recipients and their partners. Responses to four qualitative survey questions, pertaining to general experience with the service dog and the SD effect on the spousal relationship, were coded into pre-determined themes. Intra-couple responses were then compared to evaluate couple agreement or disagreement within content themes. Theme agreement and disagreement may reflect upon the respective experiences of recipients and their partners in the context of SD intervention. This analysis will be supplemented by quantitative survey data pertaining to caregiver burden and quality of life. Further research may inform qualifications for receiving a SD, efforts to maximize efficacy, and the role of spouses in SD intervention for PTSD.

The purpose of this research is to evaluate current urine culture protocols at Indiana’s Animal Disease Diagnostic Laboratory (ADDL) which tests roughly 1500 urine samples per year. In the current urine culture procedure, a negative result requires solid media to be incubated for 48 hours, and the enrichment broth to be incubated for 7 days with no growth. The utility of this extended protocol has been questionable, and other reference laboratories have recently limited their urine culture procedure to 24 hours on solid media. ADDL retrospective data evaluated in this study included over 854 canine and feline urine cultures with no growth on solid media at 24 hours. Growth was present in 2% of 48 hours solid media cultures, and in 6.47% of enrichment broths. The method of urine collection did not predict the presence of growth at 48 hours or in enrichment broth, and quantity of growth on the 48 hour solid media varied from significant to insignificant. The organisms present could be classified as contaminants from normal mucosal flora, feces or the environment. Culture protocols extending past the 24 hour observation on solid media may not contribute clinically useful data. The findings of this study will inform future changes to the urine culture procedure at the ADDL.

College of Science, Purdue University, West Lafayette, IN (Diab), Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Rogers, Beauclair, Shi), Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN (Rogers, Beauclair, Shi), Indiana University, School of Medicine, West Lafayette, IN (Thyen).

Roughly 30% of all injury-related deaths in the United States (U.S.) involve a Traumatic Brain Injury (TBI), impacting an estimated 10 million Americans annually. The associated financial burden is staggering, with direct and indirect costs for a single year calculated at 76.5 billion dollars. Unfortunately, due to the nature of these injuries and the type of sub-cellular investigative resolution required, these pathological mechanisms currently remain elusive.

In clinical practice, the use of alpha-synuclein elevation as a biomarker for traumatic brain injury is already established. Recently, the novel use of a Ballistic Pendulum (BPA) was proposed to mimic closed head injuries in vitro. In a preliminary study utilizing this new methodology and immunocytochemistry, murine cortical networks grown on micro electrode arrays (MEAs) were subjected to clinically-relevant g force levels (30-300g) and subsequently fixed (24 hrs post) and treated with a primary antibody capable of detecting alpha-synuclein. Compared to controls, impacted networks revealed significant increases of alpha-synuclein, which were pronounced in perinuclear, punctate patterns (n=5). While additional experiments are necessary to better quantify and describe this phenomenon, we hope that these pioneering studies will help pave the way for further investigations into trauma associated protein aggregation and possible pathway-links to neurodegeneration, in addition to supplementing this new and exciting model.

Leucine zipper-like transcriptional regulator 1 (lztr1) is a tumor suppressor gene, which belongs to BTB-kelch superfamily. The lztr1 gene mutations are associated to human genetic disorders such as Noonan Syndrome and tumors such as glioblastomas and schwannomas. Schwannomatosis occurs when the Schwann cells uncontrollably proliferate which causes benign tumors to typically form on spinal and peripheral nerves. The gene has also been demonstrated to play a role within the Golgi apparatus and it functions as an E3 ubiqutin ligase that negatively regulate RAS signaling pathway for suppressing tumor formation. However, why lztr1 mutations specifically lead to neural tumors remains largely unknown. We hypothesize that zebrafish lztr1 gene expression mimics human orthologous gene expression patterns as gene functions are evolutionarily conserved. Here, we investigate the lztr1 gene expression in developing zebrafish embryos (1-7dpf, days post fertilization).  In situ hybridization was employed as it provides gene expression in a temporal and spatial manner. We found that the zebrafish lztr1 gene is expressed ubiquitously in 22hpf (hours post fertilization) staged fish embryos. When the zebrafish embryos develop further to 60hpf, the lztr1 gene remains mainly in the brain region. At the stages of 4-7dpf, the gene is restricted to ear, gill arches and swim bladder. Based on these results, we conclude that the lztr1 gene is dynamically expressed during zebrafish embryonic development. Its expression in brain and craniofacial regions may be related to human disease tissue specificities. Our results will be helpful to understand lztr1 gene’s functions in human Noonan Syndrome and cancers.

Brain metastases of lung cancer are on the rise, and development of clinically relevant model systems is paramount to understanding this devastating disease. Evaluation of brain metastases of lung cancer is enhanced by in vivo bioluminescent imaging (BLI). The application of BLI in preclinical research has expanded due to its efficiency, sensitivity, and relatively low-cost. The overall signal intensity produced by luciferase activity within tumor cells provides a quantitative measurement of tumor burden. The signal intensity of luciferase activity is influenced by factors such as injection route, number of cells, tumor location, and the timing of image acquisition. This timing of image acquisition is critical for accurate, quantitative assessment of tumor burden in the progression of cancer. Herein, we hypothesized that following intracardiac injection of luciferase-labeled lung cancer cells, luciferase activity would predict overall tumor burden. Ten athymic nude mice were inoculated with luciferase-labeled non-small cell lung cancer cells (large cell carcinoma and squamous cell carcinoma types) via ultrasound guided intracardiac injection. Animals were injected intraperitoneally with 150 mg/kg of luciferin, and luciferase activity was measured using the Ivis Lumina II Optical Imaging System over 60 minutes. There was a 2.25-fold decrease in luciferase activity at minute 60 compared to minute 3.  The maximum signal intensity for all animals was identified at minute 6. These findings support the use of rapid evaluation of BLI in experimental models of lung cancer brain metastases and will contribute to the development of clinically relevant translational models.

 Cancer remains the leading cause of death in older dogs and the second leading cause of death in humans in the United States. Anticancer therapies targeting folate receptors (FR) may be of clinical significance to treat a number of cancers in both species. Two FR isoforms, FRα and FRβ, have been shown to be upregulated in many human and some canine cancers. In humans, FRα is overexpressed in several carcinomas while overexpression of FRβ is observed on activated macrophages and in hematopoietic cancers, particularly myeloid leukemias. In normal tissues, however, expression of FRs is limited, making them an attractive candidate for receptor-targeted chemotherapy. Naturally-occurring canine cancers appear to provide a relevant preclinical model for some human cancers due to similarities in histopathology, biological heterogeneity, frequency of metastases, and chemotherapy response; however, few cancers of potential translational relevance in dogs have been explored for their degree of FR expression. The goal of the present study was to utilize immunohistochemical analysis to assess FR expression in canine cancers of potential translational relevance. The results indicate that immunoreactivity in tumor-associated macrophages was plentiful across different cancer types; however, significant membranous expression of FR on tumor cells was only observed in select cases with squamous cell carcinoma and apocrine gland adenocarcinoma of the anal sac. Further work should include corroborating results through PCR, RT-PCR, and in vivo nuclear scintigraphy, as well as conducting IHC on more cases of interest.

Hydration treatments are frequently recommended for optimal voice even though the impact of dehydration on the vocal folds is not fully understood. Body weight loss due to water withholding is a common measurement of dehydration. However, laboratory rats have been shown to lose weight due to the stress of handling alone. The objective of this study was to: 1) determine adjunct biomarkers of systemic dehydration; and 2) determine if hydration state alters vocal fold histopathology. The dehydration biomarkers tested included hematocrit, osmolality, pack cell volume (PCV), and renal renin mRNA expression and protein synthesis. The dehydration biomarkers of hematocrit, osmolality, and PCV were subject to too much variability to be utilized. Renal renin mRNA expression and protein synthesis were significantly increased in dehydrated rats. To determine histopathologic changes in the vocal fold tissues of rats, larynges were prepared for histological staining with hematoxylin and eosin (HE), Masson’s trichrome, Verhoeff-van Gieson, and Alcian Blue (pH 2.5) pre- and post-hyaluronidase incubation. Dehydration did not significantly affect tissue morphology based on an adapted semi-quantitative assessment. Quantitative histopathologic analysis of vocal folds showed significantly lower levels of hyaluronan present in dehydrated rat vocal folds; and a quantifiable increase in percent stained area in the vocal fold lamina propria. This increase in percent stained area suggests that dehydration decreased the non-stained (i.e. fluid) area of the lamina propria. Collectively, these results indicate that systemic dehydration may induce changes in the rat vocal fold potentially contributing to function.

Systemic, long-lasting therapies are required to effectively combat metastatic prostate cancer. Immune-stimulation therapy can be used to treat tumors and their microenvironment by recruiting immune effectors to tumors. Prior studies have shown that a plasmid encoding for the cytokine interleukin 27 (IL-27), delivered to muscle cells via sonoporation gene delivery (sonodelivery), can reduce tumor growth. Preliminary work has shown that a peptide referred to as pep6b (LSLITRL) targets the IL-6Rα, which is upregulated on prostate cancer cells. Prior work led to the creation of a plasmid encoding for a combination of IL-27 and pep6b, separated by a flexible linker at the C-terminus of IL-27. This work suggested that the IL-27-pep6b compound exhibited improved antitumorigenic effects compared to IL-27 with a non-specific peptide. The focus of the present study was to create another IL-27-pep6b construct with a modified flexible linker in a vector conducive to in vivo use. The efficiency of the new plasmids was tested in vitro by qPCR analysis of cDNA derived from TC2Ras cells that had been transfected with the new plasmids. Genes commonly upregulated by IL-27 (Tbx21, XCL1, and IFNg) were the qPCR targets. An in vivo study was then initiated in which twenty-one C57BL/6 mice received TC2Ras cells subcutaneously to produce tumors. The treatment group received intramuscular IL-27-pep6b plasmid sonodelivery. We expect to see decreased tumor growth and enhanced immune effector infiltration in tumors of mice injected with IL-27-pep6b compared to mice “treated” with vectors without IL-27 or with IL-27-untargeted peptide as sonodelivery leads to cytokine expression and pep6b targets the cytokine to distant tumors.

Listeria monocytogenes (Lm) is a foodborne pathogenic bacterium and the primary route of infection is via translocation across intestinal epithelium. The Listeria adhesion protein (LAP) interacts with the host receptor; Hsp60 to facilitate Lm translocation. Interaction induces intestinal barrier dysfunction via redistribution of major cell-cell junctional proteins and stimulation of proinflammatory cytokine production. Thus, blocking of the LAP-Hsp60 interaction may prove effective in the prevention of listeriosis, reducing the need for antibiotic use. Several probiotic bacteria have been examined for their potential to control Lm, but their effectiveness remains uncertain. Next-generation probiotic bacteria are engineered to be pathogen-specific, overcoming the unpredictability of traditional probiotics. Here, the potential for Lactobacillus casei (LbcWT) expressing LAP to competitively exclude pathogen interaction was investigated. A/J mice were supplied with drinking water containing bioengineered LAP- expressing Lactobacillus probiotic (BLP) strains for 10 days prior to Lm challenge. Over 80% of BLP-fed mice survived while only 50% of LbcWT-fed survived. BLP-fed mice showed significant reductions in pathogen loads by ~99.99% in liver and spleen tissues and Lm was undetectable in blood and kidney samples. BLP strains decreased proinflammatory cytokines and T-cells and increased regulatory T-cells. Finally, BLP maintained barrier function by preventing mislocalization of junction proteins. This study demonstrates that BLP expressing LAP prevented listeriosis in mice via competitive exclusion, protection of gut barrier integrity, and immunomodulation, thus elucidating a novel approach in preventing infectious disease.

Antimicrobial drugs play an important role in reducing morbidity and mortality caused by infectious diseases. Use of antimicrobial drugs is also associated with increased antimicrobial resistance (AMR), now acknowledged as a global threat to health. Thus, judicious antimicrobial prescribing practices is an important way to help control AMR. The purpose of this work was to develop hospital-specific antibiograms to guide empiric antimicrobial usage for the Purdue University Veterinary Teaching Hospital (PUVTH). Bacterial culture results with antibiotic susceptibility profiles were extracted from the PUVTH database for all samples submitted during a one-year timeframe. These data were utilized in the construction of antibiograms based upon characteristics of the host (including species, hospital department of admission, and anatomic location of obtained sample) as well as the bacteria (Gram-stain classification, and species). A total of 626 susceptibility profiles were completed during the specified timeframe. The most common isolates obtained from dogs and cats were Escherichia coli, Staphylococcus spp., Enterococcus spp., and Streptococcus spp. Results indicated that between bacterial organisms with the same Gram-staining characteristics, there was a notable difference in susceptibility to different antibiotics. Further, differences in susceptibility were identified among isolates obtained from different species of animals and among isolates obtained from different anatomical locations within a single species. The variability in population-level antimicrobial susceptibilities identified in this project inform the construction of antibiograms that can be used as an aid in selecting appropriate empiric antibiotic therapy.

Transitional cell carcinoma (TCC) is a malignant tumor that develops from epithelial cells that line the bladder. Approximately 2% of all tumors in dogs is TCC making it the most common bladder cancer. TCC is frequently located in the trigone of the bladder but it can occur anywhere in the urinary tract. In dogs, TCC can be either intermediate- to high-grade but most dogs develop the high grade, invasive form that grows quickly and can metastasize to the lungs, liver, and lymph nodes. Most of dogs with TCC are presented with difficulty or pain to urinate, which are the same clinical signs of inflammatory or infectious lower urinary tract disease (LUTD) and in many cases both conditions are present. In the presence of inflammation, cytology of the urine sediment cannot differentiate reactive from neoplastic transitional cells. Diagnosis of TCC is made late once dogs have developed advanced clinical signs and tissue biopsy via surgery, cystoscopy or urinary catheter is the gold standard. A non-invasive alternative for diagnosis of TCC is through microRNA expression signatures in biofluids, like urine. In this study, urine sediment samples where obtained and classified as TCC or LUTD based on the patient’s medical records. Total RNA extraction was conducted using miRNeasy Serum/Plasma Advanced kit by QIAGEN and concentration and purity where determined by spectrophotometry. Samples with adequate concentration and purity where used to perform qRT-PCR to determine specific microRNA expression that may serve as biomarkers for non-invasive diagnostic test for TCC, which could be performed at the first presentation when dogs are suspect of having TCC by examination of urine sediment, despite the presence of inflammation.

The value of the human-animal bond (HAB) is well recognized. Even immunocompromised individuals, who have a greater risk for zoonotic disease and animal-related injury, take advantage of the benefits of the HAB via pet ownership. Communication between veterinarians and these individuals is critical for the promotion of healthy and safe human-animal interactions. The goals of this project were to investigate perceptions and practices veterinary professionals have toward immunocompromised clients and to determine the prevalence of documenting pet-related human health concerns in veterinary medical records. We hypothesized that there is an opportunity for improvement in communication between veterinarians and immunocompromised clients. A survey was sent to 36 Purdue University Veterinary Teaching Hospital (PUVTH) employees to evaluate their experiences with immunocompromised pet owners, and the overall response rate was 33%. Respondents were comfortable advising immunocompromised clients about zoonotic disease risk (median score = 4; scale = 1 (very uncomfortable) - 5 (very comfortable)). However, respondents were not familiar with the legal aspects of documenting client health conditions (median score = 1; scale = 1 (not familiar at all) – 5 (very familiar)). Additionally, PUVTH records from small animal patients throughout 2018 (n = 19,522) were analyzed. Preliminary analysis suggests only 1.5% of records included information about at-risk individuals or human health concerns. We recommend further research focus on the most effective methods to improve the communication gap between veterinarians and immunocompromised individuals and to encourage healthy and safe human-animal interactions in these vulnerable populations.

Myelin is an important component of both the central nervous system (CNS) and peripheral nervous system (PNS), where it surrounds nerve cells, protecting them and aiding the perpetuation of signal conduction. An absence of, or decrease in, myelin can thus lead to deficits in nerve signal transduction. Myelin deficits can occur either by demyelination – myelin that initially formed properly and was then degraded, or hypomyelination, where myelin never correctly/fully formed at all. In 1989, Braund, et al., described two cases of littermate, full-sibling Golden Retriever puppies with very young-onset (i.e., congenital) neurological deficits. Peripheral nerve biopsies revealed myelin sheath changes consistent with a predominantly hypomyelinating neuropathy. Uniquely, this syndrome affected only the PNS. Previously reported hypomyelination syndromes in veterinary patients had either exclusively involved the CNS, or both the CNS and the PNS together. We acquired DNA from four Golden Retrievers presenting with clinical signs similar to those in the 1989 Braund report; histopathological examination of nerve biopsies confirmed these dogs to have a congenital PNS hypomyelinating neuropathy. Physical exam of these four dogs uncovered no CNS signs. We performed whole genome sequencing on DNA from each of these dogs, and evaluation for causal variants is ongoing. Literature reviews of similar conditions in human and mouse have provided several candidate genes of interest: MPZ, PMP22, EGR2, SOX10, CNTNAP1, MTMR2, Cx32, MRS2, PRKN, and PACRG, which are being scrutinized closely. Identification of a causal variant would allow owners and breeders to test for, and thus help eliminate, this disease from the breed.

Department of Food Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Commercially grown piglets are exposed to a variety of stressors, including weaning, food deprivation, and heat stress, as they are transitioned onto grower farms. These stressors cause long-lasting changes to the secretory and barrier functions of the piglet intestine, leaving them highly susceptible to bacterial and viral diseases. Furthermore, as concerns over antibiotic resistance grow, probiotics are being increasingly used to reduce the risk of infection and promote protective immunomodulatory effects on the intestine. One set of recombinant probiotics being investigated include those that possess Listeria adhesion protein (LAP), an adhesion protein of Listeria monocytogenes that may be used to prevent infection and provide general gut-health benefits. IPEC-J2 cells, a porcine jejunal enterocyte cell line, were exposed to several strains of Lactobacillus casei to test efficacy of adhesion and subsequently infected with L. monocytogenes to elucidate the protection provided by the probiotics. These include a wild-type strain, a recombinant strain expressing LAP from pathogenic L. monocytogenes, and a recombinant strain expressing LAP from nonpathogenic L. innocua under both ideal (37°C) and heat stress (41°C) conditions. Probiotic adherence was enumerated by plating the bacteria and the level of cytotoxicity to the enterocytes was measured via an LDH assay. Development of effective probiotic interaction with host cells is a critical initial step in preventing the significant economic losses intestinal disease causes through the increase of piglet mortality and the reduction of piglet growth. The swine intestine also bears the most similarity to the human intestine, setting the future framework for human studies.

Research Grant: Biomatrix

Student Grant: Boehringer Ingelheim and Purdue Veterinary Medicine

¹Department of Animal Sciences, College of Agriculture, ²Department of Comparative Pathobiology, ³ Department of Veterinary Administration, ⁴Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN

The accurate assessment of the severity and extent of liver diseases is beneficial for determining treatment. Glutamate dehydrogenase (GDH) is an enzyme found in liver mitochondria. In rats, increased activity of GDH in serum has been shown to indicate liver damage in some diseases when a currently used biomarker, ALT did not. The purpose of this study is to determine if serum GDH could be useful as a biomarker for detecting liver damage in dogs and horses. Cast off serum samples were obtained from dogs (n = 8) and horses (n = 7) presented to the Purdue Veterinary Teaching Hospital. Standard serum chemistries for dogs (ALT, ALP, GGT) and horses (AST, ALP, GGT) were performed in the Clinical Pathology Laboratory of the VTH. GDH activity was measured spectrophotometrically using a commercially available test kit. In dogs, when ALT, ALP or GGT activities were within normal reference ranges, GDH activities generally ranged from 38 to 108 U/L. When ALT activities were slightly elevated, GDH activities also increased except in two cases. In horses, there were no apparent relationship between GDH and the other enzyme activities. Further studies are needed to establish ranges of normal GDH activity in various species and to relate increases of serum GDH activity to clinical liver injury. 

Research Grant: None 

Student Support: Purdue Veterinary Medicine Veterinary Scholars Summer Research Program

Praise Benson, ¹ Kathleen Ivester, ² Laurent Couetil²

¹Department of Agriculture, Prairie View A&M University, Prairie View, TX

²College of Veterinary Medicine, Purdue University, West Lafayette, IN 

Introduction:

Mild equine asthma, also known as inflammatory airway disease (IAD), is a common problem in athletic horses. Horses with mild asthma are categorized into different types of inflammations most commonly neutrophilic or mast cell inflammation. Inflammations in these horses are due to dust exposure especially in hay and this dust exposure varies with different forages. Presumably forage releases dust that contain protein when inhaled and this triggers inflammatory response. We hypothesize that healthy horses and horses with asthma react differently to these inhaled antigens. Also, horses with neutrophilic inflammation may react differently than horses with mast cell inflammation. The objective of this research is to compare the antigenicity of respirable dust released from hay, steamed hay, and haylage when probed with Broncho alveolar lavage fluid (BALF) from Thoroughbred horses with and without mild equine asthma. 

Materials

Samples: respirable dust released from hay, haylage, steamed hay; BALF from horses racing in Indiana (3 groups: healthy= <5% neutrophils, <2% mast cells, <1% eosinophils; neutrophilic asthma: >6% neutrophils; mast cell asthma: >3% mast cells)

Methods:

• Bicinchoninic acid (BCA) - This assay measures protein concentrations. Also compared BALF total protein concentrations between groups with one-way ANOVA.
• Western blot- SDS PAGE followed by electrophoretic transfer to nitro cellulose, Probed with BALF (primary) and horseradish peroxidase conjugated goat anti-horse IgG (secondary) and visualized with CCD camera. Protein binding patterns visually compared. 

Results:

• Protein concentration in BALF did not vary between the groups (P=0.27). Healthy horses = Mean 410±151SD, neutrophils, mean 302±188SD and mast cell ,mean 379±209SD for each group.
• Respirable dust has antigenic proteins recognized by BALF antibodies with apparently greater intensity in dust from hay. 

Conclusions: The protein concentration compared with the groups had no significant difference. Antigenic proteins recognized the BALF antibodies. Regardless of diseases group, antigens of interest appears to have higher molecular weight. However, blot needs to run for a longer time for better separation of the bands. Also hay antigens showed much greater intensity compared other groups.

Levi Smith, Audrey Ruple

Department of Veterinary Clinical Sciences (Smith), Department of Comparative Pathobiology (Ruple), College of Veterinary Medicine, Purdue University, West Lafayette, Indiana
The two-toed sloth (Choloepus spp.) has become a popular species utilized in zoological and wildlife institutions. Thus, their contact with the public has increased, but not much is known about what health risks they may pose to humans, nor which diseases they are susceptible to. This study reviewed all published literature from 1873 to 2018 that examined infectious agents affecting two-toed sloths in both captive and wild populations. Online databases were selected and electronically searched for relevant articles using strings of inclusion and exclusion terms, resulting in identification of 1,242 articles. After conducting two relevance screenings, 36 articles were deemed appropriate for inclusion in the review. A total of 1,676 two-toed sloths were accounted for in the included studies, with evidence of infection in 577 individual sloths. Approximately 77% of those identified were cryptic fungal, parasitic, and viral infections, and the remainder presented with clinical cases of bacterial, fungal, and viral infections. The infectious agents reported were parasitic (12), viral (8), bacterial (4), and fungal (3). Of the parasitic infections reported, only one was of clinical significance to the animal; however, in geographical areas where human populations have shown a high prevalence of leishmaniasis, coexisting sloths have exhibited a corresponding prevalence of subclinical Leishmania infections, indicating they are likely vectors for the disease. Significant gaps remain regarding clinical and subclinical infectious disease prevalence in sloths in captivity, which should be further investigated while sloths continue to be an important species in the eyes of the public.

Student Support: Boehringer-Ingelheim Veterinary Scholars Program and Purdue Veterinary College

Research Support: None

Alina S. Ibrahim, Melinda Anderson, Andrew Woolcock, George Moore, Sandra D. Taylor

The combined effects of ascorbic acid (AA) and hydrocortisone (HC) have been documented to alleviate inflammation associated with sepsis. This dysregulated host immune response to microbial infections can result in compromised endothelial barrier integrity, hypotension, and organ dysfunction. Systemic inflammation can be induced by intravenous administration of lipopolysaccharide (LPS). Ascorbic acid is an important antioxidant, enzyme cofactor and anti-inflammatory agent which also induces production of other antioxidants like α-tocopherol (Vitamin E). Similarly, HC is a corticosteroid that strengthens vascular resistance. This study aims to determine if administration of AA and low-dose HC inhibits inflammation in an equine model of sepsis. We utilized a randomized, blinded, placebo-controlled design for our experimental trials. Horses were randomly assigned to 1 of 4 groups consisting of 8 horses each. Group 1 (control) received saline, Group 2 received AA and HC, Group 3 received only AA and Group 4 received only HC. Serum AA and α-tocopherol concentrations were evaluated before and after IV LPS infusion, and after AA treatment. The horses were assessed through physical examinations, indirect mean arterial blood pressure measurements, and pain scoring. Blood was collected at various time points for WBC differential and serum biochemical analysis, as well as pro-inflammatory cytokine and acute phase protein evaluation. We hypothesize that therapy with AA and HC will result in reduced inflammation compared to AA or HC alone, following induction of gram negative sepsis in adult horses. Data analysis is pending. 

Purdue Veterinary Medicine Veterinary Scholars Summer Research Program

Dhara Richardson¹, Malathi Raghavan², DVM, MS, PhD

¹ Grambling State University, Grambling, LA; ² Purdue University College of Veterinary Medicine, West Lafayette, IN

Obituary studies have been used to analyze mortality patterns of health professionals. Veterinarians graduate from their professional program as a relatively homogenous group, but little is known about differences in longevity based on specialty, geography and demographic factors. Using recent obituary listings in the Journal of the American Veterinary Medical Association (JAVMA), we sought to expand earlier findings in literature on mortality patterns in veterinarians. For the purpose of this study, six years (2012-2018) of JAVMA obituary data were retrieved, entered into an Excel database, and analyzed using SAS statistical software.

From January 2012 to July 2018, 2,161 deaths were recorded in the JAVMA database. Of these decedents, 1,998 were male, and 163 were female. The mean or average age at death (AAD) of all 2,161 deceased veterinarians was 78.5 years with a standard deviation (SD) around the mean of 14.4 years. The AAD differed significantly between males (80.3 years) and females (55.7 years) (p<.0001). The AAD was also influenced by region of USA residence, employment sector prior to death/retirement, and number of surviving children.

Osteoarthritis (OA) affects over 27 million Americans, and knee OA is the most common joint affected.  OA causes progressive breakdown of articular cartilage leading to disability, decreased quality of life and substantial economic burden. Social stress identified by low socioeconomic status (SES) is a risk factor for worse OA, and periodontitis is correlated with both SES and OA, but neither causal relationship nor mechanism have been established. The aim of the study was to evaluate if chronic social stress or periodontitis change the progression of post-traumatic OA in mice. Chronic social stress was modeled using the resident-intruder chronic social defeat (SD) paradigm, and periodontitis was induced with periodontal ligature (PL). Destabilization of the medial meniscus (DMM) induced OA with additional SD or PL were hypothesized to exacerbate OA compared to DMM alone.  With IACUC approval, 14 week old male C57BL6/J mice were divided into 4 groups: 1) sham surgery control, 2) DMM alone, 3) DMM+PL and 4) DMM+ 8 weeks of SD.  DMM and PL were performed at 16 weeks, and mice were sacrificed at 24 weeks of age. Knees were scanned by micro-computed tomography and the following parameters quantified: total joint bone volume (TJBV), subchondral bone thickness, subchondral bone volume, subchondral bone density, trabecular bone mineral density, trabecular bone volume, and bone fraction (BV/TV). Based on previous studies, these values are expected to be increased in groups with worse OA, particularly in the medial tibia and medial femur, due to bone remodeling from altered weight bearing. Evaluating risk factors such as periodontitis and chronic social stress will increase the understanding of the complex phenotype of OA.

Research Support: National Institute of Health; Duke University; Purdue University

Stipend Support: Boehringer Ingelheim Veterinary Research Scholars Program 

Lauren Thompson, Kara Negrini, Thomas Jenkins, Dr. Dianne Little 

Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 

The villi and crypts in the intestine are important in digestion and absorption. Changes in their morphology or in cell types can be indicative of underlying health or disease. Previous studies identified disruption in the microbiome due to knee injury, social stress or periodontal disease, however, the effect of knee injury and underlying comorbidities on the gastrointestinal mucosa is not described. Cross-sections of ileum, jejunum, and colon from mice (n=10-14/group, 2 experimental batches) subjected to Sham knee surgery, destabilization of the medical meniscus (DMM) to induce knee osteoarthritis, DMM + chronic social defeat (SD) induced social stress, or DMM + periodontal ligature (PL) to induce periodontal disease were evaluated for intestinal villus and crypt morphology and inflammatory cell infiltration. The data were evaluated for batch effect and treatment effect using nonparametric methods (Wilcoxon). There was an effect of batch on ileum villus width, and on jejunal height, and jejunal villus:crypt ratio (p<0.05). Treatment group effects were primarily observed in the colon, with colon mucosal thickness being significantly increased in DMM+SD and DMM+PL compared to DMM alone or to Sham. There was an increase in crypt depth in the ileum in DMM+SD compared to DMM. There were no significant differences in jejunal parameters between treatment groups. When compared to other in vivo functional outcome measures, differences were observed between jejunal parameters for the four treatment groups. F4/80 immunostaining for macrophages in the submucosal and epithelial layers will be evaluated using the “Count and Measure” feature in CellSens (Olympus) software.  

Student Support: Purdue University College of Veterinary Medicine Veterinary Scholars Summer Research Program

Research Grant: Department of Basic Medical Sciences Start-Up Funds

Caitlin Smith, Grant Burcham, Nancy Boedeker

College of Veterinary Medicine, Purdue University, West Lafayette, IN (Smith)

Heeke Animal Disease Diagnostic Laboratory, Purdue University, Dubois, IN (Burcham)

Division of Fish and Wildlife, Indiana Department of Natural Resources, West Lafayette, IN (Boedeker)

The Eastern hellbender (Cryptobranchus alleganiensis) has been listed as State Endangered in Indiana since 2016. To inform conservation efforts in the Blue River region, a calibrated skeletochronological protocol could provide another tool for better understanding the population’s age structure and spread. Skeletochronology has been implemented in several other amphibians to correlate the age of an individual with the number of lines of arrested growth (LAGs) observed in cortical bone. Twenty-four bones (humeri, femurs, front phalanges, hind phalanges and caudal vertebrae) were dissected from five deceased hellbenders of known age and evaluated under various conditions to determine the best overall procedure. The use of 3% nitric acid reduced decalcification time by 75% on average when compared to 5% formic acid without affecting the appearance of the LAGs. Gill’s II hematoxylin alone appeared to stain the slides more legibly than Mayer’s hematoxylin or hematoxylin with eosin, which obstructed the LAG differentiation. Additionally, 83% of histological sections cut at 8 μm thickness yielded readable LAGs, whereas accurate counts could only be obtained from 47% of samples of 4 μm thickness. For hellbenders aged 4 years, LAG counts ranged from 4-8. The median count was 6.5 in humeri, 5.75 in femurs, 7 in front phalanges, 6 in hind phalanges and 5 in caudal vertebrae. The protocol was repeated with four bones from a larger salamander of unknown age, and the median LAG counts obtained were 16.5 in the humerus, 17 in the femur, 14.5 in the front phalanx and 7 in the hind phalanx, suggesting a much older animal. Further statistical analysis will be applied to determine the strength of the age-LAG correlation.

Research Support: Heeke Animal Disease Diagnostic

Caitlyn J. Ridenour, Nelly O. Elshafie, Pierre L. Deshuillers, Deborah W. Knapp, Jose Ramos-Vara, Andrea P. Santos 

College of Veterinary Medicine (Ridenour), Department of Comparative Pathobiology (Elshafie, Deshuillers, Ramos-Vara, Santos), Department of Veterinary Clinical Sciences (Knapp), Purdue University, West Lafayette, IN 

MicroRNAs (miRs) are molecular regulators of cell development and fate; their dysregulation can lead to carcinogenesis. Studies in human urothelial carcinomas (UC) have shown altered expression of miRs, which can be used as biomarkers for diagnostic testing, prognostic classification, disease monitoring, and as therapeutic targets. However, only a few studies in dogs are available. UC are the most common bladder tumors in dogs; deciphering the role of miRs in canine UC would provide new tools for biomarker discovery and insights into its cancer biology. The goals of this study were to confirm that amplifiable miRs can be isolated from formalin-fixed, paraffin-embedded (FFPE) bladder tissue and to compare the expression of miRs in non-cancerous and UC specimens. Tissue cores were acquired by selecting areas of interest on H&E-stained tissue sections and punching out corresponding areas in the FFPE tissue blocks using an Illinois biopsy needle. MiRs were isolated using miRNeasy FFPE kit (QIAGEN, CA). RNU6B, mir-21, 103, 155, let-7a and let-7b were quantified by RT-qPCR using the miScript PCR system (QIAGEN) on a QuantStudio3 thermocycler (ThermoFisher Scientific, MA). Relative expression was calculated by the 2^-ΔΔCt method with RNU6B as the normalizer, and compared by t-test. Let-7a is downregulated while let-7b is upregulated in canine UC tissue, as seen in human UC. The let-7 family is involved in cell cycle and tumor suppression. No expression changes were detected for miR-21, 103, and 155. This study shows that miRs can be isolated from FFPE bladder tissue allowing miR expression studies in archived samples. Moreover, two dysregulated miRs were identified in canine UC, which will be used for future biomarker research. 

Research grant: Boehringer Ingelheim and Purdue Veterinary Medicine

Student Support: Boehringer Ingelheim and Purdue Veterinary Medicine

Field of Research: Comparative Pathology

Carly N. Gundlach, Kerri E. Rodriguez, Marguerite E. O’Haire 

Center for the Human-Animal Bond, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana 

Introduction: Service dogs play an important role in assisting individuals with disabilities. Often, a strong bond is formed between the service dog and handler. Pet dogs also form a bond with their owner, but how this relationship may differ from the bond with a service dog is not well researched. The purpose of this study was to compare the strength of the human-animal bond among individuals with disabilities who have service dogs or pet dogs. Methods: Study participants were recruited through Canine Assistants, an organization that places mobility and medical service dogs across the United States. Participants had either already received a service dog (n=96) or were on the waitlist (n=38) to receive one in the future but had a pet dog in the home. The strength of the human-animal bond was assessed with The Monash Dog-Owner Relationship Scale (MDORS) subscales of Emotional Closeness and Dog-Owner Interaction. Results: Results indicated that a stronger human-animal bond existed between service dogs and their handlers when compared to pet dogs and their owners. Specifically, service dog handlers reported higher Emotional Closeness scores (t=4.31, p<0.001) and higher Dog-Owner Interaction scores (t=4.14, p<0.001) than pet dog owners. Analyses of individual questions also revealed that specific aspects of the human-animal bond were stronger in the service dog-handler relationship. Discussion: Results show that in addition to the physical assistance they provide, service dogs have a unique relationship with their handlers that elevate their role beyond that of a pet dog. Future research should examine how bonding with service dogs varies by age and disability severity and explore potential drawbacks of this relationship. 

Research Grant: Elanco Animal Health

Student Support: PetSmart Charities and Purdue Veterinary Medicine

Weldon School of Biomedical Engineering (Main) and Department of Basic Medical Sciences, Musculoskeletal Biology and Mechanics Lab, College of Veterinary Medicine, Purdue University, West Lafayette, IN.

Osteocytes, the primary cells within the bone matrix, are postulated to be the sensors of mechanical load in bone and, consequently, direct the bone's response to the load by stimulating growth through the WNT pathway. Male and female TCF/LEF:H2B-GFP transgenic mice were used to access skeletal osteocytes' molecular response to load, specifically examining the canonical WNT pathway that ends in activation of the TCF-LEF promoter. The mice underwent tibial loading in one limb and after 24 hours were euthanized and the tibiae were removed from the loaded and contralateral, non-loaded control. Cryo-sections were produced from the bone samples. Microscopic evaluation of osteocytes was performed and the total number and percentage of fluorescent cells from the loaded and control limbs, as well as between sexes, were compared. Validating the use of these reporter mice for detecting mechanical activation of the WNT pathway is a key step in developing experimental approaches for examining the effects of targeted gene knockout, sex hormones, or pharmacologic agents, like parathyroid hormone, on mechanosensitive WNT pathway activation. 

Research Grant: Purdue EVPRP New R01 Grant

Student Support: Boehringer Ingelheim

Christa Cheatham¹, My Hahn Hoang¹, Gozde Uzunalli¹, Seth Herr², Alix Dieterly¹, Riyi Shi^3,4, Tiffany Lyle^1,3 

¹Department of Comparative Pathobiology, ²Department of Basic Medical Sciences,

³Purdue University College of Veterinary Medicine, West Lafayette, IN;

⁴Purdue University Weldon School of Biomedical Engineering, West Lafayette, IN 

The signature injury of modern warfare is blast-induced neurotrauma (BINT) due to the use of explosive devices. The clinical symptoms of BINT have been correlated with neuropathology including edema, hemorrhage, neuronal necrosis, and increased paracellular permeability of the blood-brain barrier (BBB). Herein, we aim to investigate the pathological alterations of the BBB in rats following single or repeated blast exposure.  The BBB is composed of endothelial cells with tight junctions, a basement membrane, pericytes, and astrocyte endfeet.  BINT was induced by using an open-ended blast apparatus to deliver single or triple 150 kPA shockwaves to 3-month-old male Sprague Dawley rats. After 24 hours, the brains were excised, flash frozen, and cryosectioned into 5μm thick sections. Qualitative and quantitative evaluation of the BBB was accomplished using immunofluorescence microscopy and Zen blue analysis software. We observed alterations in expression with endothelial cells, tight junction proteins, tight junction adaptor proteins, basement membrane, and pericytes. Our preliminary findings demonstrated a 1.82-fold (p=0.037) increase in the expression of claudin-5, a tight junction protein, in the single blast model compared to the control. There was a 2.01-fold (p=0.018) increase in the expression of PDGFR-β, a pan-pericyte protein, in the single blast model compared to the control. The triple blast model demonstrated a 1.87-fold decrease (p=0.028) in PDGFR-ꞵ expression compared to that of the single blast. This is the first comprehensive pathologic analysis of the BBB in BINT in an experimental model. These data will support the development of a robust and reproducible experimental model of blast-induced neurotrauma.   

Research Grant: None

Student Support: Boehringer Ingelheim Veterinary Research Scholars Program and Purdue University College of Veterinary Medicine

Blair N Hooser, Dayna L Dreger, Angela M Hughes, Balasubramanian Ganesan, Lauren Holtvoigt, Kari J Ekenstedt

Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Hooser, Dreger, Ekenstedt), Wisdom Health, Vancouver, WA (Hughes, Ganesan, Holtvoigt)

Few species match the physical diversity of Canis lupus familiaris, the domestic dog. There are hundreds of recognized breeds, defined by genetically-determined physical characteristics. Epistasis, the masking of a gene’s expression by the presence of a different gene variant, results in some alleles being “hidden” or not phenotypically visible, despite being present. Data from over 12,500 purebred dogs, representing over 250 breeds, was evaluated at 7 coat color (full and dilute expression of pheomelanin and eumelanin, white spotting, and color patterns) and 5 physical characteristic (ear set, skull shape, tail length and coat type) genes. Allele frequencies at each locus were calculated by breed and related breed groups, revealing hidden variation. For example, American Eskimo Dogs, UK Schipperkes, and Flat Coated Retrievers carry the “e” allele (yellow) 92.2, 41.7 and 7.3 percent of the time, respectively. In American Eskimo Dogs, yellow is masked by other genes and persists because it is not under selection pressure. In both Schipperkes and Flat Coated Retrievers, yellow is undesirable, so may go unreported in litter records. Unexpected alleles were occasionally observed; for instance, based on physical appearance, the Polish Greyhound was expected to be more closely related to UK sighthound breeds. However, we found it more likely developed from Mediterranean sighthounds due to the high allele frequency of a coat color trait that has higher frequency in those breeds. Hidden allele frequencies provide information about the history and relatedness of breeds, and allows estimation of when specific coat color variants were first observed.

Research Grant: Data provided by Wisdom Health and the commercially available Wisdom Panel canine DNA test

Student Support: Supported by a donation from Wisdom Health, a subsidiary of Mars, Inc.

Ashley Hopkins, Kwaku Kyei-Baffour, Daniela Peña, Abhijit Mukhopadhyay, Lynn Guptill 

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, and Purdue Institute for Drug Discovery, Purdue University, West Lafayette, Indiana

This study tested the hypothesis that aryl isonitrile compounds will significantly reduce Staphylococcus pseudintermedius replication and survival in planktonic culture and biofilm, and are not toxic to  mammalian cells. Aryl isonitrile compounds were recently shown to have potent activity against drug-resistant Staphylococcus aureus. A panel of 38 compounds synthesized in Dr. Mingji Dai’s laboratory at the Purdue Institute for Drug Discovery was screened against two S. pseudintermedius isolates; 13 compounds with minimum inhibitory concentration (MIC) ≤ 4 uM were evaluated further against a panel of 13 isolates. Staphylococcus pseudintermedius isolates from animals treated at Purdue University’s Veterinary Teaching Hospital were obtained from the Indiana Animal Disease Diagnostic Laboratory. Minimum bactericidal concentration (MBC) and MIC were tested using Clinical and Laboratory Standards Institute methods. Efficacy against biofilm was tested using the 96 well plate method. Toxicity for mammalian cells (Madin-Darby canine kidney (MDCK) cells) was assessed using MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4- sulfophenyl]-2H-tetrazolium, inner salt, Promega) assay. MBC data suggest that the compounds are bacteriostatic. Time kill assays are underway to further address this issue. Compounds were not toxic to MDCK cells at concentrations well above MIC (256 uM) and showed good ability to disrupt established biofilm. In summary, aryl isonitrile compounds had potent activity against planktonic S. pseudintermedius, were not toxic for mammalian cells, and effectively disrupted established biofilms.  Further analysis is currently being conducted to investigate structure-activity relationships of the compounds. These compounds hold promise for further development as treatments for S. pseudintermedius.

Research Grant: American Veterinary Medical Association (AVMA) and American Veterinary Medical Foundation (AVMF)

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

Chris Mapes, M. Preeti Sivasankar, and Abigail Durkes 

Department of Comparative Pathobiology, College of Veterinary Medicine (Mapes, Durkes), and Speech, Hearing & Language Sciences Department (Sivasankar), College of Health and Human Sciences, Purdue University, West Lafayette, IN 

Women report a variety of voice changes associated with their menstrual cycle including breathiness, changes in pitch, unsteady pitch and loss of high notes. These voice changes are termed dysphonia premenstruales. Erythema and edema have also been reported in women experiencing dysphonia premenstruales. This study set out to determine if female rats experience gross or microscopic changes in the larynx depending on their estrous stage. To determine estrous stage, cytology of daily vaginal smears was stained with Modified wright stain and evaluated by light microscopy. Estrous stages (proestrus, estrus, and diestrus) were determined based on vaginal cell type proportions. Once a rat reached the desired estrous stage, she was culled and her larynx was harvested. Larynges were fixed in neutral buffered formalin and embedded in paraffin according standard methods. Larynges were sectioned in the coronal plane and stained with hematoxylin and eosin (HE), Masson’s trichrome, Verhoeff-van Gieson, Alcian blue, and Movat’s pentachrome. Differential staining pattern and morphology will be reported at poster presentation. Results of this study will determine the viability of female rats as comparative animal models of dysphonia premenstruales. 

Research Grant: National Institute of Deafness and Communications Disorders

Student Support: Purdue Veterinary Medicine’s 2018 Veterinary Scholars Summer Research Program

Jessica E. Linder, Stephanie Thomovsky, Jessica Bowditch, Kris Kazmierczak, Claudine Auld, Melissa Lewis 

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 

Acute intervertebral disc herniation (IVDH) occurs commonly in dogs, and physical rehabilitation is increasingly utilized to promote recovery. Objective outcome measures capable of tracking different aspects of functional recovery are needed to optimize rehabilitation protocols. The aim of this study was to determine static body weight (BW) distribution in neurologically normal small breed dogs predisposed to Hansen type I IVDH. Healthy dogs between 1 and 10 years of age weighing less than 20 kilograms were recruited. Static BW distribution between the thoracic and pelvic limbs was acquired using commercially available digital bathroom and kitchen scales and compared to a validated pressure-sensitive Tekscan^TM animal walkway system. Reproducibility between methods was determined using a paired t-test. Twenty five dogs were enrolled including 6 Dachshunds, 4 Beagles, 4 Corgis, 1 Basset Hound, 1 French Bulldog, 1 Shih Tzu, 1 Pug and 7 chondrodystrophic mixed breeds. Mean age was 4.56 years (2.69) and mean BW was 12.05 kg (3.28). Measurements could be obtained in all dogs on the digital scales and in all but one dog on the pressure sensitive walkway. Combining values for the digital scales, mean thoracic to pelvic limb BW distribution was 63% to 37% of total BW while thoracic and pelvic limb values for the pressure sensitive walkway were 68% and 32%, respectively (p<0.0001). Results demonstrated that static BW distribution in a population of healthy small breed dogs was simple to obtain using digital scales but differed from values on the pressure sensitive walkway. This study generated weight distribution data for subsequent comparison to dogs recovering severe acute IVDH. 

Student Support: Boehringer Ingelheim Veterinary Scholars Program, Purdue University College of Veterinary Medicine

Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Canada, Dreger, Ekenstedt). Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Breur).

Pseudoachondroplasia (PSACH) is a skeletal dysplasia that causes dwarfism. All human cases of PSACH have been traced exclusively to the COMP gene, encoding cartilage oligomeric matrix protein. To date, 111 PSACH-causing mutations have been described in human COMP. PSACH is clinically unrecognizable at birth, but becomes evident by 2 years, and is characterized by gait changes and decelerated growth ultimately resulting in disproportionate short stature. PSACH has been described in Scottish Deerhounds (SD) with clinical, radiologic, and pathologic findings in striking parallel to human PSACH. The mode of inheritance in humans is autosomal dominant, with occasional reports of autosomal recessive; the latter appears to be the SD pattern of inheritance based on pedigree analysis. However, human families with phenotypically normal parents and PSACH offspring, initially suspected to have autosomal recessive inheritance, have instead revealed germline/somatic mosaicism in one of the parents. This scenario should also be considered in dogs. In the present study, we used a candidate gene approach to investigate the cause of PSACH in a family of SDs. DNA was extracted from archival bone samples of 3 dwarf and 2 normal SD littermates. We Sanger sequenced all COMP exons, as well as flanking intronic sequence, in one dwarf and one normal SD to screen for mutations. Variants observed in the dwarf SD, for which the normal SD was identical to the canine reference genome, were sequenced on all other SDs to confirm or exclude the variant as a potential cause of PSACH. 

Research Grant: Funding provided by start-up funds of Dr. Kari Ekenstedt

Student Support: Boehringer Ingelheim, Purdue University College of Veterinary Medicine

Cancer is a collection of diseases in which cells lack proper regulation of growth and death. It is currently the second leading cause of death in the USA following heart disease, prostate cancer being the second leading cause of cancer death in American men. Among the different areas of cancer research, increased attention has been paid in recent years to the study of the components of the tumor microenvironment (TME) as potential targets for cancer therapeutics, because tumors are complex groups of both neoplastic cells and non-neoplastic cells recruited from other tissues to the tumor microenvironment to establish an overall favorable location for the growth of tumors. One non-neoplastic cell group present in the TME are mesenchymal stromal cells (MSCs).  MSCs, among other characteristics, can secrete paracrine factors and immunomodulatory factors into the tumor microenvironment, and dependent on previous stimulation, can have a role in the repression or promotion of tumor progression. An example of MSC modulation can be achieved by stimulation of toll-like receptors (TLRs) in these cells. Upon stimulation or priming of TLR4, human bone-marrow derived MSCs (BM-MSCs) exhibit a pro-inflammatory phenotype called MSC1 shown to have a role in tumor repression; on the other hand, upon TRL3 stimulation, MSCs show an immunosuppressive phenotype MSC2 that has been associated with the promotion of tumor progression. Similar characteristic gene expression changes have been observed in our lab with adipose-derived MSCs (ASCs) towards MSC1 and MSC2. However, these phenotypes appear to be temporary, and are dependent on environmental factors to the cells. In Lu et al 2015, it was reported that MSCs obtained from tumors showed differences in TLR expression patterns, particularly of TLR4 when compared to MSCs from healthy individuals. If intending to use MSC1 for cancer therapeutics, it is important to understand how the exposure to factors secreted by cancer cells affect the priming of MSCs to MSC1 and MSC2, and consequently the effect of these cells in tumor progression. The primary objective of the summer project will study the effects of exposure of MSC, MSC1 and MSC2 phenotypes of ASCs to factors secreted by prostate cancer cells like PC3.

Reference: Cosette Rivera-Cruz

C. Auld¹, H.Y. Weng², K. Kazmierczak³, G.J. Breur³

¹College of Veterinary Medicine, Purdue University, West Lafayette, IN

²Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN

³Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 

During a normal walk or trot there is an asymmetry between the left and right thoracic limb (LTL and RTL) and the left and right pelvic limb (LPL and RPL) that is reflected in differences in kinetic and kinematic gait variables. The physiological and clinical significance of this asymmetry is poorly understood. The goal of this study is to determine whether the asymmetry is due to true dominance or a random event. Eight dogs were started in a square stance and then moved forward on a Tekscan Walkway, while peak vertical force (PVF) and sequence of limb movement were recorded. Twelve valid trials were analyzed per dog. First, we tested the hypothesis of dominance in terms of sequence of limb movement. Overall, the RTL was used most as first limb (34%), followed by LPL (31%), LTL (19%) and RPL (16%). Limb preference, that is, the most frequently used limb for walk initiation, was 57% (ranging from 33 to 92%), which was significantly different from 25% (i.e., a random event; P=0.027). Next, we tested the hypothesis that the limb to take the first step had the lower PVF during stance. Overall, the first limb used had the lowest PVF in 67% (ranging from 25 to 92%; P=0.289) of trials. The last hypothesis was that during stand and initial step the TL and PL with the highest PVF would be contralaterally related. The results, however, showed that higher PVFs were more ipsilaterally distributed during standing (52%, ranging from 33 to 75%; P>0.05) and initial step (67%, ranging from 50 to 92%; P=0.289). The findings suggest that dogs use a preferred limb for initiating walk. The preferred limb often has the lower PVF, and the limbs with the highest PVF tend to be ipsilaterally related; however, the results are not significant.

Injection of botulinum neurotoxin creates temporary paralysis by inhibiting acetylcholine release at the neuromuscular junction.  Currently botulinum toxin is used for various treatments including, pain relief, paralysis of joints, spasticity in patients with cerebral palsy, treatment of strabismus, hemifacial spasm, and cervical dystonia.  However, due to its short acting capability, frequent injections must be utilized to maintain its effect. The efficacy of the toxin after injection is measured in a mouse model using the Digit Scoring Assay (DAS), an assay based on the degree of digit abduction due to the startle response. Based on an on-going study of a slow release vehicle for the delivery of a long-term therapeutic neuromuscular blockade, there was a growing concern about changes in scoring over time. In this mouse study, we hypothesized that changes in the DAS scoring occur over time. We evaluated 16 mice once a day for 4 weeks using a previously published grading scale. Observations were made directly by 2 observers and indirectly using the Slo-mo feature of an iPhone 7.  Several factors that may affect DAS scoring were identified. An important factor causing variation in the degree of digit abduction may be due to differences in support while handling mice.  The more support the mouse had while being held by the base of the tail, the smaller degree of digit abduction compared to a mouse with less support held by the tip of the tail. There was no evidence for change in toe response and recommendations to further standardize the DAS were made.

Research Grant: Akina Inc

Student support: Boehringer Ingelheim Veterinary Scholars Program and Purdue College of Veterinary Medicine.

Laryngopharyngeal reflux (LPR) disease is believed to involve chronic backflow of gastric refluxate, resulting in damage to the laryngopharyngeal epithelium. Gastric refluxate has been shown to contribute to many laryngological conditions, including laryngitis, sore throat, ulcers, and globus pharyngeus. Approximately 10% of laryngologic patients and over 50% of patients with voice disorders have symptoms of LPR. Despite the prevalence of LPR, there is no suitable animal model. The pig provides a unique opportunity to study laryngeal disease as porcine and human vocal folds are very similar in terms of architectural, biochemical, neuromuscular, and cellular properties. In this pilot study, a novel in vivo pig model was developed to simulate the clinical condition of human LPR more closely by challenging healthy, uninjured laryngeal epithelia with acidified pepsin. An indwelling esophagostomy catheter was placed surgically and positioned near the aryepiglottic folds. The pigs were randomly assigned to a reflux group (n=3) and a sham (n=1). The reflux group received a continuous rate infusion of acidified pepsin solution, and the sham received saline. Autopsies were completed on days 8 and 12. Laryngeal tissues were examined grossly and histologically for differences between treatment groups. Immunohistochemistry for CD3+ T-cells was quantified via digital pathology. Grossly, cloudy fluid was observed in the larynges of the reflux group. Differences between treatment groups in histopathology and CD3+ T-cell quantification and localization were not statistically significant. This pilot study paves the way for future LPR studies, in which an improved surgical approach should allow for increased study duration.

Research Grant: None 
Student Support: Merial Veterinary Scholars Program

Schistosomiasis is a helminth infection that affects 200 million people worldwide, with 20
million life-threatening cases existing predominantly in Africa. Chronic infection results in
eosinophilic activation, portal hypertension, and fibrosis, which can result in organ failure. As
such, schistosomiasis presents a critical public health concern in the developing world where
there is little to no control of disease transmission and limited access to healthcare.
Importantly, previous studies have reported that maternal S. mansoni infection leads to fetal
acquisition of S. mansoni antigens in utero, which may be linked to reductions in childhood
vaccine efficacy often seen in helminth endemic regions. The immune effects of in
utero exposure to S. mansoni antigens, however, are not yet understood. Here, we investigate
the immunomodulatory pathways through which maternal S. mansoni infection impacted
neonatal immunization in mice using the vaccine tetanus/diphtheria. We found that at day 14
post immunization, pups born to infected mothers had significantly decreased germinal centers
in popliteal lymph nodes as compared to pups from uninfected mothers. Future studies should
examine the memory and recall response and identify aberrant stimulatory/survival signals that
might underlie the immunomodulation.

Research Grant: American Heart Association, Showalter Trust
Student Support: Purdue University College of Veterinary Medicine

Around 30,000 human deaths are associated with Clostridium difficile infections each year. In addition, virulent C. difficile has been implicated in causing diarrhea and colitis in a wide array of mammals including dogs, horses, pigs and non-human primates. The main virulence factors produced by C. difficile are two large toxins: toxin A and toxin B. Accurate detection of these toxins is necessary to differentiate between non-toxigenic strains and disease causing strains. Current detection methods include cytotoxicity assays (CTA) and enzyme-linked immunosorbent assays (ELISA) which are both labor-intensive and expensive. The aim of this study is to develop a novel method of C. difficile toxin detection using MALDI-TOF Mass Spectrometry. Commercially available, purified C. difficile toxin A and concentrated supernatants from bacterial isolates underwent proteolytic digestion to generate fragments small enough to be recognized by MALDI-TOF MS. Toxin production by the bacterial isolates was verified using ELISA prior to being ran on MALDI-TOF MS. Mass to charge (m/z) ratios and spectra for the purified toxin A were compared to the bacterial isolates. Results showed no matching spectra between purified toxin A and toxigenic bacterial isolates. Additionally, toxigenic and non-toxigenic strains generated congruent spectra and m/z ratios. These findings suggest that the proteolytic digestion was unsuccessful in generating fragments that were detected by MALDI-TOF MS.

Research Support: Indiana Animal Disease Diagnostic Laboratory

Student support: Boehringer Ingelheim and Purdue Veterinary Medicine

There is an urgent need for discovery of effective antimicrobial drugs and therapeutic strategies to combat important microbial pathogens of animals and humans. Staphylococcus pseudintermedius is a gram-positive bacterium that is a predominant cause of skin and soft tissue infections (SSTI) of animals including pyoderma, otitis, surgical site infections, and urinary tract infections. Emergence of S. pseudintermedius clinical isolates resistant to beta lactam antibiotics (methicillin-resistant S. pseudintermedius (MRSP)) and/or many other antibiotic classes (multiple drug resistance), underscores the critical need for effective treatments that do not induce antimicrobial resistance. The hypothesis tested was that novel antimicrobial compounds will result in significant reduction of Staphylococcus replication and survival in planktonic culture and biofilm. A panel of 68 compounds was screened against planktonic bacteria; 16 compounds with high efficacy were evaluated further. Bacterial isolates used were obtained from the Indiana Animal Disease Diagnostic Laboratory; isolates were cultured from animals treated at Purdue University Veterinary Teaching Hospital. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested using Clinical and Laboratory Standards Institute (CLSI) methods. Efficacy against biofilm was tested in 96 well plates via previously published methods. Compounds were initially screened using 1 MSRP and 1 methicillin-susceptible (MSSP) isolate; those with MIC of ≤2 mM or 2 mg/ml were further tested against a total of 8 isolates. Highly effective compounds will be studied further including evaluation of toxicity for mammalian cell lines and efficacy in infected animals.     

Research Grant: Purdue College of Veterinary Medicine

Student Support: Morris Animal Foundation

Neurofibromatosis Type II (NFII) is an autosomal dominant human genetic disease, characterized by the growth of tumors in the nervous system, such as vestibular schwannoma, meningioma, ependymomas, astrocytomas, and neurofibromas. Mutations of the NF2 gene which encode for the cytoskeletal protein neurofibromin 2, or merlin, lead to the NFII syndrome. Moreover, mutations of NF2 are often found in several types of human cancer, suggesting it acts as a tumor suppressor gene. The tumor suppression mechanism of this gene has been difficult to delineate, partly due to the lack of a model that closely resembles human NFII. Hippo signaling is known to play important roles in animal organ size regulation and tumorigenesis, and NF2 was found to be an important regulator for this pathway. We recently created zebrafish models of NFII.  Zebrafish have two homologs of NF2, nf2a and nf2b, and they develop malignant peripheral nerve sheath tumors when either gene is mutated.  We hypothesize the downstream genes of the hippo signaling pathway will be altered in the zebrafish nf2 mutants.  Using whole mount in situ hybridization, we found cgtfa and yap1 genes are upregulated in nf2a mutants compared to wildtype zebrafish embryos (1 day post fertilization). Results of these genes in zebrafish embryos 2 days post fertilization were inconclusive. Our results demonstrate that nf2 may regulate hippo signaling in our new zebrafish NFII models.  More experiments are needed to explore the relationship of nf2 and the hippo signaling pathway. Experiments on other pathways, such as RAC-PAK signaling, may be helpful in better characterizing our zebrafish models.

Research Support: Hayward Foundation

Student Support: Boehringer Ingelheim Veterinary Scholars Program and Purdue College of Veterinary Medicine

The link between animal abuse and domestic violence is well recognized phenomenon, which has led to a call for adoption of mandatory reporting of animal abuse by veterinarians. The purpose of this study was to explore the application of visual analytics in assessing this overlap between human and animal violence and its proximity to local veterinary clinics in an urban environment. Forty-five reports of animal cruelty and 11,508 reports of family violence occurring in Lafayette, IN were extracted from police records from January 1st, 2011 through June 20th, 2017. These cases were imported and analyzed via geospatial mapping software developed by VACCINE, Purdue University’s Department of Homeland Security Center of Excellence. This software, Visual Analytics Law Enforcement Toolkit (VALET), was used to create and compare heat maps of animal cruelty, child abuse, and domestic violence. Spatial analysis revealed a high degree of correlation in certain census tracts. Sixty percent of the animal cases occurred at the same street address as reported family violence. Six addresses contained at least one involved person present in both a human and animal case, though this may be an underestimate as not every report had associated involved persons information available. Geospatial mapping methods may help encourage reporting of abuse by veterinarians and focus interventions in areas of strategic need. Although data analysis would benefit from increased reporting of animal cruelty cases, visual analytics allows for identification of areas at high risk for household violence, which would enable animal welfare personnel to more effectively reach underserved areas of the community.

Research Grant: None
Student Support: Merial Veterinary Research Scholars Program

The skeleton is sensitive to mechanical loading and understanding the cellular mechanisms by which bone forms in response to load could be important in developing anabolic therapies for bone-wasting diseases. This in vivo loading study was conducting on mice that endogenously express GFP in association with the activation of the DMP1 or TCF/LEF promoters to assess the anabolic response of the tibia to mechanical load. Both DMP1 and TCF/LEF are highly expressed in osteocytes and are known to be responsive to mechanical stimuli. Osteocytes are the most abundant cells in the bone and are regarded as the mechanosensory cells responsible for communication with other bone cells to initiate bone formation and remodeling. Following loading, the tibiae were sectioned by means of cryosectioning and analyzed under a fluorescence-ready microscope. The number of fluorescent osteocytes will be indicative of the anabolic response of the tibia to applied mechanical load.

Merial Veterinary Research Scholars Program

Asthma affects millions of people worldwide. The standard therapy is inhaled corticosteroids and bronchodilators. However, patients with severe cases may not be well-controlled by this therapy. Also, long-term use of corticosteroids can affect the body negatively, including reduced growth rate in children. This makes it essential to discover new therapeutic methods for asthma patients. Tocotrienol, a type of vitamin E, is effective in mouse models with induced allergic asthma. It is critical to test their in vivo effects in models that closely resemble human asthma. Horses are more suitable models for severe asthma than rodents that poorly mimic human inflammatory airway diseases. Horses commonly develop a naturally occurring, asthma-like disease that is responsive to glucocorticoid and bronchodilator therapy.

We hypothesized that inhaled tocotrienols will reduce airway inflammation in asthmatic horses with a lower concentration of inflammatory cytokines in bronchoalveolar lavage (BAL) secretions. The lung function and BAL cytology of healthy and asthmatic horses were evaluated. Horses were then nebulized with a mixture of γ- and δ-tocotrienols or a placebo. Two hours after a second nebulization, the horses were challenged with a high, medium, or low dose of mold, Aspergillus terreus. Twenty-four hours after, the lung function and BAL cytology were evaluated again. The BAL cytology showed that tocotrienol significantly reduced the amount of neutrophils in horses that were administered a low dose of mold and there was a trend towards reduction in horses administered medium or high dose of molds. BAL samples will be analyzed for interleukin-4 (IL-4), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) by ELISA.   

Funding source: The Purdue Institute for Inflammation, Immunology and Infectious Disease (PI4D)

The blood-brain barrier (BBB) is the tightest and most effective biological barrier in the body. This unique barrier protects the brain from pathogens and toxins by controlling the efflux and influx of nutrients and waste products into and out of the neuroparenchyma. Herein, we aim to evaluate the functional components of the BBB using immunofluorescence analysis in two immunocompromised mouse models, athymic nude mice and the NOD-SCID-IL2 receptor γ^null (NSG) mice. Our findings will determine the most suitable model for analysis of BBB alterations in experimental models of brain metastasis. Athymic nude mice are T-cell deficient and are most commonly used as xenograft models.  NSG mice, which lack B, T, and natural killer (NK) cells, permit engraftment of human immune cells, and facilitate rapid growth of human tumor cells. We evaluated functional components of the BBB, including endothelial cells, pericytes, basement membranes, and astrocyte endfeet, of five 6-week old (2 male, 3 female) athymic nude mice and NSG mice using indirect immunofluorescence staining.  Following immunofluorescence staining, all images were procured using a Zeiss M2 fluorescence microscope and positive stained areas was measured using ZEN analysis software. No significant difference in immunofluorescence expression was identified in our preliminary evaluation of the BBB functional components in athymic nude mice compared to NSG mice. Thus, our findings support the use of NSG mice in analysis of the blood-tumor barrier in analysis of brain metastases of lung cancer. The use of NSG mice will facilitate the development of a reliable model system to understand changes to the blood-brain barrier in neoplastic disease.  

Research Funding: Indiana Clinical and Translational Sciences Institute

Student Support: Purdue University Veterinary Scholars Program

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used to provide pain relief in horses. A previously established, reversible foot lameness model has been used to demonstrate analgesic efficacy among NSAIDs; however, the mechanism by which NSAIDs reverse pain induced by this model has not been determined. We hypothesized that this model induces acute inflammation in the affected (lame) foot by application of pressure to the frog by a set screw in a custom-made shoe. Inflammatory markers measured in this study included foot temperature (via thermography), serum amyloid A (SAA) and ipsilateral cephalic vein eicosanoid production (PGE2 and TNF-a). We expected that increased foot temperature, SAA and eicosanoids would be associated with the presence of foot inflammation. A randomized crossover study was done using six healthy, adult horses. All horses received each of three intravenous NSAID treatments (flunixin meglumine, phenylbutazone, and ketorolac) as well as a placebo control (saline). Treatment was administered one hour after lameness was induced, and lameness was assessed hourly for 12 hours. For each of the four trials, thermography, as well as cephalic blood collection for SAA and eicosanoid testing, was done prior to lameness induction (baseline), immediately following lameness induction (T=-1h), immediately prior to treatment (T=0h), two hours after treatment (T=2h) and 12 hours after treatment (T=12h). Thermographic images were taken of both the left and right front foot coronary bands, both front foot heel bulbs, and the apex of the left front frog. Results to date show that for all horses, temperatures in the left front (affected) foot were higher than those in the right front (unaffected) foot, but SAA concentrations did not increase throughout each trial, regardless of treatment. The results of PGE2 and TNF-  concentrations are pending. 

Research Grant: 2017 Competitive Equine Research Funds 

Student Support Fund: 2017 Summer Research Fellows Program

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is one of the most economically important diseases of swine in both North America and Europe. While signs of PRRSV illness can vary, in general, they are fever, pneumonia, and lethargy, and in gestating sows, there may be abortions and stillborn piglets. Piglets that are born successfully often struggle to thrive. PRRSV in serum is frequently detected using an ELISA test. Typically, veterinarians send several milliliters of blood or serum to the diagnostic laboratory. If sent through the mail, it must be kept cool on ice packs. To minimize the amount of blood needed, to simplify and speed up separation of plasma from blood, and to provide a way to send the sample in an envelope through the mail without refrigeration, a membrane separation technique has been developed using Noviplex cards. These cards only require 120µL of whole blood which, when placed on the cards, allow plasma to be separated, dried, stabilized, and stored on the card. They can then be transported at room temperature. We hypothesized that serum stored on Noviplex cards for variable periods of time would be able to detect PRRSV antigen comparable to standard methods. In this study, plasma from pigs was tested for the presence of PRRS virus antibodies using the IDEXX PRRS X3 ELISA testing protocol. The blood samples (120µL) from 6 known negative and 6 known positive pigs were individually placed on Noxiplex cards according to manufacturer’s instructions. The cards dried for three minutes and then the top membrane was removed. The plasma on the cards dried for another fifteen minutes. To reconstitute the plasma, 7.6 µL of diluent from the PRRS ELISA kit was added on top of the membrane. The reconstituted samples were then diluted 1:40 to remain consistent with the protocol for the ELISA test. Serum samples prepared from blood using a standard centrifugation protocol and plasmas from cards were analyzed using the PRRS X3 ELISA test kit. Optical densities from each serum/plasma test were read on an ELISA plate reader. The normalized optical densities from positive plasma stored on Noviplex cards were statistically the same (p = 0.05) as positive serum prepared by centrifugation. Negative plasma/sera from cards and centrifugation were also statistically the same(p = 0.05), and significantly different (p ≤ 0.05) from the positive samples.  Plasma prepared on cards and stored at room temperature for 7 or 14 days was stable and provided the same results. These results confirm that Noviplex cards serve as a reliable separation, storage and transport method for PRRSV antigens in plasma.

The diseases that affect wolves are diverse and the impacts varied. Disease in wolves can affect ecosystem, livestock and human health, give insight into the genetic background of dogs, and assist in conservation planning and implementation. As a keystone predator, wolves have a large impact on the health and diversity of the surrounding habitat and disease that impacts wolf survival will, in turn, affect the entire ecosystem. Infectious and parasitic diseases carried by wolves can be transmitted to other wildlife species, livestock, and humans. Also, conservation and re-population efforts may be impacted by the presence of heritable congenital defects and it is therefore important to report the prevalence of their occurrence within wolf populations. Systematic review methods were utilized to identify published reports of health outcomes in either grey (canis lupus) or red (canis rufus) wolf populations worldwide. Search terms included pertained to both wolf populations as well as multiple health outcomes including infectious, parasitic, neoplastic, congenital, and metabolic diseases. Relevant data were extracted from manuscripts included in the review. A total of 3718 articles met the initial search requirements and 301 articles were retained after the first relevance screening. Health outcomes were stratified based on species, population type, and geographic regions. The majority of health outcomes described in the literature concerned parasitic infections; other outcomes described include infectious diseases, neoplasia, congenital disorders, trauma and others. To the authors’ knowledge, this is the first comprehensive systematic review of all health outcomes in wolf populations. 

Research grant funded by Merial Veterinary Research Scholars Program 

Transitional cell carcinoma (TCC) is the most common urinary tract cancer in dogs. A new form of treatment is immunotherapy which uses the body's own immune system to help fight cancer. When TCC has become resistant to chemotherapy drugs, future immunotherapies could be used as biological reagents to help stimulate the body’s own immune response. By using the body’s own immune response in dogs with TCC, we can potentially target specifically just the TCC and not harm healthy tissues. In order to develop immunotherapy reagents, an assay such as a mixed lymphocyte reaction (MLR) assay is needed to test the efficacy in vitro. In an MLR assay leukocytes from two genetically distinct individuals of the same species are co-cultured resulting in cell blast formation, DNA synthesis, and proliferation. The purpose of this project was to set up and validate an MLR assay for dogs. Blood was collected from a normal dog, laid over Ficoll-Hypaque, and then centrifuged to isolate the mononuclear cells (PBMCs). The PBMCs were washed, resuspended in media, and then incubated for 24 hours. IL-4 and GM-CSF were added to the monocytes in an attempt to inhibit macrophage maturation and promote dendritic cell maturation, and incubated for 8 days. PBMCs containing CD4+ cells were then collected from a dog, and mixed with the dendritic cells, and treated with piroxicam (1μM, 2.5μM, and 5μM, being investigated for immunomodulating effects) or vehicle control, and incubated for 5 days. An ELISA test was then used to measure the cell's response (IFN-gamma production) to the piroxicam treatment, along with a canine IFN-gamma standard. The results so far have been inconclusive. Future investigation is in progress to further trouble shoot the assay and to improve our methods involved in this study.

The acute phase protein, fibrinogen, is commonly used in horses to detect inflammation. To avoid the confounding effects of dehydration on fibrinogen interpretation, the total plasma protein to fibrinogen ratio (TPP:Fb) is often calculated, with lower values indicating inflammation. Historically, plasma fibrinogen concentration was estimated via a heat precipitation method. A more accurate clotting method, using a Diagnostica Stago STA Compact©, is now used in our lab which renders the prior interpretive guidelines for TPP:Fb obsolete. This study seeks to establish new interpretive guidelines for TPP:Fb. First, TPP:Fb was calculated for a limited reference population of 30 clinically healthy horses and a reference interval (RI) of 23.7-48.1 was calculated (mean±1.96SD). Secondly, a retrospective study was conducted using complete blood counts, serum chemistries and plasma fibrinogen concentration from the PUVTH medical records of 226 horses presented between 5/31/2016 and 5/31/2017. The horses were divided into groups of No Evidence of Inflammation (NEI), Inflammation (INF) and OTHER (glucocorticoid, epinephrine, equivocal inflammation) based on typical leukogram changes and plasma fibrinogen concentration. From one-way ANOVA, INF horses had a lower TPP:Fb (p<0.001) than NEI or OTHER horses. A TPP:Fb below RI was observed in 55.6% (75/135) of INF horses, 12.8% (6/47) NEI horses and 15.9% (7/44) of OTHER horses. Thus, the TPP:Fb supports inflammation in 55.6% of INF cases as classified by other markers. Conversely, TPP:Fb suggests a diagnosis of inflammation in 28.7% of cases missed by other markers. The results suggest that both leukogram and TPP:Fb are needed to provide a robust identification of inflammation in horses.

Service dogs are well-known for the functional tasks they perform to help individuals with physical disabilities. However, these dogs’ impact on functioning, quality of life, and other psychosocial areas of health has not been examined quantitatively. The objective of this research was to empirically evaluate the effects of service dogs on overall psychosocial functioning including social, emotional, and work/school functioning within age categories. Analyses included 157 individuals enlisted through a national service dog provider, Canine Assistants, including those paired with a service dog (n=98) and those on the waitlist to receive one (n=59). Primary outcomes were measured with the Pediatric Quality of Life Inventory between those with and without a service dog within two age groups. For individuals 25 and younger (n=96) those with a service dog had significantly improved overall and work/school functioning compared to those on the waitlist (t=2.196, p=0.031; t=2.389, p=0.019). Improvement in social functioning approached significance (t=1.910, p=0.059), while there was no difference in emotional functioning (p=0.434). For individuals older than 25 (n=61) those with a service dog had significantly higher overall, work/school, social, and emotional functioning than those on the waitlist (t=3.573, p=0.002; t=2.647, p=0.011; t=2.076, p=0.043; t=4.404, p<0.001). The findings suggest that service dogs have significant psychosocial effects on recipients with physical disabilities, and that age may play a role in perceived social and emotional functioning. This is important for further research on the psychosocial benefits of service animals and the individuals they assist.

Research grant: Elanco Partnership: Canine Assistants

Student support: Boehringer Ingelheim Veterinary Scholars Program

Hepatocellular cancer (HCC) is the fifth leading cause of cancer deaths worldwide with chronic hepatitis B virus (HBV) infection posing an increased risk of developing HCC. Epithelial Cell Adhesion Molecule (EpCAM), a transmembrane glycoprotein found in epithelial tissue and hepatic progenitor cells, is re-expressed in a subset of hepatocytes that exhibit stem-cell-like properties. EpCAM has been studied as a biomarker for poor prognosis HCC and in activating the Wnt/β-catenin pathway. Regulated intramembrane proteolysis of EpCAM in HBV replicating cells activates Wnt/β-catenin pathway via cleavage of EpCAM by γ-secretase to release an intracellular domain (EpICD). EpICD complexes with β-catenin and other proteins to transcribe Wnt-induced oncogenes. The purpose of this study is to determine the effectiveness of diallyl trisulfide (DATS), a bioactive compound in Allium vegetables previously shown to have anticancer properties, as a potential inhibitor of γ-secretase and its downstream effects. Co-transfection of Wnt-responsive plasmid, TOPFlash, and Renilla plasmids expressing luciferase activity into HepAD38 cell lines were used to measure β-catenin transcriptional activity. Treatment with 100 µM DATS showed 80% reduced luciferase activity in comparison to cells not treated with DATS, indicating reduced cleavage of EpCAM. Western blot analyses were also performed to confirm these findings. The results of this in vitro study demonstrate the efficacy of DATS as an EpCAM signaling inhibitor; and, suggest its potential use as a preventive agent in the development of HCC in chronically infected HBV patients. Further research should study the effects of DATS on EpCAM signaling in other types of cancer.

Support of NIH Grant 5R01DK044533/19 to OA

Student Support: Merial Veterinary Scholars Program

Noviplex™ duo plasma prep cards are a new micro-sampler product that separates serum from whole blood.  They allow 3.5µL of serum to be collected into two small disks that can be used for a variety of testing.  These cards only require two drops of whole blood and hold potential clinical value for blood testing in anemic and smaller patients.  Usually, blood tests require milliliters of blood to perform selective, routine diagnostic tests.  A study was conducted to determine the usefulness of these cards in veterinary clinical practice.  Canine heart worm antigen and lactate dehydrogenase (LDH), a potential prognostic biomarker for lymphoma in dogs, were chosen to assess the efficacy of the Noviplex™ duo plasma prep cards.  Serum samples collected with Noviplex™ cards were compared to serum collected by centrifugation of whole blood.  Serum extraction from the cards was achieved using phosphate-buffered saline solution, and the activity of LDH and concentration of heartworm antigen were measured by enzyme-linked immunoabsorbent assays (ELISA).  Both tests are colorimetric assays in which the optical densities were quantitated with a microplate reader.  Using Noviplex™ cards a positive color change can be observed in both tests but the intensity of the color is usually less than that of a direct serum sample.  However the optical densities have been consistently higher than all negative controls so it is possible for a reference interval to be established.  

Student Support:  Merial Veterinary Scholars Program

Despite many advances in cancer research, in vitro models that accurately represent the environment in which cancer develops have not been established. One example of the environment’s role is the link between increased stromal density and increased risk for onset and progression of aggressive forms of breast cancer. Our hypothesis is that stromal stiffness and cells affect the phenotypes of non-neoplastic and preinvasive mammary epithelial cells by influencing the morphology of the cell nucleus. In a first approach, we are building a model that includes cancer cells, non-neoplastic epithelial cells, basement membrane component (laminin), and stromal components (collagen I and fibroblasts). First, non-neoplastic and preinvasive cells were cultured separately in the presence of collagen I matrix of adjustable stiffness. The nuclear morphology was assessed based on DAPI staining (for nuclear areas and circularity) and SC35 immunostaining (for normal differentiation) using ImageJ. The non-neoplastic cells showed a significant difference in nuclear area and circularity as well as differentiation, while the preinvasive cells displayed a significant difference in nuclear circularity depending on stiffness. Next, the non-neoplastic and preinvasive cells were cocultured on top of the collagen I matrix. A significant alteration in nuclear area for preinvasive cells was measured as stiffness changed. In future experiments, fibroblasts will be embedded in the collagen matrix underneath the coculture of epithelial cells. The data collected from these experiments will be compared with data from real breast tissue to determine which local environment allows cells to establish a phenotype that most closely resembles the real tissue. 

Research Grant

Trask Innovation Fund to SAL 

Student Support

Merial Veterinary Scholars Program 

Field of Research

Cell Biology and Cancer Cell Biology

Military veterans are increasingly seeking alternative and complementary treatments for posttraumatic stress disorder (PTSD). One emerging complementary treatment is the provision of a PTSD service dog. These specially trained service dogs live inside the home and thus are exposed to the entire family, including spouses. There is currently an absence of literature examining how the spouse copes with the costs of owning a PTSD service dog and how this relates to their caregiver burden. This study assessed how a spouse’s perceived cost of a PTSD service dog can affect the spouse’s mental health and wellbeing.

Military veterans and their spouses were recruited from an accredited service dog provider, K9s for Warriors. Couples with a service dog (n=36) and couples on the waitlist to receive one (n= 29) consented to participate in a survey with standardized self-report measures. The perceived costs of dog ownership were assessed through the Monash Dog Owner Relationship Scale Perceived Costs subscale (MDORS-PC). Spouses and veteran’s MDORS-PC scores were then used to calculate a difference score. Three groups were created: (1) Couples in which the spouse had lower perceived costs than the veteran and therefore less caregiver burden (2) couples in which the spouse had higher perceived costs than the veteran and therefore more caregiver burden, and (3) couples in which the spouse and veteran perceived roughly equal costs in owning the service dog. Measures of mental health and wellbeing were assessed with the National Institutes of Health (NIH) Patient Reported Outcomes Measurement Information System (PROMIS) short forms of anxiety, anger, social participation, sleep disturbance, social isolation, and companionship. Sleep quality was assessed with the Pittsburgh Sleep Questionnaire (PSQI) and depression was assessed with the Patient Health Questionnaire (PHQ).

Analyses indicated significantly decreased mental health and wellbeing in spouses with higher perceived costs than the veteran compared to the other two groups. Specifically, spouses perceiving more costs of owning a PTSD service dog than the veteran had decreased sleep quality (F(2,32) = 4.85, p = .014) with increased sleep disturbance (F(2,32) = 6.07, p = .006), increased depression (F(2,32) = 8.23, p = .001), and increased social isolation (F(2,31) = 11.07, p < .001). However, with the exception of sleep quality (t(40)= -2.30, p = .027), spouses who had higher perceived costs did not exhibit poorer mental health and wellbeing than the spouses in the waitlist group, indicated that there was not a significant decrease in quality of life after receiving a service dog.

Results provide initial evidence to suggest that the perception of care burden matters and a closer examination is necessary in order to fully understand the secondary impacts of a PTSD service dog on members of the entire household.

Canine Influenza Virus (CIV) H3N2 is a subtype of Influenza A Virus (IAV) of avian lineage that originated in Asia. Recently, CIV H3N2 has been of interest due to a widespread, sudden outbreak in the Chicago, IL region in April of 2015. The aim of this study was to investigate the associated risk factors and prevalence of antibodies against IAV and CIV H3N2 in serum samples collected from randomly selected dogs and cats native to various U.S. states. These samples were obtained from the Purdue University Small Animal Hospital. In order to measure the seroprevalence of antibodies against CIV H3N2 in the 458 canine and 67 feline samples, a commercial enzyme-linked immunosorbent assay (ELISA) and in-house hemagglutination inhibition (HI) test were utilized. Using ROC Analysis of the results, it was determined at a 100.00% sensitivity and 98.07% specificity that the optimal HI cutoff titer was 1:32. From the HI results, it was found that dogs had a 2.18% seropositivity for CIV H3N2 while 8.96% of cats were seropositive. At this time, there were no apparent trends found between seroprevalence and associated risk factors of the animals. With this, it was concluded that CIV H3N2 is not too widespread in the Chicago area and Midwest region as a whole, but the virus should still be kept under surveillance because of its versatile ability to re-assort and spread as a more virulent strain.

Lawn chemical exposure has been linked to the development and progression of bladder cancer. The purpose of this study is to evaluate the effects of the active ingredients of lawn herbicides on the proliferation of normal canine urothelial cells in vitro. Normal canine urothelial cells were exposed to varying concentrations of two active ingredients found in lawn herbicides: 2, 4-Dichlorophenoxyacetic acid (2, 4-D, 1μM, 2μM, 10μM, 20μM) and dicamba (1μM, 2μM, 10μM, 20μM). Once cells were treated, immunofluorescence was utilized to visualize the expression of Ki-67, a cellular marker for proliferation. Cells were counter-stained with DAPI, a fluorescent stain that binds to DNA making the nuclei visible. An increase in proliferation was noted when cells were treated with 2,4-D at concentrations of 1 or 2 μM. Increased proliferation of cell line G was noted when cells were treated with dicamba at concentrations of 1 or 2μM. In cell line K, minimal effects of the chemicals were noted. Further investigation is warranted to determine the role of herbicide exposure in pathway activation and mutational events that could contribute to the development of bladder cancer.

Dogs can form close relationships with their owners. Previous studies suggest that dogs seem to use their owners as a “secure base” for exploring their environment and are more relaxed when their owners are present. Although the dogs that were diagnosed for separation-related anxiety exhibited more stress when their owners were absent, intensity of the behavior or physiological signs have not been investigated in a quantitative manner. Separation-related anxiety in dogs is suspected as a sustained fear and is manifested in individuals with trait anxiety. The results of a previous study demonstrated that dogs with separation-related anxiety exhibit stress responses across the board. However, limited research focuses on quantitative measurements of behavioral and physiological responses for dogs at risk of separation-related anxiety. To understand characteristics contributing to separation-related anxiety, we planned to establish quantitative measurements in a laboratory setting. In this pilot study, we conducted the modified behavior test that consisted of a baseline, 5 minute-separation from the owner and greeting to the owner in an unfamiliar environment to collect both behavior and physiological data (e.g. salivary cortisol). Sixteen owners and their clinically healthy dogs that were not under any medical or behavioral treatments were recruited. All behaviors of the dogs during the separation test as well as salivary cortisol of the dogs before and after the separation test were collected. The recorded videos were analyzed using focal sampling, while the salivary cortisol levels were measured using an enzyme-linked immunosorbent assay (ELISA). Eleven dogs had both behavior and salivary cortisol data that were used for further analysis. The results showed that the mean salivary cortisol levels before and after separation were 2.1 ± 0.11 ng/mL and 2.8 ± 0.15 ng/mL, respectively. Salivary cortisol levels before and after the separation test were strongly correlated each other, however, when it compared with behavior categories dogs that whined during separation showed lower cortisol level after the separation test. Dogs that behaved actively (e.g. walking and sniffing in the room) during separation were physically closer to the owners when they returned. This study demonstrated the measurable differences between behavioral and physiological responses in dogs during the behavior test that provoked anxiety being separated from the owner. Our results will be confirmed further with an increased number of dogs including clinically affected population with separation-related anxiety.

Inflammatory airway disease (IAD) is the second most common cause of poor performance in athletic horses. The etiology of IAD is incompletely understood but assumed to be multifactorial. Viral respiratory pathogens have the potential to cause airway inflammation. Therefore, we hypothesized that respiratory viruses play a role in the development of IAD. The purpose of this study was to validate the efficiency of multiplex real-time PCR to detect equine respiratory viruses. Fluorogenic TaqMan® probe assays were performed to detect and differentiate equine herpesviruses (EHV)-1, 2, 4, and 5, equine coronavirus (ECoV), equine influenza virus (EIV), and equine arteritis virus (EAV). Serial dilution of plasmid DNA template was used to optimize singleplex reactions. Duplex reactions were performed to simultaneously detect EHV-1 and 4 and EHV-2 and 5. A triplex reaction allowed detection of ECoV, EIV, and EAV. Singleplex assay efficiency ranged from 91 to 109%, while multiplex assay efficiency ranged from 107 to 121%. Similar efficiencies of both assays indicated that multiplex assays will be a useful research tool for further investigation of the relationship between viral pathogens and IAD. The validated assays presented here will conserve up to 57% of clinical sample volume. Establishing the relationship between viruses and IAD using the designed assays will lay the groundwork for future investigations to target prevention and treatment strategies. 

Approximately 7.5 million Americans suffer from difficulty using their voice. Phonatory difficulties are often multifactorial with Reinke’s edema being one of the most common underlying pathologies. Common causes of Reinke’s edema include smoking, voice overuse, and reflux of stomach acids into the larynx. The purpose of this study was to develop an in vivo model of Reinke’s edema by challenging two group of six pigs with cigarette smoke in a customized inhalation chamber. The first group of pigs was exposed to three cigarettes per day for 20 treatments (1 time per day, 5 days per week, 4 weeks). The second group of pigs was exposed to five cigarettes per treatment for 60 total treatments (3 times per day, 5 days per week, 4 weeks). A week prior to treatment, each group of pigs was habituated to the chamber and human contact to reduce stress and ease treatment administration. Behavior modification was accomplished using positive reinforcement. At the end of the four-week study, pigs were humanely euthanized and vocal fold, tracheal, and nasal mucosal samples were obtained for histologic evaluation, RT-qPCR, and TEM. The week of habituation for each group of pigs successfully increased the efficiency and reliability of treatments. The pigs willingly entered in the chamber and tolerated enclosure for the allotted time. RT-qPCR and TEM are currently in progress. The data obtained from this study offers a potential novel experimental methodology to simulate the development of Reinke’s edema in healthy pigs where there is currently no reproducible animal model. The success of this methodology could impact future research on prevention, improving early diagnosis, and therapeutic options for Reinke’s edema.

Student support: Merial Veterinary Scholars Program

Interleukin-4(IL-4) is a critical cytokine for the differentiation of naïve helper T cells to Th2 cells and in isotype switching of IgG4 and IgE in humans. However, the role of IL-4 in the development of lymph nodes is still relatively unexplored. To better understand the role that IL-4 plays in the organization of lymph nodes, we utilized 4get IL-4Rα KO mice (these have a GFP reporter of IL-4 transcription (4get)) and 4get homozygous mice as controls. We compared peripheral lymph nodes (PLNs) through confocal microcopy and observed an absence of well-defined B cell follicles and T cell regions in the 4get IL-4RαKO mice. In addition, our confocal microscopy results showed a decrease in CD11c+ dendritic cells. Stromal cells are known to closely interact with lymphocytes and dendritic cells, presenting antigens and supporting the function and organization of these cell types. In this study we show that CD31+ cells tend towards disorganization in the absence of IL4R. Moreover, follicular dendritic cell (FDC) networks were found mostly distributed along the paracortex of the lymph nodes in contrast to our controls, suggesting a possible role of IL4R in the maintenance of stromal-lymphocyte axis. Lymphotoxin alpha (LTα) and lymphotoxin beta (LTβ) are TNF family cytokines necessary for lymph organogenesis; importantly, our data suggests that IL4R is fundamental for LTβ gene expression in steady state lymph nodes, thus providing a possible mechanism for IL4R-mediated lymph node organization. These results will contribute our understanding of the role that IL-4 plays in lymph node architecture and development, as well as provide better understanding of the mechanisms involved in this process.

Gait maturation is the developmental process by which young animals develop the pattern of walking that is considered normal in adults. This is an area that has been explored in human medicine but much less is known about gait maturation in animals. This information is valuable to determine if a deviation from what is considered normal in healthy adults is pathologic or simply a product of development. In human medicine this information has allowed for early intervention and better outcome in diseases that cause gait abnormalities in children due to early detection and treatment. The goal of this study is to identify the differences, if any, between the gait of healthy mature dogs and that of puppies and to determine when gait has fully matured. We hypothesized that gait will be mature before the age at which we begin testing. Kinetic and kinematic data was gathered on five puppies starting as young as possible continuing until 32 weeks of age. Trends were examined in the data set and compared to reference intervals (RIs) previously established on adult dogs. Most parameters examined fell within the reference ranges for healthy adults. The most obvious and well supported trend seen in the data is a negative association between the coefficient of variation (CV) of many measured variables and the puppies’ age. At 20 weeks of age all parameters appear to have normalized. A similar trend has been seen in children up to 3-4 years of age and has been attributed to continued neural development. Given these preliminary findings it seems justified to conduct further research into canine gait development and possibly establish reference ranges for healthy puppies to better identify when pathology is present in young animals.

Research Grant: None

Student Support: Purdue University Summer Research Fellowship

Background: Cancer is essentially a genetic/genomic disease, as there are various gene mutations in cancer cell genomes. Only a small proportion of gene mutations, called cancer drivers, are responsible for cancer initiation and development. Cancer driver genes are key for development of new, targeted cancer therapies and novel markers for diagnosis and prognosis. Using zebrafish-human comparative oncogenomics, we have identified mdm1 as a cancer driver gene. Approach: To elucidate molecular mechanisms of this new cancer gene, we have created a new zebrafish model for mdm1 using CRISPR. In this study we aim to investigate apoptosis in the mdm1 mutant fish embryos. UV irradiation was employed to induce apoptosis through DNA damage. Apoptosis was compared between mdm1 mutant and wildtype fish embryos using acridine orange staining. To better track apoptosis in vivo, a reporter transgenic fish line was created using the apoptosis biosensor, LSSmOrange-mKate2 caspase-3. Results: Fewer apoptotic cells were found in the mutant embryos compared to wild-type after UV-irradiation. In addition, the new reporter transgenic fish line reliably detects apoptosis in vivo. Conclusions: Mdm1 mutants may be less sensitive to UV-induced DNA damage. The LSSmOrange-mKate2 caspase-3 reporter transgenic fish can be used for further investigation of the roles of mdm1 in DNA damage response and apoptosis in the future.

Clostridium difficile is an important cause of diarrhea and enterocolitis in horses, pigs, dogs, cats and humans.  Two large exotoxins, toxin A (TcdA), an enterotoxin, and toxin B (TcdB), a cytotoxin, are essential virulence factors and account for the clinical signs of Clostridium difficile-associated diseases. Since not all C. difficile strains are toxigenic, accurate diagnosis relies on the detection of toxin from fecal samples or bacterial isolates. Previously, our laboratory has assessed the use of matrix assisted laser desorption ionization – time-of-flight mass spectrometry (MALDI-TOF MS) to efficiently detect proteolytic digests of toxin A and toxin B from commercially available, purified toxins. In the present experiment, toxins were purified from two ATCC C. difficile strains by two methods: (1) using a centrifugation filter devise for size exclusion; or (2) via binding to galactose agarose. Subsequently, masses obtained by MALDI-TOF MS analysis were compared to expected masses available from an online database and to the masses generated from commercially available, purified toxins. Few matches were found between our results and expected peptide masses of TcdA and TcdB. In addition, the masses from sample isolates were poorly matched with masses generated from purified, commercially available toxins. Our results therefore suggest that MALDI-TOF MS may be unsuitable as a method for the diagnosis of C. difficile infections. Further studies regarding toxin purification methods and MALDI-TOF MS methods of analysis are required.

Student Support: Merial Veterinary Scholars Program

Following diagnosis of Influenza A, rapid antigenic characterization of virus isolates and/or antibody is vitally important in order to make informed decisions regarding control measures.  The current characterization techniques include hemagglutinin inhibition (HI) and virus neutralization (VN). These tests yield good results but HI is not as sensitive or specific, and VN is substantially slower than the microneutralization assay. The microneutralization assay is used to detect virus-specific antibodies of serum to influenza virus or to antigenically characterize influenza viruses using a reference panel. The aim of this research, was to adapt the microneutralization assay so that it can be used to identify which vaccine is the best to prevent the spread of Influenza A. The microneutralization test consists of three stages; (1) virus titration, (2) virus microneutralization, and (3) ELISA. Variables that had to be taken into account and modified included media, viruses, incubation times, Madin-Darby Canine Kidney (MDCK) prep, and ELISA plates. To establish the test in the lab, control serum was tested against H3N2 and H1N1. The results have been varied and further testing is required. Additionally, the control serum will be tested against H3N2 and H1N1 using HI. The results of the MN and HI will be compared and analyzed. The results of our testing so far indicate that the microneutralization assay is achievable in the lab setting and can be utilized in the laboratory for determining which vaccine is best suited to prevent an Influenza outbreak.

Research Grant: None

Student Support: Merial Veterinary Scholars Program 

Osteocytes are the primary cells composing mature, healthy bone tissue. Osteocytes play key roles in bone modeling and remodeling throughout the body. The cytoplasmic processes of osteocytes connect and communicate with other osteocytes in the bone to allow for response to mechanical stimulation or injury. The purpose of this study is to quantify and compare osteocyte densities and the number of primary canaliculi per osteocyte in the femoral midshafts of representative species of birds, mammals, and reptiles. Femoral bone samples were harvested from multiple guinea fowl, mice, and monitor lizards. Each bone was fixed, dehydrated, and stained with fluorescein isothiocyanate isomer I (FITC). Bones were embedded, sectioned, and placed on microscope slides. Images were taken with an inverted confocal microscope and analyzed using digital image software. The number of osteocytes and primary canaliculi will be counted and compared between the three species examined. Because bone is an ancient tissue, one would expect homogeneous osteocyte density and canaliculi counts between species. Differences in these baseline values could relate to species differences in the skeletal response to mechanical load and injury. Future research projects will study the response to mechanical stimulation when taking osteocyte density and primary canaliculi numbers into account.

Research Support: National Science Foundation

Student Support: Merial Veterinary Scholars Program

Accurate assessments of dog behavior and welfare are important to determine the wellness of kenneled dogs and to evaluate the adequacy of their environments. These evaluations are especially important in facilities where dogs are housed for extended periods of time such as breeding kennels or shelters. In order for the results of canine welfare assessments to be valuable, they must be proven valid and reliable. In this study, a canine welfare assessment was developed to evaluate the overall wellness of breeding dogs at three Amish breeding facilities in southwest Indiana. Two novice raters evaluated the behavior and welfare of 20 dogs at each breeding facility in order to assess the inter-rater reliability (IRR) of the tool. IRR was calculated by using Cohen’s Kappa in SPSS. When all facilities were included in the analysis, raters gave the same score 81.13% of the time. When chance agreement was accounted for by use of kappa, there was moderate agreement, kappa= 0.626, p<0.005. Agreement improved when dogs were evaluated with their primary caretakers present as raters gave the same score 85.71% of the time and when chance agreement was accounted for there was moderate agreement, kappa= 0.728, p<0.005. Because there was moderate agreement, training of raters is recommended to further improve reliability results. Altogether, this canine welfare assessment is a useful tool for evaluating the wellness of kenneled dogs in breeding facilities and may be helpful in developing corresponding recommendations for individual dogs.

Research Grant: World Pet Association, Pet Food Institute

Student Support: Merial Veterinary Scholars Program, Purdue University CVM

Osteoarthritis affects over 27 million Americans age 25 and older and is a debilitating disease in which articular cartilage degradation leads to inflammation and pain in the joint. Medical management has not been very successful, as cartilage is avascular and has a difficult time regenerating itself. In joints, there are laminin receptors (LR) which bind to PEDF (pigment epithelial derived factor). When PEDF is bound to a LR, it has anti-inflammatory, pro-chondrogenic and anti-angiogenic characteristics. However, the relatively large size and low stability of PEDF at 25^oC indicates that smaller, more stable molecules could be an alternative for therapeutic purposes. We developed seven small compounds that bind to the PEDF docking zone at the LR, with the goal to mimic the effects of PEDF. In order to test our drugs in a pro-inflammatory environment, THP-1 macrophage cultures were stimulated with lipopolysaccharide. Quantitative real-time polymerase chain reaction (qPCR) was used to assess up- and down-regulation of inflammatory genes, including IL-1β. Compound 3 (C3) appears to be the best PEDF mimic as it reduced IL-1β expression by ~8-fold. A cell pellet chondrogenesis assay using adipose-derived stem cells (ASCs) was used to examine the effect of compounds on upregulating cartilage-specific genes at day 8 of differentiation. In the future, ASC pellets will be harvested at differentiation days 7, 14 and 21 to determine the timing of cartilage-specific gene expression following treatment with the small compounds. From our results, C3 appears to be the most promising drug for future development since it is able to reduce expression of pro-inflammatory gene IL-1β and promote ASCs to express cartilage-specific genes. 

Departments of Veterinary Clinical Sciences and Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Jones, Ruple); Morris Animal Foundation (Simpson); Flint Animal Cancer Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO (Page)

Longitudinal cohort studies are a valuable way to obtain a large amount of information about a population over a period of time. In veterinary medicine, few large cohort studies have been undertaken due primarily to the great amount of time and expense required to complete them. The Golden Retriever Lifetime Study (GRLS) currently being conducted by Morris Animal Foundation is the largest dog cohort study of its kind ever initiated in the U.S., and understanding factors that affect owner compliance with research requirements will help improve cohort study design and participant recruitment in the future. Thus, the objective of this study was to determine factors affecting dog owner non-compliance by the end of the first year of GRLS based on information provided by owners at the time of enrollment. The study population consisted of Golden Retrievers (n=3044) whose owners elected to participate in GRLS. Logistic regression modeling was used to determine factors associated with owner non-compliance at the end of the first year post-enrollment. The results showed that owners of dogs that sleep in areas other than their bedroom are at increased risk for non-compliance, especially those with dogs that sleep in a garage at night. Owners who do not bathe and/or groom their dogs at home and who do not vaccinate their dogs are also at increased risk for non-compliance. This study shows that survey questions about a dog’s sleeping location at night, home grooming habits, and vaccination status are important indicators of an owner’s likelihood of compliance in a cohort study. Similar questions could be used in future cohort studies to screen for owners likely to be compliant, thus increasing the likelihood of success of the study.

Aberrant immunity, genetic predisposition, and host environment all factor into the complex condition of inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) is an important inflammatory cytokine known to play a damaging role in IBD as it can exacerbate inflammation and cause downstream tissue damage. Murine models of IBD have been critical for a better understanding of the pathogenesis of human forms of IBD, such as Crohn’s disease (CD). Here, we use the haptenizing agent, trinitrobenzenesulfonic acid (TNBS) to induce CD-like colitis. In this model, there is a Th1/Th17 mediated response with patchy, transmural inflammation in the colon, similar to what is seen with CD. We used wild-type (WT) and tumor necrosis factor knockout (TNF KO) mice to evaluate the role of TNF in this model of IBD. Because of the pro-inflammatory and tissue damaging effects of TNF, we hypothesized that TNF KO mice have less severe colitis with less weight loss and higher levels of protective IgA post-colitis compared to WT. Colitis was induced by sensitization with TNBS on day 0 and an intra-rectal (IR) injection on day 7. Fecal IgA levels and colon tissue cytokine levels were evaluated using ELISA. There was no significant difference in weight loss between the groups. TNF KO mice had a significant increase in fecal IgA post-colitis. Compared to WT, TNF KO had significantly less IgA both pre and post-colitis. The IgA findings suggest TNF may play a protective role during acute colitis. The low levels of pre-colitis IgA may increase susceptibility to developing colitis.

Research Grant: None

Student Support: Purdue University Veterinary Scholars Summer Research Program

Endotoxaemia is an important cause of death in adult horses and foals. Flunixin meglumine (FM) is currently the most commonly used NSAID to treat inflammation in equine endotoxemia. Identification of a more effective NSAID for anti-inflammatory and analgesic properties could decrease the morbidity and mortality associated with equine endotoxaemia. Ketorolac tromethamine (KT) is a widely used NSAID in human medicine for analgesia and inflammation. KT has also been studied in other veterinary species, but information is lacking in the horse.  In this study we compared the in vitro efficacy of FM and KT in reducing LPS-induced production of cytokines and eicosanoids by equine monocytes. Monocytes were isolated from sterile equine blood collected from healthy donors by centrifugation over a density gradient followed by adherence to tissue culture plates for xx h. The cells were incubated in separate wells for one hour with different concentrations of either FM or KT followed by the addition of LPS. Supernatants were collected after incubation for 24 hours. Equine-specific ELISAs were used to measure the concentrations of TNF-a, IL-6, IL-8, PGE₂, and TXB₂. FM was more efficient in reducing the secretion of TNF-a.  Both FM and KT failed to suppress monocyte production of PGE₂ and TXB₂.  These preliminary results suggest that KT is not more effective than FM in treating endotoxemia-associated inflammation.

Research Grant: Purdue University College of Veterinary Medicine Competitive Equine Research Fund, 2014.

Student Support: Summer Research Fellows Program, 2015.

Chronic bacterial infections with antibiotic resistance are commonly reported but the association with biofilm is rarely considered in veterinary medicine. No drug is currently available to selectively target bacterial biofilm-associated diseases in animals, and antibiotics are only tested for treatment of bacteria in liquid suspension (planktonic bacteria). Previous work showed that Tris-EDTA improved gentamicin efficacy against planktonic bacteria and Triton X-100 (TX100) reversed multidrug resistance in tumor cells. However, the combination of Tris or TX100 with antibiotics against biofilm has not yet been evaluated. Hence, the objectives of this in vitro study were to study the association between biofilm and clinical cases of chronic eye conjunctivitis in dogs, and to investigate the efficacy of combining antibiotics with Tris or TX100 against bacterial biofilm. Three clinical pathogenic bacterial isolates from chronic conjunctivitis in dogs were used: 2 isolates of methicillin-resistant Staphylococcus pseudintermedius and 1 isolate of Neisseria spp. Formation of biofilm by these isolates was each confirmed by crystal violet staining. Antibiotic-susceptibility test was used to determine the effective antibiotics with or without combination of Tris or TX100 against planktonic and biofilm bacteria. Tris and TX100 enhanced activity of antibiotics and enhanced clearance of biofilm. Cytotoxicity test with 2 mammalian cell lines showed the effective antibiofilm concentrations of Tris (0.3%) and TX100 (0.0016%) were non-toxic in vitro.  These results suggest a novel potential therapeutic strategy against biofilm-related chronic bacterial infection by enhancing the efficacy of antibiotics with combination of Tris of TX100. 

Research Grant: None

Student Support: Merial and the Purdue University College of Veterinary Medicine Veterinary Scholars Summer Research Program

Staphylococcus pseudintermedius is an important cause of opportunistic infections in dogs and also infects other species, including humans. Methicillin-resistant S. pseudintermedius (MRSP) is a concern due to its widespread occurrence and potential for zoonosis; rapid identification is essential in clinical cases. Normally, Staphylococcus sp. express a set of penicillin binding proteins, including PBP1, PBP2, PBP3, and PBP4, to which beta-lactam antibiotics bind to inhibit cell wall formation and execute their bactericidal effects. In contrast, mecA positive strains have an altered PBP known as PBP2a. Methicillin and other beta-lactam antibiotics have a high affinity for PBP1-4, but a low affinity for PBP2a. Beta-lactam antibiotics are not effective against MRSP because they bind poorly to PBP2a and as a result are unable to inhibit cell wall formation. An identification method for MRSP, more rapid than PCR, is needed. Matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial species identification, and discriminates bacterial species based on unique protein spectral patterns. Since mecA positive and mecA negative strains differ in their cell wall protein composition, we hypothesized that MALDI-TOF MS is a viable tool for discriminating between these populations. The MALD-TOF MS was used to analyze purified PBP2a and both mecA positive and mecA negative S. pseudintermedius canine isolates over three different molecular weight ranges: low (2680-30,000 Da), mid (2000-80,000 Da), and high (26-101 kDa). Further investigation of MALDI-TOF MS as a method for discriminating methicillin resistant from sensitive strains of S. pseudintermedius is warranted.  

Research Support – Indiana Animal Disease Diagnostic Laboratory; Merial Veterinary Scholars Program 

Student Support – Merial Veterinary Scholars Program

Outbreaks of nosocomial salmonellosis in hospitalized animals have been responsible for the closure of multiple veterinary teaching hospitals resulting in significant financial loss and devastating health consequences for patients. Environmental surveillance for S. enterica can be used for early detection of contamination, which can help prevent the occurrence of outbreaks. Though there are several different surveillance methods currently in use in veterinary teaching hospitals, there have been few reports comparing their efficacy. The current protocol for environmental surveillance used at Purdue was put into practice in 2000 and incorporates the use of gauze sponges for environmental sampling. An alternative sampling method utilized in other veterinary teaching hospitals uses Swiffer® brand electrostatic wipes for environmental sample collection. For this study, it was hypothesized that use of Swiffer® wipes for sample collection would provide a more efficient environmental sampling method in terms of time, labor, and cost. A head-to-head comparison was performed in Purdue’s large animal hospital with matched samples being collected using both the gauze sponge and Swiffer® collection techniques. Statistical analysis showed the sampling techniques have fair agreement (Kappa coefficient=0.3697) in terms of ability to detect S. enterica in the environment. However, the Swiffer® wipes required fewer workers, less time, and less physical effort to collect samples; a financial analysis also showed the Swiffer® technique to be more cost-effective. These results suggest that use of electrostatic wipes would be preferable to gauze sponges for routine environmental surveillance in the large animal hospital at Purdue University. 

Research Funding: Purdue University College of Veterinary Medicine

Student Support: Merial-NIH Veterinary Scholars Program

Transfixation pin casting is a technique utilized in the repair of equine distal limb fractures.  This fixation technique distributes the horse’s weight through the pins and down the cast to alleviate stress placed on the limb distal to the pins.  While this technique is frequently used by clinicians, horses can experience fracture through pin holes both when pins are in place and with the removal of pins.  There are many factors during drilling and pin placement that lead to the development of stress risers and increase the likelihood for fracture but no attempts have been made to establish how imprecise drilling can compromise the third metacarpal bone (MCIII) ultimately leading to elevated fracture risk.  The objective of this study was to determine if removing 1mm of the dorsal cortex bordering the medullary cavity during imprecise drilling will decrease bone strength and predispose the limb to fracture.  Pairs of bones were collected and radiographed prior to drilling.  Left MCIII’s were measured and drilled placing the pin accurately through the center of the medullary cavity, while right MCIII’s were measured and drilled more dorsally, removing 1mm of the dorsal cortex bordering the medulla.  Pilot holes (3.2mm) were drilled through the marked location followed by the final 6.2mm holes.  Holes were then tapped and 6.3mm pins placed.  Each bone was placed in a custom made testing jig and aligned with the load cell of a uniaxial materials testing system.  First, the bones were loaded to 7500N with the pins in place and strain was measured around the pin hole.  The pins were then removed and the bones were again loaded to 7500N with the strain measurement repeated.  Finally, the bones were loaded to failure and the ultimate strength of the bone was determined.  Comparison of strain with pins in place and when pins were removed were compared between the groups as well as the ultimate strength of the bone.  Paired t-tests will be used to compare the accurately drilled holes with those that compromise the dorsal cortex.  Upon completion of the research, this study intends to verify that imprecise drilling can predispose the equine third metacarpal bone to fracture through pin holes placed during transfixation pin casting and make recommendations to assist clinicians in precise pin placement.

Cells are traditionally cultured in two dimensions.  However, certain cell types, such as osteocytes, which are terminally differentiated osteoblasts trapped in calcified bone, thrive for three dimensional culture conditions due to their in vivo nature.  In vivo, mineral and collagen make up the extracellular matrix (ECM) that surround osteocytes.  Through this matrix, osteocytes extend dendrites in all dimensions to communicate with other cells.  As such, a three dimensional culture method is better suited to studying osteocytes in vitro.  The goal of this study is to characterize the properties of some individual components of a bone cell 3D culture system: the mineral (hydroxyapatite, HA), and the cellular component.  HA is synthesized from its component parts based on previous publications.  The shape and size of the resulting HA crystals are analyzed under light microscopy, particle size analyzer, and transmission electron microscopy.  The cells to be used in this culture system are extracted from mice long bones according to an established protocol consisting of serial enzymatic, or chemical, digestion of the bone tissue. Different genetically modified mouse strains in which osteoblasts and/or osteocytes express fluorescent proteins are used to identify the specific cell type (osteoblast or osteocyte) and percent of cell type for each bone digestion. Identification of osteoblasts and osteocytes using these fluorescent proteins will allow for the right type of cell to be selected for in future studies to be placed in the collagen-mineral matrix.  This will allow for the creation of a three dimensional culture system to study osteocytes and possibly de novo bone formation in future studies.

NIH Grant (AR065659)

Morris Animal Foundation

Forward genetic screens in zebrafish using retroviral insertional mutagenesis have identified a mutant strain, hi2059, which has an elongated paired and median fin phenotype as well as a high risk of cancer development. According to our genetic mapping, the genes potentially affected in this mutant are kcnj13, which encodes an inwardly rectifying potassium channel, and efhd1, which encodes an adaptor protein. Whole mount in situ hybridization is a method by which gene specific mRNA can be detected in morphologically preserved cells of embryos. This allows the spatial and temporal expression patterns of a particular gene to be visualized in vivo. Using the in situ hybridization, we investigated expression kcnj13 and efhd1 at various developmental stages in both hi2059 mutant and wild type zebrafish embryos. We found that kcnj13 was misexpressed in somites at the 24 and 36 hour post-fertilization stages of hi2059 fish, while there appears to be no difference in efhd1 expression in the corresponding developmental stages when compared to the wild type. This suggests that the disruption of kcnj13 spatial and temporal gene expression was the most likely genetic factor for the long-fin phenotype and spontaneous tumors in the zebrafish mutant. In the future, further examination of the fin development organization will be needed to elucidate the detailed molecular and cellular mechanisms. 

Research Grant: Hayward Foundation

Student Support: Merial 

Feline red blood cells (RBC) are particularly sensitive to oxidative stress; however, the reason remains uncertain. In feline diabetes mellitus, the presence of Heinz bodies, a sign of RBC oxidative damage, is frequently noted, yet the cause remains unknown. MicroRNA is essential for erythropoiesis in humans. More specifically, miR-144 and miR-451 are necessary for erythroid differentiation and maturation as well as the development of RBC antioxidant defenses. Alteration in microRNA expression results in changes in the RBC antioxidant capacity. An altered microRNA expression in diabetic cats could be responsible for the increased RBC oxidative damage observed microscopically. To test this hypothesis, the microRNA profile from mature red blood cells isolated from clinically healthy cats and diabetic cats with microscopic evidence of oxidative damage will be compared. The goal of this preliminary study is to establish a protocol for mature RBC isolation and microRNA extraction. Whole blood was collected from cats and mature red blood cells were partially purified by centrifugation. Good quality microRNA was obtained using a modified extraction protocol for the miRNeasy mini kit (Qiagen). Several antibodies were tested by flow cytometry and immunocytochemistry; the antibodies will be used to further purify mature red blood cells using an immunodepletion kit adapted to cat blood. Comparison of mature RBC microRNA profiles has the potential to significantly improve our understanding of the molecular basis for increased sensitivity of feline red blood cells to oxidative stress and may result in the identification of new biomarkers for chronic, systemic oxidative stress. Student support: Purdue Veterinary Scholars Summer Research Program.

Hepatitis B virus is a double stranded DNA enveloped virus that replicates via reverse transcriptase. Its 3.2 kb genome is very compact, encoding seven different proteins. One of these proteins is the X protein (HBx). HBx activates cellular signaling cascades and is essential in the viral life cycle. One of these signaling molecules that HBx activates is polo-like kinase 1 (Plk1). Plk1 is an enzyme that regulates progression of the cell from G2 phase into mitosis. It is usually not active in hepatocytes as liver cells do not divide often. When HBx activates Plk1, cells begin to divide much faster than normal, resulting in cancer. Plk1 may also regulate the levels of HBV core antigen (HBc), the protein that forms the viral capsid. The levels of Plk1 may inversely correlate with those of HBc. We hypothesize that Plk1 acts as a signal to degrade core proteins. To address this hypothesis, a plasmid encoding HBc was prepared in bacteria. The plasmid was harvested from the bacteria and introduced into mammalian HEK 293 cells by transfection. Nocodazole and BI2536 were added to different samples of HEK 293 cells. Nocodazole arrests the cell cycle between G2 and mitosis, the same time that Plk1 is active. BI2536 inhibits Plk1. The cells were harvested for the preparation of lysates and the levels of HBc will be determined by western blotting with anti-HBc and anti-phospho-Plk1 antibodies. An inverse correlation between the levels of Plk1 and the levels of HBc would suggest that Plk1 plays a role in the degradation of HBc. This work may identify a possible new method for the treatment of HBV infection.

Plant toxins are the most common type of poisoning in livestock second to pesticides, with poison hemlock (Conium maculatum) and jimson weed (Datura stramonium) among the most frequently ingested plants. Poison hemlock and jimson weed alkaloids cause toxicity via their effects on acetylcholine receptors. These alkaloids can cause large economic losses within the livestock industry; therefore, it is imperative to have rapid, sensitive methods for diagnostic cases. The methods currently involve tedious sample preparation and long analysis times. We hypothesized that MALDI-TOF MS could be used to more rapidly detect and diagnose exposure to poison hemlock and jimson weed in livestock. To prepare samples for MALDI-TOF MS analysis, coniine, atropine and scopolamine were extracted from plant, rumen, and urine samples using liquid-liquid extraction or solid-phase extraction method protocols. Prior to analysis, the extracts were co-crystallized with matrix (1:1), 1.0 µL of the sample spotted onto a MALDI target plate, and the samples analyzed in reflector positive mode. Coniine (128 m/z) was detected in concentrations as low as 0.01% in rumen contents containing poison hemlock. MALDI analysis identified scopolamine (304 m/z) and atropine (290 m/z) at concentrations as low as 10% and 50%, respectively, in rumen samples. In addition, sheep and cattle urine samples containing poison hemlock and jimson weed alkaloid standards yielded positive identification of coniine, scopolamine, and atropine. This study revealed that MALDI-TOF MS can be used as a diagnostic tool for the detection of poison hemlock and jimson weed alkaloids in antemortem and postmortem samples, providing veterinarians with rapid results in suspect cases.

Student Support:  Merial Veterinary Scholars Program

Inflammatory Airway Disease (IAD) affects horses of all ages, causing poor performance, particularly in racehorses. To study the role of bacteria in the disease, exercise-induced contamination of the airway must be considered; however, the duration of contamination is unknown. We hypothesize that the increased bacterial contamination in healthy horses will resolve between 2-3 hours after exercise due to the, but will not resolve as quickly in horses with IAD. Nasopharyngeal, tracheal, and bronchial cytology brush samples were collected endoscopically before exercise and each hour after exercise for four hours, followed by a bronchoalveolar lavage (BAL), in training racehorses. The samples were analyzed using quantitative PCR (qPCR) of the 16S rRNA gene. Twelve horses completed the sampling protocol. The average length and maximum speed of exercise was 2.10 ± 0.357 miles and 26.10 ± 2.505 miles per hour. IAD was diagnosed in 9 out of 12 horses. The data from the qPCR will provide an estimate of bacterial burden of the respiratory tract hourly post-exercise in healthy horses and IAD horses. Knowledge of the clearance of bacteria from the airway following exercise will facilitate the design of future studies into the microbiome of the equine airway in health and disease.

Interleukin-4 (IL-4) is a cytokine that is a marker of both type 2 helper T cells (Th2) and T follicular helper cells (Tfh), and plays an integral part in B cell differentiation and immunoglobulin class switching. Although IL-4 is known as a key regulator of type 2 immune responses, a detailed understanding of its influence on lymph node organization is still lacking.  In order to investigate this problem, the effects of induced type 1 and type 2 immunity on the organization and population of lymphocytes was observed in normal (C57/B6) mice and mice that lacked IL-4 (IL4KO). Stag, a soluble Ag from Toxoplasma gondii, was used to induce a type 1 response and SEA, a soluble extract of Schistosoma mansoni eggs, was used to induce a type 2 response. Eight days post injection, draining and nondraining popliteal lymph nodes and mesenteric lymph nodes were removed and frequencies of IgG1 and IgG2a B cells as well as Tfh cells were determined. Confocal microscopic imaging was used to examine the organization of the lymph nodes by determining the location of B and T cell regions as well as germinal centers and stromal cells present.

Skeletal muscle cells can be grown in 3-dimensional (3D) scaffold constructs; however, these tissue-engineered muscle constructs lack innervation, organization, and synchronous firing after implantation. Myoblast alignment has been achieved by constant tension and/or stimulation, but viability and desired morphology is achieved only on edges of 3D constructs. Our study defines time-dependent changes in viability, morphology, and organization of human adipose-derived mesenchymal stem cells or rat myoblasts in self-assembled type I collagen oligomer matrix prepared in unaligned- and aligned-fibril formats.  Cells were put in polymerizable collagen matrices (1.5 mg/ml; 400 Pa) and plated in standard well-plates (unaligned) or flow-induced alignment formats. Following culture for up to 14 days, tissue constructs were fixed, stained with phalloidin (F-actin) and draq5 (nuclear), and imaged by confocal microscopy. The extent of fibril and cell alignment was quantified using ImageJ plugin Directionality. Results indicated that collagen fibrils formed within the flow-injection apparatus were more aligned than those that used conventional well-plate methods. Cells seeded within aligned-fibril matrices were viable throughout the construct, displaying long, spindle-shaped morphologies arranged in parallel to the fibrils. In contrast, cells seeded within unaligned matrices displayed more randomly-organized stellate morphologies. By aligning the collagen-fibril matrix, increased alignment, organization, and viability of encapsulated muscle cells were achieved. This approach may provide improved methods for making functional tissue-engineered skeletal muscle constructs for reconstruction of dysfunctional muscle in animals and human. 

Research Funding:  National Institute on Deafness and Other Communication Disorders R01DC014070 (Halum (PI) and Voytik-Harbin (Co-PI)) 

Student Support: Veterinary Scholars Summer Research Program, Merial and Purdue University 

Field of Research: Tissue Engineering

Observational and instrumented gait analysis (OGA and IGA) are techniques used by clinicians and researchers to explore the canine gait cycle. Both OGA and IGA have been used to evaluate the gait of dogs with a T3-L3 myelopathy, but their results have not been compared. To compare the two methods, fifteen ambulatory dogs, recuperating from T3-L3 disk surgery, were walked over 2 x 5 m Pressure Sensing Walkway (PSW; Walkway™ HRV4, Tekscan, Boston). Each dog was walked until 6 valid trials were for OGA and IGA were obtained. A dog’s gait on the PSW was recorded using two orthogonal cameras. Two neurologists used the collected videos to score the OGA (grade 0-10). The data from the PSW were used for IGA. Descriptive statistics were obtained for all OGA scores and IGA variables and the correlation between OGA scores and IGA variables was determined. The mean coefficient of correlation of OGA score and IGA variables with the OGA score was 0.11 and ranged from -0.13 to 0.38. The highest coefficient of correlation was between the coefficient of correlation of the Peak Vertical Force and OGA score (0.38). The correlations between OGA scores and IGA variables were unexpectedly low. A possible explanation is that the technique of OGA used in this study is insufficient for complete neurological evaluation. Alternatively, it may be that the behavioral criteria used with OGA do not reflect the kinetic and kinematic changes detected with OGA. 

Mice have a thermoneutral zone between 26°-34°C however, laboratory environments routinely maintain room temperatures between 20°-24°C for human comfort. We therefore hypothesize that laboratory mice maintained within standard laboratory conditions face an inescapable challenge to homeostasis, which is by definition stressful, and may impact many aspects of behavior and physiology. Our previous work has demonstrated the behavioral preferences of mice to increased volumes of nesting material when housed in common laboratory temperatures; which allows them to construct a nest that is thermally insulating. Further, the type of nesting environment impacts reproductive success of breeders and weaning weights of pups. A thermally insulating nest changes the metabolic demands of the individual and mice show impaired growth rates at temperatures below 18°C.  Behavioral responses to thermal stress in laboratory mice include thermotaxis (movement away from thermally stressful location), nest building, and huddling behaviors. However, little is understood on how the provision of nesting material and its thermal benefits modify maternal/paternal behavior, and the subsequent effects on offspring. This study aims to evaluate the impact of nesting material (3g or 13g) in three common laboratory strains, CD-1, BALB/c, and C57BL/6 on reproductive success of monogamous breeder pairs, nest quality, maternal/paternal investment in offspring, and the immune function of trio housed offspring to vaccination at 5 weeks of age.  Maternal/Paternal interaction was evaluated through 24 hour continuous video analysis of the frequency of exits from the nest, sex exiting the nest, and the duration outside of the nest.  

Student Support: Merial Veterinary Research Award

Invasive transitional cell carcinoma (InvTCC), also known as urothelial carcinoma, is the most common form of bladder cancer in dogs. Canine InvTCC serves as an excellent biological model for the high-grade, muscle-invasive form of InvTCC in humans. InvTCC is lethal in 50% of human cases, and in the majority of dogs. Targeted therapies are expected to improve the outcome of treatment, with folate-targeted therapy showing promise in dogs with InvTCC and humans with other cancers. In humans, folate receptors (FRs) are not present on neutrophils and platelets, thus folate-targeted therapy does not cause myelosuppression. In dogs, however, dose-limiting toxicity has commonly included neutropenia. The goal of this research project was to determine the cause of decreased circulating neutrophil numbers in these dogs with the hypothesis that canine neutrophils express FRs on their cell surface. FR expression was determined by immunocytochemistry (ICC) on acetone-fixed peripheral blood smears with an anti-FR rabbit polyclonal antibody (PU17, Endocyte, West Lafayette) from 3 tumor-bearing and 3 non-tumor bearing dogs. FRs were detected in both the membrane and cytoplasm in greater than 85% of neutrophils in 5 out of 6 cases. The immunoreactivity noted by ICC provides a molecular explanation for uptake of the chemotherapy drug into neutrophils and the resulting myelosuppression. Although some uptake of folate occurs in neutrophils, folate-targeted therapy is still showing promise in dogs with bone marrow-sparing doses having antitumor activity. 

Acknowledgements

1. Merial Veterinary Scholars Program
2. All members of the Purdue Comparative Oncology Group
3. Carol Bator

Clinical analysis of dogs with orthopedic gait abnormalities has historically involved visual observation of a patient walking and trotting and an associated orthopedic exam.  The objectives of this study were to evaluate the usefulness of kinetic and temporo-spatial gait values to identify dogs with a unilateral hind limb lameness.  In this study, dogs (n=10) that presented to the Purdue Veterinary Teaching Hospital (PVTH) with a history of acute or chronic hind limb lameness were evaluated by visual gait observation, recording of kinetic and temporo-spatial variables while walking and trotting on a pressure sensing walkway (PSW), and a complete orthopedic examination.  Variables from control dogs, matched to the experimental dogs based on breed and weight, were selected from previously collected normal dogs who were orthopedically and neurologically sound. Dogs were walked across the PSW and 3 trials in each direction were utilized to obtain values for Duty Factor (DF), Peak Vertical Force (PVF), Symmetry Index (SI), Gait Cycle Duration (GCD), and swing and stance phase durations.  The PVF for each limb was normalized for body weight and used as a measure of weight distribution.  Results of this study identified significant changes (p<0.05) in DF, PVF, weight distribution, stance phase, and swing phase between the affected and unaffected pelvic limb in the experimental group.  When the experimental dogs were compared to the control dogs, significant changes (p<0.05) were found in DF, PVF, PVF-SI, weight distribution, and stance phase between the groups.  The results of this pilot study indicate that computerized gait analysis can detect changes in kinetic and temporo-spatial parameters in dogs with hind limb lameness.  Computerized gait analysis allowed hind limb lameness parameters to be quantified, which can be especially useful in mildly lame patients where visual gait analysis may be more difficult.

Student Support: Merial & Purdue College of Veterinary Medicine

Research Support: The American College of Veterinary Surgeons (ACVS)

Toxigenic Clostridium difficile is an important cause of enterocolitis in many veterinary species, and can be identified by the presence of one or both toxins: toxin A, an enterotoxin, and toxin B, a cytotoxin.  C. difficile strains commonly have genes for both toxins, neither toxin, or only for toxin B. Detection of toxins A and/or B is important to differentiate a disease-causing toxigenic strain from a non-toxigenic strain. The “gold standard” for identification of C. difficile toxin B in feces is the tissue culture cytotoxicity assay (CTA). A commercially available enzyme linked immunosorbent assay (ELISA) has been proven useful for toxin A and B identification in feces. Polymerase chain reaction (PCR) assays can detect the toxin genes, but may not correlate well with the production of toxin. In this study, toxin detection methods using fecal isolates as samples were evaluated for improved sensitivity and specificity over toxin detection in fecal samples. To determine if the ELISA performs as well on isolates as it has been shown to perform on fecal samples, we compared the CTA and a commercially available ELISA on a set of 51 equine, canine, and porcine C. difficile isolates from the archives of the Indiana Animal Disease Diagnostic Laboratory plus three ATCC C. difficile strains with known toxin genes and toxin production status. Isolates were cultivated in 48 (+/- 6) hour meat broth cultures. ELISA and CTA had 100% agreement, detecting 51 positive and 3 negative samples for toxins A and/or B. These results demonstrate that the ELISA is a valid means for identification of toxigenic C. difficile isolates from equine, canine, and porcine fecal samples. Additionally, because MALDI-TOF is becoming a common method for bacterial identification and accurately identifies C. difficile isolates, a MALDI-TOF-based proteolytic assay is evaluated in this study as a novel means for C. difficile toxin detection.

Student support: Merial Veterinary Scholars Program

Research support: Indiana Animal Disease Diagnostic Laboratory 

Schwannomas are slow-growing benign tumors of Schwann cells, which myelinate axons of the peripheral nervous system. For a majority of schwannomas, the genetic causes remain largely unknown, an exception being type II neurofibromatosis, which typically features bilateral schwannomas of the eighth cranial nerve caused by mutations in the tumor suppressor gene NF2. Recently, LTZR1 germline mutations were identified in several schwannoma patients, suggesting that it could be a new tumor suppressor gene in humans.  In this study, we investigate whether the lztr1 gene could function as a tumor suppressor gene and its biological role in normal development compared to tumorigenesis.  We used a Type II RNA-guided DNA nuclease system, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats—CRISPR-associated 9), to target the lztr1 gene in zebrafish and create loss-of-function mutant fish lines.  Using the currently available zebrafish CRISPR system (DR274 and ML3136), we designed and created 4 different constructs that target exon 1 and exon 8 of zebrafish lztr1 gene.   To ensure accuracy, we verified all CRISPR constructs by PCR and Sanger sequencing.  Through in vitro transcription with the plasmid constructs, we made CRISPR RNAs (crRNA) and Cas9 mRNA.  Purified Cas9 nuclease mRNA and crRNAs will be injected into the pronuclei of zebrafish embryos and we will assess mutations using a T7 endonuclease I assay.  Those embryos with the correct lztr1 mutations will be raised as founders for further developmental biology and tumorigenesis studies.  

Student support:  Merial

Lab Support: This project was supported by the Hayward Foundation

Background – Transfixation pin casts (TPCs) are used for treating fractures of the equine distal limb. TPCs consist of transcortical threaded pins above the fracture and their incorporation into a fiberglass cast. An orthopedic tap consisting of cutting threads and longitudinal flutes is used to create threads. Flutes clear bone debris thereby minimizing heat transfer to the bone and thread damage. Minimizing bone damage during pin placement procedures is critical for maintaining pin stability during fracture healing.

Objective – To compare a 3-fluted with a 4-fluted tapered tap design based on tapping insertion torque, temperature, bone microdamage, and thread form at 3 different locations of the third metacarpal bone (proximal diaphysis, distal diaphysis, and distal metaphysis).

Sample – 10 third metacarpal bones from 5 horse cadavers.

Procedure – Paired metacarpal bones were randomly assigned to a 3-fluted or 4-fluted tap. A 3.2mm-diameter pilot hole was drilled at each location. Implantable thermocouples were placed at 1 and 2mm from the anticipated final hole margin. Each pilot hole was enlarged with a 6.0mm drill bit and reamed with a tapered reamer. Each hole was tapped with either a 3- or 4-fluted tap. During tapping, temperatures were recorded by the thermocouples and an infrared thermal camera, and torque was measured with a torque wrench. Holes were sectioned to evaluate microdamage and thread form. Paired data were analyzed using a paired t-test. Significance was set at p<0.05.

Results – Mean peak insertion torque and bone temperatures for 3-fluted taps were significantly higher than for 4-fluted taps (p<0.05). Bone damage assessments will be made over the next 3-4 weeks.

Conclusion – A 4-fluted tap creates threads with less effort and heat production in equine third metacarpal bones than a 3-fluted tap. If bone damage between taps is not different, then a 4-fluted tap should be recommended for equine transfixation pin insertion.

Student Support: Merial

Research Support: IMEX, VCS Graduate Student Competitive Research Funds at Purdue University

Distal limb fractures are a major cause of euthanasia in racing horses and companion horses alike. These injuries cause significant financial losses in the racing industry annually and raise many ethical concerns about the sport of horse racing. This study aims to evaluate a variety of methods to assess the structural and material properties of third metacarpal bones (MC3s) from horses that were euthanized due to fractures in these bones and to compare these properties with horses that were euthanized for non-musculoskeletal injuries. Whole MC3s were radiographed and scanned using peripheral quantitative computed tomography (pQCT) to evaluate bone morphology, mineral density/content, and to obtain data on cortical and cancellous bone volume. Bones were also evaluated using a reference point micro-indentation tool to assess bone toughness and stiffness. Finally, Raman spectroscopy was used to assess physiochemical properties of the fractured and non-fractured limbs. Tools such as the Raman spectroscopy and pQCT will aid us in developing a fundamental understanding fracture etiology in these bones. If we are able to utilize these technologies to determine differences in bones with and without fractures this would allow for development of additional diagnostic tools. This research is part of a long-term study to develop a non-invasive set of diagnostic tools including radiography and reference point micro-indentation that can be used on live animals to predict and prevent fractures in performance horses to benefit the racing industry as well as the health of all horses.

Student Funding: Merial

Research Funding: Equine Research Advisory Board, Purdue College of Veterinary Medicine

Increasing evidence supports a link between cardiovascular disease and autoimmune diseases such as rheumatoid arthritis. CD40L is an immunomodulatory molecule classically associated with the humoral response but which has recently been shown to play an important role in inflammation and the pathogenesis of rheumatoid arthritis. CD40L released by platelets activates many types of cells, inciting responses such as cytokine release, matrix degradation, and autoantibody production, while also affecting vascular cells in such a way as to mediate atheroma initiation and progression. We were the first to show that CD40L expression is dynamically regulated in megakaryocytes. Recent data show that the acute phase cytokines tumour necrosis factor alpha (TNFα), interleukin(IL)-6 and IL-1b,  associated with most autoimmune diseases, increase CD40L in differentiated megakaryocytes in vitro. In addition, TNF-α and TNF-α receptors’ mRNA are upregulated upon megakaryocyte differentiation leading to the novel hypothesis that autocrine signaling of TNF-α may play a regulatory role in basal CD40L expression. This project will evaluate the effect of endogenous TNF-α and IL-6 on CD40L levels in human megakaryocyte cell lines and mouse primary megakaryocytes. In addition, our preliminary data show that the calcineurin-inhibitor cyclosporineA (CsA) abrogates TNF-α-induced CD40L expression suggesting TNFα mediates CD40L transcriptional regulation via calcium-dependent NFAT regulation. We will also elucidate the differentiation-induced regulatory pathway(s) of TNF-α. Confirmation of this hypothesis will identify a novel regulatory pathway of CD40L, which may suggest that both exogenous and endogenous cytokines play a major role in the initiation and continuation of chronic inflammation in diseases such as cardiovascular disease. This pathway has potential as a therapeutic target to modify inflammation in chronic autoimmune inflammatory disease processes.

Student Support: Merial Veterinary Scholars Program

Research Support: NIH R01-AI060924 and the Purdue University Center for Cancer Research

Coronaviruses are enveloped RNA viruses that can cause disease in mammals and birds. Deltacoronavirus, the fourth genera of Coronaviruses, are the most recently discovered group of coronaviruses. One of the Deltacoronavirus species, HKU15, first discovered in Hong Kong, is named porcine deltacoronavirus (PDCoV). The virus was reported in the US swine population in April 2014, and became a reportable disease in June 2014. The virus is rapidly spreading throughout North America, currently affecting 15 states in the United States and Canada. The exact pathogenesis of PDCoV infection is yet unknown, but it is thought to cause similar clinical signs as porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), both of which are also coronaviruses. In this research, PDCoV RNA was detected by a reverse transcriptase real-time PCR assay focused on a highly conserved region of the matrix protein. The objective of this research was to investigate PDCoV spread in Indiana counties from diarrheic pigs in 2014.  Clinical signs associated with PDCoV positive cases were evaluated, and the presence of other enteric viral pathogens was determined in the same samples. Currently, 10.4 % of tested cases were PDCoV positive, affecting 9 counties in Indiana. The clinical signs associated with PDCoV positive included diarrhea, enteritis, anorexia, collapse, and death. Additionally, we observed that about 18.2% of the PDCoV positive cases were co-infected with PEDV, and that the number of PDCoV viral copies was less in pigs with PEDV co-infection. These findings will guide further studies to investigate the epidemiology and pathogenesis of the PDCoV infection in Indiana as a newly emerging disease as well as its association with other viruses detected in the same animal.

Many studies have focused on risk factors associated with cat and dog bites to the public but few have investigated characteristics of shelter workers that may place them at risk for bites.  It is important to understand bite risk factors associated with animal care in a shelter to promote worker safety and animal care and welfare.  The number and severity of bites to three types of shelter workers-medical staff, care staff, and volunteers-at the Humane Society of Indianapolis were evaluated to determine the extent to which these differed between groups.  Workers’ perceptions of bite numbers and severity were compared to documented incidents. In addition, bite numbers and characteristics were compared as a function of attitudes towards cats and dogs as well as knowledge levels about animal behavior.  It was hypothesized that medical staff would report a higher number and more severe bites due to their performance of invasive and/or painful procedures.  It was also hypothesized that workers with greater knowledge of animal behavior and more positive attitudes toward working with cats and dogs would have fewer and less severe bites.  A questionnaire was developed to assess staff and volunteer recollection of bite events at the shelter between June 2013 and June 2014 and to evaluate staff knowledge and attitudes relative to cats and dogs.  It is expected that the results will indicate which category of shelter worker had the highest bite risk.  It is also anticipated that specific factors contributing to higher risk of cat and dog bites in a shelter setting will be identified.  If the data indicate that some workers are at increased risk for bites, and that these are correlated with knowledge about and attitudes toward cats and dogs, shelter employee training can be tailored accordingly to promote worker safety and to decrease the distress cats and dogs experience in shelter environments which may contribute to bite risks.

Student Support: Purdue University College of Veterinary Medicine

Research Support: Purdue University, College of Agriculture and College of Veterinary Medicine, Department of Comparative Pathobiology

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an ionization technique for the rapid detection of analytes. Due to potential matrix interferences with small molecule detection, MALDI-TOF MS is typically used for large molecules such as proteins. This study utilized MALDI-TOF MS to investigate its ability to detect a variety of toxins, including small molecules. Pure standards of the peptide toxins microcystins (MC), as well as small molecule toxicants including brodifacoum, diphacinone, and strychnine, were detected using a Bruker microflex^™ LRF high performance bench-top MALDI-TOF MS, equipped with an additional gridless reflectron. The experimental design briefly included: external calibration of the instrument using α-cyanohydroxycinnamic acid (CHCA) matrix ions or standard peptide mix ions in both positive and negative ion mode, a reflector voltage set at 19.99 kV, a detector scan range of 0 m/z to 4060 m/z, and approximately 1,000 laser shots of data summed per sample. Prior to analysis, the samples were co-crystallized with CHCA matrix in a ratio of 1:1.  MALDI-TOF MS was able to detect MC-LR (995 m/z), MC-LA (910 m/z), MC-RR (1038 m/z), and MC-YR (1045 m/z). The limits of detection were 0.01 ppm, 25 ppm, 0.01 ppm, and 0.01 ppm, respectively. Brodifacoum (524/526 m/z), diphacinone (341 m/z), and strychnine (335 m/z) were also detected at detection limits of 1 ppm, 1 ppm, and 5 ppm without matrix interference. This is an exciting development for veterinary diagnostic toxicology as the MALDI-TOF MS generates results in minutes rather than the hours that other analytical tests require. Developing a method to detect these toxicants in biological samples using the MALDI-TOF MS would provide a rapid diagnosis for clinicians and pet owners and expedite treatment and toxin exposure prevention in surviving animals.

Student and research support: Eli Lilly & Co., Lilly Grant Office

Background: The mucosal microbiome significantly impacts immune response, and derangements in the human respiratory microbiome have been linked to asthma and COPD. However, no data exists on the normal microbiome of equine airways. We hypothesize that techniques established in human medicine will allow investigation of the bacterial burden along the respiratory tract in horses. Furthermore, we hypothesize that the use of guarded cytology brushes to collect tracheal samples will provide less variability over time compared to wash samples, with greater contamination apparent with repeated tracheal wash samples. Finally, we hypothesize that quantitative PCR analysis will identify greater numbers of colony forming units (CFU) than will quantitative culture. 

Methods: In a randomized cross-over study design, airway samples were collected from six healthy horses. Each horse underwent airway sampling on two separate occasions, 2-7 days apart. Each hour for 2 hours, nasal swabs and tracheal samples were collected to examine the effect of repeated sampling. Tracheal samples were taken either by saline wash or cytology brush based upon random assignment and the opposite sampling method was performed on the second collection day. Bacterial burden of each sample was determined via real-time PCR of the 16S rRNA gene and quantitative culture. Repeated measures ANOVA was used to determine the effect of time, tracheal sample, and method of analysis upon CFU.

Results: Contrary to our hypothesis, culture data did not indicate a significant difference between the initial sample and the one taken one hour later, regardless of collection method. Tracheal washes had significantly greater CFU than tracheal brush samples (p=0.005). 

Conclusion: There was no significant contamination due to sample collection in the trachea with either the tracheal wash or the cytology brush. Future studies will include analysis of effects of IAD and exercise on bacterial presence in the equine respiratory tract.

Student funding: Purdue College of Veterinary Medicine

Research funding: Indiana Quarter Horse Racing Association and Grayson-Jockey Club Research Foundation, Inc.

Increase in core temperature under stressful conditions is known as stress induced hyperthermia (SIH) and has been identified as a reliable and repeatable physiological response in many species. The purpose of this study was to examine SIH in dogs as an assessment of anxiety and to explore the accuracy of infrared thermography (IRT) in monitoring SIH. We designed a thirty-minute protocol broken into two blocks, isolation and reunion that exposed dogs to different levels of stress in order to observe the relationship between their behavioral and physiological responses. Each test subject was filmed for the entirety of the protocol and the footage was used to assign an anxiety behavior score to each individual using an ethogram. Temperatures were taken at the beginning (T0), middle (T15), and end (T30) of the protocol using both an IRT camera and a rectal probe. Infrared analysis software was used to acquire temperatures from infrared images of the subjects’ faces. We looked for evidence of SIH by examining the temperature change between measurements. It was hypothesized that temperature would increase from T0 to T15 when separated from and ignored by the owner, and then fall from T15 to T30 when reunited with and consoled by the owner. To determine the accuracy of IRT in assessing SIH, we compared the trends in temperature change between the IRT camera and the rectal probe. Additionally, we compared temperature change and anxiety behavior score to determine if there was any association. We hypothesized that dogs with a higher anxiety score would have a greater increase in SIH. This study is a pilot study working toward the development of an objective assessment of anxiety in dogs, which could help eliminate the inconsistencies in current diagnostic methods and give more accurate assessment results.

Background:  Thromboelastography (TEG) records the rapidity and strength of in vitro clot formation from whole blood and is used to estimate the in vivo coagulability of an animal.  The amount of red blood cell concentration in whole blood (hematocrit- HCT) is the largest contributor to whole blood viscosity and a previous study has shown that reduced whole blood viscosity resulted in a presumed hypercoagulable state on TEG.  That study also showed that when viscosity was held constant, a reduction in HCT resulted in a hypocoagulable state on TEG, suggesting a reduction in HCT will yield a hypercoagulable tracing from the in vitro effect of reduced blood viscosity.  The goal of this study was to determine the effect of viscosity on the TEG parameters R-time (R), K-time (K), alpha angle (α), and Maximal Amplitude (MA) and develop a clinical algorithm to more accurately estimate the coaguable state in vivo in animals with varying HCT.

Methods:  Whole blood was collected into citrate tubes from 11 healthy dogs.  The blood was differentially centrifuged to obtain packed red cells, platelet rich plasma, and platelet poor plasma.  These were combined in variable amounts to obtain samples with HCT of approximately 10%, 20%, 40%, and 70% and normal platelet count using an IDEXX Procyte DX Hematology Analyzer.  TEG was performed on all samples in duplicate by recalcifying the whole blood.  Viscosity (cP) of the whole blood was also measured from each sample using a TA Instruments AG R2 rheometer.  Data was analyzed for a relationship between HCT and viscosity as well as HCT and each of the TEG parameters.

Results:  A very strong direct relationship was seen between HCT and viscosity.  Of the TEG parameters, α had the strongest relationship to HCT where every 5% reduction in HCT resulted in an increase in α of 3.4⁰.  The next strongest relationship was with K where every 5% reduction in HCT resulted in a 0.5 min decrease.  There was an increase of 1.8 mm in MA for every 5% decrease in HCT and the least strong relationship was with R where for every 5% decrease in HCT there was a 0.1 min decrease.

Conclusion:  HCT has a strong effect on TEG parameters which can impact clinical interpretation.  The data from this study will allow the clinician to factor this in vitro effect to allow for more accurate interpretation of the TEG assay and patient care.

Mycoplasma haemofelis is a red blood cell pathogen that causes feline infectious anemia. The acute infection is characterized by depression, fever, and severe, life-threatening hemolytic anemia. M.  haemofelis is thought to be transmitted through arthropod vectors or cat bites and may have the ability to be transmitted to humans, particularly in immunocompromised individuals.  While PCR is the preferred method for M. haemofelis identification, multiplex fluorescent microbead immunoassay (MIA) may provide a more sensitive method for M. haemofelis detection. This technology facilitates the measurement of antibodies against several antigens simultaneously in the same reaction tube using a small sample volume.  A recently developed MIA for M. suis showed high sensitivity, detecting antibodies 17 days before qPCR. Three proteins of M. haemofelis (Mhf3, Mhf9, and Mhf15), previously identified as immunogenic, in addition to GrpE, used in the M. suis MIA, were selected as target antigens for M. haemofelis. In silico analysis of the mature protein sequences was performed to identify peptides consisting of 20 amino acid residues having predicted immunogenicity. The aims of the study herein were to experimentally confirm the immunogenicity of the four synthesized peptides and develop a MIA for M. haemofelis. To accomplish this, each peptide was analyzed for its immunogenicity by Western Blot with pooled sera from M. haemofelis infected cats. These antigens were then coupled to unique MicroPlex® microspheres. Sera from noninfected and infected cats were used to set the median fluorescent intensity (MFI) cutoffs for the MIA and as positive controls, respectively. Samples collected from experimentally infected cats at various time points and cats admitted to the Purdue Veterinary Teaching Hospital were tested. We expect the MIA assay to demonstrate a high degree of sensitivity for detection of known infected cats. 

Despite significant advances in medical management, sepsis continues to be a common cause of morbidity and mortality in horses. Novel approaches to address the catastrophic cascade of pro-inflammatory events are critical to improving the outcome for horses with sepsis. Mesenchymal stem cells (MSC) inhibit production of several pro-inflammatory cytokines in rodent models of sepsis. In horses, MSC have most commonly been isolated from umbilical cord blood, umbilical cord tissue, bone marrow, and adipose tissue. We hypothesize that MSC are present in equine peripheral blood and can be isolated and differentiated into various cell types. We obtained whole blood from a healthy adult horse, followed by peripheral blood mononuclear cell isolation. Standard media containing dexamethasone was used to isolate MSC in a T75 culture flask during a 10-day incubation period. The cells were characterized by flow cytometry using monoclonal antibodies against cell surface markers specific for MSC as well as antibodies against cell surface markers that are expected to be absent on MSC. The isolated cells expressed the cell surface markers CD29 and CD44, but they were negative for CD90. Next, we will perform cell lineage differentiation to confirm the “stemness” of the cultured MSC. Cultured cells will be incubated using special media to drive them toward osteogenic or chondrogenic growth. Alizarin Red and Alcian blue staining will be used to identify the osteocytes and chondrocytes, respectively. We expect the cell staining to show that the stem cells developed into osteocytes and chondrocytes. This pilot data will be used to perform subsequent experiments investigating the ability of peripheral blood MSC to inhibit pro-inflammatory cytokine gene expression in equine monocytes in vitro.

The avian influenza virus subtypes H5, H7, and H9 emerged in the past decade as important human health concerns and continue to be potential pandemic threats.  New techniques for developing broad spectrum influenza vaccines to combat the impending pandemics are needed so they can be stockpiled for influenza pandemic preparedness. Since the nature of the next pandemic influenza virus is unknown, our efforts are directed towards the generation of universal influenza vaccines, broadly protective against H5, H7 and H9 influenza subtypes (as well as H1 and H3 subtypes).  This could significantly lower morbidity, hinder transmission and prevent mortality in a pandemic situation before a strain-matched vaccine can be produced. Our immunogenicity and protective efficacy studies showed an adenovirus (Ad) vector-based H5N1 vaccine provides excellent humoral and cell-mediated immune responses leading to complete protection against challenge with antigenically distinct strains of H5N1 viruses. To further enhance the breadth of protective efficacy against avian influenza viruses, the relatively conserved domains of influenza proteins are used for multi-epitope-based vaccines in Ad vector formulation. 6-8 week old BALB/c mouse groups were vaccinated intramuscularly at day 0 and 21, with a number of multi-epitope-based vaccines.  At day 42 the mice were euthanized under anesthesia and the spleen and serum samples were collected.  The serum samples were analyzed for humoral immune responses by ELISA, hemagglutination inhibition and virus neutralization assays.  The splenocytes were analyzed for cell-mediated immune responses by ELISpot.  We hypothesize the vaccine induces B and T cell response against the individual influenza epitopes incorporated in the recombinant Ad vector and will protect against multiple influenza subtypes. This vaccine strategy will help to design pandemic influenza virus vaccines without prior knowledge of the pandemic influenza virus strain.

Student Support by Merial

Research Support for Dr. Mittal: PVM Internal funds, Egyptian Government fellowships

Inflammatory Bowel Disease (IBD) is comprised of Crohn’s disease and ulcerative colitis. Both diseases are characterized by life-long, relapsing gastrointestinal inflammation. These diseases are thought to occur in genetically susceptible individuals due to hyper-immune responsiveness to their normal intestinal flora. Tumor Necrosis Factor (TNF) is a “proinflammatory” cytokine primarily secreted by macrophages that regulates many processes, including inflammation, apoptosis, necrosis, and angiogenesis. Anti-TNF drugs inhibit many of the deleterious effects of inflammation that TNF causes. In addition, blocking of TNF may affect the inflammation by changing the composition of the intestinal microflora. Experiments performed in our lab have shown that mice that lack the TNF gene (Tnf ^-/- mice) have decreased acute colitis and an altered luminal colonic flora, when compared to wildtype (WT) mice. In the colon, mucin forms a protective barrier that separates luminal bacteria from the epithelium, preventing attachment and invasion. Mucins shown to be involved in IBD include MUC1, MUC2, MUC3, MUC4, MUC13, and MUC17. We hypothesize that TNF affects the expression of mucins in the colon.Colonic MUC 1 and 2 protein levels were evaluated by western blots analysis in the normal intestinal tissues of Tnf ^-/- mice and WT mice to determine if differences in mucin quantity in the colon contributed to the differences in the luminal flora. Periodic Acid Schiff (PAS) staining of the goblet cells in the colons of WT and Tnf ^-/- mice was also performed as an additional method of assessing mucous production. 

Characterizing human adenoviral proteins involved in genome packaging allows us to better understand viral assembly and infection, to develop next generation viral vectors, and to discover novel nanotechnology tools. During adenovirus (AdV) replication, a capsid is first assembled and then filled with genomic material through a ring-like opening. Of particular interest in this process is AdV protein IVa2, which is thought to function as an ATPase, generating the energy for viral genome packaging. AdV protein 33K may serve a complimentary role as a conformational on/off switch on IVa2, as it has been shown that IVa2’s ATPase is active only in the presence of 33K. Additionally, 33K is present in pre-filled, empty capsids, whereas IVa2 is present on both empty and filled capsids. Based on this, we hypothesize that these proteins form multimers and interact with each other and also with the AdV genome. 

Here, we studied the comparative in vivo oligomerization of the individual and combined proteins by Western blot analysis of single and dually transfected mammalian cells. As expected, denatured proteins showed up as single bands, representing monomers; non-denatured proteins generated supershifted bands, representing oligomers. Comparison of the band densities indicated higher amount of oligomer formation in the dually transfected cells (cells expressing both IVa2 and 33K), supporting the hypothesis of either enhanced oligomerization of individual proteins, of favorable binding between the two proteins, or both. Enhanced oligomerization and binding support the theory of interaction between these proteins, which is a key element in furthering our understanding of their roles in genome packaging.

Research support provided by USDA Hatch

Student support provided by Merial

Traumatic nerve and spinal cord injuries are often accompanied by loss of sensory and motor function. Functional recovery may not occur due to the limited capacity of nerves to heal, which suggests the need for interventions that help restore the function of damaged nervous tissue. Blood vessels play an important role in spinal cord repair, and it has been proposed that neural-vascular interactions are synergistic and promote the regeneration of injured nerves. To investigate these interactions, 3D cell cultures were prepared by co-culturing dorsal root ganglia (DRGs) with endothelial colony forming cells (ECFCs) in a Type I collagen oligomer matrix. The hypothesis was that ECFCs and nerve growth factor (NGF) enhance DRG neurite outgrowth. DRGs were harvested from 10-12 day old chicken embryos and co-cultured with ECFCs in 96-well plates with one DRG per well. DRGs and ECFCs were also cultured separately to serve as controls. After incubating for two days, images were taken of each DRG using phase-contrast microscopy. ImageJ software was used to analyze the images and determine the ratio of neurite outgrowth cross-sectional area to DRG cross-sectional area. When DRGs were cultured alone, the presence of NGF did not result in a statistically significant increase in neurite growth. The DRG and neurite interactions with the collagen-fibril matrix appeared to overcome the need for NGF. However, neurite outgrowth was significantly increased when DRGs were co-cultured with ECFCs. This DRG-ECFC interaction appeared to enhance neurite extension. Higher quality imaging was also attempted with a confocal microscope, but work is currently being performed to overcome several limitations. The DRGs and neurite extensions were too large in 3D, and the fluorescent staining was not vivid enough to acquire preliminary confocal images. Obtaining higher quality images is an ongoing process and will allow quantitative analyses to be performed in 3D.

Medial patellar luxation (MPL) is one of the most common orthopedic conditions in dogs caused predominantly by anatomical stifle abnormalities that misalign the extensor mechanism. Femoral deformities are more recently being corrected through distal femoral osteotomies however, radiographic preoperational angle measurement techniques lack reliability as they are prone to parallax error. This study will attempt to refine femoral angle measurement practices by utilizing cardan angles and associated anatomical landmarks in the 3D plane.

Three-dimensional digital models of 50 femora from 25 dogs without MPL were converted from CT scans. Each femur model was aligned to the global coordinate system based on the ½ -¼ proximal femoral long axis. Distal local coordinate systems were established based on the radiographic landmark resulting from the intercondylar notch (Blumensaat’s line). The distal local coordinate system was rotated around the mediolateral axis, craniocaudal axis, and the longitudinal axis to quantify the procurvatum angle, anatomical lateral distal femoral angle (aLDFA), and anteversion angle, respectively. Three investigators (novice, intermediate, and experienced) measured these angles with the novice and experienced investigators measuring angles twice. The intra- and inter-observer intraclass correlation coefficients (ICC) were evaluated.

The mean values of the groups collective data sets were 94.5 aLDFA, 30.0° anteversion and 11.6° procurvatum. Both intra- and inter-observer ICC were greater than 0.90 demonstrating high reliability associated with angle computation the 3D plane which holds promise for femoral deformity correction in the future.

Formalin fixation with paraffin embedding (FFPE) is a method used to preserve tissues. FFPE tissues can be sampled through tissue cores or scrolls. In the study of urothelial carcinomas (UC), the use of a biopsy needle to acquire cores has the advantage of sampling the targeted superficial epithelial cell layer. In contrast, tissue scrolls are collected using a microtome, which includes surrounding tissues such as the muscularis layer. While both methods allow sample collection to extract small molecules, such as miRNAs, there is a lack of literature comparing these methods. MiRNAs are non-coding RNAs responsible for gene regulation, altered in cancer. UC is the most common malignant bladder tumor in dogs, with 20,000 cases annually. The diagnosis of UC is challenging due to the small sample size of biopsies acquired by endoscopy. This project aimed to compare miRNA expression between the FFPE collection methods in UC (n=3) and healthy bladder (n=1) from dogs to understand the effect of including tissues surrounding a lesion in scrolls. MiRNA was extracted using a commercial kit, and the expression was achieved through RT-qPCR. Cores targeting the epithelia and the muscularis layer were compared to their corresponding scrolls. MiR-214, miR-152, miR-181a, and miR-39 were differentially expressed when comparing cores with scrolls and the muscularis layer, while miR-1842 and miR-143 were not. Comparison between methods within individual samples also showed differential expression for all miRNAs. These results show that the inclusion of the muscularis layer results in misleading miRNA expression and verifies cores as a more accurate technique in miRNA extraction and expression, supporting further miRNA biomarker research.

Iliopsoas muscle (IPM) injuries are the most commonly diagnosed hind limb musculo-tendinous injury in dogs. The diagnosis IPM injury is based on a combination of a positive iliopsoas maneuver, pain on rectal palpation,  radiographs, ultrasound, CT and/or MRI. Radiographs occasionally show new bone formation in the area of the iliopubic eminence (IPE), however, the genesis and clinical significance are unknown. The hypotheses tested in this study are that 1) new bone formation in the IPE area is associated with the tendinous insertion of the psoas minor muscle (PMM), and that 2) bone formation in the tendinous insertion of the PMM is commonly seen in dogs with IPM injuries. Dissection of three canine cadavers demonstrated that the psoas minor tendon has a 25 x 0.5 mm insertional foot print on the IPE and the adjacent arcuate line of the ilium. This area corresponded with the area of bone proliferation on the radiographs of some patients suffering from iliopsoas injury. Pelvic radiographs of 13 out of 28 (46%) dogs diagnosed with IPM injury had concurrent bone proliferation of the IPE and adjacent part of the arcuate line. Eight of the 13 (62%) affected dogs also were diagnosed with hip dysplasia, while 3 of 13 (23%) also had lumbo-sacral instability. The results of this study suggest that PMM injury may be associated with IPM injury and identify a possible new cause of canine hind limb lameness. Further studies regarding clinical findings, diagnosis, treatment and prognosis of PMM injury are indicated.

Research Support: Purdue University College of Veterinary Medicine NIH Merial Summer Research Program

Student Support: Purdue University College of Veterinary Medicine NIH Merial Summer Research Program

A cats’ willingness to approach and interact with unfamiliar people of both genders may affect adoption outcomes in shelters.  Active, playful, and friendly cats are more likely to be adopted.  A three-step approach test was used to assess the response of cats to an unfamiliar male or unfamiliar female person.  Subjects were 33 singly housed adult cats age 0.5-11 years at a local animal shelter.  Data collected included latency to interact, latency to approach, time spent interacting, and an approach test score (1-5).  This data was compared to the cats’ time in the shelter to determine any effect on the cats’ willingness to approach and interact.  The hypothesis was that cats who had more time to acclimate to the shelter environment would show more affiliative behaviors to the approach tester and that the cats would be more willing to approach and interact with a female tester than a male tester.  Analysis by one-way ANOVA revealed that cats that had been at the shelter for more than two weeks interacted significantly longer than cats that had been shelter housed for two weeks or less (P=0.009).  Also, cats had a significantly lower latency to approach (P=0.06) and a higher approach test score (P=0.02) when approached by a female as opposed to a male.  These results suggest that cats are more sociable after having acclimated to the shelter environment for two weeks or longer, and that cats in a shelter setting are more likely to interact and be sociable with a potential female adopter than a male adopter.  Future research should aim to understand how to increase cats’ willingness to approach and interact with males and what other factors may influence the adoptability of cats in a shelter.

Research Support: Department of Animal Science, Purdue University, West Lafayette, IN

Student Support: Merial Veterinary Scholars Program

Bacterial sepsis is an important disease in equine neonatal medicine, as it remains a
leading cause of morbidity and mortality. Accurate and early diagnosis of sepsis is difficult due
to insensitivity of available tests. Acute phase proteins, such as C-reactive protein (CRP) and
haptoglobin (Hp) are potential biomarkers for sepsis as they rise rapidly after the onset of
infection, increase over several hours, and have been shown useful in monitoring response to
treatment. We hypothesized that plasma CRP and Hp concentrations would be increased in septic
foals compared to sick non-septic and healthy control foals, and that both proteins would be
predictive of survival. A multi-center, prospective observational clinical study was performed at
Purdue University Veterinary Teaching Hospital and Hagyard Equine Medical Institute. Jugular
venous blood was collected at admission from all suspected septic and sick non-septic foals less
than one week of age, and from clinically healthy foals less than 24 hours of age. Neonates were
classified as septic or sick non-septic based on the Sepsis Scoring System and blood culture
status. Forty each of septic, sick non-septic and healthy neonatal foals were enrolled in the study.
Plasma CRP and Hp concentrations were measured using previously validated equine ELISA.
Preliminary results for both proteins presented wide concentration ranges within each group.
Statistical analysis of the results in conjunction with clinical data and survival is needed to
clearly accept or reject the hypothesis.

Bovine growth hormone (bGH) plays an important role in the regulation of metabolism in cattle.  Our laboratory is interested in investigating the influence of bGH on calcium metabolism of the periparturient dairy cow. Previously, research laboratories used radioimmunoassays for the quantification of bGH in serum and plasma. However, due to safety and special handling requirement of radioactive reagents and waste, non-isotopic immunoassays are a much-needed alternative for experiments requiring bGH quantification. Several bovine-specific enzyme-linked immunosorbent assays (ELISA) are commercially available; however, none have been validated by independent research groups. Previous experiments in our laboratory demonstrated that commercial ELISAs were unsuccessful at quantifying a highly purified, native bGH based on spike and recovery experiments. The goal of this study was to 1) identify commercially available antibodies against bGH to identify bGH in serum using western blot (WB), and 2) attempt quantification of bGH in serum using a quantitative WB. We tested two antibodies for detection of the purified native bGH, a rabbit polyclonal anti-bGH antiserum and rabbit polyclonal anti-ovine GH IgG. Serial dilutions of the bGH standard demonstrated that it is possible to perform relative quantification of bGH in WB with the anti-bGH but not the anti-ovine GH antibody. Attempts to quantify bGH in actual serum samples were not successful. Experiments are under way to concentrate bGH in serum samples via an immunoprecipitation protocol to determine if WB can be successfully used for relative quantification purposes.

Acute abdominal disease requires quick and effective diagnosis to provide accurate medical care. One such situation is a ruptured bladder, usually due to trauma, leading to an uroabdomen. Creatinine and potassium are eliminated in urine allowing for abdominal fluid: serum ratios to be diagnostic for uroabdomens. Uroabdomens are considered a surgical emergency and unfortunately abdominal fluid analysis currently can only be done at reference laboratories. The time delay in getting fluid analysis makes this currently an inefficient way to diagnose uroabdomens. The purpose of this study was to determine if in-house Ortho Clinical Vitros 5,1 FS, Idexx Catalyst, Idexx VetLyte, and point of care analyzer Abaxis iSTAT can accurately be used to evaluate a large range of potassium and creatinine levels in abdominal fluid of the canine, feline, and equine patients.Canine, feline, and equine transudate abdominal fluid samples are being analyzed on the Vitros, Catalyst, VetLyte, and iSTAT for creatinine and potassium. Samples from each species had creatinine and potassium added at increasing intervals to provide results consistent with a positive control. These samples were also sent to Idexx to be run on the gold standard analyzer Olympus AU 5400. Bland- Altman plots and linear regression plots will be created to provide the agreement, accuracy, and bias of each analyzer. In addition the coefficient of variation be calculated to provide the precision of each analyzer.

Research Support: Purdue Veterinary Clinical Pathology Department Student Support: Purdue Veterinary Scholars program

Aluminum adjuvants included in vaccines are crucial for potentiating the immune response. Purified viral or bacterial antigens are adsorbed to aluminum containing gels in vaccines such as human papilloma virus, hepatitis B, and tetanus-diphtheria-acellular pertussis (Tdap). These aluminum adjuvants augment the antibody response without enhancing the cell-mediated response.  Previous studies have revealed important information regarding the function of these adjuvants, such as their adsorptive and chemical properties, but the mechanisms by which the adjuvants predominantly enhance the humoral immune response are still unknown. ICAM-1 (intercellular adhesion molecule-1; CD54) is a ligand on the surface of vascular endothelial cells essential for migration of leukocytes to sites of inflammation as well as on the surface of antigen presenting cells involved in T-cell activation. We hypothesized that blocking ICAM-1 with specific monoclonal antibodies reduces the accumulation of inflammatory leukocytes at the injection site and diminishes inflammation at the injection site and antibody production. We injected mice with ovalbumin (OVA) and aluminum hydroxide adjuvant and either ICAM-1 or control rat IgG. After one and three days, the injection sites and draining lymph nodes were collected to examine the inflammatory reaction using fluorescence and confocal microscopy. Treatment with anti-ICAM-1 antibodies did not significantly affect the OVA-specific IgG, IgG1, and IgG2a responses. This research provides insight into the mechanisms by which aluminum adjuvants enhance the immune response, and will be useful to determine the optimal formulation for use in preventative vaccines against infectious diseases and immunotherapy of allergies.

Recurrent Airway Obstruction (RAO), or heaves, is a chronic respiratory disease commonly seen in horses ages seven and older. It is characterized by pulmonary airway inflammation leading to increased mucus, bronchoconstriction, and airway remodeling. Clinical signs include coughing, nasal discharge, difficulty breathing, and anorexia. Heaves is caused by hypersensitivity to airborne allergens, including dust, mold, and endotoxins. Good quality hay is baled at a moisture content of approximately 15% whereas moldy hay is baled at a moisture content of greater than 15% resulting in increased allergen concentration. There is currently no standardized method to quantify the amount of airborne dust that can be generated from different types of hay. We built a dust collection device consisting of a closed cylindrical box with rotating paddles inside the box to agitate hay samples and air sampling ports for dust sampling. We hypothesized that the amount of dust originating from a hay sample will be affected by rotation speed of the paddles, duration of tumbling in the dust collector, and weight of hay sample. To address the hypothesis, we collected samples from good quality and moldy hay and rotated them in the dust collector at different speeds while measuring concentration of airborne dust.

More dust was being collected from moldy hay compared to good quality. Also, there was a positive correlation between higher rotation speed and concentration of dust, and between weight of hay and dust concentration in regards to good quality hay. Understanding the concentration of dust generated by hay can lead to improved recommendations concerning production of quality hay or evaluation of hay quality in relation to respiratory health of horses. 

Traumatic brain injuries (TBI) in survivors of explosions has increased over the last decade.  Our lab previously developed a rodent model for studying the effects of brain injuries induced with a shock tube.  To create a more accurate model, the shock tube setup was refined so that the overpressures had consistent peak values.  The experimental apparatus is an open-ended shock tube composed of two segments, a compressed nitrogen driven section and an atmospheric driven section, separated by an expandable diaphragm.  Compressed nitrogen fills the driver section, increasing the pressure until the diaphragm expands and bursts.  A supersonic blast wave is released and propagates out through the open end of the tube.  Three pressure-impulse sensors quantify the blast wave dynamics.  Two of the sensors, installed at varying distances from the diaphragm within the driven chamber, are perpendicular to the propagation of the wave.  The third sensor, oriented parallel to the shock front, is located outside the tube where the shock wave has fully redeveloped.  This is the point where the head of the rat would be placed for exposure to produce the most repeatable and predictable results.  Aspects of the wave that are believed to influence severity of a TBI include, peak overpressure, velocity, impulse, and over- and under-pressure duration.  A more through characterization of the shock wave elements produced will allow for better determination of the type or severity of TBI in this model and open doors for the evaluation of potential treatments.

Pigmentary uveitis (PU) is a late-onset, heritable ocular condition found solely in Golden Retrievers. A diagnosis is made if there is pigment deposited on the anterior lens capsule in either a radial pattern or in zones. Uveal cysts are often not visualized clinically at the time of diagnosis of pigmentary uveitis. However, prior histologic studies have shown uveal cysts in the majority of dogs with pigmentary uveitis. Therefore, we hypothesized that the presence of uveal cysts increases the likelihood of developing pigmentary uveitis. This retrospective study was designed to determine whether a prior diagnosis of uveal cysts was a risk factor for the development of pigmentary uveitis. Recent examination results (2012-2013) were compiled for 50 Golden Retrievers previously examined between 2010 and 2011.  Of the 50 cases uveal cysts were initially diagnosed in the right eye (OD) 11/50 and left eye (OS) 12/50. In the OD, uveal cysts were single 4/11 or multiple 7/11. In the OS, uveal cysts were single 7/12 or multiple 5/12. Pigmentary uveitis developed in 1/77 eyes with no uveal cysts previously, 1/11 eyes with a single uveal cyst, and 7/12 eyes with multiple uveal cysts. Fishers Exact Test revealed a significant association (p <0.05) between presence of uveal cysts and development of PU. This is a suspected heritable condition, which may be prevented in the future through identification of risk factors and development of an adequate screening process prior to breeding.

Student support: Merial Veterinary Scholars Program

Rheumatoid arthritis (RA) and other autoimmune diseases are major risk factors for cardiovascular disease (CD). Recent evidence shows that CD40L (an immunomodulatory molecule) released by activated platelets influences the pathogenesis of CD via induction of adhesion molecules, chemotactic and inflammatory cytokines, and tissue factor in vascular endothelial cells, and by enhancement of adaptive immune responses, all of which contribute to atheroma initiation and progression. We previously demonstrated that CD40L expression is dynamically regulated in megakaryocytes (precursors of platelets).  Our preliminary data shows that acute inflammatory cytokines TNF-α, IL-6, and IL-1β, (in most autoimmune/ chronic inflammatory disease) can modulate CD40L activity in primary megakaryocytes in the bone marrow. We hypothesize that these megakaryocytes further develop into “hyper inflammatory platelets” producing elevated levels of CD40L, which may represent the connection between autoimmune disease and CD. To address this hypothesis, we will evaluate the impact of inflammatory cytokines on CD40L levels in human megakaryocyte cell lines and mouse primary megakaryocytes in vitro. In addition, we will validate the expression and function of platelet CD40L in experimental mouse models of inflammatory disease. Validation of this hypothesis will identify a novel regulation mechanism of an autocrine cytokine feedback loop, which may suggest that cytokines play a significant role in the initiation and perpetuation of chronic inflammatory processes in diseases such as CD. This mechanism has the potential to be a therapeutic target that can modulate inflammation in autoimmune/ chronic inflammatory disease processes.

Research Support: National Institutes of Health (NIH) R01-AI060924 and the Purdue University

Center for Cancer Research

Student Support: Merial Veterinary Scholars Program 

Dengue hemorrhagic fever threatens over one third of the world's population and is a devastating viral disease spread by mosquitoes in tropical regions.  The disease causes prolonged fever and causes increased vascular permeability, which can lead to death. Existing mouse models of the disease are inadequate because the mice die of paralysis rather than fever or hemorrhage. We are attempting to establish an improved mouse model that utilizes three features to induce disease that more effectively mimics the human disease. First, the mice are genetically deficient for interferon α/β and γ receptor. Second, the use of 2H2 antibody will provide antibody-dependent enhancement of viral uptake by cells. Finally, the mice will be infected with a virulent strain of Dengue virus (DENV 221). Knockout and wild-type mice were each given 15 μg of 2H2 antibody and 100,000 PFU DENV 221 intraperitoneally. The mice were weighed and evaluated for clinical signs of disease daily and serum was collected at 24 and 72 hours post infection. Clinical signs were not noted. Necropsies were performed at 72 hours post infection.  Gross lesions were not observed at necropsy.  Lymphofollicular depletion was noted histologically in the spleen of 2/6 of the treated KO mice which may indicate establishment of Dengue infection in these animals, but was not statistically significant. TNF and IL-6 proteins were not detectable in the spleen or liver of any mice. These findings indicate that Dengue Fever was not effectively established in this experiment. Future directions for this project include completing RT-PCR analysis of serum and tissues to evaluate virus levels and repeating the experiment with minor modifications.

Characterizing human adenoviral proteins involved in genome packaging allows us to better understand viral assembly and infection, to develop next generation viral vectors, and to discover novel nanotechnology tools. During adenovirus (AdV) replication, a capsid is first assembled and then filled with genomic material through a ring-like opening. Of particular interest in this process is AdV protein IVa2, which is thought to function as an ATPase, generating the energy for viral genome packaging. AdV protein 33K may serve a complimentary role as a conformational on/off switch on IVa2, as it has been shown that IVa2’s ATPase is active only in the presence of 33K. Additionally, 33K is present in pre-filled, empty capsids, whereas IVa2 is present on both empty and filled capsids. Based on this, we hypothesize that these proteins form multimers and interact with each other and also with the AdV genome. 

Here, we studied the comparative in vivo oligomerization of the individual and combined proteins by Western blot analysis of single and dually transfected mammalian cells. As expected, denatured proteins showed up as single bands, representing monomers; non-denatured proteins generated supershifted bands, representing oligomers. Comparison of the band densities indicated higher amount of oligomer formation in the dually transfected cells (cells expressing both IVa2 and 33K), supporting the hypothesis of either enhanced oligomerization of individual proteins, of favorable binding between the two proteins, or both. Enhanced oligomerization and binding support the theory of interaction between these proteins, which is a key element in furthering our understanding of their roles in genome packaging.

Research support provided by USDA Hatch

Student support provided by Merial

Cancer is a complex genetic disease, as there are usually many mutations in cancer genomes. Not all of the mutations equally contribute to cancer initiation, progression, and metastasis. One of the main goals of current cancer research is to identify the key genes whose mutations could promote tumorigenesis, so that therapeutic strategies and diagnostic markers may be later developed. Given the large number of genetic alterations within each cancer genome, it is often very difficult to identify new cancer drivers, even with current deep sequencing and microarray technologies. Using zebrafish-human comparative cancer genomics, we recently identified 3 human zinc and ring finger E3 ubiquitin ligase genes (ZNRF1-3) as tumor suppressor candidate genes. In order to validate their tumorigenic roles, we will make a loss-of-function mutant zebrafish model, as the zebrafish is an emerging cancer model which mimics human cancers in many aspects. Using a newly developed genome-editing tool originating from bacteria and archaea, CRISPR (clustered, regularly interspaced, short palindromic repeats), we designed two gene-specific CRISPR RNAs in the sequence form of 5′-GG-N18-NGG-3′ for each zebrafish orthologous gene. Sequencing analysis confirms that we have successfully created the guide RNA constructs using the currently available type II CRISPR-Cas system (pDR274 and MLM3613) which has already been successfully applied in zebrafish. These constructs will be used to generate RNAs for microinjection into zebrafish embryos to create mutant zebrafish lines. In the future, we can investigate the tumorigenic roles of these ZNRF genes using the zebrafish mutants. 

Research support: John T. and Winifred M. Hayward Foundation

Student support: Merial Veterinary Scholars Program

Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), affect over a million people in the United States. The pathogenesis of these diseases remains poorly understood, but inappropriate immune system stimulation is suspected. Regulatory T cells express the transcription factor FOXP3 and play a crucial role in suppressing abnormal immune responses. By producing anti-inflammatory cytokines, IL-10 and TGF-β, regulatory T cells are able to dampen the actions of effector T cells. It has previously been shown that the number of peripheral blood regulatory T cells is decreased in IBD patients. We hypothesized that the regulatory T cells have localized in the intestinal mucosa, where the inflammation is most severe. Trinitrobenzenesulfonic acid (TNBS) was used to induce a Crohn’s disease-like colitis in regulatory T cell labeled, FOXP3+-cre-tdTomato, transgenic mice. Colonic tissue was collected and imaged using a Leica SP5 multi-photon microscope with Ti:Sapphire mode-locked lasers. The rapid low energy laser pulses allowed for deeper tissue penetration and precise imaging of the inflamed tissue. Red-fluorescent-protein labeled regulatory T cells were localized and quantified using IMARIS®3D-imaging software. Additional intestinal structures, including muscular layers and collagen, were identified by intrinsic auto-fluorescence and second harmonic generation respectively. Cellular changes noted in the multi-photon imaged tissue can provide scientists with an inside look into the immune processes occurring during inflammatory bowel diseases. 

There is a strong association between transitional cell carcinoma (TCC) of the urinary tract and bacterial urinary tract infections (UTIs) in dogs. UTIs are a frequent complication of TCC, but there is also evidence to suggest that the inflammation from UTIs increases the risk for developing TCC and other forms of bladder cancer. The inflammatory mediators interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and hydrogen peroxide (H2O2) expressed in UTIs are reported to transform the urothelium in a dose-dependent manner. However, their role in tumor cell proliferation and cancer progression remains unknown. We hypothesized that IL-6, TNF-α, and H2O2 will enhance TCC cell proliferation in a similar dose-dependent fashion. Proliferation assays were performed on three previously characterized canine TCC cell lines. Cells were incubated for 72 hrs with 0.0005-10 mM H2O2, 0.01-100 ng/ml IL-6, or 0.01-100 ng/ml TNF-α and growth was compared to untreated (vehicle only) controls. Subsequent assays were performed with the addition of vinblastine (0.001-10 µM) to the treatment groups and the percentage of growth inhibition was compared to cells grown with vinblastine alone. Initial results indicate that H2O2 does not enhance proliferation, but is cytotoxic at concentrations >0.1 mM. H2O2 did not affect the IC50 of vinblastine. Understanding the effects of inflammatory mediators expressed in UTIs on the proliferation and progression of TCC as well as their influence on chemotherapeutic efficacy is important for advancement of multimodal treatments of canine TCC.

Research Support: Private donations for transitional cell carcinoma research
Student Support: Merial veterinary scholars program

Yucatan minipigs are commonly used in biomedical research. These animals are prone to dry skin ( xerosis) accompanied by pruritus. In previous work, minipigs housed by the Purdue Laboratory Animal Program were oiled twice weekly with lanolin to mitigate this condition, however, this resulted in excessive debris along the dorsum sometimes accompanied by exfoliative lesions. In this study, alternatives (glycerol and coconut oil) were used to compare their abilities to hydrate skin appropriately. The agents were applied twice a week over a four week period in a crossover design so that each pig was treated with each agent for one week with a 4 day washout period. Skin was assessed using a corneometer and both visual and tactile scoring at various timepoints.  Results from visual and tactile scoring 24 and 48 hours after each application showed that the ideal skin condition was achieved in the following order: glycerol (83.3% visual,75% tactile), coconut oil (79.2%,64.6%), control (33.3%,39.6%), followed by lanolin (29.2%,12.5%). No significant difference was found from the results of the corneometer; it was concluded that the device was inaccurate for this purpose.  Lanolin pooled on the skin and left the pigs greasy days after the agent was applied while glycerol had the highest frequency of ideal scores.  In conclusion glycerol was found to be the best option for hydrating Yucatan minipig skin in the research setting.

Proximal sesamoid bone (PSB) fracture is the leading cause of equine musculoskeletal injury leading to humane euthanasia on racetracks in the USA. Studies have shown that exercise and training cause changes in the microstructure of equine bones. It has also been shown that microstructural values are different in the contralateral PSBs from horses with fractured PSBs compared to control horses and may be predictive of fracture. Unfortunately, these values are collected from micro CT images which currently cannot be obtained in a live horse. The recent development of equine standing CT equipment bypasses the need for horses to be under anesthesia for CT imaging and could enable routine screening of racehorses. The goal of this study is to determine if clinical CT scans give microstructural values of PSBs comparable to the gold standard micro CT imaging. We hypothesized that the current clinical CT resolution is too low to accurately identify bone microstructure, but that useful correlations exist between clinical and micro CT images. We quantitatively compared the microstructure of fractured and intact PSBs at different scan resolutions. Forelimb PSBs were collected from 20 racehorses that were euthanized on Indiana racetracks, 10 for PSB fractures and 10 for non-musculoskeletal reasons. The PSBs were imaged at a resolution of 625 µm (clinical CT) and 144, 90, and 65 µm (micro CT).  Bone volume fraction, trabecular thickness, and degree of anisotropy values were collected and the statistical significance of differences between resolutions was determined. The ability to screen horses and identify microstructural changes in PSBs prior to fracture could prevent catastrophic injuries and improve equine welfare.

Phenylpropanolamine (Proin®) is a sympathomimetic amine drug used for the treatment of urinary incontinence. This medication has been reported to cause acute glaucoma and increased intraocular pressure in humans. The aim of this study is to determine if Proin has any clinically significant effects on intraocular pressure (IOP) and tear quantity and quality as measured by Schirmer I tear test (STT-1) and tear breakup time (TBUT). Three healthy Beagles with normal eyes were administered Proin (2 mg/kg orally, twice/day). Prior to administering Proin, dogs received a complete ophthalmic examination including baseline IOP, STT-1, TBUT, and ultrasound biomicroscopy (UBM) to examine anterior segment structures such as the ciliary cleft and iridocorneal angle. Once the dogs began to receive Proin, IOP was evaluated three times per day twice per week, STT-I once per week, TBUT along with a full ophthalmic examination once every two weeks, and UBM will be measured at the end of the 4-week study period. Descriptive data are presented as mean ± SD. At of the end of the first week of Proin administration, one dog with a high normal baseline IOP (24.5 ± 0.2 mmHg) exhibited a marked elevation of IOP to the level of risk of developing glaucoma (30.6 ± 1.5 mmHg). STT-1 had not changed notably. Another dog developed mild conjunctival hyperemia in both eyes at 1 week. We expect that this study will provide information to veterinary professionals regarding the safety of administering Proin to canine patients, especially those with and at risk for glaucoma.

Avian cholera, caused by the bacterium Pasteurella multocida, is a significant infectious disease affecting birds worldwide, including commercial poultry. Carrier birds act as reservoirs for P. multocida, but the role of the environment as a reservoir has not been fully elucidated. A recent study suggested that P. multocida may exist within Acanthamoebae, which are abundant in water sources and soil, but further proof of this interaction is needed. To explore this hypothesis, we utilized a mathematical model to determine if this mechanism could play a role in avian cholera outbreaks in snow geese (Anser caerulescens). We studied two snow geese migration sites in Indiana and Nebraska, which both have had more than one outbreak in the last 10 years. Publicly available data on snow geese sightings, occurrence of avian cholera outbreaks, and hourly soil temperatures were collected from 2011-2021 and plotted to explore year-to-year trends and compare outbreak and non-outbreak years. An existing compartmental model was adapted to simulate the dynamics of Acanthamoebae and P. multocida at different temperatures. Based on the results from the model, which assumes that Acanthamoebae exist in the active trophozoite form between 15-36°C, these amoebae in the soil are unlikely to play a role in avian cholera outbreaks in our study sites because the temperatures are not warm enough for the amoebae to be infected by P. multocida. The hypothesis may be supported in warmer climates and it is possible that there are other contributing environmental factors, like biofilms, other free-living amoebae, or vectors, but more research must be conducted.

Vaccines are commonly formulated with adjuvants, which stimulate immunologic pathways to improve protective immunity against infectious diseases. The most widely used adjuvants in human and veterinary vaccines are aluminum and oil-in-water emulsion adjuvants. However, these adjuvants can cause adverse reactions and are only approved for intramuscular administration of vaccines. Intradermal (ID) and Intranasal (IN) vaccination can stimulate different types of immune responses and can be done without hypodermic needles. However, there is a need for new adjuvants that can be used in ID and IN vaccines. In this study, we investigated the immune response in mice following ID and IN vaccination with a combination adjuvant composed of Nano-11 and ADU-S100. Nano-11 is a phytoglycogen-based nanoparticle that is safe for ID and IN vaccination. It can be formulated to deliver diverse antigens and cyclic dinucleotides, such as ADU-S100. This cyclic dinucleotide bind and activates the stimulator of interferon genes (STING) receptor which leads to the production of type 1 interferons. Mice were immunized twice with ovalbumin (OVA) only or OVA with Nano-11/ADU-S100. The combination adjuvant increased the titers of OVA-specific serum IgG subclasses and serum and intrabronchial IgA, as well as plasma cells in the bone marrow. Mice immunized with the combination adjuvant had increased numbers of OVA-specific Th1 and Th17 cells as well as CD8 T cells in the spleen. Immunization via the IN, but not ID route, increased the number of resident memory T cells in the lungs. These findings demonstrate that the combination adjuvant induced robust humoral and cell-mediated immunity and support its use in ID and IN vaccines. 

Electroencephalography (EEG) provides direct monitoring of the brain activity associated with the depth of anesthesia in humans. The use of such an EEG monitor has not been explored in anesthetized horses. The aims of this study were to 1) explore the clinical usage of the EEG monitor on the anesthetized horse and, 2) evaluate if the EEG waveforms and processed EEG indices could indicate the depth of anesthesia changes in horses. We hypothesized that the human EEG monitor would be able to differentiate the depth of anesthesia in the horse. Six horses subjected to orthopedic or soft tissue surgeries were used. The horses were instrumented for standard hemodynamic monitoring for the entire anesthetic procedure. Recovery quality was scored (poor, acceptable, excellent) with a video system. A human EEG monitor (Sedline with Root®) with six needle electrodes was positioned on the Fp1, Fp2, F7, and F8 frontal lead. The SedLine monitor calculated the patient state index- PSI, spectral edge frequency 95%- SEF95%, burst suppression ratio- BS, artifact percentage, and electromyographic activity. Results showed that the EEG monitor can be easily applied to the horses. The EEG monitor was able to detect the depth of anesthesia changes before the traditional hemodynamic monitor during the surgery and recovery. In addition, it was found that horses that had a frequent and long duration of BS during the surgery had a rougher recovery quality score. In conclusion, this study demonstrated that Sedline EEG monitor could be used routinely for anesthetic depth titration in horses. Further research is warranted to correlate the duration of BS and the quality of the recovery in horses.