Research Projects

Kyle Barron¹, Aynsley Romaniuk², Shanis Barnard³, Candace Croney⁴

¹ Purdue University College of Veterinary Medicine; ² Departments of Comparative Pathobiology and Animal Science, Purdue University; ³ Center for Animal Welfare Science, Purdue University 

The postnatal period of mammals is essential for the emergence of social behaviors and responses to stress. Maternal care (e.g., contact with offspring, nursing styles) during this period plays a crucial role in the behavioral development of offspring later in life. Studies conducted on rodents, for example, demonstrated that maternal care has lasting effects on the physiology of their offspring throughout life as it can modulate the activation of the hypothalamic-pituitary-adrenal axis, which is involved in the stress responses of animals. Maternal care in dogs remains understudied with current literature showing conflicting results. A study conducted in a working dog population indicated that high levels of maternal care (e.g., increased contact with offspring, increased time spent in the whelping box, and nursing) are associated with lower levels of stress and anxiety in offspring as adults. Conversely, a study conducted in a guide dog population showed a positive association between high levels of maternal care and stress and anxiety in offspring later in life. This discrepancy could be due to the differences in populations as a result of factors such as genetic selection. It is crucial to better understand maternal care in different dog populations. In this study, we conducted behavioral observations of 2 dams and their litters from a small-scale commercial dog breeding facility during the first 3 weeks post-parturition. Characterization of maternal care in dams in diverse breeding populations will inform future investigations of the relationship between maternal care and stress in puppies, which in turn, may help support best management practices and standards of care for improved welfare. 

Research Grant: Dr Candace Croney Discretionary Funds

Student support: Purdue University College of Veterinary Medicine

Lea Gamez Jimenez^a, Ahmed AbdelKhalek^a, Harm HogenEsch^{a, b}

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

^b Purdue Institute of Inflammation, Immunology, and Infectious Diseases, Purdue University, West Lafayette, IN 

Wildlife overpopulation has detrimental consequences for the sustainability of ecosystems.  Contraceptive vaccination using native porcine zona pellucida (nPZP) proteins isolated from ovaries is among the most humane, safe, and least disruptive options to mitigate this. However, improvements in the longevity, safety, and preparation efficiency of current vaccines are needed. This study compared the humoral response and fertility outcomes in female mice immunized with different vaccine formulations. Antigens included nPZP, recombinant PZP2 and PZP3 and recombinant equine IZUMO1 derived from Chinese hamster ovary (CHO) cells, and PZP3 derived from GnTI deleted HEK293 cells. Antigens were formulated with an AS03-like emulsion adjuvant, AddaS03, or with a combination adjuvant comprised of a plant-derived nanoparticle, Nano-11, and a stimulator of interferon genes (STING) agonist, ADU-S100. Serum antibody responses to nPZP and IZUMO1 were determined by ELISA. The IgG, IgG1 and IgG2b levels were significantly increased after the third dose with the highest titer seen in mice immunized with nPZP with AddaS03. Although least abundant, IgG2a levels were highest in Nano-11/ADU-S100 groups, indicating a more balanced Th1/Th2 response. Fertility was assessed by fetal count, and only the nPZP with AddaS03 group had a significant decrease in fertility. To conclude, the nPZP with AddaS03 formulation appears to be a promising alternative contraceptive vaccine, although trials with wildlife species are necessary for further formulation refinement.

Research Grant: The Humane Society of the United States

Student support: Purdue University College of Veterinary Medicine and Boehringer Ingelheim Animal Health

Field of Research: Immunology

Lisa Hoard¹, Shin Ae Park¹, Christine D. Harman², Kelly A. Leary², Vanessa A Raphtis², Kate Jongnarangsin², András M. Komáromy²

¹Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

²Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA

The gold standard for determining disease progression of glaucoma in research settings involves optic nerve axon counting, which is performed ex vivo. With the advancement of noninvasive imaging techniques, it is possible to image details of the retina and optic nerve head (ONH) in vivo. This study sought to determine the relationship between the number of axons at the ONH and various parameters using optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO) in dogs with various stages of primary open angle glaucoma. Beagles (n=6 eyes) with open angle glaucoma and age matched non-glaucoma dogs (n=2 eyes) were included in the study. OCT and cSLO images were taken of each eye, capturing the ONH, and a built-in software was used to measure neuroretinal rim area, ONH area and diameter, and optic cup diameter. Total retinal thickness, ganglion cell complex (GCC), and outer retinal thickness were also measured. Slides with the ONH samples were scanned and axons were manually counted using Image J software. A strong positive correlation existed when comparing the following parameters to the number of axons: neuroretinal rim area (r=0.91, p<0.01), ONH area (r=0.73, p<0.04), total retinal thickness (r=0.75, p=0.02), and GCC (r=0.82, p<0.01).There was a strong negative correlation (r=-0.75, p=0.03) between the number of axons and optic cup area and a moderate negative correlation (r=-0.70, p=0.12) between number of axons and age. The strong positive and negative correlations between the number of optic nerve axons and the various parameters measured support the utility of OCT and cSLO as useful noninvasive imaging techniques to assess the progression of glaucoma in vivo in dogs with open angle glaucoma.  

Research Grant: ACVO Vision for Animals Foundation Resident Research Fund, NIH R01-EY025752, and NIH K08EY030950

Student Support: Purdue University College of Veterinary Medicine and Boehringer Ingelheim Animal Health

Sarah Kelley¹, M. Carlson² , A. Hassan², D Pillai^2,3

¹ College of Veterinary Medicine, Purdue University, West Lafayette IN, 47907, USA

²Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, 47907, USA

³Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette IN, 47907, USA

Clostridioides difficile, a Gram-positive, spore-forming, anaerobic bacterium, is an opportunistic pathogen that causes severe colitis and death in humans and animals. Antibiotic treatment-induced disturbances in the gut microbiota frequently exacerbate infections caused by this bacterium. There is substantial evidence in the literature demonstrating that host stress can lead to changes in the gut microbiota. The gut is the epicenter of hormonal exuberance during stress. Stress modifies the gut physiology while modulating the gut microbiome. Previous studies have shown that norepinephrine, an abundant hormone in the gut experiencing stress, significantly affects the growth and virulence of many Gram positive and Gram negative bacteria. This research project aims to investigate the extent to which norepinephrine influences the virulence of C. difficile. The RNA seq and RT-PCR were performed to study the changes in expression and abundance on virulence genes of C. difficile. The effect of increasing concentrations of norepinephrine on the growth and virulence of C. difficile was evaluated. Our findings provide valuable insights that can inform treatment modalities and guide patient management decision-making processes. Results from this study could help us develop a treatment strategy that could include adrenergic blockers in treating C. difficile colitis.

Research Grant- PVM funds

Student support- Purdue University College of Veterinary Medicine and Boehringer Ingelheim Animal Health

Frank Leitgeb, Aidan Horvath, Mollie Madigan, Iris Bolton, Amanda Darbyshire

Purdue University Laboratory Animal Program, West Lafayette, IN

Early detection of pathogens is imperative for the health of laboratory zebrafish and to ensure reproducible scientific results. While most pathogens are present as subclinical or chronic infections, their presence can be a confounding factor in data collection, and some infections can affect zebrafish health and reproduction. Current methods to test for pathogens sample a myriad of sources, including cage swabs, detritus, water collection or filtration, and whole sentinel fish PCR or histopathology. Sentinel mice have been used in the past for mouse health monitoring, but there has been a recent shift to replace sentinel animals with filters in rack exhausts. We wished to see if such methods could be translated from mouse racks to zebrafish systems. We placed filters in the sumps of zebrafish racks to be collected and tested for pathogens at monthly intervals using PCR, and results will be compared to those detected on filters in which water was actively vacuum pumped through, swabs of sump biofilm, and whole fish PCR. We hypothesize that the filters present in the sumps will detect more pathogens than the other methods and may detect more pathogens over time. Should the evidence support the hypothesis, the use of filters could eventually replace the need for sentinel fish for health monitoring purposes of laboratory zebrafish.

Student Support: Purdue University College of Veterinary Medicine and Boehringer Ingelheim Animal Health

It is well-known that animals in laboratory facilities require environmental enrichment to allow them to display their natural behaviors. Examples of environmental enrichment include, but are not limited to, toys, nesting materials, gnawing materials, food and treats, and additional shelters. However, when given enrichment, it is unknown whether mice actually benefit from a specific enrichment, or if they have a preference as to the specific type of enrichment they receive. To see whether mice do indeed have a preference in their enrichment, several types of commercially available enrichment were placed in cages with singly housed C57BL/6 mice. After a few days of acclimation, acute behavioral trials and physiological analyses were conducted to see how the mice reacted to their enrichment. Later, chronic behavioral trials and physiological analyses were conducted to measure long-term effects of whether the provided environmental enrichment benefited the mice. Using the results from the behavioral trials and blood samples, we will observe the behavioral markers of evident stress along with analyzing the white blood cell counts for evidence of stress. This will allow visualization of any benefits from certain types of enrichment, allowing researchers to purchase that enrichment over others in the future.

Sharon Meoli, Annapoorani Jegatheesan, Vishnu Manikantan, Uma Aryal, Sriveny Dangoudoubiyam

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Meoli, Jegatheesan, Manikantan, Dangoudoubiyam); Purdue Proteomics Facility, Purdue University, West Lafayette, IN (Aryal)

Equine protozoal myeloencephalitis (EPM) is a rare, but economically devastating, degenerative neurological disease caused by Sarcocystis neurona, an intracellular protozoan. Despite high seroprevalence of S. neurona, very few horses develop EPM or present with broad neurological signs of weakness, ataxia, and neurogenic muscle atrophy. Akin to sister genera, S. neurona relies on discharge of excretory-secretory proteins (ESPs) from its apical organelles to invade the host cell and survive intracellularly. Investigation into S. neurona ESPs may reveal important virulence factors associated with EPM progression. Therefore, the aim of this study was to evaluate the antigenicity and generate a proteomic profile of S. neurona ESPs for future studies and development of additional diagnostic tests. Cell-culture derived live S. neurona underwent induced secretion and the ESPs were collected for analysis. Sera and cerebrospinal fluid from five horses of known EPM status were tested for antibodies to S. neurona ESPs by Western blot. Reactivity at two distinct molecular weight ranges was observed and amino acid sequencing is needed to establish the identity of these unknown proteins. Bottom-up proteomics of in-gel digested ESPs was performed via Mass Spectrometry and 92 S. neurona proteins were identified. 21 proteins were found to be from secretory organelles, 22 from other cellular locations, and the remaining are unstudied with unknown localization. Further optimization of sample preparation and data analysis is required for deeper characterization. Overall, this study has provided a glimpse into S. neurona ESPs and establishes a foundation for their use in future research aimed at developing new diagnostic tools for EPM.

Research Grant: Departmental Start-up Grant, Purdue University

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

Three-dimensional (3D) modeling using computed tomography (CT) scans is becoming increasingly popular in veterinary medicine. CT scans create a series of images that can be used to generate 3D models. Error during 3D modeling has been reported. In human medicine, femurs are positioned perpendicular to the CT scan, but anatomical differences in veterinary medicine do not allow for this positioning. Standard procedures for CT scans used in the generation of 3D models have not yet been developed in veterinary medicine. The goal of this research is to examine the effect of femur angle during CT scan on 3D modeling. Soft tissue was dissected from three pairs of femurs from beagles. All six femurs were placed on a custom jig in a CT scanner and one scan was obtained at each 0, 20, 40, 60, and 80 degrees relative to the table. From these scans, 3D models were generated using open-source 3D modeling software. This yielded five models of each femur, one at each listed angle. Surface area and volume of each segmentation were calculated. The five models of each femur were overlayed and an iterative process was used to minimize error. Hausdorff distances were calculated and heat maps generated comparing models from each angle to the model from the 0 degree angle scan. Repeated measures ANOVA will be run to analyze the effect of femur angle during CT on surface area, volume, and maximum and mean Hausdorff distances. We expect a decrease in surface area and volume and an increase in maximum and mean Hausdorff distances as femur angle increases. We expect errors in the model to be localized to the proximal and distal ends of the femur where bone geometry is more complex.

Research Support: Purdue University, College of Veterinary Medicine

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

Paola G. Diaz¹, Rebecca P. Wilkes^2,3, Keith Woeste⁴, Angela Chan², Hilary Richards², Farren Osborn²,Stephen Hooser^2,3 

¹St. Olaf College, Northfield, MN, ²Indiana Animal Disease Diagnostic Laboratory, W. Lafayette, IN, ³Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, W. Lafayette, IN, ⁴Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center, Purdue University, W. Lafayette, IN

Many plants can cause poisoning or death of animals following ingestion. Currently identification of the plant is performed by visually identifying plant material in stomach/rumen contents. In instances where poisonous plant parts cannot be identified, another method is needed to identify the causative plant. The goal of this study was to develop a polymerase chain reaction (PCR) based test to identify Taxus (yew) DNA in stomach/rumen contents. Taxus and non-Taxus samples were collected around the Purdue campus. DNA from each plant was extracted utilizing Qiagen’s DNeasy Plant Pro Kit. PCR primers were designed to amplify DNA from Taxus. Sensitivity was determined by extracting decreasing concentrations of Taxus, or by decreasing the amount of Taxus extracted from spiked stomach/rumen contents. Specificity was evaluated using non-Taxus plant species, non-spiked stomach/rumen contents, or nuclease free water. Cycle threshold (Ct) values were positive for samples containing Taxus DNA. However, samples from other trees were also positive. DNA from poison hemlock, an herbaceous plant, and nuclease-free water were negative. Conventional PCR and sequencing of amplicons from each plant correctly identified that plant, including those from Taxus in rumen/stomach contents. This study indicates that PCR with sequencing can be utilized as a specific and sensitive test to identify Taxus and potentially other poisonous plants in stomach/rumen contents.

Research Grant - Purdue University research account funded by the Total Wagers Tax.

Student Support - Purdue University College of Veterinary Medicine

Vector-borne pathogens (VBPs) transmit from one host to another via a vector intermediate, many of which are zoonotic. Common zoonotic VBPs include Borrelia burgdorferi and Rickettsia rickettsii. However, across different countries, the known circulating zoonotic VBPs are limited, especially within secluded regions of the world or remote populations of people. The purpose of this study was to identify whether certain VBPs found within domestic dogs were also found within their human companions. Fifty-eight human samples (nucleic acid extracted from whole-blood) obtained from remote South American populations were tested with targeted NGS using the Ion GeneStudio S5 System to detect vector-borne pathogens found in dogs. The resulting sequences from samples were mapped to a reference file using SPAdes in the Torrent Suite Software. Aligned BAM files were opened in Geneious software to evaluate the specific pathogens from the raw sequencing data. Sequences with ≥ 100 nucleotides were subjected to BLAST search in National Institute of Health's BLAST program to confirm the sequence similarity to the target pathogens. One of the 58 human samples tested positive for  Leishmania sp., which had previously been detected in these dog populations. Infected dogs are considered to be the primary reservoir for zoonotic visceral leishmaniasis and the most significant risk factor for predisposing humans to infection. This parasite tends to be sequestered in the spleen, bone marrow, or lymph nodes, which complicates use of whole blood for detection of the organism. Splenic or bone marrow aspirates are commonly used to diagnose visceral leishmaniasis. Thus, it is likely there are more positive individuals in these populations than we detected. This necessitates continued surveillance and potential use of additional sample types or the addition of serologic testing for detection of vector-borne pathogens in these populations.

Candida auris is an emerging multi-drug resistant fungal pathogen that can give rise to life-threatening, invasive infections in humans. To date, C. auris remains resistant to the majority of FDA approved antifungal medications-many of which have the potential to cause cytotoxic effects to the patient. As such, there is a tremendous need for a safe, novel therapeutic treatment option against this devastating fungus. Unlike the majority of other Candida species, C. auris predominantly colonizes the skin and causes systemic bloodstream infections. Therefore, developing a stronger understanding of the factors regulating C. auris colonization in the skin is crucial to gain insights into this species’ pathogenesis. In this research effort, human b-defensin-2 (hbD-2), a common antimicrobial peptide expressed in human skin, was tested against C. auris in physiologically accurate conditions to assess for antifungal activity. Our results indicate that hbD-2 exhibits potent antifungal activity in vitro. Future studies will focus more in depth on the antifungal activity of hbD-2 using mice models of C. auris infection.

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

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.

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.

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.

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.

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

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. 

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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.

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

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