Research Projects

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

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

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

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

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

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

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

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

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

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

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

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

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.