Past Summer Research Projects

Carisa Fraser -Purdue University

Mentored by: Dianne Little

Effects of social status and periodontal disease on the severity of post traumatic osteoarthritis in mice

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 


Anna Hassebroek -Purdue University

Mentored by: Audrey Ruple

A systematic review of the literature on health outcomes in wild and captive wolf populations worldwide

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

Research grant funded by Merial Veterinary Research Scholars Program 


Ashley Hopkins -Purdue University

Mentored by: Lynn Guptill

Innovative approaches to the treatment of Staphylococcus pseudintermedius infections

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

Research Grant: Purdue College of Veterinary Medicine

Student Support: Morris Animal Foundation


Brooke Kline -Purdue University

Mentored by: GuangJun Zhang

Investigation of the hippo signaling pathway in zebrafish nf2 gene mutants

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

Research Support: Hayward Foundation

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


Sofía López-Valle -Purdue University

Mentored by: Kenitra Hammac

Clostridium difficile toxin detection by MALDI-TOF Mass Spectrometry

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

Research Support: Indiana Animal Disease Diagnostic Laboratory

Student support: Boehringer Ingelheim and Purdue Veterinary Medicine


Jenny Nguyen -Purdue University

Mentored by: Keke Fairfax

Effects of maternal S. mansoni infection on immune response of offspring to tetanus/diphtheria immunization

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

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


Shery Park -Purdue University

Mentored by: Gert Breur

Changes in Digit Abduction Scoring of mice over time: implications for assessment of neuromuscular activity

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

Research Grant: Akina Inc

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

 


Betsy Pray -Purdue University

Mentored by: Abigail Durkes

Developing a novel porcine model of laryngopharyngeal reflux disease

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

Research Grant: None 
Student Support: Merial Veterinary Scholars Program


Mallory Stuckwisch -Purdue University

Mentored by: Lynetta Freeman

A geospatial approach to “The Link” and its local implications

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

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


Chad Van Koot -Purdue University

Mentored by: Russell Main

Assessment of the skeleton’s mechanosensitive anabolic pathways using endogenous fluorescence in mouse models

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

Merial Veterinary Research Scholars Program


Jesse Whitfield -Purdue University

Mentored by: Marguerite O'Haire

Psychosocial effects of service dogs on individuals with physical disabilities within two age groups

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

Research grant: Elanco Partnership: Canine Assistants

Student support: Boehringer Ingelheim Veterinary Scholars Program


Sean Anderson -Purdue University

Mentored by: Deborah Knapp

Mixed lymphocyte reaction to be used in the assessment of immunotherapy in dogs with transitional cell carcinoma

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

 


Cyra Bammer -Lawrence University

Mentored by: Steve Hooser

Detection of PRRS Virus antibody from serum prepared and stored using Noviplex Cards

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


Tim Brunner -Purdue University

Mentored by: Sandy Taylor

The use of thermography, serum amyloid A, prostaglandin-E2, and TNF- to assess inflammation in a reversible equine foot lameness model

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

Research Grant: 2017 Competitive Equine Research Funds 

Student Support Fund: 2017 Summer Research Fellows Program


Grant Ford-Hodges -Howard University

Mentored by: Laurent Couetil

Effect of nebulized tocotrienols on airway inflammation in asthmatic horses

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

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

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


Makayla Wiley -Alcorn State University

Mentored by: Tiffany Lyle

Immunofluorescence analysis of the blood-brain barrier in immunocompromised mice

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

Research Funding: Indiana Clinical and Translational Sciences Institute

Student Support: Purdue University Veterinary Scholars Program


Chris Williams -Wheaton College

Mentored by: John Christian

Evaluation of equine total plasma protein: fibrinogen ratio, for a new fibrinogen assay, to detect inflammation

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


Christopher Bloom -Purdue University

Mentored by: GuangJun Zhang

Characterizing apoptosis in zebrafish mdm1 mutant

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


Sydney Byerley -Purdue University

Mentored by: Russell Main

Lacunar-canalicular morphology in tetrapod long bones

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

Research Support: National Science Foundation

Student Support: Merial Veterinary Scholars Program


Joseph Devereaux -Purdue University

Mentored by: Gert Breur

At what age does canine gait mature?

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

Research Grant: None

Student Support: Purdue University Summer Research Fellowship


Janna Draper -Purdue University

Mentored by: Sam Yingst

Microneutralization assay: an economical method for determining vaccine recommendations for swine influenza

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

Research Grant: None

Student Support: Merial Veterinary Scholars Program 


Abigail Haffner -Purdue University

Mentored by: Marxa Figueiredo

Investigating novel small compounds and their effect on inflammation and chondrogenesis in joints

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


Melissa Jones -Purdue University

Mentored by: Audrey Ruple

The Golden Retriever Lifetime Study: factors affecting owner compliance after the first year of enrollment

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

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

 


Mary Jordan -Purdue University

Mentored by: Candace Croney

Do you see what I see? Inter-rater reliability of a canine welfare assessment used in dog breeding kennels

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

Research Grant: World Pet Association, Pet Food Institute

Student Support: Merial Veterinary Scholars Program, Purdue University CVM


Dian Dian Lin -Purdue University

Mentored by: Kenitra Hammac

Two methods of Clostridium difficile toxin purification for detection by MALDI-TOF MS

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

Student Support: Merial Veterinary Scholars Program


Allison Mustonen -Purdue University

Mentored by: Abby Durkes

How to smoke your bacon: An investigation on the effects of environmental inhalants in a porcine model

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

Student support: Merial Veterinary Scholars Program

Research support: NIH R01DC011759


Brittany Rasche -Purdue University

Mentored by: Sophie A. Lelièvre

Environmental awareness: Recreating the natural tissue niche in vitro for cancer research

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

Research Grant

Trask Innovation Fund to SAL 

Student Support

Merial Veterinary Scholars Program 

Field of Research

Cell Biology and Cancer Cell Biology


Sam Smith -Purdue University

Mentored by: Christina Wilson

Do Noviplex™ cards have a future in veterinary clinics?

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

Student Support:  Merial Veterinary Scholars Program


Ellen Weigel -Purdue University

Mentored by: Ourania Andrisani

Diallyl trisulfide inhibits epithelial cell adhesion molecule signaling in hepatocellular cancer

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

Support of NIH Grant 5R01DK044533/19 to OA

Student Support: Merial Veterinary Scholars Program


Jennifer Barrett -Earlham College

Mentored by: Marguerite O'Haire

Caregiver Burden and Service Dogs: Effects on the Mental Health and Wellness of Spouses of Veterans with PTSD

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

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

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

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


Sarah Gutman -Purdue University

Mentored by: Roman Pogranichniy

Serological Investigation of Exposure to Influenza A Virus in Dogs and Cats in 2015

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


Benjamin Henrichs -Southern Illinois University

Mentored by: Keke Fairfax

The role of Interleukin-4 in peripheral lymph node organization

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


Lauren Kerestes -Purdue University

Mentored by: Laurent Couetil

Validation of multiplex PCR for the detection of equine respiratory viruses

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


Keturah Ollie-Hayes -Tougaloo College

Mentored by: Niwako Ogata

Behavioral and Physiological Responses of Dogs during Separation Test from the Owner: A Pilot Study

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


Ana Vazquez-Pagan -Northeastern University

Mentored by: Debbie Knapp

Herbicide Induced Carcinogenesis in Normal Canine Urothelial Cells

Each year in the United States, it is expected that bladder cancer will affect more than 20,000 dogs. Of those, a majority (<90%), have invasive transitional cell carcinoma (TCC), also referred to as invasive urothelial carcinoma. Many factors are thought to cause the development and progression of TCC. One of the possible causes is the exposure of dogs to herbicide-treated lawns. Lawn herbicides contain both active and inert ingredients, and the combination of these chemicals could be carcinogenic for dogs who come in contact with them. The purpose behind this study is to evaluate how varying concentrations of active chemicals found in lawn herbicides affect normal canine urothelial cells, specifically their morphology and proliferation. The two active chemicals being used are 2,4-Dichlorophenoxyacetic acid (2,4-D) and dicamba. These two chemicals are very common and can be found in most lawn herbicide products. A previous study performed by the Knapp lab suggested that varying concentrations of these two chemicals were found in urine samples coming from dogs with TCC. These findings led to questions about the role of these chemicals on bladder cancer. This study used canine urothelial cell cultures derived from normal dog bladders. Cells were seeded onto laminin coated inserts suspended in 12-well plates. The cell lines were then treated with 1μM, 2μM, 10μM and 20μM of 2,4-D and dicamba. Two negative controls were also included. Immunofluorescence was utilized to visualize the expression of Ki-67, a cellular marker for proliferation.


Elexa Baron -Purdue University

Mentored by: Christina Wilson

Rapid detection of poison hemlock and jimson weed alkaloids in urine and rumen samples by MALDI-TOF MS

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

Student Support:  Merial Veterinary Scholars Program

 


Jordan Beauchamp -Purdue University

Mentored by: Timothy Lescun

The effect of transfixation pin placement factors on compressive strength of the equine third metacarpal bone

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


Ashleigh Cournoyer -Purdue University

Mentored by: Deborah Knapp

Expression of high-affinity folate receptors on canine neutrophils

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

Acknowledgements

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


Victoria Gerber -Purdue University

Mentored by: GuangJun Zhang

Identifying kcnj13 as a gene in which mutation results in long-fin development and spontaneous tumors in zebrafish

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

Research Grant: Hayward Foundation

Student Support: Merial 


Valerie Goeman -Purdue University

Mentored by: Audrey Ruple

Evaluation environmental sampling techniques for detection of S. enterica in a large animal veterinary hospital

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

Research Funding: Purdue University College of Veterinary Medicine

Student Support: Merial-NIH Veterinary Scholars Program

 


Jessica Hanlon -Purdue University

Mentored by: Kenitra Hammac

Detection of mecA positive Staphylococcus pseudintermedius by MALDI-TOF MS

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

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

Student Support – Merial Veterinary Scholars Program


Wing Yeung Haq -Purdue University

Mentored by: Mohamed Seleem

Enhancing antimicrobial and antibiofilm activities of conventional antibiotics against bacterial conjunctivitis in dogs

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

Research Grant: None

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


Natalia Hernandez Diaz -Purdue University

Mentored by: Sandy Taylor

Comparison of ketorolac tromethamine and flunixin meglumine in reducing LPS-induced production of cytokines and eicosanoids by equine monocytes.

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

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

Student Support: Summer Research Fellows Program, 2015.

 


Tamanna Ranadive -Purdue University

Mentored by: Yava Jones-Hall

The role of tumor necrosis factor in TNBS colitis: A look at IgA and cytokine patterns

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

Research Grant: None

Student Support: Purdue University Veterinary Scholars Summer Research Program


Colleen Stevenson -Purdue University

Mentored by: Russell Main

Characterization of hydroxyapatite crystals and osteoblast lineage cells for 3D cell culture

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

NIH Grant (AR065659)

Morris Animal Foundation


Melissa Swan -Purdue University

Mentored by: Brianna Gaskill

The impact of nesting material volume on nest building and maternal/paternal investment in laboratory mice.

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

Student Support: Merial Veterinary Research Award


Alexis Zobel -Purdue University

Mentored by: Sherry Voytik-Harbin

Matrix-induced cellular alignment for engineered skeletal muscle tissue constructs

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

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

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

Field of Research: Tissue Engineering


Alessandria Aikerson-Russell -Alcorn State University

Mentored by: Gert Breur

Comparison of observational and instrumental gait analysis in dogs with thoracolumbar myelopathy

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


Alexandra Bianco -Purdue University

Mentored by: Keke Fairfax

What is the role of Interleukin-4 in lymph node organization?

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


Carly Gundlach -Wittenberg University

Mentored by: Joanne Messick

Preliminary study: isolation of microRNA from mature feline red blood cells

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

 


Luiza Placheta -Greenville College

Mentored by: Laurent Couetil

A study of the microbiome of the respiratory tract in racehorses

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


Rebecca Smith -Purdue University

Mentored by: Ourania Andrisani

Does activation of polo-like kinase regulate levels of the hepatitis B virus core antigen?

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


Alyssa Booker -Purdue University

Mentored by: Scott Crist

Role of Acute Phase Cytokines on the Expression of CD40L in Megakaryocytes

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

Student Support: Merial Veterinary Scholars Program

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


Hee-Yun Cha -Purdue University

Mentored by: Roman Pogranichniy

Investigation of Porcine Deltacoronavirus in Indiana swine

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


Michelle Hanenburg -Purdue University

Mentored by: Sherry Harbin

Vasculogenesis as an influencing factor of matrix-guided nerve regeneration

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


Kyle Hohu -Purdue University

Mentored by: Russell Main

Determining Diagnostic Parameters for Fractures in Equine Metacarpal Bones

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

Student Funding: Merial

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


Whitney Johnson -Purdue University

Mentored by: Tim Lescun

Comparison of Orthopedic Taps for Transfixation Pin Placement in Equine Third Metacarpal Bones

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

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

Sample – 10 third metacarpal bones from 5 horse cadavers.

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

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

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

Student Support: Merial

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


Joseph Kowal -Purdue University

Mentored by: Amy Fauber

Use of a Pressure Sensing Walkway in the Gait Analysis of Dogs with Orthopedic Unilateral Hind Limb Lameness

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

Student Support: Merial & Purdue College of Veterinary Medicine

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


Kelly Ray -Purdue University

Mentored by: Kenitra Hammac

Identification of toxigenic Clostridium difficile isolates from equine, canine, and porcine fecal samples

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

Student support: Merial Veterinary Scholars Program

Research support: Indiana Animal Disease Diagnostic Laboratory 

 


Anna Smith -Purdue University

Mentored by: GuangJun Zhang

Targeted Mutagenesis of ltzr1 gene in Zebrafish using the CRISPR-Cas9 system

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

Student support:  Merial

Lab Support: This project was supported by the Hayward Foundation


Catherine Smith -Purdue University

Mentored by: Candace Croney

Factors influencing the incidence and severity of cat and dog bites to animal shelter workers

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

Student Support: Purdue University College of Veterinary Medicine

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


Olivia Swailes -Purdue University

Mentored by: Christina Wilson

Use of MALDI-TOF mass spectrometry to detect toxins in veterinary diagnostic toxicology

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

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


Victoria Thomas -Purdue University

Mentored by: Suresh Mittal

Evaluation of Broad Spectrum Nature of Multi-epitope-based Avian Influenza Virus Vaccines

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

Student Support by Merial

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


Jayda Bussey-Spratling -North Carolina Agricultural and Technical State University

Mentored by: Yava Jones-Hall

Evaluation of Mucin Production in Tnf -/- and WT Mice

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


Sarah Garst -Purdue University

Mentored by: Sandy Taylor

Isolation of equine peripheral blood mesenchymal stem cells

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


Leslie Lundewall -Indiana University

Mentored by: Joanne Messick

A multiplex microbead immunoassay to detect antibodies to Mycoplasma haemofelis

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


Delaney Patterson -University of Connecticut

Mentored by: Laurent Couetil

A pilot study of the microbiome of the equine respiratory tract in health

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

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

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

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

Student funding: Purdue College of Veterinary Medicine

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


Connor Sholtis -Amherst College

Mentored by: Niwako Ogata

Using Stress Induced Hyperthermia To Assess Anxiety in Dogs

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

 


Erik Wegner-Clemens -Indiana University

Mentored by: Dan Hogan

In vitro effect of viscosity on thromboelastographic parameters in healthy dogs

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

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

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

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


Shannon Arnold -Purdue University

Mentored by: GuangJun Zhang

Generating CRISPR constructs for inducing mutations in zebrafish zinc and ring finger (znrf) genes

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

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

Student support: Merial Veterinary Scholars Program


Jaime Ashmore -Purdue University

Mentored by: Wendy Townsend

Uveal cysts as a risk factor for development of Golden Retriever pigmentary uveitis

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

Student support: Merial Veterinary Scholars Program


Corina Collins -Purdue University

Mentored by: Scott Crist

Regulation of platelet inflammatory activity in autoimmune disease

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

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

Center for Cancer Research

Student Support: Merial Veterinary Scholars Program 


Kritstyn Howe -Purdue University

Mentored by: Yava Jones-Hall

Establishment and characterization of the AG129 mouse model of Dengue Fever

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

 


Erin Katz -Purdue University

Mentored by: Debbie Knapp

The effects of inflammatory mediators IL-6, TNF-a, and H2O2 on the proliferation of canine transitional cell carcinoma

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

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


Maura Lehmann -Purdue University

Mentored by: Chang Kim

Option 1: Quantification and localization of FOXP3-positive regulatory T cells in a murine colitis model using multi-photon microscopy

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


Mariann Lempert -Purdue University

Mentored by: Suresh Mittal

Interaction between adenoviral proteins 33K and IVa2 in viral genome packaging

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

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

Research support provided by USDA Hatch

Student support provided by Merial


Holly O'Hara -Purdue University

Mentored by: Niwako Ogata

Sociability of shelter cats approached by an unfamiliar male versus an unfamiliar female

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

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

Student Support: Merial Veterinary Scholars Program

 


Brittany Rayburn -Purdue University

Mentored by: Gert Breur

Is Psoas Minor muscle injury in dogs associated with Iliopsoas muscle injury?

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

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

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


Anita Richert -Purdue University

Mentored by: Craig Thompson

Evaluation of in-house and point-of-care analyzers for analysis of equine, feline, and canine abdominal fluid creatinine and potassium

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

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


Kelsey Heron -Purdue University

Mentored by: Laurent Couetil

Quantification of airborne dust originating from hay

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

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


Colleen Stevenson -Rose-Hulman Institute of Technology

Mentored by: Riyi Shi

Validation of shock tube overpressures for rodent models of traumatic brain injuries

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


Amy Trey -Duke University

Mentored by: Harm HogenEsch

Investigation of the role of inflammation in the immune response to an aluminum adjuvanted vaccine by blocking ICAM-1 (CD54)

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


Kristin Zabrecky -Purdue University

Mentored by: Sandy Taylor

Equine CRP and haptoglobin ELISA analysis in critically ill neonatal foals

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


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