PVM Research Day Abstracts

PVM Research Day highlights the research conducted by our faculty, residents, postdoctoral, and student community to enhance the well-being of animals and people.

Marwa Alhashimi - Basic

Development of Next Generation of Adenoviral vectored Vaccine to Combat SARS-CoV-2 Variants

Marwa Alhashimi1, Ekramy E Sayedahmed1, Ahmed Elkashif1, Shubhada chothe2, Suresh V. Kuchipudi2, Andrea P. Santos1, Suresh K. Mittal1

1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN; 2Department of Veterinary and Biomedical Sciences, Huck Institutes of the Life Sciences, Penn State University, University Park, PA

The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prioritized the rapid development of safe, efficacious, and deployable vaccines in record time. Despite valiant efforts made through the rollout of billions of doses of vaccines, the emergence of highly divergent variants have compromised the efficacy of most first-generation vaccines. Adenoviral (Ad) vector platforms have been at the forefront of vaccine research to prevent a range of highly pathogenic viral infections, including SARS-CoV-2. This is primarily due to the unique characteristics of these Ad vectors and their ability to elicit robust and balanced humoral and cell-mediated immune responses, which makes them an excellent candidate for the development of the next generation of COVID-19 vaccines to combat the variant strains. We developed Ad vector-based vaccines (HAd-Spike-C5 or HAd-Spike) expressing the full-length of SARS-CoV-2 spike (S) protein with or without a 22 amino acid residues Autophagy-Inducing Peptide (AIP) C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis. Mice or Syrian hamsters vaccinated intranasally (i.n.) with HAd-Spike-C5 or HAd-Spike developed similar levels of systemic or mucosal humoral immune responses. However, HAd-Spike-C5 immunized animals elicited significantly higher levels of cell-mediated immune responses compared to the HAd-Spike vaccinated groups. Both vaccines induce comparable levels of neutralizing antibody titers against SARS-CoV-2 variants. We anticipate that immunization and challenge study will determine the superiority of the HAd-Spike-C5 vaccine.

Brock Beauclair - Basic

bTBI-On-A-Chip: A Novel, In Vitro Model for Morphological and Electrophysiological Investigations of Blast Injuries

AUTHORS: T. B. Beauclair, E. A. Rogers, Z. Zhang, S. Mufti, J. Martinez, J. Crodian, D. Kim, L. Pracht, R. Shi

Annually, over 20,000 soldiers experience traumatic brain injuries (TBI), resulting in significant loss of income and quality of life. Further, up to 95% of these highly-heterogeneous injuries are caused by explosive blasts, or bTBI. Unfortunately, there are no current treatments available and the underlying mechanisms remain elusive. However, oxidative stress and inflammation are thought to play critical roles. By merging our novel in vitro concussion model with our established in vivo bTBI device, we have begun developing the “bTBI-on-a-chip” platform, a cellular blast-injury model with morphological and electrophysiological recording capabilities. This exciting new system utilizes neuronal networks generated from dissociated E-16 murine cortices and seeded onto microelectrode arrays (MEAs). After 7-days, networks are aseptically transferred to our previously established miniature-incubator system, and exposed to a high-pressure blast-wave delivered via vertical stainless-steel shock tube. Pilot studies verify life-support stability throughout the procedure while revealing the maintenance of cellular adhesion during blasts up to 160kPa, with ~95% viability. Further, we combine this system with standard immunocytochemical methods to demonstrate blast-induced increases of acrolein-lysine adducts (a clinically-established marker of oxidative stress) and the pro-inflammatory cytokine TNFα at 24h post-injury, when compared to procedurally and age matched control networks. In addition, we show that these increases can be significantly mitigated with Hydralazine, an FDA approved antihypertensive drug. These preliminary results illustrate the effectiveness of our bTBI-on-a-chip system, opening the door for future mechanistic studies while offering a semi-throughput device to investigate potential pharmaceutical interventions.

Nayan Bhowmik - Basic

Identifying Selective Sweeps Associated with Brachycephaly and Screw tail in Brachycephalic Dogs: A Proof-of-Concept Study with Runs of Homozygosity Islands

N. Bhowmik, K. J. Ekenstedt

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

The short muzzle with a flattened facial profile characterizes brachycephalic dogs. In addition, some brachycephalic dog breeds also present shortened and kinked tails referred to as screw tails. Both brachycephaly and screw tail traits were purposely selected by breeders, deliberately preserving these genetic deformities for their appealing looks. Such continuous selection of dogs for morphological features can leave unique genetic patterns on their genome, called “selective sweeps”, which often demonstrate low genetic diversity and frequent runs of homozygosity (ROH). Therefore, this study aimed to detect genomic regions related to positive selection of morphologic traits such as brachycephaly and screw tail by using ROH islands as proof-of-concept. A total of 501 dogs from 4 brachycephalic breeds (46 Boston Terriers, 102 English Bulldogs, 101 French Bulldogs, and 252 Boxers) were genotyped and, after quality control, had 504,699 autosomal imputed SNP markers available for ROH analysis, which was performed using PLINK. Genomic regions under putative selection were identified as ROH islands. We observed genomic regions that were common across the breeds sharing the brachycephalic and screw tail phenotypes. These regions included many candidate genes, with a subset having been previously identified, via association analysis, with the brachycephalic condition (BMP3 and SMOC2) and screw tail (DVL2). Therefore, we have demonstrated that the ROH islands method should be ideal for detecting genomic regions subjected to high selective pressures on specific traits in dogs; this method will now be applied in ongoing and new investigations.

Keywords Brachycephaly, Screw Tail, Runs of Homozygosity, Dogs

Jeanna Blake - Basic

Heritability of Vaccine-induced Immune Response in Beagles

Jeanna M. Blake1, James Thompson2, Harm HogenEsch3, Kari J. Ekenstedt1

1Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA

2Zoetis, Veterinary Medicine Research and Development, Kalamazoo, MI, USA
3Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA

Both genetic and non-genetic factors give rise to individual variation in the immune response to vaccination. Identifying how genetic background influences variation in both magnitude and persistence of vaccine-induced immunity is vital for improving vaccine development and understanding possible sources of vaccine failure. The objective of this study was to estimate the heritability of the antibody response to vaccination against viral and bacterial pathogens in Beagles. Sixty Beagle puppies were immunized on a standard vaccination schedule with an attenuated combination vaccine containing antigens for canine adenovirus type 2, canine distemper virus, canine parainfluenza virus, canine parvovirus, and four strains of Leptospira bacteria. Vaccines were administered on days 0, 21, and 42, and serum antibody measurements for each viral and bacterial component were taken on days 21, 42, 56, and 63. Serum antibody levels for BSA and fibronectin, common vaccine formulation by-products, were measured (days 25, 42, 56, and 63). Serum antibody titers from days 21, 25, and 63 were utilized in heritability estimations. Each dog was genotyped on the Axiom CanineHD SNP array (n = ~710,000 markers). After quality control, 57 dogs and 274,898 SNPs contributed to estimating heritability of the immune response. The heritability estimates were: 1) to Leptospira antigens, ranging from 0.179 to 0.627; 2) to viral antigens, ranging from 0.200 to 0.589; and 3) to vaccine formulation by-products, ranging from 0.250 to 0.343. This study suggests that genetic regulation of the immune response to vaccination may be antigen specific and influenced by many genes of small effect.

Yueyi Chen - Basic

Sex-Dependent Effects of Alcohol and Oxycodone Polysubstance Use

Yueyi Chen1, Salvador Huitron Resendiz2, Amanda Roberts3, Adam Kimbrough1

1. Purdue University, Department of Basic Medical Sciences, West Lafayette, IN, 47907, USA
2. The Scripps Research Institute, Department of Neuropharmacology, La Jolla, CA, 92122, USA
3. The Scripps Research Institute, Animal Models Core, La Jolla, CA, 92122, USA

People tend to use multiple drugs in the real world; however, preclinical models of concurrent
polysubstance use disorder (PUD) have not been adequately explored. We designed two novel preclinical
models to explore concurrent alcohol and oxycodone PUD. We hypothesized that withdrawal from one
drug would increase the intake of the other.

In the first study, male and female mice received several weeks of chronic intermittent ethanol
vapor to become alcohol dependent (AO), while a control group remained alcohol naïve (O). Mice from
both groups self-administered oxycodone intravenously for 2 weeks (during alcohol withdrawal). We
found a significant increase in self-administration of oxycodone during the last three sessions in the male
AO mice compared to O mice, suggesting an effect of alcohol withdrawal on oxycodone intake. However,
female AO mice did not show the same increase.

In the second study, male and female mice were made oxycodone dependent (OA) via i.p.
injection every other week. A control group (A) was injected with saline. In between injection weeks
(during oxycodone withdrawal), mice were given 2-hour 2 bottle choice drinking sessions with alcohol
and water. Female OA mice showed a significant increase in alcohol intake compared to A mice during
oxycodone withdrawal. Male mice did not show the same effect. These data suggest a sex-dependent
effect of oxycodone withdrawal on alcohol intake.

Together, these data established concurrent alcohol and oxycodone use models in mice. We
found opposing sex-dependent effects of the drug being used and associated with withdrawal.

Zhixia Chi - Basic

PRMT5 Promotes PTEN-Deficient Prostate Tumor Proliferation via AR Pathway

Zhixia Chi, Qi Shen, Hye Seung Nam, Andrew Asberry, Jogendra Pawar, Xuehong
Deng, Guangjun Zhang, Chang-Deng Hu

Purdue University Department of Medicinal Chemistry and Molecular Pharmacology.
Purdue University Department of Comparative Pathobiology, West Lafayette, IN.
These authors contributed equally to this work.

Background: According to the latest cancer statistics, prostate cancer is the second most
malignant cancer in men in the U.S. and worldwide. Androgen receptor (AR) is expressed
in nearly all primary prostate cancer (PCa). Inhibiting AR expression via androgendeprivation
treatment (ADT) or anti-androgen agents, is the mainstream treatment for
clinical localized and metastatic PCa. However, currently, those methods inevitably lead
to castration-resistant prostate cancer (CRPC). Protein arginine methyltransferase 5
(PRMT5) is a type II methyltransferase, and it plays a critical and versatile role in cell
growth. PRMT5 is overexpressed in intermediate and high-risk prostate cancer tissues,
and its over-expression positively correlates with AR expression. However, it remains
vague whether PRMT5 regulates AR expression in mouse PCa models or mouse Pca
cells. Methods: Prmt5/Pten double knockout mice model and Prmt5 L/L/Pten L/L with
castration mice model were generated. Besides, PRMT5 inhibitor BLL3.3 was utilized to
investigate PRMT5 function in AR pathway in PtenL/L cells line PTEN CaP8. Results and
conclusion: Prmt5 L/L/Pten L/L knockout mice inhibited PIN and adenocarcinoma
formation, with less cell proliferation, decreased AR and PSA expression, and larger and
heavier GU organ, comparing with Prmt5wt/Pten L/L mice. In addition, inhibition of PRMT5
with BLL3.3 significantly suppressed PTEN CaP8 cell growth.

Jessica Clark - Basic

Global Frequency Analysis of Canine prcd-PRA Using Commercial Genetic Testing Data

Jessica Clark, Heidi Anderson, Jonas Donner, Susan Pearce-Kelling, Kari J Ekenstedt

Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Clark, Ekenstedt)

Wisdom Panel Research Team, Wisdom Panel, Kinship, Portland, OR (Anderson, Donner, Pearce-Kelling)
In dogs, hundreds of genetic variants associated with traits and disorders have been identified, with DNA tests being offered commercially. However, most studies have lacked the resolution to track the geographic allelic distributions and changes in allele and genotype prevalence over a prolonged, continuous period of time. This study utilized a large dataset (n = 82,276 dogs) from a commercial genetic testing company; results span fourteen years, represent 75 countries from 6 continents, and include 61 breeds and mixed-breed dogs. Each dog was tested for the c.5G>A missense variant in the PRCD gene identified in more than 50 breeds and known to result in progressive rod-cone degeneration, a late-onset progressive retinal atrophy (prcd-PRA). Prcd-PRA is inherited in an autosomal recessive, Mendelian fashion; dogs with two copies of the variant will typically progress to complete blindness. The expectation that genetic test availability would decrease the frequency of affected and carrier animals, and result in an overall decrease of the disease-causing allele frequency over time, was not universally observed in the data. Indeed, some breeds and/or countries showed the opposite and more detrimental trend of unchanging or increasing frequencies. While our data does not capture every dog tested globally and is biased towards the population of dogs having undergone genetic testing, it is clear that some breeding programs are not successfully improving the genetic health of their breed by using such tests. It may take increased pressure from registering bodies to ultimately steer breeders toward the health of these breeds.

Shawna Cook - Basic

SH3TC2, MTMR2, and MPZ Variants in Golden Retrievers with Congenital Hypomyelinating Polyneuropathy

Shawna Cook1, Blair N Hooser1, G. Diane Shelton2, Katie Minor3, Jim Mickelson3, Jennifer Koziol4, Steven G. Friedenberg5, and Kari J Ekenstedt1

1 Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN; cook311@purdue.edu, ORCID 0000-0001-7967-9607; bhooser@purdue.edu; kje0003@purdue.edu, ORCID 0000-0002-3213-7883
2 Department of Pathology, School of Medicine, University of California San Diego, La Jolla, CA; gshelton@ucsd.edu
3 Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN
4 School of Veterinary Medicine, Texas Tech University, Amarillo, TX; jkoziol@ttu.edu
5 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN; fried255@umn.edu

Congenital hypomyelination restricted to the peripheral nervous system was first reported in domestic animals as two Golden Retriever (GR) littermates with unknown cause for their hypomyelinating polyneuropathy (HPN). Recently, four new GR cases of congenital HPN were diagnosed via neurological examination and biopsies. These four dogs were whole-genome sequenced, and each dog’s variants were compared to variants found across >1,000 other dogs, representing 158 breeds, all presumably unaffected with HPN. Presumptive causative variants were identified for each HPN-affected GR. One case had a homozygous SH3TC2 nonsense variant resulting in a premature stop codon. Two cases each had a homozygous splice donor site variant in MTMR2, with a stop codon introduced within 6 codons following the inclusion of the intron. The last dog had a heterozygous MPZ missense mutation leading to an isoleucine to threonine substitution. Haplotype analysis using 524 GR established the novelty of the identified variants, with all three appearing to have emerged recently. Each variant occurs within genes that are associated with human Charcot-Marie Tooth (CMT), a heterogeneous group of diseases affecting the peripheral nervous system. Specifically, variants within SH3TC2 are associated with CMT type 4C, and the MTMR2 and MPZ canine variants identified in this study have analogous causative variants of CMT types 4B1 and 1B, respectively. Testing a large GR population (n = >200) did not identify any dogs with these variants, confirming their nascent emergence. Although these variants are rare within the general GR population, breeders should be cautious to avoid propagating these alleles.

Ahmed Elkashif - Basic

Generation of Bovine Adenoviral Vectors using Cre/lox Recombination System

Ahmed Elkashif, Ekramy E Sayedahmed, Marwa Alhashimi, Wen-Chien Wang, Suresh K. Mittal1

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

Adenoviral (Ad) vectors offer a versatile tool for gene delivery applications, including recombinant vaccines and gene therapy. The high prevalence of preexisting Ad vector immunity in humans has prompted the development of less prevalent human or nonhuman Ad vectors. We have demonstrated that the bovine Ad (BAd) vector-based H5N1 influenza vaccine did not impact the induction of humoral or cell-mediated immune responses even in the presence of exceptionally high levels of human Ad (HAd) vector immunity. Besides, we have found that immunogenicity and efficacy of protection conferred by the BAd vector-based H5N1 influenza vaccine were significantly better than the HAd vector-based H5N1 vaccine, signifying the importance of the BAd vector platform for vaccine delivery.

We currently rely on homologous recombination in Escherichia coli BJ5183 (recBC and sbcBC) to generate the full-length infectious genomic plasmid. This process requires repeated efforts to create infectious BAd vectors. To improve the process of homologous recombination, we have developed the Cre/lox-mediated site-specific recombination system for BAd vectors. It involved the construction of a shuttle plasmid for insertion of the required gene cassette and the genomic plasmid. Each of these plasmids contains a loxP site. We have also generated a bovine cell line that expresses the early E1 genes and Cre recombinase. The transfection of the Cre-expressing cell line with the shuttle and genomic plasmid will result in the generation of infectious vectors, simplifying the production of BAd vectors.

Nelly Elshafie - Basic

Canine Diffuse Large B-Cell Lymphoma Endogenous miRNA Controls for RT-qPCR Data Analysis

Nelly O. Elshafie1, Ekramy. E. Sayedahmed1, Michael O. Childress2, and Andrea P. dos Santos1

1 Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA 

2 Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, Indiana, USA 

MicroRNAs (miRNAs) are short non-coding RNAs of approximately 22-24 nucleotides that regulate gene expression. The use of miRNAs as biomarkers is challenged by pre and post-analytic variations, which affect the results' biological interpretation. Reverse -transcription-quantitative PCR (RT-qPCR) is a quantification method used to quantify the expression levels of miRNAs. Accurate data normalization is crucial for significant, reproducible results. According to recent studies, an endogenous miRNA control is recommended to be stable and moderately expressed in the tested samples for a reliable normalization method for quantifying miRNA relative expression. This study aimed to find endogenous miRNAs controls for canine diffuse large B-cell lymphoma that corrects for discrepancies in RNA extraction or reverse transcription to qPCR data normalization. A set of miRNA candidates based on the lowest coefficient of variation were selected. The selected miRNA set was analyzed by RefFinder, a comprehensive web-based tool, to screen and determine reference genes in a specific dataset. This user-friendly tool incorporates the most extensively used computational algorithms (geNorm, NormFinder, the comparative Delta-Cq method, and BestKeeper) to assess and order the pre-defined reference miRNAs according to their stable expression. Each algorithmic program ranks and ascribes weight to the tested miRNAs then the geometric mean of those weights is calculated for a complete expression evaluation. We have found that miR-361-5p, miR-101, and miR-29c-3p with geomean ranking values (1.32, 1.86, and 2.06) are the most stably expressed miRNAs. Finally, we emphasize the need for adopting a reliable normalization method suited to each experiment to avoid misleading data analysis.

Ahmed Hassan - Basic

Effect of Cdc14 Phosphatase in the In Vivo Host Response to Candida albicans Infections

Ahmed Hassan*1, Sanjeev Narayanan1 and Mark Hall2

Department of Comparative Pathobiology1 and Biochemistry2

Purdue University, West Lafayette, IN 47907, USA

Systemic Candida infections, especially those caused by multidrug resistant strains, are major challenges that require novel therapeutic interventions. Cdc14 is a highly conserved fungal phosphatase that was recently shown to have a crucial role in Candida cell wall integrity and echinocandin antifungal resistance. In the current study, a collection of Candida albicans CAI4 strains with various levels of Cdc14 activity was generated and evaluated in a mouse model of systemic Candida infection. Our results showed that a wild-type (WT) strain of C. albicans killed 50% of the infected mice at a dose of 1 x 105 CFU/mouse (LD50). A cdc14-deleted mutant could not establish a severe form of the infection up to 5 x 106 CFU/mouse. In a second study, infecting mice with 5 x 105 CFU/mouse of the WT C. albicans strain caused severe morbidity to all the infected mice within four days post-infection, an effect that was not observed with cdc14-deleted or cdc14-hypomorphic mutants. Histopathological examination revealed a vascular shock targeting lung and spleen in mice infected with high doses of the wild-type C. albicans strain. However, surviving mice infected with lower doses of the wild-type strain and all mice infected with cdc14-deleted mutant had inflammatory profiles. Interestingly, typical hyphal formation was observed with wild-type fungal cells but shorter hyphae were present in cdc14-deleted mutant. In summary, Cdc14 was found to be crucial for in vivo fungal virulence of C. albicans and warrant further investigation as a novel drug target for various fungal infections.

Juan Hernandez-Franco - Basic

Immune Activation Pathways Elicited by a Novel Nanoparticle/poly(I:C) Combination Adjuvant in Porcine Dendritic Cells

Juan F Hernandez-Franco1, Shaojun Xie2, Jyothi Thimmapuram2, Darryl Ragland3, and Harm HogenEsch1,4.

1Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907; 2Purdue University Bioinformatics Core West Lafayette, IN 47907; 3Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN 47907; 4Purdue Institute for Immunology, Inflammation and Infectious Diseases (PI4D), West Lafayette, IN 47907.

Profiling the immune response elicited by vaccine adjuvants at the transcriptomic level is crucial for the rational design of immunization strategies. In this work, flow cytometric immunophenotyping, immunoassays, and transcriptomics were used to investigate the effects of a novel phytoglycogen-based nanoparticle (Nano-11) combined with the TLR3 agonist poly(I:C) on porcine monocyte-derived dendritic cells (Mo-DCs). Porcine Mo-DCs were generated from domestic pig peripheral blood mononuclear cells and treated with Nano-11 or Nano-11/poly(I:C). RNA sequencing was used to analyze immune-related differentially expressed genes. The gene expression of CD40, CD80, CD83, OX40L, ICAM1, and ICOSLG was elevated by the combination of Nano-11 and poly(I:C). Flow cytometry confirmed that both Nano-11 and Nano-11/poly(I:C) increased CD80 expression. As the mechanism of action of adjuvants is critical to understanding their efficacy, we investigated immunological signaling pathways that may be triggered by Nano-11 and Nano-11/poly(I:C). The NF-ĸB target genes IL1A, IL1B, TNF, PTGS2, CCL4, CXCL2, and VCAM1 were deferentially expressed following adjuvant treatment. When Nano-11 was combined with poly(I:C), the expression of multiple genes, including IL1B and TNF, was significantly enhanced. This was confirmed by quantifying secreted TNF and IL-1β in the cell culture supernatant. Furthermore, TNF production induced by Nano-11 and Nano-11/poly(I:C) was abrogated by inhibiting NF-ĸB. This data suggest that Nano-11 and Nano-11/poly(I:C) activate DCs via the NF-ĸB program. Together, Nano-11 alone and with adsorbed poly(I:C) enhanced the activation of porcine Mo-DCs with minimal cytotoxicity. These results provide support for the development of Nano-11 and Nano-11/poly(I:C) as vaccine adjuvants that are both safe and efficacious.

Clare Jensen - Basic

Psychiatric Assistance Dog and Military Veteran Partnerships: Association of Time Together and Trained Tasks with PTSD Symptom Severity

Clare L. Jensen1, Kerri E. Rodriguez1,2, Evan L. MacLean3, Hakeem A. Wahab4, Arman Sabbaghi4, & Marguerite E. O’Haire1

1Center for the Human-Animal Bond, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN
2Human-Animal Bond in Colorado, School of Social Work, Colorado State University, Fort Collins, CO
3Arizona Canine Cognition Center, School of Anthropology, University of Arizona, Tucson, AZ
4Department of Statistics, Purdue University, West Lafayette, IN

Research suggests benefits to military veterans partnered with psychiatric assistance dogs, yet the critical components for explaining benefits are still unknown. Thus, this study sought to quantify 1) time veterans and assistance dogs spent together, 2) daily use of trained assistance dog tasks, and 3) relation of these components to PTSD symptom severity.

Participants included N=65 military veterans with PTSD (82% male, Mage=37±8) and their psychiatric assistance dogs (65% male, Mage=2±0.5 years). The PTSD Checklist (PCL-5) measured symptom severity before assistance dog partnership (baseline) and at three-months follow-up. Time together was measured objectively using Bluetooth proximity monitoring. Ecological momentary assessment (EMA) was administered twice daily to measure which trained tasks veterans had used in the preceding four hours. Tasks included interrupt/alert to anxiety, calm/comfort from anxiety, block (create space), cover (watch back), and social greeting (“make a friend”). Both EMA and Bluetooth proximity monitoring took place over 14 days at follow-up. Linear regression analyses were conducted.

Veterans and service dogs were together 82% of the time, but this was not related to veterans’ PTSD symptom severity at follow-up (p=.267). The most-used assistance dog task was to calm anxiety (53% of EMAs vs. 16-18%). Symptom severity was not associated with use of most tasks (p’s=.205–.956). However, greater use of the task to interrupt anxiety was associated with less PTSD symptom severity at follow-up (p<.05).

Findings elucidate the role of assistance dogs’ anxiety-related tasks for veterans’ PTSD symptoms and suggest a future avenue for researching potential mechanisms in veteran-assistance dog partnerships.

Zhili Li - Basic

Role of DDX5 in Sorafenib Responsiveness of HBV-Related HCCs

Zhili Li1,3, Jiazeng Sun1,3, Naimur Rahman1,3, Majid Kazemian2,3 and Ourania Andrisani1,3
1Department of Basic Medical Sciences and 2 Departments of Biochemistry and Computer Science
Purdue University and 3Purdue Center for Cancer Research, West Lafayette, IN 47907, USA

Chronic Hepatitis B virus (HBV) infection is linked to hepatocellular carcinoma (HCC) development. Despite recent progress, HCC treatment remains difficult. In advanced HCC, multi-tyrosine kinase inhibitors (mTKIs) including sorafenib (SOR) or lenvatinib, followed by regorafenib or cabozantinib offer few survival benefits, due to resistance.

In our work, the focus is the RNA helicase DDX5 acting as a central regulator of HCC-cell intrinsic pathways associated with response to mTKIs. We found that mTKIs downregulate DDX5 in various human HCC cell lines, and DDX5 downregulation enables evasion from a non-apoptotic regulated cell death called ferroptosis. How DDX5 contributes to mTKI responsiveness is unknown. To this end, we have performed RNA-seq in DDX5-knockdown (DDX5KD) vs. wild type (WT) HCC cells treated with SOR. We have found that a large number of genes induced by SOR, are repressed by DDX5, and identified 313 common genes. The top-most pathways associated with these common genes are Wnt/β-catenin and non-canonical NF-κB inflammatory pathways. Thus, we hypothesize loss of DDX5 exerts multipronged effects regulating mTKI responsiveness.

Herein, we focus on how DDX5KD mediates ferroptosis escape. Nuclear factor erythroid 2-related factor 2 (NRF2) plays a central role in protecting HCC cells against ferroptosis. DDX5KD hepatocytes expressed higher NRF2. DDX5 interacts directly with p62/SQSTM1 promoting p62 degradation to affect protein level of NRF2. In addition, DDX5KD increased NRF2 transcription through activation of non-canonical NF-KB signaling. Conclusion: our data reveal the mechanism that how HCC-cells become ferroptosis resistant upon treatment with sorafenib/mTKIs. Supported by NIH grant DK044533 to OA and 5K22HL125593 to MK. Shared Resources (Computational Genomics Facility) supported by NIH grant P30CA023168, Pilot grants (Phase I and II) supported by the Purdue Center for Cancer Research to OA.

Rodrigo Mohallem - Basic

Phosphoproteomic Analysis Reveals Dynamic Regulation of PML-Nuclear Body Proteins During Senescence

Rodrigo Mohallem1,2 & Uma K Aryal1,2

1.  Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University
2. Bindley Bioscience Center, Purdue University

Oncogene induced senescence (OIS) serves as a primary layer of protection against tumorigenesis. During OIS, cells enter a state of permanent cell cycle arrest in response to proto-oncogene activation. The promyelocytic leukemia tumor suppressor protein (PML) is an important modulator of OIS and the key organizer of multi-protein nuclear sub-structures called PML-nuclear bodies (PML-NBs). Recent studies have implicated PML and its PML-NB partners in the induction of cellular senescence upon oncogenic transformation, however, little is known about the complex regulatory mechanisms that PML-NB proteins exploit to manipulate OIS. To reveal underlying regulatory mechanisms of OIS, we developed a stable IMR90 cell line, that can be induced to enter OIS upon ER:RasG12V activation via (Z)-4-Hydroxytamoxifen (4-OHT) treatment. Nuclear phosphoproteomic analysis of these cells revealed 5641 phosphosites that were significantly regulated upon the induction of OIS. Our data highlights 103 PML-NB phosphorylated proteins that were significantly regulated OIS, constituting the largest reported number of PML-NB proteins that are regulated upon OIS. The PML protein was phosphorylated at 8 different sites, sites which control its protein stability, function, and localization. Our analysis also revealed that the regulated PML-NB proteins are involved in several processes, including regulation of chromosome organization, chromatin remodeling, transcription regulator complex and aging. Interestingly, our results show that half of the PML-NB proteins identified were downregulated in OIS, indicating that site specific phosphorylation likely induces protein degradation. Thus, our data provides preliminary evidence that PML-NB proteins are involved in regulating OIS via phosphorylation.

Milton Ortiz Rivera - Basic

Comparative Analysis of Osteocyte Canalicular Networks Among Vertebrates and their Role in Bone Adaptation

Authors: Milton J. Ortiz Rivera, Russell P. Main

Affiliations: Department of Basic Medical Science, College of Veterinary Medicine, Purdue University
Weldon School of Biomedical Engineering, Purdue University

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

Sun Jung Park - Basic

Developmental Patterning Roles of the kcnj10a Gene in Zebrafish Long Fin Mutants, Dhi862 and Dhi4458

Authors: Sung Jun Park, GuangJun Zhang

Affiliations: Department of Comparative Pathobiology, Purdue University 

Background: The vertebrate diversity mainly comes from the developmental patterning mechanism, which dictate cell types, differentiation, and organogenesis. While the patterning mechanisms remain largely unexplored, through retroviral insertional forward genetic screening, two long-fin mutant zebrafish; Dhi862 and Dhi4458 were generated. kcnj10a gene was identified as causing genetic changes of the two mutants. Methods: Here, we performed morphological characterization of the two mutants by measuring adult and larva fin and body size and whole mount in situ hybridization on zebrafish mutant embryos. To further validate the function of kcnj10a transient and ectopic expression, we utilized CRISPR-Cas9 system to mutate the kcnj10a coding region. Result: We found that one and two copy transllelic of Dhi862 and Dhi4458 mutant developed elongated fins and barbel compared to wildtype siblings. The fin ray segments on Dhi862 and Dhi4458 were altered in number and length. We also identified a transient ectopic expression of kcnj10a gene in the whole somite and notochord and Dhi862 long-finned phenotype was only able to be rescued by a kcnj10a loss-of-function mutation in an allelic-specific manner. Conclusion: We characterized the fin morphological changes of the two mutants and discovered a transient activation and ectopic expression of kcnj10a gene in somite and notochord, suggesting these ectopic expression domains could be important for long fin development.

Jennifer Peterson - Basic

In Vitro Elution of Silver Nanoparticles from Three Carrier Media

Authors: Jennifer L Peterson1, DVM and Marije Risselada1, DVM, PhD, DECVS, DACVS-SA

Affiliations: 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, Indiana 47907

Body of Abstract: The objective of this study was to determine and compare the rate, pattern, and completeness of silver nanoparticle (AgNP) elution in vitro over 7 days from 3 carrier media (calcium sulfate hemihydrate (CSH) beads, poloxamer 407 gel, and a gelatin compresed sponge) in phosphate buffered saline (PBS). The carrier media were used to create AgNP constructs containing 8500 ng AgNP. All constructs were evaluated in quadruplicate, submerged in PBS, and stored at 38°C for 7 days. Samples were collected at 13 time points over 7 days. AgNP concentration was measured using inductively coupled plasma mass spectrometry. Sustained release of AgNPs was seen from all constructs for a minimum of 72 hours. Release from all constructs was incomplete with an initial burst during the first 24 hours followed by a time dependent decline in elution rate for up to 168 hours. A Mixed Effects Model showed a significant difference in AgNP release over time (P<0.0001) and among media (P<0.0003). AgNP-gel constructs released the largest quantity of AgNPs (8401.022 ng, 98.84%), followed by AgNP-sponge constructs (1503.454 ng, 17.69%). AgNP release from AgNP-CSH beads was 87.824 ng (1.03%), with no additional release after 72 hours. AgNP release was incomplete for all carriers, with minimal additional release occurring after the first 72 hours. AgNP release was highest from AgNP-gel and lowest from AgNP-CSH beads. Sustained release of AgNPs is possible in vitro, but efficacy against bacterial infections needs to be investigated prior to clinical use.

Naimur Rahman - Basic

RNA Helicase DDX5 via Interaction with IFI16 Forms a Silencing Epigenetic Complex Targeting Hepatitis B Virus cccDNA Transcription

Naimur Rahman1,3, Jiazeng Sun1,3, Rodrigo Mohallem2,3, Uma Aryal2,3 and Ourania Andrisani1,3

1Department of Basic Medical Sciences, 2Department of Comparative Pathobiology and 3Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA

This study aims to understand the role of the RNA helicase DDX5 in the innate immune response. Our earlier studies have identified DDX5 as an important player in hepatitis B virus (HBV) mediated hepatocarcinogenesis as well as a host restriction factor in suppressing HBV biosynthesis. We have shown, DDX5 is downregulated during HBV replication and in poor prognosis liver cancer. To gain further insight into the mechanism of DDX5 action and virus biosynthesis, we performed a proteomics study to identify cellular interacting partners of DDX5. Interestingly, the most prominent cellular factor interacting with DDX5 is IFI16, a cellular nuclear DNA sensor of the innate response. CORUM analysis of the DDX5 interacting proteins revealed that DDX5 associates with the MeCP1 histone deacetylase and the chromatin modifying PRC2 complexes. We validated these results by native size exclusion chromatography followed by label-free quantitative MS profiling. Indeed, IFI16 co-eluted with the PRC2 complex, the auxiliary PRC2 subunits RBPP4/7, HDAC1, 2 and DNMT3. Treatment of nuclear lysates with Benzonase to degrade associated RNA, showed reduced association of DDX5 with the IFI16 complex, suggesting RNA is an important component in the formation of this complex. To determine the significance of this IFI16 complex in cccDNA transcription, we generated a Cre/loxP-mediated rcccDNA production system. Our data show that overexpression of IFI16 inhibits transcription from cccDNA, evidenced by reduced pgRNA and HBc expression. Interestingly, knockdown of DDX5, RBBP4 and EZH2, the catalytic component of the PRC2 complex, abrogates the inhibitory IFI16 effect on cccDNA transcription. Ongoing studies include RIP-Seq to identify the RNA that associates with the IFI16/DDX5 complex, and chromatin immunoprecipitation (ChIP) assays to link the epigenetic complex associated with IFI16/DDX5 to cccDNA transcription.

Supported by NIH grant DK044533 to OA

Janet Roque-Torres - Basic

Effect of N-Acetylcysteine, Ascorbic Acid, and Alpha-Tocopherol on Oxidative and Storage Lesions in Canine Packed Red Blood Cells

Authors: Janet Roque-Torres, Andrew Woolcock, Andrea Santos, George Moore

Affiliations: Purdue University College of Veterinary Medicine

Background: Storage of canine packed red blood cells (pRBC) leads to time-dependent lesions which may increase risk of transfusion reactions. Oxidative stress is a contributor of storage lesions, but supplementation of antioxidants in canine pRBC has not been investigated.

Objective: Describe storage and oxidative lesions in canine pRBC during routine storage when supplemented with saline (control, Group 1), N-acetylcysteine (NAC) and ascorbic acid (AA; Group 2), and AA and a-tocopherol (VE; Group 3).

Hypothesis: Storage and oxidative lesions in canine pRBC [measured by glutathione (GSH), thiobarbituric acid reactive substances, and flow cytometry for intraerythrocytic reactive oxygen species (ROS)] will be decreased with antioxidant supplementation.

Methods: Nine leukoreduced units of canine pRBC were aseptically separated into three aliquots (Groups 1, 2, 3). Antioxidants were supplemented on Day 1 after baseline samples collected. Additional samples were collected on days 7, 28, and 42. Units were collected in 3 batches, with assays performed at the end of each storage period. Type 3 tests of fixed effects compared the impact of group and time on each measurement.

Results: All groups showed storage lesions and GSH depletion by day 42 compared to baseline, regardless of antioxidant supplementation. Intraerythrocytic ROS accumulation was lower in Group 3 (AA & VE) compared to other groups at all time points after baseline (p < 0.0001).

Conclusions and Clinical Importance: Supplementation of canine pRBC with AA and VE reduced oxidative stress but not storage lesions. Future studies should evaluate the clinical use and incidence of transfusion reaction with AA/VE-supplemented pRBC.

Kamrun Naher Sharmin - Basic

Strain-Specific Distinctive Impact of Norepinephrine on the Growth and Virulence of Clostridioides Difficile

Kamrun Naher Sharmin, Ahmed A. Hassan, Deepti Pillai, Sanjeev Narayanan
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA

The United States Centers for Disease Control and Prevention (CDC) classified infections with Clostridioides difficile (formerly known as Clostridium difficile) as an urgent threat to the public. The CDC’s decision was based on the considerable number of infections that exceeded 223,000 hospitalizations and 12,800 death cases in 2017, with estimated attributable healthcare costs of $1 billion in the US solely. Norepinephrine (NE) is a stress-associated neuroendocrine hormone. It is a well-known fact that this stress hormone can modulate bacterial behavior such as growth and virulence. This mechanism has been reported in several pathogenic bacteria such as Staphylococcus, Escherichia coli, Salmonella, and Vibrio cholera. Our study investigates the strain-specific response of six different C. difficile strains to NE in growth and virulence. In the present study, overnight bacterial culture was grown (+/- )NE (5μM & 50 μM) in Brain Heart Infusion broth supplemented with yeast extract. RNA was extracted and treated with DNase for genomic DNA removal. Finally, cDNA was synthesized, and qPCR was performed to observe virulence gene expression. The virulence genes- tcdA, tcdB, flagellin were upregulated in NR 49290, NR 49277 & ATCC 43255 strains in both concentrations of NE and down-regulated in NR 49282. Concentration-specific up & downregulation was observed in P8 & P13. Interestingly, Pilin genes were always upregulated in NR 49277 (pilA1, pilA3, PilA5), NR 49290 (pilA1, PilA5), ATCC 43255 & P13 (pilA1, PilA3) strains. Based on the above information, the response of C. difficile to NE warrants further investigation for reducing the virulence.

Martin Silic - Basic

Uncovering the Dynamic Bioelectric Signals During Zebrafish Embryogenesis

Martin Silic, GuangJun Zhang

Department of Comparative Pathobiology, Purdue University

Background: One newly emerging area of cell signaling during embryonic and tissue development is bioelectricity. Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence reveals that cellular electric signaling is vital for regulating embryonic development. However, in vivo dynamic electrical activity monitoring of whole organisms has been limited. Methods: We utilized a genetically tractable zebrafish model to generate a stable ubiquitin driven ASAP1, genetically encoded voltage reporter, mutant line to study the endogenous bioelectricity of embryogenesis. Embryos were mounted in agarose from 1-16 hours post fertilization and imaged using light sheet microscopy. Membrane potential changes were observed throughout zebrafish embryonic development. Results: During cleavage, hyperpolarization of the cleavage furrow was evident, and proceeded the formation of the furrow. Furthermore, these signals were not completely stabilized, but highly dynamic as cytokinesis progressed. Additionally, the order of which cell starts and follows this signal was not always consistent. During blastula and gastrula stages, rapid and frequent cellular hyperpolarizations of whole cells are visible. Intercellular signaling is apparent, as adjacent cells show hyperpolarizations directly following those in contact. During somitogenesis, many frequent hyperpolarizations are evident over the whole embryo, but certain tissues become more hyperpolarized, such as the brain, notochord, and somites. These areas also show strong transient signals. Conclusion: We demonstrate bioelectric signaling is a deeply integrated part of zebrafish embryogenesis. This evidence is significant, as specific electrical signals of developing tissues can help further scientific understanding of channelopathy congenital diseases.

Dingxun Wang - Basic

PRMT5 Inhibition Confers an Impaired Cell Viability in Malignant Peripheral Nerve Sheath Tumors

Dingxun Wang, Han Han, GuangJun Zhang
Department of Comparative Pathobiology, Purdue University

Background: Malignant peripheral nerve sheath tumor (MPNST) is a rare but highly aggressive
cancer, which originates from peripheral nerve sheath Schwann cells. Current traditional surgery
and radiation treatments for MPNST are always along with relatively low efficacy, poor prognosis
and high rate relapse. More importantly, no effective targeted therapy is available to this type of
malignancy as well. Thus, a new therapeutic drug target is urgently desired. PRMT5, a type II
protein arginine methyltransferase that symmetrically dimethylates arginine residues of numerous
substrates including but not limited to histone and transcript factors, has recently been reported as
a promising therapeutic target for widely varied types of human cancers. As the extremely high
frequency of simultaneous loss of CDKA2NA and MTAP, we hypothesized that inhibition of
PRMT5 decreases MPNST cell growth. Methods: We suppressed PRMT5 function in MPNST
cell lines (STS26T and T265) utilizing shRNA knockdown and effective PRMT5 specific
inhibitors (BLL3.3/CMP5 and JNJ-64619178). Results and conclusion: We discovered that
MPNST cell growth was reduced significantly upon PRMT5 inhibitions by both genetic and
chemical methods, suggesting that PRMT5 is an efficient therapeutic target for MPNST.

Tiange Xiao - Basic

Changes in Protein Signaling Profiles of Brain Regions Involved in Drinking After a History of Chronic Binge-Like Drinking

Tiange Xiao, Yueyi Chen, Alyssa Boisvert, Emily Knorr, Adam Kimbrough

Basic Medical Sciences, Purdue University, West Lafayette, IN, USA.

Binge drinking is a significant societal problem that is defined as a pattern of drinking that brings blood alcohol levels (BALs) to 80 mg/dL or above. A history of chronic binge drinking may produce long term changes in the brain that result in increased susceptibility to alcohol and drug dependence. Recently the posterior cortical amygdala (pCOA), the ventrolateral periaqueductal gray (vlPAG), and the lateral hypothalamus (Lh) have been identified as important in alcohol drinking behavior. Thus, we sought to examine protein signaling changes in the pCOA, vlPAG, and Lh after 12 weeks of chronic binge-like drinking, using the ‘Drinking in the Dark’ (DID) mouse model, followed by Liquid Chromatography (LC)/Mass Spectrometry (MS) analysis of brain tissue. ‘Drinking in the Dark’ (DID) is push mouse have consisted of 3 days 2-hr single bottle drinking sessions (20% w/v alcohol) beginning 3 hours into the dark cycle, followed by 1 day of 4-hour 20% w/v alcohol access. After 12 weeks of chronic binge-like alcohol drinking, brains from the DID mice(C57BL/6J mice ; n=20; 10 male, 10 female) underwent 12 weeks of DID behavior and age-matched alcohol naive control mice (n=16; 8 male, 8 female) were collected and snap frozen. Brain tissue from each target brain region (pCOA, vlPAG, and Lh) was then punched in order to process with LC/MS. We expect to identify several proteins of interest that have had protein signaling significantly altered by binge-like drinking. The identified proteins of interest will be ideal targets for future binge-like drinking studies.

Jenni Auvinen - Clinical/Applied

Bioavailability of Rectal Metronidazole in Healthy Adult Horses

Jenni Auvinen, Janice Kritchevsky, Wilson Gwin, Alex Gochenauer, Bruce Cooper

Department of Veterinary Clinical Sciences and Bindley Bioscience Center,
Purdue University, West Lafayette, IN

The objective of the study was to determine the bioavailability of metronidazole when formulated with different solvents. Our goal was to investigate a gel preparation of metronidazole (RG) with a hypothesis of improved bioavailability when compared to the typical rectal administration (RW) with tap water or dissolved in DMSO (DMSO).

Six horses were given 20 mg/kg bodyweight of metronidazole preparations orally (NG) or rectally in a cross-over study. Samples were collected for pharmacokinetic analysis at 10, 20, 30, 45, 60, 80 and 100 minutes and 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 24 and 48 hours following the administration. Plasma metronidazole concentrations were measured using high performance liquid chromatography with mass spectrometry detection and the results were reported as μg/ml. Mean Cmax and Tmax were reported and the relative bioavailability following rectal metronidazole administration was calculated in relation to oral administration.
The pharmacokinetic parameters for NG, RW, RG and DMSO, respectively, were as follows: mean Cmax (±SE) 14.82 ±2.20 μg/ml; 3.17 ±0.26 μg/ml; 0.35 ±0.06 μg/ml; and 1.97 ±0.58 μg/ml; mean Tmax (±SD) 29±20 min; 91±78 min; 38±11 min; and 40±23; and mean AUC(0-∞) 3759±1026 min*ug/mL; 1291±478 min*ug/mL; 101±41 min*ug/mL; and 687±327 min*ug/mL;. The relative bioavailability of RW, RG and DMSO compared to oral absorption were 38±18%, 3±1% and 14±8%, respectively.

Metronidazole rectal gel produced the lowest plasma concentrations. DMSO did not improve rectal bioavailability and was irritating. The p.o. dose of metronidazole (20 mg/kg) administered rectally dissolved in tap water did not yield therapeutic plasma concentrations.

Prabha Bista - Clinical/Applied

Could Outer Membrane Vesicles of Fusobacterium necrophorum be a Vaccine Candidate?

Bista, P.K.1*; Pillai, D.1,2; Narayanan, S.K.1
1Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, USA;
2Indiana Animal Disease Diagnostic Laboratory (ADDL), Purdue University, West Lafayette, IN
47907, USA

Fusobacterium necrophorum, a Gram-negative obligate anaerobe, is the causative agent for liver
abscesses and necrotic infections in cattle. Outer membrane vesicles (OMVs) are extruded
nanostructures shed by pathogenic bacteria, and contain periplasmic contents, including toxins,
virulence factors, lipoproteins, and sometimes genetic materials. Understanding the
immunogenicity and pathogenicity of these vesicles could help identify the vaccine potential of
OMVs against fusobacterial infections. In this study, the OMVs from the late log phase F.
necrophorum culture were concentrated using ultrafiltration and ultracentrifugation. The extracted
OMVs were purified by OptiPrep density gradient method and were analyzed by electron
microscopy (EM). OMV preps were then subjected to proteomics and lipid profile analysis. The
proteomics study identified major virulence factors such as leukotoxins, adhesins (43kDa-FomA,
22kDa-OmpA, 17kDa-OmpH, FadA, and 66kDa-CSP), and other autotransporter domaincontaining
proteins in abundance than in vegetative bacteria. Some of these were confirmed
through western blot analysis using corresponding antibodies.

Similarly, lipid profile study found that phospholipids and acetylcarnitine (AC) are the major lipid
components of OMVs. We performed a differential study of OMV production in iron-deficient
versus enriched conditions. The study showed a relative increase in the yield of small-sized OMVs
with high protein content in iron limiting conditions. The functional aspects of OMVs released by
F. necrophorum have not been studied yet. We plan to investigate the immunogenic response in a
mouse challenge model and the pathogenicity effect through co-culture and cytotoxicity assays.
To sum up, insights into OMVs contents could help develop potent vaccines against fusobacterial

Jessica Craig - Clinical/Applied

Interventions to Improve Antimicrobial use and Stewardship in the Human and Animal Health Sectors in High- and Low-Resource Contexts: A Systematic Literature Review and Meta-Analysis

Jessica Craig1, Felix Bahati2,3, Yewande Habitat-Alimi4, Wendy Beauvais1
1Purdue University, College of Veterinary Science, Indiana, USA
2Center for Disease Dynamics, Economics & Policy, Washington, DC, USA
3Jomo Kenyatta University, Nairobi, Kenya
4Africa Centres for Disease Control and Prevention, Addis Ababa, Ethiopia

Antimicrobial resistance (AMR) is an emerging global health threat. The World Health Organization estimates that drug-resistant bacteria cause at least 700,000 deaths per year. AMR is driven by several factors including the misuse and overuse of antimicrobials in the human health, animal health, and agricultural sectors. Non-pharmaceutical antimicrobial stewardship (AMS) interventions such as education and training, the development of clinical treatment guidelines, and the implementation of diagnostic and other technology-driven solutions, may reduce antimicrobial use (AMU) and mitigate AMR; however, there remains no consensus on best AMS practices for the human health, animal health, and agricultural sectors. Therefore, the purpose of this study was to conduct a systematic literature review and meta-analysis to identify interventions aimed at improving AMS and AMU across the various sectors and to begin to piece together best practices in this area.

We conducted a systematic literature review of PubMed, Web of Science, CABI, and the Cochrane database for peer-reviewed articles published before June 2021 (time of search) that described provider- and consumer-based behavior change interventions aimed at improving antimicrobial stewardship (AMS), consumption (AMC), and/or use (AMU) in the human and animal health (including agricultural) sectors. The following keywords, connected by the stated Boolean operators, were used to search the title and abstracts of published entries within each database: “intervention” OR “trial” AND “antimicrobial” OR “antibiotic” AND “use,” OR “stewardship,” OR “consumption,” OR “prescription.” Reviews of returned entries were conducted in two stages; a review of the title and abstract was followed by full-text review. Papers published in any language and written on interventions conducted in any country were included. All study types including randomized controlled trials (RCTs) and observational studies (i.e. pre/post study designs) were considered. Studies were included for final review and data extraction if they reported on at least one of this review's primary outcomes which fell into four general categories including overall rates of AMU or AMC, rates of adherence or compliance to facility, national, or international guidelines, intravenous to oral conversion rates, stewardship practices such as the frequency of obtaining specimen cultures for pathogen identification and/or antimicrobial susceptibility tests [AST].

The literature review returned a total of 154,106 entries of which 4,893 were duplicates, 148,639 were found to be irrelevant based on the title and abstract review, and 531 entries underwent full-text review. Following full-text review, a total of 305 studies were included and underwent data extraction. A meta-analysis examining various interventions’ impact and summary conclusions will be presented.

Nelly Elshafie - Clinical/Applied

miRNome Expression Analysis in Canine DLBCL

Nelly O. Elshafie1, Michael Gribskov2, Nathanael I. Lichti3, Ekramy. E. Sayedahmed1, Michael O. Childress4, and Andrea P. dos Santos1

1 Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA 

2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA

3 Department of Statistics, Purdue University, West Lafayette, Indiana, USA

4 Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, Indiana, USA

Lymphoma is a prevalent malignancy in dogs. Diffuse large B-cell Lymphoma (DLBCL) is the most common subtype, representing about 50% of the clinically seen lymphoma cases. Thus, the search for additional biomarkers capable of early detection and monitoring of DLBCL is important for improving and sustaining remission rates. The next-generation sequencing technology provides innovative information about biomarkers and can be used to characterize the differential expression of genes. This study broadens the knowledge on the molecular biogenesis of DLBCL by investigating the role of gene expression regulators called microRNAs (miRNA). Noncoding miRNAs negatively regulate gene expression by binding to the 3'-untranslated region of protein-coding mRNA, leading to either targeted RNA degradation or translational repression. MiRNAs' stability and easy accessibility make them promising biomarkers for identifying and sub-classifying lymphoma patients. We isolated and sequenced miRNAs from ten fresh-frozen lymph node tissue samples and compared them to healthy controls (six DLBCL and four healthy). Small RNA sequencing (sRNA-Seq) analysis identified a total of 35 differentially expressed miRNAs (DEMs). RT-qPCR confirmed 23/35 DEMs (14 upregulated and 9 downregulated) in DLBCL. The unpaired parametric Welch's 2-sample t-test and false discovery rate (FDR) were used to determine significant differences in average expression fold-change (2-∆∆Cq) of each miRNA in the DLCBL and healthy groups. The results were normalized using the geometric mean of the expression level of miR-361-5p, miR-101, and miR-29c-3p, the most stably expressed reference candidates. Ultimately, our results demonstrate the potential to harness miRNAs as unique diagnostics and therapeutics targeting DLBCL.


Hamideh Esmaeilzadeh - Clinical/Applied

A Case of an Eosinophilic Leukemia in a Hedgehog

Hamideh Esmaeilzadeh1, Priscila Beatriz da Silva Serpa2, Courtney Sweeney1, Margaret (Peg) Miller1,
John A. Christian1, Andrea Pires Dos Santos1
1. Department of Comparative Pathobiology, Purdue University
2. Department of Clinical Pathology, Virginia-Maryland College of Veterinary Medicine

A one year and two-months-old male hedgehog was presented to Purdue University Veterinary Teaching
Hospital with a history of anorexia and constipation. Fecal flotation reported no parasites. Complete blood
count showed mild neutrophilia (21,700 cells/μL), eosinophilia (650 cells/μL), basophilia (2,380 cells/μL)
and “other cells” (1,080 cells/μL) that appeared immature eosinophil precursors. Erythron and thrombon
were within reference intervals. After four months, he became lethargic and weak in his hindlimb, and
developed hematochezia. Follow-up blood work revealed a regenerative anemia with increased numbers
of eosinophils, basophils, and eosinophilic precursors. Due to the grave prognosis, the owner opted for
humane euthanasia. Histopathologic findings were consistent with a systemic eosinophilic neoplasm and
a suspicion of eosinophilic leukemia. Bone marrow evaluation showed excessive numbers of eosinophils
and eosinophilic precursors. Widespread eosinophilic infiltration was found in the lungs, liver, spleen, and
intestines. Reactive eosinophilia occurs in response to an underlying cause, such as infectious agent,
hypersensitivity or as a paraneoplastic condition. Clonal eosinophilia can be seen with chronic eosinophilic
leukemia and myelodysplastic syndrome. Idiopathic eosinophilia is a diagnosis of exclusion. In
eosinophilic leukemia, left-shift is present as far as myeloblasts, with some dysplasia. Given the bone
marrow evaluation (presence of excessive eosinophilic precursors), and the presence of more immature
forms in the peripheral blood and several organs, the diagnosis of this case is eosinophilic leukemia with
paraneoplastic basophilia. Hedgehogs with eosinophilic leukemia usually have mild to moderate anemia
or low normal hematocrit. In the case herein, the regenerative anemia was likely due to blood loss.

Ahmed Khairoun - Clinical/Applied

Electrohydraulic Shockwave for Treatment of Superficial Digital Flexor Tendinitis and Suspensory Desmitis in Horses

Ahmed Khairoun, DVM, Jan F. Hawkins, DVM, DACVS, George Moore, DVM, PhD, DACVIM, Timothy B. Lescun, BVSc, PhD, DACVS, Stephen B. Adams, DVM, MS, DACVS
From the Department of Veterinary Clinical Sciences and the Department of Pathobiology (Moore), 625 Harrison Street, Lynn Hall of Veterinary Medicine, Purdue University, West Lafayette, IN 47907.

Lameness in horses is often caused by superficial digital flexor tendonitis (SDFT) and suspensory desmitis (SD), which can limit their athletic performance. Current treatment options include rest, controlled exercise, anti-inflammatories, intralesional injections, corrective shoeing, surgery, and Extracorporeal Shock Wave (ESW) therapy. ESW therapy is safe, noninvasive, and is used to treat a variety of osseous and soft tissue abnormalities.

Our objective was to examine the effect of the number of ESW treatments on SDFT and SD, and to compare short- and long-term outcomes.

Medical records between 2010 and 2019 were reviewed. Horses were separated into two categories: (Group1: ≥ 3 treatments; Group2: < 3 treatments). Group 1 had a lameness exam and ultrasound performed at admission and before the third treatment whereas group 2 had a lameness exam and ultrasound performed at admission only.

There were 80 horses diagnosed with 86 tendon or ligament lesions. In-group 1, lameness scores and ultrasonographic severity grades were significantly reduced in both SD and SDFT (p< 0.0005) and (p<0.016), respectively. In regards to follow-up comparisons, there was a significant positive relationship between performing 3 or more ESW treatments and the 3 and 6-month lameness for SD cases (p=0.001) and (p<0.001), respectively, but not for SDFT cases. At 12 months, there was no significant difference between SD and SDFT in the different treatment groups (p=0.804).

Clinically, horses with SD that had 3 or more ESW treatments had better lameness scores up until 6 months, however, SDFT did not differ between groups at any time point.

Eric Kontowicz - Clinical/Applied

Mathematical Modeling Framework to Simulate the Transmission of Influenza Virus between Swine and the Workforce on US Hog Farms

Authors: Eric Kontowicz1, Darryl Ragland3, Max Moreno-Madrinan2, Wendy Beauvais1

Affiliations: 1.Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, 625 Harrison Street, West Lafayette, IN 479072.Department of Global Health, Indiana University Fairbanks School of Public Health, 1050 Wishard Blvd. Suite 6114, Indianapolis, IN 462023.Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine, 625 Harrison Street, West Lafayette, IN 47907

The purpose of this study was to evaluate improved hog influenza vaccine efficacy impacts on influenza transmission between hogs and human workers on a simulated US indoor hog growing farm. Influenza A virus’s (IAV) potential to evolve as a zoonotic pathogen with pandemic potential poses a public health threat. It was found swine workers in Iowa were at increased odds (54.9 times compared to a non-swine industry worker control population) of H1N1 infection.

Our Susceptible-Exposed-Infectious-Recovered (SEIR)— model based on a system of ordinary differential equations (ODEs)— incorporated stochastic hog-to-workforce transmission. The model was parameterized through literature review and expert consultation. Sensitivity analyses were performed to explore the impact of uncertainty in parameter values on results. Control measures such as vaccination, sick leave policies and temperature monitoring were evaluated.

Assuming a single hog infected with a single influenza strain was introduced onto the farm, the model predicts the first workforce infection within an average of 30 days across all iterations. For a highly-transmissible strain the average days to first workforce infection across all iterations was 7.23, 8.05 or 8.82, based on a hog vaccination effectiveness of 0%, 40% or 80% respectively.

Our results are consistent with empirical studies suggesting that hog workers are likely to become infected by IAV rapidly during an outbreak in hogs. Emergence of a highly-pathogenic and transmissible strain of IAV (to hogs or humans) could create significant workforce shortages in a sector where relatively few workers are responsible for large swine populations (approximately 1:2000 workers:hogs).

Sarah Leighton - Clinical/Applied

Assistance Dogs for Military Veterans with PTSD: A Systematic Review, Meta-Analysis, and Meta-Synthesis

Authors: Leighton, Sarah C.1, Nieforth, Leanne O.1, O’Haire, Marguerite E.1

1Department of Comparative Pathology, College of Veterinary Medicine, Purdue University

We conducted a systematic review of the literature relating to the placement of psychiatric assistance dogs for veterans with posttraumatic stress disorder (PTSD), with specific aims to (1) summarize their characteristics, (2) evaluate the quality of existing evidence, and (3) summarize outcomes. A total of 432 records were independently screened by authors SL and LN (Cohen’s kappa=.90); 41 articles (29 peer-reviewed publications and 12 unpublished dissertations) met inclusion criteria. Data extraction was conducted to address the research aims, including a meta-analysis (quantitative outcomes) and meta-synthesis (qualitative outcomes). We found that all peer-reviewed publications examining psychiatric assistance dogs for veterans with PTSD were published within the last five years. The majority of included articles were quantitative (53%), 41% were qualitative, and 6% employed mixed methods. Mean methodological rigor scores were 78% for peer reviewed articles and 71% for dissertations, where higher scores represent more rigorous methodology. Quantitative articles reported significant improvements in the domains of PTSD severity, mental health, and social health. Meta-analysis revealed that partnership with an assistance dog had a clinically meaningful, significant, and large effect on PTSD severity scores (g=−1.137; p<.0001). Qualitative meta-synthesis identified two third order constructs: (1) Impact on the individual: mental & physical health and (2) Impact beyond the individual: building relationships & connection. This synthesis of increasingly prevalent research on assistance dogs for veterans with PTSD provides support for the impact of this complementary intervention on PTSD symptom severity, and signs of meaningful improvements in adjacent domains including mental and social health.

Kerstin Muner - Clinical/Applied

Comparison of Acid-fast Bacilli Staining and qPCR to Detect Mycobacterial Infection in Human Autopsied Patients with a History of Tuberculosis and Coinfection with Tuberculosis and HIV-1

Kerstin Muner1, Naíla Cannes do Nascimento1,2, Amaro Nunes Duarte Neto3, Ana Marcia de Sá Guimarães4, Andrea Pires dos Santos1
1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA 2Department of Speech, Language, and Hearing Sciences, Purdue University, West Lafayette, IN, USA 3Department of Clinical Emergencies, Pathology Department, University of São Paulo, Brazil 4Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Brazil

Tuberculosis (TB) is an infectious disease distributed worldwide alongside the Human Immuno-Deficiency Virus (HIV-1), representing a global burden with high mortality rates in developing countries. The accurate diagnosis of TB is essential for early treatment and prevention of disease spread, especially for HIV-1 patients. Acid-fast bacilli staining (AFBS) of sputum is the first diagnostic approach for TB patients due to its fast results and affordability. Histopathological evaluation of AFBS in lung tissue is also a diagnostic tool. Nevertheless, molecular techniques such as qPCR have been increasingly performed for TB diagnosis because of its higher sensitivity and specificity. They also allow the differentiation from tuberculous and non-tuberculous mycobacterial infections, which is essential to prevent unnecessary long-term treatment for TB and avoid the development of drug resistance. Here, we compared two AFBS diagnostic tools, the sputum microscopy and histopathological evaluation of the lungs, with the molecular approach of qPCR in Formalin-Fixed Paraffin-Embedded (FFPE) multi-organ tissue to diagnose mycobacterial infection in archived samples from human autopsied patients with a history of tuberculosis (11) and coinfection of tuberculosis and HIV-1 (8). Ziehl-Neelsen stained sputum smear and FFPE lung tissue were compared with the qPCR assay targeting the IS1611 and IS1311 regions. AFBS detected 79% and 74% of cases as positive for mycobacterial infection in sputum and lung tissue, respectively, while qPCR showed 95% positivity for tuberculous mycobacterial infection. No non-tuberculous mycobacterial infection was identified. The molecular diagnosis allowed higher detection rates of mycobacteria and identified the absence of mixed infection in our archived samples.

Carla Murillo - Clinical/Applied

Electroencephalographic Character Changes During Propofol Anesthesia in Young Healthy Dogs

Carla Murilloa, Jeff C. Koa, George E. Mooreb, Ann B. Weila, Matthias Kreuzer c
a Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, United States of America
b Department of Veterinary Administration, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, United States of America
c Matthias Kreuzer, Department of Anesthesiology and Intensive Care, Technical University of Munich, School
of Medicine, München, Germany

Propofol induces characteristic electroencephalography (EEG) changes which can be used to manage the depth of propofol anesthesia. These EEG changes have not been described in dogs. The goal of this study was to characterize the propofol-induced EEG changes during different depths of anesthesia. We hypothesize that EEG changes can be used to track different depths of propofol anesthesia.
A total of six 2-year-old male dogs were induced (10 mg/kg propofol in 10 min) and maintained (deep- 2.4 mg/kg/min, moderate-1.6 mg/kg/min, and light- 0.8 mg/kg/min) in different anesthetic planes of 15- minute duration via propofol constant rate of infusion (CRI). The frontal EEG was obtained with a Sedline brain monitor. Cardiorespiratory variables and Guedel’s clinical anesthesia classification signs were monitored simultaneously with EEG. The EEG data and its processed indices were compared with repeated measures ANOVA with a p-value set at 0.05.

The patient state index (PSI), an index of conscious level, significantly changed during the different study phases (p=0.0001). The burst suppression ratio significantly (p=0.001) increased during the deep plane of propofol anesthesia. Delta (0.5-4 Hz) and Beta-1 (13-20 Hz) waves increased and persisted during the entire maintenance of anesthesia. There was a shift from Beta-1 dominance to Beta-2 (21-30 Hz) waves during recovery. The anesthetic depth changes were best described by an increase of the Beta: Delta ratio (β:δ). The cardiorespiratory and clinical signs supported the EEG observations.

We concluded that the depth of propofol anesthesia can be tracked by the use of Sedline monitor with the β:δ ratio.

Leanne Nieforth - Clinical/Applied

The Influence of Psychiatric Service Dogs for Posttraumatic Stress Disorder (PTSD) on Military Spouses

Authors: Leanne Nieforth1, Elise Miller1, Shelley MacDermid Wadsworth2, Marguerite E. O’Haire1

Affiliations: 1Comparative Pathobiology, College of Veterinary Medicine, 2Human Development and Family Studies, College of Health and Human Sciences

The purpose of the current study is to explore clinically validated and empirical survey measures in a longitudinal study of veterans and their spouses to provide insight regarding psychiatric service dogs for veterans with posttraumatic stress disorder (PTSD) and veteran families. Previous literature suggests that the benefits and challenges associated with psychiatric service dogs for veterans with PTSD may extend beyond veterans and effect the broader veteran family. A total of 88 United States military veteran spouses completed a survey composed of multiple standardized measures at baseline and three months later. In the intervention group (n=48), veterans received service dogs shortly after baseline while the waitlist control group (n=40) did not receive a service dog until after the study was completed. Linear regression was used to analyze the standardized survey responses. Analyses demonstrated significantly lower caregiver satisfaction (p=0.046, d=-0.46), higher caregiver burden (p=0.048, d=0.38), and higher participation in life activities (p=0.014, d=0.59) among spouses who had service dogs in their homes compared to those on the waitlist. Though not significant, small effect sizes were present among additional measures. Results suggest that although previous literature demonstrates psychiatric service dogs may offer significant improvements for veterans, spouses and children may not experience those same benefits. Clinicians should consider how to prepare veteran spouses and families for integrating psychiatric service dogs into their home. Future studies should explore family-focused approaches for service dog integration, defining an optimal strategy for the benefit of the entire family.

Daniela Peña - Clinical/Applied

Comparison of a Commercial Kit and Traditional Culture Method for Detection of Salmonella spp. on Spiked and Environmental Samples

Title: Comparison of a commercial kit and traditional culture method for detection of Salmonella spp. on spiked and environmental samples

Authors: Daniela Peña1,2, G. Kenitra Hendrix1,2

Affiliations: Comparative Pathobiology department, Purdue University College of Veterinary Medicine1, Indiana Animal Disease Diagnostic Laboratory2

Body: Nosocomial salmonellosis in hospitalized animals is a recognized hazard, especially in large animal clinics. Mitigation of outbreaks, as well as active surveillance efforts, require an effective and time-sensitive diagnosis. A standardized culture method for detecting Salmonella spp. in environmental samples using a 48-hours enrichment step, results in a 5-days long turnaround time for negative results. RapidChek® SELECT ™ Salmonella Test Kit (Romer Labs®) offers presumptive detection of organisms in 22-44 hours.
For this study, electrostatic wipes (Swiffers®), routinely used in the LA-PVH for environmental sampling, were artificially spiked (n=20) with Salmonella Typhimurium with either 100 or 101 CFU per sample. Additionally, Swiffer® samples submitted by the LA-PVH for detection of Salmonella spp. were collected in pairs (n=10) from the same areas within stalls.

A spiking level of 10 CFU (101 CFU) of Salmonella per sample was consistently detected across all replicates (5/5) using the RapidChek® test kit. It also allowed the detection of 1 CFU per sample in 2/5 replicates. Standard Salmonella culture failed to recover organisms from all samples inoculated with 1 CFU (100 CFU) and recovered only 40% (2/5) of replicates with a 101 CFU total inoculum. PCR confirmed both positive and negative results from both methods.

The test kit presents a clear time advantage over the traditional culture-based detection method by reducing the turnaround time from 5 days to only two days for negative results. A reduction in the limit of detection, as low as one single Salmonella organism per sample, was also obtained, suggesting higher sensitivity.

Ana Pinto - Clinical/Applied

Computed Tomography (CT) Attenuation Value and Contrast Enhancement Analyses of Neoplastic and Non-Neoplastic Lung Lesions in Dogs and Cats

Authors: Ana Carolina Fonseca Pinto1, DVM, MS, PhD (presenting author) George E. Moore1, DVM, PhD, DACVIM, DACVPM Chee Kin Lim2, DVM, BVSc (Hons), MMedVet, FMCVS, DECVDI Masahiro Murakami1, BVSc, PhD, DACVR Caroline V. Fulkerson1, DVM, DACVR

Affiliations: 1 Veterinary Clinical Sciences (VCS), Purdue University College of Veterinary Medicine, 2 VetCT Specialists Ltd

Information about CT texture analysis and contrast enhancement quantitative analysis (CEQA) for characterization of neoplastic or non-neoplastic pulmonary lesions in companion animals is lacking. This study evaluated if the pre-contrast CT attenuation value (CTAV) and CEQA will allow this differentiation. A total of 44 dogs and cats with confirmed pulmonary soft tissue attenuating lesion are included in this retrospective study. Different CT image measurements were performed. Receiver operating characteristic curves (ROC), area-under-the-curve (AUC) and a multivariable logistic regression were used to describe the best numerical variables. Of the 44 patients, 26 (59%) had pulmonary neoplasia. The pre-contrast mean CTAV of the entire area of the lesion (pre-contrast MeanCTAV), post-contrast minimum CTAV of the entire area of the lesion (post-contrast MinCTAV), and mean CTAV of the small ROI over the most vascularized area of the lesion (MeanCTAV-ROI) have the highest AUC values (95% CI) at 0.74 (0.58-0.90), 0.71 (0.55-0.87) and 0.71 (0.54-0.87) respectively. The cut-point (HU), sensitivity, and specificity were 26.5, 73% and 83% for the pre-contrast MeanCTAV; -104.0, 69% and 78% for post-contrast MinCTAV; 66.1, 77% and 67% for MeanCTAV-ROI. Neoplastic pulmonary lesions tend to have a higher pre-contrast MeanCTAV, post-contrast MinCTAV and MCTAV-ROI than non-neoplastic pulmonary lesions, similar to human pulmonary neoplasia. In conclusion, the pre-contrast MeanCTAV, and two of the CEQA parameters including the post-contrast MinCTAV and MeanCTAV-ROI showed potential to discriminate neoplastic from non-neoplastic pulmonary lesions in dogs and cats.

Aynsley Romaniuk - Clinical/Applied

Dam (Canis Familiaris) Welfare Throughout the Peri-Parturient Period in Commercial-Breeding Kennels

Aynsley Romaniuk 1, Shanis Barnard 1, Jennifer Weller 2, Hsin-Yi Weng 1, Sriveny Dangoudoubiyam 1 and Candace Croney 1

1 Department of Comparative Pathobiology, Purdue University, Indiana, USA
2 Agri-Food & Biosciences Institute, Northern Ireland, UK

Bitches in commercial breeding (CB) kennels spend a large proportion of time in the peri-parturient period. Throughout this time, their health may impact their own welfare and that of their offspring. The aim of the current study was to assess changes in physical, physiological, and behavioral health that may be indicative of dam welfare throughout the peri-parturient period in CB kennels. Dams (n=59) from eight US CB kennels were tested at 6- and 1-week prepartum, and 4- and 8-weeks postpartum. They underwent a stranger approach test, physical health assessment, hair collection for hair cortisol concentration (HCC), and fecal collection for fecal glucocorticoid metabolites (FGM), fecal secretory immunoglobulin A (sIgA), and intestinal parasite analyses. Linear mixed-effects models indicated dams exhibited more affiliative behavior in response to stranger approach at 4-weeks postpartum than 6-weeks prepartum (p=0.03), an increase in HCC from 4- to 8-weeks postpartum (p=0.02), and in FGM from 1-week prepartum to 8-weeks postpartum (p=0.04). At each respective time point, the percentage of intestinal parasites was 11%, 4%, 23% and 15%. This is the first study to explore changes in dam welfare throughout the peri-parturient period, and may have implications for dam management throughout this period. Findings likely reflect biological changes associated with changing dam state that occur during this time. However, future studies should explore the effects of changing environmental and management factors on these and other metrics of dam welfare to ensure optimal conditions for dams and their puppies.

Bushra Zaidi - Clinical/Applied

Serum Thymidine Kinase 1 Activity as a Prognostic Biomarker in Dogs with CHOP-Treated Diffuse Large B-Cell Lymphoma

Bushra Zaidi, Abhijit Mukhopadhyay, José A. Ramos-Vara, Deepika Dhawan, Audrey Ruple, Michael O. Childress

Introduction: Diffuse large B-cell lymphoma (DLBCL) is frequently treated with CHOP-based chemotherapy, which induces remission in 80% to 95% of cases. However, not all dogs derive meaningful benefit from CHOP and prognostic factors for dogs with DLBCL are poorly defined. Serum thymidine kinase 1 (TK1) activity, a marker of tumor cell proliferation, has shown promising initial results as a prognostic biomarker in dogs with multicentric lymphomas. The purpose of this study was to determine if baseline serum TK1 activity is associated with clinical outcome in dogs with CHOP-treated DLBCL.

Methods: Baseline serum TK1 activity was measured in banked sera from 100 dogs with CHOP-treated DLBCL using a commercially available ELISA kit. Data on other potential prognostic factors were obtained retrospectively from electronic medical records. Multivariable statistical methods were used to find associations between potential prognostic factors and progression-free survival (PFS) and attainment of complete remission.

Results: TK1 activity at baseline was not associated with reduced PFS (P = 0.299) or attainment of complete remission (P = 0.910) following CHOP chemotherapy. Of the other prognostic factors analyzed, only purebred (vs. mixed breed) status (HR 8.81, 95% CI 1.68-46.30, P = 0.010), attainment of complete (vs. partial) remission (HR 0.09, 95% CI 0.02-0.49, P = 0.006), and baseline serum C-reactive protein concentration (HR 1.19, 95% CI 1.07-1.32, P = 0.001) were independently associated with PFS.

Conclusion: Based on these findings, baseline serum TK1 activity does not appear to be a useful prognostic biomarker in dogs with CHOP-treated DLBCL.

Abigail Clifford - DVM

Comparison of Microstructural Values of Equine Proximal Sesamoid Bones at Different CT Resolutions

Abigail Clifford,1 Jesus Hermida,1 Hsin-Yi Weng,2 Timothy Lescun1

[1] Department of Veterinary Clinical Sciences, Purdue College of Veterinary Medicine, Purdue University, West Lafayette, Indiana
[2] Department of Comparative Pathobiology, Purdue College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

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

Research Grant: Purdue College of Veterinary Medicine Competitive Equine Research Funds and American College of Veterinary Surgeons Zoetis Dual Training Research Grant
Student support: Purdue College of Veterinary Medicine, Boehringer Ingelheim

Camryn Davis - DVM

Comparison of Agents to Maintain Hydration of Yucatan Minipig Skin

Camryn Davis1, Julie Brown RVT RALAT2 , Lee Matthews DVM2, Amanda Darbyshire DVM DACLAM2,3

1College of Veterinary Medicine; 2Department of the Executive Vice President for Research and Partnerships, Laboratory Animal Program; 3Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

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

Heather Fumia - DVM

Mathematical Modelling to Explore the Role of Environmental Factors in Avian Cholera Outbreaks in Snow Geese

Heather Fumia, Jess Dennehy, Harrison Clark, Wendy Beauvais

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana (Fumia, Clark, Beauvais); London School of Hygiene and Tropical Medicine, University of London, London, United Kingdom (Dennehy)

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

Student Support: Purdue College of Veterinary Medicine, Boehringer Ingelheim

Adrianne Glaser - DVM

Sexual Dimorphism in the Expression of Estrogen Receptor Beta in Rat Vocal Fold Tissues

Authors: Adrianne Glaser1, Abigail Cox1, Preeti M. Sivasankar2

Affiliations: Department of Comparative Pathobiology, College of Veterinary Medicine, and Speech, Language and Hearing Sciences, College of Health and Human Sciences, Purdue University, West Lafayette, IN

The sex hormones, especially estrogen, play an integral role in the maintenance of systemic hydration. This study’s purpose was to explore the inherent differences between male and female rats in estrogen receptor beta expression throughout the vocal fold and related structures within the larynx. This was accomplished by obtaining coronal sections of laryngeal tissues of ten female and six male rats, followed by immunohistochemistry staining for estrogen receptor beta throughout the sections. These areas were analyzed using digital slide analysis software to determine the percentage of positive staining for each identified area of interest. The areas chosen included true vocal fold epithelium, true vocal fold lamina propria, true vocal fold muscle, subepiglottic and submucosal glands within a single section. The results are pending statistical analysis however the raw data indicates that male rats have a lower level of expression of estrogen receptor beta in the true vocal fold epithelium based on the percentage of positive staining compared to females. There was no large variation between the percentage of strong positive staining in the lamina propria between males and females however females had more total positive staining. Within the true vocal fold muscle males had a higher percentage of strong positive staining. Within the subepiglottic and submucosal glands there was no large difference seen in the positive staining. These results indicate that there may be a difference in the level of expression of estrogen receptor beta throughout the vocal fold which may impact the response these structures have to dehydration challenges.

Malaycia Goldsmith - DVM

Increased Immune Responses by Intradermal and Intranasal Immunization with a Novel Adjuvant in Mice

Malaycia Goldsmith, Juan F. Hernandez-Franco, Harm HogenEsch

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

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

Kami Graber - DVM

Exploring Clinical use of Electroencephalography to Differentiate Anesthesia Depth in Horses - A Pilot Study

Kami Graber, Jeff Ko, Carla Murillo

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

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

Samantha Hatter - DVM

Pathogenic Bacterial Species in Porcine Hearts: Identifying Sources of Environmental Contamination for the Development of Superior Tissue Collection Protocols

Samantha Hatter, Stephanie Prahlow, Dr. Abigail Cox

Purdue University, College of Veterinary Medicine, Department of Comparative Pathobiology, IN, USA

Background: Microbial contamination is a barrier for medical professionals seeking animal tissue from reputable sources. A local tissue sourcing business provides swine hearts to a research laboratory, and recent heart samples collected at a local slaughterhouse from these animals revealed higher-than-average numbers of colony forming units. These large “bioburden counts” on the hearts make them unsuitable for research.

Objective: The study goal is to identify sources of contamination on biomedical porcine heart samples within an Indiana slaughter facility to minimize microbial contaminants.

Methods: Using sterile cotton tipped applicators, samples of equipment such as knives, gloves, collection pans, and swine are taken before, during, and after slaughter procedures. Samples from swine are isolated from the nasal and oral cavities of the specific animals utilized for biomedical research. The swabs are then placed into sterile bags, labelled appropriately, and transported to Purdue University for further processing. Following the growth period, sample colonies are identified utilizing in-house laboratory techniques and are recorded.

Results: Many of the bacterial contaminants found are common within the environment and porcine microflora, and do not necessarily indicate disease causing pathogens. Since the heart material swabbed at the time of slaughter contained minimal contaminants, it is likely that environmental contamination was the sole cause of high bioburden levels found by the research facility.

Conclusion: The repercussions if this issue went unresolved reach far beyond a simple research project: biomedical research is on the cutting edge of treatments for many diseases today and can improve the lives of real patients.

Alexander Rahn - DVM

The Safety and Efficacy of Extra-Label Nocita Use in Gastrointestinal Foreign Body Surgery

Authors: Alexander Rahn1, 3rd Year Veterinary Student, George Moore2, DVM, MS, PhD, DACVIM (SAIM), ACVPM (Epi), Marije Risselada1, DVM, DECVS, DACVS-SA, PhD

Affiliations: 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, Indiana 47907 2Department of Veterinary Administration, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, Indiana 47907

Despite its widespread use, limited data exists on safety and efficacy of Nocita use in small animal surgery. Gastrointestinal foreign body (GIFB) surgery was chosen as a well-defined, single surgery type. The OR Log at Purdue University Veterinary Hospital was used to identify all canine GIFB procedures conducted between May 2017 to August 2021. Additional patient information was obtained via electronic medical records (HIS), and included: recovery time, analgesic use and duration, other medications, complications. Patients were divided into: Nocita and non-Nocita. Patients that did not survive to discharge or lacked two-week follow-up were excluded. 205 dogs were included: 65 received Nocita, and 140 did not. Dogs that received Nocita were heavier (P=.005). Analgesic requirement (P=0.002 at 24hr; P<0.001 at 36, 48, and 60hr; P=0.006 at 72hr) and total dose required (12-24hr, 24-36hr, and 36-48hr (P<0.0001, P<0.0001, and P=0.0103) was less in the Nocita group. Postoperative wound complications were seen in 7/65 (Nocita, 10.77% CI=.044-.21) and 4/140 (non-Nocita, 2.86%, CI=.008-.072) dogs. While not statistically significant, increased wound complications could hold clinical significance. The main limitations to this investigation are the small cohort sizes impacting statistical power and variation in anatomic location of Nocita injection. Nocita reduces post-operative pain and analgesic requirements, however the risk for postoperative wound complications is higher after Nocita use in gastrointestinal surgery. Nocita is a viable adjunct to analgesic protocols, but caution should be used due to an increased complication rate associated with administration in GIFB surgery.

Max Rowley - DVM

Characterization of Immunoreactive cDNA Expression Library Clones of a Zoonotic Roundworm, Baylisascaris Procyonis

Max Rowley, Vishnu Manikantan, and Sriveny Dangoudoubiyam

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

Baylisascaris procyonis is an emerging zoonosis in North America, Europe, and Asia. Adult B. procyonis reside in the small intestine of raccoons and the eggs are shed in raccoon feces. Following accidental ingestion of eggs, the released larvae migrate within the host resulting in larva migrans in humans and over 150 different species of mammals and birds. Migrating B. procyonis larvae travel through somatic tissues (visceral larva migrans - VLM), the brain (neural larva migrans – NLM), and eyes (ocular larva migrans – OLM), inflicting serious damage. While several cases occur as covert disease, infection can lead to irreversible vision loss, encephalitis, and death. Larval proteins play a crucial role in the pathogenesis of larva migrans and this study aims to characterize the immunoreactive clones identified via screening of the larval cDNA expression library. Sequencing and bioinformatic analyses were performed to establish putative identities for these cDNA clones (Bp3.1-Astacin, Bp4- C type Lectin, Bp13- Nicalin and Bp14-Galectin). We also determined their protein parameters and presumed function(s) based on their molecular similarity to related nematodes. These clones were over-expressed in a bacterial expression system and the recombinant proteins were purified using metal-affinity chromatography. Western blot assay was used to determine their sero-reactivity. Experiments are ongoing to establish differential expression between various B. procyonis life cycle stages to determine if any of these is a larva-specific antigen. Overall, this study is part of a larger project aimed at discovery of B. procyonis larval proteins that play a role in host-pathogen interaction during larva migrans.

Research Grant: Departmental Start-up Grant, Purdue University
Student Support: Purdue College of Veterinary Medicine, Boehringer Ingelheim

Alaunie Smiley - DVM

Grape Toxicosis in Dogs: In Vitro Studies

Alaunie Smiley, Arthur Armstrong, Stephen B Hooser

Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, IN

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

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

Emily Willis - DVM

Stereotactic Radiation Therapy (SRT) Outcomes on the Treatment of Canine Nasal Tumors

Emily Willis, Isabelle Vanhaezebrouck

Department of Radiation Oncology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47906

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

Research Grant: None
Student Support: Boehringer Ingelheim, Purdue College of Veterinary Medicine

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