PVM Research Day Abstracts

Marwa Alhashimi - Basic

An adenoviral Vector-Based Combinational COVID-19 Vaccine Expressing Spike, Membrane, and Nucleocapsid Proteins Provides Enhanced T-Cell Response and Protection Against SARS-CoV-2

Marwa Alhashimi¹, Ahmed Elkashif¹, Shubhada K Chothe^2,3, Ekramy E Sayedahmed¹, Wen-Chien Wang¹, Padmaja Jakka^2,3, Abhinay Gontu^2,3, Santhamani Ramasamy^2,3, Lindsey Labella^2,3, Meera Surendran Nair^2,3, Ruth Nissly^2,3, Suresh V. Kuchipudi^2,3, and Suresh K. Mittal¹ 

¹Department of Comparative Pathobiology, Purdue Institute of Inflammation, Immunology and Infectious Disease, and Purdue University Center for Cancer Research, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.

²Department of Veterinary and Biomedical Sciences, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA.

³Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA, USA. 

The continuous emergence of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease-2019 (COVID-19), remains a major global health threat. The efficacy of the first-generation COVID-19 vaccines has been seriously undermined by the emergence of immune escape and highly transmissible variants, including the recent Omicron subvariants BA.2, BA.4, & BA.5. To develop the next-generation of the COVID-19 vaccine, we designed human adenoviral (HAd)-based vectors expressing spike (S), membrane (M), or nucleocapsid (N) proteins of SARS-CoV-2 with or without an autophagy-inducing peptide C5 (AIP-C5) (HAd-S/C5, HAd-S, HAd-M/C5, HAd-M, HAd-N/C5, and HAd-N). Mice were immunized intranasally (i.n.) with HAd vectored vaccines alone or in various combinations leading to the induction of significant levels of antigen-specific humoral (mucosal and systemic) and cell-mediated immune responses. The inclusion of AIP-C5 resulted in significantly higher levels of cell-mediated immune responses. Immunization with vectors expressing S-C5, M-C5 & N-C5 in a vaccine formulation resulted in considerably higher levels of antigen-specific cellular immune responses compared to the groups immunized with the vector expressing a single antigen. Vaccinated animals showed high virus-neutralizing antibody titers against delta and omicron variants. Immunized ACE2 transgenic mice were challenged with SARS-CoV-2 and monitored for morbidity and mortality for two weeks to investigate the protective efficacy. Significant reductions in morbidity and mortality were observed in the groups immunized with vectors expressing S-C5, M-C5 & N-C5. Our results suggest that the inclusion of M and N antigens with S correlated with improved protection and, therefore, can serve as a promising strategy for the next-generation SARS-CoV-2 vaccines.

 

---

Miriam Bates - Basic

In Vitro Antimicrobial Efficacy of Silver Nanoparticles Incorporated into Carriers for Sustained Release

Nanoparticulate silver (AgNP) is a versatile product with established bactericidal activity against a range of bacteria. The study had two objectives: 1) establish the in vitro bactericidal activity of AgNP against Methicillin-resistant Staphylococcus pseudintermedius (MRSP) and Escherichia coli using commercially available AgNP and 2) compare the in vitro bactericidal activity of free AgNP and AgNP incorporated into carriers for sustained release (SR-AgNP). It was hypothesized 1) antibacterial activity would be demonstrated against both MRSP and E. coli by free AgNP and 2) antibacterial activity would not differ between free AgNP and SR-AgNP. Meuller-Hinton broth was inoculated with bacteria at a variety of concentrations and incubated with 10 µg/ml AgNP in a microdilution assay plate, sub-cultured to blood agar, and incubated for determination of colony forming units (CFU). Results showed reduced bacterial growth up to a bacterial concentration of 10¹ CFU/ml for MRSP and 10³ CFU/ml for E. coli. Then, Meuller-Hinton broth was inoculated with bacteria at concentrations determined in objective one and incubated with 10 ug/ml AgNP incorporated into gelatin sponge, calcium sulfate hemihydrate (CSH) beads, or poloxamer 407 gel, sub-cultured to blood agar plates, and incubated for CFU determination. Free AgNP had greater antibacterial activity compared to SR-AgNP gelatin sponge and SR-AgNP CSH beads, but not SR-AgNP poloxamer 407 gel. This study did not assess the effect of wound bed characteristics. AgNP exhibits antimicrobial activity against MRSP and E. coli and has the potential for use in veterinary wound care.

 

---

Nayan Bhowmik - Basic

Two DLL3 Gene Mutations are Associated with Short Spine Syndrome in Dogs

N. Bhowmik, K. J. Ekenstedt Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN

Canine short spine syndrome is a collection of rare malformations affecting vertebral development, causing shortening and compression of various spinal segments; it has occurred sporadically since at least the 17th century. Two Coonhounds and one mixed breed dog were diagnosed with “short spine” via gross and radiographic examinations. Whole-genome sequencing (WGS) was performed in three related Coonhounds (two affected and one unaffected) and one affected mixed breed dog. The private variants of all four dogs were identified via comparison to 728 WGS control dogs representing 64 breeds. These variants were next filtered based on their predicted impacts (high, moderate, low) and how they altered protein sequences. VarElect software was then used to rank and prioritize disease-causing candidate genes. Both affected Coonhounds had a homozygous A to G missense variant in exon 7 of the DLL3 (Delta like Canonical Notch Ligand 3) gene. The related, unaffected Coonhound was heterozygous for this variant. Thirty-four unrelated and unaffected Coonhounds were also all homozygous wild type. The mixed breed dog had a splice acceptor variant (T>G) at the beginning of exon 5, also in the DLL3 gene. An additional 722 dogs representing 158 breeds, including mixed breeds, wild canids, and village dogs, were homozygous wild-type for both discovered DLL3 variants. The DLL3 gene has been previously associated with spondylocostal dysostosis, a heterogeneous group of axial skeletal disorders in humans [1]. The DLL3 protein is involved in the embryonic segmentation clock (part of the Notch signaling pathway); dysfunction affects segmentation of the vertebral column [2].

References

1) Bulman MP, Kusumi K, Frayling TM, McKeown C, Garrett C, Lander ES, Krumlauf R, Hattersley AT, Ellard S, Turnpenny PD (2000) Mutations in the human delta homologue, DLL3, cause axial skeletal defects in Spondylocostal dysostosis. Nature Genet 24: 438-441.
2) Online Mendelian Inheritance in Man (OMIM), https://www.omim.org. Entry #602768. Accessed March 21, 2023.

---

Jeanna Blake - Basic

A Splice Site Donor Variant in RBCK1 is Associated with Glycogen Storage Disease in Basset Hounds

Jeanna M Blake1, Andrew D. Miller2, Kari J. Ekenstedt1

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

2Department of Population Medicine and Diagnostics Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Glycogen storage diseases (GSDs) are rare, typically inherited, disorders caused by various defects in glycogen metabolism enzymes, generally resulting in the accumulation of glycogen in various tissues. Recently, postmortem histopathology of two adult Basset Hound (BH) littermates resulted in a diagnosis of GSD, manifesting in cardiac and smooth muscle. Using whole genome sequencing, a splice site donor variant was identified in a homozygous form in both littermates and in a heterozygous form in their unaffected sire; this splice donor variant is in exon 8 of RBCK1, a gene which encodes for an E3 ubiquitin ligase. The presumptive loss of the splice site donor introduces a stop codon within 22 codons following the inclusion of the intron. Screening for the variant in related (n = 6) and unrelated (n > 40) BHs identified one additional affected littermate and five additional familial heterozygous carriers. No variant alleles were present in the unrelated BH population, establishing the novelty of the identified mutation. Variants in RBCK1 have been associated with polyglucosan body myopathy 1, a type of GSD characterized by skeletal muscle myopathy and cardiomyopathy, as well as GSD due to glycogen branching enzyme deficiency in humans. To date, no reported variants in RBCK1 have been identified in dogs associated with GSD. The findings of this study add to the mutational spectrum of GSDs in dogs, permitting broader genetic testing for early diagnosis and for disease prevention through targeted breeding strategies.

---

Garrett Bryak - Basic

Human Beta Defensin-3, A Potential Therapeutic Antifungal Agent Against Candida auris Emerging Fungal Pathogen

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

---

Ying Cheng Chen - Basic

Examining Antitumor Potential of Small Molecule C3, a Mimic of the Glycoprotein Pigment Epithelium-Derived Factor

Ying Cheng Chen1, Lucas D. Johnson 1, Charles S. Umbaugh 1, Herman O. Sintim 2, and Marxa L. Figueiredo 1
1Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA

2Department of Chemistry, Purdue University, West Lafayette, IN, USA

Laminin receptor, 67 LR, and its precursor, 37 LR (collectively LR), are upregulated in various tumor types, including in prostate cancer, where LR is thought to aid in proliferation, metastasis, and reduced survival. LR has been shown to interact with the glycoprotein pigment epithelium-derived factor (PEDF), an anti-angiogenic factor, and this interaction is promising in reducing tumor growth. However, current delivery of PEDF in recombinant form involve peptide formulation or gene delivery which are expensive, unscalable, and complicated. Thus, a small molecule that could mimic the function of PEDF and interacts with LR to promote anti-tumorigenic and anti-angiogenic phenotypes could be of high interest and potential for cancer therapy. In our previous in silico work, a novel small-molecule, C3, showed promise as a candidate PEDF mimic. In vitro studies were conducted to analyze the efficacy of C3 through the criteria of anti-viability, anti-wound healing, anti-cancer signaling properties, and activation of PEDF signaling-related genes. In the present project, we validated the efficacy of C3 in vivo against prostate tumor growth using a syngeneic model of immune competent C57BL/6 male mice. Mice were implanted with TRAMP-C2Ras (TC2R), murine prostate cancer cells, subcutaneously and C3 efficacy was assessed by reduction in tumor size relative to the vehicle control group (2.5% DMSO) delivered intraperitoneally at two doses, 5 mg/kg (Low) and 10 mg/kg (High). The overall health of mice was continuously monitored through qualitative observations as well as quantitative measurements such as weight as a function of time.

---

Zihxia Chi - Basic

Repurposing of HJL-1 as a Novel PRMT5:pICln Protein-Protein Interaction Inhibitor for Prostate Cancer Therapy

Zhixia Chi1,2, Xuehong Deng1, Guangjun Zhang2, Michael K. Wendt1.

1. Purdue University Department of Medicinal Chemistry and Molecular Pharmacology, West Lafayette, IN.
2. Purdue University Department of Comparative Pathobiology, West Lafayette, IN.

Background: Prostate cancer (PCa) is the second most common cancer-related death in men
worldwide. Androgen receptor (AR) is expressed in nearly all primary prostate cancer. Inhibiting
AR function via androgen-deprivation treatment (ADT) or androgen receptor antagonists is the
first-line treatment for clinically localized and metastatic PCa. However, those methods
inevitably lead to castration-resistant prostate cancer (CRPC). Protein arginine methyltransferase
5 (PRMT5) is a type II methyltransferase. Increased expression of PRMT5 is positively
correlated to the progression of prostate cancer tissues. In recent decades, targeting the
enzymatic active site or the S-adenosyl methionine (SAM) binding pocket of PRMT5 has been
utilized to develop compounds and circulated them in clinical trials. Nevertheless, these
compounds could potentially impose irreversible damage to normal tissues, given the fact that
the catalytic and substrate-binding function of PRMT5 is critical for normal cell survival.
Cumulative evidence suggests developing inhibitors for PRMT5 protein-protein interaction (PPI)
could be attractive therapies for cancer treatment. We recently demonstrated that PRMT5
collaborated with pICln to promote prostate cancer growth via epigenetic activation of AR
expression. Utilizing the near-atomic cryo-EM structure of PMRT5:pICln and the structuralbased
virtual screen, our collaborators identified a small molecular therapeutic inhibitor (HJL-1)
of PRMT5:pICln protein-protein interaction. However, whether HJL-1 could provide a
promising treatment for prostate cancer therapy remains to be determined.

Methods: Utilizing prostate cancer cell lines, xenograft mouse model, Co-immunoprecipitation
(Co-IP) and bimolecular fluorescence complementation (BiFC), we investigated if HJL-1 could
decrease AR expression and imped prostate cell proliferation by disrupting PRMT5:pICln PPI.

Results and conclusion: Our CO-IP and BiFC results indicated HJL-1 impeded PRMT5:pICln
PPI in prostate cancer cells. Moreover, the proliferation and target gene expression of prostate
cancer cells that express the wild type of AR and drug-resistant AR-V7 splice variant was
decreased by HJL-1 in a time- and dosage-sensitive manner. Thus, our data suggest HJL-1 could
be a novel inhibitor of PRMT5:pICln PPI for prostate cancer therapy.

---

Diprasom Das - Basic

Understanding Microbiota and Host Immune Response to Develop Therapeutics against Candida auris Infections

Diprasom Das1, Harm HogenEsch1,2 and Shankar Thangamani1,2*
1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47906

2Purdue Institute for Immunology, Inflammation and Infectious Diseases (PI4D), West Lafayette, IN 47906

Candida auris, an emerging multi-drug resistant fungal pathogen, causes invasive infections in humans. The factors regulating the colonization of C. auris in host niches are not well understood. In this study, we examined the effect of antibiotic-induced gut dysbiosis on C. auris intestinal colonization, dissemination, microbiome composition and the mucosal immune response. Our results indicate that mice treated with cefoperazone alone had a significant increase in C. auris intestinal colonization compared to untreated control groups. A significant increase in the dissemination of C. auris from the intestine to internal organs was observed in antibiotic-treated immunosuppressed mice. Intestinal colonization of C. auris alters the microbiome composition of antibiotic-treated mice. Relative abundance of firmicutes members mainly Clostridiales and Paenibacillus were considerably increased in the cefoperazone-treated mice infected with C. auris compared to cefoperazone-treated uninfected mice. Next, we examined the mucosal immune response of C. auris infected mice and compared the results with C. albicans infection. The number of CD11b+ CX3CR1+ macrophages was significantly decreased in the intestine of C. auris infected mice when compared to C. albicans infection. On the other hand, both C. auris and C. albicans infected mice had a comparable increase of the number of Th17 and Th22 cells in the intestine. Significant increase in the C. auris-specific IgA was observed in the serum of C. auris infected mice compared to uninfected groups. However, no significant increase in the C. albicans-specific IgA was observed in the C. albicans infected mice compared to uninfected groups. Taken together, treatment with broad-spectrum antibiotic increased the colonization and dissemination of C. auris from the intestine. Furthermore, findings from this study for the first time revealed the microbiome composition, innate and adaptive cellular immune response to intestinal infection with C. auris.

---

Abhishek Datta - Basic

Modulating Bile Acids to Control Fungal Infections

Candida albicans (CA), a commensal gut fungus (mycobiome) and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract. Analysis of the gut microbiota so far has predominantly focused attention on the microbiome, while failing to underscore the critical role of the mycobiome in health and disease. Importantly, the factors regulating the colonization of mycobiome components in the GI tract and the role of gut fungi in the health and disease remain poorly understood. To address this gap in knowledge, our lab utilizes a combination of targeted metabolomics, 16S ribosomal RNA amplicon gene sequencing, and in-vivo mouse models to divulge the complex interaction between mycobiome, the metabolome and the microbiome. Our findings indicate that taurocholic acid (TCA), a major bile acid present in both humans and mice control the balance between commensalism and invasive CA infection originating from the gut. Oral administration of TCA through drinking water is sufficient to induce colonization and dissemination of CA in mouse models. Recent findings indicate that TCA regulates CA by controlling immune cells in the intestine. Here, we report that oral administration of TCA to CA-infected mice significantly decreased the number of mononuclear phagocytes and CD4+ IL17A+ T helper 17 cells that play a critical role in controlling CA in the intestine. Collectively, our results indicate that TCA modulates mucosal innate and adaptive immune responses to promote CA colonization in the intestine. Future studies to elucidate the mechanism(s) by which TCA regulates host defense will provide in-depth understanding of bile-mediated regulation of CA colonization in the intestine. Taken together, the gut mycobiome is gaining recognition as a fundamental part of our gut microbiota. Mycobiome have been increasingly found to contribute and play an active role in inflammatory bowel disease, microbiota-gut-brain axis, colonization and pathogenesis of enteric pathogens. Our findings will have broad implications for our understanding of the gut mycobiome in health and disease which is an underappreciated aspect of microbiome research.

---

Ziyu Dong - Basic

Characterization of Piezo Genes Expression in Zebrafish Early Embryogenesis

Authors: Ziyu Dong, GuangJun Zhang

Affiliations: Department of Pathobiology, PVM, Purdue University

Mechanosensing is a fundamental physiological process, including touch sensation, blood
pressure regulation, bladder function, etc. Recently, it is more evident that mechanosensing
play instructional roles during early embryogenesis. Piezo are a group of known ion channels
that transduce mechanic stimuli to bioelectric signals. Dysregulation of piezo channels has also
been linked to various diseases such as hypertension. However, the detailed function of piezo
genes remains poorly understood. Gene temporal and spatial expression are good indicators of
their functions. In this project, we took advantage of the zebrafish model, and examined piezo
gene expression in early staged embryos. We cloned 4 piezo genes from zebrafish and our
phylogenetic analysis revealed that piezo2 was duplicated as piezo2a and piezo2b, and we
isolated new piezo gene, piezo3, which is only found in the teleost, but not the tetrapod.
Moreover, we performed whole mount in situ hybridization to comprehensively characterize
the expression pattern of the four piezo genes. All the four piezo genes have distinct patterns,
suggesting each gene might specialized functions during zebrafish embryogenesis. Future
research will aim to manipulate piezo genes in vivo in zebrafish to further understand their
roles in development and regulation.

---

Said Elshafae - Basic

CART Cell Immunotherapy Targeting Cancer-Associated Receptors

Prostate cancer (PCa) is considered one of the highly frequent diagnosed malignancies among males global wide. PCa is considered a cold tumor with only few T and NK immune cells infiltrating tumor microenvironment (TME). PCa cells eventually evade CD8 T cell cytotoxicity by losing MHC I antigen presentation. Chimeric antigen receptor T (CART) cells (engineered T cells) could target an antigen on cancer cell surface independently of MHC-I engagement but their efficiency on solid tumors, i.e. PCa, is still not clear.
In this collaborative study with Dr. Philip Low, we used universal mouse Anti-FITC CART cells to target mouse PCa cells expressing human PSMA (MyC Cap PSMA). We performed LDH cytotoxicity assay, cytokine array analysis and RT-PCR to study the effect of CART cells on PCa cells in vitro. To assess the efficacy of CART cells on prostate tumor growth, MyC Cap and MyC Cap PSMA cells were injected subcutaneously in FVB immunocompetent mice. The mice were treated with Dupa-FITC (adaptor) for 1 month, and the body weight of mice, tumor volume and bioluminescence were measured. Dupa-FITC activated CART cells induced cytotoxicity of MyC Cap PSMA in a time and dose dependent manner compared to control MyC Cap cells. There was an elevation in the mRNA level of IL2 and protein level of IFNγ, IL13 and TNFα with downregulation of GM-CSF, VEGFA and CXCL1 upon incubation of Dupa activated CART cells with MyC Cap PSMA cells. Importantly, CART cells decreased MyC Cap PSMA bioluminescence and tumor volume and weight in FVB mice compared to control MyC Cap tumor. In addition, CART induced remarkable apoptosis of MyC Cap PSMA cancer cells. Blood and spleen of tumor implanted mice showed CART cells expansion compared to control mice. We concluded that targeting PSMA using universal CART cells could be a promising avenue
in treatment of local PCa.

---

Emily Hartman - Basic

The Shelf Life of Antibiotic Impregnated Calcium Sulfate Beads

E.A Hartman, DVM¹
D. Pena-Hernandez, DVM²
M. Risselada, DVM, PhD, DECVS, DACVS-SA¹
H.Y Weng, BVM, MPH, PhD²
M.G. Papich, DVM, MS³
S.Y. Kim, DVM, MS, DACVS-SA¹

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

^{2Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA}

^{3Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA} 

The objective of this project was to compare antimicrobial activity as demonstrated by zone of inhibition (ZOI) produced by antibiotic-impregnated calcium sulfate (CaSO₄) beads after storage for 0, 3, 6, 9 and 12 months. Three millimeter diameter CaSO₄ beads impregnated with vancomycin (50mg/mL) or amikacin (250mg/mL) or without antibiotic (control) were created at the onset of the study. Beads were separated for storage (light, temperature and humidity protected) for 3,6,9, or 12 months until testing. The ZOI against methicillin-resistant Staphylococcus pseudintermedius, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa was recorded with serial re-plating on a fresh lawn of bacteria every 24 hours until beads dissolved, fractured or failed to produce a ZOI. Elution samples were collected at 24 hours for objective measurement of antibiotic concentration via high-performance liquid chromatography (HPLC) The ZOIs and their changes were compared with mixed-effect linear models. Descriptive statistics of eluted concentrations of vancomycin measured via HPLC were reported. Zones of inhibition were consistent at 24 hours regardless of time since formulation, except vancomycin against P. aeruginosa which did not generate a ZOI at any point. The daily changes of ZOI and duration of activity of antibiotics did not vary between ages (p > 0.05). There was no consistent change in eluted concentration of vancomycin between different ages of beads. In conclusion, storage for up to 12 months did not impair the in-vitro activity of antibiotic-impregnated CaSO₄ beads as demonstrated through ZOIs.

 

---

Juan Franco-Hernandez - Basic

Cytotoxic T Cells Drive Antitumor Immunity Mediated by Intradermal Vaccination with a Nanoparticle Adjuvant and a STING Agonist

Juan F Hernandez-Franco¹ and Harm HogenEsch^1,2 

¹ Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University; ² Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907. 

Cancer vaccines are being developed for preventive and therapeutic applications to target and eliminate tumors. Cancer vaccines activate dendritic cells (DCs) and promote their migration to lymph nodes, where they stimulate CD8⁺ T cells to generate tumor-specific immunity. New adjuvants for cancer vaccines are needed as clinical trials have demonstrated only modest success thus far. This study investigated whether a combination adjuvant (NanoS100) containing cationic plant-derived adjuvant nanoparticles (Nano-11) and the synthetic STING agonist ADU-S100 (MIW815) enhances cross-presentation and antitumor immunity. NanoS100 increased CD80 and CD86 expression on DCs and stimulated cross-presentation of ovalbumin (OVA) as indicated by activation of SIINFEKL- specific B3Z CD8⁺ T cells in vitro. To demonstrate the induction of cytotoxic T lymphocyte (CTL) activity by NanoS100, we injected CFSE-stained donor splenocytes pulsed with SIINFEKL into vaccinated mice. A reduction of CFSE-positive cells confirmed the cross-presentation of tumor antigen by NanoS100-mediated CTL lysis of SIINFEKL-bearing target cells. Intradermal vaccination with NanoS100+OVA induced prophylactic immunotherapy by preventing the development of subcutaneously inoculated B16-OVA (melanoma) and E.G7-OVA (lymphoma) tumor cells in mice. The antitumor immunity of NanoS100 was confirmed by depletion of CD8⁺ T cells. To mimic therapeutic immunotherapy, subcutaneous inoculation of B16-OVA and E.G7-OVA tumor models were established in mice, followed by intradermal vaccination with NanoS100+OVA. The NanoS00+OVA immunotherapy significantly reduced tumor size and increased the survival rate of mice. The antitumor immunity produced by NanoS100 was validated by the establishment of potent humoral and cell-mediated responses, as shown by the induction of germinal center B cells, follicular helper T cells (Tfh), antigen-specific plasma cells, IgG, IgG1, and IgG2c production, and the differentiation of antigen-specific Th1 and CD8⁺ IFN-γ⁺ T cells. This study supports the further development of NanoS100 as a novel adjuvant platform for the design and delivery of STING-target immunotherapies, as it protected mice against tumorigenesis both prophylactically and therapeutically.

 

---

Annapoorani Jegatheesan - Basic

Characterization of SnROP9, a Rhoptry Protein of Sarcocystis Neurona

Annapoorani Jegetheesan1, Daniel K. Howe2 and Sriveny Dangoudoubiyam1

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West
Lafayette, IN. 2Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY.

Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis is a unique
apicomplexan lacking rhoptries in both merozoite (extracellular) and schizont (intracellular)
development stages. In apicomplexans, secretions from organelles viz., micronemes,
rhoptries and dense granules are crucial for successful intracellular parasitism. Rhoptry
proteins (ROPs) released via rhoptries facilitate invasion and establish a niche inside the host
cell. Surprisingly, despite lacking rhoptries, several ROP homologs were identified in S.
neurona proteome, of which SnROP9 was abundant. In this study, we have performed invitro
assays to determine protein characteristics of SnROP9 and its expression in S. neurona
developmental stages. For this purpose, CRISPR-Cas9 aided SnROP9-HA expressing S.
neurona line was successfully generated. The native (SnROP9) and fusion (SnROP9-HA)
forms were used as required. Analyses of SnROP9 protein demonstrated that is soluble and
secreted by the merozoites. In the intracellular schizont stages, SnROP9 showed consistent
expression throughout the schizont development. Immunolocalization revealed that SnROP9
localizes to either poles of the merozoite while it is scattered throughout the schizont body
during intracellular development. Also, SnROP9 did not localize to the micronemes and
therefore unlikely to be trafficked this organelle. Lack of dense granule markers precludes colocalization
assays to determine if SnROP9 homes to dense granules. Interestingly, both
SnROP9 and SnROP9-HA proteins were observed as multiple bands across all stages of
development. Presence of intact HA in all these forms is indicative of potential N-terminus
processing of SnROP9, not uncommon in apicomplexans. Additional experiments are
required to assess its biological role in S. neurona development.

---

Shreya Kumar - Basic

Combinatorial Theapy for Bone-Metastatic Prostate Cancer: A Chemo-Immunotherapeutic Approach

Shreya Kumar and Marxa L. Figueiredo

Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA

Prostate cancer is the second leading cause of cancer-related death among American men. It exhibits significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, patient sensitivity remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. We hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27) can treat bone-metastatic prostate cancer and can exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which recruits immune cells to the tumor, and promotes bone repair. To test this hypothesis, we performed in vivo experiments where syngeneic C57BL/6J mice were implanted with intratibial (cortical bone) TRAMP-C2ras tumors (TRAMP-C2 cells were infected with Lv-HrasG12V to make them suitable for growth in bone). Immunotherapy was administered in the form of gene therapy. Plasmid expressing IL-27, or an empty plasmid control, was injected intramuscularly. Ultrasound was used to aid gene delivery. Various gene-delivery methods were tested and optimized through in vivo studies with microbubbles in combination with ultrasound emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging (BLI) was used to monitor tumor size. Based on a pilot study, we anticipate that this combinatorial therapy will inhibit tumor growth. Further, RNA sequencing and cytokine arrays will be used to investigate the mechanisms involved. We hope that such chemo-immunotherapy will emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer while reducing chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and promoting healthy musculoskeletal tissue repair.
Source of research support: R01CA196947

---

Vishnu Manikantan - Basic

Insights into the Larval Excretory-Secretory Proteome of Baylisascaris procyonis, the Causative Agent of Eosinophilic Meningitis

Vishnu Manikantan1, Uma Aryal1,2 and Sriveny Dangoudoubiyam1

1Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, Purdue University, West Lafayette, IN. 2Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN

Baylisascaris procyonis is one of the three important parasitic causes of eosinophilic meningitis in humans that either ends fatally or causes life-long neurological deficits. Infection occurs upon ingestion of infective B. procyonis eggs from environmental sources contaminated with excreta of raccoons harboring adult worms in their intestine. In humans, ingested eggs hatch in the intestine, releasing larvae that undergo extra-intestinal migration, and 5-7% of larvae reach the brain. Migrating larvae release a repertoire of proteins, excretory-secretory products (ESPs) that facilitate penetration, destruction and inflammation of the host-tissue. In this study, aseptically hatched B. procyonis larvae were maintained in standard tissue culture medium to enable release of ESPs by metabolically active larvae. This larval ESP rich culture medium was collected, dialyzed, concentrated and analyzed by mass spectrometry. The Baylisascaris larval proteomics data was processed using MaxQuant and various bioinformatics tools. In the absence of B. procyonis “-Omics” data, putative identities of 269 larval ESPs were established by matching with proteome data of related nematodes. Proteins belonging to transthyretin-like family and those with calcium binding domains were predominant of the secreted proteins. Potential immunomodulatory proteins like macrophage migration inhibitory factor, glutathione S-transferase, galectins and peroxiredoxins were identified. Homologs of nematode-specific proteins viz., polyprotein allergens (immunodominant antigens), calreticulin (vaccine candidate) and calumenin (drug target) were found in Baylisascaris larval ESPs. Using B. procyonis larval transcriptome assembly currently under generation, we envision to further analyze B. procyonis larval ESPs that will be fundamental for development of strategies to mitigate harmful effects of eosinophilic meningitis.

---

Rodrigo Mohallem Ferreira - Basic

Prolyl Isomerase PIN1 Modulates Oncogene Induced Senescence by Regulating PML-Nuclear Body Dynamics

Rodrigo Mohallem1,2 & Uma K Aryal1,2
1Department of Comparative Pathobiology, Purdue University, USA; 2Bindley Bioscience Center, Purdue University, USA.
Somatic cells accumulate several mutations during an organism’s life span. Cells, however, have developed intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, most notably, oncogene-induced senescence (OIS). OIS is a process in which cells enter a state of permanent cell cycle arrest in response to the activation of proto-oncogenes, such as RasG12V. In this study, we explore the mechanisms of OIS using an integrated proteomics and molecular genetics approaches. As a model of OIS, we use IMR90 human diploid fibroblasts that were transduced with the inducible protein ER:RasG12V lentivirus. OIS was induced by treating IMR90-ER:RasG12V cells with 100nM (Z)-4-Hydroxytamoxifen (4-OHT) for 0, 2, 4 or 6 days. Purified nuclear proteins and phosphoproteins were analyzed by LC-MS/MS. Our results demonstrate that protein phosphorylation is significantly increased after Ras activation, and a majority of differentially regulated phosphosites during OIS were ERK1/2 target sites. Interestingly, phosphorylation at these motifs is recognized by the Prolyl Isomerase Pin1 protein, which isomerizes phosphorylated proteins and regulates their localization, function and interactions. Our results show that Pin1 is a key regulator PML-NB dynamics, and it is required for the senescence-dependent increase in PML-NB foci numbers. We have found that the constitutive PML-NB protein SP100 protein levels increase only in the control cells but not when Pin1 is depleted, with an observed increased cell proliferation and loss of senescence phenotype. Our data provides new evidence that Pin1 acts as a tumor suppressor in fibroblast cells during OIS.

---

Grace Mulia - Basic

Development of Interleukin-27 (IL-27) Based Targeted Gene Therapy to Treat and Prevent Covid-19

Since 2020, COVID-19 pandemic has forever changed millions of lives worldwide, with over 600 million positive cases and 6 million deaths globally. Preventative measures, such as vaccines have since been developed and distributed, and yet breakthrough infections are seen even in vaccinated individuals. Moreover, current therapies are mostly treating symptoms, thus there is still a dire need of targeted therapies that can prevent lung damage and mortality. COVID-19 patients are shown to have an imbalance in their cytokine expression, mainly upregulation in pro-inflammatory cytokines such as IL-6, IL-8, IL-1 and TNF-α leading to cytokine storm, organ damage and even death. Therefore, perhaps a therapeutic that can both reduce SARS-CoV-2 infection and rebalance the cytokine profiles through immunomodulation is crucial to prevent and treat SARS-CoV-2 infection. Interleukin-27 (IL-27) is a multifunctional cytokine involved in multiple inflammatory signaling pathways and has shown promise in its ability to rebalance lung and other tissue microenvironments. We hypothesize that the multifunctional IL-27 biologic agent can rebalance the COVID-19 inflammatory microenvironment tissue culture models. Our previous in silico data analysis showed the potential of IL-27 in inducing antiviral genes and rebalancing cytokine imbalance. We now test our hypothesis in an in vitro setting by examining the potential of our ACE2-targeting IL-27 in preventing the entry of GFP-expressing SARS-CoV-2 Spike pseudotyped lentivirus in epithelial cells with overexpression of ACE2 receptor (HEK293-ACE2). We subjected HEK293-ACE2 in various ACE2 blocking conditions prior to the administration of lentivirus, followed by analysis of GFP expression using IncuCyte. Our results shows promise of ACE2 targeting IL-27 in human adipose stromal cell conditioned media to reduce lentivirus entry compared to control and muscle cell conditioned media. However, no significant difference is yet to be seen between non-targeting and ACE2 targeting IL-27 conditioned media. Thus, we will continue to improve our current assay, particularly using alternative means to increase baseline GFP expression and ACE2 targeting peptides used in our therapy. We hope to continue determining the potential of ACE2-targeting IL-27 therapy in reducing SARS-CoV-2 infection and restoring cytokine imbalance, which could lead to novel approaches to reduce the progression and severity of COVID-19.

---

Sung Jun Park - Basic

Transient and Ectopic Expression of Kcnj10a May Cause Elongated Fins in Two Adult Zebrafish Insertional Mutants

Authors: Sung Jun Park, GuangJun Zhang

Affiliations: Department of Comparative Pathobiology, Purdue University 

Background: Vertebrate limb/fin is a great model for us to understand how organ size and shape are determined. Despite the significant impact of patterning mechanisms in vertebrate zebrafish fins, the detailed patterning mechanisms remained largely unexplored. A retroviral insertional forward genetic screening generated two long-fin zebrafish mutants, Dhi862 and Dhi4458. Genetic analysis revealed that changes in the kcnj10a gene are responsible for their long fins in both mutants. Methods: First, we performed morphological characterization of the two mutants by measuring adult and larva fin, body size and whole mount in situ hybridization on zebrafish mutant embryos to access the kcnj10a gene expression. To further validate the function of kcnj10a transient and ectopic expression, we utilized the CRISPR-Cas9 system to mutate the kcnj10a coding region. Result: We found that one or two copy transgenic alleles in Dhi862 and Dhi4458 mutants developed elongated fins compared to wildtype siblings. The fin ray segments of Dhi862 and Dhi4458 were altered in segment number and length. In addition, we identified transient ectopic expression of kcnj10a in the whole somite and notochord in mutant fish embryos. Removal of the ectopic expression using the CRISPR-Cas9 system, Dhi862 long-finned phenotype can be rescued. Conclusion: Here, 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 cause the long-fin development.

---

Worapat Sawatwong - Basic

Bone Strain Distribution on (Re)modeling Transcriptome-Level Responses to Two Different Loading Modes

Worapat Sawatwong1,2, and Russell P. Main1,2

1Musculoskeletal Biology and Mechanics Lab, Department of Basic Medical Sciences, Purdue
University, West Lafayette, IN, USA
2Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA

Osteocytes (Ots) sense and transduce mechanical signals, and mediate bone
(re)modeling in response to mechanical loading. Previous studies showed that loadinduced
strain distribution influences the osteogenic response of the skeleton. Among
various in vivo loading models, bone appears to be more sensitive to non-physiological
strain distribution compared to strains elicited during normal locomotion. However, the
effect of tissue-level strain distribution on the activity of Ots remains unclear. This
study aims to reveal the effects of differences in strain distribution on the gene
expression-level of the (re)modeling response. Four female C57BL/6 mice with strain
gauges attached to the tibiae were subjected to the tibial axial compressive (AC,
physiological) and medial-lateral cantilever-type (ML, non-physiological) loading
modes. Finite element analysis was performed to calibrate the strain at the posteriorlateral
(PL) region where Ots are exposed to opposite strain directions in AC and ML
models. The AC model requires a relatively higher load (3.5N) than the ML model (2N)
to generate the absolute strain magnitude of +1000 μe in the PL region. Our next step
is to acquire 16wk old C57Bl/6J mice subjected to either tibial AC or ML loading for
three days (n=4/group). The tibial cross-sectional cortical bone will be collected and
subjected to spatial transcriptomics analysis to reveal the strain distribution effects on
different Ot gene expressions and potential causes for bone (re)modeling. The
outcome of this study is essential for understanding mechanosensation and
mechanotransduction at the Ot, which could contribute to better osteoporosis clinical
intervention through mechanical stimulation or drug development.

---

Dingxun Wang - Basic

MTAP-Deficient Malignant Peripheral Nerve Sheath Tumors are Sensitive to PRMT5 Inhibition

Dingxun Wang1, Chang-Deng Hu2, GuangJun Zhang1

1 Department of Comparative Pathobiology, Purdue University
2 Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive sarcoma
with a poor prognosis and high relapse rate. Currently, surgical removal is still the
mainstay of the treatment, which is always limited due to tumor size and location. No
effective targeted therapy is available for this type of malignancy. Thus, a novel and
efficient therapeutic drug target is urgently desired. PRMT5, a type II protein arginine
methyltransferase, has recently been reported as a promising therapeutic target for
various types of human cancers with methylthioadenosine phosphorylase (MTAP)
deleted. The loss of MTAP caused cancer cells to become heavily dependent on the
PRMT5 arginine methyltransferase. The high reported rate of MTAP deletion led us to
hypothesize that PRMT5 is an effective therapeutic target for MPNST. We performed
immunohistochemistry on 19 human MPNST patient samples and found that PRMT5
expression was significantly increased compared to the adjacent normal tissue.
Consistently, we observed relatively high expression of PMRT5 in most human
MPNST cell lines compared to the immortalized Schwann cells. Furthermore, we
inhibited PRMT5 using shRNA and potent chemical inhibitors (JNJ-64619178 and
MRTX1719) in the MPNST cell lines with high or low MTAP levels. We discovered
that cell growth of MTAP-low MPNST cell lines was significantly suppressed while
limited effects on cell growth of MTAP-proficient MPNST cell lines were observed.
Our results suggested that PRMT5 potentially be a therapeutic target for
MTAP-deficient MPNST patients.

---

Wen-Chien Wang - Basic

Role of the Stem Region of Hemagglutinin and Ectodomain of Matrix Protein 2 in Protection Against Influenza Virus

Wen-Chien Wang1, Ekramy E. Sayedahmed1, Marwa Alhashimi1, Ahmed Elkashif1, Suryaprakash Sambhara2, Suresh K. Mittal1

1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN; 2Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA

Influenza viruses are responsible for millions of cases globally and significantly threaten public health. Since pandemic and zoonotic influenza viruses have emerged in the last 20 years and some of the viruses have resulted in high mortality in humans, a universal influenza vaccine is needed to provide comprehensive protection against a wide range of influenza viruses. Currently available seasonal influenza vaccines provide strain-specific protection and thus are less effective against mismatched strains. In this study, we target the conserved influenza domains, including the stem region of hemagglutinin (HA2) and the ectodomain of matrix protein 2 (M2e), to evaluate their roles in developing a broadly protective influenza vaccine. The secretory or anchored form of HA2 stem and tandem M2e linked with the autophagy-inducing protein (AIP) C5 (AIP-C5) were expressed in bovine or human adenoviral (Ad) vectors. The prime-boost immunization of mice with bovine and human Ad showed a comparable increase in the expression of interferon-gamma (IFN-γ) and interleukin 2 (IL-2) in splenocytes, mediastinal lymph node cells, and lung lymphocytes, indicating the development of HA2-specific cellular immunity. Besides, the comparable antibody level against HA was shown in all the candidate vaccines, signifying the induction of humoral immunity. Immunization and challenge study with an H5N1 reassortant influenza virus demonstrated a comparable decrease in lung viral titers in all candidate vaccine groups. Further challenge studies with other influenza subtypes will determine the broadly protective efficacy of HA2- and M2e-based Ad vaccines.

---

Marwa Ali - Clinical/Applied

Unraveling the Interactions Between Free-living Amoebae and Mycobacterium avium subsp. Paratuberculosis: A Novel Framework

Free-living amoeba (FLA) can act as vectors for maintaining and transmitting pathogens in the environment. This study uses mathematical modeling to investigate the population dynamics of FLA and Mycobacterium avium subsp. paratuberculosis in drinking water troughs used for cattle. A novel compartmental model was developed, and numerical simulations were run in R, to explore the impacts of different parameter values. 

Preliminary results show that carrying capacity and water flow rates are important drivers of amoebal growth in drinking water troughs and other factors. These findings suggest that this new mathematical model canbe used to explore the complex relationships between microorganisms and their environments. In conclusion, this study provides a foundation for future research efforts to investigate the growth and dynamics of amoebae-bacterial systems using mathematical modeling techniques.

---

Jessica Craig - Clinical/Applied

Understanding Perspectives on Health, Disease, Biosecurity, and Antimicrobial Use Among Poultry Owners in Rural and Urban Lilongwe, Malawi: A Qualitative Study

In Malawi, poultry are the most commonly owned livestock among households nationally; in Lilongwe, the capital city, around 50% of households own poultry. Small-scale poultry farming plays an important role in meeting household nutrition requirements and mitigating food insecurity while also reducing gender inequality and poverty. However, contact between backyard poultry and their owners presents an avenue for the transmission of infectious diseases including foodborne, zoonotic, and drug-resistant pathogens. We hypothesize that small-scale poultry owners may have relatively high rates of direct contact with domestic poultry which in turn have high rates of direct contact with wild birds, increasing the risk of infectious diseases transmission between wild birds, domestic poultry, and humans posing potential societal risks including zoonotic transmission to human owners, transmission to commercial farms which may result in regional economic impacts and food supply challenges, and the emergence of pathogens with outbreak and pandemic potential. Biosafety and biosecurity, antimicrobial use, and animal husbandry practices in small-scale backyard poultry systems in urban and peri-urban settings in Lilongwe have not been well described; our aim was to fill this gap and to qualitatively describe backyard poultry owners’ perceptions of poultry health and disease. We utilized purposive sampling to interview 13 poultry farmers across 3 communities in Lilongwe; study recruitment continued until thematic saturation was reached. We analyzed data using thematic analysis and summarize findings across 7 thematic areas.

 

---

Nelly Elshafie - Clinical/Applied

MicroRNAs in Canine Diffuse Large B-Cell Lymphoma: Implications for Prognosis

Nelly O. Elshafie1, Ekramy. E. Sayedahmed1, Marwa Ali1, Michael O. Childress2, Andrea P. dos Santos1

1Purdue University, Department of Comparative Pathobiology , 2Purdue University, Department of Veterinary Clinical Sciences

Lymphoma is one of the most communal malignancies in dogs and occurs in various forms, including multicentric, thymic, gastrointestinal, cutaneous, and solitary forms. Canine diffuse large B-cell lymphoma (DLBCL) is the highest reported and aggressive Non-Hodgkin lymphoma (NHL) subtype in dogs, with much-shared similarities with its equivalence in humans. The disease progression is hasty, and the treatment can be overwhelming. Investigators are studying using microRNAs as a prognostic indicator to enhance outcomes. In this research, we examined the correlation between progression-free survival (PFS) in dogs with DLBCL and the level of miRNA expression. MicroRNAs (miRNAs), small non-coding RNA molecules, are crucial for downstream gene transcription regulation. They participate in several pathophysiologic processes, including the growth and progression of cancer. Based on the analysis of small RNA sequencing (sRNA-seq) data using PFS, we validated and quantified the expression of the microRNAs using quantitative PCR from 44 archived lymph nodes retrieved from dogs with DLBCL with known outcomes. We then correlated these expression values with PFS. According to our findings, a particular miRNA subset is strongly linked to lower PFS in dogs with DLBCL. MiR-16-5p, miR-125a, miR192-5p, and miR-187-3p, in particular, were markedly upregulated in dogs with lower PFS. This result suggests that miRNA profiling offers promise for foreseeing PFS in DLBCL-affected dogs. Creating individualized treatment plans for canine DLBCL patients may eventually result from identifying particular miRNAs that correlate with the course of the disease.

---

Hamideh Esmaeilzadeh - Clinical/Applied

Evaluation of Folate Receptor Beta in Activated Synovial Macrophages of Dogs with Stifle Osteoarthritis Associated with Cranial Cruciate Ligament Rupture

Hamideh Esmaeilzadeh1*, Camila Benaduce Emanuelli Mello1, Nelly O Elshafie1, Sarah Malek2, Andrea
Pires dos Santos1

Purdue University College of Veterinary Medicine, 1 Department of Comparative Pathobiology, 2
Department of Veterinary Clinical Sciences

Osteoarthritis (OA) is a progressive and destructive joint disease. The activated phenotypes of
macrophages with folate receptor-ß (FRβ) are involved in OA-related inflammation in humans
and are potential targets for early detection and therapeutic interventions. The presence of FRβ
in activated synovial macrophages in dogs with OA has not been previously demonstrated. Our
objectives were to evaluate the expression of FRβ in synovial macrophages and determine the
difference in polarization of macrophages into inflammatory phenotype (M1) and antiinflammatory
phenotype (M2) between OA and control dogs. Synovial fluid from the knee was
collected from ten dogs with OA due to cranial cruciate ligament rupture and ten controls.
Straight preparations were made to estimate total nucleated cell count, and cytocentrifuge
preparations fixed with acetone. Immunocytochemical staining for FRβ, M1 marker inducible
nitric oxide synthase (iNOS), and M2 marker arginase 1 (ARG1) were performed. Positive large
mononuclear cells were counted. The number and percentage of FRβ-positive cells in the OA
samples were increased compared to controls (p < .05). The number of iNOS-positive cells was
increased in the OA compared to control samples (p < .05). The percentage of iNOS-positive
cells was higher than ARG1-positive cells in the OA samples. In conclusion, activated
macrophages with FRβ are present and detectable in synovial fluid of dogs. Higher expression
of FRβ in activated macrophages in canine OA may be further explored as potential therapeutic
targets. A higher percentage of iNOS-positive cells in canine OA indicates a polarization of
synovial macrophages toward a proinflammatory phenotype.

---

Samrin Habbani - Clinical/Applied

Identification of Critical Protein Biomarkers in Cervical Cancer Screening

Cervical cancer is a lethal gynecological malignancy and the fourth most frequent malignancy
affecting women worldwide. According to a World Health Organization estimate, there were
604,000 new cases and 342,000 mortalities from cervical cancer in 2020. There is an unequal
cervical cancer burden between high-income and low- and middle-income countries, and 90% of
cervical cancer fatalities yearly occur in low-to-middle-income nations. Due to the lack of
resources in these areas, applying existing cervical cancer screening methods is difficult because
of their limitations, including being expensive, time-consuming, technician-dependent, and
producing results with low sensitivity and selectivity. To address these challenges, there is a great
need for an accessible and low-cost screening test with sensitive, accurate, and quick results. Thus,
in collaboration with the biomedical engineering department, we are developing a simple pointof-
care test based on four critical biomarkers for screening high-risk pre-cancer and invasive
cervical cancer. This part of the work focuses on validating the four protein markers' expression
and the positive control Beta-actin in cervical cancer cell lines, cervical clinical swabs, cervical
cancer tissues, and precancerous tissues and quantifying the amount of the four proteins and β-
actin in clinical swab samples. To achieve our goal, we initially validated the expression levels of
the four protein biomarkers in cervical cancer cell lines using a western blot. Then
Immunohistochemistry was done to detect the four markers in cervical tissues (normal, low, and
high-grade cervical intraepithelial neoplasia, invasive cervical cancer). The four markers were
expressed in cervical cancer cell lines, cervical cancer tissues, and high-grade cervical
intraepithelial neoplasia. Finally, we measured our target proteins and Beta-actin concentrations in
clinical cervical swabs using commercially available enzyme-linked immunosorbent assay kits.

---

Stefanie Hansen - Clinical/Applied

An Ex Vivo Comparison of Cortex Screw Sizes to Create a Tension Band for Arthrodesis of the Equine Metacarpophalangeal Joint

Authors: Stefanie H. Hansen¹ and Timothy B. Lescun¹

Affiliations: ¹Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine

Abstract: The biomechanical strength of the tension band created by screw fixation of proximal sesamoid bones (PSB) as used in arthrodesis of equine metacarpophalangeal joints is unknown. The aim of this study was to compare the biomechanical properties of two screw sizes (4.5-mm and 5.5-mm cortex screws) inserted into the PSB. Ten pairs of skeletally normal forelimbs were used in the study. Two cortex screws were placed using lag technique through the third metacarpal condyles into the center of each PSB. Following screw insertion, 5 limb pairs were loaded in axial compression from 250N – 1.5kN for 3600 cycles in non-destructive cyclic testing. Subsequently 10 pairs underwent single cycle to failure under controlled axial loading. Mean stiffness (kN/mm) and failure load (kN) were compared between groups. Cyclic testing showed no difference in construct stiffness between the first and last 5% of cycles. All constructs failed by transverse fracture of the PSBs. There was no difference in construct stiffness between the 4.5-mm (0.448 ± 0.278kN/mm) and 5.5-mm (0.437 ± 0.197kN/mm) screw groups (P = 0.907). Mean failure load was similar between groups ((2.932 ± 0.485kN; 4.5-mm vs 3.385 ± 0.761kN;5.5-mm, P= 0.103). Limitations of this study include small sample size and lack of cyclic testing to failure. Our results show that in this mode of loading, screw size is not a critical determinant of tension band strength. The suspensory apparatus and PSBs are the weakest part of the construct when lag technique of the PSB is used in metacarpophalangeal joint arthrodesis. Results supports reported successful use of both screw sizes in clinical cases.

 

---

Jobin Kattoor - Clinical/Applied

Use of Targerted NGS for Pathogen Surveillance in Dogs from Indigenous Commuities in Brazil

Jobin Kattoor1, Andrea dos Santos1, Rebecca Wilkes1

1Department of Comparative Pathobiology, Purdue University.

Pathogen surveillance has been performed with next-generation sequencing (NGS) metagenomics because of its potential to detect any pathogen in a sample; however, it lacks the sensitivity needed for effective diagnostic use. Targeted NGS is the selective capture or amplification of nucleic acids of interest in a sample prior to sequencing, and if targeting pathogens, it can be used for clinical diagnostics because of the sensitivity and specificity of this method rival that of real-time PCR. Given the ability to include thousands of primers in a targeted method, it can be expanded for detecting pathogens for multiple syndromes or pathogens from multiple animal species, making it also ideal for surveillance.
We developed a targeted NGS panel for detecting 75 canine and feline pathogens using AmpliSeq custom-designed primers, automated library prep, and an Ion GeneStudio S5. This panel detected vector-borne pathogens from nucleic acids extracted from whole blood samples from 149 dogs from nine Indigenous communities in southeast and southern Brazil.
This test method detected nine different vector-borne disease pathogens in these dogs. Co-infection with multiple vector-borne agents was standard in this population. Though vector-borne diseases were of specific interest, we were able to detect five additional unrelated pathogens of clinical significance (including canine parvovirus and canine distemper virus) because of the comprehensiveness of the panel, and sequences obtained from the samples with this method confirmed the results.
Targeted NGS is an effective method for surveillance use for canine populations with unknown infectious disease history.

---

Eric Kontowicz - Clinical/Applied

Risk Assessment Framework for Interspecies Influenza Transmission on an Indoor Hog Grower Unit

Authors: Eric Kontowicz1, Daryl Ragland2, Max Moreno-Madrinan3, Wendy Beauvais1

Affiliations: 1. Department of Comparative Pathobiology College of Veterinary Medicine, Purdue University, 2. Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 3. Global Health Program, DePauw University
This work funded by the National Pork Board Grant NFB #21-100

The purpose of our study was to assess risks of interspecies influenza transmission (pig-to-worker or worker-to-pig) within a typical indoor pig grower unit in the midwestern United States.
We adopted the World Organization for Animal Health (WOAH) risk assessment framework and conducted separate risk assessments for H1N1, H1N1pdm09, H3N2, H3N2v, H1N2, and H1N2v. Semi-quantitative probability assessments (ranging from negligible to highly likely) were based on literature review, influenza surveillance data, and author agreement where data was lacking. We assumed a single production cycle (~26 weeks) on an indoor pig grower farm with 4,000 pigs, two workers, and no influenza vaccination. Uncertainty categories (ranging from low to high) were based on data availability and agreement between sources.

We assessed the following conditional risk steps: What is the likelihood that…1a) at least one piglet brought onto the farm is infected with influenza?; 1b) at least one worker is infected with influenza from an infected pig on the farm;1c) an infected worker develops influenza symptoms?; 2a) at least one swine worker is infected with influenza outside of swine farm?; 2b) at least one pig is infected by a worker; 2c) the newly infected pig will develop symptoms of influenza?

The probability of interspecies transmission events during a production cycle was found to be high for most influenza subtypes and associated with high uncertainty levels due to an overall lack of empirical data. Further studies are needed to improve the precision in estimates for frequency of interspecies transmission in an indoor growing pig setting.

---

Victor Oppenheimer - Clinical/Applied

Trends and Risk Factors for Multidrug-Resistant Canine Urinary Tract Infections (2009–2013; 2017–2021)

Oppenheimer Lúgaro V, Weng HY, Hui TY, Guptill L

Background – Recent evidence suggests the proportion of multidrug-resistant (MDR) urinary tract infections (UTIs) in dogs is increasing.

 Objective – To characterize antimicrobial resistance trends in canine UTIs and determine risk factors associated with development of MDR UTIs.  

 Animals – 1,106 bacterial isolates from 759 dogs treated at a veterinary teaching hospital

 Methods – Isolate susceptibility data from urine cultures were retrospectively evaluated over 2 time periods (2009–2013; 2017–2021). Information extracted from medical records included signalment, urinalysis findings, classification of sporadic or recurring UTI, presence of lower urinary tract signs, urolithiasis, neoplasia, chronic kidney disease, endocrinopathies, urinary tract anatomical disorders, micturition disorders, previous antimicrobial or immunosuppressive drug use, and history of indwelling catheterization. Chi-square tests and random-effect logistic regression were used to assess temporal changes and risk factors.

 Results – There were increases in MDR isolates between the 2 time periods (P < .001) and extensively-drug resistant (XDR) isolates from 2017–2021 (P < .001). Common isolates included Escherichia coli (40.1%), Staphylococcus spp. (14.3%), and Enterococcus spp. (12.9%), all with an increasing trend in antimicrobial resistance from 2009–2021 (P < .001). Significant risk factors for MDR UTIs included previous antimicrobial and immunosuppressive therapy (P < .001 and P = .044 respectively), micturition disorders (P = .005), and previous catheterization (P < .001).

 Conclusions and Clinical Importance – The increase in MDR/XDR UTIs emphasizes the urgent need for antimicrobial stewardship and the importance of urine culture and susceptibility, especially in dogs with previous antimicrobial or immunosuppressive drug use, micturition disorders, or previous catheterization.

---

Arielle Ostrager - Clinical/Applied

Survival in Dogs with Meningoencephalomyelitis of Unknown Etology with and without Magnetic Resonance Imaging Lesions

Arielle Ostrager VMD1; George E. Moore DVM, PhD2; R. Timothy Bentley BVSC (Dist),
MRCVS, DACVIM (Neurology)3

1, 3: Department of Veterinary Clinical Sciences, Purdue University College of Veterinary
Medicine
2: Department of Veterinary Administration, Purdue University College of Veterinary Medicine

Background: The prognosis of dogs with meningoencephalomyelitis of unknown etiology
(MUE) is difficult to predict. The purpose of this study was to determine the frequency of normal
MRI patients with MUE, and to determine whether the absence of MRI lesions affects prognosis
in patients with MUE.

Methods: 73 client-owned dogs with a clinical diagnosis of MUE presenting to Purdue
University Veterinary Hospital from 2010-2020 had their medical records reviewed. Patient
clinical data (presenting complaint, MRI findings, CSF results, MRI findings, presence of mass
effect, administration of prednisone and immunosuppressive medications, survival in months,
and cause of death were documented. Survival due to disease was compared between normal-
MRI and abnormal-MRI dogs.

Results: Death due to MUE occurred in 5% of normal-MRI dogs and 33% in abnormal-MRI
dogs. The incidence of death due to MUE significantly differed between normal-MRI and
abnormal-MRI dogs (p= 0.016)

Conclusions: Survival due to disease exceeded the 10-year study period in >50% of patients in
both groups, suggesting improved survival time than has been previously reported in patients
with MUE. Normal-MRI patients with MUE have improved survival compared to abnormal-
MRI patients with MUE.
.

---

Daniela Pena Hernandez - Clinical/Applied

Evaluation of RapidChek™ SELECT® for the detection of Salmonella spp. in environemental Samples From a Veterinary Hospital

Daniela Peña1,2,3, Joneson Jessica2, G. Kenitra Hendrix1,2

Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine1, Indiana Animal Disease Diagnostic Laboratory2 correspondence: dpeahern@purdue.edu

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 protocol (SCP) for detecting Salmonella spp. in environmental samples using a 48-hour enrichment step results in a 5-day turnaround time for negative results. The RapidChek® SELECT™ Salmonella (RCSS) test system offers detection of organisms in 22-44 hours through a double enrichment and a lateral-flow immunoassay (LFIA). Negative results are reported within 48 hours. At the Indiana Animal Disease Diagnostic Laboratory (IN-ADDL), we compared the performance of RCSS to a SCP on recovering Salmonella spp. from environmental samples. The study included ten environmental samples collected at the large animal Purdue Veterinary Hospital (LA-PVH) using electrostatic (Swiffer®) wipes and 20 wipes artificially spiked with two different concentrations of ATTC25923 S. Typhimurium. After completion of comparison experiments, a clear time advantage and higher sensitivity led to the adoption of RCSS as the primary method for testing environmental samples submitted by the LA-PVH. RCSS was used according to manufacturer instructions, including confirmation of positive LFIA results by culture in a selective and differential agar. For morphologically compatible isolates, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was chosen as the confirmation method. Eight-hundred and seventy-two environmental samples were tested over 12 months. Here, we report the performance data of RCSS in term of sensitivity, specificity, and positive predictive value. We also provide guidelines on reporting results obtained using this system.

---

Abby Reising - Clinical/Applied

Veterinary Oncologists' and Pet Owners Perception of Acceptable Chemotherapy-Related Adverse Events

Abby J. Reising1, Christopher M. Fulkerson1, Cleveland G. Shields2, Michael O. Childress1

Affiliations:
1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University
2Department of Human Development and Family Science, College of Health and Human Sciences, Purdue University

Chemotherapy is widely used in veterinary oncology, but carries real and perceived risks of adverse events (AEs). Human cancer patients perceive AEs from chemotherapy as more severe than do their attending physicians. It is currently unknown if this discrepancy also exists in veterinary oncology. This survey study’s aim was to assess differences in the ways that dog owners and veterinary oncologists perceive the acceptability of chemotherapy-related AEs. We hypothesized that oncologists would accept higher grade AEs and a greater risk of AEs of any grade than owners.
Two surveys were generated for owners and veterinarians. Respondents were asked to define maximally acceptable AE scores and risks of AEs given three hypothetical outcomes of treatment: 1) cure; 2) extension of life in the absence of cure; and 3) improved quality of life in the absence of cure or extension of life. T-tests were used to compare mean response scores between groups.
Responses from 56 dog owners and 73 veterinarians were analyzed. Owners accepted higher grade AEs if chemotherapy would improve quality of life (p = 0.004). Owners accepted greater risk of moderate (p = 0.0001) or severe (p = 0.0001) AEs if chemotherapy was expected to cure their pet’s cancer.
This is the first study to assess how owners and veterinary oncologists differ in their perception and acceptance of chemotherapy-related AEs. These preliminary results may help to frame discussions with dog owners on the goals and expectations of chemotherapy.

---

Laura Ribas Machado - Clinical/Applied

Evaluation of Selected miRNAs Expression in Myocardial Tissues of Boxer Dogs with Arrhythmogenic Right Ventricular Cardiomyopathy

Laura Machado Ribas1, Jasmine Aggarwal2, Kerstin Muner2, Nelly Elshafie2, Andrea Pires dos Santos2, Suzanne Cunningham3, Luis Felipe Neves dos Santos1

1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA
2 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA 3 Department of Clinical Sciences, Cummings Veterinary Medical Center, Tufts University, North Grafton, MA, USA

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease reported in dogs, cats, and humans. It manifests as malignant ventricular arrhythmias, cardiac dilation, and sudden death. Although multiple mechanisms have been implicated in the pathogenesis of the disease, there is no reference standard ante mortem diagnostic test in dogs or people and, since limited diagnostic criteria are available to characterize ARVC in Boxers, additional biomarkers for this disease are needed. MicroRNAs (miRNAs) are known to regulate the expression of target genes and have been implicated in cardiac diseases, attracting particular interest as potential non-invasive biomarkers. Their presence has been recently reported in the myocardium and serum of end-stage ARVC patients. This study aimed to investigate miRNAs expression in the cardiac tissue of Boxer dogs. We hypothesized that miRNAs would correlate with the clinical outcomes of the disease. Cardiac samples (right and left ventricles) from twelve dogs were analyzed. Four Boxer dogs presented with sudden cardiac death (SCD group) and a definite histopathological diagnosis of ARVC, and another four Boxers presented for non-cardiac related death (NCRD group); cardiac tissues from four non-Boxer dogs were also evaluated (control group). Four miRNAs (miR-133b, miR-145-5p, miR-185-5p, and miR-494-3p) were chosen among those known to be expressed in humans with ARVC, but their expression in cardiac tissues was not significant among groups and did not correlate with clinical outcomes between Boxer dogs’ groups. The absence of consistent connections could be related to the analytical approaches and possible different associated pathways from humans. Further investigation is needed into the potential use of miRNAs as clinical biomarkers and their involvement in the pathogenesis of ARVC in Boxers.

---

Jannatul Shabnam - Clinical/Applied

Recombinant BpRAG1 Protein-Based Serodiagnostic Assays for Detection of Baylisascaris Larva Migrans in Birds

Jannatul Shabnam and Sriveny Dangoudoubiyam
Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47906

Baylisascaris procyonis, a zoonotic nematode responsible for larva migrans in at least 70 avian species. Both free-range and captive birds may risk contracting infections by ingesting infective eggs from their surroundings contaminated with feces of raccoons harboring adult B. procyonis. Infected birds display clinical signs such as difficulty perching or flying, torticollis, wing paralysis, disorientation, and may eventually die. Antemortem diagnostic tests are currently unavailable to perform timely diagnosis or determine prevalence of this infection in birds. In our study we have optimized recombinant B. procyonis RAG1 protein (rBpRAG1)–based ELISA and Western blot assay for detection of Baylisascaris infection in birds. Sera was collected at multiple time points from cockatiels experimentally exposed to B. procyonis larval antigens either by hyperimmunization with B. procyonis larval excretory-secretory proteins (group I, n=5, HI) or by oral infection with infective eggs (group IIa, n=9, 1400 eggs and group IIb, n=9, 2200 eggs). Both groups had their own control cockatiels. On day 28, all five HI cockatiels showed high serum antibody levels with mean OD values ranging from 1.027 to 1.930. These values were 9-16 fold higher than the pre-exposure mean ODs (0.094 to 0.126). Cockatiels in groupII showed variable antibody response and seroconversion was evident in only 56% (5 of 9) of groupIIa and 67% (6 of 9) of groupIIb cockatiels. Western blot results were congruent with ELISA results. The results suggested that rBpRAG1-based serological assays will be useful to assess antibody response in birds exposed to B. procyonis.

---

Go Togawa - Clinical/Applied

Clinical Outcome in Paraplegic Dogs With or Without Pain Perception Due to Thoracolumbar Fibrocartilaginous Embolic Myelopathy or Acute Non-Compressive Nucleus Pulposus Extrusion

Authors: Go Togawa, DVM, PhD1; Melissa Lewis, VMD, PhD, DACVIM (Neurology)1; Dillon
Devathasan DVM, MS, DACVIM (Neurology)2

Affiliations: 1. Purdue University College of Veterinary Medicine, 2. Auburn University College
of Veterinary Medicine

Background: Fibrocartilaginous embolic myelopathy (FCEM) and acute non-compressive
nucleus pulposus extrusion (ANNPE) are common causes of canine spinal cord injury with
clinical similarities. Limited information is available regarding the prognosis of paraplegic dogs
with FCEM and ANNPE.

Hypothesis/Objectives: To describe the clinical features and outcome of paraplegic deep pain
positive (DPP) and deep pain negative (DPN) dogs with FCEM and ANNPE.
Animals: Thirty-one client-owned paraplegic dogs with thoracolumbar FCEM or ANNPE
presenting to university hospitals between 2012-2022.

Methods: Multi-center retrospective study. Paraplegic dogs with a clinical and magnetic
resonance imaging diagnosis of FCEM or ANNPE were included. Logistic regression analysis was
performed to investigate associations between diagnosis, clinical and imaging variables, and
outcome (recovery of independent ambulation or not).

Results: On initial presentation, 14 dogs were paraplegic DPP (8 FCEM, 6 ANNPE), 17 dogs were
paraplegic DPN (11 FCEM, 6 ANNPE). Outcome was available for 26 dogs (14 DPP, 12 DPN) with
a median follow-up time of 182 days (range, 0 – 2311). Excluding 2 DPN dogs euthanized at
diagnosis, 1/10 DPN dogs (10%) regained independent ambulation, whereas 9/14 DPP dogs
(64%) regained independent ambulation. DPN dogs had a significantly higher risk of not
regaining independent ambulation compared to DPP dogs (OR: 29.3, CI: 2.1-419.2 P = 0.013).
No other variables were associated with outcome (P > 0.05).

Conclusions and clinical importance: While the recovery of ambulation is possible, the absence
of pain perception appears to be a useful negative prognostic indicator in dogs with severe
thoracolumbar FCEM or ANNPE.

---

Zsofia Vigh - Clinical/Applied

Comparison of an Interstitial Glucose Monitor Against a Portable Blood Glucose Monitor in Juvenile Dogs

Glucose monitoring is crucial in a critical patient, especially in young animals, as those have less capacity to maintain blood glucose levels. However, repeated venipunctures in a juvenile veterinary patient can be challenging due to patient size, stress, and the potential iatrogenic anemia.

Interstitial glucose monitors are routinely used in diabetes care. It provides continuous real time glucose readings of the interstitial fluid - which correlates with the blood glucose - without the need of any needle stick. These monitors have not been evaluated in juvenile dogs in a clinical setting.

The aim of this study is to compare an interstitial glucose monitor (IGM) against a portable blood glucose monitor (PBGM) in sick juvenile dogs in a veterinary intensive care unit.

Paired interstitial and blood glucose measurements (n=159) were collected from 16 client owned dogs under one year of age with systemic illness. Pearson’s correlation, mean absolute relative difference (MARD), bias, 95% limits of agreement and Parkes Consensus Error Grid (CEG) analysis was performed.

Between the interstitial and blood glucose correlation was r = 0.65, MARD 15.4%, bias -2.6%, with the 95% limits of agreement ranging from -42.5% to 37.4%. 100% of the pairs fell into clinically acceptable zones (A+B) on CEG analysis.

Our results indicate good clinical accuracy with no or minimal differences in clinical outcome between IGM and PBGM in the target population. Further clinical studies with larger sample size, particularly in the hypoglycemic range are needed to assess the IGM performance in the lower glucose range.

---

Allyson Jones - DVM

Spatio-Temporal Changes in Avian Cholera Outbreaks in the United States and its Association with the Weather

Allyson Jones, Jessica Craig, Eric Kontowicz, Wendy Beauvais

Department of Comparative Pathobiology (Craig, Kontowicz, Beauvais), College of Veterinary Medicine (Jones), Purdue University, West Lafayette, IN

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

Research Grant: Morris Animal Foundation
Student Support: College of Veterinary Medicine, Purdue University
1

---

Brooke Lenters - DVM

Diabetes Mellitus Drug Discovery: Insights into Targeting Feline and Human Amylin with Small Molecules

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

---

Mary Nowak - DVM

The Prognostic Potential of microRNAs in Canine Splenic Hemangiosarcoma

Mary Nowak¹, Nelly Elshafie¹, Kerstin Muner¹, Thiago Macedo Lopes Correia^1,2, Jasmine Aggarwal¹, Andrea Pires dos Santos¹

¹Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana.

²Universidade Federal da Bahia, Vitória da Conquista, Bahia, Brazil

Background: Canine  hemangiosarcoma  (HSA) commonly manifests as a visceral  tumor that constitutes approximately 5% of cancers in dogs. The prognosis for visceral HSA is poor due to the aggressive nature of the tumor and lack of specific clinical signs until significant infiltration has occurred. Hence, most dogs present with metastatic  disease that responds poorly to standard surgical and chemotherapeutic intervention. Grading systems for HSA have poor prognostic significance. Thus, improved markers  are imperative to guide a patient's course of treatment. Non-coding microRNAs  regulate gene expression and may serve as predictive biomarkers  for HSA. Objective: To  investigate the potential of microRNAs in the prognostic assessment of canine splenic HSA. Methods: Retrospective study using archived splenic biopsies from 18 cases of canine splenic HSA divided into three groups based on survival times (G1: <90 days, G2: 90-180 days, and G3: >180 days). Expression of four microRNAs (miR-126, miR-150, miR-214, miR-456) with documented roles in canine hemangiosarcoma was assessed by quantitative PCR. Quantification cycle (Cq) values were normalized using the exogenous control UniSp6 and results expressed as fold change. Differences  in expression were determined via one-way ANOVA, followed by Tukey's test. Results: From the four microRNAs analyzed, miR-214 was significantly upregulated in G1 and G2 compared to G3 (p<0.05). Conclusions: Our findings suggest miR-214 may serve as a prognostic marker for canine HSA  as upregulation was associated with decreased survival times. miR-214 has been associated with regulating pro-tumorigenic processes in canine HSA and other cancers. Confirmatory studies are needed to evaluate this marker.

---

Marissa Ramón - DVM

The Role of Aldehyde Dehydrogenase-2 in Modulating Acrolein-Mediated Damage Following Spinal Cord Injury

Marissa Ramón, Siyuan Sun, and Riyi Shi

Center for Paralysis Research, Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

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

Student Support: Purdue University College of Veterinary Medicine Summer Research Scholarship

 

---

Krysten Schmidt - DVM

LARYNGEAL HISTOPATHOLOGY OF THE GONADECTOMIZED AND DEHYDRATED RAT

Background: Estradiol has a role in the body’s response to systemic dehydration. Vocal folds are negatively affected by systemic dehydration. Additionally, they are a known target organ for estradiol. This research sets the groundwork for future studies investigating the role of sex hormones in influencing the adverse effects of dehydration and the voice.

Objective: This study investigates the histopathology of the larynx in gonadectomized rats undergoing systemic dehydration.

Methods: Twelve female and 12 male Sprague Dawley rats were divided into dehydrated (n=12) and euhydrated groups (n=12). Each hydration group had intact (n=6) and gonadectomized (n=6) rats. Blood was collected at beginning and end of experiment to measure and compare serum estradiol levels (plus androgen for males), packed cell volume, and total protein. Body weight and water intake were measured daily. The dehydrated rat groups received 4 ml water/100 g of baseline body weight (approximately 35% less than baseline average intake) compared with ad lib water in the euhydrated group. Sections of larynx were stained with hematoxylin and eosin and immunohistochemically for β-estrogen receptors.

Results: On HE staining, no difference in the vocal fold morphology was identified between euhydrated and dehydrated groups, intact or gonadectomized. β-estrogen receptor labeling was variable between individuals; however, no significant differences between groups were identified.

 

---

Kathryn Wolfert - DVM

Characterization of Tumor Cell-Intrinsic PD-1 Receptor in Canine Urothelial Carcinoma Cells

Kathryn A. Wolfert, Deepika Dhawan, Seung-Oe Lim, Kimaya Bakhle, Alexander W. Enstrom, and Deborah W. Knapp

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Wolfert, Dhawan, Bakhle, Enstrom & Knapp)

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN (Lim)

Immune checkpoint inhibition has become a promising treatment option in a number of canine and human cancers, such as invasive urothelial carcinoma (InvUC). As one of these checkpoint molecules, the programmed cell death 1 receptor (PD-1) is primarily expressed on mature cytotoxic T lymphocytes (CTLs), with ligands PD-L1 and PD-L2 expressed on tumor cells and antigen-presenting cells. The interaction between PD-1 and its ligands on tumor cells leads to CTL inactivation and immune tolerance of the tumor, making the PD-1/PD-L1 axis a key immunotherapeutic target for both veterinary and human oncology.  PD-1 has also been documented to be present on the surface of a number of tumor cell types, though its function in this context is unclear. To investigate the influence of this tumor cell-intrinsic PD-1 and PD-L1 interaction on cancer cell growth, a canine InvUC cell line that overexpresses canine PD-1 (K9TCC-PU-Nk-cPD1) was developed via lentiviral transduction. Surface expression of canine PD-1 (cPD-1) in this line was confirmed via flow cytometry. Using CellTiter-Glo, soft agar, and Western blot assays, we assessed the proliferation, colony formation, and downstream signaling in the MAPK/ERK and PI3K-PKB/Akt pathways of this engineered cell line in the presence of varying amounts of cPD-L1-Fc protein. We further characterized the influence of cPD-1 on immune evasion via T-cell killing assay using activated canine PBMCs. This work in understanding the role of tumor-cell intrinsic PD-1 has significant implications for prognosis and treatment recommendations for PD-1 expressing cancers of all species.

Research Grant: Bladder Cancer Research fund

Student Support: Morris Animal Foundation

---

Maria Yañez Diaz - DVM

Ex Vivo Biomechanical and Microscopic Comparison of Two Cortical Screw Sizes in Fetlock Joint Arthrodesis

Ex vivo biomechanical and microscopic comparison of two cortical screw sizes in fetlock joint arthrodesis.

Authors:

Maria X. Yañez, Timothy B. Lescun, and Stefanie Hansen.

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

Abstract:

In horses, osteoarthritis (OA) is especially prevalent in the metacarpophalangeal (MCP) joint. The current treatment for refractory OA of the MCP joint is surgical arthrodesis using locking compression plates in combination with a palmar tension band. One method of palmar tension band application is lag screw fixation of the proximal sesamoid bones (PSBs) to the third metacarpal bone condyles. The aim of this study was to compare the biomechanical properties and microscopic damage of two cortex screws (4.5-mm and 5.5-mm diameter) inserted into the PSB after loading. We hypothesize that increased screw diameter decreases construct failure load and that microscopic damage will be greater in medial lag screws due to horses bearing greater weight in the medial side of the limb. Screws were inserted in five pairs of cadaver forelimbs. After single cycle to failure axial loading, screws were removed and evaluated by low power stereomicroscopy and high resolution scanning electron microscopy. All constructs failed by transverse fracture of the PSB through the screw holes. There was no significant difference in construct stiffness or mean failure load between the 4.5-mm and 5.5-mm screw groups. These results reveal how screw size is not a critical determinant for tension band strength. Surgeons can be confident that either screw size will provide proper strength to the construct. This allows them to prioritize other factors for screw selection such as patient size, screw availability, or price.

 

---

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

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

Affiliations
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.

Body:
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 Mohallem^1,2 & Uma K Aryal^1,2

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:Ras^G12V 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
infections.

---

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

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

Body:
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

Abstract
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

Body:
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

Body:
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

---