Research Projects

Kyle Barron¹, Aynsley Romaniuk², Shanis Barnard³, Candace Croney⁴

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

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

Research Grant: Dr Candace Croney Discretionary Funds

Student support: Purdue University College of Veterinary Medicine

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

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

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

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

Research Grant: The Humane Society of the United States

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

Field of Research: Immunology

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

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

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

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

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

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

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

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

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

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

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

Research Grant- PVM funds

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

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

Purdue University Laboratory Animal Program, West Lafayette, IN

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

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

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

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

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

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

Research Grant: Departmental Start-up Grant, Purdue University

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

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

Research Support: Purdue University, College of Veterinary Medicine

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

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

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

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

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

Student Support - Purdue University College of Veterinary Medicine

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

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

Introduction: Atherosclerosis is characterized by calcification in the walls of arteries that can lead to constriction and ultimately blockage of the whole vessel. Since the most common clinical sign for atherosclerosis in birds is sudden death and the diagnosis is usually determined post-mortem^{[1, 2]}, further diagnostic imaging is needed to improve early diagnosis. High-resolution magnetic resonance imaging (MRI) is a promising modality for assessment of arterial atherosclerotic plaques. This pilot study aims to investigate MRI as a potential technique to diagnose and quantify atherosclerosis in birds.  

Methodology: A total of 5 cadaver of birds with unknown causes of death from different species were used. These birds were collected by the community practice service at Purdue University College of Veterinary Medicine. The cadavers were scanned with a 7T MRI system (Bruker Biospec). The T₂ turbo rare scan was performed without fat suppression with the voxel volume of 0.5*0.5*0.5mm². This implies that the field of view was 50*50mm² and the slice thickness was also 50mm. For the bigger birds like owls and macaws, for ease of scanning, the outer gradient was used, and two separate scans were done to look at the heart and the aorta. Whereas in smaller birds like doves and parakeets, the inner gradient of the MRI was used and only one scan was done to observe the whole area at once. There were no gaps between slices for precision. Morphologic assessment of atherosclerotic plaques (area measurements of calcification) will be performed at the end of the study using ImageJ® software. The birds, after scanning were sent for a necropsy to confirm the presence of atherosclerotic lesions. 

Observations and Results: Preliminary evaluation demonstrates that MRI is feasible and reproducible in cadavers of birds. Given the wide range of body size among the enrolled birds (30g-500g), we plan to enroll a larger sample of subspecies of birds. This will also give us a better representative assessment of atherosclerosis in different sizes of birds before scanning alive animals. By the end of the study, we hope to be able to quantify atherosclerotic lesions in birds which would help to assess the severity of the disease. 

References:

1. Bavelaar, F. J., & Beynen, A. C. (2004). Atherosclerosis in parrots. A review. The veterinary quarterly, 26(2), 50–60. https://doi.org/10.1080/01652176.2004.9695168
2. Bertozzi, G., Cafarelli, F. P., Ferrara, M., Di Fazio, N., Guglielmi, G., Cipolloni, L., Manetti, F., La Russa, R., & Fineschi, V. (2022). Sudden Cardiac Death and Ex-Situ Post-Mortem Cardiac Magnetic Resonance Imaging: A Morphological Study Based on Diagnostic Correlation Methodology. Diagnostics (Basel, Switzerland), 12(1), 218. https://doi.org/10.3390/diagnostics12010218
3. Michaud, K., Genet, P., Sabatasso, S., & Grabherr, S. (2019). Postmortem imaging as a complementary tool for the investigation of cardiac death. Forensic sciences research, 4(3), 211–222. https://doi.org/10.1080/20961790.2019.1630944

Mild equine asthma is a common disease in racehorses developed in response to particulate matter (PM) exposure and is associated with poor performance.  PM smaller than or equal to 2.5 µm (PM2.5) is one size of dust defined as a health hazard by the EPA. Feeding hay and bedding horses on straw are associated with higher levels of PM exposure in the breathing-zone, in comparison to pelleted feed and wood shavings, respectively. However, particulate exposure varies significantly between horses, despite similar management practices. We hypothesize that within different racetracks, PM2.5 concentrations in the breathing-zone vary based on management practices and horse activity while stalled. . Therefore, real-time particulate monitors and video cameras were placed on Thoroughbred racehorses' halters to quantify breathing-zone PM exposure associated with an activity. Horses were stalled under normal management conditions and monitored with a custom-made personal PM monitor (Plantower PMS 7003) and activity was recorded (Hawkeye Firefly Q6) over 20-30 minutes. Video and PM data were uploaded to the EVADE software (NIOSH) to quantify dust exposure during various activities. We expect that this approach will identify activities and management practices associated with higher PM exposure and provide new solutions to lessen the burden of mild equine asthma.

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

This study investigated the effect of the covid pandemic on the relationship between owners and their pets and the behavior issues of the pet cats and dogs. Longitudinal surveys were conducted with participants who were 18 years and older and recruited via Amazon’s crowdsourcing platforms between June 2020 and December 2021. Participants were asked pet-related questions including perceived closeness of pet-owner relationship, behavior issues, and interactions during four different phases of the pandemic: pre-pandemic, lockdown, reopening, and recovering. Pet-owner relationship was quantified using the validated instruments, including the Cat-Owner Relationship Scale (CORS), the Monash Dog Owner Relationship Scale (MDORS), and the Inclusion of Other in the Self (IOS) diagrams, all with a higher score indicating a closer relationship. Both CORS and MDORS have human-animal interaction (HAI), Emotion, and Cost subscales. A total of 1,761 dog owners, 1,849 cat owners, and 657 non-pet owners completed one to six surveys during the study period. The results showed an increase in all pet-owner relationship measures starting from the lockdown phase and maintained the levels in the following phases. The most common behavior issues in cats and dogs are signs of aggression, fear of noise, and excess licking. This study suggests that the initial shock of the pandemic forced pet owners to adapt to the extended periods of staying at home, leading to increased interactions and commitment to both pet cats and dogs, and ultimately positively affecting pet-owner relationships. This study also supports that pet behavior changes during the pandemic resulting from their owners spending more time at home.

Aluminum-containing adjuvants have been widely used for over 90 years in many human and veterinary vaccines to enhance immune response. Today, the most commonly used aluminum adjuvants are aluminum hydroxide (AH) and aluminum phosphate (AP). While often collectively referred to as “alum”, these adjuvants possess different physical and chemical properties. In spite of these differences, most of the current literature on aluminum adjuvants reports only on AH. We are investigating if there are different mechanisms of action between AH and AP. To do this, human THP-1 cells, differentiated with phorbol 12-myristate-13-acetate (PMA), were used as a model system for antigen-presenting cells. The secretion of IL-1α, IL-1β and IL-18 was measured by enzyme-linked immunosorbent assay (ELISA). Priming of the cells with lipopolysaccharide (LPS) followed by treatment with the aluminum adjuvants markedly increased the secretion of IL-1α and IL-1β and had less effect on IL-18.  AP induced greater secretion of  IL-1α, IL-1β, and IL-18 than AH. Incubation of LPS-primed THP-1 cells with inhibitors of different signaling pathways revealed differences in the regulation of IL-1α and IL-1β secretion, and differences between AH and AP.  This indicates that these adjuvants may activate different signaling pathways in antigen-presenting cells. Pointing out the differences between these two aluminum adjuvants may allow for better manipulation of immune response, as well as aid in making informed decisions when choosing an adjuvant for vaccine formulation.

Laboratory mice will interact with intracage shelters when these are provided. However, even though mice spend a significant amount of time in their shelters, our pilot studies suggest that markers associated with chronic stress are elevated. Chronic stress in laboratory rodents is a welfare issue that can alter behavior and physiology, introducing uncontrolled variables and reducing comparability to human disease. In this study, mice were given access to clear, red, yellow, or blue intracage shelters. The control group was not provided an intracage shelter. Once the mice acclimated to the shelters, anxiety was measured using the open field test. A blood sample was also collected to compare serum corticosterone (ELISA) and complete blood count (neutrophil:lymphocyte ratio) between groups. Behavioral and physiological assessments were combined to determine how the provision of an intracage shelter and its color impact well-being. Comparing the no-shelter group to the groups that have shelters will assess the impact of intracage shelter presence on stress whereas comparing the groups with intracage shelters by color will assess the impact of light intensity on stress. As the mice will be acclimating to the shelters until late July, there is no data to report yet. Cage-side observations have shown more mice using the intracage shelters as the acclimation period progresses, with a current average of 30% of mice using the shelters when observed. Therefore, we are hopeful this project may help guide the recommendations for the use of intracage shelters for the enrichment of laboratory rodents.

Zebrafish (Danio rerio) are useful in scientific research due to their close genetic similarity to the human genome and fast reproductive lifecycle. Their increased use in scientific research calls for improved methods of monitoring their health, as current methods involve multiple types of testing including submission of whole fish to identify various pathogens. This study aims to create a novel sampling technique by exposing nitrocellulose filters to sump water over the course of twelve weeks. The filter was compared against other known testing methods of swabbing biofilm from the sump and passing sump water through a vacuum filter.  It was hypothesized that the nitrocellulose filter would identify more pathogens over time, reducing the need for multiple testing methods. Weekly PCR testing was conducted to detect Mycobacterium chelonae, zebrafish picornavirus, Myxidium streisingeri, Mycobacterium fortuitium, and Pseudoloma neurophilia. Nitrocellulose filters were the most consistent in identifying pathogens every week as their sensitivity to identify pathogens increased over time. The vacuum filter was also a consistent and sensitive method, but to a lesser degree than the nitrocellulose filters over time. Sump tank swab samples were the least sensitive in pathogen detection as its positive pathogen identification results were inconsistent. In preliminary results, none of the methods have been able to detect P. neurophilia. This suggests that the nitrocellulose filters may be a useful method of monitoring the health of laboratory zebrafish colonies for most of the agents tested.

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

[1] Retrieved May, 31, 2022, from the Wildlife Health Information Sharing Partnership event reporting system on-line database, https://whispers.usgs.gov

Many adults struggle with over consumption and binge-like drinking of alcoholic beverages. Almost always these beverages are not pure alcohol but have other taste and flavor components. In preclinical research alcohol is often studied in a pure form in order to avoid confounding effects. However, flavor may be a significant risk factor leading to increased binge-like drinking and alcohol intake. In the present study, we first determined a preferred concentration of flavor (cherry Kool-Aid) to be consumed with 20% alcohol. Mice(8 females, 8 males/group) underwent two bottle choice (2BC) drinking sessions with pure water in one bottle and alcohol flavored with 7 concentrations(0-5%) of Kool-Aid in the other bottle. We tested concentrations for two 24-hour sessions each and measured the amount of alcohol consumed and preference to water. A concentration of .1% was determined to be the preferred concentration based on preference and intake. We next compared the .1% flavored alcohol to regular alcohol in two 2-hour 2BC sessions to determine if flavored alcohol was preferred over regular alcohol. Both male and female mice showed greater intake of flavored alcohol compared to regular alcohol. A second cohort of male and female mice are currently undergoing several weeks of binge-like drinking either consuming .1% flavored alcohol or regular alcohol using a drinking in the dark model. These mice are being tested for binge-like drinking behavior for 6 total weeks to see if flavored alcohol promotes a higher level of binge-like drinking and blood alcohol levels. 

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

As many as 1 in 5 military veterans are diagnosed with posttraumatic stress disorder (PTSD). Service dogs have recently emerged as a complementary intervention, but additional scientific evidence supporting their use is needed. In this study, we examined veterans with PTSD involved in a service dog program, gathering feedback from them and their spouses to (1) understand their day-to-day experiences, (2) understand the influence of service dog partnership, and (3) assess whether the response rate was associated with demographic characteristics. This study used ecological momentary assessment to obtain real-time information from n=170 veteran and n=87 spouse participants longitudinally (baseline and 3 months later), comparing veterans with a service dog to veterans receiving usual care alone. Open-ended feedback prompts occurred four times daily through a smartphone app and were analyzed using a mixed-methods approach. Four themes were identified through qualitative content analysis: life events, technical issues, health, and sleep. Compared to veterans on the waitlist, veterans with service dogs mentioned having less overall anxiety, more resilience, and a more positive outlook. Service dog partnership did not affect spouse feedback. On average, feedback response rates were 8% for spouses and 11% for veterans. Time of day and demographics were not found to be significantly associated with response frequency. While the quantity of feedback received was relatively low, veteran and spouse responses were rich in detail and showed a meaningful influence of the service dog on day-to-day well-being. Based on these results, service dogs may be a useful adjunctive intervention for military-connected PTSD.

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

The integrity of the laryngeal vocal folds is important in the creation of the voice. They are a known target organ for estradiol, and their function is negatively affected by systemic dehydration. Estradiol in humans has been shown to increase capillary permeability by vasodilation, modulate vasopressin release, and respond to changes in blood osmolarity. The objective of this study was to determine if the presence of estradiol influences vocal fold histology during systemic dehydration. Twelve female Sprague Dawley rats were divided into dehydrated (n=6) and euhydrated groups (n=6). Each hydration group had intact (n=3) and ovariectomized (n=3) rats to simulate the loss of sex hormones. Rats were ovariectomized 14 days prior to the start of the study to allow hormone levels to stabilize. Serum estradiol levels and packed cell volume (PCV) were determined at the start of the study. During the 5-day baseline period, all rats were given ad lib food and water; the rats were weighed, and water intake was measured daily. Following the baseline period, a 5-day dehydration period in which the dehydrated group received 4 ml water/100 g of baseline body weight daily and euhydrated continued to receive ad lib water. At the end of the dehydration period all rats were euthanized, blood was collected to measure PCV and estradiol levels, and the larynx was dissected and placed in 10% neutral buffered formalin for histological processing. Larynges were sectioned at 4um in the coronal plane and stained with hematoxylin and eosin for routine morphological assessment. Immunohistochemical labeling of laryngeal estrogen receptors will be quantified using digital software.