The intersection of Interprofessional Education and One Health: A qualitative study in human and veterinary medical institutions.

Interprofessional Education (IPE) and One Health are two common and overlapping frameworks for teaching collaborative practice. IPE is common at human medical institutions, while One Health is more common in graduate and veterinary programs. The connection between IPE and One Health is still being explored both in scholarship and in real-world professional settings. This prospective, qualitative research study examines the intersection of IPE and One Health at institutions that are members of the Clinical and Translational Science Award (CTSA) One Health Alliance (COHA). COHA consists of veterinary schools partnered with medical institutions through the National Institutes of Health CTSA funding mechanism with the specific goal of advancing the understanding of diseases shared by humans and animals. Twenty-four interviews were conducted with professionals across eight professions. Subjects noted that some of the biggest barriers to IPE education were awareness, accessibility, efficacy, and implementation beyond the classroom. Competency across multiple institutions and a consistent, validated evaluation tool were noted to be lacking. Interviews highlighted a lack of a shared mental model for IPE and One Health across the medical professions, major hurdles for implementation in professional curricula, and a disconnection between bridging IPE and One Health to the workforce and global challenges. Future work in this area may be focused on assessing the IPE and One Health offerings beyond COHA institutions, giving a more holistic understanding on how IPE and One Health are being deployed. One Health can be operationalized through the adoption of IPE principles and practices into curriculum. This research is critical to educate others on current applications, role, and definitions of One Health and IPE. The ultimate goal of this work is to help cultivate transdisciplinary leaders in the human and animal medicine who will have the skills to solve systemic problems.

Impacts of Antiretroviral Therapy on the Oral Microbiome and Periodontal Health of Feline Immunodeficiency Virus Positive Cats.

Feline immunodeficiency virus (FIV) is the domestic cat analogue of HIV infection in humans. Both viruses induce oral disease in untreated individuals, with clinical signs that include gingivitis and periodontal lesions. Oral disease manifestations in HIV patients are abated by highly effective combination antiretroviral therapy (cART), though certain oral manifestations persist despite therapy. Microorganisms associated with oral cavity opportunistic infections in patients with HIV cause similar pathologies in cats. To further develop this model, we evaluated characteristics of feline oral health and oral microbiome during experimental FIV infection over an 8-month period following cART. Using 16S metagenomics sequencing, we evaluated gingival bacterial communities at four timepoints in uninfected and FIV-infected cats treated with cART or placebo. Comprehensive oral examinations were also conducted by a veterinary dental specialist over the experimental period. Gingival inflammation was higher in FIV-infected cats treated with placebo compared to cART-treated cats and controls at study endpoint. Oral microbiome alpha diversity increased in all groups, while beta diversity differed among treatment groups, documenting a significant effect of cART therapy on microbiome community composition. This finding has not previously been reported and indicates cART ameliorates immunodeficiency virus-associated oral disease via preservation of oral mucosal microbiota. Further, this study illustrates the value of the FIV animal model for investigations of mechanistic associations and therapeutic interventions for HIV oral manifestations.

Study of feline immunodeficiency virus prevalence and expert opinions on standards of care.

OBJECTIVE: The purpose of this study was to identify knowledge gaps in the global prevalence of feline immunodeficiency virus (FIV) and to obtain professional opinions and experiences regarding FIV in selected countries. We conducted a literature review of abstracts that reported the prevalence of FIV and interviewed experts in feline medicine and retroviruses from different countries to determine regional perspectives. METHODS: A total of 90 articles reporting FIV prevalence as a primary unbiased population-level analysis between 1980 and 2017 were indexed. FIV prevalence, demographics, year and location were analyzed. Statistics were evaluated and compared. In total, 10 experts were interviewed. Results were analyzed for congruence with the findings of the literature review. RESULTS: FIV prevalence was typically in the range of 5-8%, with a global prevalence of 4.7%, and remained largely constant over the reporting period (1980-2017). Over 90% of articles reported greater prevalence in older male cats. More studies were conducted in North America and Europe and reported the lowest prevalence. Expert-estimated prevalence approximated literature review prevalence. Attitudes and recommendations for management were consistent among experts. The limitations of the present review include varying inclusion criteria of cats tested in different studies, variation in testing modalities and the inability to conduct summary statistics across dissimilar cohorts. CONCLUSIONS AND RELEVANCE: The global prevalence of FIV has not changed since its discovery 40 years ago. Prevalence is higher in older male cats and is lower in North America and Europe than other continents. Experts agree that FIV is not typically a disease of high concern and is often associated with infections of the oral cavity. Vaccination is not typically recommended and has been discontinued in North America. The evaluation of risk factors for FIV progression is useful in managing infections. Recommendations for future research include analyses to determine copathogen and environmental factors that impact progression, assessment of life span impacts and investigations of treatment efficacy and side effects.

Estimating cutoff values for diagnostic tests to achieve target specificity using extreme value theory.

BACKGROUND: Rapidly developing tests for emerging diseases is critical for early disease monitoring. In the early stages of an epidemic, when low prevalences are expected, high specificity tests are desired to avoid numerous false positives. Selecting a cutoff to classify positive and negative test results that has the desired operating characteristics, such as specificity, is challenging for new tests because of limited validation data with known disease status. While there is ample statistical literature on estimating quantiles of a distribution, there is limited evidence on estimating extreme quantiles from limited validation data and the resulting test characteristics in the disease testing context. METHODS: We propose using extreme value theory to select a cutoff with predetermined specificity by fitting a Pareto distribution to the upper tail of the negative controls. We compared this method to five previously proposed cutoff selection methods in a data analysis and simulation study. We analyzed COVID-19 enzyme linked immunosorbent assay antibody test results from long-term care facilities and skilled nursing staff in Colorado between May and December of 2020. RESULTS: We found the extreme value approach had minimal bias when targeting a specificity of 0.995. Using the empirical quantile of the negative controls performed well when targeting a specificity of 0.95. The higher target specificity is preferred for overall test accuracy when prevalence is low, whereas the lower target specificity is preferred when prevalence is higher and resulted in less variable prevalence estimation. DISCUSSION: While commonly used, the normal based methods showed considerable bias compared to the empirical and extreme value theory-based methods. CONCLUSIONS: When determining disease testing cutoffs from small training data samples, we recommend using the extreme value based-methods when targeting a high specificity and the empirical quantile when targeting a lower specificity.

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats.

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

Subtilisin 3 production from Microsporum canis is independent of keratin substrate availability.

Dermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. We investigated a critical adhesion protein, subtilisin 3, utilized by Microsporum canis during initial stages of infection, analyzing its production and expression under varying growth conditions. Additionally, as this protein must be expressed and produced for dermatophyte infections to occur, we developed and optimized a diagnostic antibody assay targeting this protein. Subtilisin 3 levels were increased in culture when grown in baffled flasks and supplemented with either l-cysteine or cat hair. As subtilisin 3 was also produced in cultures not supplemented with keratin or cysteine, this study demonstrated that subtilisin 3 production is not reliant on the presence of keratin or its derivatives. These findings could help direct future metabolic studies of dermatophytes, particularly during the adherence phase of infections.

Seasonal changes in network connectivity and consequences for pathogen transmission in a solitary carnivore.

Seasonal variation in habitat use and animal behavior can alter host contact patterns with potential consequences for pathogen transmission dynamics. The endangered Florida panther (Puma concolor coryi) has experienced significant pathogen-induced mortality and continues to be at risk of future epidemics. Prior research has found increased panther movement in Florida's dry versus wet seasons, which may affect panther population connectivity and seasonally increase potential pathogen transmission. Our objective was to determine if Florida panthers are more spatially connected in dry seasons relative to wet seasons, and test if identified connectivity differences resulted in divergent predicted epidemic dynamics. We leveraged extensive panther telemetry data to construct seasonal panther home range overlap networks over an 11 year period. We tested for differences in network connectivity, and used observed network characteristics to simulate transmission of a broad range of pathogens through dry and wet season networks. We found that panthers were more spatially connected in dry seasons than wet seasons. Further, these differences resulted in a trend toward larger and longer pathogen outbreaks when epidemics were initiated in the dry season. Our results demonstrate that seasonal variation in behavioral patterns-even among largely solitary species-can have substantial impacts on epidemic dynamics.

Combination Antiretroviral Therapy and Immunophenotype of Feline Immunodeficiency Virus.

Feline Immunodeficiency Virus (FIV) causes progressive immune dysfunction in cats similar to human immunodeficiency virus (HIV) in humans. Although combination antiretroviral therapy (cART) is effective against HIV, there is no definitive therapy to improve clinical outcomes in cats with FIV. This study therefore evaluated pharmacokinetics and clinical outcomes of cART (2.5 mg/kg Dolutegravir; 20 mg/kg Tenofovir; 40 mg/kg Emtricitabine) in FIV-infected domestic cats. Specific pathogen free cats were experimentally infected with FIV and administered either cART or placebo treatments (n = 6 each) for 18 weeks, while n = 6 naïve uninfected cats served as controls. Blood, saliva, and fine needle aspirates from mandibular lymph nodes were collected to quantify viral and proviral loads via digital droplet PCR and to assess lymphocyte immunophenotypes by flow cytometry. cART improved blood dyscrasias in FIV-infected cats, which normalized by week 16, while placebo cats remained neutropenic, although no significant difference in viremia was observed in the blood or saliva. cART-treated cats exhibited a Th2 immunophenotype with increasing proportions of CD4+CCR4+ cells compared to placebo cats, and cART restored Th17 cells compared to placebo-treated cats. Of the cART drugs, dolutegravir was the most stable and long-lasting. These findings provide a critical insight into novel cART formulations in FIV-infected cats and highlight their role as a potential animal model to evaluate the impact of cART on lentiviral infection and immune dysregulation.

Habitat connectivity and host relatedness influence virus spread across an urbanising landscape in a fragmentation-sensitive carnivore.

Spatially heterogeneous landscape factors such as urbanisation can have substantial effects on the severity and spread of wildlife diseases. However, research linking patterns of pathogen transmission to landscape features remains rare. Using a combination of phylogeographic and machine learning approaches, we tested the influence of landscape and host factors on feline immunodeficiency virus (FIVLru) genetic variation and spread among bobcats (Lynx rufus) sampled from coastal southern California. We found evidence for increased rates of FIVLru lineage spread through areas of higher vegetation density. Furthermore, single-nucleotide polymorphism (SNP) variation among FIVLru sequences was associated with host genetic distances and geographic location, with FIVLru genetic discontinuities precisely correlating with known urban barriers to host dispersal. An effect of forest land cover on FIVLru SNP variation was likely attributable to host population structure and differences in forest land cover between different populations. Taken together, these results suggest that the spread of FIVLru is constrained by large-scale urban barriers to host movement. Although urbanisation at fine spatial scales did not appear to directly influence virus transmission or spread, we found evidence that viruses transmit and spread more quickly through areas containing higher proportions of natural habitat. These multiple lines of evidence demonstrate how urbanisation can change patterns of contact-dependent pathogen transmission and provide insights into how continued urban development may influence the incidence and management of wildlife disease.

Paradoxes and synergies: optimizing management of a deadly virus in an endangered carnivore.

Pathogen management strategies in wildlife are typically accompanied by an array of uncertainties such as the efficacy of vaccines or potential unintended consequences of interventions. In the context of such uncertainties, models of disease transmission can provide critical insight for optimizing pathogen management, especially for species of conservation concern. The endangered Florida panther experienced an outbreak of feline leukemia virus (FeLV) in 2002-04, and continues to be affected by this deadly virus. Ongoing management efforts aim to mitigate the effects of FeLV on panthers, but with limited information about which strategies may be most effective and efficient.We used a simulation-based approach to determine optimal FeLV management strategies in panthers. We simulated use of proactive FeLV management strategies (i.e., proactive vaccination) and several reactive strategies, including reactive vaccination and test-and-removal. Vaccination strategies accounted for imperfect vaccine-induced immunity, specifically partial immunity in which all vaccinates achieve partial pathogen protection. We compared the effectiveness of these different strategies in mitigating the number of FeLV mortalities and the duration of outbreaks.Results showed that inadequate proactive vaccination can paradoxically increase the number of disease-induced mortalities in FeLV outbreaks. These effects were most likely due to imperfect vaccine immunity causing vaccinates to serve as a semi-susceptible population, thereby allowing outbreaks to persist in circumstances otherwise conducive to fadeout. Combinations of proactive vaccination with reactive test-and-removal or vaccination, however, had a synergistic effect in reducing impacts of FeLV outbreaks, highlighting the importance of using mixed strategies in pathogen management.Synthesis and applications: Management-informed disease simulations are an important tool for identifying unexpected negative consequences and synergies among pathogen management strategies. In particular, we find that imperfect vaccine-induced immunity necessitates further consideration to avoid unintentionally worsening epidemics in some conditions. However, mixing proactive and reactive interventions can improve pathogen control while mitigating uncertainties associated with imperfect interventions.

Rapid evolution of SARS-CoV-2 in domestic cats.

SARS-CoV-2 (SARS2) infection of a novel permissive host species can result in rapid viral evolution. Data suggest that felids are highly susceptible to SARS2 infection, and species-specific adaptation following human-to-felid transmission may occur. We employed experimental infection and analysis of publicly available SARS2 sequences to observe variant emergence and selection in domestic cats. Three cohorts of cats (N = 23) were inoculated with SARS-CoV-2 USA-WA1/2020 or infected via cat-to-cat contact transmission. Full viral genomes were recovered from RNA obtained from nasal washes 1-3 days post-infection and analyzed for within-host viral variants. We detected 118 unique variants at ≥3 per cent allele frequency in two technical replicates. Seventy of these (59 per cent) were nonsynonymous single nucleotide variants (SNVs); the remainder were synonymous SNVs or structural variants. On average, we observed twelve variants per cat, nearly 10-fold higher than what is commonly reported in human patients. We observed signatures of positive selection in the spike protein and the emergence of eleven within-host variants located at the same genomic positions as mutations in SARS2 variant lineages that have emerged during the pandemic. Fewer variants were noted in cats infected from contact with other cats and in cats exposed to lower doses of cultured inoculum. An analysis of ninety-three publicly available SARS2 consensus genomes recovered from naturally infected domestic cats reflected variant lineages circulating in the local human population at the time of sampling, illustrating that cats are susceptible to SARS2 variants that have emerged in humans, and suggesting human-to-felid transmission occurring in domestic settings is typically unidirectional. These experimental results underscore the rapidity of SARS2 adaptation in felid hosts, representing a theoretical potential origin for variant lineages in human populations. Further, cats should be considered susceptible hosts capable of shedding virus during infections occurring within households.

Feline Leukemia Virus Frequently Spills Over from Domestic Cats to North American Pumas.

Feline leukemia virus (FeLV) is a gammaretrovirus with horizontally transmitted and endogenous forms. Domestic cats are the primary reservoir species, but FeLV outbreaks in endangered Florida panthers and Iberian lynxes have resulted in mortalities. To assess prevalence and interspecific/intraspecific transmission, we conducted an extensive survey and phylogenetic analysis of FeLV infection in free-ranging pumas (n = 641) and bobcats (n = 212) and shelter domestic cats (n = 304). Samples were collected from coincident habitats across the United States between 1985 and 2018. FeLV infection was detected in 3.12% of the puma samples, 0.47% of the bobcat samples, and 6.25% of the domestic cat samples analyzed. Puma prevalence varied by location, with Florida having the highest rate of infection. FeLV env sequences revealed variation among isolates, and we identified two distinct clades. Both progressive and regressive infections were identified in cats and pumas. Based on the time and location of sampling and phylogenetic analysis, we inferred 3 spillover events between domestic cats and pumas; 3 puma-to-puma transmissions in Florida were inferred. An additional 14 infections in pumas likely represented spillover events following contact with reservoir host domestic cat populations. Our data provide evidence that FeLV transmission from domestic cats to pumas occurs widely across the United States, and puma-to-puma transmission may occur in genetically and geographically constrained populations. IMPORTANCE Feline leukemia virus (FeLV) is a retrovirus that primarily affects domestic cats. Close interactions with domestic cats, including predation, can lead to the interspecific transmission of the virus to pumas, bobcats, or other feline species. Some infected individuals develop progressive infections, which are associated with clinical signs of disease and can result in mortality. Therefore, outbreaks of FeLV in wildlife, including the North American puma and the endangered Florida panther, are of high conservation concern. This work provides a greater understanding of the dynamics of the transmission of FeLV between domestic cats and wild felids and presents evidence of multiple spillover events and infections in all sampled populations. These findings highlight the concern for pathogen spillover from domestic animals to wildlife but also identify an opportunity to understand viral evolution following cross-species transmissions more broadly.

Prevalence and Genomic Sequence Analysis of Domestic Cat Hepadnavirus in the United States.

Hepadnaviruses are partially double-stranded DNA viruses that infect a variety of species. The prototypical virus in this family is the human hepatitis B virus, which chronically infects approximately 400 million people worldwide and is a risk factor for progressive liver disease and liver cancer. The first hepadnavirus isolated from carnivores was a domestic cat hepadnavirus (DCH), initially identified in Australia and subsequently detected in cats in Europe and Asia. As with all characterized hepadnaviruses so far, DCH infection has been associated with hepatic disease in its host. Prevalence of this infection in the United States has not been explored broadly. Thus, we utilized conventional and quantitative PCR to screen several populations of domestic cats to estimate DCH prevalence in the United States. We detected DCH DNA in 1 out of 496 animals (0.2%) in the U.S. cohort. In contrast, we detected circulating DCH DNA in 7 positive animals from a cohort of 67 domestic cats from Australia (10.4%), consistent with previous studies. The complete consensus genome of the U.S. DCH isolate was sequenced by Sanger sequencing with overlapping PCR products. An in-frame deletion of 157 bp was identified in the N-terminus of the core open reading frame. The deletion begins at the direct repeat 1 sequence (i.e., the 5' end of the expected double-stranded linear DNA form), consistent with covalently closed circular DNA resultant from illegitimate recombination described in other hepadnaviruses. Comparative genome sequence analysis indicated that the closest described relatives of the U.S. DCH isolate are those previously isolated in Italy. Motif analysis supports DCH using NTCP as an entry receptor, similar to human HBV. Our work indicates that chronic DCH prevalence in the U.S. is likely low compared to other countries.

Current Topics in Dermatophyte Classification and Clinical Diagnosis.

Dermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. Classification of many of these species has recently changed due to genetic analysis, potentially affecting clinical diagnosis and disease management. In this review, we discuss dermatophyte classification including name changes for medically important species, current and potential diagnostic techniques for detecting dermatophytes, and an in-depth review of Microsporum canis, a prevalent zoonotic dermatophyte. Fungal culture is still considered the "gold standard" for diagnosing dermatophytosis; however, modern molecular assays have overcome the main disadvantages of culture, allowing for tandem use with cultures. Further investigation into novel molecular assays for dermatophytosis is critical, especially for high-density populations where rapid diagnosis is essential for outbreak prevention. A frequently encountered dermatophyte in clinical settings is M. canis, which causes dermatophytosis in humans and cats. M. canis is adapting to its primary host (cats) as one of its mating types (MAT1-2) appears to be going extinct, leading to a loss of sexual reproduction. Investigating M. canis strains around the world can help elucidate the evolutionary trajectory of this fungi.

Apathogenic proxies for transmission dynamics of a fatal virus.

Identifying drivers of transmission-especially of emerging pathogens-is a formidable challenge for proactive disease management efforts. While close social interactions can be associated with microbial sharing between individuals, and thereby imply dynamics important for transmission, such associations can be obscured by the influences of factors such as shared diets or environments. Directly-transmitted viral agents, specifically those that are rapidly evolving such as many RNA viruses, can allow for high-resolution inference of transmission, and therefore hold promise for elucidating not only which individuals transmit to each other, but also drivers of those transmission events. Here, we tested a novel approach in the Florida panther, which is affected by several directly-transmitted feline retroviruses. We first inferred the transmission network for an apathogenic, directly-transmitted retrovirus, feline immunodeficiency virus (FIV), and then used exponential random graph models to determine drivers structuring this network. We then evaluated the utility of these drivers in predicting transmission of the analogously transmitted, pathogenic agent, feline leukemia virus (FeLV), and compared FIV-based predictions of outbreak dynamics against empirical FeLV outbreak data. FIV transmission was primarily driven by panther age class and distances between panther home range centroids. FIV-based modeling predicted FeLV dynamics similarly to common modeling approaches, but with evidence that FIV-based predictions captured the spatial structuring of the observed FeLV outbreak. While FIV-based predictions of FeLV transmission performed only marginally better than standard approaches, our results highlight the value of proactively identifying drivers of transmission-even based on analogously-transmitted, apathogenic agents-in order to predict transmission of emerging infectious agents. The identification of underlying drivers of transmission, such as through our workflow here, therefore holds promise for improving predictions of pathogen transmission in novel host populations, and could provide new strategies for proactive pathogen management in human and animal systems.

Genetic Characterization of Microsporum canis Clinical Isolates in the United States.

Microsporum canis is the primary agent causing dermatophytosis in cats, and also infects humans, dogs, and other species. Assessment of genetic variation among M. canis isolates in the United States has not been conducted. Further, M. canis mating type and assessment of disease severity associated with genotypic characteristics have not been rigorously evaluated. We therefore isolated M. canis from 191 domestic cats across the US and characterized genotypes by evaluation of ITS sequence, MAT locus, and microsatellite loci analysis. The genes SSU1 and SUB3, which are associated with keratin adhesion and digestion, were sequenced from a subset of isolates to evaluate potential genetic associations with virulence. Analysis of microsatellite makers revealed three M. canis genetic clusters. Both clinic location and disease severity were significant predictors of microsatellite variants. 100% of the M. canis isolates were MAT1-1 mating gene type, indicating that MAT1-2 is very rare or extinct in the US and that asexual reproduction is the dominant form of replication. No genetic variation at SSU1 and SUB3 was observed. These findings pave the way for novel testing modalities for M. canis and provide insights about transmission and ecology of this ubiquitous and relatively uncharacterized agent.