Department of Pathology, Microbiology, and Immunology

American black bears (Ursus americanus) can be considered vertebrate pests when they come in conflict with humans and create potential public safety situations that require intervention by local wildlife or animal control departments. Black bears are both omnivorous and highly intelligent, and this combination of characteristics make these animals prone to human habituation and developing an association between humans and easy sources of food. Foraging behaviors can put black bears in undesired or unsafe contact with humans when bears root through garbage on private and public lands, consume crops, prey upon small livestock or pets, cause significant property damage, and wander onto roadways leading to traffic accidents. An increasingly recognized cause of bear-human conflict is neurologic disease, such as that caused by infectious pathogens or toxicity, leading to cognitive or behavioral changes. A unique neurologic disease affecting black bears was first observed in 2014 near Lake Tahoe on the border between California and Nevada. Since then, it has been reported in eight counties in California and two counties in Nevada. Affected bears exhibit a range of neurologic symptoms including tremors, seizures, head tilts, and loss of fear of humans (i.e., “dog-like” behavior). Affected animals are typically young (<3 years old), underweight, and most often present in the spring after early emergence from hibernation. Histopathologic examination of brain tissue from these bears revealed varying degrees of inflammation in the brain (encephalitis), but the cause of this encephalitis was not apparent. Testing for common infectious causes of encephalitis (e.g., rabies, canine distemper virus, canine adenovirus, West Nile virus) and common neurotoxins did not identify a cause of the inflammation. A novel gammaherpesvirus, circovirus, parvovirus, and anellovirus were isolated from subsets of affected bears; however, a direct correlation between viral infection and encephalitis has not been established to date. The goal of this study is to investigate the potential role of protozoal parasites in this encephalitis of unknown origin, as members of this group of pathogens are known causes of encephalitis in humans and animals. Brain tissue from bears with encephalitis (n = 21) and unaffected bears (n = 15) from California and Nevada was screened via PCR targeting two loci commonly used to identify protozoal parasites (ITS1 and 18S). Protozoal parasite DNA was detected in 47.6% of bears with encephalitis (10/21) and 26.7% of unaffected bears (4/15). Parasites detected in bears with encephalitis included Sarcocystis neurona (4/21), S. canis (1/21), S. felis (1/21), an uncharacterized Sarcocystis sp. (1/21), Toxoplasma gondii (1/21), and an uncharacterized Cystoisospora-like species (2/21). Sarcocystis neurona, S. canis, and T. gondii are known to cause encephalitis in humans and animals. Sarcocystis canis is an increasingly recognized pathogen of bears and has been reported to cause inflammation in the brain, liver, and muscle in other geographical regions. The clinical significance of the poorly characterized protozoal species is uncertain. Parasites detected in unaffected bears included T. gondii (1/15), an uncharacterized Sarcocystis species (1/15) and an uncharacterized Cystoisospora-like species (2/15). These results suggest that protozoal parasites, particularly Sarcocystis species, may contribute to encephalitis in juvenile black bears, creating a public safety hazard at the human-wildlife interface.

Wide-ranging large carnivores pose myriad challenges for conservation, especially in highly fragmented landscapes. Over a 13-year period, we combined monitoring of radio collared pumas with complementary multi-generational genetic analyses to inform puma conservation in southern California. Our goals were to generate survivorship estimates, determine causes of mortality, identify barriers to movement, and determine the genetic and demographic challenges to puma persistence among >20,000,000 people and extensive urban, suburban, and exurban development. Despite protection from hunting, annual survival for radio collared pumas was surprisingly low (55.8%), and humans caused the majority of puma deaths. The most common sources of mortality were vehicle collisions (28% of deaths), and mortalities resulting from depredation permits issued after pumas killed domestic animals (17% of deaths). Other human-caused mortalities included illegal shootings, public safety removals, and human-caused wildfire. An interstate highway (I-15) bisecting this study area, and associated development, have created a nearly impermeable barrier to puma movements, resulting in severe genetic restriction and demographic isolation of the small puma population (~17-27 adults) in the Santa Ana Mountains west of I-15. Highways that bisect habitat or divide remaining “conserved” habitat, and associated ongoing development, threaten to further subdivide this already fragmented puma population and increase threats to survival. This study highlights the importance of combining demographic and genetic analyses, and illustrates that in the absence of effective measures to reduce mortality and enhance safe movement across highways, translocation of pumas, such as was done with the endangered Florida panther, may ultimately be necessary to prevent further genetic decline and ensure persistence of the Santa Ana Mountains population.

Foothill abortion, also known as epizootic bovine abortion (EBA), has been a long-standing problem for California beef cattle producers. It is a major source of economic loss for California cow and calf producers, and in the 1990s it was estimated that 5% to 10% (45,000 to 90,000 calves) of the California beef calf crop may be lost each year ( Bushnell et al. 1991 ). UC Cooperative Extension (UCCE) farm advisors, specialists and UC Davis School of Veterinary Medicine (SVM) faculty have worked on this disease for nearly 50 years. This long research process finally moved forward in 2005, when the causative agent was identified.

Equine squamous cell carcinomas (eSCCs) are common, and a proportion are likely induced by Equus caballus papillomavirus 2 (EcPV-2). Accurate prediction of clinical outcomes is challenging with no recognized prognostic criteria or consistent histopathological classification scheme for eSCC. The aims of this study were to histopathologically subtype a large case series of eSCCs (genital and ocular) and correlate them with p16 and HER-2 expression, equine papillomavirus infection status, and various clinical and histopathological parameters to predict tumour behavior and prognosis. One hundred and eighty-five samples were examined and subtyped histologically. HER-2 and p16 immunohistochemistry (IHC) and in situ hybridization (ISH) for the EcPV-2 E6/E7 oncogenes were performed on a subset of cases, and follow-up survival data were analyzed. The results were compared and correlated with published guidelines on the categorization of human SCC. Six histopathological subtypes of SCC, according to the WHO, were identified for the first time in horses: usual/invasive (most common), verrucous, pseudoglandular, papillary, warty, and basaloid, with different histological subtypes demonstrating prognostic significance. HER-2 and EcPV-2 statuses were not associated with prognosis in horses with SCC. p16 expression is not associated with EcPV-2 status but could be a potential prognostic factor. ISH demonstrated EcPV-2 genetic material in the majority of eSCCs, except for the papillary subtype, which includes mature, not just pre-cancerous, eSCCs. Widespread HER-2 expression in eSCCs could suggest a role for this cell receptor as a potential therapeutic target.

The human pathogenic orthoflavivirus St. Louis encephalitis virus (SLEV) reemerged in the western United States in 2015 after more than a decade of absence and has since expanded throughout California with sustained interannual transmission. This shift from the historically sporadic pattern of SLEV activity before 2003 raises the question of whether contemporary strains differ in fitness from earlier strains. To assess whether reemerging SLEV possesses enhanced infectivity or transmissibility, we compared five contemporary genotype III strains from California (2016-2023) with a historical genotype V strain from 2003. Growth kinetics were evaluated in mammalian, duck, and mosquito cells; vector competence was assessed in laboratory colonies of Culex tarsalis and Culex quinquefasciatus vectors; and SLEV RNA levels in blood were measured in collaborative cross recombinant intercross mice. Some genotype III strains produced higher titers than the historical genotype V strain in avian, but not mammalian or mosquito cells. Several genotype III strains infected and transmitted SLEV RNA more efficiently than the historical strain in both mosquito species, although no temporal trend in fitness was observed. SLEV fitness was comparable or greater in Culex quinquefasciatus than in Culex tarsalis. Sequencing identified no shared amino acid substitutions associated with vector infection phenotypes. Although genotype III strains exhibited a delayed peak relative to the historical strain, murine SLEV RNA levels in blood were comparable across strains. These findings show some contemporary strains exhibit equal or greater fitness than the historical strain, which may contribute to SLEV persistence and spread in California, underscoring the need for continued surveillance and targeted vector control.IMPORTANCESt. Louis encephalitis virus (SLEV) reemerged in California in 2015 after more than a decade of absence and has since established sustained transmission and expanded geographically. The factors underlying this reemergence remain poorly understood. By comparing contemporary genotype III SLEV strains with a historical genotype V strain, we found that several contemporary strains exhibit equal or greater fitness compared to the historical strain in avian cells and are transmitted more efficiently by the two principal California vector species, Culex tarsalis and Culex quinquefasciatus. We also demonstrate that Cx. quinquefasciatus can transmit infectious genotype III SLEV, supporting its role in SLEV maintenance and spread. Despite differences in mosquito infection and transmission, we found no evidence that fitness in mosquito vectors or mice has continued to rise among strains detected more recently, suggesting that enhanced transmission is not driven by ongoing directional adaptation in these models. These findings indicate that contemporary genotype III SLEV strains possess transmission competence in mosquito vectors that may have contributed to successful reestablishment and persistence of SLEV in California. Improved understanding of the characteristics of reemerging SLEV strains can inform surveillance, risk assessment, and vector control efforts aimed at reducing human exposure to prevent disease caused by SLEV.

BACKGROUND: Evaluation of current antimicrobial prescribing practices can inform antimicrobial stewardship efforts. HYPOTHESIS/OBJECTIVES: Our primary objective was to describe current antibiotic prescribing practice in veterinary schools. ANIMALS: Medical records from 1800 animals (1403 dogs, 397 cats) at 31 US and Canadian veterinary schools were included. METHODS: Hospitals collected data from medical records of animals evaluated on a single date in August 2022. Data included patient signalment, hospitalization status, clinical condition(s), diagnostic testing, antibiotics prescribed, route of administration, evidence of infection, and intended use. RESULTS: Overall, 30.4% (95% CI, 28.3%-32.6%) of animals (32.0% of dogs, 24.9% of cats) were prescribed an antibiotic. The most common conditions for which antibiotics were prescribed were surgical (27.3%, 191/699), urinary (11.9%, 83/699), and respiratory (11.6%, 81/699). The most prescribed classes of antibiotics included aminopenicillins with beta-lactamase inhibitors, first-generation cephalosporins, and fluoroquinolones. First-line antibiotics were not prescribed for sporadic bacterial cystitis. For all prescriptions intended for the treatment of infection, the median duration was 10 days, whereas for those intended for prophylaxis, the median duration was 7 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Opportunities for antimicrobial stewardship in veterinary schools include guidance for use of fluoroquinolones, decreased usage of antibiotics for surgical prophylaxis, and prioritization of first-line antibiotics for sporadic bacterial cystitis. Tracking of antibiotic use data is important to benchmark prescribing and track progress.

Introduction: Animals with intentional genomic alterations (IGAs) hold promise for meeting increasing worldwide demand for animal-source proteins. As part of regulatory risk assessment for introducing animals with IGAs into the food chain, monitoring commensal bacterial microbiota is recommended due to concern that antimicrobial resistance genes used during IGA selection could be transferred, via horizontal gene transfer, to gastrointestinal or environmental bacterial populations, potentially contributing to antimicrobial resistance. The objective of this study was to assess the antimicrobial susceptibility patterns in commensal bacteria isolated from fecal samples of GalSafe™ pigs that have an IGA that includes the aminoglycoside resistance gene nptII. Methods: Antimicrobial resistance rates observed in Escherichia coli, Salmonella, Campylobacter and Enterococcus isolated from GalSafe™ pigs were compared to resistance rates observed in conventional pigs at slaughter. Bacterial isolates were tested for antimicrobial resistance genes by PCR and one isolate underwent whole genome sequencing. Results: In total, 137 bacterial isolates recovered from 55 fecal samples collected from 47 individual adult GalSafe™ pigs were evaluated. Prevalence of antimicrobial resistance in GalSafe™ pigs was generally similar to, or lower than, resistance prevalence reported from conventional pigs at slaughter, based on National Antimicrobial Resistance Monitoring System (NARMS) data. Higher resistance rates in GalSafe™ pigs were observed only for quinolones in Campylobacter coli (ciprofloxacin and nalidixic acid) and nitrofurantoin in Enterococcus spp. One isolate (E. coli) was positive for nptII neomycin resistance gene, the same gene used for IGA selection in GalSafe™ pigs, and the remaining 136 isolates were negative for nptII. However, the positive isolate did not appear to contain nptII derived from the GalSafe™ pig genome as the sequences flanking the gene did not match the IGA. Discussion: We did not detect evidence of nptII gene transformation into bacterial species of potential human health importance in this population of GalSafe™ pigs.

A six-banded armadillo (Euphractus sexcinctus) from a zoo collection in central California developed acute neurologic signs followed by death. Postmortem examination revealed multisystemic bacterial disease (fibrinosuppurative meningoencephalomyelitis, splenitis, and endocarditis) with fibrinous necrotizing vasculitis and thrombosis. Streptococcus gallinaceus was isolated from the heart, lung, and meninges. To our knowledge, this microorganism has not been reported previously in association with systemic disease in an armadillo. Although the source of infection remains unknown, the animal was housed in the same building as chickens, a species that historically is associated with outbreaks of this bacterial infection. This case further broadens our understanding of the possible host range of S. gallinaceus and emphasizes the risk of interspecies pathogen transmission in captive environments.

Urinary tract infections (UTIs) are diagnosed based on symptoms and confirmed by urine culture, despite its limitations in sensitivity. False-negative cultures can lead to inappropriate antimicrobial use or urosepsis in high-risk patients. Next-generation sequencing (NGS)-based metagenomics offers a comprehensive and precise alternative but is rarely applied clinically. We developed and validated BIOTIA-ID, a clinical-grade NGS-based diagnostic pipeline for pathogen detection in urine. Remnant clinical and spiked urine samples underwent extraction, metagenomic library preparation, and Illumina NextSeq 550 sequencing. We trained and applied a bioinformatic pipeline that uses machine learning to identify pathogens and resistance markers. BIOTIA-DX was intentionally designed and trained to increase stringency and reduce false positive detection of urogenital commensals or opportunistic microbes present at colonization levels. Internal controls ensured standardized, high-stringency results. The assay was validated on 1,470 urine specimens evaluating over 14.5k analytes. The clinical validation achieved a 97.2% sensitivity and 99.6% specificity with a limit of detection (LoD) of <15,000 CFU/mL for most bacterial species and <5,000 CFU/mL for fungal species. Discordant results were reconciled by target-specific qPCR or 16S Sanger sequencing, and 87% of the NGS results were concordant with the comparator. A subset of 332 clinical specimens was tested and validated for antimicrobial resistance (AMR). sul and blaSHV genes were commonly associated with Escherichia coli and Klebsiella pneumoniae, while cfxA was found in Prevotella and Pseudomonas spp. detected by BIOTIA-ID. Overall, these data demonstrate that BIOTIA-ID is a comprehensive, highly accurate end-to-end diagnostic assay with notable advantages over current culture-based diagnostics.IMPORTANCEUrinary tract infections (UTIs) are among the most common infections, yet current diagnostic methods, including urine culture, often fail to detect pathogens accurately, leading to delayed treatment and inappropriate antimicrobial use. Clinical metagenomics offers a powerful alternative, especially in complicated cases. BIOTIA-ID is a validated, clinical-grade next-generation sequencing (NGS)-based assay that provides highly accurate pathogen identification and antimicrobial resistance profiling. By incorporating machine learning and stringent quality controls, BIOTIA-ID minimizes false positives and enhances diagnostic precision. Our study demonstrates its superior performance over culture, with potential to improve UTI diagnostics, guide targeted therapy, and support antimicrobial stewardship. The implementation of urine metagenomic diagnostics could support recurrent and complicated UTI patient management, providing a more reliable alternative to traditional methods.