Rat Hepatitis E Virus in Norway Rats, Ontario, Canada, 2018-2021.

We tested liver samples from 372 Norway rats (Rattus norvegicus) from southern Ontario, Canada, during 2018-2021 to investigate presence of hepatitis E virus infection. Overall, 21 (5.6%) rats tested positive for the virus. Sequence analysis demonstrated all infections to be rat hepatitis E virus (Rocahepevirus ratti genotype C1).

Ophidiomycosis in Red Cornsnakes (Pantherophis guttatus): Potential Roles of Brumation and Temperature on Pathogenesis and Transmission.

Ophidiomycosis (snake fungal disease) is caused by the fungus Ophidiomyces ophiodiicola. As ophidiomycosis is difficult to study in free-ranging snakes, a reliable experimental model is needed to investigate transmission, pathogenesis, morbidity, and mortality, and the effects of brumation and temperature on disease development. Our objective was to develop such a model via subcutaneous injection of O. ophiodiicola conidia in red cornsnakes (Pantherophis guttatus). The model was used to evaluate transmission and the effects of brumation and temperature in co-housed inoculated and noninoculated snakes. All 23 inoculated snakes developed lesions consistent with ophidiomycosis, including heterophilic and granulomatous dermatitis, cellulitis, and myositis, and embolic fungal granulomas throughout the liver and the coelomic connective tissue in 21/23 (91%). In the inoculated snakes, 21% of skin swabs, 37% of exuvia, and all liver samples tested positive by qPCR (quantitative polymerase chain reaction) for O. ophiodiicola. A post brumation skin swab from 1/12 noninoculated snakes that brumated in contact with inoculated snakes tested positive by qPCR, suggesting possible contact transmission. That snake had microscopic skin lesions consistent with ophidiomycosis, but no visible fungal elements. Of the 23 inoculated snakes, 20 (87%) died over the 70-day experiment, with ophidiomycosis considered the primary cause of death; 12 (52%) of the inoculated snakes died during brumation. Overall, this experimental model of ophidiomycosis reproduced skin lesions analogous to those of many natural cases, and internal lesions similar to the most severe natural cases. The study provides tentative experimental evidence for horizontal transmission in brumation, and offers a tool for future studies of this widespread snake disease.

Pathology associated with ophidiomycosis in wild snakes in Ontario, Canada.

Ophidiomycosis (snake fungal disease) is the most common cause of skin lesions in free-ranging snakes in North America. Naturally infected snakes with ophidiomycosis (9 carcasses, 12 biopsies) were examined grossly and histologically. These cases comprised 32% of the 66 snake cases submitted to the Canadian Wildlife Health Cooperative-Ontario/Nunavut Node in 2012 through 2018. Affected species included the eastern foxsnake (Pantherophis vulpinus; n = 15), gray ratsnake (Pantherophis spiloides; n = 3), eastern massasauga (Sistrurus catenatus; n = 2), and queensnake (Regina septemvittata; n = 1). Severity of disease varied widely from mild microscopic skin lesions to fatal, necrotizing, and ulcerative facial lesions. Key clinical message: Ophidiomycosis should be the primary differential diagnosis for skin lesions in wild snakes, particularly in southern Ontario.

L'ophidiomycose (maladie fongique du serpent) est la cause la plus fréquente de lésions cutanées chez les serpents en liberté en Amérique du Nord. Les serpents infectés naturellement avec l'ophidiomycose (9 carcasses, 12 biopsies) furent examinés macroscopiquement et histologiquement. Ces cas comprenaient 32 % des 66 cas de serpents soumis au Réseau canadien pour la santé de la faune ­ Centre régional de l'Ontario et du Nunavut entre 2012 et 2018. Les espèces affectées incluaient la couleuvre fauve de l'est (Pantherophis vulpinus; n = 15), la couleuvre obscure (Pantherophis spiloides; n = 3), la massasauga (Sistrurus catenatus; n = 2) et la couleuvre royale (Regina septemvittata; n = 1). La sévérité de la maladie variait grandement allant de lésions cutanées microscopiques à une forme fatale, nécrosante et lésions faciales ulcératives.Message clinique clé :L'ophidiomycose devrait être le diagnostic différentiel primaire pour les lésions cutanées chez les serpents sauvages, particulièrement dans le sud de l'Ontario.(Traduit par Dr Serge Messier).

Echinococcus multilocularis Infection, Southern Ontario, Canada.

Alveolar echinococcosis, the disease caused by infection with the intermediate stage of the Echinococcus multilocularis tapeworm, is typically fatal in humans and dogs when left untreated. Since 2012, alveolar echinococcosis has been diagnosed in 5 dogs, 3 lemurs, and 1 chipmunk in southern Ontario, Canada, a region previously considered free of these tapeworms. Because of human and animal health concerns, we estimated prevalence of infection in wild canids across southern Ontario. During 2015-2017, we collected fecal samples from 460 wild canids (416 coyotes, 44 foxes) during postmortem examination and analyzed them by using a semiautomated magnetic capture probe DNA extraction and real-time PCR method for E. multilocularis DNA. Surprisingly, 23% (95% CI 20%-27%) of samples tested positive. By using a spatial scan test, we identified an infection cluster (relative risk 2.26; p = 0.002) in the western-central region of the province. The cluster encompasses areas of dense human population, suggesting zoonotic transmission.

Epizootic Hemorrhagic Disease in White-Tailed Deer, Canada.

Epizootic hemorrhagic disease affects wild and domestic ruminants and has recently spread northward within the United States. In September 2017, we detected epizootic hemorrhagic disease virus in wild white-tailed deer, Odocoileus virginianus, in east-central Canada. Culicoides spp. midges of the subgenus Avaritia were the most common potential vectors identified on site.

The Expectations and Challenges of Wildlife Disease Research in the Era of Genomics: Forecasting with a Horizon Scan-like Exercise.

The outbreak and transmission of disease-causing pathogens are contributing to the unprecedented rate of biodiversity decline. Recent advances in genomics have coalesced into powerful tools to monitor, detect, and reconstruct the role of pathogens impacting wildlife populations. Wildlife researchers are thus uniquely positioned to merge ecological and evolutionary studies with genomic technologies to exploit unprecedented "Big Data" tools in disease research; however, many researchers lack the training and expertise required to use these computationally intensive methodologies. To address this disparity, the inaugural "Genomics of Disease in Wildlife" workshop assembled early to mid-career professionals with expertise across scientific disciplines (e.g., genomics, wildlife biology, veterinary sciences, and conservation management) for training in the application of genomic tools to wildlife disease research. A horizon scanning-like exercise, an activity to identify forthcoming trends and challenges, performed by the workshop participants identified and discussed 5 themes considered to be the most pressing to the application of genomics in wildlife disease research: 1) "Improving communication," 2) "Methodological and analytical advancements," 3) "Translation into practice," 4) "Integrating landscape ecology and genomics," and 5) "Emerging new questions." Wide-ranging solutions from the horizon scan were international in scope, itemized both deficiencies and strengths in wildlife genomic initiatives, promoted the use of genomic technologies to unite wildlife and human disease research, and advocated best practices for optimal use of genomic tools in wildlife disease projects. The results offer a glimpse of the potential revolution in human and wildlife disease research possible through multi-disciplinary collaborations at local, regional, and global scales.

First report of ranavirus mortality in a common snapping turtle Chelydra serpentina.

An adult male snapping turtle with marked palpebral edema and multifocal skin ulceration was found alive in a marsh in southern Ontario in summer 2017. The turtle was transported to a rehabilitation facility and died 4 d after arrival. The carcass was submitted to the Canadian Wildlife Health Cooperative for post-mortem examination. Gross lesions included ulcerative conjunctivitis, necrotizing stomatitis, and splenomegaly. Microscopically, this corresponded to multisystemic fibrinonecrotizing vasculitis and severe fibrinous splenic necrosis. Liver tissue tested positive for frog virus 3-like ranavirus and negative for herpesvirus via polymerase chain reaction. The gross and microscopic lesions were consistent with previous reports of ranavirus infection in turtles and were severe enough to have been the cause of death in this case. This is the first report of morbidity and mortality in a common snapping turtle with a ranavirus infection, and the first reported case of ranavirus infection in a reptile in Canada. Ranaviruses are considered to be an emerging infectious disease in chelonians as they are increasing in distribution, prevalence, and host range.

Red Fox as Sentinel for Blastomyces dermatitidis, Ontario, Canada.

Blastomyces dermatitidis, a fungus that can cause fatal infection in humans and other mammals, is not readily recoverable from soil, its environmental reservoir. Because of the red fox's widespread distribution, susceptibility to B. dermatitidis, close association with soil, and well-defined home ranges, this animal has potential utility as a sentinel for this fungus.

Longitudinal study of Clostridium difficile shedding in raccoons on swine farms and conservation areas in Ontario, Canada.

BACKGROUND: Clostridium difficile is an important enteropathogen affecting humans, domestic animals, and wildlife. The objectives of this study were to 1) compare the prevalence and characteristics of C. difficile isolated from the feces of raccoons trapped on swine farms and conservation sites, and 2) investigate the role of raccoons as potential reservoirs for host-adapted strains of C. difficile using a longitudinal study. Fecal swabs were collected from raccoons at 5 conservation sites and 5 swine farms, once every five weeks, from May to November, 2012. RESULTS: Clostridium difficile was isolated from 9 % (38/444) of samples, from 12 % (37/302) of raccoons, from all 10 sites. A total of 19 different ribotypes were identified, including 5 ribotypes that matched recognized international designations and which are also found in humans (001, 014, 056, 078, and 103). Location type (farm or conservation area) was not associated with C. difficile status (P = 0.448) but only 3 ribotypes (014, 056, and 078) were found in both location types. The prevalence of ribotype 078 was significantly higher on farms (4 %; 9/220) compared to conservation sites (1 %; 2/225) (P = 0.034). Only one of 108 raccoons caught in multiple sessions was positive on more than one occasion. CONCLUSIONS: We found no evidence to support the hypothesis that raccoons harbour host-adapted strains of C. difficile; rather, it appears that raccoons transiently acquire C. difficile from the environment. Raccoons are unlikely to be maintaining C. difficile, but because we detected C. difficile strains that have the potential to cause illness in humans and livestock, and because raccoons can move relatively large distances, they may play a role in the dissemination of pathogenic ribotypes of C. difficile throughout the environment.

Carriage of Clostridium difficile by wild urban Norway rats (Rattus norvegicus) and black rats (Rattus rattus).

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats (Rattus spp.) from Vancouver, Canada, carry C. difficile. Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 "novel" ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.

Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies.

OBJECTIVE: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. RESULTS: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Social and environmental risk factors for canine leptospirosis: A scoping review.

BACKGROUND: Canine leptospirosis is a zoonotic disease of global importance. The identification of social and environmental risk factors remains limited and is needed for planning interventions. Our objectives were to summarise the global trends in social and environmental risk factors for canine leptospirosis and to identify knowledge gaps. METHODS: We searched peer-reviewed and grey literature for observational studies on canine leptospirosis written in English. Variables assessed via formal statistical tests were recorded and categorised into thematic risk factor groups. The risk factors were summarised by the number of unique studies and their direction of association. RESULTS: Of the 3184 articles identified, 66 met the inclusion criteria. Eight environmental and three social risk factor groups were identified. Exposure to animals and water was commonly studied, but social risk factors were infrequently examined. Most studies were conducted on populations in higher-income countries and countries in the Americas. LIMITATIONS: Several limitations were encountered, including inconsistent study designs and measurement and control of variables as well as disproportionate representation of studies from the Americas and higher-income countries. CONCLUSION: The weight and quality of evidence for social and environmental risk factors were highly variable. Additional research following standard reporting guidelines to improve transparency and increase generalisability to different contexts is needed.

How do life history and behaviour influence plastic ingestion risk in Canadian freshwater and terrestrial birds?

Plastic ingestion presents many potential avenues of risk for wildlife. Understanding which species and environments are most exposed to plastic pollution is a critical first step in investigating the One Health implications of plastic exposure. The objectives of this study were the following: 1) Utilize necropsy as part of ongoing passive disease surveillance to investigate ingested mesoplastics in birds collected in Ontario and Nunavut, and examine the relationships between bird-level factors and ingested debris; 2) evaluate microplastic ingestion compared to ingested mesodebris in raptors; and 3) identify potential sentinel species for plastic pollution monitoring in understudied freshwater and terrestrial (inland) environments. Between 2020 and 2022, 457 free-ranging birds across 52 species were received for postmortem examination. The upper gastrointestinal tracts were examined for mesoplastics and other debris (>2 mm) using standard techniques. Twenty-four individuals (5.3%) retained mesodebris and prevalence varied across species, with foraging technique, food type, and foraging substrate all associated with different metrics of debris ingestion. The odds of ingesting any type of anthropogenic mesodebris was nine times higher for non-raptorial species than for raptors. For a subset of raptors (N = 54) across 14 species, the terminal portion of the distal intestinal tract was digested with potassium hydroxide and microparticles were assessed using stereo-microscopy. Although only one of 54 (1.9%) raptors included in both analyses retained mesodebris in the upper gastrointestinal tract, 24 (44.4%) contained microparticles in the distal intestine. This study demonstrates that a variety of Canadian bird species ingest anthropogenic debris in inland systems, and suggests that life history and behaviour are associated with ingestion risk. For raptors, the mechanisms governing exposure and ingestion of mesoplastics appear to be different than those that govern microplastics. Herring gulls (Larus argentatus) and ring-billed gulls (Larus delawarensis) are proposed as ideal sentinels for plastic pollution monitoring in inland systems.

Additional diagnoses of Echinococcus multilocularis in eastern chipmunks (Tamias striatus) from southern Ontario - results from ongoing surveillance for E. multilocularis in intermediate hosts in Ontario, Canada.

Echinococcus multilocularis, a cestode with zoonotic potential, is now known to have a high prevalence in wild canid definitive hosts of southern Ontario. The distribution of E. multilocularis across this region in red foxes (Vulpes vulpes) and coyotes (Canis latrans) is widespread yet heterogenous. In contrast, confirmed diagnoses of E. multilocularis in wild free-ranging intermediate hosts within Ontario are currently limited to a single eastern chipmunk (Tamias striatus). These findings prompted ongoing surveillance efforts in intermediate host species, primarily rodents. Our report describes the results of passive surveillance through wildlife carcass submissions to the Canadian Wildlife Health Cooperative (CWHC) and targeted active sampling of small mammal species from 2018 to 2023; a second and third eastern chipmunk were found to be infected with E. multilocularis. However, these were the only occurrences from surveillance efforts which collectively totaled 510 rodents and other small mammals. Continued surveillance for E. multilocularis in intermediate hosts is of high importance in light of the recent emergence of this parasite in Ontario.

Assessing avian influenza surveillance intensity in wild birds using a One Health lens.

Wildlife disease surveillance, particularly for pathogens with zoonotic potential such as Highly Pathogenic Avian Influenza Virus (HPAIV), is critical to facilitate situational awareness, inform risk, and guide communication and response efforts within a One Health framework. This study evaluates the intensity of avian influenza virus (AIV) surveillance in Ontario's wild bird population following the 2021 H5N1 incursion into Canada. Analyzing 2562 samples collected between November 1, 2021, and October 31, 2022, in Ontario, Canada, we identify spatial variations in surveillance intensity relative to human population density, poultry facility density, and wild mallard abundance. Using the spatial scan statistic, we pinpoint areas where public engagement, collaborations with Indigenous and non-Indigenous hunter/harvesters, and working with poultry producers, could augment Ontario's AIV wild bird surveillance program. Enhanced surveillance at these human-domestic animal-wildlife interfaces is a crucial element of a One Health approach to AIV surveillance. Ongoing assessment of our wild bird surveillance programs is essential for strategic planning and will allow us to refine approaches and generate results that continue to support the program's overarching objective of safeguarding the health of people, animals, and ecosystems.

Environmental and sociodemographic factors associated with zoonotic pathogen occurrence in Norway rats (Rattus norvegicus) from Windsor, Ontario.

AIMS: Rat-associated zoonotic pathogen transmission at the human-wildlife interface is a public health concern in urban environments where Norway rats (Rattus norvegicus) thrive on abundant anthropogenic resources and live in close contact with humans and other animal species. To identify potential factors influencing zoonotic pathogen occurrence in rats, we investigated associations between environmental and sociodemographic factors and Leptospira interrogans and Bartonella spp. infections in rats from Windsor, Ontario, Canada, while controlling for the potential confounding effects of animal characteristics (i.e., sexual maturity and body condition). METHODS AND RESULTS: Between November 2018 and June 2021, 252 rats were submitted by collaborating pest control professionals. Kidney and spleen samples were collected for L. interrogans and Bartonella spp. PCR and sequencing, respectively. Of the rats tested by PCR, 12.7% (32/252) were positive for L. interrogans and 16.3% (37/227) were positive for Bartonella species. Associations between infection status and environmental and sociodemographic variables of interest were assessed via mixed multivariable logistic regression models with a random intercept for social group and fixed effects to control for sexual maturity and body condition in each model. The odds of L. interrogans infection were significantly higher in rats from areas with high building density (odds ratio [OR]: 3.76; 95% CI: 1.31-10.79; p = 0.014), high human population density (OR: 3.31; 95% CI: 1.20-9.11; p = 0.021), high proportion of buildings built in 1960 or before (OR: 11.21; 95% CI: 2.06-60.89; p = 0.005), and a moderate number of reports of uncollected garbage compared to a low number of reports (OR: 4.88; 95% CI: 1.01-23.63; p = 0.049). A negative association was observed between median household income and Bartonella spp. infection in rats (OR: 0.26; 95% CI: 0.08-0.89; p = 0.031). CONCLUSIONS: Due to the complexity of the ecology of rat-associated zoonoses, consideration of environmental and sociodemographic factors is of critical importance to better understand the nuances of host-pathogen systems and inform how urban rat surveillance and intervention efforts should be distributed within cities.

Correction: Using whole-genome sequence data to examine the epidemiology of Salmonella, Escherichia coli and associated antimicrobial resistance in raccoons (Procyon lotor), swine manure pits, and soil samples on swine farms in southern Ontario, Canada.

[This corrects the article DOI: 10.1371/journal.pone.0260234.].

Comparing Canadian Lyme disease risk area classification methodologies.

Lyme disease risk areas have increased across Canada in recent decades with the ongoing range expansion of Ixodes scapularis and Borrelia burgdorferi. Different methodologies are used by federal and provincial governments to determine local Lyme disease risk, which can make comparisons between regions challenging. In this study, seven Canadian Lyme disease risk classification methodologies were compared with each other to highlight the strengths and limitations of how each definition measured I. scapularis and B. burgdorferi risk. Each methodology was applied to active surveillance data from Ontario, and per cent agreement and kappa statistics were calculated. The methodologies varied in their measurements of the risk of exposure to I. scapularis and B. burgdorferi based on their use of active surveillance techniques, multiple types of collected surveillance data and laboratory confirmation of B. burgdorferi. Most initial Lyme disease risk site classifications were maintained over time. Kappa and per cent agreement statistics highlighted large differences between 8 of the 15 methodology pairings, indicating the presence of inconsistencies between most methodologies. Accurate, consistent surveillance and assessment of the spread of I. scapularis and its pathogens will aid with communicating Lyme disease risk to the public and preventing tick-borne pathogen transmission.

USING WILDLIFE REHABILITATION AND POSTMORTEM DATA TO IDENTIFY KEY CAUSES OF MORBIDITY AND MORTALITY IMPACTING THE HEALTH AND WELFARE OF FREE-LIVING WILD ANIMALS IN CANADA.

The health and welfare of wild animals are of increasing concern, yet there are very few large-scale data syntheses examining how causes of wildlife morbidity and mortality vary across time, space, and taxa. Records for 18,540 animals submitted to the Canadian Wildlife Health Cooperative (CWHC) (2009-19) and 144,846 animals admitted to 19 wildlife rehabilitation centers (WRCs) (2015-19) were evaluated to 1) identify the main causes of morbidity and mortality for Canadian wildlife and 2) assess the utility and complementarity of these two data sources to further our understanding of wildlife health. The CWHC cases (mortality) were examined by pathologists and grouped by the presence or absence of five diagnostic categories: trauma, emaciation, infection or inflammation, toxicity, and other. These CWHC animals were also classified as "killed due to real or perceived human-wildlife conflict" based on finder history. The WRC admissions were categorized by health issue (according to intake records) and based on reported or observed situational reasons for admission: parental loss, unsafe or unsuitable location, nest or habitat disturbance, illegal possession, and abnormal behavior. For both datasets, the main reason for submission or admission was trauma (44 and 48%, respectively), especially vehicle collisions (7 and 11%) and window or building strikes (5 and 7%). Many other WRC admissions were due to parental loss (28%), cat attacks (6%), and immature animals being found in unsafe or unsuitable locations (6%). Most other CWHC mortalities were caused by infections (27%) and emaciation (23%). Relatively few birds, amphibians, and reptiles submitted to CWHC were killed due to human-wildlife conflict, but 22% of mammals were killed for this reason, highlighting the taxonomic differences in the perceived threat of wildlife to finders, and therefore their response. Together, these data sources highlight key issues impacting the health and welfare of wild animals in Canada.