Disease and Mortality in Free-Ranging Leporids in Canada, 1990-2019: A Retrospective Study.

Passive surveillance of wildlife disease is a valuable tool for the identification of emerging and changing disease patterns. Free-ranging leporids play an important role in their ecosystem and in the culture and diet of Canadians; however, little is known about their health status and the zoonotic pathogens they may carry. We summarized major causes of mortality and morbidity, as well as incidental infections and lesions, of free-ranging leporids submitted to the Canadian Wildlife Health Cooperative (CWHC) between 1990 and 2019. We identified Canadian leporids as competent hosts for several zoonotic pathogens, most notably Francisella tularensis (20/569; 3.5%). Trauma was the most frequent cause of mortality or morbidity among leporids, accounting for 46.0% of cases submitted to the CWHC, followed by bacterial infections (13.7%) and emaciation (5.1%). Human-mediated mortalities, such as those involving machines (23.7%), were the most common trauma case type, with apparently healthy individuals overrepresented within this mortality group. Harvesters proved to be a valuable resource for the monitoring of diseased and infected animals, as more than half (69.6%) of the animals submitted by this group had an incidental infection or lesion. The results from this study provide a scientific understanding the cause of mortality in free-ranging leporids in Canada with relevance to public health, wildlife biologists, veterinarians, and potential future surveillance programs.

Development of Highly Efficient Universal Pneumocystis Primers and Their Application in Investigating the Prevalence and Genetic Diversity of Pneumocystis in Wild Hares and Rabbits.

Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about Pneumocystis has originated from studies of merely 4% of extant mammalian species. Further studies of Pneumocystis epidemiology across a broader range of animal species require the use of assays with high sensitivity and specificity. To this end, we have developed multiple universal Pneumocystis primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of Pneumocystis in free-living hares (Lepus townsendii, n = 130) and rabbits (Oryctolagus cuniculus, n = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of Pneumocystis from hares and rabbits, respectively, with significant sequence divergence between the variants from hares. Based on phylogenetic analysis using nearly full-length sequences of the mitochondrial genome, nuclear rRNA operon and dihydropteroate synthase gene for the two most common variants, Pneumocystis in hares and rabbits are more closely related to each other than either are to Pneumocystis in other mammals. Furthermore, Pneumocystis in both hares and rabbits are more closely related to Pneumocystis in primates and dogs than to Pneumocystis in rodents. The high prevalence of Pneumocystis in hares (P. sp. 'townsendii') suggests its widespread transmissibility in the natural environment, similar to P. oryctolagi in rabbits. The presence of multiple distinct Pneumocystis populations in hares contrasts with the lack of apparent intra-species heterogeneity in P. oryctolagi, implying a unique evolution history of P. sp. 'townsendii' in hares.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.

An integrative and multi-indicator approach for wildlife health applied to an endangered caribou herd.

Assessing wildlife health in remote regions requires a multi-faceted approach, which commonly involves convenient samplings and the need of identifying and targeting relevant and informative indicators. We applied a novel wildlife health framework and critically assessed the value of different indicators for understanding the health status and trends of an endangered tundra caribou population. Samples and data from the Dolphin and Union caribou herd were obtained between 2015 and 2021, from community-based surveillance programs and from captured animals. We documented and categorized indicators into health determinants (infectious diseases and trace elements), processes (cortisol, pathology), and health outcomes (pregnancy and body condition). During a recent period of steep population decline, our results indicated a relatively good body condition and pregnancy rates, and decreasing levels of stress, along with a low adult cow survival. We detected multiple factors as potential contributors to the reduced survival, including Brucella suis biovar 4, Erysipelothrix rhusiopathiae and lower hair trace minerals. These results remark the need of targeted studies to improve detection and investigations on caribou mortalities. We also identified differences in health indicators between captured and hunter sampled caribou, highlighting the importance of accounting for sampling biases. This integrative approach that drew on multiple data sources has provided unprecedented knowledge on the health in this herd and highlights the value of documenting individual animal health to understand causes of wildlife declines.

MUSKRATS (ONDATRA ZIBETHICUS) ARE COMPETENT INTERMEDIATE HOSTS OF ECHINOCOCCUS MULTILOCULARIS IN NORTH AMERICA.

The tapeworm Echinococcus multilocularis is an emerging pathogen of significance to human and animal health in Canada, yet little is known about key rodent intermediate hosts in local urban ecosystems. In Europe, invasive muskrats (Ondatra zibethicus) are an important indicator intermediate host species; however, the role of this semiaquatic rodent in the ecology of E. multilocularis is undetermined in the North American context. We examined 93 muskrats that were livetrapped in the spring of 2017 within Calgary, Alberta, Canada, for the presence of E. multilocularis infection. The objectives of this study were to 1) diagnose alveolar echinococcosis using macroscopic assessment, histopathology, and molecular analyses; 2) quantify infection severity; and 3) assess host demographic risk factors for infection. Macroscopic cysts consistent with alveolar echinococcosis were present in 24% of muskrats (22/93). Most individuals had hepatic cysts; however, cysts were also occasionally detected in the mesentery, reproductive organs, omentum, peritoneum, spleen, diaphragm, lung, or kidney. The mean number of cysts per liver was 2.1 (range, 1-4). We examined hepatic cysts from 18 individuals using histology; all had lesions compatible with alveolar echinococcosis. Protoscoleces, indicative of patent infections, were present in 14/18 (78%). No demographic risk factors (sex, body condition, body mass) were significantly associated with infection. Muskrats in the North American context are competent intermediate hosts with high infection prevalence and may play an important role in the ecology of this emerging parasite.

Hepatic fibrosis and mineralization in a free-ranging barren-ground caribou (Rangifer tarandus groenlandicus) from the Northwest Territories.

Due to concerns about the appearance of portions of liver from a harvested adult, male barren-ground caribou (Rangifer tarandus groenlandicus), samples were submitted for diagnostic investigation. The gross and histologic findings were consistent with severe hepatic fibrosis and mineralization. Concentrations of vitamin E in the liver were also deficient. Disease investigations in wildlife of detectable abnormalities such as this provide important information for understanding the role of disease as populations change, as well as for safety of human food sources.

Fibrose hépatique et minéralisation chez un caribou de la toundra ( Rangifer tarandus groenlandicus ) en liberté provenant des Territoires du Nord-Ouest. En raison de préoccupations concernant l'apparence de portions de foie provenant d'un caribou de la toundra mâle adulte (Rangifer tarandus groenlandicus), des échantillons ont été soumis à des fins d'enquête diagnostique. Les résultats macroscopiques et histologiques étaient compatibles avec une fibrose hépatique sévère et une minéralisation. Les concentrations de vitamine E dans le foie étaient également déficientes. Les enquêtes sur les maladies de la faune sauvage portant sur des anomalies détectables telles que celle-ci fournissent des informations importantes pour comprendre le rôle des maladies à mesure que les populations changent, ainsi que pour la sécurité des sources de nourriture humaine.(Traduit par Dr Serge Messier).

Molecular characterization of Sarcocystis spp. as a cause of protozoal encephalitis in a free-ranging black bear.

A free-ranging juvenile male black bear (Ursus americanus), found dead in Alberta, Canada, had severe nonsuppurative encephalitis. Lesions in the brain were most severe in the gray matter of the cerebral cortex, and included perivascular cuffs of lymphocytes and plasma cells, areas of gliosis that disrupted the neuropil, and intralesional protozoan schizonts. The left hindlimb had suppurative myositis associated with Streptococcus halichoeri. Immunohistochemistry and molecular analyses (PCR and sequencing of 4 discriminatory loci: 18S rDNA, ITS-1 rDNA, cox1, rpoB) identified Sarcocystis canis or a very closely related Sarcocystis sp. in the affected muscle and brain tissues. The main lesion described in previously reported cases of fatal sarcocystosis in bears was necrotizing hepatitis. Fatal encephalitis associated with this parasite represents a novel presentation of sarcocystosis in bears. Sarcocystosis should be considered a differential diagnosis for nonsuppurative encephalitis in bears.

Congenital cataract and spherophakia leading to starvation in a free-ranging muskox neonate from the Northwest Territories, Canada.

A muskox neonate (Ovibos moschatus) that died of starvation was diagnosed with congenital lenticular anomalies that included spherophakia and hypermature cataract associated with probable lens-induced lymphocytic uveitis and neutrophilic keratitis. Impaired sight as a result of cataract and associated inflammation likely contributed to abandonment and starvation, although maternal death cannot be excluded definitively. Ocular lesions, such as congenital cataracts and spherophakia in neonates, may be important factors affecting survival in free-ranging animals.

Corynebacterium freneyi Bacterial Septicemia Secondary to Contagious Ecthyma in a Wild Muskox (Ovibos moschatus).

A wild muskox (Ovibos moschatus) with dermatitis typical of contagious ecthyma had secondary bacterial septicemia with Corynebacterium freneyi that included laminitis, hepatitis, and suppurative encephalitis. This case supports the association between orf virus infection and fatal secondary infections, which may have contributed to population declines on Victoria Island, Canada.

Discovery and comparative genomic analysis of elk circovirus (ElkCV), a novel circovirus species and the first reported from a cervid host.

The complete genome sequence of a novel circovirus (elk circovirus (ElkCV) Banff/2019) was determined via high throughput sequencing of liver tissue from a euthanized Rocky Mountain elk (Cervus canadensis nelsoni) from Alberta, Canada. The genome is circular and 1,787 nucleotides long, with two major ORFs encoding predicted proteins. Comparative genomic analysis to 4,164 publicly available complete and near complete circovirus genomes showed that ElkCV shares approximately 65% pairwise genome-wide nucleotide identity with the most closely related circovirus species, porcine circoviruses (PCV) 1 and 2 and bat-associated circovirus (BatACV) 11. ElkCV features a stem-loop within the origin of replication region characteristic of circoviruses. However, it differs from those found in PCV1, PCV2 and BatACV11 since it has a longer stem and contains hexamer repeats that overlap the stem in opposing orientations. Interestingly, stem-loop structures of similar length featuring repeats in a similar position and orientation are also seen in some avian circoviruses. Based on the demarcation threshold established by the International Committee on Taxonomy of Viruses (ICTV) for members of Circoviridae (80% pairwise genome-wide nucleotide identity), ElkCV represents a novel species and is the first complete circovirus genome reported from a cervid host.

Lesions of Mycobacterium avium spp. hominissuis Infection Resembling M. bovis Lesions in a Wild Mule Deer, Canada1.

We used molecular analyses to confirm Mycobacterium avium spp. hominissuis infection in lung granulomas and pyogranulomas in the tracheobronchial lymph node in a wild mule deer in Banff, Canada. These lesions are similar to those found in M. bovis-infected animals, emphasizing the critical need for disease surveillance in wildlife populations.

Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Pneumocystis Species.

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.IMPORTANCEPneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

The devil is in the details-Host disease and co-infections are associated with zoonotic pathogen carriage in Norway rats (Rattus norvegicus).

Traditionally, zoonotic pathogen ecology studies in wildlife have focused on the interplay among hosts, their demographic characteristics and their pathogens. But pathogen ecology is also influenced by factors that traverse the hierarchical scale of biological organization, ranging from within-host factors at the molecular, cellular and organ levels, all the way to the host population within a larger environment. The influence of host disease and co-infections on zoonotic pathogen carriage in hosts is important because these factors may be key to a more holistic understanding of pathogen ecology in wildlife hosts, which are a major source of emerging infectious diseases in humans. Using wild Norway rats (Rattus norvegicus) as a model species, the purpose of this study was to investigate how host disease and co-infections impact the carriage of zoonotic pathogens. Following a systematic trap and removal study, we tested the rats for the presence of two potentially zoonotic bacterial pathogens (Bartonella tribocorum and Leptospira interrogans) and assessed them for host disease not attributable to these bacteria (i.e., nematode parasites, and macroscopic and microscopic lesions). We fitted multilevel multivariable logistic regression models with pathogen status as the outcome, lesions and parasites as predictor variables and city block as a random effect. Rats had significantly increased odds of being infected with B. tribocorum if they had a concurrent nematode infection in one or more organ systems. Rats with bite wounds, any macroscopic lesion, cardiomyopathy or tracheitis had significantly increased odds of being infected with L. interrogans. These results suggest that host disease may have an important role in the ecology and epidemiology of rat-associated zoonotic pathogens. Our multiscale approach to assessing complex intrahost factors in relation to zoonotic pathogen carriage may be applicable to future studies in rats and other wildlife hosts.

Tracheobronchopathia osteochondroplastica: a rare tracheal lesion in a free-ranging grizzly bear.

A mature male grizzly bear (Ursus arctos) that died of blunt-force trauma had numerous hard 1-3-mm nodules protruding from tracheal rings into the lumen of the distal trachea. Histologically, these were round aggregates of mature cartilage within the submucosa. Such lesions are consistent with tracheobronchopathia osteochondroplastica, a rare tracheal disease in humans and animals.

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.

Pathology of wild Norway rats in Vancouver, Canada.

To achieve a contemporary understanding of the common and rare lesions that affect wild, urban Norway rats ( Rattus norvegicus), we conducted a detailed pathology analysis of 672 rats from Vancouver, British Columbia, Canada. Grossly evident lesions, such as wounds, abscesses, and neoplasms, were present in 71 of 672 rats (11%) and tended to be severe. The most common and significant lesions were infectious and inflammatory, most often affecting the respiratory tract and associated with bite wounds. We assessed a subset of rats (up to n = 406 per tissue) for the presence of microscopic lesions in a variety of organ systems. The most frequent lesions that could impact individual rat health included cardiomyopathy (128 of 406; 32%), chronic respiratory tract infections as indicated by pulmonary inducible bronchus-associated lymphoid tissue (270 of 403; 67%), tracheitis (192 of 372; 52%), and thyroid follicular hyperplasia (142 of 279; 51%). We isolated 21 bacterial species from purulent lesions in rats with bacterial infections, the most frequent of which were Escherichia coli, Enterococcus sp., and Staphylococcus aureus. Parasitic diseases in rats resulted from infection with several invasive nematodes: Capillaria hepatica in the liver (242 of 672; 36%), Eucoleus sp. in the upper gastrointestinal tract (164 of 399; 41%), and Trichosomoides crassicauda in the urinary bladder (59 of 194; 30%). Neoplastic, congenital, and degenerative lesions were rare, which likely reflects their adverse effect on survival in the urban environment. Our results establish a baseline of expected lesions in wild urban rats, which may have implications for urban rat and zoonotic pathogen ecology, as well as rat control in cities worldwide.

Observational Study Design in Veterinary Pathology, Part 2: Methodology.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

Observational Study Design in Veterinary Pathology, Part 1: Study Design.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.

Livestock-associated methicillin-resistant Staphylococcus aureus and Clostridium difficile in wild Norway rats (Rattus norvegicus) from Ontario swine farms.

Methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile are important human pathogens that are also carried by animals. The role of wild mammals on farms in their maintenance and transmission, however, is poorly understood. To determine if Norway rats (Rattus norvegicus) are potential carriers of these bacteria on Canadian farms, we tested 21 rats from swine farms in Ontario. The MRSA spa type t034 was isolated from 1 (4.8%) rat. This livestock-associated strain often colonizes pigs and pig farmers, suggesting that transmission among rats and pigs or environmental transmission is possible on pig farms. Clostridium difficile ribotype 078 was isolated from 1 rat from a different farm. This strain is associated with infection in piglets, calves, and humans. The identification of MRSA and C. difficile in Norway rats on farms in Canada adds to the growing knowledge about the role of rats in the ecology of these pathogens. Further studies are required to determine if rats play a part in the epidemiology of these pathogens on farms.

Les bactéries Staphylococcus aureus résistants à la méthicilline (SARM) et Clostridium difficile sont des agents pathogènes importants chez l'humain et sont également retrouvées chez des animaux. Le rôle des animaux sauvages sur les fermes en lien avec leur maintenance et transmission est toutefois peu compris. Afin de déterminer si les rats (Rattus norvegicus) sont des porteurs potentiels de ces bactéries sur les fermes canadiennes, nous avons testé 21 rats provenant de fermes porcines en Ontario. Du SARM spa type t034 fut isolé à partir de un rat (4,8 %). Cette souche associée au bétail colonise souvent les porcs et les éleveurs de porcs, suggérant ainsi que la transmission entre les rats et les rats ou la transmission environnementale est possible sur les fermes porcines. Le ribotype 078 de Clostridium difficile fut isolé de un rat sur une ferme différente. Cette souche est associée à l'infection chez des porcelets, des veaux, et les humains. L'identification de SARM et de C. difficile chez des rats sur des fermes au Canada accroit les connaissances sur le rôle des rats dans l'écologie de ces agents pathogènes. Des études supplémentaires sont requises afin de déterminer si les rats jouent un rôle dans l'épidémiologie de ces agents pathogènes sur les fermes.(Traduit par Docteur Serge Messier).

Environmental Factors Associated with the Carriage of Bacterial Pathogens in Norway Rats.

Worldwide, Norway rats (Rattus norvegicus) carry a number of zoonotic pathogens. Many studies have identified rat-level risk factors for pathogen carriage. The objective of this study was to examine associations between abundance, microenvironmental and weather features and Clostridium difficile, antimicrobial-resistant (AMR) Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) carriage in urban rats. We assessed city blocks for rat abundance and 48 microenvironmental variables during a trap-removal study, then constructed 32 time-lagged temperature and precipitation variables and fitted multivariable logistic regression models. The odds of C. difficile positivity were significantly lower when mean maximum temperatures were high (≥ 12.89°C) approximately 3 months before rat capture. Alley pavement condition was significantly associated with AMR E. coli. Rats captured when precipitation was low (< 49.40 mm) in the 15 days before capture and those from blocks that contained food gardens and institutions had increased odds of testing positive for MRSA. Different factors were associated with each pathogen, which may reflect varying pathogen ecology including exposure and environmental survival. This study adds to the understanding of how the microenvironment and weather impacts the epidemiology and ecology of zoonotic pathogens in urban ecosystems, which may be useful for surveillance and control activities.