Widespread Exposure to Mosquitoborne California Serogroup Viruses in Caribou, Arctic Fox, Red Fox, and Polar Bears, Canada.

Northern Canada is warming at 3 times the global rate. Thus, changing diversity and distribution of vectors and pathogens is an increasing health concern. California serogroup (CSG) viruses are mosquitoborne arboviruses; wildlife reservoirs in northern ecosystems have not been identified. We detected CSG virus antibodies in 63% (95% CI 58%-67%) of caribou (n = 517), 4% (95% CI 2%-7%) of Arctic foxes (n = 297), 12% (95% CI 6%-21%) of red foxes (n = 77), and 28% (95% CI 24%-33%) of polar bears (n = 377). Sex, age, and summer temperatures were positively associated with polar bear exposure; location, year, and ecotype were associated with caribou exposure. Exposure was highest in boreal caribou and increased from baseline in polar bears after warmer summers. CSG virus exposure of wildlife is linked to climate change in northern Canada and sustained surveillance could be used to measure human health risks.

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).

Large-scale prion protein genotyping in Canadian caribou populations and potential impact on chronic wasting disease susceptibility.

Polymorphisms within the prion protein gene (Prnp) are an intrinsic factor that can modulate chronic wasting disease (CWD) pathogenesis in cervids. Although wild European reindeer (Rangifer tarandus tarandus) were infected with CWD, as yet there have been no reports of the disease in North American caribou (R. tarandus spp.). Previous Prnp genotyping studies on approximately 200 caribou revealed single nucleotide polymorphisms (SNPs) at codons 2 (V/M), 129 (G/S), 138 (S/N), 146 (N/n) and 169 (V/M). The impact of these polymorphisms on CWD transmission is mostly unknown, except for codon 138. Reindeer carrying at least one allele encoding for asparagine (138NN or 138SN) are less susceptible to clinical CWD upon infection by natural routes, with the majority of prions limited to extraneural tissues. We sequenced the Prnp coding region of two caribou subspecies (n = 986) from British Columbia, Saskatchewan, Yukon, Nunavut and the Northwest Territories, to identify SNPs and their frequencies. Genotype frequencies at codon 138 differed significantly between barren-ground (R. t. groenlandicus) and woodland (R. t. caribou) caribou when we excluded the Chinchaga herd (p < .05). We also found new variants at codons 153 (Y/F) and 242 (P/L). Our findings show that the 138N allele is rare among caribou in areas with higher risk of contact with CWD-infected species. As both subspecies are classified as Threatened and play significant roles in North American Indigenous culture, history, food security and the economy, determining frequencies of Prnp genotypes associated with susceptibility to CWD is important for future wildlife management measures.

Granulomatous Inflammation of the Muzzle in White-Tailed Deer (Odocoileus virginianus) and Mule Deer (Odocoileus hemionus) Associated With Mannheimia granulomatis.

Since 2002, reports of deer with swollen muzzles from throughout the United States have resulted in significant interest by wildlife biologists and wildlife enthusiasts. The condition was identified in 25 white-tailed deer (Odocoileus virginianus) and 2 mule deer (O. hemionus). Microscopic lesions consisted of severe, granulomatous or pyogranulomatous inflammation of the muzzle, nasal planum, and upper lip, as well as similar but less severe inflammation of the hard palate. Lymphadenitis of regional lymph nodes was common and granulomatous pneumonia was present in one individual. Splendore-Hoeppli material was typical in the center of inflammatory foci. Other than the single instance of pneumonia, systemic disease was not evident. Various bacterial species were isolated in culture, most of which were not morphologically consistent with the colonies of small, gram-negative bacteria observed in the center of the granulomas. Amplification and sequencing of the bacterial 16S rRNA gene from tissues of affected deer resulted in the identification of Mannheimia granulomatis. Laser capture microdissection was used to confirm that the colonies in the inflammatory foci were M. granulomatis. The cases described here are reminiscent of a bovine disease in Brazil and Argentina, locally called lechiguana. Although the inflammation of lechiguana is mostly truncal, the microscopic lesions are very similar and are also attributed to M. granulomatis. It is unclear if this is an emerging infectious disease of deer, or if it is a sporadic, uncommon condition that has only recently been recognized.

Multicentric Round Cell Neoplasms and Their Viral Associations in Wild Turkeys (Meleagris gallopavo) in the Southeastern United States.

Multiple oncogenic viruses, including lymphoproliferative disease virus (LPDV) and reticuloendotheliosis virus (REV), have been detected in wild turkeys (Meleagris gallopavo). The prevalence of infection with these viruses appears to be more common than overt disease; thus, data on the manifestation of associated disease in wild turkeys are scarce. Diagnostic records from wild turkeys submitted to the Southeastern Cooperative Wildlife Disease Study from 1980 to 2017 were reviewed to identify cases of neoplasia. Neoplasia was reported in 59 of 851 (6.9%) wild turkeys submitted. Of the cases of neoplasia tested by polymerase chain reaction, LPDV was detected in 34 of 58 (59%), REV in 10 of 39 (26%), both viruses in 3 of 39 (8%), and no retroviruses detected in 5 of 39 (13%) turkeys. The most common gross lesions observed among turkeys with neoplasms were emaciation (30/40; 75%); nodules in the skin (26/59; 44%), liver (17/59; 29%), or spleen (9/59; 15%); and splenomegaly (14/59; 24%). Microscopically, nodules were composed of pleomorphic round cells with large eccentric nuclei and prominent nucleoli resembling lymphocytes or lymphoblasts (57/59; 97%) except for 2 cases, one of myeloid cell origin and the other with primarily spindloid cells. This study indicates the need to characterize the pathogenesis and potential health threat posed by REV and LPDV to wild turkeys. Experimental infection studies and the development of additional diagnostic tests to confirm the role of retroviruses in lymphoproliferative disease are warranted.

Whole-genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S.

BACKGROUND: Newcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1, is commonly isolated from columbids in the United States and worldwide. Complete genomic characterization of these genotype VI viruses circulating in wild columbids in the United States is limited, and due to the genetic variability of the virus, failure of rapid diagnostic detection has been reported. Therefore, in this study, formalin-fixed paraffin-embedded (FFPE) samples were subjected to next-generation sequencing (NGS) to identify and characterize these circulating viruses, providing valuable genetic information. NGS enables multiple samples to be deep-sequenced in parallel. When used on FFPE samples, this methodology allows for retrospective studies of infectious organisms. METHODS: FFPE wild pigeon tissue samples (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016 were analyzed using NGS to detect and sequence NDV genomes from randomly amplified total RNA. Results were compared to the previously published immunohistochemistry (IHC) results conducted on the same samples. Additionally, phylogenetic analyses were conducted on the complete and partial fusion gene and complete genome coding sequences. RESULTS: Twenty-three out of 29 IHC-positive FFPE pigeon samples were identified as positive for NDV by NGS. Positive samples produced an average genome coverage of 99.6% and an average median depth of 199. A previously described sub-genotype (VIa) and a novel sub-genotype (VIn) of NDV were identified as the causative agent of 10 pigeon mortality events in the U.S. from 2010 to 2016. The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S. CONCLUSIONS: This work reports the first successful evolutionary study using deep sequencing of complete NDV genomes from FFPE samples of wild bird origin. There are at least two distinct U.S. lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad. These findings support the hypothesis that columbids are serving as reservoirs of virulent NDV in the U.S.

Congenital Ocular Abnormalities in Free-Ranging White-Tailed Deer.

Congenital ocular abnormalities in cervids have been previously reported as individual cases from various regions of the United States and include microphthalmia, anophthalmia, congenital cataracts, dermoids, and colobomata. A common underlying cause for these abnormalities, such as nutritional deficiencies, environmental toxin exposures, or genetic mutations, has not been established. This retrospective study summarized and compared cases of suspected congenital ocular abnormalities in free-ranging white-tailed deer ( Odocoileus virginianus) submitted to the Southeastern Cooperative Wildlife Disease Study (SCWDS) in Athens, Georgia, to the preexisting literature. Of 3645 accessions of white-tailed deer submitted to SCWDS, 15 qualifying case records were found. An additional 15 cases were reported previously in the literature. Conditions described in SCWDS cases included microphthalmia (8/15), congenital cataracts (3/15), anophthalmia (2/15), colobomata (1/15), anterior segment dysgenesis (1/15), ectopic lacrimal gland tissue (1/15), and congenital blindness with corneal opacity (1/15). Most (11/15; 73%) of the SCWDS cases were male fawns with an average age of 4 months at presentation, consistent with previously described cases. Most animals had bilateral abnormalities with few extraocular congenital abnormalities, also consistent with existing reports. Cases were variably tested for various infectious agents at the time of submission; 2 cases were seropositive for bluetongue virus. Spatiotemporal clustering of cases was not evident. This study provided a concise and systematic summary of known existing cases of congenital ocular defects in fawns but did not identify a cause.

Causes of mortality of harbor porpoises Phocoena phocoena along the Atlantic and Pacific coasts of Canada.

There is increasing public interest in the overall health of the marine environment. Harbor porpoises Phocoena phocoena have a coastal distribution, and stranded animals function as sentinels for population and ecosystem health. The goal of this retrospective study was to join datasets from the western Atlantic and eastern Pacific coasts of Canada to investigate causes of morbidity and mortality in this species. A total of 241 necropsy records were reviewed including 147 (61%) from the Pacific region and 94 (39%) from the Atlantic region from 1988 to 2011. A cause of death could be determined with confidence in 118 (49%) of these cases. Of these 118 cases, the leading cause of mortality for both regions, together and separately, was infectious disease. In the Pacific region, this was followed by traumatic and anthropogenic causes, whereas in the Atlantic region, it was followed by emaciation/starvation, mortality of dependent calves, and anthropogenic causes. Pathogens of potential zoonotic significance or indicative of environmental contamination, e.g. Salmonella sp. and Cryptococcus gattii, were identified. Numerous parasitic species were observed within the lungs, liver, stomach, middle ear, and subcutaneous tissues, although they were usually interpreted as incidental findings. Anthropogenic causes may be underrepresented as they are notoriously difficult to diagnose with certainty, thereby making up a proportion of the 'unknown causes of death' (51%) category. Improved standardization of data collection and documentation is required to better understand harbor porpoise and ecosystem health.

Poorly differentiated cutaneous carcinoma of non-sebaceous origin in a 3-year-old Mongolian gerbil (Meriones unguiculatus).

A 3-year-old female gerbil developed a non-healing skin wound due to a malignant neoplasm. Histology, immunohistochemistry (cytokeratin 19 positive; vimentin, estrogen, and progesterone receptor negative), and electron microscopy (no desmosomes or melanosomes) revealed an undifferentiated carcinoma with pulmonary metastasis. Unlike in previous reports, it did not arise from the abdominal pad's sebaceous gland.

Carcinome cutané d'origine non sébacée peu différencié chez une gerbille de Mongolie âgée de 3 ans(Meriones unguiculatus). Une gerbille femelle âgée de 3 ans a développé une plaie cutanée qui ne guérissait pas en raison d'un néoplasme malin. Des examens histologiques, par immunohistochimie (positif pour la cytokératine 19; négatif pour les récepteurs de vimentine, d'œstrogène et de progestérone) et par microscopie électronique (pas de desmosomes ni de mélanosomes) ont révélé un carcinome indifférencié avec métastase pulmonaire. Contrairement aux rapports antérieurs, il n'était pas causé par la glande sébacée du coussinet abdominal.(Traduit par Isabelle Vallières).

Absolute polycythemia in a bald eagle (Haliaeetus leucocephalus).

An approximately 6-mo-old female bald eagle (Haliaeetus leucocephalus) was presented for an inability to fly and bilateral drooped wings. Pectoral muscle atrophy with a moderate polycythemia was present. Over the course of 3 wk, there were no improvements in flight capacity, although the bird gained substantial weight. Further investigation revealed a prominent cyanosis that was responsive to oxygen therapy, a chronic respiratory acidosis with hypoxia, a cardiac murmur, and a persistent polycythemia. No obvious antemortem etiology for the clinical findings was discovered on computerized tomography, angiography, or echocardiography. The bird was euthanatized as a result of the poor prognosis. Necropsy and histopathology revealed no significant cardiovascular or pulmonary pathology. No myopathy was evident on electron microscopy of formalin-fixed tissues. Based on these diagnostics, a neuromuscular disorder is suspected as the cause for the blood gas abnormalities, with a resulting polycythemia from the hypoxia.

Case report: Disseminated larval trematodiasis caused by Clinostomum marginatum in a green tree frog (Hyla cinerea).

Clinostomum spp. are common parasites of piscivorous birds. Metacercaria are typically observed in the muscles or just under the skin of fish and rarely amphibians. We describe an unusually severe case of Clinostomum marginatum infection in an adult female green tree frog (Hyla cinerea) from Georgia (USA). The frog was found in November 2015 with a high number (>250) of widely disseminated, raised, subcutaneous nodules. The frog died in December. At necropsy, it was emaciated, and the skin was covered in raised uniform, tan-green, subcutaneous, ∼2-3 mm diameter nodules. Each nodule contained 1-3 C. marginatum metacercariae. Microscopically, high numbers of trematodes were within subcutaneous tissues and in coelomic and oral cavities, lung, liver, kidney, ovary, orbit and calvarium. Small to large numbers of lymphocytes and melanomacrophages were in connective tissues and epidermis. A 732 bp region of COI was 98.8-99.8% similar to numerous sequences of C. marginatum and, phylogenetically it grouped with these C. marginatum sequences. The ITS-1 region was 100% similar to a C. marginatum sample from a great egret (Ardea alba) from Mississippi. This report represents a novel finding of severe trematodiasis in a free-ranging amphibian with C. marginatum infection.

In-nest mortality and pathology of hawksbill sea turtle (Eretmochelys imbricata) embryos and hatchlings in St. Kitts and Nevis.

Strategies to improve the hatch success and survival of critically endangered hawksbill turtles (Eretmochelys imbricata) require knowledge of threats to them including pathological conditions. The objective of this study was to describe the mortality and pathology of embryos and dead-in-nest hatchlings on St. Kitts and Nevis. Over the 2019/20 and 2020/21 nesting seasons, the combined mean (SD) hatch success for the two islands was 81.9% (13.2%) and deceased individuals within excavated nests were early-stage embryos (70.7%), late-stage embryos (17.7%), pipped-hatchlings (8.2%) or dead-in-nest hatchlings (3.4%). From 2017 to 2021, a post-mortem examination was performed on 183 turtles, including histology for 116. Anatomical malformations affected 77 (42.1%) examined turtles and included abnormal scute shape or number (22.4%), dysmelia (8.7%), schistosomus reflexus (7.7%) and compressed carapace (7.7%). Microscopic lesions were found in 49.1% of turtles and included tissue mineralization (26.7%, including renal, fetal membrane, liver, heart or muscle), chorioallantoitis (16.2%) and skeletal muscle degeneration and necrosis (10%). Inflammatory lesions associated with fungal or bacterial infections were in the skin (n = 3), chorioallantois (n = 4), lung (n = 3) or yolk sac (n = 1). These lesions may reflect non-specific terminal conditions but their presence in-nest helps explain some of the mortality and pathology documented in hatchlings that die during rehabilitative care. All of the gonads adequately represented for histological determination of sex were female (n = 62), supporting concern for feminization of Caribbean hawksbill turtle nests. The study identifies lesions that could affect hatch and emergence success. The high frequency of skeletal malformations indicates the need for investigations addressing regional impact and pathogenesis, especially genetic and environmental aetiologies including nest temperature. Immediate examination of live hatchlings on nest emergence is warranted to better determine the prevalence of non-fatal malformations that could impact fitness and population genetics.

Causes of morbidity and mortality in wild cottontail rabbits in the eastern United States, 2013-2022.

Interest in causes of mortality of free-ranging, native North American lagomorphs has grown with the emergence of rabbit hemorrhagic disease virus 2 (RHDV2). Over the years 2013-2022, the Southeastern Cooperative Wildlife Disease Study received 119 Sylvilagus spp. case submissions from the central and eastern United States, comprising 147 rabbits. Most (86%) of these submissions occurred after detecting RHDV2 in the United States in 2020. Laboratory data from these rabbits were retrospectively evaluated for major causes, contributors to mortality, and pathogen detections. Gross and histologic examination was performed for 112 rabbits. Common primary causes of death included trauma (n = 49), bacterial disease (n = 31), emaciation (n = 6), and parasitism (n = 6). Among the 32 rabbits with bacterial disease, 12 were diagnosed with tularemia and 7 with pasteurellosis. Rabbits with pasteurellosis had disseminated abscessation, septicemia, and/or polyserositis. Less commonly, cutaneous fibroma (n = 2), notoedric mange (n = 2), encephalitozoonosis (n = 2), neoplasia (round-cell sarcoma; n = 1), and congenital abnormalities (n = 1) were diagnosed. RHDV2 was not detected in 123 rabbits tested. Although RHDV2 has not been detected in wild lagomorphs in the eastern United States, detections in domestic rabbits from the region emphasize the need for continued surveillance. Furthermore, continued surveillance for Francisella tularensis informs public health risk. Overall, increased knowledge of Sylvilagus spp. health furthers our understanding of diseases affecting these important prey and game species.

APPARENT PREVALENCE, DIVERSITY, AND ASSOCIATED LESIONS OF PERIORBITAL NEMATODES IN A POPULATION OF BARRED OWLS (STRIX VARIA) FROM NORTHERN CALIFORNIA, USA.

Over the last four decades, Barred Owls (Strix varia) have expanded their range to include much of western North America, including California. This expansion is suspected to have contributed to declining populations of a closely related species, the federally threatened Northern Spotted Owl (Strix occidentalis caurina). As a result, understanding potential health threats to Barred Owls has implications for Spotted Owl health and recovery. From 2016 to 2020, 69 Barred Owls were collected to determine the apparent prevalence of periorbital nematode infection, to identify the parasite species present, and to investigate the potential pathologic effects on their hosts. The nematodes were morphologically identified as Oxyspirura and Aprocta spp. On the basis of phylogenetic analyses, they were clearly divergent from published sequences of other species within these genera. Overall, 34 (49%) Barred Owls were infected with periorbital nematodes, with Oxyspirura sp. infections being much more common (94%) than Aprocta sp. (18%). Histopathology revealed varying severity of conjunctivitis in infected owls. Despite the frequency of infection and subsequent inflammation, parasite burden was not associated with reduced body weight in these owls. As a result, the potential health effect of these nematodes is unclear. Further taxonomic characterization is needed to determine potential novelty of these nematodes.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Co-circulation of five species of dog parvoviruses and canine adenovirus type 1 among gray wolves (Canis lupus) in northern Canada.

Several viruses can infect wild carnivores but their impact on wildlife health is poorly understood. We investigated the presence, diversity and distribution of various DNA viruses in 303 wolves inhabiting a vast area of the Northwest Territories, Canada, over a period of 13 years. We found evidence for the presence of canine bufavirus (CBuV, 42.6%), canine parvovirus 2 (CPV-2, 34.0%), canine bocavirus 2 (CBoV-2, 5.0%), cachavirus (CachaV-1, 2.6%), canine adenovirus 1 (CAdV-1, 1%) and minute virus of canines (MVC, 0.3%). To our knowledge, this is the first detection of CBoV-2, MVC and CachV-1 in wild animals. We also demonstrate that CBuV and CachaV-1 were already circulating among wild animals at least 11 and 10 years, respectively, before their discoveries. Although CBuV prevalence was higher, CPV-2 was the most prevalent virus among juveniles, while CBuV infection was associated with poor nutrition conditions. Even if its prevalence was low, CachaV-1 had the highest multiple infection rate (87.5%). CadV-1 and MVC sequences were highly identical to reference strains, but we observed a high diversity among the other viruses and detected three new variants. One CPV-2 variant and one CBuV variant were endemic since the beginning of the 2000s in the entire investigated region, whereas one CBuV variant and two CBoV-2 variants were found in a more restricted area over multiple years and CachaV-1 was found only in one region. Two CPV-2 variants and one CachaV-1 variant were observed only once, indicating sporadic introductions or limited circulation. Different patterns of endemicity might indicate that viruses were introduced in the wolf population at different timepoints and that mixing between wolf packs may not be constant. Different epidemiological behaviors depend on viral factors like infectivity, transmission routes, pathogenicity and tissue-tropism, and on host factors like proximity to densely populated areas, carnivory and pack density and mixing.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Heavy Rainfall, Sewer Overflows, and Salmonellosis in Black Skimmers (Rynchops niger).

Extreme weather events, particularly heavy rainfall, are occurring at greater frequency with climate change. Although adverse human health effects from heavy rainfall are often publicized, impacts to free-ranging wildlife populations are less well known. We first summarize documented associations of heavy rainfall on wildlife health. We then report a novel investigation of a salmonellosis outbreak in a colony of black skimmers (Rynchops niger) in Florida, USA. During June-September 2016, heavy rainfall resulted in the discharge of millions of gallons of untreated wastewater into the Tampa Bay system, contaminating the water body, where adult skimmers foraged. At least 48 fledglings died, comprising 39% of the colony's nesting season's offspring. Of eight examined deceased birds from the colony, six had a systemic salmonellosis infection. Isolates were identified as Salmonella enterica serotype Typhimurium. Their pulsed-field gel electrophoresis patterns were identical to each other and matched those from several human Salmonella sp. infections. Differences among whole-genome sequences were negligible. These findings and the outbreak's epidemic curve suggest propagated transmission occurred within the colony. A multidisciplinary and One Health approach is recommended to mitigate any adverse effects of climate change-driven stochastic events, especially when they place already imperiled wildlife at further risk.

Distinctive Gross Presentation in Free-Ranging White-Tailed Deer (Odocoileus virginianus) with Rabies.

The white-tailed deer (Odocoileus virginianus) is a popular game species in North America and often lives in close proximity to humans and domestic animals. Deer with neurologic signs are of high interest to the general public and wildlife managers because of disease and safety concerns. Our aim was to describe diagnostic findings from free-ranging white-tailed deer diagnosed with rabies from across the eastern US from 2000 to 2021, with emphasis on gross lesions in the skin and soft tissue overlying the skull. We reviewed diagnostic reports of white-tailed deer cases submitted to the Southeastern Cooperative Wildlife Disease Study for those diagnosed with rabies from 2000 to 2021. Rabies virus infection was confirmed by immunohistochemistry or fluorescent antibody test of brain, or both. Nine adult deer from five states were diagnosed with rabies, including seven (78%) females and two (22%) males. Three (33%) deer were found dead, and six (67%) were humanely dispatched for abnormal behavior. Six deer heads were examined grossly and had lesions, including forehead or periorbital alopecia, cutaneous erythema, abrasions and ulcers, and subcutaneous edema. Histologic examination was performed for eight of nine cases, all of which had intraneuronal eosinophilic inclusion (Negri) bodies in cerebrum, cerebellum, or both. Most (6/8; 75%) had perivascular lymphoplasmacytic encephalitis. Rabies should be considered a differential diagnosis in deer with this pattern of head lesions, suggestive of head rubbing or head pressing.

Combining deep sequencing and conventional molecular approaches reveals broad diversity and distribution of fleas and Bartonella in rodents and shrews from Arctic and Subarctic ecosystems.

BACKGROUND: Bartonella are intracellular bacteria that are transmitted via animal scratches, bites and hematophagous arthropods. Rodents and their associated fleas play a key role in the maintenance of Bartonella worldwide, with > 22 species identified in rodent hosts. No studies have addressed the occurrence and diversity of Bartonella species and vectors for small mammals in Arctic and Subarctic ecosystems, which are increasingly impacted by invasive species and climate change. METHODS: In this study, we characterized the diversity of rodent fleas using conventional PCR targeting the mitochondrial cytochrome c oxidase II gene (COII) and Bartonella species in rodents and shrews (n = 505) from northern Canada using conventional PCR targeting the ITS (intergenic transcribed spacer) region and gltA (citrate synthase) gene. Metagenomic sequencing of a portion of the gltA gene was completed on a subset of 42 rodents and four rodent flea pools. RESULTS: Year, total summer precipitation the year prior to sampling, average minimum spring temperature and small mammal species were significant factors in predicting Bartonella positivity. Occurrence based on the ITS region was more than double that of the gltA gene and was 34% (n = 349) in northern red-backed voles, 35% (n = 20) in meadow voles, 37% (n = 68) in deer mice and 31% (n = 59) in shrews. Six species of Bartonella were identified with the ITS region, including B. grahamii, B. elizabethae, B. washoensis, Candidatus B. rudakovii, B. doshiae, B. vinsonii subsp. berkhoffii and subsp. arupensis. In addition, 47% (n = 49/105) of ITS amplicons had < 97% identity to sequences in GenBank, possibly due to a limited reference library or previously unreported species. An additional Bartonella species (B. heixiaziensis) was detected during metagenomic sequencing of the gltA gene in 6/11 rodents that had ITS sequences with < 97% identity in GenBank, highlighting that a limited reference library for the ITS marker likely accounted for low sequence similarity in our specimens. In addition, one flea pool from a northern red-backed vole contained multiple species (B. grahamii and B. heixiaziensis). CONCLUSION: Our study calls attention to the usefulness of a combined approach to determine the occurrence and diversity of Bartonella communities in hosts and vectors.