Limited Outbreak of Highly Pathogenic Influenza A(H5N1) in Herring Gull Colony, Canada, 2022.

In summer 2022, highly pathogenic influenza A(H5N1) virus reached the herring gull (Larus argentatus subspecies smithsonianus) breeding colony on Kent Island, New Brunswick, Canada. Real-time monitoring revealed a self-limiting outbreak with low mortality. Proactive seabird surveillance is crucial for monitoring such limited outbreaks, protecting seabirds, and tracing zoonotic transmission routes.

Echinococcus multilocularis infection in a red fox (Vulpes vulpes) on Prince Edward Island, Canada.

Objective: Molecular identification of small cestodes, morphologically consistent with Echinococcus multilocularis, recovered at necropsy from the gastrointestinal tract contents of a red fox, was accomplished by PCR using published species-specific n ad1 primers and methods. Animal: Red fox (Vulpes vulpes). Procedure: Small cestodes recovered from intestinal contents of a red fox trapped on Prince Edward Island in December 2020 (frozen at -20°C before being processed for parasite recovery in June 2021) were morphologically identified. Species identity confirmation and haplotyping of the cestodes were done via PCR and DNA sequencing of the n ad1, nad2, and cob genes. Results: Small cestodes morphologically consistent with E. multilocularis were detected in the gastrointestinal tract contents of a red fox trapped near Montague, PEI. The species identity was confirmed via PCR. Haplotyping revealed that they were of the European E1 haplotype. Conclusion: In Canada, E. multilocularis has been reported as far east as Québec, with most reports being in central and western provinces and territories. This is the first report of E. multilocularis infection in a canid host east of Ontario, Canada and illustrates the need for regular wildlife disease surveillance to enhance our understanding of emerging pathogens of veterinary and medical importance. Clinical Relevance: Echinococcus multilocularis is a highly pathogenic zoonotic cestode from the family Taeniidae that can cause alveolar echinococcosis (AE) when rodents, dogs, horses, pigs, non-human primates, or humans ingest its eggs. Alveolar echinococcosis is challenging to treat, and survival rates for untreated individuals are low.

Objectif: L'identification moléculaire de petits cestodes, morphologiquement compatibles avec Echinococcus multilocularis, récupérés à l'autopsie du contenu du tractus gastro-intestinal d'un renard roux, a été réalisée par PCR à l'aide d'amorces nad1 spécifiques à l'espèce et de méthodes publiées. Animal: Renard roux (Vulpes vulpes). Procédure: De petits cestodes récupérés du contenu intestinal d'un renard roux piégé à l'Île-du-Prince-Édouard en décembre 2020 (congelés à −20 °C avant d'être traités pour la récupération des parasites en juin 2021) ont été morphologiquement identifiés. La confirmation de l'identité des espèces et l'haplotypage des cestodes ont été effectués par PCR et séquençage de l'ADN des gènes nad1, nad2 et cob. Résultats: De petits cestodes morphologiquement compatibles avec E. multilocularis ont été détectés dans le contenu du tractus gastro-intestinal d'un renard roux piégé près de Montague, Î.-P.-É. L'identité de l'espèce a été confirmée par PCR. L'haplotypage a révélé qu'ils étaient de l'haplotype européen E1. Conclusion: Au Canada, E. multilocularis a été signalé aussi loin à l'est que le Québec, la plupart des signalements ayant été rapportés dans les provinces et territoires du centre et de l'ouest. Il s'agit du premier rapport d'infection à E. multilocularis chez un canidé hôte à l'est de l'Ontario, au Canada, et illustre la nécessité d'une surveillance régulière des maladies de la faune pour améliorer notre compréhension des agents pathogènes émergents d'importance vétérinaire et médicale. Pertinence clinique: Echinococcus multilocularis est un cestode zoonotique hautement pathogène de la famille des Taeniidae qui peut provoquer une échinococcose alvéolaire (EA) lorsque des rongeurs, des chiens, des chevaux, des porcs, des primates non humains ou des humains ingèrent ses oeufs. L'échinococcose alvéolaire est difficile à traiter et les taux de survie des personnes non traitées sont faibles.(Traduit par Dr Serge Messier).

First report of Angiostrongylus vasorum (French heartworm) in red foxes (Vulpes vulpes) on Prince Edward Island.

Objective: To identify first-stage nematode larvae (L1) recovered from a red fox scat sample and adult female worms recovered from 2 red fox lungs at necropsy, using published molecular methods to confirm a morphological diagnosis of Angiostrongylus vasorum (French heartworm). Animal: Red fox (Vulpes vulpes). Procedure: Nematode larvae recovered from a Baermann examination survey of wild canid scats (n = 101) conducted from January 2017 to August 2020, were identified by size and morphology and subjected to PCR and DNA sequencing of the small subunit (SSU) rRNA gene, the large subunit (LSU) rRNA gene, or the second internal transcribed spacer (ITS2). In addition, these techniques were applied to adult female worms recovered from the heart/lungs of 2 red foxes (obtained from PEI trappers and stored frozen at -20°C since December of 2018 and 2020). Results: Size and morphology of L1 recovered by Baermann examination from a wild canid scat sample (presumed to be red fox) collected near Montague, PEI and adult female worms recovered at necropsy from 2 red fox carcasses were identified as A. vasorum. Molecular analysis confirmed the larvae and adult worms were A. vasorum. Conclusion: These findings indicated that A. vasorum has become endemic in the red fox population on PEI. Clinical relevance: Angiostrongylus vasorum infection is potentially fatal in dogs. Veterinarians and regional diagnostic laboratories in the Maritime provinces should consider the possibility of A. vasorum infection in dogs with clinical signs of cardiopulmonary and/or central nervous system disease or bleeding disorders.

Objectif: Identifier les larves de nématodes de premier stade (L1) récupérées à partir d'un échantillon d'excréments de renard roux et les vers femelles adultes récupérés à partir de deux poumons de renard roux à l'autopsie, en utilisant des méthodes moléculaires publiées pour confirmer un diagnostic morphologique d'Angiostrongylus vasorum (ver du coeur français). Animal: Renard roux (Vulpis vulpis). Procédure: Les larves de nématodes récupérées lors d'une enquête sur des excréments de canidés sauvages (n = 101) par examen Baermann menée de janvier 2017 à août 2020, ont été identifiées par taille et morphologie et soumises à la PCR et au séquençage de DNA de la petite sous-unité (SSU) du gène de rRNA, de la grande sous-unité (LSU) du gène de rRNA ou du deuxième espaceur interne transcrit (ITS2). De plus, ces techniques ont été appliquées à des vers femelles adultes récupérés du coeur/poumons de deux renards roux (obtenus auprès de trappeurs de l'Î.-P.-É. et conservés congelés à −20 °C depuis décembre 2018 et 2020). Résultats: La taille et la morphologie de L1 récupérées par examen Baermann à partir d'un échantillon d'excréments de canidés sauvages (présumé être du renard roux) prélevé près de Montague, Î.-P.-É. et des vers adultes femelles récupérés des carcasses lors de la nécropsie de deux renards roux ont été identifiés comme étant A. vasorum. L'analyse moléculaire a confirmé que les larves et les vers adultes étaient A. vasorum. Conclusion: Ces résultats indiquent qu'A. vasorum est devenu endémique dans la population de renards roux de l'Î.-P.-É. Pertinence clinique: L'infection à A. vasorum est potentiellement mortelle chez le chien. Les vétérinaires et les laboratoires de diagnostic régionaux des provinces maritimes devraient envisager la possibilité d'une infection à A. vasorum chez les chiens présentant des signes cliniques de maladie cardio-pulmonaire et/ou du système nerveux central ou de troubles de la coagulation.(Traduit par Dr Serge Messier).

Acute toxoplasmosis and pox-viral dermatitis in a juvenile bald eagle (Haliaeetus leucocephalus) in New Brunswick, Canada.

Although birds of prey are commonly subclinically infected by Toxoplasma gondii tissue cysts, clinical disease is relatively rare in these species. The present report describes a rare case of fatal toxoplasmosis in a juvenile bald eagle in New Brunswick. Necropsy investigation revealed severe emaciation and poxviral dermatitis which partially obscured the palpebral fissures. Microscopically there was severe lymphoplasmacytic inflammation and necrosis of the lung that was associated with abundant protozoal tachyzoites. Infection with T. gondii was confirmed in the lung via immunohistochemistry and DNA sequencing. Key clinical message: Wildlife rehabilitation centers should be aware of the potential occurrence of acute clinical toxoplasmosis in stressed malnourished raptors.

Toxoplasmose aigu ë et dermatite à poxvirus chez un pygargue à tête blanche ( Haliaeetus leucocephalus ) au Nouveau-Brunswick, Canada. Bien que les oiseaux de proie soient fréquemment infectés de manière subclinique par des kystes tissulaires de Toxoplasma gondii, la maladie clinique est relativement rare chez ces espèces. Le présent rapport décrit un rare cas de toxoplasmose fatale chez un pygargue à tête blanche juvénile au Nouveau-Brunswick. La nécropsie a révélé une émaciation sévère et une dermatite à poxvirus qui obstruait partiellement les fissures palpébrales. L'examen microscopique révéla une inflammation lympho-plasmocytaire sévère et une nécrose du poumon qui fut associé à une abondance de tachyzoïtes d protozoaires. L'infection par T. gondii fut confirmée dans le poumon via immunohistochimie et séquençage de l'ADN.Message clinique clé :Les centres de réhabilitation de la faune devrait être averti de l'existence de toxoplasmose clinique aiguë chez des rapaces stressés et mal nourris.(Traduit par Dr Serge Messier).

De novo transcriptome reconstruction and annotation of the Egyptian rousette bat.

BACKGROUND: The Egyptian Rousette bat (Rousettus aegyptiacus), a common fruit bat species found throughout Africa and the Middle East, was recently identified as a natural reservoir host of Marburg virus. With Ebola virus, Marburg virus is a member of the family Filoviridae that causes severe hemorrhagic fever disease in humans and nonhuman primates, but results in little to no pathological consequences in bats. Understanding host-pathogen interactions within reservoir host species and how it differs from hosts that experience severe disease is an important aspect of evaluating viral pathogenesis and developing novel therapeutics and methods of prevention. RESULTS: Progress in studying bat reservoir host responses to virus infection is hampered by the lack of host-specific reagents required for immunological studies. In order to establish a basis for the design of reagents, we sequenced, assembled, and annotated the R. aegyptiacus transcriptome. We performed de novo transcriptome assembly using deep RNA sequencing data from 11 distinct tissues from one male and one female bat. We observed high similarity between this transcriptome and those available from other bat species. Gene expression analysis demonstrated clustering of expression profiles by tissue, where we also identified enrichment of tissue-specific gene ontology terms. In addition, we identified and experimentally validated the expression of novel coding transcripts that may be specific to this species. CONCLUSION: We comprehensively characterized the R. aegyptiacus transcriptome de novo. This transcriptome will be an important resource for understanding bat immunology, physiology, disease pathogenesis, and virus transmission.

Rift Valley fever virus clearance and protection from neurologic disease are dependent on CD4+ T cell and virus-specific antibody responses.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. Human RVFV infections generally manifest as a self-limiting febrile illness, but in some individuals, the disease can progress to a fatal encephalitis or hemorrhagic syndrome. Little is known about the host characteristics that predispose development of more severe disease. Early in infection, interferon-mediated antiviral responses are critical for controlling RVFV replication, but the roles of downstream adaptive immune responses in determining clinical outcome have not been examined. Here, using a C57BL/6 mouse disease model, we evaluated the roles of B cells and T cells in RVFV pathogenesis. Given the profound inhibition of the innate response by the viral NSs protein and rapid course of wild-type infection, we utilized an attenuated RVFV lacking NSs to examine host responses following primary infection. Experiments utilizing B-cell-deficient mice or targeted T cell depletions of wild-type mice demonstrated that B cells and CD4(+) T cells, but not CD8(+) T cells, were critical for mediating viral clearance, even in the presence of a functional innate response. One-third of CD4-depleted mice developed severe neurologic disease following infection, in contrast to virus-infected mock-depleted mice that showed no clinical signs. CD4(+) T cells were required for robust IgG and neutralizing antibody responses that correlated with RVFV clearance from peripheral tissues. Furthermore, CD4-depleted mice demonstrated significantly stronger proinflammatory responses relative to controls, suggesting CD4(+) T cells regulate immune responses to RVFV infection. Together, these results indicate CD4(+) T cells are critical determinants of RVFV pathogenesis and play an important role in preventing onset of neurologic disease.

Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.

Multiple transatlantic incursions of highly pathogenic avian influenza clade 2.3.4.4b A(H5N5) virus into North America and spillover to mammals.

Highly pathogenic avian influenza (HPAI) viruses have spread at an unprecedented scale, leading to mass mortalities in birds and mammals. In 2023, a transatlantic incursion of HPAI A(H5N5) viruses into North America was detected, followed shortly thereafter by a mammalian detection. As these A(H5N5) viruses were similar to contemporary viruses described in Eurasia, the transatlantic spread of A(H5N5) viruses was most likely facilitated by pelagic seabirds. Some of the Canadian A(H5N5) viruses from birds and mammals possessed the PB2-E627K substitution known to facilitate adaptation to mammals. Ferrets inoculated with A(H5N5) viruses showed rapid, severe disease onset, with some evidence of direct contact transmission. However, these viruses have maintained receptor binding traits of avian influenza viruses and were susceptible to oseltamivir and zanamivir. Understanding the factors influencing the virulence and transmission of A(H5N5) in migratory birds and mammals is critical to minimize impacts on wildlife and public health.

Avian influenza viruses in wild birds in Canada following incursions of highly pathogenic H5N1 virus from Eurasia in 2021-2022.

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners. IMPORTANCE: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners.

Renal Coccidial Infection in North American Northern Gannets (Morus bassanus).

We identified by light microscopy micro- and macrogametes and oocysts of renal coccidia in 78 of 220 (35.5%) Northern Gannets (Morus bassanus) from the western North Atlantic population. This infection was not considered clinically significant in any of the affected birds, although the potential effect of this parasite in breeding colonies, particularly among nestlings, is unknown. Analysis of the 18S rRNA gene from frozen renal tissue by PCR and subsequent sequencing revealed 95.6% identity with Eimeria auritusi from Double-crested Cormorants (Phalacrocorax auritus), suggesting a novel Eimeria sp. in the Northern Gannets.

Recurring Trans-Atlantic Incursion of Clade 2.3.4.4b H5N1 Viruses by Long Distance Migratory Birds from Northern Europe to Canada in 2022/2023.

In December 2022 and January 2023, we isolated clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) viruses from six American crows (Corvus brachyrhynchos) from Prince Edward Island and a red fox (Vulpes vulpes) from Newfoundland, Canada. Using full-genome sequencing and phylogenetic analysis, these viruses were found to fall into two distinct phylogenetic clusters: one group containing H5N1 viruses that had been circulating in North and South America since late 2021, and the other one containing European H5N1 viruses reported in late 2022. The transatlantic re-introduction for the second time by pelagic/Icelandic bird migration via the same route used during the 2021 incursion of Eurasian origin H5N1 viruses into North America demonstrates that migratory birds continue to be the driving force for transcontinental dissemination of the virus. This new detection further demonstrates the continual long-term threat of H5N1 viruses for poultry and mammals and the subsequent impact on various wild bird populations wherever these viruses emerge. The continual emergence of clade 2.3.4.4b H5Nx viruses requires vigilant surveillance in wild birds, particularly in areas of the Americas, which lie within the migratory corridors for long-distance migratory birds originating from Europe and Asia. Although H5Nx viruses have been detected at higher rates in North America since 2021, a bidirectional flow of H5Nx genes of American origin viruses to Europe has never been reported. In the future, coordinated and systematic surveillance programs for HPAI viruses need to be launched between European and North American agencies.

Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Granulomatous myositis associated with a novel alveolate pathogen in an adult southern leopard frog (Lithobates sphenocephalus).

Since 1999, infections with an incompletely characterized alveolate protozoan variously reported as a Dermocystidium-like organism, a Perkinsus-like agent, and Dermomycoides sp. have been associated with mortality events in tadpoles of ranid frogs from the USA. However, disease or mortality events due to this organism have not been described in post-metamorphic animals. We describe infection with a morphologically similar protozoan presenting itself as a leg mass in a free-ranging adult southern leopard frog Lithobates sphenocephalus. Using histological examination, we found a mass within skeletal muscle; this mass was composed of macrophages with intracytoplasmic, thick-walled, 4 to 6 µm in diameter, spherical basophilic protozoal organisms that exhibited green autofluorescence with epiflorescence illumination. Using transmission electron microscopy, organism cell walls were found to have electron-dense plates that, when viewed by scanning electron microscopy, were reminiscent of the thecal plates of dinoflagellates. Additional morphologic and molecular phylogenetic research is needed to resolve the taxonomic status of this organism.

Treatment of chytridiomycosis with reduced-dose itraconazole.

Effective treatment methods to eliminate infection with Batrachochytrium dendrobatidis (Bd) are required for development of sustainable captive survival assurance populations of amphibians and to reduce the risk of introducing Bd to new locations as part of amphibian trade or reintroduction programs. Treatment with itraconazole baths at 100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages. Naturally occurring outbreaks of chytridiomycosis in Wyoming toads Anaxyrus baxteri and White's tree frogs Litoria caerulea were treated with lower-dose itraconazole baths (e.g. 50 mg l-1 for White's tree frogs) and followed post-treatment with serial Taqman PCR testing to confirm elimination of Bd infection. Post-treatment PCR tests were consistently negative for the presence of Bd and treatment was deemed successful. Although this was not a controlled clinical trial, results suggest that lower doses of itraconazole may be effective for treatment of chytridiomycosis with resulting cost savings to amphibian conservation programs and a potential for a reduction in dose-related side effects from itraconazole treatment. Prospective clinical trials of alternative itraconazole treatment protocols are encouraged.

IN SITU HYBRIDIZATION AND VIRUS CHARACTERIZATION OF SKUNK ADENOVIRUS IN NORTH AMERICAN WILDLIFE REVEALS MULTISYSTEMIC INFECTIONS IN A BROAD RANGE OF HOSTS.

Skunk adenovirus-1 (SkAdV-1) has been reported infecting several North American wildlife species; however, lesions associated with disease have not yet been completely characterized, particularly in porcupines. We describe and characterize the tissue distribution and lesions associated with SkAdV-1 infection in 24 wildlife diagnostic cases submitted between 2015 and 2020, including 16 North American porcupines (Erethizon dorsatum), three striped skunks (Mephitis mephitis), and five raccoons (Procyon lotor), which constitute a new host species. The most common lesion in all species was severe necrotizing bronchopneumonia with (n=12) or without (n=10) interstitial involvement. Intranuclear inclusion bodies were common in respiratory epithelium (n=21) and less often in renal tubular (n=6) and biliary epithelium (n=1). Several cases (n=4) had secondary bacterial infections, including Bordetella bronchiseptica, Pasteurella multocida, and Streptococcus zooepidemicus. In situ hybridization in porcupine (n=6), raccoon (n=1), and skunk (n=1) revealed SkAdV-1 DNA in multiple tissue types, including lung, trachea, turbinates, liver, kidney, lymph node, and brain, and multiple cell types including epithelial, endothelial, and mesothelial cells. These findings were consistent across species. Comparison of viral genomes from a porcupine and a raccoon with that originally isolated from a skunk demonstrated DNA point mutations affecting several viral genes, including the fiber protein gene. Our findings show the spectrum of disease associated with SkAdV-1 infection in a broad host range of wildlife species.

A SYNDROME OF ISCHEMIC LEG NECROSIS IN NORTHERN GANNETS (MORUS BASSANUS).

The Northern Gannet (Morus bassanus) is a large marine bird whose whole North American population breeds in waters of eastern Canada. Opportunities to identify causes of morbidity and mortality in recently hatched birds of this species are therefore limited to this region of North America. During the three decades since 1990 of wildlife health surveillance at the Atlantic regional center of the Canadian Wildlife Health Cooperative, what appears to be a previously undescribed syndrome of ischemic leg necrosis affecting mainly hatch-year Northern Gannets has emerged, which may relate to some unique aspects of the life history of these birds. This syndrome, observed in 14 birds, is characterized by severe necrosis and fibrinopurulent inflammation of soft tissues of the feet extending along the whole tarsometatarsus. An infectious cause is proposed to explain the pattern of lesions observed in these birds, possibly favored by a specialized and rich vascular system in their legs and feet. An acute or subacute cardiomyopathy, thought to be secondary to the severe leg lesions, was also observed microscopically in six of these birds.

Clinical, Histopathologic, and Immunohistochemical Characterization of Experimental Marburg Virus Infection in A Natural Reservoir Host, the Egyptian Rousette Bat (Rousettus aegyptiacus).

Egyptian rousette bats (Rousettus aegyptiacus) are natural reservoir hosts of Marburg virus (MARV), and Ravn virus (RAVV; collectively called marburgviruses) and have been linked to human cases of Marburg virus disease (MVD). We investigated the clinical and pathologic effects of experimental MARV infection in Egyptian rousettes through a serial euthanasia study and found clear evidence of mild but transient disease. Three groups of nine, captive-born, juvenile male bats were inoculated subcutaneously with 10,000 TCID50 of Marburg virus strain Uganda 371Bat2007, a minimally passaged virus originally isolated from a wild Egyptian rousette. Control bats (n = 3) were mock-inoculated. Three animals per day were euthanized at 3, 5⁻10, 12 and 28 days post-inoculation (DPI); controls were euthanized at 28 DPI. Blood chemistry analyses showed a mild, statistically significant elevation in alanine aminotransferase (ALT) at 3, 6 and 7 DPI. Lymphocyte and monocyte counts were mildly elevated in inoculated bats after 9 DPI. Liver histology revealed small foci of inflammatory infiltrate in infected bats, similar to lesions previously described in wild, naturally-infected bats. Liver lesion severity scores peaked at 7 DPI, and were correlated with both ALT and hepatic viral RNA levels. Immunohistochemical staining detected infrequent viral antigen in liver (3⁻8 DPI, n = 8), spleen (3⁻7 DPI, n = 8), skin (inoculation site; 3⁻12 DPI, n = 20), lymph nodes (3⁻10 DPI, n = 6), and oral submucosa (8⁻9 DPI, n = 2). Viral antigen was present in histiocytes, hepatocytes and mesenchymal cells, and in the liver, antigen staining co-localized with inflammatory foci. These results show the first clear evidence of very mild disease caused by a filovirus in a reservoir bat host and provide support for our experimental model of this virus-reservoir host system.

Fatal pulmonary hemorrhage associated with RTX toxin producing Actinobacillus equuli subspecies haemolyticus infection in an adult horse.

A case of fatal pulmonary hemorrhage in a 6-year-old American Paint mare with a 2-week history of intermittent coughing, fever, and epistaxis is described. Significant macroscopic abnormalities at postmortem examination were restricted to the respiratory system, and microscopically, severe pulmonary hemorrhage with suppurative bronchopneumonia was found. Actinobacillus equuli subsp. haemolyticus was cultured from a transtracheal wash performed antemortem as well as from the lungs at necropsy. The presence of airway-associated hemorrhage in conjunction with bacterial bronchopneumonia suggested endothelial damage caused by a locally elaborated bacterial toxin, possibly produced by the A. equuli strain isolated from the lungs. The objective of this report was to indirectly document the presence of hemolysin repeat in structural toxin (RTX) in the lungs of the reported mare. A real-time polymerase chain reaction (PCR) assay targeting the recently described aqx gene of A. equuli subsp. haemolyticus was established and validated. Transcriptional activity of the aqx gene was used as a surrogate method to document toxin production. Real-time PCR analysis of the transtracheal fluid and lung tissue of the affected mare confirmed the presence and the transcriptional activity of the aqx gene at the genomic (gDNA) and complementary DNA (cDNA) levels, respectively. The presence of pneumonia associated with hemorrhagic pulmonary fluid and the culture of large numbers of hemolytic A. equuli should prompt the clinician to consider endothelial damage caused by bacterial toxins.

Modelling filovirus maintenance in nature by experimental transmission of Marburg virus between Egyptian rousette bats.

The Egyptian rousette bat (ERB) is a natural reservoir host for Marburg virus (MARV); however, the mechanisms by which MARV is transmitted bat-to-bat and to other animals are unclear. Here we co-house MARV-inoculated donor ERBs with naive contact ERBs. MARV shedding is detected in oral, rectal and urine specimens from inoculated bats from 5-19 days post infection. Simultaneously, MARV is detected in oral specimens from contact bats, indicating oral exposure to the virus. In the late study phase, we provide evidence that MARV can be horizontally transmitted from inoculated to contact ERBs by finding MARV RNA in blood and oral specimens from contact bats, followed by MARV IgG antibodies in these same bats. This study demonstrates that MARV can be horizontally transmitted from inoculated to contact ERBs, thereby providing a model for filovirus maintenance in its natural reservoir host and a potential mechanism for virus spillover to other animals.

Experimental Inoculation of Egyptian Rousette Bats (Rousettus aegyptiacus) with Viruses of the Ebolavirus and Marburgvirus Genera.

The Egyptian rousette bat (Rousettus aegyptiacus) is a natural reservoir for marburgviruses and a consistent source of virus spillover to humans. Cumulative evidence suggests various bat species may also transmit ebolaviruses. We investigated the susceptibility of Egyptian rousettes to each of the five known ebolaviruses (Sudan, Ebola, Bundibugyo, Taï Forest, and Reston), and compared findings with Marburg virus. In a pilot study, groups of four juvenile bats were inoculated with one of the ebolaviruses or Marburg virus. In ebolavirus groups, viral RNA tissue distribution was limited, and no bat became viremic. Sudan viral RNA was slightly more widespread, spurring a second, 15-day Sudan virus serial euthanasia study. Low levels of Sudan viral RNA disseminated to multiple tissues at early time points, but there was no viremia or shedding. In contrast, Marburg virus RNA was widely disseminated, with viremia, oral and rectal shedding, and antigen in spleen and liver. This is the first experimental infection study comparing tissue tropism, viral shedding, and clinical and pathologic effects of six different filoviruses in the Egyptian rousette, a known marburgvirus reservoir. Our results suggest Egyptian rousettes are unlikely sources for ebolaviruses in nature, and support a possible single filovirus-single reservoir host relationship.