Lesions associated with Bartonella taylorii-like bacterium infection in a free-ranging, young-of-the-year raccoon from Prince Edward Island, Canada.

A young-of-the year, female raccoon was presented with clinical signs of weakness and tremors. The raccoon was euthanized, and autopsy findings included poor body condition, diffuse lymphadenopathy, and pale, firm kidneys with petechial hemorrhages throughout the renal cortex. Histologic lesions included systemic fibrinoid vascular necrosis and severe renal lesions, including lymphoplasmacytic interstitial nephritis and fibrinosuppurative glomerulonephritis. Inflammatory vascular lesions were also present within the uvea, heart, lymph nodes, and the lamina propria of the gastric wall. Ancillary testing was negative for Borrelia burgdorferi, Leptospira sp., Aleutian disease virus, canine distemper virus, feline coronavirus, porcine circovirus 2, and rabies virus. Transmission electron microscopy revealed large numbers of ~1.3 × 0.35 µm bacterial rods surrounded by a trilaminar cell wall located within the glomeruli and associated with aggregates of fibrin and vascular damage. Analysis of partial citrate synthase gene and 16S-23S ribosomal RNA intergenic spacer region sequences from kidney tissue confirmed that the organism was a Bartonella spp. that was related to numerous Bartonella spp. from shrews in Europe. This group formed a sister clade to the genetically diverse Bartonella taylorii group that has been reported from a wide range of Eurasian rodent and flea species.

Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest.

The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C. gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C. gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10-8, 1.59 × 10-8, and 2.70 × 10-8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10-9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species' slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C. gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100 years. While the cause of C. gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., <25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events.

Profiling the immunome of little brown myotis provides a yardstick for measuring the genetic response to white-nose syndrome.

White-nose syndrome (WNS) has devastated populations of hibernating bats in eastern North America, leading to emergency conservation listings for several species including the previously ubiquitous little brown myotis (Myotis lucifugus). However, some bat populations near the epicenter of the WNS panzootic appear to be stabilizing after initial precipitous declines, which could reflect a selective immunogenetic sweep. To investigate the hypothesis that WNS exerts significant selection on the immunome of affected bat populations, we developed a novel, high-throughput sequence capture assay targeting 138 adaptive, intrinsic, and innate immunity genes of putative adaptive significance, as well as their respective regulatory regions (~370 kbp of genomic sequence/individual). We used the assay to explore baseline immunogenetic variation in M. lucifugus and to investigate whether particular immune genes/variants are associated with WNS susceptibility. We also used our assay to detect 1,038 putatively neutral single nucleotide polymorphisms and characterize contemporary population structure, providing context for the identification of local immunogenetic adaptation. Sequence capture provided a cost-effective, "all-in-one" assay to test for neutral genetic and immunogenetic structure and revealed fine-scale, baseline immunogenetic differentiation between sampling sites <600 km apart. We identified functional immunogenetic variants in M. lucifugus associated with WNS susceptibility. This study lays the foundations for future investigations of rangewide immunogenetic adaptation to WNS in M. lucifugus and provides a blueprint for studies of evolutionary rescue in other host-pathogen systems.

Persistence of Trichomonas gallinae in Birdseed.

Trichomonas gallinae has emerged worldwide as a cause of mortality in songbirds (passerines). The congregation of numerous birds, including the reservoir hosts, pigeons and doves (columbids), at backyard feeding and watering sources has been suggested as a potential driver for the outbreaks. Evidence supporting a role for water in transmission has been established, but the role of birdseed in the transmission of trichomoniasis remained to be investigated. We assessed the survival of T. gallinae in three commercial birdseeds (mixed seed, black-oil sunflower seed, and niger seed) routinely used to attract passerine birds to local properties. Trichomonad suspensions were inoculated (low dose: 1 × 103; high dose: 1 × 105) into each of the three seed types in petri dishes, using both dry and moist (water-soaked) conditions, in triplicate. Petri dishes were incubated at 37 C and monitored for T. gallinae survival for 48 hr by wet-mount microscopy and by InPouch™ TF medium culture for 10 days. Surviving trichomonads were not detected in any of the dry birdseed treatments. In moist conditions, however, trichomonads were found to survive ≤24 hr in all three seed types and ≤48 hr in the mixed seed that contained organic debris. We demonstrate that T. gallinae has the ability to survive in moist birdseed, which suggests that public bird-feeding sites may play a significant role in the transmission of trichomoniasis.

Molecular Detection of Pseudogymnoascus destructans (Ascomycota: Pseudeurotiaceae) and Unidentified Fungal Dermatitides on Big Brown Bats ( Eptesicus fuscus ) Overwintering inside Buildings in Canada.

Big brown bats ( Eptesicus fuscus ) overwintering outside the underground environment are not believed to play a role in the epidemiology of the disease white-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Pd). Using quantitative real-time PCR (qPCR), we provide molecular evidence for Pd on four big brown bats overwintering in heated buildings in New Brunswick, Canada. Two of the affected individuals also had very mild, focal, pustular, fungal dermatitis identified microscopically. A third bat, which was qPCR Pd-negative, had similar fungal lesions. Despite determining that these fungal lesions were caused by a suspected ascomycete, the intralesional fungi were not confirmed to be Pd. These findings demonstrate that bats overwintering in heated buildings and other above-ground sites may have subclinical or preclinical WNS, or be contaminated with Pd, and could play a role in local dispersal of Pd. Our inability to determine if the ascomycetes causing pustular lesions were Pd highlights the need for ancillary diagnostic tests, such as in situ hybridization or immunohistochemistry, so that Pd can be detected directly within a lesion. As the host-pathogen relationship for Pd evolves, and where bat species are exposed to the fungus under varying temperature regimes, lesions may become less stereotypic and such tests could help define these changes.

A PELAGIC OUTBREAK OF AVIAN CHOLERA IN NORTH AMERICAN GULLS: SCAVENGING AS A PRIMARY MECHANISM FOR TRANSMISSION?

Avian cholera, caused by the bacterium Pasteurella multocida , is an endemic disease globally, often causing annual epizootics in North American wild bird populations with thousands of mortalities. From December 2006 to March 2007, an avian cholera outbreak caused mortality in marine birds off the coast of Atlantic Canada, largely centered 300-400 km off the coast of the island of Newfoundland. Scavenging gulls ( Larus spp.) were the primary species detected; however, mortality was also identified in Black-legged Kittiwakes ( Rissa tridactyla ) and one Common Raven ( Corvus corax ), a nonmarine species. The most common gross necropsy findings in the birds with confirmed avian cholera were acute fibrinous and necrotizing lesions affecting the spleen, air sacs, and pericardium, and nonspecific hepatomegaly and splenomegaly. The etiologic agent, P. multocida serotype 1, was recovered from 77 of 136 carcasses examined, and confirmed or probable avian cholera was diagnosed in 85 cases. Mortality observed in scavenging gull species was disproportionately high relative to their abundance, particularly when compared to nonscavenging species. The presence of feather shafts in the ventricular lumen of the majority of larid carcasses diagnosed with avian cholera suggests scavenging of birds that died from avian cholera as a major mode of transmission. This documentation of an outbreak of avian cholera in a North American pelagic environment affecting primarily scavenging gulls indicates that offshore marine environments may be a component of avian cholera dynamics.

Molecular characterization of Trichomonas gallinae isolates recovered from the Canadian Maritime provinces' wild avifauna reveals the presence of the genotype responsible for the European finch trichomonosis epidemic and additional strains.

Finch trichomonosis, caused by Trichomonas gallinae, emerged in the Canadian Maritime provinces in 2007 and has since caused ongoing mortality in regional purple finch (Carpodacus purpureus) and American goldfinch (Carduelis tristis) populations. Trichomonas gallinae was isolated from (1) finches and rock pigeons (Columbia livia) submitted for post-mortem or live-captured at bird feeding sites experiencing trichomonosis mortality; (2) bird seed at these same sites; and (3) rock pigeons live-captured at known roosts or humanely killed. Isolates were characterized using internal transcribed spacer (ITS) region and iron hydrogenase (Fe-hyd) gene sequences. Two distinct ITS types were found. Type A was identical to the UK finch epidemic strain and was isolated from finches and a rock pigeon with trichomonosis; apparently healthy rock pigeons and finches; and bird seed at an outbreak site. Type B was obtained from apparently healthy rock pigeons. Fe-hyd sequencing revealed six distinct subtypes. The predominant subtype in both finches and the rock pigeon with trichomonosis was identical to the UK finch epidemic strain A1. Single nucleotide polymorphisms in Fe-hyd sequences suggest there is fine-scale variation amongst isolates and that finch trichomonosis emergence in this region may not have been caused by a single spill-over event.

Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

Detection and characterization of a Trichomonas isolate from a rehabilitated bald eagle (Haliaeetus leucocephalus).

A hatching-year bald eagle (Haliaeetus leucocephalus) was presented for clinical examination after being found unable to fly. Upon admission, routine wet-mount microscopy detected no trichomonads. Five months later, oral cavity inspection found no abnormalities, but the eagle was swabbed for research on trichomonosis in maritime birds. The swab was used to inoculate an InPouch TF culture and trichomonads were visible within 24 hr. Genotyping (ITS) revealed a Trichomonas isolate that was 100% identical to an isolate from a bearded vulture (Gypaetus barbatus) from the Czech Republic. The eagle was treated with metronidazole (50 mg/kg q 12h PO for 5 consecutive days). Following treatment, the eagle was swabbed and the inoculated InPouch TF culture was monitored daily for 1 wk. No trichomonads were observed. Rehabilitation centers interested in surveillance should consider combining the InPouch TF technique with clinical inspection of live birds to confirm trichomonosis and for future research.

Babesia (Theileria) annae in a red fox (Vulpes vulpes) from Prince Edward Island, Canada.

A 4-6-mo-old female red fox (Vulpes vulpes) was presented to the Atlantic Veterinary College (AVC) Teaching Hospital, Prince Edward Island, Canada. On presentation, the fox was weak and had pale mucous membranes. A complete blood count and a serum biochemistry profile were performed. Blood smear examination revealed low numbers of erythrocytes containing centrally to paracentrally located, single, rarely multiple, approximately 1 x 2 microm, oval to round organisms with morphology similar to Babesia microti. Polymerase chain reaction testing and DNA sequencing of the Babesia species 18S rRNA gene were performed on DNA extracted from whole blood. Results were positive for a Babesia microti-like parasite genetically identical to Babesia (Theileria) annae. The fox was euthanized due to poor prognosis for recovery. Necropsy examination revealed multifocal to locally extensive subacute nonsuppurative meningoencephalitis, an eosinophilic broncho-pneumonia, a moderate diffuse vacuolar hepatopathy, and lesions associated with blunt trauma to the left abdominal region. This is the first reported case of a red fox in Canada infected with a piroplasm. It remains uncertain whether the presence of this hemoparasite in this fox was pathogenic or an incidental finding. The potential for competent vectors of Babesia species on Prince Edward Island, the potential for this Babesia microti-like parasite to infect other wild and domestic canids, and the significance of this parasite to the health of infected individuals are yet to be determined.

Trichomoniasis in finches from the Canadian Maritime provinces--An emerging disease.

Trichomoniasis was diagnosed in multiple incidents of mortality in wild purple finch (Carpodacus purpureus) and American goldfinch (Carduelis tristis) in the Canadian Maritimes. Birds exhibited regurgitation, emaciation, and hyperplastic oropharyngitis, ingluvitis, and esophagitis. Trichomonas gallinae was identified by histopathology and polymerase chain reaction (PCR). Trichomoniasis (trichomonosis) is an emerging disease in wild finches of eastern Canada.

Canine distemper virus-associated encephalitis in free-living lynx (Lynx canadensis) and bobcats (Lynx rufus) of eastern Canada.

Between 1993 and 1999, encephalitis caused by morbillivirus was diagnosed by immunohistochemistry and histology in six lynx (Lynx canadensis) and one bobcat (Lynx rufus) in the eastern Canadian provinces of New Brunswick and Nova Scotia. Five of the six cases in lynx occurred within an 11-mo period in 1996-97. A second bobcat with encephalitis caused by unidentified protozoa and a nematode larva also had immunohistochemical evidence of neurologic infection by morbillivirus. The virus was identified as canine distemper virus (CDV) by reverse transcriptase polymerase chain reaction and nucleotide sequencing in four of five animals from which frozen tissue samples were available, and it was isolated in cell culture from one of them. To our knowledge, this is the first report of disease caused by CDV in free-living felids in North America.

Tularemia, plague, yersiniosis, and Tyzzer's disease in wild rodents and lagomorphs in Canada: a review.

Information related to infection of wild rodents or lagomorphs in Canada by Francisella tularensis, Yersinia pestis, other Yersinia spp., and Clostridium piliforme was searched for this study. Reports on tularemia in humans linked to these species came from diagnostic databases, literature, wildlife health specialists, and public health agencies. Tularemia has been diagnosed in 8 species of wild rodent and 2 species in the genus Lepus in Canada. Tularemia occurred in wild animals, or in humans associated with these species, in all jurisdictions except the Yukon and Nunavut. Tularemia was diagnosed most frequently in beaver, muskrats, and snowshoe hares, and although tularemia is closely linked to cottontail rabbits in the USA, it has not been reported in cottontails in Canada. Tularemia in humans was associated with muskrats and hares more commonly than with beaver. Plague was diagnosed in bushy-tailed woodrats in British Columbia in 1988. Based on surveys, Y. pestis may occur enzootically in southern Alberta, Saskatchewan, and British Columbia. Infection with Yersinia pseudotuberculosis and Y. enterocolitica has been diagnosed in beaver, muskrats, and snowshoe hares in many provinces. Tyzzer's disease has been diagnosed in muskrats in British Columbia, Saskatchewan, Ontario, and Quebec and in snowshoe hares in Ontario. Infection with these bacteria is likely much more frequent than indicated by diagnostic records.

Ethylene glycol toxicosis in a free-ranging raccoon (Procyon lotor) from Prince Edward Island.

A juvenile free-ranging raccoon (Procyon lotor) was presented for acute onset of abnormal mentation and seizures. Ethylene glycol toxicosis was diagnosed on postmortem examination. This report highlights the importance of including ethylene glycol toxicosis on the list of differential diagnoses for abnormal mentation and seizures in free-ranging raccoons.