Using transcriptomics to predict and visualize disease status in bighorn sheep (Ovis canadensis).

Increasing risk of pathogen spillover coupled with overall declines in wildlife population abundance in the Anthropocene make infectious disease a relevant concern for species conservation worldwide. While emerging molecular tools could improve our diagnostic capabilities and give insight into mechanisms underlying wildlife disease risk, they have rarely been applied in practice. Here, employing a previously reported gene transcription panel of common immune markers to track physiological changes, we present a detailed analysis over the course of both acute and chronic infection in one wildlife species where disease plays a critical role in conservation, bighorn sheep (Ovis canadensis). Differential gene transcription patterns distinguished between infection statuses over the course of acute infection and differential correlation (DC) analyses identified clear changes in gene co-transcription patterns over the early stages of infection, with transcription of four genes-TGFb, AHR, IL1b and MX1-continuing to increase even as transcription of other immune-associated genes waned. In a separate analysis, we considered the capacity of the same gene transcription panel to aid in differentiating between chronically infected animals and animals in other disease states outside of acute disease events (an immediate priority for wildlife management in this system). We found that this transcription panel was capable of accurately identifying chronically infected animals in the test dataset, though additional data will be required to determine how far this ability extends. Taken together, our results showcase the successful proof of concept and breadth of potential utilities that gene transcription might provide to wildlife disease management, from direct insight into mechanisms associated with differential disease response to improved diagnostic capacity in the field.

Seroprevalence of Toxoplasma gondii in American Black Bears (Ursus americanus) in Nevada, USA, using an Enzyme-linked Immunosorbent Assay.

Archived serum samples taken between 1997 and 2017 from 170 American black bears (Ursus americanus) in the Lake Tahoe area between California and Nevada, US, were tested for Toxoplasma antibodies to assess the seroprevalence of this agent. Samples were screened using a commercial porcine Toxoplasma (enzyme-linked immunosorbent assay [ELISA]) modified with Protein A/G peroxidase and compared to a traditional fluorescent antibody test. Results were analyzed to determine if there were differences in seroprevalence based on the test used, sex of bears, or habitat usage (urban-suburban vs. wildland). No significant differences in seroprevalence were attributable to any of these parameters. The overall seropositivity for bears was 36% (62/170), with urban-suburban bears scoring lower (31%; 37/119) than rural-wildland bears (40%; 18/45). Our results strongly support the use of a Protein A/G-modified ELISA for determining Toxoplasma exposure in black bears. We found somewhat lower levels of Toxoplasma antibodies in black bears from this region than in several reports from populations in the eastern US.

Traceback of the Psoroptes outbreak in British Columbian bighorn sheep (Ovis Canadensis).

Psoroptes are a non-burrowing, ectoparasitic, mange-causing mite that has been documented in American bighorn sheep populations throughout the 19th and 20th centuries; however, it was not seen on Canadian bighorn sheep until 2006. The aim of this study was to determine the potential source of the Psoroptes outbreak in Canadian bighorn sheep. Morphological and molecular analyses were used to compare mites recovered from outbreak-associated bighorn sheep, pet rabbits in Canada, and on historically infested bighorn sheep in the USA. The results revealed that Psoroptes acquired from the Canadian and outbreak-associated American bighorn sheep were morphologically more similar to those collected from rabbits than mites on historically infested bighorn sheep. Outer opisthosomal setae lengths measured an average of 81.7 µm (±7.7 µm) in outbreak associated bighorn mites, 88.9 µm (±12.0 µm) in rabbit mites and 151.2 µm (±16.6 µm) in historically infested bighorn mites. The opisthosomal lobe morphology of bighorn mites in the outbreak herds was also more similar to that of rabbit mites, previously described as P. cuniculi, than historically infested bighorn mites, which match previous descriptions of P. ovis. This finding was supported by DNA sequence data of the mitochondrial cytochrome B gene. This is the first report of Psoroptes of the rabbit ecotype on bighorn sheep. The morphological and molecular data therefore support the hypothesis that the source of Psoroptes outbreak in Canadian bighorn sheep represented a disease spillover event from rabbits rather than transmission from infested American bighorn sheep populations.

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus).

Viral infections were investigated in American black bears (Ursus americanus) from Nevada and northern California with and without idiopathic encephalitis. Metagenomics analyses of tissue pools revealed novel viruses in the genera Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. The circovirus and parvovirus were of particular interest due to their potential importance as pathogens. We characterized the genomes of these viruses and subsequently screened bears by PCR to determine their prevalence. The circovirus (Ursus americanus circovirus, UaCV) was detected at a high prevalence (10/16, 67%), and the chaphamaparvovirus (Ursus americanus parvovirus, UaPV) was found in a single bear. We showed that UaCV is present in liver, spleen/lymph node, and brain tissue of selected cases by in situ hybridization (ISH) and PCR. Infections were detected in cases of idiopathic encephalitis and in cases without inflammatory brain lesions. Infection status was not clearly correlated with disease, and the significance of these infections remains unclear. Given the known pathogenicity of a closely related mammalian circovirus, and the complex manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears.

Comparison of multilocus sequence types found among North American isolates of Mycoplasma bovis from cattle, bison, and deer, 2007-2017.

A prior multilocus sequence typing (MLST) study reported that Mycoplasma bovis isolates from North American bison possess sequence types (STs) different from those found among cattle. The 42 bison isolates evaluated were obtained in 2007 or later, whereas only 19 of 94 (~20%) of the available cattle isolates, with only 1 from North America, were from that same time. We compared STs of additional, contemporary, North American cattle isolates with those from bison, as well as isolates from 2 North American deer, all originating during the same timeframe, to more definitively assess potential strain-related host specificity and expand our understanding of the genetic diversity of M. bovis. From 307 isolates obtained between 2007 and 2017 (209 from cattle, 96 from bison, 2 from deer), we identified 49 STs, with 39 found exclusively in cattle and 5 exclusively in bison. Four STs were shared between bison and cattle isolates; one ST was found in cattle and in a deer. There was no clear association between ST and the health status of the animal of origin. An MLST-based phylogeny including 41 novel STs identified in our study reveals that STs found in bison fall within several divergent lineages that include STs found exclusively in cattle.

The genetic legacy of 50 years of desert bighorn sheep translocations.

Conservation biologists have increasingly used translocations to mitigate population declines and restore locally extirpated populations. Genetic data can guide the selection of source populations for translocations and help evaluate restoration success. Bighorn sheep (Ovis canadensis) are a managed big game species that suffered widespread population extirpations across western North America throughout the early 1900s. Subsequent translocation programs have successfully re-established many formally extirpated bighorn herds, but most of these programs pre-date genetically informed management practices. The state of Nevada presents a particularly well-documented case of decline followed by restoration of extirpated herds. Desert bighorn sheep (O. c. nelsoni) populations declined to less than 3,000 individuals restricted to remnant herds in the Mojave Desert and a few locations in the Great Basin Desert. Beginning in 1968, the Nevada Department of Wildlife translocated ~2,000 individuals from remnant populations to restore previously extirpated areas, possibly establishing herds with mixed ancestries. Here, we examined genetic diversity and structure among remnant herds and the genetic consequences of translocation from these herds using a genotyping-by-sequencing approach to genotype 17,095 loci in 303 desert bighorn sheep. We found a signal of population genetic structure among remnant Mojave Desert populations, even across geographically proximate mountain ranges. Further, we found evidence of a genetically distinct, potential relict herd from a previously hypothesized Great Basin lineage of desert bighorn sheep. The genetic structure of source herds was clearly reflected in translocated populations. In most cases, herds retained genetic evidence of multiple translocation events and subsequent admixture when founded from multiple remnant source herds. Our results add to a growing literature on how population genomic data can be used to guide and monitor restoration programs.

Detection of Mycoplasma ovipneumoniae in Pneumonic Mountain Goat ( Oreamnos americanus) Kids.

We documented bronchopneumonia in seven mountain goat ( Oreamnos americanus) kid mortalities between 2011 and 2015 following a pneumonia epizootic in bighorn sheep ( Ovis canadensis) and sympatric mountain goats in the adjacent East Humboldt Range and Ruby Mountains in Elko County, Nevada, US. Gross and histologic lesions resembled those described in bighorn lambs following all-age epizootics, and Mycoplasma ovipneumoniae was detected with real-time PCR in the lower and upper respiratory tracts of all kids. Mannheimia haemolytica, with one isolate being leukotoxigenic, was cultured from the upper respiratory tract of five kids, and in one kid, a leukotoxigenic strain of Mannheimia glucosida was isolated from both upper and lower respiratory tracts. During this same period, 75 mountain goats within the two populations were marked and sampled for respiratory pathogens, and M. ovipneumoniae, leukotoxigenic Bibersteinia trehalosi, and Mannheimia haemolytica were identified. The M. ovipneumoniae recovered from the kid mortalities shared the same DNA sequence-based strain type detected in the adult goats and sympatric bighorn sheep during and after the 2009-10 pneumonia outbreak. Clinical signs in affected kids, as well as decreased annual kid recruitment, also resembled reports in bighorn lambs from some herds following all-age pneumonia-associated die-offs. Mycoplasma ovipneumoniae, Pasteurellaceae spp., and other respiratory bacterial pathogens should be considered as a cause of pneumonia with potential population-limiting effects in mountain goats.

Identification of gammaherpesvirus infection in free-ranging black bears (Ursus americanus).

Herpesvirus infection was investigated in black bears (Ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. Visible cytopathic effects (CPE) could only be observed on days 3-5 post-infection in HrT-18G cell line inoculated with bear tissue extracts. The observed CPE in HrT-18G cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. Herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60 nm to 100 nm in size were often observed in viral cultures within the first two passages of propagation. Herpesvirus infection in the bear tissues and tissue cultures were detected by PCR using degenerate primers specific to the DNA polymerase gene (DPOL) and glycoprotein B gene (gB). DNA sequencing of the amplicon revealed that the detected herpesvirus has 94-95% identity to Ursid gammaherpesvirus 1 (UrHV-1) DNA sequences of DPOL. Phylogenetic analysis of DPOL sequences indicates that black bear herpesviruses and UrHV-1 are closely related and have small distances to members of Rhadinovirus. Interestingly, black bear herpesvirus infections were also found in bears without neurological signs. The DPOL DNA sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. However, the gB DNA sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%-70% identity to each other. It is possible that at least two different types of gammaherpesviruses are present in the U. americanus population or several gammaherpesviruses exist in ursine species.

Lipoidal corneal degeneration in aged falcons.

OBJECTIVE: To present a case series of idiopathic lipoidal corneal degeneration in falcons. ANIMALS STUDIED: Five falcons including three peregrine falcons (Falco peregrinus), one prairie falcon (Falco mexicanus), and one red-naped shaheen (Falco peregrinus babylonicus) were observed to develop slowly progressive corneal opacification that began at the temporal limbus and extended centripetally across the cornea over a period of years. Four of the birds were over 20 years old. PROCEDURES: All animals underwent complete ophthalmic examinations. A red-naped shaheen underwent ocular imaging via spectral-domain optical coherence tomography. Two peregrine falcons were euthanized due to declining health, and their eyes were examined histologically. RESULTS: The opacities were pale and granular, with frequent vascularization associated perilimbally. Diffuse neutral lipid was observed in stromal cells throughout the corneal stroma of both clear and opaque areas of the cornea, sparing only the acellular anterior limiting lamina. Clusters of cholesterol crystals surrounded by macrophages were present in the mid-stroma. Fibrosis was evident in a subepithelial location, which separated the epithelium from the anterior limiting lamina. Ultrastructurally, diffuse vacuolization of the keratocytes was observed. No other ophthalmic or systemic abnormalities were noted. CONCLUSIONS: Results suggest that lipid degeneration occurs rarely in captive falcons of advanced age. The underlying cause is unclear. Though unsubstantiated, possible contributing factors include dyslipoproteinemia, corneal trauma, diet, and age-related alterations in corneal metabolism. The initiation of pathology at the temporal limbus, as well as slow progression, suggests that exposure contributes to the onset and progression of this unique keratopathy.

Isolation and characterization of a novel cervid adenovirus from white-tailed deer (Odocoileus virginianus) fawns in a captive herd.

A novel adenovirus, CeAdV1, was isolated from buffy coat and nasal swab samples collected from two captive white-tailed deer (Odocoileus virginianus) fawns. The isolation was an incidental finding in the course of screening animals for use in a research study on an unrelated pathogen. In the screening process, virus isolation was performed on both nasal swabs and buffy coat samples and cytopathic effect was observed. Electron microscopy revealed viral particles with the shape and morphology of an adenovirus. Next generation sequencing followed by phylogenetic analysis classified this virus to the Mastadenovirus genus. Its sequence was genetically distinct from all other recognized species in this genus, with only 76% sequence identity to its closest genetic match, bovine adenovirus 3 (BAdV3). The virus could be propagated in bovine derived cells but grew to a higher titer in cervid derived cells. Inoculation of white-tailed deer fawns with the isolated virus resulted in pyrexia, depletion of thymus tissue and mild respiratory disease. Comparative serology performed using convalescent sera revealed distinct antigenic differences between the novel cervid adenovirus and BAdV3. A retrospective serological survey of the captive deer herd indicated that this virus had been circulating in the herd for at least 14 years with no report of clinical disease. A survey of serum collected from free ranging mule deer residing in Nevada revealed high serum titers against this novel adenovirus.

Evidence of Bovine viral diarrhea virus Infection in Three Species of Sympatric Wild Ungulates in Nevada: Life History Strategies May Maintain Endemic Infections in Wild Populations.

Evidence for bovine viral diarrhea virus (BVDV) infection was detected in 2009-2010 while investigating a pneumonia die-off in Rocky Mountain bighorn sheep (Ovis canadensis, canadensis), and sympatric mountain goats (Oreamnos americanum) in adjacent mountain ranges in Elko County, Nevada. Seroprevalence to BVDV-1 was 81% (N = 32) in the bighorns and 100% (N = 3) in the mountain goats. Serosurveillance from 2011 to 2015 of surviving bighorns and mountain goats as well as sympatric mule deer (Odocoileus hemionus), indicated a prevalence of 72% (N = 45), 45% (N = 51), and 51% (N = 342) respectively. All species had antibody titers to BVDV1 and BVDV2. BVDV1 was isolated in cell culture from three bighorn sheep and a mountain goat kid. BVDV2 was isolated from two mule deer. Six deer (N = 96) sampled in 2013 were positive for BVDV by antigen-capture ELISA on a single ear notch. Wild ungulates and cattle concurrently graze public and private lands in these two mountain ranges, thus providing potential for interspecies viral transmission. Like cattle, mule deer, mountain goats, and bighorn sheep can be infected with BVDV and can develop clinical disease including immunosuppression. Winter migration patterns that increase densities and species interaction during the first and second trimester of gestation may contribute to the long term maintenance of the virus in these wild ungulates. More studies are needed to determine the population level impacts of BVDV infection on these three species.

Persistent Bovine Viral Diarrhea Virus Infection in Domestic and Wild Small Ruminants and Camelids Including the Mountain Goat (Oreamnos americanus).

Bovine viral diarrhea virus (BVDV) is a pestivirus best known for causing a variety of disease syndromes in cattle, including gastrointestinal disease, reproductive insufficiency, immunosuppression, mucosal disease, and hemorrhagic syndrome. The virus can be spread by transiently infected individuals and by persistently infected animals that may be asymptomatic while shedding large amounts of virus throughout their lifetime. BVDV has been reported in over 40 domestic and free-ranging species, and persistent infection has been described in eight of those species: white-tailed deer, mule deer, eland, mousedeer, mountain goats, alpacas, sheep, and domestic swine. This paper reviews the various aspects of BVDV transmission, disease syndromes, diagnosis, control, and prevention, as well as examines BVDV infection in domestic and wild small ruminants and camelids including mountain goats (Oreamnos americanus).

PCR assay detects Mannheimia haemolytica in culture-negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010.

Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep (Ovis canadensis) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009-2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae, an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009-2010.

A review of episodes of zinc phosphide toxicosis in wild geese (Branta spp.) in Oregon (2004-2011).

Epizootic mortality in several geese species, including cackling geese (Branta hutchinsii) and Canada geese (Branta canadensis), has been recognized in the Willamette Valley of Oregon for over a decade. Birds are generally found dead on a body of water or are occasionally observed displaying neurologic clinical signs such as an inability to raise or control the head prior to death. Investigation of these epizootic mortality events has revealed the etiology to be accidental poisoning with the rodenticide zinc phosphide (Zn(3)P(2)). Gross and histologic changes are restricted to acute pulmonary congestion and edema, sometimes accompanied by distension of the upper alimentary tract by fresh grass. Geese are unusually susceptible to this pesticide; when combined with an epidemiologic confluence of depredation of specific agricultural crops by rodents and seasonal avian migration pathways, epizootic toxicosis may occur. Diagnosis requires a high index of suspicion, appropriate sample collection and handling, plus specific test calibration for this toxicant. Interagency cooperation, education of farmers regarding pesticide use, and enforcement of regulations has been successful in greatly decreasing these mortality events since 2009.

Bighorn sheep pneumonia: sorting out the cause of a polymicrobial disease.

Pneumonia of bighorn sheep (Ovis canadensis) is a dramatic disease of high morbidity and mortality first described more than 80 years ago. The etiology of the disease has been debated since its initial discovery, and at various times lungworms, Mannheimia haemolytica and other Pasteurellaceae, and Mycoplasma ovipneumoniae have been proposed as primary causal agents. A multi-factorial "respiratory disease complex" has also been proposed as confirmation of causation has eluded investigators. In this paper we review the evidence for each of the candidate primary agents with regard to causal criteria including strength of association, temporality, plausibility, experimental evidence, and analogy. While we find some degree of biological plausibility for all agents and strong experimental evidence for M. haemolytica, we demonstrate that of the alternatives considered, M. ovipneumoniae is the best supported by all criteria and is therefore the most parsimonious explanation for the disease. The strong but somewhat controversial experimental evidence implicating disease transmission from domestic sheep is consistent with this finding. Based on epidemiologic and microbiologic data, we propose that healthy bighorn sheep populations are naïve to M. ovipneumoniae, and that its introduction to susceptible bighorn sheep populations results in epizootic polymicrobial bacterial pneumonia often followed by chronic infection in recovered adults. If this hypothesized model is correct, efforts to control this disease by development or application of vectored vaccines to Pasteurellaceae are unlikely to provide significant benefits, whereas efforts to ensure segregation of healthy bighorn sheep populations from M. ovipneumoniae-infected reservoir hosts are crucial to prevention of new disease epizootics. It may also be possible to develop M. ovipneumoniae vaccines or other management strategies that could reduce the impact of this devastating disease in bighorn sheep.

Causes of pneumonia epizootics among bighorn sheep, Western United States, 2008-2010.

Epizootic pneumonia of bighorn sheep is a devastating disease of uncertain etiology. To help clarify the etiology, we used culture and culture-independent methods to compare the prevalence of the bacterial respiratory pathogens Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae in lung tissue from 44 bighorn sheep from herds affected by 8 outbreaks in the western United States. M. ovipneumoniae, the only agent detected at significantly higher prevalence in animals from outbreaks (95%) than in animals from unaffected healthy populations (0%), was the most consistently detected agent and the only agent that exhibited single strain types within each outbreak. The other respiratory pathogens were frequently but inconsistently detected, as were several obligate anaerobic bacterial species, all of which might represent secondary or opportunistic infections that could contribute to disease severity. These data provide evidence that M. ovipneumoniae plays a primary role in the etiology of epizootic pneumonia of bighorn sheep.

Detection of relapsing fever spirochetes (Borrelia hermsii and Borrelia coriaceae) in free-ranging mule deer (Odocoileus hemionus) from Nevada, United States.

Surveillance of mule deer (Odocoileus hemionus, Rafinesque, 1917) populations for tick-borne diseases has helped define the distribution of these pathogens and their subsequent risk of transmission to humans and domestic animals. We surveyed three mule deer herds across the state of Nevada for infection with relapsing fever Borrelia spp. spirochetes. Bacterial prevalence varied by the county where deer were sampled but Borrelia spirochetes were detected in 7.7% of all deer sampled. Infected deer were identified in every location from which mule deer samples were obtained. Sequencing of the Borrelia intergenic spacer gene (IGS) revealed that one individual was infected with Borrelia coriaceae and all others were infected with Borrelia hermsii. The vector of B. hermsii, Ornithodoros hermsi (Acari: Argasidae, Wheeler, Herms, and Meyer, 1935), feeds primarily on wild rodents and has not been identified infesting deer. Additionally, Ornithodoros coriaceus (Acari: Argasidae, Koch, 1844), which readily feeds on deer and is frequently infected with B. coriaceae, has not been shown to be a competent vector for B. hermsii. Our data represent the first sylvatic evidence of B. hermsii infection in mule deer. Additionally, our data provide evidence that infection with relapsing fever spirochetes in Nevada is wide ranging in the state's deer populations.

Experimental deerpox infection in black-tailed deer (Odocoileus hemionus columbianus).

The pathogenic potential of deerpox virus was investigated via an experimental study utilizing seven black-tailed deer fawns (Odocoileus hemionus) between June and August of 2007. Successful transmission was achieved via intracutaneous and intravenous routes, and by commingling an uninoculated animal with experimentally infected fawns. One fawn became depressed and reluctant to eat but systemic clinical signs in the other fawns were confined to mild transient pyrexia. Typical multifocal poxviral cutaneous lesions of erythema, papules, pustules, ulceration, and crusting were observed. Two locally extensive ulcerative lesions also developed at inoculation sites. Oral lesions were seen in one commingled fawn, with some palatine epithelial cells containing intracytoplasmic inclusions. Microscopic cutaneous lesions included epithelial cell hyperplasia with hydropic change, intraepithelial pustules, erosions, folliculitis, and dense leukocytic dermal infiltrates. Transmission was confirmed by one or more of virus isolation, polymerase chain reaction or serum neutralization tests. Significant internal lesions were not seen at necropsy.