Prevalence of Mycoplasma spp. in the Respiratory Tract of Healthy North American Bison (Bison bison) and Comparison with Serum Antibody Status.

Mycoplasma bovis is a primary cause of respiratory and reproductive diseases in North American bison (Bison bison), with significant morbidity and mortality. The epidemiology of M. bovis in bison is poorly understood, hindering efforts to develop effective control measures. Our study considered whether healthy bison might be carriers of M. bovis, potentially serving as unrecognized sources of exposure. We used culture and PCR to identify mycoplasmas in the nasal cavity or tonsil of 499 healthy bison from 13 herds and two abattoirs in the US and Canada. Mycobacterium bovis was detected in 15 bison (3.0%) representing two herds in the US and one in Canada, while M. bovirhinis, M. bovoculi, M. arginini, or M. dispar was identified from an additional 155 bison (31.1%). Mycoplasma bovirhinis was identified most frequently, in 142 bison (28.5%) representing at least 10 herds. Of the 381 bison for which serum was available, only 6/13 positive for M. bovis (46.2%) tested positively with an M. bovis ELISA, as did 19/368 negative for M. bovis (5.2%). Our data reveal that M. bovis can be carried in the upper respiratory tract of healthy bison with no prior history or clinical signs of mycoplasmosis and that a large proportion of carriers may not produce detectable antibodies. Whether carriage of other mycoplasmas can trigger cross-reactive antibodies that may confound M. bovis serology requires further study.

Serological evidence for historical and present-day exposure of North American bison to Mycoplasma bovis.

BACKGROUND: Mycoplasma bovis causes mastitis, otitis, pneumonia and arthritis in cattle and is a major contributor to bovine respiratory disease complex. Around the year 2000, it emerged as a significant threat to the health of North American bison. Whether healthy bison are carriers of M. bovis and when they were first exposed is not known. To investigate these questions we used a commercially available ELISA that detects antibodies to M. bovis to test 3295 sera collected from 1984 through 2019 from bison in the United States and Canada. RESULTS: We identified moderately to strongly seropositive bison from as long ago as the late 1980s. Average seroprevalence over the past 36 years is similar in the United States and Canada, but country-specific differences are evident when data are sorted by the era of collection. Seroprevalence in the United States during the pre-disease era (1999 and prior) was significantly higher than in Canada, but was significantly lower than in Canada during the years 2000-2019. Considering individual countries, seroprevalence in the United States since the year 2000 dropped significantly as compared to the years 1985-1999. In Canada the trend is reversed, with seroprevalence increasing significantly since the year 2000. ELISA scores for sera collected from free-ranging bison do not differ significantly from scores for sera from more intensively managed animals, regardless of the era in which they were collected. However, seroprevalence among intensively raised Canadian bison has nearly doubled since the year 2000 and average ELISA scores rose significantly. CONCLUSIONS: Our data provide the first evidence that North American bison were exposed to M. bovis many years prior to the emergence of M. bovis-related disease. Patterns of exposure inferred from these results differ in the United States and Canada, depending on the era under consideration. Our data further suggest that M. bovis may colonize healthy bison at a level sufficient to trigger antibody responses but without causing overt disease. These findings provide novel insights as to the history of M. bovis in bison and will be of value in formulating strategies to minimize the impact of mycoplasmosis on bison health and production.

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing.

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.

A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses.

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

The global distribution of Bacillus anthracis and associated anthrax risk to humans, livestock and wildlife.

Bacillus anthracis is a spore-forming, Gram-positive bacterium responsible for anthrax, an acute infection that most significantly affects grazing livestock and wild ungulates, but also poses a threat to human health. The geographic extent of B. anthracis is poorly understood, despite multi-decade research on anthrax epizootic and epidemic dynamics; many countries have limited or inadequate surveillance systems, even within known endemic regions. Here, we compile a global occurrence dataset of human, livestock and wildlife anthrax outbreaks. With these records, we use boosted regression trees to produce a map of the global distribution of B. anthracis as a proxy for anthrax risk. We estimate that 1.83 billion people (95% credible interval (CI): 0.59-4.16 billion) live within regions of anthrax risk, but most of that population faces little occupational exposure. More informatively, a global total of 63.8 million poor livestock keepers (95% CI: 17.5-168.6 million) and 1.1 billion livestock (95% CI: 0.4-2.3 billion) live within vulnerable regions. Human and livestock vulnerability are both concentrated in rural rainfed systems throughout arid and temperate land across Eurasia, Africa and North America. We conclude by mapping where anthrax risk could disrupt sensitive conservation efforts for wild ungulates that coincide with anthrax-prone landscapes.

Investigation of the utility of feces and hair as non-invasive measures of glucocorticoids in wild black-tailed prairie dogs (Cynomys ludovicianus).

Non-invasive measures of glucocorticoid (GC) hormones and their metabolites, particularly in feces and hair, are gaining popularity as wildlife management tools, but species-specific validations of these tools remain rare. We report the results of a validation on black-tailed prairie dogs (Cynomys ludovicianus), a highly social engineer of the grasslands ecosystem that has experienced recent population declines. We captured adult female prairie dogs and brought them into temporary captivity to conduct an adrenocorticotropic hormone (ACTH) stimulation test, assessing the relationship between plasma GC and fecal glucocorticoid metabolite (FGM) levels following a single injection of a low (4 IU/kg) or high dose (12 IU/kg) of ACTH, compared to a single injection of saline. We also gave repeated injections of ACTH to adult females to assess whether this would result in an increase of hair cortisol concentrations, compared with control individuals repeatedly injected with saline. A single injection of ACTH at either low or high dose peaked plasma cortisol levels after 30 min, and thereafter the cortisol levels declined until 120 min, where they returned to pre-treatment levels comparable to those of the saline injected group. Despite the significant elevation of plasma cortisol in the treatment groups following ACTH injection, the elevation of FGM levels in the treatment groups were not significantly different from those in the control group over the following 12 h. Repeated injection of a high dose of ACTH failed to increase hair cortisol concentration in treatment animals. Instead, hair cortisol levels remained comparable to the pre-treatment mean, despite an increase in post-treatment hair cortisol levels seen in the saline-injected group. The magnitude of increase in the saline control group was comparable to natural seasonal variation seen in unmanipulated individuals. These results highlight that while measurement of GCs and their metabolites in feces and hair are potentially valuable conservation tools for black-tailed prairie dogs, further validation work is required before these matrices can be to real-world conservation applications.

Fascioloides magna in Moose ( Alces alces) From Elk Island National Park, Alberta.

Thirty-seven adult female moose ( Alces alces) from 2 distinct but adjacent populations in Elk Island National Park (EINP), Alberta, Canada (19 in north EINP and 18 in south EINP), were fitted with mortality-sensing VHF radio-collars, and radio signals were acquired daily to ascertain mortality status. At capture, serum, whole blood, and feces were collected; pregnancy was determined; teeth were aged by visual inspection; and a portion of liver was assessed by ultrasound examination. Postmortem examination was conducted on 20 suitable carcasses. Clinical pathological abnormalities, including eosinophilia, polycythemia, elevated levels of liver enzymes in serum, hemoglobin, hematocrit, and red blood cell distribution, and liver damage as seen in ultrasound images occurred only in moose from north EINP. Infected moose had 4.7 ± 4.8 Fascioloides magna flukes per liver (mean ± SD). The proportion of moose pregnant at capture was similar in both populations (74% in north EINP, 61% in south EINP). Proportional mortality was significantly higher in moose from the north (68%) than the south (32%). Fascioloides magna was associated as a cause of death in 7 of 14 (50%) moose in the north where cause of death was determined, while predation ( n = 1), acute toxemic syndrome ( n = 3), dystocia ( n = 1), and roadkill and undetermined causes ( n = 3) were additional causes of mortality. F. magna was associated with poor body condition and was a major cause of mortality in north EINP but not south EINP, despite very similar habitat and proximity, suggesting a significant role for these flukes in affecting health and viability of naturally infected moose populations.

TUBERCULOSIS AND BRUCELLOSIS IN WOOD BISON (BISON BISON ATHABASCAE) IN NORTHERN CANADA: A RENEWED NEED TO DEVELOP OPTIONS FOR FUTURE MANAGEMENT.

Effective, long-term strategies to manage the threat of bovine tuberculosis and brucellosis spillback from northern, diseased bison to the Canadian cattle herd and adjacent disease-free wood bison (Bison bison athabascae) herds have eluded policy makers in recent decades. A controversial plan to depopulate infected herds and repopulate them with disease-free wood bison was rejected in 1990 because of significant public concern. Since then, technical advances in vaccine technology, genetic salvage, selective culling, and diagnostic test development have occurred. Containment strategies to reduce further spread of these diseases are a necessary first step; recent progress has been made in this area, but challenges remain. This progress has produced more options for management of these herds in northern Canada, and it is time to consider wood bison conservation and long-term disease eradication as equally important goals that must satisfy concerns of conservation groups, agriculture sectors, aboriginal groups, and the general public. Management of wildlife disease reservoirs in other areas, including Yellowstone and Riding Mountain national parks, has demonstrated that effective disease management is possible. Although combinations of different strategies, including vaccination, genetic salvage techniques, and selective culling, that use sensitive and specific diagnostic tests may offer alternatives to depopulation/repopulation, they also have logistic constraints and cost implications that will need consideration in a multistakeholder, collaborative-management framework. We feel the time is right for this discussion, so a long-term solution to this problem can be applied.

Sylvatic plague in a Canadian black-tailed prairie dog (Cynomys ludovicianus).

In 2010, a black-tailed prairie dog (Cynomys ludovicianus) was found dead in Grasslands National Park, Saskatchewan, Canada. Postmortem gross and histologic findings indicated bacterial septicemia, likely due to Yersinia pestis, which was confirmed by molecular analysis. This is the first report of Y. pestis in the prairie dog population within Canada.

Field evaluation of three blood-based assays for elk (Cervus canadensis) naturally infected with Mycobacterium bovis.

Diagnosis of Mycobacterium bovis in wild populations is very challenging due to complications imposed by the use of traditional skin tests, poor sensitivity of gold standard tests which rely on culture of M. bovis from tissues and wide variations in severity of disease. Various combinations of a lymphocyte stimulation test (LST), fluorescence polarization assay (FPA) and the Cervid TB Stat-Pak were evaluated using two different validation approaches: a latent class analysis and classical statistical approach using culture as a gold standard. A validation subsample consisting of animals culled for population control and mortalities from capture provided an unbiased estimate of test performance for comparison. The sensitivity of the LST (0.83, 95% CI: [0.70-0.97] as a single test was similar to existing tuberculin skin tests, but the sensitivity of the FPA (0.40, 95% CI: [0.22-0.58]) and Cervid TB Stat-Pak (0.62, 95% CI: [0.41-0.83]) were lower in this population. Test performance of the LST and Cervid TB Stat-Pak in parallel was similar to the use of all three tests in parallel and inclusion of the FPA did not greatly enhance test performance. Prevalence of M. bovis in elk varied substantially between the high risk area of southern Manitoba (9.1%, 95% CI: [6.09-12.1%]) and lower risk areas outside this zone (0.76%, 95% CI: [0-2.26%]). Bayesian latent class analysis indicated lack of covariance between the two antibody tests (FPA and Cervid TB Stat-Pak) while the classical two-stage analysis indicated there was conditional dependence between the tests. All three tests when used in parallel resulted in 100% NPV using all three validation methods, indicating few elk were misclassified as false negative by post mortem culture. Similar to previous studies, this study found that combinations of blood tests that utilize cell mediated responses along with humoral antibody responses maximize the sensitivity of tests for diagnosis of M. bovis in wild cervid populations.

Lesion Distribution and Epidemiology of Mycobacterium bovis in Elk and White-Tailed Deer in South-Western Manitoba, Canada.

Surveillance for Mycobacterium bovis in free-ranging elk (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) from south-western Manitoba was carried out from 1997 to 2010 to describe the lesions, epidemiology, and geographic distribution of disease. Tissues were cultured from animals killed by hunters, culled for management, blood-tested, or found opportunistically. Period prevalence in elk was approximately six times higher than deer, suggesting a significant reservoir role for elk, but that infected deer may also be involved. Prevalence was consistently higher in elk compared to deer in a small core area and prevalence declines since 2003 are likely due to a combination of management factors instituted during that time. Older age classes and animals sampled from the core area were at significantly higher risk of being culture positive. Positive elk and deer were more likely to be found through blood testing, opportunistic surveillance, and culling compared to hunting. No non-lesioned, culture-positive elk were detected in this study compared to previous studies in red deer.

Intranasal naltrexone and atipamezole for reversal of white-tailed deer immobilized with carfentanil and medetomidine.

Carfentanil and medetomidine were used to immobilize 8 captive female white-tailed deer (Odocoileus virginianus) using mean dosages [+/- standard deviation (s)] of 14.2 +/- 1.11 microg/kg carfentanil and 17.8 +/- 2.03 microg/kg of medetomidine. Deer were reversed by intranasally or intramuscularly administered naltrexone and atipamezole. Dosages of carfentanil and medetomidine proved reliable for immobilization of most, but not all deer, with a mean induction time of 13.3 +/- 3.13 min. Effective and reliable immobilization will require higher dosages of carfentanil and possibly medetomidine than were used in this study. No significant differences in recovery times were observed for deer given reversal agents intranasally (9.45 +/- 5.37 min) versus intramuscularly (7.60 +/- 4.42 min). Naltrexone and atipamezole can be administered intranasally at 1.5 mg/kg and 0.1 mg/kg, respectively to safely and quickly reverse the effects of carfentanil and medetomidine in immobilized white-tailed deer. This route could potentially be useful for other reversal agents.

Suspected cervical spinal cord vascular anomaly in an African warthog (Phacochoerus africanus).

Vascular myelopathies of the spinal cord have not been described in Suidae, and are a rare finding in companion animals. An 8.5-yr female African warthog (Phacochoerus africanus) presented with an acute onset of tetraparesis. Based on neurologic findings, a cervical spinal cord lesion between C7-T2 was suspected. Magnetic resonance imaging revealed severe intramedullary hemorrhage with suspected abnormal vessels in the spinal cord at the level of the seventh cervical vertebrae. The acute onset of clinical signs and rapid deterioration of neurological status precluded surgical managements. A vascular anomaly was suspected on gross pathology and histology. Immunohistochemistry identified the lesion as a spontaneous intramedullary hematoma. Spontaneous intramedullary hematomyelia should be considered as a differential for acute onset of paresis in suid species.