Evaluating Two Sampling Methods for Mycoplasma Bovis Diagnosis in American Bison (Bison Bison).

Mycoplasma bovis is a bacterial pathogen endemic to cattle. In the early 2000s, M. bovis emerged as a cause of respiratory disease in American bison (Bison bison), causing significant morbidity and mortality. Bison herds that experience an outbreak of M. bovis are at higher risk for subsequent outbreaks, suggesting that chronic, subclinical infections can be established. Antemortem testing is therefore crucial to disease management; however, the precise sampling method to maximize detection of M. bovis in bison is unknown. We evaluated two sample types-superficial nasal swabs and deep nasopharyngeal swabs-collected from apparently healthy or symptomatic bison from January 2021 through December 2022. We used real-time PCR to detect M. bovis in 76/938 bison (8.1%) from 11 herds. For bison testing positive on at least one swab type, M. bovis was detected in 63/76 (82.8%) deep nasopharyngeal swabs and 29/73 (38.1%) superficial nasal swabs. Agreement between swabs for positive bison was 21% (n=16, kappa coefficient 0.319). We conclude that deep nasopharyngeal swabbing is more sensitive than superficial nasal swabbing for detection of M. bovis in bison and that low agreement between methods may be related to stage of infection. We further tested pooled samples by PCR and found that pooling of up to five samples can be effective to increase throughput and minimize costs. Management of wild bison relies on the ability to relocate animals to maintain gene flow and healthy populations. Sensitive and specific diagnostic tests are needed to inform decisions and minimize risk of transmission, especially from subclinical carriers. This study provides valuable insight that will inform best practices for M. bovis testing, thereby supporting the conservation of bison as healthy wildlife, which in turn promotes ecological restoration, safeguards cultural practices of Tribal Nations, and upholds the bison as a unique American icon.

SOURCE AND SEASONALITY OF EPIZOOTIC MYCOPLASMOSIS IN FREE-RANGING PRONGHORN (ANTILOCAPRA AMERICANA).

Mycoplasma bovis is an economically important bacterial pathogen of cattle (Bos taurus) and bison (Bison bison) that most commonly causes pneumonia, polyarthritis, and mastitis. It is prevalent in cattle and ranched bison; however, infections in other species are rare. In early 2019, we identified M. bovis in free-ranging pronghorn (Antilocapra americana) in northeastern Wyoming. Here, we report on additional pronghorn mortalities caused by M. bovis, in the same approximately 120-km2 geographic region 1 yr later. Genetic analysis by multilocus sequence typing revealed that the mortalities were caused by the same M. bovis sequence type, which is unique among all sequence types documented thus far in North America. To explore whether pronghorn maintain chronic infections and begin assessing M. bovis status in other sympatric species, we used PCR testing of nasal swabs to opportunistically survey select free-ranging ungulates. We found no evidence of subclinical infections in 13 pronghorn sampled from the outbreak area (upper 95% binomial confidence limit [bCL], ∼24.7%) or among 217 additional pronghorn (upper 95% bCL, ∼1.7%) sampled from eight additional counties in Wyoming and 10 in Montana. All mule deer (Odocoileus hemionus; n=231; upper 95% bCL, ∼1.6%) sampled from 11 counties in Wyoming also were PCR negative. To assess the potential for environmental transmission, we examined persistence of M. bovis in various substrates and conditions. Controlled experiments revealed that M. bovis can remain viable for 6 h in shaded water and 2 h in direct sunlight. Our results indicate that environmental transmission of M. bovis from livestock to pronghorn is possible and that seasonality of infection could be due to shared resources during late winter. Further investigations to better understand transmission dynamics, to assess population level impacts to pronghorn, and to determine disease risks among pronghorn and other ungulate taxa appear warranted.

City sanitation and socioeconomics predict rat zoonotic infection across diverse neighbourhoods.

Rat-associated zoonoses transmitted through faeces or urine are of particular concern for public health because environmental exposure in homes and businesses may be frequent and undetected. To identify times and locations with greater public health risks from rats, we investigated whether rat characteristics, environmental features, socioeconomic factors, or season could predict rat infection risk across diverse urban neighbourhoods. In partnership with a pest management company, we sampled rats in 13 community areas along an income gradient in Chicago, a large city where concern about rats has increased in recent years. We collected kidneys for Leptospira spp. testing and colon contents for aerobic bacteria such as Salmonella spp. and Escherichia coli. Of 202 sampled rats, 5% carried Leptospira spp. and 22% carried E. coli. Rats were significantly more likely to carry Leptospira spp. on blocks with more standing water complaints in higher-income neighbourhoods (OR = 6.74, 95% CI: 1.54-29.39). Rats were significantly more likely to carry E. coli on blocks with more food vendors (OR = 9.94, 2.27-43.50) particularly in low-income neighbourhoods (OR = 0.26, 0.09-0.82) and in the spring (OR = 15.96, 2.90-88.62). We detected a high diversity of E. coli serovars but none contained major virulence factors. These associations between environmental features related to sanitation and infection risk in rats support transmission through water for Leptospira spp. and faecal-oral transmission for E. coli. We also found opposing relationships between zoonotic infection risk and income for these two pathogens. Thus, our results highlight the importance of sanitation for predicting zoonotic disease risks and including diverse urban areas in pathogen surveillance to mitigate public health risks from rats.

Development and validation of a real-time PCR specific for the leukotoxin gene of Bibersteinia trehalosi.

A real-time PCR assay for the leukotoxin gene of Bibersteinia trehalosi was developed and validated to better identify this pathogen, which is a cause of respiratory disease in bighorn sheep. The specificity of the PCR primers was evaluated with DNA from 59 known isolates of the Pasteurellaceae family. For validation, 162 field samples were compared using both the new assay and an indirect method using 2 sets of published protocols. The real-time PCR assay was found to be specific for the leukotoxin gene of B. trehalosi and provides a rapid and direct approach for detecting leukotoxin-producing forms of this organism from samples containing mixed species of leukotoxin-positive Pasteurellaceae.

Respiratory pathogens and their association with population performance in Montana and Wyoming bighorn sheep populations.

Respiratory disease caused by Mycoplasma ovipneumoniae and Pasteurellaceae poses a formidable challenge for bighorn sheep (Ovis canadensis) conservation. All-age epizootics can cause 10-90% mortality and are typically followed by multiple years of enzootic disease in lambs that hinders post-epizootic recovery of populations. The relative frequencies at which these epizootics are caused by the introduction of novel pathogens or expression of historic pathogens that have become resident in the populations is unknown. Our primary objectives were to determine how commonly the pathogens associated with respiratory disease are hosted by bighorn sheep populations and assess demographic characteristics of populations with respect to the presence of different pathogens. We sampled 22 bighorn sheep populations across Montana and Wyoming, USA for Mycoplasma ovipneumoniae and Pasteurellaceae and used data from management agencies to characterize the disease history and demographics of these populations. We tested for associations between lamb:ewe ratios and the presence of different respiratory pathogen species. All study populations hosted Pasteurellaceae and 17 (77%) hosted Mycoplasma ovipneumoniae. Average lamb:ewe ratios for individual populations where both Mycoplasma ovipneumoniae and Pasteurellaceae were detected ranged from 0.14 to 0.40. However, average lamb:ewe ratios were higher in populations where Mycoplasma ovipneumoniae was not detected (0.37, 95% CI: 0.27-0.51) than in populations where it was detected (0.25, 95% CI: 0.21-0.30). These findings suggest that respiratory pathogens are commonly hosted by bighorn sheep populations and often reduce recruitment rates; however ecological factors may interact with the pathogens to determine population-level effects. Elucidation of such factors could provide insights for management approaches that alleviate the effects of respiratory pathogens in bighorn sheep. Nevertheless, minimizing the introduction of novel pathogens from domestic sheep and goats remains imperative to bighorn sheep conservation.

A Survey of Bacterial Respiratory Pathogens in Native and Introduced Mountain Goats ( Oreamnos americanus).

In contrast to broad range expansion through translocations, many mountain goat ( Oreamnos americanus) populations have shown signs of decline. Recent documentation of pneumonia in mountain goats highlights their susceptibility to bacterial pathogens typically associated with bighorn sheep ( Ovis canadensis) epizootics. Respiratory pathogen communities of mountain goats are poorly characterized yet have important implications for management and conservation of both species. We characterized resident pathogen communities across a range of mountain goat populations as an initial step to inform management efforts. Between 2010 and 2017, we sampled 98 individuals within three regions of the Greater Yellowstone Area (GYA), with a smaller sampling effort in southeast Alaska, US. Within the GYA, we detected Mycoplasma ovipneumoniae in two regions and we found at least two Pasteurellaceae species in animals from all regions. Mannheimia haemolytica was the only pathogen that we detected in southeast Alaska. Given the difficult sampling conditions, limited sample size, and imperfect detection, our failure to detect specific pathogens should be interpreted with caution. Nonetheless, respiratory pathogens within the GYA may be an important, yet underappreciated, cause of mountain goat mortality. Moreover, because of the strong niche overlap of bighorn sheep and mountain goats, interspecific transmission is an important concern for managers restoring or introducing mountain ungulates within sympatric ranges.

Assessing Timing and Causes of Neonatal Lamb Losses in a Bighorn Sheep ( Ovis canadensis canadensis ) Herd via Use of Vaginal Implant Transmitters.

We evaluated the use of vaginal implant transmitters (VITs) as a means of detecting, capturing, and radio collaring Rocky Mountain bighorn sheep ( Ovis canadensis canadensis) lambs to estimate survival and to facilitate carcass recovery to assess causes of mortality. We focused on one of several bighorn herds in Colorado, US, suffering from depressed recruitment that was not preceded by a classic all-age die-off. We captured, radio-collared, diagnosed pregnancy by ultrasound examination, and inserted VITs into 15 pregnant ewes from a herd residing near Granite, Colorado. We were subsequently able to collar a lamb from each of 13 VITs, and two additional lambs opportunistically from ewes without transmitters. As lambs died, we recovered and submitted carcasses for necropsy and laboratory assessment. All lambs captured and one additional lamb (carcass found opportunistically) were dead by about 130 d of age: 11 died of apparent pneumonia (all within 8-10 wk of age), one died from trauma after being kicked or trampled, one was killed by a mountain lion ( Puma concolor ), and three died of starvation likely caused by abandonment after capture. Pneumonic lambs had involvement of Mycoplasma ovipneumoniae and leukotoxigenic Bibersteinia trehalosi . The use of VITs and lamb collars enabled us to efficiently identify pneumonia as the predominant cause of depressed lamb recruitment in this herd; however, we urge care in neonatal lamb handling to minimize abandonment.

How Respiratory Pathogens Contribute to Lamb Mortality in a Poorly Performing Bighorn Sheep ( Ovis canadensis ) Herd.

We evaluated bighorn sheep ( Ovis canadensis ) ewes and their lambs in captivity to examine the sources and roles of respiratory pathogens causing lamb mortality in a poorly performing herd. After seven consecutive years of observed December recruitments of <10%, 13 adult female bighorn sheep from the remnant Gribbles Park herd in Colorado, US were captured and transported to the Thorne-Williams Wildlife Research Center in Wyoming in March 2013. Ewes were sampled repeatedly over 16 mo. In April 2014, ewes were separated into individual pens prior to lambing. Upon death, lambs were necropsied and tested for respiratory pathogens. Six lambs developed clinical respiratory disease and one lamb was abandoned. Pathology from an additional six lambs born in 2013 was also evaluated. Mycoplasma ovipneumoniae , leukotoxigenic Mannheimia spp., leukotoxigenic Bibersteinia trehalosi , and Pasteurella multocida all contributed to lamb pneumonia. Histopathology suggested a continuum of disease, with lesions typical of pasteurellosis predominating in younger lambs and lesions typical of mycoplasmosis predominating in older lambs. Mixed pathology was observed in lambs dying between these timeframes. We suspected that all the ewes in our study were persistently infected and chronically shedding the bacteria that contributed to summer lamb mortality.

Assessing respiratory pathogen communities in bighorn sheep populations: Sampling realities, challenges, and improvements.

Respiratory disease has been a persistent problem for the recovery of bighorn sheep (Ovis canadensis), but has uncertain etiology. The disease has been attributed to several bacterial pathogens including Mycoplasma ovipneumoniae and Pasteurellaceae pathogens belonging to the Mannheimia, Bibersteinia, and Pasteurella genera. We estimated detection probability for these pathogens using protocols with diagnostic tests offered by a fee-for-service laboratory and not offered by a fee-for-service laboratory. We conducted 2861 diagnostic tests on swab samples collected from 476 bighorn sheep captured across Montana and Wyoming to gain inferences regarding detection probability, pathogen prevalence, and the power of different sampling methodologies to detect pathogens in bighorn sheep populations. Estimated detection probability using fee-for-service protocols was less than 0.50 for all Pasteurellaceae and 0.73 for Mycoplasma ovipneumoniae. Non-fee-for-service Pasteurellaceae protocols had higher detection probabilities, but no single protocol increased detection probability of all Pasteurellaceae pathogens to greater than 0.50. At least one protocol resulted in an estimated detection probability of 0.80 for each pathogen except Mannheimia haemolytica, for which the highest detection probability was 0.45. In general, the power to detect Pasteurellaceae pathogens at low prevalence in populations was low unless many animals were sampled or replicate samples were collected per animal. Imperfect detection also resulted in low precision when estimating prevalence for any pathogen. Low and variable detection probabilities for respiratory pathogens using live-sampling protocols may lead to inaccurate conclusions regarding pathogen community dynamics and causes of bighorn sheep respiratory disease epizootics. We recommend that agencies collect multiples samples per animal for Pasteurellaceae detection, and one sample for Mycoplasma ovipneumoniae detection from at least 30 individuals to reliably detect both Pasteurellaceae and Mycoplasma ovipneumoniae at the population-level. Availability of PCR diagnostic tests to wildlife management agencies would improve the ability to reliably detect Pasteurellaceae in bighorn sheep populations.

Bighorn sheep (Ovis canadensis) sinus tumors are associated with coinfections by potentially pathogenic bacteria in the upper respiratory tract.

Bighorn sheep (Ovis canadensis) sinus tumors are hyperplastic to neoplastic, predominantly stromal masses of the paranasal sinuses that expand the sinus lining and obstruct the sinus cavities. Obstruction of the sinus cavities and disruption of normal sinus lining anatomy may interfere with clearance of bacterial pathogens from the upper respiratory tract. To examine this possibility, we explored whether the presence of sinus tumor features (tumor score) affected the likelihood of detecting potentially pathogenic bacteria from upper respiratory sinus lining tissues in bighorn sheep. We developed or used existing PCR assays for the detection of leukotoxigenic Pasteurellaceae and Mycoplasma ovipneumoniae in sinus lining tissues collected from 97 bighorn sheep in Colorado, US from 2009 to 2012. With the use of logistic regression analyses we found that tumor score was a good predictor of the probability of detecting potentially pathogenic bacteria in sinus lining tissues; we were more likely to detect potentially pathogenic bacteria from samples with high tumor scores. These findings add to our understanding of possible mechanisms for the maintenance and shedding of bacterial agents from the upper respiratory tracts of bighorn sheep.

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.

Phylogenetic and epidemiologic relationships among Pasteurellaceae from Colorado bighorn sheep herds.

We used 16S rRNA sequencing and leukotoxin gene (lktA) screening via PCR assay to clarify phylogenetic and epidemiologic relationships among Pasteurellaceae isolated from bighorn sheep (Ovis canadensis). Only six of 21 bighorn isolates identified as "Mannheimia haemolytica" in original laboratory reports appeared to be isolates of M. haemolytica sensu stricto based on 16S rRNA sequence comparisons; the remainder grouped with M. glucosida (n=8) or M. ruminalis (n=7). Similarly, 16S rRNA sequence comparisons grouped only 16 of 25 trehalose-fermenting bighorn isolates with reference strains of Bibersteinia trehalosi; nine other trehalose-fermenting bighorn isolates formed a clade divergent from B. trehalosi reference strains and may belong to another species. Of the 16 bighorn isolates identified as B. trehalosi by 16S rRNA sequences, only nine carried detectable lktA and thus seemed likely pathogens; none of the Bibersteinia clade isolates yielded detectable lktA despite reportedly showing ß hemolysis in culture. Our findings suggest that traditional metabolism-based methods for identifying Pasteurellaceae isolates lack sufficient accuracy and resolution for reliably discerning bacterial causes of respiratory disease in bighorn sheep. Consequently, these traditional methods should minimally be augmented by molecular techniques to improve epidemiologic relevance. Streamlined surveillance approaches focused primarily on detecting pathogenic Pasteurellaceae (e.g., M. haemolytica sensu stricto and lktA-positive B. trehalosi) and other select pathogens may be most informative for investigating and managing bighorn respiratory disease.