Evaluating the transmission dynamics and host competency of aoudad (Ammotragus lervia) experimentally infected with Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae.

Feral populations of aoudad (Ammotragus lervia) occur in Texas bighorn sheep (Ovis canadensis) habitat and pose several conceptual ecological threats to bighorn sheep re-establishment efforts. The potential threat of disease transmission from aoudad to bighorn sheep may exacerbate these issues, but the host competency of aoudad and subsequent pathophysiology and transmissibility of pneumonic pathogens involved in the bighorn sheep respiratory disease complex is largely unknown. Because the largest population-limiting diseases of bighorn sheep involve pathogens causing bronchopneumonia, we evaluated the host competency of aoudad for Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae. Specifically, we described the shedding dynamics, pathogen carriage, seroconversion, clinical patterns, and pathological effects of experimental infection among wild aoudad held in captivity. We found that aoudad are competent hosts capable of maintaining and intraspecifically transmitting Mycoplasma ovipneumoniae and Pasteurellaceae and can shed the bacteria for 53 days after exposure. Aoudad developed limited clinical signs and pathological findings ranged from mild chronic lymphohistiocytic bronchointerstitial pneumonia to severe and acute suppurative pneumonia, similarly, observed in bighorn sheep infected with Mycoplasma spp. and Pasteurellaceae bacteria, respectively. Furthermore, as expected, clinical signs and lesions were often more severe in aoudad inoculated with a combination of Mycoplasma ovipneumoniae and Pasteurellaceae as compared to aoudad inoculated with only Mycoplasma ovipneumoniae. There may be evidence of interindividual susceptibility, pathogenicity, and/or transmissibility, indicated by individual aoudad maintaining varying severities of chronic infection who may be carriers continuously shedding pathogens. This is the first study to date to demonstrate that aoudad are a conceptual disease transmission threat to sympatric bighorn sheep populations due to their host competency and intraspecific transmission capabilities.

Antibiotic treatment of Mycoplasma ovipneumoniae in domestic sheep (Ovis aries): Working at the livestock-wildlife interface in Yukon, Canada.

Domestic sheep (Ovis aries) can carry the bacterium Mycoplasma ovipneumoniae (M. ovipneumoniae) in their upper respiratory tract, often with little effect on health and productivity. However, for bighorn sheep (Ovis canadensis) populations, there is a link between M. ovipneumoniae infection and pneumonia, poor lamb recruitment, and high fatality rate. Because of these outcomes, preventing transmission of M. ovipneumoniae to free-ranging wild sheep has garnered interest from both the livestock and wildlife sectors. We hypothesized that treatment with intranasal and systemic enrofloxacin would reduce the prevalence of M. ovipneumoniae-positive animals in a flock of domestic sheep. Initially, the prevalence decreased in the treated group; but by 34 d post-treatment, the number of M. ovipneumoniae-positive sheep returned to near pretreatment prevalence. Key clinical message: Test-and-slaughter is a method used to reduce the risk of transmission of pneumonia-causing M. ovipneumoniae from domestic sheep and goats to free-ranging wild sheep. In an effort to find an alternative, we used enrofloxacin to treat a flock of M. ovipneumoniae-positive domestic sheep; however, long-term reduction of M. ovipneumoniae prevalence in the flock was not achieved.

Traitement antibiotique de Mycoplasma ovipneumoniae chez le mouton domestique (Ovis aries): travail à l'interface bétail-faune au Yukon, Canada. Les moutons domestiques (Ovis aries) peuvent être porteurs de la bactérie Mycoplasma ovipneumoniae (M. ovipneumoniae) dans leurs voies respiratoires supérieures, avec souvent peu d'effets sur la santé et la productivité. Cependant, pour les populations de mouflons d'Amérique (Ovis canadensis), il existe un lien entre l'infection à M. ovipneumoniae et la pneumonie, un faible recrutement d'agneaux et un taux de mortalité élevé. En raison de ces résultats, la prévention de la transmission de M. ovipneumoniae aux moutons sauvages en liberté a suscité l'intérêt des secteurs de l'élevage et de la faune sauvage. Nous avons émis l'hypothèse qu'un traitement par enrofloxacine intranasale et systémique réduirait la prévalence d'animaux positifs à M. ovipneumoniae dans un troupeau de moutons domestiques. Initialement, la prévalence a diminué dans le groupe traité; mais 34 jours après le traitement, le nombre de moutons positifs à M. ovipneumoniae est revenu à une prévalence proche de celle précédant le traitement.Message clinique clé :L'essai et l'abattage sont une méthode utilisée pour réduire le risque de transmission de M. ovipneumoniae, responsable de la pneumonie, des moutons et chèvres domestiques aux moutons sauvages en liberté. Dans le but de trouver une alternative, nous avons utilisé l'enrofloxacine pour traiter un troupeau de moutons domestiques positifs à M. ovipneumoniae; cependant, aucune réduction à long terme de la prévalence de M. ovipneumoniae dans le troupeau n'a été obtenue.(Traduit par Dr Serge Messier).

Assessing shared respiratory pathogens between domestic (Ovis aries) and bighorn (Ovis canadensis) sheep; methods for multiplex PCR, amplicon sequencing, and bioinformatics to characterize respiratory flora.

Respiratory disease is responsible for dramatic population declines in bighorn sheep (Ovis canadensis), and respiratory pathogen diagnostics contribute to the management of bighorn populations. To create a comprehensive and consistent approach to bighorn sheep respiratory diagnostics, we created a culture-independent assay to detect and strain type Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae. The assay also detects and characterizes the Pasteurellaceae leukotoxin A gene, and broadly assesses the bacterial composition of each sample based on 16S rRNA sequences. The assay is based on a three-step approach: 1) Multiplex PCR to amplify targets including eight loci for each bacterial species, the Pasteurellaceae lktA gene, and the 16S rRNA gene 2) Library preparation, barcoding, and short-read Illumina sequencing to determine the genetic sequences of each target, and 3) Bioinformatics in the form of automated software to analyze genetic sequences. The assay was designed to assess shared pathogens between domestic and bighorn sheep, but could be useful for many applications in bighorn sheep respiratory disease research and management.

How sure are you? A web-based application to confront imperfect detection of respiratory pathogens in bighorn sheep.

The relationships between host-pathogen population dynamics in wildlife are poorly understood. An impediment to progress in understanding these relationships is imperfect detection of diagnostic tests used to detect pathogens. If ignored, imperfect detection precludes accurate assessment of pathogen presence and prevalence, foundational parameters for deciphering host-pathogen dynamics and disease etiology. Respiratory disease in bighorn sheep (Ovis canadensis) is a significant impediment to their conservation and restoration, and effective management requires a better understanding of the structure of the pathogen communities. Our primary objective was to develop an easy-to-use and accessible web-based Shiny application that estimates the probability (with associated uncertainty) that a respiratory pathogen is present in a herd and its prevalence given imperfect detection. Our application combines the best-available information on the probabilities of detection for various respiratory pathogen diagnostic protocols with a hierarchical Bayesian model of pathogen prevalence. We demonstrated this application using four examples of diagnostic tests from three herds of bighorn sheep in Montana. For instance, one population with no detections of Mycoplasma ovipneumoniae (PCR assay) still had an 6% probability of the pathogen being present in the herd. Similarly, the apparent prevalence (0.32) of M. ovipneumoniae in another herd was a substantial underestimate of estimated true prevalence (0.46: 95% CI = [0.25, 0.71]). The negative bias of naïve prevalence increased as the probability of detection of testing protocols worsened such that the apparent prevalence of Mannheimia haemolytica (culture assay) in a herd (0.24) was less than one third that of estimated true prevalence (0.78: 95% CI = [0.43, 0.99]). We found a small difference in the estimates of the probability that Mannheimia spp. (culture assay) was present in one herd between the binomial sampling approach (0.24) and the hypergeometric approach (0.22). Ignoring the implications of imperfect detection and sampling variation for assessing pathogen communities in bighorn sheep can result in spurious inference on pathogen presence and prevalence, and potentially poorly informed management decisions. Our Shiny application makes the rigorous assessment of pathogen presence, prevalence and uncertainty straightforward, and we suggest it should be incorporated into a new paradigm of disease monitoring.

Bighorn sheep gut microbiomes associate with genetic and spatial structure across a metapopulation.

Studies in laboratory animals demonstrate important relationships between environment, host traits, and microbiome composition. However, host-microbiome relationships in natural systems are understudied. Here, we investigate metapopulation-scale microbiome variation in a wild mammalian host, the desert bighorn sheep (Ovis canadensis nelsoni). We sought to identify over-represented microbial clades and understand how landscape variables and host traits influence microbiome composition across the host metapopulation. To address these questions, we performed 16S sequencing on fecal DNA samples from thirty-nine bighorn sheep across seven loosely connected populations in the Mojave Desert and assessed relationships between microbiome composition, environmental variation, geographic distribution, and microsatellite-derived host population structure and heterozygosity. We first used a phylogenetically-informed algorithm to identify bacterial clades conserved across the metapopulation. Members of genus Ruminococcaceae, genus Lachnospiraceae, and family Christensenellaceae R7 group were among the clades over-represented across the metapopulation, consistent with their known roles as rumen symbionts in domestic livestock. Additionally, compositional variation among hosts correlated with individual-level geographic and genetic structure, and with population-level differences in genetic heterozygosity. This study identifies microbiome community variation across a mammalian metapopulation, potentially associated with genetic and geographic population structure. Our results imply that microbiome composition may diverge in accordance with landscape-scale environmental and host population characteristics.

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.

Exposure of bighorn sheep to domestic goats colonized with Mycoplasma ovipneumoniae induces sub-lethal pneumonia.

BACKGROUND: Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis) that has been associated with contact with domestic Caprinae. The disease is polymicrobial but is initiated by Mycoplasma ovipneumoniae, which is commonly carried by both domestic sheep (O. aries) and goats (Capra aegagrus hircus). However, while previous bighorn sheep comingling studies with domestic sheep have resulted in nearly 100% pneumonia mortality, only sporadic occurrence of fatal pneumonia was reported from previous comingling studies with domestic goats. Here, we evaluated the ability of domestic goats of defined M. ovipneumoniae carriage status to induce pneumonia in comingled bighorn sheep. METHODOLOGY/PRINCIPAL FINDINGS: In experiment 1, three bighorn sheep naïve to M. ovipneumoniae developed non-fatal respiratory disease (coughing, nasal discharge) following comingling with three naturally M. ovipneumoniae-colonized domestic goats. Gross and histological lesions of pneumonia, limited to small areas on the ventral and lateral edges of the anterior and middle lung lobes, were observed at necropsies conducted at the end of the experiment. A control group of three bighorn sheep from the same source housed in isolation during experiment 1 remained free of observed respiratory disease. In experiment 2, three bighorn sheep remained free of observed respiratory disease while comingled with three M. ovipneumoniae-free domestic goats. In experiment 3, introduction of a domestic goat-origin strain of M. ovipneumoniae to the same comingled goats and bighorn sheep used in experiment 2 resulted in clinical signs of respiratory disease (coughing, nasal discharge) in both host species. At the end of experiment 3, gross and histological evidence of pneumonia similar to that observed in experiment 1 bighorn sheep was observed in both affected bighorn sheep and domestic goats. CONCLUSIONS/SIGNIFICANCE: M. ovipneumoniae strains carried by domestic goats were transmitted to comingled bighorn sheep, triggering development of pneumonia. However, the severity of the disease was markedly milder than that seen in similar experiments with domestic sheep strains of the bacterium.

Myocardial Necrosis Associated with Clostridium novyi Infection in a Bighorn Sheep ( Ovis canadensis ).

We describe a case of myocardial emphysema and necrosis in a bighorn sheep ( Ovis canadensis ), associated with infection by Clostridium novyi , diagnosed through necropsy, histopathology, and fluorescent antibody testing. We documented rapid onset of disease in an apparently healthy wild sheep and discuss our findings in the context of reported clostridial infections in humans, domestic animals, and wildlife.

Contact and contagion: Probability of transmission given contact varies with demographic state in bighorn sheep.

Understanding both contact and probability of transmission given contact are key to managing wildlife disease. However, wildlife disease research tends to focus on contact heterogeneity, in part because the probability of transmission given contact is notoriously difficult to measure. Here, we present a first step towards empirically investigating the probability of transmission given contact in free-ranging wildlife. We used measured contact networks to test whether bighorn sheep demographic states vary systematically in infectiousness or susceptibility to Mycoplasma ovipneumoniae, an agent responsible for bighorn sheep pneumonia. We built covariates using contact network metrics, demographic information and infection status, and used logistic regression to relate those covariates to lamb survival. The covariate set contained degree, a classic network metric describing node centrality, but also included covariates breaking the network metrics into subsets that differentiated between contacts with yearlings, ewes with lambs, and ewes without lambs, and animals with and without active infections. Yearlings, ewes with lambs, and ewes without lambs showed similar group membership patterns, but direct interactions involving touch occurred at a rate two orders of magnitude higher between lambs and reproductive ewes than between any classes of adults or yearlings, and one order of magnitude higher than direct interactions between multiple lambs. Although yearlings and non-reproductive bighorn ewes regularly carried M. ovipneumoniae, our models suggest that a contact with an infected reproductive ewe had approximately five times the odds of producing a lamb mortality event of an identical contact with an infected dry ewe or yearling. Consequently, management actions targeting infected animals might lead to unnecessary removal of young animals that carry pathogens but rarely transmit. This analysis demonstrates a simple logistic regression approach for testing a priori hypotheses about variation in the odds of transmission given contact for free-ranging hosts, and may be broadly applicable for investigations in wildlife disease ecology.

Potential disease agents in domestic goats and relevance to bighorn sheep (Ovis canadensis) management.

Domestic goats are raised for meat, milk and hair production, in herds for rangeland weed control, and as pack animals. Domestic sheep, goats and wild bighorn sheep are all susceptible to a multifactorial pneumonia. We sampled 43 herd goats from 7 herds and 48 pack goats from 11 herds for viral and bacterial serology, parasitology, and Pasteurellaceae microbiology. The goats in this study were in generally good health, although most goats did harbor various pathogens and parasites including several bacteria, specifically Pasteurellaceae, which have been associated with pneumonia in free-ranging bighorn sheep. It is not known if domestic goats can transmit the Pasteurellaceae or other pathogens found in this study readily to wild bighorn sheep. However, due the possibility of transmission, domestic goats in areas in or near bighorn sheep habitat should be managed to minimize the risk of spreading disease agents to bighorn sheep.

Comparison of Two Bacterial Transport Media for Culture of Tonsilar Swabs from Bighorn Sheep ( Ovis canadensis ) and Mountain Goats ( Oreamnos americanus ).

Duplicate tonsilar swabs were collected from 77 bighorn sheep ( Ovis canadensis ) and 19 mountain goats ( Oreamnos americanus ) in Utah. Swabs were refrigerated in bacterial transport medium or frozen in cryopreservation medium prior to bacteriologic culture. The cryopreservation medium yielded comparable or superior bacterial growth while permitting more flexibility in specimen shipment to the laboratory.

A chimeric protein comprising the immunogenic domains of Mannheimia haemolytica leukotoxin and outer membrane protein PlpE induces antibodies against leukotoxin and PlpE.

Mannheimia haemolytica is a very important pathogen of pneumonia in ruminants. Bighorn sheep (BHS, Ovis canadensis) are highly susceptible to M. haemolytica-caused pneumonia which has significantly contributed to the drastic decline of bighorn sheep population in North America. Pneumonia outbreaks in wild BHS can cause mortality as high as 90%. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, an experimental vaccine containing leukotoxin and surface antigens of M. haemolytica developed by us induced 100% protection of BHS, but required multiple booster injections. Vaccination of wild BHS is difficult. But they can be vaccinated at the time of transplantation into a new habitat. Administration of booster doses, however, is impossible. Therefore, a vaccine that does not require booster doses is necessary to immunize BHS against M. haemolytica pneumonia. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation. As the first step towards developing a herpesvirus-vectored vaccine, we constructed a chimeric protein comprising the leukotoxin-neutralizing epitopes and the immuno-dominant epitopes of the outer membrane protein PlpE. The chimeric protein was efficiently expressed in primary BHS lung cells. The immunogenicity of the chimeric protein was evaluated in mice before inoculating BHS. Mice immunized with the chimeric protein developed antibodies against M. haemolytica leukotoxin and PlpE. More importantly, the anti-leukotoxin antibodies effectively neutralized leukotoxin-induced cytotoxicity. Taken together, these results represent the successful completion of the first step towards developing a herpesvirus-vectored vaccine for controlling M. haemolytica pneumonia in BHS, and possibly other ruminants.

Fusobacterium necrophorum in North American Bighorn Sheep ( Ovis canadensis ) Pneumonia.

Fusobacterium necrophorum has been detected in pneumonic bighorn sheep (BHS; Ovis canadensis ) lungs, in addition to the aerobic respiratory pathogens Mannheimia haemolytica , Bibersteinia trehalosi , Pasteurella multocida , and Mycoplasma ovipneumoniae . Similar to M. haemolytica , F. necrophorum produces a leukotoxin. Leukotoxin-induced lysis and degranulation of polymorphonuclear leukocytes (PMNs) and macrophages are responsible for acute inflammation and lung tissue damage characteristic of M. haemolytica -caused pneumonia. As one approach in elucidating the role of F. necrophorum in BHS pneumonia, we determined the frequency of the presence of F. necrophorum in archived pneumonic BHS lung tissues, and susceptibility of BHS leukocytes to F. necrophorum leukotoxin. A species-specific PCR assay detected F. necrophorum in 37% of pneumonic BHS lung tissues (total tested n=70). Sequences of PCR amplicons were similar to the less virulent F. necrophorum subsp. funduliforme. Fusobacterium necrophorum leukotoxin exhibited cytotoxicity to BHS PMNs and peripheral blood mononuclear cells. As with the M. haemolytica leukotoxin, F. necrophorum leukotoxin was more toxic to BHS PMNs than domestic sheep PMNs. It is likely that F. necrophorum enters the lungs after M. haemolytica and other aerobic respiratory pathogens enter the lungs and initiate tissue damage, thereby creating a microenvironment that is conducive for anaerobic bacterial growth. In summary, Fusobacterium leukotoxin is highly toxic for BHS leukocytes; however, based on the PCR findings, it is unlikely to play a direct role in the development of BHS pneumonia.

Costs and benefits of group living with disease: a case study of pneumonia in bighorn lambs (Ovis canadensis).

Group living facilitates pathogen transmission among social hosts, yet temporally stable host social organizations can actually limit transmission of some pathogens. When there are few between-subpopulation contacts for the duration of a disease event, transmission becomes localized to subpopulations. The number of per capita infectious contacts approaches the subpopulation size as pathogen infectiousness increases. Here, we illustrate that this is the case during epidemics of highly infectious pneumonia in bighorn lambs (Ovis canadensis). We classified individually marked bighorn ewes into disjoint seasonal subpopulations, and decomposed the variance in lamb survival to weaning into components associated with individual ewes, subpopulations, populations and years. During epidemics, lamb survival varied substantially more between ewe-subpopulations than across populations or years, suggesting localized pathogen transmission. This pattern of lamb survival was not observed during years when disease was absent. Additionally, group sizes in ewe-subpopulations were independent of population size, but the number of ewe-subpopulations increased with population size. Consequently, although one might reasonably assume that force of infection for this highly communicable disease scales with population size, in fact, host social behaviour modulates transmission such that disease is frequency-dependent within populations, and some groups remain protected during epidemic events.

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.

Free-ranging Rocky Mountain bighorn sheep and an outbreak of inflammatory bowel disease along the Clark Fork River in Plains, Montana.

Nine individuals with ulcerative colitis or Crohn disease grew up or lived in Plains, Montana, a 1,200-person community adjacent to the Clark Fork River near herds of free ranging Rocky Mountain bighorn sheep. This inflammatory bowel disease outbreak is similar to others that have occurred along rivers contaminated by animal feces.

Proximity-dependent inhibition of growth of Mannheimia haemolytica by Pasteurella multocida.

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 µm) but not when they were separated by a membrane that limited contact (pore size, 0.4 µm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.

Evaluation of management treatments intended to increase lamb recruitment in a bighorn sheep herd.

We administered a suite of treatments to a herd of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) that was experiencing poor lamb recruitment and showing signs of respiratory disease. Despite 3 yr of treatment with various combinations of anthelmentics, antibiotics, vaccines, and hyperimmune serum products, recruitment was not improved.

Detection of Mycoplasma ovipneumoniae and M. arginini in bighorn sheep using enrichment culture coupled with genus- and species-specific polymerase chain reaction.

Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul™ tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.