Glen C. Weiser

Association of Mycoplasma ovipneumoniae Infection with Population-Limiting Respiratory Disease in Free-Ranging Rocky Mountain Bighorn Sheep (Ovis canadensis canadensis)

ABSTRACT Bronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniae was detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniae in the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniae was isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniae PCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P < 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniae antibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P < 0.001]). M. ovipneumoniae is strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.

Bibersteinia trehalosi Inhibits the Growth of Mannheimia haemolytica by a Proximity-Dependent Mechanism

ABSTRACT Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time (∼10 min versus ∼27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-μm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 μm that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.

CHARACTERIZATION OF PASTEURELLA MULTOCIDA ASSOCIATED WITH PNEUMONIA IN BIGHORN SHEEP

Pasteurella multocida is a highly diverse group of bacteria recognized as important pathogens. Although P. multocida is not ordinarily associated with disease in Rocky Mountain bighorn sheep (Ovis canadensis canadensis), numerous isolates were cultured in high numbers from free-ranging bighorn sheep in the Hells Canyon area of Idaho, Washington, and Oregon (USA) during the winter of 1995–96. Animals captured in Hells Canyon and held in captivity, and their offspring, also harbored P. multocida. Biochemical utilization tests on 90 isolates identified three subspecies: P. multocida multocida a (n=54); P. multocida multocida b (n=13); and P. multocida gallicida (n=15); and a non-speciated biotype, U6 (n=8). Genomic DNA digestion with restriction endonuclease Hha I separated the isolates into 62 unique restriction fragment length polymorphism profiles. Capsular type A was predominant (72% of isolates). Only one isolate type, which may have been transmitted from a feral goat, was capsular type D, possessed the structural gene, toxA, for dermonecrotoxin detected by polymerase chain reaction, and produced toxin as determined by monoclonal antibody immunoblot. In conclusion, bighorn sheep in this study carried diverse types of generally non-toxigenic P. multocida associated with epizootic pneumonia.

MICROORGANISMS ASSOCIATED WITH A PNEUMONIC EPIZOOTIC IN ROCKY MOUNTAIN BIGHORN SHEEP (OVIS CANADENSIS CANADENSIS)

Abstract A comprehensive study of a pneumonic epizootic was initiated when the first signs of disease were noted in a metapopulation of bighorn sheep inhabiting Hells Canyon, bordering Idaho, Oregon, and Washington. A total of 92 bighorn sheep were tested for etiologic agents during the following 6-mo study period. The study population included bighorn sheep believed to be the subpopulation in which disease was first noted, and these sheep were translocated to a holding facility in an effort to contain the disease (group A1, n = 72); bighorn sheep in other subpopulations (group A2) with evidence of clinical disease were captured, sampled, given antibiotics, and released (n = 8) and those that were found dead were necropsied (n = 12). Samples, including oropharyngeal and nasal swabs, and lung and liver tissue were collected from the bighorn sheep identified above. Tissue was collected at necropsy from 60 group A1 bighorn sheep that died following translocation, and samples were cultured for bacteria and viruses. Blood samples were tested for antibodies against known respiratory viruses, and histopathology was conducted on tissue samples. The major cause of death in both group A1 and group A2 bighorn sheep was a rapidly developing fibrinous bronchopneumonia. Multiple biovariants of Pasteurella were isolated from oropharyngeal and nasal samples from both groups, and Mycoplasma ovipneumonia was isolated from five group A1 oropharyngeal samples. Organisms isolated from lung tissue included Pasteurella multocida multocida a and Pasteurella trehalosi, both of which differentiated into multiple strains by restriction enzyme analysis, and parainfluenza-3 virus (PI-3). Paired serum samples revealed >fourfold increases in titers against PI-3 and bovine respiratory syncytial viruses. It was concluded that this epizootic resulted from a complex of factors including multiple potential respiratory pathogens, none of which were identified as a primary pathogen, and possible stress factors.

MICROBIAL DIVERSITY IN BIGHORN SHEEP REVEALED BY CULTURE-INDEPENDENT METHODS

We investigated the effectiveness of culture-independent molecular methods for determining host-associated microbial diversity in bighorn sheep (Ovis canadensis). Results from bacterial culture attempts have been the primary source of information on host-associated bacteria, but studies have shown that culture-based results significantly underestimate bacterial diversity in biological samples. To test the effectiveness of culture-independent methods, we extracted DNA from nasal and oropharyngeal swab samples collected from bighorn sheep in four different populations. From these samples, we amplified, cloned, and sequenced small subunit (16S) ribosomal DNA (rDNA) to identify the scope of microbial diversity in bighorn respiratory tracts. Phylogenetic analysis of these rDNA gene sequences revealed organismal diversity an order of magnitude higher than was determined by culture methods. Pasteurellaceae bacteria were the most diverse phylogenetic group in live bighorn sheep, and members of bacterial genera often associated with respiratory disease were found in all the samples. Culture-independent methods were also able to directly detect leukotoxin (lktA) gene sequences in swab and lung tissue samples. Overall, our results show the power of culture-independent molecular methods for identifying microbial diversity in bighorn sheep and the potential for these methods to detect the presence of virulence genes in biological samples.

An ecologic study comparing distribution of Pasteurella trehalosi and Mannheimia haemolytica between Sierra Nevada bighorn sheep, White Mountain bighorn sheep, and domestic sheep.

The prevalence and phenotypic variability of Pasteurella and Mannheimia isolates from Sierra Nevada bighorn sheep (Ovis canadensis sierrae), White Mountain bighorn sheep (Ovis canadensis nelsoni), and domestic sheep (Ovis aries) from California, USA, were compared. The White Mountain bighorn sheep population had a recent history of pneumonia-associated mortality, whereas the Sierra Nevada bighorn sheep population had no recent history of pneumonia-associated mortality. The domestic sheep flocks were pastured in areas geographically near both populations but were not known to have direct contact with either bighorn sheep population. Oropharyngeal swab samples were collected from healthy domestic and bighorn sheep and cultured to characterize bacterial species, hemolysis, biogroups, and biovariants. Pasteurella trehalosi and Mannheimia haemolytica were detected in all of the study populations, but the relative proportion of each bacterial species differed among sheep populations. Pasteurella trehalosi was more common than M. haemolytica in the bighorn sheep populations, whereas the opposite was true in domestic sheep. Mannheimia haemolytica was separated into 11 biogroups, and P. trehalosi was characterized into two biogroups. Biogroup distributions for M. haemolytica and P. trehalosi differed among the three populations; however, no difference was detected for the distribution of P. trehalosi biogroups between the Sierra Nevada bighorn sheep and domestic sheep. The prevalence odds ratios (pOR) for the distribution of M. haemolytica biogroups suggested little difference between White Mountain bighorn sheep and domestic sheep compared with Sierra Nevada bighorn sheep and domestic sheep, although these comparisons had relatively large confidence intervals for the point estimates. Hemolytic activity of the isolates was not different among the sheep populations for M. haemolytica but was different for P. trehalosi. No clear evidence of association was found in the Pasteurellaceae distribution between the White Mountains bighorn sheep and domestic sheep.

A Review of Hypothesized Determinants Associated with Bighorn Sheep (Ovis canadensis) Die-Offs

Multiple determinants have been hypothesized to cause or favor disease outbreaks among free-ranging bighorn sheep (Ovis canadensis) populations. This paper considered direct and indirect causes of mortality, as well as potential interactions among proposed environmental, host, and agent determinants of disease. A clear, invariant relationship between a single agent and field outbreaks has not yet been documented, in part due to methodological limitations and practical challenges associated with developing rigorous study designs. Therefore, although there is a need to develop predictive models for outbreaks and validated mitigation strategies, uncertainty remains as to whether outbreaks are due to endemic or recently introduced agents. Consequently, absence of established and universal explanations for outbreaks contributes to conflict among wildlife and livestock stakeholders over land use and management practices. This example illustrates the challenge of developing comprehensive models for understanding and managing wildlife diseases in complex biological and sociological environments.

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 CO2-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-CulTM tubes, cryoprotectant in liquid N2 and Fisher Transport System) gave similar results under our study conditions.

Variation in Pasteurella (Bibersteinia) and Mannheimia spp. Following Transport and Antibiotic Treatment in Free-Ranging and Captive Rocky Mountain Bighorn Sheep (Ovis canadensis canadensis)

Abstract Morbidity and mortality associated with respiratory disease following capture and translocation of bighorn sheep (Ovis canadensis canadensis) is a significant concern, particularly when establishing new or augmenting existing bighorn populations. Administration of prophylactic antibiotics at the time of capture is often done to minimize the risk of respiratory disease, but the efficacy of this practice is unknown. The effects of oxytetracycline and florfenicol on the Pasteurella (Bibersteinia) and Mannheimia spp. isolated from samples collected from the oropharynx at the time of capture and 3 or 42 day later were evaluated in two groups of bighorn sheep. The most evident change in the isolation rates or types of Pasteurella (Bibersteinia) spp., Mannheimia spp., or both was an increase of β-hemolytic strains isolated from bighorn sheep 3 day following oxytetracycline treatment. Both groups of bighorn sheep carried Pasteurella (Bibersteinia) trehalosi identified as the same biovariants, but they did not share biovariants of Mannheimia spp. No animals had signs of respiratory disease. Isolates representative of all biovariants present in cultures from the two bighorn sheep groups were sensitive to in vitro tests to both oxytetracycline and florfenicol and the majority were also sensitive to seven other antibiotics tested. The administration of neither oxytetracycline nor florfenicol eliminated Pasteurella (Bibersteinia) or Mannheimia from the oropharyngeal mucosa. Resistance to either antibiotic used in these animals was not noted. Although the prophylactic benefits of these drugs in preventing disease are uncertain, therapeutic levels of antibiotics in lung tissue during times of stress may reduce the risk of disease. Representative sampling of the oropharyngeal microflora of bighorn sheep source and recipient populations prior to being intermingled should be considered as one of the tools to minimize exposure of naïve populations to potentially pathogenic bacteria.

Development of a multilocus sequence typing (MLST) scheme for Mannheimia haemolytica and assessment of the population structure of isolates obtained from cattle and sheep.

Mannheimia haemolytica is an important veterinary pathogen affecting cattle and sheep. Previous typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. Multilocus sequence typing (MLST) has now almost replaced MLEE and is a definitive and portable typing method, allowing global data exchange. The purpose of this study was to develop a MLST scheme for M. haemolytica. A collection of isolates from 10 countries, including the type strain and reference strains for all recognized serotypes were included in the study. Partial sequences of the housekeeping genes adk, aroE, deoD, gapDH, gnd, mdh and zwf were used to define the MLST scheme. The 95 isolates demonstrated 34 different sequence types (ST) of which 19 were connected in three clonal complexes (CC). ST1 constituted more than one-third of the isolates and was most frequently demonstrated among isolates from bovine sources. The analysis indicated a common evolutionary origin of 33 isolates from the French alps, collected from domestic and wild animals and demonstrating several related STs. An analysis of 17 isolates from the USA demonstrated the same ST in 14 of the isolates. In conclusion, an unambiguous typing scheme is presented for M. haemolytica and results obtained confirm previous observations of a clonal population of the organism.