Alton C. S. Ward

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.

Teat skin normal flora and colonization with mastitis pathogen inhibitors

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial flora became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow teats with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.

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.

Characterization of Pasteurella spp isolated from healthy domestic pack goats and evaluation of the effects of a commercial Pasteurella vaccine.

OBJECTIVE To characterize Pasteurella spp isolated from healthy pack goats and evaluate the effects of administration of a commercial Pasteurella vaccine. ANIMALS 45 goats. PROCEDURE Pharyngeal swab specimens and blood samples were collected on day 0 before vaccination with a Pasteurella (Mannheimia) haemolytica serotype A1 bacterin. Samples were also collected from 17 goats on days 21 and 35. Isolated Pasteurella spp were assigned to biovariant groups on the basis of results of biochemical utilization tests and serotyped. Serum antibody titers were determined. RESULTS Multiple strains of Pasteurella spp were isolated from swab specimens and assigned to 30 nonhemolytic and 14 beta-hemolytic biovariant groups. The most common biovariant isolated was nonhemolytic P trehalosi belonging to group 2. This strain was isolated from 41 goats. Nonhemolytic P haemolytica strains were isolated from 31 goats, whereas beta-hemolytic strains of P trehalosi and P haemolytica were isolated from 8 and 35 goats, respectively. Vaccination with the A1 serotype did not affect the proportion of goats from which we isolated each biovariant group or the number of biovariant groups isolated. CONCLUSIONS AND CLINICAL RELEVANCE Multiple strains of P haemolytica and P trehalosi belonging to nonhemolytic and beta-hemolytic biovariant groups were isolated from the pharynx of healthy domestic pack goats. Because hemolytic activity correlates with leukotoxin production, beta-hemolytic strains may have a greater potential to cause disease in naive populations of wild ruminants. However, vaccination with an A1 serotype bacterin did not decrease the proportion of culture-positive goats.

Isolation of piliated Escherichia coli from diarrheic foals.

Escherichia coli was isolated from the feces and intestines of foals with and without diarrhea. Piliation of isolates was demonstrated by electron microscopy and agglutination in antisera having specificity for K88, K99, P987 and F41 pili. Piliation was also demonstrated by electron microscopy on organisms which did not react with any of the antisera.

Pasteurellaceae isolated from bighorn sheep (Ovis canadensis) from Idaho, Oregon, and Wyoming.

OBJECTIVE To elucidate the species and biovariants of Pasteurellaceae isolated from clinically normal bighorn sheep (Ovis canadensis) or bighorn sheep with evidence of respiratory disease. SAMPLE 675 Pasteurellaceae isolates from 290 free-ranging bighorn sheep in Idaho, Oregon and Wyoming. PROCEDURES Nasal and oropharyngeal swab specimens were inoculated onto selective and nonselective blood agar media. Representatives of each colony type were classified via a biovariant scheme. The association of respective β-hemolytic isolates with respiratory disease was evaluated via χ(2) analyses. RESULTS Bacterial isolates belonged to 4 species: Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia (Pasteurella) trehalosi. Within the latter 3 species, 112 subspecies, biotypes, and biovariants were identified. Bibersteinia trehalosi 2 and B trehalosi 2B constituted 345 of 675 (51%) isolates. Most (597/618 [97%]) isolates from adult sheep were from clinically normal animals, whereas most (47/57 [82%]) isolates from lambs were from animals with evidence of respiratory disease. Twenty-two Pasteurellaceae biovariants were isolated from sheep with respiratory disease; 17 of these biovariants were also isolated from clinically normal sheep. The ability of isolates to cause β-hemolysis on blood agar was associated with respiratory disease in adult bighorn sheep (OR, 2.59; 95% confidence interval, 1.10 to 6.07). CONCLUSIONS AND CLINICAL RELEVANCE Bighorn lambs appeared more susceptible to respiratory disease caused by Pasteurellaceae than did adult sheep. β-Hemolytic Pasteurellaceae isolates were more likely to be associated with respiratory disease than were non-β-hemolytic isolates in adult sheep. Identification of Pasteurellaceae with the greatest pathogenic potential will require studies to estimate the risk of disease from specific biovariants.

Habitat Quality in Association with Herd Health in Two Bighorn Sheep (Ovis canadensis canadensis) Populations

Multiple factors have been identified as elements of the respiratory disease complex in Rocky Mountain bighorn sheep (Ovis canadensis canadensis). We hypothesized that differences in health factors and Pasteurellaceae in the upper respiratory tracts could exist between populations of bighorn sheep with and without histories of pneumonic epizootics. We evaluated bighorn sheep in the Big Creek area of Central Idaho which had a history of respiratory disease, and hunter-harvested bighorn sheep from the Montana Spanish Peaks Range which had no history of respiratory disease. Factors evaluated included Pasteurellaceae, indicators of exposure to respiratory viruses, lungworm (Protostongylus spp.) loads, nutritional status and forage nutrient availability. Pasteurellaceae were isolated from both populations; however, significant differences (P < 0.01) in β-hemolysis and cytotoxicity were detected between isolates from the two populations; 64% of isolates cultured from Idaho were β-hemolytic and 38% of those produced >50% cytotoxicity. In contrast, 19% of the isolates from Montana were βhemolytic and none of those produced cytotoxicity >50%. Low serum antibody titers against bovine viral diarrhea, and bovine respiratory syncytial virus were detected in Idaho bighorn sheep following a pneumonic epizootic and in both herds against parainfluenza-3 virus. Low lungworm loads were detected in both herds. Forage nutrient values were similar for the two populations; however, the selenium level present in the Montana forage was significantly higher (P = 0.06) than that detected in the Idaho forage. It is concluded that variances in Pasteurellaceae virulence factors and dietary selenium, important in immune function, may be critical factors to consider when identifying conditions favoring a pneumonic epizootic.