Isolation of Pasteurella haemolytica from tonsillar biopsies of Rocky Mountain bighorn sheep

Isolations of Pasteurella haemolytica were compared from tonsillar biopsies versus nasal passages for 29 free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from central Idaho. Overall, P. haemolytica was isolated from 11 (38%) of 29 sheep. Two (18%) of the 11 positive samples were from only nasal passages compared to eight (73%) from tonsillar biopsies. Pasteurella haemolytica biotype T was isolated from tonsils of nine sheep and from nasal biopsies. Pasteurella haemolytica biotype T was isolated from tonsils of nine sheep and from nasal passages of only one sheep. Two sheep were positive for P. haemolytica biotype A from nasal passages. Culturing tonsillar biopsies as compared to nasal swab samples was a more reliable technique in detecting P. haemolytica, especially biotype T, in bighorn sheep.     

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.     

Isolation of a chlamydial agent from Rocky Mountain bighorn sheep

A total of 53 clinical specimens from both healthy and diseased Rocky Mountain bighorn sheep (Ovis canadensis) were examined for Chlamydia. An agent consistently lethal for chicken embryos was recovered from a nasal swab taken from a normal ewe. This agent, designated BHS-15, possesses antigens which fix complement in the presence of anti-chlamydial serum, is susceptible to chlortetracycline, and is resistant to sodium sulfadiazine and streptomycin. Attempts to culture the isolate in quality control media, including blood agar, thioglycolate broth, and PPLO broth and agar were unsuccessful. A recommendation is made for classification of agent BHS-15 as a member of the species Chlamydia psittaci. The possible relationship of the isolate to the pneumonia complex in bighorn sheep is discussed.     

Detecting nonhemolytic Pasteurella haemolytica infections in healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis): influences of sample site and handling

Effects of sampling procedures on ability to culture Pasteurella spp. from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were examined experimentally. Sample site influenced (P less than 0.0001) recovery of P. haemolytica in adult bighorn sheep. We isolated nonhemolytic P. haemolytica from 18 of 19 tonsillar swabs and 18 of 19 tonsillar biopsies from adult sheep, yet only four of 19 nasal swabs yielded isolates. Sample handling also affected (P less than 0.0001) recovery of P. haemolytica. Nonhemolytic P. haemolytica was cultured from 14 of 19 tonsillar swabs plated directly onto blood agar, but from only two of 19 swabs stored for 24 hr in modified Stuart's medium. We detected nonhemolytic P. haemolytica at least once in bronchial aspirates from four and in nasal swabs from three of six bighorn lambs. Based on direct cultures of tonsillar swabs and/or biopsies, all 26 bighorn sheep (seven lambs, 19 adults) sampled were infected with nonhemolytic P. haemolytica; only two lambs developed pneumonia during the study period. Thirty-four of 37 nonhemolytic P. haemolytica isolates tested were biotype T; three were biotype A. Serotypes 3; 4; 3, 4 and 3, 4, 10 were identified in a subsample of 17 isolates. Our data suggest tonsillar swabs or biopsies plated directly onto blood agar and incubated immediately offer the greatest probability of recovering nonhemolytic P. haemolytica from health bighorn sheep.     

Toxoplasmic encephalitis in a free-ranging Rocky Mountain bighorn sheep from Washington

A 4-mo-old free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from the Hells Canyon area (Washington, USA) was diagnosed with encephalitis associated with Toxoplasma gondii infection. The sheep had concurrent pneumonic pasteurellosis and resided in a geographic area with endemic Pasteurella-associated pneumonia and mortality in bighorn sheep. The brain had multifocal necrotizing and nonsuppurative encephalitis with intralesional protozoa. The protozoa were identified as T. gondii by immunohistochemistry. To our knowledge, this is the first report of T. gondii infection in a Rocky Mountain bighorn sheep.     

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.     

Reproductive costs of sons and daughters in Rocky Mountain bighorn sheep

Differential maternal investment theory predicts that in sexuallydimorphic and polygynous species mothers should invest morein sons than in daughters. We tested the hypothesis that bighornewes that raise sons incur greater reproductive costs than ewesthat raise daughters. Although ewe mass gain during lactationand subsequent winter body mass loss were independent of lambsex, lambs born the year following the weaning of a son hadlower survival than lambs born after a daughter. The effectsof lamb sex on subsequent reproductive success of ewes becamemore evident at high population density. Lamb sex did not affectmaternal survival. Population density, weather, and ewe agedid not alter the relationship between lamb sex and subsequentreproductive success of the ewe. The year after weaning a son,ewes were more likely to have a daughter than a son, while ewesthat had previously weaned a daughter had similar numbers ofsons and daughters. Our results show that for bighorn sheepewes, sons have a greater life-history cost than daughters,suggesting a differential maternal investment in the sexes.     

Comparison of pulmonary defense mechanisms in Rocky Mountain bighorn (Ovis canadensis canadensis) and domestic sheep

Alveolar macrophages were obtained from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and domestic sheep for the purpose of comparing pulmonary host defense mechanisms in the two species. Specific variables studied included (1) characterization of the cell types present in the lung, (2) alveolar macrophage phagocytic and bactericidal functions, (3) measurement of protein levels in lavage fluid, and (4) measurement of cortisol levels in lavage fluid. While phagocytic cell populations were similar between bighorn and domestic sheep, a significantly higher percentage of lymphocytes were present in bighorns than domestics (20% in bighorn versus 6% in domestic sheep). Significant differences were not observed in the phagocytic or bactericidal functions of macrophages between the two species. Significant differences were not observed in either lavage fluid protein levels or in cortisol levels.     

Psoroptic scabies in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Wyoming

Thirteen Rocky Mountain bighorn sheep (Ovis canadensis canadensis) with clinical signs of psoroptic scabies were captured in Wyoming. Signs included droopy ears, depilation on the head and neck, and exudate in the ears. Mites were identified as either Psoroptes cervinus or P. equi. Two ewes with scabies at the time of original capture had no clinical signs of mite infection 1 and 2 yr later.     

Seroprevalence of Toxoplasma gondii in Rocky Mountain bighorn sheep (Ovis canadensis)

Serum samples from 697 Rocky Mountain bighorn sheep (Ovis canadensis) from North America were examined for antibodies to Toxoplasma gondii by the modified agglutination test incorporating mercaptoethanol and formalin-fixed tachyzoites. Antibodies to T. gondii were found in 25 of 697 (3.6%) sheep in titers of 1:25 (8 sheep), 1:50 (4 sheep), 1:100 (7 sheep), 1:200 (1 sheep), 1:400 (1 sheep), 1:800 (1 sheep), and 1:1,600 (3 sheep). This is the first record of T. gondii exposure in bighorn sheep.     

Using ribosomal RNA gene restriction patterns in distinguishing isolates of Pasteurella haemolytica from bighorn sheep (Ovis canadensis).

Pasteurella haemolytica isolates (n = 31) from two isolated captive herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were characterized and compared phenotypically (biotype, serotype, hemolytic activity) and by a genomic fingerprinting method known as ribotyping. Seven to nine distinct phenotypes were observed. Depending on the method used for serotyping, one to three phenotypes were common to both herds. Eighteen isolates, recovered from both herds, were non-hemolytic, biotype T, indirect hemagglutination assay serotype 4. Ribotyping, a method for highlighting genetically conserved deoxyribonucleic acid restriction site heterogeneity with a 32P-labelled Escherichia coli ribosomal ribonucleic acid probe, produced six to eight distinct ribotype pattern groups within the 31 P. haemolytica isolates, depending on the restriction enzyme used. In contrast to phenotypes, ribotypes appeared unique to each herd, and ribotyping helped to further differentiate some isolates of the same biotype and serotype. In addition, ribotyping provided an alternative means for evaluating relationships between isolates differing in hemolytic activity but which were otherwise phenotypically identical. We propose that ribotyping may be a useful adjunct to other bacterial characterization methods in studying the epizootiology of pasteurellosis in bighorn sheep.     

Freemartinism in a captive herd of Rocky Mountain bighorn sheep (Ovis canadensis).

Freemartinism in two animals from a captive herd of Rocky Mountain bighorn sheep (Ovis canadensis) at the Denver Zoological Gardens (Denver, Colorado, USA) is described. A young ewe had female external genitalia, a masculine appearance, and demonstrated male behavior as she matured. Another ewe with female external genitalia died as a yearling. Necropsy revealed a non-patent vagina and internal male genitalia. Both females were chimeric with karyotypes containing XX and XY sex chromosomes.