Differential pulmonary immunopathology of domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) with Mycoplasma ovipneumoniae infection: A retrospective study

Mycoplasma ovipneumoniae is a respiratory pathogen that impacts domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). BHS are reported to be more susceptible than DS to developing polymicrobial pneumonia associated with M. ovipneumoniae infection. Using formalin-fixed paraffin-embedded tissues, we performed a retrospective study investigating the pulmonary immune response of DS and BHS to M. ovipneumoniae infection. M. ovipneumoniae infected DS exhibited a more robust and well-organized BALT formation as compared to BHS. Digital analysis of immunohistochemical chromogen deposition in lung tissue was used to quantitate T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. A significant interaction of species and infection status was identified for CD3, CD163, and IL-17. BHS had a greater increase in bronchiolar CD3 and bronchiolar and alveolar CD163 with infection, as compared to DS. BHS had an increase in bronchiolar associated lymph tissue (BALT) and alveolar IL-17 with infection, while these remained similar in DS regardless of infection status. IL-17 in respiratory epithelium of bronchi and bronchioles comparatively decreased in DS and increased in BHS with infection. These data begin to define the interspecies differential immune response to pulmonary M. ovipneumoniae infection in DS and BHS and provide the first investigations of respiratory epithelium-associated IL-17 in ovine.     

Respiratory infections in housed sheep,with particular reference to mycoplasmas

A commercial housed flock with an annual occurrence of pneumonia was investigated. The organism most commonly isolated from the respiratory tract of lambs up to 6 months old was Mycoplasma ovipneumoniae. Mycoplasma arginini, Mycoplasma conjunctivae, Acholeplasma laidlawii, ureaplasmas and Pasteurella haemolytica biotype A were also isolated: viruses were not isolated, but infection with parainfluenza virus type 3 (P13) was indicated by serology. Colostrum derived antibodies to M. ovipneumoniae and M. arginini declined to minimum levels by 50 days. The development of active immunity to mycoplasmas and P13 virus was associated with an increased incidence of clinical respiratory disease. Histopathological examination of the lungs from 34 lambs showed that 15 had lesions of a proliferative exudative (P.E.) pneumonia, a further 11 showed lymphoid hyperplasia sometimes associated with interstitial thickening, and eight showed no significant pathological changes. Isolations of M. ovipneumoniae were highest from animals with P.E. pneumonia, while M. arginini did not appear to be associated with any specific lung changes. P. haemolytica biotype A was isolated from all cases of P.E. pneumonia. M. ovipneumoniae, M. arginini and P. haemolytica were also isolated from the lower respiratory tract of a proportion of 31 ewes examined post-mortem, but P.E. pneumonia was not observed in these animals.     

Expression of functions by normal sheep alveolar macrophages and their alteration by interaction with Mycoplasma ovipneumoniae

Normal sheep alveolar macrophages collected by bronchial lavage were exposed to live or heat-killed Mycoplasma ovipneumoniae organisms, and their capability to ingest Staphylococcus aureus and to elicit antibody-dependent cellular cytotoxicity against sensitized chicken red blood cells was tested. Controls consisted of non-infected macrophages in M199 medium. In addition, the effect of M. ovipneumoniae on expression of surface molecules on these sheep alveolar macrophages was determined. The percentage of S. aureus ingested by nontreated sheep alveolar macrophages was significantly higher than that of infected macrophages. Live mycoplasmas were more effective in suppressing the ingestion of S. aureus by these macrophages than killed mycoplasmas. Both live and killed mycoplasma suppressed the cytolytic effect of the sheep alveolar macrophages to a similar degree. About 78% and 45% of the normal sheep alveolar macrophages had IgG and complement receptors, respectively. Infection of these macrophages with M. ovipneumoniae decreased significantly the expression of IgG receptors but had no effects on complement receptors. There were substantial increases in the expression of both MHC class I and class II by the mycoplasma-induced macrophages as compared with unstimulated macrophages. Live mycoplasmas were more effective in inducing expression of both classes than killed mycoplasmas. The results, taken together, suggest that M. ovipneumoniae induced alterations in macrophage activities and this may be a contributing factor in the pathogenesis of respiratory disease induced by the organism.     

Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a bacterium that specifically infects sheep and goat and causes ovine infectious pleuropneumonia. In an effort to understand the pathogen–host interaction between the M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory response using a primary air–liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (Ovis aries). The ALI culture of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. Exposure of ALI cultures to M. ovipneumoniae led to increased expression of Toll-like receptors (TLRs), and components of the myeloid differentiation factor 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, infection with M. ovipneumoniae failed to induce the expression of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of the MyD88-independent signaling pathway. These results suggest that the MyD88-dependent TLR pathway may play a crucial role in sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating pathogen–host interactions between M. ovipneumoniae and airway epithelial cells.     

Chronic non-progressive pneumonia of sheep in New Zealand – a review of the role of Mycoplasma ovipneumoniae

Chronic non-progressive pneumonia (CNP) is a common disease which affects lambs in New Zealand during late summer and autumn. Mycoplasma ovipneumoniae can be recovered from a high proportion of lesions but it is also present in some normal lungs. Bacteria, especially Pasteurella haemolytica, can also be recovered from more than half the lungs of affected animals.

Isolates of M. ovipneumoniae are genetically heterogeneous, as demonstrated by examination of their DNA or total cellular proteins, and are serologically heterogeneous as shown by metabolic inhibition tests. The number of strains present in New Zealand is large and several distinguishable strains can be recovered from each affected lung. Mycoplasma ovipneumoniae has pathogenic potential as indicated by its ability to produce hydrogen peroxide, cause ciliostasis and by its possession of a capsule.

Chronic non-progressive pneumonia can be transmitted consistently to over 50% of lambs by inoculation of pooled pneumonic lung homogenate and transmission can be suppressed by broad spectrum antibiotics. In contrast, penicillin does not prevent the development of lesions but diminishes their severity. Pooled lung homogenate treated with digitonin, which inactivates mycoplasmas, has failed to transmit CNP. Pure cultures of M. ovipneumoniae produce only mild lesions in some animals, whereas inoculation with pooled lung homogenate (from which no viruses were isolated) containing mixed strains of M. ovipneumoniae and free from bacteria, is more effective in producing lesions.

Research work to date suggests that CNP may be initiated by colonisation of the lung by M. ovipneumoniae which causes ciliostasis and elicits an exudate allowing colonisation of the lungs by bacteria especially P. haemolytica and by other strains of M. ovipneumoniae. The immune response to the initial strain of M. ovipneumoniae may inhibit its replication but would be less effective in inhibiting heterologous strains of the organism allowing their sequential replication. Eventually production of a broad immune response to M. ovipneumoniae would lead to its elimination which in turn would facilitate the elimination of other microorganisms and the resolution of lesions. As natural immunity to CNP occurs within the first year, it may be possible to develop an effective and useful vaccine. Such a vaccine may need to include multiple strains of M. ovipneumoniae.     

Atypical non-progressive pneumonia in goats

An outbreak of severe respiratory disease in a goat herd was associated with Mycoplasma ovipneumoniae, Mycoplasma arginini, Mannheimia haemolytica and Pasteurella multocida with mortality rates exceeding 20% in kids. Post mortem features in affected kids included severe pleuropneumonia, lung consolidation, large quantities of pleural fluid and pericarditis. This is the first report of atypical proliferative pneumonia in goats in Portugal.     

A spatial risk assessment of bighorn sheep extirpation by grazing domestic sheep on public lands

Bighorn sheep currently occupy just 30% of their historic distribution, and persist in populations less than 5% as abundant overall as their early 19th century counterparts. Present-day recovery of bighorn sheep populations is in large part limited by periodic outbreaks of respiratory disease, which can be transmitted to bighorn sheep via contact with domestic sheep grazing in their vicinity. In order to assess the viability of bighorn sheep populations on the Payette National Forest (PNF) under several alternative proposals for domestic sheep grazing, we developed a series of interlinked models. Using telemetry and habitat data, we characterized herd home ranges and foray movements of bighorn sheep from their home ranges. Combining foray model movement estimates with known domestic sheep grazing areas (allotments), a Risk of Contact Model estimated bighorn sheep contact rates with domestic sheep allotments. Finally, we used demographic and epidemiologic data to construct population and disease transmission models (Disease Model), which we used to estimate bighorn sheep persistence under each alternative grazing scenario. Depending on the probability of disease transmission following interspecies contact, extirpation probabilities for the seven bighorn sheep herds examined here ranged from 20% to 100%. The Disease Model allowed us to assess the probabilities that varied domestic sheep management scenarios would support persistent populations of free-ranging bighorn sheep.     

Characterization of Mannheimia haemolytica biofilm formation in vitro

Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M. haemolytica required at least 36 h to form robust biofilms on plastic in vitro when incubated in RPMI-1640 tissue culture medium at 37 °C, with maximal biofilm formation being evident at 48 h. Biofilm formation was inhibited by adding the monosaccharides d(+) galactose and d(+) mannose to the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric substance we found it contained 9.7 μg/cm² of protein, 0.81 μg/cm² of total carbohydrate, and 0.47 μg/cm² of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms (P < 0.05) while α-amylase and micrococcal nuclease decreased biofilms to a lesser extent. M. haemolytica biofilm cells were more resistant than planktonic cells to the antibiotics florfenicol, gentamicin, and tulathromycin. These results provide evidence that M. haemolytica can form biofilms, which could contribute to its ability to persist as a commensal in the bovine upper respiratory tract.     

Phenotypic characterisation of Australian sheep and cattle isolates of Mannheimia haemolytica, Mannheimia granulomatis and Mannheimia varigena

Objective To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella‐Actinobacillus and obtained from cattle and sheep. Design The 35 isolates that had been obtained from Australian animals, 30 from cattle and five from sheep, were compared with reference strains of the five recognised species of the genus Mannheimia ‐ M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena. Results Thirty‐four of the isolates could be confidently assigned to three species of the genus Mannheimia. Twenty‐nine were M haemolytica, with 25 being isolated from cattle and four from sheep. All but three of the bovine M haemolytica were isolated from pneumonic lungs. Of the three remaining bovine M haemolytica isolates, one was obtained in pure culture from a bovine milk sample and the other two as part of a mixed flora associated with a middle ear infection of a calf suffering mucosal disease. Of the four ovine M haemolytica isolates, two were isolated in pure culture from milk and two, also in pure culture, from pneumonic lungs. Three bovine isolates were identified as M granulomatis ‐ one from a tongue abscess, one from a jaw abscess and one from a lung showing suppurative bronchopneumonia. Two bovine isolates were identified as M varigena‐ one coming from an udder and the other from a spleen. The available diagnostic records provided no information on whether these isolates were associated with a disease process. The remaining isolate was obtained from an ovine tongue abscess and could not be assigned to a recognised species within the genus Mannheimia. Conclusion The study represents the first time that M haemolytica, M granulomatis and M varigena have been recognised as being present in cattle and sheep in Australia. Veterinary laboratories that encounter Pasteurella‐Actinobacillus‐like organisms from cattle and sheep should attempt as complete a characterisation as possible to help improve our knowledge of the disease potential of these organsims.     

Diversity of Mannheimia haemolytica and Pasteurella trehalosi Serotypes from Apparently Healthy Sheep and Abattoir Specimens in the Highlands of Wollo,North East Ethiopia

The prevalence and serotypic diversity of Mannheimia [Pasteurella] haemolytica and Pasteurella trehalosi from nasal swabs, sera and abattoir specimens from sheep in the highlands of Wollo, North East Ethiopia was investigated. Prevalence rates of 83% and 75% of these microorganisms were found in the serum samples and nasal swabs, respectively, from apparently healthy sheep. In a local abattoir, 205 lungs were investigated, 34% of which showed pneumonia, from which samples were collected from 51 lungs and the same number of corresponding tonsils. Mannheimia and Pasteurella species were isolated from 59% of these pneumonic lungs and 69% of the respective tonsils. M. haemolytica serotypes accounted for 41 (59%) and P. trehalosi for 11 (32%) of the isolates from the abattoir specimens. The majority (67%) of isolates from nasal swabs were P. trehalosi, M. haemolytica being isolated f rom 4 (13%) of the swabs. M. glucosida was isolated only from the tonsils. The predominant serotypes of the isolates from both the nasal swabs and the abattoir specimens were M. haemolytica A1 (17%) and P. trehalosi T4 (16%) and T3 (13%). P. trehalosi T15 was less commonly encountered, while M. haemolytica A9 and A13 were not isolated. Studies on sera from 100 sheep indicated that antibodies against M. haemolytica serotype A1 (14%) were most common, followed by A5 and A8 (each 10%) and A9 and P. trehalosi T3 (each 9%) and T4 (8%). Antibodies against M. glucosida or serotype A11 occurred in 2% of the sera. Multiple serotypes were common in all types of samples. The importance of including in vaccines the most prevalent serotypes involved in the pneumonia of sheep in the area is discussed.     

A Real-Time PCR for Detection and Quantification of Mycoplasma ovipneumoniae

A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 10² to 2.2 × 10⁷ genomes.     

Cytotoxic effect of Pasteurella haemolytica on ovine bronchoalveolar macrophages in vitro

Live Pasteurella haemolytica A1 was shown to have a cytotoxic effect on suspensions of sheep bronchoalveolar macrophages. Cytotoxic activity was also demonstrable in bacteria-free supernatants from suspensions containing P. haemolytica. Heat-killed and ultraviolet killed organisms of P. haemolytica and live Staphylococcus aureus were not toxic to sheep BAM. These results suggest that a bacterial cell-free cytotoxin is produced by metabolically active P. haemolytica. Guinea-pig peritoneal macrophages, McCoy and pig kidney epithelial cell suspensions were unaffected by live P. haemolytica and supernatant from P. haemolytica cultures, indicating that the cytotoxin may only affect phagocytic cells of ovine or bovine origin.     

Transmission dynamics of Mannheimia haemolytica in newly-received beef bulls at fattening operations

The primary objective of this study was to determine, at the lung level, whether single or multiple clones of Mannheimia haemolytica are present within a pen during a bovine respiratory disease (BRD) episode. A secondary objective was to assess whether M. haemolytica isolates obtained from nasal swabs (NS) are identical to those isolated deeper within the respiratory tract. Sixteen BRD episodes that naturally occurred in 12 pens of eight to 12 bulls (n = 112) newly-received at three fattening operations were investigated. One hundred and seventy five M. haemolytica isolates were collected from 239 pairs of trans-tracheal aspirations (TTA) and NS performed during these 16 BRD episodes. M. haemolytica isolates were characterized by pulsed-field gel electrophoresis (PFGE). PFGE types obtained from NS and TTA were then compared. M. haemolytica was isolated during 14 BRD episodes. Two to three different clones of M. haemolytica were recovered during 10 episodes whereas only one clone was recovered in four episodes. A moderate agreement (kappa = 0.50) between NS and TTA for M. haemolytica isolation was observed. Identical PFGE types were only observed in 77% of matched NS-TTA pairs. The significant within-pen diversity of M. haemolytica during BRD episodes indicates that the disease is not primarily due to the spread of a single virulent clone among cattle and highlights the importance of predisposing factors that enable the resident flora to overcome the cattle's immune system. The results also demonstrate that isolates recovered from NS are not always representative of the isolates present deeper within the respiratory tract.     

Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey

This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.     

Cloning and comparison of bighorn sheep CD18 with that of domestic sheep, goats, cattle, humans and mice

Previously, we have shown that CD18, the beta-subunit of beta(2)-integrins, serves as a receptor for leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica on bovine leukocytes. Anti-CD18 monoclonal antibodies (mAbs) inhibit Lkt-induced cytolysis of bighorn sheep (Ovis canadensis) leukocytes suggesting that CD18 may serve as a receptor for Lkt on the leukocytes of this species as well. Confirmation of bighorn sheep CD18 as a receptor for Lkt, and elucidation of the enhanced Lkt-susceptibility of bighorn sheep polymorphonuclear leukocytes (PMNs), necessitates the cloning and sequencing of cDNA encoding bighorn sheep CD18. Hence, in this study we cloned and sequenced the cDNA encoding CD18 of bighorn sheep, and compared with that of other animal species. The cDNA of bighorn sheep CD18 has an open reading frame (ORF) of 2310bp. CD18 sequences obtained individually from peripheral blood mononuclear cells (PBMCs) and PMNs were identical to each other. Comparison of the deduced 770-amino acid sequence of CD18 of bighorn sheep with that of domestic sheep, goats, cattle, humans and mice revealed 99, 98, 95, 82 and 80% identity, respectively. Availability of cloned bighorn sheep CD18 cDNA should allow the molecular characterization of M. haemolytica Lkt-receptor interactions in bighorn sheep and other ruminants that are susceptible to this disease.     

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-016-0378-1) contains supplementary material, which is available to authorized users.     

Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China

In the present study, the seroprevalence and genetic identification of Mycoplasma ovipneumoniae infection in goats were investigated in Hainan Province, tropical China between October 2012 and October 2013. A total of 1,210 serum samples collected from 16 herds in various administrative regions in tropical China were evaluated using indirect hemagglutination assay (IHA). Antibodies to M. ovipneumoniae were tested in (31.7 %, 95 % confidence interval (CI) 29–34.3) 383 of 1,210 serum samples (IHA titer ≥1:16). The M. ovipneumoniae seroprevalence ranged from 26.8 % (95 % CI 20.8–32.9) to 39 % (95 % CI 30.8–47.2) among different regions in tropical China, and the difference was statistically significant (P?<?0.01). The seroprevalence of M. ovipneumoniae infection in goats was higher in winter (46.1 %, 95 % CI 39.6–52.5) and spring (33.8 %, 95 % CI 28.3–39.3) than in autumn (27.5 %, 95 % CI 22.6–32.3) and summer (24.7 %, 95 % CI 20.3–29.1), and the difference was statistically significant (P?<?0.01). In addition, DNA was extracted from nasal swab; lung samples and the 16S rRNA gene sequences were amplified by polymerase chain reaction (PCR) and then sequenced. Twenty-four of 329 (7.3 %) nasal swab samples and 73 of 280 (26.1 %) pneumonic lung tissues were found to contain M. ovipneumoniae, respectively. The results of the present survey indicate that M. ovipneumoniae infection is highly prevalent in goats in tropical China. This is the first report of the comprehensive survey of M. ovipneumoniae prevalence in goats in China.     

Microorganisms associated with a pneumonic epizootic in Rocky Mountain bighorn sheep (Ovis canadensis canadensis).

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.     

Haemophilus somnus (Histophilus somni) in bighorn sheep.

Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host-parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection.