Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.     

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.     

Proteomic analysis using an unfinished bacterial genome: the effects of subminimum inhibitory concentrations of antibiotics on Mannheimia haemolytica virulence factor expression

Here we identify, using nonelectrophoretic proteomics, effects of subminimum inhibitory concentrations (subMIC) of two antibiotic preparations, chlortetracycline (CTC), and chlortetracycline-sulfamethazine (CTC + SMZ), on protein expression in the bovine respiratory pathogen Mannheimia haemolytica. The M. haemolytica genome is currently in draft form, and annotation is incomplete. Relying on the principle of gene sequence conservation across species, we used annotated genomes from closely related species to identify, confirm, and functionally annotate 495 M. haemolytica proteins. To conduct quantitative comparative proteomics, we developed a protein quantitation method based on the cross correlation function of the SEQUEST algorithm. When M. haemolytica was cultivated in the presence of 1/4 MIC of CTC and CTC + SMZ, expression of proteins involved in energy production, nucleotide metabolism, translation, and the bacterial stress response (chaperones) were affected. The most notable subMIC effect was a significant decrease in the expression of leukotoxin A, which is an important M. haemolytica virulence factor. Reduction in leukotoxin expression could be one of the molecular mechanisms responsible for the efficacy of these antibiotics against bovine respiratory disease.     

Bighorn sheep beta2-integrin LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin

Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.     

Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.     

Evaluation of ewe vaccination as a tool for increasing bighorn lamb survival following pasteurellosis epizootics

We conducted field and laboratory experiments to evaluate whether treating pregnant bighorn ewes with a combination of an experimental Pasteurella trehalosi and Mannheimia haemolytica (formerly P. haemolytica) vaccine and a commercially-available bovine P. multocida and M. haemolytica vaccine would increase lamb survival following a pneumonia epidemic. Three free-ranging bighorn herds affected by pasteurellosis outbreaks between November 1995 and June 1996 were included in the field experiment. Post-epidemic lamb survival was low in all three herds in 1996, with November lamb:ewe ratios of < or = 8:100. In March 1997, thirty-six ewes (12/herd) were captured and radiocollared. Half of the ewes captured in each herd were randomly selected to receive both vaccines; the other half were injected with 0.9% saline solution as controls. Lambs born to radiocollared ewes were observed two or more times per week and were considered to have survived if they were alive in October 1997, about 6 mo after birth. Lamb survival differed among herds (range 22% to 100%), and survival of lambs born to vaccinated ewes was lower (P = 0.08) than survival of lambs born to unvaccinated ewes. Bronchopneumonia (pasteurellosis) was the dominant cause of mortality among lambs examined. We concurrently evaluated vaccine effects on survival of lambs born to seven captive ewes removed from the wild during the 1995-96 epidemic. Antibody titers were high in captive ewes prior to vaccination, and vaccines failed to enhance antibody titers in treated captive ewes. None of the captive-born lambs survived. These data suggest that, using existing technology, vaccinating bighorn ewes following pneumonia epidemics has little chance of increasing neonatal survival and population recovery.     

The outer membrane protein OmpA of Mannheimia haemolytica A1 is involved in the binding of fibronectin

An enzyme-linked immunosorbent assay using bovine fibronectin as the substrate was used to demonstrate that Mannheimia haemolytica A1 binds to fibronectin. This binding to fibronectin was specific as no binding was observed with bovine fibrinogen. The binding to fibronectin was not observed if the M. haemolytica A1 cells were pretreated with trypsin or proteinase K, suggesting that it involved a protein molecule on the cell surface. Interestingly, the fibronectin-binding activity was found to be higher in an acapsular mutant compared with its parent strain. The fibronectin-binding protein was shown to be present in the outer membrane fraction of M. haemolytica A1. A 45 kDa outer membrane protein that binds to fibronectin was identified by Far-Western immunoblot analysis. This protein was purified and subjected to MS matrix-assisted laser desorption ionization time-of-flight analysis. The results identified it to be outer membrane OmpA based on comparison with the M. haemolytica A1 genomic sequence.     

Coinfection with BHV-1 modulates cell adhesion and invasion by P. multocida and Mannheimia (Pasteurella) haemolytica

Viruses are thought to facilitate bacterial infections of the respiratory tract. The present study shows the effect of BHV-1 on Pasteurella multocida and Mannheimia haemolytica adherence and invasion of MDBK cells. The virus-infected MDBK cells become more susceptible to the adherence of both species of Pasteurella. The observed adherence increase depends on the length of virus pre-incubation time and on virus concentration. When MDBK cells are not infected with virus, they are only invaded by P. multocida, while M. haemolytica is not able to penetrate. The viral infection favours also the invasion by M. haemolytica.     

Characterization of a restriction endonuclease, PhaI, from Pasteurella haemolytica serotype A1 and protection of heterologous DNA by a cloned PhaI methyltransferase gene.

Pasteurella haemolytica is the leading cause of economic loss to the beef cattle industry in the United States and an important etiologic agent worldwide. Study of P. haemolytica is hindered by researchers' inability to genetically manipulate the organism. A new restriction endonuclease, PhaI, an isoschizomer of SfaNI (R. J. Roberts, Methods Enzymol. 65:19-36, 1980), was isolated from P. haemolytica serotype 1, strain NADC-D60, obtained from pneumonic bovine lung. PhaI recognizes the 5-base nonpalindromic sequences 5'-GCATC-3' and 5'-GATGC-3'. Cleavage occurs 5 bases 3' from the former recognition site and 9 bases 5' from the latter recognition site. A gene encoding a methyltransferase which protects against PhaI cleavage was cloned from P. haemolytica NADC-D60 into Escherichia coli. Whereas unmethylated plasmid DNA containing a P. haemolytica origin of replication was unable to transform P. haemolytica when introduced by electroporation, the same plasmid DNA obtained from E. coli which contained a cloned PhaI methyltransferase gene could do so. The data indicate that PhaI is an effective barrier to the introduction and establishment of exogenous DNA in P. haemolytica.     

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.     

An Investigation of Ovine Pneumonia in Four Herds from Central Norway

In a study of 4 sheep herds, 1 apparently healthy and 3 having respiratory problems, lesions typical of subacute or chronic pneumonia were found in 3–36 % of slaughtered lambs. Occurrence appeared to be related to certain environmental factors such as pasture, whereas moderate lungworm invasion was not found to contribute to subacute or chronic pneumonia. Relation between pneumonia and low carcass weight was established only in 1 herd. Lungs were subjected to microbiological examination. Mycoplasma ovipneumoniae was isolated from both normal and pneumonic lungs from all 4 herds. The prevalence was far higher in pneumonic (98 %) than in normal ones (28 %). Bacteria, mostly Pasteurella haemolytica, were also found in both pneumonic (49 %) and normal (18 %) lungs from all 4 herds. These results confirm the conclusions of a previous study that M. ovipneumoniae is of etiological significance in subacute or chronic pneumonia, whereas bacteria mainly occur as secondary invaders. M. ovipneumoniae appears, however, only to be a potential pathogen. Examinations for Mycoplasma arginini and virus were negative and these agents are considered to be of less significance in subacute or chronic pneumonia under Norwegian conditions.     

Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi

The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394-1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M. haemolytica. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M. glucosida and P. trehalosi. In contrast, the lktD gene is highly conserved in M. haemolytica. The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M. haemolytica. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5' and 3' ends of the operon are highly conserved in M. haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.     

Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate inflammation

The severity of bovine respiratory infections has been linked to a variety of factors, including environmental and nutritional changes, transportation, and social reorganization of weaned calves. Fatal respiratory infections, however, usually occur when a primary viral infection compromises host defences and enhances the severity of a secondary bacterial infection. This viral-bacterial synergy can occur by a number of different mechanisms and disease challenge models have been developed to analyse host responses during these respiratory infections. A primary bovine herpesvirus-1 (BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolytica results in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model to demonstrate that stress significantly altered the viral-bacterial synergy resulting in fatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-like receptors (TLR) expression and increased pro-inflammatory responses which contribute to the severity of a Mannheimia haemolytica infection. TLRs play a critical role in detecting bacterial infections and inducing pro-inflammatory responses. It is difficult to understand, however, how stress-induced corticosteroids could enhance this form of viral-bacterial synergy. Nuclear translocation of the glucocorticoid receptor activates cell signalling pathways which inhibit both TLR signalling and pro-inflammatory responses. The apparent conundrum between stress-induced corticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease.     

Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi

Mannheimia haemolytica and Bibersteinia (Pasteurella) trehalosi are the most common bacterial isolates that cause pulmonary diseases in ruminants worldwide. The disease is determined by specific serotypes found in cattle and small ruminants. The molecular epidemiology of strains involved in disease is important in the control of outbreaks as well as in the preparation of vaccines. This study aimed to detect the instability and variations of bacterial strains that may affect the analysis of epidemic strains, or the stability of vaccinal strains. Eight strains of M. haemolytica belonging to serotypes A1 and A2 and three B. trehalosi strains of the T3 and T4 serotypes were used. Strains were subjected to pulsed field gel electrophoresis (PFGE) and capsular and phenotypic typing at each round of a total of 50 successive subcultures. Remarkable stability was found in all selected strains of B. trehalosi in contrast to M. haemoltyica, in which strains of both serotypes showed pattern variations produced by PFGE and capsular and phenotypic analysis. Objective criteria for M. haemolytica and B. trehalosi typing are consequently addressed.     

Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage

The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.     

Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.     

p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis

To analyse the role of Pasteurella haemolytica Leukotoxin (LKT) in the mechanism of apoptotic cell death of bovine lymphocytes, we evaluated DNA fragmentation and p53 and c-myc expression. P. haemolytica strain ATCC 14003 was cultivated for LKT production. DNA fragmentation was analysed by electrophoresis on Agarose gel. DNA strand breaks in individual apoptotic cells were also detected by an in situ Terminal deoxy nucleotidyl Transferase (TdT). The Polymerase Chain Reaction (PCR) procedure was used for verified p53 and c-myc activation by P. haemolytica LKT. LKT was able to induce DNA fragmentation in a dose and time-dependent fashion. The greatest apoptotic effect was obtained using LKT at a concentration of 0.25 U. The results show that p53 and c-myc activation by LKT is correlated with apoptosis of bovine lymphocytes and monocytes. Our data suggest that LKT may have an important role in the bacterial virulence of Pasteurella haemolytica.     

Spontaneous pasteurellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis canadensis): clinical, laboratory, and epizootiological observations.

We observed clinical signs, compared adrenal responses, and performed diagnostic tests on 12 captive Rocky Mountain bighorn sheep (Ovis canadensis canadensis) during a spontaneous outbreak of pasteurellosis. Cortisol in urine and feces was measured for bighorns sampled three times between 20 October and 1 November 1986. By 6 November, four of these had developed pneumonia, four showed only mild rhinitis, and four remained clinically normal. Bighorns that ultimately developed pneumonia showed elevated mean urinary (P = 0.003) and fecal (P = 0.046) cortisol excretion over the 12-day sampling period. Twenty-four hour mean urine cortisol: creatinine ratios ranged from 10 to 57 ng/mg dry matter for affected and 5 to 22 ng/mg for healthy individuals; 24 hr mean fecal cortisol concentrations ranged from 7.2 to 20 ng/g dry matter for affected and 3.6 to 9.1 ng/g dry matter for healthy individuals. Elevated cortisol excretion preceded clinical pneumonia in affected bighorns by less than or equal to 16 days. Beta-hemolytic Pasteurella haemolytica biotype T, serotype 3 or 4, was isolated from nasal and pharyngeal swabs from all eight bighorns with pneumonia or mild rhinitis. We detected no evidence of parainfluenza 3, bovine respiratory syncytial virus, or Chlamydia psittaci using fluorescent antibody and/or serologic tests. Although elevated cortisol excretion was associated with pneumonia, we also believe age, reproductive physiology, and/or prior recovery from clinical pasteurellosis may have influenced individual susceptibility to pneumonia during this epizootic.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.