Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1

Sixteen 3-week-old calves were intratracheally inoculated with Mycoplasma bovis. Follow-up consisted of regular bronchoalveolar lavages (BALs) and clinical examinations. Animals were slaughtered from 4 to 21 days after inoculation. Counts were made of the mycoplasmas and other bacteria systematically isolated from the BAL liquids and lung lobes after slaughter. On the 6th day, spectinomycin 20mg/kg was given intramuscularly in three repeated doses at 24h intervals to six randomly chosen calves. All animals had developed a persistent M. bovis infection with a maximum BAL count on the 6th day (start of treatment). Co-occuring Pasteurella multocida infection was found in most animals with a maximum rate on the 14th day. The extent of lung surface lesions varied widely (0-64%) but was greater in the later slaughtered calves. Average counts of M. bovis and P. multocida in the BAL liquids were lower in treated calves than in untreated ones but the difference was not statistically significant. However, M. bovis and P. multocida counts in the lungs of the treated group were significantly lower than in the untreated group (p=0.003 and 0.009, respectively).     

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Survey of restriction-modification systems and transformation in Mannheimia haemolytica and Pasteurella trehalosi

A significant obstacle to molecular studies of Mannheimia (Pasteurella) haemolytica, has been its resistance to genetic transformation. The lack of competence of many M. haemolytica strains has been attributed to the presence of restriction modification systems. In this study, representative strains of 12 M. haemolytica serotypes and four Pasteurella trehalosi serotypes were successfully transformed by electroporation using a recombinant vector derived from the native M. haemolytica A1 serotype plasmid pNSF2176. Transformation was achieved despite PCR-based evidence for the presence of genes encoding a type I restriction enzyme, phaI, and a type II restriction enzyme hsdM, in each of the M. haemolytica strains.     

beta(2) integrin Mac-1 is a receptor for Mannheimia haemolytica leukotoxin on bovine and ovine leukocytes

Pneumonia caused by Mannheimia haemolytica is an important disease of cattle (BO), domestic sheep (DS, Ovis aries) and bighorn sheep (BHS, Ovis canadensis). Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these three species. Although it is certain that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of leukocytes, whether CD18 of all three beta(2) integrins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and CR4 (CD11c/CD18), mediates Lkt-induced cytolysis of BO, DS and BHS leukocytes remains a controversy. Based on antibody inhibition experiments, earlier studies suggested that LFA-1, but not Mac-1 and CR-4, serves as a receptor for M. haemolytica Lkt. PMNs express all three beta(2) integrins, and they are the leukocyte subset that is most susceptible to Lkt. Therefore we hypothesized that all three beta(2) integrins serve as the receptor for Lkt. The objective of this study was to determine whether Mac-1 of BO, DS and BHS serves as a receptor for Lkt. cDNAs for CD11b of BO, DS and BHS were transfected into a Lkt-non-susceptible cell line along with cDNAs for CD18 of BO, DS and BHS, respectively. Transfectants stably expressing BO, DS or BHS Mac-1 specifically bound Lkt. These transfectants were lysed by Lkt in a concentration-dependent manner. Increase in intracellular [Ca(2+)](i) was observed in transfectants following exposure to low concentrations of Lkt indicating signal transduction through secondary messengers. Collectively, these results indicate that Mac-1 from these three species serves as a receptor for M. haemolytica Lkt.     

Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins

Fibrinogen-binding proteins were found in the culture supernatants of Mannheimia haemolytica serotype 1 (ATCC 43270) and Pasteurella trehalosi serotype 10 (ECO-100). Sheep fibrinogen was biotinylated and shown to bind to proteins in the culture supernatants by modified western blot. Fibrinogen-binding proteins in the culture supernatant may be important virulence factors leading to the characteristic fibrinous pneumonia caused by these organisms and may be critical antigenic targets for immune prophylaxis.     

Development of an in vitro fluorometric assay to study adherence of Pasteurella haemolytica to bovine cells

OBJECTIVE: To develop an in vitro fluorometric assay to assess Pasteurella haemolytica adherence to bovine respiratory and epithelial cells and compare adherence of single strains of P. haemolytica serovars A1 and A2 (PhA1 and PhA2, respectively). SAMPLE POPULATION: Monolayers of bovine turbinate and Madin Darby bovine kidney (MDBK) cells. PROCEDURE: To determine optimal inoculum concentration and incubation time, various concentrations of P. haemolytica were labeled with fluorescein isothiocyanate and incubated with monolayers of bovine cells for various times. Bovine cells were washed to remove nonadherent bacteria, and percentage of bacteria adhered (percentage of adherence) was estimated fluorometrically. Percentage of adherence of PhA1 was compared with that of PhA2. RESULTS: The optimal inoculum concentration that resulted in measurable fluorescence of adherent bacteria was 1 x 10(8) colony-forming units/ml, and the optimal incubation time was 45 minutes. Percentage of adherence of PhA1 to MDBK and turbinate cells was significantly greater than that determined for PhA2. CONCLUSIONS: The in vitro fluorometric assay is a time-efficient, inexpensive, and labor-saving method for evaluation of P. haemolytica adherence to bovine cells. The concentration of bacteria used to inoculate bovine cells in this assay is similar to that typically used in other types of in vitro adherence assays. The predominance of PhA1 over PhA2 during the early stages of bovine respiratory disease may be attributable to the ability of PhA1 to adhere more avidly to nasopharyngeal tissue.     

Relationships between clinical and pathological signs of disease in calves infected with Mannheimia (Pasteurella) haemolytica type A1.

Respiratory disease was induced in 16 calves, and the terminal clinical signs of disease and postmortem pathological observations were recorded. By Spearman's correlation coefficient, the respiratory rate, rectal temperature and clinical scores of the calves were significantly correlated with the extent of lung consolidation. The respiratory rate was the clinical sign most consistently correlated with the other clinical and pathological signs of respiratory disease.     

Quantification of Mannheimia haemolytica leukotoxin by indirect ELISA

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.     

Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung

The potential synergistic effect of bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica in the production of pneumonia after aerosol/intranasal infection of conventionally reared lambs was evaluated. A mild clinical response was observed in lambs given virus and/or bacteria. Gross pulmonary lesions were seen in 3 of 6 lambs given RSV and then P haemolytica 3 or 6 days later, respectively (groups D and E), and in 1 lamb of 5 given virus and bacteria simultaneously (group G). Gross lesions were not seen in control sheep (group A), in lambs given virus or bacteria alone (groups B and C), or in lambs exposed to bacteria and then virus 3 days later (group F). Bovine RSV and P haemolytica were recovered from the lungs of 5 of 7 lambs with macroscopic lesions. Gross pulmonary lesions were cranioventral firm areas of red consolidation. Microscopically, the predominant lesion was a suppurative bronchopneumonia. Bovine RSV was recovered from the nasal cavity of 8 of 27 (30%) lambs given RSV during days 3 to 6 after viral inoculation, including 1 lamb in group B, 2 in groups D, E, and F, and 1 in group G. Pasteurella haemolytica was recovered from the nasal cavity of 9 of 28 (32%) inoculated lambs, including 2 lambs from groups C and E, 3 in group D, and 1 in groups F and G. Viral antigen, as determined by immunofluorescence, was concentrated mainly in individual cells in alveolar walls, some alveolar macrophages, and a few bronchiolar epithelial cells. In vitro alveolar macrophage assays indicated decreased numbers of Fc receptors on those macrophages collected from lambs given RSV 6 days before P haemolytica infection, as compared with that in the other groups. These cellular defects disappeared after 24 hours of culture. Seemingly, bovine RSV does facilitate P haemolytica pulmonary infection in conventional, immuno-competent lambs and provides evidence for decreased Fc receptors on alveolar macrophages.     

Tilmicosin does not inhibit interleukin-8 gene expression in the bovine lung experimentally infected with Mannheimia (Pasteurella) haemolytica.

The expression of the interleukin-8 (IL-8) gene was examined by in situ hybridization in lung tissues from calves experimentally infected with Mannheimia (Pasteurella) haemolytica and treated with tilmicosin. Interleukin-8 mRNA expression was detected in alveolar areas, particularly along interlobular septa, in the lumen, and in the epithelial cells of some bronchioles. In lesional lung tissues from animals that had received tilmicosin, we found large areas with limited inflammation. There was no staining for IL-8 mRNA in these areas. In contrast, in strongly inflamed areas, the same patterns and intensities of staining for IL-8 mRNA were detected in tilmicosin- and sham-treated animals. We conclude that tilmicosin does not affect the expression of IL-8 mRNA in tissue showing microscopic signs of inflammation. Together with previous reports, this supports the view that the pro-apoptotic properties of tilmicosin on neutrophils do not compromise the host defense mechanisms required to control the infection.     

The bovine alveolar macrophage. II. In vitro studies with Pasteurella haemolytica.

Bovine alveolar macrophages were cultured in vitro and challenged with suspensions of live and dead bacteria. These cells showed severe cytotoxic morphological changes and a low rate of phagocytosis after exposure to live Pasteurella haemolytica type I was readily phagocytosed and produced only mild cytotoxic changes.     

Serotypes and electrophoretic protein profiles of Pasteurella haemolytica isolated from pneumonic ovine lungs.

Serotypes and SDS-PAGE protein profiles of P. haemolytica isolated from pneumonic ovine lungs were investigated. Of 268 P. haemolytica isolates, 232 (86.6%) were serotypable. A total of 12 serotypes were recognized in 20 different geographic origins of central Turkey. The most common serotype was A2, followed by A7, A1 and T4. Serotypes A13, A14, A16 and T15 could not be detected. In SDS-PAGE, marked differences between major bands of biotype A and T strains were found. In numerical analysis of protein profiles, biotype A and T strains were separated at 58% similarity level. Biotype A isolates produced a cluster at 80% similarity level, and biotype T isolates at 92% similarity level. No single cut off level was able to discriminate between each serotype studied and isolates could not be clustered on the basis of their geographic origins.     

Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia

Mannheimia (M.) haemolytica (formerly Pasteurella [P.] haemolytica) is the primary aetiological agent of pneumonic pasteurellosis--one of the most important respiratory diseases in cattle and sheep. While bovine pneumonic pasteurellosis is regarded to be mainly caused by M. haemolytica serotype A1, and in Germany during the last years also by serotype A6, sheep can be infected by all serotypes although there is an increased prevalence of serotypes A2 and A5-7. The obligate pathogenicity of M. haemolytica is proven by isolation of pure cultures from pneumonic lungs as well as by infection studies. Knowledge about the virulence mechanisms of M. haemolytica and their molecular basis are fragmentary, most probably due to the complex gene regulation of virulence associated factors in lung tissues. This review summarizes the current literature covering virulence factors to substantiate a model of pathogenesis. After serotype A1 strains have colonized the bovine upper respiratory tract they replace other serotypes by mechanisms unknown to date. After fulminant proliferation in the upper respiratory tract the microorganisms colonize the lower respiratory tract, finally entering alveolar spaces. An inflammatory cascade is initiated by M. haemolytica LPS and Leukotoxin, causing activation of the complement system and release of cytokines. Pathognomonic for bovine pneumonic pasteurellosis is the strong influx of neutrophiles accompanied by accumulation of fibrin, finally causing necrosis of alveolar spaces. Depending on lesion size this fibronecrotizing pneumonia can result in death of the animals. In addition, possible protective antigens are discussed. There is still a great effort in the development of efficacious vaccines against pneumonic pasteurellosis in cattle and sheep caused by various M. haemolytica serotypes worldwide. The scarce knowledge concerning presence and distribution of virulence associated factors in M. haemolytica strains and their role in pathogenesis made it difficult to determine a suitable vaccine candidate in the past. In addition, there is lack of knowledge concerning the variability of virulence factors in individual isolates. Genome sequence analysis of M. haemolytica, enabling proteomics and transciptomics, hopefully will give new insight into the pathogenesis of pneumonic pasteurellosis.     

The effects of danofloxacin and tilmicosin on neutrophil function and lung consolidation in beef heifer calves with induced Pasteurella (Mannheimia) haemolytica pneumonia

Pneumonia caused by Pasteurella (Mannheimia) haemolytica was induced in weaned beef heifer calves, approximately 6 months of age. Calves were treated at 20 h after challenge with therapeutic doses of danofloxacin or tilmicosin. Peripheral blood neutrophils were collected at 3, 24 and 48 h after treatment. The ex vivo effects on neutrophil function, neutrophil apoptosis, and hematological parameters were examined, as was the effect on percentage lung consolidation. Neutrophil function assays included random migration under agarose, cytochrome C reduction, iodination, Staphylococcus aureus ingestion, chemotaxis, and antibody-dependent and antibody-independent cell-mediated cytotoxicity. Apoptosis was determined using a cell death detection kit. Killing was performed at 72 h after treatment. Statistical comparisons were made among the three groups of challenged-treated animals: saline, danofloxacin, and tilmicosin. Comparisons were also made between nonchallenged nontreated animals (NCH) and challenged saline-treated animals. There were no significant differences for any of the neutrophil function assays or neutrophil apoptosis among the challenged-treated groups. This suggests that danofloxacin and tilmicosin have no clinically significant effects on neutrophil function or apoptosis. There were also no significant differences in percentage lung consolidation among the challenged-treated groups. Significant differences were found between the NCH calves and the challenged saline-treated calves in several neutrophil assays, which were attributed to effects of P. haemolytica infection.     

Health and performance of young dairy calves vaccinated with a modified-live Mannheimia haemolytica and Pasteurella multocida vaccine.

OBJECTIVE: To evaluate the health and performance of young dairy calves vaccinated with a commercial Mannheimia haemolytica and Pasteurella multocida vaccine. DESIGN: Randomized clinical trial. ANIMALS: 358 Holstein dairy calves between 14 and 20 days of age on 8 farms. PROCEDURE: Calves were randomly assigned to a control or vaccinated group. The vaccine used was a commercial modified-live M. haemolytica and P. multocida vaccine that was administered on days 0 and 14. Calf weight was measured on day 0 and monthly for 3 months. Farmers were asked to record any treatment given to the calves and the reason for treatment during the 4 months of the study. Blood was collected from all calves on days 0 and 28, and titers of antibodies to M. haemolytica were determined by means of direct bacterial agglutination. RESULTS: Mean daily gain was not significantly different between vaccinated and control calves. Vaccinated calves had a significantly greater increase in antibody titers (5.3-fold increase), compared with control calves (3.6-fold increase). There was no significant difference between vaccinated and control calves for any of the treatment outcomes (number and duration of treatments and age at first and last treatments). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the M. haemolytica and P. multocida vaccine, given twice 2 weeks apart, was effective in increasing titers of antibodies against M. haemolytica in young dairy calves but did not improve calf performance or health.     

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.     

Lysis of bovine platelets by Pasteurella haemolytica leukotoxin

Pasteurella haemolytica A1 culture supernatants caused rapid cytolysis (less than 5 minutes) of isolated bovine platelets as measured by leakage of the cytoplasmic enzyme lactate dehydrogenase (LD). The platelet lytic factor had several features similar to P haemolytica leukotoxin. Like P haemolytica leukotoxin, the platelet lytic factor was produced by P haemolytica during logarithmic growth phase, was heat-labile, and was active against target cells (platelets) from ruminant species (cattle and sheep), but not from non-ruminant species (horses, pigs, and human beings). Additionally, the platelet lytic factor was neutralized with antileukotoxin rabbit serum. The amount of LD leaked by a fixed concentration of bovine platelets was proportional to the amount of toxin added at low toxic doses and became maximal at 88 +/- 11% of the total platelet LD activity for high doses of toxin. When a fixed dose of toxin was used and the platelet concentration was varied, LD leakage was initially proportional to the platelet concentration, but plateaued at higher platelet concentrations. The platelet lytic factor required Ca2+ and was inhibited by addition of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. Toxin-mediated platelet damage may be important in thrombi formation and fibrin exudation typically associated with P haemolytica pleuropneumonia of cattle.     

Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin

Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the beta(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.     

In vitro assessment of the efficacy of erythromycin in combination with oxytetracycline or spectinomycin against Pasteurella haemolytica

The effects of combining erythromycin (Ery) with oxytetracycline (Oxy) or spectinomycin (Sp) on Pasteurella haemolytica were evaluated in vitro using the chessboard (checkerboard) technique. These combinations were selected because all are drugs widely used in bovine respiratory disease treatment, and they represent possible sequential or complementary mechanisms of action. Using the recommended breakpoints of greater than 4 micrograms/ml for Ery, 16 micrograms/ml for Oxy, and 32 micrograms/ml for Sp, of the 33 P. haemolytica isolates, 32 were resistant to Oxy, 27 to Sp, and 14 to Ery. Based on the fractional inhibitory concentration index, Ery and Oxy in combination were synergistic or additive against 32 of 33 isolates. The combination of Ery and Sp was synergistic or additive against 27 of 33 isolates. No instances of antagonism were seen. When the effects were considered within the context of therapeutically achievable serum/tissue concentrations, the effects of Ery and Oxy in combination were only marginal. Thus, against P. haemolytica isolates, Ery and Sp appeared to represent an effective antimicrobial combination, whereas Ery and Oxy were only of marginal efficacy as a combination.     

The effect of Pasteurella haemolytica and the leukotoxin of Pasteurella haemolytica on bovine lung explants.

Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neisseria subflava, or the crude leukotoxin of P. haemolytica on alveolar macrophages and lung parenchymal cells. Concentrated, purified peripheral blood neutrophil suspensions were added with the bacteria to some explants. Duplicate pairs of cultures from each treatment group were fixed at regular intervals up to 24 hours after seeding and morphological changes were assessed by light and electron microscopy. Pasteurella haemolytica caused deterioration of alveolar macrophages within one hour but did not affect parenchymal cells for more than 12 hours. Neisseria subflava did not affect alveolar macrophages initially, but caused an accelerated deterioration after four hours. After 24 hours, bacterial overgrowth caused similar deterioration of all cells in explants seeded with either bacterium. Alveolar macrophages phagocytosed large numbers of N. subflava but rarely ingested P. haemolytica. Added neutrophils did not have any discernible effect on any of the explants and did not potentiate bacterial effects. Addition of crude leukotoxin of P. haemolytica to the culture medium significantly accelerated alveolar macrophage deterioration without apparent effect on parenchymal cell survival. These results support the hypothesis that the severe tissue destruction of fulminant pneumonic pasteurellosis is not a direct result of bacterial infection.