Investigations on the species specificity of Mannheimia (Pasteurella) haemolytica serotyping

Eleven serotypes (1, 2, 5-9, 12-14 and 16) have been demonstrated within Mannheimia haemolytica. Subsequent serotyping of 166 Mannheimia haemolytica-like strains, genetically and phenotyphically distinct from Mannheimia haemolytica, and isolated from ruminants, pigs, hares and rabbits showed that 13.2% were typeable, 19 of which were serotype 11 representing strains now being classified as M. glucosida. In addition, three strains belonged to serotypes 6, 9 and 16, respectively. Additionally, the serotyping results of 98 (P.) haemolytica-like isolates from non-ruminant sources collected by the UK Veterinary Investigation Centres during the period 1982-1996 were investigated. None of these isolates have been kept, making further genetic characterization impossible. Among these isolates, 25.5% were typeable representing serotypes 1, 2, 3, 5, 6, 9, 10, 13 and 15. Substantial evidence has been reported indicating that M. haemolytica-like isolates from non-ruminant sources represent species different from M. haemolytica. The present investigation underlines that serotyping does not represent a reliable method for the identification of M. haemolytica or M. glucosida. These observations emphasize that extended phenotypic and genetic characterization is necessary for the proper identification of these organisms.     

Survival of Mannheimia (Pasteurella) haemolytica in tracheobronchial washings of sheep and cattle

The growth, morphology and long-term survival of a representative isolate of Mannheimia haemolytica serotypes A1 and A2 were monitored in ovine and bovine tracheobronchial washings. Both strains survived for at least 244 days in ovine tracheobronchial washings and 156 days in bovine tracheobronchial washings. The addition of fresh washings at these times prompted an increase for serotype A2 but no change in viability for serotype A1 in ovine tracheobronchial washings and an increase for both serotypes in bovine tracheobronchial washings. When growth and survival was compared using tracheobronchial washings from ruminant and non-ruminant species there was a trend towards longer survival in ruminant fluids.Long-term survival was associated with temporary or permanent change from normal size colonies to 'micro-colonies' on sheep blood agar. Subculture allowed reversion to normal colony morphology. Analysis showed these micro-colonies to consist of chains of elongated bacteria. M. haemolytica serotype A2 was more robust in its ability to withstand nutrient deprivation for long periods of time. These survival mechanisms may have important implications for pathogenesis.     

A putative iron-regulated TonB-dependent receptor of Mannheimia (Pasteurella) haemolytica A1: possible mechanism for phase variation.

A recombinant plasmid that codes for a novel iron receptor protein (Irp) of Mannheimia (Pasteurella) haemolytica A1 was isolated by the partial complementation of an Escherichia coli fur mutant. The deduced amino acid sequence of Irp exhibited characteristics typical of TonB-dependent receptors. These include: a TonB-box at the N-terminal; a 50 amino acid region homologous to the "plug" domain of the E. coli FhuA and FepA receptors; and a C-terminal TonB-dependent signature which likely functions as an outer membrane anchoring domain. Previously uncharacterized Irp homologues were detected by BLAST analysis of available databases and incomplete microbial genomes. When the irp homologues from Neisseria gonorrhoeae and N. meningitidis were cloned by PCR and expressed in E. coli, novel proteins of the predicted size (84kDa) were detected in cell lysates, demonstrating that these are functional genes. The M. haemolytica A1 irp gene undergoes phase variation at a nucleotide region which contain the sequence AAAAAAATTAAAA (7A-2T-4A) flanked by a short inverted repeat. Site-specific mutagenesis of the 7A-2T-4A sequence as well as replacement of the inverted repeats resulted in a stable construct that expressed the Irp protein without phase variation. The expression of irp in M. haemolytica A1 was regulated by iron concentrations and most likely a Fur homologue, consistent with the proposed function of Irp in iron metabolism. The irp genes may represent contingency loci that play a role in iron acquisition during infection.     

Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States.

Mannheimia (Pasteurella) haemolytica biotype A serotype1 (A1) is the primary bacterial agent responsible for the clinical signs and pathophysiologic events in bovine pneumonic pasteurellosis. The goal of this study was to determine the prevalence of other serotypes of M. haemolytica biotype A organisms obtained from the upper Midwest diagnostic laboratories. A total of 147 M. haemolytica isolates were collected from Minnesota, South Dakota, and Michigan. Isolates were tested against M. haemolytica antisera obtained from the National Animal Disease Center, Ames, Iowa. Results indicated that M. haemolytica serotype 1 represented approximately 60%, serotype 6 represented 26%, and serotype 2 represented 7% of the total examined isolates. In addition, 7% of the isolates were serotype 9, 11, or untypable. This finding suggests that M. haemolytica serotypes other than serotype 1 can be isolated from the lung lesions of diseased cattle and seem to be capable of causing the pathologic changes observed in the lung with pneumonic pasteurellosis.     

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.     

Construction and analysis of a Mannheimia haemolytica A1 luxS mutant

Mannheimia haemolytica A1 is the causative agent of bovine pneumonic pasteurellosis, a major cause of sickness, death, and economic loss to the feedlot cattle industry. M. haemolytica A1 produces autoinducer-2 (AI-2) like molecules that are capable of inducing quorum sensing system 2 of Vibrio harveyi. This interspecies quorum sensing system has been shown to regulate the expression of virulence genes in several pathogenic bacteria. The protein central to the production of AI-2 is LuxS. To determine if quorum sensing is involved in the regulation of virulence genes in M. haemolytica A1, a luxS mutant was constructed by replacing luxS with a cat cassette. This mutant was verified by PCR analysis, Southern hybridization, as well as its inability to induce bioluminescence in the V. harveyi reporter strain. RT-PCR analysis showed there was no difference in leukotoxin (lktC) mRNA levels, however there were increased mRNA levels of putative virulence associated genes, transferrin binding protein B (tbpB), adhesin (ahs) and capsule biosynthesis (nmaA). Electron microscopy showed that the level of encapsulation in the mutant is higher than the parent. Additionally, the mutant was slightly more adherent to bovine tracheal cells than the parent. In vitro competition assays showed the mutant out-competed the parent under iron-restricted conditions. However, in a calf challenge, the parent was the dominant isolate recovered.     

Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage beta2 integrins

Mannheimia (Pasteurella) haemolytica, the etiologic agent of bovine pneumonic mannheimiosis, produces an exotoxic leukotoxin. The leukotoxin (LktA) is a member of the RTX (repeats in toxin) family of bacterial cytolysins and is distinguished from other toxins by its unique target cell specificity to ruminant leukocytes occurring through binding to a specific receptor. We have demonstrated previously that the beta2 integrin LFA-1 is a receptor for LktA in bovine leukocytes and is involved in leukotoxin-induced biological effects. However the subunits within LFA-1 involved in binding to LktA, and post-binding signaling leading to cellular activation have not been well characterized. The purpose of our study was to pinpoint these precise subunits on bovine alveolar macrophages and to characterize their interaction with LktA. The results in this study indicate that although LktA can efficiently bind to the CD18 subunit of both LFA-1 and Mac-1, post-binding signaling events including elevation of intracellular calcium and CD18 tail phosphorylation are only observed through LFA-1. Furthermore, LktA also binds to the CD11a subunit of LFA-1. LktA binding to CD11a could be inhibited by a small molecule inhibitor of the I(inserted)-domain, the major ligand binding interface on CD11a. I-domain inhibition significantly blunts LktA-induced intracellular calcium elevation and tyrosine phosphorylation of the CD18 tail. Based on our results we suggest that LFA-1 serves as the functional leukotoxin receptor on bovine alveolar macrophages.     

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.     

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.     

Typing and virulence testing of Pasteurella haemolytica field strains

The greater part of 145 typed Pasteurella haemolytica strains from calf could be attributed to Type A 1. Hence, in the context of virulence testing, this is the most important type at present for calf. Clearly manifest pneumonia was caused in calf by other strains of Types A 2, A 6, A 12, and T 10 which were also tested for their virulence parameters. The same applied to a number of strains which had so far been characterised merely as T or A/T strains.     

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.     

Maternally and naturally acquired antibodies to Mannheimia haemolytica and Pasteurella multocida in beef calves

The dynamics and duration of maternally derived antibodies as well as the onset of acquired immunity against Mannheimia haemolytica and Pasteurella multocida in range-pastured beef calves were investigated. Two groups of unvaccinated cattle were used in this study. Serum antibody responses were measured by enzyme-linked immunoassay for antibodies of the IgG1, IgG2 and IgM isotypes binding M. haemolytica whole cells (WC) or leukotoxin (LKT) and P. multocida outer membrane proteins (OMPs). Comparisons of mean antibody responses to M. haemolytica LKT and WC and P. multocida OMPs were made within each group. Maternally derived antibodies against M. haemolytica and P. multocida reached lowest levels at 30-90 days after birth. Calves began production of antibodies against M. haemolytica and P. multocida between 60 and 90 days of age in both groups. Based on the results of this study, in beef herds vaccinated against M. haemolytica and/or P. multocida, it may be best to vaccinate calves around 3 months of age. In contrast, beef calves from unvaccinated herds might benefit from vaccination at 4 months of age.     

Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1)

Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.     

Pharmacodynamics of amoxicillin against Mannheimia haemolytica and Pasteurella multocida and pharmacokinetic/pharmacodynamic (PK/PD) correlation in sheep

Silicone-made tissue cages were implanted in sheep. Blood serum (SBS) and tissue cage fluid (TCF) samples were collected after amoxicillin intravenous and intramuscular administrations, at the dose of 15 mg/kg. Amoxicillin pharmacodynamics were studied in an artificial culture medium, SBS and TCF with use of a Mannheimia haemolytica and a Pasteurella multocida strain. A concentration-independent antimicrobial activity of amoxicillin was confirmed for levels higher than 0.79–1.75 × MIC. This result favored the use of the percentage of the 24 h dosing interval during which drug levels remain above MIC as the appropriate pharmacokinetic/pharmacodynamic index. The subsequent correlation revealed that intravenous administration could be considered effective against “deep” infections caused by bacteria with MICs < 1 μg/mL or “shallow” infections caused by bacteria with MICs < 0.1 μg/mL. Intramuscular administration could be safely considered effective against both “deep” and “shallow” infections when the MICs of the targeted pathogens are lower than 1 μg/mL.     

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.     

Pharmacokinetic and pharmacodynamic integration and modelling of marbofloxacin in calves for Mannheimia haemolytica and Pasteurella multocida

The pharmacokinetics (PK) and pharmacodynamics (PD) of marbofloxacin were established in calves for six strains of each of the pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. The distribution of marbofloxacin into inflamed (exudate) and non-inflamed (transudate) tissue cage fluids allowed comparison with the serum concentration–time profile. To establish the PD profile, minimum inhibitory concentration (MIC) was determined in Mueller–Hinton broth (MHB) and calf serum.Moderately higher MICs were obtained for serum compared to MHB. An initial integration of PK–PD data established C_max/MIC ratios of 45.0 and AUC_24h/MIC values of 174.7 h, based on serum MICs, for both bacterial species. Using bacterial time-kill curves, generated ex vivo for serum marbofloxacin concentrations, PK–PD modelling established three levels of growth inhibition: AUC_24h/MIC ratios for no reduction, 3 log₁₀ and 4 log₁₀ reductions in bacterial count from the initial inoculum count were 41.9, 59.5 and 68.0 h for M. haemolytica and 48.6, 64.9 and 74.8 h for P. multocida, on average respectively. Inter-strain variability for 3 log₁₀ and 4 log₁₀ reductions in bacterial count was smaller for P. multocida than for M. haemolytica. In conjunction with literature data on MIC₉₀ values, the present results allowed prediction of dosages for efficacy for each organism for the three levels of growth inhibition.