Mannheimia Haemolytica A1 Research Articles

Association of LPS chemotype of Mannheimia (Pasteurella) haemolytica A1 with disease virulence in a model of ovine pneumonic pasteurellosis

Host responses during pneumonic pasteurellosis were compared in sheep infected with strains of Mannheimia (Pasteurella) haemolytica A1 differing in their O-antigen type. Nine-week-old, specific pathogen-free lambs were infected intratracheally with parainfluenza type 3 virus (108 TCID50) followed 7 days later by 5—6 . 107 CFU of M. haemolytica A1 possessing rough (group R, 6 lambs) or smooth (group S, 6 lambs) lipopolysaccharide, or saline (group C, 4 lambs). Group C lambs remained afebrile with no evidence of endotoxaemia or bacteraemia and biochemical parameters were normal. Group R and group S lambs became febrile within 2—3 h postinfection and the response was higher and more prolonged in group R lambs. Four group R and 2 group S lambs developed clinical pneumonic pasteurellosis within 24—48 h and the severity of disease correlated with episodes of endotoxaemia, bacteraemia and elevated eicosanoid concentrations. At postmortem, M. haemolytica (107—109 CFU/g) was isolated from the lungs of all 6 group R lambs but from only 1 group S lamb. The results indicate an association between the incidence and severity of ovine pneumonic pasteurellosis and LPS chemotype and suggest an important role for LPS chemotype in determining host-species susceptibility to lung infection.

Efficacy of recombinant sialoglycoprotease in protection of cattle against pneumonic challenge with Mannheimia ( Pasteurella) haemolytica A1

Secreted recombinant sialoglycoprotease fusion protein (Gcp-F) of Mannheimia ( Pasteurella) haemolytica A1 was examined for its ability to protect cattle from experimental challenge with M. haemolytica A1. Five M. haemolytica vaccines were compared including Gcp-F, logarithmic phase culture supernate (Presponse ®) and Presponse ® enriched with Gcp-F, recombinant leukotoxin (rLkt) or both. All calves receiving Gcp-F had significant serum antibody responses to this antigen, measured by ELISA, prior to challenge. Those vaccinated with Gcp-F alone had significantly lower percent pneumonic tissue than unvaccinated controls and a trend ( P=0.085, one-tailed test) to lower clinical scores. Calves receiving Presponse ® with Gcp-F and rLkt had lower percent pneumonic tissue than those receiving Presponse ® alone, and calves receiving Presponse ® enriched with Gcp-F and/or rLkt had lower mean clinical scores, but the differences were not significant. This trial demonstrates the protective capacity of sialoglycoprotease. While, remarkably, recombinant Gcp-F provided some protection alone the results support its practical potential as a component of a multiple antigen vaccine.

Edible vaccine development: stability of Mannheimia haemolytica A1 leukotoxin 50 during post-harvest processing and storage of field-grown transgenic white clover

Mannheimia haemolytica is the principal microorganism responsible for bovine pneumonic pasteurellosis, or shipping fever. We have previously expressed a fragment of leukotoxin, a major virulent factor of M. haemolytica A1, as a fusion protein with green fluorescent protein (GFP) in transgenic white clover and demonstrated that this antigen was immunogenic and elicited toxin neutralizing antibodies in rabbits. These previous results showed that using plants to produce M. haemolytica antigen for use as a vaccine against this disease is a viable strategy. In this present study, we examined the stability of the truncated leukotoxin GFP-fusion protein (Lkt50-GFP) in field-grown transgenic white clover. Transgenic clover expressing Lkt50-GFP was clonally propagated and a confined field trial was established. Western immunoblotting showed that the level of Lkt50-GFP expression in field plants was the same as in transgenic plants maintained under optimal conditions in the greenhouse. We also observed that after harvesting and oven drying at 50 °C, the antigen was still present in the dried clover after 1 year of storage at ambient temperature. As special post-harvest conditions (e.g., refrigeration) are not required, the use of transgenic plants to deliver an oral vaccine against shipping fever appears to be economically feasible.

Studies on the production of quorum-sensing signal molecules in Mannheimia haemolytica A1 and other Pasteurellaceae species

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS M.h. was able to complement AI-2 production in the Escherichia coli DH5α luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.

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 (84 kDa) 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.

Studies on the production of quorum-sensing signal molecules in Mannheimia haemolytica A1 and other Pasteurellaceae species.

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS(M.h.) was able to complement AI-2 production in the Escherichia coli DH5alpha luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.

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 β 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 β 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.

Reduction of Pulmonary Mast Cells in Areas of Acute Inflammation in Calves with Mannheimia (Pasteurella) haemolytica Pneumonia

Mast cells in the left cranial pulmonary lobe of colostrum-deprived neonatal calves were quantified 2 and 6 h after intrabronchial inoculation with Mannheimia (Pasteurella) haemolytica A1. The mast cells were detected (1) immunohistochemically with a mouse anti-human mast cell tryptase monoclonal antibody, and (2) by metachromatic staining with low pH toluidine blue. A greater number of mast cells was demonstrated by the second method than by the first. At 6 h after inoculation, but not at 2 h, the number of mast cells was significantly reduced at the site of the main lesions. Treatment of calves with a sialyl Lewis mimetic (TBC1269) did not appreciably affect the results at 6 h.

Response from Rutz et al.

Using a molecular genetic approach, the genes that code for the various virulence factors of Mannheimia haemolytica A1 have been cloned for detailed characterizations. These included analysis of the encoded proteins, their biological activities, secretion of the molecules from the bacterium as well as their use in a vaccine component.Two newly characterized antigens of M. haemolytica A1 have been identified. The first one is a TonB-dependent iron regulated outer-membrane receptor that is distinct from the transferrin binding proteins. The 84 kDa Irp protein exhibits features including a TonB box and a 50 amino acid region that can adopt occluded β-barrel structures similar to the “plug” domain of the Escherichia coli FhuA and FepA crystal structures. Homologues of Irp were identified by analysis of the genome sequences of a number of Gram negative mucosal pathogens, including Neisseria meningitidis and N. gonorrhoeae. The Neisserial irp genes were cloned by PCR and expressed the 84 kDa protein as expected, demonstrating that they are functional genes. In addition to being regulated by iron and Fur, irpMh undergoes phase variation by a slipped-strand mispairing mechanism and may represent a contingency locus for iron acquisition during an infection.Another locus that codes for a putative adhesin molecule has also been partially characterized. This putative adhesin protein is highly homologous with the high-molecular-weight adhesin proteins of non-piliated non-typable strains of Haemophilus influenzae (NTHi) including Hia, Hsf, HMW1, HMW2. Currently, we have cloned the DNA that codes for 2223 amino acids (225 kDa) and is still missing the stop codon. It is anticipated that when complete, the protein could be close to 240 kDa, similar to the molecular mass of Hsf. Though incomplete, analysis of the adhesin showed that it exhibits characteristics of autotransporter (AT) proteins. The role of this high-molecular-weight adhesin in infection is being investigated.