Isolation of immunogenic outer membrane proteins from Mannheimia haemolytica serotype 1 by use of selective extraction and immunoaffinity chromatography

OBJECTIVE: To use antibodies produced by calves in response to infection with Mannheimia haemolytica in immunoaffinity chromatography for the identification and subsequent isolation of the dominant immunogenic antigens from bacteria grown in iron-deficient media. SAMPLE POPULATION: Serum from 10 calves actively infected with M haemolytica. PROCEDURE: An outer membrane protein fraction was obtained from sonicated salt-extracted M haemolytica cells by extraction with N-lauroyl sarcosinate. The immunoglobulin fraction of serum from calves actively infected with M haemolytica was used to prepare an immunoaffinity column. The immunoaffinity column was used to isolate the dominant immunogenic proteins from the outer membrane protein fraction. The resultant immunogenic protein fraction was subjected to ELISA and immunoblot methods as well as carbohydrate quantification. Sequencing of the N-terminal was performed on the most prominent protein. RESULTS: 5 immunogenic proteins with molecular weights of 42, 30, 24, 20, and 15 kd were isolated. The immunogenic protein fraction was found to contain 51% carbohydrate. The immunoaffinity column capacity was 1 microg of immunogenic protein/mL of gel. The N-terminal sequence of the 42-kd protein was Tyr-Gln-Thr-Tyr-Gln-Ser-X-Leu-Gln, where X could not be identified. CONCLUSIONS AND CLINICAL RELEVANCE: lmmunogenic proteins were isolated by use of immunoaffinity chromatography. A substantial amount of carbohydrates was co-purified in the process. Additional experiments are needed to determine whether the carbohydrates would hinder or enhance development of vaccine preparations. This method could potentially allow a more rapid production of antigens for use in vaccines.     

Mannheimia haemolytica and bovine respiratory disease

Mannheimia haemolytica is the principal bacterium isolated from respiratory disease in feedlot cattle and is a significant component of enzootic pneumonia in all neonatal calves. A commensal of the nasopharynx, M. haemolytica is an opportunist, gaining access to the lungs when host defenses are compromised by stress or infection with respiratory viruses or mycoplasma. Although several serotypes act as commensals, A1 and A6 are the most frequent isolates from pneumonic lungs. Potential virulence factors include adhesin, capsular polysaccharide, fimbriae, iron-regulated outer membrane proteins, leukotoxin (Lkt), lipopolysaccharide (LPS), lipoproteins, neuraminidase, sialoglycoprotease and transferrin-binding proteins. Of these, Lkt is pivotal in induction of pneumonia. Lkt-mediated infiltration and destruction of neutrophils and other leukocytes impairs bacterial clearance and contributes to development of fibrinous pneumonia. LPS may act synergistically with Lkt, enhancing its effects and contributing endotoxic activity. Antibiotics are employed extensively in the feedlot industry, both prophylactically and therapeutically, but their efficacy varies because of inconsistencies in diagnosis and treatment regimes and development of antibiotic resistance. Vaccines have been used for many decades, even though traditional bacterins failed to demonstrate protection and their use often enhanced disease in vaccinated animals. Modern vaccines use culture supernatants containing Lkt and other soluble antigens, or bacterial extracts, alone or combined with bacterins. These vaccines have 50-70% efficacy in prevention of M. haemolytica pneumonia. Effective control of M. haemolytica pneumonia is likely to require a combination of more definitive diagnosis, efficacious vaccines, therapeutic intervention and improved management practices.     

Identification of immunogenic, surface-exposed outer membrane proteins of Pasteurella haemolytica serotype 1

Pasteurella haemolytica serotype 1 (S1) is the bacterium most frequently recovered from the lungs of cattle that have succumbed to shipping fever pneumonia. P. haemolytica outer membrane proteins (OMPs) are important immunogens in the development of resistance to pneumonic pasteurellosis. The purpose of this study was to identify the repertoire of immunogenic, surface-exposed P. haemolytica (S1) OMPs, that could be important in the development of protective immunity. We determined surface exposure of OMPs by (1) their susceptibility to protease treatment and (2) their ability to adsorb out antibodies from bovine immune sera. For a comprehensive identification of immunogenic, surface-exposed OMPs, we used bovine antisera from calves that were resistant to experimental P. haemolytica challenge after (1) natural exposure to P. haemolytica, (2) vaccination with live P. haemolytica, or (3) vaccination with P. haemolytica OMPs. We identified 21 immunogenic, surface-exposed P. haemolytica OMPs. Most were recognized by all three immune sera. However, some were recognized by one or two of the three antisera. Our analyses identified surface-exposed, immunogenic proteins that were not identified in previous studies.     

Sequence diversity of the immunogenic outer membrane lipoprotein PlpE from Mannheimia haemolytica serotypes 1, 2, and 6

Mannheimia haemolytica serotype 1 (S1), S6 and S2 are the most common bacterial isolates found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. Recombinant PlpE (rPlpE) is highly immunogenic in cattle, and the acquired immunity markedly enhanced resistance to experimental challenge. We previously demonstrated that the immunodominant epitope (R2) is located between residues 26 and 76 on the N-terminus of PlpE from a reference S1 strain (). This region consists of eight hexapeptide repeats. The potential of this epitope as a vaccine or supplement to commercial vaccines is dependant on its state of conservation amongst isolates of the three serotypes. To determine this, we sequenced plpE genes from 32 isolates. The sequences from S1 and S6 were identical with one exception. Substantial variation was observed among sequences from S2 strains, particularly in the R2 region of the protein. These variations in S2 isolates range from 3 to 28 hexapeptide repeats. Calculated molecular weight of PlpE from S1 and S6 isolates was 37 kDa, where as PlpE from S2 strains ranged from 30 to 50 kDa. These similarities and differences were demonstrated by western blot. Competitive binding assay was used to determine that antibody against rPlpE from S1 binds native PlpE on surfaces of both S1 and S2 cells.     

Investigation of outer membrane proteins of Pasteurella. 2: Iron-regulated outer membrane proteins of Pasteurella multocida and Pasteurella haemolytica

Pasteurella multocida and Pasteurella haemolytica produce specific proteins in the outer membrane under iron-depleted conditions. Pasteurella multocida serovar A expresses these proteins of molecular masses of 76 and 96 kDa as determined by electrophoresis. The analogous serovar D produces a further iron-regulated protein of 85 kDa. The Pasteurella haemolytica strains of serovar A1, A6 and T contain iron-regulated outer membrane proteins of molecular masses of 71, 77 and 100 kDa. These proteins possess binding positions for iron ions. Both Pasteurella multocida and Pasteurella haemolytica strains utilize iron from porcine and bovine transferrin, but not from haemin and haemoglobin.     

In vivo gene expression in Mannheimia haemolytica A1 during a time-course trial in the bovine host

The objective of this study is to examine the expression of Mannheimia haemolytica genes over time during the early stage of infection. In addition, gene expression at different sites of infection in the bovine host was examined. A time-course experiment was designed to collect pharyngeal swabs and lung washings from the same animals over two time points. Six calves were experimentally challenged with M. haemolytica A1; pharyngeal swabs were collected from all animals 5h post infection. Three calves were euthanized at 6h; pharyngeal swabs were collected from the remaining 3 calves at 12h and the calves were euthanized. Lung washings were recovered from all animals at necropsy. Total RNA was prepared from the pharyngeal swabs and lung washings and primers for eight well characterized virulence-associated genes were used in qRT-PCR to examine mRNA levels. The expression of key virulence genes such as lktA, gcp and tbpB was higher in vivo compared to in vitro with the highest changes observed from 6 to 12h. The expression of lktA and gapA increased while expression of fbpA, gs60, nmaA and tbpB was found to decreased over time in the 6h period. Gene expression profiles in the lungs versus the pharynx also differed, with most genes (fbpA, tbpB, nmaA, gs60, lktA and narP) showing higher expressing in lung washings. This is the first study to follow gene expression by M. haemolytica in the same animal over time during an infection.     

Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.     

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.     

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.     

Serotyping of Mannheimia haemolytica isolates from bovine pneumonia: 1987-2006

Over a period of 20 years, a total of 207 Mannheimia haemolytica samples were isolated from calves affected with pneumonic pasteurellosis and serotyped by the indirect haemagglutination test. Serotypes A1 (102 isolates), A2 (47 isolates) and A6 (42 isolates) were most common; in addition, 16 isolates were serotypes A7, A13, A14 or untypable. The relative prevalence of serotype A6 has increased recently in Japan, as has been reported from other countries. The results of this study provide useful information towards the design of efficient vaccines for the prevention of M. haemolytica infection in Japan.     

The N-acetyl-D-glucosamine specific adhesin from Mannheimia haemolytica activates bovine neutrophils oxidative burst

In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.     

Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.     

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).     

Effects of sub-minimum inhibitory concentration antibiotic levels and temperature on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus

The objective of this study was to determine the effects of sub-minimum inhibitory concentrations (sub-MICs) of 2 veterinary antibiotic preparations, chlortetracycline (CTC) and chlortetracycline-sulfamethazine (CTC + SMZ), on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus at normal and febrile body temperatures. Sub-minimum inhibitory concentrations of both antibiotics reduced the growth rates of M. haemolytica and H. somnus. Growth of both species was not inhibited when grown at 41 degrees C compared to 37 degrees C. There was no detectable consistent effect of antibiotic or temperature on outer membrane protein expression for either species. Our study indicates that sub-MIC levels of CTC and CTC + SMZ markedly impair growth of clinical M. haemolytica and H. somnus isolates, potentially allowing more effective host clearance during infection.     

Mannheimia haemolytica vegetative endocarditis in a Suffolk wether

A 12-week-old Suffolk wether was diagnosed with bacterial endocarditis associated with Mannheimia haemolytica. The wether had shown signs of lethargy, inappetance, fever, and a grade 5 of 6 holosystolic murmur. Mannheimia haemolytica was cultured from blood premortem and the valvular lesion postmortem.     

Adenosine-5'-triphosphate release by Mannheimia haemolytica, lipopolysaccharide, and interleukin-1 stimulated bovine pulmonary epithelial cells

Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1.     

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.     

Comparison of antibody responses in cattle to outer membrane proteins from Pasteurella haemolytica serotype 1 and from eight untypeable strains.

Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.     

Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates.