Iron-regulated Outer Membrane Proteins Research Articles

Inactivated Aeromonas salmonicida impairs adaptive immunity in lumpfish (Cyclopterus lumpus).

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A-layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 104 A. salmonicida cells/dose at 8 weeks-post immunization (wpi). Immunized and non-immunized fish died within 2 weeks post-challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc-ii and cd4) were down-regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin-based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

A Chimeric Vaccine Consisting of Highly Immunogenic Regions Form Escherichia coli Iron Regulated Outer-Membrane Proteins: An In Silico Approach

Background: Six pathogen-associated Outer Membrane Iron receptors (OMPs) reside in Uropathogenic strains of E. coli (UPEC): haem-utilization gene (ChuA), Heme acquisition protein (Hma), IrgA homologue adhesin (Iha), Iron-regulated virulence gene (IreA), IroN, and IutA. Cumulative concern over the prevalence of this bacteria in hospital environments, especially in Intensive Care Units (ICUs), highlights the significance of vaccination against this pathogen. In this study, we aimed to develop 3D models of ChuA, Hma, IutA, IreA, Iha, and IroN proteins by invoking various in silico methods and design a chimeric immunogen composed of highly immunogenic regions from these six Escherichia coli antigens as a chimeric vaccine. Materials and Methods: In the present study, homology modeling, fold recognition, Ab initio approaches, and their combination were invoked to determine the Three-Dimensional (3D) structures of ChuA, Hma, Iha, IreA, IroN, and IutA. Next, a set of biochemical, immunological, and functional properties were predicted using various bioinformatics tools. Results: The obtained results indicated that all six modeled proteins fold to a β-barrel structure. The results of biochemical, immunological, and functional analysis determined the regions of each antigen carrying the best immunogenic properties. These regions are employed to construct the final vaccine linked via flexible GGGGS linkers. Intriguingly, re-analyzing the properties of the final vaccine indicated its immunological advantage over individual proteins. Conclusion: The strategy of this study to predict the protein 3D structure, followed by epitope prediction, could be adapted to design efficient vaccine candidates. Applying this approach, we designed a vaccine candidate harboring the most promising regions of six OMPs. This approach could lead to better functional, structural, and therapeutic outcomes in the context of vaccine design investigations.

In Silico Analysis of the Antigenic Properties of Iron-Regulated Proteins against Neisseria meningitidis

Neisseria meningitidis is a commensal pathogen that causes infectious cerebrospinal disease in people of all ages. The multivariate role of six disease-causing polysaccharide serotypes is found to play a crucial role in developing vaccines (or general treatment strategies) to treat this emerging pathogen. Iron is a crucial transition metal for N. meningitidis. Proteomic analysis data could be valuable for vaccine design. Here, we conduct a comparative study using computational bioinformatic tools to identify the most effective iron-regulated outer membrane proteins (OMPs) as immunogenic targets for a potential vaccine against N. meningitidis. The basic properties of N. meningitidis OMPs are explored for flexibility, solubility, hydrophilicity, beta-turns, and overall antigenic probability. Results of our study suggest that iron-regulated OMPs are flexible and soluble in water with high densities of conformational B-cell epitopes. As such, they can be recommended as a novel candidate for a vaccine against N. meningitidis both in vitro and in vivo.

Comparision of iron-regulated outer membrane proteins (IrOMP) and iron-sufficient outer membrane proteins (IsOMP) of Pasteurella multocida strains of porcine origin

An investigation was carried out to compare the effect of growth of P. multocida type A of pig origin in iron restricted and in iron sufficient media on the basis of their outer membrane protein extract. Pasteurella multocida serotype A was cultured in two ways. In one process only BHI medium was used and on the other process same medium was used which was supplemented with 2, 2’- Dipyridyl (iron-restricted medium). Both these cultures were used to obtain Outer Membrane Proteins (OMPs) by extraction of bacterial cells with 1% Sarcosyl. Separation of the OMPs in SDS-PAGE showed that OMPs were mixture of protein fraction with molecular weight ranging from 110 to 22.6 kDa in case of iron-restricted OMP and 47.3 to 29.9 kDa in case of iron sufficient OMP. The OMP with molecular masses 29.9 kDa grew in both iron-restricted as well as in iron sufficient medium.

Adaptation to host in Vibrio vulnificus, a zoonotic pathogen that causes septicemia in fish and humans.

Vibrio vulnificus is a siderophilic pathogen spreading due to global warming. The zoonotic strains constitute a clonal-complex related to fish farms that are distributed worldwide. In this study, we applied a transcriptomic and single gene approach and discover that the zoonotic strains bypassed the iron requirement of the species thanks to the acquisition of two iron-regulated outer membrane proteins (IROMPs) involved in resistance to fish innate immunity. Both proteins have been acquired by horizontal gene transfer and are contributing to the successful spreading of this clonal-complex. We have also discovered that the zoonotic strains express a virulent phenotype in the blood of its main susceptible hosts (iron-overloaded humans and healthy eels) by combining a host-specific protective envelope with the common expression of two toxins (VvhA and RtxA1), one of which (RtxA1) is directly involved in sepsis. Finally, we found that both IROMPs are also present in other fish pathogenic species and have recently been transmitted to the phylogenetic lineage involved in human primary sepsis after raw seafood ingestion. Together our results highlight the potential hazard that the aquaculture industry poses to public health, which is of particular relevance in the context of a warming world.

Antigenic Properties of Iron Regulated Proteins in Acinetobacter baumannii: An In Silico Approach

Acinetobacter baumannii, an emerging nosocomial pathogen, causes multi-drug and pan-drug resistant infections. This phenomenon necessitates the development of new treatment strategies or vaccines against this pathogen. Iron acquisition systems are important factors for the virulence of pathogenic organisms. Antibodies against iron regulated outer membrane proteins (IROMPs) showed bactericidal and opsonizing activities against A. baumannii in vitro. Data obtained from proteomic studies could be valuable for vaccine design. Comparative proteomic analysis of total lysate and outer membrane fractions isolated from A. baumannii ATCC 19606 cells cultured under iron-rich and chelated conditions resulted in the identification of protein spots differentially produced. Need for bioinformatic tools is imperative for analysis of these data as well as to design novel proteins bearing desired properties. This study undertakes in silico identification of the most effective immunogenic target amongst these iron regulated proteins in A. baumannii which can be employed as a vaccine candidate. Here a screening was carried out on iron- regulated A. baumannii OMPs, based on hydrophilicity, flexibility, beta-turns, solubility and overall antigenic probability. 3D structure of the proteins was modeled. Linear and conformational B cell epitopes predicted and their densities were used for comparison of their immunogenicity. CarO was selected as an efficient immunogenic IROMP in A. baumannii which contributes to sidrophore mediated iron uptake.

Identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against Brucella melitensis through reverse vaccinology and immunoinformatics approach

Brucella melitensis is an intracellular pathogen resides in the professional and non-professional phagocytes of the host, causing zoonotic disease brucellosis. The stealthy nature of the Brucella makes it's highly pathogenic, and it is hard to eliminate the bacteria completely from the infected host. Hitherto, no licensed vaccines are available for human brucellosis. In this study, we identified potential antigens for vaccine development from non-classically secreted proteins through reverse vaccinology approach. Based on the systemic screening of non-classically secreted proteins of B. melitensis 16M, we identified nine proteins as potential vaccine candidates. Among these, Omp31 and Omp22 are known immunogens, and its role in the virulence of Brucella is known. Roles of other proteins in the pathogenesis are yet to be studied. From the nine proteins, we identified six novel antigenic epitopes that can elicit both B-cell and T-cell immune responses. Among the nine proteins, the epitopes were predicted from Omp31 immunogenic protein precursor, Omp22 protein precursor, extracellular serine protease, hypothetical membrane-associated protein, iron-regulated outer membrane protein FrpB. Further, we designed a multitope vaccine using Omp31 immunogenic protein precursor, Omp22 protein precursor, extra cellular serine protease, iron-regulated outer membrane protein FrpB, hypothetical membrane-associated protein, and LPS-assembly protein LptD and polysaccharide export protein identified in the previous study. Epitopes were joined using amino acid linkers such as EAAAK and GPGPG. Cholera toxin subunit B, the nontoxic part of cholera toxin, was used as an adjuvant and it was linked to the N-terminal of the multitope vaccine candidate. The designed vaccine candidate was modeled, validated and the physicochemical properties were analyzed. Results revealed that the vaccine candidate is soluble, stable, non-allergenic, antigenic and 87% of residues of the designed vaccine candidate is located in the favored region. In conclusion, the computational analysis showed that the newly designed multitope protein could be used to develop a promising vaccine for human brucellosis.

Quantitative proteomic analysis of iron-regulated outer membrane proteins in Aeromonas hydrophila as potential vaccine candidates

The iron-regulated outer membrane protein (OMP) of Aeromonas hydrophila is an effective vaccine candidate, but its intrinsic functional components are largely unknown. In this study, we compared the differentially expressed sarcosine-insoluble fractions of A. hydrophila in iron-limited and normal medium using tandem mass tag labeling-based quantitative proteomics, and identified 91 upregulated proteins including 21 OMPs and 83 downregulated proteins including 10 OMPs. Subsequent bioinformatics analysis showed that iron chelate transport-related proteins were enriched in increasing abundance, whereas oxidoreductase activity and translation-related proteins were significantly enriched in decreasing abundance. The proteomics results were further validated in selected altered proteins by Western blotting. Finally, the vaccine efficacy of five iron-related recombinant OMPs (A0KGW8, A0KFG8, A0KQ46, A0KIU8, and A0KQZ1) that were increased abundance in iron-limited medium, were evaluated when challenged with virulent A. hydrophila against zebrafish, suggesting that these proteins had highly efficient immunoprotectivity. Our results indicate that quantitative proteomics combined with evaluation of vaccine efficacy is an effective strategy for screening novel recombinant antigens for vaccine development.

IN SILICO DETERMINATION AND VALIDATION OF OMR STRUCTURE AND LIGAND BINDING SITE AS A VACCINE CANDIDATE IN ACINETOBACTER BAUMANNII

Acinetobacterbaumannii is an emerging nosocomial pathogen, causes multi-drug and pan-drug resistant infections. This phenomenon necessitates the development of new treatment options and vaccines against this pathogen. Iron acquisition systems are important factors for the virulence of pathogenic organisms. Antibodies against iron Regulated Outer Membrane Proteins (IROMPs) showed bactericidal and opsonizing activities against A. baumanniiin vitro . Data obtained from proteomic studies could be valuable for vaccine design. Comparative proteomic analysis of total lysate and outer membrane fractions isolated from A. baumannii ATCC 19606 cells cultured under iron-rich and chelated conditions resulted in the identification of protein spots differentially produced. Among iron-regulated A. baumannii OMPs, carried out a primary screening based on hydrophilicity, flexibility, beta-turns, solubility and overall antigenic probability. Bioinformatic tools are imperative for analysis of these data as well as to design novel proteins bearing desired properties. This study undertakes in silico identification of the OMR 3D structure in A.baumannii which can be employed as vaccine candidate.

Computational modeling and preliminary iro N, fep A, and cir A gene expression in Salmonella Enteritidis under iron-deficiency-induced conditions

Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from the poultry industry. Salmonella grown under iron-limiting conditions has the capability to increase concentration of several iron-regulated outer-membrane proteins to augment the acquisition of the metal. These proteins have been proved to have immunogenic properties. Our aim was to increase the relative expression of iroN, fepA, and cirA in Salmonella Enteritidis domestic strain. Furthermore, we proposed a 3-dimensional structure model for each protein to predict and locate antigenic peptides. Our eventual objective is to produce an effective vaccine against regional avian salmonellosis. Two simple factorial designs were carried out to discriminate between 2 nitrogen sources and determine chelating-agent addition timing to augment relative gene expression. Two antigenic peptides located at the external face of each protein and 2 typical domains of iron-regulated outer-membrane proteins, plug and TonB-dep-Rec, were identified from the 3-dimensional models. Tryptone was selected as the best nitrogen source based on growth rate (μx = 0.36 h(-1)) and biomass productivity (Px = 0.9 g•h(-1)•L(-1)) as determined by a general factorial design. Optimum timing for chelating agent addition was in the middle of the log phase, which allowed relative expressions at 4 h of culture. Increase in iroN, fepA, and cirA relative expression was favored by the length of log phase and the addition of chelating agent, which decreased chelating toxicity and enhanced cell growth rate.

In SilicoDetermination and Validation of Baumannii Acinetobactin Utilization A Structure and Ligand Binding Site

Acinetobacter baumannii is a deadly nosocomial pathogen. Iron is an essential element for the pathogen. Under iron-restricted conditions, the bacterium expresses iron-regulated outer membrane proteins (IROMPs). Baumannii acinetobactin utilization (BauA) is the most important member of IROMPs in A. baumannii. Determination of its tertiary structure could help deduction of its functions and its interactions with ligands. The present study unveils BauA 3D structure via in silico approaches. Apart from ab initio, other rational methods such as homology modeling and threading were invoked to achieve the purpose. For homology modeling, BLAST was run on the sequence in order to find the best template. The template was then served to model the 3D structure. All the models built were evaluated qualitatively. The best model predicted by LOMETS was selected for analyses. Refinement of 3D structure as well as determination of its clefts and ligand binding sites was carried out on the structure. In contrast to the typical trimeric arrangement found in porins, BauA is monomeric. The barrel is formed by 22 antiparallel transmembrane β-strands. There are short periplasmic turns and longer surface-located loops. An N-terminal domain referred to either as the cork, the plug, or the hatch domain occludes the β-barrel.

Analysis of a ferric uptake regulator (Fur) knockout mutant in Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis; a serious infectious disease in aquaculture raised salmonids. Iron acquisition has been shown to be critical for the survival of pathogenic bacteria during the course of infection. Previous work has demonstrated that A. salmonicida expresses iron-repressible IROMP proteins, suggesting the presence of iron acquisition systems that are under the control of a ferric uptake regulator (Fur). In this study, the A. salmonicida fur has been sequenced and a fur deletion strain generated. The A. salmonicida fur gene has an open reading frame of 428bp, coding for a protein of 143 amino acids, and with high homology to previously described Fur proteins. The Fur protein product had a 94% sequence identity and 96% sequence similarity to the Aeromonas hydrophila Fur protein product. Transcription of the A. salmonicida fur gene was not regulated by the iron status of the bacterium and is not autoregulated, as in Escherichia coli. Proteomic analysis of the A. salmonicida fur mutant, fails to repress iron-regulated outer membrane proteins in the presence of iron. The A. salmonicida fur::KO mutant shows significantly reduced pathogenicity compared to the wild-type parental strain. In addition, the A. salmonicida fur mutant provides an important tool for further investigation of the iron acquisition mechanisms utilized by A. salmonicida.

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum)

The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole-cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron-regulated outer membrane proteins (IROMP) of c.90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.

Siderophore Receptor IroN Is an Important Protective Antigen Against Salmonella Infection in Chickens

In iron-limiting environments, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium synthesize and secrete several types of siderophore to trap trivalent ferric ions; these bacteria then express siderophore receptors called iron-regulated outer membrane proteins (IROMPs). In this study, we experimentally reproduced iron-limiting environments using a divalent metal chelator. IroN, one of the IROMPs, was purified by affinity chromatography with an anti-IroN-MAb-immobilized column. Thirty-day-old chickens were immunized intramuscularly with purified IroN from Salmonella Typhimurium mixed with Freund's incomplete adjuvant; the chickens were then challenged intravenously with Salmonella Enteritidis. The mortality rate of immunized chickens was 10%. On the other hand, that of control chickens was 80%. By Western blot analysis, specific IgG antibody responses against IroN of Salmonella Enteritidis were identified in chickens immunized with purified IroN. These results indicate that IroN might be promising as an important vaccine component against Salmonella infection in chickens.

Molecular, Antigenic, and Functional Characteristics of Ferric Enterobactin Receptor CfrA in Campylobacter jejuni

The ferric enterobactin receptor CfrA not only is responsible for high-affinity iron acquisition in Campylobacter jejuni but also is essential for C. jejuni colonization in animal intestines. In this study, we determined the feasibility of targeting the iron-regulated outer membrane protein CfrA for immune protection against Campylobacter colonization. Alignment of complete CfrA sequences from 15 Campylobacter isolates showed that the levels of amino acid identity for CfrA range from 89% to 98%. Immunoblotting analysis using CfrA-specific antibodies demonstrated that CfrA was dramatically induced under iron-restricted conditions and was widespread and produced in 32 Campylobacter primary strains from various sources and from geographically diverse areas. The immunoblotting survey results were highly correlated with the results of an enterobactin growth promotion assay and a PCR analysis using cfrA-specific primers. Inactivation of the cfrA gene also impaired norepinephrine-mediated growth promotion, suggesting that CfrA is required for C. jejuni to sense intestinal stress hormones during colonization. Complementation of the cfrA mutant with a wild-type cfrA allele in trans fully restored the production and function of CfrA. A growth assay using purified anti-CfrA immunoglobulin G demonstrated that specific CfrA antibodies could block the function of CfrA, which diminished ferric enterobactin-mediated growth promotion under iron-restricted conditions. The inhibitory effect of CfrA antibodies was dose dependent. Immunoblotting analysis also indicated that CfrA was expressed and immunogenic in chickens experimentally infected with C. jejuni. Amino acid substitution mutagenesis demonstrated that R327, a basic amino acid that is highly conserved in CfrA, plays a critical role in ferric enterobactin acquisition in C. jejuni. Together, these findings strongly suggest that CfrA is a promising vaccine candidate for preventing and controlling Campylobacter infection in humans and animal reservoirs.

The gene frpB2 of Helicobacter pylori encodes an hemoglobin-binding protein involved in iron acquisition

Human hemoglobin (Hb) is a metalloprotein used by pathogens as a source of iron during invasive process. It can support the Helicobacter pylori growth and several proteins are induced during iron starvation. However, the identity of those proteins remains unknown. In this work, by in silico analysis we identified FrpB2 in H. pylori genome. This protein was annotated as an iron-regulated outer membrane protein. Multiple amino acid alignment showed the motifs necessary for Hb-binding. We demonstrate the ability of FrpB2 to bind Hb by overlay experiments. In addition, the overexpression of this gene allowed the cell growth in media without free iron but supplemented with Hb. All these results support the idea that frpB2 is a gene of H. pylori involved in iron acquisition when Hb is used as a sole iron source.

Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants

Pasteurella multocida is a Gram-negative bacterium responsible for diseases affecting a broad range of farm-reared animals. Although there is an association between capsular serotype and disease, the molecular basis of host specificity is poorly understood. Outer membrane proteins (OMPs) are at the interface of bacterium and host and are likely to play important roles in host specificity and disease. Two classes are of particular importance—adhesins that are adapted for colonization of specific host niches and iron-acquisition proteins that allow pathogens to acquire iron from host-specific iron complexes. A comparative analysis of the outer membrane (OM) proteome of eight P. multocida isolates associated with disease of avian, bovine, ovine and porcine species was performed to identify putative host-specificity determinants. Isolates were cultured in iron-replete media, and also in iron-limited conditions to mimic the iron-limited host environment, and induce expression of iron-regulated OMPs expressed in vivo. The OMP-rich sarcosyl-insoluble cell fraction was isolated and the OMPs were separated by SDS-PAGE and identified by matrix-assisted light desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The expressed proteome was compared with the in silico predicted proteome from the genome sequence of P. multocida strain Pm70, using PSORTb and Proteome Analyst subcellular localization software. In iron-rich conditions isolates were clustered into three groups based on high molecular weight (HMW) OMP similarity. Isolates responsible for invasive disease were clustered into a single group. Putative colonization OMPs were present in isolates recovered from different host species, but showed molecular weight heterogeneity. Such proteins are good candidates for further study as disease or host-specificity determinants, as variation between these proteins may be a consequence of adaptation to different host niches. HMW OMPs were identified as being involved in iron-uptake. However, isolates associated with different diseases and host species expressed different iron-uptake proteins, or regulated expression differently, suggesting adaptation to specific host niches.

Screening and identification of natural antisense transcripts in Helicobacter pylori by a novel approach based on RNase I protection assay

Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. Studies have shown that NATs may participate in a broad range of gene regulatory events. The identification of NATs in human, mouse and Escherichia coli has revealed their widespread occurrence in both eukaryotic and prokaryotic life. However, little is known about NATs in Helicobacter pylori (H. pylori), a human pathogen which is associated with gastric diseases. Here we systematically screened NATs in H. pylori by a novel experimental strategy based on RNase I protection assay. We successfully constructed a cDNA library of NATs and developed a novel poly(A)-tailed RT-PCR method to monitor the expression of NATs. After sequencing, bioinformatic analysis and expression detection, two novel NATs (NAT-39 and NAT-67) were confirmed. They were, respectively, complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). Taken together, the results suggest that NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. Pylori in a sequence complementary manner with target mRNAs.

Heterologous protective immunization elicited in mice by Pasteurella multocida fur ompH.

Different strategies have been developed to produce vaccines against Pasteurella multocida. The approach described herein involves overexpression on the bacterial cell surface of Fur-regulated IROMPs (iron-regulated outer-membrane proteins). Accordingly, the ability of fur mutants to promote heterologous protection was examined in a Swiss mouse animal model. Twofur mutants derived from P. multocida were isolated, one of which was also defective in the OmpH protein. In mice challenged with virulent P. multocida, outer-membrane protein (OMP) extracts of fur cells conferred the same protection as obtained with wild-type cells grown in iron-depleted medium. Total protection was achieved with 40 microg of OMP extract from the fur ompH mutant. Mice administered heat-inactivated fur ompH cells were 60% cross-protected. The presence of a galE mutation in these cells did not further increase the protection level. Additionally, cell disruption by sonication provoked a higher level of protection than conferred by heat-treated cells. Taken together, the results showed that P. multocida fur ompH cells offer a simple and suitable approach for cross-protecting animals against infection with P. multocida.

Mannheimia haemolyticaand 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.