Robert E. Briggs

Response of calves persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) subtype 1b after vaccination with heterologous BVDV strains in modified live virus vaccines and Mannheimia haemolytica bacterin-toxoid.

Abstract Seronegative persistently infected (PI) calves with bovine viral diarrhea virus (BVDV) subtype 1b were vaccinated with each of four modified live virus (MLV) BVDV vaccines and a Mannheimia haemolytica bacterin-toxoid. Nasal swabs and peripheral blood leukocytes (PBL) were collected for virus isolation and serums were collected after vaccination and tested for BVDV1a, BVDV1b, BVDV2, bovine herpesvirus-1 (BHV-1), bovine parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV) antibodies. M. haemolytica and Pasteurella multocida antibodies were detected using ELISA procedures. None of the PI calves developed mucosal disease (MD) after MLV vaccination. None of the BVDV PI calves seroconverted to BVDV1b after MLV vaccination. Calves receiving MLV vaccines seroconverted to the respective type/subtype in the vaccine. Calves receiving a MLV vaccine with noncytopathic (NCP) BVDV1 (subtype not designated) did not seroconvert to BVDV1a, BVDV1b, or BVDV2. The PI calves were positive for BVDV subtype 1b, in the PBL and nasal swabs throughout the study. Calves receiving each of three vaccines with known BVDV1a strains had BVDV1a positive samples after vaccination, in some but not all calves, up to Day 28. The PI BVDV1b calves did not respond with increased M. haemolytica antibodies after vaccination compared to BVDV negative calves receiving the same M. haemolytica vaccine.

Biological effects of two genetically defined leukotoxin mutants of Mannheimia haemolytica

Mannheimia(Pasteurella)haemolytica serotype 1 is the primary causative agent responsible for bovine pneumonic mannheimiosis, also known as shipping fever in cattle. The bacterium produces a variety of virulence factors, foremost of which is the exotoxic leukotoxin. The leukotoxin is a calcium-dependent cytolysin that is a member of the RTX (repeats in toxin) family and exhibits a narrow cell-type and species specificity and has biological effects only on ruminant leukocytes and platelets. The genetic organization of the leukotoxin is comprised of four genes: lktC, lktA, lktB and lktD. The lktA structural gene encodes the protoxin (pro-LktA) and lktC encodes a transacylase that post-translationally modifies the inactive pro-LktA to a biologically active wild-type leukotoxin (LktA). The LktA has been implicated as the key factor that contributes to the pathogenesis of lung injury associated with the disease and considerable efforts have been employed in abrogating toxin function while retaining immunogenicity, with an eye towards design of attenuated vaccines. We hypothesized that the pro-LktA retains the ability to cause biological effects on target cells as has been reported in the case of the closely related RTX toxin alpha-hemolysin (HlyA). We also examined the biological effects of an amino-terminal truncation mutant leukotoxin DeltaLktA on target cells. Thus the objectives of our study were to investigate whether two different mutant leukotoxins, one a nonacylated pro-LktA, and the other lacking 344 amino acids at the N-terminal end of the LktA protein; DeltaLktA, are capable of (i). binding to the beta2-integrin leukotoxin receptor, (ii). inducing the elevation of second messenger intracellular calcium ([Ca(2+)](i)), and (iii). inducing inflammatory gene expression, reactive oxygen metabolites (ROMs) and cytolysis in target cells. Our results demonstrate that neither acylation nor the amino terminal 344 amino acids are required for LktA binding but are essential for LktA-induced [Ca(2+)](i) elevation, generation of ROM, generation of the inflammatory cytokine IL-8 and cytolysis in target cells.

Sialic acid uptake is necessary for virulence of Pasteurella multocida in turkeys.

Many pathogenic bacteria employ systems to incorporate sialic acid into their membranes as a means of protection against host defense mechanisms. In Pasteurella multocida, an opportunistic pathogen which causes diseases of economic importance in a wide range of animal species, sialic acid uptake plays a role in a mouse model of systemic pasteurellosis. To further investigate the importance of sialic acid uptake in pathogenesis, sialic acid uptake mutants of an avian strain of P. multocida P-1059 (A:3) were constructed, characterized, and an in-frame sialic acid uptake deletion mutant was assessed for virulence in turkeys. Inactivation of sialic acid uptake resulted in a high degree of attenuation when turkeys were challenged either intranasally or intravenously. Resistance of the sialic acid uptake mutant to killing by turkey serum complement was similar to that of the parent, suggesting other mechanisms are responsible for attenuation of virulence in turkeys.

Comparative genome analysis of an avirulent and two virulent strains of avian Pasteurella multocida reveals candidate genes involved in fitness and pathogenicity

BackgroundPasteurella multocida is the etiologic agent of fowl cholera, a highly contagious and severe disease of poultry causing significant mortality and morbidity throughout the world. All types of poultry are susceptible to fowl cholera. Turkeys are most susceptible to the peracute/acute forms of the disease while chickens are most susceptible to the acute and chronic forms of the disease. The whole genome of the Pm70 strain of P. multocida was sequenced and annotated in 2001. The Pm70 strain is not virulent to chickens and turkeys. In contrast, strains X73 and P1059 are highly virulent to turkeys, chickens, and other poultry species. In this study, we sequenced the genomes of P. multocida strains X73 and P1059 and undertook a detailed comparative genome analysis with the avirulent Pm70 strain. The goal of this study was to identify candidate genes in the virulent strains that may be involved in pathogenicity of fowl cholera disease.ResultsComparison of virulent versus avirulent avian P. multocida genomes revealed 336 unique genes among the P1059 and/or X73 genomes compared to strain Pm70. Genes of interest within this subset included those encoding an L-fucose transport and utilization system, several novel sugar transport systems, and several novel hemagglutinins including one designated PfhB4. Additionally, substantial amino acid variation was observed in many core outer membrane proteins and single nucleotide polymorphism analysis confirmed a higher dN/dS ratio within proteins localized to the outer membrane.ConclusionsComparative analyses of highly virulent versus avirulent avian P. multocida identified a number of genomic differences that may shed light on the ability of highly virulent strains to cause disease in the avian host, including those that could be associated with enhanced virulence or fitness.

Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae

ABSTRACT Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42°C in M. hemolytica but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organisms restriction enzyme HsoI (recognition sequence 5′-GCGC-3′) characterized herein.

Construction of In-Frame aroA Deletion Mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus by Using a New Temperature-Sensitive Plasmid

ABSTRACT A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus. TS replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30°C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.

Protection Against Fowl Cholera Conferred by Vaccination with Recombinant Pasteurella multocida Filamentous Hemagglutinin Peptides

Abstract Three gene fragments, derived from Pasteurella multocida strain P-1059 (serotype A:3), encoding approximately the 5′ one-third of fhaB2 were overexpressed individually in Escherichia coli. The recombinant peptides were purified, pooled, and administered to turkey poults to evaluate immunity. The results showed that turkeys immunized twice with the recombinant peptides were significantly protected against intranasal challenge with P. multocida strain P-1059. Vaccination elicited antibody responses, based on Western blotting, that were reactive with a wild-type P-1059 cellular product approximately 170 kDa in size and multiple high molecular weight products in culture supernatant. These antibodies did not react with cell or supernatant blots of a P-1059 fhaB2 isogenic mutant. Pasteurella multocida fhaB2 genes of a bovine strain (A:3) and an avian strain (F:3) are highly conserved as is the portion of P-1059 fhaB2 examined here (>99% identities). These findings suggest that broad cross-protection against this heterogeneous pathogen may be achievable through immunization with specific recombinant FHAB2 peptides.

Monoclonal Antibodies Bind A SNP-Sensitive Epitope that is Present Uniquely in Mycobacterium avium Subspecies Paratuberculosis

Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non-paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mismatch) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the specificity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen.

Genome Sequences of Mannheimia haemolytica Serotype A2 Isolates D171 and D35, Recovered from Bovine Pneumonia

ABSTRACT Here, we report two genomes, one complete and one draft, from isolates of serotype A2 Mannheimia haemolytica recovered from pneumonic bovine lung.

Draft Genome Sequences of Two Virulent Serotypes of Avian Pasteurella multocida.

ABSTRACT Here we report the draft genome sequences of two virulent avian strains of Pasteurella multocida. Comparative analyses of these genomes were done with the published genome sequence of avirulent P. multocida strain Pm70.