Molecular characterization of a secreted enzyme with phospholipase B activity from Moraxella bovis.

A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized from Moraxella bovis. The plb gene encodes a protein of 616 amino acids (molecular mass of ~65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

The immune response to a DNA vaccine can be modulated by co-delivery of cytokine genes using a DNA prime-protein boost strategy.

A large-scale DNA vaccination trial was performed in sheep to investigate whether co-delivery of the cytokine genes IL-4, IL-5, IL-15, GM-CSF or IFN-gamma could modulate the immune response generated to an antigen, in a DNA prime-recombinant protein boost regime. Vaccination with the recombinant EG95 protein has been shown to induce protection in sheep from Echinococcus granulosus infection, the causative agent of hydatid disease. Here we demonstrate that vaccination with DNA encoding EG95 effectively primed the humoral response, as judged by high IgG anti-EG95 titres detected one-week after a boost with the recombinant protein. However, by two weeks after protein-boost the titres in the control group had reached levels similar to the groups primed with EG95 DNA. Priming with two doses of DNA vaccine followed by boosting with recombinant protein induced a predominantly IgG1 response. In contrast, priming and boosting with the protein vaccine generated a strong IgG2 response. Co-delivery of the EG95 DNA vaccine with DNA encoding GM-CSF enhanced the antibody titre to EG95 while co-delivery of IFN-gamma or IL-4 encoding DNA appeared to reduce the ability of the DNA vaccine to prime an IgG antibody response. This study has demonstrated the efficacy of the co-delivery of cytokines to modulate immune responses generated in a DNA prime-protein boost strategy.

Induction of lymphocyte recruitment in the absence of a detectable immune response.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.

Identification of type 4 fimbriae in Actinobacillus pleuropneumoniae.

Type 4 fimbriae have been identified on the cell surface of Actinobacillus pleuropneumoniae by electron microscopy and N-terminal sequencing analysis. A. pleuropneumoniae type 4 fimbrial subunit protein, purified from cell cultures and from outer membrane preparations, reacted with polyclonal antibody raised against type 4 fimbriae of Moraxella bovis on Western blots. N-terminal sequence analysis of the purified 17 kDa type 4 fimbrial subunit protein, named ApfA, revealed the first 12 amino acids to be identical to those of other type 4 fimbrial subunit proteins.

Ovine interleukin-12: analysis of biologic function and species comparison.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.

Characterization of hemolysin of Moraxella bovis using a hemolysis-neutralizing monoclonal antibody.

A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the pathogenesis of infectious bovine keratoconjunctivitis, while neutralization of hemolytic and cytotoxic activities by MAb G3/D7 implies that these activities are related or have common epitopes. The action of M. bovis hemolysin was further characterized in sheep erythrocyte preparations with a binding step and Ca(2+) required for lysis to proceed, similar to the RTX family of bacterial exotoxins. Neutralization of lytic activity in vitro is evidence for the presence of M. bovis antigens, which may be capable of protecting cattle from the development of infectious bovine keratoconjunctivitis.

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

Identification, purification, and characterization of the type 4 fimbriae of Pasteurella multocida.

The presence of fimbriae on Pasteurella multocida has been reported, but there have been no prior studies aimed at conclusively characterizing these structures. We now report on the identification and characterization of type 4 fimbriae on serogroup A, B, and D strains of P. multocida. Under microaerophilic conditions P. multocida showed an increased expression of the fimbriae, which were observed to form bundles. Fimbriae purified by high-performance reverse-phase liquid chromatography constituted a single 18-kDa subunit, the first 21 amino acids of which shared very high similarity with the N-terminal amino acid sequence of other type 4 fimbrial subunits. Antiserum against the P. multocida 18-kDa protein immunostained the type 4 fimbrial subunit of Moraxella bovis and Dichelobacter nodosus. Based on these observations we conclude that P. multocida possesses type 4 fimbriae and have designated the P. multocida fimbrial subunit PtfA.

The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK).

Calves were vaccinated with cloned Moraxella bovis pili of serogroup C (experiment 1) or B (experiment 2) either as a monovalent formulation or as part of a multivalent preparation with pili of six other serogroups. Within 4 weeks of the second vaccine dose vaccinated calves and non-vaccinated controls were challenged via the ocular route with either virulent M. bovis strain Dal2d (serogroup C) or M. bovis strain 3WO7 (serogroup B) in experiments 1 and 2, respectively. Calves vaccinated with multivalent vaccines had significantly lower antibody titres than those vaccinated with monovalent preparations. Nevertheless, the levels of protection against infectious bovine keratoconjunctivitis (IBK) achieved with multivalent vaccines were 72% and 83% for the groups challenged with M. bovis strains of serogroups B and C, respectively. The serogroup C monovalent vaccine gave 100% protection against experimentally induced IBK and M. bovis isolates cultured from the eyes 6 days post-challenge were identified as belonging solely to serogroup C. Unexpectedly, only 25% protection was achieved against homologous strain challenge of calves that received the monovalent serogroup B vaccine. Furthermore, the majority of M. bovis isolates recovered from calves in this group belonged to serogroup C, as did half of those isolates cultured from the multivalent vaccinates. The remaining bacterial isolates from the latter group, together with all isolates from the non-vaccinated controls, belonged to serogroup B. Results are consistent with the hypothesis that derivatives of the serogroup B challenge inoculum had expressed serogroup C pilus antigen within 6 days of the challenge, possibly as a result of pilus gene inversion occurring in response to the presence of specific antibody in eye tissues and tears.

A haemolytic cell-free preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis.

Protection conferred by a cell-free preparation from a haemolytic Moraxella bovis isolate, UQV 148NF, was compared to an equivalent fraction from a non-haemolytic M. bovis isolate, Gordon 26L3, and to a recombinant DNA-derived pili vaccine. Three groups of ten calves were vaccinated twice with one of the three preparations and, together with ten non-vaccinated calves, challenged with virulent M. bovis isolate Dal 2d. Compared to the control group, significant protection was observed in the group receiving the pili vaccine and the group receiving the preparation from haemolytic isolate, UQV 148NF.

Characterization of pilin genes from seven serologically defined prototype strains of Moraxella bovis.

Numerous field isolates of Moraxella bovis have previously been classified by serological techniques into seven serogroups, each defined by homologous cross-reaction with antisera prepared against purified pili of a single prototype strain. The gene encoding pilin from each of the prototype strains has been characterized by nucleotide sequence determination. The coding sequences show extensive homology (70 to 80%) while the proximal downstream sequences show a dichotomy into nonhomologous sets. The pilin genes of three more strains were also characterized. The presence of an additional, partial pilin gene in each prototype strain was confirmed by Southern blot analysis, and the partial pilin genes from two strains of one serogroup were characterized by sequence determination. Features of the pilin gene sequences are considered in relation to pilin gene inversion and the serological variants of strains which may arise from gene inversion events.

Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci.

The occurrence of resistance to antiseptics and disinfectants in clinical isolates of coagulase-negative staphylococci (CNS) was examined. Of 164 clinical strains of CNS isolated in the early 1980s, 65 were resistant to cationic antimicrobial compounds such as cetyltrimethylammonium bromide. Further characterisation of 40 resistant isolates by DNA-DNA hybridisation analysis and phenotypic resistance studies revealed that this resistance was mediated by the multidrug export genes qacA and qacC, characterised previously in Staphylococcus aureus. Of the resistant CNS isolates, 50% contained only qacA, 10% contained only qacC, and the remaining 40% contained both qacA and qacC. Both qacA and qacC genes resided on plasmids in all cases, with qacA located on plasmids of > 10 kb, whereas qacC was located primarily on plasmids of 2-3 kb. Representative qacA and qacC plasmids were characterised by restriction endonuclease mapping, and were found to be similar in some cases, but different in others, to those plasmids on which these genes are found in S. aureus.

A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis.

Pili (fimbriae) were prepared from Moraxella bovis strain Dalton 2d (Dal2d) and from a derivative of Pseudomonas aeruginosa K/2PfS that contained a plasmid-borne Dal2d pilin gene and produced pili having serogroup-specific identity to Dal2d. Nine calves were vaccinated with two doses each of 30 micrograms authentic M. bovis Dal2d pili in oil adjuvant and 10 calves were vaccinated with a similar dose of P. aeruginosa-derived Dal2d pili in the same formulation. All 19 calves and 10 non-vaccinated controls were challenged by instillation of 1 x 10(9) virulent M. bovis Dal2d cells into both conjunctival sacs 19 days after the second vaccine dose. The serological response to vaccination and the degree of protection against experimentally induced infectious bovine keratoconjunctivitis (IBK) were assessed. None of the nine calves vaccinated with authentic M. bovis Dal2d pili developed IBK while two of those vaccinated with P. aeruginosa-derived Dal2d pili developed lesions which accounted for a mean group lesion score of 0.3. In contrast, 9 of the 10 non-vaccinated calves developed IBK lesions, the majority of which were progressive, required early treatment and accounted for a mean group lesion score of 1.5. These results demonstrate the potential of a relatively low dose of pili produced by recombinant DNA technology for development of an effective vaccine against IBK.

The protective efficacy of pili from different strains of Moraxella bovis within the same serogroup against infectious bovine keratoconjunctivitis.

Three groups of ten calves were each immunised with a total of 400 micrograms pili prepared from three separate strains of Moraxella bovis in Alhydrogel-oil adjuvant as two divided, equal doses 21 days apart. Groups 1 and 2 each received a monovalent vaccine made from strain 4L and S276R respectively, which belonged to pili serogroup A. Group 3 received vaccine made from pili of strain Maff1, belonging to serogroup F. A further group of ten calves served as non-vaccinated controls. Calves in groups 1 and 2 had developed serogroup A-specific antibody and those in group 3 developed serogroup F-specific antibody, and some evidence of cross-reacting antibody was also detected when measured by an agglutination test using formalin-killed piliated cells of serogroup A strain 4L. Although antibody titres measured against purified pili by ELISA were highest with homologous serogroup antigens, cross-reactive titres to shared epitopes of M. bovis pili were also detected by this method. Ocular challenge of the 40 calves with virulent M. bovis of serogroup A strain S276R was carried out 14 days after the second vaccine dose. All non-vaccinated calves developed infectious bovine keratoconjunctivitis (IBK). The percentage protection in groups 1 (strain 4L) and 2 (strain S276R) was 60% and 80% respectively (P less than 0.05), with mean lesion scores of 0.7 and 0.3 out of a possible 6.0. The percentage protection of calves in group 3 (strain Maff1) was only 30%, with a mean lesion score of 1.4 compared with 2.2 for non-vaccinated controls. The present findings, together with other evidence indicating that immunity to IBK is serogroup-specific, suggest that inclusion of pili from one representative strain from each of the seven Australian and British serogroups in a polyvalent, subunit vaccine should effectively protect the majority of cattle against IBK caused by most field strains of M. bovis encountered in Australia and the United Kingdom.

Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus.

Clinical isolates of Staphylococcus aureus carry various antiseptic and disinfectant resistance determinants (qac genes) on a variety of plasmids. The biochemistry and specificity of these resistance genes in S. aureus is the subject of this report. The qac genes were separated into two families on the basis of resistance profiles and DNA homology. Isotopic and fluorimetric assays demonstrated that the qac genes encode efflux systems that rely on proton motive force.

Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties.

Escherichia coli strains bind to Gal alpha 1-4Gal-containing glycolipids via P pili-associated G-adhesins. Three functional classes of adhesins with different binding specificities are encoded by conserved G-alleles. We suggest that the Class I papG-allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain. Closely related strains in the ECOR collection of natural E. coli isolates carry either a Class II or a Class III G-adhesin. Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual pap/prs genes have occurred. We propose that the retention and spread of pap/prs DNA among E. coli is the result of selection pressure exerted by mammalian intestinal isoreceptors.

Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ.

The papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Gal alpha (1-4)Gal-binding P pili. Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells. In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from internal breakages to the pilus. The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide-binding proteins. PapJ may function as a 'molecular chaperone' directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.

The PapG adhesin of uropathogenic Escherichia coli contains separate regions for receptor binding and for the incorporation into the pilus.

Most uropathogenic strains of Escherichia coli produce heteropolymeric organelles, known as P pili, that bind to the globoseries of glycolipids present in the urinary tract. The formation of a P pilus is the result of a family of related proteins being coordinately assembled into the structure in a defined order with the adhesin located exclusively at the tip. The preassembled digalactoside alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose-binding adhesin was purified to homogeneity from the periplasmic space in a complex with the periplasmic assembly protein PapD by affinity chromatography to alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose-Sepharose. A receptor-binding domain was mapped to the amino-terminal half of the adhesin. The interaction of PapD with PapG, which was required for the incorporation of the adhesin into the pilus, was found to protect PapG from proteolytic cleavages and enhanced the processing of the PapG signal peptide. A preassembly domain necessary for forming a complex with PapD was mapped to the carboxyl terminus of PapG.