Janet I. MacInnes

The first description of a (1 → 6)-β-D-glucan in prokaryotes: (1 → 6)-β-D-glucan is a common component of Actinobacillus suis and is the basis for a serotyping system.

Abstract The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1→6)-β- d -glucan homopolymer, β- d -Glc p -(1-[→6)-β- d -Glc p -(1-] n →, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1→6)-β- d -glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1→6)-β- d -glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1→6)-β- d -glucan, but, instead, produced a different O-chain (designated serotype O2); these three strains expressed their own characteristic CPSs. (1→6)-β- d -Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1→6)-β- d -glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the β- d -Glc p -(1→6)-β- d -Glc p linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1→6)-β- d -glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1→6)-β- d -glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis . In addition, these (1→6)-β- d -glucan background antibodies may be protective against A. suis infection. The characterization herein of (1→6)-β- d -glucan is the foundation for the development of a serotyping system for A. suis .

Genome Sequencing and Analysis of a Type A Clostridium perfringens Isolate from a Case of Bovine Clostridial Abomasitis

Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ∼3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (∼18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified.

Characterization of colonization-deficient mutants of Actinobacillus suis

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P=0.02) and to PBMEC (P=0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.

The microbiome of the soft palate of swine

Abstract The tonsil of the soft palate in pigs is a secondary lymphoid tissue that provides a first line of defense against foreign antigens entering by the mouth or nares. It has been known for a long time to be the site of colonization of important swine and zoonotic bacterial pathogens. Initially our understanding of microbes present at this site came from culture-based studies. Very recently, sequence-based approaches have been used to identify the core microbiome of the swine tonsil. Although animal to animal and herd to herd variation was detected in these studies, >90 of the organisms detected belonged to the phyla Proteobacteria and Firmicutes. Members of the family Pasteurellaceae appeared to be predominate in the tonsil; however, the relative proportions of Actinobacillus, Haemophilus, and Pasteurella varied. Members of the families Moraxellaceae, Fusobacteriaceae, Veillonellaceae, and Neisseriaceae were also seen as frequent residents of the tonsil.

Enhanced Resistance to Bacterial Infection in Protegrin-1 Transgenic Mice

ABSTRACT Antibiotic-resistant bacteria have become a public health concern. It was suggested that one source of resistant pathogens may be food-producing animals. Alternative approaches are therefore needed to enhance the resistance of farm animals to bacterial infection. Protegrin-1 (PG-1) is a neutrophil-derived antimicrobial peptide that possesses activity against a wide range of bacteria and enveloped viruses. Here we report on the production of transgenic mice that ectopically expressed PG-1 and compare their susceptibilities to Actinobacillus suis infection with those of their wild-type (WT) littermates. Of the 126 mice that were challenged with A. suis, 87% of the transgenic mice survived, whereas 31% of their WT littermates survived. The PG-1 transgenic mice had significantly lower bacterial loads in their lungs and reduced numbers of pulmonary pathological lesions. The antimicrobial function of PG-1 was confirmed in vitro by using fibroblast cells isolated from the transgenic mice but not the WT mice. Moreover, differential blood cell counts in bronchoalveolar lavage fluid indicated greater number of neutrophils in PG-1 transgenic mice than in their WT littermates after bacterial challenge. Our data suggest that the ectopic expression of PG-1 in mice confers enhanced resistance to bacterial infection, laying the foundation for the development of livestock with improved resistance to infection.

Identification of Actinobacillus suis Genes Essential for the Colonization of the Upper Respiratory Tract of Swine

ABSTRACT Actinobacillus suis has emerged as an important opportunistic pathogen of high-health-status swine. A colonization challenge method was developed, and using PCR-based signature-tagged transposon mutagenesis, 13 genes belonging to 9 different functional classes were identified that were necessary for A. suis colonization of the upper respiratory tract of swine.

Analysis of Actinobacillus pleuropneumoniae and Related Organisms by DNA-DNA Hybridization and Restriction Endonuclease Fingerprinting

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.

Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyX:FNR hybrids.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae is homologous to FNR, an anaerobic transcriptional regulator of Escherichia coli. It endows a haemolytic phenotype upon E. coli, and will complement the anaerobic respiratory deficiencies of fnr mutants of E. coli. The coding region of the hlyX gene was expressed in E. coli and the HlyX protein was purified by using an assay based on its immunological cross-reactivity with anti-FNR antibodies. The HlyX protein had the predicted N-terminal sequence, and resembled the isolated FNR protein in size ( Mr 29000) and monomeric organization. It had no detectable haemolysin activity per se, and is therefore presumed to confer a haemolytic phenotype by activating a latent haemolysin gene in E. coli. Studies with gene fusions showed that HlyX, like FNR, can function as an anaerobic activator and repressor of FNR-regulated genes in vivo. Plasmids that express hybrid HlyX : FNR proteins in which the 189/190-residue N-terminal segments and the remaining 50/60-residue C-terminal segments are exchanged, retained their FNR-specific functions but failed to confer a haemolytic phenotype. This suggests that the specificity for activating the haemolytic response requires the participation of unique features in both the N- and C-terminal segments of HlyX.

Identification of putative adhesins of Actinobacillus suis and their homologues in other members of the family Pasteurellaceae.

BackgroundActinobacillus suis disease has been reported in a wide range of vertebrate species, but is most commonly found in swine. A. suis is a commensal of the tonsils of the soft palate of swine, but in the presence of unknown stimuli it can invade the bloodstream, causing septicaemia and sequelae such as meningitis, arthritis, and death. It is genotypically and phenotypically similar to A. pleuropneumoniae, the causative agent of pleuropneumonia, and to other members of the family Pasteurellaceae that colonise tonsils. At present, very little is known about the genes involved in attachment, colonisation, and invasion by A. suis (or related members of the tonsil microbiota).ResultsBioinformatic analyses of the A. suis H91-0380 genome were done using BASys and blastx in GenBank. Forty-seven putative adhesin-associated genes predicted to encode 24 putative adhesins were discovered. Among these are 6 autotransporters, 25 fimbriae-associated genes (encoding 3 adhesins), 12 outer membrane proteins, and 4 additional genes (encoding 3 adhesins). With the exception of 2 autotransporter-encoding genes (aidA and ycgV), both with described roles in virulence in other species, all of the putative adhesin-associated genes had homologues in A. pleuropneumoniae. However, the majority of the closest homologues of the A. suis adhesins are found in A. ureae and A. capsulatus—species not known to infect swine, but both of which can cause systemic infections.ConclusionsA. suis and A. pleuropneumoniae share many of the same putative adhesins, suggesting that the different diseases, tissue tropism, and host range of these pathogens are due to subtle genetic differences, or perhaps differential expression of virulence factors during infection. However, many of the putative adhesins of A. suis share even greater homology with those of other pathogens within the family Pasteurellaceae. Similar to A. suis, these pathogens (A. capsulatus and A. ureae) cause systemic infections and it is tempting to speculate that they employ similar strategies to invade the host, but more work is needed before that assertion can be made. This work begins to examine adhesin-associated factors that allow some members of the family Pasteurellaceae to invade the bloodstream while others cause a more localised infection.

Restriction endonuclease map of endogenous mouse mammary tumor virus loci in GR, DBA, and NFS mice

Mouse mammary tumor virus (MMTV) is integrated in the genome of most mice as an endogenous provirus. Two of these MMTV proviral loci (Mtv-1 and Mtv-2) are associated with virus expression and tumorigenicity. We prepared restriction endonuclease maps of the endogenous MMTV proviruses in two strains, DBA and GR, which contain the Mtv-1 and Mtv-2 loci, plus a third strain, NFS, which has a low mammary tumor incidence. We find that all these mouse strains have certain MMTV loci in common even though their origins are widely divergent. We also find that some integrated MMTV proviruses appear to have undergone alterations or deletions when compared with MMTV exogenous proviral DNA. We have thus made a comprehensive characterization of MMTV loci in these mouse strains which could serve as a basis for the study of their differences in expression.