Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory ciliated epithelial cells. Cilium adherence is mediated by P97, a surface protein containing a repeating element (R1) that is responsible for binding. Here, we show that the cilium adhesin is proteolytically processed on the surface. Proteomic analysis of strain J proteins identified cleavage products of 22, 28, 66, and 94 kDa. N-terminal sequencing showed that the 66-and 94-kDa proteins possessed identical N termini and that the 66-kDa variant was generated by cleavage of the 28-kDa product from the C terminus. The 22-kDa product represented the N-terminal 195 amino acids of the cilium adhesin preprotein, confirming that the hydrophobic leader signal sequence is not cleaved during translocation across the membrane. Comparative studies of M. hyopneumoniae strain 232 showed that the major cleavage products of the cilium adhesin are similar, although P22 and P28 appear to be processed further in strain 232. Immunoblotting studies using antisera raised against peptide sequences within P22 and P66/P94 indicate that processing is complex, with cleavage occurring at different frequencies within multiple sites, and is strain specific. Immunogold electron microscopy showed that fragments containing the cilium-binding site remained associated with the cell surface whereas cleavage products not containing the R1 element were located elsewhere. Not all secreted proteins undergo multiple cleavage, however, as evidenced by the analysis of the P102 gene product. The ability of M. hyopneumoniae to selectively cleave its secreted proteins provides this pathogen with a remarkable capacity to alter its surface architecture.Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, significantly impacts swine production (28). During colonization, M. hyopneumoniae forms an intricate association with the ciliated epithelial lining of the porcine respiratory tract, leading to chronic respiratory disease. Colonization disrupts the normal function of the mucociliary escalator through ciliostasis, loss of cilia, epithelial cell death, and acute inflammation. This results in a purulent exudate (composed primarily of neutrophils and mononuclear cells) in the airways (17). Disease resolution occurs only after a prolonged period (if at all). M. hyopneumoniae colonization also predisposes the host to more-severe infections from secondary pathogens (2). For example, it is now clear that colonization by M. hyopneumoniae leads to more-severe and longer-lasting disease with the porcine respiratory and reproductive syndrome virus (34). Thus, the impact of M. hyopneumoniae on swine production has not been fully realized.It is known that the initial event in colonization by M. hyopneumoniae is binding to swine respiratory cilia (19,32). In the absence of binding activity, colonization does not occur (38). Identification of the molecules involved in cilium binding occurred only after the discovery of adherence-blocking monoclonal antibod...

SummaryMycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 ( and F3P216 adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.

The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolyticuremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (␣1, ␣2, ␤, ␥, , , , , , , and ) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int-, Int-, and Int-. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx ؊ ) and ehxA-positive (ehxA ؉ ) isolates, and 42 were stx ؊ and ehxA-negative isolates. Int-␤, the most commonly identified eae subtype (82 of

A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 andstx2 ), intimin (eaeA), and enterohemolysin A (hlyA) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx1 and hlyA were the most commonly encountered virulence factors.

Class 1 integrons play a role in the emergence of multi-resistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance genes. 512 E. coli strains sourced from humans (n = 202), animals (n = 304) and the environment (n = 6) were screened for the presence of the intI1 gene. In 31/79 integron positive E. coli strains, the gene cassette regions could not be PCR amplified using standard primers. DNA sequence analysis of 6 serologically diverse strains revealed atypical integrons harboured the dfrA5 cassette gene and only 24 bp of the integron 3′-conserved segment (CS) remained, due to the insertion of IS26. PCR targeting intI1 and IS26 followed by restriction fragment length polymorphism (RFLP) analysis identified the integron-dfrA5-IS26 element in 27 E. coli strains of bovine origin and 4 strains of human origin. Southern hybridization and transformation studies revealed the integron-dfrA5-IS26 gene arrangement was either chromosomally located or plasmid borne. Plasmid location in 4/9 E. coli strains and PCR linkage of Tn21 transposition genes with the intI1 gene in 20/31 strains, suggests this element is readily disseminated by horizontal transfer.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena kucocephala var. Peru or siratro plants. The ability of these Exo-mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod-Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo-mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.A complex multistep interaction between the soil bacterium Rhizobium and specific leguminous plants results in the induction of nitrogen-fixing nodules on legume roots (19 and references therein). The early steps of the interaction are characterized by the distortion or curling of the root hair cells. The cell walls of the root hairs are penetrated after 24 h by a compatible Rhizobium strain, and an infection thread is synthesized by the plant after the nucleus of this cell has migrated to the infection site (4, 17). The bacteria are carried toward the root cortex inside the infection thread, where they actively divide. Shortly before or concurrent with initiation of infection thread synthesis, cortical cell division is thought to be induced by the Rhizobium strain, presumably by diffusible substances released by the bacterium (1, 2).Another feature of the Rhizobium-legume interaction is the host specificity displayed. Fast-growing ("temperate") Rhizobium strains, for example, usually nodulate only one plant species effectively, whereas slow-growing Bradyrhizobium strains typically have a broad host range. In contrast, the fast-growing Rhizobium sp. strain NGR234 (28) possesses an unusually extensive host range, which includes a variety of tropical and temperature legumes as well as the nonlegume tropical tree Parasponia andersonii (29).Since the initial interaction between the symbionts occurs at the surface of the two organisms, cell surface molecules may be important in determining the outcome of the infection. Rhizobia characteristically produce large amounts of exopolysaccharides (EPSs) on various laboratory media, and the colonies formed are mucoid (Muc+) Rhizobium polysaccharides, particularly EPSs and lipopolysaccharides, have been postulated to be involved in the infection and nodulation of legumes (24, 25), including specific adhesion to the root hair surfaces (9) and the determination of host specificity (13). Exo-mutants of Rhizobium meliloti are apparently affected at an early stage of infection (15). In other species, EPS m...

A pair of oligonucleotide primers (MP1 and MP2) were used for the polymerase chain reaction (PCR) amplification of a 486 base pair (bp) fragment of the 16S rRNA gene of 26 geographically diverse Australian Melissococcus pluton (causative agent of European foulbrood) isolates. PCR primers spanning a region of the 16S rRNA gene from position 893-1377 failed to amplify a product when template DNA from a wide range of pathogenic and saprophytic bacteria were used including Paenibacillus larvae, Paenibacillus alve~ Enterococcus faecium and Spiroplasma melliferum. The PCR did, however; reliably amplify a 486 bp fragment (when the annealing temperature was lowered by 5°C) using template DNA isolated from the phylagenetically-related bacterium Enterococcus faecalis. PCR amplicons generated from E faecalis and M. pluton were readily distinguished by digestion with the restriction endonuclease Hinfl and electrophoresis in 1.5% agarose or by electrophoresis in 1% agarose containing bisbenzidene/polyethylene glycol. A hemi-nested PCR requiring a combination of primers MP1 and a third primer, MP3, which spanned 25 nucleotides from position 1168--1144 and internal to the 486 bp amplicon generated by primers MP1 and MP2 was developed. The hemi-nested PCR amplified a 276 bp M. plutonspecific product that was not amplified with E faecalis DNA. In sensitivity studies, the PCR assay could reliably detect approximately 1-10 organisms/mi. This level of sensitivity was achieved using crude DNA templates (boiled cell lysate) prepared using lnstagene matrix. The PCR assay could also detect M. pluton in brood with European foulbrood. Downloaded by [University of Birmingham] at 08

Full genome sequences of 20 strains of Clostridium chauvoei, the etiological agent of blackleg of cattle and sheep, isolated from four different continents over a period of 64 years (1951–2015) were determined and analyzed. The study reveals that the genome of the species C. chauvoei is highly homogeneous compared to the closely related species C. perfringens, a widespread pathogen that affects human and many animal species. Analysis of the CRISPR locus is sufficient to differentiate most C. chauvoei strains and is the most heterogenous region in the genome, containing in total 187 different spacer elements that are distributed as 30 – 77 copies in the various strains. Some genetic differences are found in the 3 allelic variants of fliC1, fliC2 and fliC3 genes that encode structural flagellin proteins, and certain strains do only contain one or two alleles. However, the major virulence genes including the highly toxic C.chauvoei toxin A, the sialidase and the two hyaluronidases are fully conserved as are the metabolic and structural genes of C. chauvoei. These data indicate that C. chauvoei is a strict ruminant-associated pathogen that has reached a dead end in its evolution.