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.

A group of 1,623 ovine fecal samples recovered from 65 geographically distinct mutton sheep and prime lamb properties across New South Wales, Australia, were screened for the presence of Shiga toxin-producing Escherichia coli (STEC) virulence factors (stx 1 , stx 2 , eaeA, and ehxA). A subset was cultured for STEC isolates containing associated virulence factors (eaeA and/or ehxA), which were isolated from 17 of 20 (85%) and 19 of 20 (95%) tested prime lamb and mutton sheep properties, respectively. STEC isolates containing stx 1 , stx 2 , and ehxA were most commonly isolated (19 of 40 flocks; 47.5%), and this profile was observed for 10 different serotypes. Among 90 STEC isolates studied, the most common serotypes were O91:H Although isolates belonging to serogroup O157 are regarded as the most clinically significant Shiga toxin-producing Escherichia coli (STEC) strains, the number of non-O157 serotypes recovered from episodes of hemorrhagic colitis and hemolyticuremic syndrome (HUS) continues to increase. Currently over 160 serotypes of E. coli have been isolated from human sources (5,6,13,14,25). Ruminants, in particular (6,7,16,24), but also other domestic animals, including pigs, poultry, cats, and dogs (1,6,8), are natural reservoirs of STEC. Although more than 200 different STEC serotypes have been isolated from cattle (reference 13 and references therein), few studies have extensively examined the presence of STEC in sheep. Existing studies have been performed on comparatively small numbers of sheep or have focused intensively on a single flock or only examined the presence of O157 serotypes (3,12,16,17). Kudva et al. (17) investigated the presence of E. coli O157:H7 in a single flock over a 16-month period and described the presence of non-O157 STEC isolates of serotypes O128:NM, O5:NM, O6:H49, O88:NM, and O91:NM, with various combinations of the virulence-associated genes stx 1 , stx 2 , and eaeA. In Australia, one of the largest sheep-producing countries in the world, recent studies focusing primarily on the southeastern parts of Queensland have suggested that the prevalence of stx in fecal cultures ranges from 69 to 88% (12,22).In this study, we investigated the presence of STEC in fecal enrichment broths derived from 65 geographically diverse flocks of slaughter-age sheep (mutton sheep and prime lambs) in New South Wales, Australia, using a multiplex PCR which detects stx 1 , stx 2 , eaeA, and ehxA (11) and vancomycin-cefixime-cefsulodin blood agar (BVCCA) (18). Mutton sheep and prime lambs represent two different genetic lines of meatproducing animal and comprise sheep of different slaughter ages and production systems, parameters which have been reported to influence STEC colonization in ruminant species (26). Our aim was to isolate STEC strains that contained at least one other virulence factor (eaeA and/or ehxA). These STEC isolates were serotyped and examined for the ability to express Shiga toxins using Vero cell cultures. Commercial properties from 29 prime lamb and 36 mutton sheep flocks we...

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.

Unlike Shiga toxin 2 (stx 2 ) genes, most nucleotide sequences of Shiga toxin 1 (stx 1 ) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx 1 among STEC isolates derived from human or animal sources. However stx 1OX3 , originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx 1 subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx 1c . In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx 1c from other stx 1 subtypes. The PCR-RFLP assay was used to study 378 stx 1 -containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx 2 ); the combination stx 1 , stx 2 , and ehxA was the most common (175 of

-Polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis and microscopy were used to test 307 adult bee and 37 honey samples collected in Australia for the presence of two microsporidia, Nosema ceranae and Nosema apis. N. ceranae was detected in samples from 4 states (Queensland, New South Wales, Victoria and South Australia) and was most commonly found in samples from Queensland where 28 (33.7%) of 83 samples were positive. New South Wales had the second highest prevalence with 15 (15.8%) of 95 samples positive. South Australia and Victoria had 4 (16%) of 25 and 2 (4.5%) of 44 samples positive respectively. N. ceranae was not detected in samples from Western Australia and Tasmania. N. apis was detected in samples from all states. Three honey samples (8.1%) were PCR positive for N. ceranae. These positive honey samples originated from beekeepers in Queensland. Six imported honey samples tested were negative for both Nosema spp.Nosema ceranae / Nosema apis / nosemosis / Apis mellifera / PCR / RFLP

Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx 2 subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans.

European foulbrood (EFB) is a severe bacterial honey bee brood disease caused by the Gram-positive bacterium Melissocccus plutonius. The disease is widely distributed worldwide, and is an increasing problem in some areas. Although the causative agent of EFB was described almost a century ago, many basic aspects of its pathogenesis are still unknown. Earlier studies were hampered by insensitive and unspecific methods such as culture based techniques. Recent advances in molecular technology are making it increasingly easy to detect and characterize microbes, and nucleic acid detection technologies are quickly displacing the traditional phenotypic assays in microbiology. This paper presents selected methodologies which focus on EFB and its causative agent M. plutonius. Métodos estándar para la investigación sobre la loque europea ResumenLa loque europea (LE) es una grave enfermedad bacteriana de la cría de la abeja de la miel causada por la bacteria Gram-positiva Melissococcus plutonius. La enfermedad se encuentra ampliamente distribuida en todo el mundo y es un problema creciente en algunas áreas.Aunque el agente causante de la LE fue descrito hace casi un siglo, muchos aspectos básicos de su patogénesis son aún desconocidos.Estudios anteriores se vieron obstaculizados por métodos poco sensibles e inespecíficos, tales como las técnicas basadas en cultivos. Los recientes avances en la tecnología molecular están haciendo cada vez más fácil la detección y caracterización de los microbios, y las tecnologías de detección de ácidos nucleícos están desplazando rápidamente a los ensayos fenotípicos tradicionales de microbiología. Este artículo presenta algunas metodologías seleccionadas que se centran en LE y en su agente causal M. plutonius. 欧洲幼虫腐臭病研究的标准方法 欧洲幼虫腐臭病是蜜蜂幼虫病中较为严重的细菌病，由革兰氏阳性细菌Melissocccus plutonium 引起。欧洲幼虫腐臭病在世界范围内广泛分布， 成为一些地区日益突出的主要病害。虽然一个世纪前就已有相关病原体的描述，但早期研究受实验技术不灵敏、特异性不好的限制(比如细菌培 养的技术)，其发病机理至今未能全面揭示。近年来随着分子技术的发展检测微生物日益容易，核酸检测技术快速替换了传统的微生物学表型实 验。本文展示了近期开展欧洲幼虫腐臭病及其病原体 M. plutonius方面的常用技术。