The prevalence of Shiga toxin-producing Escherichia coli (STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with stx-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested stx-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony stx hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic E. coli (EHEC) hlyA genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.

A sudden outbreak of epidemic diarrhoea of piglets occurred in Japan, the principal features being watery diarrhoea, dehydration and high mortality in newborn animals. The microscopical lesions were villous atrophy in the small intestine, the villous enterocytes being vacuolated and cuboidal in shape. The villus-crypt ratio was severely reduced, varying from 1:1 to 3:1. Transmission electron microscopy showed numerous coronaviruses within the cytoplasm of enterocytes and among microvilli. Specific antigens of porcine epidemic diarrhoea (PED) virus were detected in the cytoplasm of enterocytes by the streptavidin-biotin (SAB) technique. Infected cells, which were most abundant in the villous epithelia of the jejunum and ileum, were present in small numbers in the large intestine, the crypt epithelia, the lamina propria and Peyer's patches. The study suggests that the SAB technique is useful for the diagnosis of PED.

Many bacterial pathogens encode ADP-ribosyltransferase toxins. The authors identified an ADP-ribosyltransferase toxin homologue (ArtA, ArtB) in Salmonella enterica serovar Typhimurium (S. typhimurium) DT104. ArtA is most homologous to a putative pertussis-like toxin subunit present in Salmonella typhi (STY1890) and Salmonella paratyphi A (SPA1609), while ArtB shows homology to a hypothetical periplasmic protein of S. typhi (STY1364) and S. paratyphi A (SPA1188), and a putative pertussis-like toxin subunit in S. typhi (STY1891) and S. paratyphi A (SPA1610). The artA gene was detected from the phage particle fraction upon mitomycin C induction, and the flanking region of artAB contains a prophage-like sequence, suggesting that these putative toxin genes reside within a prophage. Southern blotting analysis revealed that artA is conserved in 12 confirmed DT104 strains and in four related strains which are not phage-typed but are classified into the same group as DT104 by both amplified-fragment length polymorphism and pulsed-field gel electrophoresis. Except for one strain, NCTC 73, all 13 S. typhimurium strains which were classified into different groups from that of DT104 lacked the artA locus. The results suggest that phage-mediated recombination has resulted in the acquisition of art genes in S. typhimurium DT104 strains.

Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P ؉) ETEC strains from piglets, K99 ؉ ETEC strains from calves, and K99 ؉ F41 ؉ or F41 ؉ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab ؉ or K88ac ؉ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88 ؉ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type. Escherichia coli is a normal inhabitant of the intestinal tracts of animals. However, particular E. coli strains such as enterotoxigenic E. coli (ETEC) (10, 18, 32) and Vero cytotoxinproducing E. coli (30) are associated with diarrhea; ETEC has been proved to be a dominant bacterial agent of diarrheal diseases in piglets and calves (1, 14). Adherence to mucosa is the initial, essential step in the development of pathogenicity for diarrhea-associated E. coli (16). Most ETEC strains from piglets with diarrhea possess the pilus adherence factor K88 (including K88ab, K88ac, and K88ad), 987P, K99, or F41 (10, 32); of those, K88 is the predominant adherence factor (27, 34, 36). In contrast, ETEC strains from calves with diarrhea, in most cases, possess K99 alone or a combination of K99 and F41 (10, 32). ETEC strains elaborate heat-labile enterotoxin (LT) that is similar to cholera toxin and/or heat-stable enterotoxin I (STI) or II (STII) (3, 11). K88-positive (K88 ϩ) ETEC strains are usually LT ϩ , LT ϩ , STI ϩ , or LT ϩ STII ϩ , and K99 ϩ or 987P ϩ ETEC strains are STI ϩ in most cases (12, 22, 34). Some strains of enteroaggregative E. coli (EAggEC), the most recently recognized category of human diarrheagenic E. coli, elaborate an enterotoxin of 38 amino acids named EAggEC heat-stable enterotoxin 1 (EAST1) (28, 29). EAST1 of EAggEC and STI of ETEC are genetically and immunologically distinct enterotoxins (29). EAST1 is detected in the rabbit intestinal model (28), and STI is detected in suckling mice (4). EAST1 activates guanylate cyclase, as does STI (28). The EAST1 gene was also found in ETEC strains pathogenic for humans (37) or in a subgroup of diffusely adhering E. coli (DAEC) strains isolated from patients with diarrhea (39). In this study, we investigated the presence of the EAST1 gene in ETEC strains isolated from piglets and calves with diarrhea and determined the EAST1 gene sequences distributed a...

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.Salmonella infections in livestock have been a concern for both animal and human health. In particular, a common serotype causing salmonellosis in humans is Salmonella enterica serotype Typhimurium, a globally distributed zoonotic serotype that is common in both cattle and poultry. In order to study the epidemiology of its outbreaks and determine the source of contamination so that a recurrence can be avoided, detailed characterization is necessary. Although the majority of outbreaks in livestock are caused by a select number of serotypes, serotyping is not an adequate method for determination of the source of contamination during an outbreak. One subtyping method for epidemiological investigations of human and animal salmonellosis outbreaks is phage typing (3), which discriminates phenotypically at the intraserotype level. However, phage typing requires access to special reagents and a specialized laboratory and fails to reflect evolutionary relationships of bacterial strains. In the last decade, with the development of new techniques in molecular biology techniques, new approaches have become available. Widely used are plasmid analysis (29, 39), chromosomal fingerprinting by Southern hybridization (12,16,31,36,37), and macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) (...

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Isolates of the Salmonella enterica serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.Recently, Salmonella enterica serotype Typhimurium multidrug-resistant strain definitive phage type 104 (DT104) has emerged and spread over many countries (4,9,14,15). The organism has a core pattern of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. Previously, we used fluorescent amplified-fragment length polymorphism fingerprinting (FAFLP) analysis for molecular epidemiological investigation of serotype Typhimurium (13). Among 120 isolates from cattle, there were 17 FAFLP profiles that formed four distinct clusters (A to D). The isolates belonging to cluster A, in which all of the isolates of DT104 were included, have become increasingly common since 1992 in the northernmost island of Japan. The sequence of a polymorphic marker that is common to the strains of FAFLP cluster A has homology with the segment of the eae gene of phage P22. In this study, we isolated prophage ST104, which is common to isolates of DT104, and determined the whole sequence of this phage. The genomic architecture is similar to that of P22, and a number of regions are very similar to those of P22.Isolation of prophage common to serotype Typhimurium DT104. Forty-two serotype Typhimurium strains, including the 12 isolates of DT104 used in this study, were described previously (13). To investigate whether lysogenic prophages are present in serotype Typhimurium, we cultured the strains in the presence of mitomycin C at a concentration of 0.5 g/ml as previously described (17). Thirty-four out of 42 strains released phages that produced plaques on lawns of serotype Typhimurium strain LT2. To characterize the isolated phages, the restriction patterns of their DNAs were compared. Endonuclease digestion with EcoRI revealed five DNA types, designated a1, a2, b, c, and d (Fig. 1). All of the phages isolated from strains that belong to FAFLP cluster A (13), including 12 isolates of DT104, show the same restriction pattern, type a1 (Fig. 1). We have named this phage ST104. The restriction patterns of the prophages from strains NET25 and NET26, both of which belong to FAFLP cluster B, are similar to that of ST104; however, an additional EcoRI site was observed (Fig. 1). Therefore, the DNA type of these phages was designated a2. The restriction patterns of the phages isolated from strains belonging to FAFLP cluster B or C are different from those of ST104. No phage was detected in the two strains that belong to FAFLP cluster D. Schicklmaier et al. (10) suggested that prophage restriction patterns in natural isolates of serotype Typhimurium could serve as markers for the epidemiologic classification of pathogenic strains. Our resul...

A total of 46 Escherichia coli O157:H7 isolates were obtained from sequential faecal samples from seven cattle collected over periods of 2 months. Nine closely related genetic subtypes, determined by pulsed-field gel electrophoresis types using three kinds of restriction endonuclease were observed among the isolates. Distinguishable, but closely related genetic subtypes can be isolated from one farm, or from one cow, should be considered when undertaking an epidemiological survey.