f For exhaustive detection of diarrheagenic Escherichia coli, we previously developed a colony-hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR. To assess the role of domestic animals as the source of atypical enteropathogenic E. coli (aEPEC), a total of 679 samples (333 from foods, fecal samples from 227 domestic animals, and 119 from healthy people) were examined. Combining 48 strains previously isolated from patients and carriers, 159 aEPEC strains were classified by phylogroup, virulence profile, and intimin typing. Phylogroup B1 was significantly more prevalent among aEPEC from patients (50%) and bovine samples (79%) than from healthy carriers (16%) and swine strains (23%), respectively. Intimin type 1 was predominant in phylogroup B1; B1-1 strains comprised 26% of bovine strains and 25% of patient strains. The virulence profile groups Ia and Ib were also observed more frequently among bovine strains than among porcine strains. Similarly, virulence group Ia was detected more frequently among patient strains than strains of healthy carriers. A total of 85 strains belonged to virulence group I, and 63 of these strains (74%) belonged to phylogroup B1. The present study suggests that the etiologically important aEPEC in diarrheal patients could be distinguished from aEPEC strains indigenous to humans based on type, such as B1, Ia, and 1/␥1, which are shared with bovine strains, while the aEPEC strains in healthy humans are different, and some of these were also present in porcine samples.
The public health importance of Kudoa infection in fish remains unclear. Recently in Japan a Kudoa species, K. septempunctata, was newly implicated as a causative agent of unidentified food poisoning related to the consumption of raw olive flounder. Other marine fishery products are also suspected as causative raw foods of unidentified food poisoning. For this study, we detected kudoid parasites from sliced raw muscle tissues of a young Pacific bluefin and an adult yellowfin tuna. No cyst or pseudocyst was evident in muscles macroscopically, but pseudocysts were detected in both samples histologically. One substitution (within 1100 bp overlap) and ten substitutions (within 753 bp overlap) were found respectively between the partial sequences of 18S and 28S rDNAs from both isolates. Nucleotide sequence similarity searching of 18S and 28S rDNAs from both isolates showed the highest identity with those of K. neothunni from tuna. Based on the spore morphology, the mode of parasitism, and the nucleotide sequence similarity, these isolates from a Pacific bluefin and a yellowfin tuna were identified as K. neothunni. Phylogenetic analysis of the 28S rDNA sequence revealed that K. neothunni is classifiable into two genotypes: one from Pacific bluefin and the other from yellowfin tuna. Recently, an unidentified kudoid parasite morphologically and genetically similar K. neothunni were detected from stocked tuna samples in unidentified food poisoning cases in Japan. The possibility exists that K. neothunni, especially from the Pacific bluefin tuna, causes food poisoning, as does K. septempunctata.
Classification and identification of muscle-parasitizing didymozoids found in marine fish is difficult because of their novel parasitism and morphology. Recent sequence analysis has helped, but only seven sequences are available. Therefore, the usefulness of molecular methods for differentiation of muscle-parasitizing didymozoids, as well as genetic differences between the muscle and the other site-parasitizing didymozoids are quite unclear. In the present study, six unidentified didymozoid isolates from the trunk muscles of four marine fish species (Diagramma pictum, Plectorhinchus cinctus, Pagrus major and Cypselurus heterurus) were examined genetically using sequence analysis (18S rDNA, 28S rDNA, ITS-2 and coxI). All isolates were placed phylogenetically as a lineage independent of other site-parasitizing didymozoids at 18S rDNA, ITS-2 and coxI. They were grouped into three distinct lineages. The present and the previous unidentified or identified didymozoids from trunk muscles were found to differ clearly for every host species by sequence analysis, suggesting that muscle-parasitizing didymozoids are host-specific. This report is the first describing the molecular characteristics of muscle-parasitizing didymozoids by sequence analysis targeting the nuclear and mitochondrial DNA loci, which is proposed as a superior method for didymozoid differentiation.
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