The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n ‫؍‬ 124), while no PCR amplicons were found in other vibrios or related genera (n ‫؍‬ 50). High levels (10 6 to 10 10 CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10 8 V. parahaemolyticus cells or 10 9 E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35؇C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.

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Enteroviruses can be introduced into the water environment as a result of human activity. Contaminations within hot tubs, spas and public baths are also possible. We investigated the distribution of enteroviruses at six hot spring recreation areas throughout Taiwan. Spring water was collected from 34 sites and enteroviruses were detected in 13 (38.2%). The most frequently detected was coxsackievirus A2, followed by echovirus 11. Enterovirus 71 (EV 71) and porcine enterovirus 9 were detected once. Water quality indicators were not statistically associated with the occurrence of enteroviruses, although the enterovirus-positive samples were positive for a greater number of microbiological indicators and showed a link to pH and water temperature. The results confirm the ubiquity of enteroviruses in Taiwan spring recreation areas. Coxsackievirus A2, echovirus 11 and EV 71, the enteroviruses responsible for disease outbreaks identified at these sites, should be considered a potential public health threat in spring recreation areas of Taiwan.

Municipal wastewater treatment plants play a crucial role in reducing the microbial and pathogen load of human wastes before the end-products are discharged to surface waters (final effluent) or land spread (biosolids). This study investigated the occurrence frequency of noroviruses,

BackgroundThe human norovirus (NV) circulates worldwide and is a major cause of epidemics, which have increased in Taiwan since 2002. NV in acute gastroenteritis (AGE) and non-acute gastroenteritis (asymptomatic) patients, including children and adults, have not been previously examined in Taiwan; therefore, we examined the epidemiology and phylogeny of NV in AGE and asymptomatic patients of all ages.Methods253 stool samples were collected from August 2011 to July 2012 (including 155 AGE and 98 asymptomatic samples in Taiwan) and analyzed using reverse transcription-polymerase chain reaction (RT-PCR) for NV. Primers targeting the RNA-polymerase gene were used for RT-PCR to allow DNA sequencing of Taiwan NV strains and phylogenetic analyses.ResultsNV was detected in 24 (9.5%) of 253 stool specimens using RT-PCR. NV was isolated from all age groups (1 to 86 y) and those NV-positive samples were major identified from inpatients (79.2%, 19/24). Statistical analysis showed that the NV infectious rate of AGE patients was statistically significant (P < 0.05) for age, season and water type, respectively. Phylogenetic analyses of the RdRp region sequence showed that 24 NV isolates belonged to Genogroup II Genotype 4 (GII.4). They were closely related to the epidemic strain in Taiwan in 2006, the GII.4-2006b pandemic strain in 2006, and the GII.4-New Orleans strain in 2010.ConclusionThis study is the first to examine NV in sporadic AGE and asymptomatic patients in Taiwan. Furthermore, epidemic strains of isolated GII.4 were predominant in Taiwan during 2011 and 2012.

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