Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples

Babesia microti-Group Parasites Compared Phylogenetically by Complete Sequencing of the CCT Gene in 36 Isolates

Babesia microti, the erythroparasitic cause of human babesiosis, has long been taken to be a single species because classification by parasite morphology and host spectrum blurred distinctions between the parasites. Phylogenetic analyses of the 18S ribosomal RNA gene (18S rDNA) and, more recently, the β-tubulin gene have suggested inter-group heterogeneity. Intra-group relationships, however, remain unknown. This study was conducted to clarify the intra- and inter-group phylogenetic features of the B. microti-group parasites with the η subunit of the chaperonin-containing t-complex polypeptide l (CCTη) gene as a candidate genetic marker for defining the B. microti group. We prepared complete sequences of the CCTη gene from 36 piroplasms and compared the phylogenetic trees. The B. microti-group parasites clustered in a monophyletic assemblage separate from the Babesia sensu stricto and Theileria genera and subdivided predominantly into 4 clades (U.S., Kobe, Hobetsu, Munich) with highly significant evolutionary distances between the clades. B. rodhaini branched at the base of the B. microti-group parasites. In addition, a unique intron presence/absence matrix not observable in 18S rDNA or β-tubulin set the B. microti group entirely apart from either Babesia sensu stricto or Theileria. These results have strong implications for public health, suggesting that the B. microti-group parasites are a full-fledged genus comprising, for now, four core species, i.e., U.S., Kobe, Hobetsu, and Munich species nova. Furthermore, the CCTη gene is an instructive and definitive genetic marker for analyzing B. microti and related parasites

Intron sequences from the CCT7 gene exhibit diverse evolutionary histories among the four lineages within the Babesia microti-group, a genetically related species complex that includes human pathogens

Babesia microti, the primary causal agent of human babesiosis in North America, was thought to distribute in Europe in association with ixodid ticks and rodents. Recent analyses of β-tubulin and the eta subunit of the chaperonin-containing t-complex protein 1 (CCT7) genes revealed discrete clusters (a species-complex comprised of at least 4 taxa for the U.S., Kobe, Munich, and Hobetsu). To further assess the micro-evolutionary history and genetic variability within the taxon, we combined a set of 6 introns from the CCT7 gene to use as a rapidly evolving DNA marker. Phylogenetic and comparative sequence analyses subdivided the U.S. taxon into 3 geographic subclades-North America, western to central Eurasia, and northeastern Eurasia (≥98% bootstrap supports for each node). The Kobe taxon, which occurs only in a few geographic foci of Japan, could further be subdivided into 2 subgroups (100% support). The Munich and Hobetsu taxa, common to Europe and Japan, respectively, exhibited little or no pairwise sequence divergence among geographically diverse samples, suggesting an extreme population bottleneck during recent history. Despite the small sample size, this study provides a better understanding of the micro-evolutionary relationships and the genetic variability present within each lineage of the B. microti-group

Molecular evidence of the multiple genotype infection of a wild Hokkaido brown bear (Ursus arctos yesoensis) by Babesia sp. UR1

A frozen-stored blood clot of a wild brown bear cub Ursus arctos yesoensis that had been captured in Hokkaido, Japan was examined for piroplasma infection using polymerase chain reaction (PCR). Two 18S ribosomal RNA gene (SSU rDNA) sequences were generated. One 1565-bp sequence showed the highest similarity with B. gibsoni (95.9% identity) but, phylogenetically, was found to belong to a distinct lineage. The other sequence (1709-bp) could not be definitively assigned to a described taxon, sharing only limited homology to the closest named species (90.1% identity with C. felis). In order to enhance information obtained from the SSU rDNA sequence, further detection and sequence analysis of the CCTη gene sequence were done revealing the simultaneous presence of three closely related genotypes (all in a monophyletic lineage) within a single bear host. This finding suggested the possibility that a new Babesia species (Babesia sp. UR1) might have been maintained in nature in wild brown bears. While the parasite\u27s biology is yet unknown, to our knowledge, this is, excepting the single case documentation in 1910 of a hemoparasite in a bear at Russian zoo, the first reported case of piroplasms inhabiting a bear species