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.

Longitudinal study on respiratory viral co-infections in the presence or absence of clinical manifestation in infants aged 0-2 years.

We conducted a long-term follow-up study between December 2005 and February 2007 on 4 immunocompetent infants, who repeatedly presented with respiratory symptoms, using PCR-based techniques targeting 14 viruses related to acute respiratory tract infection. Of 38 specimens, 30 were collected from symptomatic infants and 8 were collected when respiratory symptoms were absent. Overall, one or more respiratory viruses were detected in 94.7% (36/38) of the specimens. Of the 36 PCR-positive specimens, 77.8% (28/36) were positive for more than one virus. Most of these co-infections were double infections (55.6% or 20/36). Of note, co-infections with 4 and 3 viruses were observed in 3 (8.3% or 3/36) and 5 (13.9% or 5/36) specimens, respectively. Of the 8 specimens collected from the 4 infants when apparent respiratory symptoms were absent, 7 (87.5%) were positive for respiratory viruses. Respiratory viral co-infections were also frequent and found in 5 of the specimens (62.5%). However, apparent correlation between disease severity and co-infection was undetectable due to the limit of the number of cases studied. Taken together, this longitudinal study revealed that respiratory viral co-infections were not infrequent in infants aged 0-2 years, regardless of the presence of respiratory symptoms (62.5-77.8%).

A coprological survey of intestinal helminthes in stray dogs captured in osaka prefecture, Japan.

This study aimed to investigate intestinal helminth infection in stray dogs in Osaka Prefecture by surveying coprological samples from dogs captured from 2006-2011. Of 212 fecal samples collected, overall prevalence of infection was 39.2%. The most common species was Toxocara canis (25.0%), followed by Trichuris vulpis (8.0%), Spirometra erinaceieuropaei (3.3%), Taeniidae (2.4%), Ancylostoma caninum (1.9%) and Toxascaris leonine (0.5%). In the molecular analysis, all of the taeniid eggs were negative for Echinococcus multilocularis and were identified as other taeniid species (e.g., Taenia pisiformis). Our results suggest that stray dogs remain important infection reservoirs of zoonotic parasites in Osaka Prefecture. Therefore, control of stray dogs is crucial for reducing the risk of public health problems due to parasitic infections.

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 ß-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 CCTeta 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's 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.

Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan.

Here, we carried out a survey to determine the prevalence of free-living amoebae (FLA) in tap-water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 374 raw water samples were collected from 113 sampling points. The samples were filtrated and transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli and incubated for 2 to 7 days at 30 degrees C or 42 degrees C. The plates were examined by microscopy to morphologically identify FLA families, and polymerase chain reaction and sequence analysis were then performed to define the species of the detected Naegleria and Acanthamoeba isolates. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy, and among these there were 800 FLA isolates, including Acanthamoeba and Naegleria species. Sequence analysis identified five Acanthamoeba spp. isolates of the known pathogenic T4 genotype and 43 Naegleria australiensis isolates, a reported pathogen to mice and also of concern as a potential pathogen to humans. Our results suggest a wide distribution of FLA, including potential pathogenic species, in tap-water sources of western Japan.

Molecular evidence for the presence of new Babesia species in feral raccoons (Procyon lotor) in Hokkaido, Japan.

We recently reported that feral raccoons (Procyon lotor) with splenomegaly native to Japan were carriers of a Babesia microti-like parasite identical to that found in the United States, which was likely introduced to Japan from North America via raccoons imported as pets. Thus, we attempted extensive molecular survey for piroplasma infections of feral raccoon with normal spleen in Hokkaido, Japan using nested PCR that target broadly to 18S ribosomal RNA gene (SSU-rDNA) of all the parasites in the genus Babesia, Theileria, Cytauxzoon and B. microti group. Of the 348 raccoon samples analyzed, 9 gave positive signals. Cloning and phylogenetic analysis on SSU-rDNA sequences revealed that six of nine positives were found to be infected with Babesia and the remaining three with previously unreported Sarcocystis. Babesia sequences were further separated into two distantly related groups, those that reside in a novel phylogenetic group were consisted solely of four parasites found in this study, while those which included one identical sequence found in the three of our specimens were assembled together with both Babesia parasites of tick's in Japan and of raccoon's in U.S. These results may indicate that not only a B. microti-like parasite but also at least two yet undescribed Babesia species are being established in their new life cycles in the feral raccoon populations in Japan.

Babesia microti-group parasites compared phylogenetically by complete sequencing of the CCTeta 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 beta-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 eta subunit of the chaperonin-containing t-complex polypeptide l (CCTeta) gene as a candidate genetic marker for defining the B. microti group. We prepared complete sequences of the CCTeta 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 beta-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 CCTeta gene is an instructive and definitive genetic marker for analyzing B. microti and related parasites.