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.

Aims:  To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results:  Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 105 to 2·6 × 1011 cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions:  Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study:  More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.

We investigated the prevalence and genotypes of Cryptosporidium parasite in 50 brown rats (Rattus norvegicus) inhabiting an urban area of Japan. Fecal samples collected from the animals were examined by an immuno-fluorescence assay (IFA). Genomic DNA was extracted directly from fecal sample of each animal and nested PCR was performed to amplify part of the 18S ribosomal RNA (18SrRNA) of the Cryptosporidium species. The detection rate was 8% by IFA and 38% by nested PCR. The sequence and phylogenetic analyses of 13 PCR products showed that the Cryptosporidium from brown rats were clustered into four distinct genotypes. Interestingly, one of the four genotypes was significantly distinct from the C. parvum and C. hominis genotypes. Our results suggest the existence of a new genotype of Cryptosporidium in brown rats.

Legionnaires’ disease, predominantly caused by the bacterium Legionella pneumophila, has increased in prevalence worldwide. The most common mode of transmission of Legionella is inhalation of contaminated aerosols, such as those generated by cooling towers. Simple, rapid and accurate methods to enumerate L. pneumophila are required to prevent the spread of this organism. Here, we applied a microfluidic device for on-chip fluorescent staining and semi-automated counting of L. pneumophila in cooling tower water. We also constructed a portable system for rapid on-site monitoring and used it to enumerate target bacterial cells rapidly flowing in the microchannel. A fluorescently-labelled polyclonal antibody was used for the selective detection of L. pneumophila serogroup 1 in the samples. The counts of L. pneumophila in cooling tower water obtained using the system and fluorescence microscopy were similar. The detection limit of the system was 104 cells/ml, but lower numbers of L. pneumophila cells (101 to 103 cells/ml) could be detected following concentration of 0.5–3 L of the water sample by filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip staining and counting), and portable for on-site measurement, and it may therefore be effective in the initial screening of Legionella contamination in freshwater.

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.

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.

No abstract

In order to understand the habitation conditions of the bacteria of the genus Legionella in Noyu (unattended natural hot springs in mountains and fields) in Japan, isolation of Legionella spp. was attempted in the Noyu samples from 11 prefectures nationwide between May and September 2012, and the following results were obtained. Overall, Legionella spp. was isolated from 16 of 43 samples (37.2%). The species was isolated from the Hokkaido region to the Chugoku region but not from the Shikoku region to the Kyushu region. The number of bacteria detected was usually small, less than 5.0 × 10(1) CFU/100 ml, as found in 11 samples (68.8%), while counts of 10(2) or more to 10(3) or less CFU/100 ml were found in two samples (12.5%). Legionella pneumophila was the most commonly found strain, with 19 strains (90.5%) found, and was the dominant species. Regarding the serogrouping, four strains (21.1%) fell under group 1, the most common grouping, followed by three strains (15.8%) in group 3, two strains (10.5%) in group 5, etc. Moreover, the detected bacterial strains other than L. pneumophila included two strains (9.5%) of L. londiniensis. The temperature of the Noyu from which Legionella spp. was isolated was between 33.1°C and 41.5°C with a pH ranging from 5.2 to 8.1. The present report is the first report to clarify the habitation conditions of strains of Legionella spp. isolated from Noyu in Japan.