We surveyed Rickettsiales bacteria, including Rickettsia, Ehrlichia, Anaplasma, and Neoehrlichia, in wild sika deer (Cervus nippon nippon) from Shizuoka prefecture, Japan. In spleen samples from 187 deer, Anaplasma phagocytophilum (deer type), A. bovis, and A. centrale were successfully detected by PCR assay targeting to 16S rDNA or p44/msp2, and their positive rates were 96.3% (180/187), 53.5% (100/187), and 78.1% (146/187), respectively. Additionally, 2 or 3 Anaplasma species could be detected from a single deer in 165 spleen samples (88.2%), indicating dual or triple infection. In contrast, A. phagocytophilum (human type) 16S rDNA, Rickettsia gltA, Ehrlichia p28/omp-1, and Neoehrlichia 16S rDNA could not be amplified. The serological test of 105 deer serum samples by immunofluorescence assay showed that the detection of antibodies against antigens of A. phagocytophilum HZ (US-human isolate) and Rickettsia japonica YH were 29.5% (31/105) and 75.2% (79/105), respectively. These findings suggest that A. phagocytophilum (deer type), A. centrale, and A. bovis are highly dominant and prevalent in wild sika deer from Shizuoka, a central region of Japan, and that the antibodies against some Rickettsiales bacteria have also been retained in deer blood.
Abstract Three strains of spirochetes (IKA1 to 3) were isolated from the midgut of Ixodes ovatus collected in the Ikawa region of the northern part of Shizuoka, Japan. These isolates had eight flagella, and their size and other morphological features were similar to Borrelia burgdorferi . They showed similar motility and reacted with monoclonal antibody (MAb) H9724 against borrelial flagella and with MAb H5332 against the outer surface protein A. These strains showed similar SDS‐PAGE profiles to that of B. burgdorferi strain B31 and P/Bi isolated in the U.S.A. and Europe, respectively. Immunoblot with Lyme disease patient serum showed positive reactions with the flagella (41 Kilodalton, kDa), protein C (20 to 22 kDa), and outer surface protein A (29 kDa) of the isolates. Immunological properties, morphological characteristics, and epidemiological features revealed that these isolates were B. burgdorferi .
Using the Tullgren funnel method, we surveyed for trombiculid larvae around dwellings and in mandarin orange groves on the slopes of Sengenyama and Maruyama, Yamakita Town, Kanagawa Prefecture, Japan, during the period between 8 October 1992 and 8 January 1993. In total, 6,690 trombiculid larvae, consisting of 6,669 Leptotrombidium scutellare (Nagayo, Mitamura, Tamiya & Tenjin), 13 L. fuji Kuwata, Berge & Philip, two L. himizu (Sasa, Kumada, Hayashi, Enomoto, Fukuzumi & Obata), two Neotrombicula japonica (Tanaka, Kaiwa, Teramura & Kagaya), one Gahrliepia saduski Womersley, and three Walchia ogatai Sasa & Teramura, were taken from 240 soil samples (40 samples, six times at intervals of 2–4 wk). From the above species composition, L. scutellare is suspected to be the sole vector of scrub typhus in the survey area, where the disease is known to be caused by two serotypic strains of Rickettsia tsutsugamushi Ogata, Kawasaki and Kuroki. L. scutellare larvae rapidly increased in number in late October to form an acuminate peak of abundance in early November, followed by a gradual wane.
Based on the previous data (Uchikawa et al., 1994), we presumed that Leptotrombidium scutellare larvae were distributed widely in man-made environments around densely populated areas inwestern Kanagawa Prefecture, Japan. Employing modified Suzuki's visual sampling method (Uchikawa et al., 1993), this assumption was examined during the period between November 4 and 6, 1993, in Yamakita Town and Minamiashigara City, Kanagawa Prefecture. The above assumption was verified, indicating at the same time that the method was appropriate for our purpose. Many L. scutellare larvae were frequently found on shaded and humid places but not on dry soil surface. Very heavily infested places were encountered in mandarin orange groves and, unexpectedly, in a young kiwi fruit orchard surrounding the center of Yamakita Town with crowded dwellings, and in mandarin orange groves on gently rolling hills of Minamiashigara City. As many as 9, 658 unfed larvae to use in other analyses were taken on sampling and collecting pieces of black cloth during the survey. These larvae were observed to have stayed still in clusters on the cloths sealed in plastic bags that had been kept in a container since the previous day.
Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.
The multilocus variable-number tandem repeat analysis (MLVA) method to target eight variable-number tandem repeat loci, based on agarose gel electrophoresis separation of multiplexed PCR products, and the PFGE method were applied to clinical isolates of Escherichia coli O157 : H7 with the aim of comparing their performance as methods of typing this bacterium. Using MLVA, a total of 57 isolates from patients in Shizuoka prefecture, Japan, were divided into 20 types and classified into 23 PFGE types. Twenty-four isolates from four sporadic infections, four household contact infections and one outbreak that occurred in central parts of Shizuoka prefecture during August to November in 2005 were shown to be the same MLVA type, and most of the isolates had identical PFGE banding patterns, suggesting the diffuse outbreak in these parts of Japan. Thus, there was a good correlation between MLVA types and PFGE types, with both methods displaying broadly similar discriminatory powers. However, the MLVA typing proved to be a much easier and more rapid method for the analysis of E. coli O157 : H7 strain relatedness to identify transmission routes. Hence, our MLVA method would be a suitable technique for routine typing in many laboratories, including public health agencies, and even in hospitals.
Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.
Latin American countries produce more than a quarter of the world’s beef and are a major global supplier of livestock protein. Tick-borne diseases (TBDs) are a major constraint to the livestock industry worldwide, including in Latin America. The aim of this study was to detect and characterise tick-borne pathogens in cattle from Santa Cruz, Bolivia, where no detailed epidemiological data are available. Blood samples were collected from 104 cattle. Apicomplexan parasites were detected by nested PCR amplification of the 18S ribosomal RNA gene (rDNA), and Anaplasmataceae was screened by the PCR amplification of 16S rDNA, followed by characterisation based on the heat shock protein and citrate synthase gene sequences. Babesia infection was observed in nine cattle (one Babesia bovis and eight Babesia bigemina), while Anaplasmataceae infection was detected in thirty-two cattle. A sequencing analysis confirmed the presence of Anaplasma marginale and Anaplasma platys-like. These results provide the first molecular evidence for the four above-mentioned tick-borne pathogens in cattle in Bolivia. This information improves our understanding of the epidemiology of TBDs and will help in formulating appropriate and improved pathogen control strategies.
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