Genetic diversity of cervid Trypanosoma theileri in Honshu sika deer (Cervus nippon) in Japan.

The taxonomy of ruminant Trypanosoma theileri and its relatives (Kinetoplastida: Trypanosomatidae) is controversial, with recent phylogenetic studies segregating T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers. In the present study, T. theileri-like trypanosomes isolated from Honshu sika deer (Cervus nippon) in the western Japan (YMG isolate) were genetically characterized using a number of genetic markers. Sika deer trypanosomes of the YMG isolate were genetically different from the Trypanosoma sp. TSD1 isolate previously recorded from Hokkaido sika deer in northern Japan, with the former trypanosome isolate being genetically closer to European cervid trypanosomes and the bovine T. theileri TthII lineage. In contrast, the latter isolate exhibited greater relatedness to North American cervid trypanosomes and the bovine T. theileri TthI lineage, although a clear genetic distinction between these was apparent. Furthermore, trypanosomes in Honshu sika deer from the central part of Japan harboured additional genetic diversity and were closer to either TSD1 or YMG isolates, while distinct from known T. theileri-related genotypes. Importantly, cervids and wild ruminants worldwide might harbour divergent descendants of a T. theileri ancestor, which exhibit rigid host specificity to either bovines or cervid species.

Integrative taxonomic approach of trypanosomes in the blood of rodents and soricids in Asian countries, with the description of three new species.

Trypanosoma lewisi (Kinetoplastea: Trypanosomatida: Trypanosomatidae) with a cosmopolitan distribution is the type species of the subgenus Herpetosoma, which includes ca. 50 nominal species isolated mainly from rodents. Since members of Herpetosoma in different host species have an almost identical morphology of bloodstream forms, these trypanosomes are referred to as 'T. lewisi-like', and the molecular genetic characterization of each species is necessary to verify their taxonomy. In the present study, we collected blood samples from 89 murid rodents of 15 species and 11 soricids of four species in Indonesia, Philippines, Vietnam, Taiwan, and mainland China for the detection of hemoprotozoan infection. T. lewisi and T. lewisi-like trypanosomes were found in the blood smears of 10 murid animals, which included Bandicota indica (two rats), Rattus argentiventer (one rat), and Rattus tiomanicus (two rats) in Indonesia; Rattus rattus (one rat) in the Philippines; and Niviventer confucianus (four rats) in mainland China. Furthermore, large- or medium-sized non-T. lewisi-like trypanosomes were detected in two soricids, Crocidura dracula in Vietnam and Anourosorex yamashinai in Taiwan, respectively. Molecular genetic characterization of the small subunit (SSU) ribosomal RNA gene (rDNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene indicated that the trypanosomes from all the murid hosts had identical SSU rDNA or gGAPDH gene nucleotide sequences except for those in N. confucianus in mainland China. These N. confucianus-infecting trypanosomes also showed several unique morphological features such as smaller bodies, anteriorly positioned nuclei, and larger rod-shaped kinetoplasts when compared with T. lewisi trypomastigotes. Trypanosoma (Herpetosoma) niviventerae n. sp. is erected for this new species. Similarly, based on morphological and molecular genetic characterization, Trypanosoma sapaensis n. sp. and Trypanosoma anourosoricis n. sp. are proposed for the trypanosomes in C. dracula in Vietnam and A. yamashinai in Taiwan, respectively. More effort directed toward the morphological and molecular genetic characterization of the trypanosomes of rodents and soricids is required to fully understand the real biodiversity of their hemoflagellates.

Molecular detection of Babesia and Hepatozoon species and morphological characteristics of Babesia species in Japanese wild boars.

We investigated intraerythrocytic Babesia parasites in 21 Japanese wild boars, Sus scrofa leucomystax, captured in Wakayama Prefecture on the mainland from 2008 to 2009 and in 31 Japanese wild boars from 2011 to 2013 in Kochi Prefecture on Shikoku Island, Japan. We detected small subunit ribosomal RNA (18S rRNA) gene (SSUrDNA) fragments of a Babesia species in 17 boars from Wakayama and 18 boars from Kochi. The nearly full SSUrDNA sequence (1669 bps) of this species was determined. A FASTA search revealed that the SSUrDNA sequence of the Babesia sp. in Japanese wild boars was the most homologous to those of several Babesia isolates reported as Babesia gibsoni. Phylogenetic analysis showed that the Babesia sp. found in Japanese wild boars was the closest relative to B. gibsoni but made a different clade from B. gibsoni. The Babesia sp. in Japanese wild boars was completely different from Babesia sp. Suis found in a European domestic pig, Sus scrofa domesticus. By microscopic examination, ring-shaped, oval and pear-shaped small sized intraerythrocytic parasites were observed on blood smears of 12 of 18 Japanese wild boars whose blood smears could be examined in Wakayama. We also detected SSUrDNA fragments of a Hepatozoon species in 6 of the 21 wild boars from Wakayama. The nearly full SSUrDNA sequence (1774 bps) of the Hepatozoon sp. was shown to be identical to that of Hepatozoon apri.

Development of absolute quantification method for genotype-specific Babesia microti using real-time PCR and practical experimental tips of real-time PCR.

Babesia microti, a rodent babesia, is known as a pathogen of zoonosis, human babesiosis, is composed of several genotypes of small subunit ribosomal RNA gene (SSUrDNA) and different genotypes have been suggested to have different infectivity and pathogenicity to humans. We established a real-time PCR assay using SYBR Green I, which allows specific detection and absolute quantification for each SSUrDNA-type-B. microti of four SSUrDNA-types found in Japanese rodents even in mixed infection. In this assay, four genotype-specific primer pairs targeted on internal transcribed spacer 1 or 2 sequences were used. Primer pairs have the characteristics for a high specificity for homologous genotype DNA. The calibration curves of cycle threshold (Ct) values versus log concentrations of DNA for all four genotypes were linear over 107 fold range of DNA concentrations with correlation coefficient from 0.95 to 1 and sufficient amplification efficiency from 90% to 110%. The standard curves for all four genotypes were not changed even in the presence of heterologous DNA. In this paper, we introduce how to establish and perform the genotype-specific real-time PCR and our practical experimental tips to be recommended.

Continuous in vivo culture and indirect fluorescent antibody test for zoonotic protozoa of Babesia microti.

Babesiosis has attracted attention as a zoonotic disease. The disease is caused in immunocompetent individuals almost solely by Babesia microti, a rodent babesia. Most cases of human babesiosis by B. microti have been reported in the endemic foci of the Northeastern coastal areas and upper Midwest regions of the United States, while some sporadic cases have recently been reported in several Asian countries including Japan. Our previous surveys identified that four small subunit ribosomal RNA gene (SSUrDNA) types of B. microti parasitize Japanese rodents. Indirect fluorescent antibody test (IFAT) is often performed for the diagnosis of babesiosis together with microscopical examination of thin blood smears and PCR. We established IFAT against four SSUrDNA-types of B. microti using erythrocytes of SCID mice or Syrian hamsters infected with each SSUrDNA-type B. microti. The results of IFAT for sera of ICR mice or Syrian hamsters infected with each SSUrDNA-type B. microti demonstrated that the four SSUrDNA-types of B. microti have different serotypes. Here, we report technical or practical procedures of IFAT, which gains sufficiently stable results, including procedures of continuous in vivo culture of B. microti.

Development of real-time PCR assay for differential detection and quantification for multiple Babesia microti-genotypes.

We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA. The standard curves of cycle threshold (Ct) values versus amount of target DNA per reaction (log) for all four genotypes were linear and the correlation coefficient (Rsq) values for the curves were from 0.970 to 0.997. The standard curves were almost identical even in the presence of heterologous genotype DNA. This assay could detect 10-30 fg purified DNA (equivalent to the amount of 1-5 parasite DNA) of each genotype B. microti. This assay could also detect each genotype B. microti infection in blood with 3×10(-6)%-1×10(-5)% parasitemia. This assay was applicable to field rodent and tick samples to reveal mixed infection in several samples, for which a single genotype of B. microti had been detected by direct sequencing analyses in our previous studies. This assay also seemed to be applicable to clinical human samples, showing Kobe-type positive results for the first Japanese babesiosis patient and the asymptomatic donor, both infected with Kobe-type B. microti.

Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati, Bangladesh.

A combination of chloroquine (CQ) and primaquine (PQ) had been used as the first-line treatment of uncomplicated Plasmodium falciparum malaria in Rangamati, Bangladesh until the end of 2004. Doctors or medical staffs had felt that CQ plus PQ had become less effective against uncomplicated falciparum malaria patients, but that it was more effective against the minority-indigenous patients than the Bengali patients. The efficacy of CQ plus PQ and the mutation status of the CQ resistance transporter (pfcrt) gene of infecting P. falciparum were, thus, investigated for 45 uncomplicated falciparum malaria patients in Rangamati in 2004. The total failure rate was 57.8%. One or two pfcrt sequences (CIETH and SMNTH at positions 72, 74-76, and 97, mutation underlined) with K76T mutation known to be related to CQ-resistant phenotype were detected in 38 patients' blood samples. Of the 38 patients, in total 15 patients (14/25 minority-indigenous and 1/13 Bengali patients) resulted in adequate clinical and parasitological response (ACPR). There was a statistically significant difference in ACPR rate between the minority-indigenous patients and the Bengali patients. P. falciparum with mutant or resistant pfcrt (pfcrt-resistant) was detected by PCR in blood samples on day 28 for 10 ACPR minority-indigenous patients but not for the only one Bengali ACPR patient, who all were infected with pfcrt-resistant P. falciparum on day 0. The minority-indigenous patients, but not Bengalis, are suggested to be often cured by CQ plus PQ, leaving a very few parasites detectable only by PCR, even when they are infected with pfcrt-resistant P. falciparum.

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test.

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.

Survey of Babesia microti infection in field rodents in Japan: records of the Kobe-type in new foci and findings of a new type related to the Otsu-type.

Of 247 rodents comprising 5 genera and 7 species collected at 17 sites throughout Japan from 2003 to 2005, Babesia microti was detected microscopically and by polymerase chain reaction (PCR) in 36 rodents comprising 2 genera and 3 species from 12 sites. Based on the analysis of small subunit ribosomal RNA gene (SSUrDNA) sequences, the Kobe-type, the etiological type of the first Japanese case of human infection was found in Apodemus speciosus and Apodemus argenteus in Aomori, the northernmost prefecture of the Japanese mainland, while the U.S.-type was found on Hokkaido Island and the Otsu-type was widely distributed. In addition, a new Otsu-related type was detected exclusively in Eothenomys andersoni in Nagano, a prefecture in central Japan. The sequences of internal transcribed spacer 1 to 2 (ITS1/2) of the present Kobe- and Otsu-types were almost identical to those of the same types previously identified. The ITS1/2 sequence of the U.S.-type identified in Hokkaido in this survey was somewhat different from that of the U.S.-type strain originating from the U.S.A., with approximately 95% identity. This value was similar to the 94% identity found between the ITS1/2 sequences of the Otsu-type and the new Otsu-related type. The new Otsu-related type of B. microti was isolated as the Nagano strain, which was serologically differentiated from the other type strains of B. microti. The divergence and distribution of genotypes are important factors in investigating the epidemiology of human B. microti infection in Japan.

Japanese Babesia microti cytologically detected in salivary glands of naturally infected tick Ixodes ovatus.

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field-collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporoblast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood-sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector-pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.

Identification of a new type of Babesia species in wild rats (Bandicota indica) in Chiang Mai Province, Thailand.

A new type of rodent babesia, which resembled Babesia microti but was phylogenetically placed closest, with the highest level of statistical support, to Babesia canis, a canine babesia, was identified in Thai Bandicota indica in Thai provinces to which malaria is endemic. Close watch should be kept on human babesiosis in Thailand.

Survey of rodents and ticks in human babesiosis emergence area in Japan: first detection of Babesia microti-like parasites in Ixodes ovatus.

Babesia microti-like parasites were detected for the first time in Ixodes ovatus in Hyogo Prefecture, Japan, where two reported types of B. microti-like parasites were recognized in many rodents. Of 80 adult I. ovatus ticks collected, 5 possessed the reported type and 1 possessed a new type of B. microti-like parasite.