Molecular survey of Babesia microti, Ehrlichia species and Candidatus neoehrlichia mikurensis in wild rodents from Shimane Prefecture, Japan.

A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.

Babesia microti-like parasites detected in Eurasian red squirrels (Sciurus vulgaris orientis) in Hokkaido, Japan.

Six Eurasian red squirrels (Sciurus vulgaris orientis), victims of road traffic found during 2002 and 2004 near the Noppro Forest Park in Ebetsu, Hokkaido, Japan, were examined for the presence of Babesia parasites. Three of the six squirrels exhibited positive signals by nested PCRs targeting both the 18S rRNA and beta-tubulin genes. Three squirrels proved to be infected with a B. microti-like parasite as evidenced by sequencing the amplified DNAs and by the morphology of the intraerythrocytic parasites. Genotypically, however, the parasite appeared to be of a new type, as it was clearly distinguishable from any of the known types that have previously been reported in various wild animals. This is the first report showing molecular evidence for the presence of B. microti-like parasites in Sciuridae.

Isolation of a human erythrocyte-adapted substrain of Babesia rodhaini and analysis of the merozoite surface protein gene sequences.

Babesia rodhaini is a rodent hemoparasite closely related to B. microti, the major causative agent of human babesiosis. We tested the infectivity of B. rodhaini for human erythrocytes by using the SCID mouse model in which the circulating erythrocytes were replaced with those of humans. Initially, parasites grew very poorly in the mouse model, but a variant capable of propagating in human erythrocytes emerged after an adaptation period of three weeks. In an attempt to identify parasite proteins involved in the alteration of host cell preference, an expression cDNA library of B. rodhaini was constructed and screened with immune mouse sera. Although we were able to obtain three merozoite surface protein (MSP) genes, sequences of these genes from both the parental strain and human erythrocyte-adapted substrain were identical. Our results suggest that B. rodhaini has potential ability to infect human erythrocytes, but development of this ability may not be brought about by an amino acid change in MSPs.

Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan.

Raccoons (Procyon lotor), which have recently become feral in Japan, were examined for the presence of Babesia microti-like parasites. Out of 372 raccoons captured in the west-central part of Hokkaido, 24 animals with splenomegaly were selected and tested by nested PCR targeting the babesial 18S rRNA gene. B. microti-like parasites were detected in two of the 24 individuals, and their DNA sequences were identical to that of the B. microti-like parasite reported from raccoons in the United States, suggesting that the parasites were probably imported into Japan and that the life cycle of the parasite has already been established in the country. The potential risk of this B. microti-like parasite spreading among dogs and foxes in Japan will need to be carefully monitored, as parasitization by phylogenetically very close parasites has been reported from such animals.

U.S.-type Babesia microti isolated from small wild mammals in Eastern Hokkaido, Japan.

Our previous report demonstrated that small wild rodents in Japan harbored two types of novel Babesia microti-like parasites (Kobe and Hobetsu types), but not the type widely distributed throughout the temperate zones of North American and Eurasian Continents (U.S. type). In this study, we surveyed small wild mammals collected at various places in the northern part of Japan, seeking for U.S.-type B. microti. A total of 197 small mammals comprising 10 species, Apodemus speciosus, A. argenteus, Clethrionomys rufocanus, C. rutilus, Eothenomys andersoni, Microtus montebelli, Tamias sibiricus, Sorex unguiculatus, S. caecutiens, and Urotrichus talpoides, were examined. Babesia parasites were detected in A. speciosus, C. rufocanus, C. rutilus, M. montebelli, S. unguiculatus, and S. caecutiens by microscopy of blood smears and by PCR targeting babesial nuclear small-subunit rRNA (rDNA) and beta-tubulin genes. Inoculation of their bloods into experimental animals gave rise to 23 parasite isolates, which included 16 from A. speciosus, 4 from C. rufocanus, and 1 each from C. rutilus, M. montebelli and S. unguiculatus. Sequencing analyses of their rDNA and beta-tubulin genes revealed that, of the 23 isolates, 20 and 3 were of Hobetsu and U.S. types, respectively. The U.S.-type B. microti strains isolated in Japan, however, were distinguishable from the isolates in the United States when their beta-tubulin gene sequences and antigen profiles in Western blots were compared. We conclude that U.S.-type B. microti exists in Japan although it has been genetically and antigenically diversified from that distributed in the United States. The results also suggest that not only rodents, but also some insectivores may serve as reservoirs for the agent of human babesiosis.