Human babesiosis in Japan: epizootiologic survey of rodent reservoir and isolation of new type of Babesia microti-like parasite

Abstract

We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species, Apodemus speciosus, Apodemus argenteus, Clethrionomys rufocanus, Eothenomys smithii, Crocidura dsinezumi, and Sorex unguiculatus, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring Babesia microti-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from A. speciosus, and the other 1 was from C. rufocanus. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States B. microti (U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of B. microti-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that A. speciosus serves as a major reservoir for both Kobe- and Hobetsu-type B. microti-like parasites, and that C. rufocanus may also be an additional reservoir on Hokkaido Island.

FIG. 1

Map of Japan showing the locations of field survey points (●).

FIG. 2

Phylogenetic tree constructed by the neighbor-joining method with rDNA sequences of various apicomplexan parasites. A portion corresponding to bases 22 to 1715 of the rDNA sequence of strain Ho226 (GenBank accession no. AB050732) was included for analysis. The number on each branch shows the percent occurrence in 1,000 bootstrap replicates.

FIG. 3

Western blot analyses of four closely related Babesia parasites. Lanes 1 through 4 contained strains Aw1 (Kobe type), Ho234 (Hobetsu type), and Gray (U.S. type) and B. rodhaini, respectively. Parasite antigens were probed with convalescent-phase sera from hamsters (anti-Aw1 and anti-Ho234) or mice (anti-Gray-Mo and anti-B. rodhaini) that were infected with each parasite.

FIG. 4

Western blot analyses of Hobetsu-type B. microti-like parasites isolated from various places in Japan. The respective parasite strains analyzed were Ho226, Ho232, Ho233, Ho234, Ho235, Ho236, Ho237, and Ho240 from Hobetsu in Hokkaido Prefecture (lanes 1 to 8); Ya501 from Yamanaka in Shiga Prefecture (lane 9); An2 and An3 from Anan in Tokushima Prefecture (lanes 10 and 11); Da111, Da112, and Da116 from Daito in Shimane Prefecture (lanes 12 to 14); Ot1 and Ot2 from Ohtaki in Chiba Prefecture (lanes 15 and 16); and Aw3 and Aw6 from Awaji in Hyogo Prefecture (lanes 17 and 18). Parasite antigens were probed with a convalescent-phase serum from a hamster that was infected with strain Ho234.

FIG. 5

Photomicrograph of Giemsa-stained thin-smear blood films showing various forms of piroplasms in hamster erythrocytes. (Upper Panels) a, dot form; b, ring form; c, ovoid form; d, pyriform; e, crescent arch form; f to i, ameboid forms; j, Maltese cross form. (Lower Panels) Selected microscopic views of strains Aw7 (k) and Ho234 (l), emphasizing the difference between Kobe- and Hobetsu-type parasites, respectively.