Human babesiosis in Japan: isolation of Babesia microti-like parasites from an asymptomatic transfusion donor and from a rodent from an area where babesiosis is endemic

Abstract

To determine the source of infection for the Japanese index case of human babesiosis, we analyzed blood samples from an asymptomatic individual whose blood had been transfused into the patient. In addition, we surveyed rodents collected from near the donor's residence. Examination by microscopy and PCR failed to detect the parasite in the donor's blood obtained 8 months after the donation of the blood that was transfused. However, we were able to isolate Babesia parasites by inoculating the blood sample into SCID mice whose circulating red blood cells (RBCs) had been replaced with human RBCs. A Babesia parasite capable of propagating in human RBCs was also isolated from a field mouse (Apodemus speciosus) captured near the donor's residential area. Follow-up surveys over a 1-year period revealed that the donor continued to be asymptomatic but had consistently high immunoglobulin G (IgG) titers in serum and low levels of parasitemia which were microscopically undetectable yet which were repeatedly demonstrable by inoculation into animals. The index case patient's sera contained high titers of IgM and, subsequently, rising titers of IgG antibodies, both of which gradually diminished with the disappearance of the parasitemia. Analysis of the parasite's rRNA gene (rDNA) sequence and immunodominant antigens revealed the similarity between donor and patient isolates. The rodent isolate also had an rDNA sequence that was identical to that of the human isolates but that differed slightly from that of the human isolates by Western blot analysis. We conclude that the index case patient acquired infection by transfusion from a donor who became infected in Japan, that parasitemia in an asymptomatic carrier can persist for more than a year, and that A. speciosus serves as a reservoir of an agent of human babesiosis in Japan.

FIG. 1

Isolation of a Babesia parasite from an asymptomatic blood donor with hu-RBC-SCID mice. A dose of 6 × 10⁹ RBCs from the donor was inoculated into splenectomized NOD/shi-scid mice on days 0, 1, and 3 (open arrowheads), and thereafter, the mice were repeatedly transfused with an equal dose of Babesia-free RBCs from a healthy volunteer on the days indicated by closed arrowheads. Two mice (open and closed symbols, respectively) were used, and peripheral blood samples from these mice were periodically examined for the rate of replacement with human RBCs (⋄ and ♦ on dotted lines) and for levels of parasitemia (○ and ● on solid lines).

FIG. 2

Propagation of Babesia parasites derived from a field mouse (A. speciosus) in a hamster, an SCID mouse, and an hu-RBC-SCID mouse. The blood sample from the Apodemus mouse, which contained approximately 2 × 10⁷ parasitized RBCs, was divided into three portions and inoculated on day 0 into a hamster (□), an NOD/shi-scid mouse (▵), and an hu-RBC-SCID mouse (○). All animals had been splenectomized prior to the experiment.

FIG. 3

Western blot analysis of Babesia parasites isolated from the index case patient, the donor, and an Apodemus mouse (lanes 1, 2, and 3, respectively). Insoluble membrane fractions of the lysates of infected hamster RBCs were analyzed with sera obtained from the patient, the donor, and the Apodemus mouse. The relative mobilities of the molecular mass marker proteins are indicated.

FIG. 4

Western blot analysis of Babesia parasites isolated from the patient and the donor at various sampling times. Isolates were obtained from the patient's blood samples obtained on 24 May 1999, 27 July 1999, and 31 August 1999 (lanes 1, 2, and 3, respectively) and from the donor's blood samples obtained on 27 July 1999, 31 August 1999, 2 October 1999, 11 November 1999, 31 January 2000, and 6 March 2000 (lanes 4 through 9, respectively). Parasite antigens were immunostained with pooled sera obtained from the donor.