Detection of Kobe-type Babesia microti associated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China.

Field rodent surveys for Babesia infection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China. Babesia microti was identified by microscopical examination and/or PCR in 1 Rattus coxinga and 1 Crocidura horsfieldii in central Taiwan and in 13 Niviventer confucianus and 1 Apodemus agrarius in Zhejiang and Fujian Provinces of southeastern China. Of 15 B. microti samples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2-3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.

Distinct distribution of four Ca2+-dependent subtypes of protein kinase C in rat olfactory bulb; definite expression of betaII-subtype in the accessory olfactory bulb.

The localization of four subtypes of Ca2+-dependent protein kinase C (PKC) in the main and accessory olfactory bulb was examined by immunocytochemistry by using specific antibodies against each PKC subtype. In the main olfactory bulb, alpha-PKC was densely localized in a large number of granule cells and in a few tufted cells, and faint immunoreactivity was seen in some periglomerular cells. betaI-PKC was intensely found in periglomerular cells and tufted cells. gamma-PKC immunoreactivity was present in the external plexiform layer, the internal plexiform layer, and the granular layer, but the immunoreactivity was found only in the neuropils. Little, if any, betaII-PKC was seen in the main olfactory bulb. On the other hand, the intense immunoreactivity for betaII-PKC was seen in periglomerular cells of the accessory olfactory bulb. The betaI-PKC and gamma-PKC were also present in periglomerular cells of the accessory olfactory bulb, while alpha-PKC was localized only in granule cells. Double staining study in the accessory olfactory bulb showed that betaII-PKC was present in the GABAergic periglomerular cells, while betaI-PKC localized to the non-GABAergic periglomerular cells; gamma-PKC was expressed in both GABAergic and non-GABAergic cells. These findings suggest that four calcium-dependent subtypes of PKC play different roles in the olfactory bulb and definite expression of betaII-PKC strongly suggested the involvement of this subtype in a specific function in the accessory olfactory bulb.

Transfusion-acquired, autochthonous human babesiosis in Japan: isolation of Babesia microti-like parasites with hu-RBC-SCID mice.

We have isolated piroplasms from a patient who developed the first case of human babesiosis in Japan by using NOD/shi-scid mice whose circulating erythrocytes (RBCs) had been replaced with human RBCs (hu-RBC-SCID mice). Following inoculation of the patient's blood specimen into hu-RBC-SCID mice, parasites proliferated within the human RBCs in the mice, resulting in a high level of parasitemia. Parasite DNA was prepared from blood samples of the patient and the mice, and the nuclear small-subunit rRNA gene (rDNA) was amplified and sequenced. Both DNA samples gave rise to identical sequences which showed the highest degree of homology (99.2%) with the Babesia microti rDNA. Because the patient had received a blood transfusion before the onset of babesiosis, we investigated the eight donors who were involved. Their archived blood samples were analyzed for specific antibody and parasite DNA; only a single donor was found to be positive by both tests, and the parasite rDNA sequence from the donor coincided with that derived from the patient. The donor's serum exhibited a high antibody titer against the isolate from the patient, whereas it exhibited only a weak cross-reaction against B. microti strains isolated in the United States. We conclude that the first Japanese babesiosis case occurred due to a blood transfusion and that the etiological agent is an indigenous Japanese parasite which may be a geographical variant of B. microti. Our results also demonstrated the usefulness of hu-RBC-SCID mice for isolation of parasites from humans and for maintenance of the parasite infectivity for human RBCs.

[First documentation of transfusion-associated babesiosis in Japan].

A 40-year-old man received blood transfusion in December 1998 because of gastric bleeding from a peptic ulcer. One month later, he developed febrile hemolytic anemia. Administration of high doses of glucocorticoid significantly reduced the hemolysis, but did not cure the disease. To investigate the cause of the hemolysis, the patient was transferred to our hospital in May 1999. Giemsa-stained peripheral blood smears showed Babesia parasites in the red blood cells (RBC), and PCR analysis confirmed the presence of Babesia microti DNA. The parasitemia disappeared hematologically after 2 weeks of quinine and clindamycin therapy. However, parasite DNA was still detectable in the RBC. Although treatment with oral atovaquone was given for 2 weeks, parasitemia and febrile hemolysis recurred within a month after the last treatment. Fortunately, complete remission was obtained after a second 12-week course of therapy with quinine and clindamycin. PCR analysis revealed asymptomatic Babesia infection in one of eight samples from the original blood donor. The initial steroid therapy given to the patient without an accurate diagnosis seemed to have delayed augmentation of the specific antibodies (IgG) against Babesia microti, thus prolonging the parasitemia after the initial acute stage of babesiosis.

Modulation of the growth of Plasmodium falciparum in vitro by protein serine/threonine phosphatase inhibitors.

To elucidate the physiological roles of the protein serine/threonine phosphatases of P. falciparum, first we identified and characterized phosphatase activities of Plasmodium falciparum enzymologically and pharmacologically. We have demonstrated that P. falciparum possesses phosphatase-1-like activities predominantly over phosphatase-2A-like activities, while erythrocytes possess mainly phosphatase-2A-like activities. Then, we examined the effects of okadaic acid and calyculin A, potent inhibitors of protein phosphatase 1 and 2A, on the growth of P. falciparum in vitro. Both of the drugs inhibited parasite growth dose dependently. The manner of growth inhibition by calyculin A and okadaic acid suggested that these drugs inhibit parasite growth mainly by inhibiting parasite phosphatase-1-like activities. Both drugs were shown to inhibit the growth of three different developmental stages of parasites--ring forms, trophozoites, and schizonts--and inhibit trophozoites the most. This is the first report on P. falciparum protein serine/threonine phosphatase activities, which are essential to regulate the erythrocytic stage of parasite growth.

A PCR method for molecular epidemiology of Plasmodium falciparum Msp-1.

Merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a strong malaria vaccine candidate. However, MSP-1 exhibits extensive antigenic polymorphism, an issue which may compromise the development of effective vaccine based on this molecule. Since polymorphic nature of MSP-1 has not been fully understood in endemic areas of malaria, variation of the MSP-1 gene (Msp-1) must be studied in detail in natural parasite populations. Here, a PCR-based method for determination of P. falciparum Msp-1 haplotype is described, which can detect up to 24 different haplotypes per infected person. The method can be applied to various purposes of molecular epidemiology of not only Msp-1 haplotype but the genetic structure of P. falciparum populations.

Cloning and structural analysis of the gene for cAMP-dependent protein kinase catalytic subunit from Plasmodium yoelii.

We isolated the gene encoding the cAMP-dependent protein kinase catalytic subunit (cAPK[C]) from Plasmodium yoelii by screening a genomic library for the DNA fragment as produced by the polymerase chain reaction. The deduced protein of 341 amino acids conserves residues that are important for the function of serine/threonine protein kinases and shows the highest homology to cAPK[C]s of other organisms. However, P. yoelii cAPK[C] has 8 residues, which are perfectly conserved in cAPK[C]s of other organisms, radically replaced with residues having different side-chain properties. It is stressed that two radical replacements occur in regions for the binding with a regulatory subunit and/or a heat-stable inhibitor protein.