Atsuko Saito-Ito

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.

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.

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.

Survey of Rodents and Ticks in Human Babesiosis Emergence Area in Japan: First Detection of Babesia microti-Like Parasites in Ixodes ovatus

ABSTRACT 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.

Identification of a New Type of Babesia Species in Wild Rats (Bandicota indica) in Chiang Mai Province, Thailand

ABSTRACT 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.

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.

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 sporo‐blast 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.

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.

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.

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