Heat-shock proteins (HSPs) are evolutionarily highly conserved polypeptides. They are immunodominant antigens in a wide variety of bacteria and parasites. We reported that the induction of 65 000 MW HSP (HSP65) plays an important role in developing protective immunity against infection with Toxoplasma gondii. Here, we examined the role of T cells in the expression of HSP65 and in the acquisition of protective immunity in SCID mice transplanted with fetal thymus or liver cells from syngeneic C.B-17 mice and infected with T. gondii. When SCID mice lacking mature T and B cells were transplanted with fetal liver cells (FLT-SCID mice), T and B lymphopoiesis were reconstituted to the level of normal mice, whereas only T lymphopoiesis was reconstituted in SCID mice grafted with a fetal thymus (TG-SCID mice). These mice were infected with the Beverley strain bradyzoites of T. gondii 7 days after immunization with Toxoplasma homogenate. Immunization effect did not occur in untreated SCID mice (UT-SCID mice), all of which died within 2 weeks after infection. HSP65 was not expressed in their macrophages. The survival of TG-SCID mice was significantly prolonged by immunization as compared with that of the immunized UT-SCID mice, and all the immunized FLT-SCID mice acquired complete resistance as well as the immunized C.B-17 mice. HSP65 was expressed in macrophages of TG-SCID mice after immunization, and much higher in those of FLT-SCID mice. These results indicate that T cells play a crucial role in the expression of HSP65 and in the induction of protective immunity against infection with T. gondii.

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Nine strains of the free-living amoeba Willaertia magna and one of Naegleria fowlen were compared by detection of whole-cell and small subunit ribosomal DNA (ssurDNA) restriction fragment length polymorphisms (RFLPs). Ail strains of W. magna showed identical Pst I whole-cell RFLPs, although variation in Bg1 II, Hae III and Kpn I RFLPs were found. These RFLPs did not correlate with the geographic origin of the strains. Phylogenetic analysis of the whole-cell RFLPs separated the W. magna strains into three distinct clusters. However, these were not sufficiently diverse to indicate interspecies variation. Homologous ssurDNA RFLPs were found for ail strains of W. magna regardless of the restriction endonuclease used. Both the whole-cell and ssurDNA RFLPs were different for W. magna and N. fowleri and can be used to identify the species. On the basis of homologous whole-cell Pst I and ssurDNA RFLPS, the findings of this study confirm that W. magna is a single species genus.

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We recently reported that the presence of a Babesia ovata-like large intraerythrocytic parasite, Babesia sp.1 in cattle population of Hokkaido area in Japan, and the development of DNA probe (SpS7) for the parasite. A nucleic acid probe is a powerful tool for direct detection and identification of parasites in animals and vector insects. However, polymerase chain reaction (PCR) technique has advantages over hybridization technique. In this paper we report the development of PCR and nested-PCR techniques for amplification of the SpS7 sequence from Babesia sp.1 DNA. The PCR with one set of primers was specific for Babesia sp.1, since no amplification detected with DNA from bovine white blood cell, Theileria sergenti, Anaplasma marginale, A. centrale, Eperythrozoon wenyoni, B. bovis, B. bigemina, and B. ovata. The nested-PCR, which reamplified 470-base-pair (bp) in the internal region of the first primer set (670 bp), enabled the detection of less than 10 parasites on calculation. The technique was 10-fold more sensitive than the one-step PCR, and detected Babesia sp.1 in larval ticks that had been generated from the parasite-infected adults Haemaphysalis longicoronis.

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The antigenic differences between Blastocystis hominis and Blastocystis sp. (isolated from a human and a chicken sources, respectively) were investigated by using polyclonal and monoclonal antibodies. Polyclonal antibodies did not reveal distinct antigenic differences between the strains by Western blot analysis, whereas monoclonal antibodies (MAbs) produced against B. hominis did detect antigenic polymorphism between B. hominis and Blastocystis sp. Six out of 8 MAbs were specific to B. hominis antigens and 2 MAbs reacted with both B. hominis and Blastocystis sp. antigens. Immunoelectron microscopy using these MAbs, revealed labeling on the filamentous layer or the central vacuole. Interestingly, the 6 MAbs reacted only with B. hominis antigens on the filamentous layer, whereas the other 2 MAbs labeled on the central vacuole. Therefore, the antigenic components of the filamentous layer may be strain or species specific. In contrast, the antigenic components of the central vacuole are shared by both B. hominis and Blastocystis sp. suggesting the metabolic substances in the central vacuole are antigenically common.

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