Motohiro Iseki

Infectivity of Cryptosporidium muris (strain RN 66) in various laboratory animals

The infectivity ofCryptosporidium muris (strain RN 66), originally isolated from the house rat (Iseki 1986), to various laboratory animals was studied by transmission experiments. After oral inoculation with 1×106 oocysts, mice, guinea pigs, rabbits, dogs, and cats all discharged endogenously produced oocysts in their feces. Among these host species, mice and cats were highly susceptible to the parasite. The prepatent period for six 3-week-old specific pathogen-free (SPF) mice was 5 days postinoculation (PI), the patent periods varied between 34 and 75 days for each mouse, and the number of oocysts discharged per individual per day (OPD) was 11–46×106 at the maximum on days 16–26 PI. The total number of oocysts discharged per mouse during the patent period was estimated to be 170–560×106. Three inoculated cats (1–2 months old) also discharged a large number of oocysts for a long period. Guinea pigs, rabbits, and dogs showed low susceptibility to this strain; the OPD was extremely small and the patent periods were less than 3 weeks. The entire endogenous development of this parasite occurred in the stomach and not in the small and large intestines of these experimental animals. Because of this lack of host specificity, it is suspected thatC. muris could be infective to humans, especially immunocompromised patients such as those with AIDS.

Ultrastructure of Cryptosporidium muris (strain RN 66) parasitizing the murine stomach.

The ultrastructure of Cryptosporidium muris, which parasitizes the stomach of mice, was studied by transmission electron microscopy. The entire development of the parasite occurred in the microvilli of the surface mucus cells in the gastric glands. The ultrastructural features of the attachment site of C. muris to the host cell differed remarkably from those of C. parvum and its closely related species, which parasitize the intestine of various animals. The size of C. muris was greater at almost every developmental stage than that of C. parvum. These findings confirmed that C. muris and C. parvum are distinct species. The mitochondria, subpellicular microtubules, and Golgi complex were demonstrated in detail. A small invagination in the meront and intravacuolar tubules were found in Cryptosporidium. The wall of each developing oocyst in the parasitophorous vacuole was composed of three layers: the outermost layer was considered to be a true oocyst wall, whereas the middle and innermost layers were assumed to develop into the sporocyst wall. The outermost layer was fragile and disintegrated as the oocyst matured. In excystation in vitro, a suture was seen in a thick layer of the two-layered sporocyst wall of an oocyst (sporocyst wall; see Discussion) that enveloped four sporozoites. The fine structure of the attachment site of the present species to the host cell appears to reveal a unique mode of hostparasite interaction in Cryptosporidium infection.

RECOMBINANT BOVINE HERPESVIRUS-1 EXPRESSING p23 PROTEIN OF CRYPTOSPORIDIUM PARVUM INDUCES NEUTRALIZING ANTIBODIES IN RABBITS

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.

Molecular Characterization of a Cryptosporidium Isolate From a Banded Mongoose Mungos mungo

Cryptosporidium spp. has been found in more than 150 species of mammals, but there has been no report in mongooses. In this study, we report the isolation of Cryptosporidium sp. in a banded mongoose Mungos mungo, which was brought from Tanzania to Japan; the isolate was analyzed genetically to validate the occurrence of a new, host-adapted genotype. Cryptosporidium diagnostic fragments of 18S ribosomal RNA and 70-kDa heat shock protein genes were amplified from this isolate and compared with the other Cryptosporidium species and genotypes reported previously. Analyses showed that the mongoose isolate represents a new genotype, closely related to that of bears.

Heterogeneous Distribution of Membrane Cholesterol at the Attachment Site of Cryptosporidium muris to Host Cells

ABSTRACT. Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze‐fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris, the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin‐cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.