Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

Two Eimeria species isolated from wild Japanese rock ptarmigans (Lagopus mutus japonicus) in Japan.

Fecal samples were collected from 64 Japanese rock ptarmigans (Lagopus mutus japonicus) at 13 locations in the alpine zone of Japan and examined for internal parasites. We found 2 morphologically different types of eimerian oocysts. Based on morphological characteristics, one was identified as Eimeria uekii, which had already been found from Japanese rock ptarmigans in 1981, and the other was likely a new type of Eimeria. The new type of eimerian oocysts required a longer sporulation time than E. uekii. The prevalence of both eimerian oocysts in Japanese rock ptarmigans was 62.5%, while that of E. uekii was 60.9% and the new type 29.7%. Mixed infection of both types was found in 28.1%.

Cryptosporidium infection in juvenile pet rabbits.

Cryptosporidium infection was confirmed by fecal examination for the first time in pet rabbits in a wholesale store located in Kanagawa Prefecture, Japan. Fecal samples were obtained postmortem from juvenile rabbits (n=66), which had died after developing diarrhea. Feces from healthy rabbits (n=30) were also collected and examined as controls. Two types of Cryptosporidium oocysts distinctive in size and shape were found (Type A and B). Types A and B oocysts were detected from 16.7% and 13.6% of the diarrheic, and 3.3% and 0% of the normal feces, respectively. Since Cryptosporidium oocysts were detected at a higher rate in the diarrheic rabbits than in the healthy rabbits, special caution should be taken when handling a pet rabbit presenting with diarrhea.

A novel method for development of malaria vaccines using full-length cDNA libraries.

We describe a novel method to screen malaria DNA vaccine candidates using a full-length cDNA library and a murine malaria infection model. For the development of effective malaria vaccines, much effort has been made with meager success. The completion of genome sequencing of Plasmodium falciparum has provided invaluable information for achieving this goal. We have been studying full-length cDNA libraries of malaria parasites as a part of genome analysis. Mice vaccinated with a DNA vaccine consisting of 2000 pooled clones showed significantly prolonged survival after challenge infection. In addition, spleen cells of vaccinated mice produced augmented levels of IL-2 and IFN-gamma when incubated with the crude parasite antigens, indicating that cellular immunity plays an important role in the protection. This approach will not only form the basis for development of malaria vaccines but will also be applicable to other parasites and pathogenic microorganisms.

Short report: Lethal malaria in cytosolic phospholipase A2- and phospholipase A2IIA-deficient mice.

Lipid mediators play important roles in the pathogenesis of malaria. Phospholipase A2s are enzymes involved in the production of these mediators, and they function in inflammation. Among them, cytosolic phospholipase A2 (cPLA2) is a key enzyme in the metabolism of arachidonic acid, the first intermediate in the production of lipid mediators. Plasmodium berghei ANKA causes cerebral malaria in CL57B/6 mice, and we recently produced cPLA2-deficient mice with this background. With the expectation of reduced pathogenicity, we performed experimental infection in these mice. Unexpectedly, the infected mice developed cerebral malaria and died at the same time as the control mice, while the parasitemia progressed similarly in both groups. These observations suggest that secretory PLA2s rather than cPLA2 may be involved in the aggravation, although possible compensation by the induction of other enzymes has not been excluded. The present findings are expected to help clarify the involvement of various phospholipase A2s in malaria.

Prevalence of anti-Toxoplasma gondii antibody in hunter-killed wild boars, Sus scrofa leucomystax, on Amakusa Island, Kumamoto Prefecture, Japan.

The prevalence of Toxoplasma gondii was surveyed in wild boars (Sus scrofa leucomystax) and domiciled cats obtained in various areas of Amakusa Island, Kumamoto Prefecture, Japan. The antibody titers against T. gondii were measured with a latex agglutination test. Among specimens taken from 90 wild boars, 1 (1.1%) was positive and 3 (3.3%)were doubtfully positive. Among the specimens from 50 cats, none were positive and 1 (3.3%) was doubtfully positive. These results suggest that the wild boars and cats on Amakusa Island have quite low prevalence of the T. gondii infection. Continuous surveys will be needed to monitor the prevalence of Toxoplasmosis and other zoonoses in game animals.

The first record of sucking louse, Neohaematopinus callosciuri, infesting pallas squirrels in Japan.

A species of sucking louse, Neohaematopinus callosciuri, was found for the first time in Japan. The species was found on an invasive species of squirrel, Pallas squirrel, Callosciurus erythraeus, in the Kamakura district, Kanagawa Prefecture, Japan. A total of 52 lice were obtained from 22 of 104 squirrels captured. The lice were about three times more prevalent in male squirrels than in females and were detected most frequently in the winter. As N. callosciuri has never been reported on wild animals in Japan, this species probably was introduced into Japan along with their host, Pallas squirrels.