池 和憲

As the comparative study was carried out on the susceptibility by the pursuit of parasitemia among the Djungarian, Syrian, and Chinese hamsters as well as BALB/c mice infected with the Syrian hamster-adapted Babesia microti strain, and Djungarian hamsters showed the highest parasitemia among them. Then, the other hematological parameters were pursued in the Djungarian hamsters infected with the hamster-adapted B. micron strain. Remarkable symptoms observed were hemoglobinuria clinically, anemia hematologically, and splenomegaly macroscopically during all over the observation period for 24 weeks post infection (PI). Parasitemia began to rise at 2 weeks and peaked at 4 weeks PI. After that, parasitemia decreased gradually but was maintained with a level of about 10% on average until 24 weeks PI at the end of the experiment. A decrease in the RBC count, Hb, and PCV, and an increase in the reticulocyte and WBC counts due to the development of immature neutrophils, lymphocytes and monocytes were recognized together with a rise of parasitemia. The hamsters had macrocytic hypochromic anemia due to the increase of MCV and the decrease of MCHC in the growth phase of the parasite. It was considered that the Djungarian hamsters will be useful for the infection examination, isolation, maintenance, and passage of B. microti in laboratory.

Mice were inoculated intraperitoneally wih 34 different types of vegetable juices, and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured as markers for the induction of Th1 and Th2 cells, respectively. Serum IFN-gamma level was markedly increased in mice inoculated with bitter gourd (Momordica charantia) juice, but IL-4 levels were not increased with any of the 34 vegetable juices. Testing of the various components of bitter gourd, including peel, pulp, and seed, showed that the pulp induced the highest levels of IFN-gamma. Trial immunogen including the heat extract of the pulp induced specific IgG(2a) antibody of the mice serum inoculated with this immunogen. These results demonstrate that bitter gourd pulp induced IFN-gamma production and show its promise as a means of effective imnunostimulatory therapy specific for Th1 cells and IFN-gamma production.

Interleukin 10 (IL-10) genes of Djungarian, Chinese, and Syrian hamsters were cloned. The clones of IL-10 consisted of 537 bp nucleotides and 178 amino acids in full length, and the nucleotide and amino acid sequences exhibited a high degree of homology with those of the mouse and human. Since the number and position of signal sequences, N-glycosylations and cysteine sites in the IL-10 amino acid sequences of the hamsters were the same as those of the mouse, we suggest that the IL-10 molecular structures of the hamster are closer to that of the mouse than human.

Hematological findings during the course of infection and the antibody response in Syrian hamsters infected with Babesia microti were examined. A macrocytic hypochromic anemia with an increase of the reticulocyte count was detected as a rise in the parasitized erythrocyte rate. White blood cell counts also remarkedly increased with the increases of both neutrophils and active-shaped monocytes, and thus they particularly play an important role in eliminating the parasite. In Western blotting with the sera from the hamsters infected with B. microti, a 38 kDa protozoan antigen reacted to the early-term sera, and additionally 28, 32, and 34 kDa antigens also reacted to the medium- and latter-term, and convalescent sera. These antigens were immunodominant and the antibodies against these antigens had also important roles for inhibition of this parasite.

Chinese hamsters were examined for the susceptibility to the infection with Babesia microti based on the hematological parameters during the course of infection. A marked decease in the RBC count, Ht value, Hb concentration, and an increase in WBC count due to the development of neutrophils or monocytes were recognized with the progress of parasitemia. Remarkable clinical findings were anemia and persistent infection with a low level of parasite burden in the chronic and convalescent stages. From these findings, it was concluded that Chinese hamsters were susceptible to infection with B. microti and would be useful for infection examination with the parasite.

Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells.

During a survey of the ciliate protozoal composition of the stomach contents of nine dromedary camels of Egypt, fourteen morphotypes of Entodinium ovumrajae, which has been considered as a species peculiar to camels, were found in six camels. Except for five morphotypes including one originally described as an independent species and its forms, these were newly detected. These morphotypes, divided into three groups, can be identified mainly by the morphology of their ectoplasmic processes. Each camel had on average, about five morphotypes of this species.

Rumen ciliate species and composition were surveyed on the reindeer kept in Inner Mongolia, China. As a result of survey, all the reindeer had the same 18 species of 8 genera of the ciliates in their rumen. Appearance of Entodinium parvum is the first record in this host species. Of the species detected, 15 species including Entodinium anteronucleatum, E. bicornutum, Enoploplastron confluens and Epidinium gigas which have been detected only from reindeer were common with those in the reindeer kept in the other areas, such as Alaska, Russia and Finland, indicating that every rumen ciliate faunae in reindeer has high similarity to each other, and suggesting that these hosts have been isolated from the other ruminants for long time and established characteristic ciliate fauna. The ciliate density was estimated as 1.1-2.5 x 10(6)/ml, which value was fairly higher than those in domestic ruminants. Surface structures of characteristic species were observed by SEM.

Djungarian (Phodopus sungorus) and Chinese (Cricetulus griseus) hamster IFN-gamma genes were cloned and sequenced. The Djungarian and Chinese hamster genes were both 525bp nucleotides, resulting in 174 amino acids in full length with a predicted molecular weight (MW) of 19,560 dal and 19,775 dal, respectively. The first 23 amino terminal amino acids consisted of a hydrophobic signal sequence when cleavaged, which would result in a mature 151 amino acid polypeptide with a predicted MW of 17,115 dal in the Djunaarian hamster IFN-gamma and 17,255 dal in the Chinese hamster one.

Djungarian (Phodopus sungorus) and Chinese (Cricetulus griseus) hamster IFN-gamma genes were cloned and sequenced. The Djungarian and Chinese hamster genes were both 525bp nucleotides, resulting in 174 amino acids in full length with a predicted molecular weight (MW) of 19,560 dal and 19,775 dal, respectively. The first 23 amino terminal amino acids consisted of a hydrophobic signal sequence when cleavaged, which would result in a mature 151 amino acid polypeptide with a predicted MW of 17,115 dal in the Djunaarian hamster IFN-gamma and 17,255 dal in the Chinese hamster one.

Parasitologic and histopathologic examinations were performed on a wild Japanese serow (Capricornis crispus) with severe dermatitis from Tochigi, Japan in February 1999. The serow was infested with a large number Chorioptes bovis. Marked parakeratotic hyperkeratosis was characteristic of the lesions induced by these mites. Japanese serow is a new host of C. bovis.

Djungarian hamsters were examined for the susceptibility to Neospora caninum infection. After 29 Djungarian hamsters were intraperitoneally inoculated with 5 x 10(6) N. caninum tachyzoites of JPA1 strain, some animals showed symptoms such as ataxia, and many tissue cysts were detected in the brain and a cyst in the muscular tunics of stomach. Especially, more than 100 cysts per head were observed after 5 weeks post inoculation. It is suggested that the Djungarian hamster is a model useful to examine neosporosis.

Dermonecrotic toxin (DNT) produced by Pasteurella multocida was purified by anion- exchange chromatography, gel filtration, and high-performance liquid chromatography. Pigs were given an intramuscular injection of P. multocida dermonecrotic toxin (Pm-DNT), which produced necrȯtic lesions of the liver. Liver sections were taken at intervals of 2, 12, and 24 hours after Pm-DNT injection. We examined the sections histopathologically, immunopathologically, and electron microscopically. The earliest alterations observed were loss of glycogen from hepatocytes, which became more severe during later intervals, vacuolation and single-cell necrosis of hepatocytes, as well as desquamation and fragmentation of the sinusoidal endothelium. The vacuoles subsequently increased in quantity and size with the passage of time and were found to contain homogeneous fine material and/or fibrillar material. These materials were thought to be fibrin and blood serum. The vacuoles penetrated the cytoplasm of the hepatocytes. We considered that liver necrosis caused by Pm-DNT probably resulted from the principal effects of vascular injury, although the membrane of the hepatocytes was more or less damaged by the toxin.

A total of 115 strains of Escherichia coli isolated from chickens with colisepticemia in Japan were examined for chicken lethality and virulence factors. It was found that serum resistance and aerobactin-mediated iron uptake are the most prevalent characteristics in these strains. Among them, S-20, a representative virulent strain of serotype O2, was further studied. S-20 harbored a conjugative 100-megadalton (Mdal) plasmid, designated pKI100. Curing and reintroduction experiments showed that pKI100 encodes both serum resistance and aerobactin-mediated iron uptake, and the diminished virulence of the pKI100-cured strain was fully restored by the reintroduction of the plasmid. These results demonstrated that pKI100 is the virulence plasmid of the S-20 strain, and that serum resistance and aerobactin-mediated iron uptake are the virulence factors in E. coli strains which cause avian colibacillosis.

A total of 115 strains of Escherichia coli isolated from chickens with colisepticemia in Japan were examined for chicken lethality and virulence factors. It was found that serum resistance and aerobactin-mediated iron uptake are the most prevalent characteristics in these strains. Among them, S-20, a representative virulent strain of serotype O2, was further studied. S-20 harbored a conjugative 100-megadalton (Mdal) plasmid, designated pKI100. Curing and reintroduction experiments showed that pKI100 encodes both serum resistance and aerobactin-mediated iron uptake, and the diminished virulence of the pKI100-cured strain was fully restored by the reintroduction of the plasmid. These results demonstrated that pKI100 is the virulence plasmid of the S-20 strain, and that serum resistance and aerobactin-mediated iron uptake are the virulence factors in E. coli strains which cause avian colibacillosis.

ブタヒゼンダニに対するin vitroでの薬剤試験法の確立を試みるとともに, 本法を用いて有機リン系殺虫剤であるナレドの殺虫効果について検討した. 豚疹癬罹患豚より痂皮, 皮膚片を集め, 37℃ で1時間以上放置したのち, 脱出したダニを有柄針で拾い上げ試験に供した. 薬剤試験用容器には, 底部外側に1cm間隔の画線を描いた直径9cmのガラスシャーレを用い, これにアセトンで所定濃度に希釈したナレドを1mlずつ滴下した. これを均等に拡散させ, 乾燥したのち, 採取した虫体を投入した. 採取した虫体には幼ダニ, 若ダニ, 成ダニの各ステージのものが見られたが, 数量的にこれらを区別して集めることは困難で, 本試験ではいずれのステージのものも1個体として計数した. 試験の結果, 対照群において, 6時間後には平均13.5%, 24時間後には100%の虫体が死亡したことから, 本法では6時間程度の試験時間が適当であると考えられた.本試験によるナレドの殺虫効果は0.2μl/dishでKT₅₀=0.78時間, 0.1μl/dishでKT₅₀=1.24時間で, 本剤はブタヒゼンダニに対し, 比較的速効性をもつ有効な薬剤であることが示唆された.