Comparison Of Spore Proteins And Infectivity Of Two Kinds Of Microsporidia

Microsporidia are obligatory intracellular parasites found almost in all members of animal phyla, and constitute most important pathogens in insects, fish and human. As well microsporidia are considered as promising protozoa for use in pest control. The variation in morphology and infectivity of microsporidia and their invasion mechanism are important theoretic problems, which need in depth study. Spore germination is a prerequisite process when the invasion occurs, and spore surface protein plays an important role in the spore germination. The comparison of the microsporidian proteins, especially of microsporidia with different infectivity to the same host is an effective way to explore proteins crucial for invasion. During the present study, three kinds of microsporidia namely Nosema bombycis, its variant obtained after passing through mulberry looper Hemerophila atrilineata, and third a small microsporidian isolated from Bombyx mori were used, consequently three kinds of spores of two different species were used. The morphology, infectivity, and spore protein of three microsporidia were studied, some proteins possibly related to spore morphology and invasion mechanism were found, which is useful in analyzing crucial information to elucidate the mechanism of microsporidian variation and invasion.1. A new method of microsporidia spore purification by high-speed centrifugation was established. In comparison with the ultra-speed centrifugation method (4℃, 73 000.g, 30 min) by Sato et a/.(1980), nearly 100% pure and mature spores were obtained by the high-speed centrifugation method (4℃, 46 000 .g, 90min) using Percoll. Thus the high-speed centrifuge can be used as an alternative purification method. Similar purity was achieved even when a small microsporidium were purified using this method. After comparing the durations of centrifuge, the results suggest, 90 min is appropriate.2. The infection of TV. bombycis influenced the midgut microfauna of Bombyx mori, the population of enterococci increased greatly, while the diversity of enterococci decreased. Two hundred strains of enterococci were isolated from the intestine of healthy, pebrine and bacterial flacherie diseased silkworms. These isolates were classified by using API STREP (V.5.0) system based on numerical taxonomy. The results showed that, in comparison with that of the healthy silkworms, the number of the enterococci in silkworms infected with N.bombycis to newly moulted 4th instar and 5th instar increased to 5.5 X 105 and 0.74 .102 times respectively, and the number of the enterococci species decreased from 6 in healthy to 396Ph. D Dissertation of Zhejiang Universityin 4th instar and 4 in 5th instar. The biochemical phenotypic diversity decreased from 27 to 13 and 14 respectively. The distribution frequency of Enterococcus avium decreased from 56% to 20% and 38% respectively; the distribution frequency of Ent. faecium increased from 2% to 40% and 16% respectively. There are 59 biochemical phenotypes within the 200 enterococci stains.3. Passing through different host induced the variation of spore morphology and infectivity. N.bombycis was inoculated into the microsporidia-free larvae of mulberry looper (H. atrilineatd). The spores thus isolated was designated as INbh and fed to the microsporidia-free H. atrilineata again. By continuously passing for 24 times, the spores obtained were designated as 24Nbh spores. Comparing the morphology of mature spores among Nb and several Nbh showed that, after 15 times of passage through H. atrilineata, the Nb spores became significantly short and wide. Compared with Nb, the length of 15Nbh spore decreased significantly (p<0.05), and that of 24Nbh decreased more significantly (p<0.01). The infectivity tests conducted on second instar silkworm larvae showed that IC50 of 24Nbh was 1.98xl04 spores/mL and of Nb was 1.72.103spores/mL, which indicated that the infectivity of Nb decreased 11.5 times after multiplying in mulberry looper for 24 times. The ICso of spores from silkworm infected with 24Nbh was 6.9 time...