Survey Of Epidemiology And Study On Molecular Characters Of Microsporidia In Sheep And Goats

Microsporidia are unicellular eukaryotic, obligate intracellular protozoan parasites that infect a broad range of invertebrates and vertebrates. The first Microsporidia identified was Nosema bombycis, which infects silkworms in1857. The first mammalian infections with Microsporidia were reported in rabbits in1922. The first reported human Microsporidia infection was published in1959. First found microsporidia in a AIDS patient was in1985. To date, more and more publishments reported that Microsporidia infects mammals (includes people), there are at least eight of these generas, including14species, cause disease in humans. Enterocytozoon bieneusi, Encephalitozoon cuniculi, E.intestinalis and E.hellem are the most commonly reported Microsporidia infect both man and nonhuman mammals. As an important parasite both in humans and mammals Microsporidia gradually attracted many researchers’attention. Microsporidia has been recognized as an opportunistic infection associated with chronic persistent diarrhea in immunocompromised individuals, but in immunocompetent persons with acute, self-limited diarrhea. Microsporidia can also cause human’s keratoconjunctivitis. In the world, there were many reports about Mircosporidia infected in humans, pigs and cattles, but rarely in sheep or goats.In order to understand the epidemiology of Microsporidia in sheep and goats in Henan province, we used Chromotrope2R stain and PCR to detect Microsporidia and study on molecular characteristics of it.1. In order to understand the infection of intestinal parasites in sheep in some areas of China, we collected1109fresh fecal specimens from sheep in six cities of Henan province(they are Zhengzhou, Dengfeng, Anyang, Luoyang, Sanmenxia, Shangqiu) and Shuangliao city of Jilin province, Chaoyang city of Liaoning province, Dongying city of Shandong province between March2011and February2012. We examined the presence of oocysts, cysts or eggs in all the samples using the Centrifugation method、the Lugol’s iodine-solution staining method and the Sheather’s sugar flotation. Nine intestinal parasite species were discovered in1078samples and the overall infection rate was97.20%; they are Eimeria spp., Cryptosporidium, Trichuris, Nematodirus, Strongylus spp., Moniezia, Trematoda, Giardia and Entamoeba. Eimeria spp. were found with the highest infection rate of93.51%.80.70%(895/1109) of the positive samples were mixed infections (the number of coinfection are between2and5).The highest coinfection rates are41.90%(2) and 44.47%(3).2. E.bieneusi, E.cuniculi and E.hellem had been reported in sheep and goats. In order to understand the infection of Microsporidia in sheep and goats in six cities of Henan province, Chromotrope2R staining method was used to detected spores of Microsporidia from802sheep (goats) between July2011and December2011.15positive samples were detected, and the infection rate was1.78%. After staining the spores were stained pink which were oval while bacteria and fecal residue dyed green. Many spores also showed a typical band structure, that was, diagonal or vertical line of the belt-like surrounding the spores. In order to identified the infection rate and species of Microsporidia in sheep (goats), Nested PCR was used to detect the fecal specimens. As a result,23positive samples were amplified (607bp) from802samples, and the infection rate was2.87%, they are all E.bieneusi. The infection rate of Microsporidia in sheep (3.90%) was higher than that in goats (1.47%); the infection rate of Microsporidia in juvenile (3.16%) was higher than that in adult (2.44%); the infection rate of Microsporidia in sheep/goats (4.59%) between July and September was higher than that in sheep/goats (0.82%) between October and December; the infection rate of Microsporidia in sheep/goats which were grazing (4.55%) was higher than that in sheep/goats which were domestic (1.94%).3. To study the molecular characteristics of Microsporidia in sheep (goats),23positive samples amplified by nested PCR were sequenced. Subsequently, Blast methods was used to search homological sequences in NCBI and14sequences were download.15sequences were alignmented. Phylogenetic tree and homological analysis were made by some biological softwares such as Clustal X1.81, Phylip3.64and DNAstar4.0. The results showed that the homoloty between the positive samples (JQ769381) and AF024657(found in AIDS patients) was up to100%, which showed that the isolate from sheep (goats) was E.bieneusi. There is only one different base between JQ769381and AF023245(which was isolated from Macaca mulatta), AY257180(which was isolated from Bos taurus), the homoloty was99.8%;4different bases between JQ769381and AY298728(which was isolated from AIDS patient), the homoloty was99.3%;5different bases between JQ769381and AY315717(which was isolated from AIDS patient), the homoloty was98.7%;14different bases between JQ769381and DQ793212(which was isolated from falcon), the homoloty was97.5%;21different bases between JQ769381and AH012971(which was isolated from AIDS patient), the homoloty was95.5%.This was the first survey of epidemiology and molecular characters of Microsporidia in sheep and goats of several areas in Henan province, the result showed that the the homoloty between Microsporidia found in sheep (goats) and AF024657(found in AIDS patients) was up to100%, indicated that the Microsporidia isolated from sheep (goats) may infect humans Microsporidia is one of the most important zoonosis, so it is fairly important significance of public health to research the Microsporidia in sheep and goats...