| The coccidiosis in pigs is a common protozoal disease caused by Isospora suis and some species of Eimeria coccidian which can cause diarrhea. This disease is widely distributed all over the world. In some western countries,I. suis is the main pathogen of causing diarrhea in 7-14 days old piglets. Because the clinical symptoms of I. suis were similar to other piglets diarrhea, it is difficult to diagnose the disease for veterinary. In addition, due to the strict host specificity, animal model only limited in pigs. However, because of the selfish characteristics of parasite and the relatively large difficulty of experimental management, as well as the lower reproductive rate of oocysts and the uncertainty of artificial infection, thus, there are few studies on pig coccidian. Clinical treatment of this disease is to choose some sulfa drugs, but the treatment effect is not stable; according to the traditional classification, I. suis belongs to the Eimeriidae and Isospora, however with the study of coccidium in other mammals, the coccidium in mammals is considered as coccidium of encystations that is different from coccidium of birds. In this paper, through the study and the analysis of biological characteristics and phylogenetic relationship of I. suis, the author conducted artificial infection experiment to evaluate the effect of toltrazuril towards/. suis. All these provide the theoretical and experimental basis for the prophylaxis and treatment of I. suis, and provide the evidence to the classification of I. suis.I The dentification and biological characteristics of I. suis,To explore the biological characterization and the regulation of oocysts shedding of piglets. The oocyst isolation method were used to obtain the pure oocyst of I. suis. Then the 24-old-day coccidia-free piglets were infected with 400 or 1×104 the sporulated oocysts of I. suis respectively. After infection the clinical symptoms, the period of prepatent, patent period and the amounts of oocysts were observed. The result showed that the piglets infected with 400 sporulated oocysts did not show obviously clinical symptoms. The piglets infected with 1×104 sporulated oocysts showed obviously clinical symptoms at six days post infection(DPI), including depression, anorexia, and emaciation, pale skin. The piglets recovered at twelve DPI. The regulation of oocysts shedding in the two groups was similar. The prepatent period was 5 days in both groups, but the patent period was 7 days in pigs infected with 400 oocysts and 18 days infected with 1×104, respectively. The peak of oocysts shedding were in 7-11 DPI and 7-12 DPI, respectively. A total of 1.27x106 oocysts per piglet was shedded in the group infected with 400 oocysts, and 6.47x 106 and 6.37x106 oocysts per piglet were shedded in the two group infected with 1×104, respectively. The results showed that the strain 20 mg/kg I. suis( named as I.suis(Yangzhou))was pathogenicity to piglets and could be used to study pig coccidiosis.2 Cloning and phylogenetic analysis of the 18S rRNA gene of I. suisIn order to learn about the classification status and characteristics of I. suis at molecular level, explore the relationship among coccidian, the 18S rRNA gene of I. suis was obtained by PCR, cloning and sequencing. The homology of 18S rRNA gene was analyzed and the phylogenetic tree was constructed, through compared with the similar species 18S rRNA gene of parasites from GenBank. The results shows that the sequence similiarity of 18S rRNA gene between I. suis and some other isospora spp, which the host were mammals, was among the range of 98%?99.8%. The 18S rRNA gene of I. suis was relatively conservative, which can be used as a molecular diagnostic target gene. At the same time, the phylogenetic tree showed I. suis and other isospora spp, which have no Stieda bodies and take mammals as hosts, is more closely to Toxoplasma, Neospora and Sarcocystis spp. than Eimeria indicating that Isospora belongs in the family Sarcocystidae instead of Eimeriidae. This is different from the traditional classification methods.3 Cloning and phylogenetic analysis of the ITS gene of I. suisIn order to take the further study on the category of I. suis, the ITS gene was obtained by PCR, Cloning and sequencing. The homology of ITS gene was analyzed and the phylogenetic tree was constructed, through compared with the similar species 18S rRNA gene of parasites from GenBank. The results shows the similarity between different cysto-isospora species ranged from 88.3%?91.6%. The similarity between I. suis and T.gondi and N.caninum was 51.0%-51.5%. The similarity between I. suis and Hammondia was 45.8%?49.2%. Whereas the similarity between I. suis and Eimeria Species was 43.4%?48.0%. At the same time, the homology of ITS-1 gene was analyzed and the phylogenetic tree was constructed, through compared with the similar species 18S rRNA gene of parasites from GenBank. The similarity between two I. suis was 99.0%, with other Isospora ranged from 74.5%?94.5%, whereas the similarity with T. gondi was 32.6%?35.3%. Conclusion:This results are consistent with 18S rRNA gene, the coccidium of mammals on the molecular level belong to the Sarcocystidae, and the coccidium of birds belong to Eimeriidae.4 Efficacy of toltrazuril thixotropic suspension for coccidiosis of pigletsIn order to evaluate the effectiveness of Toltrazuril thixotropic suspension against Isospora suis. the piglets were divided into 6 groups:the high dose group(40 mg/kg), the middle dose group(20 mg/kg), the low dose group(10mg/kg), the control group of Totrazuril suspension(20mg/kg), the positive and the negative control group. The piglets infected with 10000 sporulated oocysts of I. suis orally 4-day-old,2 days later, the dosage of Toltrazuril was given orally. The parameters of clinical symptoms, viscosity of feces, diarrhea rate, the numbers of oocysts shedding of piglets, oocysts decrement rate and the changes of piglet weight and histopathology were observed. The diarrhea phenomenon was not found in the process of high dose groups, low dose groups, control group, negative groups experiments, the oocysts shedding was not detected in the feces, also the differences between the weight gain of groups was not significant; while slight diarrhea and a small amount of oocysts shedding appeared in the low dose groups experiment process. In contrast, the pathological changes in the positive groups and the weight gain comparing to the negative groups was significant. All these indicate that the toltrazuril thixotropic suspension and toltrazuril suspension have a good efficacy in anti -I. suis. The clinical recommended dose of toltrazuril thixotropic suspension was 20 mg/kg. |
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