Development And Application Of Molecular ELISA Method For Diagnosing Bovine Schistosomiasis

Schistosomiasis is a serious worldwide amphixenosis, which is also one of the six tropical diseases defined by WHO and meanwhile also one of the five serious diseases prevalent in China. The great varieties and quantities as well as the large amplitude for movements of the susceptible domestic and wild animals make it hard to control the transmission of schistosomiasis only employing chemotherapy. Specifically, farm cattle, who is susceptible to schistosoma japonicum, with a long growh cycle, large quantities of diachoresis and more chances to be involved in pestilential water during grazing or working, also have increased the difficulties for the prevention and control of bovine schistosomiasis. Diagnosis is central to the control of schistosomiasis. But at present, there havn't any available stardard kit for diagnosing bovine schistosomiasis, so the development for quick, accurate and simple diagnosing agents is one of the most important contents of the prevention and control of schistosomiasis. The 23KD integral membrane protein of schistosome and the signaling protein 14-3-3 of Schistosoma japonicum were presumed as potential diagnosing antigens. Thus in this research, the recombinant LHD-Sj23-GST and Fusion-GST were highly expressed in E.coli; the recombinant expressed fusion proteins were employed to develop indirect ELISA methods for detecting antibodies of bovine schistosomiasis. Subsequently, the established rLHD-Sj23-GST indirect ELISA method was employed to detect the antibody of experimental cattle from 0 to 54 weeks after the infection with cercarial of schistosoma japonicum and the the antibody of experimental cattle from 0 to 28 weeks after the reinfection with cercarial of schistosoma japonicum. Therefore the variation law of the antibody in the experimental cattle that infected and reinfected with schistosoma japonicum was preliminarily elucidated.In this research, the primers were derived from the DNA sequences that have been published in GenBank, and the RNA template was extracted from the worms with a kit, then the RT-PCR method was employed to amplify the LHD-SJ23 gene fragment and the Sj14-3-3 gene fragment of Schistosoma japonicum. It is the first time that a splicing overlap extension PCR method was used to fuse the LHD-Sj23 and the Sj14-3-3 gene fragment of Schistosoma japonicum. Subsequently, the E.coli expressing system was employed to highly express the rLHD-Sj23-GST, rSjl4-3-3-GST and rFusion-GST protein, and all of the expressed products were proved good immunoreactive activity by western-blot.The indirect ELISA diagnosing methods were established employing the purified rLHD-Sj23-GST and rFusion-GST as an antigen respectively. The established indirect ELISA methods presented negative results when detecting the positive sera of Babesia orientalis and Theileria sp. Serum dilutions for detecting the specific antibody with the indirect ELISA methods could be as high as 1:320. The diagnosing agents tests within and between batches demonstrated that the coefficient of variability was under 10%. The shelf life tests showed that the batches coated with rLHD-Sj23-GST stored at 4℃for 6 months could still keep good condition. All of the reults above demonstrated that the established indirect ELISA methods had high specificity, good sensitivity, stability and longer shelf life of products at 4℃(rLHD-Sj23-GST).The cercarial of schistosoma japonicm emitting from the positive Oncomelania hupensis was employed to infect and reinfect experimental cattle, and the Schistosomiasis of cattle was produced. After the infection we monitored the infection process of the experimental cattle in four aspects as follows: the clinical syndrome, hatching of miracidium, the variations of physiological and biochemical indexes of cattle after infection and the results of circumoval precipitin test. After the infection, we observed the variations of the clinical syndrome of the experimental cattle; the miracidium hatching test observed the hatching of the miracidium in five experimental cattle 49 days after the infection; the physiological and biochemical indexes of cattle from 0 to 14 weeks after the infection exhibited certain variations; the circumoval precipitin test employed with the sera from 1 to 16 weeks indicated that the sera from 7 to 11 weeks were positive; all of the results above demonstratd that the experimental cattle has successfully infected with schistosoma japonicum. The established rLHD-Sj23-GST indirect ELISA method was employed to detect the antbody of experimental cattle from 0 to 54 weeks after the infection with schistosoma japonicum and the the antibody of experimental cattle from 0 to 28 weeks after the reinfection with schistosoma japonicum. The results indicated that after the first infection the antibody increased to a positive level in 5 to 8 weeks; the peak of the antibody was in 9 or 10 weeks after infection; then the antibody decreased gradually, the antibody of most cattle decreased to a negative level in 18 to 22 weeks. But the variations of the cattle number 4 was distinct, the antibody of who decreased to a negative level in 34 weeks after the infection. However the situation of variations in reinfection was different. In the early stage of reinfection (2w or 3w), the antibody increased to a positive level, but the increasing was rather slow and the peak of the antibody was in 14 to 18 weeks after reinfection, which is obviously distinct from the case of the first infection. Subsequently the antibody of cattle number 1 and number 3 decreased gradually to a negative level, but the antibody of cattle number 4 decreased very slowly, it hasn't decreased to a negative level in 28 weeks after reinfection.In this research the established rLHD-Sj23-GST and the rFusion-GST indirect ELISA methods will pave a road for the development of a quick, accurate and simple diagnosing kit for bovine schistosomiasis. Futhermore it is also helpful for the diagnosis and epidemiologic survey, which would be of great significance to the prevention and control of bovine schistosomiasis. In addition, the elucidation of the variation law of the antibody in the experimental cattle infected and reinfected with schistosoma japonicum was important for us to master the characteristic of the antibody variation after the infection and reinfetion of schistosoma japonicum, and it also provided evidences for the prevention and control of bovine schistosomiasis.