Establishment Of Detection Methods For Neosporiasis And Epidemiological Investigation On Three Kinds Of Animals

The host range of Neospora caninum(Neospora caninum)is very extensive.Final host contain dog,cattle,sheep,dogs,horse,pigs,rabbits,livestock,wild animals and meiofauna.Neosporiasis is a protozoan disease caused by parasitic neosporacaninum in a variety of mammalian cells,contain dogs,cows,sheep,etc..The disease has particularly serious harm for cattle and sheep,and it is one of the main causes of world cattle abortion,and it caused huge economic losses to animal husbandry.At present there is no effective prevention methods,only be taken out of the sick livestock method to prevent and control Neospora caninum disease.The aim of this study was to establish a sensitive,rapid and accurate detection methods for Neospora caninum disease,and investigate epidemiological of the disease of the new spore for many kinds of animals at the same time,provide basis for the prevention and treatment of the disease.According to the sequence of the NcSRS2 of the new spore bug,a pair of PCR primers were designed and synthesized,and establish the PCR detection method of the new spore.PCR reaction conditions and reaction system were optimized to determine the optimal reaction conditions,and proceed sensitivity and specificity detection.Cloning NcSRS2 gene,connected to pMD 18-T cloning vector.The recombinant plasmid was 10 times of gradient dilution,the dilution of the plasmid as a template,doing PCR amplification,with good sensitivity when the concentration of the template is 10~3copies/μL.To testing the positive nucleic acid by using the primers for Neospora caninum,Toxoplasma gondii,theleria sergenti,theileria annulata,B.bigemina,Cattle Trypanosoma evansi,can only amplify Neospora caninum,and other positive nucleic acids were not expanded,which explained that the method had good specificity.According to the sequence of NcSRS2,a pair of fluorescent PCR primers and probes were designed and synthesized.With the recombinant plasmid as template,the detection method of fluorescence PCR was established.The reaction conditions and reaction system were optimized to determine the optimal reaction conditions of PCR.Then the sensitivity,specificity and repeatability were detected.The plasmid after dilution as a template when the template is10~2copies/μL,doing fluorescence PCR amplification.Can only amplify the positive gene of Neospora caninum,and other positive nucleic acids were not expanded,which showed that the method had good specificity.The recombinant plasmid 10~8copies/L~10~6copies/L was amplified by three times,results showed that its amplification time and the amount of amplification were similar,which explained that have a good repeatability.According to the sequence of Nc5,4 pairs of the specificity primers were designed,and the rapid detection method of LAMP(loop mediated isothermal amplification)were established.Through the optimization of LAMP amplification system and reaction temperature,and the sensitivity,reproducibility and specificity test,the result showed that the reaction temperature of LAMP is 63℃,its sensitivity can be detected in the lowest concentration is 10~2copies/L.It is proved that the LAMP method has high sensitivity and specificity.The recombinant plasmid10~6copies/L~10~4copies/L was amplified by three times,and the time of each amplification is basically the same,when can amplify the positive curve.It is proved that the LAMP detection method has good stability.To establish new Neospora caninum PCR detection method to detected of brain tissue of200 rabbits,the result showed there are 14 samples amplified NcSRS2 gene fragment,the positive rate was 7%.Comparative analysis of the 14 strains new Neospora caninum gene and13 strains SRS2 gene have finding that the homology of all genes were relatively close,the homology of SRS2 gene with the isolated NC1 strain was 99%,and the homology between Rabbit(1/3/6/7/8/10/12/14)SRS2 strain and Nc-Liv SRS2 JL is 100%.Comparative analysis of the SRS2 Nc-Liv strains gene and other genes have finding that there are individual base mutations.The base mutation of JL Rabbit 2/5/9/13 affected the translation of amino acid.Other genes also have base mutations,but does not affect the translation into amino acids.Using N.caninum VMRD competitive ELISA kit for the detection of 1630 samples of cattle serum,the cattle serum contain Jilin,Shanxi,Shandong,Henan,Xinjiang,Inner Mongolia and Qinghai regions.At same time,578 sheep sera and 200 rabbits sera were detected.The results showed that the positive rate of the infection of the cattle in Jilin was 18.09%,Shandong 15.39%,Shanxi35.94%,Henan 15.57%,Inner Mongolia 6.87%,Xinjiang 8.75%,Qinghai 10.92%.The positive rate of sheep N.caninum infection were 20.71%in Jilin,Shanxi 23.81%,Xinjiang 16.31%,Qinghai 7.98%.The positive rate was 24.5%of rabbits.