Investigation Of Eimeria Spp.of A Rex Rabbit Farm In Fujian And Developement Of Real-time PCR For Detecting Tow Rabbit Eimeria Spp.

Eimeria spp.of rabbit is one of the major parasitosis in domestic rabbits and cause great economic lose.Investigation of Eimeria spp.of rabbits from different ages,species and and seasons should be an essential input to prevent and remedy of Eimeria spp..And we should focus on developing and validating an effective test to explore and control these infections in rabbits.1.Epidemiological investigation and identification of Eimeria spp.of a Rex rabbit farm in Fujian province.In order to investigate Eimeria spp.infection of a Rex rabbit farm in the northeastern Fujian province,658 faucal samples were collected from different age of Rex rabbits and different seasons from year,and examined by McMaster counting and the method of saturated saline flotation.The results showed average prevalence of infection was 84.80%and the mean oocyst per gram(OPG)value were 1.78×104.The infection rates of Eimeria spp.among each rabbit age were different,and it was highest at the age of 30 to 50(97.14%)rabbits and the mean oocyst per gram(OPG)value was 4.17×104.The age over 150d rabbit’s infection rates(62.5%)and OPG(1213)were significantly lower.Infection rates and OPG of Autumn’s were all the highest one and winter was the lowest one around the year.The distribution pattern of OPG values was negative binomial distribution in rabbits by using fitting of distributions.In total,eleven species of Eimeria spp.were identified from oocyst-positive samples.The infection of Eimeria spp.was mixed infection with more than two species,and mostly of which were infected by three to seven Eimeria spp..The dominant species of Eimeria spp.were E.perforans,E.magna,E.exigua and E.media,and their prevalences were 32.90%,21.02%,12.77%and 10.12%,respectively.The infection of species at different ages and seasons were different.2.Establishment of SYBR Green I Real-time PCR to assay E.flvescens,E.magna and E.intestinalis.According to the biological of Eimeria spp.oocysts,we lysised oocysts by repeated freezing and melting,we extracted DNA template from Eimeria spp.oocysts by the methods of repeated freezing and melting and operational approach by the kit.Found the especial primer sequences of F,M and I to amplify the ITS1/ITS2 sequences.Clone the insert DNA into pMD-18-T vector which should be transformed into E.coli DH5α and checkout them(pMD-18-T-F,pMD-18-T-M and pMD-18-T-I).The efficiency of standard curve of three genes were both between 97%～105%,and the R2(coefficient correlation)were all above 0.995.Daul SYBR Green I Real-time PCR of M and I have been set up and its melt curve was 84.5℃ and 81.3℃.The melt curve of negative control have no amplification.Application of this method which can offer higher specificity and sensitivity could make great contribution to detect and identify the Eimeria spp.of rabbit.