Development Of An Indirect ELISA For The Detection Of Anaplasma Ovis Infection And Its Application

Anaplasma ovis is a tick-borne obligatory intraerythrocytic bacterium,which infects sheep and goats to cause ovine anaplasmosis.The clinical signs of the disease is fever,anaemia,haemoglobinuria and even death.The disease is distributed worldwide and seriously harms to the sheep and goats industry.At present,the commonly used diagnosis methonds are blood smear examinatioin by microscopy and PCR.Smear examination requires sophiscated expertise for the examiners and the PCR requires well equipped laboratory to conduct.In contrast,serological tests are more common in local laboratory.In this study,we investigated the specific Anaplasma ovis Appendage-Associated Protein(AAAP)by means of Mass spectrometric analysis and bioinformation analysis,and an indirect ELISA diagnostic technique was established.Then the ELISA was applied to detect the prevalence of ovine anaplasmosis in 54 different regions in 23 provinces in China.The major outcomes were presented as following:1.Immunoprecipitation assays was used to find the differential protein bands by incubation of A.ovis lysate with pre-infection and post-infection antibody from experimental sheep.The differential protein bands were identified by MS analysis combinding with genome date and BLAST searches in NCBI,eventually a candidate gene,AAAP was selected.2.The recombinant AAAP protein was Prokaryotic expressed and purified.Westeron blot analysis showed reactivity with A.ovis positive sera.And there is no cross-reactivity was seen with the serum samples of A.bovis,M.ovipneumoniae;M.capricolum subsp.capricolum;Babesia motasi.Lintan;Babesia sp.Xinjiang;T.uilenbergi;T.luwenshuni.The results indicated that the rAAAP was suitable to be used as a diagnostic antigen for ovine anaplasmosis.3.The specificity of the rAAAP indirect ELISA was estabished.The thresholed value,sensitivity,and specificity were determined by testing 597 sera(434 negative sera and 163 positive sera)and analyzed using MedCalc statistical software.When the thresholed was set to be 6.00 %,the sensitivity and specificity were 91.4 % and 94.5 %,respectively.4.The ELISA was applied to test 3138 sera collected from 54 different regions in 23 provinces.The highest prevalence of A.ovis infection was 66.67 %(66/99)in Yunnan province,the lowest prevalence was 9.41 %(8/85)in Henan province,and the results indicated that the average positive rate was 35.25 %(1106/3138).The results of this study provide a basic clues for integrate control of ovine anaplasmosis in the future.