| Akabane disease,caused by akabane virus,is an insect-borne infectious disease in ruminants such as cattle and sheep,and is clinically usually characterized by abnormal reproduction or abnormal production of fetuses and congenital joint curvature-hydrocephalic syndrome(AH).Nowadays,with the continuous rise of the animal husbandry,animal diseases have also to be a problems of clinical diseases for healthful aquaculture.Mass abortion is one of the common diseases in the breeding process,which has caused huge economic losses to the aquaculture industry,so this study were conducted epidemiological investigation on the pathogen of clinical mass abortion and conduct related research mainly on akabane disease.1.Detection of the cause of mass abortion:A total of 1395 samples including suspected abortion samples and random samples from cattle and sheep in different regions was collectted,to detect abortion pathogens such as akabane disease,chlamydia,Q fever,brucellosis,by etiological methods,and detect AKAV antibodies by serological methods.The etiological test results showed that samples from Zoige in Sichuan and Gannan in Gansu were detected AKAV in bovine samples,with detection rates of 26%and 17%.The total detection rate of chlamydia was 26%,then the total detection rate of Q fever was 22%,and the total detection rate of brucellosis was 21%.Serological tests also detected AKAV antibodies in Zoige,Sichuan Province and Gannan in Gansu Province,with detection rates of 18%and 4.6%.None of the sheep samples detected Akabane virus.The overall detection rate of chlamydia was 22%.The total detection rate of Q fever was 23%.The overall detection rate of brucellosis was 14%.Analysis of the data shows that the infection rate of mass abortion in cattle is chlamydia,Q fever,brucellosis,and akabane disease.In the flock of sheep,Q fever,chlamydia,brucellosis were usually occurred,but akabane disease was not detected.2.Molecular epidemiological analysis of Akabane disease:Bovine whole blood and serum samples detected by etiology suspected of infection with akabane disease were treated and inoculated into bovine kidney(MDBK)subcellular cells for virus isolation,and the 6 generations after blind transmission of virus was identified,and specific primers were designed for amplification sequencing.When the sequencing results were consistently compared,a genetic evolutionary tree and nucleotide homology analysis were constructed,and their genetic evolution relationship and homology with other akabane virus strains in other regions were determined.The results showed that F1 and F2 generation cells had cytopathy and PCR identification was positive,but F3-F6 generation had no cytopathy because of PCR identification was negative.F2 generation PCR product sequencing comparison results were akabane disease,and the affinity with KU375443.1 Jilin isolate was high,and the homology with KU375443.1 gene sequence reached more than 99%.It shows that the isolate is likely to come from Jilin,China.3.Establishment of indirect ELISA method for akabane disease:G1 gene fragment with good antigenicity was selected as the target fragment,pET-32a(+)-G1 prokaryotic expression vector was constructed,the induction expression and antigenicity detection of recombinant protein were carried out,the recombinant protein was purified by nickel column to obtain G1 protein for coating as a diagnostic antigen,the indirect ELISA method was established by the control variable method,and the specificity,sensitivity and repeatability of the method were verified,and the compliance rate was calculated by comparing with the commercial kit.The results showed that the recombinant protein was expressed in the form of inclusion bodies,the purified concentration was 0.75mg/mL,with the purity was 93%,and the western blot verified that it had good responsiveness to AKAV-positive serum.The protein was coated at 1:80,the concentration of protein per well was 9.375μg/mL,and the serumdiluted 1:100 was the optimal condition;The optimal blocking solution was 1%BSA,and the best enzyme-labeled secondary antibody was diluted 1:20000 as the optimal condition;The cut-off value of seronegative positivity is 0.25;through verification,this method has good specificity,sensitivity and reproducibility,and the compliance rate of clinical sample detection at the same time as foreign competitive ELISA kit is 80%.In summary,this study collected samples from cattle and sheep in different regions,used etiological and serological methods to detect the causes of brucellosis,chlamydia,Q fever,akabane disease and other mass abortion diseases.The results revealed the distribution and infection of these four pathogens,and amplified the M fragment gene of akabane disease virus by specific primers for genetic evolution analysis and nucleotide homology analysis,and clarified the kinship and homology between the obtained sequences and domestic and foreign reference plant sequences.The indirect ELISA method was established by selecting the G1 protein of akabane disease,which had a high compliance rate with foreign commercial kits and could be used for the detection of clinical samples. |
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