| Bluetongue disease(BT) is a non-contagious, insect-borne, viral disease of ruminants, mainly sheep and less frequently cattle, goats, buffalo, deer, and wild animals. It is caused by the bluetongue virus(BTV) which is transmitted by the midge Culicoides imicola, Culicoides variipennis, and other culicoids. BTV is prototype members of the Orbivirus genus in Reoviridae family, in which 27 distinct serotypes bluetongue virus(BTV) have been recognized. The major signs are high fever,excessive salivation, swelling of the face and tongue and cyanosis of the tongue, as well as oral inflammation edema, and dental ulcer accompanied. Oftenly BT causes pregnant ruminant animals with abortions, stillbirth, especially, in sheep BTV causes an acute disease with high morbidity and mortality, hence resulting in the big losses in animal production.This experiment according to the Xinjiang bluetongue epidemiological investigation of historical data and the life of insect vectors library midge, Yuli county were choosen as monitory point, and five sheep, five goats and ten cows were placed as the sentinel animals. Blood samples were taken regularly every week. From May 25, 2014, there were 21 times in total, 420 parts serum and heparin anticoagulant were collected. Using the BTV competetive ELISA to detect the serum of sentinel animals, testing the BT infection serological. The serum of sentianel animals with negative turn positive bluetongue antibody were inoculated BHK-21 and conducted bluetongue virus isolation. The samples with cell pathology were detected by antigen-capture ELISA, RT-PCR amplification,performed electron microscopy morphology, samples of the CPE were identified by BTV serogroup.Then using BTV VP2 fragment RT-PCR amplification, 1~24 type of serum neutralization test and virus neutralization test to identify the serum type of the BTV isolated strains.Results showed that 8 sentianel animals serum were detected to positive by the method of competitive ELISA, among them the turned positive rate of sheep were 15%(3/20), goat were25%(5/20), the turned positive time of sheep were from June to July, the goat were from September to October, the cows did not occur. According to the time of the turned positive could be seen that the susceptibility of sheep were earlier than the goat. The anticoagulant of turned positive inculated BHK-21 cells, 7 blood samples appeared cytopathic effect(CPE). The cytopathic effect mainly manifestied cell shrinkage, cell turn to round and cell agglomeration, after the pathological changes, the cell would began to fall and the gap increased, eventually form plaques. The isolated strains were positive by antigen capture ELISA test. The morphological characteristics of typical BTV observed by electron microscope virus: there were clear boundaries between particles center virus and the nucleocapsid, the strains were surrounded by fluff layer surface of the virus, the size were 70~80 nm; 7 strains of bluetongue virus isolated strain TCD50 between 10-3.33/0.1 m L~10-5/0.1m L; The highest homology of specific fragment VP7 compared with bluetongue virus sequences were 80%~82% by gene amplification, sequencing alignment genetic evolution analysis. By the above identification the 7 isolated strains were belong to bluetongue virus. Virus neutralization test results showed that existing BTV 1~24 typet, the isolated strains and corresponding serum had no neutralization protect ability, every hole occurred cell pathology; sequecing results of type specific fragment VP2 showed that the homology between the existing BTV and the isolated strains were nothigh from 64%~70%. Thus, the 7 isolated strains were belong to BTV serum group, but not belong to the present BTV serum type.Results: This experiment first isolated non BTV 1~24 type of new BTV strains from Xinjiang,provisional named XJ/1/2014~XJ/7/2014 strains. Preliminary determined as new BTV serotype, but the specific serotype identification requires further study to determine. |
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