| Pseudorabies (PR) , also known as Aujeszky's disease, is an acute and high contagious disease of a variety of domestic and wild animals caused by Pseudorabies virus (PRV). The disease is characterized with fever, extreme itch (except for pig) and encephalomyelitis. The infection in swine is most common. The sow usually display the reproductive disorders such as miscarriage, fetal death and mummy foetus. The mortality rates of newly born piglets are up to 100%. In addition, the infection in growth pigs usually leads to the decrease of semen quality, even the loss of species value. Therefore, the occurrence of the disease will result in enormous economics loss of swine industry. At present, the disease has been listed as class B animal loimia by the world health organization (OIE), and listed as the secondary kind animal loimia in our country.In recent years, the occurrence of the disease showed a rising trend. At present, the disease had became an important loimia which composes a threat to swine industry. Currently, the extreme majority of farms carried on immune prevention by the use of PRV gE gene deletion vaccine, so the use of PRV gE-ELISA antibody detection kit could quickly and accurately detect the antibody of PRV wild isolate, thus could effectively monitor the infection of PRV wild isolate. In order to understand the infection status of PRV wild isolate in the farms of different areaes in Jilin province in recent years, the PRV serum antibodies of 3849 serum samples from different batches collected from 152 different scale immunized farms of Jilin province were detected by PRV gE-ELISA antibody detection kit. The results would provide important theoretical basis for the formulation of scientific prevention and control measures. The serological survey result showed that the antibody positive rates in different areaes of Jilin province ranged from 9.47% to 40.00%, and the average positive rates was 22.66%. There were 103 PRV antibody positive farms in 153 detected farms, and the positive rates of farms ranged from 37.50% to 87.50%. The result showed that the infection of PRV wild isolate remained more serious. In addition, we carried on investigation and analysis for the PRV wild infection status in different ages, sexualities and breeds. The serosurvey result would provide important theoretical basis for the prevention and control work in Jilin province.In November 2008, the PRV natural infection occurred in a farm in Siping area of Jilin province. The pregnant sows showed the symptoms of abortion and dead foetus, and newly born piglets more than 50 showed vomit, diarrhoea and the neurological symptoms such as muscle trembling, hind limb parlysis and four limbs swimming sliding. The treatment had no effects by a variety of antibiotic. The sick pigs began death one after another after 2 to 3 days. The mortality rate was up to 100%. Necropsies were performed on died piglets. The main pathological changes of the samples showed meningeal congestion , pulmonary congestion and edema, liver congestion and canous necrosis focuses observed in the surface, however, no obvious pathological changes were observed in the other organs. According to the clinical symptoms and pathological changes, the disease was preliminarily diagnosed as Pseudorabies. Because the disease was easily confused with porcine reproductive and respiratory syndrome, porcine parvovirus and porcine brucellosis, the disease was aggregate analyzed from the pathology and etiology aspects. First, the histological section were performed on the inner organs of heart, liver, spleen, lungs, kidney, and brain tissue. The dead reason of piglets was analyzed by histological examination. Microscopic lesions in the brain tissue showed the non-suppurative encephalitis changes including perivascular cuffing and neuronophagia. So we concluded that the pathological changes were caused by viral infection. And the bacteriology detection result also exclused the possibility of bacterial infection. In addition, part of liver occurred necrosis and formed the focal cellular necrosis focus. The pathological change was an important directive change of PRV infection. In order to final diagnosis for the disease, the systemic isolation and identification of pathogen was performed on BHK-21 cells, and one PRV isolate was successfully obtained which be named PRV JL/08/SP isolate.In order to further understand the proliferation ability and dynamic infextation process of PRV JL/08/SP isolate on BHK-21 cells, the morphology development process of PRV JL/08/SP isolate and the ultrastructural changes of infected cells were observed by the manufacture of ultrathin sections. After adsorption to the cell membrane surface, PRV entered in the endochylema via membrane fusion way. The viral replication were carried out in the cytoplasm. The assembled virus left nucleus into cytoplasm by budding way. Then, the virions in the cytoplasm synthetized the second peplos using the membrane structure of Golgi's body and further formed complete virions. Eventually, Golgi vesicles packaging with intact virions occurred fusion with cellular membrane and the virons were released to the ecto-cell. The proliferation process and the release form were basic concordance with the report in previous literature. This indicated that the morphology development process of different PRV isolate were very similar, but exist certain differences only in the gain way of peplos. In addition, the virulence of PRV JL/08/SP isolate was determined using BHK-21 cell. The result showed that the titre of PRV JL/08/SP isolate on BHK-21 cells was 107.2/0.1mL. This indicated the PRV isolate had more stronger virulence. In order to further identify its pathogenicity, animal regression experiments were performed on rabbits and piglets, and duplicated the similar clinical symptoms with the natural infection. Above on, PRV JL/08/SP isolate was systemic identified in the study, and its pathogenicity was preliminary analyzed. Thus, the disease was final diagnosed as PRV wild virus infection.Because PRV wild virus infection especially inapparent infection was still very common in pig farms, the thorough eradication of PR confronted with a hard problem. Considering the reason, it was necessary to eatablish a quick, specific and sensitive diagnostic method for the detection of PRV wild isolate. In the process of controlling PR, PRV gene deleted vaccine had played a very important role, and the use of PRV gE gene deleted vaccine was most wide. Thus, the foundation of diagnostic method which could effectively distinguish from wild virus infection and vaccine strains would play extremely important role for the elimination of positive pigs and the cleaning of farms. At present, all PRV gE gene deleted vaccine strains deleted partial even complete gE gene, and the gE gene could stable expression in the wild virus isolate and its genetic variability was small, so gE gene could as a marker gene of differentiation for PRV wild virus and vaccine strains which used for PRV molecular level diagnosis. On the basis of obtaining PRV JL/08/SP, we designed specific primers according to the gE gene conservative sequences which published in Genbank database. Then, gE gene of PRV JL/08/SP isolate was amplified and cloned by PCR, and the gE gene was performed on sequencing analysis. The analytic result indicated that gE gene had individual nucleotide mutations compared with other isolates, but gE gene of PRV JL/08/SP isolate was still highly conserved from the the results of homology and genetic evolution analysis. So, the gE gene of PRV JL/08/SP isolate could be used as the diagnostic antigens for the identification of PRV wild isolate and vaccine strains. In addition, a rapid PCR detection method was established which used for the detection of clinical samples, and the method had strong specificity, high sensitivity and good reproducibility. A total of 68 samples including spleen, lung, liver and brain tissue collected from suspected PRV pigs which comed from the farms of different areaes in Jilin province were detected by PCR method. The positive rate of the samples was 27.94%, and it was consistent with the seroepidemiological survey result. This indicated that PRV infection was still very common in Jilin province. In the study, the PCR method based on gE gene of PRV JL/08/SP isolate not only could rapidly and efficiently detect the clinical samples, but also efficiently distinguish PRV wild virus and vaccine strains, which largely improved the detection rate and accuracy of PRV. In addition, the method had very important meaning for the diagnosis of earlier period PRV infection and latent infection persistent infection, so could provide a powerful technical support for the cleansing and eradication of PR.In addition, in view of the current severe situation of PR occurrence, it was necessary to performed on cleaning and eradication in different pig farms. Therefore, we applied PRV gE gene deleted vaccine combined with PRV gE-ELISA antibody detection kit to perform on immunization, monitoring and elimination in the six different scale pig farms in Jilin province from 2009 to 2010 years, thus achieved cleaning and eradication, and obtained the expectant cleaning effect. This would provide reference basis for PR cleaning and eradication in Jilin province even whole country. |
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