Study On The Isolation And Identification Of Sheep PRV And On The Prevention And Control Of The Disease

Isolation and identification of the virus and Pathological observation from a suspected sheep pseudorabies case were studied.BHK-21 cells inoculated by tissue samples showed lesions.Pseudorabies virus(PRV)in BHK-21 cells was confirmed by specific fluorescence of indirect immunofluorescence staining and fluorescence PCR,and named as SH1311 strain.The morphology of virus particles under electron microscope was round and there were a lot of fibers on the surface of the capsule.The histopathological observation of infected sheep tissues showed extensive neuronal degeneration and necrosis occurred in the brain tissue accompanied by neurotropic phenomenon and gliosis.In this study,g D gene of SH1311 strain was amplified by PCR and sequenced,sequence alignments of gD genes indicated that SH1311 strain shared 99.8% to 100% nucleotide homology with the isolated PRV strains from Shanghai since 2012,but shared lower nucleotide homology(only 80.5%)with the ones from Shanghai in 2010.The evolutionary analysis showed that SH1311 strain had the same evolutionary branch with the isolated PRV strains from Shanghai during 2012-2015 and domestic epidemic stains HNB,HN1201,JS-2012,HLJ8,BJRD,ZJ01 and had a close relationship,but was in different branches with the strains isolated from Europe,America and South Korea.In order to evaluate the immune protection efficacy of Bartha-K61 vaccine resistance to SH1311 strain,14 6-month-old sheep were used to test.Of these,6 were inoculated with Bartha-K61 vaccine,5 were used as non-immune group,and 3 were used as blank control.Two weeks after vaccination,the immune group and non-immune group sheep were challenged intramuscularly with SH1311 strain,and then observed for 14 days to check clinical signs,rectal temperature,nasal virus shedding and infected antibody level.For non-immune group,4 sheep showed typical cutaneous pruritus symptoms of pseudorabies,the morbidity was 80%,and the fatality rate was 100%.Nasal virus shedding were negative for all sheep,but PRV in internal organs and brains of dead sheep were positive by fluorescent PCR detection.Among sheep vaccinated with Bartha K61 strain,no typical clinical signs were observed in all animals 14 days after challenge with SH1311 strain and nasal virus shedding were also negative for all sheep by fluorescent PCR detection.It confirms that porcine pseudorabies vaccine(Bartha K61 strain)can provide good protection efficiency against sheep PRV.In order to further study the pathogenic mechanism of SH1311 strain in sheep,Seven healthy sheep were randomly divided into test group(5 sheep)and control group(2 sheep),the test group were challenged by SH1311 strain,the tissue samples of infected sheep were collected for histopathological examination and immunohistochemical analysis.The infected sheep showed the clinical symptoms at day 3 post infection,and typical sysmptoms such as depression,anorexia,pruritus and an increase in body temperature were observed at day 4.Four sheep died within 3 days of the onset of sysmptoms and one survived,the mortality rate reached as much as 80%.Gross lesions showed dural congestion,pulmonary congestion and consolidation.Microscopically,the major histopathological findings were neuron degeneration and necrosis in brain and medulla,interstitial pneumonia,spleen and kidney hemorrhage.Immunohistochemical staining displayed that PRV exists widely in liver,lung,brain,spleen and other organs.The virus in liver mainly exists in Kupffer cells,type II cells in lung,macrophages in spleen,and neurons in brain tissue.The results showed that PRV resulted in degeneration and necrosis of the neurons in brain,interstitial pneumonia and renal hemorrhage.