Isolation And Identification Of Porcine Epidemic Diarrhea Virus PEDV-YX Strain And Efficacy Of The Inactivated Vaccine

Porcine epidemic diarrhea(PED) is a severe intestinal infectious disease which is caused by Porcine epidemic diarrhea virus(PEDV) with diarrhea, vomit, and dehydration.Porcine epidemic diarrhea disease is one of the most widely epidemic diseases around the world. As a fulminating infectious disease, PED has brought the enormous economic loss to our breeding industry in recent years. PED was found in Guangdong, Guangxi, Jiangxi,Hunan, Fujian, Zhejiang, Anhui, Jiangsu, Hubei, Hebei, Shandong and other provinces in china,and it gradually spreaded in our country. The investigation among 26 provinces showed that the mortality of PED accounted for 1.74% of total mortality of 36 kinds of diseases. The disease usually occurs in winter and spring.But due to the environment of high humidity throughout the year in Guangdong, PED may continues year-round,which shows a trend of high incidence.The 7-days-newborn piglets suffering diarrhea would usually die within 3 or 4 days because of dehydration,and the mortality rate was as high as100%. At present, in West Guangdong, PED showed a high trend. Once entering winter or spring, it would be a large-scale outbreak, with the high death rate. Although vaccination could be taken to control the disease epidemic, but immune effect of the classical vaccine of strain cv777 was not ideal, for the vaccinated pigs with vaccine was still infected. This research is mainly divided into the following three parts:1.Isolation and identification of Porcine Epidemic Diarrhea Virus PEDV-YX strain180 copies of tissue samples were collected from 9 industrialized pig farm with suspected diarrhea outbreak in West Guangdong. 9 copies of tissue samples were identificated as PEDV positive by the colloidal gold rapid diagnostic technology, PCR amplification and gene sequencing technology, including four HS, four YC and one KP strains. Through the pig regression test, YC strain with the most obvious clinicalsymptoms and the strongest toxicity was selected as the research object and was named PEDV-YX. Under the action of 7.5μg/ml trypsin, the strain was incubated to the 8th generation.The cytopathic effect(CPE) was observed obviously,such as pyknosis, aggregation and increased vacuolization,2. Biology, neutralization test and determination of TCID50 of PEDV – YX.The results of biology, neutralization test and determination of TCID50 of PEDV – YX showed that : the isolated strains was RNA virus with membrane vesicles;insensitive to5-Brd U;sensitive to ether and chloroform; easy deactivation at p H 3.0 and more sensitive to heat, which was inactivated in 50℃ for 1h; the result of RT-PCR was as expected and the neutralizing titer of the antiserum with PEDV was 120.23; the TCID50 of the PEDV-YX F10 was 10-5.67/0.1ml.3. Preparation of PEDV-YX strain aluminum hydroxide gel inactivated vaccinePEDV-YX F10 was inactivated by 0.3% formaldehyde solution and the PED inactivated vaccine was formulated with 40 ml seedlings virus liquid sample,40 ml PBS,20 ml alhydrogel.The piglets were vaccinatied through orally and houhai acupoint with different dosages of 2ml, 1ml and 0.5ml and the dynamical changes of antibody induced by the PED inactivated vaccine was observed. The antibody titer of Houhai acupoint injection immunization is higher than orally, and the antibody maintained longer,when immuned with same dosage;The overall antibody level when immuned with2 ml inactivated vaccine is much higher than that of with 1ml, neutralizing antibody leve was the lowest when immuned with 0.5ml.It is helpful for us to know PEDV epidemic strain characteristics and epidemic situation in West Guangdong through separating PEDV. This study provides theory basises for the establishment of a fast and accurate diagnostic method of PEDV and its noval vaccine. so as to make effective prevention and control measures and promote the steady development of pig industry in the region.