Epidemiological Investigation Of Porcine Viral Diarrhea In Parts Of Shandong Province In 2016-2018

Porcine epidemic diarrhea(PED)is an acute intestinal contact infection in pigs,which mainly causes vomiting,severe diarrhea and dehydration in pigs.Infections caused by viruses and bacteria and improper feeding can cause diarrhea in pigs,but viral infection is a major factor.Porcine epidemic diarrhea virus(PEDV),Porcine rotavirus(RV),Porcine transmissible gastroenteritis virus(TGEV)and Porcine delta coronavirus(PDCoV),etc.are common viruses causing diarrhea.Being very similar in the transmission route and clinical symptoms,the four viruses could not be distinguished in clinical symptoms and pathological anatomy.Recently,more and more studies have shown that the four viruses are often mixed infections in the clinic,so it is a huge challenge for the differential diagnosis of diseases.Some differential testing methods were needed to be established in the laboratory.Obviously,single-detection technology can not meet the needs of clinical large-scale detection.Multiple RT-PCR technology was especially suitable for mixed infection for simultaneously detecting multiple clinical samples in one reaction system and identifing several pathogens in a short time.In this study,a multiple RT-PCR detection technology that can simultaneously detect and distinguish four viruses was established and initially used for clinical sample testing.Based on the conserved sequences of four viruses in Shandong's main epidemic strains in recent years,the specific primers were designed for simultaneous detection of PEDV,RV,TGEV and PDCoV.Sensitivity experimental data showed that the multiplex PCR method established in this study can amplify the minimum template amount of PEDV,RV,TGEV and PDCoV were 5copies,50 copies,5 copies,and 5 copies respectively.The PCR method is specific for it could not amplify other common pathogens.It can be used for the detection of clinical samples.The clinical samples,collected from Dezhou,Qingdao and Binzhou cities in Shandong Province in 2016-2018,were tested by the established multiple PCR technique.The total number of samples about feces of intestinal contents in the clinically-infected pigs were1212.The test results showed that the positive rates of PEDV,RV,TGEV and PDCoV were15.51%(188/1212),4.54%(55/1212),1.40%(17 /1212),and 2.06%(25/1212)respectively;while the positive rates of double mixed infections such as PEDV and TGEV,PEDV and RV,TGEV and RV,and TGEV and PDCoV were 3.63%,1.24%,1.07%,and 1.65%,respectively;the positive rates of triple infections such as PEDV,TEGV and RV,PEDV,TEGV and PDCoV,and PEDV,PDCoV and RV were 1.65% and 1.82%,0.83% respectively.The results showed that the total infection rates of PEDV,TGEV,RV and PDCoV were 24.68%,14.36%,7.18%,and 6.36% respectively,and the total mixed infection rate was 11.89%,but no four viruses mixed infection was found.To understand the specific differences in S gene between PEDV vaccine strains and the current epidemic strains in some areas of Shandong Province,8 strains were selected from the positive drug samples of different cities from 2016 to 2018.The S gene of the 8 wild strains were amplified,cloned and sequenced,and the genetic evolution were analyzed.The results showed that the S gene of 8 wild strains had 12-nucleotide insertion and 3-nucleotide deletion compared with the domestic vaccine strain CV777,and the SDJN171115 strain had a another3-nucleotide deletion in 3592-3594.In the 8 PEDV wild strains,8 amino acid mutations were identified in the neutralizing epitope region of the S1 region of the major mutation region of the S gene.The homology alignment revealed that the nucleotide homology between the 8strains of Shandong Province was 93.7%-99.9%,and the nucleotide homology was93.1%-98.9% between the the 8 strains and the reference strains.Genetic evolution analysis showed that the identified strains were divided into two different groups,and five of the sample strains were closely related to the main vaccine strains,but they were still related to the epidemic strains in China in recent years.The results indicated that the PEDV strains in some parts of Shandong Province in recent years had a large variation compared with the main vaccine strains in China,and it might be necessary to develop a more effective vaccine for the prevention and control of PED.