The Epidemiology Of Porcine Circovirus Type2in Southeast China And A Multiplex RT-PCR Assay For Rapid And Differential Diagnosis Of Four Porcine Diarrhea Associated Viruses (PCV2/PEDV/TGEV/GAR)

Porcine circovirus disease is the general term for a series of pig diseases caused by porcine circovirus type2alone or secondary/mixed infection with other pathogenic microorganisms which mainly include postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome(PDNS), congenital tremors-ALL,(CT-ALL), proliferative and necrotizing pneumonia(PNP), proliferative and necrotizing enterocolitis and reproductive failure and so on. The first outbreak of the disease was in Canada in1991, and now the disease is widely spread worldwide and has a catastrophic economic impact on the global pig industry, Lang Hong Wu first reported on this disease in our country in2001.An outbreak of diarrhea in pigs started in China in October2010and caused heavy economic losses to the pig industry in China. TGEV, PEDV, GAR and PCV2are dominant causes of enteric disease in swine. They replicate in differentiated enterocytes, resulting in diarrhea and malabsorption in pigs, the clinical course of PEDV disease was markedly affected by transplacental infection of PCV2. They are often give rise to simple or mixed infection. In this study, the serological survey and molecular epidemiology of PCV2were explored in Southeast China. At the same time, a multiplex RT-PCR assay was developed and applied to four porcine diarrhea associated viruses(TGEV, PEDV, GAR and PCV2). The contents of this paper contain three parts as following:1. Serological survey of porcine circovirus type2in Southeast China from2008to2012A total of2626(1990copies were not immuned by commercialized PCV2vaccine, 636were immunized by commercialized PCV2vaccine)pig serum samples were collected from120large pig farms in six provinces in Southeast China from2008to2012for detection of porcine cirovirus type2(PCV2) antibodies using commercialized diagnostic kit (PCV2-ELISA). Among the1990samples tested,1417samples were were diagnosed as PCV2positive (71.21%). The positive rates were53.40%,35.33%,80.63%,86.37%and78.43%for samples collected in2008,2009,2010,2011and2012respectively. The positive rates were80.94%,68.02%,68.63%and55.79%for samples collected in spring, summer, autumn, winter, which suggested that PCV2might occured in four seasons of a year and the winter and spring was the epidemical season for PCVD. The positive rates varied among different growth stages, i.e.90.61%in sows,81.51%in piglets,74.12%in fattening pigs and58.33%in nursery pigs. For pigs immunized by PCV2vaccine, the positive rate of PCV2antibodies was significantly higher than that of pigs unimmunized by PCV2vaccine.2. The molecular epidemiology and genome sequence analysis of porcine circovirus type2in Southeast ChinaA total of441pig Lung and lymph node samples were collected from large pig farms in six provinces in Southeast China from2009to2012for detection of porcine cirovirus type2(PCV2) using PCR.37isolates of the determination in this study and15isolates of GenBank PCV2whole genome sequence were analyzed from2009to2012in Southeast China using molecular biological software, the results show that the total positive rate of PCV2was38.10%(168/441), of which48.31%(43/89) in Anhui,36.95%(109/295) in Jiangsu,The positive rates were35.77%,54.17%and29.34%;3of52isolates belonged to PCV2a,49of52isolates belonged to PCV2b, of which40isolates were grouped into PCV1C and9isolates into PCV1A/1B. PCV1C has been a dominant subgenotype in China since2009. A comparison of the deduced amino acid sequences of these PCV2isolates revealed specific mutation sites within PCV2subgroups, and the amino acid sequence analysis of3isolates of the determination in this study revealed unique variable sites or fragment (S47, L80, FPKS130-133, NR(K)I185-187, K191).They belong to a new PCV2a subsets.3. Development and preliminary application of a multiplex RT-PCR assay for simultaneous detection of four porcine diarrhea associated viruses(TGEV, PEDV, GAR and PCV2)Since October2010, clinical outbreaks of diarrhea in suckling piglets emerged in pig-producing areas of China and acute infectious increased morbidity and mortality in pigs. porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine group A rotavirus (GAR), and Porcine circovirus2(PCV2) are major viruses causing enteric diseases of piglets. A novel multiplex reverse transcription-polymerase chain reaction(mRT-PCR) was developed for simultaneous detection of four viruses in field samples from piglets. A mixture of four pairs of published primers were used for amplification of viralnucleic acids, yielding four different amplicons with sizes of481bp,651bp,859bp and309bp for PCV2, PEDV, TGEV and GAR, respectively. The sensitivity of the mRT-PCR usingplasmid constructs containing the specific viral target fragments were2.17×103,2.1×103,1.74×104and1.26×104copies for PCV2, PEDV, TGEV and GAR, respectively. A total of144field samples were collected from suckling piglets with diarrhea in Jiangsu and Anhui province from October to December2012, and detected by mRT-PCR. The positive rates of PEDV, PCV2, TGEV and GAR were50.00%,22.22%,11.11%and8.30%, respectively. It was suggested that PEDV was a majorpathogen in these diarrhea outbreaks. The compliance rate of multiplex PCR and uniplex PCR was above98.63%, Taken together, all data proved that this mRT-PCR assay was a simple, rapid, sensitive, and cost-effective detection method for clinical diagnosis of mixedinfections of porcine diarrhea associated viruses.