Molecular Epidemiology On Multi-Agent Infection In Post-Weaning Multisystemic Wasting Syndrome

Post-weaning multisystemic wasting syndrome (PMWS) was firstly found in Canada at1991, which was firstly reported by Harding and Clark at1996. Soon afterwards, American, European and Asian countries have reported this disease sequentially. Initially, people thought PMWS was caused by porcine circovirus type2(PCV2). The clinical manifestations of typical PMWS consist of progressive loss of body condition, visibly enlarged lymph nodes, difficulty in breathing, and sometimes diarrhea, pale skin, and jaundice. The morbidity in piglets7-15weeks old were3%-30%, but the mortality could reach up to80%-100%. In recent years, this disease brings great harm to the world pig farms due to its more and more popular. It has now been confirmed that the multi-pathogen mixed infection is the major risk factors for PMWS, among them, PCV2is an important pathogen. The others include common pathogens and uncommon pathogens, such as PRRSV, PTTV and PBoV, PHoV.Form the point of view of the epidemiological investigation, we detect the infection rate of single pathogen and the mixed infection rate of PCV2with other pathogen in clinical samples by PCR. Then DNA sequencing of the major pathogens is used for measuring genetic variation. Thereby, we expect to clarify the law of molecular epidemiology and genetic variation about the major pathogen. This study includes:1. The epidemiological investigation of multi-pathogens co-infection for the PMWSRefer to the specific primers which have been published in literatures or designing a couple of primers based on virus gene sequence deposited into GenBank, which is used to develop a PCR assay for detection of one pathogen has been listed following. We detected1183clinical samples from several provinces of China collected from2009to2012by PCR. The results show the positive rate of PCV2, PBoV1, PBoV3-4, PHoV, TTV1, TTV2、PPV4and PRRSV are47.00%(556/1183),22.49%(266/1183),15.56%(75/482),19.86%(235/1183),17.58%(208/1183),19.86%(235/1183),17.89%(102/570) and28.32%(145/512), respective. The positive rate of PCV2and PPV4in the incidence samples was significantly higher than the rate in the healthy samples; the co-infection rate of PC V2with other pathogens was different significantly in the incidence samples and the healthy samples; the detection rate of PCV2was the highest in the samples collected from30-60days’pigs; the positive infection rate was high in the diffecent type of incidence samples.2. The DNA sequencing and humology comparison of ORF2partical gene of PCV2The objective was to study the genetic variation of PCV2isolated in Jiangsu province which is a major pathogen of PMWS. In this study, we amplify ORF2partical nucleotide sequences of PCV2by PCR with the DNA extracted from clinical samples collected from2009to2012as template. The PCR products are cloned after purification, sequenced, sequence alignmented by using DNAman and DNAStar software. The result show the26strains were closely related to each other displaying87.1%～100%, compared with4domestic and overseas PCV2strains displaying83.3%～97.1%. Simultaneously, the genome sequence of PCV2JSTZ-4strain isolated from stool samples of a piglet was acquired and deposited in GenBank under accession NO. JQ413808. This is the first time to analyze the genome sequence of PCV2from stool sample in China. The comparison of genome sequence revealed PCV2strain JSTZ4exhibited95.0%～99.9%nucleotide identity with other PCV2strains and shared94.9%～97.9%nucleotide identity with other PCV2genome from stool samples in other countries.3. The DNA sequencing and humology comparison of NS1partical gene of PBoV3-4Porcine bocavirus(PBoV), an emerging DNA virus, was first identified in2009in Swedish swine herds suffering from post-weaning multisystemic wasting syndrome (PMWS). To evaluate the prevalence and the genetic variation of PBoV in China, we amplify NS1partial nucleotide sequences of PBoV3-4by PCR which has been reported in literatures. The PCR products are cloned after purification, sequenced, sequence alignmented by using DNAStar software. The comparison of nucleotide sequence revealed sectional NS1gene of the18PBoV3-4strains exhibited89.3%-95.8% nucleotide identity with PBoV3(JG512472) strain and shared86.8%-91.1% nucleotide identity with PBoV4(JF512473). The phylogenetic tree analysis revealed9PBoV3-4strains in the same branch with PBoV3(JG512472) and the other9PBoV3-4strains in the same branch with PBoV4(JF512473).4. The DNA sequencing and humology comparison of ORF5gene of PRRSV PRRSV as an RNA virus, which genome is prone to mutation, so it is very meaningful to master the variation trend of PRRSV for controlling this disease. In this study, we select ORF5as a target gene, analyze the genetic variation of PRRSV isolated in Jiangsu Province from2011to2012. The result showed the11strains were closely related to each other displaying86.5%～98.7%overall ORF5gene. Compared with5domestic and overseas PRRSV strains indicated these strains shared the highest homology with JXA1which is the representative of highly pathogenic, shared the lowest homology with the Americas strain VR-2332which is the representative of low pathogenic. The phylogenetic tree analysis also revealed11PRRSV strains in the same branch with highly pathogenic.In this study, we detect the infection rate of single pathogen and the mixed infection rate of PCV2with other pathogen in clinical samples by PCR. With the help of DNA sequence work, we grasp the genetic variation of the main pathogens and deep research the etiology of PMWS, which is able to provide theoretical and scientific basis for the control of the disease.