Investgation And Study On Porcine Circovirus Type2(PCV2) Infections In Pigs Farms In Liaoning

In order to find out the epidemic situation of PCV2in Liaoning province and provideimportant first-hand materials and scientific technology for the Diagnosis， comprehensiveprevention and control of PCV2，this research projects applied the new technology of modernmolecular biology to establish PCV2PCR and LAMP test methods, according to the geneconserved sequences of PCV2ORF2and ORF1. On the basis of PCV2molecular diagnosticmethods above, the prevalence of PCV2was investigated and studied systematically combiningwith immunology test methods, in swine herds in Liaoning province. The main results were asfollows：1. Established PCR method according to the conserved sequences of PCV2ORF2geneReferring to the published PCV2and PCV1sequences in GenBank and the determinationsequences of PCV2in this study, PCR primers were designed by selecting the PCV2relativeconservative sequences but different from the sequences of PCV1. The optimal concentration ofprimers was0.4μmol/L in the reaction system. And the best annealing temperature was53℃.This method could only detect PCV2virus nucleic. But for PPV, PRV and PCV1virusesnucleic, there was no amplification banding. At the same time, through analysing the determinationsequences found that the sequences homology reached95.6%~100%between amplificationpurpose fragments sequences and PCV2sequences in GenBank. The sensitivity of PCR methodwas2853copies/μL of PCV2genome DNA. This method could finish the sample detection in4h。2. Established realtime-PCR method according to the conserved sequences of PCV2ORF1geneReferring to the published PCV2and PCV1sequences in GenBank and the determinationsequences of PCV2in this study, a set of primers and probe was designed. Then primersamplification products were inserted into pMD-18T carriers to prepare the positive recombinantplasmid as standards. The experiments showed that the optimum of primers and probe were0.5μmol/L and0.6μmol/L. Only the amplification curve of PCV2nucleic was as “S” form. Andthere was no amplification curve for detection of PPV、PRV and PCV1nucleic, which indicatedthat this method had a good specificity.The limit copies quantity that the method could detect, was15copy/μL of PCV2genomeDNA, more than190times higher sensitivity than common PCR. The standard curve plotting slope (k) was3.004973; its intercept was6.631623; and the correlation coefficient (R2) was0.999212. Itindicated that the primers and probe had high detection efficiency. The repeatability test resultsshow that the method had good repeatability as the template concentration between1.50×103and108copies/μL.3. Established LAMP method according to the conserved sequences of PCV2ORF1geneApplying the Primer Explorer4.0online software, two sets of primers for LAMP weredesigned according to the conserved sequences of PCV2ORF1gene. Each set has four primers,including two internal primers (FIP and BIP) and two outer primers (F3and B3). Then two sets ofprimers were screened preliminary. Through parameters optimization, such as the proportion ofinternal primer and outer primers, the concentration of Mg2+, reaction temperature and reactiontime, finally a rapid, sensitive, specific and visualization of PCV2LAMP method was set up.The sensitivity of LAMP method was1.4copy/μL of PCV2genome DNA,10times higherthan real-time PCR method establishing in this study. From the nucleic extraction to the finalresults report only need1.5h. It was suitable for grass-roots application. And it provided a new,quick, easy and practical detection method for clinical diagnosis and epidemiological investigationof PCV2.4. Investigation and analysis the clinical infection situation of PCVD in swine herdsThe clinical morbidity rate of PCVD was42.90%(145/338) in scale pigs farm of Liaoningprovince. With the enlargement of farm scale， the Clinical morbidity of PCVD showedascendant tendency. The PCVD clinical morbidity rate was highest（54.76%）in swine herdswhich basic of sows reached500heads above. And it was obviously higher than average level(42.90%)(P<0.05). But basic of sows between51and100swine herds had the lowest clinicalmorbidity rate of PCVD(33.33%), which was obvious lower than average level（P<0.05）. Thestudy also found that the clinical morbidity rate of PCVD in Eastern Liaoning (32.81%)was notonly significantly lower than the average level of the province (42.90%)(P<0.05), but also lowerthan any other areas(P<0.05). And it was extremely significant lower than Mid-Liaoning(48.28%)(P<0.01). It suggested that the clinical morbidity rate of PCVD should have obviousregionality in Liaoning province.5. Investigation and analysis of the serum PCV2antibody positive rate of swine herds inLiaoning ProvinceELISA was used for a comprehensive investigation for the serum PCV2antibody positive rateof swine herds in Liaoning Province. It found that the PCV2antibody positive rate of swine herdswas ascending year by year. In2010, the average positive rate of the province was as high as96.77%；the positive rate had reached100%in Mid-Liaoning and western Liaoning. The PCV2antibody positive rate of swine herds was significant difference (P<0.05) year by year, from2008 to2010. It indicated that the herds quantities which were infected with PCV2, were not onlyenlarging but also existing regional differences in Liaoning province.Serum PCV2antibody monitoring results of different scale herds found that the averagepositive rate of PCV2antibody was68.98%(776/1125) in2010，was76.19%(1280/1680) in2011in Liaoning, which presented significantly increasing trend year by year(P<0.05). The serum PCV2antibody positive rate of different scale herds from different regions was65.78%-71.11%in2010,and was74.33%-78.33%in2011, which was also increasing significantly year by year(P<0.05).The positive rate of PCV2antibody in different regions had no statistical difference (P>0.05); andthere was also no statistical difference in different scale herds (P>0.05). The results aboveindicated that herds infected with PCV2had spreaded year by year.The investigation of the pigs in different growth periods found that the total positive rate ofPCV2antibody was66.28%; And the weaned piglets was the lowest (47.22%); The multiparoussows was the highest. The total positive rate of pathogenic was36.17%; And the suckling pigletswas the lowest (18.33%); The fattening pigs was highest. The variation of PCV2antibody positiverate among different growth periods was significant (P<0.05); And the variation of PCV2positiverate among different growth periods was extremely significant (P<0.01). The results suggested thatinfection of PCV2was quite serious in swine herds in Liaoning Province. And it might involve allcomponent elements of pig production. With the feeding cycle extended, the infection rates ofPCV2was increasing.The investigation showed that the variation PCV2antibody positive rate among differentstrains of breed was significant(P<0.05).The serum PCV2antibody positive rate of landrace pigswas the highest(67.91%); And three crossbred pigs was the lowest. The serum PCV2antibodypositive rate of duroc pigs was lower than landrace and large white pigs, but higher than twocrossbred pigs and three crossbred pigs. The results above suggested that different strains of breedpigs have difference susceptibility to the infection of PCV2.6. PCV2pathogenic surveillance and co-infection with other dominating viruses in swineherdsReal-time PCR method was applicated for PCV2pathogenic surveillance. The results showedthat the average pathogen positive rate of PCV2was54.20%from2008to2010. The tendency ofthe average pathogen positive rate was ascending year by year. The ascending tendency inSouthern Liaoning and the Western Liaoning was very obvious (P<0.05). The results abovesuggested that the infection of PCV2was prevalent in swine herds in Liaoning.The pathogenic co-infection detection results of suffering swine herds indicated that thedominating type was PCV2and PRRSV co-infection from2008to2010in Liaoning province. Theco-infection rate of PCV2and PRRSV was20.83%. And the co-infection rate of PCV2and CSFVwas6.54%. A total of630swine tissue samples from slaughterhouse were detected by PCV2real-timePCR method from2010to2012. The total positive rate is53.81%. The tendency of the pathogenpositive rate was ascending obviously year by year (P<0.05). The tendency of positive rate in theEastern Liaoning, the Western Liaoning and the Central Liaoning was ascending obviously year byyear(P<0.05), and was ascending extremely obviously in Southern Liaoning and the NorthernLiaoning(P<0.01).But there was no significant difference among swine herds from differentgeographical regions. The results suggested that PCV2inapparent infection or/and sub clinicalinfection were prevalent s, and increasing year by year in assumed healthy swine herd. But therewas no significant difference among swine herds from different regions in Liaoning.The total co-infection rate of PVC2and PRRSV was13.97%in assumed healthy swine herds.The co-infection rate of PVC2and PRRSV in the Western Liaoning was20.74%, and in theEastern Liaoning was7.86%.7. The epidemic situation of different genotypes PCV2in swine herds in LiaoningA total of170PCV2positive tissue samples of suffering swine herds were detected by PCV2PCR method. The results showed that the detectable rate of PCV2a was55.29%; the PCV2b was87.06%. The PCV2a single infection rate was12.94%; the PCV2b single infection rate was44.71%; the co-infection rate was42.35%. It suggested the predomination genotype was PCV2bin suffering swine herds in2010.A total of360PCV2positive tissue samples from slaughterhouse from2010to2012, thedetectable rate of PCV2a was50.28%; and the PCV2b was89.72%. The PCV2a single infectionrate was10.28%; single infection rate was49.72%; and the co-infection rate was40%. It indicatedthat PCV2b was the predomination genotype in assumed healthy swine herds in Liaoning.8. Complete genome sequence determination and the molecular phylogenetic analysis ofPCV2strainsPCR method and molecular cloning technique was used to determination the completegenome sequence of41strains of PCV2from2009to2012. Sequence homologous analysis amongthe41strains of PCV2showed that the homology of41strains of PCV2was94.3%~100%; thehomology of the different year and different geographical PCV2strains were both94.3%~99.9%.Itindicated that the prevalent strains of PCV2has no obvious regional and temporal characteristicsin recent four years. The homology between41strains of PCV2in Liaoning Provence and strainsof domestic was94.5%~100%; The homology between41strains of PCV2in Liaoning Provenceand abroad strains was92.5%~99.6%. It indicated that domestic and foreign PCV2strains variationdegree was nor so far. That was to say the global strains had no obvious regional difference.There were38strains of PCV2belonging to PCV2b genotype,19strains belonging to1A/1Bsubtype and another19strains belonging to1C subtype the19strains. There were3strains ofPCV2belonging to PCV2a genotype, but not belonging to the2A,2B,2C,2D and2E subtype. And PCV2a and PCV2b recombinant strains virus was found in this research. It indicated thatthere was sequence recombination or drift phenomenon among different PCV2genotypes strains inswine herds in under natural conditions.9. Preliminary study on the dynamics of PCV2infection in swine herdsApplication of ELISA and real-time PCR methods, the dynamics of PCV2infection waspreliminary studied in swine herds. The results found that the antibody positive rate of PRRSV,CSFV and PRV were all lower in piglets, which inapparent infection or/and sub clinical infectionwas severe. Correlation analysis indicated that the correlations between PCV2nucleic positive rateand PRRSV and CSFV immune antibody positive rate in piglets serum were extremely significant.And the correlation between PCV2nucleic positive rate and PRV immune antibody positive rate inpiglets serum were significant. It suggested that PCV2infection might reduce humoral immunefunction in some extent.Through analysis the correlation of the nucleic positive rate between PCV2and PRRSV inassumed healthy swine herds. It showed that the correlation above was significant indifferentregions of assumed healthy swine herds. It suggested that there was a coordination effect betweenPRRSV and PCV2infection in swine herds.The viral load results show that boar might play an important role in the prevalence of PCV2.Besides lymphoid organs and lymph tissue, intestine maybe another important target organ ofPCV2infection. The tissue tropism of PCV2was general. But lymphoid tissue might be the mainreplication sites. Pigs suffered PMWS smaller, the clinical symptom was more obvious, andnecropsy more remarkable. It was closely related with high titers of virus load.There was apositively correlation between viral load and the degree of visible pathological injury in variousorgans and tissues of pigs suffered PMWS.