辽宁省规模化养猪场PCV2感染流行病学调查及其研究
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摘要
本项目应用现代分子生物学新技术，在成功建立针对PCV2基因保守序列检测的PCR、荧光定量PCR和LAMP方法，并在对各检测方法进行优化和比较的基础上，结合免疫学和传统病理学检测等手段，对辽宁省猪群PCV2感染进行了较全面系统的调查和研究，主要结果如下：
一、参照GenBank中PCV2全基因序列，针对PCV2基因保守序列建立了分子生物学检测技术，包括普通PCR、荧光定量PCR和LAMP方法，并对其条件进行优化和比较。
1.普通PCR方法反应体系中引物最佳浓度为0.4μmol/L，最佳退火温度为53℃，灵敏度为2853拷贝/μL PCV2基因组DNA，可在4h内完成对样品的检测。
2.荧光定量PCR方法引物和探针最佳浓度分别为0.5μmol/L和0.6μmol/L，可检测的极限拷贝量为15拷贝/μL，较普通PCR方法灵敏度高出190倍；绘制的标准曲线斜率（k）为-3.004973，截距为6.631623，其相关系数（R2）为0.999212，表明该套引物和探针检测效率极高；模板浓度在1.50×103～108拷贝/μL范围内，具有良好的重复性。
3. LAMP方法灵敏度达1.4拷贝/μL，较荧光定量PCR方法高出10倍以上。从样品的核酸提取到最终结果报告仅需1.5h，特别适合现地和基层应用，为猪群PCV2感染的临诊检测和流行病学调查提供了一个快速、简便、实用的新技术。
二、辽宁省规模养殖场猪群PCV2感染及其分子流行病学调查与分析
4.辽宁省规模养猪场PCVD临诊发病情况调查及其分析
2010年度辽宁省规模养殖场猪群PCVD临诊场发病率为42.90%(145/338)，随规模场养猪规模的扩大，PCVD临诊场发病率呈上升趋势。其中，基础母猪存栏500头以上规模养殖猪群PCVD临诊场发病率最高（54.76%），显著高于总体平均水平（42.90%）（P<0.05）；基础母猪存栏51～100头规模养殖猪群，PCVD临诊场发病率最低（33.33%），明显低于猪群总体平均水平（42.90%）（P<0.05）。辽宁省内规模化养猪场PCVD临诊发病率具有明显的地域性。其中，辽东地区PCVD临诊场发病率（32.81%）不但明显低于全省总体平均水平（42.90%）（P<0.05），而且显著低于其他地区（P<0.05），与辽中地区（48.28%）差异极其显著（P<0.01）。
5.辽宁省规模化养殖场猪群PCV2血清阳性率的调查与其分析
应用ELISA检测方法，对2008年-2010年，辽宁省猪群PCV2血清阳性率进行了全面系统的调查研究。结果发现，辽宁省猪场（或猪群）PCV2抗体场阳性率呈现逐年上升趋势，至2010年，全省PCV2抗体场阳性率高达96.77%；其中，2009年辽西和辽中地区PCV2抗体场阳性率均达到100%。被分析的全省不同年度间PCV2抗体场阳性率差异显著（P<0.05），进一步表明，近年来PCV2在辽宁省规模化养猪场的感染范围逐年扩大，并且存在地域性差异。
不同规模养猪场猪群血清PCV2监测结果表明，2010年和2011年全省猪群血清PCV2抗体总体阳性率分别为68.98%（776/1125）和76.19%（1280/1680），呈显著上升趋势（P<0.05）；不同地域规模养猪场猪群血清PCV2抗体阳性率分别在65.78%-71.11%和74.33%-78.33%之间，也呈明显上升趋势（P<0.05）。表明辽宁省猪群PCV2感染范围逐年扩大。但不同地域间和不同规模养猪场间猪群血清PCV2抗体阳性率均未见统计学差异（P>0.05）。
对不同生长时期猪群调查发现，PCV2抗体总体阳性率为66.28%，其中，断奶仔猪最低（47.22%），经产母猪最高（82.67%）；病原总体阳性率为36.17%，哺乳仔猪最低（18.33%），育肥猪最高（47.89%）。不同生长时期猪群血清PCV2抗体阳性率差异显著（P<0.05），而其PCV2病原阳性率差异极其显著（P<0.01）。表明辽宁省猪群PCV2感染相当严重，其涉及养猪生产的各个环节；猪群随着饲养周期的延长，其PCV2感染率也随之升高。
不同年龄、不同品系猪群血清样品检测结果发现，不同品系猪群间PCV2抗体阳性率差异显著（P<0.05）。其中，长白猪群血清PCV2抗体阳性率最高（67.91%），三元杂交猪群血清PCV2抗体阳性率最低（46.43%），杜洛克猪群血清PCV2抗体阳性率低于长白和大白，但又高于二元杂交和三元杂交。表明辽宁省规模化养猪场猪对PCV2的易感性存在明显的品系差异。
6.猪群PCV2病原学监测及与猪其他主要病毒性疫病混合感染的研究
应用荧光PCR检测方法，对2008年-2010年患病猪群组织样品PCV2病原学监测结果表明，3年平均病原阳性率为54.20%，全省病原学阳性率呈逐年上升趋势，其中，辽南和辽西上升趋势尤为明显（P<0.05）；表明PCV2感染在辽宁省患病猪群中十分普遍。患病猪群病毒性疫病混合感染情况监测结果表明，2008年-2010年，辽宁省猪群以PCV2与PRRSV混合感染为主，其混合感染率高达20.83%；其次是PCV2与CSFV混合感染，混合感染率为6.54%。
对2010年-2012年的630份来自屠宰场的猪组织样品进行了病原学监测，结果总体阳性率为53.81%，呈逐年明显上升趋势（P<0.05）；随时间推移，辽东、辽西和辽中地区病原学阳性率均呈明显上升趋势（P<0.05），辽南地区和辽北地区上升趋势极其明显（P<0.01）；但不同地域间未见统计学差异。上述结果表明，辽宁省假定健康猪群中，PCV2隐性感染或/和亚临诊感染普遍存在，且感染率逐年上升，但地域间未见明显差异。假定健康猪群的混合感染监测还发现，PCV2与PRRSV总体混合感染率达13.97%，其中，辽西地区混合感染率高达20.74%，最低的辽东地区也达到7.86%。
7.不同基因型PCV2在辽宁猪群中的流行情况及其分析
应用PCR方法，对2010年度来自门诊的170份PCV2阳性组织病料进行不同基因型PCV2分析，结果发现，PCV2a单纯感染占12.94%，PCV2b单纯感染率占44.71%，二者混合感染率占42.35%。表明2010年度辽宁省内各地区发病猪群中PCV2b为优势流行基因型，其形式包括CV2b单纯感染和PCV2a与PCV2b混合感染。对2010年-2012年，360份来自屠宰猪PCV2阳性组织样品检测发现，PCV2a单纯感染率占10.28%，PCV2b单纯感染率占49.72%，二者混合感染率占40%。表明辽宁省假定健康猪群中广泛流行的PCV2也以2b基因型为主。由此可见，辽宁省各地猪群中流行的优势基因型为PCV2b。
8.41株PCV2毒株全基因序列测定及分子遗传演化分析
应用PCR方法和分子克隆技术，扩增并测定了2009年-2012年41株辽宁省PCV2毒株的全基因序列，并对其分子遗传演化进行了系统分析。同源性分析表明，本研究测定的41株PCV2毒株之间同源性为94.3%~100%；不同年度和不同地域毒株间同源性均为94.3%-99.9%。表明近4年来，辽宁省内各地流行的PCV2毒株序列没有明显的地域和时序特征。被分析的41株辽宁PCV2毒株与国内毒株间同源性高达94.5%-100%，与国外毒株间同源性为92.5%-99.6%。表明国内、外PCV2毒株至今变异程度均不大，全球范围内毒株序列均没有明显的地域差异。进化分析发现，41株辽宁PCV2毒株中有38株属于PCV2b基因型，其中，1A/1B和1C亚群（型）各占19株；3株属于PCV2a基因型，但不属于PCV2a基因型2A、2B、2C、2D及2E任一亚群；发现了PCV2a与PCV2b重组型病毒。表明在自然条件下，PCV2不同基因型毒株间在猪体内存在序列重组或漂移现象。
9. PCV2感染猪的动力学初步研究
应用ELISA和荧光PCR方法，对PCV2感染猪的动力学进行了初步研究，结果发现，PCV2隐性或/和亚临诊感染严重的仔猪群，其PRRSV、CSF和PRV抗体阳性率均低。相关性分析表明，仔猪血清PRRS、CSF免疫抗体阳性率与PCV2核酸阳性率相关性均极显著；仔猪血清PR免疫抗体阳性率与PCV2核酸阳性率相关性显著。表明PCV2感染在一定程度上降低了机体免疫系统的体液免疫功能。
假定健康猪群PCV2核酸阳性率与PRRSV核酸阳性率相关性分析发现，不同地域假定健康猪群PRRSV核酸阳性率与PCV2核酸阳性率双侧相关性显著。表明PRRSV感染与PCV2感染间存在相互协同作用。
病毒载量测定结果表明，种公猪在PCV2流行中可能发挥着重要作用；除淋巴器官和淋巴组织外，肠道可能为PCV2感染的另一重要靶器官。PCV2具有广泛的组织嗜性，但淋巴组织仍为其主要复制场所。PMWS患猪日龄越小，不论其临诊症状，还是病理剖检变化均越明显，且其变化与组织器官中病毒载量密切相关。PMWS患猪群各组织器官病毒载量与其眼观病理损伤程度呈正相关。
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 PCV2ORF2gene
Referring 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 PCV2ORF1gene
Referring 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 PCV2ORF1gene
Applying 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 herds
The 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 Province
ELISA 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 swineherds
Real-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 Liaoning
A 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 ofPCV2strains
PCR 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 herds
Application 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.
一、参照GenBank中PCV2全基因序列，针对PCV2基因保守序列建立了分子生物学检测技术，包括普通PCR、荧光定量PCR和LAMP方法，并对其条件进行优化和比较。
1.普通PCR方法反应体系中引物最佳浓度为0.4μmol/L，最佳退火温度为53℃，灵敏度为2853拷贝/μL PCV2基因组DNA，可在4h内完成对样品的检测。
2.荧光定量PCR方法引物和探针最佳浓度分别为0.5μmol/L和0.6μmol/L，可检测的极限拷贝量为15拷贝/μL，较普通PCR方法灵敏度高出190倍；绘制的标准曲线斜率（k）为-3.004973，截距为6.631623，其相关系数（R2）为0.999212，表明该套引物和探针检测效率极高；模板浓度在1.50×103～108拷贝/μL范围内，具有良好的重复性。
3. LAMP方法灵敏度达1.4拷贝/μL，较荧光定量PCR方法高出10倍以上。从样品的核酸提取到最终结果报告仅需1.5h，特别适合现地和基层应用，为猪群PCV2感染的临诊检测和流行病学调查提供了一个快速、简便、实用的新技术。
二、辽宁省规模养殖场猪群PCV2感染及其分子流行病学调查与分析
4.辽宁省规模养猪场PCVD临诊发病情况调查及其分析
2010年度辽宁省规模养殖场猪群PCVD临诊场发病率为42.90%(145/338)，随规模场养猪规模的扩大，PCVD临诊场发病率呈上升趋势。其中，基础母猪存栏500头以上规模养殖猪群PCVD临诊场发病率最高（54.76%），显著高于总体平均水平（42.90%）（P<0.05）；基础母猪存栏51～100头规模养殖猪群，PCVD临诊场发病率最低（33.33%），明显低于猪群总体平均水平（42.90%）（P<0.05）。辽宁省内规模化养猪场PCVD临诊发病率具有明显的地域性。其中，辽东地区PCVD临诊场发病率（32.81%）不但明显低于全省总体平均水平（42.90%）（P<0.05），而且显著低于其他地区（P<0.05），与辽中地区（48.28%）差异极其显著（P<0.01）。
5.辽宁省规模化养殖场猪群PCV2血清阳性率的调查与其分析
应用ELISA检测方法，对2008年-2010年，辽宁省猪群PCV2血清阳性率进行了全面系统的调查研究。结果发现，辽宁省猪场（或猪群）PCV2抗体场阳性率呈现逐年上升趋势，至2010年，全省PCV2抗体场阳性率高达96.77%；其中，2009年辽西和辽中地区PCV2抗体场阳性率均达到100%。被分析的全省不同年度间PCV2抗体场阳性率差异显著（P<0.05），进一步表明，近年来PCV2在辽宁省规模化养猪场的感染范围逐年扩大，并且存在地域性差异。
不同规模养猪场猪群血清PCV2监测结果表明，2010年和2011年全省猪群血清PCV2抗体总体阳性率分别为68.98%（776/1125）和76.19%（1280/1680），呈显著上升趋势（P<0.05）；不同地域规模养猪场猪群血清PCV2抗体阳性率分别在65.78%-71.11%和74.33%-78.33%之间，也呈明显上升趋势（P<0.05）。表明辽宁省猪群PCV2感染范围逐年扩大。但不同地域间和不同规模养猪场间猪群血清PCV2抗体阳性率均未见统计学差异（P>0.05）。
对不同生长时期猪群调查发现，PCV2抗体总体阳性率为66.28%，其中，断奶仔猪最低（47.22%），经产母猪最高（82.67%）；病原总体阳性率为36.17%，哺乳仔猪最低（18.33%），育肥猪最高（47.89%）。不同生长时期猪群血清PCV2抗体阳性率差异显著（P<0.05），而其PCV2病原阳性率差异极其显著（P<0.01）。表明辽宁省猪群PCV2感染相当严重，其涉及养猪生产的各个环节；猪群随着饲养周期的延长，其PCV2感染率也随之升高。
不同年龄、不同品系猪群血清样品检测结果发现，不同品系猪群间PCV2抗体阳性率差异显著（P<0.05）。其中，长白猪群血清PCV2抗体阳性率最高（67.91%），三元杂交猪群血清PCV2抗体阳性率最低（46.43%），杜洛克猪群血清PCV2抗体阳性率低于长白和大白，但又高于二元杂交和三元杂交。表明辽宁省规模化养猪场猪对PCV2的易感性存在明显的品系差异。
6.猪群PCV2病原学监测及与猪其他主要病毒性疫病混合感染的研究
应用荧光PCR检测方法，对2008年-2010年患病猪群组织样品PCV2病原学监测结果表明，3年平均病原阳性率为54.20%，全省病原学阳性率呈逐年上升趋势，其中，辽南和辽西上升趋势尤为明显（P<0.05）；表明PCV2感染在辽宁省患病猪群中十分普遍。患病猪群病毒性疫病混合感染情况监测结果表明，2008年-2010年，辽宁省猪群以PCV2与PRRSV混合感染为主，其混合感染率高达20.83%；其次是PCV2与CSFV混合感染，混合感染率为6.54%。
对2010年-2012年的630份来自屠宰场的猪组织样品进行了病原学监测，结果总体阳性率为53.81%，呈逐年明显上升趋势（P<0.05）；随时间推移，辽东、辽西和辽中地区病原学阳性率均呈明显上升趋势（P<0.05），辽南地区和辽北地区上升趋势极其明显（P<0.01）；但不同地域间未见统计学差异。上述结果表明，辽宁省假定健康猪群中，PCV2隐性感染或/和亚临诊感染普遍存在，且感染率逐年上升，但地域间未见明显差异。假定健康猪群的混合感染监测还发现，PCV2与PRRSV总体混合感染率达13.97%，其中，辽西地区混合感染率高达20.74%，最低的辽东地区也达到7.86%。
7.不同基因型PCV2在辽宁猪群中的流行情况及其分析
应用PCR方法，对2010年度来自门诊的170份PCV2阳性组织病料进行不同基因型PCV2分析，结果发现，PCV2a单纯感染占12.94%，PCV2b单纯感染率占44.71%，二者混合感染率占42.35%。表明2010年度辽宁省内各地区发病猪群中PCV2b为优势流行基因型，其形式包括CV2b单纯感染和PCV2a与PCV2b混合感染。对2010年-2012年，360份来自屠宰猪PCV2阳性组织样品检测发现，PCV2a单纯感染率占10.28%，PCV2b单纯感染率占49.72%，二者混合感染率占40%。表明辽宁省假定健康猪群中广泛流行的PCV2也以2b基因型为主。由此可见，辽宁省各地猪群中流行的优势基因型为PCV2b。
8.41株PCV2毒株全基因序列测定及分子遗传演化分析
应用PCR方法和分子克隆技术，扩增并测定了2009年-2012年41株辽宁省PCV2毒株的全基因序列，并对其分子遗传演化进行了系统分析。同源性分析表明，本研究测定的41株PCV2毒株之间同源性为94.3%~100%；不同年度和不同地域毒株间同源性均为94.3%-99.9%。表明近4年来，辽宁省内各地流行的PCV2毒株序列没有明显的地域和时序特征。被分析的41株辽宁PCV2毒株与国内毒株间同源性高达94.5%-100%，与国外毒株间同源性为92.5%-99.6%。表明国内、外PCV2毒株至今变异程度均不大，全球范围内毒株序列均没有明显的地域差异。进化分析发现，41株辽宁PCV2毒株中有38株属于PCV2b基因型，其中，1A/1B和1C亚群（型）各占19株；3株属于PCV2a基因型，但不属于PCV2a基因型2A、2B、2C、2D及2E任一亚群；发现了PCV2a与PCV2b重组型病毒。表明在自然条件下，PCV2不同基因型毒株间在猪体内存在序列重组或漂移现象。
9. PCV2感染猪的动力学初步研究
应用ELISA和荧光PCR方法，对PCV2感染猪的动力学进行了初步研究，结果发现，PCV2隐性或/和亚临诊感染严重的仔猪群，其PRRSV、CSF和PRV抗体阳性率均低。相关性分析表明，仔猪血清PRRS、CSF免疫抗体阳性率与PCV2核酸阳性率相关性均极显著；仔猪血清PR免疫抗体阳性率与PCV2核酸阳性率相关性显著。表明PCV2感染在一定程度上降低了机体免疫系统的体液免疫功能。
假定健康猪群PCV2核酸阳性率与PRRSV核酸阳性率相关性分析发现，不同地域假定健康猪群PRRSV核酸阳性率与PCV2核酸阳性率双侧相关性显著。表明PRRSV感染与PCV2感染间存在相互协同作用。
病毒载量测定结果表明，种公猪在PCV2流行中可能发挥着重要作用；除淋巴器官和淋巴组织外，肠道可能为PCV2感染的另一重要靶器官。PCV2具有广泛的组织嗜性，但淋巴组织仍为其主要复制场所。PMWS患猪日龄越小，不论其临诊症状，还是病理剖检变化均越明显，且其变化与组织器官中病毒载量密切相关。PMWS患猪群各组织器官病毒载量与其眼观病理损伤程度呈正相关。
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 PCV2ORF2gene
Referring 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 PCV2ORF1gene
Referring 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 PCV2ORF1gene
Applying 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 herds
The 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 Province
ELISA 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 swineherds
Real-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 Liaoning
A 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 ofPCV2strains
PCR 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 herds
Application 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.
引文
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