华东地区猪圆环病毒病流行病学调查与防治技术研究
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摘要
猪圆环病毒(porcine circovirus)是一种小型无囊膜的单股环状DNA病毒,依据基因的差异可分为PCVl和PCV2。PCV1最先发现于PK15细胞(CCL-33),尽管血清学调查表明其广泛流行于猪群,但至今没有致病证据。PCV2对猪有致病性,可导致一系列疫病综合征,统称为猪圆环病毒病(PCVDs)。PCVDs包括仔猪断奶后多系统衰竭综合征(PMWS)、猪肾病皮炎综合征(PNDS)和繁殖障碍征。此外,PCV2还与猪呼吸疾病综合征有关。但是,到目前只有PCV2与PMWS感染有明确的因果关系。
PMWS是一种多因素疾病,PCV2被认为是其必须非充分病因。临床上大部分猪群或猪群中的多数个体都是PCV2抗体阳性,但只有少数猪群或猪群中的少数个体会发生PMWS。PMWS大多发生于2至4月龄的猪群,即保育的前期和育肥的后期。PMWS的临床表现为死淘率增加、消瘦、淋巴结肿大、呼吸困难,有时会表现皮肤苍白、黄疸和腹泻。激活免疫应答对于PCV2感染猪发生PMWS起重要作用。PMWS猪表现出广范的混合感染,猪细小病毒、猪繁殖与呼吸综合征病毒和猪肺炎支原体均可加重PCV2感染导致的病变。尽管早期的研究没有发现PCV2基因与其致病性的关系,但流行病学调查结果表明近年来PMWS发病率的升高与新型PCV2的流行有关,PCV2b的致病性强于PCV2a。
PCV2母源抗体能够保护仔猪不发生PMWS,这种保护与仔猪PCV2抗体水平密切相关。从临床的角度看,疫苗的接种应在猪群易感期前,PCV2母源抗体消退后。目前,全球共有4种商品化的猪圆环病毒疫苗,1种用于种猪,3种用于仔猪。接种疫苗能够显著提高猪体血清或初乳中PCV2抗体的滴度,有效保护仔猪不发生PMWS。仔猪一般在3周龄接种猪圆环病毒疫苗,此时母源抗体开始消退,产生的PCV2中和抗体能够减少和延迟猪群在保育晚期和育肥早期感染PCV2。商品化的猪圆环病毒疫苗都是基于PCV2a,但可保护接种猪抗PCV2a和PCV2b感染致病。由于PCV2b与PCV2a存在抗原的差异,基于PCV2b的疫苗是否能更有效保护猪群抗PCV2b感染致病仍有待研究。目前,我国刚刚上市德国进口的猪圆环病毒亚单位疫苗,其在国内猪场应用的效果仍是未知。本研究对华东地区的规模化猪场进行了PCVD的流行病学调查并对接种疫苗和饲养管理等防治措施进行了效果评价。具体研究内容如下。
1.华东地区规模化猪场猪圆环病毒2型血清流行病学调查。为了解华东地区猪圆环病毒2型(PCV2)的血清流行病学特征,本研究应用单层过氧化物酶技术(IPMA)对2008年1月至6月期间收集自福建省、浙江省、上海市、江苏省、安徽省和山东省的36个500头母猪以上规模猪场的620份健康猪血清样品和60份疑似PMWS猪血清样品进行了PCV2抗体检测。检测结果表明华东地区规模化猪场广泛存在PCV2的流行,健康猪血清样品和疑似PMWS猪血清样品中PCV2抗体的阳性率分别为74%和100%。健康猪群中经产母猪的PCV2抗体阳性率为100%,仔猪群在8周龄PCV2抗体阳性率最低,8周龄至12周龄期间PCV2抗体阳性率升高最为显著,提示为猪群感染PCV2的高峰时期。为了进一步分析猪群感染PCV2的关键时期,本研究对采集自华东规模华猪场保育猪和不同日龄育肥猪的血清样品进行了PCV2特异的IgM检测,结果同样表明保育后期和育肥早期是猪群感染PCV2的高峰时期。为了进一步研究PMWS猪产生PCV2抗体的特性,本研究随机选取PCV2抗体阳性的健康猪和疑似PMWS猪血清样品各8份进行了PCV2总抗体与中和抗体的定量测定。疑似PMWS猪血清样品中的PCV2中和抗体水平(平均值约为1：64)普遍低于健康猪群(平均值约为1：256)。健康猪群血清样品中PCV2中和抗体水平与IPMA水平有较好的线性关系,约为1：5-10；而疑似PMWS猪血清样品中PCV2中和抗体水平与IPMA水平的对应关系存在较大的差异,PMWS猪不能产生有效的中和抗体来清除体内的PCV2感染。
2.华东地区仔猪断奶后多系统衰竭综合征病原流行病学研究。为了解华东地区仔猪断奶后多系统衰竭综合征的病原流行病学特征,本研究对2008-2009年间收集自华东地区规模化猪场的疑似仔猪断奶后多系统衰竭综合征(PMWS)病猪的182份组织样品和血清样品进行了病原学检测。发现疑似PMWS猪样品中猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)、猪伪狂犬病毒(PRV)和猪源Torque teno virus (TTV1和TTV2)的阳性率分别为90%、51%、8%、12%、32%和46%。PCV2和PRRSV混合感染率高达43%,为华东地区规模化饲养猪群PMWS的主要病因。猪源TTV与PCV2混合感染的比例为37%,因此,猪群感染TTV与发生PMWS具有较强的相关性,值得关注本研究还对来源于华东地区6个规模化猪场的300份不同日龄健康猪血清样品进行了PCV2的检测,发现健康猪群中哺乳仔猪、保育猪、育肥猪、经产母猪、后备母猪和种公猪的血清样品PCV2阳性率分别为15%、35%、76%、12%、27%和18%,表明猪群感染PCV2主要发生在保育的后期和育肥期。这些调查结果对于华东地区规模化猪场PMWS的防治提供了理论依据。
3.华东地区猪圆环病毒2型分子流行病学研究。为了探明华东地区规模化猪场猪圆环病毒病2型(PCV2)的分子流行病学特征,本研究对2009年收集自华东地区10个规模化猪场的疑似仔猪断奶后多系统衰竭综合征(PMWS)病猪和健康猪的共200份血清样品进行了PCV2检测分析。结果发现疑似PMWS猪和健康猪血清样品中PCV2阳性率分别96%和49%。PCV2样品随机取样进行PCR-RFLP基因分型发现PCV2b为优势流行毒株,但也存在疑似PMWS猪感染PCV2a;多数疑似PMWS样品和健康猪样品中PCV2为同一基因型,但也发现一个猪场中疑似PMWS猪感染PCV2b,而健康猪感染PCV2a;猪场疑似PMWS猪血清样品中PCV2基因拷贝数显著高于健康猪样品。比较猪场PMWS发病率和猪群出栏率等生产指标发现PCV2b流行猪场PMWS发病率显著高于PCV2a流行猪场,提示PCV2b可能具有更强的致病性。
4.猪圆环病毒疫苗防治效果初探。为了试验国内新上市进口猪圆环病毒亚单位疫苗的田间免疫效果,本研究在山东某规模化猪场进行了2批猪群免疫试验,该猪场在2006-2008年期间一直受到仔猪断奶后多系统衰竭综合征的困扰。2批猪群免疫试验都是在3周龄接种一次CircoFLEX疫苗,纵向跟踪试验猪群至出栏。猪群的日增重、死淘率和料肉作为试验的主要评价指标。免疫猪群在疫苗接种后4周(7周龄)平均PCV2抗体水平显著高于对照组的猪群,并一直保持至11周龄。猪群在11周龄左右出现PCV2的病毒血症,有的个体表现出消瘦、咳嗽、呼吸困难、皮肤苍白和死亡的症状。疫苗免疫组猪群在试验期间PCV2阳性率和高病毒血症阳性率显著低于对照猪群。试验猪群在育肥阶段生产性能指标差异最显著,疫苗免疫组和对照组猪群的死淘率分别为3.03%和13.53%,差异极显著；疫苗免疫组和对照组猪群的日增重分别为713.59g/day和685.92g/day差异极显著。这些结果表明,一次免疫CircoFLEX疫苗疫苗能有效降低PCV2引起的PMWS等多因素疫病,显著改善猪群的生产性能。
5.猪圆环病毒病综合防治措施研究。猪圆环病毒2型(PCV2)被认为是引发PMWS的主要病原,但通常感染PCV2的仔猪需要其它病原或免疫刺激或应激因素共同作用才会表现出PMWS的典型症状。发生PMWS的猪群中仔猪的死亡率5%-30%不等。现阶段对该病的控制主要依赖于改进饲养管理措施。本研究对30个规模化猪场进行了PMWS疫情相关的猪群饲养和管理调查与分析。结果表明：执行全进全出的饲养操作规程、保持合适的饲养密度、将不同年龄组的猪只饲养在不同的猪舍或分区里、控制共同感染疾病、早期鉴别发病猪只并加以隔离对PMWS的预防和控制最为有益。此外,猪场的生物安全管理也是非常重要,猪舍的设施在批次间的闲置期要彻底消毒去除有机污垢；应激也是临床发生PMWS的一个重要诱导因素,猪场要关注应激因素并采取适当方法降低应激的影响；科学的营养和舒适的猪舍环境也可减少PMWS的发生,有的猪场发现补充抗氧化剂Ve有效果；供给足量清洁的饮水也非常重要,供水系统至少每批猪要清理消毒一次。
Porcine circovirus (PCV) is a small, non-enveloped virus with a circular, single-stranded DNA genome. Two genotypes of PCV, PCV1and PCV2, have been recognized. PCV type1(PCV1) was first detected as a contaminant of the porcine kidney PK15cell line (CCL-33) distributed by the American Type Culture Collection. Serologic surveys indicated that PCV1is widespread in swine, but no known animal disease has been associated with PCV1. The PCV2is found world widely in domestic pigs and has been demonstrated to be pathogenic. PCV2has been recently associated with a number of disease syndromes which have been collectively named porcine circovirus diseases (PCVDs). PCVDs include postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PNDS) and reproductive disorders. PCV2has been also involved in the porcine respiratory disease complex (PRDC). However, a clear causal association between PCV2and the development of disease has only been demonstrated for PMWS while its association with other diseases is still a matter of controversy.
PMWS is now recognized as a multifactorial disease for which the presence of the virus is a necessary but not sufficient cause. Actually, most herds and pigs are PCV2seropositive but only a few suffer PMWS outbreaks or have individual PMWS cases. The disease most commonly affects pigs from2to4months of age, the late-nursery and early-fattening phases of production. Clinical signs include a marked increase in the mortality rate, wasting, generalized enlargement of lymph nodes, respiratory signs, and occasionally pallor, jaundice and diarrhea. For the production of PMWS activation of the immune system has been shown to be the pivotal event. PMWS-affected pigs exhibit a wide spectrum of concomitant infections. Porcine parvovirus, porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae all may enhance PCV2-associated lesions and increase the incidence of PMWS under experimental and field conditions. Although initial studies did not find any link between PCV2sequences and the occurrence of PMWS, later work suggested an association between novel epizootics and a new variant of PCV2. Epidemiological studies indicate that genotype PCV2b may be more virulent than PCV2a.
Maternally-derived immunity against PCV2can protect pigs against the development of PMWS, and this protection is considered to be antibody titre-dependent. From a practical perspective, piglet vaccination should occur some weeks prior to the spread of PCV2, when maternally-derived immunity has waned, in order to avoid any interference with vaccine uptake. Four commercial vaccines against PCV2(one registered for use in breeding stock and three for use in piglets) are now available. The vaccination of sows and gilts increases PCV2antibody titres in serum and colostrum and protects piglets against the development of PMWS. Piglets are vaccinated after3weeks of age, as maternal antibodies wane. This elicits a neutralising antibody response and reduces or delays PCV2infection during weaning or fattening. Commercially available PCV2vaccines are all based on PCV2a but protect against both genotypes. However, given the antigenic differences between genotypes, it would be interesting to assess whether a vaccine based on PCV2b would have a higher protective efficacy.
The aim of this work was to investigate the epidemelogical character of PCV2infection in east China during2008to2009and to evalutate the prophylactic measures. The details contents are as follows.
1. To characterization of the serum epidemolgy of porcine circovirus type2on large scale swine farms in East China.180serum samples from health pigs and60serum samples from PMWS-affected pigs were collected during2008from breeding farms with sows over500in East China. Serum samples were analyzed for antibodies to PCV2by an immunoperoxidase monolayer assay (IPMA). The result indicated that PCV2was widely prevalence in the investigated pig farms. The total positive rate of serum samples from health pigs and PMWS-affected pigs were74%and100%respectively. In the health pig herds, the PCV2antibody positive rate of sows and breast-feeding piglets were100%and the8weeks growing pigs declined to the lowest. PCV2antibody positive rate increased most significant during the8weeks to12weeks old, suggesting that the peak period of pigs infected with PCV2. In order to further analyze the critical period of PCV2infection, pig serum samples selected from different ages of pigs and PCV2-specific IgM were detected. PCV2-specific IgMs seem to be short-lived and decreased below the detection limit2-3weeks after seroconversion. The results also show that the late nurseries and early fattening period is the key window times for pig infection PCV2. In order to further study the characteristics of PCV2antibodies of PMWS-affected pigs,16positive serum samples from healthy pigs and PMWS-affected pigs were randomly selected for total PCV2antibody (TA) and neutralizing antibody (NA) quantitative ananlysis. Within the PCV2-infected animals a positive correlation was detected between NA and TA titres in health herds, some PMWS-affected pigs had high titres at IPMA but low or absent NA. It was shown that PMWS-affected pigs developed low NA titres to PCV2, while the majority of subclinical infected animals had significantly higher titres. These results reinforce the notion that NA are essential to cope with viral dissemination through blood. Thus, the inability of some pigs to develop a strong neutralizing antibody response could be interpreted either as a cause or consequence of the development of the PMWS.
2. Since late2006, the swine industry in the Southeast China has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). The objective of the present investigation was to ascertain the etiological character of postweaning multisystemic wasting syndrome (PMWS) in large scalce swine farms in Southeast China. During2008and2009,182tissure and serum samples from PMWS cases were collected and PCV-2was the only virus that could be found consistently by PCR in all samples. The PCR detection results obtained for other viruses revealed the following:51%were positive for porcine reproductive and respiratory syndrome virus (PRRSV),8%for porcine parvovirus (PPV),12%for pseudorabiaes virus (PrV),32%for swine torque teno virus type1(swTTV1) and46%for swine torque teno virus type2(swTTV2). The2viruses that were found in higher proportion by PCR in PCV-2positives cases were PRRSV and swTTV with43%and37%positive samples, respectively. It is well known that PRRSV is a major pathogen that can lead to PMWS when present in PCV-2infected swine. It indicated that PCV2and PRRSV coinfection were the main pathogeny of PMWS in large scalce swine farms in Southeast China during2008and2009. The results suggest that swine TTV may be widespread in post-weaning pigs and could play aetiological roles in pig diseases in China even though the potential role and effect of swTTV during coinfection with PCV-2is even more obscure.300serum samples of healthy pigs at different ages from six large-scale pig farms in the East China were also analysis in the present study. As the result PCV2antibody positive rate of breeding piglets, nursery piglets, finishing pigs, sows, younger sows and boars were3%,35%,76%,12%,27%and18%respectively. Which indicating that PCV2infection in pigs occurs mainly during the late of nursery and early fattening period. These above findings provide a theoretical basis for prevention and treatment of PMWS in large scale swine farms.
3. The aim of the present study was to clarify the molecular epidemiologic character of PCV2prevalent in intensive pig farms in East China.200serum samples from suspected PMWS pigs and healthy pigs from10large-scale pig farms were collected for PCV2analysis in2009. As the result, PCV2positive for samples from suspected PMWS pigs was96%, and49%for samples from health pigs.20PCV2positvie samples were randomly selected for futher PCR-RFLP genotyping. The result revealed that both PCV2a and PCV2b are prevalent in East China and genotype2b PCV2has become dominant on these farms. Majority of the pig farms contain one certain genotype of PCV2, but it was also found that in a pig farm suspected PMWS pigs infected PCV2b and healthy pigs infected PCV2a. The PCV2genome copy number of samples of suspected PMWS was significantly higher than samples of healthy pigs. The incidence of PMWS of PCV2b infected pigs were significantly high than pig herds infected PCV2a. The data of PMWS status and pig slaughter rates from different pig farms revealed that PCV2b may have greater pathogenicity.
4. It was the purpose of this field study to evaluate the efficacy of a newly marketing recombinant ORF2subunit vaccine for active immunization of pigs against PCV2. Two bathes of triales were carried out in a farm with about1800sows which suffering from post-weaning multi-systemic wasting syndrome (PMWS). In the trial, piglets were allocated randomly into two treatment groups at approximately21days of age. Groups received either recombinant PCV2Cap protein vaccine by single intramuscular injection or left uninoculated. Primary parameters such as Average daily weight gain, mortality and FCR were evaluated. Study animals were followed from the time of vaccination until the end of finishing. Onset of PCV2viraemia and clinical signs of PMWS (wasting, cough, dyspnoea, pallor and lethargy) were observed when animals were approximately11weeks old. Vaccinated pigs had a significantly higher mean PCV2antibody titre4weeks after vaccination and a considerably higher mean PCV2antibody titre at the time of seroconversion (11weeks of age) compared to un-treated animals. Compared to untreated pigs, vaccinated pigs had a significantly reduced PCV2viral load and duration of viraemia. During the period of study (from3to25weeks of age) vaccinated animals exhibited a reduced mortality rate (3.03%vs.13.53%, difference-10.5%; p<0.001), an improved average daily weight gain (713.59g/day vs.685.92g/day; difference+27.67g/day; p<0.001). These data demonstrate that vaccination against PCV2alone can significantly improve the overall growth performance of pigs in a multi-factorial disease.
PMWS是一种多因素疾病,PCV2被认为是其必须非充分病因。临床上大部分猪群或猪群中的多数个体都是PCV2抗体阳性,但只有少数猪群或猪群中的少数个体会发生PMWS。PMWS大多发生于2至4月龄的猪群,即保育的前期和育肥的后期。PMWS的临床表现为死淘率增加、消瘦、淋巴结肿大、呼吸困难,有时会表现皮肤苍白、黄疸和腹泻。激活免疫应答对于PCV2感染猪发生PMWS起重要作用。PMWS猪表现出广范的混合感染,猪细小病毒、猪繁殖与呼吸综合征病毒和猪肺炎支原体均可加重PCV2感染导致的病变。尽管早期的研究没有发现PCV2基因与其致病性的关系,但流行病学调查结果表明近年来PMWS发病率的升高与新型PCV2的流行有关,PCV2b的致病性强于PCV2a。
PCV2母源抗体能够保护仔猪不发生PMWS,这种保护与仔猪PCV2抗体水平密切相关。从临床的角度看,疫苗的接种应在猪群易感期前,PCV2母源抗体消退后。目前,全球共有4种商品化的猪圆环病毒疫苗,1种用于种猪,3种用于仔猪。接种疫苗能够显著提高猪体血清或初乳中PCV2抗体的滴度,有效保护仔猪不发生PMWS。仔猪一般在3周龄接种猪圆环病毒疫苗,此时母源抗体开始消退,产生的PCV2中和抗体能够减少和延迟猪群在保育晚期和育肥早期感染PCV2。商品化的猪圆环病毒疫苗都是基于PCV2a,但可保护接种猪抗PCV2a和PCV2b感染致病。由于PCV2b与PCV2a存在抗原的差异,基于PCV2b的疫苗是否能更有效保护猪群抗PCV2b感染致病仍有待研究。目前,我国刚刚上市德国进口的猪圆环病毒亚单位疫苗,其在国内猪场应用的效果仍是未知。本研究对华东地区的规模化猪场进行了PCVD的流行病学调查并对接种疫苗和饲养管理等防治措施进行了效果评价。具体研究内容如下。
1.华东地区规模化猪场猪圆环病毒2型血清流行病学调查。为了解华东地区猪圆环病毒2型(PCV2)的血清流行病学特征,本研究应用单层过氧化物酶技术(IPMA)对2008年1月至6月期间收集自福建省、浙江省、上海市、江苏省、安徽省和山东省的36个500头母猪以上规模猪场的620份健康猪血清样品和60份疑似PMWS猪血清样品进行了PCV2抗体检测。检测结果表明华东地区规模化猪场广泛存在PCV2的流行,健康猪血清样品和疑似PMWS猪血清样品中PCV2抗体的阳性率分别为74%和100%。健康猪群中经产母猪的PCV2抗体阳性率为100%,仔猪群在8周龄PCV2抗体阳性率最低,8周龄至12周龄期间PCV2抗体阳性率升高最为显著,提示为猪群感染PCV2的高峰时期。为了进一步分析猪群感染PCV2的关键时期,本研究对采集自华东规模华猪场保育猪和不同日龄育肥猪的血清样品进行了PCV2特异的IgM检测,结果同样表明保育后期和育肥早期是猪群感染PCV2的高峰时期。为了进一步研究PMWS猪产生PCV2抗体的特性,本研究随机选取PCV2抗体阳性的健康猪和疑似PMWS猪血清样品各8份进行了PCV2总抗体与中和抗体的定量测定。疑似PMWS猪血清样品中的PCV2中和抗体水平(平均值约为1：64)普遍低于健康猪群(平均值约为1：256)。健康猪群血清样品中PCV2中和抗体水平与IPMA水平有较好的线性关系,约为1：5-10；而疑似PMWS猪血清样品中PCV2中和抗体水平与IPMA水平的对应关系存在较大的差异,PMWS猪不能产生有效的中和抗体来清除体内的PCV2感染。
2.华东地区仔猪断奶后多系统衰竭综合征病原流行病学研究。为了解华东地区仔猪断奶后多系统衰竭综合征的病原流行病学特征,本研究对2008-2009年间收集自华东地区规模化猪场的疑似仔猪断奶后多系统衰竭综合征(PMWS)病猪的182份组织样品和血清样品进行了病原学检测。发现疑似PMWS猪样品中猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)、猪伪狂犬病毒(PRV)和猪源Torque teno virus (TTV1和TTV2)的阳性率分别为90%、51%、8%、12%、32%和46%。PCV2和PRRSV混合感染率高达43%,为华东地区规模化饲养猪群PMWS的主要病因。猪源TTV与PCV2混合感染的比例为37%,因此,猪群感染TTV与发生PMWS具有较强的相关性,值得关注本研究还对来源于华东地区6个规模化猪场的300份不同日龄健康猪血清样品进行了PCV2的检测,发现健康猪群中哺乳仔猪、保育猪、育肥猪、经产母猪、后备母猪和种公猪的血清样品PCV2阳性率分别为15%、35%、76%、12%、27%和18%,表明猪群感染PCV2主要发生在保育的后期和育肥期。这些调查结果对于华东地区规模化猪场PMWS的防治提供了理论依据。
3.华东地区猪圆环病毒2型分子流行病学研究。为了探明华东地区规模化猪场猪圆环病毒病2型(PCV2)的分子流行病学特征,本研究对2009年收集自华东地区10个规模化猪场的疑似仔猪断奶后多系统衰竭综合征(PMWS)病猪和健康猪的共200份血清样品进行了PCV2检测分析。结果发现疑似PMWS猪和健康猪血清样品中PCV2阳性率分别96%和49%。PCV2样品随机取样进行PCR-RFLP基因分型发现PCV2b为优势流行毒株,但也存在疑似PMWS猪感染PCV2a;多数疑似PMWS样品和健康猪样品中PCV2为同一基因型,但也发现一个猪场中疑似PMWS猪感染PCV2b,而健康猪感染PCV2a;猪场疑似PMWS猪血清样品中PCV2基因拷贝数显著高于健康猪样品。比较猪场PMWS发病率和猪群出栏率等生产指标发现PCV2b流行猪场PMWS发病率显著高于PCV2a流行猪场,提示PCV2b可能具有更强的致病性。
4.猪圆环病毒疫苗防治效果初探。为了试验国内新上市进口猪圆环病毒亚单位疫苗的田间免疫效果,本研究在山东某规模化猪场进行了2批猪群免疫试验,该猪场在2006-2008年期间一直受到仔猪断奶后多系统衰竭综合征的困扰。2批猪群免疫试验都是在3周龄接种一次CircoFLEX疫苗,纵向跟踪试验猪群至出栏。猪群的日增重、死淘率和料肉作为试验的主要评价指标。免疫猪群在疫苗接种后4周(7周龄)平均PCV2抗体水平显著高于对照组的猪群,并一直保持至11周龄。猪群在11周龄左右出现PCV2的病毒血症,有的个体表现出消瘦、咳嗽、呼吸困难、皮肤苍白和死亡的症状。疫苗免疫组猪群在试验期间PCV2阳性率和高病毒血症阳性率显著低于对照猪群。试验猪群在育肥阶段生产性能指标差异最显著,疫苗免疫组和对照组猪群的死淘率分别为3.03%和13.53%,差异极显著；疫苗免疫组和对照组猪群的日增重分别为713.59g/day和685.92g/day差异极显著。这些结果表明,一次免疫CircoFLEX疫苗疫苗能有效降低PCV2引起的PMWS等多因素疫病,显著改善猪群的生产性能。
5.猪圆环病毒病综合防治措施研究。猪圆环病毒2型(PCV2)被认为是引发PMWS的主要病原,但通常感染PCV2的仔猪需要其它病原或免疫刺激或应激因素共同作用才会表现出PMWS的典型症状。发生PMWS的猪群中仔猪的死亡率5%-30%不等。现阶段对该病的控制主要依赖于改进饲养管理措施。本研究对30个规模化猪场进行了PMWS疫情相关的猪群饲养和管理调查与分析。结果表明：执行全进全出的饲养操作规程、保持合适的饲养密度、将不同年龄组的猪只饲养在不同的猪舍或分区里、控制共同感染疾病、早期鉴别发病猪只并加以隔离对PMWS的预防和控制最为有益。此外,猪场的生物安全管理也是非常重要,猪舍的设施在批次间的闲置期要彻底消毒去除有机污垢；应激也是临床发生PMWS的一个重要诱导因素,猪场要关注应激因素并采取适当方法降低应激的影响；科学的营养和舒适的猪舍环境也可减少PMWS的发生,有的猪场发现补充抗氧化剂Ve有效果；供给足量清洁的饮水也非常重要,供水系统至少每批猪要清理消毒一次。
Porcine circovirus (PCV) is a small, non-enveloped virus with a circular, single-stranded DNA genome. Two genotypes of PCV, PCV1and PCV2, have been recognized. PCV type1(PCV1) was first detected as a contaminant of the porcine kidney PK15cell line (CCL-33) distributed by the American Type Culture Collection. Serologic surveys indicated that PCV1is widespread in swine, but no known animal disease has been associated with PCV1. The PCV2is found world widely in domestic pigs and has been demonstrated to be pathogenic. PCV2has been recently associated with a number of disease syndromes which have been collectively named porcine circovirus diseases (PCVDs). PCVDs include postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PNDS) and reproductive disorders. PCV2has been also involved in the porcine respiratory disease complex (PRDC). However, a clear causal association between PCV2and the development of disease has only been demonstrated for PMWS while its association with other diseases is still a matter of controversy.
PMWS is now recognized as a multifactorial disease for which the presence of the virus is a necessary but not sufficient cause. Actually, most herds and pigs are PCV2seropositive but only a few suffer PMWS outbreaks or have individual PMWS cases. The disease most commonly affects pigs from2to4months of age, the late-nursery and early-fattening phases of production. Clinical signs include a marked increase in the mortality rate, wasting, generalized enlargement of lymph nodes, respiratory signs, and occasionally pallor, jaundice and diarrhea. For the production of PMWS activation of the immune system has been shown to be the pivotal event. PMWS-affected pigs exhibit a wide spectrum of concomitant infections. Porcine parvovirus, porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae all may enhance PCV2-associated lesions and increase the incidence of PMWS under experimental and field conditions. Although initial studies did not find any link between PCV2sequences and the occurrence of PMWS, later work suggested an association between novel epizootics and a new variant of PCV2. Epidemiological studies indicate that genotype PCV2b may be more virulent than PCV2a.
Maternally-derived immunity against PCV2can protect pigs against the development of PMWS, and this protection is considered to be antibody titre-dependent. From a practical perspective, piglet vaccination should occur some weeks prior to the spread of PCV2, when maternally-derived immunity has waned, in order to avoid any interference with vaccine uptake. Four commercial vaccines against PCV2(one registered for use in breeding stock and three for use in piglets) are now available. The vaccination of sows and gilts increases PCV2antibody titres in serum and colostrum and protects piglets against the development of PMWS. Piglets are vaccinated after3weeks of age, as maternal antibodies wane. This elicits a neutralising antibody response and reduces or delays PCV2infection during weaning or fattening. Commercially available PCV2vaccines are all based on PCV2a but protect against both genotypes. However, given the antigenic differences between genotypes, it would be interesting to assess whether a vaccine based on PCV2b would have a higher protective efficacy.
The aim of this work was to investigate the epidemelogical character of PCV2infection in east China during2008to2009and to evalutate the prophylactic measures. The details contents are as follows.
1. To characterization of the serum epidemolgy of porcine circovirus type2on large scale swine farms in East China.180serum samples from health pigs and60serum samples from PMWS-affected pigs were collected during2008from breeding farms with sows over500in East China. Serum samples were analyzed for antibodies to PCV2by an immunoperoxidase monolayer assay (IPMA). The result indicated that PCV2was widely prevalence in the investigated pig farms. The total positive rate of serum samples from health pigs and PMWS-affected pigs were74%and100%respectively. In the health pig herds, the PCV2antibody positive rate of sows and breast-feeding piglets were100%and the8weeks growing pigs declined to the lowest. PCV2antibody positive rate increased most significant during the8weeks to12weeks old, suggesting that the peak period of pigs infected with PCV2. In order to further analyze the critical period of PCV2infection, pig serum samples selected from different ages of pigs and PCV2-specific IgM were detected. PCV2-specific IgMs seem to be short-lived and decreased below the detection limit2-3weeks after seroconversion. The results also show that the late nurseries and early fattening period is the key window times for pig infection PCV2. In order to further study the characteristics of PCV2antibodies of PMWS-affected pigs,16positive serum samples from healthy pigs and PMWS-affected pigs were randomly selected for total PCV2antibody (TA) and neutralizing antibody (NA) quantitative ananlysis. Within the PCV2-infected animals a positive correlation was detected between NA and TA titres in health herds, some PMWS-affected pigs had high titres at IPMA but low or absent NA. It was shown that PMWS-affected pigs developed low NA titres to PCV2, while the majority of subclinical infected animals had significantly higher titres. These results reinforce the notion that NA are essential to cope with viral dissemination through blood. Thus, the inability of some pigs to develop a strong neutralizing antibody response could be interpreted either as a cause or consequence of the development of the PMWS.
2. Since late2006, the swine industry in the Southeast China has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). The objective of the present investigation was to ascertain the etiological character of postweaning multisystemic wasting syndrome (PMWS) in large scalce swine farms in Southeast China. During2008and2009,182tissure and serum samples from PMWS cases were collected and PCV-2was the only virus that could be found consistently by PCR in all samples. The PCR detection results obtained for other viruses revealed the following:51%were positive for porcine reproductive and respiratory syndrome virus (PRRSV),8%for porcine parvovirus (PPV),12%for pseudorabiaes virus (PrV),32%for swine torque teno virus type1(swTTV1) and46%for swine torque teno virus type2(swTTV2). The2viruses that were found in higher proportion by PCR in PCV-2positives cases were PRRSV and swTTV with43%and37%positive samples, respectively. It is well known that PRRSV is a major pathogen that can lead to PMWS when present in PCV-2infected swine. It indicated that PCV2and PRRSV coinfection were the main pathogeny of PMWS in large scalce swine farms in Southeast China during2008and2009. The results suggest that swine TTV may be widespread in post-weaning pigs and could play aetiological roles in pig diseases in China even though the potential role and effect of swTTV during coinfection with PCV-2is even more obscure.300serum samples of healthy pigs at different ages from six large-scale pig farms in the East China were also analysis in the present study. As the result PCV2antibody positive rate of breeding piglets, nursery piglets, finishing pigs, sows, younger sows and boars were3%,35%,76%,12%,27%and18%respectively. Which indicating that PCV2infection in pigs occurs mainly during the late of nursery and early fattening period. These above findings provide a theoretical basis for prevention and treatment of PMWS in large scale swine farms.
3. The aim of the present study was to clarify the molecular epidemiologic character of PCV2prevalent in intensive pig farms in East China.200serum samples from suspected PMWS pigs and healthy pigs from10large-scale pig farms were collected for PCV2analysis in2009. As the result, PCV2positive for samples from suspected PMWS pigs was96%, and49%for samples from health pigs.20PCV2positvie samples were randomly selected for futher PCR-RFLP genotyping. The result revealed that both PCV2a and PCV2b are prevalent in East China and genotype2b PCV2has become dominant on these farms. Majority of the pig farms contain one certain genotype of PCV2, but it was also found that in a pig farm suspected PMWS pigs infected PCV2b and healthy pigs infected PCV2a. The PCV2genome copy number of samples of suspected PMWS was significantly higher than samples of healthy pigs. The incidence of PMWS of PCV2b infected pigs were significantly high than pig herds infected PCV2a. The data of PMWS status and pig slaughter rates from different pig farms revealed that PCV2b may have greater pathogenicity.
4. It was the purpose of this field study to evaluate the efficacy of a newly marketing recombinant ORF2subunit vaccine for active immunization of pigs against PCV2. Two bathes of triales were carried out in a farm with about1800sows which suffering from post-weaning multi-systemic wasting syndrome (PMWS). In the trial, piglets were allocated randomly into two treatment groups at approximately21days of age. Groups received either recombinant PCV2Cap protein vaccine by single intramuscular injection or left uninoculated. Primary parameters such as Average daily weight gain, mortality and FCR were evaluated. Study animals were followed from the time of vaccination until the end of finishing. Onset of PCV2viraemia and clinical signs of PMWS (wasting, cough, dyspnoea, pallor and lethargy) were observed when animals were approximately11weeks old. Vaccinated pigs had a significantly higher mean PCV2antibody titre4weeks after vaccination and a considerably higher mean PCV2antibody titre at the time of seroconversion (11weeks of age) compared to un-treated animals. Compared to untreated pigs, vaccinated pigs had a significantly reduced PCV2viral load and duration of viraemia. During the period of study (from3to25weeks of age) vaccinated animals exhibited a reduced mortality rate (3.03%vs.13.53%, difference-10.5%; p<0.001), an improved average daily weight gain (713.59g/day vs.685.92g/day; difference+27.67g/day; p<0.001). These data demonstrate that vaccination against PCV2alone can significantly improve the overall growth performance of pigs in a multi-factorial disease.
引文
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[1].曹胜波,陈焕春,肖少波,等.猪圆环病毒2型的PCR检测方法的建立与应用[J].华中农业大学学报,2001(1)：53-56.
[2]. Mamkertz A, Caliskan R, Hattermann K, et al. Molecular biology porcine circovirus:analyses gene expression and viral diplication[J]. Veterinary Microbiology,2004,98:81-88.
[3]. Olvera A, Cortey M, Segales J, Molecular evolution of porcine circovirus type 2 genomes:Phylogeny and clonality[J]. Virology,2007,35 (7):175-185.
[4]. Liu Q, Willson P, Attoh-poku S, et al. Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein[J]. Pro Exp Purif,2001,21:115-120.
[5]. Yuna K, Jinhyun K. Characterization of the recombinant protein of porcine circovirus type 2 field isolate expressed in the baculovirus system[J]. Vet Sci, 2002.3(1):19-23.
[6]. Hamel A L, Lin L L, Nayar G P. Nucleotide sequence of porcine circovirus associated with postwemfing multisystemic wasting syndrome in pigs[J]. J. Virol., 1998,72:5262
[7].-5267.
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