广西种猪场猪瘟监控方法的建立及其应用
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摘要
猪瘟(Hog Cholera,HC)又称古典猪瘟(Classical Swine Fever,CSF),是由猪瘟病毒(Classical Swine Fever Virus,CSFV)引起的一种高度接触性传染病,也是世界范围内危害最严重的猪病之一,被国际兽疫局(OIE)列为必须通报的动物传染病,我国将其列为一类传染病。猪和野猪是其唯一宿主。目前CSF仍在我国持续流行,免疫失败现象时有发生,给CSF防控带来困难。本试验旨在建立一套适合广西猪群的CSF防控模式,为大面积控制和消灭该病提供科学依据。
一、猪瘟病毒抗体检测
用阻断ELISA方法从不同免疫剂量、母猪的不同胎次和母源抗体消长规律及抗体持续期4个方面对猪瘟病毒抗体进行检测,同时将正向IHA和阻断ELISA两种抗体检测方法在不同猪场应用并比较,结果如下：
A.分别用CSF(脾淋源和细胞源)活疫苗(各一个批次)以1、1.5、2和3头份4种不同的剂量分别免疫同一个猪场的80只断奶仔猪,检测仔猪体内CSF抗体上升速度。结果显示,免疫剂量2头份的仔猪抗体上升速度最快,其次是剂量为3头份的仔猪,最慢是1头份和1.5头份的仔猪。CSF脾淋源活疫苗免疫效果略优于CSF细胞源活疫苗。
B.对某个猪场的产后1个月的1-5胎次的母猪用CSF脾淋源活疫苗以2头份剂量各免疫6头,检测免疫前后各胎次母猪CSF抗体变化情况。结果5胎次母猪抗体上升最快,其次是4胎次母猪,3胎次母猪第三,1胎次母猪抗体上升最慢。
C.通过对同一个猪场的1-5胎次母猪产后40d和其所产仔猪出生后40d内母源抗体消长情况作比较,得知母猪产后40d的抗体滴度平均比仔猪高6.02-11.2%,仔猪的抗体水平与母猪的抗体水平呈正相关。母猪和仔猪的抗体均随着哺乳期的推移逐渐下降,并且仔猪母源抗体衰减速度比母猪抗体衰减速度快。产后10d、20d、30d和40d的母猪的抗体阳性率分别为100%、96%、85%和76%,胎次越高,抗体水平也越高。仔猪在出生10d、20d、30d和40d的抗体阳性率分别82%、74%、66%和46%。抗体表现阴性的猪不能阻止CSF野毒的侵袭,且有排毒和散毒危险。在产后20d时有4%母猪不能阻止CSF野毒的侵袭,到产后40d时有24%的母猪不能阻止CSF野毒的侵袭；对仔猪来说,40d时CSF抗体阳性率仅为46%,需进行CSF疫苗免疫。
D.用脾淋源CSF活疫苗以1头份的剂量免疫一个猪场25日龄的断奶仔猪20头,在免疫后4d,没有1份猪血清抗体达到阳性,免疫后10d～80d(间隔10d)的抗体阳性率平均分别是为43%、58%、76%、100%、85%、66%、48%和32%,免疫后40d抗体达到最高峰。
E.用CSF正向IHA检测广西5个种猪场800份不同生长发育阶段的猪血清CSF抗体水平,用阻断ELISA方法检测对2个种猪场304份不同阶段的猪血清中CSF抗体水平,并比较两种方法在CSF抗体检测上的差异,结果显示,两种方法均可用于猪场常规CSF抗体监测,将这两种抗体滴度值之间进行平行比较,两者复合性不高,仅为62%左右,但ELISA的敏感性明显高于IHA。
二、血清中猪瘟野毒感染调查及血清抗体与猪瘟病毒Erms之间的关系探讨
用CSFV糖蛋白Erns ELISA试剂盒检测2006年6个种猪场1614份猪血清,发现广西种猪场CSF野毒感染情况严重,平均阳性率达25.22%(407/1614)；同时将CSF两种抗体(IHA和阻断ELISA)滴度值按高低分别糖蛋白Erms阳性率之间进行比较,将IHA和阻断ELISA抗体滴度值按高低,分别与糖蛋白Ems阳性率之间进行比较,发现在两种检测方法的任何一个抗体滴度均可出现糖蛋白Ems阳性,抗体滴度值与糖蛋白Ems阳性率没有直接的线性关系,在抗体滴度值偏低或偏高时,糖蛋白Ems的阳性率会高些。
三、猪群猪瘟病毒(CSFV)检测
分别用CSFV糖蛋白Ems (gp44/48)ELISA、CSF抗原ELISA、RT-PCR和荧光抗体染色法四种方法对广西种猪群进行CSFV检测和比较,结果如下：
2006-2007年用CSF抗原ELISA共检测850份活种猪的全血样品,阳性76份,阳性率为8.94%(76/850)；2005-2007年用RT-PCR方法检测病死猪病料434份,阳性44份,阳性率为10.14%(44/434),公猪精液1317份,阳性51份,阳性率为3.87%(51/1317),检测母猪扁桃体867份,阳性23份,阳性率为2.65%(23/867)；2006年用荧光抗体染色法检测250份猪扁桃体,阳性43份,阳性率为17.2%(43/250),2007检测250份猪扁桃体全部为阴性。
四、几种检测活猪感染猪瘟病毒的方法比较
分别用CSFV糖蛋白Ems (gp44/48)ELISA、CSF抗原ELISA、RT-PCR和荧光抗体染色法四种方法这四种方法对2个猪场的血清、扁桃体、全血样品进行一一对应CSFV检测和比较,结果显示,荧光抗体染色法检出率最高,RT-PCR方法的次之,CSF抗原ELISA方法第三,但重复性最好,CSFV糖蛋白Erms检出率最低,但仍可筛选出感染CSF野毒猪,与CSF抗原ELISA方法的符合性达75%；三种样品中用扁桃体检测重复性最好。
通过淘汰CSFV阳性猪,CSF的发病率逐渐下降,病料中CSFV阳性率从2004年的27.45%下降到2007年的2.25%；母猪CSFV阳性率从2005年的8.11%下降到2007年的0.3%,种公猪精液中CSFV的阳性率从2005年的6.67%下降到2007年的O。
Hog cholera (HC), also known as the classical swine fever (CSF), is a highly contagious disease of pigs caused by classical swine fever virus (CSFV). It is a listed disease of the World Organization for Animal Health (OIE). The only natural hosts of CSF include the domestic pigs and wild boars. At present CSF is still prevalent in China, and immunity failures have been reported frequently, which makes it difficult to defend the CSF. The objective of this study was to establish a prevention and control model against CSFV spread in the pig farms in Guangxi.
Firstly, CSFV antibodies levels in the sera of pigs from the different immunity dosages and births, as well as maternal antibodies, were investigated to establish the reasonable immunization procedure with the blocking ELISA method. At the same time, two methods, blocking ELISA and forward IHA, were applied in different pig farms and the results were as follows:
A. CSF live vaccines derived from spleens and cells were used to immunize 80 weaned pigs in the same farm with 4 different dosages (1,1.5,2 and 3 shares), respectively and CSFV antibody levels in piglets were examined. The results showed that piglets with the fastest increasing speed of antibodies were those immunized with the dosage of 2 shares, followed by the piglets immunized with the dosage of 3 shares, and piglets with the slowest increasing speed of antibodies were those immunized with 1 and 1.5 share of dosages. And CSF live vaccines derived from spleen were better than that of cell origin.
B. Six one-month-old sows with 1-5 births received the dosages of 2 shares of the CSF live vaccines derived from spleens.The antibody changes around the immune time were detected in the sows with different births. The result indicated that the increasing rate of antibodies in the sows with 5 births was fastest, the second was sows with 4 births, the third was sows with 3 births, and that in the sows with one birth was the slowest.
C. The antibody levels in the sows with 1-5 births postpartum 40d and 40-days-old piglets in the same pig farm were detected. It is found that the former was higher by 6.02～11.2% than the latter. The levels of antibodies between piglets and sows showed direct correlation. Antibodies of both were dropped along with the duration of lactation period and the dropping speed of maternal antibodies in piglets was much faster than that in sows. In the sows positive rates of antibody were 100%,96%,85% and 76%, respectively at postpartum 10d,20d,30d and 40d. The sows with the higher birth, got higher level of antibody. In the piglets the positive rates of antibody were 82%,74%,66% and 46%, accordingly. Pigs with negative antibody could not prevent against wild CSFV infection and there was a risk of excreting and diffusing CSFV. Only 4% sows couldn't prevent against wild CSFV infection at the postpartum 20d and 24% at 40d postpartum. The positive rate of antibody against CSFV was only 46% in piglets at 40d postpartum, and therefore CSF vaccines for piglets were in need.
D. Twenty 25-days-old weaned pigs were injected CSF live vaccines derived from spleens with doseage of 1 share in a farm. The antibody changes around the immune time were detected in piglets. It were found that there was no positive antibody at 4d.The positive rates of antibody were 43%,58%,76%,100%,85%,66%,48%and 32% respectively from 10d to 80d at regular intervals(an interval of 10 days) and it was highest at 40d.
E. Eight hundreds of sera samples from the different growth stages of pigs in five pig farms of Guangxi were collected and antibody levels were detected using IHA method. Three hundreds and four samples from two pig farms in Guangxi of that were detected by the blocking ELISA test. The difference between the two methods was compared at the same time. The results showed that both methods could be used for detection of antibodies against CSFV, and there was only about 62% coherence between the antibodies titers by the two methods on parallel. The sensitivity of blocking ELISA was higher than that of IHA.
Secondly,1614 sera samples from six boar farms in 2006 were detected for CSFV glycoprotein Erns by ELISA kit to discuss relationship between serum antibodies and the positive rate of CSF Glycoprotein Erns (gp44/48).It was found that pig farms in Guangxi were infected by wild CSFV, and the average positive rate was 25.22%(407/1614). Comparing the IHA and ELISA antibodies with positive rate of glycoprotein Erns, it was found that any antibody titer of the two methods without exception might appear positive glycoprotein Ems and there was no direct linear relationship between them, but when sera antibody titers were lower or higher, it was also higher to the positive rate of glycoprotein Ems.
Thirdly,CSFV was detected in Guangxi herd, and the results by using CSFV glycoprotein Ems (gp44/48) ELISA, CSFV antigen ELISA, RT-PCR and fluorescent antibody dying technique were compared. At the same time the glycoprotein Ems antibody levels by forward IHA and blocking ELISA were compared. The results were as follows:
850 whole blood samples were detected by CSFV antigen ELISA test from 2006 to 2007 and the positive rate was 8.94%(76/850). From 2005 to 2007, by using RT-PCR method 434 samples of organic tissues from pigs died of illness were detected and got 10.14% positive rate (44/434).Besides, the positive rate was 3.87%(51/1317) in 1317 samples of boar semens and 2.65% in the sow tonsils (23/867).250 samples detected with fluorescent antibody dying method and the positive rate wasl7.2%(43/250) in 2006 and 200 samples of pig tonsils were negative in 2007.
Fourthly,the samples of sera, tonsil and the whole blood from two pig farms were detected, respectively, by using CSFV glycoprotein Ems (gp44/48) ELISA, CSFV antigen ELISA, RT-PCR and fluorescent antibody dying technique. The results showed that fluorescent antibody dying technique got the highest positive percentage, and RT-PCR was the second. CSF antigen ELISA method got the third, but had the best reproducibility. Though CSFV glycoprotein Ems ELISA was the lowest positive percentage, it could determine the pigs infected by wild CSFV and there was 75% consistency with the CSF antigen ELISA. Tonsil got the best repeatability among the three samples for testing.
The incidence of CSF decreased gradually by eliminating CSFV-positive pigs. The positive rates decreased from 27.45% in 2004 to 2.25% in 2007 in the pathological samples, from 8.11% in 2005 to 0.3% in 2007 in sows and from 6.67% in 2005 to 0 in 2007 in boar semens.
一、猪瘟病毒抗体检测
用阻断ELISA方法从不同免疫剂量、母猪的不同胎次和母源抗体消长规律及抗体持续期4个方面对猪瘟病毒抗体进行检测,同时将正向IHA和阻断ELISA两种抗体检测方法在不同猪场应用并比较,结果如下：
A.分别用CSF(脾淋源和细胞源)活疫苗(各一个批次)以1、1.5、2和3头份4种不同的剂量分别免疫同一个猪场的80只断奶仔猪,检测仔猪体内CSF抗体上升速度。结果显示,免疫剂量2头份的仔猪抗体上升速度最快,其次是剂量为3头份的仔猪,最慢是1头份和1.5头份的仔猪。CSF脾淋源活疫苗免疫效果略优于CSF细胞源活疫苗。
B.对某个猪场的产后1个月的1-5胎次的母猪用CSF脾淋源活疫苗以2头份剂量各免疫6头,检测免疫前后各胎次母猪CSF抗体变化情况。结果5胎次母猪抗体上升最快,其次是4胎次母猪,3胎次母猪第三,1胎次母猪抗体上升最慢。
C.通过对同一个猪场的1-5胎次母猪产后40d和其所产仔猪出生后40d内母源抗体消长情况作比较,得知母猪产后40d的抗体滴度平均比仔猪高6.02-11.2%,仔猪的抗体水平与母猪的抗体水平呈正相关。母猪和仔猪的抗体均随着哺乳期的推移逐渐下降,并且仔猪母源抗体衰减速度比母猪抗体衰减速度快。产后10d、20d、30d和40d的母猪的抗体阳性率分别为100%、96%、85%和76%,胎次越高,抗体水平也越高。仔猪在出生10d、20d、30d和40d的抗体阳性率分别82%、74%、66%和46%。抗体表现阴性的猪不能阻止CSF野毒的侵袭,且有排毒和散毒危险。在产后20d时有4%母猪不能阻止CSF野毒的侵袭,到产后40d时有24%的母猪不能阻止CSF野毒的侵袭；对仔猪来说,40d时CSF抗体阳性率仅为46%,需进行CSF疫苗免疫。
D.用脾淋源CSF活疫苗以1头份的剂量免疫一个猪场25日龄的断奶仔猪20头,在免疫后4d,没有1份猪血清抗体达到阳性,免疫后10d～80d(间隔10d)的抗体阳性率平均分别是为43%、58%、76%、100%、85%、66%、48%和32%,免疫后40d抗体达到最高峰。
E.用CSF正向IHA检测广西5个种猪场800份不同生长发育阶段的猪血清CSF抗体水平,用阻断ELISA方法检测对2个种猪场304份不同阶段的猪血清中CSF抗体水平,并比较两种方法在CSF抗体检测上的差异,结果显示,两种方法均可用于猪场常规CSF抗体监测,将这两种抗体滴度值之间进行平行比较,两者复合性不高,仅为62%左右,但ELISA的敏感性明显高于IHA。
二、血清中猪瘟野毒感染调查及血清抗体与猪瘟病毒Erms之间的关系探讨
用CSFV糖蛋白Erns ELISA试剂盒检测2006年6个种猪场1614份猪血清,发现广西种猪场CSF野毒感染情况严重,平均阳性率达25.22%(407/1614)；同时将CSF两种抗体(IHA和阻断ELISA)滴度值按高低分别糖蛋白Erms阳性率之间进行比较,将IHA和阻断ELISA抗体滴度值按高低,分别与糖蛋白Ems阳性率之间进行比较,发现在两种检测方法的任何一个抗体滴度均可出现糖蛋白Ems阳性,抗体滴度值与糖蛋白Ems阳性率没有直接的线性关系,在抗体滴度值偏低或偏高时,糖蛋白Ems的阳性率会高些。
三、猪群猪瘟病毒(CSFV)检测
分别用CSFV糖蛋白Ems (gp44/48)ELISA、CSF抗原ELISA、RT-PCR和荧光抗体染色法四种方法对广西种猪群进行CSFV检测和比较,结果如下：
2006-2007年用CSF抗原ELISA共检测850份活种猪的全血样品,阳性76份,阳性率为8.94%(76/850)；2005-2007年用RT-PCR方法检测病死猪病料434份,阳性44份,阳性率为10.14%(44/434),公猪精液1317份,阳性51份,阳性率为3.87%(51/1317),检测母猪扁桃体867份,阳性23份,阳性率为2.65%(23/867)；2006年用荧光抗体染色法检测250份猪扁桃体,阳性43份,阳性率为17.2%(43/250),2007检测250份猪扁桃体全部为阴性。
四、几种检测活猪感染猪瘟病毒的方法比较
分别用CSFV糖蛋白Ems (gp44/48)ELISA、CSF抗原ELISA、RT-PCR和荧光抗体染色法四种方法这四种方法对2个猪场的血清、扁桃体、全血样品进行一一对应CSFV检测和比较,结果显示,荧光抗体染色法检出率最高,RT-PCR方法的次之,CSF抗原ELISA方法第三,但重复性最好,CSFV糖蛋白Erms检出率最低,但仍可筛选出感染CSF野毒猪,与CSF抗原ELISA方法的符合性达75%；三种样品中用扁桃体检测重复性最好。
通过淘汰CSFV阳性猪,CSF的发病率逐渐下降,病料中CSFV阳性率从2004年的27.45%下降到2007年的2.25%；母猪CSFV阳性率从2005年的8.11%下降到2007年的0.3%,种公猪精液中CSFV的阳性率从2005年的6.67%下降到2007年的O。
Hog cholera (HC), also known as the classical swine fever (CSF), is a highly contagious disease of pigs caused by classical swine fever virus (CSFV). It is a listed disease of the World Organization for Animal Health (OIE). The only natural hosts of CSF include the domestic pigs and wild boars. At present CSF is still prevalent in China, and immunity failures have been reported frequently, which makes it difficult to defend the CSF. The objective of this study was to establish a prevention and control model against CSFV spread in the pig farms in Guangxi.
Firstly, CSFV antibodies levels in the sera of pigs from the different immunity dosages and births, as well as maternal antibodies, were investigated to establish the reasonable immunization procedure with the blocking ELISA method. At the same time, two methods, blocking ELISA and forward IHA, were applied in different pig farms and the results were as follows:
A. CSF live vaccines derived from spleens and cells were used to immunize 80 weaned pigs in the same farm with 4 different dosages (1,1.5,2 and 3 shares), respectively and CSFV antibody levels in piglets were examined. The results showed that piglets with the fastest increasing speed of antibodies were those immunized with the dosage of 2 shares, followed by the piglets immunized with the dosage of 3 shares, and piglets with the slowest increasing speed of antibodies were those immunized with 1 and 1.5 share of dosages. And CSF live vaccines derived from spleen were better than that of cell origin.
B. Six one-month-old sows with 1-5 births received the dosages of 2 shares of the CSF live vaccines derived from spleens.The antibody changes around the immune time were detected in the sows with different births. The result indicated that the increasing rate of antibodies in the sows with 5 births was fastest, the second was sows with 4 births, the third was sows with 3 births, and that in the sows with one birth was the slowest.
C. The antibody levels in the sows with 1-5 births postpartum 40d and 40-days-old piglets in the same pig farm were detected. It is found that the former was higher by 6.02～11.2% than the latter. The levels of antibodies between piglets and sows showed direct correlation. Antibodies of both were dropped along with the duration of lactation period and the dropping speed of maternal antibodies in piglets was much faster than that in sows. In the sows positive rates of antibody were 100%,96%,85% and 76%, respectively at postpartum 10d,20d,30d and 40d. The sows with the higher birth, got higher level of antibody. In the piglets the positive rates of antibody were 82%,74%,66% and 46%, accordingly. Pigs with negative antibody could not prevent against wild CSFV infection and there was a risk of excreting and diffusing CSFV. Only 4% sows couldn't prevent against wild CSFV infection at the postpartum 20d and 24% at 40d postpartum. The positive rate of antibody against CSFV was only 46% in piglets at 40d postpartum, and therefore CSF vaccines for piglets were in need.
D. Twenty 25-days-old weaned pigs were injected CSF live vaccines derived from spleens with doseage of 1 share in a farm. The antibody changes around the immune time were detected in piglets. It were found that there was no positive antibody at 4d.The positive rates of antibody were 43%,58%,76%,100%,85%,66%,48%and 32% respectively from 10d to 80d at regular intervals(an interval of 10 days) and it was highest at 40d.
E. Eight hundreds of sera samples from the different growth stages of pigs in five pig farms of Guangxi were collected and antibody levels were detected using IHA method. Three hundreds and four samples from two pig farms in Guangxi of that were detected by the blocking ELISA test. The difference between the two methods was compared at the same time. The results showed that both methods could be used for detection of antibodies against CSFV, and there was only about 62% coherence between the antibodies titers by the two methods on parallel. The sensitivity of blocking ELISA was higher than that of IHA.
Secondly,1614 sera samples from six boar farms in 2006 were detected for CSFV glycoprotein Erns by ELISA kit to discuss relationship between serum antibodies and the positive rate of CSF Glycoprotein Erns (gp44/48).It was found that pig farms in Guangxi were infected by wild CSFV, and the average positive rate was 25.22%(407/1614). Comparing the IHA and ELISA antibodies with positive rate of glycoprotein Erns, it was found that any antibody titer of the two methods without exception might appear positive glycoprotein Ems and there was no direct linear relationship between them, but when sera antibody titers were lower or higher, it was also higher to the positive rate of glycoprotein Ems.
Thirdly,CSFV was detected in Guangxi herd, and the results by using CSFV glycoprotein Ems (gp44/48) ELISA, CSFV antigen ELISA, RT-PCR and fluorescent antibody dying technique were compared. At the same time the glycoprotein Ems antibody levels by forward IHA and blocking ELISA were compared. The results were as follows:
850 whole blood samples were detected by CSFV antigen ELISA test from 2006 to 2007 and the positive rate was 8.94%(76/850). From 2005 to 2007, by using RT-PCR method 434 samples of organic tissues from pigs died of illness were detected and got 10.14% positive rate (44/434).Besides, the positive rate was 3.87%(51/1317) in 1317 samples of boar semens and 2.65% in the sow tonsils (23/867).250 samples detected with fluorescent antibody dying method and the positive rate wasl7.2%(43/250) in 2006 and 200 samples of pig tonsils were negative in 2007.
Fourthly,the samples of sera, tonsil and the whole blood from two pig farms were detected, respectively, by using CSFV glycoprotein Ems (gp44/48) ELISA, CSFV antigen ELISA, RT-PCR and fluorescent antibody dying technique. The results showed that fluorescent antibody dying technique got the highest positive percentage, and RT-PCR was the second. CSF antigen ELISA method got the third, but had the best reproducibility. Though CSFV glycoprotein Ems ELISA was the lowest positive percentage, it could determine the pigs infected by wild CSFV and there was 75% consistency with the CSF antigen ELISA. Tonsil got the best repeatability among the three samples for testing.
The incidence of CSF decreased gradually by eliminating CSFV-positive pigs. The positive rates decreased from 27.45% in 2004 to 2.25% in 2007 in the pathological samples, from 8.11% in 2005 to 0.3% in 2007 in sows and from 6.67% in 2005 to 0 in 2007 in boar semens.
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