陕西关中地区奶牛结核病调查与PCR检测方法研究
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摘要
牛结核病(Bovine tuberculosis)是由牛分枝杆菌(Mycobacterium bovis)和结核分枝杆菌(Mycobacterium tuberculosis)引起的一种人畜共患的慢性消耗性传染病。世界动物卫生组织(OIE)将其列为B类动物疾病,我国将其列为二类动物疫病。该病不仅给畜牧业造成巨大的经济损失,而且严重威胁着人类的身心健康。随着近年来陕西关中地区奶业的迅速发展,牛结核病的对奶业健康发展具有越来越严重的威胁,奶牛结核病的防控也愈来愈受到重视。但是近期陕西关中地区牛结核病的流行状况却十分不清楚,背景也不明是该地区对牛结核病的防控遇到的最大障碍之一。由于牛结核病没有有效的疫苗,多数国家都采取检疫加扑杀的措施对牛结核病进行控制,因此确立快速、经济和准确的检疫方法对该病的防治尤为重要,牛型结核菌素皮内变态反应法是OIE推荐的唯一方法,也是我国的法定检疫方法,然而,该方法试验周期长、成本较高,而且易受其他因素干扰出现假阳性和假阴性反应,寻找该法的替代或辅助诊断方法是目前牛结核病的一个研究热点。
本研究对陕西关中地区进行了牛结核病调查,建立了牛结核病的PCR诊断技术,并将其和牛型结核菌素皮内变态反应法和IFN-γ测定法进行比较研究,主要获得以下结果:
1.2006～2008年对陕西关中部分地区(西安,宝鸡,咸阳和渭南)奶牛结核病依据国家标准《GB/T118645-2002》“结核菌素试验”进行调查,调查数据显示2006年~ 2008年检测奶牛只分别为6090,5328头和4286头,阳性率分别为0.77 %、1.05 %和1.42 %,陕西关中地区奶牛结核病阳性率有明显的上升趋势。西安、宝鸡、咸阳和渭南地区的检测头数分别为4129头、4680头、3797头和3058头,阳性率分别为0.90%、0.80%、1.18%和1.43%,其中渭南地区的阳性率最高。从养殖规模上来看,10头以下养殖户奶牛结核阳性率最高,为1.48%。调查结果表明,陕西省关中地区奶牛养殖业的迅速发展和牛结核病检疫工作发展缓慢之间的矛盾是造成该地区奶牛结核病阳性率升高的重要原因。
2.根据GENBANK上发表的牛结核杆菌和结核分枝杆菌的IS1081基因的序列设计1对引物,建立了牛结核病的PCR检测方法。IS1081基因引物可以从结核分枝杆菌(M.tuberculosis)国际参考株H37RV和牛分枝杆菌(M.bovis)国际参考株Vallee菌可扩增出324bp大小的目的条带而副结核分枝杆菌、鸟分枝杆菌和草分枝杆菌的DNA不能扩增出目的条带。该方法的敏感性分别为500fg。采用本方法对10肺脏培养阳性样品和10份血液培养阳性样品进行检测,结果阳性符合率分别为90%(9/10)和90%(9/10)。
3.以杨凌某场200头奶牛为研究对象,采用建立的PCR方法进行检测,并与提纯结核菌素(PPD)皮内变态反应法和IFN-γ测定法进行比较研究。采用3种方法对200头奶牛检测结果表明,结核菌素法的阳性率为5.50%,PCR检测法的阳性率为5.50%,IFN-γ检测法的阳性率为6.50%。IFN-γ检测法的阳性率大于结核菌素法的阳性率和PCR检测法的阳性率。PCR方法作为辅助PPD试验的快速检测方法用于牛结核病的调查,具有重要的实用价值和应用前景。
Bovine tuberculosis caused by Mycobacterium. bovis and Mycobacerium tuberculosis is one of chronic consumption of infectious zoonotic diseases. OIE Listed it to B type of animal disease, while classified as the 2nd kind of quarantive animal disease in China.The disease didn’t only bring great economic losses to the livestock industry, but also seriously threat to health of human being.In recent years, with the rapid development of dairy industry in Guanzhong Arear of Shaanxi province. Bovine tuberculosis has inscreasingly become a serious threat to the development of dairy industry,so much more attention on its prevention and control were paid.But the unclear bovine tuberculosis epidemic situation of Guanzhong Area in Shaanxi and its unknown background become one of the biggest encountered obstacles to the prevention and control of bovine tuberculosis.So far, that’s no effective vaccine for bovine tuberculosis, most countries of the world have taken measures of quarantine and culling to prevent and control it,for this reason, rapid, economic and accurate quarantine method for preventing and curing the disease is particularly important; bovine tuberculin intradermal allergy(PPD ,tuberculin test) is the only method for testing bovine tuberculosis which was recommended by OIE, as well as the statutory Quarantine method in china, however, this method is not only long cycle test, higher costed, but also vulnerable to other factors that interfered with false positive and false negative reaction.,thence seeking alternative or supplementary diagnostic methods for bovine tuberculosis becomes a research hotspot.
In our study, bovine tuberculosis investigations have been carried out, the PCR diagnostic techniques of bovine tuberculosis has been established, and comparation of PCR, intradermal tuberculin-type hypersensitivity and IFN-γassay has been researched. Conclusions drawed as following:
1.The tuberculin test based on the P.R.C. animal tuberculosis diagnostic technique standard GB/T118645-2002 had been applied in the epidemiological investigation of dairy tuberculosis in part of Guanzhong Area(Xi-an, Baoji, Xianyang, Weinan) of Shaanxi province from 2006 to 2008. 6090, 5328 and 4286 dairy had been quarantined for the 3 years respectively. Survey data shows that dairy tuberculosis positive rate of Guanzhong Area in Shaanxi has obvious upward trend from 0.77% of 2006, 1.05 % of 2007, to 1.42 % of 2008, Weinan city has a highest positive rate among this 4 citys with 1.43% in 3058 dairy, in other 3 city,the dairy tuberculosis positive rate were: Xi-an, 0.90% in 4129; Baoji, 0.80% in 4680, Xianyang, 1.18% in 3797. For different scale herds, the ten-dairy size and its below has a highest positive rate up to 1.48%. Survey results suggests that increasing gap between the rapid development of dairy industry in Guanzhong Area of Shaanxi province and the slow development of bovine tuberculosis quarantine level, as a important reason, made increasing higher dairy tuberculosis positive rate.
2. A PCR detection method for bovine tuberculosis were established, due to one pairs of designed primers based on bovine tuberculosis and Mycobacterium tuberculosis IS1081 gene sequence published in the GENBANK IS1081 gene primers from Mycobacterium tuberculosis (M.tuberculosis) international reference strains of Mycobacterium bovis H37RV and (M.bovis) international reference strains of bacteria can be amplified Vallee size of 324bp with the target bands, while DNA of M.paratuberculosis, M.avium, and M.phlei cann’t amplified purpose bands.The sensitivity of the method were 500fg. Samples from 10 lungs culture-positive and 10 blood culture-positive were detected by using this method. As a result, dairy tuberculosis positive rate were 90% (9 / 10) and 90% (90/10) respectively.
3.Comparison study on tuberculosis diagnosis of 200 dairys from PCR method established, purification protein of tuberculin (PPD) intradermal allergy and IFN-γassay in a field showed that the positive rate of PCR detection with 4.50 %, tuberculin test with 5.50%, IFN-γtest positive with 6.00%. The result of IFN-γtest has a higher tuberculosis positive rate than other two methods. The PCR method has important practical value and application prospects, as a supplementary method of the PPD test for the rapid detection of bovine tuberculosis investigations.
本研究对陕西关中地区进行了牛结核病调查,建立了牛结核病的PCR诊断技术,并将其和牛型结核菌素皮内变态反应法和IFN-γ测定法进行比较研究,主要获得以下结果:
1.2006～2008年对陕西关中部分地区(西安,宝鸡,咸阳和渭南)奶牛结核病依据国家标准《GB/T118645-2002》“结核菌素试验”进行调查,调查数据显示2006年~ 2008年检测奶牛只分别为6090,5328头和4286头,阳性率分别为0.77 %、1.05 %和1.42 %,陕西关中地区奶牛结核病阳性率有明显的上升趋势。西安、宝鸡、咸阳和渭南地区的检测头数分别为4129头、4680头、3797头和3058头,阳性率分别为0.90%、0.80%、1.18%和1.43%,其中渭南地区的阳性率最高。从养殖规模上来看,10头以下养殖户奶牛结核阳性率最高,为1.48%。调查结果表明,陕西省关中地区奶牛养殖业的迅速发展和牛结核病检疫工作发展缓慢之间的矛盾是造成该地区奶牛结核病阳性率升高的重要原因。
2.根据GENBANK上发表的牛结核杆菌和结核分枝杆菌的IS1081基因的序列设计1对引物,建立了牛结核病的PCR检测方法。IS1081基因引物可以从结核分枝杆菌(M.tuberculosis)国际参考株H37RV和牛分枝杆菌(M.bovis)国际参考株Vallee菌可扩增出324bp大小的目的条带而副结核分枝杆菌、鸟分枝杆菌和草分枝杆菌的DNA不能扩增出目的条带。该方法的敏感性分别为500fg。采用本方法对10肺脏培养阳性样品和10份血液培养阳性样品进行检测,结果阳性符合率分别为90%(9/10)和90%(9/10)。
3.以杨凌某场200头奶牛为研究对象,采用建立的PCR方法进行检测,并与提纯结核菌素(PPD)皮内变态反应法和IFN-γ测定法进行比较研究。采用3种方法对200头奶牛检测结果表明,结核菌素法的阳性率为5.50%,PCR检测法的阳性率为5.50%,IFN-γ检测法的阳性率为6.50%。IFN-γ检测法的阳性率大于结核菌素法的阳性率和PCR检测法的阳性率。PCR方法作为辅助PPD试验的快速检测方法用于牛结核病的调查,具有重要的实用价值和应用前景。
Bovine tuberculosis caused by Mycobacterium. bovis and Mycobacerium tuberculosis is one of chronic consumption of infectious zoonotic diseases. OIE Listed it to B type of animal disease, while classified as the 2nd kind of quarantive animal disease in China.The disease didn’t only bring great economic losses to the livestock industry, but also seriously threat to health of human being.In recent years, with the rapid development of dairy industry in Guanzhong Arear of Shaanxi province. Bovine tuberculosis has inscreasingly become a serious threat to the development of dairy industry,so much more attention on its prevention and control were paid.But the unclear bovine tuberculosis epidemic situation of Guanzhong Area in Shaanxi and its unknown background become one of the biggest encountered obstacles to the prevention and control of bovine tuberculosis.So far, that’s no effective vaccine for bovine tuberculosis, most countries of the world have taken measures of quarantine and culling to prevent and control it,for this reason, rapid, economic and accurate quarantine method for preventing and curing the disease is particularly important; bovine tuberculin intradermal allergy(PPD ,tuberculin test) is the only method for testing bovine tuberculosis which was recommended by OIE, as well as the statutory Quarantine method in china, however, this method is not only long cycle test, higher costed, but also vulnerable to other factors that interfered with false positive and false negative reaction.,thence seeking alternative or supplementary diagnostic methods for bovine tuberculosis becomes a research hotspot.
In our study, bovine tuberculosis investigations have been carried out, the PCR diagnostic techniques of bovine tuberculosis has been established, and comparation of PCR, intradermal tuberculin-type hypersensitivity and IFN-γassay has been researched. Conclusions drawed as following:
1.The tuberculin test based on the P.R.C. animal tuberculosis diagnostic technique standard GB/T118645-2002 had been applied in the epidemiological investigation of dairy tuberculosis in part of Guanzhong Area(Xi-an, Baoji, Xianyang, Weinan) of Shaanxi province from 2006 to 2008. 6090, 5328 and 4286 dairy had been quarantined for the 3 years respectively. Survey data shows that dairy tuberculosis positive rate of Guanzhong Area in Shaanxi has obvious upward trend from 0.77% of 2006, 1.05 % of 2007, to 1.42 % of 2008, Weinan city has a highest positive rate among this 4 citys with 1.43% in 3058 dairy, in other 3 city,the dairy tuberculosis positive rate were: Xi-an, 0.90% in 4129; Baoji, 0.80% in 4680, Xianyang, 1.18% in 3797. For different scale herds, the ten-dairy size and its below has a highest positive rate up to 1.48%. Survey results suggests that increasing gap between the rapid development of dairy industry in Guanzhong Area of Shaanxi province and the slow development of bovine tuberculosis quarantine level, as a important reason, made increasing higher dairy tuberculosis positive rate.
2. A PCR detection method for bovine tuberculosis were established, due to one pairs of designed primers based on bovine tuberculosis and Mycobacterium tuberculosis IS1081 gene sequence published in the GENBANK IS1081 gene primers from Mycobacterium tuberculosis (M.tuberculosis) international reference strains of Mycobacterium bovis H37RV and (M.bovis) international reference strains of bacteria can be amplified Vallee size of 324bp with the target bands, while DNA of M.paratuberculosis, M.avium, and M.phlei cann’t amplified purpose bands.The sensitivity of the method were 500fg. Samples from 10 lungs culture-positive and 10 blood culture-positive were detected by using this method. As a result, dairy tuberculosis positive rate were 90% (9 / 10) and 90% (90/10) respectively.
3.Comparison study on tuberculosis diagnosis of 200 dairys from PCR method established, purification protein of tuberculin (PPD) intradermal allergy and IFN-γassay in a field showed that the positive rate of PCR detection with 4.50 %, tuberculin test with 5.50%, IFN-γtest positive with 6.00%. The result of IFN-γtest has a higher tuberculosis positive rate than other two methods. The PCR method has important practical value and application prospects, as a supplementary method of the PPD test for the rapid detection of bovine tuberculosis investigations.
引文
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