牛结核γ-干扰素ELISA检测方法在检疫工作中的应用

为评价牛γ-干扰素ELISA检测方法检测牛结核的效果及国产试剂盒的检测效果,本试验首先将国产试剂盒与Prionics试剂盒对42份相对阳性的样品和105份相对阴性的样品进行对比研究。然后对5个规模化牛场的3000头奶牛首先进行国产单纯结核菌素颈部皮内变态反应试验,3天后选取皮内变态反应阳性和可疑及部分阴性牛共418头,进行牛γ-干扰素试验。结果国产试剂盒对阳性和阴性样品的检测敏感性和特异性分别为95.2%和100%,与Priobics试剂盒的符合率为99.3%。表明国产试剂盒与进口试剂盒的检测能力一致,牛γ-干扰素检测方法准确可靠。5个牛场的3000头奶牛单纯结核菌素颈部皮内变态反应试验阳性为138头,可疑105头。γ-干扰素试验对418头奶牛的检测,其中阳性74头,与颈部皮内变态反应（可疑牛暂时视为阴性）的符合率为60.5%。     

牛型结核菌素皮内变态反应试验和γ-干扰素ELISA试验检测奶牛结核病的比较研究

本研究采用PPD皮内变态反应试验和γ-干扰素ELISA试验,对甘肃省3个地区的1585头奶牛进行结核病检测.结果表明,PPD皮内变态反应共检出7份阳性样品;经γ-干扰素ELISA检测2份为阳性、其余5份为假阳性或禽型阳性,假阳性或禽型阳性样品再经细菌分离鉴定表现为阴性;PPD皮内变态反应检出的21份疑似样品再经γ-干扰素ELISA检测,表现为禽型阳性、假阳性或阴性;PPD皮内变态反应阴性样品经γ-干扰素ELISA试验检测,结果为阴性或禽型阳性.在检测奶牛结核病时,PPD皮内变态反应试验特异性较差,γ-干扰素ELISA试验结果与牛结核分枝杆菌细菌分离鉴定结果一致,而且该技术敏感性、特异性和鉴别假阳性均优于PPD皮内变态反应试验.     

两种奶牛结核病检测方法的比较研究

牛结核病是由牛分枝杆菌引起的一种人兽共患的慢性消耗性传染病,严重威胁着人类的健康。因此,对该病的定期检疫对公共卫生和食品安全具有重大意义。本研究采用提纯结核菌素(PPD)皮内变态反应试验和γ-干扰素(IFN-γ)ELISA试验,对扬州市的892头奶牛进行结核病检测。结果表明,PPD皮内变态反应检出的31头牛型结核阳性牛和可疑牛中,经IFN-γELISA试验检测仅检出9头禽型结核可疑牛。PPD皮内变态反应试验特异性较差,出现了较高的假阳性率,其原因可能与禽结核分枝杆菌感染或其他非特异性因素的干扰有关。     

对奶牛结核病平行检测的不同方法

[目的]综合分析奶牛的健康状况,评价不同检测方法的优缺点,提出实际应用建议。[方法]同时采用单纯牛结核菌素（PPD）皮内变态反应试验（TST,使用国产PPD简称国产TST、使用进口PPD简称进口TST）、欧盟比较变态反应试验（SICTT）和牛结核γ-干扰素酶联免疫吸附试验（IFN-γ-ELISA）,对河北省唐山市某奶牛场存栏的115头牛进行结核病平行检测。[结果]经综合判定牛型结核阳性15头（其中5头为牛型、禽型双阳性）、阴性100头（其中1头为禽型阳性）,牛型结核阳性率13.04%、禽型结核阳性率5.22%、二者混合感染率为4.35%。试验表明,对于伴有非结核分枝杆菌感染的牛群,仅靠一种活体检测方法难以做出牛结核病准确诊断。[结论]因此,可采用进口TST初筛,阳性和可疑牛用IFN-γ-ELISA复检以排除部分假阳性,对于早期感染牛场可直接采用IFN-γ-ELISA方法检测。     

γ-干扰素法检测奶牛结核病的试验报告

本试验对26头经结核菌素皮内变态反应检测出6头阳性的牛群进行γ-干扰素检测,结果只有2头是阳性。进一步验证使用γ-干扰素释放试验对结核菌素皮内变态反应阳性的牛群进行筛查和确诊可以排除假阳性牛。省去3次牛型结核菌素皮内变态反应检测的繁琐,既减少养殖场户经济损失,又节省人力、物力。     

牛γ-干扰素EIA与TST检测牛结核病的比较

采用牛γ-干扰素EIA（酶免疫分析）与结核菌素皮内试验（TST）两种方法对某奶牛场中182头奶牛进行牛结核病的检测,并对牛γ-干扰素EIA与TST两种检测方法进行比较。结果：TST检出阳性123份、可疑20份、阴性39份,牛γ-干扰素EIA检出阳性58份、阴性124份,TST的阳性率大于牛γ-干扰素EIA,EIA与TST相比,敏感性为30.30%、特异性为82.05%、符合率为39.56%。     

γ-干扰素检测技术和皮内变态反应在奶牛结核病检测中的应用比较

笔者对牛型提纯结核菌素(PPD)皮内变态反应试验和新型牛结核杆菌γ-干扰素检测技术进行比较,分析其优劣。试验结果表明,γ-干扰素检测技术特异性好、敏感性高、操作简便、易于标准化,但价格较高。该方法可作为PPD皮内变态反应的平行试验,用于复核检查目前国家标准方法 PPD皮内变态反应阳性牛。     

两种奶牛结核病检测方法与PPD检测符合率比较

对新疆某规模化奶牛场的2 106头奶牛用PPD进行普检,共检出结核病阳性牛26头;随机采集20头阴性牛和26头阳性牛抗凝血和全血,进行牛结核γ-干扰素ELISA试验和胶体金检测,然后将检测结果与PPD检测结果进行比对。结果显示:3种方法检测均为阳性的16份,均为阴性的10份,总符合率为57%。PPD和γ-干扰素ELISA相比,均为阳性的23份,阳性符合率为88%(23/26),均为阴性的11份,阴性符合率为55%(11/20);PPD和胶体金检测相比,均为阳性的19份,阳性符合率为73%(19/26),均为阴性的19份,阴性符合率为95%(19/20)。结果表明:3种检测方法的符合率较低;每种检测方法各有优缺点,γ-干扰素ELISA敏感性较高,而胶体金特异性较高。因此,在实际操作中应根据需要,合理选择相应检测方法。     

变态反应检测水牛结核病标准的探索

通过比较水牛与黄牛、黑白花牛的皮厚,探索出水牛皮厚与结核菌素(PPD)敏感性的关系,进而说明应用现行牛结核检测标准检疫水牛阳性率过高,需建立水牛PPD检测标准。本试验对广西不同地区牛场不同年龄段的水牛、相同年龄段的水牛与黄牛、黑白花奶牛的皮厚及注射结核菌素前后皮厚差进行比较,水牛PPD不同用量单位的对比,并将PPD检测判为可疑或阳性的水牛样品应用γ-干扰素方法及PCR法进行对比检测。结果对1～2岁、3～4岁和5～7岁组的水牛共1 240头、黄牛487头、黑白花牛1 284头进行测试比较,水牛的皮厚及注射结核菌素后的皮厚差远远大于黄牛和黑白花牛的皮厚差,差异显著(P0.05);对不同年龄段的水牛共1 467头的皮厚及注射结核菌素后的皮厚差进行测试,不同年龄段的水牛的皮厚及注射结核菌素后的皮厚差差异不显著(P0.05);对注射不同用量的PPD进行比较检测,水牛最佳PPD用量为2 000 U;对经PPD变态反应检测判为可疑或阳性的水牛共148头,采用γ-干扰素ELISA和PCR方法进行检测,结果只有1份皮厚差超过11.0 mm的样品应用γ-干扰素ELISA检测为阳性,其余应用这两种方法检测都为阴性,进一步证明现行牛结核检测标准不适用于水牛,急需针对水牛的结核检测标准的出台。     

牛结核病两种活体诊断方法检测结合病原学诊断的研究

本文报道应用两种活体诊断方法检测牛结核病的试验结果。2010～2011年度,应用PPD皮内变态反应方法分3批次对5156头奶牛和奶水牛实施监测,监测阳性数57头,阳性率1.11%。采集该57头牛抗凝全血,应用γ-干扰素酶联免疫吸附试验进行检测,检出阳性样品3份,阳性率5.26%。对14头第一批PPD皮内变态反应阳性牛进行病理检验和进行细菌分离培养和PCR检测,结果3份样品分离培养呈阳性,其中1份PCR鉴定为结核分枝杆菌,2份为非分枝杆菌。PCR方法检测的14份组织样品中,2份为结核分枝杆菌阳性,1份为牛结核分枝杆菌阳性。结合病理剖检和病原PCR诊断,分析比较PPD皮内变态反应试验和γ-干扰素酶联免疫吸附试验的敏感性和特异性,由于PPD纯度、非特异性反应、结果判定的主观性等因素,导致PPD皮内变态反应监测检出假阳性率较高。     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.