两种方法检测奶牛结核的比较

采用变态反应与ELISA同时对奶牛结核病进行检测．并以剖检进行验证。结果表明：变态反应和ELISA对奶牛结核的检出率分别为2．0％和0．33%,变态反应阳性并不能全包括ELISA阳性。变态反应与剖检的符合率为3．14％；而ELISA与剖检的符合率为75%.ELISA检测具有较好的特异性。     

皮试法与γ-干扰素ELISA法在奶牛结核病检疫中的对比试验

为制定奶牛结核病净化方案提供参考,通过颈部皮内变态反应(SICT)检测上海某奶牛场的483头奶牛,然后用γ-干扰素ELISA方法检测皮试法阳性牛及与阳性牛密切接触牛共50头。结果发现:颈部皮内变态反应与γ-干扰素ELISA的阳性符合率100%;在变态反应阴性牛中,仍然检出了部分ELISA阳性牛。我们考虑在高风险牛场增加皮内变态反应试验频率,再随之实施抗体检测,会更有利于结核牛场的净化与防制。     

ESAT-6及PPD ELISA检测疫区和非疫区牛结核病的比较研究

本实验克隆、表达了牛结核早期分泌靶抗原蛋白6（ESAT-61抗原,建立了ESAT-6酶联免疫吸附检测方法（ELISA）。同时应用牛结核提纯菌素（PPD）ELISA和ESAT-6-ELISA对采自疫区的641头牛和非疫区的324头牛进行检测,其中PPD-ELISA检测中,疫区有581头阳性牛,阳性率为90．64％,非疫区有2头阳性牛,阳性率为0、62％;ESAT-6-ELISA检测中,疫区有567头阳性牛,阳性率为88．45％,非疫区没有阳性牛。对采自疫区的23例变态反应阳性牛和非疫区的7例变态反应阳性牛进行检测,其中PPD-ELISA检测中,疫区有20头为阳性。与变态反应的符合率为86．90％,非疫区有1头为阳性,与变态反应的符合率为14％;在ESAT-6-ELISA检测中,疫区有18头为阳性,与变态反应的符合率为78．30％,非疫区没有阳性。这些结果表明：ESAT-6-ELISA的敏感性要低于PPD-ELISA;牛结核ELISA在非疫区应用效果较差,在疫区更具有应用价值。     

皮试法与ELISA法在奶牛结核病检测中的对比试验

通过单纯颈部皮内变态反应(SICT)、比较变态反应(SICCT)检测方法、γ-干扰素ELISA和ELISA 4种方法,同时检测上海2个奶牛场的222头奶牛。结果发现:阳性检出率以颈部皮内变态反应为最高(67.6%)、ELISA(44.1%)和γ-干扰素ELISA居中(31.1%),比较变态反应最低(13.1%);单纯的颈部皮内变态反应与2种ELISA的符合率较差,而比较变态反应与2种ELISA具有较好的符合率;在变态反应阴性牛中,仍然检出了部分ELISA阳性牛。由此认为,先以变态反应检疫牛群,再随之实施抗体检测,会更有利于结核病牛场的净化与防制。     

牛结核ELISA及其抗原的比较研究

作者采用4种抗原建立了ELISA检测牛结核血清抗体的方法,并对其进行了比较。以结核清净声场1026头牛血清建立了ELISA阴性平均值(X),以X±3SD为临界值检测了结核菌素变态反应阳性牛118头,各抗原ELISA阳性率分别为:PPD53％,KCl浸出抗原64％,Triton X-100浸出抗原60％,聚合OT56％。在屠宰牛中对6头变态反应阳性,39头变态反应阴性牛进行了符合试验,ELISA与病变及细菌培养有高的符合率。通过对其它疾病和分枝杆菌免疫血清的检测,证明ELISA法检测牛结核有较好的特异性。4种抗原中以Triton x-100浸出抗原最佳。     

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%。     

三种变态反应判定标准检测牛结核病的比较

同时应用牛型PPD和禽型PPD皮内变态反应，对300头牛进行了检测；并分别采取OIE、中国和欧共体的判定标准进行判定。结果发现：阳性数分别为48头、19头和7头；检出率分别为16％、6．3％和2．3％。从变态反应强度而言，牛结核变态反应的平均皮厚差为1．38mm，禽结核变态反应的平均皮厚差为1．46mm。可看出，由于判定标准不同。欧共体阳性检出率最小，其次是中国，而OIE最高。禽结核变态反应强度比牛结核变态反应强度略高。说明禽结核分枝杆菌对牛结核变态反应有很大的干扰。同时欧共体比较试验法能够排除禽结核分枝杆菌干扰。     

牛结核病污染场4种检测方法评估

为寻找最适合牛结核病污染场的检测方法,加快牛结核病净化,采用牛结核菌素(PPD)皮内变态反应试验(TST)、比较皮内变态反应试验(SICTT)、牛结核病ELISA-γ-干扰素试验(IFN-γ-ELISA)、牛结核病抗体ELISA试验4种方法,对某结核病污染牛场300头奶牛进行检测,并以TST为参考标准,对不同检测方法的检测结果进行对比分析。结果显示,这4种方法中,IFN-γ-ELISA的阳性检出率最高,与TST相比,差异显著(P0.05),而其他方法的阳性检出率均低于TST。结果表明,IFN-γ-ELISA方法的检测敏感性较高,在牛结核病污染场净化初期使用,可以尽量避免阳性牛漏检,从而加快牛结核病净化进程。     

新疆石河子地区牛结核病检测方法的比较研究

采用比较变态反应检测方法对石河子地区6家规模牛场牛型提纯结核菌素(PPD)皮内变态反应检出的475头阳性牛进行了检测,按照国家标准-动物结核病诊断技术(GB/T18645-2002)进行结果判定,结果表明:牛型结核阳性457头,符合率在93.18%～100%之间,平均96.21%。经反复试验对比分析采用牛型提纯结核菌素皮内变态反应进行结核病的检测,结果准确,既经济实用又便于操作,还可避免再采用比较变态反应要隔离阳性牛而导致牛结核病传染散播风险。项目组通过在四个牛场的对比分析,在未建立符合防疫条件的独立隔离圈舍的牛场检测阳性牛及可疑牛在隔离45d后再次检测时新增阳性牛数量增加;而采用牛型提纯结核菌素检测后对阳性牛采取扑杀、无害化处理、消毒等综合性措施后,间隔45d对全群再次检测时新增阳性牛数量明显减少。因此牛结核病检疫采用适宜基层实际的牛型提纯结核菌素皮内变态反应对及时检出阳性牛,并尽快进行扑杀、无害化处理、净化牛群、控制结核的传播扩散,实现公共卫生安全具有重要意义。     

应用斑点酶联免疫吸附试验快速检测猪弓形虫抗体

将斑点酶联免疫吸附试验(Dot—ELISA)用于检测猪弓形虫抗体,并与常规ELISA和IHA法进行了比较。结果,对102份滴度下降的猪阳性血清检测,弓形虫抗体阳性检出率,Dot—ELISA为66.67％(68/102),常规ELISA为48.04％(49/102),IHA为27.45％(28/102);对675头商品猪血清检测,弓形虫抗体阳性检出率,Dot—ELISA为48.15％(325/675),常规ELISA为41.93％(283/675),IHA为33.80％(228/675);与3种寄生虫(猪囊虫、猪旋毛虫、住肉孢子虫)阳性血清无交叉反应;对123份弓形虫抗体阳性和158份阴性猪血清进行3次重复性试验,结果完全一致。结果证明,该法敏感性高,特异性强,操作简便快速(于接到病料后2h报告结果),便于在基层推广使用。     

Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis

The sensitivity and specificity of an ELISA for the detection of bovine IgG anti-Mycobacterium bovis antibodies were 73.6% and 94.1%, respectively, as determined in 53 bacteriologically confirmed tuberculous cattle and 101 healthy cattle from a tuberculosis-free area. In addition, the results of ELISA and tuberculin tests in 149 cattle were compared with those of subsequent necropsy studies. Both tests failed to detect 2 animals with tuberculous lesions and positive culture; 3/12 cattle with M. bovis isolation and no lesions, and 2/7 with atypical mycobacterial infection reacted to tuberculin, but none had antibodies; in 128 cattle with neither lesions nor mycobacterial isolation, 6 were tuberculin reactors and 7 others had antibodies. Negative results were obtained by ELISA in 21/22 paratuberculous cattle. Antibodies were not detected in 88.9% to 96.4% of 697 cattle from two tuberculin negative herds of an endemic area. In a herd with proved M. bovis infection, distribution of seropositive animals in tuberculin and non-tuberculin reactors was similar. Antibody responses to cutaneous tuberculin stimuli were observed in 4 experimentally infected cattle, but only in 2/10 healthy controls after repeated PPD stimuli. Nine controls which had either received a single tuberculin dose or none showed no increase in antibody levels. The low sensitivity of this ELISA limits its usefulness as a diagnostic tool for bovine tuberculosis eradication campaigns. However, it could be helpful in epidemiological surveillance if its efficiency to identify infected herds is demonstrated.     

排除牛结核检疫中非特异性干扰的研究

本文采用变态反应和ＥＬＩＳＡ相结合的方法对牛结核进行检疫，并以剖检、组织学和细菌学证实。通过对３２３０头奶牛的检测结果表明：采用结核变态反应和ＥＬＩＳＡ相结合的方法有助于提高结检疫的特异性，排除非典型分枝杆菌引起的非特异性干扰。     

Identification of Mycobacterium bovis in bovine clinical samples by PCR species-specific primers.

Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.     

Evaluation of an antigen-capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle

A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.     

An ELISA for the detection of anergic tuberculous cattle

An enzyme-linked immunosorbent assay (ELISA) for bovine antibody to antigens in unheated Mycobacterium bovis culture filtrate was standardised against a reference serum from an experimentally infected cow. Two Northern Territory herds with a total of 561 cattle were tested. All cattle reacting in the caudal fold tuberculin test, those giving strong reactions in the ELISA and those with visible lesions of tuberculosis were subjected to a detailed bacteriological examination. Of the 19 cattle which yielded isolates of M. bovis, only 4 were positive to the tuberculin test. Serum samples from 5 cattle gave ELISA values greater than 7.0 units. None of these 5 reacted in the tuberculin-test and 2 had no visible lesions. Of the 10 remaining cattle from which M. bovis was isolated, 3 had ELISA values between 6.5 and 7.0 units and were also without visible lesions. The ELISA values for the remaining 7 infected cattle ranged down to 4.6 units. Forty cattle yielded no M. bovis on culture of their tissues. They included 7 which were reactors in the tuberculin test and 23 with ELISA values of 7.0 units or more. The evident low specificity and sensitivity of the ELISA make it of little value as an alternative to the tuberculin test, but it can detect some anergic cattle at the cost of increasing the number of false positive reactors. This may be acceptable in some circumstances and would justify the use of the ELISA as a complement to the tuberculin test or to an in vitro assay of T-cell immunity. In the 2 Northern Territory herds described, the removal of 5 of the anergic cattle would have required a cull of 28 animals of 5% of the total. A cut off value of 6.5 units would have eliminated 3 more, but at the cost of culling 80 animals or nearly 15% of the cattle. Even so, 7 cattle from which M. bovis was isolated would have remained undetected by either test.     

Preparation and evaluation of the specificity of Parafilaria bovicola antigen for detection of specific antibodies by ELISA

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occurred. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.     

Serological and cutaneous testing of bovine tuberculosis with the A60 antigen complex from Mycobacterium bovis, strain Calmette-Guérin

A60, a major thermostable macromolecular antigen complex of Mycobacterium bouis strain Calmette-Guérin (BCG), is immunodominant in tuberculosis and able to elicit both humoral and cellular immune reactions in infected hosts. A60-based ELISA and cutaneous tests have been used, in conjunction with the PPD-based skin reaction, in a control group of healthy animals, and in a herd including tuberculous animals. Cutaneous testing with A60 yielded results comparable with those with PPD: both were negative with control cattle and positive with infected animals. Moreover, comparative cutaneous testing with avian tuberculin yielded similar results with PPD and A60. When animals from the infected herd were tested with both avian and bovine sensitins, 54% of cattle were diagnosed as fully positive, 26% suspect, and 20% negative. Serological analysis with the A60-ELISA of part of the infected herd yielded 74% positive, 21 % suspect and 5% negative results. Thus, positivity was 74% for Serological analysis and 54% for cutaneous testing, whereas positive plus suspect results were 95% for serological analysis and 80% for cutaneous testing. It can be concluded that A60 can be used in place of PPD for cutaneous testing in cattle, and that the diagnostic value of the A60-ELISA is superior to that of the PPD-cutaneous test.     

三抗体间接Dot-ELISA检测犬旋毛虫病IgG抗体的研究

本试验成功地建立了检测旋毛虫病犬血清中的特异性抗体的一种新的免疫学诊断法 -三抗体间接 Dot-EL ISA法 ,并与压片镜检法 (TSM)和琼脂扩散试验 (AGID)进行了比较。试验结果表明 ,Dot- EL ISA和 AGID对2 4 5份被检血清的阳性检出率为 2 2 .86 % (5 6 / 2 4 5 )和 6 .94 % (17/ 2 4 5 ) ,两者符合为 30 .36 %。该法的检出率比AGID高 15 .92 % ,比 TSM高 18.37%。Dot- EL ISA的相对灵敏度为 AGID的 6 7.4 7倍     

Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis

The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD, and lipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bovis infected cattle and 223 cattle from a TB free herd. ELISA results were analyzed using receiver operating characteristic (ROC) curves in relation to culture results. The areas under the three ROC curves were 71 ± 49% SE (MPB70), 71 ± 27% SE (bovine PPD), and 56 ± 4% SE (LAM).