Validation of an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy.

The sensitivity and specificity of an immunoperoxidase monolayer assay (IPMA) was evaluated in a blind serologic study of a group of disease-free pigs and a group of pigs experimentally infected with intestinal homogenate containing Lawsonia intracellularis organisms. Sixty pigs from the control group were kept in the source farm, and another 60 animals were transferred to an isolation unit aid challenged intragastrically. All animals were bled before and 21 days after challenge. Fecal samples were collected on the same dates. The IPMA results were tested for sensitivity and specificity in a 2 x 2 table using the challenged and nonchallenged status as gold standard. Sensitivity and specificity were evaluated for different cutoff points (serum dilutions). Specificities of 100% were obtained for all the serum dilutions tested (1:15, 1:30, 1:60, and 1:120). The sensitivity levels were 90.7%, 88.9%, 81.5%, and 75.9% for the serum dilutions 1:15, 1:30, 1:60, and 1:120, respectively. The sensitivity of the dilution 1:15 was slightly, but not significantly, higher than the dilution currently used as the cutoff point (1:30). Cross-reactivity of the IPMA test was evaluated using sera from pigs experimentally inoculated with Brachyspira pilosicoli and various Campylobacter species. All these samples were negative. Sera samples from 3 porcine proliferative enteropathy known negative populations, 40 growing pigs from 2 commercial farms and a group of 6 cesarean-derived and colostrum-deprived pigs, also tested negative by IPMA. The IPMA serologic test with the cutoff point of 1:30 showed specificity of 100% and sensitivity close to 90% and, therefore, is an appropriate diagnostic test for herd screening but not for diagnosing PPE on the individual level.     

Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy.

The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) using a sonicated pure culture of Lawsonia intracellularis as the antigen (So-ELISA). A total of 332 serum samples, consisting of 232 experimentally infected animals and 100 animals naturally infected with L. intracellularis, were used to assess the diagnostic sensitivity. Three hundred and fifty-five sera from uninfected animals were used to determine the diagnostic specificity. The receiver operating characteristic and mean +3 standard deviation of optical density (OD) values from uninfected animals were used for selecting cut-off points. The diagnostic accuracy of So-ELISA was considered to be high as the area under the curve index was 0.991 with 0.0029 standard error. The optimal cut-off for So-ELISA was set at 0.45 OD with 89.8% sensitivity and 99.4% specificity based on a combination of good sensitivity and high specificity. No cross-reactivity was found in sera from pigs exposed to Brachyspira pilosicoli, B. hyodysenteriae, Campylobacter mucosalis, C. jejuni, or C. coli. Inter- and intracoefficient of variation of all control sera tested with So-ELISA was less than 10%. The observed agreements between So-ELISA and the immunoperoxidase monolayer assay tested with experimental challenge animals and field samples were 95.08% with 0.88 kappa and 90.65% with 0.74 kappa value, respectively. So-ELISA was able to detect the seroconversion of infected animals at 2 to 4 weeks after exposure to L. intracellularis. Based on the validation results, So-ELISA could be used as an alternative serology for proliferative enteropathy diagnosis.     

PRV-IPMA抗体检测试剂盒的研制及其应用

本研究建立了一种免疫过氧化物酶单层细胞试验法（IPMA）,用于猪伪狂犬病病毒（PRV）血清抗体检测。通过对IPMA反应条件的优化,组装成PRV-IPMA诊断试剂盒,并对该试剂盒检测的敏感性、特异性、重复性及保存期等进行了试验。结果表明,IPMA检测相对于SN的敏感性为96．2％,特异性为97．7％,两者的总符合率为96．9％;该试剂盒检测的重复性好,与其它病毒参考血清无交叉反应;试剂盒可在-20℃保存12个月,用该试剂盒检测PRV人工感染猪血清,于感染后2周抗体全部阳转,健康对照组猪血清抗体检测均为阴性结果。对来自黑龙江、吉林、上海、内蒙古、河北等地采集的5个猪场后备母猪150份血清样本进行检测,PRV血清抗体阳性检出率为16．7％～50％。检测结果表明这些猪场后备猪群仍需加强疫苗免疫。该试剂盒的研制为我国PRV流行病学调查和疫苗免疫效果的评价提供了技术手段。     

Evaluation of a novel enzyme-linked immunosorbent assay for serological diagnosis of porcine proliferative enteropathy

A specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the porcine pathogen Lawsonia intracellularis was developed and evaluated using sera from na?ve, naturally infected as well as experimentally infected pigs. On the basis of 37 serum samples collected from experimentally infected pigs and 62 serum samples from naturally infected pigs the sensitivity of the ELISA was calculated to 98.0%. The specificity of the test was 99.3%, calculated on the basis of 273 serum samples collected in six herds free of L. intracellularis after medicated eradication. The novel ELISA was a specific and sensitive method for detecting specific antibodies, and may be a good alternative to the existing serological tests for L. intracellularis. It may be usable for diagnosis of proliferative enteropathy and for determination of a herd's epidemiologic status.     

Literature review of the diagnosis of porcine proliferative enteropathy

Lawsonia (L.) intracellularis is the etiologic agent of ileitis, which occurs in different forms in pigs. The infection can be demonstrated by several methods. In routine diagnosis the indirect detection of L. intracellularis is conducted by measuring antibodies performing an indirect IFT or ELISA. Furthermore, an IFT and PCR on faecal and/or tissue samples are well established for the direct detection. The suitability of PCR on faecal samples is frequently discussed. Problems are related to various inhibitors that are component of faeces. When interpreting laboratory results, the past medical history of pigs, i.e. antimicrobial treatment, has to be considered. Since 2004 a new vaccine against ileitis is licensed. A possible influence of vaccination on diagnostic testing, especially PCR and ELISA, is hardly investigated. Therefore, the interpretation of laboratory results from vaccinated animals is, considering the current literature, restrained.     

Evaluation of an indirect fluorescent antibody test, enzyme-linked immunosorbent assay and quantification of immunoglobulins in the diagnosis of bovine trypanosomiasis.

An indirect fluorescent antibody test (IFAT), a microscale version of the enzyme-linked immunosorbent assay (microELISA) and determination of IgM levels in serum were assessed for their comparative diagnostic value in the detection of bovine trypanosomiasis. Serum samples from drug-treated N'dama cattle and untreated N'dama and Zebu cattle from Liberia were examined for the presence fo antibodies to trypanosomes. In the untreated Zebu cattle, infections with T. vivax predominated and the prevalence of infection was higher than that found in untreated N'damas in which infections with T. congolense predominated. The proportion of animals which showed serological evidence of trypanosomiasis in the untreated Zebus was slightly higher than that found in the untreated N'damas. The prevalence of infection was low in N'dama cattle which had been treated with diminazene aceturate and homidium chloride but 50% of the animals showed serological evidence of trypanosomiasis. More serologically positive animals were detected by microELISA than IFAT, but both tests were equally sensitive in detecting antibodies in cattle in which trypanosomes were demonstrated by examination of peripheral blood. With both IFAT and microELISA it was necessary to carry out tests using antigens prepared from T. brucei, T. vivax and T. congolense in order to detect all serologically positive animals. Increases in serum IgM occurred in both N'dama and Zebu cattle but the levels were raised in only approximately half of the known infected animals. Overall, more animals gave positive reactions with IFAT and microELISA than showed raised IgM levels.     

Comparison of different methods for diagnosis of porcine proliferative enteropathy

The objectives of this study were: (1) to compare 2 methods of serology; (2) to compare 3 histologic techniques; and (3) to compare 2 methods of detecting shedding in pigs experimentally challenged with Lawsonia intracellularis. The sensitivities of these tests were determined by the detection of infection. Forty 5-week-old pigs were inoculated on day 0 with intestinal homogenate from pigs with proliferative enteropathy (PE). Clinical evaluation was done on day 7 and daily from day 14 to 28 postinoculation. Fecal shedding of L. intracellularis was monitored by use of polymerase chain reaction (PCR) analysis and immunoperoxidase staining at 7-day intervals. Serum was obtained on days 0 and 28 for serologic testing by glass slide and tissue culture indirect fluorescent antibody tests. At euthanasia on day 28, gross intestinal lesions were evaluated and ileum samples collected for histologic analyses. Ileal histologic sections from each animal were stained by hematoxylin and eosin, Warthin-Starry silver stain, and immunohistochemistry (IHC). Of the 40 pigs, 36 had gross lesions typical of PE at necropsy. The percentage of agreement between the 2 serologic methods was 94.4%. Immunoperoxidase stain of fecal smears was more sensitive than PCR for detecting fecal shedding, especially on day 21 (89.5% and 60.5%, respectively) and day 28 (59.4% and 37.5%, respectively) post-inoculation. The IHC stain was much more sensitive for detecting infection than the routinely used hematoxylin and eosin and Warthin-Starry silver stains. In conclusion, in experimentally infected pigs, both serologic methods were appropriate techniques for detecting infection. For fecal samples, PCR has low sensitivity. Immunohistochemistry is the best diagnostic tool for formalin-fixed samples.     

PRRSV-IPMA抗体检测试剂盒的研制及其应用

建立了一种检测猪生殖与呼吸综合征病毒（PRRSV）血清抗体的免疫过氧化物酶单层细胞试验法（IPMA），并组装成PRRSV-IPMA抗体检测试剂盒。对该试剂盒的敏感性、特异性、重复性及保存期等进行了试验，并与国外IDEXX ELISA试剂盒进行了比较。结果显示，用该试剂盒检测PRRSV人工接种猪血清样品，第1周抗体阳性检出率为50％，从第2周起抗体阳性检出率均为100％，而对照组猪血清抗体检测均为阴性。该试剂盒重复性好，-20℃保存18个月稳定，与其他病毒参考血清无交叉反应，与国外IDEXX ELISA试剂盒的符合率为83．3%。用该试剂盒对采自黑龙江、吉林、辽宁、河北、上海等地13个猪场的520份正常猪血清样品进行了抗体检测，结果，4个猪场阳性率高达90％以上，仅有2个猪场为阴性，其余猪场阳性率为10％～57%。     

Comparison of 2 serologic tests for the diagnosis of porcine proliferative enteropathy

The purpose of this study was to compare results from 2 serological assays at the individual- and herd-level for porcine proliferative enteropathy diagnosis. Cohen’s kappa coefficient (k) was used to measure agreement. The tests tend to show better agreement when used at the herd level.     

猪圆环病毒2型免疫过氧化物酶单层细胞试验抗体检测试剂盒的研制及应用

本研究建立了一种免疫过氧化物酶单层细胞试验（Immunoperoxidase monolayer assay,IPMA）,用于猪圆环病毒2型血清抗体检测,通过对IPMA反应条件的优化,组装了诊断试剂盒。研究结果表明,用IPMA检测猪圆环病毒2型人工感染猪血清,于感染后3周抗体阳转,第3周～10周抗体阳性检出率为92.8%（52/56）,对照组猪血清抗体检测均为阴性（33/33）。试剂盒在-20℃稳定保存18个月与其他几种猪病毒参考血清无交叉反应,与用重组蛋白抗原建立的rcELISA符合率为89.2%。对来自黑龙江、吉林、河北、上海、内蒙古、云南、江西等地猪场健康成年猪血清480份和发病猪血清424份进行了检测,抗体检出率分别为91.7%和79.2%,表明我国猪群中猪圆环病毒2型污染相当严重。该试剂盒的研制为我国PCV2流行病学调查和疫苗免疫效果的评价提供了技术手段。     

Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test

An indirect fluorescent antibody test was used for detection of serum antibodies to the platelet-specific rickettsial organism that is the causative agent of infectious cyclic thrombocytopenia (ICT) in dogs. The test converted from negative to positive in 7 of 7 experimentally inoculated dogs. One of 2 attempts to recover the rickettsial agent of ICT from naturally occurring seropositive dogs, by blood inoculation of experimental dogs, was successful. Seemingly, the test did not detect antibodies to Ehrlichia canis, nor did a similar test, using E canis antigen slides, detect antibodies to the rickettsial agent of ICT. The rickettsial agent of ICT has been classified tentatively as E platys. When applied to sera from a group of healthy random-source dogs, the test revealed a relatively low (5%) occurrence of positive reactions. A higher occurrence of positive reactions (35%) was noticed in sera from a group of thrombocytopenic dogs from the University of Florida. A majority of these positive sera were also positive for antibodies to E canis. The highest occurrence of positive reactions was found (greater than 50%) in E canis-positive sera from dogs at the University of Florida, as well as from dogs from 9 other states.     

Prevalence of antibodies to porcine adenovirus in swine by indirect fluorescent antibody test

An indirect fluorescent antibody test was developed for routine identification of a porcine adenovirus and its specific antibody. Two specific-pathogen-free young pigs were inoculated with the viral antigen prepared in continuous porcine kidney cell cultures, and their sera were used as an antibody reagent to standardize the test. Sera of adult pigs with respiratory problems, obtained from pig farms in Quebec, were tested for antibodies to this virus; 83 of 540 sera tested (15.2%) were found to be positive.     

Comparative evaluation of the fluorescent antibody test and microtiter immunoperoxidase assay for detection of bovine viral diarrhea virus from bull semen.

An indirect immunoperoxidase staining technique (IP) is described for the detection of bovine viral diarrhea virus (BVDV) in bovine semen. The performance of the IP was compared to the reference immunofluorescent staining test in its ability to detect BVDV in 23 coded field semen samples. The IP assay which can be applied with ease to a large number of samples and does not require expensive fluorescence microscope equipment, appears to be an alternative method for BVDV detection. The IP assay can be strongly recommended for certification of BVDV-free bovine semen for artificial insemination and trading purposes and for laboratories which are not equipped for performing the immunofluorescent test.     

检测猪捷申病毒抗体的间接免疫荧光方法的建立与初步应用

为建立检测猪捷申病毒(PTV)抗体的方法,本研究利用PTV-8 Jilin/2003株为杭原,建立了检All PTV杭体的的间接免疫荧光技术(IFA)方法.该方法工作浓度分别是:一抗最适稀释度为1:10:FITC标记二抗最适稀释度为1:100;二杭中伊文氏兰最适稀释度为1:30 000.敏感性试验、特异性试验表明该杭原板可以检出包含PTV-8型和1型在内的的所有PTV阳性血清,方法敏感、特异.与SN符合率94.0.用该方法对黑龙江、山东、天津和河南等地的1 500份临床血清样品进行检测,阳性检出率为55.8,表明我国猪群中PTV的感染已经非常普遍.该方法做为PTV的血清学诊断新技术,为开展PTV分子流行病学调查莫定了基础.     

An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine.

The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transmissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20 degrees C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advantages over the virus neutralization test.     

Development of an indirect fluorescent antibody test, using microfluorometry as a diagnostic test for bovine anaplasmosis

The indirect fluorescent antibody test for the diagnosis of Anaplasma marginale infection in cattle was modified for use with microfluorometry. The test was standardized by use of a fluorometer that measures intensity of fluorescence. Standardization included A marginale-infected blood smears on microscope slides as antigen, serum from an inoculated calf as a positive control containing specific antibody, and an affinity-purified fluorescein-conjugated anti-bovine immunoglobulin as 2nd antibody. The modified test and microfluorometry allowed for titration of sera from A marginale (Florida isolate)-inoculated cattle with a degree of accuracy exceeding visual determinations. In addition, the fluorometric test was more sensitive than the complement fixation or card agglutination tests in identifying cattle that had previous Anaplasma infections.     

Evaluation of the fluorescent antibody test for diagnosis of paratuberculosis

Results of indirect fluorescent-antibody microscopy did not differ significantly from complement-fixation test results in diagnosing paratuberculosis (Johne's disease) in cattle. Neither test had acceptable sensitivity or specificity for detecting subclinical cases.     

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.