Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

Establishment of an efficient enzyme-linked immunosorbent assay for the detection of Eperythrozoon sius antibody in swine

The Eperythrozoon suis (E. suis) antigen was purified using a Sephadex G-200 chromatograph, and thereby, a high-affinity, specific E. suis antigen was collected and confirmed with Western blotting. Using this antigen, an enzyme-linked immunosorbent assay (ELISA) system to detect the antibody against E. suis in swine was established. There was no cross-reaction with swine sera, which were affected with Mycoplasmal pneumonia, swine fever, swine colibacillosis, or toxoplasmosis. A comparison of this ELISA system with an indirect hemagglutination (IHA) test using 78 swine samples revealed that the ELISA system significantly improved the sensitivity, specificity, and stability for the serodiagnosis of swine E. suis.     

贵州省猪弓形虫病的血清学调查

为全面了解贵州省猪弓形虫感染情况,对采自9个市（州、地）264个养猪场（户）的2 906份血清用酶联免疫吸附试验（ELISA）检测猪弓形虫抗体,总体阳性率为65.83%,变化范围为27.88%~85.42%。采集65份猪血清做ELISA和间接血凝试验（indirect heamagglutination assay,IHA）比较测定,两种方法总体符合率为58.46%。IHA方法补充检测177份猪血清,弓形虫抗体阳性率为27.68%。血清学调查结果与国内部分省（市）报道相符。     

Evaluation of the indirect hemagglutination assay as a practical serodiagnostic test for mycoplasmal pneumonia of swine

Sera from swine experimentally or naturally infected with Mycoplasma hyopneumoniae (the etiological agent of mycoplasmal pneumonia of swine, MPS) were tested by the indirect hemagglutination assay (IHA), the enzyme-linked immunosorbent assay (ELISA) and the complement fixation (CF) test. The IHA detected antibody at comparable times and levels to the other 2 serological tests following experimentally-induced infection. In the late antibody response (greater than or equal to 86 days post-infection), the ELISA titres were higher than either the IHA or the CF test. The IHA appeared least satisfactory when it was used to test sera from commercial swine herds. When 1000 sera were tested, the IHA was positive for only 30 (22%) of 135 sera which were positive by the ELISA and the CF test. The IHA titres were low; 20 of the 30 sera had a titre of only 10. The end-points for the IHA were difficult to read for sera of this low titre. The relationship between positive IHA results for the herd sera obtained at necropsy, and the occurrence of gross or microscopic lesions typical of MPS was poor (41 and 50% agreement, respectively). An agreement of 39% was noted between positive IHA results and the localization of mycoplasmal antigens by an indirect immunofluorescence (IIF) test. However, IHA results correlated significantly (P less than 0.05) with gross and microscopic lesions, but not with the IIF test. No significant correlation was noted between the IHA (or the other 2 serologic tests) and the cultural isolation of M. hyopneumoniae or M. flocculare. On the basis of these results, the IHA appears to have limited promise as a practical test for the diagnosis of MPS in commercial swine herds because of the low titres observed, poor correlation of the IHA and other indicators of MPS, the necessarily subjective determination of end-points, and other inherent technical limitations of the test.     

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.     

猪衣原体病的诊断

对湖北省2个猪场的母猪发生流产和返情的疾病进行诊断,采集母猪血样和组织样,对衣原体病、猪瘟等采用IHA、ELISA、PCR等方法进行实验室检验,衣原体IHA抗体、ELISA抗体分别为84.6%(22/26)、88.5%(23/26),PCR检测阳性率为69.2%(18/26)。根据实验室检验结果,结合临床症状分析,诊断为猪衣原体感染,经采取综合防控措施,疫情得到了有效控制。     

Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus

In order to establish an indirect ELISA to detect antibody of porcine epidemic diarrhea virus (PEDV).The experiment using the recombinant and purified truncated N protein as antigen expressed in E.coli BL21(DE3),the indirect ELISA was named rnPED-ELISA.The recombinant truncated N protein antigen showed no cross-reaction with the positive sera of other 7 kinds of swine diseases,CV%of intro-batch duplicativity test and inter-batch duplicativity test were less 13%;Sensitivity and specificity of rnPED-ELISA relative to SN were 93.33% and 90.00%,respectively;rnPED-ELISA compared with TSZ PEDV antibody diagnosis Kit,91.67% concordance was obtained.200 serum samples were detected by this method,the total masculine ratio was 69.5%.Therefore,this rnPED-ELISA based on recombinant truncated N protein antigen had good sensitivity and specificity,could afforded a simple and rapidmeans for assessment of vaccination in the field and investigation of PED epidemiology.     

PEDV流行株重组截短N蛋白间接ELISA检测方法的建立

为了建立猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)抗体检测的间接ELISA方法,本研究以纯化的原核表达的PEDV截短N蛋白作为包被抗原,建立了PEDV抗体检测的间接ELISA方法,将该方法命名为rnPED-ELISA。该抗原不与其他常见的7种猪病的阳性血清发生交叉反应,批内和批间重复性试验的变异系数均小于13%;rnPED-ELISA相对于血清中和试验(SN)试验的敏感性为93.33%,特异性为90.00%;rnPED-ELISA与TSZ全病毒抗体检测试剂盒的符合率达91.67%。采用rnPED-ELISA方法检测200份临床样品,PEDV抗体阳性检出率为69.5%。本试验建立的rnPED-ELISA方法具有良好的敏感性和特异性,可为免疫猪群抗体监测和猪流行性腹泻流行病学调查提供一种快速、简便的血清学诊断方法。     

集约化养猪场嗜流产衣原体病流行状况的初步调查

本试验对北京郊区5个规模化猪场断奶仔猪、育成猪、育肥猪和怀孕母猪采集血清共507份,同时采集密切接触饲养人员、仔猪、育肥猪的喉头拭子、母猪阴道拭子及公猪的精液共183份。分别使用间接血凝诊断试剂盒、法国ELISA试剂盒检测抗体;利用直接荧光染色法检测抗原,包括166份猪喉头拭子、阴道拭子和精液以及17份饲养人员喉头拭子。结果发现间接血凝诊断试剂盒、ELISA试剂盒抗体阳性率分别为4.14%和2.17%;抗原平均阳性率为14.8%,其中精液阳性率达到37.5%,母猪阴道拭子阳性率为27.5%,饲养人员阳性率为23.5%。本试验证实北京郊区猪嗜流产衣原体在种公猪、母猪群和饲养员呈高流行趋势。同时研究结果显示,5个被调查的规模化猪场嗜流产衣原体抗体阳性率显著低于有报道的其他省市,这与间接血凝诊断试剂盒检测结果高于ELISA试剂有关。针对发现的问题,必须采取有效措施控制种公猪精液污染衣原体现象,阻断向母猪和饲养人员传播,以降低人兽共患病发生的风险。     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

猪繁殖与呼吸综合征病毒重组M蛋白间接ELISA抗体检测方法的建立及应用

为建立一种敏感、特异、快速、高通量的猪繁殖与呼吸综合征病毒(PRRSV)抗体血清学检测方法,本研究利用原核表达技术表达了PRRSV M蛋白,将纯化后的重组M蛋白作为包被抗原建立了检测PRRSV抗体的间接ELISA方法。参照已发表的PRRSV基因组M基因序列,设计合成1对特异性引物,RT-PCR扩增了长约435 bp的M基因片段,将目的片段亚克隆至pET32a(+)表达载体中,经IPTG诱导获得了以包涵体形式表达的重组M蛋白,重组蛋白纯化后,免疫印迹检测结果表明具有良好的抗原性和特异性。以重组M纯化蛋白为包被抗原,经间接ELISA反应条件的优化,建立了检测PRRSV抗体的间接ELISA方法,该方法检测猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、猪伪狂犬病病毒(PRV)、猪传染性胃肠炎病毒(TGEV)、猪圆环病毒2型(PCV2)其他6种常见猪病病原的阳性血清均为阴性;该方法批内与批间重复性试验的变异系数分别小于5%和10%;该方法与商品化ELISA试剂盒的符合率为95.3%。本研究建立的M-ELISA检测方法将为猪群免疫PRRS疫苗后抗体水平监测及PRRSV野毒感染的快速诊断与流行病学调查等提供了一种简便易行、快速、高通量的血清学抗体检测方法。     

Evaluation of a monoclonal blocking ELISA and IHA for antibodies to Mycoplasma hyopneumoniae in SPF-pig herds.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) and an indirect haemagglutination assay (IHA) were applied to serum samples from 124 specific pathogen-free (SPF) breeding and multiplying herds, which participate in the routine serological surveillance of the Danish SPF programme. Clinical and pathological observations of the herds and microbiological culturing of Mycoplasma hyopneumoniae were used to calculate herd sensitivity, herd specificity and herd predictive values for the two serological assays. The ELISA was superior to the IHA in herd sensitivity and herd specificity, with values of 93 per cent and 96 per cent, respectively, for the ELISA, and 61 per cent and 92 per cent for the IHA. During the six month period of evaluation 2.5 per cent of the herds were infected with M hyopneumoniae each month. At this level the IHA was found to have a positive herd predictive value of 16 per cent, compared with 39 per cent for the ELISA. The negative herd-predictive value on the same level was 99.8 per cent for the ELISA and 98.9 per cent for the IHA. If the assays were applied to a group of herds with a herd prevalence of M hyopneumoniae infection of 30 per cent (as is the case with the production herds in the Danish SPF programme) the predictive value of a positive herd diagnosis would be 91 per cent for the ELISA and 76 per cent for the IHA, and the predictive value of a negative herd diagnosis would be 97 per cent with the ELISA and 85 per cent with the IHA.(ABSTRACT TRUNCATED AT 250 WORDS)     

牛病毒性腹泻病毒感染对猪瘟免疫的影响

猪瘟病毒（CSFV）与同属的牛病毒性腹泻病病毒（BVDV）同源性较高,抗原性上有交叉。本次调查对368份猪瘟免疫猪血清样本进行BVDV抗原检测,其中7份呈阳性,阳性率1.90%。对7份BVDV阳性血清采用ELISA和IHA两种方法检测猪瘟（CSFV）抗体水平,抗体合格率偏低,两者的结果符合率为71%。研究表明：BVDV在一定程度上干扰了猪瘟疫苗的免疫效果,影响抗体水平。     

基于E^rns蛋白的猪非典型瘟病毒间接ELISA检测方法的建立

猪非典型瘟病毒(APPV)是新近发现的仔猪先天震颤的病原体,其血清学检测方法亟待建立.E^rns蛋白是APPV诱导机体产生中和抗体的重要保守性抗原,是血清学检测的特异性靶标之一.本研究制备了猪非典型瘟病毒原核表达的Erns蛋白,以其作为包被抗原,建立了间接ELISA检测方法.结果表明,最佳抗原包被浓度为8μg/mL,最佳血清稀释度为1:10,阴阳性临界值D450=0.318,灵敏度达到1:64,特异性好,与猪瘟、猪伪狂犬、猪繁殖与呼吸障碍综合征、猪流感及圆环病毒病的阳性血清均不发生交叉反应.该检测方法重复性好,批内变异系数最大值为7.84%,批间变异系数最大值为8.57%.利用建立的间接ELISA方法对50份猪临床血清样本进行了检测,其阳性率为10%.基于Erns蛋白间接ELISA检测方法的建立为APPV的流行病学调查和临床诊断提供了有效的工具.     

Application of recombinant antigens in serodiagnosis of swine toxoplasmosis and prevalence of Toxoplasma gondii infection among pigs in Poland

In this study, to determine the prevalence of swine toxoplasmosis, 1754 pigs from different regions of Poland were tested for IgG antibodies by an in-house ELISA technique based on native Toxoplasma lysate antigen. Seropositive individuals were found in 19.2% of the examined population. The diagnostic usefulness of three T. gondii recombinant antigens (rMAG1, rSAG1, and rGRA7), either individually or in cocktails (M1: rMAG1 + rSAG1; M2: rMAG1 + rGRA7; M3: rSAG1 + rGRA7; M4: rMAG1 + rSAG1 + rGRA7) were also assessed with serum samples from naturally infected pigs by ELISA analysis. Both rSAG1 and rGRA7 antigens detected specific IgG antibodies with a similar sensitivity (85.3% and 81.3%, respectively), whereas the lower sensitivity was obtained for rMAG1 (only 64%). Better results of reactivity were obtained for mixtures of two antigens: M1 (86.7%), M2 (89.3%) and M3 (92%). Furthermore, the reactivity of three antigens cocktail M4 (97.3%) was much higher than that of individual proteins and combinations containing two antigens. These results suggest that the combination of three recombinant antigens might be useful for the serological detection of T. gondii infection in pigs.     

ELISA和IHA检测弓形虫抗体的比较试验研究

目的:分析ELISA方法和IHA方法检测弓形虫病的可靠性。方法:本文应用酶联免疫吸附试验(ELISA)和间接血凝试验(IHA)两种方法,平行检测来自龙岩市某个猪场的32份猪血清中的抗弓形虫特异性抗体。结果:ELISA和IHA对猪弓形虫抗体阳性检出率分别为62.5%(20/32)、53.13%(17/32),这两种方法的阳性检出符合率较高,为71.88%(23/32)。其中,ELISA方法的敏感性(93.33%)、检测效率(81.25%)以及Youden指数(63.92%)都在IHA方法之上,但ELISA方法的特异性(12/17,70.59%)略低于I-HA方法(13/17,76.47%)。结论:ELISA和IHA两种方法均可用于猪弓形虫病的诊断和血清学调查,但ELISA方法更适用于猪弓形虫病的血清学调查。     

A sandwich enzyme-linked immunosorbent assay for the detection of Streptococcus suis.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and the identification of Streptococcus suis capsular types 1, 2, 1/2, 3 and 22. The specificity of this test was first evaluated using reference strains of S. suis capsular types 1 to 28 and 1/2 as well as 15 different bacterial species susceptible to be isolated from swine. The ELISA developed was very specific for capsular types 1, 3 and 22 but it could not discriminate between capsular types 2 and 1/2. In a second study, S. suis isolates from 328, 493, 368 and 76 diseased pigs were used to detect capsular types 1, 2 or 1/2, 3 and 22 respectively. The relative specificity and sensitivity varied between 98% and 100%. The ELISA results were in excellent agreement with the standard techniques (biochemical tests, coagglutination and capsular reaction tests) in detecting both positive and negative strains. Kappa values were 0.80, 0.99, 0.97 and 1.00 for detecting S. suis capsular types 1, 2 or 1/2, 3, and 22 respectively. To evaluate the relative-sensitivity of the test, primary cultures from 73 diseased pigs and tissue samples from 67 diseased pigs were used directly for detecting these capsular types. With primary cultures, the relative specificity and sensitivity (95.9% and 91.6% respectively) remained high and the test was very suitable (Kappa = 0.87). The ELISA using tissue samples gave a good specificity (97.6%), a moderate sensitivity (62.5%) and a low agreement with standard tests (Kappa = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a serodiagnostic test using Ascaris suum haemoglobin for the detection of roundworm infections in pig populations

The significant economical consequences of infections with Ascaris suum in pigs are already well documented. However, due to the subclinical nature of the disease and the lack of practical diagnostic means, ascariasis often remains undiagnosed. Here we describe the development and evaluation of a novel indirect ELISA using the purified A. suum haemoglobin (AsHb) molecule as an antigen. Initial validation using sera from 190 pigs experimentally infected twice a week with A. suum and Trichuris suis (25 and 5eggskg(-1)day(-1) respectively) demonstrated that the AsHb ELISA is able to detect long-term exposure to A. suum with a high sensitivity and specificity (99.5% and 100.0% respectively). Furthermore, this serological technique proved to be more sensitive than faecal examination on week 7 and 14 of the experiment (99.5% and 100% compared to 59.5% and 68.4% respectively). Cross-reactivity caused by T. suis infection was shown to be limited after analysing sera from pigs with an experimental T. suis mono-infection. Seroconversion was shown to occur from week 6 onwards in pigs receiving 100 A. suum eggs 5 times a week. Preliminary testing of the ELISA on six randomly selected farms confirmed the results obtained in the artificial infection trials, showing a higher sensitivity of the serologic method compared to faecal examination. Finally, the ELISA was used to investigate Ascaris infection rates on 101 conventional Flemish pig farms. The results showed that on 38.6% of the farms less than 20% of the tested samples were seropositive, while in 19.8% of the farms 80-100% of all pigs were seropositive. The results of this study suggest that the AsHb ELISA could provide pig farmers and veterinarians with an easier and more sensitive way to estimate the overall prevalence of A. suum on their farm.     

Quantitative and Qualitative Study of Enzyme-linked Immunosorbent Assay to Detect IgG against Japanese Encephalitis Virus in Swine Sera

This study was designed to investigate the application of indirect enzyme-linked immunoassay (ELISA) in detecting IgG against Japanese encephalitis virus in swine sera and the qualitative nature of this test. The attenuated strain SA14-14-2 of Japanese encephalitis virus (JEV) was inoculated into 9-day-old chicken embryos and virus was harvested, purified and suspended in 0.9% saline as JEV antigen. The control antigen was prepared by the same method as for the antigen. In the ELISA, the optimal concentrations of antigen coated and dilution factor were selected using chi2 test. Ninety-two swine sera negative to haemagglutination inhibition (HI) were tested by this assay and the positive threshold was determined. The results of this study indicate that indirect ELISA has high specificity, sensitivity and reproducability. Simultaneous testing of 74 serum samples from nine pig farms was carried out to compare the existing HI test and the indirect ELISA. The coincidence rate of the two assays was 85.1% (63/74) and no significant difference was observed between them (p > 0.05). This ELISA test can detect 46 swine serum samples qualitatively and the titre of eight swine serum samples through endpoint dilution quantitatively within one 96-well plate.     

Brucellosis due to Brucella suis in a swine herd associated with a human clinical case in the State of S?o Paulo, Brazil

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (S?o Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480?IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.