Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests.

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.     

Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum

An enzyme-linked immunosorbent assay (ELISA) was evaluated for detection of antibodies (Ab) against Mycoplasma hyopneumoniae and M. flocculare in sera from swine experimentally infected with these agents. In addition, the ELISA was compared with the complement fixation test (CFT), and radial immunodiffusion enzyme assay (RIDEA) for the demonstration of Ab against M. hyopneumoniae. Twenty two 6-week-old swine from a respiratory disease-free herd were divided into five groups. Two or three pigs from each of the four groups were inoculated, respectively, with M. hyopneumoniae or with M. flocculare while two pigs in each group were contact exposed to the inoculated penmates. A fifth group, consisting of three pigs, served as inoculated controls. Pigs inoculated with M. hyopneumoniae began coughing 13 days post inoculation (PI). Antibodies were first detected 2 weeks PI with the CFT, 3 weeks PI with the ELISA, and 5 weeks PI with the RIDEA. With the ELISA and RIDEA, Ab were still detectable one year PI at a very low level. With the CFT, Ab were not detectable in sera from any swine beyond 5 months PI. At necropsy 1 year PI, no lesions were detected in lungs of any of the animals nor were mycoplasmas detected. M. flocculare inoculated or contact-exposed pigs never evidenced clinical signs. Antibodies against M. flocculare were first detected 5 to 12 weeks PI with CFT, and 6 to 12 weeks PI with the ELISA. Peak optical density (OD) values obtained in the ELISA with M. flocculare Ab were as high as the values obtained with peak M. hyopneumoniae Ab titers. Levels of Ab against M. flocculare were at relatively higher OD at 1 year PI than Ab against M. hyopneumoniae. Sera with high levels of Ab against M. flocculare cross-reacted slightly with M. hyopneumoniae antigen in immunoblotting and ELISA.     

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.     

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 abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. III. Serological findings and their relationship to pathomorphological and microbiological findings

Blood samples from 777 pigs, originating from 9 different herds, were collected at slaughter and examined for antibodies to Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae by the indirect hemagglutination assay (IHA) and the complement fixation (CF) test, respectively. Results were compared to pathological and microbiological findings. Antibodies to M. hyopneumoniae in positive titers of 1/80 or higher were found in 62% of the samples. The relationship between positive IHA titers to M. hyopneumoniae and gross findings indicative of enzootic pneumonia of pigs (EPP), histological findings indicative of EPP, the isolation of M. hyopneumoniae and the demonstration of M. hyopneumoniae by indirect immunofluorescent testing ranged from 64% to 68%. No correlation was noted between positive IHA titers and the isolation of Mycoplasma flocculare. Positive antibody titers to A. pleuropneumoniae of 1/10 or higher were detected in 5% to 85% of the samples from individual herds. Positive titers to A. pleuropneumoniae serotype 2 were found in 71% to 79% of the sampled animals from herds with high frequencies of pneumonic lesions indicative of pleuropneumonia. In herds with low frequencies of pleuropneumonia, positive titers were recorded in from 0 to 4% of the tested pigs. However, no statistical association was found between pleuropneumonia and positive titers to A. pleuropneumoniae serotype 2 in individual animals. Twenty-one per cent of samples with positive CF titers to A. pleuropneumoniae showed antibodies to more than one serotype.     

Comparison of serological tests for the detection of antibody to natural and experimental murine cytomegalovirus.

Three serological tests, i.e. complement fixation test, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA) were compared for sensitivity in the detection and titration of murine cytomegalovirus antibody. The three tests were compared using sera from experimentally inoculated and naturally infected mice bled at intervals from 3 to 140 days postinfection. In the acute infection, complement fixation and indirect immunofluorescent assay tests were of comparable sensitivity for early detection of antibody, whereas the ELISA was less sensitive. In persistent infection, higher titers were recorded with ELISA. Since murine cytomegalovirus has been shown to exert significant effects on the immune response of infected mice, this antigen should be included routinely in viral antibody screening programs.     

Enzyme-linked immunosorbent assay for the detection of toxoplasmosis in swine: interpreting assay results and comparing with other serologic tests

An enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of swine toxoplasmosis. The results of the ELISA were interpreted as percentage (% ELISA) and ELISA titer and were compared with results of the dye test, indirect fluorescent antibody test, and indirect hemagglutination test. The results obtained from both the % ELISA and ELISA titer directly correlated with the dye test results. The ELISA is a rapid, sensitive, and readily performed serologic test that should be ideal for examining (screening) swine for toxoplasmosis.     

Comparison of the enzyme-linked immunosorbent assay and complement fixation test for detecting Brucella ovis antibodies in sheep

A solid-phase, indirect, enzyme-linked immunosorbent assay (ELISA) was compared with the microtitre complement fixation test for detecting Brucella ovis antibodies in 220 ram sera. The ELISA was more sensitive than the complement fixation test; it demonstrated antibodies in 11 sera from known infected or vaccinated rams that were complement fixation test negative. No false positives were recorded with the ELISA and, in 36 sera positive to both tests, the ELISA titres were consistently higher than the corresponding complement fixation test titres.     

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of anti-chlamydial antibodies in pig sera

The evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against chlamydiae in pig sera is described. The most widely used serological test is the complement fixation test (CFT). The CFT has a lack of sensitivity and specificity because of low antibody titers and unspecific reactions. Eight conventionally raised pigs were exposed to a pathogenic strain of Chlamydia suis, four controls were mock infected. The immune responses was monitored by CFT and indirect ELISA. There was no agreement between CFT and ELISA data. These results were confirmed by a study with 191 sera from nine pig farms. As shown by ELISA and PCR chlamydiae are widespread in swine.     

Detection of pseudorabies virus antibodies: time of appearance by two serotests

This report describes a time-course comparison of detection of pseudorabies virus antibodies in experimentally infected swine by the virus-neutralization (VN) and indirect hemagglutination tests. Specific antibody titers were observed by the IHA test at 5 days after swine were inoculated, but not until 12 days by the VN test. The predominant immunoglobulin (Ig) class present in the serums of the swine at 5 and 7 days after inoculation was IgM, as determined by sulfhydryl reductions. The VN test lacked sensitivity to early Ig levels (IgM) in these experimentally infected swine, while the indirect hemagglutination test was highly sensitive to these same levels. On the basis of these results, it is possible that the VN test may read early infections as pseudorabies virus negative, due to the low presence of IgG in these samples.     

An enzyme-linked immunosorbent assay for detection of antibodies against Escherichia coli: association between indirect hemagglutination test and survival

An enzyme-linked immunosorbent assay (ELISA) was modified for detection of antibodies against the two main pathogenic serotypes of Escherichia coli: serotypes O78:K80 and O2:K1. The ELISA was a more sensitive and repeatable test than the indirect hemagglutination test (IHT), which is a common method for detecting antibodies against E. coli. Cross-reactivity between the two strains was measured by reacting antisera of each serotype against homologous and heterologous antigens. The results suggest that aside from similar determinants expressed by the two serotypes, serotype O2:K1 expresses more strain-specific determinants than does O78:K80. Comparison of mean antibody titers of immunized chicks by IHT and ELISA along the primary response revealed that during the first 15 days after immunization with inactivated E. coli, the titers in both tests were parallel. After 15 days post-immunization, antibody titers measured by IHT decreased rapidly, whereas titers measured by ELISA decreased only slightly. In addition, a higher correlation was found between titers detected by ELISA and survival through challenge with E. coli than between titers detected with IHT and survival through challenge. The results suggest that the ELISA is a better test for detection of antibody in flocks suspected of being infected with E. coli.     

猪传染性胸膜肺炎研究进展

猪传染性胸膜肺炎(PCP)已成为危害各国养猪业的主要传染病之一,其病原体胸膜肺炎放线杆菌主要定居在猪的呼吸道,有较高的宿主特异性。国内外已建立起补体结合试验、酶联免疫吸附试验、乳胶凝集试验、间接荧光抗体技术等用于PCP的检测和流行病学研究。该文就其疫苗的研制、分类、应用等进行概述。     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.     

猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立

猪肺炎支原体是猪喘气病的病原体,本研究选择猪肺炎支原体P46膜蛋白基因亲水区序列进行克隆,并将其内3个编码Trp的TGA突变成TGG,然后再克隆到pET28a(+)载体中,在大肠杆菌BL21 (DE3)细胞内实现了高效表达,表达产物相对分子质量约为31 ku,约占菌体总蛋白35％,表达形式为包涵体,通过Western blotting 证明表达产物与猪肺炎支原体高免血清具有很好的反应原性和特异性.将大肠杆菌表达的猪肺炎支原体P46重组蛋白经过洗涤、过柱纯化后,作为间接ELISA包被抗原用于检测猪血清中猪肺炎支原体抗体,通过对各参数和试剂的优化建立了rP46-ELISA方法,获得了较好的效果,通过与现有ELISA检测方法的比较,结果表明二者间具有较高的符合率.     

A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and reproducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.     

Identification of Mycoplasma hyopneumoniae in formalin-fixed porcine lung, using an indirect immunoperoxidase method

An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae.     

猪金属硫蛋白抗体的制备及应用

以自猪肝提取的Zn—MT与BSA的偶联物为抗原，免疫昆明小鼠制备了多克隆抗体。获得的抗体经鉴定后用于建立检测猪MT的间接竞争EI。ISA。结果表明：制备的IgG达到电泳纯，是猪MT的特异性抗体。所建立的间接竞争ELISA的灵敏度为4．1ng／mL，批内变异系数是9．976％，批间变异系数为14．3858％，可用于猪体MT的定性、定量测定。     

Enzyme-linked immunosorbent assay for the detection of porcine epidemic diarrhea coronavirus antibodies in swine sera

An enzyme-linked immunosorbent assay (ELISA) for detecting serum antibodies to the porcine epidemic diarrhea coronavirus (PEDV) was established by using cell culture-grown PEDV as antigen for coating. Ultracentrifugation through 20 and 45% (w/w) sucrose cushions proved to be the best antigen purification method. Examination of 1024 swine sera showed a high specificity and a greater sensitivity of the ELISA, when compared with indirect immunofluorescence. Reference sera with high antibody titers to PEDV originated from two pigs experimentally infected with PEDV. Three different antigen purification methods and the advantages of the ELISA compared with an immunofluorescence test are discussed.