Use of an enzyme-linked immunosorbent assay for the detection of IgG antibodies to rinderpest virus in epidemiological surveys

A comparison was made between an indirect enzyme-linked immunosorbent assay (ELISA) and the virus neutralisation test (VNT) for the detection of antibodies to rinderpest virus in field sera. The results did not agree for 6 per cent of the sera tested where 3 per cent of the samples gave ELISA positive/VNT negative and 3 per cent gave ELISA negative/VNT positive. The latter sera all had high levels of IgM antibody, which may indicate animals being at an early stage of infection or detection of a non-specific reaction. The ELISA results give a representative picture of the immune status for field surveys and a greater number of sera can be assayed with relative ease, compared to the traditional serum neutralisation test.     

Pestiviral infections in sheep and pigs in Northern Ireland

Serological surveys were carried out to determine the prevalence of pestiviral infections in sheep and pigs in Northern Ireland. Sera from 918 ewes in 92 flocks from 10 regions were tested by ELISA for antibodies to border disease virus and positive results were obtained from 49 ewes (5.3 per cent) in 28 flocks (30.4 per cent). There were highly significant geographical variations in its flock prevalence ranging from 0 per cent in the Enniskillen region to 70 per cent in the Coleraine region. There was no significant association between the proportion of seropositive flocks and the presence of cattle on the farm (P = 0.583). In the positive flocks, the average rate of seroprevalence was 17.5 per cent, and the highest was 40 per cent. Comparative neutralisation studies on 14 positive sera with bovine viral diarrhoea virus type I (BVDV I) and border disease virus revealed higher titres (> or = four-fold) to BVDV I in all cases. Only one positive result was obtained when fluids from 186 aborted ovine fetuses were tested for border disease virus by ELISA. Serum samples from 680 pigs in 46 herds were tested for virus neutralising antibodies to border disease virus. Twenty sera (2.9 per cent) were cytotoxic, and only one of the remaining 660 sera gave a positive result. This sample tested negative for classical swine fever by ELISA, and comparative neutralisation studies showed that it had a four-fold higher titre to BVDV I than to border disease virus.     

An enzyme-linked immunosorbent assay for the detection of antibody to avian reovirus by using protein sigma B as the coating antigen

An enzyme-linked immunosorbent assay using the expressed protein sigma B as the coating antigen (sigma B-ELISA) for detecting antibody to avian reovirus (ARV) in chickens was developed and compared with a conventional ELISA. Both ELISA s and a serum neutralisation (SN) test were used to test the sera from experimentally vaccinated and farm chickens. The sigma B-ELISA could clearly distinguish the SN-positive and -negative sera in 38-week-old chickens. The correlation rate between SN and a sigma B-ELISA was 100 per cent (65/65), and that between SN and conventional ELISA was 84 per cent (55/65). With the sigma B-ELISA, all SN-negative sera had low absorbance values (below 0.06), and the absorbance values correlated closely with the SN titres. However, the sera which were antibody-negative by SN had various absorbance values, ranging from 0.07 to 0.39 in the conventional ELISA. Hence, the sigma B-ELISA had lower non-specific binding reactions than the conventional ELISA against sera from ARV -negative birds. Antibody against ARV could be detected by sigma B-ELISA after vaccination. Absorbance values peaked 4 weeks after vaccination at 2 weeks of age and were maintained until the birds were 27 weeks old. The results suggest that the presence of antibody against viral protein sigma B in birds may be used as a good indicator by the sigma B - ELISA for detecting immune status of a chicken flock or to detect chickens infected with ARV.     

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological studies of transmissible gastroenteritis in Great Britain, using a competitive ELISA.

A competitive ELISA which differentiates between transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was used to detect non-neutralising antibodies to the peplomer protein of TGEV in porcine sera. The test was shown to be TGEV specific, having a relative specificity of 100 per cent, and to have a relative sensitivity of 94.9 per cent when compared with the virus neutralisation test. The prevalence of TGEV in Great Britain is low; only 0.6 per cent of sows sampled in 1990 were seropositive to TGEV. Seroconversion to the virus neutralisation test occurred in a closed herd in 1984, with no apparent spread, but later testing by the ELISA did not detect any blocking antibodies. The possibility of the existence of a less contagious strain of PRCV is discussed. All British isolates of TGEV tested by the indirect fluorescent antibody test were recognised by the monoclonal antibody 1D.B12, the indicator antibody in the ELISA.     

Detection of canine distemper virus antigen in canine serum and its application to diagnosis

Canine distemper virus (CDV) antigen was detected in the serum of dogs by an ELISA and the results of this assay were compared with an anti-CDV immunoglobulin M (IgM) antibody test. In paired sera from 26 naturally infected dogs, the antigen-positive rate was 26.9 per cent at the first examination and 11.5 per cent at the second examination two to three weeks later. The antigen was detected in three of the 10 dogs which were negative for anti-CDV IgM antibody at the first examination. It could also be detected in the serum of between eight and two of 40 specific pathogen-free dogs vaccinated against CDV, for up to four weeks after they were vaccinated.     

Detection of antibodies to Borna disease virus in Turkish cats by using recombinant p40.

Recombinant p40 produced by baculovirus was used in an ELISA to screen samples of serum taken from 80 cats in Istanbul. The sera were also analysed for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Antibodies to Borna disease virus- (BDV) p40 were detected in 34 (42-5 per cent) of the 80 cats. Seventy-three per cent of the sera which were positive for FIV and 26 per cent of the sera which were negative for FIV had antibodies to BDV. There was no difference in the percentage of sera which were positive for BDV between the cats that were positive or negative for FeLV. Three of the cats had neurological disease and two of these had antibodies to BDV. Six sera with low, medium or high optical densities (ODS) by ELISA were analysed by Western blotting. Only the sera with medium and high ODS reacted specifically with p40 at a dilution of 1 in 1,000.     

Serological survey of encephalomyocarditis virus infection in pigs in France

Samples of serum from 76 gilts, 1440 sows, 1473 piglets and 3093 finishing pigs from 96 farrow-to-finish herds were tested for antibodies to encephalomyocarditis virus (EMCV) in microtitre serum neutralisation tests employing two strains of virus, one associated with myocarditis and the other with reproductive failure. The total seroprevalence of EMCV infection was 2.48 per cent. There was no significant difference between the seroprevalence of the reproductive failure strain (1.6 per cent) and the myocardial strain (1.85 per cent). The seroprevalence was higher in the gilts (6.57 per cent) and sows (5.13 per cent) than in the piglets (1 per cent) and finishing pigs (1.84 per cent), and the highest titres were observed in the sows (1:540) and finishing pigs (1:640). In the gilts, the difference in seroprevalence between the reproductive failure strain (3.95 per cent) and the myocardial strain (5.33 per cent) was wider than in the other groups.     

Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders

The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard test. Highest sensitivity was shown for the CIDC antigen (100 per cent) followed by the c.c.pro antigen (99.39 per cent). However, the USDA antigen showed substantially less (p<0.05) sensitivity (62.19 per cent). Highest specificity was found for the USDA antigen (100 per cent) followed by the CIDC (97.5 per cent) and c.c.pro antigen (96.5 per cent). Positive and negative predictive values (assumed glanders prevalence of <0.1 per cent) for each antigen were calculated to be 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33 per cent (USDA), respectively. Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB.     

Comparison of the enzyme-linked immunosorbent assay (ELISA) and serum neutralisation test for the serodiagnosis of Aujeszky's disease

The serum neutralisation test (SNT) and the indirect enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease were compared, utilising 3202 sera from Aujeszky's disease free pig herds, and 304 SNT reactor and 245 non-reactor sera from Aujeszky's disease infected piggeries. ELISA was found to give good discrimination between positive and negative sera, results showing 96.9% and 99.7% agreement with the SNT in classifying positive and negative reactor pigs respectively. The ELISA appeared to detect a slightly higher proportion of reactors than did the SNT. Absorbance values obtained with ELISA showed a high degree of overall correlation with SW titres (r = 0.916), though correlations were lower when applied to individual sera. The ELISA was considered to be a rapid and convenient procedure, offering many advantages over the SNT for routine use.     

Detection of foot-and-mouth disease antigen in bovine epithelial samples: comparison of sites of sample collection by an enzyme linked immunosorbent assay (ELISA) and complement fixation test

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.     

牛副结核ELISA诊断方法研究

本文建立了牛副结核病ELISA诊断方法。采用亲和层析抗原。被检血清以高压粉碎草分枝杆菌吸收原进行吸收。该方法的敏感性76％,特异性97％,通过对2483头奶牛进行检测,检出率为6.1％,并与常规的补反和变态反应进行了比较。作者认为:牛副结核ELISA可以替代补反,做为检测牛副结核的一种有力手段。     

Detection of antibodies against infectious bursal disease virus: a comparison of three serological methods

Four different oil emulsion infectious bursal disease virus (IBDV) vaccines were inoculated into four-week-old specific pathogen-free chickens. At weekly intervals for five weeks, sera were obtained from the vaccinated birds and from uninoculated control birds and examined for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA), the quantitative agar gel precipitin (QAGP) test and the virus neutralisation (VN) test. There was a highly significant correlation between the mean responses to all tests; the highest correlation (0.818) was between VN and QAGP and the lowest (0.573) between QAGP and ELISA. Generally the ELISA detected positive sera earlier than the VN test which in turn was more sensitive than the QAGP test. The ELISA and QAGP test were less variable, more reproducible and easier to perform than the VN test.     

An enzyme-linked immunosorbent assay for enzootic bovine leukosis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to enzootic bovine leukosis (EBL) virus is described and its sensitivity compared with that of the agar gel immunodiffusion test (AGIDT) using 198 sera collected in Great Britain. There was 95 per cent agreement between the ELISA and AGIDT, when sera with positive/negative ratio (P/N) values of 1 . 5 or greater were considered positive. A total of 259 out of 264 sera (98 per cent) collected in Northern Ireland had P/N values of less than 1 . 5, the remaining sera having P/N values of 1 . 5 and 1 . 6. As Northern Ireland is clinically and serologically free of EBL infection it is proposed that sera with P/N values of 1 . 5 and 1 . 6, which account for approximately 3.5 per cent of the total sera tested, are considered doubtful and should be tested by another serological test.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

Diagnostic investigation into the role of Chlamydiae in cases of increased rates of return to oestrus in pigs

Cervical swabs and serum samples were taken from Swiss herds of sows with high rates of irregular return to oestrus (group A) and from control herds without reproductive problems (group B. The genital tracts of 21 slaughtered sows of group A were also examined. The swabs and genital tracts were screened for Chlamydiae by a new 16S rRNA PCR and the sera by an ELISA for Chlamydiaceae lipopolysaccharide. Chlamydophila (Cp) abortus was isolated from seven of the 65 swabs taken from group A but from none of the 128 swabs taken from group B. Chlamydia suis was present in swabs from both groups A (1.5 per cent) and B (2.3 per cent). In addition, Cp abortus was detected in 33.3 per cent of the genital tracts. Of the 193 sera tested, 61.7 per cent were positive, with no significant difference between group A (52.3 per cent) and group B (66.4 per cent). Chlamydia-like organisms were detected in 28.2 per cent of the swabs from group A and in 22 per cent of those from group B.     

Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.     

Evidence of Brucella infection in marine mammals in the North Atlantic Ocean.

Between 1983 and 1996 a total of 1386 samples of serum were taken from four species of seal and three species of whale in the waters west of Iceland, the area of pack-ice north-west of Jan Mayen, the northern coast of Norway and the Kola Peninsula, the waters west of Svalbard, and the Barents Sea; they were tested for the presence of anti-Brucella antibodies with an indirect ELISA (protein G conjugate). The positive sera were re-tested with classical brucellosis serological tests, such as the serum agglutination test, the EDTA-modified serum agglutination test, the Rose Bengal test, and the complement fixation test, as well as an anti-complement ELISA. Anti-Brucella antibodies were detected in all the species investigated, except for the bearded seal (Erignathus barbatus), with the following prevalences: hooded seals (Cystophora cristata) 35 per cent; harp seals (Phoca groenlandica) 2 per cent; ringed seals (Phoca hispida) 10 per cent; minke whales (Balaenoptera acutorostrata) 8 per cent; fin whales (Balaenoptera physalus) 11 per cent; and sei whales (Balaenoptera borealis) 14 per cent. An isolate belonging to the genus Brucella was obtained from the liver and spleen of one of the seropositive minke whales. The findings suggest that antibodies against the surface lipopolysaccharide of Brucella species are widely distributed among marine mammals in the North Atlantic Ocean.     

Detection of antibodies to Eperythrozoon ovis by the use of an enzyme-linked immunosorbent assay

Specific antibody to Eperythrozoon ovis was detected by an enzyme-linked immunosorbent assay (ELISA) in the sera of infected sheep. In the presence of parasite antigen, positive control serum showed a reaction approximately eight times that of negative serum. When compared to an immunofluorescent antibody test (IFAT), the ELISA was eight times more sensitive. Positive control sera gave a titre of 1:3200 by IFAT and 1:25,600 by ELISA. Through the use of a reference titration curve ELISA could be used as a semi-quantitative system to determine antibody levels in test sera.