Application of latex beads agglutination test for the detection of the antibody against virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus

Latex beads agglutination (LA) for the detection of the antibody against virus infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus was estimated using experimentally infected animals. The VIA antibody titer by the LA test were compared with the neutralization titer and the titer by agarose gel diffusion (AGD) test, which has been used as a standard method for VIA antibody titration. The latex beads were coated with VIA antigen in carbonate buffer solution (0.5 M, pH 9.6) for the test. The sensitivity of the LA test was clearly higher than that of the AGD test in the results for cattle and swine infected experimentally. The antibody was detected in the bovine serum obtained at the 13th week after inoculation by the LA but not by the AGD test. The LA test appears to be simple, rapid and sensitive for the detection of the antibody of FMD virus in the surveillance of FMD and the FMD quarantine of imported animals.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

Detection of foot-and-mouth disease virus infection in vaccinated cattle

The aim of this study was to evaluate the value of commercially available kits for the detection of foot-and-mouth disease (FMD) virus infection in vaccinated cattle. The cattle were vaccinated with a commercial aqueous FMD vaccine type A24 and subsequently challenged 28 days post vaccination with homologous FMD virus. Seven of eight animals were protected from clinical disease and all became carriers. They were bled sequentially for up to 130 days post infection and samples of sera were tested with three ELISA kits: CHEKIT FMD-3ABC, Ceditest FMDV-NS and SVANOIR FMDV 3ABC-Ab ELISA. The Ceditest kit appears to be relatively higher sensitive than the others. When examined with this ELISA, all cattle developed of FMDV nonstructural proteins (NSPs) antibodies and remained positive throughout the period of the experiment. The response of antibodies against 3ABC antigen delayed in two cattle challenged with FMDV A24 virus. One of the cattle reacted negatively in Svanoir ELISA kit and sera from two animals were found negative in CHEKIT ELISA. It can be concluded that all tested kits can be a promising tool for FMD control and eradication campaigns in situation where emergency vaccination was applied.     

Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.     

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.     

Prevalence of seroreagents to FMDV in the cattle population in Poland: results of 9-year monitoring studies

The aim of this study was to evaluate the occurrence of antibodies to foot-and-mouth disease virus (FMDV) in sera of cattle in Poland. The examinations were performed using the virus neutralization (VN) test and ELISA methods: liquid-phase blocking ELISA (LPBE) and 3ABC-ELISA. During 1993-2001, about 681,000 samples of sera collected from animals held on the territory of Poland were tested. Of about 600,000 sera taken from animals exported to the European Union, 963 samples (0.16%) were found to be positive to FMDV types A, O and/or C. During 1996-2001 out of 85,000 sera tested as part of the national serological surveillance program for FMD, the FMDV antibodies were recorded in 51 (0.06%) samples. Persistence of FMD antibodies was observed in sera of cattle from the region around Zduńska Wola, which had been vaccinated annually during 1985-1985 with trivalent FMD vaccine. The results of the serological studies of 550 animals from this region indicates the presence of FMDV antibodies in sera of 240 (44%) cattle. A half-life of maternal antibodies in sera of calves born to seropositive dams was estimated; the highest level of FMDV antibodies was detected in sera taken from new-born calves aged 5-10 days. The level of FMDV antibodies in beestings of dams was highest during the first 10 hours after parturition; after 24 hours a significant decrease (3-5 times) was found and in two weeks post parturition FMDV antibodies were undetectable in the milk. It was established that all LPBE/VN positive sera of cattle exported from Poland, from the vaccination zone around Zduńska Wola as well as those tested as part of the national serological surveillance program for FMD, were taken exclusively from vaccinated animals or calves born to vaccinated dams.     

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.     

Indirect immunofluorescence and immunodiffusion tests in the detection of antibodies to foot-and-mouth disease virus

The antibody response detected by indirect immunofluorescence (IIF) as well as that directed against 140 S and virus infection associated antigen (VIA), as detected by agar immunodiffusion, was studied in three mammal species susceptible to Foot and Mouth Disease Virus, after challenge with living virus, immunization and hyperimmunization with inactivated virus, and immunization followed by challenge. By spot indirect immunofluorescence, antibodies were detected only in animals undergoing an active infection, and were not detected in immunized or hyperimmunized animals. This behaviour was similar to that of the anti-VIA antibodies in the same groups of animals and differed from that of anti-140 S antibodies. It appeared that spot indirect immunofluorescence for the detection of VIA antigen is comparable to the immunodiffusion test, but the speed of IIF and the possibility of handling many samples make it more practical.     

Une technique ELISA pour la detection des anticorps anti-virus maedi-visna. Etude comparative avec l'immunodiffusion en gelose et la fixation du complementAn ELISA test for detection of maedi-visna antibodies. Comparative study with gel immunodiffusion and complement-fixation test

An indirect microELISA test was performed for detection of maedi-visna antibodies in ovine and caprine species. The antigen consisted in viral particles, highly purified by successive ultracentrifugations.

By comparative testing of 934 sera in ELISA and gel immunodiffusion, we found a good correlation between these two te ts, and moreover, ELISA revealed another 11.3% of positive samples. The precocity of this ELISA was shown by experimental infection of sheep with different strains of maedi-visna: positive sera were detected 7 weeks post-infection, instead 4–5 months with gel immunodiffusion. The complement fixation test was compared with gel immunodiffusion and was found the less sensitive.

This ELISA test appeared to be satisfactory, and may be used for early diagnosis of maedi-visna infection.     

Une technique ELISA pour la detection des anticorps anti-virus maedi-visna. Etude comparative avec l'immunodiffusion en gelose et la fixation du complement

An indirect microELISA test was performed for detection of maedi-visna antibodies in ovine and caprine species. The antigen consisted in viral particles, highly purified by successive ultracentrifugations.By comparative testing of 934 sera in ELISA and gel immunodiffusion, we found a good correlation between these two teṡts, and moreover, ELISA revealed another 11.3% of positive samples. The precocity of this ELISA was shown by experimental infection of sheep with different strains of maedi-visna: positive sera were detected 7 weeks post-infection, instead 4–5 months with gel immunodiffusion. The complement fixation test was compared with gel immunodiffusion and was found the less sensitive.This ELISA test appeared to be satisfactory, and may be used for early diagnosis of maedi-visna infection.     

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia antibody to purified P26 viral protein

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine infectious anemia (EIA) antibody in horse sera. Purified P26 viral protein was the antigen; alkaline phosphatase linked to rabbit anti-horse immunoglobulin G was the conjugate. The ELISA detected EIA antibodies in horse sera as early as 11 to 14 days after experimental inoculations. There was full agreement between the results of ELISA and the agar-gel immunodiffusion tests on EIA proficiency test sera. The ELISA readily detected EIA antibody in horse sera that had weak positive reactions on agar-gel immunodiffusion.     

Evaluation of humoral immunity to Brucella sp in cattle by use of an agar-gel immunodiffusion test containing a polysaccharide antigen

Results of a double agar gel immunodiffusion (Ouchterlony) test that contained a polysaccharide (poly-B) antigen of Brucella melitensis strain B115 were compared with those of 5 other serotests. To determine the sensitivity and specificity of the immunodiffusion, standard tube, 2-mercaptoethanol, Rivanol, card, and complement fixation tests, sera obtained from 1,328 vaccinated, infected, and seronegative cattle, 56 of which had been examined bacteriologically, were used to evaluate the humoral response to Brucella sp. The poly-B antigen confirmed infection in 87.5% of the 56 cattle from which Brucella abortus biotype 1 had been isolated, and in 96.6% (205/212) of a group of cattle suspected to be infected on the basis of results of conventional serotests. Likewise, sera from 4 groups of vaccinated cattle did not react with poly-B antigen, whereas they did react in conventional tests. The poly-B antigen was more specific in detecting infected cattle even in a group of vaccinated adults. A useful strategy to identify infected cattle might be screening, using a combination of the Rivanol and card tests together with the agar-gel immunodiffusion test containing poly-B antigen.     

Serological evidence of FMD subclinical infection in sheep population during the 1999 epidemic in Morocco.

During 1999, 11 outbreaks of foot and mouth disease (FMD) were declared in the east and central part of Morocco. All the FMD clinical cases reported were cattle. In order to analyse the serological status of sheep from the FMD outbreak areas, 598 sheep sera were tested using a liquid-phase blocking ELISA (LPBE) to detect antibodies against FMDV structural proteins. The study confirmed the presence of FMDV specific antibodies in 77 clinically normal sheep, indicating that unrecognised FMDV-infected sheep could represent a potential risk of FMD dissemination in Morocco.Subsequently, sera from flocks of sheep that had been exposed to FMD outbreaks were assayed by an indirect ELISA using the recombinant FMDV non-structural protein 3ABC expressed in E. coli to evaluate the potential use of this serological test in future epidemiological studies and the development of FMD control strategies. The results indicated that the 3ABC-ELISA was able to detect antibodies indicative of infection with FMDV in asymptomatic sheep in field conditions.     

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.     

Development of solid phase antigen for indirect ELISA for the detection of specific antibody responses to infection with Newcastle disease virus

A simple and inexpensive method of antigen preparation by ultrafiltration was investigated using the V4 strain of Newcastle disease virus. The antigen designated XM300 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Newcastle disease virus in chicken serum. The assay was evaluated using both experimental and field sera, as well as reference control reactor and non-reactor sera. Antigen prepared by the ultrafiltration method was compared with antigen prepared by ultracentrifugation and the ultrafiltration antigen was found to react specifically with Newcastle disease virus antiserum in this ELISA system. This antigen preparation technique is also suitable for use in developing countries. The ELISA provides an excellent method for measuring antibodies in the early stages of infection in serum samples from experimentally infected chickens. More than 14.58 % of the total serum samples which failed to be recognized as reactors by the conventional haemagglutination inhibition test were detected in the ELISA.     

An enzyme-linked immunosorbent assay for the detection of antibodies to avian reticuloendotheliosis virus using whole cell antigen

An enzyme-linked immunosorbent assay (ELISA) using reticuloendotheliosis virus-infected chick embryo fibroblasts as coating antigen is described for the detection of antibodies to reticuloendotheliosis virus in chicken sera. The ELISA was specific and during the early stages of infection more sensitive than an indirect fluorescent antibody test.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

Isolation and purification of a diagnostic antigen for bovine cysticercosis by hydrophobic chromatography

An ammonium sulfate fraction of Taenia hydatigena cyst fluid (ThFAS) was further fractionated by hydrophobic interaction chromatography, using alkylagarose and omega-amino alkylagarose columns, in an effort to isolate and purify a specific diagnostic antigen in the ThFAS preparation. The less than 12 kDa antigen was found to have an affinity for immobilized alkanes with chain length of six carbons or greater. The antigen was recovered in an ethylene glycol eluate from a hexylagarose column then analyzed by Western blot; it reacted with bovine and human cysticercosis infection sera and with specific monoclonal antibodies but not with control sera or fascioliasis infection sera. When the eluate was used as coating antigen in a plate ELISA assay no false positive reactions were seen in sera from cattle infected with Fasciola hepatica; false positive reactions were observed for the unfractionated ThFAS antigen preparation.     

Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus.

The O/Taiwan/99 foot-and-mouth disease virus (FMDV), a South Asian topotype of serotype O, was introduced into Taiwan in 1999. The Chinese yellow cattle infected by the virus did not develop clinical lesions under experimental and field conditions. A blocking enzyme-linked immunosorbent assay (ELISA) kit with the 3AB antigen, a polypeptide of FMDV non-structural (NS) proteins, was used to evaluate the development and duration of anti-3AB antibodies, proving active viral replication, in the Chinese yellow cattle. The specificity of the assay was 99%, as was established with negative sera from regularly vaccinated and from na?ve cattle. The sensitivity tested with sera from naturally infected animals was approximately 64% and it was lower than that obtained by serum neutralization (SN) test. Under experimental infection, the Chinese yellow cattle developed lower anti-3AB antibodies than that developed in other species. Duration of anti-3AB antibodies was traced in two herds of naturally infected animals, indicating that anti-3AB antibodies persisted for approximately 6 months after outbreaks. On the basis of this study, we propose that the Chinese yellow cattle may have natural resistance, which limits viral replication and reduces the development of anti-3AB antibodies.