The quantitative estimation of antibody to Pasteurella haemolytica in sheep sera using a micro-enzyme-linked immunosorbent assay (ELISA)

The standardization of a micro-method of enzyme-linked immunosorbent assay (ELISA) coupled to a flow system of reading is described for the quantitation of antibodies to Pasteurella haemolytica in sheep. This indirect ELISA is simple to perform, highly sensitive and possesses a degree of reproducibility superior to more commonly used tests. A capsular extract of P. haemolytica biotype A serotype 1 was adsorbed to the surface of microplate wells and levels of specific immunoglobulin G (IgG) in test sera were assayed using a pig anti-sheep IgG serum conjugated with alkaline phosphatase. After reaction of the enzyme, substrate colour intensities in individual wells were measured using a “flow-through” system resulting in accurate, objective assessments.     

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.     

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.     

Latest developments in the enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assays (ELISAs) for the serologic detection of both antigen and antibody in monitoring programs of commercial poultry flocks have begun to be recognized as an improvement over more conventional diagnostic procedures. The feasibility of employing double-antibody sandwich assays for the detection of virus without prior isolation of virus has been demonstrated and shows promise as the method of choice for the detection of lymphoid leukosis virus shedding. The versatility of indirect ELISA for the measurement of antibody induced by a wide variety of potential pathogens using a single basic overlapping ELISA system has also been demonstrated. It shows potential as a likely candidate to replace some of the more costly and time-consuming or less sensitive conventional serologic methods that do not overlap. Although some aspects of the two major types of immunoassays currently used in poultry health may need some modifications or improvements before delivery for routine use, it is likely that the use of computer-assisted ELISA will gain increased acceptance and use as the preferred way to efficiently and accurately monitor the health of poultry flocks on a broad scale.     

Infection of specific-pathogen-free lambs with parainfluenza virus type 3, Pasteurella haemolytica and Mycoplasma ovipneumoniae

Groups of specific-pathogen-free lambs were inoculated with combinations of parainfluenza virus type 3 (PI₃), Pasteurella haemolytica (P.h.) and Mycoplasma ovipneumoniae ( (M.o.). Acute, necrotising bronchopneumonia developed in 8/9 lambs inoculated with PI₃ followed by P.h. whereas only 1/5 lambs inoculated with PI₃ followed by a combination of M.o. and P.h. developed a pneumonic lesion. When M.o. was inoculated 29 days before PI₃ and P.h., pneumonia developed in 3/4 lambs but M.o. was not reisolated from any of the lungs. Pneumonia was observed in 1/5 lambs inoculated with P.h. alone and in 1/5 inoculated with M.o. plus P.h. In addition, one lamb in the latter group died of acute septicaemic pasteurellosis. None of the lambs inoculated with M.o. alone, PI₃ alone or PI₃ followed by M.o. had any gross or microscopic evidence of pneumonia although the virus alone, or in combination, did produce minor pulmonary lesions.These data suggest that M.o. is not an important primary or secondary lung pathogen.     

Detection of IgM antibodies against canine distemper virus in dog and mink sera employing enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against canine distemper virus (CDV) in canine and mink serum is described. The diagnostic potential of this technique was evaluated by analyzing sera from natural or experimental infections in dog and mink and negative control sera. These results were compared with results obtained in the developed CDV IgG ELISA and in the virus neutralization test. The IgM test, which requires only a single serum specimen, is a useful method for diagnosing current or recent CDV infections in dog and mink.     

Serodiagnosis of porcine cysticercosis by enzyme-linked immunosorbent assay (ELISA) using fractionated antigens

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Taenia solium cysticercosis was evaluated in experimentally and naturally infected pigs, using T. solium larval scoleces and its fractionated 1st and 2nd peaks on Sephadex G-200 as antigens. First peak antigen gave maximum sensitivity and highest antibody titres. The overall sensitivity of this test was found to be 91.5, 95.8 and 70.8% with scolex, 1st and 2nd peak antigens, respectively. False positive reactions occurred in 9.09% of uninfected pigs with scolex and 1st peak antigens and cross-reactions occurred in 25% of Taenia hydatigena-infected animals using scolex and 2nd peak antigens. No cross-reaction was observed using 1st peak antigen. The specificity of the test was 92.3, 96.2 and 92.3% with scolex, 1st and 2nd peak antigens, respectively.     

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (mvv) in sheep is described, in which microtitre plates are with a partly purified preparation of mvv. The antibodies bound are detected by a horseradish peroxidase conjugate.The results obtained with ELISA on a total of 493 serum samples from several commercial flocks were compared to those of a routine agar gel precipitation test (AGPT) and a complement fixation test (CFT).All samples which scored positive in AGPT, CFT or both (20.8%) were also found positive by ELISA. In addition, with ELISA a further 11.5% of the samples were positive. Serum samples from maedi-free flocks, from sheep suffering from sheep pulmonary adenomatosis and from lambs immunized against other viruses were all negative by ELISA. The assay has been used routinely for some years and proved to be specific, sensitive and suited for screening of large numbers of serum samples.     

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.     

Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona

Monoclonal antibodies (mAb) were produced by fusing Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c and ND4 mice that were immunized with killed Leptospira interrogans serovar pomona whole cells. Thirty hybridomas which produced antibodies (of the IgG1, IgG2a, IgG2b, or IgG3 isotype) that bound to epitopes on the serovar pomona whole cell antigen were identified by an indirect enzyme-linked immunosorbent assay (ELISA). Twenty-eight of these 30 mAbs cross-reacted in the indirect ELISA with at least one whole cell antigen prepared from 12 other pathogenic Leptospira serovars, and/or with whole cell antigen from the non-pathogenic Leptospira biflexa serovar patoc. The two serovar pomona-specific mAbs, which were designated M897 and M898, were obtained from the ND4 mouse and were both of the IgG1 isotype. In competitive ELISAs, M897 and M898 were inhibited from binding to the pomona antigen by bovine sera with anti-serovar pomona microscopic agglutination test (MAT) titres ranging from 100 to 6400. No significant inhibition was observed with pomona MAT-negative sera or with sera from animals experimentally infected with serovars canicola, copenhageni, grippotyphosa, hardjo type hardjobovis or sejroe. The epitopes recognized by M897 and M898 were both highly susceptible to sodium meta-periodate oxidation, indicating a carbohydrate composition. Neither of these mAbs reacted in immunoblots with the separated components of the serovar pomona whole cell antigen.     

An indirect enzyme-linked immunosorbent assay (ELISA) for measuring antibodies in chickens infected with infectious bursal disease virus

An immuno-enzyme assay for measuring infectious bursal disease antibodies in chickens is described. The test is performed rapidly after coating plates overnight with partially purified antigen prepared in cell culture. Coated plates can be stored for at last 4 months. The chromatographically purified rabbit anti-chicken immunoglobulin-G, conjugated to horseradish peroxidase, was used optimally at a dilution of 1:3,000. It could be stored for at least 10 months without a reduction in titer. The test is safe, highly reproducible, specific, and sensitive. Results can be read visually or by spectrophotometry. Antibodies could be detected as early as 4 days postinfection. Serum titers rose rapidly to high levels, ranging from 1:1,600 to 1:25,600 by one week postinfection. High titers persisted for up to one year. The results of this assay compare favorably with results obtained with the agar-gel precipitin and virus-neutralization tests.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.     

Comparison of the enzyme-linked immunosorbent assay (ELISA) with three other methods for the detection of Trichinella spiralis infections in pigs

Four methods employed in the diagnosis of trichinellosis [trichinoscopy, digestion method, immunofluorescence techniques and Enzyme-Linked Immunosorbent Assay (ELISA)] were compared by laboratories in eight countries of the European Economic Community. Material from 24 pigs infected with 10 000, 5000, 500, 150 and 0 T. spiralis larvae was examined during a period from 17 days to 12 weeks post infection. ELISA was more sensitive than immunofluorescence during the onser of the infection in groups in infected with higher numberts of larvae (1500, 5000 and 10 000 larvae). In general, however, results of both ELISA and immunofluorescence were comparable with regard to reliability. In pigs infected with a lower number of T. spiralis larvae both serological assays were more sensitive than the direct methods (trichinoscopy and digestion method).It was concluded that not enough evidence was available to suggest ELISA as an alternative to the direct methods for slaughterhouse control. Both the ELISA and the immunofluorescence technique may prove to be applicable for epidemiological surveys.