Evaluation of four serological tests for the diagnosis of caprine melioidosis

A complement fixation (CF) test, 2 indirect haemagglutination (IHA-A; IHA-L) tests which differed in antigen preparation and technique, and a microtitre agglutination (MA) test were compared in the serodiagnosis of melioidosis in goats. One hundred and eighteen experimental serums and 3143 field serums from goats in endemic and non-endemic areas of north Queensland were used in the evaluation. Culture of samples for Pseudomonas pseudomallei from 112 goats provided substantiating evidence of infection. The IHA-A test was the most sensitive, and the CF test the most specific. We advocate the use of the IHA-A as a screening test followed by the CF test for confirmation of active melioidosis. The IHA-A test is the better indicator of past infection.     

Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.     

A microplate enzyme-linked immunorsorbent assay for measuring antibody to Anaplasma marginale in cattle serum

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.     

A STANDARDISED HAEMAGGLUTINATION INHIBITION TEST FOR PORCINE PARVOVIRUS ANTIBODY

Basic variables of the haemagglutination inhibition (HI) test for porcine parvovirus antibody were investigated. Nonspecific serum inhibitors were satisfactorily removed without loss of specific antibody when undiluted serum was adsorbed with 25 percent kaolin in borate saline at pH 9.0. Natural haemagglutinins in test serums could be completely removed using 0.1 ml of packed erythrocytes to 0.6 ml of kaolin treated serums. Adsorption of prediluted serum resulted in a depression of specific antibody titres. Highest HI titres were obtained using guinea pig erythrocytes, following incubation of virus-serum mixtures for 18 hours at 4 degrees C, 3 hours at 25 degrees C or 2 hours at 37 degrees C. Micro- and macro-tests gave comparable HI titres.     

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.     

Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections

An enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specific mouse monoclonal antibodies, which recognise different epitopes of the haemagglutinin of CPV and which also neutralise the virus. A double antibody sandwich (DAS) ELISA for the detection of CPV in dog faeces was compared with the haemagglutination (HA) test. The DAS-ELISA proved to be more specific, sensitive and easier to perform than the HA assay. An indirect ELISA and a competitive ELISA for the detection of CPV-specific antibodies in dog sera were compared with the haemagglutination inhibition (HI) test. Both ELISA systems proved to be specific and easy-to-use methods for the detection of CPV-specific antibodies. The indirect ELISA, specially, proved to be more sensitive than the HI test. The higher sensitivity and specificity of the ELISA's as compared to HA and HI tests, and their ease of use, make them suitable for routine use in the serology and diagnosis of CPV infections.     

Detection of fish antibody against protein antigen of Aeromonas salmonicida by enzyme-linked immunosorbent assay using biotin-avidin system

Antibody against Aeromonas salmonicida was detected in sera from immunised or experimentally infected rainbow trout by enzyme-linked immunosorbent assay (ELISA) using the biotin-avidin system. The ELISA titre correlated well with the agglutinin titres of the sera, but the ELISA was found to be more sensitive than the agglutination test. When the rainbow trout serum was separated by column chromatography, antibody activity (determined by ELISA and agglutination test) was detected in the IgM fractions. Minimum cross reaction was observed in the ELISA system between antigen prepared from A salmonicida and antibodies against Vibrio species and other species of Aeromonas. The specificity of the ELISA was also confirmed by inhibition test. Immunisation of rainbow trout with a virulent strain of A salmonicida provided good protection, though no correlation was observed between the protection and the ELISA titres of sera.     

Bovine rotavirus diagnosis: comparison of various antigens and serological tests

Agar gel immunodiffusion (AGID) and counter-immunoelectrophoresis (CIEP), complement fixation (CF), radio-immunoassay (RIA), haemagglutination (HA) and haemagglutination inhibition (HI) tests were compared in their efficiency for the detection of bovine rotavirus antigens and antibodies. As a test for antigen using hyperimmune serum, CIEP was found to have advantages over AGID by being more rapid as well as approximately four times more sensitive regardless of whether the antigen was of faecal or tissue culture origin. The CF test was more sensitive than either of the immunodiffusion procedures studied for antigen detection, but was more tedious to perform and of limited use as some faecal samples exhibited anti-complementary activity. For measurement of rotavirus antibody the radio-immunoassay (RIA) was the most sensitive technique and the CIEP least sensitive. Using the RIA a limited survey of cattle demonstrated that approximately 75% of the animals tested possessed specific antibody to rotavirus.     

Application of an enzyme-linked immunosorbent assay in the final stages of a bovine brucellosis eradication program

Bovine field serums from the Australian brucellosis eradication program were used to compare 2 enzyme linked immunosorbent assays (ELISA) with the complement fixation test (CFT) and Rose Bengal test (RBT). One ELISA used an anti-bovine IgG horseradish peroxidase conjugate (ELISA 1) and the other a monoclonal anti-bovine Ig alkaline phosphatase conjugate (ELISA 2). When compared with the CFT, the ELISA 2 like the ELISA 1 lacked specificity in B. abortus vaccinated herds but the ELISA 2 was more specific than the ELISA 1 in previously infected herds and equally as specific as the ELISA 1 in nonvaccinated Brucella free herds. In this study the ELISA 2 proved more sensitive than the CFT, RBT and ELISA 1 particularly in herds where B. abortus biotype 2 was present. The value of using the ELISA 2 in conjunction with the CFT in an eradication program is discussed.     

Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens

Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of parameters for detecting antibodies against infectious bronchitis virus using an enzyme-linked immunosorbent assay: temporal response to vaccination and challenge with live virus

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for avian infectious bronchitis virus (IBV) were evaluated and optimized. The use of purified IBV as antigen at 50 ng protein/well and high-ionic-strength serum dilution buffer has resulted in a test with minimal nonspecific binding of chicken immunoglobulins and very high sensitivity. Optimum conditions for serum dilution, conjugate dilution, and substrate incubation were determined for minimizing background and nonspecific reactions. The use of this test in a controlled challenge study with chickens vaccinated with live IBV demonstrated its effectiveness in monitoring circulating antibody levels to infectious bronchitis. The IBV ELISA, which is rapid, inexpensive, highly sensitive, and capable of handling very large numbers of samples, should provide the poultry industry with a reliable means for IBV flock monitoring.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine virus diarrhea virus in sera from border disease virus-infected sheep.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.     

Evaluation of the critical parameters of a sensitive ELISA test using purified infectious bovine rhinotracheitis virus antigens

An enzyme-linked immunosorbent assay (ELISA) for the survey or titration of bovine sera for the presence of IgG antibodies against infectious bovine rhinotracheitis (IBR) virus was developed. The optimal conditions of serum dilution, antigen concentration, conjugate dilution, substrate concentrations, and reaction time were established using the signal/ noise (S/N) ratio as the determining criterion. Equilibrium density gradient purified IBR virus was used as antigen at an optimal concentration of 0.60 μg/cuvette. The use of purified antigen allowed the testing of sera at a 1 : 10 dilution without nonspecific reaction.The conditions of conjugate dilution, substrate concentration and reaction time were shown to have significant effects on the ELISA test. Results from 35 sera showed this optimized ELISA procedure to be as much as 1000-fold more sensitive than the serum neutralization plaque reduction assay. Numerous sera showing no neutralizing titer to IBR virus were found to be positive when examined by this ELISA method.     

鹦鹉热嗜性衣原体感染SPF鸡和污染相关疫苗的初步调查

为初步调查SPF鸡感染鹦鹉热嗜性衣原体状况及相关SPF鸡胚源疫苗是否出现污染,本试验通过采集不同日龄的SPF鸡血清70份、SPF种蛋卵黄膜30份,收集市场上销售的SPF鸡胚源疫苗共41支,利用国产间接血凝试剂盒检测抗体,进口免疫荧光试剂盒分别测定其抗体、抗原阳性率,以评价SPF鸡鹦鹉热嗜性衣原体的流行状况和相关疫苗的污染状况。本试验结果显示,SPF鸡血清阳性率分别为31.4%(荧光法)、5.7%(间接血凝法);SPF种蛋阳性率33.3%,SPF鸡胚源疫苗平均阳性率31.7%。SPF鸡已经感染了鹦鹉热嗜性衣原体,且发现经鸡胚卵黄膜而传播病原的新途径,进而造成SPF鸡胚源疫苗出现衣原体污染。因此,加强SPF鸡鹦鹉热嗜性衣原体监测已势在必行。     

Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.     

Dot-enzyme linked immunosorbent assay for demonstration of Newcastle disease virus infection

Dot-enzyme linked immunosorbent assay (ELISA) was standardised to detect Newcastle disease virus (NDV) specific antigen in chicken tissues, embryos and allantoic fluid samples. Samples positive by virus isolation were also found positive by haemagglutination (HA) and haemagglutination inhibition (HI) tests and by dot-ELISA but negative samples were found negative by all the serological tests used. Dot-ELISA was able to detect 0.25-0.50 HA units of virus. The emerging utility of dot-ELISA for diagnosis of Newcastle disease virus infection has been discussed.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.     

The use of a selective medium in the growth-agglutination test for chronic Erysipelothrix insidiosa infections in swine

The use of a phosphate-buffered horse-meat infusion broth (pH 7.8) containing neomycin sulphate 50 pg/ml, kanamycin 400xg/ml and vancomycin 25 pg/ml was compared with the medium without antibiotics in the E. insidiosa growth-agglutination test on sterile and contaminated, positive and negative swine and rabbit serums.The addition of the inhibitory agents to the medium caused no visible growth depression on the 2 antigen strains employed — regardless of the percentage (S 40 %) of specific hyperimmune serum content.The use of the selective medium in testing hyperimmune serum revealed no significant difference in the recorded antibody titer as compared to the ordinarily used medium. The presence of some contaminants in the agglutinating system did not influence the results — provided that the multiplication of these organisms was inhibited (Table 1).The inhibitory efficiency was tested on 161 contaminated sw’ine serum samples and on 12 contaminated rabbit serums. None of the selective broth dilutions of these serums showed multiplication of contaminants during a 24 hrs. preincubation period or in a control tube after 48 hrs. incubation.The inhibitors allow bacteriologic aseptic technique in the procedure of the growth-agglutination test to be deviated to a certain limit. This limit was not exceeded in this material by applying a serum dilution technique otherwise used in agglutination tests with killed antigen.As the described method eliminates the demand of complete aseptic bleeding and serum preparation procedures or an elaborious sterile filtration procedure, and since the test can be used by any laboratory without previous preparation and testing of a killed antigen, the growth-agglutination test is recommended as a fast and reliable aid in the differential diagnosis of erysipelas arthritis on herd basis in swine.     

A COMPARISON OF 4 SEROLOGICAL TESTS IN THE DETECTION OF HUMORAL ANTIBODIES TO ANAPLASMOSIS IN CATTLE

A capillary agglutination (CA), a complement fixation (CF), a plate agglutination (PT) and an indirect fluorescent antibody (IFA) test to detect humoral antibodies to Anaplasma marginale are described. Serums from 3, 4 or 5 groups of cattle were used to examine the efficiency of the tests. Agreement between all 4 tests was 86.6%. Agreement between pairs of tests was greater. The CF test was the most sensitive while the PT test was the least sensitive. However the PT could be carried out very rapidly and was suggested as the best screening test, providing improved antigen preparation techniques could increase sensitivity. The CA, CF and IFA tests all showed a stronger homologous antibody reaction when A. marginale antigen was tested against serums obtained from cattle infected with either A. marginale or A. centrale. Antibodies in the A. marginale serums were first detected by day 7 post-inoculation, rose to peak around day 29 and were still present on day 200. Antibodies in the A. centrale serums were first detected by day 29 rose to a peak around day 50 and had disappeared by day 150.     

The cultural and pathological examination of bulls serologically positive for brucellosis

The genitalia from 34 bulls were examined for the presence of Brucella lesions using histological, bacteriological and serological methods. Tissue fluids extracted from the various genital organs and the serums were examined using the serum agglutination test (SAT), the Rose Bengal plate agglutination test (RBPT), the complement fixation test (CFT) and the indirect haemagglutination test (IHLT) for the presence of Brucella antibody. Titres to one or more of these tests were found in 17 bulls and B. abortus was cultured from 5 of these. The testing of tissue fluids from genital organs did not increase the number of serologically positive animals.