An indirect haemagglutination test for the detection of Vibrio fetus antibodies

Abstract

Extract

Indirect bacterial haemagglutination was first reported by Keogh et al. (1947 Keogh, E. V., North, E. A. and Warburton, M. F. 1947. Nature (Lond.), 160: 63–63.  [Google Scholar]). It depends on the adsorption of bacterial antigens to the surface of red blood cells rendering them agglutinable in the presence of homologous bacterial antibody. A comprehensive review by Neter (1956 Neter, E. 1956. Bact. Rev., 20: 166–166.  [Google Scholar]) summarizes the methods used and results achieved with various bacterial antigens. Biberstein (1955 Biberstein, E. L. 1955. Cornell Vet., 46: 144–144.  [Google Scholar]) used an adaption of Neter's method of antigen preparation in studying the antigenic relationships of Vibrio species. The objects of the present studywere to determine whether an erythrocyte adsorbable antigen could be obtained from Vibrio fetus, to compare its sensitivity with a formalinized bacterial antigen, and to study its application to the detection of antibodies in bovine vaginal mucus. The work will be described in two sections, the first dealing with the preparation and properties of sheep red cells modified with material derived :from V. fetus, and the second with the detection of antibodies in bovine vaginal mucus.     

Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.     

An indirect hemagglutination test for the detection of antibodies to Clostridium chauvoei

An indirect hemagglutination (IHA) test using sonicated extract as the antigen was developed for the detection of antibodies to Clostridium chauvoei. This antigen can be adsorbed onto glutaraldehyde-fixed sheep red blood cells treated with tannic acid and can be destroyed by trypsin and heat treatment. It corresponded well with the flagella of the organism, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gel diffusion test. No serological cross-reactivity was found in the IHA test when the antigen was tested against 4 species of clostridial antibodies. Our results suggest that the IHA test mainly detects antibodies against the flagella of C. chauvoei.     

An indirect flow cytometric test for detection of anti-neutrophil antibodies in dogs

OBJECTIVE: To develop a clinically applicable assay for detection of serum anti-neutrophil antibodies in dogs. SAMPLE POPULATION: Serum samples of 20 healthy dogs and 20 sick dogs. PROCEDURES: An indirect immunofluorescence assay was developed in which canine serum was incubated with paraformaldehyde-fixed neutrophils and subsequently incubated with fluorescein-conjugated rabbit anti-dog IgG. Neutrophil median fluorescence intensity and the percentage of neutrophils with an increase in fluorescence intensity were determined by use of a flow cytometer. RESULTS: Neutrophils incubated with serum from healthy and sick dogs had a normally distributed curve when displayed as a histogram. Alloantibodies or immune complexes that significantly affected test results were not detected. Hyperglobulinemia did not appear to affect test results. The neutrophil donor did not significantly affect test results. With 1 exception, results for the sick dogs did not differ appreciably from those for healthy dogs. Serum from a dog with steroid-responsive neutropenia had a greater neutrophil fluorescence value and percentage of neutrophils with an increase in fluorescence intensity, compared with either healthy or sick dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The indirect immunofluorescence test gave consistent results for healthy and sick dogs and detected anti-neutrophil antibodies in a dog with steroid-responsive neutropenia. Definitive evaluation of the test will be dependent on evaluation of persistently neutropenic dogs and correlation of test results with a response to immunosuppressive therapy.     

副猪嗜血杆菌血清5型间接血凝试验和间接ELISA抗体检测方法的建立

为建立副猪嗜血杆菌（HPS）的血清学诊断方法，通过探索HPS荚膜多糖产生的最适体外培养条件，提取了HPS血清5型菌株的荚膜多糖（CPS），并以之为抗原分别建立了间接血凝试验（IHA）和间接ELISA两种抗体检测方法，对其特异性、敏感性和符合率进行了比较研究。结果表明，两种检测方法的特异性良好，但ELISA的敏感性是IHA的5～10倍，二者的阳性符合率、阴性符合率和总符合率分别为79．7％、55．2％和65．3％。用这两种方法检测了320份临床送检猪血清，IHA和ELISA的阳性率分别为40％和59％。结果证实，这两种方法适用于不同实验室条件下HPS的诊断和流行病学调查。     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

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.     

Comparison of different antigenic preparations for the detection of ovine serum antibodies against Brucella ovis by ELISA

An enzyme-linked immunosorbent assay has been developed for the detection of antibodies against Brucella ovis using serum from control rams (Con-S), naturally infected rams (Inf-S), rams inoculated intravenously with B. ovis (IV-S) and rams vaccinated intramuscularly (IM-S). The serum was titrated by serial double dilutions from 1/25 to 1/25,600 against whole bacteria, B. ovis lipopolysaccharide and a detergent-extracted component of the outer membrane complex of B. ovis as antigens immobilised on microtitre plates. Sheep antibodies bound to antigen were assayed with rabbit anti-sheep gammaglobulin and alkaline phosphatase conjugated protein A. A high level of antibody activity against intact B. ovis cells was detected in Inf-S and IM-S. When lipopolysaccharide was the immobilised antigen, only IM-S yielded significant antibody activity. The component from detergent extracts of the outer membrane complex of B. ovis reacted best with serum (up to 1/6,400) from field-infected rams, while serum from vaccinated and intravenously inoculated rams registered significant titres up to a serum dilution of 1/800 and 1/200 respectively. These results indicate that ELISA is a very sensitive test but its value as a serodiagnostic procedure is dependent upon the choice of antigen used in the assay.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

Use of enzyme-linked immunosorbent assay for detection of antibodies to Brucella ovis in sheep: field trial

An enzyme-linked immunosorbent assay (ELISA), using an autoclave-extracted soluble antigen, for the detection of serum antibodies to Brucella ovis in sheep was developed. The test seemed to be both sensitive and specific, on the basis of the control groups studied. The antigen showed no deterioration in prepared plates stored at -70 C for up to a year. The ELISA was used in conjunction with palpation of rams for epididymal lesions as a means to detect and control B ovis infection in a naturally infected flock. All rams were evaluated by the ELISA. At the time that the first blood sample was obtained, all positive and suspicious reactors were removed. With subsequent blood sample collections, only the positive reactors were removed. Brucella ovis was not isolated from any rams during the following year, and none of the mature breeding rams developed epididymal lesions.     

The detection of Brucella ovis infection in rams

Abstract

Extract

Each year in New Zealand, many rams are palpated for lesions of the epididymes and testes. Although regarded initially as a method of detecting infertile rams (Crawford and James, 1950 Biberstein, E. L. and McGowan, B. 1958. Cornell Vet., 48: 31–31.  [Google Scholar]), the technique is now regarded primarily as a method of detecting infection with the organism for which Buddie (1956 Buddle, M. B. 1956. J.Hyg., 54: 351–351.  [Google Scholar]) proposed the name Brucella ovis.