Enzyme-linked immunosorbent assay using solubilised antigen for detection of antibodies toAnaplasma marginale

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against Anaplasma marginale. A marginale bodies were separated from parasitised erythrocytes by a modified nitrogen decompression method, sonicated, then solubilised with Triton X-100 and used as the ELISA antigen. In this ELISA system the required amount of antigen protein was 16.2 ng for each well. In the course of experimental infections, of calves, significant antibody levels were detected by ELISA and the complement fixation test (CFT) at almost the same time. Antibodies against A. marginale were detectable for longer periods using the ELISA than using the CFT. Sera from calves infected with Babesia bigemina, B. bovis, B. ovata, Theileria sergenti and Eperythrozoon wenyoni gave no reaction; however, antisera against A. centrale did react with the A. marginale ELISA antigen.     

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.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

A microplate enzyme immunoassay for detecting and measuring antibodies to Babesia bovis in cattle serum

A microplate enzyme immunoassay (EIA) is described for measuring IgG antibody to Babesia bovis in cattle serum. B. Bovis antibody status (whether positive or negative) and the amount of B. Bovis antibody (EIA score), were measured by comparison with reference serums. The EIA was shown to be specific for B. Bovis, and EIA score correlated well with EIA titre. Comparison of EIA with the Indirect Fluorescent Antibody Test (IFAT) showed more than 95% agreement between the methods and disagreement in only 1.6% of serum samples tested. The remaining 3.2% were positive by EIA and suspected positive by IFAT. The EIA was shown, by titrating positive serums, to be more sensitive than IFAT, which explained its tendency to detect more positive serums than IFAT. EIA detected B. bovis antibody in experimentally infected cattle by day 14 post infection (pi) and for at least 268 days pi. EIA score for B. bovis antibody in immune cattle increased significantly (p less than 0.05) following heterologous strain challenge.     

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

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.     

THE PREVALENCE OF ANTIBODIES TO MEMBERS OF LEPTOSPIRA INTERROGANS IN CATTLE

A total of 1,355 random samples taken from bovine serums submitted for brucellosis testing in Victoria were submitted to the microscopic agglutination test for the presence of antibody to 12 serovars of Leptospira interrogans . The most common reaction obtained was to serovar hardjo , although the percentage of reactors varied from 24.8% in the metropolitan area to 56.3% in north-eastern Victoria (mean 40.6%). A total of 86.3% of farms from which 3 or more samples were taken had at least one reactor to serovar hardjo . The prevalence of antibody to other serovars was tarassovi (7.8% of reactors), ballum (3.7%), pomona (2.4%), autumnalis (1.8%) and bataviae (1.2%). Reactions to other serovars were observed in serums of less than 1% of cattle tested; serums from 50.8% of cattle did not react to any antigen.     

检测牛边缘无浆体抗体的竞争抑制ELISA方法的建立

为建立检测牛边缘无浆体(Anaplasma marginale)抗体的方法,本研究以牛A.marginale膜表面重组MSP5蛋白作为包被抗原,抗MSP5单克隆抗体(MAb)作为竞争抗体,建立一种用于检测牛A marginale抗体的重组MSP5蛋白竞争抑制ELISA(CI-ELISA)方法.经优化确定CI-ELISA的最佳反应条件为:抗原包被浓度为2μg/孔,封闭液为2%脱脂乳,MAb的稀释度为1:400,酶标二抗的稀释度为1:1000,阴性和阳性血清临界值分别为33%和40%;该方法具有良好的特异性和重复性;2 348份临床血清样品的检测结果表明,217份为阳性,阳性率为9.2%,与IDEXXA marginale抗体检测试剂盒的阳性符合率为95.3%,阴性符合率为100%.本实验建立的ELISA方法具有较高的特异性和重复性,可用于流行病学调查研究.     

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.     

Epidemiology of tick-borne diseases of cattle in Botshabelo and Thaba Nchu in the Free State Province

A seroepidemiological study was conducted on 151 cattle from the Botshabelo and Thaba Nchu areas in the central Free State Province of South Africa, two areas where small scale, peri-urban cattle farming is practised. An indirect fluorescent antibody test was used to test for Babesia bigemina and B. bovis antibodies. To test for Anaplasma marginale antibodies a competitive inhibition enzyme-linked immunosorbent assay method was used. There were no significant differences in serological test results between the cattle from Botshabelo and those from Thaba Nchu. The herd (two areas combined) had an average seroprevalence of 62.42% to B. bigemina, 19.47% to B. bovis and 98.60% to A. marginale. Based on the percentage of cattle that were seropositive to B. bigemina the immune status of cattle in the Botshabelo-Thaba Nchu area is approaching a situation of endemic stability. With reference to A. marginale, the high seroprevalence is indicative of a situation of endemic stability. The occurrence of B. bovis antibodies in the cattle is difficult to explain as Boophilus microplus ticks do not occur in the area in which the study was conducted.     

Use of the dot enzyme-linked immunosorbent assay with isolated Anaplasma marginale initial bodies for serodiagnosis of anaplasmosis in cattle

Isolated Anaplasma marginale initial bodies were successfully used in a dot ELISA for rapid detection of antibodies to Anaplasma organisms. The enzyme immunoassay used only 25 ng of antigen dotted onto nitrocellulose disks. Antigen-antibody complexes were detected by use of alkaline phosphatase-conjugated protein A, and reactions were read visually after addition of a precipitable, chromogenic substrate. The test allowed the processing of multiple sera, either for screening or for titer determination, in less than 3 hours and was found to be as sensitive as the indirect fluorescent antibody test. The overall performance of the dot ELISA, using isolated A marginale initial bodies, for 580 bovine serum samples was as follows: sensitivity, 93%; specificity, 96%; and predictive value, 95%. Cross-reactivity was not observed with sera positive to Babesia bovis and B bigemina, Trypanosoma vivax, or common bacteria or viruses infecting cattle. The antigen dotted onto nitrocellulose disks was stable when stored at -20, 4, or 25 C. Compared with the indirect fluorescent antibody test, the dot ELISA allowed easier, faster, and more objective interpretation of results. Its simplicity and low cost combined with high sensitivity and specificity indicate that this assay could effectively replace serologic assays currently used for diagnosis of anaplasmosis in cattle.     

Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica

A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%).     

Immunization of cattle with Anaplasma marginale derived from tick cell culture.

Anaplasmosis is a hemolytic disease of cattle caused by the ehrlichial tick-borne pathogen Anaplasma marginale. Killed vaccines used for control of anaplasmosis in the US used antigen harvested from infected bovine erythrocytes which was often contaminated with bovine cells and other pathogens. In this study, we performed an initial cattle trial to test A. marginale harvested from tick cell culture as an immunogen for cattle. Eleven yearling Holstein cattle were immunized with the cell culture-derived A. marginale and 11 cattle were non-immunized contact controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale in an oil-based adjuvant. Two immunizations were administered subcutaneously 4 weeks apart and the cattle were challenge-exposed 10 weeks after the second immunization with A. marginale infected blood. Maximum antibody levels as determined by an A. marginale specific competitive ELISA were observed 2 weeks after the last immunization. Antibody responses against major surface proteins (MSPs) 1a and 1beta1 were also characterized and immunized cattle demonstrated a preferential recognition for MSP1beta1. Cattle immunized with the cell culture-derived A. marginale had a significantly lower percent reduction in the packed cell volume (P<0.05) after challenge exposure as compared with the controls and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.     

A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.     

Prevalence of Anaplasma marginale, Babesia bigemina and B. bovis in Nigerian cattle using serological methods

Serum samples collected randomly from 500 cattle from the 10 northern states of Nigeria were tested for antibodies against Anaplasma marginale by indirect fluorescent antibody (IFA), card agglutination (CT) and capillary tube-agglutination (CA) tests. The serum samples were also examined for antibodies to Babesia bigemina and B. bovis by the IFA test only. Of the serum samples tested, 79.4% had antibodies against A. marginale by the IFA test, 40 and 25% in the CT and CA tests, respectively. The IFA test results for B. bigemina and B. bovis were 29.4 and 14.1%, respectively.     

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.     

Development of an indirect fluorescent antibody test, using microfluorometry as a diagnostic test for bovine anaplasmosis

The indirect fluorescent antibody test for the diagnosis of Anaplasma marginale infection in cattle was modified for use with microfluorometry. The test was standardized by use of a fluorometer that measures intensity of fluorescence. Standardization included A marginale-infected blood smears on microscope slides as antigen, serum from an inoculated calf as a positive control containing specific antibody, and an affinity-purified fluorescein-conjugated anti-bovine immunoglobulin as 2nd antibody. The modified test and microfluorometry allowed for titration of sera from A marginale (Florida isolate)-inoculated cattle with a degree of accuracy exceeding visual determinations. In addition, the fluorometric test was more sensitive than the complement fixation or card agglutination tests in identifying cattle that had previous Anaplasma infections.     

Seroprevalence survey for Anaplasma marginale-infection of Austrian cattle.

A serologic survey study of 5,076 Austrian cattle farming herds was carried out in the period of December 1988 till March 1990. One animal was randomly selected from each herd and the antibody titer against Anaplasma marginale in blood serum samples was evaluated by means of the complement fixation test. The number of these tested blood samples was 3.6% of 140,081 cattle herd farms of Austria. 109 (2.1%) of the tested animals showed positive titers (1:10) against Anaplasma marginale, in relation to the 140,081 cattle herds 0.08%, 4,786 (94.3%) blood serum samples were sero-negative, 188 (3.8%) reacted anticomplementary. The highest number of antibody-positive animals of 8 tested Austrian districts could be found in Carinthia (46 = 5.7%). In Burgenland all tested sera turned out to be negative. Concerning the distribution of sero-positive animals in Austria it can be stated that a decrease of positive reactors from southern to northern region is evident. A connection between the occurrence of anaplasmosis in Italy, Yugoslavia, Switzerland and Hungary, is postulated as a result of the different systems of keeping cattle in the provinces and the regional increase of tick invasion. Possibly an intensive animal transportation is of importance due to the introduction of the disease mentioned before. The results obtained show that anaplasmosis does occur in different areas of Austria. For control of this disease in Austria it is proposed that all imported cattle should be tested serologically for antibodies against Anaplasma marginale. Other diseases in connection with anemia should be excluded by clinical, serological, blood-, as well as pathological examinations.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.