Evaluation of three competitive ELISAs and a fluorescence polarisation assay for the diagnosis of bovine brucellosis.

Bovine brucellosis is an infectious disease of worldwide public health and economic importance. The usual tests for the diagnosis of this disease include the Rose-Bengal test (RBT), complement fixation test (CFT), serum agglutination test (SAT) and indirect ELISA. New tests such as competitive ELISAs (C-ELISA) and fluorescence polarisation assay (FPA) have been developed. However, C-ELISA may correspond to different protocols and a wide variation may exist in their diagnostic performance. The aim of this study was to evaluate three commercially available C-ELISA kits (C-ELISA1-3) and FPA for the diagnosis of bovine brucellosis and compare test performance with RBT, CFT, indirect ELISA and FPA. Sera submitted to EU laboratories in 2011 from 5111 adult cattle were tested. Individual test sensitivities (Se) and specificities (Sp) were estimated. Threshold assessment using the receiver operating characteristic method was also performed. The most sensitive tests were FPA (99.0%; 95% confidence interval [CI], 97.9-100%), C-ELISA1 (98.4%; 95% CI, 97.0-99.8%) and RBT (97.7%; 95% CI, 95.9-99.3%). The most specific tests were CFT (99.98%; 95% CI, 99.93-100%), SAT (99.98%; 95% CI, 99.93-100%) and RBT (99.89%; 95% CI, 99.79-99.99%). Among the new tests, none of the three C-ELISA kits studied could be recommended as a single screening test because of their low specificity, especially when used in a herd. C-ELISA3 could not be recommended as confirmatory test on individual animals to determine whether false positive serological test results had occurred.

Validation of a fluorescence polarization assay (FPA) performed in microplates and comparison with other tests used for diagnosing B. melitensis infection in sheep and goats.

Fluorescence polarization assay (FPA) is a relatively new test for the serological diagnosis of Brucella spp. infection in animals. FPA, carried out in 96-well microplate format, was validated here for diagnosing B. melitensis infection in sheep and goats. This study included sera from 1933 sheep and goats, from animals reared in naturally infected flocks (verified by culture) and showing a positive reaction to two different tests conducted in parallel. In addition, 2154 sera originating from healthy sheep and goats, reared in areas where B. melitensis had never been isolated, were assayed. The optimum cut-off value offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 15 mP over the mean value of the buffer control used in each microplate as determined by receiver operating characteristic analysis. The DSn and DSp of the FPA for small ruminants carried out in microplates at this cut-off value were calculated to be 95.9% and 97.9% with 95% confidence intervals (95% CI) of 94.9-97.7% and 97.2-98.4%, respectively. The accuracy of the FPA, as expressed by determination of the area under the curve, was 0.991. Indirect ELISA and FPA tests offered the highest DSn when compared with the Rose Bengal test, the complement fixation test, the modified Rose Bengal test and competitive ELISA. The parallel or serial combination of FPA with indirect ELISA offers the highest DSn and DSp. As temperature can affect the results of the FPA, all reagents must be at the same temperature and the standard for comparison must always be read under the same conditions as the sera under test. FPA performed in microplates is a promising assay; the DSn and accuracy are better than those of the tests currently approved for diagnosing B. melitensis in small ruminants. Because of its simplicity, speed, and accuracy, this test can improve capacity for laboratory testing and the efficacy of an eradication program based on a test-and-slaughter policy.

Assessment of serological response of young and adult sheep to conjunctival vaccination with Rev-1 vaccine by fluorescence polarization assay (FPA) and other serological tests for B. melitensis.

The serological response of young and adult sheep vaccinated conjunctivally with Rev-1 vaccine was assessed by fluorescence polarization assay (FPA), Rose Bengal test (RBT), complement fixation test (CFT), modified Rose Bengal test (m-RBT), indirect ELISA (i-ELISA) and competitive ELISA (c-ELISA), at different post vaccination intervals. One hundred and thirty six adult sheep and 64 lambs were used in the study. The vaccinated animals were bled prior to vaccination (0 day) and thereafter at 21st, 42nd, 35th, 63rd, 91st, 125th, 159th, and 223rd and 330th day post vaccination. The majority of animals (young and adult) showed positive reaction by FPA, RBT, CFT, m-RBT and c-ELISA 21 days post vaccination, whereas by i-ELISA at 42 days. All tests perform equal when animals vaccinated as young are tested 125 days (4 months) post vaccination. In case of animals vaccinated at adulthood, FPA, RBT, CFT and c-ELISA perform equal if the animals are tested 223 days (approximately 8 months) post vaccination. I-ELISA and m-RBT show low specificity if ewes vaccinated at adulthood are tested 330 days (11 months) post vaccination. If control of brucellosis in sheep is based on conjunctivally vaccination of lambs with Rev-1, the vaccinated animals can be tested by any test used for diagnosis of B.melitensis infection accurately at least 4 months post vaccination. If brucellosis control is based on mass vaccination the use of m-RBT and i-ELISA is not recommended for testing adult animals at least for 330 days (11 months) post vaccination due to tests low specificity. Further research is needed so the appropriate cut-offs to be established for FPA, c-ELISA or i-ELISA to become valuable tools for the eradication of Brucella spp. infection in small ruminants in areas where vaccination is practiced.

Validation of fluorescence polarization assay (FPA) and comparison with other tests used for diagnosis of B. melitensis infection in sheep.

Fluorescence polarization assay (FPA) is a new test for the serological diagnosis of Brucella spp. infection in animals. The FPA is validated for the diagnosis of B. melitensis infection in sheep. For this purpose, 166 sera originated from natural infected sheep (verified by culture) and 851 sera originated from healthy animals (reared in areas where B. melitensis was never been isolated) were tested. The optimum cut-off value that offers the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 87mP with the use of ROC analysis. The DSn and DSp of FPA using this cut-off value are calculated at 97.6 and 98.9% with a 95% confidence interval (CI) of 93.9-99.3% and 98.0-99.5%, respectively. The DSn and DSp of FPA have been assessed also using as positive reference (n=587), sera that gave positive results at least in two tests used for diagnosis of B. melitensis in sheep as Rose Bengal Test (RBT), modified Rose Bengal Test (m-RBT), complement fixation test (CFT), indirect Elisa (i-Elisa) and competition Elisa (c-Elisa) originated from animals reared in flocks infected by B. melitensis. The optimum cut-off value using the above panel of positive reference sera was the same offering a DSn of 95.9% with a 95% CI, 94.0-97.4%, since the DSp remains the same. The DSn and DSp as well as performance, accuracy and agreement of FPA's result were compared with those of other tests used. The accuracy of FPA is very high, similar with that of i-Elisa. FPA is a promising assay, which offers a DSn and accuracy better that of those of the tests currently approved for the diagnosis of B. melitensis in sheep and goats. Due to its simplicity, the sort time that results can be obtained and its accuracy it can be used and improve the laboratory testing capacity as well as the efficacy of the eradication program based on test-and-slaughter policy.

The "effects" of Rev-1 vaccination of sheep and goats on human brucellosis in Greece.

Vaccination of young animals (3-6-month-old sheep and goats) with Rev-1 vaccine for 15 years in Greece, importantly decreased the abortions in sheep and goats as well as the incidence of brucellosis in humans. After the stop of vaccination in 1994, all over Greece, the prevalence of brucellosis in animals and the incidence in humans quickly increased. It was a positive rank correlation (0.90) among these variables. Once an emergency mass-vaccination programme of young and adult animals with Rev-1 vaccine was started in 1998, the human incidence again decreased. The association of the vaccination coverage of animals and incidence of brucellosis in humans was not linear; the decrease in human brucellosis incidence was observed when the vaccination coverage of animals was >30%.