Effectiveness of Rose Bengal test and fluorescence polarization assay in the diagnosis of <Emphasis Type="Italic">Brucella</Emphasis> spp. infections in free range cattle reared in endemic areas in Zambia

The effectiveness of Rose Bengal test (RBT) and fluorescence polarization assay (FPA) in diagnosing cattle brucellosis in endemic areas was assessed and RBT and FPA test agreement was compared (n = 319). The sensitivity of RBT and FPA in detecting low Brucella titres were evaluated in paired sera (n = 34). A logistic regression model was constructed to predict cattle test result in FPA using RBT as the main predictor and incorporating bio-data and animal history. There was 79.3% agreement between the RBT and FPA (Kappa = 0.59; Std error = 0.05; p = 0.000) and a high correspondence between high RBT scores and positive FPA results suggesting that sera with high RBT score may not require confirmation with tests such as competitive-ELISA or CFT. High FPA cut-off points were more likely to miss animals with low antibody titres. The RBT had a reduced ability in detecting low antibody titres compared to the FPA. FPA test interpretation was improved if a priori information, such as sex and age was used. Under the challenging disease surveillance conditions prevailing in rural Africa, field-testing methods that are sensitive and specific; allow single animal contact, low technical skills in data interpretation are suitable.     

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.     

Comparison of fluorescence polarization assay with card and complement fixation tests for the diagnosis of goat brucellosis in a high-prevalence area

An evaluation of fluorescence polarization assay (FPA) to detect antibodies against Brucella melitensis according to the Mexican Official Norm (NOM) was performed. In this study, a total of 2582 goat serum samples from a high-prevalence area in northeast Mexico where vaccination is applied, were used. Of these, 1094 were classified as NOM negatives (card test (CT) negatives or CT positives/complement fixation test (CFT) negatives) and 1488 as NOM positives (CT and CFT positives). The receiver operator characteristics (ROC) curve analysis was used to obtain the FPA sensitivity (83.5%), specificity (82.2%) and accuracy (88.2%) compared with NOM criteria, using a cut-off value of 89mP for positive samples. In addition, FPA produced 84.1% of negative results versus 65.7% of CT using 1094 CFT negative samples, which indicated that FPA performance was better than CT to detect negative samples or differentiate samples from vaccinated animals. Finally, FPA showed 95.8% sensitivity when using 702 negative non-vaccinated samples. Taken together, these results suggested that FPA might replace CT as a screening test for its better performance compared with CFT, its adjustable cut-off useful in different epidemiological situations, and for its reliability, ease of performance, comparable cost with CT regimen, and potential application in field and high-throughput laboratories. The use of FPA as screening test will help to reduce the percentage of goats wrongly slaughtered because of brucellosis misdiagnosis. More studies on FPA are required for its approval as diagnostic tool for goat brucellosis.     

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.     

Validation of the Fluorescence Polarisation Assay (FPA) for the serological diagnosis of brucellosis

In order to evaluate suitability of Fluorescence Polarisation Assay (FPA) for serological Brucella diagnostic, 1739 samples of sera from cattle, pigs, sheep and goats (65 Brucella-positive, 960-negative and 714 false-positive sera) were investigated at a dilution of 1:10. The cut-off was adjusted by means of ROC analysis. Furthermore, the serum samples were examined for Brucella antibodies using SAT, CFT and ELISA and the results were evaluated regarding sensitivity and specificity. FPA, SAT, CFT and ELISA attained a sensitivity of 92.3, 98.5, 84.6 and 86.2%. In comparison, specificity varied with 87.8, 72.6, 92.5 and 85.8%, respectively. Accordingly, FPA is a suitable test for serodiagnosis of brucellosis.     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

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 an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.     

3种奶牛布鲁氏菌病血清学检测方法的贝叶斯评估

在无"金标准"的情况下,为评估虎红平板凝集试验(RBT)、荧光偏振分析法(FPA)和间接酶联免疫吸附试验(I-ELISA)用于奶牛布鲁氏菌病的抗体检测能力,作者分别以RBT、FPA、I-ELISA3种方法对采自某奶牛场的血清样品进行检测,通过贝叶斯分析法对检测结果进行分析,研究3种方法检测奶牛布鲁氏菌血清抗体的敏感性(Se)和特异性(Sp)。结果显示,RBT、FPA、I-ELISA3种检测方法的敏感性(Se)分别为91.81%、95.92%和95.98%;特异性(Sp)分别为98.80%、86.54%和98.65%。本研究表明,3种血清学检测方法中以I-ELISA的检测能力最好。     

Improved performance of Brucella melitensis native hapten over Brucella abortus OPS tracer on goat antibody detection by the fluorescence polarization assay

The current method for goat brucellosis diagnosis is based on the World Organization for Animal Health (OIE) using the screening card test (CT), with antigen at 8% (CT8) or 3% (CT3) of cell concentrations, and the confirmatory complement fixation test (CFT). However, these tests do not differentiate antibodies induced by vaccination from those derived from field infections by Brucella species or other bacterial agents; in places like Mexico, where the prevalence of brucellosis and the vaccination rates are high, there is a considerable percentage of false positive reactions that causes significant unnecessary slaughter of animals. Furthermore, results of the fluorescence polarization assay (FPA) using the Brucella abortus O-polysaccharide (OPS) tracer in goats are poorer than those with cattle. The present study was undertaken to investigate a tracer prepared from the native hapten (NH) of the Rev. 1 strain of Brucella melitensis to improve FPA performance on goat brucellosis diagnosis. Evaluation of 48 positive samples and 96 negative samples showed that the NH tracer was more accurate (p<0.01) than the OPS tracer (97.2% vs. 93.8% accuracy, respectively). On the diagnostic performance evaluation, the NH tracer performed better (87.5% accuracy, 79.5% sensitivity, 84.3% specificity, and 163.8 performance index) than the OPS tracer (83.5%, 75.9%, 81.0%, and 156.9, respectively) using 1009 positive and 2039 negative Mexican field goat sera samples selected by test series approved by the OIE (card test 3% and CFT). We demonstrated a new application for the NH lipopolysaccharide on detecting antibodies against Brucella using the FPA, which may yield faster results (minutes vs. 24-72h) than the immunodiagnosis assays frequently used in bovine brucellosis. In addition, NH tracer produces similar or better performance results than the conventional OPS tracer, using the FPA in goat sera samples.     

Evaluation of sensitivity and specificity of RBT, c-ELISA and fluorescence polarisation assay for diagnosis of brucellosis in cattle using latent class analysis

The sensitivity (Se) and specificity (Sp) of the Rose Bengal test (RBT), competitive ELISA (c-ELISA), serum (sFPA) and blood (bFPA) fluorescence polarisation assay for brucellosis were evaluated using latent class analysis using sera and whole blood collected from infected cattle reared in smallholder dairy farms of Zimbabwe. The latent class model allowed estimation of Se and Sp in the absence of a gold standard test. The c-ELISA had the highest Se (99.0%; 95% credible posterior interval (CPI): 94.8; 100%), while the RBT and sFPA had the highest Sp (99.0%; 95% CPI: 98.0; 99.6%). The bFPA had the lowest Se (71.3%; 95% CPI: 56.2, 83.5%), while its Sp (96.3%; CPI: 93.9; 98.0%) was marginally higher than that of the c-ELISA (95.4% CPI: 93.7; 96.8%). Therefore based on these data, test regimen using the RBT and c-ELISA could be suitable for diagnosis of brucellosis in smallholder dairies in Zimbabwe. Based on cost and ease of performance, the sFPA may be adopted as a confirmatory test, but its performance may be optimised by altering cut-off points to suit the Zimbabwean conditions. Thus, latent class models provide an alternative method for evaluating Se and Sp of diagnostic tests, which could be used to optimise test performance in different cattle populations.     

Evaluation of three serological tests for brucellosis in naturally infected cattle using latent class analysis

Serological methods are traditionally used in diagnosis of brucellosis. However, the comparative performance of these tests and their accuracy under the local environment in Zambia has not been assessed. Thus, the objective of our study was to evaluate the diagnostic performance of three serological tests for brucellosis; Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and Fluorescence Polarisation Assay (FPA) in naturally infected cattle in Zambia without an appropriate reference test to classify animals into truly infected and non-infected. Serological test results from a study to determine sero-prevalence were used to compare the performance of RBT, c-ELISA and FPA in diagnosing brucellosis in traditional cattle. Since none of the tests can be seen as a perfect reference test or gold standard, their performance in a population of naturally infected cattle was evaluated using latent class analysis which allows the sensitivity (Se) and specificity (Sp) to be estimated in the absence of a gold standard. The highest Se was achieved by the c-ELISA (97%; Credible Posterior Interval (CPI)=93-100%) and the highest Sp by the FPA (93%; CPI=85-99%), conversely these tests also had the lowest Sp and Se, respectively, with the RBT performing well in both the Se (93%; CPI=84-98%) and Sp (81%; CPI=61-97).     

Validation of enzyme-linked immunosorbent assays for the diagnosis of bovine brucellosis

The purpose of this study was to evaluate the performance of the indirect enzyme immunoassay (IELISA) and the competitive enzyme immunoassay (CELISA) for the diagnosis of bovine brucellosis in comparison to conventional serological tests routinely used in Argentina. Serum samples (n = 3500), from Brucella-free herds, from vaccinated cattle and from naturally infected cattle, were tested by the following tests: buffered antigen agglutination test (BPAT), rose bengal test (RBT), 2-mercaptoethanol test (2-ME), complement fixation test (CFT), IELISA and CELISA. Sensitivity and specificity of the BPAT, RBT, IELISA and CELISA were determined relative to the 2-ME and the CFT. The CELISA was considered suitable for eliminating most serological reactions of vaccinated animals and was more specific than the other tests. The results indicate the potential use of the CELISA as a complementary assay in the brucellosis control and eradication program in Argentina and other countries, where Brucella abortusstrain 19 vaccination is mandatory.     

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n = 118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA).The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.     

Comparison of three serological tests in the diagnosis of Brucella infection in unvaccinated cattle in Eritrea

Three serological methods, the Rose-Bengal test (RBT), the complement-fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (I-ELISA) were compared for the detection of Brucella-infected animals in unvaccinated cattle herds in Eritrea. In this study, 71 herds first were classified as positive or negative for Brucella infection on the basis of at least one animal being seropositive by RBT and CFT. All the 159 RBT-positive samples from the 26 seropositive herds and 214 RBT-negative samples randomly selected from the seropositive herds and from the 45 negative herds were tested further by CFT and I-ELISA. Using the ELISA titer as main predictor, and incorporating the RBT results, a logistic model was built to predict the CFT-negative or -positive status of individual sera and to estimate sensitivity and specificity. Whilst the ELISA titers (< or =20) accurately predicted all the negative sera in herds that were also negative by the CFT, the number of seropositive animals was higher by ELISA in herds that had positive animals. Serum samples which give higher degrees of agglutination with the RBT need not be re-tested with CFT; consideration of the seropositive status of a herd should be taken into consideration on defining the cut-off optical density readings for ELISA.     

Brucellae through the food chain: the role of sheep, goats and springbok (Antidorcus marsupialis) as sources of human infections in Namibia

A confirmed case of human brucellosis motivated an investigation into the potential source of infection in Namibia. Since domestic animals are principal sources of Brucella infection in humans, 1692 serum samples were screened from sheep, goats and cattle from 4 presumably at-risk farms and 900 springbok (Antidorcas marsupialis) serum samples from 29 mixed farming units for Brucella antibodies by the Rose-Bengal test (RBT) and positive cases confirmed by complement fixation test (CFT). To assess the prevalence of human brucellosis, 137 abattoir employees were tested for Brucella antibodies using the standard tube agglutination test (STAT) and by enzyme linked immunosorbent assay (ELISA). Cattle and sheep from all 4 farms were negative by RBT and CFT but 2 of the 4 farms (Ba and C) had 26/42 and 12/285 seropositive goats, respectively. Post mortem examination of seropositive goats revealed no gross pathological lesions typical of brucellosis except enlarged mesenteric and iliac lymph nodes seen in a single buck. Culture for brucellae from organs of seropositive animals was negative. None of the wildlife sera tested positive by either RBT or CFT. Interviews revealed that besides the case that prompted the investigation, a family and another person from other farms with confirmed brucellosis shared a common history of consumption of unpasteurised goat milk, home-made goat cheese and coffee with raw milk and prior contact with goats, suggesting goats as the likely source of infection. All 137 abattoir employees tested negative by STAT, but 3 were positive by ELISA. The 3 abattoir workers were clinically normal and lacked historical connections with clinical cases. Although goats are often associated with B. melitensis, these studies could not explicitly implicate this species owing to cross-reactivity with B. abortus, which can also infect goats. Nevertheless, these data reinforce the need for a better National Control Programme for brucellosis in Namibia.     

Assessment of milk ring test and some serological tests in the detection of <Emphasis Type="Italic">Brucella melitensis</Emphasis> in Syrian female sheep

Brucella melitensis infection prevalence among Syrian female sheep, to evaluate a number of serological tests and to discuss some epidemiological aspects of brucellosis, was studied. A total of 2,580 unvaccinated Syrian female sheep sera samples were tested for B. melitensis antibodies detection using four serological methods: the Rose Bengal test (RBT), the serum agglutination test (SAT), the complement fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (iELISA). In addition, 2,375 milk samples were collected, then milk ring test (MRT) and bacterial isolation test were employed to evaluate the natural organism shedding. The samples were considered positive in 66%, 64%, and 60% when we employed the RBT, SAT, and iELISA tests, respectively. Whereas, the CFT test revealed the smallest number of positive samples. By using the MRT, the total prevalence of brucellosis was nearly 38% of samples. A large variation was observed concerning the studied areas, ranging from 24% in Tartous to 44% in both Damascus and Damascus rural areas. Brucella was isolated from only 677 samples out of the 2,375 female sheep milk samples.     

Comparative evaluation of Rose Bengal plate agglutination test, mallein test, and some conventional serological tests for diagnosis of equine glanders.

The Rose Bengal plate agglutination test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic efficiency was compared with that of mallein and other serological tests, including indirect hemagglutination test (IHAT), complement fixation test (CFT), and modified counter immunoelectrophoresis test (mCIET). Sera from 70 naturally infected culture-positive, 96 potentially exposed cohorts, and 110 healthy equines were tested. All tests but mCIET showed 100% specificity when testing the sera from glanders-negative equines. The calculated sensitivities of RBT, IHAT, CFT, mCIET, and mallein test when testing culture-positive equines were 90.0, 97.1, 91.4, 81.4, and 75.7%, respectively. The RBT was significantly (P < 0.05) more sensitive than the mallein test and mCIET. The positive and negative predictive values of each test (RBT, IHAT, CFT, mallein test, and mCIET) were as follows: 100 and 94, 100 and 98.2, 100 and 96.7, 100 and 86.6, and 90.5 and 88.6, respectively. On comparing glandered and nonglandered animals, the highest agreement (0.987) was found between RBT and CFT followed by RBT and IHAT (0.940), RBT and mallein test (0.871), and RBT and mCIET (0.852). Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.     

A fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in cattle sera

A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.     

Bayesian estimation of the true prevalence,sensitivity and specificity of the Rose Bengal and indirect ELISA tests in the diagnosis of bovine brucellosis

Serology is the most convenient method for detecting brucellosis but the efficient use of such tests in disease control requires evaluation of diagnostic performance and discriminative ability. The objective of this study was to assess the performance of the Rose Bengal test (RBT) and an indirect ELISA (iELISA) in diagnosing brucellosis in 995 serum samples collected from cattle in the Ivory Coast between 2005 and 2009. A Bayesian approach was used to evaluate the two tests by estimating their sensitivities and specificities.The correlation-adjusted sensitivity of the iELISA was estimated to be 96.1% (credibility interval [CrI], 92.7–99.8), whereas that of the RBT was 54.9% (CrI, 23.5–95.1). High correlation-adjusted specificities were found for both tests (95.0%; [CrI, 91.1–99.6] for the iELISA and 97.7%; [CrI, 95.3–99.4] for the RBT, respectively). The true prevalence of brucellosis was estimated from the serum samples to be 4.6% (95%; [CrI, 0.6–9.5]). The level of agreement between the two tests was evaluated using indices of agreement (n = 995). Good agreement was found for negative results (96.6%; confidence interval [CI], 95.7–97.4), a finding supported by an estimated significant correlation of 0.37 (95%; CI, 0.01–0.73) within the sera testing negative. Agreement was lower for sera testing positive (52.2% CI: 41.9–62.5). The findings highlight the importance of using these two tests in combination as part of any brucellosis control programme.