Development and Bayesian validation of a competitive inhibition ELISA for detection of antibodies against Brucella abortus in cattle.

Bovine brucellosis is a zoonotic disease caused by Brucella abortus, responsible for abortions in cows. It is endemic in low- and middle-income countries, where the brucellosis control and eradication programs are based on compulsory vaccination, detection of infected cattle through serologic assays, and culling of infected animals at slaughterhouses. The development of high sensitivity and specificity, and low-cost serologic assays guarantee their implementation in countries where the disease is endemic. The aim of the present study was to develop and validate a competitive inhibition enzyme-linked immune assay (ciELISA) to detect anti-B. abortus antibodies in sera from cattle. The developed ciELISA was validated using 2833 serum samples from dairy and beef cattle. From these, 1515 sera were from uninfected cows that belonged to free of brucellosis herds and 1318 were from infected cows that belonged positive to brucellosis herds. Sera were analyzed with the developed ciELISA, the buffer plate antigen (BPA) test, and the complement fixation test (CFT). The brucellosis status of the herds was officially established according to the country legislation and consistent for at least 5 years and was defined for each cow using the CFT as gold standard. The cutoff for the ciELISA was calculated using a ROC curve and its sensitivity and specificity were analyzed using the Bayesian Latent Class Model (BLCM) approach. The agreement among tests was calculated using the kappa (κ) value. In addition, 15 calves were vaccinated with 3 × 1010 viable cells of B. abortus Strain 19 vaccine, and the dynamics of antibodies were measured by CFT, buffered plate antigen (BPA) test, and the developed ciELISA. The obtained cutoff for ciELISA was ≥ 47 percentage of inhibition (% I), at the BLCM approach the sensitivity was 99.01 % (95 % CI: 97.55-100) and the specificity 98.74 % (95 % CI: 97.68-99.8). The κ between the ciELISA and BPA was κ = 0.88 and between the ciELISA and CFT κ = 0.95. Antibodies against B. abortus were detected in all the vaccinated calves 7 days after vaccination (AV) by the three assays, at day 135 AV all the calves were negative to CFT (15/15), 93.3 % (14/15) to ciELISA and 73.3 % (11/15) to BPA, and at day 190 AV all the calves were negative to the three assays. The developed ciELISA showed a very good performance, could detect the majority of vaccinated animals as negative after 135 days and could be used for the detection of anti-B. abortus antibodies in serum samples for the brucellosis control and eradication program.

Evaluation of frequency of antibodies against Neospora caninum, Toxoplasma gondii and Brucella melitensis, risk factors and spatial distribution of infection in goat and sheep flocks from Argentina.

Neospora caninum, Toxoplasma gondii and Brucella melitensis are pathogens that cause abortion in small ruminants. Besides, B. melitensis and T. gondii are zoonotic pathogens. The aim of this study was to describe the frequency of antibodies against N. caninum, T. gondii and B. melitensis in sheep and goats from three provinces of the center region of Argentina. In addition, the spatial distribution of the infected flocks/herds and risk factors were evaluated. A cross-sectional study was conducted from 2015 through 2016. Serum samples from 4783 goats and 1524 sheep from 186 goat, 51 sheep and 38 mixed flocks/herds were analyzed. Competitive inhibition enzyme-linked immunosorbent assay (ciELISA) and indirect fluorescent antibody test (IFAT) were performed for detection of antibodies against N. caninum and IFAT for T. gondii. The buffered plate antigen test and complement fixation test were performed for detection of antibodies against B. melitensis. The frequency of anti-T. gondii antibodies was 41.2% and 29.7% for sheep and goats, respectively. The frequency of anti-N. caninum antibodies was 17.2% and 14% for sheep and goats, respectively. About 97.1% of the sheep flocks, 79.4% of the goat herds and the 91.3% of the mixed flocks had seropositive animals to T. gondii. About 61.8% of the sheep flocks, 58% of the goat herds and the 82.6% of the mixed flocks had seropositive animals to N. caninum. All the analyzed animals were negative to anti-B. melitensis antibodies. For T. gondii, a significant cluster of high risk of seropositive flocks/herds was detected in the littoral of the Parana River. The province of origin of the flock/herd was the only variable associated to T. gondii positivity (p = 0.003). Animals from Santiago del Estero and Santa Fe Provinces had 3.48 and 1.77 times more risk to be positive to T. gondii than animals from Entre Ríos Province, respectively. For N. caninum, a cluster of high risk of seropositive flocks/herds was detected in the north of Santa Fe Province. The only explanatory variable associated to N. caninum positivity was animal species (p = 0.003). Sheep had 1.73 times more risk to be positive to N. caninum than goats. The absence of antibodies against B. melitensis in all the analyzed animals is an important finding for the public health of the region. Since bordering provinces have infected flocks/herds, brucellosis in small ruminants should be under epidemiologic surveillance in the region.

Development, validation and field evaluation of an indirect ELISA for the detection of antibodies against Brucella abortus in bulk and individual milk samples in dairy cattle.

Brucellosis is an abortigenic and zoonotic disease. In cattle, it is mainly caused by Brucella abortus. The disease is endemic in low- and middle-income countries, being considered a neglected zoonotic disease. In these countries, it is of high importance to develop and validate sensitive, specific and low-cost diagnostic assays for brucellosis. The aim of the present study was the development of an indirect enzyme-linked immune assay (iELISA) to detect anti-B. abortus antibodies in milk samples. We purified the lipopolysaccharide antigen from B. abortus and produced an anti-bovine IgG monoclonal antibody to develop an iELISA (iELISAINTA). The iELISAINTA was validated using 1730 bulk milk samples and 1734 individual milk samples. The sampled dairy herds had at least 3 years of consistency at their positive or negative official brucellosis status. Individual milk samples were taken in parallel with serum samples from the cows. The status of the cows was defined by the result of the complement fixation test (CFT) performed with their serum sample. The reproducibility of the assay was evaluated in two laboratories. In addition, we evaluated the performance of the assay in the field, using 4385 bulk milk samples and 968 individual milk samples. The results of the iELISAINTA were compared with those obtained using the officially accepted brucellosis techniques: iELISA from Canada (iELISACFIA) in milk samples, and the buffered plate antigen (BPA) and the CFT in serum samples. At validation, the sensitivity (Se) of the iELISAINTA in bulk milk samples was 98.61 %, and the specificity (Sp) 98.79 % with a ≥ 10 % of positivity (PP) cutoff. In individual milk samples, the Se was 98.04 %, and the Sp 98.56 % with a ≥ 16 PP cutoff. The chance-corrected agreement kappa value (κ) between the results obtained in the different laboratories was κ = 0.87. In the field evaluation, in bulk milk samples the κ value between the iELISAINTA and the iELISACFIA was κ = 0.86. On individual milk samples, the κ values were: between the iELISAINTA and the iELISACFIA κ = 0.79, between the iELISAINTA and BPA was κ = 0.85, and between the iELISAINTA and CFT κ = 0.82. The developed iELISAINTA showed a very good performance and it could be used as a screening assay for anti-B. abortus antibodies detection in individual milk samples and for epidemiologic surveillance in bulk milk samples.

Fluorescent polarization assay, two versions of indirect and competitive enzyme- linked immunoassay for brucellosis diagnosis in vaccinated buffaloes (Bubalus bubalis) / Teste de polarização fluorescente, duas versões de imunoensaio enzimático indireto e imunoensaio competitivo para diagnóstico de brucelose em búfalos vacinados (Bubalus bubalis)

ABSTRACT: The fluorescence polarization assay (FPA), two variants (V) of the indirect enzyme-linked immunosorbent assay (I-ELISA) and the competitive enzyme-linked immunosorbent assay (C-ELISA) were evaluated in buffaloes to detect antibodies against Brucella spp. The V1 of I-ELISA identifies them through the monoclonal (M23) anti-bovine IgG (I-ELISAM23) and the V2 through the ProteinA / G (I-ELISA-A/G). Serum samples of 862 buffaloes (Bubalus bubalis) from the Northeast of Argentina (NEA) were analyzed using the complement fixation test (CFT) as the reference. Receiving Operator Characteristic (ROC) analysis defined for the area under the curve (AUC) determined the cutoff points, sensitivity (Se) and specificity (Sp) for each test. CFT identified 107 positive and 755 negative sera. The best AUC (0.986), Concordance with CFT (96.3%) and kappa value (0.843) was obtained by I-ELISA A/G test. This assay showed the highest Se (95.33%) and C-ELISA the highest Sp (97%). FPA failed to measure the antibodies in 23 (2.65%) serum samples due to unsuccessful reading. I-ELISA M23 proved to be ineffective to diagnose brucellosis in bubaline sera. The four serological tests showed cutoff points lower than those standardized for bovines. As conclusion, I-ELISA A/G, C-ELISA and FPA with its limitations would be effective techniques for the diagnosis of brucellosis in buffaloes in the NEA, requiring an appropriate cut-off point to guarantee their maximum performance in this species.

RESUMO: O ensaio de polarização de fluorescência (FPA), duas variantes (V) do ensaio imunoenzimático indireto (I-ELISA) e o ensaio imunoenzimático competitivo (C-ELISA), foram avaliados em búfalos para detectar anticorpos contra Brucella spp. O V1 do I-ELISA os identifica através do IgG monoclonal (M23) anti-bovino (I-ELISAM23) e o V2 ​​através da Proteína A / G (I-ELISA-A / G). Amostras de soro de 862 búfalos (Bubalus bubalis) do Nordeste da Argentina (NEA) foram analisadas usando o teste de fixação do complemento (CFT) como referência. A análise Receiving Operator Characteristic (ROC) definida pela área sob a curva (AUC) determinou os pontos de corte, sensibilidade (Se) e especificidade (Sp) de cada teste. A CFT identificou 107 soros positivos e 755 soros negativos. Os melhores valores de AUC (0.986), concordância com CFT (96.3%) e kappa (0.843) foram obtidos pelo teste I-ELISA A / G. Este ensaio mostrou a maior Se (95.33%) e C-ELISA a maior Sp (97%). O FPA falhou em medir os anticorpos em 23 (2,65%) amostras de soro devido à falha na leitura. O I-ELISA M23 provou ser ineficaz para o diagnóstico de brucelose em soros bubalinos. Os quatro testes sorológicos mostraram pontos de corte inferiores aos padronizados para bovinos. Em conclusão, I-ELISA A / G, C-ELISA e FPA com suas limitações seriam técnicas eficazes para o diagnóstico de brucelose em búfalos no NEA, exigindo um ponto de corte adequado para garantir seu desempenho máximo nesta espécie.

Validation and field evaluation of a competitive inhibition ELISA based on the recombinant protein tSAG1 to detect anti-Neospora caninum antibodies in sheep and goats.

Neospora caninum is a protozoan parasite that causes abortion and reproductive failure in small ruminants. We validated and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1) from N. caninum for the detection of anti-N. caninum antibodies in sheep and goat flocks. The assay was validated using 80 positive and 142 negative serum samples from sheep and goats analyzed by IFAT and immunoblot (IB). ciELISAtSAG1 was then used to evaluate the prevalence of anti-N. caninum antibodies in 1449 goats from 143 flocks and 385 sheep from 40 flocks and compared to IFAT. The prevalence of anti-Toxoplasma gondii antibodies was evaluated by IFAT. The ciELISAtSAG1 cut-off was ≥ 36 percent inhibition, with a diagnostic sensitivity of 100.0 % (95 % CI = 95.4-100.0 %) and a diagnostic specificity of 98.6 % (95 % CI = 95.0-99.8 %) relative to the agreement between IFAT and IB. The field evaluation revealed a concordance between ciELISAtSAG1 and IFAT of 97.4 %, with an agreement (κ) of 0.90 for sheep sera, and a concordance of 96.5 % with κ = 0.85 for goat sera. The overall prevalence of anti-N. caninum antibodies in sheep was 14.3 % by IFAT and 15.8 % by ciELISAtSAG1. In goats, prevalence was 12.9 % by IFAT and 14.6 % by ciELISAtSAG1. The overall prevalence of anti-T. gondii antibodies was 28.8 % in goats and 43.8 % in sheep. The ciELISAtSAG1 could be useful for large-scale detection of anti-N. caninum antibodies in sheep and goats, and for seroepidemiological investigations due to its appropriate sensitivity and specificity, and the simplicity of production.

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA.

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.