RESUMO
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.
Assuntos
Anticorpos Antibacterianos , Teorema de Bayes , Brucella abortus , Brucelose Bovina , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Animais , Bovinos , Brucella abortus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Antibacterianos/sangue , Brucelose Bovina/diagnóstico , Brucelose Bovina/prevenção & controle , Brucelose Bovina/imunologia , FemininoRESUMO
Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.
Assuntos
Brucelose Bovina , Brucelose , Doenças dos Bovinos , Feminino , Humanos , Bovinos , Animais , Brucella abortus , Teorema de Bayes , Análise de Classes Latentes , Sensibilidade e Especificidade , Testes de Aglutinação/veterinária , Brucelose/epidemiologia , Brucelose/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Brucelose Bovina/diagnóstico , Brucelose Bovina/epidemiologia , Anticorpos Antibacterianos , Testes Sorológicos/veterináriaRESUMO
The direct methods for diagnosis of bovine brucellosis have several limitations, therefore serological tests are the basis for the diagnosis of the disease. However, a meta-analysis estimating the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) on the main tests used in bovine brucellosis control programs worldwide has not been performed. This systematic review and meta-analysis aimed to estimate the DSe, DSp and thereby accuracy of serological tests individually used in the diagnosis of bovine brucellosis. The databases CABI, Cochrane Library, PubMed/MEDLINE, SciELO, Scopus and Web of Science were used to select articles. The search resulted in 5308 studies, of which 71 were selected for systematic review using quality assessment tools and 65 studies were included in the meta-analysis. For the meta-analysis, 178 assays and 11 different serological tests were considered. To estimate DSe and DSp of the tests, studies were divided according to animal selection for the studies: (1) studies that carried out a random or consecutive selection of participants (noncasecontrol studies) and (2) all studies, including casecontrol studies. Considering only the non-case-control studies to estimate the DSe, the tests that exhibited the best and worst performance were the iELISA test (indirect enzyme immunoassay - bacterial suspension as antigen - BS) (96.5%, 95% CI: 94.1-97.9%) and 2ME (2- mercaptoethanol test) (85.0%, 95% CI: 79.6-89.1%), respectively; while for DSp, the FPA (fluorescence polarization assay) (99, 7%, 95% CI: 99.5-99.8%) and PCFIA tests (protein concentration fluorescence immunoassay) (78.5%, 95% CI: 70.0-85.1%) showed better and worse performance, respectively. Overall, our results showed an overestimation in the DSe and DSp of the eleven serological tests assessed when casecontrol studies were included in the meta-analysis, which is a concern considering its impacts on the time and costs associated with populational diagnosis of the diseases, since several of these tests are routinely used in the control and eradication programs of bovine brucellosis worldwide. Furthermore, the tests that exhibited the best DSe and DSp, iELISA (BS) and FPA, respectively, are relatively easy to perform and interpret and the test which showed the best overall accuracy was FPA.
Assuntos
Brucelose Bovina , Brucelose , Doenças dos Bovinos , Bovinos , Animais , Sensibilidade e Especificidade , Brucelose Bovina/diagnóstico , Imunoensaio de Fluorescência por Polarização/métodos , Imunoensaio de Fluorescência por Polarização/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Testes Sorológicos/veterinária , Brucelose/diagnóstico , Brucelose/veterinária , Anticorpos AntibacterianosRESUMO
Bovine brucellosis diagnosis is a major problem to be solved; the disease has a tremendous economic impact with significant losses in meat and dairy products, besides the fact that it can be transmitted to humans. The sanitary measures instituted in Brazil are based on disease control through diagnosis, animal sacrifice, and vaccination. Although the currently available diagnostic tests show suitable quality parameters, they are time-consuming, and the incidence of false-positive and/or false-negative results is still observed, hindering effective disease control. The development of a low-cost, fast, and accurate brucellosis diagnosis test remains a need for proper sanitary measures at a large-scale analysis. In this context, spectroscopy techniques associated with machine learning tools have shown great potential for use in diagnostic tests. In this study, bovine blood serum was investigated by UV-vis spectroscopy and machine learning algorithms to build a prediction model for Brucella abortus diagnosis. Here we first pre-treated the UV raw data by using Standard Normal Deviate method to remove baseline deviation, then apply principal component analysis - a clustering method - to observe the group formation tendency; the first results showed no clustering tendency with a messy sample score distribution, then we properly select the main principal components to improve clusterization. Finally, by using machine learning algorithms (SVM and KNN), the predicting models achieved a 92.5% overall accuracy. The present methodology provides a test result in an average time of 5 min, while the standard diagnosis, with the screening and confirmatory tests, can take up to 48 h. The present result demonstrates the method's viability for diagnosing bovine brucellosis, which can significantly contribute to disease control programs in Brazil and other countries.
Assuntos
Brucella abortus , Brucelose Bovina , Animais , Bovinos , Humanos , Brucelose Bovina/diagnóstico , Testes Sorológicos , BrasilRESUMO
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.
Assuntos
Brucelose Bovina , Brucelose , Doenças dos Bovinos , Feminino , Bovinos , Animais , Brucella abortus , Leite/química , Reprodutibilidade dos Testes , Lipopolissacarídeos , Anticorpos Antibacterianos/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Brucelose/veterinária , Imunoglobulina G , Anticorpos Monoclonais , Zoonoses , Sensibilidade e Especificidade , Brucelose Bovina/diagnóstico , Brucelose Bovina/epidemiologia , Doenças dos Bovinos/diagnósticoRESUMO
A brucelose é uma doença bacteriana de grande importância para a economia pecuária e para a saúde pública por se tratar de uma zoonose. É uma doença infecto-contagiosa que tem com agente etiológico bactérias do gênero Brucella. Em bovinos, as espécies do gênero é a Brucella abortus, que são cocobacilos gram negativo, intracelulares facultativos, imóveis e não esporulado. A infecção apresenta evolução crônica e acomete animais de todas as idades, sendo mais frequente em indivíduos sexualmente maduros. O objetivo desse trabalho é investigar, por meio da sorologia para brucelose bovina, utilizando a técnica do ELISA indireto, amostras de animais reagentes abatidos em frigoríficos inspecionados no estado da Bahia. Foram utilizados 666 animais, selecionados aleatoriamente no momento do abate. O sangue foi coletado com finalidade de obtenção de soro, todas as amostras foram submetidas à prova de triagem do Antígeno Acidificado Tamponado (AAT), prova do 2mercaptoetanol (2-ME) e ELISA Indireto. Das amostras reagentes no teste do AAT, obteve-se uma prevalência estimada em 1,2%. A prevalência no teste do ELISA foi de 13,21% (n=86). Esse resultado sugere a ocorrência de falsos negativos quando se utiliza a prova do antígeno acidificado tamponado.
Brucellosis is a bacterial disease of great importance to the livestock economy and to public health because it is a zoonosis. It is an infectious disease that has etiologic agent with bacteria of the genus Brucella. In cattle, the species of the genus Brucella is Brucella abortus that are gram negative, facultative intracellular, real estate and not sporulated. The infection presents chronic and affects animals of all ages, being more frequent in sexually mature individuals. This study aimed to investigate through serology for brucellosis, using the technique of indirect ELISA, samples from positive animals slaughtered in slaughterhouses inspected in the state of Bahia. A total of 666 animals were used, randomly selected at the time of slaughter. Blood was collected in order to obtain serum, all samples were subjected to a screening test Antigen Buffered Acidified (AAT), proof of 2-mercaptoethanol (2-ME) and Indirect ELISA. Of reagents in the test samples of AAT obtained an estimated prevalence of 1.2%. The prevalence in the ELISA test was 13.21% (n = 86). This result suggests the occurrence of false negatives when using the buffered acidified antigen test.
Assuntos
Animais , Bovinos , Brucella abortus , Brucelose Bovina/diagnóstico , Bovinos/anormalidades , Ensaio de Imunoadsorção Enzimática/veterinária , Zoonoses Bacterianas/diagnóstico , PrevalênciaRESUMO
AIMS: Bovine brucellosis is a worldwide zoonotic disease that causes important economic losses and public health concerns. Because control of the disease depends on vaccination, serodiagnosis and isolation of the infected animals, affordable, rapid and accurate point of care (POC) tests are needed. METHODS AND RESULTS: We developed and evaluated a novel glycoprotein-based immunochromatographic test for the detection of IgG antibodies against the O-polysaccharide of Brucella in bovine serum samples. Brucella GlycoStrip combines the power of immunochromatographic and bacterial glycoengineering technologies for the diagnosis of bovine brucellosis. The analysis of positive and negative reference samples indicated that the test has a diagnostic sensitivity and specificity of 96.9% (95% CI: 92.7%-100.0%) and 100%, respectively. CONCLUSIONS: Due to the recombinant glycoprotein-based antigen OAg-AcrA, which consists of the O-side chain of Brucella smooth lipopolysaccharide (sLPS) covalently linked to the carrier protein AcrA, the test is highly accurate, allows the differentiation of infected animals from those vaccinated with a rough strain or with a single dose of a smooth strain and fulfil the minimum diagnostic requirements established by the national and international regulations. SIGNIFICANCE AND IMPACT OF STUDY: This strip test could provide a rapid (10 min) and accurate diagnosis of bovine brucellosis in the field contributing to the control of the disease.
Assuntos
Brucella , Brucelose Bovina , Brucelose , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias , Brucelose/diagnóstico , Brucelose Bovina/diagnóstico , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/veterináriaRESUMO
Cervical bursitis is a suggestive lesion of bovine brucellosis. Diagnostic sensitivity and specificity of two brucellosis serological tests, Rose Bengal (RB) and serum agglutination test with 2-mercaptoethanol (SAT/2-ME), and of isolation and identification (bacteriology) were evaluated through Bayesian latent class analysis (BLCA). A total of 165 paired serum and cervical bursitis samples detected at inspection by Brazilian federal meat inspection services were analyzed. The best model fit to the data occurred when accounting for the conditional dependence between serological tests. According to this model, RB and SAT/2-ME had almost the same sensitivity, 0.960 [0.903 - 0.992] and 0.963 [0.906 - 0.994] with 95 % Credible Interval (95 %CrI), respectively. Specificities were 0.9068 [0.562 - 0.997] and 0.875 [0.546 - 0.990] for RB and SAT/2-ME, respectively, also with 95 %CrI. Bacteriology had lower sensitivity than serological tests, 0.594 (95 %CrI: [0.525 - 0.794]) and the highest specificity of all evaluated tests, 0.992 (95 %CrI: [0.961-1.00]). Prevalence of infected animals was 0.829 (95 %CrI: [0.700-0.900]). BLCA showed that both RB and SAT/2-ME fitted to the purpose of initial screening the brucellosis suspect in carcasses with cervical bursitis in a reliable way. The results of RB or SAT/2-ME can guide the sanitary actions for brucellosis control and help the implementation of a risk-based surveillance system in the meat production chain. This strategy is especially true in remote areas with large beef cattle herds, raised extensively, where in vivo tests are rarely performed due to logistic and management constraints, as in the Northern region of Brazil.
Assuntos
Brucelose Bovina , Brucelose , Doenças dos Bovinos , Testes de Aglutinação/veterinária , Animais , Anticorpos Antibacterianos , Teorema de Bayes , Brasil/epidemiologia , Brucelose/diagnóstico , Brucelose/epidemiologia , Brucelose/veterinária , Brucelose Bovina/diagnóstico , Brucelose Bovina/epidemiologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Testes Diagnósticos de Rotina , Carne , Sensibilidade e EspecificidadeRESUMO
A cross-sectional study was conducted in Colombia to recover Brucella spp. DNA from bovine whole-blood samples through probe-based real-time PCR (qPCR). By an SNP-based assay, vaccine strains were differentiated from field strains. The associated factors were evaluated using logistical regression models. A total of 656 random cows from 40 herds were selected and analyzed using serology and PCR. The qPCR assay detected 9.5% (n = 62/656; 95% CI: 7.3, 12.0) of the animals with Brucella-DNA presence, while the serological test detected a 6.6% (n = 43/656; CI: 4.8, 8.7). 62.5% (n = 25/40; 95% CI: 45.8, 77.3) of positive cases were detected at the herd-level by the qPCR, while only 27.5% (n = 11/40; 95% CI: 14.6, 43.9) were detected by the serological test. All positive samples were identified as field Brucella strains employing the SNP-based assay. In the final regression model at the animal-level, five variables were associated with Brucella-DNA presence: the use of bulls for mating recorded history of reproductive problems, pregnant cows, parlor milking, and cows belonging to farms ≤200 m from the main road. At the herd-level, two variables were associated with Brucella-DNA presence: recorded history of reproductive problems and the use of bulls for mating. Given the fluctuant brucellosis prevalence in endemic areas, updated epidemiological studies are necessary to evaluate the disease dynamic and if established prevention and control measures have been effective or need to be adjusted. The increase in the prevalence of brucellosis in animal reservoirs creates an important risk of transmission in humans.
Assuntos
Brucella , Brucelose Bovina , Animais , Anticorpos Antibacterianos , Brucella/genética , Brucelose Bovina/diagnóstico , Brucelose Bovina/epidemiologia , Bovinos , Colômbia/epidemiologia , Estudos Transversais , Feminino , Masculino , Gravidez , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Fatores de RiscoRESUMO
Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.
Assuntos
Vacina contra Brucelose/análise , Brucella abortus/isolamento & purificação , Brucelose Bovina/diagnóstico , Brucelose Bovina/prevenção & controle , Proteínas de Fluorescência Verde/análise , Animais , Vacina contra Brucelose/uso terapêutico , Bovinos/microbiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fluorescência , Proteínas de Fluorescência Verde/uso terapêutico , Camundongos , Reação em Cadeia da Polimerase Multiplex , Vacinação/veterináriaRESUMO
This study aimed to determine whether cows detected as tuberculosis (bTB) reactors and seropositive to brucellosis (bBR), as well as co-positive to bBR and bTB (bBR-bTB) and with a complete lactation before slaughter, were associated with reduced milk production and fertility. A total of 8068 productive and reproductive records of high-yielding Holstein cows from a single large dairy herd with a high prevalence of bTB and bBR were collected from 2012 to 2015. Lactation derived either from calving (n = 6019) or hormonally induced lactation (n = 2049), and all cows received growth hormone throughout lactation. For cows not induced into lactation, pregnancy rate to first service for healthy cows (C; 26.6%) was higher (P < 0.01) than bBR (15.2%), bTB (15.8%), and bBR-bTB (1.3%) cows. For induced cows, pregnancy rate to first service did not differ significantly among C, bBR, and bTB (14.5-17.3%) cows, but the percentage success of first service was extremely low (1.3%; P < 0.01) in bBR-bTB cows. Services per pregnancy (only pregnant cows) were lowest for C (3.3 ± 2.9; P < 0.01) and highest (6.4 ± 3.4) for bBR-bTB non-induced cows. This variable was lowest for C (2.9 ± 2.5; P < 0.01) and highest for bBR-bTB non-induced cows (6.3 ± 3.1). Pregnancy rate to all services did not differed for C (79.5%), bBR (76.7%), and bTB (75.9%) but was lower (58.9%; P < 0.01) for bBR-bTB non-induced cows. For induced cows this variable was highest for bBR (53.3%) and lowest for bBR-bTB (34.1%; P < 0.01) non-induced cows. 305-d milk production was increased by 4%, and total milk yield by 7% in TB-positive cows compared to that of the negative cows non-induced hormonally into lactation. This study showed the negative impact of the co-positivity for bTB and bBR on the reproductive efficiency of Holstein cows, although positive bTB and bBR tests enhanced milk yield.
Assuntos
Brucelose Bovina/diagnóstico , Doenças dos Bovinos , Fertilidade , Leite , Tuberculose Bovina/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Feminino , Lactação , GravidezRESUMO
Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12.
Assuntos
Vacina contra Brucelose/genética , Vacina contra Brucelose/imunologia , Brucelose Bovina/diagnóstico , Brucelose Bovina/prevenção & controle , Marcadores Genéticos , Vacinas Sintéticas , Animais , Vacina contra Brucelose/administração & dosagem , Brucelose Bovina/imunologia , Brucelose Bovina/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Citocinas/metabolismo , Diagnóstico Diferencial , Modelos Animais de Doenças , Engenharia Genética , Imunização , Imunogenicidade da Vacina , Camundongos , Avaliação de Resultados em Cuidados de Saúde , Proteômica/métodos , Espectrometria de Massas em Tandem , VirulênciaRESUMO
Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Brucella/imunologia , Brucelose Bovina/diagnóstico , Brucelose/diagnóstico , Brucelose/veterinária , Doenças das Cabras/diagnóstico por imagem , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Reações Cruzadas , Doenças das Cabras/diagnóstico , Cabras , Humanos , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologiaRESUMO
Objetivou-se avaliar a ocorrência de animais soro reagentes à brucelose bovina em fazendas localizadas no Estado de Mato Grosso do Sul, por meio de exame sorológico utilizando o Antígeno Acidificado Tamponado (AAT) e discutir as possíveis diferenças entre as soroprevalências de fêmeas e machos. Foram avaliados, a partir do teste de triagem com Antígeno Acidificado Tamponado (AAT), 724 bovinos da raça Nelore, sendo 274 machos e 450 fêmeas, provenientes de oito propriedades com histórico de problemas reprodutivos. O teste foi procedido conforme o protocolo determinado pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA). Os resultados demonstraram baixa soroprevalência da doença nos bovinos testados, sendo detectada prevalência para a doença de 1,10% nos machos e 2,88% nas fêmeas.Quando se considera o touro isoladamente nos rebanhos, pode-se perceber que a fertilidade é muito mais importante nos machos do que nas fêmeas individualmente, uma vez que os touros podem se acasalar com um número muito maior de fêmeas, seja na monta natural ou na inseminação artificial, demonstrando a importância do inquérito epidemiológico na população geral, principalmente nos machos. A maior frequência da doença foi encontrada nas fêmeas podendo estar relacionada à infecção por Brucella spp. no ambiente decorrente de parto ou aborto tornando as fêmeas transmissoras permanentes da doença.
The objective of this study was to evaluate the occurrence of seroreactive animals to bovine brucellosis in farms located in the State of Mato Grosso do Sul, by means of a serological examination using the Acidified Buffered Antigen (AAT) and to discuss the possible differences between the seroprevalence of females and males. A total of 724 Nellore cattle, 274 males and 450 females, from eight farms with a history of reproductive problems, were evaluated using the screening test with Acidified Buffered Antigen (AAT). The test was carried out according to the protocol determined by the Ministry of Agriculture, Livestock and Food Supply (MAPA).The results showed a low seroprevalence of the disease in the tested cattle, with a prevalence of 1.10% in males and 2.88% in females.When considered the bull alone in herds, it can be shown that fertility is much more important in males than in females individually, since bulls can mate with a much larger number of females, either in natural mating or in artificial insemination, demonstrating the importance of epidemiological survey in the general population, especially in males. The highest frequency of the disease was found in females and may be related to infection by Brucella spp. in the environment from childbirth or abortion making females permanent transmitters of the disease.
Assuntos
Animais , Bovinos , Brucella abortus/patogenicidade , Brucelose Bovina/diagnóstico , Testes Sorológicos/veterinária , Doenças dos Bovinos/diagnóstico , Estudos Soroepidemiológicos , Inquéritos Epidemiológicos/métodos , Aborto Animal/patologia , FazendasRESUMO
Brucellosis is a zoonotic disease of great animal welfare and economic implications worldwide known since ancient times. The emergence of brucellosis in new areas as well as transmission of brucellosis from wild and domestic animals is of great significance in terms of new epidemiological dimensions. Brucellosis poses a major public health threat by the consumption of non-pasteurized milk and milk products produced by unhygienic dairy farms in endemic areas. Regular and meticulous surveillance is essentially required to determine the true picture of brucellosis especially in areas with continuous high prevalence. Additionally, international migration of humans, animals and trade of animal products has created a challenge for disease spread and diagnosis in non-endemic areas. Isolation and identification remain the gold standard test, which requires expertise. The advancement in diagnostic strategies coupled with screening of newly introduced animals is warranted to control the disease. Of note, the diagnostic value of miRNAs for appropriate detection of B. abortus infection has been shown. The most widely used vaccine strains to protect against Brucella infection and related abortions in cattle are strain 19 and RB51. Moreover, it is very important to note that no vaccine, which is highly protective, safe and effective is available either for bovines or human beings. Research results encourage the use of bacteriophage lysates in treatment of bovine brucellosis. One Health approach can aid in control of this disease, both in animals and man.
Assuntos
Brucella , Brucelose Bovina/diagnóstico , Brucelose Bovina/prevenção & controle , Animais , Vacinas Bacterianas , Brucelose Bovina/epidemiologia , Brucelose Bovina/transmissão , Bovinos , Humanos , MicroRNAs , ZoonosesRESUMO
INTRODUCTION: a cross-sectional study was conducted to determine the seroprevalence and associated factors of brucellosis and Q-fever among cattle in a rural setting in Oyo State, Nigeria. METHODS: one hundred and fourty nine serum samples (24 males; 125 female) from 16 cattle herds were collected and screened. The Rose Bengal Plate test (RBPT) and competitive Enzyme-Linked Immunosorbent Assay (cELISA) were used for brucellosis while indirect Enzyme-Linked Immunosorbent Assay (iELISA) was used for Q-fever. Further, a checklist was used to collect data on cattle sampled. Data were analyzed using STATA 12. RESULTS: serum analysis revealed that 11.4% (17/149) and 6.7% (10/149) were seropositive by RBPT and cELISA respectively for brucellosis, while 23.5% (35/149) were seropositive by iELISA for Q-fever. A significant association was detected between cattle age (OR=27.7; 95% CI: 2.34-449.86), herd size (OR=10.53; 95% CI: 1.85-60.53) and seropositivity to Brucella infection. Also, there was a significant association between breed (OR=6.69; 95% CI: 1.7-28.74), herd size (OR=4.25; 95% CI: 1.31-13.85) of cattle and seropositivity to Coxiella burnetii infection. Importantly, the only significant associated factor to cattle herd seropositivity to Brucella and C. burnetii infections was the method of handling aborted foetuses. CONCLUSION: the study revealed that brucellosis and Q-fever are prevalent among cattle in the study area. Thus, there is a need for further studies to provide better insight into the epidemiology of both diseases and particularly Q-fever. This becomes imperative in the study area and generally in Nigeria given the dearth of information about the diseases in pastoralist communities who are at grave risk of infection at the human-animal-ecosystem interface.
Assuntos
Brucelose Bovina/epidemiologia , Febre Q/epidemiologia , Animais , Brucelose Bovina/diagnóstico , Bovinos , Estudos Transversais , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Masculino , Nigéria/epidemiologia , Febre Q/diagnóstico , Febre Q/veterinária , Rosa Bengala , Estudos SoroepidemiológicosRESUMO
This study was conducted to evaluate the sensitivity (Se) and specificity (Sp) of the Rose Bengal test (RBT), complement fixation test (CFT), the serum lateral flow assay (LFAserum) and the blood lateral flow assay (LFAblood) for the detection of antibodies to Brucella spp. using Bayesian latent class models (BLCMs). Sera and whole blood were collected from naturally infected cattle reared in smallholder, small-scale commercial and large-scale commercial farms in Zimbabwe (n = 1022) and Botswana (n = 770). The BLCMs were fitted under the assumption that conditional dependences existed between the tests. Based on the conditional dependence model, the RBT had the highest Se of 0.897 (95 % Probability Intervals: 0.854; 0.932) compared to 0.827 (0.773; 0.872), 0.812 (0.76; 0.858) and 0.809 (0.785; 0.832) for the LFAserum, LFAblood and CFT, respectively. The CFT recorded a higher Sp of 0.999 (0.995; 1.000) than the LFAserum 0.996 (0.99; 1.000), the LFAblood 0.984 (0.976; 0.991) and the RBT 0.969 (0.959; 0.978). The data indicated that both the Se and Sp of RBT and CFT and the Sp of LFAserum and LFAblood were conditionally independent, while the Se appeared to be conditionally dependent. These results indicated that none of the evaluated tests had perfect Se and Sp and consequently could not be used alone for the diagnosis of brucellosis in cattle from the studied farming sectors. Thus, based on high Se and Sp, respectively, a brucellosis testing regimen using the RBT (screening) and the LFA (confirmatory) may be considered.
Assuntos
Análise Química do Sangue/veterinária , Brucelose Bovina/diagnóstico , Testes de Fixação de Complemento/veterinária , Rosa Bengala/química , Animais , Teorema de Bayes , Botsuana , Bovinos , Análise de Classes Latentes , Sensibilidade e Especificidade , ZimbábueRESUMO
This study was designed to compare a milk I-ELISA with a serum ELISA for the diagnosis of brucellosis in dairy cattle and then to use the milk I-ELISA to determine the prevalence and incidence of brucellosis in dairy herds in Hubei Province, China. The two tests were shown to have good agreement with a Cohen's kappa statistic of 0.747 (p < 0.001) when 147 animals originating from 4 dairy herds in the province were tested. The results of Bayesian Latent Class Analysis estimated that the sensitivity and specificity of the milk I-ELISA under field conditions were 87.2 % and 92.0 %, respectively. An epidemiological survey based on the milk I-ELISA was then conducted in 3091 cows from 15 commercial dairy herds from January to July 2018 in Hubei Province. The animal level real prevalence varied from 34.9 % (95 % CI: 28.5, 41.8) to 51.4 % (95 % CI: 48.2, 54.6) in the 15 herds tested. Most farms (93.3 %) tested contained at least one test-positive animal. As only ten farms met the inclusion criteria for the calculation of incidence risk, the overall real incidence risk in 10 of these farms was 0.4 % (95 % CI: 0.1, 1.2) per 3 months, which highlights the potential for spread of the disease within infected herds. It is concluded that the milk I-ELISA test could be used as a rapid screening test for brucellosis in unvaccinated dairy cows and, given the high occurrence of bovine brucellosis in this study, an effective prevention and control program needs to be developed and implemented in Hubei Province, China.
Assuntos
Brucelose Bovina/epidemiologia , Testes Diagnósticos de Rotina/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Leite/química , Soro/química , Animais , Brucelose Bovina/diagnóstico , Bovinos , China/epidemiologia , Indústria de Laticínios , Testes Diagnósticos de Rotina/instrumentação , Ensaio de Imunoadsorção Enzimática/instrumentação , Incidência , PrevalênciaRESUMO
Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.
Assuntos
Vacina contra Brucelose , Brucelose Bovina , Brucelose , Animais , Anticorpos Antibacterianos , Brucella abortus , Brucelose Bovina/diagnóstico , Brucelose Bovina/prevenção & controle , Bovinos , Escherichia coli , Testes SorológicosRESUMO
BACKGROUND: Brucellosis is a zoonosis whose incidence is not declining worldwide despite the global effort to control the disease. Accurate and precise diagnosis is a crucial step in any prophylaxis program but single tests to unequivocally detect animals infected with Brucella spp. are currently unavailable. In Italy, serological diagnosis of bovine brucellosis is performed with two official tests: a rapid agglutination test (i.e., Rose Bengal Plate test, RBPT) and a complement fixation test (CFT) that detect antibodies directed mainly to the smooth lipopolysaccharide (S-LPS). Neither of the two tests is able to avoid the detection of false positive serological reactions (FPSRs) caused by bacteria sharing S-LPS components with Brucella spp. and responsible for the single reactors (SR) phenomenon. A B. melitensis R strain-based ELISA showed a good diagnostic performance in unravelling FP animals; however, since a limited number of animals were analyzed in that study, a large field study was conducted here to discriminate between Brucella-infected from FP animals, with the final aim of reducing the unnecessary slaughter of the latter. An ELISA based on a R strain of Brucella, i.e., Brucella melitensis B115, was employed to measure specific IgG responses in a collection of bovine sera (n = 648). Sera were obtained from 180 farms (either officially brucellosis-free or not brucellosis-free) recruited during an extended period of time (2014-2018) and were preliminarily assayed with the official tests by the Italian Reference Centers and then subjected to the ELISA. RESULTS: Negative sera, when subjected to the ELISA, gave O.D. values below the cutoff; SR sera, i.e. RBPT positive and CFT negative, as well as double positive (DP) sera, i.e. RBPT and CFT positive, gave O.D. values that were below the cutoff. All positive sera, i.e. from Brucella-infected animals, were RBPT positive and CFT positive (ICFTU ranging from 20 to 1280) and gave ELISA O.D. values above the cutoff. CONCLUSIONS: The B. melitensis B115-based ELISA systematically unravelled all false positive (FP) sera while confirming the diagnosis in Brucella-infected animals. Thus, the test employed in the present study may complement the official assays to avoid the costly slaughter of FP animals.