Diagnosis Of Bovine Brucellosis Research Articles

Bovine brucellosis in Pakistan; an analysis of engagement with risk factors in smallholder farmer settings.

A cross‐sectional study was conducted to estimate the seroprevalence of bovine brucellosis in smallholder farms in seven regions in Pakistan, identify herd and individual level risk factors for seropositivity and assess the level of engagement of farmers with risk factors. In total, 1063 cattle and buffalo belonging to 420 herds in seven districts were sampled. The Rose Bengal test (RBT), indirect enzyme‐linked immunosorbent assay (I‐ELISA) and competitive enzyme‐linked immunosorbent assay (C‐ELISA) were used for the serological diagnosis of bovine brucellosis on all the serum samples. The associations between herd‐and animal‐level risk factors and seropositivity were investigated using logistic regression analyses. In addition, herd management practice scores, created to quantify the number of management practices undertaken that pose a risk for Brucella transmission, were calculated and compared between seropositive and negative herds within each district. Overall herd and animal prevalence were estimated to be 16.2% (95% CI, 13–20%) and 8.7% (95% CI, 7.2–10.6%), respectively, across all districts sampled. Herds with a history of last trimester abortion were found to be more likely to be positive than herds without such history (OR = 2.06, 95% CI, 1.09–3.89), providing validation of our findings and identifying that clinical disease is occurring in this region. It was also identified that herds with five to eight buffalo (OR = 3.80, 95% CI, 1.69–8.49), and those with more than eight buffalo (OR = 3.81, 95% CI, 1.51–9.58) were more likely to be positive for Brucella than those with less (one to two and three to four) buffaloes present in the herd. The presence of other domestic animals at the farm and purchasing animals in last year were found to have no association with seropositivity. The findings of this study support the need for the development of targeted intervention strategies specific to the disease status of each district.

Development and standardization of a western blotting test for detection of antibodies against B. abortus

ABSTRACT Brucellosis is an infectious disease caused by bacteria of the genus Brucella spp. with diagnosis based on use of serological techniques. The present study aimed to develop and standardize a western blotting (WB) test for detection of antibodies against B. abortus. Samples from two groups of cattle were analyzed: group I: 60 serum samples from true positive and true negative vaccinated animals (30 positive samples from infected animals according to rose bengal test (RBT), 2-mercaptoethanol, serum agglutination test (SAT) and complement fixation test (CFT) and 30 RBT negatives samples); group II: 383 field samples (90 positive and 293 CFT negative sera). The most reactive band in the western blotting, which properly identified and separated infected from non - infected had a molecular weight of ≤ 20kDa. The sensitivity, specificity and accuracy of the WB compared to RBT was 93%, 99%, 98%, respectively and k= 0.938. When compared to CFT, the sensitivity, specificity and accuracy of the WB was 97%, 98% and 97%, respectively and k= 0.929. The WB developed and standardized in the present study is a serological test with potential use as a confirmatory test for the diagnosis of bovine brucellosis.

Comparative Evaluation of Various Diagnostic Tests in Diagnosis of Bovine Brucellosis

Comparative Evaluation of Various Diagnostic Tests in Diagnosis of Bovine Brucellosis

Trials for bovine diagnosis using molecular biology

Background: Brucellosis is a major bacterial zoonosis of global importance affecting a range of different mammals including cattle, sheep, goats, swine, rodents, marine mammals and man worldwide. In order to control brucellosis, comprehensive vaccination, surveillance and quarantine programs should be implemented. Both of control and prevention procedures are highly depending on accurate diagnostic tools and effective and safe vaccination programs. idea of this ELISA kit depends on the facts that eryC gene is absent in Brucella abortus S19 only but it is present and functional in all other brucella strains and isolates. Methods & Materials: omp31 as a DNA vaccine against Brucellosis were evaluated in Balb/C mice where eryC recombinant protein as a coating antigen for accurte diagnosis of brucellosis and differention between vaccinated and infected cattle were evaluated using IELISA aganist ELISA using hot saline extract and soluble brucella proteins as a coating antigens as a control postive. Results: results of DNA vaccine in Balb/C mice revealed a 60% protection after challange using brucella melitensis 16 M where results of ELISArevealed that in case of sera of naturally infected cattle, sero-positivity was 94.7%, 100%, 100% and 100% with eryC-ELISA, HSE-ELISA, SBP-ELISA and rose Bengal test respectively where in case of sera of S19 vaccinated cattle, all samples were negative when tested with eryC-ELISA where in case of HSE-ELISA, SBP-ELISA and Rose Bengal test, sero-positivity was 92.3%, 84.6% and 100% respectively. All sera of brucella free cattle were negative when tested with Rose Bengal test and different IELISA tests. Conclusion: The Results of this study recommended the use of omp31 DNA vaccine as a safe vaccine for control of bovine brucellosis and use of eryC protein as a good diagnostic antigen to be used in serological tests for diagnosis of bovine brucellosis and differentiation between infected and Brucella abortus S19-vaccinated cattle but more studies are needed to be done on large cattle populations accompanied with bacteriological isolation to detect the sensitivity and specificity of this protein as a diagnostic antigen and also for validate this test.

Serum based polymerase chain reaction and enzyme linked immunosorbent assays for diagnosis of bovine brucellosis

Brucellosis is one of the economically important diseases in India and diagnosis of brucellosis using single test is cumbersome due to variation in sensitivity and specificity among the different test. The present study was aimed to assess the suitability of serum as clinical specimen in molecular diagnosis and evaluate the serology and molecular assays as in diagnosis of bovine brucellosis. A total of 821 bovine sera samples were subjected to indirect Enzyme Linked Immunosorbent Assay (i-ELISA) and serum based Polymerase Chain Reaction (PCR) assay. On serology 6.70 per cent positivity of brucellosis were reported and on PCR assay, 47 and 29 sera samples were positive for bcsp 31 genus specific and IS711 species specific PCR assay respectively with per cent positivity of 5.72 and 3.53. In comparison between serology and molecular test, 44 samples were positive for both assays and 11 and 3 samples were positive for serology and molecular assays individually. This study suggests that serum sample can be utilised as the choice of clinical specimen for both PCR assay and i-ELISA will be a future choice for the diagnosis of bovine brucellosis.

Comparative Assessment of Sensitivity and Specificity of Rose Bengal Test and Modified In-house ELISA by using IS711 TaqMan Real Time PCR Assay as a Gold Standard for the Diagnosis of Bovine Brucellosis

Serological approaches such as Rose Bengal Test (RBT) and enzyme linked immunosorbent assay (ELISA) are common tests, however they are generally not sensitive or specific enough for diagnosis of Brucella because of cross-reactivity with different bacterial antigens. The work objected to evaluate the sensitivity and specificity of rose Bengal and modified in-house ELISA using IS711 real time PCR as a gold test to detect Brucella in calves sera. Two hundred and thirty (n=230) blood samples were collected from (2-3) years asymptomatic male calves in two Egyptian abattoirs. Rose Bengal test (RBT) and modified in-house ELISA were applied to determine the seroprevalence of brucellosis in abattoirs animals while quantitative Taqman real-time PCRs (RT-PCR) were implemented for the identification of Brucella genus. The overall prevalence of brucellosis was (53.9 %), (75.2 %) and (79.1 %) as determined by ELISA,RBT and RT- PCR assays respectively. Regarding statistical analysis of the reported data and considering real time PCR the gold standard, the RBT recorded a sensitivity of (79.12%) and a specificity of (39.58 %) with an accuracy of (70.87%). While (83.24%) was reported as positive predictive value and (33.33 %) as a negative predictive value. The sensitivity and specificity of ELISA were (55.49%) and (52.08 %) respectively while the accuracy was (54.78%). Positive predictive value and negative predictive value for ELISA were determined as (81.45%) and (23.58 %) respectively. RBT can be routinely used for diagnosis of Brucella as cost effective , more sensitive and accurate test than ELISA However, real time PCR is highly recommend as gold test for identification and differentiation of bovine brucellosis.

Using combined recombinant protein in the diagnosis of bovine brucellosis

The aim of this research was to obtain a combined recombinant protein consisting of immunodominant regions of Brucella outer membrane proteins (Omps) with the molecular weights of 25 kDa (Omp25) and 31 kDa (Omp31). The search for candidate proteins was carried out using NCBI PubMed and NCBI GenBank databases. The two most immunodominant regions of Omps were selected. The first region was in a position from 48 to 83 amino acids of Omp31, while the second one was in the position from 180 to 224 amino acids of Omp25. This combined sequence was designated as OmpBm-Ba. The pET32 vector was used for cloning and the expression of the combined recombinant protein in E. coli BL21(DE3). The antigenicity of recombinant OmpBm-Ba (rOmpBm-Ba) as compared with rOmp25 and rOmp31 has been tested on the sera of 109 cattle with positive results to brucellosis according to classical serological tests. The rOmpBm-Ba had a higher antigenicity than the single ones, since it confirmed the presence of Brucella-antibody in all serum samples with positive results of i-ELISA/rOmp25 and/or i-ELISA/rOmp31, as well as additionally revealing 7.3% and 31.2% seropositive animals, respectively. A comparative study of the diagnostic value of i-ELISA/rOmpBm-Ba and conventional tests on blood sera of 24 cattle subjected to a bacteriological examination at slaughter showed a higher reliability of enzyme immunoassay. Further research is necessary to get a more objective evaluation of the diagnostic accuracy of Brucella rOmpBm-Ba by challenging laboratory animals. .

Comparative efficacy of serological diagnostic methods and evaluation of polymerase chain reaction for diagnosis of bovine brucellosis.

The aim of present study was to compare the diagnostic performance of the different Brucella abortus antigen based serological and molecular tests such as Rose Bengal Plate Test (RBPT), indirect enzyme linked immunosorbent assay (I-ELISA) and polymerase chain reaction (PCR). Out of 89 samples, 44 were positive by I-ELISA, 18 by RBPT and 21 by PCR. Substantial agreement was observed between PCR and I-ELISA (κ=0.48). A slight degree of agreement was observed between RBPT and I-ELISA and PCR (κ=0.18) and RBPT and PCR (κ=0.11). Indirect enzyme linked immunosorbent assay detected more samples as positive among these tests. In conclusion, I-ELISA can be routinely used for an accurate and efficient diagnosis of Brucella infection, because the chances of non-detection of an infected animal in I-ELISA are minimal. However, PCR could be used as a supplement and complement test along with I-ELISA for identification and differentiation of bovine brucellosis.

Evaluation of three serological tests for diagnosis of bovine brucellosis in smallholder farms in Pakistan by estimating sensitivity and specificity using Bayesian latent class analysis

The present study aimed to estimate the herd-level sensitivity (Se) and specificity (Sp) of three commonly used serological tests in naturally-infected cattle and buffalo in smallholder farms in Pakistan. Between February and June 2015, a cross-sectional study was carried out in five districts of Punjab (Kasur, Okara, Pakpattan, Jhelum, and Bhakkar) and two districts of Sindh (Badin and Thatta). Serum samples from mixed farms of cattle (n=441) and buffalo (n=621) were collected and tested using the Rose Bengal test (RBT), indirect ELISA (I-ELISA) and competitive ELISA (C-ELISA). A Bayesian latent class analysis (LCA) approach was used to estimate the Se and Sp of these three serological tests and the true herd-level prevalence in each district. The model was fitted under the assumption of conditional independence between three tests and also conditional dependence by including covariances between the two ELISAs. In addition, the model was fitted using three different shapes of beta distributions to incorporate prior information in the model. The test with the highest Se was the C-ELISA, with a range from 76.3% (95% PCI (Posterior Credibility Interval), 62.6–88.2%) to 81.4% (95% PCI, 68.2–92.8%). The RBT was found to have the highest Sp (99.1–99.4%) of the tests. The highest estimated herd-level prevalence, 45% (95% PCI, 32–59%), was found in Jhelum district and the lowest in Thatta district, 1.1% (95% PCI 0.04–6.0%). The results of this study identified some discrepancy between the published literature on the level of Se of these tests, especially for RBT. It appears that RBT has lower Se and higher Sp when used in the field conditions of the present study. Consequently, it is recommended that none of the evaluated tests should be performed in isolation for the diagnosis of bovine brucellosis in the field conditions of Pakistan, but the use of tests in combination, with RBT and C-ELISA used in parallel returning optimal Se and Sp, is warranted.

Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays

The present study was carried out to evaluate the conventional and molecular techniques for diagnosis of bovine brucellosis. A total of four isolates of Brucella abortus obtained from 100 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges were characterized biochemically. The isolates were confirmed as Brucella spp. by PCR using B4/B5 primer pair and as B. abortus by Bruce Ladder multiplex PCR. By Hinic Real-time PCR, all the four isolates were confirmed as Brucella spp. with Ct values between 14-16. With DNA extracted from 40 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges, Real time PCR appeared most sensitive of the three molecular and conventional techniques as it detected maximum number of positive samples.

Comparison of diagnostic tests for the detection of bovine brucellosis in the natural cases of abortion.

Rapid and precise diagnosis in natural field cases of bovine abortion caused by Brucella abortus warrants the use of the most sensitive and reliable diagnostic methods. In the present study, bacterial isolation, serology, gross, histopathology, immunohistochemistry and polymerase chain reaction technique(s) were applied. Sero-prevalence studies showed the rate of 28.86% positive cases using the competitive ELISA. Histopathological changes were mainly seen in the placenta, fetal lungs, kidney, liver and spleen. Immunohistochemical (IHC) staining of Brucella spp. was evident as brown, finely granular intracytoplasmic staining in trophoblasts of placental sections and in section(s) of liver, lung, kidney and spleen. Twenty-eight out of the 103 samples (17 from stomach contents, 3 from placental cotyledons, 2 from vaginal discharges and 6 from pooled fetal tissues) produced 193 bp amplicon specific for Brucella genus. Moreover, the species-specific primers amplified a 498 bp amplicon which corresponded to B. abortus. Comparison of diagnostic tests revealed PCR and IHC provide a reliable test for the diagnosis of bovine brucellosis in aborted fetal tissue and placental cotyledons whereas serology is most important for detection of Brucella positive animals in a herd.

Performance of microbiological, serological, molecular, and modified seminal plasma methods in the diagnosis of Brucella abortus in semen and serum of bovine bulls

Brucellosis remains as a major infectious disease of domestic animals and is considered a re-emerging zoonosis in several countries. B. abortus infections in bulls are related to reproductive tract infections, although infected animals show transient serological titers or nonreactor status. Thus, diagnosis of bovine brucellosis based exclusively on serological tests probably underestimates B. abortus infections in bulls. In this scenario, three hundred thirty-five serum samples from reproductively mature bovine bulls were subjected simultaneously to standard serodiagnosis using the rose Bengal test (RBT), 2-mercaptoethanol (2-ME), complement fixation (CFT), and fluorescence polarization assay (FPA). Furthermore, conventional semen plasma agglutination (SPA) and modified 2-ME, FC and, FPA were carried out in all bulls replaing serum by seminal plasma. Semen from all bulls was also analyzed for sperm viability, microbiological culture in Farrell media, and polymerase chain reaction (PCR). Only eight (2.38%) semen samples were considered improper for reproduction services (necrospermia and azoospermia), although none of these animals was positive in any of the diagnosis methods used. Five bulls (1.49%) were simultaneously positive in conventional RBT, 2-ME, SPA, modified 2-ME, microbiological culture in Farrell media, and in PCR for B. abortus strain 19. Two (1.67%) bulls were positive in PCR for B. abortus field strains and negative in all other tests, although semen was considered viable to reproduction service. The identification of B. abortus B19 strain in serum and semen of bulls occurred probably due to improper vaccination of males or infection by B19 strain shedding by vaccinated females that could to contaminated environment of farms. In addition, detection of B. abortus field strains only using PCR in bulls without sperm viability abnormalities indicate the need for including molecular methods to improve diagnosis of the disease in bovine bulls.

Validation of non-commercial indirect ELISA techniques in the diagnosis of bovine brucellosis.

Ninety one blood samples were collected from unvaccinated cattle with a history of Brucella melitensis biovar 3 infection and belonged to the Nile Delta region during slaughter in abattoirs. Serum samples were separated and sample size were estimated according to OIE requirements (2% errors and 95% confidence interval) for the validation of two developed indirect enzyme linked immunosorbent assay (iELISA) techniques using different antigens namely lipopolysaccharide antigen (LPS) and soluble periplasmic protein antigen (PPP) against an iELISA (LPS) commercial kit. Diagnostic performance characteristics were estimated considering complement fixation test (CFT) as the gold standard. The estimated relative sensitivities (Se) and specificities (Sp) of the three iELISA versions were as follows: iELISA (PPP) (88% and 94%), iELISA (LPS) (92% and 87%) commercial kit and home-made iELISA (LPS) (90% and 84%). The estimated κ agreement values with the CFT indicated substantial agreement in case of iELISA versions used both LPS and PPP antigens. The performance of iELISA based on both ROCs and AUCs was very good being equal to or closer to 0.9. Performance indices (PI) values were charted in ascending order. The accuracy % was calculated to be 90% for both iELISA (PPP) and iELISA (LPS) kit and 88% for home-made iELISA (LPS). Highest positive predictive values were achieved by iELISA (PPP), while the highest negative predictive values were achieved by iELISA (LPS) standard kit. The highest positive likelihood ratio (LR+) was attained by iELISA (PPP), while the negative likelihood ratio (LR-) was almost the same. Authors suggest that the home-made iELISA (LPS) is fit for its purpose as a rapid screening test. The high performance characteristics of iELISA (PPP), especially the balance of both Se and Sp, necessitates a reclassification of this version as confirmatory rather than screening test with further studies.

Determining a Diagnostic Cut-Off on Fluorescence Polarization Assay (FPA) for Bovine Brucellosis in Carchi, Ecuador

Serology is the foundation of any brucellosis control and eradication program worldwide, thus it is important to define accuracy diagnostics assays and cut-off of those assays, due to variations from country to country and even among specific areas in the country. The variation of cutoff values depended on: prevalence of disease, vaccination status, animal management, and control and eradication programs. Therefore, a cut-off for the diagnosis of bovine brucellosis through fluorescence polarization assay (FPA) in Carchi—Ecuador was determined. The survey has been carried out in Carchi province of Ecuador, who is considered a province of high prevalence of brucellosis and the vaccination status is unknown due to the lack of registers. Sera samples (n = 200) were obtained from individual cows from randomly selected herds. Blood sera were tested through Fluoresce Polarization Assay (FPA) and competitive enzyme-linked inmunosorbent assay (cELISA) as confirmatory test, and then receiver operating characteristic (ROC) analysis was done. The sensitivity and specificity values of FPA were 88.7% and 92.50% respectively using a cut-off of 89.90 mP. Moreover, the area under the curve showed that 92.2% is the probability accuracy of the test. The advantage of the FPA is that it is a test with good characteristics of sensitivity and specificity as well as a simple and quick test.

Comparative evaluation of three serological tests for the detection of Brucella antibodies from infected cattle herds

ABSTRACTThree serological methods used in the diagnosis of bovine brucellosis were conducted to assess the diagnostic sensitivity of the tests in brucellosis-infected herds. A total of 487 unvaccinated bovine serum samples collected from infected herds in the southeast region of Turkey having history of abortion with a known bacteriological status for the last three years were tested for anti-Brucella antibodies detection using the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and indirect enzyme-linked immunosorbent assay (i-ELISA). A protein A/G conjugate was used in i-ELISA in order to reduce background reactivity and to use the test in multispecies if required. The serum samples were found as positive in 74.3%, 83.7% and 81.3% when we employed the CFT, i-ELISA and RBPT, respectively. i-ELISA could be used as a single test or combined with the RBPT for serologic diagnosis for brucellosis in infected herds.

Development of Indirect ELISA (IELISA) for Diagnosis of Bovine Brucellosis, Comparison of Three Different Labeled Detection Reagents

Development of Indirect ELISA (IELISA) for Diagnosis of Bovine Brucellosis, Comparison of Three Different Labeled Detection Reagents

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

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

Bayesian Estimation of Sensitivity and Specificity of Rose Bengal, Complement Fixation, and Indirect ELISA Tests for the Diagnosis of Bovine Brucellosis in Ethiopia.

Test evaluation in the absence of a gold standard test was conducted for the diagnosis and screening of bovine brucellosis using three commercially available tests including RBPT, CFT, and I-ELISA in National Animal Health Diagnostic and Investigation Center (NAHDIC) Ethiopia. A total of 278 sera samples from five dairy herds were collected and tested. Each serum sample was subjected to the three tests and the results obtained were recorded and the test outcomes were cross-classified to estimate the sensitivity and specificity of the tests using Bayesian model. Prior information generated on the sensitivity and specificity of bovine brucellosis from published data was used in the model. The three test-one population Bayesian model was modified and applied using WinBug software with the assumption that the dairy herds have similar management system and unknown disease status. The Bayesian posterior estimate for sensitivity was 89.6 (95% PI: 79.9–95.8), 96.8 (95% PI: 92.3–99.1), and 94 (95% PI: 87.8–97.5) and for specificity was 84.5 (95% PI: 68–94.98), 96.3 (95% PI: 91.7–98.8), and 88.5 (95% PI: 81–93.8) for RBT, I-ELISA, and CFT, respectively. In this study I-ELISA was found with the best sensitivity and specificity estimates 96.8 (95% PI: 92.3–99.1) and 96.3 (95% PI: 91.7–98.8), compared to both CFT and RBPT.

Serological diagnosis of bovine brucellosis using B. melitensis strain B115

Bovine brucellosis is diagnosed by official tests, such as Rose Bengal plate test (RBPT) and Complement Fixation test (CFT). Both tests detect antibodies directed against the lipolysaccharide (LPS) of Brucella cell wall. Despite their good sensitivity, those tests do not discriminate between true positive and false positive serological reactions (FPSR), the latter being generated by animals infected with other Gram negative microorganisms that share components of Brucella LPS. In this study, an antigenic extract from whole Brucella melitensis B115 strain was used to set up an ELISA assay for the serological diagnosis of bovine brucellosis. A total of 148 serum samples from five different groups of animals were tested: Group A: 28 samples from two calves experimentally infected with Yersinia enterocolitica O:9; Group B: 30 samples from bovines infected with Brucella abortus; Group C: 50 samples from brucellosis-free herds; Group D: 20 samples RBPT positive and CFT negative; Group E: 20 samples both RBPT and CFT positive. Group D and Group E serum samples were from brucellosis-free herds.Positive reactions were detected only by RBPT and CFT in calves immunized with Y. enterocolitica O:9. Sera from Group B animals tested positive also in the ELISA assay, whereas sera from the remaining groups were all negative. The results obtained encourage the use of the ELISA assay to implement the serological diagnosis of brucellosis.

Evaluation of recombinant AspC of Brucella abortus for serological diagnosis of bovine brucellosis in Korea

Evaluation of recombinant AspC of Brucella abortus for serological diagnosis of bovine brucellosis in Korea