Diagnosis Of Bovine Brucellosis Research Articles

Use of recombinant malate dehydrogenase (MDH) and superoxide dismutase (SOD) [Cu[sbnd]Zn] as antigens in indirect ELISA for diagnosis of bovine brucellosis

The objective of this study was to validate an indirect enzyme-linked immunoassay (iELISA) using the recombinant proteins, malate dehydrogenase (MDH) and superoxide dismutase (SOD) [CuZn], as antigens and to evaluate its ability to discriminate antibodies produced by vaccination from those induced by infection in the diagnosis of bovine brucellosis. Sera from six groups were evaluated: G1 – culture-positive animals (52 serum samples) (naturally infected); G2 - non-vaccinated animals (28 serum samples) positive in RBT (Rose Bengal test) and 2ME (2-mercaptoethanol test) selected from brucellosis-positive herds; G3 - animals from a brucellosis-free area (32 serum samples); G4 - S19 vaccinated heifers (114 serum samples); G5 – RB51 vaccinated heifers (60 serum samples); G6 - animals inoculated with inactivated Yersinia enterocolitica O:9 (42 serum samples). Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were estimated using the frequentist approach and the confidence interval (CI) (95%) calculated by the Clopper-Pearson (exact) method. The DSe for iELISA_MDH in the G1 group was 71.7% (CI 95%: 57.6–83.2%) and for the G2 100.0% (CI 95%: 87.7–100.0%), whereas the DSp was 84.4% in the G3 (CI 95%: 67.2–94.7%). For the iELISA_SOD the DSe was estimated 67.3% for the G1 (CI 95%: 52.9–79.7%) and 71.4% for G2 (CI 95%: 51.3–86.8%), while the DSp for G3 was 87.5% (CI 95%: 71.0–96.5%). iELISA_MDH and iELISA_SOD showed potential to be used in the diagnosis of infected animals, increasing the range of serological tests available for the diagnosis of bovine brucellosis, with the advantage of being S-LPS-free. However, none of the tests could differentiate between infection and vaccination.

Accuracy of serological tests for bovine brucellosis: A systematic review and meta-analysis

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.

Comparison of diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 strain vaccines.

The diagnosis of bovine brucellosis in animals vaccinated with strain-19 (S19) and Rose Bengal (RB)-51 strain vaccines can be misinterpreted due to false positives. This study aimed to compare diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 vaccine strains. Two groups of 12 crossbred Holstein calves between 6 and 8 months of age were used. On day 0, blood samples were collected from the animals, and the competitive enzyme-linked immunosorbent assay was used for serological diagnosis of bovine Brucellosis. All animals tested negative. After the first blood collection, the animals were subcutaneously vaccinated: one group received the S19 vaccine and the other received the RB51 vaccine. From the 3rd month after vaccination, all animals were sampled. Sampling was repeated every 2 months until the 7th month. Serological diagnosis of bovine brucellosis was performed using RB, tube serum agglutination test (SAT), SAT with 2-mercaptoethanol (SAT-2Me), and fluorescence polarization assay (FPA). Animals vaccinated with S19 showed positive results with the RB, SAT, and SAT-2Me tests in all months of post-vaccination diagnosis. In animals vaccinated with S19, FPA showed positive results at months 3 and 5 and negative results at month 7, indicating that this test discriminates vaccinated animals from infected animals 7 months after vaccination. Rose Bengal, SAT, SAT-2Me, and FPA tests showed negative results in animals vaccinated with RB51 in all months of diagnosis. Animals vaccinated with S19 may test positive for brucellosis using RB, SAT, or SAT-2Me tests 7 months later. Fluorescence polarization assay is an optimal alternative for diagnosing animals in the field, thereby preventing false positives, and consequently, unnecessary confiscations of animals. Animals vaccinated with RB51 tested negative with RB, SAT, SAT-2Me, and FPA tests in all months of diagnosis, confirming that the tests are ineffective for diagnosing brucellosis caused by rough strains.

Comparison of Fluorescence Polarization Assay with Rose Bengal Plate Agglutination test and Competitive Enzyme-Linked Immunosorbent assay for bovine brucellosis in Tanzania

Brucella are Gram-negative, facultative, intracellular bacterial species with B. abortus, B. melitensis, and B. suis carrying the smooth-lipopolysaccharide antigen. Accurate diagnostic results of brucellosis are needed for its control and eradication, however, they are primarily based on the serological testing of brucellosis in animals. The Rose Bengal Plate Agglutination Test (RBPT) and competitive Enzyme-Linked Immunosorbent Assay (c-ELISA) are the most commonly used tests for making such diagnosis. The use of Fluorescence Polarization Assay (FPA) in Tanzania is still in nascent stage. The purpose of this study was to compare RBPT, c- ELISA, and FPA in the diagnosis of bovine brucellosis. A total of 75 serum samples from cattle that were infected with Brucella in Kagera region were obtained. The FPA showed 90% (68 samples) prevalence, RBPT revealed 93% (70 samples) prevalence, and c-ELISA revealed 81% (61 samples) prevalence of brucellosis in the farm. The RBPT test has shown an inability to distinguish antibodies from cross-reacting organisms compared to the FPA test, while the c- ELISA was unable to pick a positive sample compared to the FPA test. FPA is very quick (5 min per sample), does not require specialized staff, and may be performed under field conditions. Therefore, FPA has a potential to overcome limitations in the detection of bovine brucellosis and can be used as a confirmatory test.

Accuracy of molecular diagnostic methods for the detection of bovine brucellosis: A systematic review and meta-analysis.

Bovine brucellosis is a disease of global socio-economic importance caused by Brucella abortus. Diagnosis is mainly based on bacterial culture and serology. However, these methods often lack sensitivity and specificity. A range of molecular diagnostic methods has been developed to address these challenges. Therefore, this study aims to investigate the diagnostic accuracy of molecular tools, in comparison to gold standard bacterial isolation and serological assays for the diagnosis of bovine brucellosis. The systematic review and meta-analysis were conducted based on analyses of peer-reviewed journal articles published between January 1, 1990, and June 6, 2020, in the PubMed, Science Direct, Scopus, and Springer Link databases. Data were extracted from studies reporting the use of molecular diagnostic methods for the detection of B. abortus infections in animals according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The quality of included journal articles was assessed using the quality assessment of diagnostic-accuracy studies assessment tool and meta-analysis was carried out using Review Manager. From a total of 177 studies, only 26 articles met the inclusion criteria based on PRISMA guidelines. Data from 35 complete studies were included in the meta-analysis and used to construct 2 × 2 contingency tables. Improved diagnostic performance was observed when tissue (sensitivity 92.7% [95% confidence interval (CI) 82.0-98.0%]) and serum samples (sensitivity 91.3% [95% CI 86.0-95.0%]) were used, while the BruAb2_0168 locus was the gene of preference for optimal assay performance (sensitivity 92.3% [95% CI 87.0-96.0%] and specificity 99.3% [95% CI 98.0-100.0%]). Loop-mediated isothermal amplification (LAMP) had a higher diagnostic accuracy than polymerase chain reaction (PCR) and real-time quantitative PCR with sensitivity of 92.0% (95% CI 78.0-98.0%) and specificity of 100.0% (95% CI 97.0-100.0%). The findings of this study assign superior diagnostic performance in the detection of B. abortus to LAMP. However, due to limitations associated with decreased specificity and a limited number of published articles on LAMP, the alternative use of PCR-based assays including those reported in literature is recommended while the use of LAMP for the detection of bovine brucellosis gains traction and should be evaluated more comprehensively in future.

Rapid diagnosis of bovine brucellosis by peptide nucleic acid-fluorescence in situ hybridization assay

Brucellosis is a major economical disease of livestock and an important zoonotic disease. Rapid and accurate diagnosis is a fundamental prerequisite in the quest for control and elimination of brucellosis. The current study was conducted to evaluate a rapid molecular technique, peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for the diagnosis of brucellosis. Samples, including fetal stomach contents, vaginal discharges, and placenta collected from cattle and buffaloes experiencing abortion and other reproductive disorders were processed for isolation. Brucella isolates obtained were confirmed by biochemical tests and polymerase chain reaction (PCR), thereby subjected to PNA-FISH which yielded positive fluorescence. The clinical samples were processed and subjected directly to PNA-FISH. On performing PNA-FISH directly on the clinical samples, this test reduces the time spent on processing the sample for isolation, avoiding the risk of handling the infectious organism while going through the process of isolation. Hence, PNA FISH can be considered as a rapid and efficient diagnostic method for Brucellosis.

First report and whole-genome sequencing of Pseudochrobactrum saccharolyticum in Latin America

The Brucellaceae family comprises microorganisms similar both phenotypically and genotypically, making it difficult to identify the etiological agent of these infections. This study reports the first isolation, identification, and characterization of Pseudochrobactrum saccharolyticum (strain 115) from Latin America. Strain 115 was isolated in 2007 from a bovine in Brazil and was initially classified as Brucella spp. by classical microbiological tests and bcsp31 PCR. The antimicrobial susceptibility of strain 115 was tested against drugs used to treat human brucellosis by minimal inhibitory concentration test. Subsequently, the whole genome of the strain was sequenced, assembled, and characterized. Phylogenetic trees built from 16S rRNA and recA gene sequences enabled the classification of strain 115 as Pseudochrobactrum spp. Phylogenomic analysis using Single Nucleotide Polymorphisms and Average Nucleotide Identity allowed the classification of the strain as P. saccharolyticum. Additionally, a Tetra Correlation Search identified one related genome from the same species, which was compared with strain 115 by analyzing genomic islands. This is the first identification and whole-genome sequence of P. saccharolyticum in Latin America and highlights a challenge in the diagnosis of bovine brucellosis, which could be solved by including the sequencing of 16S rRNA and recA genes in routine diagnostics.

Some Serological Analytic Approaches and Test Execution in Determination of Bovine Brucellosis: Overview

Bovine brucellosis is a highly contagious, zoonotic, and economically significant bacterial disease. The purpose of this paper was to review available scientific data on serological tests for the diagnosis of bovine brucellosis and compare test performance based on assay sensitivity (Se) and specificity (Sp) values. Abortion, placentitis, epididymitis, and orchitis are symptoms of the disease. The clinical picture of brucellosis is not pathognomonic, and the patient's clinical history, particularly the occurrence of abortion, is crucial for diagnosis. Although the causative agent is isolated and identified for a definitive diagnosis of bovine brucellosis, serological tests are usually preferred. The host defense mechanism against Brucella is functionally divided into innate or non-specific immunity and adaptive or specific immunity. The pathogenic brucella organism has evolved a slew of mechanisms for evading and/or modulating both innate and adaptive immune responses in its host. Serological assays are based on the fact that Brucella abortus and other smooth Brucella have a unique O-polysaccharide that induces a humoral response with the production of IgM first, followed by IgG1, and then IgG2/IgA. Screening serological tests (Milk ring test, Rose Bengal Test (RBT), and Buffered plate agglutination test (BPAT)) and confirmatory serological tests (complement fixation test (CFT), serum agglutination test (SAT), 2-mercaptoethanol test (2ME), indirect-enzyme-linked immunosorbent assay (iELISA), and, more recently, competitive-ELISA (cELISA) and Brucella fluorescent pol (FPA). CFT, RBT, and iELISA tests are currently used in EU legislation for intra-community cattle trade and show comparable Se and Sp. The new FPA test demonstrated Se and Sp levels comparable to standard tests and was proposed for inclusion as a standard test in EU legislation. Similarly, the OIE considers the four tests to be a mandatory requirement for international trade. Several methods, however, produce both false-positive and false-negative results. There is no single serological test that is 100% accurate and applicable in all epidemiological situations. In general, confirmatory serological procedures involve testing each serum with multiple tests, usually a screening test with high sensitivity followed by a confirmatory test with high specificity. As a result, the development of rapid, low-cost serological tests with high sensitivity and specificity for brucellosis detection is a major issue that requires further investigation. Keywords: Bovine Brucellosis; Brucella Abortus; Diagnosis; Sensitivity; Specificity; Serological Tests, Test Performance DOI: 10.7176/JNSR/13-12-03 Publication date: June 30 th 2022

Development of a novel glycoprotein-based immunochromatographic test for the rapid serodiagnosis of bovine brucellosis.

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. 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. 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. This strip test could provide a rapid (10min) and accurate diagnosis of bovine brucellosis in the field contributing to the control of the disease.

Evaluation of lateral flow assay as a field test for sero-diagnosis of bovine brucellosis

Indirect-ELISA offers a significant advantage over conventional serological methods in the diagnosis of brucellosis in endemic geographical region. Considering iELISA as a gold standard test, RBPT was more sensitive than LFA and the concordance of iELISA with LFA was comparable. The Lateral flow assay is a rapid point-of - care diagnostic test which makes it ideal for use in resource pour countries. It is an immuno-assay and is used also for diagnosis of bovine brucellosis. It is a highly sensitive and specific test which does not require expensive equipment, electricity and or refrigeration or special training. The LFA has shown good PPV (positive predictive value) and NPV (negative predictive value) greater than RBPT in the current study suggest that the test is a simple, cost-effective and rapid that provides accurate detection of antibodies to B. abortus in bovine serum samples, thereby saving time and eliminating the need for special training. It could be used conveniently on the field even in farms located in remote areas. However, evaluation on large sample size would be required for future use. Hence, looking to the results obtained in the present study and by other workers, it is recommended that this rapid test can therefore be practically implemented in serological screening for bovine brucellosis, although evaluation on a larger scale with various sera, and blood samples is still necessary.

Bayesian Estimation of the Prevalence and Test Characteristics (Sensitivity and Specificity) of Two Serological Tests (RB and SAT-EDTA) for the Diagnosis of Bovine Brucellosis in Small and Medium Cattle Holders in Ecuador

In Ecuador, a national program for bovine brucellosis control has been in implementation since 2008. Given the costs, small- and medium-sized livestock holders are not completely committed to it. The objective of this study was to determine true prevalence (TP) of bovine brucellosis in small- and medium-sized herd populations, as well as the diagnostic sensitivity and specificity of the Rose Bengal (RB) test and the sero-agglutination test (SAT)-EDTA using a Bayesian approach. Between 2011 and 2016, 2733 cattle herds were visited, and 22,592 animal blood samples were taken in nineteen provinces on mainland Ecuador. Bayes-p and deviance information criterion (DIC) statistics were used to select models. Additionally, risk-factor analysis was used for herds according to their brucellosis test status. True prevalence (TP) in herds was estimated by pool testing. National seroprevalence of farms was 7.9% (95% CI: 6.79–9.03), and TP was 12.2% (95% CI: 7.8–17.9). Apparent prevalence (AP) in animals was 2.2% (95% CI: 1.82–2.67), and TP was 1.6% (95% CrI: 1.0–2.4). Similarly, the sensitivity of the RB was estimated at 64.6% (95% CrI: 42.6–85.3) and specificity at 98.9% (95% CrI: 98.6–99.0); for the SAT-EDTA test, sensitivity was 62.3% (95% CrI: 40.0–84.8) and 98.9% (95% CrI: 98.6–99.1) for specificity. Results of the two tests were highly correlated in infected and uninfected animals. Likewise, high spatial variation was observed, with the Coastal Region being the zone with the highest TP at 2.5%. (95% CrI: 1.3–3.8%) in individual animals and 28.2% (95% CI: 15.7–39.8) in herds. Risk factors include herd size, type of production (milk, beef, and mixed), abortions recorded, and vaccination. The results of this study serve to guide authorities to make decisions based on parallel testing at the beginning of a bovine brucellosis program for small livestock holders to increase sensitivity level of the screening tests in Ecuador.

Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples

Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 104 fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only.To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.

Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle.

Background and Aim:An accurate diagnosis of Brucella-infected animals is one of the critical measures in eradication programs. Conventional serological tests based on whole-cell (WC) antigens and detecting antibodies against pathogen-associated lipopolysaccharide might give false-positive results due to the cross-reactivity with other closely related bacteria. This study evaluated the serological potential of Brucella spp. chimeric outer membrane proteins (Omps) as antigens in an indirect enzyme-linked immunosorbent assay (i-ELISA).Materials and Methods:The chimeric gene constructs of the most immunodominant regions of Brucella Omps 25+31, 25+19, and 19+31 were cloned into the pET28a expression vectors and transformed into Escherichia coli BL21 (DE3). The serological potential of chimeric proteins compared with single recombinant Omps (rOmps)19, 25, and/or 31 were studied on blood serum samples of (i) a rabbit immunized with killed Brucella abortus 19WC, (ii) mice immunized with single rOmps, (iii) cows seropositive for brucellosis by rose Bengal test, and (iv) cattle naturally and/or experimentally infected with brucellosis.Results:E. coli BL21 actively produced Brucella chimeric rOmps, the concentration of which reached a maximum level at 6 h after isopropyl-b-D-1-thiogalactopyranoside stimulation. Target proteins were antigenic and expressed in an active state, as recognized by rabbit anti-B. abortus antibodies in an i-ELISA and western blotting. Murine antibodies against the single rOmps reacted with chimeric antigens, and conversely, antichimeric antibodies found their epitopes in single proteins. Brucella chimeric rOmps showed higher antigenicity in blood sera of seropositive cattle kept in the hotbed of the infection and/or experimentally challenged with brucellosis than single proteins.Conclusion:Brucella chimeric recombinant outer membrane proteins could be a potential antigen candidate for developing an ELISA test for accurate diagnosis of bovine brucellosis.

Studies on Diagnosis of Bovine Brucellosis by Immunocyto-chemistry and Immunohistochemistry

Background: Brucellosis is a major threat to livestock economy and an important zoonotic disease. A rapid and accurate diagnosis is a necessity to curb the spread and progress of the disease. The current study aimed to evaluate sensitivity of Immunocytochemistry and Immunohistochemistry methods for detection of Brucella spp.Methods: A total of 50 samples comprising of fetal stomach content, vaginal discharges and placenta were collected from cattle and buffaloes suffering from abortions and other reproductive disorders in and around Ludhiana, Punjab during the period 2017-2018. All the samples were processed for isolation and confirmed with biochemical analysis and Polymerase chain reaction (PCR). The isolates obtained and 43 clinical samples excluding placental samples were subjected to Immunocytochemistry (ICC). Immunohistochemistry (ICH) was performed on placental samples.Result: A total of four isolates were recovered from the screened samples. The four isolates also yielded positive results in Immunocytochemistry. Among the 43 clinical samples screened by Immunocytochemistry, five were positive, however only 3 isolates were recovered on isolation. A total of seven placental tissue samples were processed and subjected to immunohistochemistry. Of the three placental samples positive by immunohistochemistry, only one sample was isolated on culture. The results suggest that both immunocytochemistry and immunohistochemistry are sensitive diagnostic techniques in comparison to isolation.

The use of IHT with milk in the diagnosis of bovine brucellosis

The use of IHT with milk in the diagnosis of bovine brucellosis

The seroprevalence of brucellosis and molecular characterization of Brucella species circulating in the beef cattle herds in Albania.

BackgroundBrucellosis is a ubiquitous zoonotic disease globally. It is endemic among bovines, sheep, and goats in Albania. The national control and eradication programs for brucellosis has been applied on sheep and goat farms as well as large dairy cattle farms, i.e., those with more than ten milking cows. The current study aims at estimating the herd and average individual animal prevalence of brucellosis in the national beef cattle herds, the missing information that was essential to propose the most appropriate control measures for this sub-population. Rose Bengal Test (RBT), Fluorescence Polarization Assay (FPA), and Enzyme-Linked Immunosorbent Assay (ELISA) were used as serological tests and classical bacteriology for isolation. Results were also used to investigate the difference in sensitivity between the assays used.MethodologyIn total, 655 animals from 38 beef cattle herds from six southern districts of Albania were sampled. Sera were tested using RBT, FPA, and ELISA. Fifteen positive cows and a bull from eight high-prevalence positive herds were slaughtered, and particular tissue samples were collected for bacteriology.ResultsThe overall herd seroprevalence in the tested beef cattle population was 55%, while the overall average within-herd prevalence (including only positive herds) was 38.3%, 42.7%, and 45.6% determined by the RBT, ELISA, and FPA, respectively. FPA was used for the first time in the diagnosis of bovine brucellosis in Albania, and its sensitivity was higher than RBT and ELISA. Three B. abortus strains were identified, two from the supra-mammary lymph node of two cows and one from the epididymis of a seropositive bull.ConclusionBrucellosis was highly prevalent in beef cattle in the southern part of Albania, and B. abortus was isolated from this subpopulation. To the best of our knowledge, this is the first statistically based survey of bovine brucellosis in beef herds in Albania. Using the FPA in parallel with other serological tests improved overall diagnostic sensitivity. Test and slaughter policy is not a rational approach for the control of brucellosis in beef cattle in Albania, and vaccination is only applicable, including strict control of the movement of animals.

Brucella melitensis B115-based ELISA to unravel false positive serologic reactions in bovine brucellosis: a field study

BackgroundBrucellosis 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.ResultsNegative 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.ConclusionsThe 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.

Comparative evaluation of fluorescence polarization assay and competitive ELISA for the diagnosis of bovine brucellosis vis-a-vis sero-monitoring

Brucellosis is an important zoonosis that constitutes a serious public health hazard which is caused by a bacterium belonging to the genus Brucella. In the present study, two highly specific serological tests for brucellosis diagnosis, fluorescence polarization assay (FPA) and competitive ELISA (cELISA) were standardized in the laboratory, evaluated and compared with rose bengal plate test (RBPT), indirect ELISA (iELISA) and commercial cELISA kit. For test evaluation, 1386 serum samples [apparently healthy animals (n = 260), samples from Brucella infected farms (n = 701) and B. abortus S19 vaccinated animals (n = 425)] were analyzed to assess suitable diagnostic test in B. abortus S19 post vaccinated bovine population. In apparently healthy brucellosis free farms, RBPT, iELISA, in-house FPA and cELISA were found to be highly specific than commercial cELISA. Commercial cELISA kit was comparatively more sensitive than other serological tests in samples collected from infected farms. The FPA showed sensitivity nearly equal to RBPT and in-house cELISA showed greater sensitivity than RBPT in infected farms. In animals with persistent vaccinal antibodies, only in-house FPA and cELISA recorded higher specificity of 87.64 and 90.27%, respectively. The other tests, RBPT and iELISA displayed similar reactivity with vaccine antibodies to that of infection antibodies whereas commercial cELISA kit showed an intermediate specificity of 47.69%. With these findings, RBPT, iELISA and cELISA are suggested for screening infected herds, and in-house developed FPA and cELISA tests with a proven specificity can be used for confirmatory diagnosis of brucellosis in B. abortus S19 post vaccinated animal populations.

Serological and Molecular Diagnosis of Bovine Brucellosis in Menoufia Province

Brucellosis represents a serious problem in cattle relaying huge economic losses. Brucella meletensis on of brucella strains that can infect human which its usual host is sheep, might isolated from cattle. The aim of present study was detection of brucellosis among cattle population in Menoufia governorate, Egypt and analyze risk factors. Serologically brucella were detected with RBPT. Evaluation of the specificity and sensitivity of RBPT, IC assay and cELISA against CFT test were done. Molecular detection of brucella using PCR and bacteriological isolation followed by phenotypic and molecular typing of the isolated bacteria. The overall prevalence using RBT was 1.44%. Holstein Friesian cattle have highest prevalence (1.57%) while native cattle were (1.28%). Cattle over 3 years old have the highest seroprevalence (2.77%). The positive result for RBPT, cELISA, IC Assay and CFT were 91%, 60%, 91% and 88% respectively after examination 100 serum sample. Brucella melitensis biovar 3 was isolated from the tissue specimens (uterus and/or lymph nodes). PCR targeting (Immuno-dominant antigen, gene bp26) generated product of 450 bp from (16/20) tissues specimens. These findings assisting in future planning pragmatic control strategies against bovine brucellosis in Egypt and for herd health fertility.

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.