Modelling the control of bovine brucellosis in India.

Brucellosis imposes substantial impacts on livestock production and public health worldwide. A stochastic, age-structured model incorporating herd demographics was developed describing within- and between-herd transmission of Brucella abortus in dairy cattle herds. The model was fitted to data from a cross-sectional study conducted in Punjab State of India and used to evaluate the effectiveness of control strategies under consideration. Based on model results, stakeholder acceptance and constraints regarding vaccine supply, vaccination of replacement calves in large farms should be prioritized. Test and removal applied at early stages of the control programme where seroprevalence is high would not constitute an effective or acceptable use of resources because significant numbers of animals would be 'removed' (culled or not used for breeding) based on false positive results. To achieve sustained reductions in brucellosis, policymakers must commit to maintaining vaccination in the long term, which may eventually reduce frequency of infection in the livestock reservoir to a low enough level for elimination to be a realistic objective. This work provides key strategic insights into the control of brucellosis in India, which has the largest cattle population globally, and a general modelling framework for evaluating control strategies in endemic settings.

The quest for a true One Health perspective of brucellosis.

One Health is an interdisciplinary collaboration that aims at mitigating risks to human health arising from microorganisms present in non-human animal species, which have the potential to be transmitted and cause disease in humans. Different degrees of scientific collaboration and sectoral integration are needed for different types of zoonotic diseases, depending on the health and associated economic gains that can be expected from a One Health approach. Indeed, mitigating zoonotic risks related to emerging diseases with pandemic potential is different from mitigating risks related to endemic zoonotic diseases like brucellosis. Likewise, management of brucellosis at the wildlife-livestock interface in wildlife conservation areas is in essence different from mitigating transmission of a given Brucella species within its preferential host species, which in turn is different from mitigating the spillover of a given Brucella species to non-preferential host species, humans included. Brucellosis economic models often oversimplify and/or wrongly assess transmission between reservoir hosts and spillover hosts. Moreover,they may not properly value non-market outcomes, such as avoidance of human disease, consumer confidence and conservation biology issues. As a result, uncertainty is such that the economic predictions of these models can be questionable. Therefore, understanding the infection biology of Brucella species is a prerequisite. This paper reviews and highlights important features of the infection biology of Brucella species and the changing epidemiology of brucellosis that need to be integrated into a true One Health perspective of brucellosis.

New developments in the immunodiagnosis of brucellosis in livestock and wildlife.

Although relatively effective diagnostic tests for brucellosis have been in existence for more than 100 years, it remains a serious, embedded and also a re-emerging disease in many parts of the globe. There are many factors besides suboptimal diagnosis that impede the complete and sustained eradication of animal brucellosis. In this review a case for the continued improvement of diagnostic methods is made through identifying existing shortcomings and considering what impact these have upon control and eradication. The focus is on developments in immunodiagnostics as these seem more likely to yield the pragmatic solutions needed. Moreover, developments in DNA detection methods have been neatly and recently reviewed elsewhere. This article reviews issues such as test cost, mobility, sensitivity and specificity. Advances in low-cost materials, high-throughput testing, assay multiplexing and the quantification of pen-side tests are described and their relevance to disease control considered. Poor test specificity when resolving positive serology, due to infection with cross-reactive bacteria and vaccination with smooth Brucella strains, is also an impediment to efficient disease eradication. A case for the development of novel discrete epitope antigens to address this is presented alongside in silico methods of selection and tools that enable increased analytical sensitivity that may be required to detect relatively low, but potentially significant, analytes. References have been drawn from the study of brucellosis wherever possible. However, in some cases new technological developments worthy of discussion have been included via the use of pertinent alternative examples. In conclusion, despite developments and innovations the classical serological tests seem under no imminent danger of mass extinction but there is potential for significant improvement and supplementation.

An evaluation of the capability of existing and novel serodiagnostic methods for porcine brucellosis to reduce false positive serological reactions.

Porcine brucellosis is a zoonotic disease of truly global significance because even in countries without the disease the occurrence of false positive serological reactions (FPSRs) creates significant problems. Statutory diagnostic testing is required in many disease free countries or regions and is often a prerequisite for the movement of live animals. Currently this testing is dependent almost entirely on serological assays and these may result in a significant number of FPSRs. The aim of this study was to examine existing and novel serodiagnostic assays to evaluate their diagnostic sensitivity and resilience to FPSRs. The existing assays evaluated were the RBT, smooth lipopolysaccharide (sLPS) indirect (i) ELISA, sLPS competitive (c) ELISA, and the FPA. The novel assays evaluated were the sLPS TR-FRET assay, a rough (r) LPS iELISA, a recombinant protein BP26 iELISA and a cytoplasmic protein extract (Brucellergene™) iELISA. Four populations of sera were evaluated: those from Brucella suis infected swine (n=34), randomly selected samples from non-infected swine (n=161), sera from non-infected swine within herds exhibiting FPSRs (n=132) and sera from swine experimentally infected with Yersinia enterocolitica O:9 (n=4). The results show that all the assays dependent on the sLPS O-polysaccharide (OPS) for their sensitivity (the RBT, sLPS ELISAs, FPA and the sLPS TR-FRET) had significantly reduced diagnostic specificity when applied to the FPSR population, the RBT being most affected. Of the two rapid homogeneous assays, the TR-FRET was diagnostically superior to the FPA in this study. Neither of the protein based iELISAs demonstrated sufficient diagnostic sensitivity to resolve the FPSRs. The rLPS iELISA showed no cross reaction with the FPSRs and had diagnostic sensitivity similar to that of the OPS based assays.

The first International Standard anti-Brucella melitensis Serum.

The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.

Improving the specificity of immunodiagnosis for porcine brucellosis.

This report describes the use of cell mediated immunity to improve specificity of current diagnosis for Brucella suis. Diagnosis is problematic due to cross reactions that lead to false positive serological reactions (FPSR) in the standard diagnostic tests. A common cause of this cross reactivity is infection with the organism Yersinia enterocolitica O:9. Gottingen mini-pigs were experimentally infected with B. suis biovar I field strain or Y. enterocolitica serotype O:9 biotype 3. Infection was followed for 70 days. During this time whole blood stimulation assays were set up using Brucella specific antigen. IFNgamma was measured in the supernatants (SN) from these assays by ELISA. Concurrent standard serological tests were carried out. The results indicate that the IFNgamma assay is specifically able to distinguish Y. enterocolitica O:9 infection from a B. suis infection in experimentally infected mini-pigs. These results represent an improvement in diagnostic specificity compared to currently used serological tests. Thus suggesting that in a surveillance setting this test could be applied as a confirmatory test in the face of FPSR.

The improved specificity of bovine brucellosis testing in Great Britain.

The brucellosis surveillance scheme in Great Britain includes the serological testing of approximately 1 million bovine samples per year. These are screened by iELISA, positives going forward for confirmatory testing by CFT and SAT. Samples positive by confirmatory testing prompt substantial field investigations and interventions, but the animals involved are usually uninfected. Described below are a series of modifications to the screening method, which have resulted in a 10-fold reduction in false positive results whilst maintaining sensitivity. The key modifications include the introduction of blocking agents, a change in serum test dilution and the introduction of a control that directly defines the positive/negative cut-off. These simple modifications have had a large impact in reducing the cost of the surveillance programme due to reductions in confirmatory test requirements and a knock on effect of reducing costly field intervention.

Harmonisation of European tests for serological diagnosis of Brucella infection in bovines.

The principal methods for the serological diagnosis of bovine brucellosis are the complement fixation test (CFT), serum agglutination test (SAT), Rose-Bengal test (RBT), indirect enzyme-linked immunosorbent assay (iELISA) and more recently the competitive ELISA (cELISA) and the fluorescent polarisation assay (FPA). Guidelines set by the World Organisation for Animal Health (OIE) describe methods and diagnostic thresholds for each of these tests. Many countries have adopted these methods for the purposes of eradication of brucellosis and have legislated for the use of these tests (the CFT and SAT in particular) for the prevention of the spread of the disease through international trade. Within the European Union (EU) each member state has a National Reference Laboratory which regulates the quality of brucellosis diagnosis and works to the recommendations set by the OIE. This article describes the results from the first three EU ring trials assessing the harmonisation of diagnostic tests between each member state. The general level of harmony for SAT, CFT, and iELISA was found to be good, but issues of standardisation of the RBT, cELISA and FPA remain. The cELISA and FPA in particular need further work to create European harmony. The ring trials also proved successful at providing specific evidence of poor performance in some areas. The decision on whether or not to take action on the basis of these results rested with the individual laboratories concerned. The increase in the number of participants in these trials over time reflected the enlargement of the EU and increased the need for quality assurance.

Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA.

The fluorescence polarisation assay (FPA) is a recently described test for the serological diagnosis of Brucella infection. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. To validate the FPA, serum samples from 146 confirmed (by culture) Brucella-infected cattle were tested in conjunction with serum samples from 1947 noninfected cattle. The competitive ELISA (cELISA) was validated using these positive reference samples and 1440 negative samples, while data for the indirect ELISA (iELISA) was generated from 6957 negative samples plus the positive sera. Published diagnostic specificity (DSp) data for the complement fixation test (CFT) and serum agglutination test (SAT) was used in conjunction with the test results on the positive sera to obtain diagnostic specificity plus diagnostic sensitivity (DSn). After selection of a cutoff for the FPA and cELISA, the diagnostic specificity and sensitivity total for each test were compared. The results, with 95% confidence intervals, were: FPA (195.7+/-2.79), iELISA (195.0+/-2.70), cELISA (194.9+/-3.48), CFT (191.7+/-4.45), and SAT (180.4+/-6.33). The data presented supports the use of the FPA in diagnosis of brucellosis and questions the use of the SAT and CFT for either screening or confirmatory testing.