Brucellosis is a zoonotic disease imposing significant impacts on livestock production and public health worldwide. India is the world’s leading milk producer and Punjab is the state which produces the most cattle and buffalo milk per capita. The aim of this study was to investigate the epidemiology of bovine brucellosis to provide evidence for control of the disease in Punjab State, India. A cross-sectional study of dairy farms was conducted in humans and livestock in rural Ludhiana district using a multi-stage sampling strategy. The study suggests that brucellosis is endemic at high levels in cattle and buffalo in the study area with 15.1% of large ruminants testing seropositive and approximately a third of dairy farms having at least one animal test seropositive. In total, 9.7% of those in direct contact with livestock tested seropositive for Brucella spp. Persons that assisted with calving and/or abortion within the last year on a farm with seronegative livestock and people which did not assist with calving/abortion had 0.35 (95% CI: 0.17 to 7.1) and 0.21 (0.09 to 0.46) times the odds of testing seropositive compared to persons assisting with calving/abortion in a seropositive farm, respectively. The study demonstrated that persons in direct contact with cattle and buffalo in the study area have high risk of exposure to Brucella spp. Control of the disease in livestock is likely to result in benefits to both animal and public health sectors.

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.

Brucellosis is a global disease and the world’s most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.

The aim of this study was to develop a multiplex bead assay using a Brucella rLPS antigen, a Brucella suis smooth antigen, and a Yersinia enterocolitica O:9 antigen that not only discriminates Brucella-infected from Brucella-uninfected pigs and wild boar, but also overcomes the cross reactivity with Y. enterocolitica O:9. Sera from 126 domestic pigs were tested: 29 pigs were Brucella infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 Brucella-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-Brucella infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth B. suis 1330 antigen, all Brucella-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth B. suis antigen and Y. enterocolitica O:9 antigen enabled discriminating all Brucella infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with Y. enterocolitica.

Brucellosis remains a major zoonotic disease worldwide, with disproportionate burden in low- and middle-income countries where limited veterinary and healthcare infrastructure constrain effective control measures. However, its pathways of transmission are poorly understood. In pastoralist settings, we hypothesize that camels have a high burden of Brucella spp. and play a key role in spreading it to humans and other livestock. This manuscript presents a study protocol to quantify the relative contribution of various livestock species to brucellosis transmission and identify cost-effective control strategies in Kenya. Using probability-proportional-to-size sampling, we aimed to recruit a longitudinal cohort of 170 households and their herds per site in the Marsabit and Kajiado counties. Households rearing at least one livestock species (cattle, camels, goats, sheep) were eligible. Serum, milk, and vaginal swabs (from livestock), and serum (from humans) were collected for testing using Rose Bengal Test, ELISA, qPCR, and culture methods. Concurrently, surveillance for suspected brucellosis was conducted in study health facilities. A qualitative ethnographic study and livestock movement monitoring using GPS-collared animals were nested within the cohort. These data will be used to parameterize a multi-host, multi-species infectious disease model through Approximate Bayesian Computation. Through this One Health approach, our study will identify and optimize potential interventions and help inform the development of a comprehensive cost-effective national control program for brucellosis.