Quantitative assessment of the probability of introducing bovine brucellosis into English cattle herds by imported live cattle

A stochastic simulation model was developed to estimate the quarterly probability (PIntro) of introducing bovine brucellosis into English cattle herds, by at least one imported live cattle (potential carrier of Brucella abortus). The probability of spread after introduction was not included and imports from several countries were considered. Information used to parameterise model´s inputs was obtained from the literature, legislation and analysis of several national datasets (2013 to 2016), which contained information on imported cattle and testing schemes used in the English cattle population. Exporting countries were divided according to official brucellosis status “J” into: Officially Brucellosis Free (OBF), Non-Officially Brucellosis Free (Non-OBF) and in OBF-Validation (during the first five years of OBF status). The entire English cattle population was divided into eight strata “S” by combination of laboratory testing data and herd type. The only risk mitigation measure considered was the testing for antibodies on animals older than one year and imported from Non-OBF countries. Probabilities of introduction at herd and stratum level were combined into the overall national PIntro. Two scenarios were run. In the baseline scenario, the between herds prevalence BHP(OBF) in OBF countries was set according to information from EFSA and from the EU legislation. In the alternative scenario only the former was used and BHP(OBF) was set very low/negligible. For Non-OBF and OBF-Validation countries, the BHP was set with distributions based on the literature. In the baseline scenario, between 2013 and 2016, the quarterly median PIntro ranged from 1.3% to 5.5%. For the last year considered, the median of the quarterly medians PIntro was 2.8% (median of 5th percentiles = 0.4%; median of 95th percentiles =10.7%). Therefore, on average, at least one introduction could be expected each (approximated) 36 surveillance periods (9, 281), so each ≈ 9 years (2; 70). According to the alternative scenario, the PIntro was very low and on average at least one introduction could be expected each ≈ 125 years

Rapid veterinary diagnosis of bovine reproductive infectious diseases from semen using paper-origami DNA microfluidics

The health and well-being of cattle is a significant concern for global agricultural output. In dairy production within low and middle income countries (LMICs), there is a significant biosensing challenge in detecting sexually transmitted infection (STI) pathogens during animal husbandry, due in part to difficulties associated with the limited infrastructure for veterinary medicine. Here we demonstrate low-cost, multiplexed and sample-to-answer paper-origami tests for the detection of three bovine infectious reproductive diseases in semen samples, collected at a test site in rural India. Pathogen DNA from one viral pathogen, Bovine Herpes virus-1 (BoHV-1) and two bacteria (Brucella and Leptospira) was extracted, amplified (using loop-mediated isothermal amplification, LAMP) and detected fluorescently, enabling <1 pg (~ from 115 to 274 copies per reaction) of target genomic DNA to be measured. Data was collected as a fluorescence signal either visually, using a low-cost hand-held torch, or digitally with a mobile-phone camera. Limits of detection and sensitivities of the paper-origami device for the three pathogens were also evaluated using pathogen-inoculated semen samples and were as few as 50 Leptospira organisms, 50 CFU Brucella and 1 TCID50. BoHV-1. Semen samples from elite bulls at a germplasm centre were also tested in double-blind tests, as a demonstrator for a low cost, user-friendly point-of-care sensing platform, for in-the-field resource-limited regions. The sensors showed excellent levels of sensitivity and specificity, and for the first of time a demonstrated ability of the application of paper-origami devices for the diagnosis multiple infectious diseases from semen samples

Epidemiology of brucellosis in cattle and dairy farmers of rural Ludhiana, Punjab.

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

Development of a multiplex bead assay to detect serological responses to Brucella species in domestic pigs and wild boar with the potential to overcome cross-reactivity with Yersinia enterocolitica O:9

This article belongs to the Special Issue Emerging Themes in Brucella and 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.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) entitled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

Performance characteristics and costs of serological tests for brucellosis in a pastoralist community of northern Tanzania

The control of brucellosis across sub-Saharan Africa is hampered by the lack of standardized testing and the use of tests with poor performance. This study evaluated the performance and costs of serological assays for human brucellosis in a pastoralist community in northern Tanzania. Serum collected from 218 febrile hospital patients was used to evaluate the performance of seven index tests, selected based on international recommendation or current use. We evaluated the Rose Bengal test (RBT) using two protocols, four commercial agglutination tests and a competitive enzyme-linked immunosorbent assay (cELISA). The sensitivity, specificity, positive predictive value, negative predictive value, Youden’s index, diagnostic accuracy, and per-sample cost of each index test were estimated. The diagnostic accuracy estimates ranged from 95.9 to 97.7% for the RBT, 55.0 to 72.0% for the commercial plate tests, and 89.4% for the cELISA. The per-sample cost range was $0.69–$0.79 for the RBT, $1.03–$1.14 for the commercial plate tests, and $2.51 for the cELISA. The widely used commercial plate tests performed poorly and cost more than the RBT. These findings provide evidence for the public health value of discontinuing the use of commercial agglutination tests for human brucellosis in Tanzania

Brucellosis in dairy herds: a public health concern in the milk supply chains of West and Central Africa

Ten herd-level cross-sectional studies were conducted in peri-urban dairy production areas of seven West and Central African countries (Burkina Faso, Burundi, Cameroon, Mali, Niger, Senegal and Togo). The objectives were to estimate herd level Brucella spp. seroprevalence and identify risk factors for seropositivity. In each of the ten study areas, herds (between 52 and 142 per area, total = 965) were selected probabilistically and a structured questionnaire was administered to gather information on their structure and management. A bulk milk sample from each herd was tested by indirect ELISA for Brucella spp. For each area, herd seroprevalence estimates were obtained after adjusting for the assumed performance of the diagnostic test. Herd level risk factors for Brucella spp. seropositivity were identified by means of stratified logistic regression, with each peri-urban zone as a stratum. Area-specific models were also explored. Estimated herd seroprevalences were: Lomé (Togo) 62.0% (95% CI:55.0-69.0), Bamako (Mali) 32.5% (95% CI:28.0-37.0), Bujumbura (Burundi) 14.7% (95%CI:9.4-20.8), Bamenda (Cameroon) 12.6% (95% CI:7.6-21.9), Ouagadougou (Burkina Faso) 3.0% (95% CI:1.0-9.1), Ngaoundere (Cameroon) 2.3% (95% CI:1.0-7.0), Thies (Senegal) 1.3% (95% CI:0.1, 5.3), Niamey (Niger) 1.2% (95% CI:0.08-5.3), Dakar (Senegal) 0.2% (95% CI:0.01-1.7) and Niakhar (Senegal) <0.04%. Logistic regression modelling revealed transhumant herds to be at lower risk of infection (adjusted OR: 0.25, 95% CI: 0.13 - 0.5) and in one of the areas (Bamenda), regular purchase of new animals was found to be strongly associated with Brucella spp. seropositivity (adjusted OR = 5.3, 95% CI: 1.4-25.9). Our findings confirm that Brucella spp. circulates among dairy cattle supplying milk to urban consumers in West and Central Africa, posing a serious public health concern. Control programs are urgently needed in areas such as Lomé or Bamako, where more than 30% of the herds show evidence of infection

Immunoselection and structural evaluation of Brucella O-polysaccharide epitopes and their application to the serodiagnosis of bovine brucellosis

Brucellosis is one of the world’s most significant zoonosis and is caused by infection with members of the genus Brucella. These have a cell wall characteristic of Gram-negative bacteria including lipopolysaccharides and, in the most significant Brucella species, O-polysaccharide (OPS). The major economic and health impacts of the disease arise from livestock, in particular ruminants and swine, where the main clinical feature is reproductive failure. The principle source of infection in the general human population is most often via ingestion of unpasteurised dairy products. Serology is the most cost effective means of disease detection but has significant imperfections including false positive serological reactions (FPSRs) due to antibodies that are raised against other Gram-negative bacteria in possession of OPS structures similar to that of Brucella. Serology with non-OPS antigens has been largely ineffective and alternative approaches such as bacterial culture, PCR or measurements of cell mediated immunity are impractical, ineffective or unproven. The OPS from Brucella is an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyls (D-Rha4NFo) that are variably -(12) and -(13) linked. This structure contains some epitopes that are shared with the OPS from other organisms, notably Yersinia enterocolitica O:9, and some that appear to be unique. Previous attempts to harness the unique epitopes using competitive ELISA failed to resolve FPSRs because the unique and common epitopes overlap causing steric hindrance of specific antibody binding. Oligosaccharides were derived from the OPS of B. abortus, B. melitensis and Y. enterocolitica O:9 by partial acid hydrolysis. These were separated and analysed by chromatography with on-line ESI-QqToF and QqQ. OPS specific antibodies were used to select from this pool of oligosaccharides and those captured were evaluated by graphitised carbon chromatography with on-line ESI-QqQ. On the basis of the mass spectrometry evidence the synthesis of a D-Rha4NFo tetrasaccharide comprised of a single -(13) link flanked on either side by single -(12) links was commissioned. This tetrasaccharide was used to develop an indirect ELISA for the detection of specific antibodies. Equivalent indirect ELISAs were also developed using native OPS and from synthetic penta- and nonasaccharide antigens, received from a collaborating laboratory. The tetrasaccharide ELISA was the most effective assay for discriminating between bovine FPSRs and true positives. The diagnostic capability of this ELISA was significantly superior (P < 0.05) than all others except the pentasaccharide ELISA (P = 0.159). The results show that the iELISA developed with the tetrasaccharide may effectively detect antibodies from animals infected with Brucella strains with low or high abundance of -(13) links within their OPS and has a significantly improved capability to resolve FPSRs compared to antigens that include common OPS epitopes.Open Acces

Brucellosis: Improved Diagnostics and Vaccine Insights from Synthetic Glycans

ConspectusBrucellosis is a serious zoonotic bacterial disease that is ranked by the World Health Organization among the top seven “neglected zoonoses” that threaten human health and cause poverty. It is a costly, highly contagious disease that affects ruminants, cattle, sheep, goats, and other productive animals such as pigs. Symptoms include abortions, infertility, decreased milk production, weight loss, and lameness. Brucellosis is also the most common bacterial disease that is transmitted from animals to humans, with approximately 500 000 new human cases each year. Detection and slaughter of infected animals is required to eradicate the disease, as vaccination alone is currently insufficient. However, as the most protective vaccines compromise serodiagnosis, this creates policy dilemmas, and these often result in the failure of eradication and control programs. Detection of antibodies to the Brucella bacterial cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is used in diagnosis of this disease, and the same molecule contributes important protective efficacy to currently deployed veterinary whole-cell vaccines. This has set up a long-standing paradox that while Brucella OPS confers protective efficacy to vaccines, its presence results in similar antibody profiles in infected and vaccinated animals. Consequently, differentiation of infected from vaccinated animals (DIVA) is not possible, and this limits efforts to combat the disease. Recent clarification of the chemical structure of Brucella OPS as a block copolymer of two oligosaccharide sequences has provided an opportunity to utilize unique oligosaccharides only available via chemical synthesis in serodiagnostic tests for the disease. These oligosaccharides show excellent sensitivity and specificity compared with the native polymer used in current commercial tests and have the added advantage of assisting discrimination between brucellosis and infections caused by several bacteria with OPS that share some structural features with those of Brucella. During synthesis and immunochemical evaluation of these synthetic antigens, it became apparent that an opportunity existed to create a polysaccharide–protein conjugate vaccine that would not create antibodies that give false positive results in diagnostic tests for infection. This objective was reduced to practice, and immunization of mice showed that antibodies to the Brucella A antigen could be developed without reacting in a diagnostic test based on the M antigen. A conjugate vaccine of this type could readily be developed for use in humans and animals. However, as chemical methods advance and modern methods of bacterial engineering mature, it is expected that the principles elucidated by these studies could be applied to the development of an inexpensive and cost-effective vaccine to combat endemic brucellosis in animals

Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-Brucella Antibodies in Ruminant Serum Samples▿

Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases

Evaluation of Competitive ELISA for Detection of Antibodies to Brucella Infection in Domestic Animals

Aim To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis. Methods cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested. Results Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzymelinked immunosorbent assay, and CFT were greater than 90%. Conclusion cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis