The emergence of Brucella canis as a public health threat in Europe: what we know and what we need to learn.

The zoonotic bacteria, Brucella canis, is becoming the leading cause of canine brucellosis in Europe. In dogs, it causes reproductive problems as well as non-specific lameness or discospondilitis. In humans, B. canis can be origin of chronic debilitating conditions characteristic to its genus such as undulant fever, splenomegaly, and lymphadenopathy. Although B. canis shows some pathogenic characteristics similar to B. abortus and B. melitensis, it lacks surface O-polysaccharide, like nonzoonotic B. ovis. This review shows that host-B. canis interactions are still poorly understood, with many knowledge and capability gaps, causing relatively poor sensitivity and specificity of existing diagnostic tools. Currently, there is no vaccine for this rough Brucella species. Besides, antimicrobial therapy does not guarantee bacterial elimination, and infection relapses are frequently reported, increasing the risks of antibiotic resistance development. B. canis has been detected in dogs in almost all European countries which increased human exposure, but currently there is no systematic surveillance. Moreover, B. canis caused brucellosis is not included in Animal Health Law, and therefore there is no legal framework to tackle this emerging infectious disease. To map out the diagnostic strategies, identify risks for human infections and propose management scheme for infected pet and kennel dogs, we present current understanding of canine B. canis caused brucellosis, outline major knowledge gaps and propose future steps. To address and highlight challenges veterinary and public health services encounter in Europe, we developed two B. canis infection scenarios: of a single household pet and of a kennel dog in larger group.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

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.

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.

The Tip of Brucella O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

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.

Brucella Seroprevalence and Associated Risk Factors in Occupationally Exposed Humans in Selected Districts of Southern Province, Zambia.

Background: Brucellosis is a neglected debilitating zoonosis widely recognized as an occupational health hazard. The seroprevalence of human anti-Brucella antibodies in high-risk populations, as well as their risk factors, have not been well-documented in Zambia. This study aimed at estimating the Brucella seroprevalence in herdsmen and abattoir workers and assess the associated risk factors. Methods: A cross-sectional seroepidemiological study was carried out between May and December 2020 among abattoir workers and herdsmen in Namwala, Monze and Choma districts of Southern Province in Zambia. Seroprevalence was assessed by indirect enzyme-linked immunosorbent assay (i-ELISA) or competitive enzyme-linked immunosorbent assay (c-ELISA) while a questionnaire was administered to obtain epidemiological data. Results: A total of 153 individuals were recruited in the study. The overall Brucella seroprevalence was 20.3% (95% CI: 14.6-27.5). Seropositivity among herdsmen and abattoir workers was 14.4% (95% CI: 9.2-21.8) and 46.4%, (95% CI: 28.8-65.0), respectively. Comparable seropositive results among districts showed Namwala with 26.9%, which was the highest, seconded by Monze 19.0%, and the least was Choma with 11.36%, seropositivity. The multivariate logistic regression model showed that occupation, age category, and district of residence were predictors of being seropositive to Brucella spp. antibodies. The odds of abattoir workers being seropositive to Brucella antibodies were 8.6 (95% CI: 2.6-28.2) higher than that of herdsmen being the reference group. The odds of age category 17-50 years being seropositive to Brucella antibodies were 7.0 (95% CI: 0.7-72.2) higher than being <16 years as the reference group. The odds of one having attained primary level of education being seropositive to Brucella were 1.3 (95% CI: 0.1-14.7) or secondary level of education were 6.2 (95% CI: 0.5-72.6) or tertiary level of education were 5.1 (95% CI: 0.2, 113.3) higher than that of no level of education as the reference group. Furthermore, the odds of a respondent being seropositive to Brucella antibodies were 4.5 (95% CI: 1.3-15.7) for Namwala and 4.9 (95% CI: 1.1-21.7) for Monze higher than that of Choma as the reference group. Conclusion: Anti-Brucella antibodies are prevalent among herdsmen and abattoir workers in the study areas of Zambia (20.26%), a sign of exposure to Brucella pathogens. Type of profession, age and level of education seem to influence the exposure to Brucella pathogens. This zoonosis should be considered as one of the differential diagnosis in humans presenting intermittent fever, malaria-like signs and general pain in humans.

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.

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.

Prevalence and speciation of brucellosis in febrile patients from a pastoralist community of Tanzania.

Brucellosis is an endemic zoonosis in sub-Saharan Africa. Pastoralists are at high risk of infection but data on brucellosis from these communities are scarce. The study objectives were to: estimate the prevalence of human brucellosis, identify the Brucella spp. causing illness, describe non-Brucella bloodstream infections, and identify risk factors for brucellosis in febrile patients from a pastoralist community of Tanzania. Fourteen (6.1%) of 230 participants enrolled between August 2016 and October 2017 met study criteria for confirmed (febrile illness and culture positivity or ≥four-fold rise in SAT titre) or probable (febrile illness and single SAT titre ≥160) brucellosis. Brucella spp. was the most common bloodstream infection, with B. melitensis isolated from seven participants and B. abortus from one. Enterococcus spp., Escherichia coli, Salmonella enterica, Staphylococcus aureus and Streptococcus pneumoniae were also isolated. Risk factors identified for brucellosis included age and herding, with a greater probability of brucellosis in individuals with lower age and who herded cattle, sheep or goats in the previous 12 months. Disease prevention activities targeting young herders have potential to reduce the impacts of human brucellosis in Tanzania. Livestock vaccination strategies for the region should include both B. melitensis and B. abortus.

Evaluation of the English bovine brucellosis surveillance system considering probability of disease introduction and non-random sampling.

The English surveillance system for bovine brucellosis was evaluated. The confidence in detecting at least one infected herd in the local population (surveillance system sensitivity or SSe), and the confidence in freedom from disease (PFree) adjusted (PFreeAdj) for the probability of disease introduction from abroad by imported animals (PIntro), were estimated for quarterly surveillance periods of 2016; because dairy herds were tested quarterly on bulk tank milk (BTM) with an antibody indirect ELISA. A stochastic model was developed and six surveillance components (representing also the local population strata), were evaluated. All English herds and their relative risk (RRs) of infection within each stratum were considered. The importance of each component was assessed using actual national data, which reflected non-random sampling. The contribution of the abortions testing was assessed with particular focus, because a decline in statutory submissions was observed in recent years. Beef herds without submissions (B-NoTest herds) at the laboratories were still considered as a population stratum, where infected cattle could be imported. Additionally, we evaluated the importance of different hypothetical design between-herds prevalence (Ph) values, at which the country could be classified as "infected". The potential negative effect on SSe due to the dilution of antibodies when individual samples are pooled within BTM and tested by the milk iELISA, was also investigated. The quarterly median SSe and PFreeAdj were both ≥ 95 % if at least four (0.008 %) herds were infected in the country due to independent import events. The system appeared able to substantiate Official Brucellosis Free (OBF) status frequently (on quarterly basis) using Ph=0.2 % (EU legislation). The component based only on BTM testing (M herds) showed the highest sensitivity; while the surveillance components based on abortions or post import calving (PIC) testing, had very low sensitivity at the (considered) Ph values lower than 0.2 %. In contrast, at Ph = 0.2 %, components based on abortion testing had median sensitivity between 91.3 % and 99.9 %, and the dilution effect on BTM testing did not change remarkably the SSe and PFreeAdj. When Ph was set to 1-2 infected herds (0.002-0.004 %), these were usually allocated by the model within the B-NoTest stratum (the largest stratum) and SSe reduced. Thus, if policy considers necessary increasing the SSe for low Phs (system's optimization as an early warning system); the cost efficiency of active risk based surveillance in beef herds (considering imports) could be investigated in the future.

The prevalence and risk factors for human Brucella species infection in a cross-sectional survey of a rural population in Punjab, India.

BACKGROUND: Brucellosis is an important neglected zoonosis. Effective cattle vaccines are available but are infrequently used in India, where rural households commonly own one or two cattle as sources of protein and income. We assessed the prevalence of infection and risk factors in humans. METHODS: We conducted a cross-sectional sero-survey in randomly selected individuals in 60 villages in Punjab. Infection prevalence was assessed by positive Rose Bengal testing or immunoglobulin G enzyme-linked immunosorbent assay. Risk factors were adjusted for potential confounding using multivariable analyses. RESULTS: Of the 1927 subjects who were approached, 93% agreed to participate. Age-standardised prevalence for Brucella infection was 2.24% (95% confidence interval [CI] 1.61 to 3.11). More than 60% of households kept cattle and 10% assisted with calving or abortions. Nearly all individuals consumed boiled cow/buffalo milk from their own or neighbours' cattle and 3.3% consumed goat's milk. There was a 2.18 times increased odds (95% CI 0.96 to 4.95) of infection with calving/abortions and a 4.26 times increased odds (95% CI 1.33 to 13.6) with goat's milk but not bovine milk consumption. CONCLUSIONS: An association with calving/abortions and goat's milk consumption was seen. Brucella vaccination of household livestock would reduce the risk to humans in such settings. Additional measures include biosecurity training around calving/abortions, education to boil all milk and for healthcare workers to test for brucellosis.

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.

Rapid Veterinary Diagnosis of Bovine Reproductive Infectious Diseases from Semen Using Paper-Origami DNA Microfluidics.

The health and well-being of cattle is an important issue in maintaining and increasing 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 center 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 time a demonstrated ability of the application of paper microfluidics devices for the diagnosis multiple infectious diseases from semen samples.

Brucellosis: Improved Diagnostics and Vaccine Insights from Synthetic Glycans.

Brucellosis 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.

Novel Solutions for Vaccines and Diagnostics To Combat Brucellosis.

Brucellosis is diagnosed by detection of antibodies in the blood of animals and humans that are specific for two carbohydrate antigens, termed A and M, which are present concurrently in a single cell wall O-polysaccharide. Animal brucellosis vaccines contain these antigenic determinants, and consequently infected and vaccinated animals cannot be differentiated as both groups produce A and M specific antibodies. We hypothesized that chemical synthesis of a pure A vaccine would offer unique identification of infected animals by a synthetic M diagnostic antigen that would not react with antibodies generated by this vaccine. Two forms of the A antigen, a hexasaccharide and a heptasaccharide conjugated to tetanus toxoid via reducing and nonreducing terminal sugars, were synthesized and used as lead vaccine candidates. Mouse antibody profiles to these immunogens showed that to avoid reaction with diagnostic M antigen it was essential to maximize the induction of anti-A antibodies that bind internal oligosaccharide sequences and minimize production of antibodies directed toward the terminal nonreducing monosaccharide. This objective was achieved by conjugation of Brucella O-polysaccharide to tetanus toxoid via its periodate oxidized terminal nonreducing monosaccharide, thereby destroying terminal epitopes and focusing the antibody response on internal A epitopes. This establishes the method to resolve the decades-long challenge of how to create effective brucellosis vaccines without compromising diagnosis of infected animals.

Improved serodiagnosis of bovine brucellosis by novel synthetic oligosaccharide antigens representing the capping m epitope elements of Brucella O-polysaccharide.

Members of the genus Brucella have cell wall characteristics of Gram-negative bacteria, which in the most significant species includes O-polysaccharide (OPS). Serology is the most cost-effective means of detecting brucellosis, as infection with smooth strains of Brucella leads to the induction of high antibody titers against the OPS, an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyl residues (D-Rha4NFo) that are variably α(1→2)- and α(1→3)-linked. Six d-Rha4NFo homo-oligosaccharides were synthesized, each containing a single α(1→3) link but with a varied number of α(1→2) links. After conjugation to bovine serum albumin (BSA), glycoconjugates 1 to 6 were used to develop individual indirect enzyme-linked immunosorbent assays (iELISAs). The diagnostic capabilities of these antigens were applied to panels of cattle serum samples, including those falsely positive in conventional assays, and the results were compared with those of the complement fixation test (CFT), serum agglutination test (SAT), fluorescent polarization assay (FPA), smooth lipopolysaccharide (sLPS) iELISA, and competitive enzyme-linked immunosorbent assay (cELISA) methods. Results from field serum samples demonstrated that all of the synthetic antigens had excellent diagnostic capabilities. Assays developed with the α(1→3)-linked disaccharide conjugate 1 were the best at resolving false-positive serological results. This was supported by the results from serum samples derived from experimentally infected cattle. Data from synthetic trisaccharide antigens 2 and 3 and tetrasaccharide antigen 4 identified an OPS epitope equally common to all Brucella abortus and Brucella melitensis strains but unique to Brucella. Synthetic oligosaccharide conjugates function as effective surrogates for naturally derived antigens. The creation of discrete OPS epitope antigens reveals not only the previously untapped diagnostic potential within this key diagnostic structure but also holds significance for the design of brucellosis vaccines and diagnostics that enable the differentiation of infected from vaccinated animals.

Molecular recognition of Brucella A and M antigens dissected by synthetic oligosaccharide glycoconjugates leads to a disaccharide diagnostic for brucellosis.

The cell wall O-polysaccharides of pathogenic Brucella species are homopolymers of the rare sugar 4,6-dideoxy-4-formamido-α-D-mannopyranose. Despite the apparent simplicity of the polysaccharide it appears to be a "block copolymer" composed of A and M polysaccharide sequences expressed as a single molecule. The simultaneous presence of both in the cell wall has complicated the understanding of the molecular recognition of these antigens by antibodies present in the serum of infected animals and humans and by monoclonal antibodies. Since presumptive diagnosis of brucellosis, a serious disease in domestic livestock, wild animals, and humans, is based on detection of these antibodies it is important to separate the two antigenic epitopes, one of which is also found in other bacteria. Chemical synthesis provides the only means to achieve this outcome. A series of six oligosaccharides from di to hexasaccharides 1-6 were synthesized and conjugated to proteins to provide glycoconjugate antigens and conjugate vaccines. These chemically defined antigens identified the M antigenic determinant and provided a structural basis for understanding the fine specificity of monoclonal and polyclonal antibodies that bind the M antigen. This resulted in the discovery of a disaccharide that shows considerable potential as an unambiguous diagnostic antigen for detecting brucellosis in humans and animals and two hexasaccharide conjugate vaccine candidates that produce high levels of O-polysaccharide specific antibodies in mice.

Investigating the use of protein saver cards for storage and subsequent detection of bovine anti-Brucella abortus smooth lipopolysaccharide antibodies and gamma interferon.

Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.

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 enzyme-linked 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.

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.

Competitive electrochemiluminescence wash and no-wash immunoassays for detection of serum antibodies to smooth Brucella strains.

Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISA(SP)SS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.