Economic analysis of bovine brucellosis control in the Rondônia state, Brazil.

Bovine brucellosis, mainly caused by Brucella abortus, is a worldwide distribution anthropozoonosis that causes great economic losses. In 2001, Brazil launched the National Program for the Control and Eradication of Brucellosis and Tuberculosis (PNCEBT). Contemporaneously, a great effort to characterize the epidemiology of the disease in Brazilian states was started. In the state of Rondônia, a first epidemiological study was carried out in 2004, revealing a prevalence of 35.2% of infected herds and 6.22% of seropositive females. In 2014, after a successful heifer vaccination program with strain 19 (S19), a second study detected a reduction in the prevalence of infected herds to 12.3% and of seropositive females to 1.9%. The present study aimed to quantify and compare the costs and benefits related to the control of bovine brucellosis in the state through an accounting analysis. Vaccinating heifers and performing serological tests to move animals were computed as private costs. The expenditures of the state official veterinary service for brucellosis control were considered public cost. The considered benefits of lowering prevalence were decreased cow replacement, decreased abortions, decreased perinatal and cow mortality, and increased milk production. Considering private and public costs, the net present value (NPV) was estimated at US$ 18.3 million, the internal rate of return (IRR) was calculated at 23%, and the benefit-cost ratio (BCR) was 1.7. When considering only the private costs, the NPV was US$34.9 million, the IRR was 49%, and the BCR was 3.0, meaning that the bovine producer had a return of 3 for each unit of currency invested. The results showed that the bovine brucellosis control measures implemented in the state of Rondônia, which had as its main strategy the vaccination of heifers with S19, produced highly advantageous economic results. The state should continue with its vaccination program, stimulating the use of the RB51 vaccine in addition to S19, to achieve further reductions in prevalence at low cost.

Genetic characteristics of an amikacin-resistant Brucella abortus strain first isolated from Marmota himalayana.

Brucella spp. are facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonotic brucellosis. Brucellosis affects public health and safety and even affects economic development. Our lab found that a Brucella strain isolated from Marmota himalayana exhibited amikacin resistance. To annotate and analyze the potential resistance genes in this strain, we utilized sequencing platforms in this study and cloned potential resistance genes. The findings showed that the isolated strain belonged to B. abortus biovar 1 and was similar to B. abortus 2308. The isolate had amikacin resistance genes encoding aminoglycoside 3'-phosphotransferase. Based on the results of genome analysis, the isolated strain may have obtained amikacin resistance genes from Salmonella spp. through Tn3 family transposons. Notably, this study establishes a foundation for further research on the resistance mechanism of Brucella spp. and provides data that may be useful for the prevention and control of drug-resistant Brucella strains.

High protection of mice against Brucella abortus by oral immunization with recombinant probiotic Lactobacillus casei vector vaccine, expressing the outer membrane protein OMP19 of Brucella species.

Brucellosis is a zoonotic disease threatening the public health and hindering the trade of animals and their products, which has a negative impact on the economic development of a country. Vaccination is the most effective way to control brucellosis. The recombinant vector vaccines are promising candidates for immunization in humans and animals. In this study, the gene encoding OMP19 antigen was primarily amplified and cloned into an expression vector called pT1NX, and then transformed to L. casei cell via electroporation technique. The expression was confirmed using specific antibody against the recombinant protein via immunological screening tests such as western blot and immunofluorescence assay. Finally, recombinant L. casei was orally fed to mice and the results were further recorded, indicating that the mice group which received OMP19 through L. casei based vaccine represented a very good general and mucosal immune responses protective against challenges with virulent B. abortus 544 strain compared with negative control recipient groups. Therefore, the vaccine produced in this research plan can be a very good candidate for protection against brucellosis.

Screening and identification of a human domain antibody against Brucella abortus VirB5.

Brucellosis is caused by the genus Brucella. Brucella is widely distributed in cattle, swine, sheep, goat and other mammals including human. Animal brucellosis causes severe economic losses and affects related international transportation and trade. Human brucellosis causes both acute and chronic symptoms of multi-organ dysfunction. Brucella type IV secretion system (T4SS) VirB5 was required for macrophages infection and essential for virulence in mice. VirB5 is located on the cell surface and serves as a specific adhesin targeting host cell receptors. The aim of this study was to isolate and characterize a specific human domain antibody against Brucella abortus (B. abortus) VirB5 from human single domain antibody (sdAb or VHH) phage display library. Following five rounds of screening, an sdAb named as BaV5VH4 showed the highest affinity by enzyme-linked immunosorbent assay (ELISA). Its interaction with B. abortus VirB5 was verified by binding assay, dot blot and molecular docking. These findings in this paper could greatly help elucidate the molecular mechanisms of Brucella infection, and accelerate the development of sdAbs-based vaccines and neutralizing therapeutics of brucellosis.

Assessment of fluorescence polarization assay: a candid diagnostic tool in Brucella abortus strain 19 vaccinated areas.

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals.

Methodology for the assessment of brucellosis management practices and its vaccination campaign: example in two Argentine districts.

BACKGROUND: In Argentina, vaccination with Brucella abortus Strain 19 vaccine is mandatory. The objective of the study was to develop and test a method for evaluating, in an innovative way, some farmers' and veterinarians' management practices in relation to brucellosis and to assess the vaccination campaign and coverage. The work took place in Brandsen and Navarro districts. Four questionnaires were designed (for officials from Local Sanitary Entities, vaccinators, vet practitioners and farmers). Responses were coded as "ideal" (0) and "not ideal" (1). To assess the relative weight of each question ("item"), experts ranked the items according to their impact on management practices and vaccination. A weighted score was then calculated. A higher weighted score was assigned to the worse practices. Farmers obtaining a global weighted score above the third quartile were classified as "inappropriately managed farms", to be compared per type of production system and district. To assess the immunization coverage, female calves were sampled 30 to 50 days post vaccination; they were expected to react positively to serological diagnostic tests (DT+). RESULTS: There were significantly more inappropriately managed farms and higher global scores among beef farmers and in Brandsen. Eighty three percent (83%) of female calves were DT+, significantly under the ideal immunization coverage (95%). Only 48% of farms were considered well vaccinated. DT+ results were positively associated with the Brandsen district (OR = 25.94 [4.60-1146.21] and with the farms having more than 200 cow heads ((OR = 78.34 [4.09-1500.00]). On the contrary, DT+ were less associated with vaccinators being veterinary practitioners (OR = 0.07 [0.006-0.78]). Farmers are well advised by their veterinary practitioners but they should improve some management practices. CONCLUSIONS: The vaccination campaign is globally well implemented, but the immunization coverage and some vaccinators' practices should be improved. This study leads to a better understanding of the most common used management and control practices regarding brucellosis, which affect its epidemiology. Any vaccination campaign should be periodically assessed to highlight possible fails. The described methodology can be extrapolated to other countries and different contexts.

The economic efficiency and equity of government policies on brucellosis: comparative insights from Albania and the United States of America.

Brucellosis is a zoonotic bacterial disease that causes recurring febrile illness in humans, as well as reproductive failure and reduced milk production in livestock. The cost of brucellosis is equal to the sum of lost productivity of humans and animals, as well as private and public expenditures on brucellosis surveillance, prevention, control and treatment. In Albania, Brucella abortus and B. melitensis affect humans, cattle and small ruminants. In the United States, B. abortus affects cattle and wild ungulates in the Greater Yellowstone Area. These two case studies illustrate the importance of place-specific context in developing sustainable and effective brucellosis mitigation policies. Government regulations and mitigation strategies should be designed with consideration of all costs and benefits, both to public agencies and private stakeholders. Policy-makers should, for example, weigh the benefits of a regulation that increases epidemiological certainty against the costs of compliance for producers and households. The distribution of costs and benefits amongst public agencies and private individuals can have important implications for a policy's economic efficiency and equity quite apart from their total magnitude.

La brucellose est une maladie bactérienne zoonotique responsable d'épisodes fébriles récurrents chez l'être humain ainsi que d'infertilités et d'une baisse de la production de lait chez les animaux d'élevage. Le coût de la brucellose équivaut à la somme des pertes de productivité chez l'homme et chez les animaux et des dépenses privées et publiques engagées dans la surveillance, la prévention, la prophylaxie et le traitement de la brucellose. En Albanie, Brucella abortus et B. melitensis affectent aussi bien l'être humain que les bovins et les petits ruminants. Aux États-Unis d'Amérique, B. abortus affecte les bovins et les ongulés sauvages de la région du Grand Yellowstone. Les deux études de cas présentées dans cet article illustrent l'importance de prendre en compte le contexte spécifique de chaque site particulier lors de la mise en place de politiques durables et efficaces d'atténuation de la brucellose. La réglementation et les stratégies d'atténuation mises en oeuvre par les pouvoirs publics doivent être conçues en considérant l'ensemble des coûts et des bénéfices induits pour les agences gouvernementales et les intervenants privés. Par exemple, les décideurs politiques devraient évaluer les avantages induits par une réglementation basée sur un accroissement des certitudes épidémiologiques, par rapport aux coûts supportés par les producteurs et les ménages se conformant à cette réglementation. La répartition des coûts et des bénéfices entre les agences gouvernementales et les individus privés peut avoir d'importantes répercussions sur l'efficacité économique et l'équité d'une politique sanitaire, indépendamment de leur amplitude totale.

La brucelosis es una enfermedad bacteriana zoonótica que provoca dolencias febriles recurrentes en el ser humano, así como trastornos reproductores y una menor producción lechera en el ganado. Su costo es igual a la suma de las pérdidas de productividad en personas y animales, junto con el gasto público y privado dedicado a tareas de vigilancia, prevención, control y tratamiento de la enfermedad. En Albania, Brucella abortus y B. melitensis afectan a personas, ganado bovino y pequeños rumiantes. En los Estados Unidos, B. abortus afecta al ganado bovino y a ungulados salvajes de la zona del Gran Yellowstone. Los autores presentan dos estudios monográficos que ponen de relieve la importancia del contexto geográfico a la hora de definir políticas duraderas y eficaces para mitigar la brucelosis. Al elaborar tanto reglamentos como estrategias públicas de mitigación es preciso tener en cuenta la totalidad de los costos y beneficios, a la vez para los organismos oficiales y para el sector privado. Los planificadores deben, por ejemplo, sopesar los beneficios derivados de un reglamento que aporte mayor certidumbre epidemiológica en relación con los costos que entrañe su aplicación para productores y familias. La forma en que costos y beneficios se distribuyan entre organismos públicos y personas físicas puede influir sustancialmente en los niveles de eficiencia económica y equidad de una política, con independencia de su magnitud total.

First evaluation of an influenza viral vector based Brucella abortus vaccine in sheep and goats: Assessment of safety, immunogenicity and protective efficacy against Brucella melitensis infection.

Previously we developed and evaluated a candidate influenza viral vector based Brucella abortus vaccine (Flu-BA) administered with a potent adjuvant Montanide Gel01 in cattle, which was found safe and highly effective. This study was aimed to establish a proof-of-concept of the efficacy of Flu-BA vaccine formulation in sheep and goats. We vaccinated sheep and goats with Flu-BA vaccine and as a positive control vaccinated a group of animals with a commercial B. melitensis Rev.1 vaccine. Clinically, both Flu-BA and Rev.1 vaccines were found safe. Serological analysis showed the animals received Flu-BA vaccine did not induce antibody response against Brucella Omp16 and L7/L12 proteins during the period of our study (56days post-initial vaccination, PIV). But observed significant antigen-specific T cell response indicated by increased lymphocyte stimulation index and enhanced secretion of IFN-γ at day 56 PIV in Flu-BA group. The Flu-BA vaccinated animals completely protected 57.1% of sheep and 42.9% of goats against B. melitensis 16M challenge. The severity of brucellosis in terms of infection index and colonization of Brucella in tissues was significantly lower in the Flu-BA group compared to negative control animals group. Nevertheless, positive control commercial Rev.1 vaccine provided strong antigen-specific T cell immunity and protection against B. melitensis 16M infection. We conclude that the Flu-BA vaccine induces a significant antigen-specific T-cell response and provides complete protection in approximately 50% of sheep and goats against B. melitensis 16M infection. Further investigations are needed to improve the efficacy of Flu-BA and explore its practical application in small ruminants.

Risk assessment and management of brucellosis in the southern greater Yellowstone area (II): Cost-benefit analysis of reducing elk brucellosis prevalence.

Recent cases of bovine brucellosis (Brucella abortus) in cattle (Bos taurus) and domestic bison (Bison bison) of the southern Greater Yellowstone Area (SGYA) have been traced back to free-ranging elk (Cervus elaphus). Several management activities have been implemented to reduce brucellosis seroprevalence in elk, including test-and-slaughter, low-density feeding at elk winter feedgrounds, and elk vaccination. It is unclear which of these activities are most cost-effective at reducing the risk of elk transmitting brucellosis to cattle. In a companion paper, a stochastic risk model was used to translate a reduction in elk seroprevalence to a reduction in the risk of transmission to cattle. Here, we use those results to estimate the expected economic benefits and costs of reducing seroprevalence in elk using three different management activities: vaccination of elk with Brucella strain 19 (S19), low-density feeding of elk, and elk test-and-slaughter. Results indicate that the three elk management activities yield negative expected net benefits, ranging from -$2983 per year for low-density feeding to -$595,471 per year for test-and-slaughter. Society's risk preferences will determine whether strategies that generate small negative net benefit, such as low-density feeding, are worth implementing. However, activities with large negative net benefits, such as test-and-slaughter and S19 vaccination, are unlikely to be economically worthwhile. Given uncertainty about various model parameters, we identify some circumstances in which individual management activities might generate positive expected net benefit.

A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle.

To investigate the usefulness of culture for the confirmation of brucellosis in cattle, a comparison of culture and serology was undertaken on 248 animals in four dairy herds where the disease was active. Paired supramammary (SM), retropharyngeal (RP), and internal iliac (IL) lymph nodes were cultured, and five serological tests were deployed: the microserum agglutination test (MSAT), complement fixation test (CFT), the indirect (iELISA) and competitive ELISA, and the fluorescence polarisation assay (FPA). Brucella abortus was isolated from 86.8% of animals on combined culture of all three lymph nodes. Individually, the highest isolation rate was from the RP (90.5% of culture positives). Of culture positive animals, 13.7% and 6.2% were positive from the RP and SM alone, respectively. Approximately half of the positive cultures yielded <10 colonies/culture plate. Although 80.9% of animals were positive in at least one serological test, only 45.2% were positive in all five. For culture-positive animals, the MSAT was the most sensitive test (71.8%). Of the culture-negative animals 67.7% were positive in at least one test, while 12.9% were positive in all five. Titres were higher in animals culture-positive from the SM, and there was a direct correlation between higher titres and higher colony counts in SM cultures. Only 8.9% of animals were both culture-negative and seropositive (in at least one test), while 16.5% were culture-positive and seronegative in all five tests. The results highlight and validate the sensitivity of bacteriological culture in confirming a diagnosis of bovine brucellosis. While the MSAT and FPA were the most sensitive serological tests, a significant percentage of infected animals were undetectable using these standard serological assays.

Economic losses due to bovine brucellosis in Brazil / Perdas econômicas devidas à brucelose bovina no Brasil

Brucellosis is an important zoonosis of worldwide distribution. Reliable epidemiologic brucellosis data covering approximately 90% of the cattle population in Brazil have been recently published. Therefore, considering the scarcity of information regarding the economic impact of bovine brucellosis in Brazil, the goal of this study was to estimate economic impact of brucellosis on the Brazilian cattle industry. Several parameters including abortion and perinatal mortality rates, temporary infertility, replacement costs, mortality, veterinary costs, milk and meat losses were considered in the model. Bovine brucellosis in Brazil results in an estimated loss of R$ 420,12 or R$ 226,47 for each individual dairy or beef infected female above 24 months of age, respectively. The total estimated losses in Brazil attributed to bovine brucellosis were estimated to be approximately R$ 892 million (equivalent to about 448 million American dollars). Every 1% increase or decrease in prevalence is expected to increase or decrease the economic burden of brucellosis in approximately 155 million Reais.

A brucelose é uma zoonose de importância mundial. Recentes dados epidemiológicos dessa doença foram obtidos por meio de estudos que abrangeram em torno de 90% do rebanho bovino do Brasil. Observa-se escassez de informações sobre o impacto econômico causado pela brucelose bovina, portanto, o objetivo do presente estudo foi estimar as perdas econômicas na pecuária brasileira. Gastos relacionados com as ocorrências de abortos, natimortos, subfertilidade, descartes involuntários, mortalidade, intervenções veterinárias, diminuição da produção de leite e de carne, foram considerados nos cálculos. As perdas devidas à brucelose bovina no Brasil foram estimadas em R$ 420,12 ou R$ 226,47 para cada fêmea infectada acima de 24 meses de idade em rebanhos de leite ou corte, respectivamente. O prejuízo total estimado foi de, aproximadamente, R$ 892 milhões (equivalentes a $ 448 milhões de dólares americanos). A cada 1% de variação na prevalência, estima-se a variação de 155 milhões de reais no custo da brucelose bovina no Brasil.

Preliminary assessment of binary ethylenimine inactivated and saponized cutaneous warts (BPV-2) therapeutic vaccine for enzootic bovine haematuria in hill cows.

A preliminary therapeutic vaccine trial was conducted in hill cows to evaluate the therapeutic potential of binary ethylenimine (BEI) inactivated and saponized bovine papillomavirus-2 (BPV-2) for enzootic bovine haematuria (EBH). Although the vaccine failed to show favorable clinical vaccine results in treatment of EBH affected cows at 120 days post-vaccination but immunopathological responses were encouraging. A significant difference was observed in humoral (against Brucella abortus strain 19S) and cell-mediated (in vivo phytohaemagglutination delayed type hypersensitivity (PHA DTH) test and CD4+/CD8+ T-cells ratio by FACS analysis) immune responses following vaccination. The vaccinated animals grossly failed to show regression of bladder tumours but microscopically engorgement and marked perivascular infiltration of mononuclear cells was observed which are indicative of the induction of initial stages of tumour regression. Overall results indicated that the therapeutic vaccine developed can have potentials for treating EBH in cows, for which further modifications in vaccine dose and field trial is required.

How human brucellosis incidence in urban Kampala can be reduced most efficiently? A stochastic risk assessment of informally-marketed milk.

BACKGROUND: In Kampala, Uganda, studies have shown a significant incidence of human brucellosis. A stochastic risk assessment involving two field surveys (cattle farms and milk shops) and a medical record survey was conducted to assess the risk of human brucellosis infection through consumption of informally marketed raw milk potentially infected with Brucella abortus in Kampala and to identify the best control options. METHODOLOGY/PRINCIPAL FINDINGS: In the cattle farm survey, sera of 425 cows in 177 herds in the Kampala economic zone were sampled and tested for brucellosis using a competitive enzyme-linked immunosorbent assay (CELISA). Farmers were interviewed for dairy information. In the milk shop surveys, 135 milk sellers in the urban areas were interviewed and 117 milk samples were collected and tested using an indirect enzyme-linked immunosorbent assay (IELISA). A medical record survey was conducted in Mulago National Referral Hospital for serological test results. A risk model was developed synthesizing data from these three surveys. Possible control options were prepared based on the model and the reduction of risk was simulated for each scenario. Overall, 12.6% (6.8-18.9: 90%CI) of informally marketed milk in urban Kampala was contaminated with B.abortus at purchase and the annual incidence rate was estimated to be 5.8 (90% CI: 5.3-6.2) per 10,000 people. The best control option would be the construction of a milk boiling centre either in Mbarara, the largest source of milk, or in peri-urban Kampala and to ensure that milk traders always sell milk to the boiling centre; 90% success in enforcing these two options would reduce risk by 47.4% (21.6-70.1: 90%CI) and 82.0% (71.0-89.0: 90%CI), respectively. CONCLUSION/SIGNIFICANCE: This study quantifies the risk of human brucellosis infection through informally marketed milk and estimates the incidence rate in Kampala for the first time; risk-based mitigation strategies are outlined to assist in developing policy.

Evaluation of the fluorescence polarization assay for the detection of Brucella abortus antibodies in bison in a natural setting.

Bison and elk in the greater Yellowstone area are the last-known reservoir of Brucella abortus in the United States. Diagnosis of brucellosis is challenging as there is no perfect reference test. The objectives of this study were to estimate the accuracy of the fluorescence polarization assay (FPA) for the screening of B. abortus antibodies in bison in a natural setting. Serum and tissue samples were collected and analyzed from the known brucellosis-infected bison herd in Yellowstone National Park (YNP). Additionally, serum samples from privately owned bison were serologically tested for brucellosis. While the FPA and five other tests had perfect sensitivity, all tests had substantially lower specificity in the YNP herd. However, a Bayesian analysis showed that as many as 59-74% of the culture-negative animals were most-likely truly infected. A decision-tree analysis showed that the expected cost of FPA testing was comparable to the cost of other serologic tests. The FPA was shown to be highly sensitive but may not be able to differentiate culture-positive and culture-negative animals. There is a need for long-term longitudinal studies to estimate diagnostic accuracy of tests for B. abortus in bison.

Occupational infection due to Brucella abortus S19 among workers involved in vaccine production in Argentina.

The pathological consequences of exposure to the vaccine strain Brucella abortus S19 were evaluated in 30 employees from vaccine-manufacturing plants. Active brucellosis was diagnosed in 21 subjects, of whom only five recalled an accidental exposure. Clinical manifestations were mild, and only one patient presented a complication. After antimicrobial therapy, initially symptomatic patients either experienced clinical remission or had mild persistent symptoms. This is the first study reporting infection by B. abortus S19 among workers from vaccine-manufacturing plants, which in many cases was acquired from unnoticed exposures. Measures to improve the safety of B. abortus S19 handling should be implemented.

Tailored magnetic nanoparticles for direct and sensitive detection of biomolecules in biological samples.

We developed nanoparticles with tailored magnetic properties for direct and sensitive detection of biomolecules in biological samples in a single step. Thermally blocked nanoparticles obtained by thermal hydrolysis, functionalized with specific ligands, are mixed with sample solutions, and the variation of the magnetic relaxation due to surface binding is used to detect the presence of biomolecules. The binding significantly increases the hydrodynamic volume of nanoparticles, thus changing their Brownian relaxation frequency which is measured by a specifically developed AC susceptometer. The system was tested for the presence of Brucella antibodies, a dangerous pathogen causing brucellosis with severe effects both on humans and animals, in serum samples from infected cows and the surface of the nanoparticles was functionalized with lipopolysaccharides (LPS) from Brucella abortus. The hydrodynamic volume of LPS-functionalized particles increased by 25-35% as a result of the binding of the antibodies, measured by changes in the susceptibility in an alternating magnetic field. The method has shown high sensitivity, with detection limit of 0.05 microg x mL(-1) of antibody in the biological samples without any pretreatment. This magnetic-based assay is very sensitive, cost-efficient, and versatile, giving a direct indication whether the animal is infected or not, making it suitable for point-of-care applications. The functionalization of tailored magnetic nanoparticles can be modified to suit numerous homogeneous assays for a wide range of applications.

Estimating herd prevalence of bovine brucellosis in 46 USA states using slaughter surveillance.

Making valid inferences about herd prevalence from data collected at slaughter is difficult because the observed sample is dependent on the number of animals sampled from each herd, which varies with herd size and culling practices, and the probability of a positive test result, which depends on variable within-herd prevalence levels as well as test sensitivity and specificity. In this study, brucellosis herd prevalence among beef cow-calf operations is estimated from slaughter surveillance data using a method that combines process modeling with Bayesian inference. Inferences are made for two populations; the first population comprises cow-calf beef herds in a typical U.S. state. The second population represents all beef herds in a collection of 46 low-risk states. The Bayesian Monte Carlo method used in this study links process model inputs to observed surveillance results via Bayes Theorem. The surveillance evidence across multiple years is accumulated at a discounted rate based on the probability of introducing new infection into an area. The process model's inputs include herd size, culling rate per herd, within-herd prevalence, serologic test performance, and the probability of successfully investigating positive results. The surveillance results comprise the number of cows and bulls tested at slaughter and the number of affected herds detected each year. The results find at least 95% confidence that brucellosis herd prevalence among beef cow-calf herds is less than 0.014% (3 per 21,500 herds) and 0.00081% (5 per 6,15,770) after 5 years of slaughter surveillance (with no detections of affected herds) in a typical U.S. state and across 46 low-risk U.S. states, respectively. These results were based on conservative modeling assumptions, but sensitivity analysis suggests only slight changes in the results from changing the assumed process model input values. The most influential analytic input was the probability of introducing new infection into a putatively brucellosis-free state or group of states.

Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium.

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

[Assessment of polymerase chain reaction (PCR) to diagnose brucellosis in a Brucella infected herd]. / Evaluación de la reacción en cadena de la polimerasa para el diagnóstico de la brucelosis en un rebaño lechero infectado con Brucella spp.

The diagnosis of bovine brucellosis using PCR in blood and milk samples from two dairy herds were compared to in vitro isolation, complement fixation test (CF), competitive ELISA (C-ELISA) in serum, and indirect ELISA (I-ELISA) in milk. Samples were obtained from 99 cows vaccinated with Brucella abortus strain 19, from a naturally infected herd (A), whose cows were also vaccinated with B. abortus strain RB51 as adults, and 100 from brucellosis free herd (B). In herd A, PCR identified 14 B. abortus infected cows: nine infected with wild type, and five with wild type and RB51, B. abortus S 19 was not identified. B. abortus biotype 1 was isolated from one cow. All cows infected with a wild strain of B. abortus were positive in serologic tests. Brucella was not found in herd B using PCR. Serological test showed 100% sensitivity related to PCR. The specificity for CF, C-ELISA and I-ELISA was 100%, 99% and 95% respectively. PCR could be useful to identify Brucella biotypes and to complement serologic tests.

Evaluación de la reacción en cadena de la polimerasa para el diagnóstico de la brucelosis en un rebaño lechero infectado con Brucellaspp / Assessment of polymerase chain reaction (PCR) to diagnose brucellosis in a Brucella infected herd

Para el diagnóstico de la brucelosis bovina en muestras de sangre y/o leche, se comparó la reacción en cadena de la polimerasa (PCR) con el aislamiento in vitro de Brucella abortus, las pruebas serológicas defijación del complemento (FC) e inmunoenzimáticas de competición (ELISA-C) en suero e indirecto (ELISA-I) en leche. Se analizaron muestras de vacas lecheras de un rebaño infectado “A”, vacunadas con B. abortus cepa 19 antes de los 8 meses de edad y revacunadas con B. abortus cepa RB51 como adultas (n= 99) y de otro “B”, libre de brucelosis (n=100), como control. En A, la PCR identificó 14 vacas infectadas con B. abortus: nueve con cepa silvestre y cinco con cepa silvestre y RB51. No se identificó B. abortus cepa 19. El biotipo 1 se aisló en un caso. Las 14 vacas infectadas con la cepa silvestre resultaron positivas en las tres pruebas serológicas. En B, por PCR no se identificó Brucella. Las pruebas serológicas mostraron una sensibilidad del 100% respecto de PCR. La especificidad para FC, ELISA-C y ELISA-I fue del 100%, 99% y 95%, respectivamente. Se concluye que la PCR sería útil como complemento de las pruebas serológicas o cuando no hay un resultado concluyente.

The diagnosis of bovine brucellosis using PCR in blood and milk samples from two dairy herds were compared to in vitro isolation, complement fixation test (CF), competitive ELISA (C-ELISA) in serum, and indirect ELISA (I-ELISA) in milk. Samples were obtained from 99 cows vaccinated with Brucella abortus strain 19, from a naturally infected herd (A), whose cows were also vaccinated with B. abortus strain RB51 as adults, and 100 from brucellosis free herd (B). In herd A, PCR identified 14 B. abortus infected cows: nine infected with wild type, and five with wild type and RB51, B. abortus S 19 was not identified. B. abortus biotype 1 was isolated from one cow. All cows infected with a wild strain of B. abortus were positive in serologic tests. Brucella was not found in herd B using PCR. Serological test showed 100% sensitivity related to PCR. The specificity for CF, C-ELISA and I-ELISA was 100%, 99% and 95% respectively. PCR could be useful to identify Brucella biotypes and to complement serologic tests.