Buffered Plate Agglutination Test Research Articles

Some Serological Analytic Approaches and Test Execution in Determination of Bovine Brucellosis: Overview

Bovine brucellosis is a highly contagious, zoonotic, and economically significant bacterial disease. The purpose of this paper was to review available scientific data on serological tests for the diagnosis of bovine brucellosis and compare test performance based on assay sensitivity (Se) and specificity (Sp) values. Abortion, placentitis, epididymitis, and orchitis are symptoms of the disease. The clinical picture of brucellosis is not pathognomonic, and the patient's clinical history, particularly the occurrence of abortion, is crucial for diagnosis. Although the causative agent is isolated and identified for a definitive diagnosis of bovine brucellosis, serological tests are usually preferred. The host defense mechanism against Brucella is functionally divided into innate or non-specific immunity and adaptive or specific immunity. The pathogenic brucella organism has evolved a slew of mechanisms for evading and/or modulating both innate and adaptive immune responses in its host. Serological assays are based on the fact that Brucella abortus and other smooth Brucella have a unique O-polysaccharide that induces a humoral response with the production of IgM first, followed by IgG1, and then IgG2/IgA. Screening serological tests (Milk ring test, Rose Bengal Test (RBT), and Buffered plate agglutination test (BPAT)) and confirmatory serological tests (complement fixation test (CFT), serum agglutination test (SAT), 2-mercaptoethanol test (2ME), indirect-enzyme-linked immunosorbent assay (iELISA), and, more recently, competitive-ELISA (cELISA) and Brucella fluorescent pol (FPA). CFT, RBT, and iELISA tests are currently used in EU legislation for intra-community cattle trade and show comparable Se and Sp. The new FPA test demonstrated Se and Sp levels comparable to standard tests and was proposed for inclusion as a standard test in EU legislation. Similarly, the OIE considers the four tests to be a mandatory requirement for international trade. Several methods, however, produce both false-positive and false-negative results. There is no single serological test that is 100% accurate and applicable in all epidemiological situations. In general, confirmatory serological procedures involve testing each serum with multiple tests, usually a screening test with high sensitivity followed by a confirmatory test with high specificity. As a result, the development of rapid, low-cost serological tests with high sensitivity and specificity for brucellosis detection is a major issue that requires further investigation. Keywords: Bovine Brucellosis; Brucella Abortus; Diagnosis; Sensitivity; Specificity; Serological Tests, Test Performance DOI: 10.7176/JNSR/13-12-03 Publication date: June 30 th 2022

Seroprevalence and risk factors for bovine brucellosis in the state of Paraná, Brazil: an analysis after 18years of ongoing control measures.

Seroprevalence and risk factors of bovine brucellosis (Brucella abortus) in herds and cattle were estimated by a cross-sectional study in the state of Paraná, Brazil. The state was divided into seven regions and a random, two-stage sampling was performed on properties and cattle from each region between 2018 and 2019. Serum samples were collected from 11,592 cows over 24months from 1,757 properties and a questionnaire was applied to identify potential risk factors. As recommended by the National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), serological testing for the detection of anti-Brucella antibodies included the buffered plate agglutination test (screening test) and the fluorescence polarization assay (confirmatory test). The seroprevalence of bovine brucellosis on properties and in cattle was 4.87% (95% confidence interval [CI]: 3.98-5.93%) and 2.24% (95% CI: 1.47-3.41%), respectively. Multiple logistic regression analysis identified larger herd size and failure to test for brucellosis as risk factors for the presence of anti-B. abortus antibodies. These results demonstrate no change in the prevalence when comparing initial studies conducted in 2002. Given our findings, it is recommended that policies for brucellosis control include a widespread vaccination program for higher prevalence areas and eradication approach to lower prevalence areas. All steps related to correct immunization of the herds should be verified and improved by training and education. Health education action must be carried out informing farmers about the risks of introducing animals not tested for brucellosis into their herds and the benefits of testing their herds regularly.

Identification of a small-scale pig farm infected with Brucella suis linked to a clinical case of human brucellosis in Buenos Aires province, Argentina

Here we report a case of human brucellosis due to Brucella suis in a person who worked in a small-scale pig farm. The farm had no history of clinical brucellosis, and signs of the disease were not observed upon clinical examination of the animals. Serum from all the 3 boars, 16/22 sows and 9/25 gilts was obtained for serological examination by Buffered Plate Agglutination Test (BPAT), Rose Bengal Test (RBT) and Fluorescent Polarization Assay (FPA). Bacteriological culture and Direct Fluorescence Antibody Test (DFAT) were performed in tissue samples from a seropositive boar and a sow. Specific antibodies were detected in 53 % (10/19) adult pigs, while all sampled gilts were seronegative. B. suis biovar 1 was isolated from one boar. In contrast, while the bacterium was not isolated from any tissue from a seropositive sow, it was detected by DFAT. From the bacteriological and serological evidence of B. suis endemic infection in the pig farm and the lack of preventive measures and biosecurity practices, it is concluded that the person most likely acquired the disease from the infected animals or by contact with contaminated environment in the farm.

Latex agglutination using the periplasmic proteins antigen of Brucella melitensis is a successful, rapid, and specific serodiagnostic test for ovine brucellosis

Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity.

Epidemiología de la brucelosis caprina y ovina en la provincia de Formosa, Argentina

An epidemiological study of brucellosis was carried out in 516 goats and mixed flocks (goat/sheep) from the three agro-ecological regions of Formosa province, Argentina. Serum samples from a total of 25401 goats and 2453 sheeps were analyzed using buffered plate agglutination test (BPAT) and complement fixation test (CFT). Bacteriological and PCR analyses on milk samples from goats in three flocks with a history of brucellosis and recent abortions were also performed. Brucellosis was detected in four of the nine departments of the province with an overall prevalence of 2% and an intra-flock prevalence ranging between 1% and 40%. The proportion of infected flocks was 3.6%, 12% and 36% for the eastern, central and western regions, respectively. Brucella melitensis bv. 1 was isolated efrom goats for the first time in the province. The expected fragments of 827bp from the omp2ab gene (Brucella spp.) and 731bp from the insert IS711 (B. melitensis) were amplified by PCR. Detection of antibodies by BPAT and FCT in sheep cohabiting with goats suggests that infections could have been caused by B. melitensis, posing an additional risk to public health. Control and eradication programs for brucellosis should consider mixed flocks as a single epidemiological unit. The results indicate that brucellosis by B. melitensis bv1 is highly endemic in the central and western regions of Formosa province.

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the ‘Bruce ladder’ multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.

Inquérito sorológico e molecular da brucelose canina no município de Natal, Estado do Rio Grande do Norte

O objetivo do presente trabalho foi determinar a ocorrência de anticorpos anti-Brucella rugosa e anti-Brucella lisa em cães do município de Natal, Estado do Rio Grande do Norte, Brasil, bem como identificar fatores de risco associados à positividade e realizar a detecção molecular em animais soropositivos. Foram utilizados soros sanguíneos de 416 cães atendidos em clínicas veterinárias durante o período de março a novembro de 2011. Para o diagnóstico sorológico da infecção por Brucella rugosa, foi empregada a prova de imunodifusão em gel de ágar (IDGA), utilizando antígeno de lipopolissacarídeos e proteínas de Brucella ovis, amostra Reo 198 e, para o diagnóstico da infecção por Brucella lisa, foi utilizado o teste do antígeno acidificado tamponado (AAT). De animais soropositivos, foram coletadas amostras de sangue com citrato de sódio para o diagnóstico pela reação em cadeia pela polimerase (PCR). A frequência de anticorpos anti-Brucella rugosa foi de 28,9% (120/416). Todos os animais foram negativos para anticorpos anti-Brucella lisa. Dentre 80 animais soropositivos, o DNA de Brucella spp. foi amplificado em três animais (3,8%). Não foram identificados fatores de risco associados à soropositividade. Conclui-se que a infecção por Brucella rugosa está presente no município de Natal, bem como se sugere o monitoramento sorológico de animais atendidos em clínicas visando à identificação de fontes de infecção.

Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine

Thirty-two water buffalo (Bubalus bubalis) calves aged 6–10 months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose-response study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I-III received the recommended vaccine dose (RD) twice 4 weeks apart, RD twice 18 weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16 weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson's correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P > 0.05).

Frequency of Detection of Immunoglobulins of Toxoplasma gondii, Leptospira spp., and Brucella abortus in Livestock/Farm and Abattoir Workers in Trinidad

ABSTRACT Toxoplasma gondi, Leptospira spp., and Brucella abortus are all established parasitic and bacterial zoonoses that manifest themselves in several forms of human diseases. They have been associated with occupational exposures, particularly amongst workers associated with livestock farms. The primary objectives of this study were to determine the seroprevalence of T. gondii immunoglobulin M (IgM) immunoglobulins (serum antibodies), Leptospira IgM immunoglobulins, and B. abortus IgG immunoglobulins, suggestive of acute or chronic infections, in livestock/farm and abattoir workers in Trinidad, and to relate to risk factors. Blood samples were collected from 394 consenting livestock/farm workers and 99 abattoir workers across the island of Trinidad. Serological status was determined by enzyme immunoassay (EIA) for T. gondii IgM, enzyme-linked immunosorbent assay (ELISA) for Leptospira IgM immunoglobulins, and buffered plate agglutination test (BPAT) and competitive ELISA for B. abortus IgG immunoglobulins. Of 394 apparently healthy livestock/farm workers sampled, 150 (38.1%) were seropositive for T. gondii IgM immunoglobulins, compared with 44 (44.4%) of 99 abattoir workers (p > .05; χ2 test). Five (1.3%) of 371 and 1 (1.0%) of 99 livestock/farm and abattoir workers respectively were positive for Leptospira IgM immunoglobulins. All samples from livestock/farm workers and abattoir workers were negative for B. abortus immunoglobulins. None of the risk factors investigated was statistically significantly (p > .05; χ2 test) associated with T. gondii and Leptospira spp. infections.

Anticorpos anti Brucella abortus, anti Brucella canis e anti Leptospira spp. em raposas (Pseudalopex vetulus) do semiárido paraibano, Nordeste do Brasil

O objetivo do presente trabalho foi determinar a ocorrência de anticorpos anti Brucella abortus, anti Brucella canis e anti Leptospira spp. em raposas (Pseudalopex vetulus). Para tanto, foram utilizadas 60 raposas atropeladas em rodovias no semiárido da Paraíba, Nordeste do Brasil. Para a detecção de anticorpos anti Brucella abortus, o teste do antígeno acidificado tamponado (AAT) foi empregado como teste de triagem, e a prova do 2-mercaptoetanol foi empregada como método confirmatório. Para o diagnóstico sorológico das infecções por Brucella canis e Leptospira spp., foram utilizados os testes de imunodifusão em gel de agar (IDGA) e soroaglutinação microscópica, respectivamente. Todas as amostras foram negativas na pesquisa de anticorpos anti Brucella canis e anti Leptospira spp. Das 60 raposas testadas, 16 (26,6%) foram positivas para anticorpos anti Brucella abortus no teste de AAT, e quatro (6,7%) amostras foram confirmadas no teste de 2-mercaptoetanol, sendo duas amostras com título 100 e duas com título 50.

Use of the Brucella melitensis Native Hapten To Diagnose Brucellosis in Goats by a Rapid, Simple, and Specific Fluorescence Polarization Assay

The performance of the fluorescence polarization assay (FPA) using the recently described Brucella melitensis native hapten and the Brucella abortus O-polysaccharide tracer was evaluated and compared with those of The World Organization for Animal Health tests related to indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from a high-prevalence area where vaccination was performed; test series were also evaluated to increase the final specificity of the tests. Our results showed that the respective relative sensitivity and specificity were 99.7% and 32.5% for the rose Bengal test with a 3% cell concentration (RBT3), 92.8% and 68.8% for the rose Bengal test with 8% cell concentration (RBT8), 98.4% and 84.9% for the Canadian complement fixation test (CFT), 83.7% and 65.5% for the Mexican CFT, 98.4% and 81.0% for the buffered plate agglutination test (BPAT), and 78.1% and 89.3% for the fluorescence polarization assay (FPA). The use of the FPA as the secondary test significantly increased the final specificities of test combinations; the screening tests BPAT, RBT3, and RBT8 plus FPA resulted in 90%, 91.2%, and 91.3% final specificities, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, the specificities were 65.5%, 63.2%, and 91.7%, respectively. The results suggested that the FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis, since its cutoff can be adjusted to improve its sensitivity or specificity, it is a rapid and simple test, it can be the test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of goats wrongly slaughtered due to misdiagnosis.

Infecção em cão por Brucella abortus: relato de caso

Brucella abortus infection is reported in a dog from a rural area that presented at clinical evaluation left testicular enlargement and right testicular decrease. Serum resulted negative to rapid agglutination test and agar gel immunodifusion with Brucella ovis antigen but positive to buffered plate agglutination test, tube agglutination test and 2- Mercapthoetanol with B. abortus antigen. Brucella isolation was negative in blood, testicular material, semen and urine. Brucella DNA was detected in PCR from urine and blood.

Application of the Fluorescence Polarization Assay for Detection of Caprine Antibodies to Brucella melitensis in Areas of High Prevalence and Widespread Vaccination

The screening Rose Bengal test (RBT), the buffered plate agglutination test (BPAT), and the confirmatory complement fixation test (CFT) are currently approved by the World Organization for Animal Health (OIE) for diagnosis of goat brucellosis. However, RBT (at 3% or 8% cell concentration) is known to be affected by vaccinal antibodies. In the present study, Mexican and Canadian OIE tests were compared with the fluorescence polarization assay (FPA), alone or in combination, using indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from an area of high prevalence and widespread vaccination. The relative sensitivities and specificities were, respectively, 99.7% and 32.5% for RBT3, 92.8% and 68.8% for RBT8, 98.4% and 84.8% for Canadian CFT, 83.7% and 65.5% for Mexican CFT, and 78.1% and 89.3% for FPA. The use of FPA as the confirmatory test in combination with other tests significantly increased the final specificities of the screening tests alone; BPAT, RBT3, and RBT8 plus FPA resulted in final specificities of 90%, 91.2%, and 91.3%, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, specificities were 65.5%, 63.2%, and 91.7%, respectively. We suggest that FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis and as a confirmatory test for screening test series. Some advantages of FPA are that its cutoff can be adjusted to improve its sensitivity or specificity, it is a low-cost and easy-to-perform test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of misdiagnosed and killed goats.

Toxoplasma gondii and Chlamydophila abortus in Caprine Abortions in Tobago: a Sero‐Epidemiological Study

A sero-epidemiological study was conducted on a goat farm that experienced an abortion epidemic in the 2005 breeding season in Tobago. Serum samples of goats (aborting and non-aborting) and cats were collected, in addition to the use of stored sera from the farm sampled in 2003 and 2004. Farm records on the reproductive and mortality rates for year 2003, 2004 and 2005 were also reviewed. The sera were screened for Toxoplasma gondii antibodies using the latex agglutination test (LAT), Chlamydophila abortus with an enzyme-linked immunosorbent assay (ELISA) and Brucella abortus using the buffered plate agglutination test (BPAT). Farm records revealed that for the period 2003-2005, the average kid per doe rate decreased from 2.1 to 1.5, the mortality rate increased from 6.3% in 2002 to 19.4% in 2004 and the fertility rate decreased from 98-99% (2002-2004) to 89% (2005). There was a dramatic increase in the abortion rate from <1% (2002, 2003 and 2004) to 29.2% (2005). Of a total of 161 sera tested comprising 12 from 2003, 89 from 2004 and 70 from 2005, 0 (0.0%), 21 (23.6%) and 45 (64.3%) were positive for T. gondii agglutinins (i.e. titres > or =1 : 64) and the differences were statistically significant (P < 0.05; chi(2)). Of all serum samples tested, only 1 (1.1%) of 89 from 2004 was positive for C. abortus while all the sera tested were negative for B. abortus. Amongst the 24 does which aborted in 2005 and were available for testing in mid-2005, 15 (62.5%) had reciprocal titres of > or =1 : 2048, three (12.5%) each had titres of 1 : 1024, 1 : 256 and < or =1 : 16 i.e. negative. The seroprevalence and titres of does that aborted, 20 (87.0%) of 23, all with titres > or =1 : 256 suggesting current infection, were statistically significantly (P < 0.05; chi(2)) higher than was detected amongst does that delivered normal kids, 25 (53.25) of 47 with 22 (48.8%) having titres of > or =1 : 256. One (50.0%) of two cats caught and tested was seropositive with a reciprocal titre of 128. This is considered the first documentation of T. gondii agglutinins in caprine abortion as well the detection of C. abortus antibodies from livestock in Trinidad. It is concluded that of the three zoonotic abortifacient pathogens tested for, T. gondii appeared to have played some aetiological role in the abortion epidemic investigated.

Serologic responses, biosafety and clearance of four dosages of Brucella abortus strain RB51 in 6–10 months old water buffalo ( Bubalus bubalis)

Thirty water buffalo were obtained from a brucellosis-free farm in order to evaluate antibody responses, bacterial clearance and safety to Brucella abortus strain RB51 vaccine in a dose response study. The animals were randomly divided into five treatment groups. Groups I–V received the recommended dose of RB51 vaccine (RD) once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Antibody responses to RB51 were monitored at 2, 4, 6, 8, 10, 12, 16 18, 22, 24 and 27 post-initial-inoculation weeks (PIW). Clearance of RB51 from the prescapular lymph node was evaluated at 2, 4, 6, 12, 18 and 24 PIW for groups 1, III and V and at 6, 8, 10, 16, 22 and 27 PIW for groups II and IV. To evaluate shedding of the RB51 strain, nasal, conjunctival, vaginal or preputial swabs were taken from all experimental animals at 1, 2, 3, 4, 6, 8 and 12 PIW. Sera taken at all PIW were negative for field strain B. abortus by both the buffered plate agglutination test (BPAT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibody responses to RB51 were demonstrated in all vaccinates but not in the controls, up to 12 PIW, by complement fixation test (CFT) and the dot-blot assay with an 83.7% agreement for both tests. Clearance of RB51 occurred between 6 and 12 PIW in group I but less than 2 weeks after booster vaccinations in groups II and IV and between 4 and 6 PIW in group III. RB51 was not recovered at any time from swabs obtained from either RB51-vaccinates or non-vaccinates. The results of this study indicate that serologic responses to RB51 vaccination can be monitored by both CFT and dot-blot assay in water buffalo. Our data also indicates that RB51 vaccination does not interfere with brucellosis sero-surveillance and is safe (no serological and bacteriological evidence of spread to non-vaccinates, no adverse clinical signs or detectable abnormalities on haematology and serum biochemistry) for use in water buffalo.

Evaluation of cattle for experimental infection with and transmission of Brucella suis biovar 4.

To determine whether cattle can become persistently infected with Brucella suis biovar 4, whether the organism can be transmitted vertically or horizontally, and whether tests for bovine brucellosis are diagnostic. Observational study. 24 pregnant cows and their calves and 6 bulls. Cows and bulls were housed separately in groups of 6 with each group consisting of 3 cattle experimentally infected with B suis biovar 4 and 3 naïve animals. Cattle were observed for clinical signs daily; blood samples were collected weekly. Clotted blood from each sample was submitted for bacterial culture. Serum was tested with an indirect ELISA and the standard tube agglutination test (STAT), buffered plate agglutination test, brucellosis card test (BCT), and complemen't fixation test (CFT). Tissues collected at necropsy were submitted for bacterial culture and histologic examination. All 15 inoculated cattle seroconverted on 2 or more serologic tests, and bacteria were isolated from 4 inoculated cows at necropsy. There was no bacteriologic evidence of vertical or horizontal transmission, and none of the cattle developed clinical abnormalities or gross or histologic lesions. Results of the indirect ELISA were positive for all inoculated cattle. The other tests gave variable results; the CFT, STAT, and BCT yielded negative results for at least 1 of the 4 cattle from which the organism was isolated. Results suggest that cattle-to-cattle transmission of B suis biovar 4 is unlikely. Serologic tests for bovine brucellosis should be used cautiously when attempting to identify cattle with rangiferine brucellosis, as they do not discriminate between the 2 diseases and vary in their ability to detect exposed cattle.

Evaluation of brucellosis RB51 vaccine for domestic water buffalo ( Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo ( Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10 10 colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals ( P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.