[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.

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

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.

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200. Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.

Comparison of cattle responses to mono-specific or combined inoculations with Babesia bigemina and Babesia bovis vaccine strains.

Combined inoculation of cattle with vaccine strains of Babesia bigemina and Babesia bovis induced lower antibody titers to B. bigemina than to B. bovis (previous study). Three groups of heifers were used to detect if the low antibody level was due to competition between Babesia species: individuals of G1 and G2 were inoculated with 10 million B. bigemina and B. bovis, respectively, and those of G3 with 10 million of each parasite. The prepatent periods, maximum parasitaemias and antibody titers (indirect immunofluorescent antibody test) were evaluated. The mean prepatent periods (days) for B. bigemina was of 5.6 (G1) and 5.2 (G3) and 7.0 (G2) and 6.7 (G3) for B. bovis (P > 0.05, "t" test). No differences were found in the parasitaemias. The only difference was found in the antibody titers to B. bovis, that were lower (P < 0.05 "t" test) from week 7 onwards when B. bovis was used in combination. The biological significance of this difference is unclear.

Evaluation of an indirect ELISA for the diagnosis of bovine brucellosis in milk and serum samples in dairy cattle in Argentina.

An indirect enzyme-linked immunosorbent assay (ELISA) for Brucella abortus antibodies detection in bovine milk and serum samples was validated. The assay use B. abortus smooth lipopolysaccharide as antigen, immobilized on a polystyrene matrix; milk diluted 1:2 or serum diluted 1:50, in a buffer containing divalent cation chelating agents EDTA and EGTA (ethyleneglycol-bis-aminoether-N,N,N',N'-tetraacetic acid) to reduce non-specific reactions; and a mouse monoclonal antibody specific for an epitope of bovine IgG1, conjugated with horseradish peroxidase. A total of 2646 sera and 2119 milk samples from cows older than 24 months were obtained from 12 brucellosis-free herds for at least the previous 5 years. Milk samples were obtained in parallel with serum samples. The remaining 527 serum samples were from dry cows. All cattle were vaccinated with B. abortus strain 19 between 3-10 months of age. Five hundred and fifty-two milk samples and 562 serum samples were obtained from 6 infected herds with abortions where B. abortus was isolated at least once no more than 6 months before sampling. The complement-fixation test (CFT) on serum samples was considered the gold standard. Serum samples were also tested with the official screening test: the buffered plate antigen (BPA) test. The cut-off point was determined using receiver-operating characteristic (ROC) analysis. For milk samples, it was fixed at 36 percent positivity (PP) giving a sensitivity of 99.6% with a 95% confidence interval (CI) of 98.6-99.9%. The specificity was 99.1% (CI 98.9-99.4%). For serum samples, the cut-off was fixed at 53 PP giving a sensitivity of 99.6% (CI 98.6-99.9%) and a specificity 98.6% (CI 98-99%). The BPA test showed a relative sensitivity of 99.6% (CI 98.6-99.9%) and a relative specificity of 98.6% (CI 98.1-99%). Our results indicate that the indirect ELISA is a highly sensitive and specific test and can be adapted to process a large number of samples.

Babesial antibody dynamics after cattle immunisation with live vaccines, measured with an indirect immunofluorescence test.

The efficacy of vaccination of Argentinean cattle against babesiosis and anaplasmosis using live immunogens was tested to detect specific antibodies in samples obtained about 60 days after vaccination. Under these conditions a higher than expected proportion of cattle failed to show antibodies against Babesia bigemina. Therefore, a study was designed to evaluate if this failure was due to insensitivity of the routine test to detect antibodies to B. bigemina or to lack of infectivity of the live vaccine. Four groups (G) of cattle were each inoculated subcutaneously with 10 million Babesia bovis (vaccinal strain R1A), 10 million B. bigemina (vaccinal strain S1A) and 10 million Anaplasma centrale (strain M1). G1 and G2 consisted of ten Angus bulls 20-24 months old and ten Angus bulls 15-18 months old, respectively; G3 and G4 consisted of ten and 16 Holstein 1-month-old male calves, respectively. Blood samples were obtained on days 0, 10, 20, 30, 40, 50 and 60 after vaccination and the sera were analysed with an indirect immunofluorescent (IFA) test to detect antibodies to B. bovis (baseline dilution for a positive result 1:60) and B. bigemina (baseline dilution 1:120). Positive IFA titres were considered as evidence of the infectivity of the Babesia vaccinal strains contained in the vaccine. All Angus bulls were found positive to antibodies against both Babesia species, by day 20 (B. bovis) and day 30 (B. bigemina), whereas 10-25% of Holstein calves were negative throughout. The partial lack of vaccine infectivity in the calves was considered to be a consequence of innate resistance of young calves to Babesia. Antibody titres to B. bovis and B. bigemina declined by day 60 after vaccination. However, all cattle that were positive to B. bovis antibodies on day 50 were still positive to the IFA test 10 days later while 10%, 30% and 12% of cattle of G1, G2 and G3 that were positive to B. bigemina antibodies on day 50 after vaccination were found negative to the IFA test on day 60. In future, samples taken on days 40-50 will be used for detection of B. bigemina antibodies in vaccinated cattle, on day 60 for A. centrale and on either occasion for B. bovis. The reaction to the inoculation of B. bigemina S1A strain appears to lag behind the reaction to B. bovis R1A strain. It is not certain if this is a normal reaction to this B. bigemina strain or the result of interaction with the B. bovis strain.

Cross-sectional estimation of Babesia bovis antibody prevalence in an area of Argentina used for extensive cattle breeding as an aid to control babesiosis.

The prevalence of Babesia bovis antibodies was estimated by using an ELISA (98% sensitivity and 95% specificity). Sera were obtained from 165 calves (mean age and standard deviation: 9.7 +/- 2.7 months) from an area in Argentina known to be unfavourable for the development of the vector tick, Boophilus microplus. The area comprised about 300,000 ha used for cattle breeding. The cattle population of 55,000 included 12,000 cattle under 1 year of age. Cattle were maintained mainly on natural grasses in communal lands. The true prevalence of antibodies to Babesia bovis was 12.2% with a confidence interval of 7.6% to 18.2%, and an inoculation rate (h; daily probability of infection) of 0.0004. This confidence interval has its lower boundary in the area of endemic stability due to low h of Babesia bovis by the vector tick and the upper limit in the area of endemic instability. This type of analysis could help to decide the implementation of preventive measures (e.g. vaccination) rationally, even in remote areas (such as the one of the present study) with an extensive cattle industry.

Chronic lymphocytic leukaemias and non-Hodgkin's lymphomas by histological type in farming-animal breeding workers: a population case-control study based on a priori exposure matrices.

OBJECTIVES: A population based case-control study was conducted in a highly agricultural area in Italy to investigate the association between chronic lymphocytic leukaemias (CLLs) and non-Hodgkin's lymphomas (NHLs), and subtypes, and exposure to pesticides in farming-animal breeding workers. METHODS: 187 cases of CLLs and NHLs and 977 population controls were interviewed on medical, residential, family, and occupational history. Detailed information was collected about cultivated crops and animals bred from subjects who worked in farming and animal breeding. Information on crop diseases and pesticides used (and their quantity and duration) was also obtained. A priori job-exposure matrices were applied when a crop disease was reported, estimating the most probable pesticide and, when possible, an estimate of the cumulative dose. Odds ratios (ORs) were calculated by unconditional logistic analysis with adjustment for relevant confounders in farmers who bred animals and in farmers alone, for the main crops, types of animals, and pesticides categories. First recall and then the matrices were used for defining exposure, as it affected CLLs and NHLs and then separately on CLLs and low grade NHLs. Finally, the dose-response was investigated for those pesticides which had shown some association. RESULTS: No variable under study was associated with work in farming alone. In farming and animal breeding, no crop or animal showed an association with CLLs and NHLs when adjusted by exposure during childhood to farming and animal breeding (an indicator of life in a farming and animal breeding environment before the age of 13, which behaved as an independent risk variable). A non-significant association was found with stannates, arsenates, phosphates, and dichloro-diphenyl-trichloroethane (DDT) based on recall, and for stannates, arsenates, and DDT after the application of the matrices. When CLLs together with low grade NHLs were considered, the association with insecticides in general, carbamates, and phosphates became significant according to personal recall (ORs and 95% confidence intervals (95% CIs) 2.46, 1.07-5.63; 3.08, 1.05-9.00; 2.97, 1.28-6.91, respectively). The application of the matrices also showed a risk of borderline significance for stannates and dithiocarbamates. A significant dose-response effect was found for phosphates (for logarithmic unit increase, OR 1.17, 95% CI 1.00-1.57); a strong trend for stannates and carbamates did not reach significance. CONCLUSION: The association of CLLs and NHLs with work in farming-animal breeding is partially explained by exposure to pesticides-namely insecticides (carbamates, phosphates, and DDT) and stannates-possibly related to their use in animal breeding. The association is limited to cases of CLL and low grade NHL. The independent effect of the variable exposure during childhood suggests that early exposures, including possible contact with animals, may play a part in the pathogenic process of these neoplasms.

Iterative use of a priori exposure matrices to improve the characterization of chemical exposures in agricultural work studies.

To assess pesticide exposures of agricultural workers, a priori exposure matrices based on "circumstantial determinants" of pesticide use were incorporated into the questionnaire of a case-referent study. Circumstantial determinants (crops cultivated, their surface areas and crop infestations) were recalled more frequently than specific chemicals. After the matrices were applied, the proportion of missing values fell from 44 to 9% for specific chemicals, from 97 to 17% for the dose for each treatment, and from 80 to 16% for number of treatments per year in a random sample of 40 questionnaires. The number of workers changed from 19 to 30 for parathion use, from 4 to 10 for mancozeb use, and from 4 to 12 for DDT (dichlorodiphenyltrichloroethane) use when the matrix was applied. The matrix enabled exposure levels to be assigned in each case. Provided that the matrix used is valid, this approach could increase the efficiency of case-referent studies on agricultural exposure to chemicals.