Summary
The ovine production is economically important in the agricultural system of Argentina and brucellosis, caused by Brucella ovis, is considered the main factor causing reproductive problems in this species.Although it has not been reported as a disease in humans, there are studies showing that antibodies where detected in workers in contact with ovines. The disease is found in all the areas of the country where ovines are bred, with an incidence varying from 3 to 50%.
To date, there is no national or provincial programme for its control, and very few laboratories offer a diagnostic service with high sensitive and specific techniques.
The clinical diagnoses of B. ovis infection by palpating the testicles and epididymides are not sensitive enough and it must be considered presumptive. Although bacteriological diagnosis is recommended as a confirmatory test, this method is inadequate for detection of the disease in large numbers of ovines, since it fails to detect all the infected animals. Indirect methods of serological testing are preferred for routine diagnosis, among which, agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA test are considered the most efficient.
The first objective of the present study was to identify simple and practical serological tests with high sensitivity and specificity.
Since B. ovis shares antigenic components with Brucella canis, similar results are expected if either strain is used as an antigen. However, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination test.
We present data (testing 225 animals) on AGID and ELISA test using B. ovis antigen, and rapid screening agglutination test (RSAT), 2-mercapto-ethonol RSAT (2ME-RSAT) and ELISA using B. canis antigen. The cut-off value was adjusted by ROC analysis using 51 negative and 32 positive sera. The ELISA-B. canis cut-off value was 39 (%P) and ELISA-B. ovis 51 (%P), with 100% sensitivity and specificity. Considering the 32 positive sera from the infected flocks, RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Taken into account the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, ELISA-B. canis and ELISA-B. ovis, 20
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positive only with RSAT and 2 positive only by both ELISAs. RSAT is a very sensitive screening test that due to its simplicity and easy interpretation, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis, with large number of samples. ELISA-B. canis or ELISA-B ovis tests could be used as confirmatory tests, since they show equal specificity and sensitivity.
A second objetive was to investigate the presence of antibodies in sheep milk. Milk is a clinical sample not enough studied in the diagnosis of ovine brucellosis caused by B. ovis, although it has the advantage that samples may be taken quickly in a non invasive manner. Thus, we studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using B. canis and B. ovis antigens.
ELISA was done on milk samples with B. canis and B. ovis antigens, while serum samples were studied following RSAT, IDGA and ELISA tests using the same antigens. A group of 75 ewes in an uninfected flock were serologically negative to RSAT, ELISA-B. canis, AGID and IELISA-B. ovis.
The cut-off values were adjusted by ROC analysis using 75 uninfected sheep and 23 positive samples from 5 infected sheep. The results showed cut off values of (%P) 33 for milk-IELISA-B. canis and (%P) 26 for a milk-IELISA-B. ovis, with 100% sensitivity and specificity. Considering the 64 samples from the suspect flock (data not shown) 11 were positive by milk-IELISA-B. canis and milk-IELISA-B. ovis.
Based on our results, we suggest that with large number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis Brucellosis in lactating ewes.
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