An hemolysis-in-gel test for bovine brucellosis

An hemolysis-in-gel test (HIGT) for bovine antibody against Brucella abortus was developed and evaluated. Sera to be tested were placed in wells in an agarose gel containing guinea pig complement and J-negative bovine erythrocytes coated with lipopolysaccharide prepared from B. abortus biotype 1. After incubation, zones of hemolysis were produced by positive sera. The activity of some positive sera was heat labile, but was restored to heated sera by addition of a crude preparation of the first component of bovine complement. The HIGT was compared with the complement fixation test (CFT), the buffered antigen plate test (BPAT), the standard tube agglutination test (STAT) and the standard plate agglutination test (SPAT), using sera from 1041 brucellosis-free cattle, from 51 cattle infected with B. abortus biotype 1 or biotype 4, and from six heifers vaccinated with strain 19. Three of 1041 sera (0.29%) from brucellosis-free cattle were HIGT-positive, ten (0.96%) were BPAT-positive, and none were positive in the CFT, STAT, or SPAT. The HIGT was more sensitive, and detected infection earlier than the other tests in the case of B. abortus biotype 1 infection, but was less sensitive for biotype 4 infection. In vaccinated heifers, HIGT reactions appeared later than reactions to the other tests, and persisted as long as 565 days. Further studies are needed to standardize the HIGT test conditions and to improve its sensitivity for B. abortus biotype 4 infections. Attempts to determine the effects of LPS antigen prepared from different biotypes of B. abortus are in progress.