Efficacy of different Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats

Abstract

Fifteen different Rose Bengal antigens showed large differences with respect to pH, cell concentration and agglutination with the international standard anti-Brucella abortus serum, demonstrating the lack of international standardisation. Their sensitivity and specificity, compared with that of the complement fixation test, were evaluated for the diagnosis of B melitensis infection in culture-positive sheep, brucella-free ewes, and sheep and goats belonging to field flocks under different epidemiological conditions. All the Rose Bengal antigens and the complement fixation test had 100 per cent specificity when testing brucella-free sheep or animals belonging to flocks in unvaccinated brucellosis-free areas, but there were large differences in sensitivity between the Rose Bengal antigens with sera from culture-positive sheep or from animals belonging to infected flocks. When using the most sensitive antigen, no difference was observed in Rose Bengal sensitivity between animals infected with either biovar 1 or biovar 3 of B melitensis. The relationship between the sensitivity of the Rose Bengal antigens and cell concentration was unclear, but their sensitivity was related to the standardisation of the antigens with the international standard serum. The complement fixation test was less sensitive than the Rose Bengal test when testing culture-positive sheep. When testing sera from animals belonging to infected flocks with antigens standardised according to European Union rules, no great differences were observed in the sensitivities of the two tests. However, great differences in sensitivity between the Rose Bengal antigens were observed with sera from animals belonging to flocks with low levels of prevalence.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a modified Rose Bengal test and an indirect Enzyme-Linked Immunosorbent Assay for the diagnosis of Brucella melitensis infection in sheep

“…As in other EU countries [4], in the current B. melitensis eradication programme conducted in Portugal, a relatively high percentage of sheep which are negative in the RB screening test but positive in the CF confirmatory test has been identified. Tables II and III, figures as high as 3% (in infected animals) and 7% (in suspected animals) resulted in RB negative but CF positive.…”

“…However, it has been reported that a relatively high proportion of sheep and goats from B. melitensis-infected flocks show negative results in the standard RB test but positive in the CF test, questioning the sensitivity of the former as a screening test [4]. Thus, the simultaneous use of both is recommended to obtain a maximal sensitivity [4,12].…”

“…Thus, the simultaneous use of both is recommended to obtain a maximal sensitivity [4,12]. To improve the sensitivity of the RB test a simple modification consisting in increasing the volume of sera to be tested has been recommended [4,5]. This RB modified test (mRB) significantly increases the sensitivity of the RB standard procedure and considerably reduces the problem of sera being RB negative but CF positive (Blasco J.M., personal communication).…”

“…This test was performed following the procedure described by Blasco et al [4], mixing 75 mL of sera and 25 mL of the antigen. The plates were shaken for 4 min and any agglutination that appeared within this time was recorded as a positive reaction.…”

“…In fact, it has been clearly shown that this antigen standardisation procedure significantly limits the sensitivity of the RB test in sheep and goats and explains the existence of many animals that are negative in the RB test but positive in the CF test, particularly in moderate to low prevalence conditions [4]. Suitable alternatives could be based on increasing the sensitivity limits against the OIEISS without affecting the specificity or, alternatively, modifying the antigen standardisation procedure using a wide sera panel of B. melitensis culturepositive and Brucella-free animals rather than a single reference serum [4]. Moreover, the current standard RB test procedure using equal volumes of serum and antigen is considered to be suitable (despite no rigorous existing evidence) for the diagnosis of B. abortus infection in cattle, but is not sensitive enough for B. melitensis diagnosis in sheep [4].…”