ContentsThe most widely used screening test for the diagnosis of brucellosis in the dog is the rapid slide agglutination test in the presence of 2-mercaptoethanol (2ME-RSAT). The diagnosis is partially confirmed by the agar gel immunodiffusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases not detected by these tests may be detected by ELISA tests. The use of 2ME-RSAT in routine clinical practice requires a microscope and an experienced operator. An immunochromatographic diagnostic test for canine brucellosis (FASTest ® Brucella c., Megacor, Ho¨rbranz, Austria) has been recently released. In this study, we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs. Sera from 17 healthy dogs used as negative controls yielded negative results by FASTest, indicating a 100% specificity in this sample. Among 27 sera of dogs with acute or subacute brucellosis confirmed by B. canis isolation, all of which were positive by RSAT and ELISAs, the FASTest was positive in 24 cases and AGID in 23. In acute and subacute cases, the sensitivity of FASTest was 89%. Sera from six dogs with bacteriologically confirmed chronic brucellosis, which were positive by ELISAs but negative by 2ME-RSAT, were also tested; 1 was positive by FASTest and 4 were positive by AGID. These preliminary results indicate a good specificity of the FASTest (100% in this sample) but an unacceptable sensitivity as a screening test. In cases with chronic brucellosis, the sensitivity of the FASTest was lower than that of ELISAs but this assay could make a good intermediate test to be run after a positive RSAT and before running an AGID.

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Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.