BRUCELLIN — BRUCELLIN

Brucellae are highly contagious zoonotic bacteria, causing great losses in animal husbandry and wildlife preservation. The coreBrucella species are characterised by surface smooth lipopolysaccharide (S-LPS) and lack of specific pathogen-associated molecular patterns. The majority of serological tests use Brucella LPS as an antigen, although it cross-reacts with antibodies against other bacteria, triggering strong humoral false-positive serological reactions (FPSR) which lowers test specificity. To avoid economically devastating culling of FPSR animals, the confirmatory Brucellin skin test (BST), based on the innate allergic type three or type four reaction is used. This test uses LPS-free mixture of inner highlyBrucella spp. specific bacterial proteins, which does not cause formation of specific antibodies. However, standardizing Brucellin is a complex due to its components’ variability, and batch controls in sensitized guinea pigs. Moreover, repeated inoculations can elicit antibodies production and anergy of immune system. Several specific Brucella intracellular proteins have been shown to stimulate individually humoral immune responses in the same manner, making them promising replacements. In addition, concentrations of interferon gamma and other pro-inflammatory cytokines are high duringBrucella infection in many species, stimulating Th1 and Th2 immune responses. Our project aims to investigate the efficacy of various Brucellin batches and standardizein vitro batch potency testing, removing reliance on experimental animals. Furthermore, using advanced shotgun proteomics and single-cell analyses on Brucellin antigens and host blood samples, interactions will be modelled to identify innate immune infection markers, such as IFN-gamma. These markers will be used for new ex vivo stimulation assays of peripheral blood mononuclear cells to confirm Brucella infected animals and reduce FPSR.