WOAH Terrestrial manual (chapter 3.1.4) states that no single serological test is appropriate in all epidemiological situations and all animal species, especially because the FPSRs impede accurate serological diagnosis (1, 17). One of the EFSA recommendations has been to conduct specific studies to elaborate testing protocols to reduce FPSRs. Current opinion is that the FPSR problem may only be reduced, in pigs in particular, by I-ELISAs using extracts from rough strains of Brucellae, but in cattle neither the use of chaotropic I-ELISAs or procedures using heterologous extracts or Brucella cytosolic proteins resolve this problem (3, 4). Therefore, the most specific diagnostic procedure remains the BST, whichrelies on the delayed-type hypersensitivity reaction (2, 13). The results are interpreted 48 - 72 hours post injection. The BST is a cell-mediated immune assay, which uses purified Brucella genus specific intra-bacterial proteins as allergens, almost completely devoid of outer membrane components, and can differentiate theirinfections from other bacteria with similar LPS such as Y. enterocolitica O:9, E. coli O:116 and O:157, Salmonella serotypes of Kauffmann-White group N, or Pseudomonas maltophilia (11, 14). Besides S-LPS, no other Brucella protein shows immunological cross-reactions with other bacteria, culprits of FPSRs (5, 6, 14, 15). In the European Union, two BST tests are registered, but none is currently available for ruminants. The Brucellinbased on R-LPS-freecytosolic extract from rough B. melitensis strain B115 (Brucellergene®, Zoetis) was used in ruminants, but the commercialization was stopped in 2023. The brucellin based on rough B. abortus mutant (Brucellin Aquilon®, Aquilon; AQ1302) has been registered only for application in pigs (3, 4). Several studies confirmed the absence of surface exposed O-polysaccharide (OPS) in either formulation, but to the best of our knowledge there is no complete comparison of diagnostic performances between these two. WOAH Terrestrial Manual stipulates that standardized brucellin, free of S-LPS, is used, as FPSR affected animals always give negative results in the BST (13, 16, 17). Various alternatives have been explored.In 2012, McGiven et al. described indirect ELISA (I-ELISA) for diagnostics of B. suis, based on combination of two antigens derived from both rough and smooth Brucella LPS, limiting FPSR (10, 12). When the ID Screen® (Innovative Diagnostics), based on the same concept, but only in pigs, B. suis indirect, double- well I-ELISA, was tested by the EURL for brucellosis,measured sensitivity was 72.7%, with specificity of 97.5%, limiting the use of the test only as a confirmatory tool. In addition to the S-LPS, several outer membrane proteins (OMP) are also exposed on the surface of bacteria, with some more specific for genus, while others for individual species. However, current results show that these proteins, as antigens, show lower sensitivity than S-LPS based diagnostic tests (11). This can be either due to the lower concentrations of OMPs compared to LPS or the accessibility of these proteins on the bacterial surface. To mitigate hyper-sensibilisation or anergy, ex vivo stimulation has been developed. Brucellae through TLR-4 and alternative pathway - myD88, stimulate Th1, Th2, Th17 and Treg cellular response by overexpression of IFN-gamma, IL-17B as well as IL-6 and TNF-alpha (6, 8, 9). With the recent advances in proteomics, single-cell analyses enable detection of specific peptides for host-pathogens interactions, even in low concentrations (7). Based on superior performances of instrumentation with the advent of the Orbitrap Astral analyzer, this technology is able to pinpoint relevant biomarkers in an innovative discovery phase with high-throughput comparison of multiple strains, proteins that could be then produced in a given cellular chassis to systematically test sera, or alternatively targeted for developing robust and cheap immune-based assays. 

Relevance to the call scope and topic This project corresponds to topic 1 of EUPAHW call. Through application of innovative technologies, we aim to improve diagnostics of Brucella in infected production animals and develop new methods to monitor this major mandatory reportable zoonotic disease, thus improving decision support tools. The project will collect data, to complete the knowledge on BST application in different production species, develop a new method for Brucellin standardization and offer alternatives to current diagnostics. The project aims also to improve animal welfare by: i) offering new ex vivo stimulation assay and thus removing the possibility of accidental allergic reactions; ii) reducing the number of experimental animals used in batch control validations of drugs. Impact of the research subject According to the AHL Reg (EU) 2020/689, the BST can only be used in sheep and goats to acquire and maintain the brucellosis free status. The project, based on innovative shotgun proteomics, will improve specificity of indirect diagnostic tools for brucellosis surveillance in cattle, pigs, sheep and goats, in line with WOAH recommendations, enhancing public health protection, reducing the FPSRs and unnecessary culling of whole herds. The new schemes of status granting, outbreak management, control of the food chain will benefit from the optimized tools and prevent further spread within the farm, protecting animal, human and environmental health. Scientific idea / research objectives To resolve the above-mentioned problems, we need to: (i) expand understanding of Brucella immune system stimulation; (ii) improve specificity of tools used in ruminants based on cell-mediated response; and (iii) develop new approaches to counteract the root cause of FPSRs due to the application of the same type of antigen (S-type LPS) in the currently approved tests. Therefore, we propose: (i) to analyse in-depth Brucellin composition and performances; (ii) perform in vivo experiments and shotgun proteomic analyses to identify, and then validate a set of specific biomarkers for in vitro standardization of Brucellin composition; (iii) perform next-generation meta-proteomic analyses on Brucella naturally infected production animals; and (iv) develop ex vivo based stimulation assay, based on innate immune response. 

Bacteria from the genus Brucella are facultative intracellular organisms that can live in environment and majority of their species are very pathogenic to animals and humans, to the extent that they can be used as a bioweapon. Currently, except anti-Brucella LPS antibody detection assays, there are no other specific serological tests neither for Brucellae nor for cross-reacting bacterial species for indirect diagnosis. In EU, an eradication strategy consists of slaughtering of serologically positive animals including potential FPSRs, and raw milk products related to that farm have to be withdrawn from markets, which is detrimental for agriculture and genetic selection of production animals. The most specific test that can eliminate FPSRs was BST. According to AHL, BST use is limited to confirmation of Brucella negative small ruminants, and manufacturers can’t economically justify Brucellin production anymore. BRU-CELL-IN aims to standardise the production of Brucellin and offer more efficient, precise ex vivo stimulation assay. To achieve this, we will use non targeted proteomic analysis to identify and quantify all Brucellin components to compare with WOAH recommended in vivo validation assays and give detailed potency map of each element that will be the basis for future diagnostic tools. This will create more precise specifications documents for future Brucellin formulations, and replace validation assays in experimental animals significantly lowering the production costs. Furthermore, we will characterise structurally, main Brucellin components by combining structure-based immunogen design and target optimisation to identify major immune response catalysts. This will map out alternative immune stimulation pathways animals use against Brucella infections, which will be used for future vaccine development. Finally, by analysing the innate immunity against Brucellae from naturally infected, FPSRs and negative animals key cellular signalling will be identified to create ex vivo stimulation assay, that can be used in concert with indirect diagnostic to eliminate FPSRs when Brucellin is not planned by law or not possible to use. This will improve the efficiency of brucellosis eradication programmes in endemic parts of Europe and facilitate the surveillance scheme in disease-free countries, where FSPRs are more frequent. BRU-CELL-IN will secure and strengthen European agriculture, veterinary medications pharmaceutical industry, while still improving public health and protection measures. Furthermore, with new ex vivo diagnostic strategy, there is no need to apply to Committee for Medicinal Products, which will save time and f inances while significantly improve indirect brucellosis diagnostics and lower the FPSRs. Hence, our project transcends between industry, animal welfare, protection of animal, public and environmental health.