Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is resolved, WGS-derived bacterial typing adds an important method to complement epidemiological surveys during outbreak investigations. This is the first report of a detailed genetic investigation of an extensive collection of B. melitensis strains isolated from human cases in Germany

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining

BACKGROUND: The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49-96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed. RESULTS: Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies. CONCLUSION: The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs