Novel diagnostic approach on the identification of Brucella melitensis Greek endemic strains‐discrimination from the vaccine strain Rev.1 by PCR‐RFLP assay

Abstract

Despite the intensive implementation of control programmes goat, sheep and human brucellosis remains endemic in Greece. As the discrimination between field endemic strains and vaccine strain Rev.1 is not feasible, it is essential to develop new diagnostic tools for brucellosis diagnosis. Moreover, effective disease control requires enhanced epidemiological surveillance in both humans and animals including robust laboratory support. Two new multiplex (duplex) polymerase chain reactions (PCRs) were developed and the results were compared with those obtained by real‐time PCR and bacteriological biotyping. A total of 71 Brucella spp. Greek endemic strains were identified at species and biovar level, using both molecular and conventional techniques. Their discrimination from the vaccine strain Rev.1 was achieved, using polymerase chain reaction‐restriction fragment length polymorphism assay (PCR‐RFLP). All 71 strains were identified as Brucella melitensis by multiplex PCR as well as by real‐time PCR and conventional biotyping. Sixty‐two (87.3%) out of 71 strains were identified as B. melitensis biovar 3, eight (11,3%) strains as biovar 1 and only one (1,4%) as biovar 2. Digestion with PstI restriction enzyme revealed that all strains were field endemic strains, as they gave different patterns from the vaccine strain Rev.1. Brucella melitensis biovar 3 appears to be the predominant type in Greece. The novel multiplex PCR produced results concordant to ones obtained by real‐time PCR and conventional biotyping. This technique could support and facilitate the surveillance of Brucellosis in Greece contributing in the control of the disease.

Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS

“…Here, we propose MALDI-TOF MS profiling as a costeffective alternative to the currently applied methods of classical microbiological testing (Elberg and Meyer, 1958) and molecular PCR-techniques (Bardenstein et al, 2002;Lopez-Goni et al, 2008;Alvarez et al, 2017;Christoforidou et al, 2018) for the differentiation of the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates. This approach will be of benefit for reference laboratories and large healthcare facilities that have already implemented MALDI-TOF MS diagnostics for the identification of bacterial pathogens.…”

Modelling Human Brucellosis Based on Infection Rate and Vaccination Coverage of Sheep and Goats

“…1 vaccine, are scarce. Identification of wild or vaccine Brucella strains from clinical specimens is not routinely performed in Greece [19]; however, previous reports indicate that the vaccine strain can be held accountable for only a small proportion of human cases (0.65%, N = 308) [8]. A causative relation of human infection with the procedure of vaccinating the animals cannot be established based on the existing data.…”

Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing

“…At the time of isolation, all colonies were analysed based on morphology, CO 2 requirement, H 2 S production, as well as oxidase, catalase, and urease activity. Further, basic fuchsin and thionin sensitivity tests, lysis by Brucella phages (Tbilisi (Tb), Berkeley (Bk), Izatnagar (Iz) and Weybridge (Wb)) were conducted and agglutination in monospecific anti-sera A and M was tested, all as described by Christoforidou et al [ 25 ].…”