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

David Kornspan,Holger Brendebach,Dirk Hofreuter,Sascha Al Dahouk,Shubham Mathur,Marcelo Fleker,Shlomo Eduardo Blum,Svetlana Bardenstein

doi:10.3389/fmicb.2021.712601

Abstract

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

Highlights

Brucellosis is a global zoonotic disease affecting domestic and wild animals as well as humans (Pappas et al, 2006)
Our study aimed to identify MALDI-TOF MS biomarkers that allow for clearly distinguishing B. melitensis wild-type strains from the B. melitensis Rev.1 vaccine strain
The resulting in silico core proteome shared by all three B. melitensis genome assemblies consisted of 2,962 translated coding sequences with identical amino acid sequences (Figure 1)

Summary

Introduction

Brucellosis is a global zoonotic disease affecting domestic and wild animals as well as humans (Pappas et al, 2006). Ovine and caprine brucellosis are endemic throughout the Middle East as well as in many countries of Africa, Asia, and Latin America (Rossetti et al, 2017) In these regions, efforts are undertaken to control brucellosis by the vaccination of sheep and goats. The most common vaccination policy for small ruminant livestock against B. melitensis infections is the application of the live attenuated B. melitensis Rev. strain to female animals aged between 2 and 6 months (Banai, 2002). This procedure has proven to be protective and to reduce abortions in treated animals, but may be contraindicated in females that are vaccinated during their last trimester of pregnancy (Banai, 2002). Laboratory methods that can distinguish B. melitensis field strains from the vaccine strain are relevant for (i) effective brucellosis control programs, (ii) for epidemiological surveillance, and (iii) for outbreak clarification

Methods

Results

Conclusion