Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of <i>Mycobacterium bovis</i> in Bovinae

Abstract

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

Comparison between generalised peritonitis and parietal fibrinous peritonitis in cows after caesarean section

“…Two readings were performed of each medium at 18–24 hours and 36–48 hours of incubation, respectively. Bacterial identification was performed by use of matrix‐assisted laser desorption ionisation‐time of flight 22…”

Atypical case of parietal fibrinous peritonitis in a Belgian Blue heifer without a history of laparotomy

“…Two readings were performed of each medium at 18–24 hours and 36–48 hours of incubation, respectively. The bacterial identification was performed by use of matrix‐assisted laser desorption ionisation‐time of flight 17 . Antibiotic sensitivity was tested by disk diffusion assay for the following molecules: penicillin G amoxicillin, amoxicillin‐clavulanate, cefquinome, ceftiofur, colistin, enrofloxacin, florfenicol, gentamycin, kanamycin, marbofloxacin, tetracycline and trimethoprim‐sulfonamide.…”

Instrumental analytical tools for mycobacteria characterisation

“…Several modifications have been developed, most of them based on cell disruption, adding sonication or bead-beating steps for liberating mycobacterial protein for analysis (Alcolea-Medina et al 2019). Recently, Bacanelli et al (2019) modified the previously mentioned MycoEx method and introduced the My-coLyser method, which includes homogenisation in tissue macerator rather than vortex. In comparison, MycoLyser showed higher scores and identification consistency with the Biotyper system (Bacanelli et al 2019).…”

“…Recently, Bacanelli et al (2019) modified the previously mentioned MycoEx method and introduced the My-coLyser method, which includes homogenisation in tissue macerator rather than vortex. In comparison, MycoLyser showed higher scores and identification consistency with the Biotyper system (Bacanelli et al 2019). However, mass spectra can also be obtained from ionised lipid molecules after lipid extraction (Kim et al 2018).…”

“…The reaction volume minimised to 1-2 µL and the deposition volume to 100 nL, thereby reducing the further cost (Haslam et al 2016). Studies conducted to analyse and identify mycobacteria usually employed sample deposition volume from 1 µL to 2 µL and matrix volume 1 µL (Alcaide et al 2018;Epperson et al 2018;Moreno et al 2018;Rodriguez-Temporal et al 2018;Bacanelli et al 2019).…”