653-2Identification of Prototheca spp. from bovine milk by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/TOF-MS)
Autores:Juliano Leonel Gonçalves (USP/FMVZ - Nutrition and animal production Dept.) ; Patrícia A.c. Braga Braga (UNICAMP - ThoMSon MS Laboratory, Institute of Chemistry) ; Juliana Regina Barreiro Barreiro (USP/FMVZ - Nutrition and animal production Dept.) ; Tiago Tomazi (USP/FMVZ - Nutrition and animal production Dept.) ; Marcos André Arcari (USP/FMVZ - Nutrition and animal production Dept.) ; Christina R. Ferreira (UNICAMP - ThoMSon MS Laboratory, Institute of Chemistry) ; Sarah Hwa In Lee (USP/FZEA - Food Engineering Dept.) ; Diego M. Assis Assis (UNIFESP - Biophysics Dept.) ; Alessandra Tata (UNICAMP - ThoMSon MS Laboratory, Institute of Chemistry) ; João Pessoa Araújo Junior (IBB-UNESP - Institute of biosciences) ; Marcos N. Eberlin (UNICAMP - ThoMSon MS Laboratory, Institute of Chemistry) ; Marcos Veiga dos Santos (USP/FMVZ - Nutrition and animal production Dept.)


Mastitis is a common and easily disseminated disease in dairy herds and causes the most part of economic losses to dairy farmers. Mastitis caused by the genus Prototheca is of special interest due to its zoonotic characteristics and by the persistent infection refractory to traditional therapeutic procedures. MALDI is a “soft” technique being already used in human microbiology clinical settings that allows analyzes bacteria and identifies microorganisms based on their protein “fingerprinting”, a group of mainly ribosomal proteins unique to each bacterial species. The objective of this work is to identify Prototheca species isolated from bovine milk by MALDI/TOF MS. Milk samples were collected from 1,282 cows of 21 dairy herds. Prototheca spp. was identified in 24 milk samples. To prepare the samples for MALDI/TOF MS, the algae strains isolated from milk were thawed and cultured for 24h in Sabouraud dextrose agar. The algae culture was prepared for MALDI-TOF MS analysis by formic acid extraction directly on plate or by using the protocol previously described by Barreiro et al., 2010 (J Dairy Sci. 93:5661-7). Cyano-4-hydroxy-cinnamic acid was used as the organic matrix. Mass spectra acquired were analysed for bacterial identification based on database search by the MALDI Biotyper 3.0 software (Bruker Daltonik) at default settings. The formic acid extraction directly on plate method was ineffective to generate good quality mass spectra and consequently microorganism identification. The protocol already reported by our group (Barreiro et al., 2010) was effective for the ribosomal protein extraction of algae, as shown by the mass spectra, but the software couldn't identify the species Prototheca due to the lack of data from this genus in the database. Therefore, we are creating our own sub-databsase in the Biotyper Software to be able to perform the diagnosis of Prototheca mastistis by MALDI-TOF MS based on the established sample preparation protocol. In conclusion, MALDI-MS was successfully applied for protein ribosomal extraction of Prototheca spp. and the spectra of these species on the database is required due to pathogen infectivity and identification features which can be made easily and quickly by this method.

Palavras-chave:  Mass spectrometry, MALDI/TOF-MS, Bovine mastitis, Prototheca