Use of Maldi-TOF mass spectrometry in veterinary mycology

Abstract

Use of MALDI-TOF mass spectrometry to identify yeast and molds in animal feed. The material for the work was animal feed samples received for research from diff erent regions of Ukraine. The presence of yeast and molds was determined according to DSTU ISO 7954:2006. To establish the general contamination of the feed with micromycetes, the fungi were fi rst isolated from the feed by planting them on Saburo medium, and the serial dilution method was used to calculate the content of fungi diaspores in 1 g of feed. The feed samples were incubated and studied at a temperature of 24 ° C for 5–7 days. The identifi cation of molds was carried out using the MALDI-TOF method. In the process of mycological examination of feed during 2018–2019. 198 animal feed samples were examined. During the study period, the largest number of feed was examined, which was 30.4% in 2018, of the total number of samples (19.6% - feed for poultry, 10.8% - for pigs). For fi ve months of 2019, we observed the same trend: in 31.1% of cases, the defi nitions of yeast and molds in compound feeds prevailed, of which 19.8% accounted for compound feeds for poultry and in 11.3% of cases for pigs. In second place in the number of studies, corn samples are 11.9 and 11.3% in 2018 and 2019, respectively. The most common types of fungi in the feed were representatives of the genera Fusarium, Penicillium, Aspergillus, Alternaria, Mucor, Rhizopus, Cladosporium. The affi liation of microscopic fungi to specifi c genera was determined by assessing the morphology of the fungal colony on media and the morphology of conidiophore structures Particular attention was paid to microscopic fungi of the Fusarium family, which are producers of various mycotoxins. Using the MALDI Biotyper software, automatic identifi cation was performed based on a comparison of the collected initial spectra of the fungus with the reference spectra of the database of the instrument itself, as well as with the library of the University of Belgium (BCCM, Belgian Co-Ordinateo collections of micro-organism). Following the results of mass spectrometry, microscopic fungi of the Fusarium family were represented by 9 species. Of these, 5 species were most often found: F. proliferatum, F. acutatum, F. subglutinans, F. verticillioides. Among the fungi of the Aspergillus family, A. fl avus, A. pseudoglaucus, A. tubingensis, and A. niger predominated. Species identifi cation of microscopic fungi using mass spectrometry helps quickly and accurately identify mold fungi and yeast. Determination of the species affi liation of microscopic organisms occurs through analysis of the protein fraction of the lysate of microscopic fungi and yeast ("direct protein profi ling"). MALDI Biotyper software includes automatic identifi cation of molds based on a comparison of the output spectra with the reference spectra of the database. Identifi cation of microorganisms using MALDI-TOF MS is based on the assessment of ribosomal proteins that are usually present in the cell. The sensitivity of the MALDI-TOF MS method is 103106 m.k./cm. In this case, the accuracy of identifi cation depends on the amount of test material. To determine the likelyhood of identifi cation, a given logarithmic indicator is the compliance coeffi cient Score, the value of which is used to evaluate the reliability and adequacy of the results. The higher the match rate, the more likely it is to get the correct identifi cation result. MALDI-TOF technology for mass spectrometric identifi cation of micromycetes has a high measurement speed, low cost of reagents and materials used, and simple preparation holes. MALDI-TOF MS has a high diagnostic sensitivity. Key words: mold identifi cation, MALDI-TOF, mass spectrometry, Fusarium, Penicillium, Aspergillus, Alternaria, Mucor, Rhizopus, Cladosporium.