P470 Determination of the virulence potential of yeast fungi isolated from cattle milk
Authors: Sokolov Vladimir , Manoyan Marina , Gabuzyan Narine , Panin Alexander 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM
It is reported about the activity of proteolytic enzymes and phospholipases of yeast fungi of the genus Candida isolated from raw milk of clinically healthy cattle in some regions of Russia. The most common species were C. kefyr (16%), C. membranifaciens (15%), C. catenulata (13%), C. rugosa (9%), C. lambica (8%), C. pararugosa (7%), C. krusei (7%), and Candida albicans (6%). Candida albicans isolates had the highest enzyme activity, C. membranifaciens had the lowest.

Objectives
Proteolytic enzymes and phospholipases are known to be pathogenic factors in yeast fungi of the genus Candida. We examined raw milk taken from clinically healthy cows to determine the diversity of yeast fungi of the genus Candida that are found in milk, and also determined the activity of proteolytic enzymes and phospholipases of isolated isolates.

Methods
Raw milk samples (50 ml) were taken after the treatment of the udder with antiseptic agents, manually into sterile plastic tubes of the Falcon type, immediately before the planned milking. Then the samples were cooled to + 4°C and transported to the laboratory in this form.
Isolation of yeast fungi: 1 ml of milk was mixed with 20 ml of melted and cooled to 45°C Sabouraud medium with the addition of an antibiotic (M063 + FL033, HiMedia), poured into Petri dishes, and cultivated for 48 h. The isolated yeast fungi were inoculated onto a tube-slant wort-agar medium (M129, HiMedia).
Identification: Yeasts were identified using MALDI-TOF MS according to the standard procedure.
Determination of enzyme activity: filter paper discs (diameter 5.0 mm) were impregnated with 10 μL of pure culture with a density of 0.5 McF and laid out on the surface of the nutrient medium, cultivated at + 37°C for 7 days.
Enzyme activity was determined as the ratio of the diameter of the substrate hydrolysis zone to the disk diameter.
The composition of the nutrient medium for determining the activity of proteolytic enzymes: YCB (HiMedia) 11.7 g, Yeast extract 1.0 g, bacteriological agar 15.0 g, 1.0 g BSA after autoclaving. To assess the zone of hydrolysis, the surface of the nutrient medium was stained after cultivation with a 0.1% solution of Amido Black.
The composition of the nutrient medium for determining the activity of phospholipases: glucose 30.0 g, bacteriological peptone 10 g, 1 M NaCl, 5 mM CaCl2, bacteriological agar 15.0 g, 80 ml egg yolk emulsion after autoclaving.

Results
As a result of the research, 257 isolates of yeast fungi of the genus Candida were segregated. The most common were tested for proteinase and phospholipase activity, the results are presented in Table 1.

Conclusion
Yeast fungi known to be pathogenic for animals, such as C. albicans and C. krusei, have been isolated from the milk of healthy cows. Isolates of C. catenulata, C. rugosa, and C. pararugosa have a high activity of proteinases and phospholipases. Based on the results obtained, it was concluded that these types of yeast fungi have the potential to infect the host.