# Molecular identification with virulence gene profiles and in-vitro antifungal susceptibility of Candida species associated with subclinical mastitis in Anatolian buffaloes and dairy cows

## TL;DR

This study identifies Candida species in subclinical mastitis in Anatolian buffaloes and dairy cows, highlighting the importance of molecular methods for accurate diagnosis and antifungal resistance.

## Contribution

The study provides the first molecular evidence of the SAP1 virulence gene in C. albicans from Anatolian buffaloes with mastitis.

## Key findings

- Non-albicans Candida species were the most frequently identified in both buffaloes and dairy cows.
- C. albicans isolates from buffaloes carried the SAP1 virulence gene but lacked ALS1 and PLB1.
- Antifungal susceptibility testing showed reduced susceptibility to tested agents in Candida isolates.

## Abstract

Mastitis is a serious multifactorial disease of dairy animals. Etiologically, bacterial mastitogens are intensively investigated. Conversely, yeast-associated mastitis, particularly Candida species, has received comparatively less attention in buffaloes. The present study aimed to perform species-level molecular identification of Candida isolates obtained from Anatolian buffaloes and dairy cows with subclinical mastitis, to characterize the selected virulence gene profiles, and to evaluate antifungal susceptibility patterns. Milk samples were collected on an udder-quarter basis from 1188 quarters of 301 buffaloes and 1321 quarters of 332 dairy cows raised in smallholder farms. Following phenotypic yeast isolation, presumptive Candida isolates were subjected to an Internal Transcribed Spacer (ITS)-based PCR approach followed by sequence-based identification. The results were compared with chromogenic Candida agar findings. The presence of ALS1, SAP1, and PLB1 virulence genes was investigated in Candida albicans isolates. Antifungal susceptibility testing via the disk diffusion method was performed. A total of 192 Candida isolates were recovered, predominantly representing non-albicans Candida species in both host species. Pichia kudriavzevii (Candida krusei) and Kluyveromyces marxianus (Candida kefyr) were the most frequently identified species. C. albicans was detected exclusively in buffalo-derived samples (2/93; 2.15%). Both isolates carried the SAP1 virulence gene, whereas ALS1 and PLB1 were not detected. Sequencing-based identification showed high agreement (94.9%) with chromogenic agar results. Antifungal susceptibility testing revealed reduced susceptibility to the antifungal agents tested. In conclusion, the predominance of non-albicans Candida species, together with the observed virulence and susceptibility-related findings, emphasizes the importance of accurate species-level identification in the diagnosis of mycotic mastitis. The data generated provide region-specific baseline information that may support improved diagnostic approaches and inform mastitis management strategies in dairy herds. This study may provide the first molecular evidence from Türkiye demonstrating the presence of the SAP1 virulence gene in C. albicans isolates originating from Anatolian buffaloes with mastitis.