Isolation, molecular typing, detection of virulence factors and antifungal resistance of Candida species from Anatolian buffaloes and dairy cows with mastitis

The thesis study aimed to isolate Candida species from Anatolian buffaloes and dairy cows with mastitis, their molecular typing, investigate the presence of agglutinin-like sequence (ALS1), secreted aspartyl proteinase (SAP1) and phospholipase enzyme (PLB1) virulence genes in the isolates identified to be Candida albicans and determine the antifungal sensitivity/resistance profiles of the Candida isolates. In this thesis, 1188 mammary quarter milk samples belong to 301 Anatolian buffaloes and 1321 mammary quarter milk samples belong to 332 dairy cows located in the center and districts of Afyonkarahisar were used. Following the phenotypic isolation of Candida species from the samples, PCR was performed on suspected isolates using genus-specific ITS primers designed for the first time in this thesis study. The isolates determined to be Candida by PCR were identified by single-direction Sanger sequencing analysis. Of Anatolian buffalo isolates, 79.59% Pichia kudriavzevii (Candida krusei, n=74), 11.82% Kluyveromyces marxianus (Candida kefyr, n=11), 4.3% Suhomyces xylopsoci (Candida xylopsoci, n=4), 2.15% Candida albicans (n=2), 1.07% Candida glabrata (n=1) and 1.07% as Candida rugosa (n=1) were typed. While of species isolated from dairy cows, 54.54% P. kudriavzevii (C. krusei, n=54), 39.4% K. marxianus (C. kefyr, n=39), 4.04% C. xylopsoci (n=4), 1.01% Candida parapsilosis (n=1) and 1.01% as C. rugosa (n=1) were identified, C. albicans could not be identified from dairy cows. In this thesis study, the species isolated at the highest rates from both buffaloes and dairy cows were determined to be P. kudriavzevii (C. krusei) and K. marxianus (C. kefyr). The presence of ALS1, SAP1 and PLB1 genes in two C. albicans isolated from Anatolian buffaloes was investigated by PCR in thesis study. While it was determined that both species carried the SAP1 gene, the ALS1 and PLB1 genes were not found in the species. In the thesis study, the antifungal resistances of Candida species isolated from Anatolian buffaloes and dairy cows were investigated using disc diffusion method. While all species isolated from Anatolian buffaloes were resistant to flucytosine, high resistance iv rates against to itraconazole (98.9%; n=92), amphotericin B %88.2%; n=82), caspofungin (86%; n=80) and fluconazole (83.9%; n=78) were also obtained in the species.). While all of 99 species isolated from dairy cows were resistant to itraconazole and flucytosine, the resistance was determined to amphotericin B in 96 (97%), to caspofungin in 75 (75.8%) and to fluconazole in 63 (63.6) of species. Buffalo isolates were susceptible to voriconazole and ketoconazole at rates of 90.3%, while 97% were found to be susceptible to ketoconazole and 92% to voriconazole of dairy cow isolates. As a result, it was shown that non-albicans Candida species were more common in the yeast-related mastitis etiology in the sampled animals. It was considered that antifungal susceptibility tests may be a significant guide in determining mastitis treatment plans against Candida species and effectively controlling the disease. In addition, this is the first thesis study typed by molecular methods the Candida species isolated from Anatolian buffaloes and shown the presence of SAP1 virulence gene in C. albicans isolates of buffalo origin in Turkey. It is thought that the findings obtained in the thesis study may be an important source of epidemiological data for future researches on the subject.