In dairy cattle, mastitis is an economically important disease in which fungi may participate as etiological agents, although their incidence is usually low as compared to other agents of mastitis (1). They are considered to be opportunist and cause infection when natural defense mechanisms in the host are lowered. Candida species are most frequent isolated from infected mammary glands. This study focused on determining the prevalence of yeasts involved in dairy cattle mastitis and subsequent phenotypic, genotypic characterization and determination of virulence traits and antifungal susceptibilities of the isolates. A total of 23 yeast strains used in this study were previously isolated from 1.602 quarter milk samples with clinical mastitis between 2015 and 2017 from 55 dairy farms of Lazio Region. Genomic DNA was isolated (2) the strains identification was performed by sequencing of variable D1/D2 domain of 26S rDNA (3) and the internal transcribed spacer (ITS) region (4). The RAPD-PCR assay was performed with primer M13 (5) to explore the genetic diversity of the strains and the resulting fingerprints were compared with the BioNumeric 5.0 software package (Applied Maths, Sint Martens-Latem, B), using the UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. The reproducibility value of the RAPD-PCR assay, calculated from two repetitions of independent amplification of type strains, was higher than 90%. Genomic DNA was used also to detect the presence of agglutinin-like sequence 1 (ALS1) and hyphal wall protein 1 (HWP1) genes. PCR reactions were performed according to Inci et al. (6). The strains were characterized at the phenotypic level for their resistance toward oxidative stress induced by H2O2 (7), invasiveness (8), Esterase activity. (9) and biofilm formation (10). Furthermore, each isolate was tested against 6 antifungal agents (Caspofungin CA, Fluconazole FL, Flucytosine FU, Itraconazole IT, Ketoconazole KE and Voriconazole VO) by the E-test method as recommended by the manufacturer (bioMerieux). Yeasts were isolated from 1.44% (n.23) of total clinical samples (n.1602). We found a low prevalence of mastitis causing by yeasts, although the frequency of yeast isolation in surveys carried out in many countries varies considerably (1). Sequence analysis of D1/D2 domain and ITS region enabled to identify 23 isolates as C. parapsilosis (n°1), C. rugosa (n°4), K. marxianus (C. kefyr) (n°3), M. capitatus (G. capitatum) (n°4), M. farinosa (P. sorbitophila) (n°2), P. kudriavzevii (C. krusei) (n°9). In agreement with other authors (1) Candida species were the organisms most frequently isolated in this study. Random amplification of the strains showed a notable genotypic heterogeneity at the species level. Considering the predominant species isolated, P. kudriavzevii, the coefficient of similarity was 64% and the genetic polymorphism detected allowed the identification of 4 different RAPD genotypes among 9 analyzed strains. No amplification occurred for ALS1 and HWP1 genes. A low resistance toward oxidative stress was detected, showing all the strains halos of inhibition ranging from 2.5 to 4.6 mm. Two strains belonging to M. capitatus showed higher grading than 2 in invasiveness (from 0 not invasive, to 4 highly invasive) and they were the only ones able to hydrolyse esters. Except for one K. marxianus, all the strains were able to produce biofilm even if at different levels: one showed strong biofilm production (P. kudriavzevii), 10 showed moderate production, and 1 displayed a weak biofilm production. With regard to antifungal resistance, the majority of isolates were resistant to FL, and FU, while all the examined strains were sensitive to CA, IT, KE and VO, in agreement with other authors (1) The results highlight a low prevalence of mycotic mastitis, mainly caused by genus Candida. The presence of multiple virulence traits our found encourage further investigations.
Phenotypic and genotypic characterization of yeasts isolated from mastitis cattle
Silvetti T;Morandi S;Brasca M;
2018
Abstract
In dairy cattle, mastitis is an economically important disease in which fungi may participate as etiological agents, although their incidence is usually low as compared to other agents of mastitis (1). They are considered to be opportunist and cause infection when natural defense mechanisms in the host are lowered. Candida species are most frequent isolated from infected mammary glands. This study focused on determining the prevalence of yeasts involved in dairy cattle mastitis and subsequent phenotypic, genotypic characterization and determination of virulence traits and antifungal susceptibilities of the isolates. A total of 23 yeast strains used in this study were previously isolated from 1.602 quarter milk samples with clinical mastitis between 2015 and 2017 from 55 dairy farms of Lazio Region. Genomic DNA was isolated (2) the strains identification was performed by sequencing of variable D1/D2 domain of 26S rDNA (3) and the internal transcribed spacer (ITS) region (4). The RAPD-PCR assay was performed with primer M13 (5) to explore the genetic diversity of the strains and the resulting fingerprints were compared with the BioNumeric 5.0 software package (Applied Maths, Sint Martens-Latem, B), using the UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. The reproducibility value of the RAPD-PCR assay, calculated from two repetitions of independent amplification of type strains, was higher than 90%. Genomic DNA was used also to detect the presence of agglutinin-like sequence 1 (ALS1) and hyphal wall protein 1 (HWP1) genes. PCR reactions were performed according to Inci et al. (6). The strains were characterized at the phenotypic level for their resistance toward oxidative stress induced by H2O2 (7), invasiveness (8), Esterase activity. (9) and biofilm formation (10). Furthermore, each isolate was tested against 6 antifungal agents (Caspofungin CA, Fluconazole FL, Flucytosine FU, Itraconazole IT, Ketoconazole KE and Voriconazole VO) by the E-test method as recommended by the manufacturer (bioMerieux). Yeasts were isolated from 1.44% (n.23) of total clinical samples (n.1602). We found a low prevalence of mastitis causing by yeasts, although the frequency of yeast isolation in surveys carried out in many countries varies considerably (1). Sequence analysis of D1/D2 domain and ITS region enabled to identify 23 isolates as C. parapsilosis (n°1), C. rugosa (n°4), K. marxianus (C. kefyr) (n°3), M. capitatus (G. capitatum) (n°4), M. farinosa (P. sorbitophila) (n°2), P. kudriavzevii (C. krusei) (n°9). In agreement with other authors (1) Candida species were the organisms most frequently isolated in this study. Random amplification of the strains showed a notable genotypic heterogeneity at the species level. Considering the predominant species isolated, P. kudriavzevii, the coefficient of similarity was 64% and the genetic polymorphism detected allowed the identification of 4 different RAPD genotypes among 9 analyzed strains. No amplification occurred for ALS1 and HWP1 genes. A low resistance toward oxidative stress was detected, showing all the strains halos of inhibition ranging from 2.5 to 4.6 mm. Two strains belonging to M. capitatus showed higher grading than 2 in invasiveness (from 0 not invasive, to 4 highly invasive) and they were the only ones able to hydrolyse esters. Except for one K. marxianus, all the strains were able to produce biofilm even if at different levels: one showed strong biofilm production (P. kudriavzevii), 10 showed moderate production, and 1 displayed a weak biofilm production. With regard to antifungal resistance, the majority of isolates were resistant to FL, and FU, while all the examined strains were sensitive to CA, IT, KE and VO, in agreement with other authors (1) The results highlight a low prevalence of mycotic mastitis, mainly caused by genus Candida. The presence of multiple virulence traits our found encourage further investigations.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
