Characterization of ß-glucan obtained from Candida albicans of caprine mastitis.

Background: Beta-glucan (ß-glucan) is a polysaccharide containing ß-glycosidic bonds that is an important structure part of different yeast cells. Aim: The purpose of the study is to characterize ß-glucan obtained from Candida albicans (C. albicans) isolated from caprine mastitis. Methods: The ß-glucan was extracted by using utilizing an Alkaline-acidic extraction technique. The dry weight of extracted ß-glucan was 7.47/150 g with 4.98%. Results: The findings demonstrated that the extracted ß-glucan had similarity in the primary peak 5.78 of liquid samples using the method of high-performance liquid chromatography when compared to the standard form of ß-glucan. However, scanning electron microscopy studies revealed that the standard of ß-glucan was distinct in morphology but similar to ß-glucan isolated from C. albicans in terms of particle sizes in the range of 1.60-2.65 m and the lack of cell wall traces. The findings of an investigation using energy-dispersive X-ray fluorescence spectroscopy (EDS/EDX) of extracted and standard ß-glucan, showed the principal elements discovered were carbon (C), oxygen (O), and nitrogen (N). Aluminum (Al), silicon (Si), nickel (Ni), and gold (Au) were also present, but in less amounts. Conclusion: The extracted ß-glucan displayed a high degree of similarity and purity to the standard ß-glucan, according to the findings of Fourier transform infrared spectroscopy (FT-IR) research.

The A756T Mutation of the ERG11 Gene Associated With Resistance to Itraconazole in Candida Krusei Isolated From Mycotic Mastitis of Cows.

Candida krusei (C. krusei) has been recently recognized as an important pathogen involved in mycotic mastitis of cows. The phenotypic and molecular characteristics of 15 C. krusei clinical isolates collected from cows with clinical mastitis in three herds of Yinchuan, Ningxia, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. In addition to sequencing analysis, the ERG11 gene that encodes 14α-demethylases, the expression of the ERG11 gene, and efflux transporters ABC1 and ABC2 in itraconazole-susceptible (S), itraconazole-susceptible dose dependent (SDD), and itraconazole-resistant (R) C. krusei isolates was also quantified by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. Sequencing analysis revealed three synonymous codon substitutions of the ERG11 gene including T939C, A756T, and T642C in these C. krusei clinical isolates. Among them, T642C and T939C mutations were detected in itraconazole-resistant and -susceptible C. krusei isolates, but the A756T substitution was found only in itraconazole-resistant isolates. Importantly, the expression of the ERG11 gene in itraconazole-resistant isolates was significantly higher compared with itraconazole-SDD and itraconazole-susceptible isolates (p = 0.052 and p = 0.012, respectively), as determined by the qRT-PCR assay. Interestingly, the expression of the ABC2 gene was also significantly higher in itraconazole-resistant isolates relative to the itraconazole-SDD and itraconazole-susceptible strains. Notably, the expression of ERG11 was positively associated with resistance to itraconazole (p = 0.4177 in SDD compared with S, p = 0.0107 in SDD with R, and p = 0.0035 in S with R, respectively). These data demonstrated that mutations of the ERG11 gene were involved in drug resistance in C. krusei. The A756T synonymous codon substitution of the ERG11 gene was correlated with an increased expression of drug-resistant genes including ERG11 and ABC2 in itraconazole-resistant C. krusei isolates examined in this study.

Chronic incurable mastitis in sheep: prevalence, identification of predisposing factors, and genotyping of fungal causative species using PCR-RFLP.

Mycotic mastitis is a neglected problem type of incurable chronic mastitis in sheep flock of many countries which associated with wide economic burden. In the current study, a total of 600 ewes at Menofia governorate, Egypt, were subjected to clinical and molecular examination using PCR-RFLP to estimate the prevalence of chronic mycotic mastitis and identify the causative agent. A structured questionnaire is distributed to shepherds in the study area to identify the risky behavioral practices being followed and lead to increase the prevalence of mycotic mastitis cases. The results showed that out of 600 ewes examined, 150 showed clinical signs of mastitis (25%). A total of 25 ewes with clinical mastitis did not respond to antibiotic treatment for long time and suffered from mycotic mastitis (16.7%, CI 11.1-23.6%). A total of 31 fungal isolates were identified: 14 yeast spp., Candida albicans, Candida parapasilosis, Candida rugosa, and Saccharomyces spp. and 17 mold spp., Alternaria spp., and Fusarium spp. Results showed also the widespread of risky practices among shepherds which could be responsible for the increase the prevalence of mycotic mastitis among ewes in the study area including presenting of decayed food to sheep, uncontrolled usage of antibiotics for mastitis treatment, lack of usage of antiseptics, and keeping of chronic infected animals in flocks for breeding. In conclusions, using of specific ITS1 and ITS4 primer sets with PCR-RFLP technique provided a suitable method for rapid identification and genotyping of Candida spp., Scaccharomyces, Alternaria, and Fusarium vertolliodes isolated from chronic mastitis in sheep. Furthermore, this study is considered up to our knowledge one of scarce estimates available on mycotic mastitis in sheep flocks in Egypt. Mycotic mastitis existed at higher prevalence estimates in the study area and educational campaigns to shepherds are much required to increase their awareness on the threat of risky of behaviors responsible for spread of the disease among their animals.

Epidemiological investigation of non-albicans Candida species recovered from mycotic mastitis of cows in Yinchuan, Ningxia of China.

BACKGROUND: Candida spp. is the vital pathogen involved in mycotic mastitis of cows. However the epidemiology and infection of Candida species in mycotic mastitis of cow in Ningxia province of China has not been explored. In the present study, the epidemiology, antimicrobial susceptibility and virulence-related genes of non-albicans Candida (NAC) species were investigated. METHODS: A total of 482 milk samples from cows with clinical mastitis in four herds of Yinchuan, Ningxia were collected and used for the isolation and identification of mastic pathogens by phenotypic and molecular characteristics, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The antimicrobial susceptibility to antifungal agents was also determined by a disk diffusion assay. The presence of virulence-related genes was determined by polymerase chain reaction (PCR). RESULTS: A total of 60 isolates from nine different Candida species were identified from 256 (60/256, 23.44%) milk samples. The most frequently identified species in cows with clinical mastitis groups were Candida krusei (n = 14) and Candida parapsilosis (n = 6). Others include Candida lipolytica, Candida lusitaniae, Cryptococcus neoformans. But no Candida albicans was identified in this study. Interestingly, All C. krusei isolates (14/14) were resistant to fluconazole, fluorocytosine, itraconazole and ketoconazole, 2 out of 14 C. krusei were resistant to amphotericin, and 8 out of the 14 were resistant to nystatin. Similarly, all six C. parapsilosis isolates were resistant to fluorocytosine, but susceptible to fluconazole, ketoconazole and nystatin; two of the six were resistant amphotericin and itraconazole. Molecularly, all of the C. parapsilosis isolates carried eight virulence-related genes, FKS1, FKS2, FKS3, SAP1, SAP2, CDR1, ERG11 and MDR1. All of the C. krusei isolates contained three virulence-related genes, ERG11, ABC2 and FKS1. CONCLUSION: These data suggested that Candida species other than C. albicans played a pathogenic role in mycotic mastitis of cows in Yinchuan, Ningxia of China. The high incidence of drug-resistant genes in C. parapsilosis and C. krusei also highlighted a great concern in public and animal health in this region.

Antifungal activity of essential oils extract from Origanum floribundum Munby, Rosmarinus officinalis L. and Thymus ciliatus Desf. against Candida albicans isolated from bovine clinical mastitis.

OBJECTIVE: The aim of this study is to limit the antibiotic use in mastitis treatment and to find other alternatives. The antifungal activity of the essential oils from Origanum floribundum Munby., Rosmarinus officinalis L. and Thymus ciliatus Desf. is studied in the present work against a Candida albicans reference strain and ten C. albicans isolated strains from bovine clinical mastitis. MATERIALS AND METHODS: Essential oils were extracted by hydrodistillation technique using Clevenger apparatus. Their chromatographic analysis was performed with a Gas Chromatograph/Mass Spectrometer (GC/MS). Antifungal activities of essential oils were investigated by macrobroth method of dilution in tubes to determine the Minimum Inhibitory Concentrations (MIC 80%). RESULTS: Analysis of the essential oil showed chemical profile dominated by thymol (50.47 and 62.41%) and P-cymene (24.22 and 15.51%) in the oregano and the thyme respectively, 1, 8-cineol (31.50%) and α-pinene (18.33%) in Rosemary. The three essential oils revealed highly effective anticandidal activity, with an MIC of 80% values ranged from 15.02 to 31.08µg/mL. CONCLUSION: These results suggest that essential oils studied can be real alternatives in the control of mastitis fungi but deserving studies more in-depth and detailed on their application in vivo.

Mastite micótica em ruminantes causada por leveduras / Mycotic mastitis in ruminants caused by yeasts

A ocorrência de casos esporádicos de mastite causados por microrganismos de origem ambiental pode ser considerada como emergente e as leveduras, os fungos leveduriformes e os filamentosos são alguns dos principais agentes envolvidos. Em relação à mastite micótica, as leveduras são freqüentemente causas de infecções da glândula mamária em animais produtores de leite. A maior parte dos casos ocorre sob a forma de surtos localizados e/ou após tratamento com antimicrobianos. Os principais gêneros envolvidos são Candida e Cryptococcus, além de outros como Geotrichum, Pichia e Trichosporon. O objetivo do trabalho é revisar os aspectos gerais sobre a mastite micótica causada por leveduras e fungos leveduriformes, tais como etiologia, causas predisponentes, patogenicidade, diagnóstico, tratamento e profilaxia por meio de uma abordagem cronológica dos primeiros relatos, mas com enfoque principal nos resultados descritos recentemente na literatura veterinária nacional e internacional. O controle da mastite micótica é fundamentado em métodos preventivos, como adequado manejo da ordenha, correta higienização dos equipamentos e das instalações, com a finalidade de reduzir o número de animais acometidos, além de garantir a qualidade e a inocuidade dos produtos lácteos.

The veterinary literature registers sporadic cases of infections caused by environmental microorganisms in which the main agents involved are yeasts, yeasts-like and filamentous fungi. In relation to dairy animals, the yeasts are more frequently incriminated as the aetiological agents of mycotic mastitis. Most cases occur under the form of localized outbreaks and / or after treatment with antimicrobial agents. The main genera involved in mastitis are Candida and Cryptococcus, besides others such as Geotrichum, Pichia and Trichosporon. The purpose of this review is to point out the main aspects of mastitis caused by yeasts and yeast-like fungi, such as etiology, predisposing factors, pathogenicity, diagnosis, treatment and prophylaxis, through a chronological approach of the first reports, with main focus on results described recently in national and international veterinary literature. The mycotic mastitis control should be mainly focused on preventive methods, especially based on adequate management of essential factors like proper milking procedures and a high level of hygiene of the environment and equipments, with the aim of reducing the number of affected animals, assuring the quality and innocuity of dairy food safety.