Recovery Rate of Fungal Pathogens Isolated from Cases of Bovine and Ovine Mycotic Mastitis………..

Mastitis is frequently brought on by a wide range of microorganisms, which results in significant economic consequences and impairment to the dairy processing industry (Bakr et al., 2015). The use of injectable tools, cannulas, or antibiotic preparations (intramammary infusions) during the treatment of animal disorders is frequently linked to mycotic mastitis. Teat injuries may also increase the risk of the development of fungal infection (Farag and El Said, 2012).

Investigating bovine and ovine mycotic mastitis was the study's goal.A total of 250 milk samples were collected from cows, ewes and does suffering from mastitis, not responding for antibiotic therapy, to determine the prevalence of fungal pathogens associated with mastitis.For the isolated yeasts, antimicrobial susceptibility testing was done through disc diffusion method, and biofilm formation was evaluated phenotypically by cultivation on Congo Red Agar (CRA) medium.Also, MDR1, CDR and ERG11 genes were detected by PCR.Out of the collected milk samples, 53 samples (21.2%) were positive for the presence of fungal pathogens.Out of them, 54 fungal pathogens were recovered, including 49 Candida species, 2 A. niger, in addition to one of A. fumigatus, A. flavus and Penicillium species.The yeast isolates included 18 C. guilliermondii, 16 C. parapsilosis, 7 C. tropicalis, 4 C. albicans, and 4 C. kefyr.Antimicrobial susceptibility testing of Candida isolates showed that all isolates were resistant to nystatin.Regarding to fluconazole, 25 isolates were sensitive, 2 isolates were intermediately sensitive and 22 isolates were resistant.Nine yeast isolates were biofilm former.All tested isolates harbored MDR1 gene, 4 isolates contained CDR gene and only 1 isolate was positive for ERG11 gene.
Biofilm, Mastitis, Molds, Resistance genes, Yeasts which also protect the microbe from the host's defenses.Additionally, because Candida biofilms are more resistant to antifungal treatment, their development has significant clinical complications.Consequently, it is thought that biofilm development is crucial to the pathophysiology of candida infections (Şeker and Erhan, 2011).
Therefore, the aim of this study is to detect the recovery rate of fungal pathogens associated with bovine and ovine mycotic mastitis, resistance, virulence and molecular characterization of Candida species.
The Institutional Animal Care and Use Committee of Cairo University (CU-IACUC) accepted this work and assigned it an approval number (Vet CU 01122022580) for animal sampling and handling for isolating the tested fungi.
A total of 250 mastitic milk samples were collected under complete aseptic conditions from clinically diseased cattle, sheep and goats (120 bovine and 130 ovine milk samples), not responding to treatment with antibiotics, for mycological examination.From January to October 2021, milk samples were collected from a variety of dairy farms in the Governorates of Beni-Suef and Fayoum.As soon as possible, the samples were carried in an ice box to the Faculty of Veterinary Medicine at Beni-Suef University's Bacteriology, Mycology, and Immunology laboratory.

2004).
Identification of isolated molds depended upon macroscopic examination (colors, consistency of growth on SDA and external general colors of their Petri dishes) (Jawetz et al., 2004) and microscopic examination by lactophenol cotton blue (LPCB) staining to observe the shape and structure of hyphae and spores (Winn et al., 2006).Yeasts were identified according to colony morphology, Gram staining as well as germ tube and chlamydospores production on new milk medium (Pincus et al., 2007).
As per the M60 protocol's instructions, the standard disc diffusion technique was used to 2 antifungal drugs, including fluconazole and nystatin, 0.5McFarland standard turbidity isolate suspensions were made, and Mueller Hinton agar plates with chloramphenicol (0.05g/l) were inoculated.On the plates, fluconazole (25mg) and nystatin (100mg) antifungal discs were placed.According to the M60 procedure, the tested isolates were classified as sensitive, intermediately sensitive or resistant (CLSI, 2020).
According to Sharma et al., (2017), Congo Red Agar (CRA) medium was made with brain heart infusion broth 37 g/l, sucrose 50 g/l, agar 10 g/l, and Congo red 8 g/l.This medium was used to identify the production of biofilms by isolated yeasts.Prior to being added to the autoclaved brain heart infusion agar with sucrose, which is cooled at 55°C, Congo red stain was first made as a concentrated aqueous solution separately from the other medium constituents and autoclaved at 121°C for 15 min.The yeasts were inoculated onto CRA plates, and then incubated for 48 hour at 37°C.Positive biofilm production is indicated by black colonies with a dry crystalline consistency.
Five Candida isolates (2 C. albicans, 2 C. guilliermondii and 1 C. parapsilosis), including the species that mostly associated with bovine and ovine mycotic mastitis phenotypically resistant to fluconazole, were chosen for genotypic characterization using PCR to identify the presence of several fluconazole-resistant genes, such as CDR, MDR1, and ERG11 genes using their particular forward and reverse primers, as indicated in Table (1).DNA used as a positive control was taken from a Candida field isolate that tested positive for RLQP (Reference laboratory for veterinary quality control on poultry production, Dokki, Giza, Egypt).A PCR mixture devoid of the DNA template served as a negative control, on the other hand.The antimicrobial susceptibility testing of Candida isolates (n = 49) showed that all isolates were resistant to nystatin.
Out of the total tested Candida isolates (n=49), 9 Candida isolates (18.4%) were grown as black colonies with a dry crystalline consistency on CRA medium and described as biofilm formers.While, 40 Candida isolates (81.6%) were grown as red colonies and described as non-biofilm formers, as illustrated in Table (3       In this study, out of the collected 250 mastitic milk samples, 53 samples were positive for fungi with the percentage of 21.2%, bovine mycotic mastitis recoded 12.5% and ovine mycotic mastitis recorded 29.2%.The present study focused on the recovery rate of fungal pathogens associated with bovine and ovine mycotic mastitis, where Candida species other than C. albicans were more prevalent, with detection of their susceptibility to fluconazole and nystatin, biofilm formation and molecular characterization of some resistance genes (MDR1, CDR and ERG11).We recommend that, taking into account the mycotic mastitis during dealing with udder infections.

Fig. 2
Fig. 2 Cultivation of Candida isolates on CRA medium.Right side: Candida colonies appeared black with dry crystalline consistency (biofilm positive).Left side: Candida colonies seemed to be red on CRA medium (biofilm negative).

Fig. 1 .
Fig. 1.Antimicrobial susceptibility testing of Candida showing sensitivity to fluconazole and complete resistance to nystatin.

Fig. 3 .
Fig. 3. PCR amplification of the CDR and MDR1 genes at 503 bp and 590 bp, respectively.On the right side: lanes (1, 2, 4 and 5) showed positive amplification of CDR gene.On the left side: lanes (1-5) showed positive amplification of MDR1 gene.L = ladder; P = Positive control; N = Negative control.

Tables, 2-3).Table 2 .
Prevalence of mycotic mastitis and recovery rates of the isolated yeasts and molds.

Table 3 .
Recovery rates of the isolated fungal species and percentage of biofilm former isolates.

Hassan et al., 2014).
of the collected samples as well as other factors including climate, sanitation, the use of antibiotics in continuous as intramammary infusion therapy and environmental bacterial mastitis, which can result in necrotic tissues in the udder and mycotic infections (Penicillium species (1.9%).Most of issued papers mentioned that C. albicans had taken the upper hand in mycotic mastitis, but in our study, C. guilliermondii and C. parapsilosis recorded the highest percentages followed by C. tropicalis, C. albicans and C. kefyr.Similar results were documented by Dworecka by Bakr et al., (2015).Also, Dworecka-Kaszak et al., (2012) recovered 55 (83.3%) fungal isolates out of 66 mastitic milk samples collected from cows.On the other hand, very lower percentages of 2, 9.4 and 14.5 % were recorded by Bekele et al., (2019), Hassan et al., (2014), and Mousa et al., (2021), respectively.The differences in prevalence of mastitis between studies may be due to the wide variety between the number

et al., (2014).
Changes in sterol biosynthesis, mutations in the drug target enzyme and sterol 14a-demethylase, overexpression of the ERG11 gene, mutations in the genes encoding membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) super families and changes in sterol biosynthesis are some of the mechanisms that can lead to fluconazole resistance.Where it has been demonstrated that C. albicans develops fluconazole resistance when CDR1, CDR2 and MDR1 expression levels are increased ( Out of the total tested Candida isolates in this study, 9 isolates (18.4%) were positive for biofilm formation that was lower than results recorded by Sharma et al., (2017), Şeker and Erhan, (2011) and SAV and Özturk, (2022).

Khosravi Rad et al., 2016), that
is agreed with our results where MDR1 and CDR genes were detected by 100% and 80%, respectively.One isolate (20%) was positive for ERG11 gene, similar results were recorded by Mohamed et al., (2022) who detected an overexpression of ERG11 gene by a percentage of 27.8%.