RARE CAUSATIVE AGENTS OF MAMMARY GLAND INFECTION: CANDIDA LAMBICA -CASE REPORT-Dubravka

A brief case report on bovine subclinical mastitis caused by yeast species Candida lambica is presented in this article. Basic cultural, microscopic and biochemical traits of this rare agent implicated in bovine mammary infection are described. Identifi cation of isolates was performed using an Integral System Yeasts Plus test, a commercial kit for identifi cation of yeasts of importance in medicine. Th e available literature off ers only sporadic reports on C. lambica infection in both humans and animals.


INTRODUCTION
Wide application of antibiotics and corticosteroids in both human and veterinary medicine throughout the period since the 1950s resulted in an increased incidence of yeast infections. Th e fungi, particularly yeasts, are considered opportunistic pathogens for mammary gland. Previous administration of antibiotics, treatment with contaminated antibiotic preparations, as well as syringes are prerequisites for the occurrence of the infection (Krukowski and Saba, 2003;Zaragoza et al., 2011). Prevalence of mycotic mastitis is usually low, 1-12% off all mastitis cases (Krukowski and Saba, 2003). However, if the infection remains unidentifi ed and the factors implicated in its development are not eliminated on time, yeast mastitis may reach even epizootic proportions (Krukowski and Saba, 2003;Costa et al., 2012). During the past several years, increased incidence of subclinical and clinical mastitis caused by yeasts from the genus Candida has been reported (Zaragoza et al., 2011;Dworecka-Kaszak et al., 2012). Microbiological examination of milk samples is inevitable in the diagnostics of mycotic mastitis, and identifi cation at species level is performed according to morphologic features (formation of chlamydoconidium, pseudohyphae and germinal tube development), growth in the presence of 0.1% cyclohexamide, acidic pH tolerance and carbohydrates assimilation and/ or fermentation.
Mastitis in dairy cows is associated with a variety of fungal species. Th ough the yeasts of the genus Candida are most commonly isolated species (Zaragoza et al., 2011;Milanov et al., 2014) there are only sporadic reports on the isolation of C. lambica (Spanamberg et

CASE REPORT
Regular monthly examination of milk sample using California Mastitis Test revealed positive fi nding in the single infected udder quarter in a Holstein-Friesian cow aged 3.9 years. Th e colour and consistency of the milk were not signifi cantly changed, but the presence of small patches was evident in the sample. Before milk sampling for microbiological examination, the cow underwent two unsuccessful antibiotic treatments that included parenteral administration of enrofl oxacin, amoxicillin and penicillin as well as intramammary administration of cefquinome, bacitracin, neomycin, tetracycline and prednisolone. Th e time period between the last antibiotic administration and collection of milk sample was more than one month. In July, when the sampling was performed, the somatic cell count per one mL of milk from infected udder was 1679000. Th e sampling was performed on 170th day of second lactation, and average seven-day milk yield was a 27 Litres.
Milk samples for microbiological examination were obtained separately from each of the four quarters. Disinfection of teats was performed using 70% ethanol, and the milk was collected into sterile plastic tubes, cooled and transported to the laboratory. To the purpose of bacterial isolation, aliquots of 50μL were inoculated onto Columbia blood agar base (Oxoid, Basingstoke, UK, CM0331) with 5% defi brinated ovine blood, MacConkey agar (Oxoid, CM0007) and onto Sabouraud dextrose agar (Oxoid, CM0041) and incubated during 2-3 days at 25°C and 37°C.
From the milk sample collected from the right anterior quarter, massive amounts of pure culture yeast were isolated. Th e growth was noticed aft er 24hour incubation at all nutritive media and at both incubation temperatures (25 o C and 37 o C), with largest colonies observed on Sabouraud dextrose agar. Colonial appearance on blood and Sabouraud dextrose agar aft er 72h incubation at 25 o C is presented in Figure 1 (A and B). Preparations made of 7-day old cultures grown on Sabouraud dextrose agar at 37 o C. Th e preparations were stained according to Giemsa method and examined using light microscopy (1000x, immersion) (Figure 1, C). B. C.

C.lambica + -------+ ---1040
Aft er the fi rst microbiological examination of the samples and isolation of the yeast, the sampling was repeated for fi nding confi rmation. Namely, C. lambica was found in dairy products, water, and fruits and thus may be present in the sample as a contaminant. Second sampling was performed under maximum aseptic conditions, at the very end of milking. Such procedure minimizes the probability of contamination with yeasts that are commonly present on the skin of the udder and teats (Krukowski and Saba, 2003). Th e repeated sampling has confi rmed the previously obtained result.
C. lambica is a rare causative agent of mastitis in dairy cattle. Similar to other yeasts of the genus Candida, it readily grows on nutritive media commonly applied in bacteriology labs, such as blood agar, MacConkey agar and Sabouraud dextrose agar. According to cultural and biochemical traits, it is similar to the species C. krusei, which is more commonly isolated from the milk of cows with mycotic mastitis (Türkyılmaz and Kaynarca, 2008; Wawron et al., 2010; Dworecka-Kaszak et al., 2012). By using chromogenic agars and a commercial phenotyping gallery, C. lambica might be misidentifi ed as Candida krusei (Vervaeke et al., 2008). Neither C. lambica, nor C. krusei grow on media containing cycloheximide (which diff erentiates them from C.lipolytica). Besides molecular methods that enable most reliable diff erentiation between these two related species, some simpler and readily available methods such as maximum growth temperature and biochemical traits determination could be successfully applied. Both species grow at incubation temperatures 25 o C and 37 o C; however, contrary to C. krusei, Candida lambica does not grow at 42 o C. Commercial test Integral System Yeasts Plus used in this research enables diff erentiation between these two species based on their assimilation abilities ( Table 2).
Some of our previous investigation of yeasts isolates from milk of cows with subclinical and clinical mastitis using the Integral System Yeasts Plus demonstrated that biochemical profi le of the achlorophyllous alga Prototheca zopfi i is identical to that of C. krusei (Milanov et al., 2014). Since this test is not applicable for the identifi cation of P. zopfi i (contrary to P. wickerhamii), the simi-larity of cultural features of these organisms makes microscopic examination of the preparation unavoidable for fi nal identifi cation at species level.
Even though there are some therapeutic options for the management of yeast mastitis, it is rarely practiced in everyday practice. Th erapeutic options in Candida infections include amphotericin B, fl uconazole, tioconazole, miconazole and nystatin (Krukowski and Saba, 2003). Regrettably, in this case as well as in the number of other cases, yeast mastitis remains undiagnosed. It enhances spread of the infection within the herd, and commonly practiced antibiotic therapies only aggravate the situation. Milk samples are submitted for microbiological examination only aft er several unsuccessful treatments with diverse antibiotic classes.

CONCLUSION
Th e incidence of yeast mastitis in dairy cattle has been showing increasing tendency. Timely identifi cation, control of infection spread within the herd and adequate management require microbiological examination of milk samples. Everyday practice in our veterinary clinical laboratories does not implicate identifi cation of yeasts to the level of species. Moreover, specifi c test designed for application in veterinary medicine are not commercially available. Integral Systems Yeasts Plus test is designed for identifi cation of yeasts of medical importance and is highly applicable for the identifi cation of Candida species involved in the mastitis of dairy cows.