A STUDY ON THE DIVERSITY OF YEASTS AND COLIFORMS ASSOCIATED WITH BOVINE SUBCLINICAL MASTITIS IN PERI-URBAN DAIRY FARMS IN KADUNA METROPOLIS, KADUNA STATE, NIGERIA
This study aimed to examine the diversity of yeasts and coliforms associated with bovine subclinical mastitis in peri-urban dairy farms in Kaduna Metropolis, Kaduna State, Nigeria, including the use of molecular techniques. A cross-sectional study was conducted on twenty-six dairy farms drawn from four local government areas in the state. A total of 300 composite milk samples were collected from 300 cows-in-milking and examined. The result show that: 37 (12.3%) fungal isolates were identified using the API 20C AUX as; (9) Candida albicans, vitro sensitivity test using five antimycotic agents showedthat 85% of the isolates were sensitive to amphotericin B, followed by griseofulvin, nystatin, variconazole and fluconazole in decreasing order. Nested PCR of the D1 / D2 domains of the 26S rRNA gene of 11 yeast isolates showed they all had a distinct band- 600bp. DNA sequencing and GenBank BLASTN of the eleven genes identified them as: (3) Candida albicans,(1) Saccharomyces cerevisiae and (7) Pichia Kudriavzevii(Candida krusei). The D1/D2 26S rRNA gene sequences were 93-100% identical for yeast isolates within the same species. Phylogenetic reconstruction based on the D1/D2 26S rRNA gene sequences grouped them into 3 clusters and showed heterogeneity in C. albicans. A 10.3% prevalence was recorded for coliform organisms using the Microgen GN-ID A+B Kit (Medica-TecTM), these were; (1) Enterobacter cloacae, (1) Enterobacter aerogenes, (2)Enterobacter gergoviae, (4) Candida famata, (4) Candida krusei, (1) Candida boidinii, (1) Candida pelliculosa, (1) (2) Citrobacterfreundii, (1) Citrobacter koseri, (11) Klebsiellapneumoniae, (5) Klebsiella oxytoca, (4) Serratia marcescens, (3) Proteus mirabilis and (1) Pantoea agglomerans. In-vitro sensitivity test using seven commonly available antibiotics showed that all the isolates were sensitive to amoxicillin followed by ciprofloxacin, gentamicin, chloramphenicol, streptomycin, tetracycline and erythromycin in decreasing order. Eighty-nine (29.7%) cow milk samples were CMT positive of these: 13(4.3%) were contaminated with yeasts only, 7(2.3%) contaminated with only coliforms while, 24(8%) were contaminated with both yeasts and coliforms. Age, parity number, stage of lactation, management system, milking hygiene and presence of lesion on udder/teat were found to be significantly associated (p<0.05) with the prevalence of mastitis in cows. The lowest prevalence (24%; 48 of 200) was recorded in cows within 3–4 years of age while, the highest (60.6%; 20 of 33) was in cows aged above 5 years. Stage of lactation was significant with the prevalence of mastitis being the highest (45.5%; 30 of 66) during the initial stage of lactation (0 to 5 month). It was concluded that, the relatively high prevalence of yeasts and coliforms in bovine subclinical mastitis in dairy herds could significantly reduce milk production and cause economic losses. The milk samples contained diverse yeast species including isolates of the pathogenic yeast C. albicans and this raises the possibility of milk and dairy products being vehicles for transmission of pathogenic yeasts. It is therefore recommended that the farmers practice good milking hygiene, milk clinically infected cows last, cull chronic mastitis cases, treat clinically infected cows and administer dry period therapy to their cows. As this will go a long way to reduce the prevalence of yeasts and coliforms in subclinical mastitis in peri-urban dairy farms in Kaduna metropolis, Kaduna State, Nigeria.
1.1 Background Information
Bovine mastitis is the inflammatory response of the udder to noxious agents that can be either infectious or non-infectious characterized by pathological alterations in mammary tissues, compositional changes in milk, elevated milk somatic cells, and pain to the affected animal resulting in reductions of milk yield and quality (Matofari et al., 2005; Radostits et al., 2007). Most frequently the aetiology are infectious and organisms as diverse as bacteria, mycoplasmas, yeasts and algae have been implicated (Smith and Hogan, 2001; Krukowski et al., 2006; Kivaria and Noordhuizen, 2007).
Bovine mycotic mastitis is usually caused by yeasts, but mastitis due to filamentous fungi mostly Aspergillus fumigatus has been reported (LasHeras et al., 2000). Mycotic mastitis occurs as sporadic cases affecting a small percentage of cows, or as outbreaks affecting the majority of animals. In both situations, however, the seriousness of infection depends on the number of organisms present in the glands and the species of yeast involved (Pengov, 2002). Generally, studieshave shown that mycotic mastitis is on the increase and the most frequently isolated organisms from affected quarters are Candida species (Malinowski et al., 2001; Spanamberg et al., 2008; Tarfarosh and Purohit, 2008) which are a group of unicellular opportunistic organisms, ever present in the natural surroundings of dairy cattle (milker‟s hands, milking machines, treatment instruments, floor, straw, feed, saw dust, soil, drug mixtures and sanitizing solutions) and are normal inhabitants of the skin of the udder and teats, where they exist in low numbers (Santos and Marin, 2005). They can invade mammary glands and cause clinical mastitis characterized by pain, prolonged fever, tenderness, inflammatory reaction in the mammary gland and associated lymph nodes,and also, reductions of milk yield and quality in animals (Şeker, 2010). Some intra-mammary fungal infections such as A. fumigatus and Candida spp. may result in death of affected animals (Krukowski et al., 2000).
Outbreaks of mycotic mastitis are generally believed to result from an ascending infection subsequent to incorrect administration of antibiotic preparations during drying-off period (Spanamberg et al., 2008),contamination of the teat end or cannulas by environmental yeasts and fungi, lack of hygiene during milking and poor equipment cleaning(Gaudie et al., 2009). Also, administration of large doses of antibiotics may cause a reduction in vitamin A leading to injury to the udder‟s epithelium and affecting the micro-flora of the mammary glands, which acts as an animal natural defense, thus facilitating the invasion of fungi and yeasts (Şeker, 2010).
The clinical signs of mycotic mastitis are non-specific and in some cases, their development may be masked by symptoms of an underlying disease. Therefore, the disease is generally diagnosed by demonstrating and identifying the etiological agent histopathologically and in culture (Krukowski et al., 2000; Santos and Marin, 2005).
Current diagnostic methods have not proven to be sufficiently sensitive and specific to enable an early and effective diagnosis of the disease, with the result that the search for an optimal diagnostic method continues (Garcia and Blanco, 2000). Among non-culture methods under investigation, Polymerase Chain Reaction (PCR) offers advantages over classical approaches, because theoretically, low level fungal infections (e.g. with Candida albicans) can be detected from minimal volumes of clinical samples such as blood, and DNA from both dead and viable organisms could serve as a target template for the amplification reaction (Polanco et al., 1999).