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).

Random use of growth promoters, antibiotics and antimycotics in animals leads to immune suppression and development of multiple drug resistant strains to compounds used in human medicine (Ström et al., 2002; Magnusson et al., 2003). Probiotic bacteria present in the alimentary tract and vagina of humans and animals prevent the overgrowth of Candida spp. and thereby decrease the occurrence of mucosal or systemic candidiasis (Drozdowicz and Kwiecien, 2006; Zwolinska-Wcislo et al., 2006).The increasing incidence of mycotic infection, the increasing resistance of mycotic species to anti-fungal agents and the rise in mortality associated with infections by Candida species demand a safe way to prevent and treat infections such as mycotic mastitis caused by opportunistic yeasts and fungi.

Therefore, the present study reports mycotic mastitis in some peri-urban dairy farms in Kaduna State, Nigeria, and the causal mycotic agents, diagnosed directly using nested PCR and DNA sequencing. This study draws attention to the importance of the disease in Nigeria, and could therefore stimulate interest for further investigations.

1.2 Statement of Research Problem

Mastitis is one of the most serious problems in the dairy cattle farms. The great majority of cases are caused by bacteria, but recently there have been an increasing number of reports about cases caused by yeasts or yeast-like organisms, following post-treatment with antimicrobial agents (Crawshaw et al., 2005; Saleh, 2005).Studies have shown that fungal organisms and their spores can survive pasteurization and that yeast intra-mammary infections were responsible for 2-3% of all clinical cases seen in a veterinary practice (Chahota et al., 2001; Spanamberg et al., 2008).

Nowadays, public health concern associated with microbial food safety has arisen. Numerous epidemiological reports have implicated non-heat treated milk and raw-milk products as the major factors responsible for illnesses caused by food-borne pathogens (Oliver et al., 2009). Some yeast may further pose threats to food safety given their association with opportunistic infections and other adverse conditions in humans (Fleet, 2007).

There are few documented cases of mycotic bovine mastitis in Nigeria prior to this study. In all forms of mastitis, a cow may not reach her full production potential as some of the milk secretory tissue would have been destroyed. Yeast infections should be suspected when there is a history of unsuccessful treatment or an intensification of clinical signs of mastitis after intra-mammary infusion of antibiotics (Krukowski et al., 2006). Although antimycotic drugs have been used for treatment of yeast mastitis, there was no clear evidence of the effectiveness of this therapy (Crawshaw et al., 2005).

1.3 Justification of the Research

Mastitis is the most widespread infectious disease of cattle and from economic aspect the most damaging (Dodd, 1985; Oliver et al., 2009). However the rate of subclinical mastitis is 15 to 40 times greater than the clinical cases (National Mastitis Council (NMC), 2000).

The normal somatic cell count (SCC) is 200,000 SCC/ml of milk, however the cow is not considered to be infected until the count rises to 300,000 SCC/ml of milk (NMC, 2000). Each time the count rises by 100,000 SCC/ml of milk, the loss of milk production is about 2.5% per lactation. For example, for a herd that has a cell count of 400,000 SCC/ml, the loss of milk due to mastitis would be 5%. At the same time as the SCC increases, the yield of processed products such as yoghurt and cheese will decline by 1%. Reducing SCC is of benefit not only to the farmer, but to the processor as well. Increased mastitis prevalence can be expected with increased milk production unless all lactating cows are tested at least monthly and treated during the dry period (Burns et al., 2000; NMC, 2000; FAO, 2008).

Mastitis presents grave public health hazards for the human populace as milk is a good vehicle for disease transmission to humans when inadequately treated. Some of the important milk borne diseases includes; candidosis (thrush), tuberculosis, Q-fever, brucellosis, salmonellosis, botulism, campylobacteriosis, Escherichia coli, diptheria, hepatitis A, histamine intoxication, Iron intoxication, milk sickness, shigellosis, staphylococcal intoxication, streptococcal sore throat, scarlet fever, etc. (El-Sharoud et al., 2009; WHO, 2010).

The increasing number of dairy farms coupled with the presence of mycosis in animals may be an indicator of its probable presence in man especially in those working with cattle or their by-products or consuming unprocessed milk and milk by-products. In most parts of the northern states of Nigeria, local customs encourage the consumption of raw bovine milk with public health safety problems (Ocholi et al., 2004).

There are increasing reports of failure of intra-mammary antibiotic treatment in bovine mastitis by farmers and veterinarians in Nigeria and Northern States in particular (Kwaga, 2012).This study was designed to provide information on fungal and coliform micro-flora of bovine milk. The in vitro susceptibility of the isolates to various antimicrobials agents was evaluated to provide comprehensive information on bovine mastitis in Kaduna metropolis, Kaduna State, Nigeria.

1.4 Aim of the Study

The aim of the study was to determine the diversity and prevalence of yeasts and coliforms associated with bovine mastitis in peri-urban dairy farms in Kaduna State, Nigeria and employing molecular techniques in yeast identification. Also the risk factors associated with bovine mastitis and the antibiograms of isolates.

1.5 Objectives of the Study

The objectives of this work are to:

i.Establish the occurrence of subclinical mastitis in cows by using the California mastitis test (CMT).

ii. Culture, isolate and identify yeasts and coliform organisms from milk of cows with subclinical and clinical mastitis in Kaduna metropolis using API 20C-AUX and Microgen GN-ID A+B Kit (Medica-TecTM).
iii. determine the in vitro susceptibility of the isolates to various antimycotic and antibiotic agents.

iv. Characterize the yeasts associated with milk of cows using polymerase chain reaction (PCR) and sequencing of the domains D1 and D2 of the 26S rRNA gene.

v. Establish association between various predisposing factors of subclinical mastitis (age, parity number, stage of lactation, management practices, teat/udder injury) and occurrence of mastitis in cows using structured questionnaires.

1.6 Research Questions

1. Are there cases of subclinical mastitis in cows in Kaduna metropolis?

2. What are the different types of yeast and coliform organisms in milk of cows with mastitis in Kaduna metropolis?

3. What antimycotic and antibiotic agents are the isolates susceptible to?

4. Are yeasts of public health importance present in milk from cows with mastitis in Kaduna metropolis?

5. Are there any associations between the risk factors of subclinical mastitis caused by coliform organisms?