PREVALENCE, ASSOCIATED RISK FACTORS AND MAJOR CAUSATIVE AGENTS OF BOVINE MASTITIS IN SELECTED DAIRY FARMS IN AND AROUND DIRE DAWA MSc THESIS HAGOS GEBRESELASSIE WELDEMARIAM NOVEMBER 2015 HARAMAYA UNIVERSITY, HARAMAYA Prevalence, Associated Risk Factors and Major Causative Agents of Bovine Mastitis in Selected Dairy Farms in and around Dire Dawa A Thesis Submitted to the Postgraduate Program (School of Animal and Range Sciences) HARAMAYA UNIVERSITY In Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE (ANIMAL PRODUCTION) BY Hagos Gebreselassie Weldemariam November 2015 Haramaya University, Haramaya ii POSTGRADUATE PROGRAM DIRECTORATE HARAMAYA UNIVERSITY As Thesis Research advisors, we here by certify that we have read and evaluated this thesis Prepared, under our guidance by Hagos Gebreselassie Weldemariam titled “Prevalence, Associated Risk Factors and Major Causative Agents of Bovine Mastitis in Selected Dairy Farms in and Around Dire Dawa.” We recommend that it can be submitted as it is fulfilling the Thesis requirement. Yitbarek Getachew (PhD) Major Advisor _______________ Signature Mengistu Urge (PhD) Co–Advisor _______________ Signature As members of the Board of Examiners of the MSc Thesis Open Defense Examination, we Certify that we have read, evaluated the Thesis prepared by Hagos Gebreselassie Weldemariam and examined the candidate. We recommend that the Thesis be accepted as fulfilling the Thesis requirement for the Degree of Master of Science in Agriculture (Animal Production). ______________________ _________________ Chairperson Signature ______________________ _________________ Internal Examiner Signature ______________________ _________________ External Examiner Signature Final approval and acceptance of the Thesis is contingent upon the submission of its final copy to the Council of Graduate Studies (CGS) through the candidate’s department or school graduate committee (DGC or SGC). iii DEDICATION Dedicated to my Mother Weizero Letebirhan Abebe who sacrificed much to bring me up to this level but not lucky to see the final fruits of my effort. iv STATEMENT OF THE AUTHOR First, I declare that this thesis is the result of my own work and that all sources or materials used for this thesis have been duly acknowledged. This thesis is submitted in partial fulfilment of the requirements for MSc degree at Haramaya University and to be made available at the University’s Library under the rules of the Library. I confidently declare that this thesis has not been submitted to any other institutions anywhere for the award of any academic degree, diploma, or certificate. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the School/Director of the Graduate Program when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. Name: Hagos Gebreselassie Weldemariam Signature_________ Place: Haramaya University, Haramaya Date of Submission: ----------------------- v BIOGRAPHICAL SKETCH The author was born in Ginner town, central zone of Tigray Region, in June 21, 1983 from his father Ato Gebreselassie Weldemariam and his mother W/o Letebirhan Abebe. He attended his primary and secondary school at Myssesela and Werei Secondary School, respectively. After completing secondary school with good results of the Ethiopian schools Leaving Certificate Examination (ESLC), he joined Addis Ababa University in 2004 academic year and graduated with DVM (Doctor of Veterinary Medicine) in 2009. After completing his study, he was employed in Army foundation of Hurso Agriculture Development. He joined Haramaya University in 2012 to pursue his graduate studies in MSc in Animal Production. Currently, he is assigned and working as a veterinarian and supervisor in Army Foundation Hurso Agriculture Development. The author is married and blessed with one child. vi ACKNOWLEDGEMENT First of all, I would like to thank the Almighty God and my wife, Sister Abirehet Hailemariam who gave me the opportunity to pursue my graduate study at Haramaya University where I gained much. I would like to thank many people and organizations who supported me in accomplishing this thesis work. My greatest thank and heartfelt appreciation goes to my major advisor Dr. Yitbarek Getachew and co-advisor Dr. Mengistu Urge for their valuable guidance, suggestions and provision of related study materials in both proposal development and thesis writing. I greatly acknowledge Dire Dawa Regional Laboratory technician Ermias Demssie and his work mates for their unreserved and valuable support in sample collection and laboratory work. Without their assistances, the completion of this paper would have been hardly possible. I would like also to express my sincere appreciation to Army Foundation for giving me chance to pursue my postgraduate study and covering study expenses. I would like also to forward my appreciation to the manager of Hurso Agriculture Development, Major Zeray Desalegn for his contribution in moral and materials. vii TABLE OF CONTENTS DEDICATION IV STATEMENT OF THE AUTHOR V BIOGRAPHICAL SKETCH VI ACKNOWLEDGEMENT VII TABLE OF CONTENTS VIII LISTS OF TABLES X LISTS OF FIGURES XI ACRONYMS AND ABBREVIATIONS XII ABSTRACT XIII 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 2.1. Definition of Mastitis 4 2.2. Clinical and Subclinical forms of Mastitis 4 2.3. Prevalence of Bovine Mastitis 5 2.4. Prevalence of Bovine Mastitis in Ethiopia 6 2.5. Pathogenesis of Bovine Mastitis 7 2.6. Aetiology of Bovine Mastitis 8 2.6.1. Staphylococcus Species 2.6.2. Streptococcus Species 2.6.3. Coliforms Bacteria 2.7. Economic Importance of Mastitis 8 9 10 10 2.8. Diagnosis of Mastitis 12 2.8.1. California Mastitis Test (CMT) 2.8.2. Culture 2.9. Classification of Mastitis Pathogens 12 12 13 2.9.1. Contagious Mastitis (cow-associated) 2.9.2. Environmental Pathogens (Mastitis) 2.10. Mastitis and Associated Risk Factors 13 14 14 2.10.1. Age and parity of the cow 2.10.2. Inherited features of the cow 2.10.3. Breed and milk yield 2.10.4. Stage of lactation 2.10.5. Mammary regression 2.10.6. Milking Machine 2.10.7. Nutrition 15 16 16 16 17 17 18 viii TABLE OF CONTENTS (Continued) 2.10.8. Weather and climate 2.10.9. Milking hygiene and procedures 3. MATERIALS AND METHODS 18 19 20 3.1. Study Area 20 3.2. Study population 20 3.3. Study Design, Sample Size and Sampling Method 21 3.4. Data Collection 22 3.4.1. Questionnaire 3.4.2. Clinical examination of udder and milk 3.4.3. California Mastitis Test (CMT) 3.4.4. Milk Sample Collection, Handling and Transportation 3.4.5. Microbiological Culture (Etiological Factors) 3.5. Data Analysis 22 22 23 23 24 25 4. RESULTS 26 4.1. Description of the Study Population and Dairy Farms 26 4.1.1. Study Animals 4.1.2. Farming System 4.2. Prevalence of Mastitis 26 26 26 4.2.1. Animal level 4.2.2. Quarter level 4.2.3. Types of Dairy Production Systems and Mastitis Prevalence 4.3. Associated Risk Factors 26 27 27 28 4.3.1. Host Factors 4.3.2. Environmental Factors 4.3.3. Microbiological Culture (Aetiological Factors) 5. DISCUSSIONS 29 30 31 32 6. SUMMERY, CONCULISION AND RECOMMENDATIONS 36 7. REFERENCES 37 8. APPENDEXES 47 ix LISTS OF TABLES Table page Table 1. prevalence of subclinical mastitis at animal level 27 Table 2. Clinical, blind and CMT scores of each quarters of lactating cow in sample farms 27 Table 3. Prevalence of bovine mastitis related to production systems in and around Dire Dawa. 27 Table 4. Prevalence of bovine mastitis in small and medium farms 28 Table 5. Association between the occurrence of subclinical mastitis and risk factors 29 Table 6. Association between subclinical mastitis and Environmental Factors 30 Table 7. Frequency distribution of isolated bacteria from mastitic dairy farms 31 x LISTS OF FIGURES Figure Page 1. Map of the study area 21 2. Bar graph for the prevalence of subclinical mastitis in the study area 28 xi ACRONYMS AND ABBREVIATIONS BMSCC Bulk Milk Somatic Cell Count CM Clinical Mastitis CMT California Mastitis Test CNS Coagulase Negative Staphylococci CPS Coagulase Positive Staphylococci CSA Central Statistics Agency DDPAIA Dire Dawa Provisional Administration Investment Agency DNA Dinuclotide Nucleic Acid DNP Days Not Pregnant DVM Doctor of Veterinary Medicine ETB Ethiopian Birr FVM Faculty of Veterinary Medicine HF Holstein Friesian IMI Intramammary Infection KPa Kilo Pascal MS Micro- Soft MWT Modified White Side Test NMC National Mastitis Council PCR Polymerase Chain Reaction SAS Statistical Analysis System SCC Somatic Cell Count xii Prevalence, Associated Risk Factors and Major Causative Agents of Bovine Mastitis in Selected Dairy Farms in and around Dire Dawa ABSTRACT A cross-sectional investigation was conducted from August, 2014 to December, 2014 to determine the prevalence of bovine mastitis, assess the association of some putative risk factors with occurrence of mastitis in lactating cows and identify the major bovine mastitis causative agents in selected dairy farms in and around Dire Dawa. A total of 403 lactating cows (331 high grade Holstein Friesian and 72 indigenous zebu breeds) were examined clinically. California Mastitis Test (CMT) was used to detect clinical and subclinical mastitis. The overall prevalence of mastitis at cow and quarter level was 91.8% and 70.85%, respectively. Three hundred twenty (79.4%) cows were positive for subclinical mastitis while only 50 (12.4%) had clinical mastitis. Among the total (1612) quarters investigated, 20 quarters (1.24%) were blind. The prevalence of mastitis was significantly (P<0.05) associated with age (X2= 30.38, P=0.000), breed (X2=3.93, P=0.047), milk yield X2=13.99, P=0.001), and previous record of mastitis (X2=12.23, P=0.000). Adult cows, Holstein breed, high milk yield and mastitis history were determined as attributing factors for higher prevalence of mastitis in the dairy farms. A total of 27 bacterial isolates were recovered from 27 pooled milk samples and gram-positive cocci were the most common pathogens. The pathogens isolated were Staphylococcus species (66.67%), Streptococcus species (14.81%), Coli-forms (Escherichia coli (11.11%) and Bacillus species (7.41%)). The present study showed that mastitis is a serious problem in dairy farms in and around Dire Dawa city. Contagious were the major causes of mastitis. Therefore, hygienic milking practice, screening and culling of chronically infected and older cows with repeated mastitis records, cow therapy and awareness creation among dairy farm owners should be practiced to reduce the risk of mastitis. Keywords: Clinical Mastitis, Hygienic milking, Prevalence, Subclinical Mastitis xiii 1. INTRODUCTION Ethiopia holds large potential for dairy development mainly due to its large livestock population and relatively favourable climate for improved high-yielding animal breeds. The country enjoys diverse topographic and climatic conditions and milk production takes place across all agro-ecological zones. In the highlands, milk is mainly produced by small scale mixed farmers, while in the lowlands, pastoralist production systems are predominant. There are also intensive and commercial dairy farms concentrated in and around major cities and towns of the country. The majority of cows kept are indigenous breeds, with a limited number of farmers keeping few crossbred grade dairy animals (Holloway et al., 2000). Dairy provides rural farmers with a way to increase assets, a method to diversify income and nutrition. Dairy is also an important tool to address poverty, enhance agricultural development, and create employment opportunities beyond an immediate household or smallholder dairy operation. Dairy is a development tool because it widens and sustains pathways out of poverty through securing assets of the poor, improving smallholder productivity and increasing market participation by the poor (ILRI, 2007). Hence, development of the dairy sector in Ethiopia can contribute significantly to poverty alleviation, improved nutrition and household income (Mohamed et al., 2004). But, the dairy sector has not been fully exploited and promoted (Tangka et al., 2002). In Ethiopia, lack of modern animal husbandry and management, limited skilled manpower in dairy technology and marketing, inadequate distribution systems and limited packaging choices have affected the sector. Severe shortages of animal feed supplies and the high cost of running a dairy farm are becoming the major bottlenecks to dairy development. Transportation cost is the other additional extra costs paid by regional farm holders, as they are buying majority of the feeds from Addis Ababa. Low productivity of cattle because of their genetic makeup, increased running and investment cost per unit (CSA, 2007) and disease, mainly udder inflammation, is becoming a serious problem. Mastitis (inflammation of mammary gland) is among important health problems in dairy cattle. It has been considered as one of the most important threats affecting the dairy 2 industry. Mastitis remains to be the most economically damaging and zoonotic potential disease for dairy industry and consumers worldwide irrespective of the species of animal (Ojo et al., 2009). It is the major concern of the dairy industry worldwide for a number of reasons, such as mastitis has deleterious effects on milk composition, yield, and quality of dairy products, it is considered a welfare concern due to the pain cows experience, especially during an episode of acute, severe mastitis (Kemp et al., 2008; Leslie and Petersson-Wolfe, 2012). Mastitis is the most common production limiting and costly disease. Factors which contribute to the economic impacts of mastitis include milk production losses, diagnostic costs, treatment costs, discarded milk, and increased risk of other diseases and culling of dairy animals (Halasa et al., 2007). In Ethiopia, the available information indicates that bovine mastitis is one of the most frequently encountered diseases of dairy cows. According to Lemma et al. (2001) of the major diseases of crossbred cows in Addis Ababa milk shed, clinical mastitis was the second most frequent disease next to reproductive diseases, in which 171 cows out of 556 were found to be affected. According to Hussein et al. (1997) the prevalence of clinical and subclinical mastitis in the central regions of Ethiopia are found to be 5.3% and 19% on cow basis and 1.9% and 7.4 % on quarter basis, respectively. In a study conducted at Repi and Debre-Zeit dairy farms, out of 186 lactating cows, forty (21.5%) were clinically affected and 71 (38%) sub-clinically infected (Workineh et al., 2002). Bishi (1998) noted that the economic losses from clinical and subclinical mastitis in Addis Ababa milk shed to be approximately 270 Ethiopian birr (ETB) per lactation. According to Mungube (2001) the estimated economic losses in terms of milk production loss, treatment cost, withdrawal and culling losses due to mastitis in the peri-urban areas of Addis Ababa were about 210.8 ETB/cow/lactation of which milk production loss contributed 38.4%. In other study conducted in the same study area (Mungube et al., 2005) a milk production loses of 17.2% was reported due to quarter infected with mastitis. In the country, mastitis has long been known (Tamirat, 2007, Bitew et al., 2010). However, the information available on the magnitude, risk factors and causative agent of the disease is inadequate. Such information is important when designing appropriate strategies that would help to reduce its prevalence and effects (Biffa et al., 2005). Most studies in Ethiopia were carried out in Addis Ababa and its surroundings, which are not representative of other regions of the country (Almaw et al., 2009). 3 Dairy farming in Dire Dawa Administration is an emerging employment sector of the youth, university graduates and women. As most of the farm owners are new to the sector, they are deficient of the basic knowledge of mastitis control and prevention strategies and these could favour mastitis to be prevalent. Moreover, heat, humidity and draught are the prevailing climatic conditions of Dire Dawa as heat and humidity increases, so does the bacterial multiplication as well as the load of pathogens in the environment. Most of the dairy farms are established around the home of owners due to lack of land and also milking practice is manual, which increases mastitis disease transmission (Yonus, 2011). These all mentioned above are the most important risk factors which contribute to the increase in intramammary infection in the dairy farms (Ranjan et al., 2011). Therefore, it was hypothesised that the poor practise in the emerging dairy industry and climatic conditions favour the presence and persistence of mastitis in the study area. Therefore, the purpose of the present study is to generate up-to-date information with the following objectives: To determine the prevalence of clinical and subclinical mastitis To identify the major mastitis causative agents and associated risk factors associated with the prevalence of mastitis 4 2. LITERATURE REVIEW 2.1. Definition of Mastitis Mastitis is inflammation of the parenchyma of the mammary gland, which can result from cow exposure to a variety of infectious agents. It is characterized by an array of physical and chemical changes in milk, pathologic changes to the glandular mammary tissue, and at times systemic changes within the affected animal. Mammary infections are divided into two categories, sub-clinical and clinical. Cases of sub-clinical mastitis are described as the presence of an infection without visually evident sign of local inflammation or systemic involvement (Erskine, 2011). Detection of sub-clinical mastitis can be accomplished by determining the somatic cell count (SCC) in milk of individual cows using a California Mastitis Test (CMT) or automated cell count methods. Somatic cells include lymphocytes, macrophages, polymorphonuclear cells and some epithelial cells, all of which reflect the inflammatory response in the udder to an intramammary infection (IMI). Somatic cell counts to monitor for sub-clinical mastitis can be conducted at the quarter, cow, and herd levels (Schukken et al., 2003). 2.2. Clinical and Subclinical forms of Mastitis Mastitis in both clinical and subclinical forms is a frustrating, costly and extremely complex disease that results in a marked reduction in the quality and quantity of milk (Harmon, 1994). Annual losses in the dairy industry due to mastitis was approximately two billion dollars in USA and 526 million dollars in India, in which subclinical mastitis are responsible for approximately 70% of these dollars losses (Varshney and Naresh, 2004). The invisible changes in subclinical mastitis can be recognized indirectly by several diagnostic methods including the California Mastitis test (CMT), the Modified White Side Test (MWT), SCC, PH, chlorine and catalase tests. These tests are preferred to be screening tests for subclinical mastitis as they can be used easily, yielding rapid as well as satisfying results (Lesile et al., 2002). 5 In clinical mastitis (CM), there are visible changes to the normal appearance of milk, which could include a colour change, a consistency change, or the presence of flakes, clots, and/or blood. Physical changes to the udder may be present in CM cases ranging from warmth, diffuse swelling, and pain to gangrene in severe cases. Chronic mastitis can result in local fibrosis and atrophy of mammary tissue. When only local signs are evident a case of mastitis is considered mild or moderate. A case of mastitis is considered severe when systemic signs of an inflammatory response are apparent including fever, anorexia, and shock (Erskine, 2011). 2.3. Prevalence of Bovine Mastitis The prevalence of mastitis pathogens varies from herd to herd. However, bacterial pathogens appear to be the most prominent contributor to worldwide mastitis (Wilson et al., 1997). The prevalence of subclinical mastitis in dairy herds is often surprising to producers. Moreover, subclinically infected udder quarters can develop clinical mastitis and the rate of new infections can be high. Cows with subclinical mastitis are those with no visible changes in the appearance of the milk and/or the udder, but milk production decreases by 10 to 20% with undesirable effect on its constituents and nutritional value rendering it of low quality and unfit for processing (Holdway, 1992). Although there are no visible or palpable external changes, the infection is present and inflammation is occurred in the udder (Blowey and Edmondson, 1995). Clinical mastitis is less likely in younger animals. Reduction in clinical mastitis has been a major success over the past 35 years, in countries with a developed dairy industry (Poelarends et al., 2001). Most mastitis occurs as a low grade infection, a subclinical state, which affects 10-15% of the cows, increasing milk leucocytes content, reducing milk production and increasing milk bacterial content. These all contribute to reduced milk value as a food and in monetary terms (Barbano, 2004). The prevalence of such infections is a significant risk to uninfected animals in the herd as many mechanisms exist to expose the animals to new infection. Most commonly these include the common lying areas in housing or at pasture, the milking machine and successive contact of different cows or teats by the milker preparing the teats for milking. The prevalence of Coagulase-negative 6 staphylococci (CNS) mastitis is higher in primiparous cows than in older cows (Tenhagen et al., 2006). Contagious mastitis is primarily transmitted at milking time and the milking process affects the patency of the teat orifice which can increase the risk of development of environmental mastitis. Mammary quarter infection prevalence ranges between 28.9-74.6% prepartum and 12.3-45.5% at parturition (Fox, 2009). Coagulase-negative staphylococci (CNS) are the most prevalent cause of subclinical intramammary infections in heifers. Coagulasepositive staphylococci (CPS) in some studies are the second most prevalent pathogens, while in other studies the environmental mastitis pathogens are more prevalent. The risk factors or subclinical mastitis appear to be season, herd location and trimester of pregnancy (Fox, 2009). 2.4. Prevalence of Bovine Mastitis in Ethiopia In Ethiopia, the available information indicated that bovine mastitis is one of the most frequently encountered diseases of dairy cows. According to Lemma et al. (2001) of the major diseases of crossbred cows in Addis Ababa milk shed, clinical mastitis was the second most frequent disease next to reproductive diseases, in which 171 cows out of 556 were found to be affected. Generally, the prevalence of clinical and subclinical mastitis in different parts of Ethiopia range from 1.2 to 21.5% and 19 to 46.6%, respectively (Kerro and Tareke, 2003). These limited studies showed that bovine mastitis is among the problems hindering dairy productivity in Ethiopia and this requires the development of methodologies of control program under the prevailing husbandry system. However, according to Hussein et al. (1997) so far efforts have been concentrated only on the treatment of clinical cases. On the other hand, losses from mastitis have been attributed mainly to decreased milk production from subclinical mastitis (DeGraves and Fetrow, 1993). Kassa et al. (1999) carried out a survey of mastitis in dairy herds of the Ethiopian central high lands. Out of 10, 908 quarters examined from 2,681 cows, they found prevalence of clinical mastitis, non-functional or blocked quarters and subclinical mastitis to be 1.2%, 7 3.8% and 38.9% on cow basis, respectively. According to Hussein et al. (1997) the prevalence of clinical and subclinical mastitis are found to be 5.3% and 19% on cow basis and 1.9% and 7.4 % on quarter basis, respectively, in the central regions of Ethiopia. In a study conducted at Repi and Debre-Zeit dairy farms, out of 186 lactating cows, forty (21.5%) were clinically affected and 71 (38%) subclinically infected (Workineh et al., 2002). The overall prevalence in this study was 59.7%. Bishi (1998) reported mastitis prevalence rates of 34.3% and 5.3% at cow level in Addis Ababa region, for subclinical and clinical mastitis, respectively. Generally, the prevalence of clinical and sub clinical mastitis in different parts of Ethiopia range from 1.2 to 21.5% and 19 to 46.6%, respectively (Workineh et al., 2002). Clinical (4.9%) and sub clinical (45.5%) was reported in Bahir Dar, Ethiopia (Mekuria, 1986). In the same study area after ten years, a prevalence of 40% sub clinical mastitis was reported (Shirmeka, 1996). Even though the disease has been known locally in Ethiopia, it has not been studied systematically, making information available on the prevalence of disease and associated with economic loss inadequate. 2.5. Pathogenesis of Bovine Mastitis Mastitis is a complex and multi factorial disease, the occurrence of which depends on variables related to the animal, environment and pathogen. Among the pathogens, bacterial agent are the most common one, the greatest share of which resides widely distributed in the environment of dairy cows, hence a common threat to the mammary. There is evidence that pathogens use various mechanisms to impinge upon cell death pathways. A number of pathogens are armed with an array of virulence determinants, which interact with key components of a host cell’s death pathways or interfere with regulation of transcription factors monitoring cell survival. These virulence factors induce cell death by a variety of mechanisms, which include, pore-forming toxins, which interact with the host cell membrane and permit the leakage of cellular components, toxins that express their enzymatic activity in the host cytosol, effect or proteins delivered directly into host cells by a highly specialized type-III secretary system, super antigens that target immune cells and other modulators of host cell death (Weinrauch and Zychlinsky, 1999). 8 2.6. Aetiology of Bovine Mastitis The primary cause of mastitis is a wide spectrum of bacterial strains. However, incidences of viral, algal and fungal-related mastitis were also reported (Pyorala, 2003). Over 135 different microorganisms (bacterial, algal or fungal) have been isolated from bovine IMI, but the majority of infections are caused by staphylococci, streptococci, and gram-negative bacteria (Watts, 1988). Microorganisms that cause mastitis are generally classified as either contagious or environmental based upon their primary reservoir and mode of transmission. Staphylococcus aureus and Streptococcus agalactiae are contagious pathogens and are commonly transmitted among cows by contact with infected milk. These pathogens are of particular importance because they cause mainly subclinical forms of IMI that are often difficult to detect. Primary environmental pathogens include different types of bacteria: species of streptococci other than Streptococcus agalactiae (Streptococcus species), coliform species (Escherichia coli, Klebsiella species, Enterobacter species) and pseudomonas species. 2.6.1. Staphylococcus Species Staphylococcus aureus is one of the most prevalent contagious mastitis pathogen that colonizes the teats during damage to the skin surface. It produces many enzymes and toxins and penetrates deep into the mammary tissue and resists phagocytosis. Some of the Staphylococcus aureus strains have antibiotic resistance and can cause the problem with the treatment (Petersson-Wolfe et al., 2010). The result of Staphylococcus aureus infections is a decrease in milk yield and increase somatic cell count. Transmission of Staphylococcus aureus infections occurs mainly through contaminated milking machines, udder wash equipment, and the hands of milking machine operators. It can survive outside of the cow for a shorter period of time. Infections caused by Staphylococcus aureus are mostly sub-clinical with periodic flare-up of clinical symptoms. Chronic infection of heifers can serve as a source of new infection in the herd. The frequency of the Staphylococcus aureus infections is related to age of the cow. Culling, grouping and dry cow therapy helps fight Staphylococcus aureus infections in a herd (Syensk Mjölk, 2003). 9 The chronic and subclinical forms predominate and on a herd basis, are the most important. Staphylococcus aureus bacteria produce toxins that destroy cell membranes and can directly damage milk-producing tissue. White blood cells (leukocytes) are attracted to the area of inflammation, where they attempt to fight the infection. Initially, the bacteria damage the tissues lining the teats and gland cisterns within the quarter, which eventually leads to formation of scar tissue. The bacteria then move up into the duct system and establish deep-seated pockets of infection in the milk secreting cells (alveoli). This is followed by the formation of abscesses that wall-off the bacteria to prevent spread but allow the bacteria to avoid detection by the immune system. The abscesses prevent antibiotics from reaching the bacteria and are the primary reason why the response to treatment is poor (Petersson-Wolfe et al., 2010). 2.6.2. Streptococcus Species Streptococcus dysgalactiae is also one of the contagious pathogens. It can spread throughout a herd from a single infected animal. The infected udder is the most important reservoir for this bacterium. They are transmitted to uninfected quarter mainly at milking time. Contaminated milking machines, udder wash cloths, and the hands of machine operator also transmits these bacteria (NMC, 1996). Breakdowns of contagious mastitis are usually due to the introduction of infected animals to the herd, or the employment of men who carry infection with them. The infections are mainly sub-clinical (NMC, 1996) and there are most frequent in the younger age groups. Streptococcus dysgalactiae is generally characterized as an environmental pathogen, but also may have characteristics of a contagious organism and appears to spread from cow to cow. This pathogen is generally responsive to teat dipping and dry cow therapy, but new infections can occur in a herd when no other udder infections by this organism are present (Harmon, 1996). Teat damage caused by contamination with sand or grit, or poorly operating milking machines is prime reasons for Streptococcus dysgalactiae mastitis. Streptococcus uberis is an important environmental pathogen particularly because it is ubiquitous in the dairy environment. Identification of Streptococcus uberis is currently based on observation of the cultural and morphological characteristics, biochemical tests determination, and enzyme activity (Khan et al., 2003). On the other hand, several 10 commercial microbial identification systems have also been used to differentiate Streptococcus uberis from other species of streptococci and enterococci isolated from bovine mastitis, and more recently, molecular tools such as PCR-based protocols have been proposed to provide an accurate identification of Streptococcus uberis isolates (Reinoso et al., 2011). 2.6.3. Coliforms Bacteria Among coliforms bacteria, E. coli is the most frequently isolated from bovine milk in cows belonging to dairy farms with intensive systems of milk production. E. coli is a member of Enterobacteriaceae family. Its primary importance is its ability for lactose fermentation. Over 700 antigenic types or serotypes of E. coli have been recognised based on O, H, and K antigens. Two classes of coliforms have to be distinguished: strains that are harmless (non-pathogenic strains) and strains that cause a wide variety of typical clinical infections (pathogenic strains). Millions of non-pathogenic E. coli bacteria are living in the humans and animals normal intestinal microflora. E. coli is ubiquitous in the cow’s environment because is massively excreted with the faeces. E. coli causes infection and inflammation of the mammary gland in dairy cows mainly around parturition and during early lactation striking local and sometimes severe systemic clinical symptoms. Clinical signs vary from very severe, even fatal forms, or mild mastitis, where cows have only local signs in the udder. During mastitis, the host defense status is a factor determining the outcome of the disease. Particularly, during E. coli mastitis, the neutrophil is a key factor in the cows’ defence against IMI. However, virulence of the involved bacterial strain may also play a role. Most of the pathogenic E. coli strains posses several kinds of pathogenic mechanisms and virulence factors. A non-specific but potent factor that is important during the pathogenesis of E. coli is the endotoxin or lipopolysaccharide, which is responsible for most pathophysiological effects (Tormo et al., 2005). 2.7. Economic Importance of Mastitis Mastitis is one of the most important diseases that cause economic loss in dairy industry worldwide (Bachaya et al., 2011). The mean annual incidence is 41.6 cases per 100 cows and affected cows suffered a mean of 1.5 cases and 16.4% of quarters suffered at least one 11 repeat case (Bradley and Green, 2001). Bennett et al. (1999) estimated the total economic impact of clinical mastitis to be £119 per cow-case in Great Britain. More than $130 million is lost by the Australian dairy industry ($A200/cow/year) every year due to poor udder health resulting in reduced milk production that is mainly associated with mastitis. A herd without an effective mastitis control programme may witness morbidity as high as 40% with infection, on an average of two quarters of the mammary gland. Of the various clinical manifestations, subclinical mastitis is economically the most important due to its long term effects on milk yields (Zafalon et al., 2007). Huge economic losses are also incurred due to unmarketable milk or milk-products contaminated with antibiotic residues originating from treatment in the developing nations as well as from the use of antibiotics as growth promoters particularly in dairy feedlots in the developed world. The prolonged use of antibiotics in the treatment of mastitis has led to the additional problem of emergence antibiotic resistant strains, hence the constant concern about the resistant strains entering the food chain (Virdis et al., 2010). Clinical and subclinical mastitis both severely affect milk yield and milk quality. Milk production of cows bearing mastitis is significantly lower than that of healthy cows. Furthermore, the nutritional quality is lower and the somatic cells count (SCC) is substantially higher (Schukken et al., 2009). The SCC of milk is regarded as the industry’s standard indicator for the general quality of produced milk. It is determined as the total count of white blood cells per millilitre of milk. Normal milk is believed to have SCC of approximately 200,000 cells/ml or less. An infection in the mammary gland of the udder causes a large influx of somatic cells, predominantly polymorphonuclear neutrophils, which can increase the SCC of milk up to 1 million cells/ml (Madouasse et al., 2010). Subclinical mastitis commonly contributes a more substantial part to high SCC’s within a herd and is usually a reliable indication of the development of clinical mastitis (Van den Borne et al., 2011). Mastitis causes a loss of over 1.7 billion dollars a year in the USA alone (Sahoo et al., 2012). It is characterised by an increase in somatic cells, especially leukocytes, in the milk and by pathological changes in the mammary tissue (Ranjan et al., 2010). 12 2.8. Diagnosis of Mastitis Clinical mastitis is recognized by the appearance of abnormal milk, gland swelling and /or illness. Subclinical mastitis is characterized by normal milk and hence requires indirect tests to detect. 2.8.1. California Mastitis Test (CMT) The California Mastitis Test (CMT) remains the only reliable screening test for subclinical mastitis that can be easily used at the cow side. The CMT was developed to test milk from individual quarters but also been used on composite and bulk milk samples. The CMT involves mixing and swirling equal parts of bromocresol violet reagent and milk in a plastic paddle with a compartment for each quarter (Quinn et al., 1999). The test results are interpreted subjectively as either a negative, trace, 1+, 2+ or 3+ inflammatory response based on the viscosity of the gel formed by mixing the reagent with milk. Fresh unrefrigerated milk can be tested using the CMT for up to 12 hours. Reliable readings can be obtained from refrigerated milk for up to 36 hours. If stored milk is used, the milk must be thoroughly mixed prior to testing because somatic cells tend to segregate with milk fat. The CMT reaction must be scored within 15 seconds of mixing because weak reactions will disappear after that time. The degree of reaction between the detergent and the DNA of nuclei is a measure of the numbers of somatic cells in milk. The threshold for CMT scores depend on the objective of the study. If it is used to minimize the rate of false negatives, the test should be read as negative versus positive with trace scores regarded as positive. If the CMT is to be used in culling decisions, a threshold with a lower rate of false positives may be desirable (Larsen, 2000). 2.8.2. Culture The microbiological examination of both individual cow and bulk tank culture are elements of mastitis control. Most mastitis control programs include the use of individual cow cultures to determine which mastitis pathogens are present on the farm. Culturing can be used in a targeted fashion for specific control programs such as segregation plans for 13 contagious mastitis or for surveillance to detect the presence of new or emerging pathogen. Culturing is also used to evaluate treatment efficacy and to establish susceptibility patterns to aid in the development of rational treatment strategies (Larsen, 2000). 2.9. Classification of Mastitis Pathogens Classically, mastitis pathogens have been classified as contagious pathogens and environmental pathogens (Burvenich et al., 2003). The contagious pathogens are adopted to survive within the host particularly within the mammary gland. They are capable of causing subclinical infections, which are typically manifest as an elevation in the somatic cell count (leukocytes, predominantly neutrophils and epithelial cells) of milk from the affected quarter; they are typically spread from cow to cow at or around the time of milking (Radostits et al., 1994). In contrast, the environmental pathogens are best described as opportunistic invaders of the mammary gland, not adapted to survival within the host; typically they invade, multiply, engender a host immune response and are rapidly eliminated. The major contagious pathogens comprise Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae; the major environmental pathogens comprise the Enterobacteriacae (particularly E. coli) and Streptococcus uberis (Dogan et al., 2006). 2.9.1. Contagious Mastitis (cow-associated) Contagious mastitis is defined as IMI transmitted directly from cow to cow (Erskine, 2001). Incidence of contagious mastitis depends on the dose and type of microbes to which a cow is exposed as well as physical barriers and the innate and acquired defense mechanisms. Although many different types of bacteria may cause mastitis, those of greatest interest are the pathogens commonly found on dairy farms and those for which the prevalence of IMI is high. The main contagious organisms are Streptococcus agalactaiae, Staphylococcus aureus, Corynebacterium bovis and Mycoplasma species. Staphylococcus aureus is generally considered to be the most prevalent cause of IMI. It has been estimated that, depending on the breed and geographical location of the herd, between 7-40% of all cows are infected with Staphylococcus aureus at any given time. 14 2.9.2. Environmental Pathogens (Mastitis) Mastitis caused by the environmental pathogens is traditionally considered to occur sporadically without long lasting effects within the host. The contagious pathogens, on the contrary, are able to persist within the host for prolonged periods of time causing continuous occurrence of mastitis and spreading between quarters and cows (Passey et al., 2008). More recently, DNA fingerprinting data suggested that some E. coli strains have adapted to survive within the udder and cause recurrent mastitis. There is, however, no apparent single factor which is responsible for the persistence of E. coli infections in the udder (Almeida et al., 2011). The main environmental organisms are gram-negative bacteria, which include the coliforms and environmental streptococci. The gram-negative bacteria include Escherichia coli, Klebsiella species, Enterobacter species, Citrobacter species, Seratia, Pseudomonas species, Proteus and Actinomyces pyogenes. The environmental streptococci include Streptococcus uberis, Streptococcus dysgaladiae, and Streptococcus equinus. The majority of infections caused by coliform pathogens result in acute mastitis when compared to infections caused by contagious pathogens and environmental streptococci, but the infections are generally of shorter duration (less than seven days). The exception to this is Actinomyces pyogenes, which generates large and persistent production losses (Smith and Hogan, 1993). 2.10. Mastitis and Associated Risk Factors Many risk factors of mastitis related to environment, the microflora and the animal have been investigated in spite of wide control efforts with little reasonable results. However, the bovine mammary gland infection (mastitis) is a frequent and important problem among livestock herds in most of the countries. The occurrence of mastitis is influenced by managemental and different environmental factors like housing of animals, type of milking and milking utensils, and type of feeding, hygienic quality of water, health of lactating animals and execution of various preventive procedures. Incidence of mastitis changes with season, its rate has been reported to be highest during the winter (Nyman et al., 2007). 15 Breen et al. (2009) investigated risk factors which were related with milk leukocyte count in different quarters of cattle. The following individual risk factors, teat end callosity, hyperkeratosis of quarters, body state, udder and leg hygiene and capacity of milk quality and production were assessed. Significant association with an increased risk of milk leukocyte count was found with increasing lactation number and lactation stage than contamination of skin of quarters and udder. Results suggested that energy status, individual quarter and different factors at animal level play important role to measure intramammary infections by measuring milk somatic cell in subsequent lactations. The large number of predisposing factors that contribute to the emergence of mastitis in dairy cattle may be physiological, genetic, pathological or environmental (Sordillo, 2005) described below: 2.10.1. Age and parity of the cow Increasing parity increased the risk of clinical mastitis in cows (Kavitha et al., 2009), although the reason for this association is not clear. Sharma et al. (2007) conducted a study on 500 lactating cows of different age, parities and stage of lactation belonging to different organized or un-organized dairy farms. Older cows (>10 years) are at more risk (44.6%), particularly for subclinical mastitis (38.6%), than younger cows (23.6%) in which clinical mastitis was predominant. Cows with many calves (>7) have about 13-times greater risk (62.9%) of developing an udder infection than those with fewer (3) calves (11.3%). It has been demonstrated that occurrence of mastitis in infected quarters increases with age in cows (Harmon, 1994), being the highest at 7 years of age. This may be due to an increased cellular response to intramammary infection or due to permanent udder tissue damage resulting from the primary infection. Efficient innate host defence mechanisms of the younger animals are one possibility that makes them less susceptible to infection (Dulin et al., 1988). However, at least one study conducted using 4133 cattle including both cross-bred and non-descriptive breeds revealed the highest risk of occurrence of mastitis to be between the ages of 4-6 years, followed by the age group between 2-4 years, with the least occurrence noted between 6-8 years of age (Mahajan et al., 2011). 16 2.10.2. Inherited features of the cow Various genetic traits may also have a considerable impact upon the susceptibility of the animal to mastitis. These genetic traits include the natural resistance, teat shape and conformation, positioning of udders, relative distance between teats, milk yield and fat content of milk. High milk yielders with higher than average fat content are reported to be more susceptible to mastitis (Grohn et al., 1990). The conformation of the udder and shape of the teat are inherited characteristics that may also affect susceptibility to mastitis. Cows with elongated teats are more vulnerable to mastitis infection than cows with inverted teat ends (Seykora and Mc Daniel, 1985). Broad udders, lower hind-quarters and teats placed widely help the infectious agent and should be selected against it (Thomas et al., 1984). 2.10.3. Breed and milk yield Risk of mastitis varies from breed to breed. High yielding cows are generally considered to be more susceptible to intramammary infection e.g. Holstein Frisian (HF), Jersey or HF and Jersey cross bred dairy cows are more susceptible to mastitis than Desi (Zebu) breeds of cows (Compton et al., 2007). It might be due to more resistance to disease and they are low milk producer than cross bred cows. Increased risk of clinical mastitis in Friesian compared with Jersey and Ayrshire heifers (Compton et al., 2007). 2.10.4. Stage of lactation The incidence of mastitis is reported to be higher immediately after parturition, early lactation and during the dry period, especially the first 2-3 weeks (Fadlelmula et al., 2009) due probably to increased oxidative stress and reduced antioxidant defence mechanisms during early lactation (Sharma et al., 2011). An increase in somatic cell numbers or count (SCC) which is mainly neutrophils is observed immediately after parturition, which remains high for a few weeks irrespective of the presence or absence of infection. This increased SCC is the cow’s natural first line of defence to prepare for the onset of the new lactation. Relatively recent studies have revealed that cows in late lactation always show a higher than average SCC than that seen at other stages of the lactation period (Peeler et al., 2000), potentially representing increased subclinical infection, leading to a fall in milk production. 17 2.10.5. Mammary regression There are significant functional changes in the udder during the early and late lactation and dry period, which affect the cow’s susceptibility to infections. Lactating cows under stress show premature mammary regression. Such a condition compromises udder’s natural defence mechanisms (Giesecke et al., 1994; Capuco et al., 2003) leading to invasion of the teat canals by potential pathogens. The same condition prevails during the healing process of lesions because the resistance to causal agents remains less effective. 2.10.6. Milking Machine Extraneous factors such as the milking habits of farmers and faulty milking machines favour the pathogens to gain access to mammary gland and proliferate, potentially leading to mastitis (Mein et al., 2004). In farms where machines are employed for milking, it is important to maintain physiologically optimal pressure [50 kPa for most machines], because pressures in excess of this may lead to injury in the teat. Fluctuations in the pressure due to inadequate vacuum reserve must be avoided to prevent occurrence of mastitis. Proper installation as well as the correct maintenance of milking machines is important to avoid an inadequate vacuum level, teat and tissue damage and incomplete milking. The vacuum level created by the vacuum pump is another important factor for complete and high quality milking. Experiments have shown that a teat subjected to a vacuum level of 10.5-12.5 inches at the time of peak milk flow results in rapid, complete and high quality milk yield, and the teat suffers minimum physical pressure (Jones, 2009). Two-chambered teat cups are found to be better than single chambered teat cups in regard to achieving complete milking as well as fewer incidences of teat injuries (Mein and Schuring, 2003). Sanitary milking habits are important to avoid the spreading of bacteria or their proliferation. Faulty milking equipment due to poor installation or maintenance can cause tissue trauma, teat damage, poor milking out, erratic vacuum levels and can also transmit infectious agents at milking time. 18 2.10.7. Nutrition The quality and plan of nutrition appears to be an important factor that influences clinical manifestation of mastitis in heifers and cows (Heinrichs et al., 2009) although no relationship between the incidence of mastitis and either high energy or high protein feed in cows has been reported (Rodenburg, 2012). Vitamin E is one of the important supplements in dairy feed to boost the immune response of cows (Spears and Weiss, 2008) as it has been reported to enhance the neutrophil function as well as the phagocytic properties of neutrophils after parturition. Vitamin E is often combined with selenium, which acts as an anti-oxidant by preventing oxidative stress. A number of investigations have demonstrated that neutrophils of selenium fed cows are more effective at killing mastitis causing microorganisms than those not supplemented with selenium (Underwood and Suttle, 1999). There are several factors both infectious and non-infectious, that can cause bovine mastitis; and there is an increase in the evidence that nutritional factors are associated with mastitis in cows and heifers (Heinrichs et al., 2009). Despite all the measures taken by dairy producers to prevent intramammary infections in their herds, it is not always clearly understood that there is a strong relationship between nutrition and susceptibility to mastitis. When the consumption of minerals (selenium) and vitamins (vitamin E) by dairy cows is not optimal, it can have negative effects on immunity. Animals become more susceptible to diseases such as mastitis, because a depressed immune system is not able to fight off bacteria that invade the udder. 2.10.8. Weather and climate The incidence of mastitis is greatly influenced by the weather conditions and prevailing climatic conditions. Heat, humidity, cold and draught are the important predisposing factors (Reneau, 2012). A higher incidence of mastitis has been reported to occur particularly during summer rainy months (Sentitula et al., 2012). As heat and humidity increases, so does the bacterial multiplication as well as the load of pathogens in the environment. Conversely, an alternative study has reported a higher incidence of coliform 19 mastitis during the cold months of the year when the temperature was reported to be less than 21°C (Ranjan et al., 2011). Housing is also a factor that aggravates the incidence of mastitis, first due to excess numbers of animals in a limited space; and also because of the use of bedding material that easily allows for bacterial survival and growth, which over exposes animals and challenges their immune defense mechanisms (Hogan and Smith, 2003). Prevention of intramammary infections leads to better milk quality, which in turn is beneficial for the dairy industry because high quality milk means extended shelf life, increased cheese yield, and increased consumption of dairy products. 2.10.9. Milking hygiene and procedures Moisture, mud, and manure present in the environment of the cow are the primary sources of exposure for environmental mastitis pathogens, and hygiene scores of cows provide visible evidence of exposure to these potential sources. Milking hygiene reduce the pathogenic organisms from inhabiting the immediate environment or skin of the animals and minimizing their spread during milking process. The practice of regular teat dipping is not much more common at house hold level but washing the udder with clean water and drying with individual cow towel, using strip cups and disinfecting the teat with germicide are some of the milking procedures used to minimize the occurrence of mastitis in the dairy farms. In addition to these, milking of heifer first, healthy cows second and mastitic cows last are also good practices to minimize the intramammary infection. Therefore, prevalence of mastitis in cows is more at unorganized dairy farms as compared to organized dairy farms. Udder hygiene significantly associated with the risk of environmental pathogen intramammary infection in cows and milking procedures (Compton et al., 2007). 20 3. MATERIALS AND METHODS 3.1. Study Area The study was conducted in and around Dire Dawa (Fig 1) from August, 2014 to December, 2014. (DDPAIA, 2005) reported that Dire Dawa is located in eastern part of Ethiopia between 9 27’ E’ and 49’N latitude and between 41 38’ and 19’ E longitude occupying about 133,000 hectare of land and the distance from Addis Ababa is 515 kilometre. The climatic condition of Dire Dawa Administrative Region seems to be greatly influenced by its topography, which lies between 950–1250 metre above sea level, and which is characterized by warm and dry climate with a relatively low level of precipitation. The region has two rainy seasons; that is, a small rain occurs from March to April, and a main shower of rain that extends from August to September. The aggregate average annual rainfall that the region gets from these two seasons is about 604 mm. On the other hand, the region is believed to have an abundant underground water resource. The monthly average maximum and minimum temperatures of the study area are 32.40C and 19.10C, respectively and the mean annual relative humidity is 48.2 %. 3.2. Study population There are 31 small (3-29 cows) and medium (30-52 cows) private dairy farms in and around Dire Dawa. The study population were, therefore, selected dairy farms in the study area. Most of the dairy farms are established around the home of owners due to lack of land and appropriate allocation of land by the city administration. For this reason, disease transmission may be very high and mastitis is one of the most prevalent economically important diseases (Yonus, 2011). In the current study, the study animals were lactating dairy cows that are found in selected dairy farms in and around Dire Dawa. About 403 lactating cows (331 high grades Holstein Friesian and 72 local (zebu)) breed were investigated. 21 Figure 1. Map of the study area 3.3. Study Design, Sample Size and Sampling Method A cross sectional study was conducted. Dairy farms were purposively selected based on accessibility. Simple random sampling technique was followed to select the dairy farms but the lactating cows in the selected dairy farms were sampled using cluster sampling method. The desired sample size was calculated according to the formula given by Thrusfield (2005). n= 1.962Pexp (1-Pexp) d2 Where: 22 n = required sample size Pexp = expected prevalence d = desired absolute precision Previously subclinical mastitis prevalence was reported as 36% in Holstein and 6% in local breeds (Adugna, 2008). As there was variation of mastitis prevalence among the breeds and thus breed specific sample size was calculated as follows: High grade Holstein breed: n= 1.962*0.36 (1-0.36) = 354 (above 80% blood level) 0.052 Local breed: n= 1.962*0.06 (1-0.06) = 87 (100% local) 0.052 3.4. Data Collection 3.4.1. Questionnaire Questionnaire was compiled to evaluate the effect of potential risk factors on the occurrence of mastitis. Farm owners having dairy cows of varied breed and cow number were interviewed through a one stop visit. Data on each sample cow was collected in a properly designed format. Risk factors considered were breed, age, parity, and stage of lactation, previous mastitis history, udder washing, towel usage, udder drying, barn washing, blind teat, good milking procedure, hand milking, housing and lesion on the udder skin or teat. The factors were categorized into host factors, management and environmental factors. The host factors to be considered were age, parity, inherited features of the cow, breed, milk yield, stage of lactation and mammary regression. The management factors were: nutrition, milking hygiene, milking machine. Housing and climate were considered as environmental factors. 3.4.2. Clinical examination of udder and milk Clinical findings like abnormalities of secretions, abnormalities of size, consistency and temperature of mammary gland were examined by visual inspection and palpation. Pain 23 reaction upon palpation, changes in the milk (blood tinged milk, watery secretions, clots, pus), and change in consistency of udder were considered as indications of the presence of clinical mastitis. The milk was examined for its colour, odour, consistency and other abnormalities. 3.4.3. California Mastitis Test (CMT) The reaction involved in the CMT is the disintegration of leukocytes when milk is mixed with the reagent (Babaei et al., 2007). According to the visible reactions, the results were classified in four scores: 0 = negative or trace 1 = weak positive, 2 = distinct positive and 3 = strong positive. Mammary glands without clinical abnormalities and with apparently normal milk that was bacteriologically negative and negative on the California mastitis test were considered to be normal milk, while those that were bacteriologically positive and with the CMT positive were considered to have subclinical mastitis. The California Mastitis Test was carried out as described by Quinn et al. (2004). A squirt of milk, about 2 ml from each half was placed in each of 2 shallow cups in the CMT paddle. An equal amount of the commercial CMT reagent was added to each cup. A gentle circular motion was applied to the mixtures in a horizontal plane for 15 seconds. The result of the test was indicated on the basis of gel formation. The interpretation (grades) of the CMT was evocated and the results graded as 0 for negative and trace 1, 2 and 3, for positive (Quinn et al., 2002). 3.4.4. Milk Sample Collection, Handling and Transportation Aseptic procedures for collecting CMT positive quarter milk samples as described by Hogan et al. (1999) and Quinn et al. (2004) were followed. About 27 pooled milk samples were collected from 15 dairy farms and five kebeles in and around Dire Dawa. This means the samples were collected from each lactating cow and were mixed in one container for microbiological culture. Each milk sample was collected under aseptic conditions in a sterile screw caped bottle containing anticoagulant which numbered to identify the particular dairy farm. All milk samples were sent directly to the laboratory, within an hour for routine cultural techniques. The time for milk sample collection was before milking. Udders and especially teats were cleaned and dried before sample 24 collection. Each teat end was scrubbed vigorously with cotton alcohol pads. A separate pledged of cotton was used for each teat. The first stream of milk was discarded and 10 ml of milk from each cattle in a given farm was pooled into clean container. After collection, the sample was placed in an icebox and transported to Dire Dawa regional laboratory for cultural examination. 3.4.5. Microbiological Culture (Etiological Factors) To prepare media for bacterial culture, the manufacturer´ s instructions were followed. All glass wares used for the preparation of media were first sterilized using appropriate equipment like autoclave, hot air oven, the appropriate amount of dehydrated media were weighed out of using sensitive balance and the required amount of distilled water was added to the powder media. Dehydrated media containing agar were dissolved in heating mantle until it boils and frothy appearance was settled (removed), then the media were sterilized by autoclave at 121C for 15 min holding time, and cooled in water bath at 50C before poured into the Petri dishes. Some media like blood agar requires addition of blood after it is cooled to 50C since RBC do not tolerate higher temperature (Quinn et al., 2002). The common media used during the study were blood agar and Mac Conkey agar. Milk samples were cultured onto 10% sheep blood agar and Mac Conkey agar plates according to Coulon et al. (2002). Culturing of milk sample collected from dairy farms, in search for mastitis producing organisms in standard of examination for mastitis (Radostits et al., 2007). The inoculated plates were incubated aerobically for 24-48 h at 37°C. The plate was examined for growth, morphology activity. Suspected colonies were sub-cultured on a new plate for further investigation. For primary identification of bacteria, once a pure culture is obtained, a gram-stained smear from the culture was used to categorize the bacteria based on gram-reaction and cellular morphology. Staphylococcus species, streptococcus species and E. coli were the targets, because, these pathogens are the major microorganisms which cause bovine mastitis (Potter, 2008). Suspected colonies were identified morphologically and microscopically. Cultures with fine bacterial growth were considered as positive and cultures with no visible growth were taken as negative, but polluted cultures with disturbed media were considered as contaminated (Shakoor, 2005). 25 3.5. Data Analysis The collected data during the study periods were entered into MS- Excel spread sheet and descriptive statistics was used to illustrate the various variables in the production system including husbandry and management variables. For data analysis, SPSS version 20 was used. The chi-square (χ2) test was used to assess the association among the hypothesised risk factors namely the breed, age, udder washing, udder drying, farm hygiene management system and stage of lactation with the occurrence of the disease. Finally, logistic regression analysis was used to determine the predicting factor of mastitis. In all the analysis, confidence level was held at 95% and statistical analysis was consider significant at p<0.05. 26 4. RESULTS 4.1. Description of the Study Population and Dairy Farms 4.1.1. Study Animals The average age of the lactating dairy cows was 8.5 years (ranged between 2 and 15 years) and the milk yield ranged between one and 35 litres per day (average 18). The average parity of the lactating cows was 6.5 (ranged between one and 12). 4.1.2. Farming System The dairy farms practised both extensive and intensive dairy production systems. Number of animals per farm ranges from one to fifty one (1-51) lactating cows in the farms surveyed. The extensive dairy production system ranged from one to three lactating cows but the intensive dairy production system ranged from two to fifty one lactating cows. The feeding status of the dairy farms was quite different between extensive and intensive dairy production systems. In extensive dairy production system no concentrate feeding; it was only roughage feeding but in intensive dairy production system, there was relatively concentrate and roughage feeding practice. The milking practice of all dairy farms was hand milking (manual). The establishment of the dairy farms (farm age) ranges from five to thirty six (5-36) years. 4.2. Prevalence of Mastitis 4.2.1. Animal level The animal level prevalence of mastitis is presented in Table1. The dairy farms were classified based on numbers of lactating cows. During the study period, 403 lactating cows were clinically examined from 20 smallholder and medium dairy farms in and around Dire Dawa. The prevalence of bovine mastitis in the study area was 50/403 (12.4%) and 320/403 (79.4%) for clinical and subclinical mastitis, respectively. Among 50 clinical mastitic cases 2 of them were negative on CMT. The prevalence of subclinical mastitis in 27 different breeds was 81.27% in Holstein, 70.8% in Zebu and the overall prevalence was 79.4%. Table 1. Prevalence of Subclinical Mastitis at animal level Animals Holstein Zebu Overall Number of cows examined 331 72 403 Mastitis +ve 269 51 320 Prevalence (%) 81.27 70.80 79.40 4.2.2. Quarter level All quarters of the lactating cows were examined, out of 1592 quarters examined 777 (48.80%) were positive for subclinical mastitis and 50 (3.14%) were detected with clinical mastitis. Among the quarters examined 28.27% were negative and 1.24% was blind. The strong positive and trace positive at quarter level were 3.27% and 16.52%, respectively (Table 2). Table 2. Clinical, blind and CMT scores of each quarters of lactating cow in sample farms CMT Scores RF RH LF LH Negative (0) 96 94 144 116 Trace (+) 72 72 67 52 Positive (2+) 204 203 167 203 Strong positive (3+) 16 11 11 14 Clinical 10 17 10 13 Blind 5 6 4 5 Total 403 403 403 403 RF= Right Front; RH= Right Hind; LF= Left Front; LH= Left Hind Prevalence% 450(28.27) 263(16.52) 777(48.80) 52(3.27) 50(3.14) 20(1.24) 1612 4.2.3. Types of Dairy Production Systems and Mastitis Prevalence Table 3. Prevalence of bovine mastitis related to production systems in and around Dire Dawa. Intensive Extensive Total No. of cows 331 72 403 Clinical 40 (12.08%) 10 (13.89%) 50 (12.4%) Subclinical 269 (81.4%) 51 (70.8%) 320 (79.4%) Total 93.35% 84.72% 91.8% 28 Table 4. Prevalence of Bovine Mastitis in small and medium farms Small farm Medium farm Total Examined cows 289 114 403 Clinical 32 (11.07%) 18 (15.79%) 50 (12.4%) Subclinical 224 (77.5%) 96 (84.21%) 320 (79.4%) Overall 88.57% 100.00% 91.8% The prevalence of bovine mastitis was 11.07% clinical, 77.5% subclinical in small farm and 15.79% clinical, 84.21% subclinical in medium farm. The overall bovine mastitis prevalence was 88.57% and 100.00% in small and medium farms, respectively. Prevalence 82 80 78 76 74 72 70 68 66 64 Prevalence Holstein Zebu Overall The prevalence of subclinical mastitis in the study area 4.3. Associated Risk Factors Many factors were considered as potential risks for the occurrence of bovine mastitis in the current study. The potential risk factors were classified as host factors (breed, age, milk yield, lactation stage, previous mastitis history, parity and teat lesion) and environmental or management factors (udder washing, towel usage, udder drying, barn washing and concrete floor). 29 4.3.1. Host Factors Age of animal was significantly associated (P=0.000) with prevalence of mastitis. The condition was four time less likely (OR: 0.24) to be detected in young animals (66.67%) compared to adult cows (89.08%). Breed was significantly associated with mastitis prevalence. The prevalence of mastitis was 81.2% in Holstein and 13.89% in zebu cattle. Holstein breeds had 1.78 [1.0, 3.1] higher odds to be positive for CMT compared to zebu breed. Significantly higher presence of mastitis was noted in high milk yielding cows (P= 0.001). Cows with previous clinical mastitis history were 3.6 time more likely to be positive on CMT (Table 5). Table 5. Association between the occurrence of subclinical mastitis and risk factors Host Factors Animal examined Mastitis Positive (%) Breed HF Zebu 331 72 174 229 P-value OR[95%CI] 0.047 1.78[1.0, 3.2] 30.38 0.000 0.24[0.14, 0.41] 13.99 0.001 0.42 0.51 2.27 0.32 12.23 0.000 0.344 0.557 269 (81.2) 51 (70.8) Age Young Adult Chisquare value 3.93 116(66.67) 204(89.08) Milk Yield (<10 lit) Low 138 100(72.46) (>10 lit) Medium 184 152(82.61) (>20 lit) High 81 75(92.59) No. parity 1-5 330 260(78.79) >=6 73 60(82.19) Lactation stage Early 164 128(78.05) Mid 129 108(83.72) Late 110 26(23.63) History of mastitis Yes 98 90(91.83) No 305 230(75.41) Teat lesion Present 25 21(84) Absent 378 299(79.10) OR= Odds ratio, CI=Confidence interval 3.66[1.7, 7.9] 30 The mastitis positive cows were 78.04% in early, 83.72% in mid and 76.36% (X2=2.27, P=0.32) in late lactation stages for subclinical mastitis. From the 320 subclinical mastitis positive cows 21(6.00%) cows had teat lesion but 299 (93.44%) were free from teat lesion. 4.3.2. Environmental Factors Presence or absence of good milking practices and environmental factors that were hypothesised to favour the existence of mastitis in a farm were cross checked but all environmental and husbandry factors considered in the study were not significantly associated with mastitis prevalence. The detail information on mastitis prevalence Vis –aVis the actors is provided in Table 6. Table 6. Association between subclinical mastitis and Environmental Factors Environmental Factors Animal examined Mastitis Positive (%) Udder Washing Yes No 331 72 Towel Usage Yes No 152 251 Chisquare value 3.94 269(81.27) 51(70.80) 2.1 P-value OR[95%CI] 0.047 1.78 [1.00, 3.18] 0.148 115(75.66) 205(81.67) Udder Drying 2.1 0.148 Yes No 152 251 115(75.66) 205(81.67) Yes No 265 138 214(80.75) 0.86 106(76.81) 0.353 1.49 0.222 Barn Cleaning Floor Type Concrete 316 Soil 87 255(80.69) 65(74.71) OR= Odds ratio, CI=Confidence interval Cows which were washed their udder had high mastitis prevalence than that of not washed, because, using of common source of washing water (bucket) and common towel usage. 31 4.3.3. Microbiological Culture (Aetiological Factors) All samples were positive for different bacterial species in microbiological culture. The bacteria isolated with high prevalence were staphylococcus species 18/27 (66.67%), streptococcus species 4/27 (14.81%), E. coli 3/27 (11.11%), and bacillus species 2 /27 (7.41%). Table 7. Frequency distribution of isolated bacteria from mastitic dairy farms Bacterial isolates Staphylococcus species Streptococcus species E. coli Bacillus species Total Frequency 18 04 03 02 27 Prevalence (%) 66.67 14.81 11.11 7.41 100.00 Among the samples examined, 18 samples were positive for Staphylococcus species. This took the highest percent of the mastitis causative agents. But Bacillus species were the least prevalent mastitis causative agents. 32 5. DISCUSSIONS This study showed overall prevalence of bovine mastitis in lactating dairy cows to be higher than that of the previous reports in and around Dire Dawa. Adugna (2008) reported the prevalence to be 36% in Holstein and 6% in local breeds in Dire Dawa. The high prevalence in the present study might be attributed to the poor management and increasing environmental temperature. In the present study, like some previous studies (Almaw et al., 2008; Getahun et al., 2008), the majority of the cases of mastitis were subclinical. Farmers have good knowledge about clinical mastitis due to the observed visible changes, thus they timely treat the disease, but subclinical mastitis is not easy to be identified by farmers and its silent effect is critical to dairy producers. The prevalence of clinical type of bovine mastitis is higher than the previous findings of Bedada and Hiko (2011) and is lower than Workineh et al. (2002), who reported the prevalence rate of 10.3 and 21.5%, respectively in different parts of Ethiopia. The clinical mastitis prevalence in high grade Holstein breed in this study is higher than that reported by Bishi (1998) who reported 5.3% prevalence in Addis Ababa. Mastitis is a complex disease and the difference in results could be due to difference in management system, topographic and climate condition between the farms. Subclinical mastitis was high in both breeds compared to clinical mastitis. The prevalence of subclinical mastitis of this finding is in agreement with 80.6% reported by Dabash et al. (2012), and it is somewhat comparable with the prevalence rate of 89.5% reported by Argaw and Tolosa (2008). However, the prevalence of sub clinical mastitis in the present study is far higher than previous reports of Moges et al. (2012 and Mekibib et al. (2010) who noted a prevalence rate of 30.6 and 25.22%, respectively in different areas of Ethiopia. The high prevalence rate is due to improper milking hygiene, lack of post milking teat dipping and poor housing facilities in the present dairy farms studied. The quarter level clinical mastitis prevalence (3.1%) recorded in the current study is lower than the finding of Haftu et al. (2012), but the subclinical mastitis prevalence is higher than that reported by Haftu et al. (2012). This could be due to poor hygiene and management system. The prevalence of blind teat in this work is higher when compared with that 33 reported by Haftu et al. (2012) and Bitew et al. (2010). The blind quarters observed in this study are an indication of a serious mastitis problem on the farms and the absence of culling that should have served to remove a source of mammary pathogens for the cows. Association of mastitis occurrence with parity was evaluated and found statistically not significant (P>0.05). The reported in the current study is contradicts with the previous reports (Tamirat, 2007; Mekibib et al., 2010; Haftu et al., 2012). This is attributed to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without mastitis control program (Radostits et al., 2007). Age of animal was highly significantly associated (P=0.000) with prevalence of mastitis. The condition was four time less likely (OR: 0.24) to be detected in young animals (89.1%) compared to adult cows (66.7%). Breed was significantly associated with mastitis prevalence. The prevalence of mastitis was 81.2% in Holstein and 13.89% in zebu cattle. Holstein breeds had 1.78 [1.0, 3.1] higher odds to be positive for CMT compared to zebu breed. Significantly higher presence of mastitis was noted in high milk yielding cows (P= 0.001). Cows with previous clinical mastitis history were 3.6 time more likely to be positive on CMT. Among the risk factors considered to have effect on the occurrence of mastitis, udder washing was found to be statistically significant (P < 0.05). In the present study, parity and lactation stage were not found to increase the occurrence of mastitis significantly (p > 0.05). According to Erskine (2001) primiparous cows have more effective defense mechanism than multiparous cows. The prevalence of subclinical infection decreases as the stage of lactation progresses. These infections are generally the result of contagious mastitis and caused by an inability of mastitis control rather than a physiologic effect (Erskine, 2001). In this study, it was observed that cows with skin lesions on their teats and/or udder had a high prevalence of mastitis. Similar observation has been recorded by Abdurahman (2006) in eastern Ethiopia. Mulei (1999) also noted that mammary gland quarters with teat lesions were 7.2 times more likely to have bacterial organisms isolated from them than those without any teat lesions in the Kiambu district of Kenya. 34 The association between concrete floor and high prevalence of subclinical mastitis recorded in this study is due to absence of barn cleaning and poor barn hygiene. Cows with previous history of mastitis were found more likely to be mastitic. This observation is supported by the findings of Biffa et al. (2005). Prevalence of mastitis was significantly (P<0.05) associated with milking hygiene practice. The results revealed that animals with poor hygiene of milking process had a high prevalence of mastitis. Poor hygiene of milking process also was identified as a risk factor for occurrence of bovine mastitis in another study in Ethiopia (Abdurahman, 2006). This might be due to absence of udder washing, milking of cows with common milkers’ which have cuts and chaps on their hands, and using of common udder cloths, which could be vectors of spread specially for contagious mastitis The common isolated genera of bacteria in the present study (Staphylococcus, Streptococcus, Bacillus, and Escherichia) agree with the findings of Abdurrahman (2006), Kalla et al. (2008), and Abera et al. (2010) who noted Staphylococcus, Streptococcus, and Escherichia as major mastitogens. Radostits et al. (2000) asserted that Satphylococcus aureus is well adapted to survive in the udder and usually establishes a mild subclinical infection of long duration, from which it shed in milk, facilitating transmission to healthy animals, mainly during milking procedures. Bacterial isolates were recorded from all the 403 clinically and sub clinically affected lactating cows. All the investigated dairy farms were positive for different bacterial species. This indicates that dairy farms have similar problems in hygiene and husbandry practices which did not able to minimize the mastitis causing micro-organisms. Staphylococcus species were the predominant pathogens constituting 66.67% of all bacterial isolates in the current study. This does not agree with that reported by Mekibib et al. (2010) percentage of which is lower than the current study. The relative high prevalence of Staphylococcus species in the current study show the absence of dry cow therapy and low culling rate of chronically infected animals in the study area. Streptococcus species were also found prevalent with 14.81% share of the total isolates. The finding in the present study is in agreement with that noted by Bitew et al. (2010) at Bahir Dar and its environs (13.9%). However, Hawari and Al-dabbas (2008) reported 26.2% relative 35 frequency of Streptococcus species in Jordan and Atyabi et al. (2006) who found 33.54% at farms around Tehran. E. coli occurred with the prevalence of 11.11% of the isolates. The findings is different from the previous reports by Mekibib et al. (2010) at Holeta (4.6%) and Sori et al. (2005) in and around Sebeta (0.75%), but is comparable with the report of Hawari and Al-dabbas (2008) in Jordan (15.6%). E. coli is an environmental contaminant and its high prevalence in the present report could be related to hygienic status practiced at the study site. Bacillus species were the least prevalent bacteria species (7.41%) from the bacterial isolates. The reason could be bacillus species are not common causative agents of bovine mastitis. The present study shows high prevalence of bovine mastitis with Staphylococcus and Streptococcus species to be the dominant bacterial isolates in the study area. Based on the results of this study, we recommend implementation of strict fortnight mastitis control program, improved milking hygiene, prevention of skin lesions, culling of chronic mastitis carriers, and treating of clinically infected cows in the study area. 36 6. SUMMERY, CONCULISION AND RECOMMENDATIONS In developing countries such as Ethiopia, cows in the dairy farms are maintained in inadequate hygienic environment, poor animal health service, and lack of proper attention to health of the mammary gland. Mastitis has been known to cause a great loss of productivity through poor milk quality, reduced milk yield, and due to culling of cows. The overall prevalence of mastitis at cow and quarter level in the current study was 91.8% and 70.85%, respectively. Three hundred twenty (79.4%) cows were positive for subclinical mastitis while only 50 (12.4%) had clinical mastitis. Among the total (1612) quarters investigated, 20 teats (1.24%) were blind. The present study shows high prevalence of bovine mastitis with Staphylococcus and Streptococcus species to be the dominant bacterial isolates in the study area. Based on the results of this study, we recommend implementation of strict fortnight mastitis control program, improved milking hygiene, prevention of skin lesions, culling of chronic mastitis carriers, and treating of clinically infected cows in the study area. Based on the above conclusion, the following points are forwarded as recommendations To reduce the prevalence of the disease, different epidemiological factors that interplay in mastitis occurrence should be studied. Extension service including health education must be launched to create public awareness about the disease, since dairy is becoming the common enterprise and owners are new to the business and not knowledgeable about udder health. Implementation of better management practices with introduction of comparatively mastitis tolerant animals. 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Milking practice: Manual______________ or using milking machine_______________ Do you wash udder before milking? yes____ no____ Do you dry after washing? yes____ no____ Do you use the same cloth for all teats? yes____ no____ Do you practice milking mastitic cows last? yes____ no____ Do you wash your hand before milking and after milking one cow? Yes___ no___ 3. Feeding Status: concentrate_______, grass________, legumes__________, feeding interval______, concentrate: roughage ratio__________, kilogram per day__________ 4. Housing: Floor concrete______stone_____soil______slopy_____leveled_____ Roof: metal sheet______ grass_____ Wall: concrete_________mud______other______ Manure removal: daily______weekly____monthly____other (specify) ______ 5. Do you practise good milking procedures? Yes___ no____ 6. Have you ever used teat dipping after milking? Yes___ no___ if yes, what type? 48 Annex 2. Interpretation of CMT Findings Source: Quinn et al. (1999) Score Interpretation Visible reaction 0 Negative Milk fluid and normal T (Trace) Trace Slight precipitation 1 Weak positive Distinct precipitation but no gel formation 2 Distinct positive Mixture thickens with gel formation 3 Strong positive Viscosity greatly increased .strong gel that is cohesive with a convex surface