ASSOSA UNIVERSITYCOLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES DEPARTMENT OF APPLIED BIOLOGY Detection of Escherichia coli & Staphylococcus aurous Enumeration from Raw Milk in Assosa Town Submitted to Department of Biology for Partial Fulfillment of Requirement of Bachelor of Science in Applied Biology By: GUTU TESFAYE……………………………………...NCR/157/07 ADDISU AYELE………………………………………NCR/016/07 ETAGEGN ABEBAYEW……………………………..NCR/086/06 SENA MOGOS……………………………………….. NCR/254/07 ELSA WOLDU……………………………………….. NCR/117/07 Advisor: Mohammed T. June, 2017 Assosa, Ethiopia I|Page Acknowledgments Firstly we are thankful to our God for his mercy & help. Next we are grateful thanks to our advisor (instructor) Mohammed Tesfaye and biology department head Mr. Asmamaw Abate for their good assistance and valuable comments starting from proposal developing until the fruitiness of the final research. We are also grateful to respondents (milk handlers) who permitted and participated to scarifying their time with us for responding our questions & helped us in collecting samples and finally to our families for their financial as well as moral support. I|Page TABLE OF CONTENT ACKNOWLEDGMENTS ...............................................................Error! Bookmark not defined. LIST OF ABBREVIATIONS .................................................................................................... IV LIST OF TABLES AND FIGURES ........................................................................................... V ABSTRACT ......................................................................................Error! Bookmark not defined. 1. INTRODUCTION..................................................................................................................... 1 1. 1. Background of the Study .................................................................................................... 1 1.2. Statement of the Problem ..................................................................................................... 2 1.3. Objective of the Study .......................................................................................................... 2 1.3.1. General Objective .......................................................................................................... 2 1.3.2. Specific Objective .......................................................................................................... 2 1.4. Significance of the study ---------------------------------------------------------------------------- 3 1.5. Limitation of the Study ........................................................................................................ 3 2. LITERATURE REVIEW ........................................................................................................ 4 2.1. Raw Milk and Its Contamination with Bacteria ................................................................... 4 2.2. The Two Main Milk Contaminants ...................................................................................... 4 2.2.1 Escherichia coli............................................................................................................... 4 2.2.2 Staphylococcus aurous ................................................................................................... 5 2.3. Safely Handling Raw Milk and Proper Milking Procedures................................................ 5 2.4. Handling Practice of Milk by Milk Handlers ....................................................................... 7 3. METHODOLOGY ................................................................................................................... 8 3.1. The Study Area Description ................................................................................................. 8 3.2. Study Design ........................................................................................................................ 8 3.3. Sample Collection ................................................................................................................ 8 3.4. Bacteriological Analysis ...................................................................................................... 8 3.4.1. Isolation Of E. coli......................................................................................................... 9 3.4.2. Isolation Of S. aurous .................................................................................................... 9 3.5. Assessments of Milk Handling Practices by Milk Handlers ................................................ 9 3.6. Data analyses and Interpretations-------------------------------------------------------------------9 3.7. Ethical Considerations -----------------------------------------------------------------------------10 II | P a g e 4. RESULTS AND DISCUSSION……………………………………………………………..11 5. CONCLUSION AND RECOMMENDATIONS .................................................................. 16 5.1. Conclusions ........................................................................................................................ 16 5.2. Recommendations .............................................................................................................. 16 6. REFERENCES ........................................................................................................................ 18 Appendix ...................................................................................................................................... 21 III | P a g e List of Abbreviations E. coli ----------------------------------------------Escherichia coli EHEC………………………………… …… Entero hemorrhagic E. coli EMB…………………………………………Eosin methylene blue agar HC……………………………………………Hemorrhagic colitis NAM……………………………………… …Nutrient agar medium S. aurous …………………………………… Staphylococcus aurous IV | P a g e List of Tables and Figures Table 1. Contamination level and percentage of E. coli from raw milk……………....11 Table 2. Enumeration of S. aurous (Cfu log/ml) ………………………………………12 Table 3. Average Contamination of S. aurous in raw milk …………………;;………13 Table 4. Assessment of milk handling practices among milk handlers and venders….14 Figure 1. Percentage of milk contamination with E. coli………… ………….……... 11 V|Page Abstract Milk is supposed to constitute a complex ecosystem for various microorganisms including bacteria since milk is reach in all types of nutrients. Contamination of milk and milk products with pathogenic bacteria is largely due to processing, handling, and poor hygienic practices in which they can cause high virulence. Among microbes in raw milk, E. coli and S. aurous are common especially in unhygienic milk and milk products and they have a great impact throughout the world on a children and young populations. The main objective of this study is to isolate and study the presence of E. coli and S.aurous in raw milks, and to assess the handling practice of raw milk handlers in Assosa town, Benishangul Gumuz regional state. A cross sectional study was done on a total 45 samples of raw cow milk and each of 15 samples which taken from cattle udder (teat), milk storage containers and milk venders (merchants) from March 2017 to June 2017. The isolation and confirmatory test of E. coli and S. aurous enumeration was conducted following the standard procedures by using EMB agar and mannitol salt agar, respectively. And finally, all of the data was organized and analyzed through Excel software in terms of frequency and percentage with tables and chart for simple reporting of the findings. The study shown that from the total of 45 samples of raw cow milk collected from three different sites (udder, storing bucket and market place), 28.89% is contaminated with E. coli and 48.89% of raw milk samples were contaminated with S. aurous. The present study revealed that there is medium contamination level of sample by S. aurous <5 log10 to pose pathogenic effect on the body of milk users. But, still the result suggests that there is a need for hygienic safety and analysis from E. coli suggests that the milkier and utensils for milking purpose should be kept clean as they are the major sources of contamination. Keywords/phrases/: Assosa, Benishangul Gumuz, E. coli, Enteropathogenic, Raw milk, S. aurous VI | P a g e 1. INTRODUCTION 1. 1. Background of the Study Milk and milk products consist of high moisture, nearly neutral in pH and are rich in carbohydrate, protein, fat and vitamins. Hence, milk easily favors the growth and multiplication of many bacteria, even pasteurized or refrigerated. These bacteria may significantly influence the quality of the milk and milk products. Milk contains relatively few bacteria when it is secreted from the udder of a healthy animal. However, during milking process, raw milk gets contaminated from the exterior of the udder and the adjacent areas, dairy utensils, milking machines or the hands of the milking man, from the soil and dust. In these way, bacteria, yeasts and molds get entry into the milk and thus constitute the normal flora of milk. Milk might also be contaminated with pathogenic bacteria or bacterial toxins which may serve as vehicle for the transmission of diseases to humans such as salmonellosis, diarrhea, food poisoning, tuberculosis etc. (Uddin et al., 2011). The milk of domesticated cattle is the most popular to human consumption in which milk is approximately contains 87% water, 4.9% carbohydrate (lactose), 3.4% fat, 3.3% protein and the remaining 1.4% is vitamins and minerals (Https://www.havemilk.com/delicious-dairy/milk- varieties). The primary carbohydrate found in the milk is lactose. Lactic acid is produced when bacteria in milk metabolize lactose (Saurav, 2013). The quality of milk is evaluated by its composition and hygienic properties. It serves as excellent culture medium for the multiplication of many different bacteria such as E. coli and S. aureus in laboratory. They could colonize diary plant premises and consequently contaminate dairy products. According to Singh and Prakash (2008), milk is supposed to constitute a complex ecosystem for various microorganisms including bacteria. Milk products like cheese and curd are widely consumed and market for them has existed in many parts of the world for many generations. Contamination of milk and milk products with pathogenic bacteria is largely due to processing, handling, and unhygienic condition in which Escherichia coli frequently contaminating food organism and it is a good indicator of fecal pollution (Diliello, 1982: Soomro et al., 2002). 1|Page Pathogenic bacteria in milk have been a major factor for public health concern, since, many diseases are transmissible via milk products. Traditionally, raw or unpasteurized milk has been a major vehicle for transmission of pathogens (http://www.sciensage.info/jasr). The health of dairy herd and milking condition basically determines the milk quality. Another source of transmission of microorganism is unclean teats. The use of unclean milking and transporting equipment also contributed to the poor hygienic quality (Hafsa et al., 2014). 1.2. Statements of the problem Milk is a host for many microorganisms like Escherichia coli and staphylococcus aurous. This two major bacterial species have a great impact throughout the world on a children and young populations who use unhygienic milk and milk products and are considerable causative agents of disease worldwide. Microbiological assessments have an important role to play in the dairy industry to protect the public health and can reduce economic losses by the early detection of inadequate processing, packaging or refrigeration. (Uddin et al., 2010; EATA, 2015). Presence of E. coli in the milk product indicates the presence of entero-pathogenic microorganisms, which constitute a public health hazard. Entero pathogenic E. coli can cause severe diarrhea and vomiting in infants and young children (anon., 1975). Particularly in Assosa area the distribution and virulence of this pathogenic organisms is believed to be higher, because of poor hygienic practices of residents. Hence the present study investigates the isolation and detection of E. coli and S. aurous enumeration from raw milk. 1.3. Objective of the Study 1.3.1. General Objective To isolate Escherichia coli and enumeration of staphylococcus aurous from raw milk. 1.3.2. Specific Objectives To isolate and determine E. coli from raw milk To isolate and enumerate S. aurous from raw milk To assess the handling practice of raw milk in Assosa town 2|Page 1.4. Significance of the Study In Ethiopia including Assosa, there is high annual milk production with large number of cattle resource. But still the handling and consumption of this milk is in a traditional and unhealthy way. To assess this problem such kind of study is very useful as they help individuals in understanding of what the milk properly handled as it is not harm to health. In addition, it helps further study by providing as base line information. Therefore, the present study investigates to isolate E. coli and Staphylococcus aurous from raw milk sold in Assosa city 1.5. Limitation of the Study In did this study we faced some problems like shortage of time, scarcity of laboratory equipment’s and media which makes unable to conduct of biochemical test. 3|Page 2. LITERATURE REVIEW 2.1. Raw Milk and Its Contamination with Bacteria Milk is among the most perishable foods and it is potentially susceptible to contamination with microorganisms because it is an excellent medium for the growth of bacteria. Some of these are harmless but some may be pathogenic to humans. Contaminated or spoiled milk is a one that causes a remarkable discomfort on individual. This contamination (spoilage) may be either by contaminated hands of workers or unsanitary utensils, contaminated with flies, and use of polluted water for cleaning the utensils of storage materials. The sanitary quality of milk can be checked by bacterial isolation and count in the milk (perihar and perihar, 2007; Dubey and Maheshwari, 2002). In the recent years there has also been a steady increase in the production and consumption of processed milk products worldwide because of their high nutritive value and convenience. However, un processed milk products may at a time constitute a public health hazard due to the possible presence of food pathogenic bacteria which cause illness, intoxication and sometimes outbreak of death (Doaa El-Hadedy ; Salwa Abu El-Nour, 2012). 2.2. The Two Main Milk Contaminants According to priyanka and alka (2012), the most important pathogens found in milk and milk products are Escherichia coli and staphylococcus species. Most strains of E. coli are harmless but several strains are extremely pathogenic like E. coli 0157:H7 causing complication and death associated with hemorrhagic colitis and acute renal failure, especially in children (Robins brown et al., 2004). The infective dose of E. coli is estimated to be about 10 cells in contrast the infective dose of S. aurous is high because the actual cause the food poisoning is not the organism itself, but rather a number of heat stable protein toxins produced by the bacterium and capable of withstanding 100c for >30 minutes (presscott et al., 2002). 2.2.1. Escherichia Coli It is considered to be a fecal contamination indicator in foods, because of its presence in the gut. The presence of E. coli in foods is a matter of concern because some strains may be pathogenic (Thaker et al., 2012). E. coli 0157;H7 serotypes, identified as enterohaemorrhagic E. coli (EHEC) and grouped as verotoxin-producing E. coli (VTEC), are recognized as the primary 4|Page cause of hemorrhagic colitis (HC) and the diarrhea associated form of hemolytic uremic syndrome (HUS) (Radii et al., 2011). Fermented dairy products made with unpasteurized milk are potential vehicle for the transmission of E. coli to consumers. It has been shown that the pathogen is present in raw milk, although it will survive proper pasteurization, if it occurs after post pasteurization the organism has been shown to survive the manufacturing and ripening stages of fermented dairy products (Coia et al., 2001). 2.2.2. Staphylococcus aurous Staphylococcus aurous is the most prevalent and economically significant pathogen causing inflammatory infections in dairy ruminants. S. aurous can get access to milk either by direct excretion from udders with clinical or subclinical Staphylococcal mastitis or by contamination from the environment during handling of raw milk. Staphylococcus aurous is a pathogenic bacterium that induces several of human illnesses. The ability to cause a wide range of diseases may be associated with its production of a large spectrum of extracellular toxic compounds and other virulence factors such as toxic shock syndrome, exfoliative toxins and Enterotoxins (mueena et al., 2015). However, despite genetic up gradation and modern methods of rearing livestock production per capita has remained low. One reason for this could be mastitis especially for the sub clinical type. Mastitis remains one of the most common economic problems of dairy industry worldwide. It is mainly caused by bacteria, and the infection results in inflammation and pathophysiological changes in the udder tissue, resulting in compromised milk quality and decreased amount of milk production. Depending on the severity of the inflammation the disease can be categorized in to sub clinical or clinical. Clinical mastitis can be readily detected whereas asymptomatic conditions make detection of sub clinical mastitis difficult nonetheless (mueena jahan, et al., 2015). 2.3. Safely Handling of Raw Milk and Proper Milking Procedures Always try and maintain a healthy animal: Sick cows will not produce healthy milk. Still, it’s important that your animal have access to fresh water, fresh grass/high-quality hay, clean living conditions, and mineral supplements/salt licks/etc. (http://theelliot thomestead.com/2014/04/ handling-raw-milk-procedures/). 5|Page Prepare a proper milking area: Wet and dirty ground is not good. So, keep the milking area clean and as free of feces as possible (http://theelliotthomestead.com/2014/04/handling-raw- milk-procedures/). Having proper and Clean equipment: In order to keep the milk clean and free of contaminates, it’s absolutely essential that the equipment is clean and stainless steel buckets for the milk storage, filter equipment (for filtering the milk). On the other hands, all of the equipment’s should be easy to maintain, inexpensive, and easily sterilized to ensure its cleanliness ( http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/) When it’s time for milking, clean animal properly: Using the warm water to dissolve anything crusted on the teat we usually only wash for 15-30 seconds. Enough time to get nice and clean and stimulate milk let down (http://theelliotthomestead.com/2014/04/handling-raw-milk- procedures/). Milk cleanly: Make sure the milker’s hands are clean. Dirty hands dirty milk (http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/). Cool the milk quickly: This is one of the most important steps in raw milk handling. As soon as a cow is milk, the bacteria in the milk beginning eating and digesting the sugar in the milk. Cooling the milk helps to deter the growth of bacteria, allowing for fresher milk for longer. We carry our bucket up to the house, where it is then filtered into the jugs. The jugs are then placed in the freezer for an hour. After an hour of speed of cooling, the milk is transferred to the refrigerator where it remains until it is drunk. The freezing step helps to get a jump start on the cooling process (http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/). Handle with care: it’s still important to handle the milk with care and respect for the product since it is a perishable product. Just like any other agriculture products, has the potential to be produced well and safely. Handled improperly also has the potential to make consumers sick. (http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/). Poor hygiene, practiced by handlers of milk and milk products, may lead to the introduction of pathogenic micro-organisms into the products. Since they do not undergo further processing before consumption, these foods may pose risk to the consumers. Therefore, provision of milk 6|Page and milk products of good hygienic quality is desirable from consumer health point of view (Yohannis and Mesfin , 2015; STTA Report – Vladimir Kokarev, 2006). 2.4. Handling Practice of Milk by Milk Handlers Whether milking by hand or machine, good hygiene is essential. This requires that: The milkers hands and clothes are clean and he or she is in good health. The milking machine and milk storage equipment such as milk churns are kept clean and are in good condition (i.e. without cracks or dents which are difficult to clean and can easily harbor bacteria. Immediately after milking, the milk must be cooled preferably to 4° C. This requires mechanical refrigeration or milk cooling tanks. These are expensive and can usually be afforded by large scale commercial farms. For small scale dairy farmers, setting up a milk cooling center centrally may be the ideal solution. Where farmers bring their milk to a cooling centre through a co-operative, they should do so as soon as milking is completed. A Milk cooling centre with a capacity of 1000 - 3000 liters will serve up to 300 small holder farmers ensuring that the quality of their milk when produced under hygienic conditions is well preserved and accepted at the processing plant. (http://www.fao.org/ag/againfo/resources/documents/mpguide/mpguide1.htm) To maintain the quality of milk during transportation, adhere to the following guidelines (Lusato, 2006): Ensure that milk containers and the transport vehicle are kept clean. Do not use milk containers for storage of other goods. Do not transport milk with other goods. The code of hygiene requires that vessels and carriers used for milk transport should be used only for that purpose and be labeled “MILK ONLY”. Keep the milk as cool as possible and avoid exposing it to high temperatures. Keep the milk covered at all times to protect it from light and dust. Transport the milk to the sales point or processing factory in the shortest time possible. Do not smoke, handle tobacco or other materials with strong odor (e.g. kerosene fuel) when handling milk. Avoid excessive agitation of the milk cans. 7|Page 3. METHODOLOGY 3.1. The Study Area Description The study was conducted in Assosa town of Benishangul gumuz regional state. The town is about 661km far from Addis Ababa which is the capital of Ethiopia. The study region located in north western part of the country between 0917’-1206’ north latitude and 3410’-374’ east longitude (moreda, 2013) and having a total area of about 50,382 kilometer square (flatei, et al., 2009). According to 2007 census report, the population size of the region was 670,847 (CSA, 2008). And the region is known by the availability of livestock resource and high milk production as most of the other regions do. 3.2. Study Design A cross-sectional study was conducted in the town from April 2017 to May 2017 to collect and determine E. coli and S. aurous from raw milk, and assess raw milk handling practice of milk venders and owners in different Keble’s of the town. Samples of raw milk was collected at morning and evening from dairy cooperative milk producer centers and farmers from purposively selected three urban Kebeles (03, 02 and Amba 5) which have large number of customers. 3.3. Sample Collection Milk sample were collected from points or milk seller who considered to be associated with contamination. The sampling point was at the teat during milking, milking buckets and at the market place. Over all 45 samples of raw milk was taken in sterile bottles using ice box, in which, 15 samples from the teat, 15 from milking bucket or storage, and the remaining 15 have been collected after arrival to the market in the market place. And all the samples were transported to Assosa university Biology laboratory for immediate bacteriological analyses. 3.4. Bacteriological Analysis The milk samples was subjected to serial dilution, where one ml of each sample was added to 9 ml of peptone bacteriological (an enrichment media) and shaken gently. All the samples were serially diluted up to 10-3 dilution factor and the test tubes were incubated for 24 h at 37° C (Hafsa, et al., 2013) for enrichment purpose. 8|Page 3.4.1. Isolation of E. coli For the isolation of E. coli, 1ml of the enriched sample from peptone bacteriological water was cultured on selective and differential MacConkey agar medium and incubated at 37˚C for 24 hours. Typical colonies having pink color from MacConkey plate agar streaked on a nutrient slant for refreshment and incubated for additional 24 hours. Then, transferred to eosin methylene blue (EMB) and E. coli is confirmed by its green metallic sheen color on the EMB media. Morphologically typical colonies having metallic green sheen was taken and confirmed as E. coli and into for further identification on nutrient agar slants (http://www.sciensage.info/jasr.media for E. coli isolation). 3.4.2. Isolation of S. aurous From each samples of prepared serial dilution, one ml was transferred into triplicate sterile Petri dishes and Mannitol salt agar (Oxoid, England) was poured and swirled gently and finally incubated at 37ºC for a maximum of 48 hours (Robers and Greenwood, 2003). Yellow and orange colonies surrounded by yellow zones due to mannitol fermentation enumerated as the presence of S. aurous. 3.5. Assessments of Milk Handling Practices by Milk Handlers The observation checklist and semi structured questioner was used for assessment of the sanitary conditions and milk hygienic practices of the handlers. A check lists covering topics on the personal hygiene of the milk seller and milker’s hygiene practices (modes of cleaning and sanitizing utensils) and hygiene of the selling area to asses weather the area exposed to flies, insects and animals, presence of solid and liquid waste in vending area. Assessment of the hygienic practices of farmers, venders and vending environment was a total of 18. Respondents were selected purposely who engage with milk handling or milk venders and also interview and a semi structured questionnaires was used. 3.6. Data Analysis and Interpretation After sample collection from milk handlers, the result was analyzed using tables and charts to show and make comparison of E. coli contamination or presence/absence among milk samples taken from different sites. In addition, descriptive statistics such as percentage and frequency 9|Page distribution was done using Microsoft excel software to describe the enumerated S. aurous and E. coli contamination rate. Finally, short tabular description was used about milk handling practice among venders and handlers in order to make conclusion 3.7. Ethical Considerations In conducting this research we put all necessary issues to be considered in to account when doing every action: all farmers and venders including those who participate in responding questionnaires had got awareness of benefits of the research. In addition, we have taken every action with the recommended license from our department and institution. Generally the present study was done by creating positive attitude with every participant and respondents. 10 | P a g e 4. RESULTS AND DISCUSSION The present study shows that (Table 1 & figure 1) among the total of 45 samples of raw cow milk collected in three different sites (udder, storing bucket and market place) 15 from each, 7 (46.66%) is contaminated with Escherichia coli (E. coli) as higher contamination recorded at storage bucket followed by udder (26.66%) and low contamination at market place (13.33%). Generally, as shown (Table 1 & figure 1), the total contamination rate of raw milk samples taken from all sources with E. coli was 13 (28.89%). Table. 1. E. coli contamination level and percentage of raw milk collected from three sources No. Source of sample Number of Number of E. coli Percentage (%) samples positive samples 1 Udder/teat 15 4 26.66 2 Bucket/storage 15 7 46.66 3 Vender/market 15 2 13.33 Total 45 13 28.89 E. coli 13.33% 26.66% udder 46.66% bucket market Figure 1. E. coli contamination percentage Presence of E. coli in milk suggests that the sample is contaminated through transportation, unhygienic processing or through fecal contamination. Similarly, in the present study, the 11 | P a g e methods of production, transportation, handling and sale of milk were entirely unhygienic and the study showed that 13 out of 45 milk samples were contaminated with E. coli. On the other hands, high incidence of E. coli was found in raw milk samples conducted by many researchers, Martin and others (1986), for instance, reported that 43% of the milk samples were contaminated with E. coli; and similarly Sharma and Joshi (1992) reported that 50% of the milk samples were contaminated with E.coli. According to the results (kumar, 2011), the 16 (29.09%) samples out of 55 showed that the contamination of E. coli. E. coli is one of the bacteria that exist in the normal micro flora of the intestinal tract of humans and warm blooded animals. Its contamination with milk and milk products, is largely due to processing, handling, and unhygienic conditions. In addition, E. coli is, furthermore, a known causative agent of diarrhea and other food-borne related illnesses through the ingestion of contaminated foodstuffs. Pathogenic members of the E. coli form group as well as the enterobacteriacae family are represented by genera such as salmonella and shigella and are found in the intestines of humans and animals (collins et al., 1995). Table 2. Enumeration of S. aurous (Cfu log/ml) in raw milk from Udder, Bucket and Market Sample site Sample code Cfu/ml Cfu log/ml Udder 2 1203 3.08 “ 4 1297 3.11 “ 8 1420 3.15 “ 9 1273 3.10 “ 11 540 2.73 “ 13 5527 3.74 Bucket 17 340 2.53 “ 19 30 1.48 “ 20 413 2.62 “ 21 23 1.36 “ 22 3387 3.53 “ 28 617 2.80 “ 29 3483 3.54 “ 30 7 0.85 12 | P a g e Market 33 280 2.45 “ 34 3420 3.53 “ 35 1950 3.30 “ 37 3197 3.50 “ 38 1200 3.08 “ 42 157 2.20 “ 43 2433 3.39 “ 45 4090 3.61 S. aurous is often found in unprocessed milk and dairy products due to contamination caused by poor hygiene conditions, or the origin of the milk, which can come from mastitis cows or through hands or skins of handlers (human being). Staphylococcus is one of the most important food borne microorganisms responsible for staphylococcal food poisoning (SFP) consumption of 100µg of staphylococcus Enterotoxins (SE ) produced by enterotoxigenic strains causes SFP (Enquebaher, 2015). When compared to our finding with the study conducted by Aftab (2010) reported that 11 (50%) raw milk samples out of 22 tested samples were contaminated with staphylococci, it is somewhat almost similar with the present findings. On the other hands, contamination of the raw milk with S. aurous is suspected to result from the origin of the milk or poor hygiene conditions which can be from mastitis cow or unclean utensils. An increase in time or temperature before consumption could lead to further proliferation of the pathogen and the production of toxins by enterotoxigenic if any (Enquebaher, 2015). Table 3. Average S. aurous contamination level of raw milk in each sources Source of No of No of positive Average log cfu/ml Percentage (%) sample samples samples Udder 15 6 3.15 40.0 Bucket 15 8 2.34 53.33 Market 15 8 3.13 53.33 Total 45 22 Average 2.874 48.89 13 | P a g e On the other hands, The present finding showed that, from the total 45 samples at three different sites; 22 (48.89%) are shown to be S. aurous positive in which 6 (40%), 8 (53.33%), 8 (53.33%) at udder, bucket and market, respectively were contaminated with S. aurous. The average number of S. aurous colonies per milliliter of samples as a form of (log cfu/ml) were 2.34, 3.13 and 3.15 at bucket, market, and udder, respectively (Table 3). Table 4: Assessment of the hygienic practice of milk handlers, venders and the environment No. Questions Total number of Percentage (%) respondents (n)= 18 1 Do you check whether your cow is healthier or not before milking? a. yes 11 61.11 b. no 7 39.89 2 Have you clean your milking bucket, teats of milked animal and wash your hands before milking? 1. Wash hands before milking a. yes 12 66.67 b. no 6 33.33 2.washing udder(teats) before milking A. yes 8 44.44 B. no 10 55.56 3.Clean bucket before milking a. yes 9 50 b. no 9 50 Stage of selling 1.raw 2 11.11 3 2.after boiling 16 88.89 4 Area of milking a. large and clean area 1 5.56 b. small corner 3 16.67 c. where ever free area 14 77.78 5 Storage condition at home: a. at refrigerator 6 33.33 b. at room temperature 12 66.67 14 | P a g e 6 Frequency of milking a. one’s per day 1 5.56 b. two times per day 17 94.44 c. more than two 0 0 7 Habit of washing the teat with______ a. detergent 1 5.56 b. hot water 1 5.56 c. tap water 6 33.33 8 Presence of separate worker a. yes 1 5.56 b. no 17 94.44 9 Presence of separate cloth for milking a. yes 1 5.56 b. no 17 94.44 10 Type of bucket a. plastic 11 61.11 b. metal 1 5.56 c. others 6 33.33 11 Practice of cleaning of milking area a. yes 3 b. no 15 16.67 83.33 12 Cleaning of vending environment a. yes 4 22.22 b. no 14 77.78 Assessments of milk handlers: A total of 18 milk handlers including venders were interested and participated in responding the questions rose to access the milk handling tradition and their knowledge against infectious diseases transmitted through milk and milk products. From 18 participants, 15 were female and the other 3 were males. The age of the respondents ranges between 24 and 52 years. 15 | P a g e 5. CONCLUSION AND RECOMMENDATION 5.1. Conclusions The result of the present study revealed that there is medium contamination level of milk samples by S. aurous, i.e., <5log10 cfu/ml (Enquibahar et al., 2015) to pose pathogenic effect. But, still the result shows that there is a need for hygiene to keep safety of milk and following up the health of the animal in order to reduce contamination of milk and its products by this microbe. In the same manner analysis from E. coli suggests that the milker and utensils for milking purpose should be kept clean as they are the major sources of contamination. As easily understood from (table 1 and figure 1) high contamination rate with E. coli is recorded at storage utensils which indicates low practice of cleaning the materials. As the data collected from milk handlers and venders, most of the respondents do not use those necessary measures such as: - a) detergents and hot water to clean the teat of their animal, b) lack of cleaning their hands before milking, c) absence of separate worker and cloth for safety and d) shortage of large and clean area which is free from insect exposure might be the main reasons of much contamination of the milk samples with E. coli and S. aurous. 5.2. Recommendations Based on the present study we will be recommended the following points: The milk handlers and the venders who do not properly protect their hygienic quality and those who supply low quality milk to the market should be undertake some quality measures and awareness creation among them about safety handling practice of milk and milk products Poor hygiene, practiced by handlers of milk and milk products, may lead to the introduction of pathogenic micro-organisms into the products. Since they do not undergo further processing before consumption, these foods may pose risk to the consumers. Therefore, provision of milk and milk products of good hygienic quality is desirable from consumer health point of view. 16 | P a g e In addition the habit of washing the animal udder by soap or warm water should be developed among the community as there is high probability of contamination of udder with those disease posing pathogens from the environment. In the present study, due to lack of reagents and culture media we cannot conduct some biochemical study regarding to E. coli. So, further researchers in the future should conduct biochemical tests and isolate other pathogens from milk samples. 17 | P a g e 6. REFERENCES 1 Akshita Sharmal, Kamal Devl (2016). Isolation and characterization of Escherichia coli producing β galactosidase from raw milk of dairy industry. 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Chand and Company LTD New Delhi. 8 Enquebaher TSiv S, Knut R, Taran S and Judith A (2015). Staphylococcus aurous and other staphylococcus species in milk and milk products from tigray region, Northern Ethiopia 9 Ethiopian agricultural transformation agency (EATA), 2014. 10 Flatie T, Gedif T, Asres k, and Gebremariam T (2009). Ethnomedical survey of berta ethnic group assosa zone Benishangul gumuz regional state, Midwest Ethiopia. J. ethnobiol. ethnomed, 5;14-24. 11 Hafsa A, Fouzia S, Facrudin MD, Kamrunnahar, Zahed U, Mahmood k, Suvamoy D, (2013). Isolation of E. coli and S. aureus from full cream powder milk and sold under market conditions at Dhaka. Bangladish, and their antibiotic susceptibility. Journal of advanced scientific research. 4 (3). 12 Lusato R. Kurwijila, (2006) Hygienic milk handling, processing and marketing: Reference guide for training and certification of small-scale milk traders in Eastern Africa Volume 1. 18 | P a g e 13 Uddin Md, Hasan Md, Motazzim-ul-Haque, 1 Rashed Noor 1 (2011). Isolation and Identification of Pathogenic Escherichia coli, Klebsiella spp. and Staphylococcus spp. in Raw Milk Samples Collected from Different areas of Dhaka City, Bangladesh. Stamford Journal of Microbiology, July 2011. Vol. 1, Issue 1 ISSN: 2074-5346. 14 Melese AR, Tesfaye WB, Ayalew NA, (2016). Bacterial contamination of raw cow’s milk consumed at jijiga city of Somali regional state, eastern Ethiopia. International journal of food contamination; 10 (1186). 15 Moreda T, (2013). Postponed local concerns, implication of land acquisitions for indigeneous local communities in benishangul gumuz regional state, Ethiopia. 16 Mueena JL, Marzia Rahman1,Md. Shafiullah PL, Shah Md. Ziqrul HC , Md. Enamul H, Md. Abdul KT and Sultan A, (2015). Isolation and characterization of Staphylococcus aureus from raw cow milk in Bangladesh. J. Adv. Vet. Anim. Res., 2(1): 49-55. 17 Parihar and parihar (2007). Diary microbiology. Student edition. Brahat printers press, jodhour. 18 Presscott LM, Doughari JP, Klein DA (2002). Textbook of microbiology 5th edition brown publishers. 19 Preethirani PL, Sloor S, Sundareshan S, Nutanala K, Shivi V, Deepthikiran K, Sinha Ay, Akhauri Y(2015). Isolation, biochemical and molecular identification and invitro antimicrobial resistance patterns of bacteria isolated from bubaine subclinical mastitis in south india. Plos one 10 (11); 1371. 20 Priyanka Sand Alka P (2008). Isolation oF Escherichia coli, Staphylococcus aureus AND Listeria monocytogene from milk products sold under market conditions at agra region. Acta agriculturae Slovenica, 92 (1), 83–88 21 Robins-brown RM, Bordun AM, Tauschek M, Bennett-wood VR, Rusell J, Oppedisano F, Lister A, Bettelhiem NA, Fairle NC, Sinctair MI, Hellard ME (2004). Infectious disease, 10; 1797-1805. 22 Saurav S, (2013).milk microbiology. Shree publisher and distributer, New delhi-110 002. 23 Soomro AH, Arain MA., Khaskheli, MB. (2002). Isolation of Escherichia coli from raw milk and milk products in relation to public health sold under market condition at Tandojam. Pak. J. Nutr., 13:151–152. 24 STTA Report – Vladimir Kokarev, August 2006. 19 | P a g e 25 Thaker HC, Brahmbhatt MN, Nayak JB. (2012). Study on occurrence and antibiogram pattern of E. coli from raw milk samples in Anand, Gujarat, India. Vet World. 5 (9):556–559. 26 Yohannis Abera and Mesfin Angaw (2015). Handling Practice and Microbial Quality of Raw Cow’s Milk Produced and Marketed In Adigrat Town, North Eastern Tigray. Journal of Biology, Agriculture and Healthcare, 5(15): 1- 3. 27 http://www.sciensage.info/jasr.media for E. coli isolation. March, 26, 2017. 28 http://www.microbe online.com procedure of methylene red test for E. coli, March, 26, 2017 29 . Https://www.havemilk.com/delicious-dairy/milk-varieties1/contents of milk, April 12, 2017. 30 http://theelliotthomestead.com/2014/04/handling-raw-milk-procedures/ 31 (http://www.fao.org/ag/againfo/resources/documents/MPGuide/mpguide1.htm),June 20,2017 32 (http://milkfacts.info/Milk%20Processing/Milk%20Processing%20Page.htm),June 20,2017 20 | P a g e APPENDIX 1. Do you check whether your cow is healthier or not before milking? A) yes b) No 2. Have you ever clean your milking bucket, teats of milked animal and wash your hands before milking? A) Yes b) No 3. At which stage you sell the milk a) At raw stage b) boil and cool back in refrigerator before feeding 4. Which areas you choose to milk the animal? a) Large b) medium c) small d) clean e) others 5. At what condition the milk is stored after milking (for owners) and after arrival (for sellers)? a) At room temperature b) in refrigerator C) Others…. if any 6. Frequency of milking per day a) Ones per day b) two times per day c) more than two times 7. The habit of washing the teat is:- a) Detergent b) hot water c) tap water D) If any, others 8. Presence of separate worker for milking a) Yes b) No 9. Presence of separate cloth for milking purpose a) Yes b) No 10. Type of milking utensil a) Plastic b) metallic c) others 11. Practice of cleaning milking environment. a) Yes b) no 12. Cleaning vending environment. a) Yes b) no 21 | P a g e