SENSITIVITY OF MASTITIS CAUSING BACTERIA TO ANTIBIOTICS USED
IN TREATMENT OF LACTATING DAIRY COWS AT MARIAKANI, KILIFI
COU...
ii
DECLARATION
This report is my original work and has not been submitted for an award in any other
institution
Signature:...
iii
DEDICATION
This report proposal is dedicated to my parents. Your support and prayers have made me
who I am.
iv
ACKNOWLEDGEMENT
I wish to acknowledge my parents for the sacrifice, blessings and encouragement they
have given me thro...
v
TABLE OF CONTENTS
DECLARATION..............................................................................................
vi
2.1.1 Spread of infection…… …………………………………………………………6
2.1.1.1 Bacterial characteristics… ………………………………………………........7
2.1....
vii
3.3.1 Culture and sensitivity test…...………………………………………………15
3.3.2 Bacterial culturing of milk……………….…………………………………….16
3...
viii
LIST OF ABBREVIATIONS
E. coli…………………………..Escherichia coli
Staph. aureus……………………Staphylococcus aureus
B. subtilis……………...
ix
ABSTRACT
Mastitis is an inflammation of the mammary glands regardless of the cause. It is
characterized by physical, ch...
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of study
Mastitis is an inflammation of the mammary glands regardless of the c...
2
Contagious pathogens that cause mastitis tend to live on the cow's udder and teat skin and
transfer from affected cow (o...
3
Emphasis will be placed on cases prevalent at Mariakani in Kilifi County and its
environment that is among one of the co...
4
1.3 Research Questions
i. Is mastitis causing bacteria sensitive against the selected antibiotics?
ii. What is the most ...
5
1.6 Justification
In Kenyan, the dairy industry and livestock farming has contributed a lot towards the
economy. The sec...
6
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 General features of mastitis
Mastitis is caused by many agents. These could be bac...
7
2.1.1.1 Bacterial characteristics
These include ability of organism to survive in the cow’s immediate environment- that ...
8
2.1.2.2 Establishment of the infection and inflammation of the damaged area
Some bacteria may proceed into the udder by ...
9
milk secretory tissue is, in effect the cow’s third line of defense to bring the infection under
control. (Halley et al....
10
is elicited but no apparent intra mammary resistance (Blood et al., 2000; Radostis et al.,
2000) Infected animals shoul...
11
Naivasha and Nyahururu) of Kenya over a seven-month period. (Shitandi et al., 2014;
Milcah et al., 2014)
2.4 Antibiotic...
12
2.4.3 Effect on nucleic acids
These prevent synthesis of DNA or RNA. This blocks the growth of cells. They include
quin...
13
Prolonged use of antibiotics and influences such as feeding of antibiotic treated milk to
calves may select for resista...
14
CHAPTER THREE
3.0 RESEARCH METHODOLOGY
3.1 Study Areas and Justification
The study will be contacted at Mariakani in Ki...
15
n = ( Z2 * σ2 ) / ME2
Where:
ME is the desired margin of error =4%
Z is the z score set at 95% confidence level
σ (alph...
16
3.3.2 Bacterial culturing of milk
The general appearance of milk samples will first be checked using an alcohol test, 1...
17
3.3.4 Sensitivity testing
Identified colonies will be sub cultured to obtain pure cultures. Further sub culturing will
...
18
Work Plan
ACTIVITY TIME IN MONTHS [2015 – 2016]
SEP OCT NOV DEC JAN FEB MAR APRIL
Literature
review
Proposal
Writing an...
19
Budget
ITEM QUATATION KSHS
Blood agar 500
Glucose broth serum 1,000
Crystal violet stain 1,000
Lugols iodine 1,000
Dilu...
20
REFERENCES
1. Banerjee, K., Ray, J., Sadhukhan, T., Das, R., and Chandra, S. (2002). Studies on
etiological agents of s...
21
8. Bilal, Q., Iqbal, U., Muhammad, G., Avais, M., and Sajid, M. (2004). Factors
affecting the prevalence of clinical ma...
22
16. Gigi, D., Plumb, G., and Donald, C. (2003). Veterinary Drug Handbook: Client
information edition, 93(7) 150.
17. De...
23
24. Getahun, K., Kelay, B., Bekana, M., and Lobago, F. (2008). Bovine mastitis and anti-
biotic resistance patterns in ...
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Nandwa Project Proposal

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Transcripts - Nandwa Project Proposal

  • 1. SENSITIVITY OF MASTITIS CAUSING BACTERIA TO ANTIBIOTICS USED IN TREATMENT OF LACTATING DAIRY COWS AT MARIAKANI, KILIFI COUNTY Nandwa Olumasai Jairus (BTMB/056J/2012) A research proposal submitted to the Pure and Applied Sciences department in partial fulfillment of the requirement for the award of a degree in bachelor of Technology in Industrial Microbiology and Biotechnology of Technical University of Mombasa DECEMBER, 2015
  • 2. ii DECLARATION This report is my original work and has not been submitted for an award in any other institution Signature:…………………………………..Date:…………………………………… Nandwa Olumasai Jairus Declaration by supervisors This research proposal has been submitted with my approval as the University supervisor. Signature:……….………………………..Date:……………………………… Dr. Huxley M. Makonde (TUM)
  • 3. iii DEDICATION This report proposal is dedicated to my parents. Your support and prayers have made me who I am.
  • 4. iv ACKNOWLEDGEMENT I wish to acknowledge my parents for the sacrifice, blessings and encouragement they have given me throughout my studies. My appreciation goes to my brother, sister and friends for the advices and prayers. I thank the Almighty God for his never-ending blessing.
  • 5. v TABLE OF CONTENTS DECLARATION................................................................................................................ii DEDICATION................................................................................................................... iii ACKNOWLEDGEMENT................................................................................................iv LIST OF ABBREVIATIONS………………..….………………………………...…..viii ABSTRACT.......................................................................................................................ix CHAPTER ONE ................................................................................................................1 1.0 INTRODUCTION........................................................................................................1 1.1 Background of study....................................................................................................1 1.2 Problem statement .......................................................................................................3 1.3 Research Questions......................................................................................................4 1.4 Objectives….................................................................................................................4 1.4.1 General objective...................................................................................................4 1.4.2 Specific objectives..................................................................................................4 1.5 Hypothesis.....................................................................................................................5 1.6 Justification..................................................................................................................5 CHAPTER TWO ...............................................................................................................6 2.0 LITERATURE REVIEW ...........................................................................................6 2.1 General features of mastitis ........................................................................................6
  • 6. vi 2.1.1 Spread of infection…… …………………………………………………………6 2.1.1.1 Bacterial characteristics… ………………………………………………........7 2.1.1.2 Transmission mechanisms……………………………………………………7 2.1.2 Course of infection………………………………………………………………7 2.1.2.1 Invasion of the teat.............................................................................................7 2.1.2.2 Establishment of the infection and inflammation of the damaged area .......8 2.1.2.3 Destruction of alveolar tissue ............................................................................8 2.2 Disease diagnosis,treatment and control……………………………………...……9 2.3 Economic importance of Mastitis.............................................................................10 2.4 Antibiotic mode of action ..........................................................................................11 2.4.1 Cell wall synthesis inhibition.............................................................................11 2.4.2 Protein synthesis inhibition...............................................................................11 2.4.3 Effect on nucleic acids .......................................................................................12 2.4.4 Cell membrane inhibitors..................................................................................12 2.5 Antibiotic resistance and causes ...............................................................................12 CHAPTER THREE .........................................................................................................14 3.0 RESEARCH METHODOLOGY .............................................................................14 3.1Study Areas and Justification ....................................................................................14 3.2 Sample size and Sampling .........................................................................................14 3.3 Laboratory procedure………………………………………………………………15
  • 7. vii 3.3.1 Culture and sensitivity test…...………………………………………………15 3.3.2 Bacterial culturing of milk……………….…………………………………….16 3.3.3 Gram staining and bacterial identification………..………………………...16 3.3.4 Sensitivity testing ..............................................................................................17 3.4 Data collection and management..............................................................................17 Work Plan……………………………………………………………………………….18 Budget…………………………………………………………………………………...19 REFERENCES…………………………….……………………………………………20
  • 8. viii LIST OF ABBREVIATIONS E. coli…………………………..Escherichia coli Staph. aureus……………………Staphylococcus aureus B. subtilis………………………..Bacillus subtilis B.cereus………………………….Bacillus cereus
  • 9. ix ABSTRACT Mastitis is an inflammation of the mammary glands regardless of the cause. It is characterized by physical, chemical and usually bacteriological changes in milk and by pathological changes in the glandular tissue. This reaction of milk secreting tissues to injury produced by physical force, chemicals introduced into the gland or most commonly bacterial toxins, is the most common and perhaps most expensive disease of dairy cattle. This study will be conducted to determine the sensitivity (to selected antibiotics) of bacteria causing mastitis in dairy cattle at Mariakani in Kilifi county of Kenya. Experiments will involve culture and sensitivity testing and results presented in tables. The isolated colonies will studied for the staining characteristics and morphology. Selected isolates will then be studied for biochemical characteristics and then subjected to selected antibiotics (Co- trimoxazole, gentamycin and kanamycin) Data will be randomly collected from 6 samples of infected milk. The results will be analyzed inferentially using ANOVA technique to test hypothesis. This will help to know the suitable antibiotic to be applied in case of mastitis infection.
  • 10. CHAPTER ONE 1.0 INTRODUCTION 1.1 Background of study Mastitis is an inflammation of the mammary glands regardless of the cause. The term is derived from a Greek word ‘mastos’ for breast and ‘itis’ for inflammation of. It is characterized by physical, chemical and usually bacteriological changes in milk and by pathological changes in the glandular tissue. (Cressier, 2002) This reaction of milk secreting tissues to injury produced by physical force, chemicals introduced into the gland or most commonly bacterial toxins, is the most common and perhaps most expensive disease of dairy cattle. (Ruegg, 2001) The most important changes in milk include discoloration, presence of clots, and presence of large numbers of leukocytes (Swarz, http://www.case- agworld.com).Mastitis may be classified according two different criteria: either according to the clinical symptoms or depending on the mode of transmission. (Eriskine, 2001). Symptoms and Mode of transmission In clinical mastitis, the infected quarter often becomes swollen, sometimes painful to touch and the milk is visibly altered by the presence of clots, flakes, or discolored serum and sometimes blood. In severe cases, the cow shows signs of generalized reaction: fever, rapid pulse, loss of appetite and sharp decline in milk production (Radostits et al., 2000.) In contrast, subclinical mastitis is subtle and more difficult to detect. The cow appears healthy, the udder does not show signs of inflammation and the milk seems normal. However, microorganisms and white blood cells that fight infections are found in elevated numbers. The vast majority of cases are subclinical (Saunders, 1984).
  • 11. 2 Contagious pathogens that cause mastitis tend to live on the cow's udder and teat skin and transfer from affected cow (or quarter) to unaffected cow (or quarter) during milking. They adhere easily to the skin, colonizing the teat end and then 'grow' into the teat canal, where infection occurs; because of this, post-milking teat disinfection and dry cow therapy play an important role in controlling contagious mastitis while environmental mastitis pathogens - present in the housing and bedding - can transfer during milking or between milking’s, when the cow is loafing, eating or lying down (Myllys et al., 1995). Many infective agents have been implicated as causes of mastitis. The common causes in cattle are Streptococcus agalactiae and Staphylococcus aureus. Escherichia coli is becoming a significant cause in housed or confined cattle (Blood et al., 2000; Henderson et al., 2000). Recorded cases include;-Corynebacterium, Klebsiella, Enterobacter aerogenes, Mycobacterium bovis, Bacillus cereus, Serratia marcescens, Norcardia asteroids, Bacillus subtilisin cattle (Makovec et al ., 2003; Ruegg et al ., 2003),in sheep and goats are Pasteurella haemolytica, Staphylococcus aureus, Actinobacillus lignieresi, Escherichia coli, Streptococcus uberis (Lim et al., 2004; Lira et al., 2004) and in pigs they include Aerobacter aerogenes, E. coli, Klebsiella sp, Pseudomonas aeroginosa (Makovec et al., 2003; Ruegg et al., 2003). The above agents have over the years been considerably controlled by introducing antibiotics on diseased organs. Most bacteria though have gained resistance to the antibiotics and thus increasing the prevalence of mastitis in dairy cattle (Getahun et al., 2008; Kelay et al., 2008). This goes a long way in affecting milk production by small and large scale farmers in Kenya. This study will seek to find out the resistance/ sensitivity of the bacteria to antibiotics.
  • 12. 3 Emphasis will be placed on cases prevalent at Mariakani in Kilifi County and its environment that is among one of the counties practicing livestock farming in Kenya. It will also seek to find out the best antibiotic of choice for controlling mastitis in the county and Kenya at large. 1.2 Problem statement Mastitis in Kenya has wreaked havoc and has become a great nuisance to small scale and large scale dairy farmers, due to its wide spectrum in terms of causative agents. A wide range of bacteria have been isolated from infected samples. (Jones et al., 1984; Heald et al., 1984) It is therefore very difficult to administer treatment as different bacterial strains respond differently to antibiotics. The causative microorganisms have shown considerable resistance to antibiotics thereby reducing control of his mastitis (Nijsten et al., 1996). This in effect reduces dairy production in Kenya and hence brings about impoverishment of dairy farmers. Large scale industries are also not spared as it leads to collapses due to lack of milk supply. The right antibiotic has to be one that accurately treats mastitis caused by a wide range of causatives. That is, effective to most microorganisms (Films, 1999).
  • 13. 4 1.3 Research Questions i. Is mastitis causing bacteria sensitive against the selected antibiotics? ii. What is the most effective antibiotic for treatment of mastitis causing bacteria? 1.4 Objectives 1.4.1 General objective To determine the sensitivity of mastitis causing bacteria against selected antibiotics at Mariakani, Kilifi County 1.4.2 Specific objectives i. To know the trends in sensitivity of the causative agents to gentamycin, co- trimoxazole and kanamycin in the area. ii. To determine the most suitable antibiotic(s) for use to control mastitis in the area. 1.5 Hypothesis Null hypothesis: Co-trimoxazole, gentamycin and kanamycin have no effect on bacteria causing mastitis in dairy cattle. Alternative hypothesis: Co-trimoxazole, gentamycin and kanamycin have an effect on bacteria causing mastitis in dairy cattle.
  • 14. 5 1.6 Justification In Kenyan, the dairy industry and livestock farming has contributed a lot towards the economy. The sector has suffered a major blow over the years due to emergence of resistant strains of bacteria causing mastitis. Knowledge of the most probable causative of mastitis and how they respond to treatment by three of the most common antibiotics in the region will enable veterinarians in the region to manage the disease. Advisory services advanced to farmers that shall be addressed by this study will greatly contribute to containment of the disease. This research will also be useful in ensuring that the antibiotics offered in the market are those that are most effective to reduce frustration of farmers, who buy expensive antibiotics without professional advice and regret later when they don’t work.
  • 15. 6 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 General features of mastitis Mastitis is caused by many agents. These could be bacterial or fungal agents. Bacterial agents include E. coli, Staph. aureus, Staph. Epidermidis, Klebsiella, Pseudomonas, B. subtilis, Corynebacteria sp., Serratia marcesens, Mycoplasma bovis, Mycoplasma Canadensis and Streptococcus pyogenes. Fungal infections include Trichosporon sp, Aspergillus fumigates, Candida sp, Cryptococcus neoformans and Saccharomyces sp (Hinton, 1986). 2.1.1 Spread of infection Infection of each mammary gland occurs via the teat canal, the infection originating from two main sources, the infected udder and the environment. In dairy cattle, the important infections are those that persist readily in the udder, especially Staph.aureus. Bacteria which are normal inhabitants of the environment, such as E. coli cause mastitis less frequently but when they do the disease is much more resistant to hygienic control measures. (Wattiaux, 2001) The contamination of milker’s hands, wash cloths and milking machine cups from infected quarters may quickly lead to the spread of infection to the teats of other animals. Frequency of occurrence of mastitis depends on the ability of bacteria or fungus to set up infection in mammary tissue. This is dependent on bacterial characteristics and transmission mechanisms (Amore, 1997).
  • 16. 7 2.1.1.1 Bacterial characteristics These include ability of organism to survive in the cow’s immediate environment- that is, its resistance to environmental influences including cleaning and disinfection procedures, its ability to colonize the teat duct, ability to adhere to mammary epithelium and its resistance to antibiotic therapy (Blood et al., 2000; Radostits et al., 2000) 2.1.1.2 Transmission mechanisms These depend on bulk of the infection in the environment including infected quarters, efficiency of milking personnel and the susceptibility of the cow. (Garg, 2001) 2.1.2 Course of infection 2.1.2.1 Invasion of the teat The teat itself is the first line of defense against the penetration of the bacteria into the udder. Normally, the sphincter muscle closes the teat canal tightly when the cow is not being milked. Invasion of the teat most often occurs during milking. Organisms present in the milk or at the teat end are propelled into the teat canal. After milking, the teat canal remains open for one or two hours; however, the canal of a damaged teat may remain partially open permanently. Organisms from the environment (manure, beddings) or those found on injured skin at the tip of the teat may easily invade an open or partially open canal (Power, 1999).
  • 17. 8 2.1.2.2 Establishment of the infection and inflammation of the damaged area Some bacteria may proceed into the udder by attaching and colonizing new tissue; others may move around via milk current produced by the cow’s movement. A bacterium first damages the tissues lining the large milk collecting ducts. The bacteria may encounter leukocytes present naturally in small numbers in the milk. These cells are the cow’s second line of defense because they can engulf and destroy bacteria. However, during this process, the leukocytes release substances that cause the movement of additional leukocytes from the blood into the milk. If bacteria are not entirely destroyed, they continue to multiply and begin to invade smaller ducts and alveolar areas. (Cressier et al., 2011; Bissonnette et al., 2011). Milk secreting cells damaged by toxins and other irritants release substances that lead to increased permeability of blood vessels. Additional leukocytes move to the site of infection. They enter the alveolar tissue in great numbers by squeezing through the damaged milk secreting cells. Fluids, minerals and clotting factors also leak into the affected area. Clotted milk may close ducts, and in effect, isolate the infected regions (Bownian, 1986). 2.1.2.3 Destruction of alveolar tissue Sometimes the microorganisms are eliminated rapidly and the infection is cleared. In this case, the clogged ducts are opened and milk composition and production return to normal in several days. However, as the infection persists and ducts remain clogged, the entrapped milk causes the secretory cells to revert to a resting (non - producing) state and the alveoli begin to shrink. Substances released by leukocytes lead to the complete destruction of alveolar structures, which are replaced by connective and scar tissues. The destruction of
  • 18. 9 milk secretory tissue is, in effect the cow’s third line of defense to bring the infection under control. (Halley et al., 1999; Hillerton et al., 1999). 2.2 Disease diagnosis, treatment and control In the diagnosis of mastitis, laboratory procedures are of value in the examination of milk samples for cells, bacteria and chemical changes, and for testing for sensitivity of bacteria to specific drugs. Methods of diagnosis include culture and sensitivity testing, California mastitis test [which is designed to measure the milk's somatic cell count as a means for detecting inflammation and infection of the udder.], Wisconsin mastitis test and catalase test (Marshall, 1992). In this research, the culture and sensitivity testing will be used. Treatment of mastitis can be systemic by parenteral injection or local by intra mammary infusion. The choice depends on the degree of response of infected animal. Parenteral treatment is advisable in all cases of mastitis in which there is a marked systemic reaction, to prevent the development of a septicemia. (Ryan, 1998) Intra mammary infusions are the preferred method of treatment. Disposable tubes containing suitable drugs are used. Strict hygiene is necessary during treatment (Blood et al., 2000; Radostis et al., 2000) after an infusion the gland is not emptied to avoid losing the antibiotic and milking is thus discouraged for some time. Practices such as good nutrition, proper milking hygiene, and the culling of chronically infected cows can help. (Saunders, 1984) Dairy workers should wear rubber gloves while milking, and machines should be cleaned regularly to decrease the incidence of transmission. Vaccination has been attempted for Streptococcal mastide where a systemic hyper immunity
  • 19. 10 is elicited but no apparent intra mammary resistance (Blood et al., 2000; Radostis et al., 2000) Infected animals should be isolated and treated promptly. 2.3 Economic importance of Mastitis Mastitis occurs sporadically in all species and has major economic importance in dairy production. Economically it is one of the most important disease with which the dairy industry has to contend. Most of the losses incurred are based on decline of milk production. In a few cases though, fatalities may occur where cattle die from disease. On average, an infected quarter suffers a 30% reduction in productivity. These losses are supplemented by a loss of about 1% of total solids by changes in milk composition. For instance, fat, casein and lactose are reduced while glycogen, whey proteins and chlorides are increased. These, coupled by an increase in pH of milk interfere with manufacturing processes (Blood et al, 2000; Henderson et al, 2000). Mastitis is a very painful condition and is one of the major welfare problems of dairy cows (Broom & Fraser, 2007; Webster, 1999). Even mild cases of clinical mastitis cause increased responsiveness to pain and affected cows become hyper sensitized to stimuli normally considered innocuous (Fitzpatrick et al., 1998).Cows having experienced one case of clinical mastitis often develop a subsequent case of clinical mastitis later in lactation (Rajala & Gröhn, 1998; Houben et al., 1993). Also, as contagious pathogens use the udder of infected cows as reservoir, having mastitis cows in a herd increases the risk of spreading infection to healthy cows. Mastitis is associated with increased risk of lameness (Peeler et al., 1994; Dohoo & Martin et al., 1984).Some of the case study include investigation of the current status of inflammatory mastitis in smallholder dairy bovine udders in the Rift Valley(Elburgon, Njoro, Nakuru,
  • 20. 11 Naivasha and Nyahururu) of Kenya over a seven-month period. (Shitandi et al., 2014; Milcah et al., 2014) 2.4 Antibiotic mode of action Antibiotics are therapeutic agents used in treatment of microbial diseases. They are naturally produced by certain groups of microorganisms (Woolcock, 1983). Antibiotics may have a cidal or static effect. They can be said to be bactericidal-killing bacteria by damaging the cell membrane that causes the cell metabolites to leak out eventually killing the organism. Bacteriostatic antibiotics stop bacterial growth and multiplication. Broad spectrum antibiotics are effective against a wide range of bacteria while narrow spectrum bacteria are effective against either gram positive or gram negative bacteria. (Courvalin et al., 2006; Guardabassi et al., 2006.)The antibiotics could work in one of the following ways; 2.4.1 Cell wall synthesis inhibition Cell wall synthesis inhibitors generally inhibit some step in the synthesis of bacterial peptidoglycan. They include beta lactams, penicillin, cephalosporin, bacitracin, cycloserine, co-trimoxazole (Woolcork, 1983) 2.4.2 Protein synthesis inhibition These owe their action to inhibition of some step in the complex process of protein synthesis. Their attack is always at one of the events occurring on the ribosome. They include tetracycline, kanamycin, macrolides aminoglycosides, gentamycin and neomycin (Saunders, 1984).
  • 21. 12 2.4.3 Effect on nucleic acids These prevent synthesis of DNA or RNA. This blocks the growth of cells. They include quinolones and rifamycin (Power, 1999). 2.4.4 Cell membrane inhibitors They disorganize the structure of bacterial membrane and thus kill the cell. An example of this is polymixin (Saunders, 1984). The mode of action to be used in this study will be cell wall synthesis and protein synthesis inhibition. 2.5 Antibiotic resistance and causes The introduction of antibiotics into clinical use has almost invariably been followed by the emergence of resistance to these drugs in bacterial populations. Resistance emerging from spontaneous mutation of chromosomal genes encoding a target site is probably of limited importance in clinical setting (Blood et al., 2000; Radostits et al., 2000). This can be combated by the inclusion of a second antibacterial in the treatment regimen. Plasmid and transposon determined drug resistance is of much more importance in clinical situations and has led to widespread multi resistance patterns in certain bacterial populations. Plasmids are extra chromosomal genetic elements that replicate independently. They can be transferred between or within bacterial species and may also act as vectors for transposons (Blood et al., 2000; Radostits et al., 2000). Plasmids encode for single or multiple patterns of resistance. With veterinary pathogens, plasmid determined resistance is particularly important in the Enterobacteriaceae, Staphylococcus aureus and to some extent Pasteurella spp. (Blood et al., 2000; Radostits et al., 2000).
  • 22. 13 Prolonged use of antibiotics and influences such as feeding of antibiotic treated milk to calves may select for resistance among organisms. These resistant organisms can persist in the animal and subsequently form part of the normal flora. Mechanisms of resistance in bacteria include change of target molecule, antibiotic inactivation, sequestering by providing alternative pathways, preventing drug from entering bacterium and pumping out the drug as quickly as it enters the bacterium (Baggot et al., 1985; Prescott et al., 1985)
  • 23. 14 CHAPTER THREE 3.0 RESEARCH METHODOLOGY 3.1 Study Areas and Justification The study will be contacted at Mariakani in Kilifi County. This will be concerned with the analysis of microorganisms causing mastitis and their major trends in sensitivity to major antibiotics. Mariakani is one of the areas in Kilifi County practicing livestock farming thus mastitis may be a major problem affecting farmers. The study will enable farmers in rural places of Mariakani to understand the best antibiotic to be administered in case of the infection. 3.2 Sample size and Sampling This research will be a random experimental study concerned with analysis of mastitis causing microorganisms in cattle and their general trends in sensitivity and resistance to major antibiotics. The study will be based at KALRO (Beef Centre, Mariakani), Regional Veterinary Investigation Laboratory (Mariakani) and Technical University of Mombasa and samples will be from the farmers. Samples will be collected from acute and chronic cases of mastitis and from the different infected quarters of the udder which will be placed in an ice containing thermo- flask and transported to the laboratory as early as possible. Control study will involve the culture of fresh milk. The formulae below will be used to determine the sample size by considering the population size is unknown.
  • 24. 15 n = ( Z2 * σ2 ) / ME2 Where: ME is the desired margin of error =4% Z is the z score set at 95% confidence level σ (alpha) is the significance level i.e. (1 – 0.95) = 0.05 n is the sample size (to be found). n = (1.962*0.052)/0.042 n = (3.8416*0.0025)/0.0016 n = (0.009604)/0.0016 n = 6.0025 Therefore the sample size will be approximately 6 3.3 Laboratory procedure When samples will be delivered to the laboratory and be subjected to culture and sensitivity tests in the bacteriology laboratory. 3.3.1 Culture and sensitivity test Biological: Milk samples Chemical: Blood agar, maconkey’s agar, Glucose broth serum, crystal violet stain, Lugols iodine, acetone, dilute carbol fuchsin, Ethanol Technical: Test tubes, petri dishes, microscope slides, inoculation loop, staining rack, centrifuge, Incubator
  • 25. 16 3.3.2 Bacterial culturing of milk The general appearance of milk samples will first be checked using an alcohol test, 10ml of Milk will be dispensed into labeled tubes according to individual quarters. The milk will be spun in centrifuge at 2500rpm for 10 minutes. The milk will be separated into layers with heavier bacteria settling at the bottom while lighter bacteria settled at the top with the cream. The cream layer will be loosened by heating with a wire loop. The liquid part will be poured out and the sediments mixed thoroughly. A loopful of the sediment will be inoculated onto blood agar and Maconkey’s agar and streaks made. The remnant will be inoculated into glucose broth serum medium. This will be done to boost bacterial growth in case the animal will have been treated thus poor growth on medium. This will be incubated for 18-24 hours at 37oc and Growth will be observed and microscopic identification done. (Cheesbrough et al., 1985) 3.3.3 Gram staining and bacterial identification A thin smear will be prepared and allowed to dry in air. The smear will be passed through the Bunsen flame three times to fix. Crystal violet will be flooded onto the fixed smear for thirty seconds. The slide will be washed thoroughly. Lugols iodine will be poured and left for another thirty minutes. The slide will then be washed again. Acetone will be poured on to differentiate and act as decoloriser. This will be done quickly followed by washing to avoid over differentiating. The slide will be drained well and counter stained with carbol fuchsin for thirty seconds. Gram positive bacteria will be stained dark blue, Gram negative bacteria will be stained pink. (Packer et al., 1967)
  • 26. 17 3.3.4 Sensitivity testing Identified colonies will be sub cultured to obtain pure cultures. Further sub culturing will be done with sensitivity discs containing small amounts of the antibiotics of selected antibiotics. After 18-24 hours, the plates will be observed and regions of inhibition will be noted. Depending on the extent of the clearance zones results will be recorded and analyzed. (Cowan et al., 1970; Steel et al., 1970). 3.4 Data collection and management The results will be recorded in diagnostic books, data will be tabulated and analyzed. Isolates will be classified as susceptible, intermediate and resistant categories based on the standard interpretation tables. Analysis of variance (ANOVA) will be used for the analysis of antibiotic susceptibility results.
  • 27. 18 Work Plan ACTIVITY TIME IN MONTHS [2015 – 2016] SEP OCT NOV DEC JAN FEB MAR APRIL Literature review Proposal Writing and Presentation Sampling and Laboratory work Data Analysis Project Writing and Submission
  • 28. 19 Budget ITEM QUATATION KSHS Blood agar 500 Glucose broth serum 1,000 Crystal violet stain 1,000 Lugols iodine 1,000 Dilute carbol fuchsin 1,000 Acetone 1,000 Petri dishes 500 Antibiotics 3,000 Maconkey’s medium 1,000 Stationary, binding and Printing 1,000 Transport 3,000 Miscellaneous 1,000 TOTAL 15,000
  • 29. 20 REFERENCES 1. Banerjee, K., Ray, J., Sadhukhan, T., Das, R., and Chandra, S. (2002). Studies on etiological agents of subclinical mastitis in dairy cows in West Bengal. Indian Journal of Animal Health, 41(2), 109-112. 2. Barbuddhe, S., Chakuyrkar, E., and Sundaram, R. (2001). Studies on incidence and etiology of bovine mastitis in Goa Region; Indian Journal of Comparative Microbiology: Immunology and infectious Diseases, 22(1), 164-165. 3. Bauer, A., Kirby, W., Sherris, J., and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology, 36(3), 493-496. 4. Begum, H., Uddin, M., Islam, J., Nasir, K., Islam, M., and Rahman, M. (2007). Detection of biofilm producing coagulase positive Staphylococcus aureus from bovine mastitis, their pigment production, hemolytic activity and antibiotic sensitivity pattern; Journal of the Bangladesh Society for Agricultural Science and Technology, 4, 97-100. 5. Bertolatti, D., O'Brien, F., and Grubb, W. (2003). Characterization of drug-resistant Staphylococcus aureus isolated from poultry processing plants in Western Australia. International Journal of Environmental Health Research, 13(1), 43-54. 6. Beytut, E., Aydn, F., Ozcan, K., and Genc, O. (2002). Pathological and bacteriological investigations on bovine mastitis in Kars Region and its surrounds, 8(2), 111-122. 7. Bhattacharya, A. (2002). Etiology and antibiotic spectra of bacterial isolates from field cases of mastitis in cows from West Tripura District. Indian Veterinary Journal, 79(9), 961-962.
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