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ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ MASTITIS, IN DAIRY COWS AND IT’S ECONOMIC IMPLICATIONS IN THE RIVER NILE STATE ( SUDAN) By ISAM MAHMOUD MOHAMED ADAM ( B.V.Sc., University of Baghdad, Iraq, 1986) (M.V.Sc., University of Khartoum, Sudan, 1997) A Thesis submitted to fulfill the requirements of the Ph.D. in veterinary science. Department of preventive medicine and public health University of Khartoum Faculty of veterinary medicine February 2007 Preface This work was undertaken at Atbara Livestock Research Station from 2003 till 2005 and part of this work has been done at Atbara Regional Veterinary Laboratory. I DIDICATION To the soul of my mother, great brothers, sisters and uncles. II father, patient wife, dear Acknowledgements Initially I would like to express my sincerest and deepest gratitude to my academic advisor, Professor Abdel Aziz ElTayeb for diligent guidance, valuable advices and continued encouragement during the course of this study. My gratitude and appreciation are extended to Dr. Ahmed ElGhali, the director of Atbara regional veterinary research laboratory, for the valuable contribution and permission of conducting my work in the laboratory. I would like to extend my dear thanks to Dr. Kamal Abdel Wahab, the director of Atbara dairy research station, and Dr. Ahmed Gamal, general director of ministry of agriculture and animal resources, River Nile State, for their valuable help and financial support. Also my gratitude is extended to Dr. Mohamed Tag El-Dien, Dr. Yassir Ahmed Hassan, Dr. Khalid Mohamed Taha, Dr. Mohamed Osman Hussein, Mr. Mohamed El-Hassan Burhan and to the staff members of Atbara dairy research station and laboratory for their great help and support. Many thanks to all of those who submitted an advice and gave a hand to accelerate the practical work in this study. III Abstract This study was conducted in River Nile State to investigate, the prevalence of mastitis in dairy cows and the economical implication of bovine mastitis, through the reduction in milk yield, milk dumped after antibiotic treatment and the cost of drugs and other costs inferred with mastitis. California mastitis test was performed in dairy farms to identify the infected quarters. The result revealed that about 87.5% of herd individuals were mastitic. The bacteria isolated from milk samples were staphylococci, streptococci, micrococci; coliform bacteria together with mycotic infection. The economical implication of mastitis was evaluated through the calculation of milk yield reduction. Reduction, which represented 67.2% from the total loss, had been assessed through the direct and indirect estimation of somatic cell count. It represented more than 12% of milk yield for one infected quarter. Also the reduction percentage had been assessed through comparison between infected and healthy quarter for the same cow. The reduction percentage of one infected quarters was about 12.5% . The reduction as average was 14.2% (234kg /cow/season). The milk reduction cost was 27325 Sudanese Dinars. The discarded milk due to treatment with antibiotic was 14.5% of the total loss, with total cost 5670 S.D. Drug cost was estimated as 5167 SD(13.2%.). Veterinary services cost 2000Sudanese Dinnars. Represented 5.1 % of total financial loss. The total financial loss was estimated as 30252 SD per cow/year. Cows were investigated to confirm the effect of mastitis on milk yield. The average of total and daily milk yield had been adopted. The IV results showed irregularity in average of total and daily milk yield for the mastitic cows compared to healthy ones. The effect of mastitis on milk yield was confirmed statistically and. The milk yield curve verified the differences in milk yield between mastitic and healthy cows. V اﻟﺨﻼﺻﺔ ﺗﻤﺖ هﺬﻩ اﻟﺪراﺳ ﺔ ﺑﻮﻻﻳ ﺔ ﻧﻬ ﺮ اﻟﻨﻴ ﻞ ﻟﺘﻘ ﺼﻲ ﻣ ﺪي اﻧﺘ ﺸﺎر ﻣ ﺮض اﻟﺘﻬ ﺎب اﻟ ﻀﺮع واﻷﺛ ﺮ اﻻﻗﺘﺼﺎدي اﻟﻨﺎﺟﻢ ﻋﻦ اﻟﻤﺮض ﻓﻲ اﻷﺑﻘﺎر ،واﻟﻤﺘﻤﺜﻞ ﻓﻲ ﻧﻘﺺ إﻧﺘﺎج رﺑﻊ اﻟﻀﺮع ﺟﺮاء اﻟﻤ ﺮض، اﻷﻟﺒ ﺎن اﻟﺘ ﻲ ﻳ ﺘﻢ إﻋ ﺪاﻣﻬﺎ ﺟ ﺮاء ﻣﺰﺟﻬ ﺎ ﺑﺎﻟﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ أﺛﻨ ﺎء اﻟﻌ ﻼج .وآ ﺬﻟﻚ ﺗﻜﻠﻔ ﺔ اﻷدوﻳ ﺔ اﻟﻤ ﺴﺘﺨﺪﻣﺔ ﻓ ﻲ اﻟﻌ ﻼج و أﻳ ﺔ ﺗﻜ ﺎﻟﻴﻒ إﺿ ﺎﻓﻴﺔ أﺧ ﺮى ﺗ ﺪﺧﻞ ﺿ ﻤﻦ داﺋ ﺮة اﻷﺛ ﺮ اﻻﻗﺘ ﺼﺎدي ﻟﻬ ﺬا اﻟﻤﺮض. ﺗﻢ اﻋﺘﻤﺎد ﻓﺤﺺ آﺎﻟﻴﻔﻮرﻧﻴﺎ ﻟﻤﻌﺮﻓﺔ اﻷﺑﻘﺎر اﻟﻤﺮﺿﻴﺔ ﺑﻤ ﺰارع اﻷﻟﺒ ﺎن ،ﺣﻴ ﺚ وﺟ ﺪت ﻧ ﺴﺒﺔ اﻹﺻﺎﺑﺔ ﺑﻤﻌﺪل %87.5ﻣﻦ ﺟﻤﻠﺔ اﻟﻘﻄﻴﻊ وان اﻟﺒﺎآﺘﺮﻳﺎ اﻟﺘ ﻲ ﺗ ﻢ اﻟﺘﻌ ﺮف ﻋﻠﻴﻬ ﺎ آﻤ ﺴﺒﺒﺎت ﻣﺮﺿ ﻴﺔ آﺎﻧ ﺖ ﻣ ﻦ ﻧ ﻮع اﻟﻤﻜ ﻮرات اﻟﻌﻨﻘﻮدﻳ ﺔ واﻟ ﺴﺒﺤﻴﺔ ،واﻟﻌ ﺼﻴّﺎت اﻟﻘﻮﻟﻮﻧﻴ ﺔ ،وآﺎﻧ ﺖ هﻨ ﺎك إﺻ ﺎﺑﺎت ﻓﻄﺮﻳﺔ. ﻋﻠ ﻰ أﺛ ﺮ إﺛﺒ ﺎت اﻹﺻ ﺎﺑﺔ ،ﺗ ﻢ ﺗﻘ ﺪﻳﺮ اﻟﻔﺎﻗ ﺪ ﻓ ﻲ اﻹﻧﺘ ﺎج ﻋ ﻦ ﻃﺮﻳ ﻖ ﺣ ﺴﺎب ﻋ ﺪد اﻟﺨﻼﻳ ﺎ اﻟﺠﺴﻤﻴﺔ ،واﻟﺬي أﺷﺎر إﻟﻰ أآﺜﺮ ﻣﻦ %ٍٍ12ﻟﺮﺑﻊ اﻟﻀﺮع اﻟﻤﺼﺎب .آﻤ ﺎ ﺗ ﻢ أﻳ ﻀًﺎ ﺗﻘ ﺪﻳﺮ اﻟﻔﺎﻗ ﺪ ﻓ ﻲ إﻧﺘﺎج رﺑﻊ اﻟﻀﺮع ﻋﻦ ﻃﺮﻳﻖ اﻟﻤﻘﺎرﻧﺔ ﺑ ﻴﻦ اﻟﺤﻠﻤ ﺎت اﻟﻤ ﺼﺎﺑﺔ واﻟ ﺴﻠﻴﻤﺔٍ .أﻇﻬ ﺮت اﻟﻨﺘ ﺎﺋﺞ أن اﻟﻔﺎﻗ ﺪ ﻓ ﻲ إﻧﺘ ﺎج رﺑ ﻊ اﻟ ﻀﺮع ﺟ ﺮاء اﻹﺻ ﺎﺑﺔ ﺑﻤ ﺮض اﻟﺘﻬ ﺎب اﻟ ﻀﺮع ﺗﺤ ﺖ اﻟ ﺴﺮﻳﺮي ﺑ ـ ،%12.5ﺗ ﻢ اﻋﺘﻤ ﺎد اﻟﻤﻌ ﺪل ﺑ ﻴﻦ اﻟﻄ ﺮق اﻟﻤ ﺴﺘﺨﺪﻣﺔ ﻓ ﻲ ﺗﻘ ﺪﻳﺮ ﻧﻘ ﺼﺎن اﻹﻧﺘ ﺎج ﺑ ـ %14.2ﺧ ﻼل اﻟﻤﻮﺳ ﻢ )234آﺠﻢ( واﻟﺘﻲ ﻗﺪرت ﺑﻤﺒﻠﻎ وﻗﺪرﻩ 27325دﻳﻨﺎر ﺳﻮداﻧﻲ وهﺬا ﻳﺸﻜﻞ ً اﻟﺠﺰء اﻷآﺒﺮ ﻣ ﻦ اﻟﻔﺎﻗ ﺪ اﻻﻗﺘ ﺼﺎدي ﺑﻤﻌ ﺪل ، % 67.2ﺛ ﻢ ﺗﻘ ﺪﻳﺮ اﻷﻟﺒ ﺎن اﻟﻤﺒﻌ ﺪة ﺟ ﺮاء ﻣﺰﺟﻬ ﺎ ﺑﺎﻟﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ﺑ ـ %14.2ﺑﻤﺎ ﻗﻴﻤﺘﻪ 5670دﻳﻨﺎر ﺳ ﻮداﻧﻲ ،أﻣ ﺎ ﺗﻜ ﺎﻟﻴﻒ اﻷدوﻳ ﺔ اﻟﻤ ﺴﺘﺨﺪﻣﺔ ﻓ ﻲ اﻟﻌ ﻼج )(%13.2 ﻗ ﺪرت ﺑ ـ 5167دﻳﻨ ﺎر ﺳ ﻮداﻧﻲ ﻓ ﻲ ﺣ ﻴﻦ آﻠﻔ ﺖ اﻟﺨ ﺪﻣﺎت اﻟﺒﻴﻄﺮﻳ ﺔ ﺑ ـ % 5.1ﻣ ﻦ ﺟﻤﻠ ﺔ اﻟﻔﺎﻗ ﺪ اﻻﻗﺘ ﺼﺎدي وﺑﻤﺒﻠ ﻎ ﻗ ﺪرﻩ 2000دﻳﻨ ﺎر ﺳ ﻮداﻧﻲ ﻟﺘ ﺼﺒﺢ ﺟﻤﻠ ﺔ اﻟﻔﺎﻗ ﺪ اﻻﻗﺘ ﺼﺎدي ﺟ ﺮاء اﻹﺻ ﺎﺑﺔ ﺑﺎﻟﺘﻬﺎب اﻟﻀﺮع ﺗﺤﺖ اﻟﺴﺮﻳﺮي ﻟﻸﺑﻘﺎر ﻣﺒﻠﻎ ﻗﺪرﻩ 30252دﻳﻨﺎر ﺳﻮداﻧﻲ ﻟﻠﺒﻘﺮة /ﻓﻲ اﻟﺴﻨﺔ. ﺗﻢ اﻋﺘﻤﺎد ﻣﻌﺎﻳﻴﺮ أﺧﺮى ﻟﺘﺜﺒﻴﺖ أﺛﺮ ﻣﺮض اﻟﺘﻬﺎب اﻟﻀﺮع ﻋﻠﻰ اﻹﻧﺘﺎج ﻋﻦ ﻃﺮﻳ ﻖ ﻣﺘﺎﺑﻌ ﺔ اﻹﻧﺘﺎج اﻟﻴﻮﻣﻲ آﻤﻌ ﺪل ﻟﻤ ﺪة ﺳ ﺒﻌﺔ ﺷ ﻬﻮر وﻣﻘﺎرﻧﺘﻬ ﺎ ﻓ ﻲ ﻇ ﻞ وﺟ ﻮد اﻹﺻ ﺎﺑﺔ واﻟﺘ ﻲ أﻇﻬ ﺮت ﻋ ﺪم اﻧﺘﻈﺎم ﻣﻌﺪل اﻹﻧﺘﺎج اﻟﻴﻮﻣﻲ وﻓﻖ ﺣﺎﻟﺔ اﻹﺻﺎﺑﺔ . ﺗﻢ اﻋﺘﻤﺎد اﻟﺘﺤﻠﻴﻞ اﻹﺣﺼﺎﺋﻲ ﻟﺪراﺳﺔ اُﺛﺮ ﻣﺮض اﻟﺘﻬﺎب اﻟﻀﺮع ﻋﻠﻰ اﻹﻧﺘﺎج ﻣﻊ ﺟﻤﻠﺔ ﻣ ﻦ اﻟﻌﻮاﻣﻞ اﻷﺧﺮى واﻟﺘﻲ أﻇﻬﺮت ﻧﺘﺎﺋﺞ ذات ﻣﻌﻨﻰ ﻋﻨﺪ ﺗﺪاﺧﻞ اﻟﻌﻮاﻣﻞ ﻣﻊ ﺑﻌﻀﻬﺎ ﻣﺜ ﺎل رﻗ ﻢ اﻟ ﻮﻻدة واﻟﻤﻮﺳﻢ ﻣﻊ ﻓﺘﺮة اﻟﺠﻔﺎف وﻃﻮل ﻓﺘﺮة اﻟﺤﻠﻴﺐ وأﺛﺮهﻢ ﻋﻠ ﻰ اﻹﻧﺘ ﺎج اﻟﻴ ﻮﻣﻲ وﺟﻤﻠ ﺔ اﻹﻧﺘ ﺎج ،اﻷﻣ ﺮ اﻟﺬي ﻳﺆآﺪ ارﺗﺒﺎط ﻋﺎﻣﻞ ﻣﺮض اﻟﺘﻬﺎب اﻟﻀﺮع ﻣﻊ رﻗﻢ اﻟﻮﻻدة واﻟﻤﻮﺳﻢ ﻓﻲ اﻟﺘﺄﺛﻴﺮ ﻋﻠﻰ اﻹﻧﺘﺎج. VI أﺧﻴ ﺮًا ﺗ ﻢ اﻋﺘﻤ ﺎد رﺳ ﻢ اﻟﻤﻨﺤﻨ ﻰ وﻣﻼﺣﻈ ﺔ اﻟﻔ ﻮارق ﺑ ﻴﻦ إﻧﺘ ﺎج اﻷﺑﻘ ﺎر اﻟ ﺴﻠﻴﻤﺔ واﻟﻤ ﺼﺎﺑﺔ ﻳﻮﻣﻴًﺎ آﻤﻌﺪل واﻹﻧﺘﺎج اﻟﺴﻨﻮي ،واﻟﺬي أﺑﺮز ﺑﻮﺿﻮح ﻗﻠﺔ ﻓ ﻲ اﻹﻧﺘ ﺎج ﻋﻨ ﺪ اﻷﺑﻘ ﺎر اﻟﻤ ﺼﺎﺑﺔ ﻣﻘﺎرﻧ ًﺔ ﺑﺎﻷﺑﻘﺎر اﻟﺴﻠﻴﻤﺔ. VII Table of content Preface Dedication Acknowledgement Abstract Arabic Abstract Table of content List of tables List of Figures Introduction 1. Literature Review 1.1. Mastitis 1.2. Classification of mastitis 1.3. Signs of mastitis 1.4. A etiology of mastitis 1.4.1. Etiology of clinical mastitis 1.4.2. Etiology of subclinical mastitis 1.5. Pathogenesis of mastitis 1.6. Factors affecting susceptibility to mastitis 1.6.1. The per partum period 1.6.2. Deficiencies of vitamins and minerals 1.7. Mastitis caused by different organisms 1.7.1. Staphylococcal mastitis 1.7.2. Streptococcal mastitis 1.7.3. Coliform mastitis 1.8. Diagnosis of mastitis 1.8.1. Clinical mastitis 1.8.2. Subclinical mastitis 1.9. Somatic cell count 1.9.1. Advantage of CMT 1.9.2. Factors affecting milk somatic cell count 1.10. Economic importance of mastitis 1.10.1. Somatic cell count and milk reduction 1.11. The milk yield curve and mastitis 1.12. Factors affecting production traits 1.12.1Morphology of the mammary gland 1.12.2.Milk yield 1.12.3. Lactation length 1.12.4. Dry period 2. Materials and Methods 2.1 Location of the study 2.2 Herd Management VIII I II III IV VI VIII XI XII 1 4 4 5 6 7 9 10 10 11 11 11 12 12 13 13 13 14 14 15 16 19 22 23 23 23 24 25 27 27 27 2.3 Data collection and manipulation 28 2.4 Milk tests and sampling 29 2.4.1 California mastitis test. (C.M.T) 29 2.4.2. The direct microscopic somatic cell count 30 2.4.3. pH test 31 2.5. Sampling procedure 31 2.6. Laboratory examination of milk 31 2.6.1. Sterilization methods 31 2.6.2. Preparation of culture media 31 2.6.2.1. Blood agar media 32 2.6.2.2. Edward's medium 32 2.6.2.3. Mac Conkey's agar medium 32 2.6.2.4. Chapman stone medium 32 2.6.3. Media for biochemical test 33 2.6.3.1. 40% bile agar 33 2.6.3.2. Hugh and Leifson's (O.F) medium 33 2.6.3.3. Motility medium 33 2.6.3.4. Aesculin broth. 33 2.6.3.5. Arginine broth 33 2.6.3.6. Mac Conkey's broth purple 34 2.6.3.7. Bromothmol blue 34 2.6.4. Reagents 34 2.6.4.1. Hydrogen peroxide 3 %( Volume10) 34 2.6.4.2. Oxidase test reagent 34 2.6.4.3. Nessler's reagent 34 2.6.5. Culture methods 34 2.6.5.1. Primary isolation 34 2.6.5.2. Examination of inoculated culture media 34 2.6.5.3. Purification 35 2.6.6. Primary identification 35 2.6.7. Identification of bacterial genera 35 2.7 Statistical analysis 37 3. Results 38 3.1. Recorded number of cases with subclinical mastitis and dead quarters in dairy farms examined 38 3.2. The number of cows with positive quarters examined by California mastitis test 38 3.3. The number of CMT performed in farm N0.1 and the number of infected quarters 38 3.4. The allocation of infected quarters 39 3.5. The grading of infected quarter 39 3.6. The relationship between CMT result and microbial isolation 39 IX 3.7. 3.8. The microbial isolated from milk samples 39 Somatic cell counts as they relate to estimated milk losses 39 3.9. The reduction percentage in milk yield 40 3.10. The percentage of infected cows in relation to the milk yield 40 3.11. The Evaluation of the average daily milk yield 40 3.12. Comparison of the milk yield of mastitic and non-mastitic cows 41 3.13. The effect of mastitis on milk yield 41 3.14. The cost of treatment and discarded milk 41 3.15. The assessment of reduction in milk yield in relation to the methods usage 42 3.16. The evaluation of economical loss 43 4. Discussion 61 Recommendations 68 References 69 X List of tables 1234567891011121314151617181920212223- 24- The estimated costs of the factors related to the occurrence of mastitis. 17 Milk loss related to CMT, WMT and somatic cell counts 20 21 The reduction in milk yield in relation to SCC/ml . Reduction of milk yield in relation to various factors 21 Subclinical mastitis and dead quarters in the dairy farms 42 Results of the California Mastitis test . 42 The result of California mastitis tests in farm No (1). 43 Allocation of infected quarters. 43 The grading of infected quarters positive to the CMT. 44 Relationship between C.M.T result and microbial isolation 44 Identification of bacteria isolated. 45 Somatic cell counts as they relate to estimate milk losses 46 Reduction percentage in milk yield. 47 The percentage of infected cows in relation to the milk yield 47 Evaluation of the average daily milk yield 48 Comparison between Mastitic and non- mastitic cows milk yield in both summer and winter (A) Non mastitic cows (B) mastitic 49 The mean effect of mastitis and season on total milk yield 50 Analysis of variance for total milk yield 50 The mean effect of mastitis and season on daily milk yield 51 The analysis of variance for daily milk yield 51 The discarded milk and treatment costs due to the mastitis treatment . 52 Reduction in milk yield as calculated by different methods 54 The economical loss evaluation /cow/season. 55 The expected cost of treatment and discarded milk (prospective) 56 XI List of figures 1- Average daily milk yield (mastitic and non- mastitic) (winter season). 57 2- Average daily milk yield (mastitic and non- mastitic) (summer season). 58 3- Mastitic and non-mastitic total milk yield/cow (summer season) 59 4- Mastitic and non-mastitic total milk yield/cow (winter season) 60 XII Introduction Milk Production System:The production of milk by dairy farmers is not only a business but also a way of life. Many of the decisions taken on the farm are consequently dictated by both financial constraints and job satisfaction. Nevertheless during recent decades there has been a continuing squeeze on profits in the milk production industry as increase in prices which have taken place in systems of milk production are mainly a response to reduced profit per cow. This has given the dairy farmer an increasing awareness of the need to produce milk more efficiently. (Leaver, 1982). River Nile State lies approximately between 22-35 longitude east and 16-22 latitude north, and extend from Elsabuloga falls on River Nile south toward Bayoda desert to the Egyptian boarder north. The River Nile passes through the state from south to north and Atbara river passes obliquely through the State from east to west where it meets the River Nile at Atbara town (Mohamed et. al, 1996). Livestock production system in Africa is classified into intensive, semi- intensive and extensive system according to husbandry practice and distribution of pasture that varies with the rainfall, season or cultivated crop (Pyne 1986). In Sudan 92% percent of livestock population is possessed by nomads that follow extensive system of husbandry in eastern, western and southern part of the Sudan (Kamal, 1983). In Nahr EL-Nil State, the system of husbandry adopted is a semi-intensive one. Among the Sudanese breeds of cattle, two breeds namely Kenana and Butana are known to show high potentiality for milk production (Alim, 1960; Osman, 1970; Osman and EL Amin, 1971). In the area 1 Introduction of the present study, cattle represent the second most important source of milk production. Butana cattle is the dominant breed. Friesian and Butana and Friesian crosses have been recently introduced in the State in order to improve milk productivity to meet the increasing demand for milk as high quality food. In the area where the study was conducted, milk production is so scanty and it is almost always sold by producers in unprocessed form. Little information is available on diseases of cattle in Nahr EL-Nil State. How ever, mastitis, pneumonia and tropical theleriosis represent the major diseases of cattle in the State (Elghali and ELHussien, 1995). Bovine Mastitis Bovine mastitis can be defined as inflammation of the udder resulting from infection or trauma. It is caused by a variety of well known microorganisms, such as Staphylococcus aureus and Streptococcus agalactia. The economical implication of Mastitis Mastitis is known as the disease that causes heavy economic losses to milk producers. The average production loss per lactation for one infected quarter is about 1.600 pounds "weight" (Schroeder, 1997). Other losses are due to discarded abnormal milk and milk withheld from cows treated with antibiotics, costs of early replacement of affected cows, reduced sale value of culled cows, costs of drugs and veterinary services and increased labour costs. Objectives of the Study The present investigation was conducted in Aedammer Province to study: 1. The prevalence of mastitis in dairy cows. 2. The economical implications of bovine mastitis through:• Reduction in milk yield. 2 Introduction • Milk dumped after antibiotic treatment. • Other expenses incurred as a result of mastitis. 3 1- Literature Review 1-1 Mastitis: During the last two decades milk production has grown into a specialized business. Economic pressures make it vital for farmers to increase the profitability of their dairy herds; a goal only to be achieved by the intelligent application such as good husbandry based on the scientific principles involved in breeding, management and feeding of cow (Leaver,1982). It is well established that bovine mastitis is of great economic importance to the dairy industry (Blosser,1979; Meyer,1980; Miller,et. al,1984).The infection of the udder with microorganisms capable of causing mastitis. Hurley and Morin (1996) define mastitis as intramammary infection, primarily bacterial infection, but also mycoplasmal, mycotic, or algal infection. Whereas Jones and Bailey (1998) consider mastitis when the udder becomes inflamed because leukocytes are released into the mammary gland in response to invasion of the teat canal usually by bacteria. These bacteria multiply and produce toxins that cause injury to milk secreting tissues and various ducts throughout the mammary gland. Elevated leukocytes, or somatic cells cause a reduction in milk production and alter milk composition (Jones, and Bailey,1998). Mastitis is also defined by Cole (1962) as inflammation of the mammary gland caused by microbial infection or undue stress or both. Some investigators believe that the mere presence of pathogens in milk is not indicative of mastitis. The increase in somatic cell count and the finding of pathogens in milk samples give positive diagnosis (McDonald, 1977). The two criteria, somatic cell count 4 Literature Review and isolation of pathogens are necessary to differentiate actual cases of mastitis from mere contamination (McDonald, 1977). According to National Mastitis Council’s (1996): mastitis is an inflammation of the mammary gland in response to injury for the purpose of destroying or neutralizing the infectious agents and to prepare the way for healing and return to normal function. Inflammation can be caused by many types of injury irritants. In the dairy cow, mastitis is nearly always caused by microorganisms, usually bacteria that invade the udder, multiply in the milk producing tissues and produce toxins that are immediate cause of injury. 1.2 Classification of mastitis:Craplet (1963) classified the disease on the following criteria: A- By symptoms differentiates between :Acute mastitis that develops rapidly with severe congestion and rapidly but is of infrequent occurrence, easily diagnosed and of reduced economic importance and chronic mastitis, the development of which is more or less obvious clinically with a high frequency in occurrence. B- By clinical characteristics which consider the mastitis condition as gangrenous, catarrhal, parenchymatous or suppurative. C- By stage of lactation :Beginning of lactation, drying off and dry period . D-By the causal organism :Contagious or chronic mastitis caused by Streptococcus agalactia and common mastitis caused by streptococcus groups, staphylococcus groups, Corynebacterium pyogenes, Escherichia.coli Pseudomonas, Proteus, yeast and bacteria species. 5 Literature Review Radostitis, Blood and Gay (1994) classified mastitis into two forms, clinical and sub clinical mastitis. Clinical mastitis is characterized by apparent changes of both milk and mammary gland and subclinical mastitis in which there are no apparent changes. clinical mastitis are classified in to: a- Peracute mastitis: In this type there is marked abnormality of milk and udder with severe systemic reaction. b- Acute mastitis: Characterized by severe inflammation of the udder without marked systemic reaction. c- Sub acute mastitis: There is mild inflammation of the mammary gland and abnormal milk secretion. d- Chronic mastitis: Most changes are detected in the milk with recurrent attacks of inflammation. In subclinical mastitis there are no visible abnormalities of milk or udder. Were subclinical mastitis is characterized by an increase in somatic cell and/ or leukocyte count (Radostitis, Blood and Gay, 1994). It is a problem of the herd rather than individual animals. 1-3. Signs of mastitis: Michel (2000) described the signs of mastitis according to the mastitis form. In clinical mastitis, the infected quarter often becomes swollen, some times painful to touch and the milk is visibly altered by the presence of clots, flakes or discolored serum and some times blood. 6 Literature Review In severe cases (acute mastitis), the cow shows signs of generalized reaction: fever, rapid pulse, loss of appetite and sharp decline in milk production. In contrast, subclinical mastitis is subtle and more difficult to detect. The cow appears healthy, the udder does not show any signs of inflammation and the milk seems normal. However, microorganisms and white blood cells (somatic cells) that fight infections are found in elevated numbers in the milk. Schroeder(1997) characterized subclinical mastitis by lack of consistent, visible and elevation of somatic cells count of the milk. Bacteriological culturing of milk will detect bacteria in milk and this form causes the greatest loss in dairy farms through lowered milk production. 1-4.A etiology of mastitis: Of the several causes of mastitis, only microbial infection is important. Although fungi, yeasts and possibly virus can cause udder infection. 1-4-1. Etiology of clinical mastitis: Microbial causes of clinical mastitis include Staphylococcus aureus (Bryson, 1973), Streptococcus agalactiae (Gonzales et. al, 1988), Escherchia coli (Schukken et. al, 1989), Pseudomonas aeruginosa (Howel, 1972) Klebsiella pneumoniae and Proteus spp ( Park, 1979), Streptococcus dysagalactiae (Schaufuss et. al, 1968), Streptococcus ubris, Streptococcus faecalis, (Schaufuss et. al, 1968), Streptococcus faecium, Streptococcus bovis (Boutrel and Runiewiez, 1984), Micrococcus spp. (Jha et al, 1994), Coagulase negative Staphylococci (Gonzalez et. al, 1988), Pasteurella multocida (Pasco, 1960, and O’sullivan et. al, 1971),Corynebacterium spp. (Costa et al, 1987) Mycobacterium spp .(Oudar et. al, 1966), 7 Literature Review Bacillus cereus (Nielsen, 1972), Yersinia pseudotubercullosis (Messerli, 1972), Serratia marcensce (Kim and Kim, 1979), and Mycoplama spp. (Blood and Henderson, 1989). However the predominant bacterium incriminated in acute bovine mastitis is Staphylococcus aureus (Bryson,1973; Pearson and Macki, 1979; Innes and Lynch, 1990) followed by Streptococcus agalactiae (Gonzalez et. al,1988; Innes and Lynch,1990) or Escherichia coli (Jha et. al, 1994). In the Sudan, Staphylococcus auerus was considered as the major bacteria isolated from bovine clinical mastitis ( Mamoun and Bakheit, 1992) followed by Streptococcus agalactiae. Other organisms isolated include Bacillus cereus (Adlan et. al, 1980), Escherichia coli (Haghour and Ibrahim, 1980) ,Klebsiella pneumoniae (Bagadi, 1972) and Staphylococcus epidermidis . 1-4-2. Etiology of subclinical mastitis: As in the case of clinical mastitis, many organisms have been isolated from cases of subclinical mastitis. These include Streptococcus lactis, and Enterococcus faecalis (Keskintepe et. al, 1992) Staphylococcus Streptococcus aureus, agalactiae, Staphylococcus Streptococcus epidermidis, dysagalactiae, Streptococcus uberis, Escherichia coli, Pseudomonas aeruginosa, (Bozkir, 1986 and Aydin et. al, 1995), Coryne-bacterium spp., Micrococcus spp. (Costa et. al, 1987). In the Sudan several bacteria have been isolated from cases of subclinical mastitis. These include Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium spp., Pseudomonas spp. Streptococcus agalctia, Streptococcus dysagalactia and Micrococcus spp.,(Shalallai et. al, 1992). High incidence of subclinical mastitis has been reported in Khartoum and the 8 Literature Review commonest species of bacteria isolated were Enterococcus faecalis, Enterococcus faecium, Streptococcus bovis, Streptococcus equi, Streptococcus lactis and Streptococcus pyogenes (Hashim et. al, 1991). 1-5. Pathogenesis of mastitis: Infections begin when microorganisms penetrate the teat canal and multiply in the mammary gland. Irritation 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 dilated for one to two hours. Organisms from the environment (Manure, bedding, etc.) or those found on injured skin at the tip of the teat may easily invade an open or partially open canal. Some bacteria may proceed into the udder by attaching and colonizing new tissues; others my move around via milk current produced. Bacteria first damages the tissues lining the large milk collecting ducts. The bacteria may encounter leukocytes (white blood cells) present naturally in small numbers in milk. These cells are the cow’s second line of defense because they can engulf and destroy bacteria. If bacteria are not entirely destroyed, they continue to multiply and begin to invade smaller ducts and alveolar areas. Milk secreting cells are damaged by toxins and other released irritants substances (chemotaxes factors) 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 between the damaged milk secreting cells . Fluid, minerals and clotting factors also leak into the affected area. Clotted milk may close milk ducts. 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 9 Literature Review several days. However as the infection persists and ducts remain clogged , the entrapped milk causes the secretory cells to revert to arresting (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. Thus as the disease progresses the number of somatic cells in the milk becomes elevated and this are associated with a permanent reduction in milk yield (Michel,2000, Hurley and Morin,1996). 1-6. Factors affecting susceptibility to mastitis: Hurley and Morin (1996) discussed the factors which lead to susceptibility of mammary gland to infection. Whether or not intramammary infection occurs depends on the interaction of host, agent and environmental factors. Host factors include, the presence or absence of natural resistance to mastitis, the state of defense mechanisms, the stage of lactation. Whereas agent factors include the number of organisms in the gland, the pathogencity of the organisms and the presence of other virulence factors. In addition the environmental factors include, the milking environment, the milking practices hygiene, the type of housing, bedding and the weather. 1-6-1. The Peripartum period:In the peripartum period, several defense mechanisms are compromised just prior to and after parturition which predispose the gland to mastitis. Fluid volume in the gland increases resulting in increased intramammary pressure and dilatation of the teat canal and sometimes leakage of colostrums. Citrate concentration rises and lactoferrin is low. Phagocytic cells are not efficient in engulfing and killing bacteria in colostrum contained in the gland at this time. 10 Literature Review High immunoglobulin concentration in the gland at this time is not effective in preventing new intramammary infection. IgG1, the major immunoglobulin isotype in cow colostrum, is not normally an effective opsonin in the mammary gland. Antibiotic concentration from dry cow antibiotic therapy is too low to combat infection and teat dipping during this period is not particularly effective in mastitis prevention. 1-6-2.Deficiencies of vitamins and minerals: Deficiencies shown to be related to increased incidence of clinical or subclinical mastitis increased severity of infection , or elevated somatic cell counts, include Selenium, Vitamin E, Vitamin A, Zinc, Cobalt and some others deficiencies.(Hurley and Morin,1996). 1-7. Mastitis caused by different organisms:1-7-1. Staphylococcal mastitis:Staphylococci are widely distributed in nature where they make up the normal bacterial flora of the mucous membranes and the skin (Zingeser et. al, 1991). Staphylococci are gram positive, catalase positive and ferment glucose. They are classified according to the coagulation of human or rabbit plasma in to coagulase postive staphylococci represented by Staphylococcus aureus and coagulase negative Staphylococci such as Staphylococcus epidermidis (Baird and Parker,1962). Staphylococci were found to be the most frequent causative agents of mastitis among cattle (Cargil and Bootas, 1970; Kapur and Singh, 1978). Staphylococcus aureus is known to cause peracute, sub acute and chronic mastitis, in addition to gangerinous mastitis (Radostitis and Gay, 1994). It also acts as contaminant of milk (VanDijk and Swanberge, 1963; Zingeser et. al, 1991; Lafont and Lafont, 1985). On the other hand coagulase negative Staphylococci were identified 11 Literature Review as primary caustive agent of cattle mastitis during first lactation (Timms and Schultz, 1987; Derieze and Keyser, 1980). 1-7-2. Streptococcal mastitis:Streptococci are most common upon skin, mucous membranes and intestine of man and animals (Garge and Mital, 1990). They are classified according to precipitation reaction of specific carbohydrate antigens into 12 groups (Merchant and Packer, 1967). Streptococci are the second most common pathogens isolated from cows' milk (Sharma and Pasker, 1970; Ahmed et. al, 1991). Streptococcus agalactiae represents the major streptococcus species that cause mastitis in cattle (Costa et. al, 1991). However that is known as environmental mastitis. Enterococci that include Enterococcus faecalis, Enterococcus faecium and Streptococcus bovis gain entry into milk and milk products through water supply, equipments and insanitary and unhygienic conditions of production and handling (Hashim et. al, 1991), They have been incriminated as direct or indirect causes of the disease ( Garge and Mital, 1991). 1-7-3. Coliform mastitis:Coliform mastitis is an udder infection of cows caused by Escherichia coli, klebsiela pneumoniae and Enterobacter aerogenes (Odongo and Ambani, 1989) . They are gram – negative bacilli, that inhabit water and soil and transmitted by flies , contaminated water and feed (Merchant and Packer, 1967). Escherichia coli was defined as the most common Gram -negative bacilli associated with clinical and subclinical mastitis (Jha 12 Literature Review et. al,1994; Elliot et al,1976) and causes sudden sharp drop in production of milk (Mustafa et al ,1977) . Escherichia coli was also isolated from udders of cattle at calving and during dry period (Timms and Schultz, 1987) . Escherich coli may cause acute and peracute form of clinical mastitis (Radostitis and gay, 1994). It has been isolated from milk, teat canal and teat wall puncture milk samples as contaminants (Preeze, 1988; Mammoun ,1981). Klebsiella is the second most common Gram-negative bacillus isolated from cattle milk infected with mastitis (McDonald et al 1970; Howel et al, 1972). Enterobacter spp. were found to cause bovine mastitis (Park, 1979; Haghour and Ibrahim ,1980). 1-8. Diagnosis of mastitis:1-8-1. Clinical mastitis:Diagnosis of clinical mastitis can be achieved by visual examination of both milk and mammary gland where the abnormality could be detected easily (Blood and Henderson , 1989) .Confirmation of diagnosis is usually done by the isolation of causative agent (Emanuelson et al,1986). 1-8-2. Subclinical mastitis:Diagnosis of subclinical mastitis presents a problem due to unapparent signs, however several screening tests have been developed beside culturing methods. Culturing method is not suitable for large scale monitoring of udder health (Emanuelson et. al, 1986). Screening tests commonly used include various inflammatory mediators such has somatic cell, cerum proteins, emzymes such as NAGas(N-acetyl-B-D-Glucosaminidase), sodium 13 Literature Review and chloride concentration were conductivity test, directly and indirectly used for somatic cell count.( Kitchen et. al, 1978; Sandholm, 1983 and Mancini et. al, 1995). 1-9. Somatic cell count:Somatic cells are defined as epithelial cells or neutrophils derived from the blood (Schalm and lasmanis ,1968).Normally milk contains somatic cells and the number of these cells in normal quarter milk is less than 10.000 cells / ml/,however , counts as great as 250.000 cell/ml .are found in normal milk samples (Blood and Henderson, 1989). The preferable method used for indirect counting of somatic cell is the California Mastitis Test (C.M.T.) (Brook banks, 1966). California Mastitis Test was developed by Schalm and Noorlander (1957).It contains Aikayl aryl sulphonate that breaks down the DNA of the cells and precipitate them, and bromo cresol purple that impart shade to the tested milk and reveals the alkalinity or acidity of milk. The test is simple and good diagnostic tool for detection of subclinical mastitis. 1-9-1. Advantage of C.M.T:a. The C.M.T .is fairly accurate in measuring somatic cell concentration in milk, correlating well with other tests. b- It is sensitive, primarily developed for sampling quarters; it can also be used on bucket and bulk tank milk samples. c. Foreign material such as hair or other matter does not interfere with the reaction. d. It is inexpensive. e. The test is simple, and little equipments is needed. f. Environmental temperature changes have little effect on the C.M.T. as long as the milk has been refrigerated and is not over two days old(Duane, 1997). 14 Literature Review 1-9-2.Factors affecting milk somatic cell count (SCC):The determination of milk SCC is widely used to monitor udder health and thus milk quality. SCC is readily available to most udder health and thus milk quality. SCC is readily available to most bacteriological culture results, the factors of greatest importance can be determined include the presence of natural resistance to maistits, the state of defense mechanisms, the stage of lactation the number of organisms and the pathogencity. When SCC is elevated , they consist primarily of leukocytes or white blood cells which include macrophages ,lymphocytes and polymorph nuclear neutrophil (PMN) .During the inflammation , the major increase in SCC is due to the influx of PMN into milk .At this time over 90% of the cells may be PMN (Jones and Bailey ,1998). Milk from uninfected quarters displays little change in SCC. As number of lactation or days in milk increase. SCC of milk from uninfected quarters rises from 83,000 at 35 day post- partum to 160,000/ml of milk by day 285. SCC of milk from quarters infected with Staphylococcus aureus was found to rise from 234,000/ml to 1 million over the same period. SCC in uninfected quarters should be less than 300,000/ml by 5 days post partum (Jones and Bailey, 1995). The interpretation of SCC records is particularly applicable to herds experiencing infection from contagious pathogens (Staphylococcus aureas, Streptococcus agalactiae. As infections by these pathogens tend to be of long duration. New infections in a herd may lead to increased prevalence of infection and are reflected by elevated SCC for bulk tank or herds average SCC scores. Well – managed herds that have controlled mastitis due to contagious pathogens and have higher average milk production can experience 15 Literature Review problems with increased cases of clinical mastitis caused by environmental pathogens, yet maintain herd average SCC below 300,000/ml. Data collected on 50,085 Finnish heifers from 1983 through 1991 revealed that, on average a greater percentage of heifers were treated in 1991 than in 1983 (27% vs. 18%). Intramammary infection by environmental pathogens tends to be shorter than those caused by contagious pathogens (Myllys and Rautala, 1995). 1-10. Economic importance of mastitis:Mastitis in developed countries is considered as the most important and main disease of dairy cows (kaneen and Bandhard, 1990). It is of great economic importance due to reduction in milk yield, change in milk quality ,the possibility of permanent damage of a quarter or even the entire udder and death of the cow (ElTayeb and Habiballa,1978). The loss of milk yield due to clinical mastitis may reach 40% whereas in subclinical mastitis it may reach 60% (Dijkhuizen and Stelwagen , 1981). In Sudan, clinical and sub clinical mastitis are lead to substantial (20%) drop in milk (Musstafa et al, 1977). Schroeder (1997) estimated the economic loss from mastitis in the United States is estimated to be approximately $ 185/cow annually. If the loss assumed, the same milk price and this value is multiplied by the total number of milking cows (9.5 million head), the total annual cost of mastitis would be about $ 1.8 billion .This is approximately 10% of the total value of farm milk sales,and about two – thirds of this loss is due to reduced milk production in sub clinically infected cows. Morin et al, 1993 and Stephen ,1999. found that ,the overall financial loss calculated , including cases of mastitis amounting 16 Literature Review $160- $344, also it has been found that the annual costs of mastitis infection were around $ 200-$300in the USA and France (Seegers et al, 1977) and 120pound in England (Fourichon et al, 1977). The National Mastitis Council estimated the annual losses due to mastitis, that the average production loss per lactation for one infected quarter is about 1,600 pounds. "weight" Other losses are due to discarded milk and milk withheld from cows treated with antibiotic , costs of early replacement of affected cows , reduced sale value of culled cows, costs of drugs and veterinary services and increased labor costs .( Schroeder ,1997) . Table No. (1): The estimated costs of the Items related to the occurrence of mastitis Loss per milk cow$ Total % 1- Reduced production 121.00 66% 2- Discarded milk 10.45 5.7% 3- Replacement cost 41.73 22.6% 4- Extra labor 1.41 0.1% 5- Treatment 7.36 4.1% 6- Veterinary services 2.77 1.5% Items involved Source: current concepts in Bovine Mastitis. (National Mastitis Council, 1996). These estimates do not include additional costs arising from mastitis associated problems related to antibiotic residues in human foods, milk quality control, dairy manufacturing, and nutritional quality of milk, degrading of milk supplies due to high bacteria or somatic cell counts, and interference with genetic improvement of 17 Literature Review dairy animals (Schroeder, 1997). Jones and Bailey (1998) found that mastitis cost the U.S. dairy industry about $1.7-2 billions annually or 11% of total U.S. milk production. Much of this cost was attributed to reduced milk production, discarded milk and replacements which are estimated at $ 102, $24 and $33 per cow per year respectively. The obvious costs for treatment labor, and veterinary services were low, can not be eliminated from a herd. However, the total cost of mastitis in the average herd enrolled in DHIA (Dairy herd improvement association) was approximately $171 per cow, which amounted to $18.6 million to the Virginia dairy industry annually (Jones and Bailey, 1998). In the Sudan Ibtisam et. al, 1995. investigated the fate of the cows suffering from the mastitis and found that, (63.6%) dried of the cows were cured, (23.3%) dried off spontaneously, and (7.1%) by management (7.1%) . The study showed that the amount of milk discarded during one year (July 1991 to June, 1992), was 54, 543 liters and this at market price of 19.67 Sudanese pounds/liter, that amount to a little more than one million Sudanese pounds. Moreover a total of 118,894 860 Sudanese pounds were spent on drugs to treat mastitis. Other losses which were not expressed in term of money included extra time spent with individual diseased cows for milking and treating (labor/hr, veterinarian/ hr), loss of genetic potential due to early culling and reduction in milk production due to subclinical mastitis. Many workers established the disease effects in the form of reduced milk production in subclinical mastitis. In subclinical mastitis, decreased milk production results in the greatest loss, representing about 75% of the total loss (Fetrow, 1980). Production loss from udder quarters with subclinical mastitis 18 Literature Review has been assessed for several different causative organisms and ranged from 10% to 26% of quarter yield. Fat content was reduced by between 3% and 12%, while non- fat solids content declined by 2% to 11% (De Graves and Fetrow, 1993) . Assume a decrease in milk production of 10-26% for an affected quarter together with above changes in the quality of the milk produced (Wood and Booth, 1983). As with sub clinical disease, the greatest economic loss in clinical mastitis is reduced milk production .Clinical mastitis has been reported as causing an overall decrease in production (covering all stages of lactation) of between 5.9% and 6.4% whereas an 11% reduction was seen if mastitis occurred before peak milk production (wood and Booth, 1983; Lucey and Rowlands , 1984). Mastitis may also decrease the length of lactation. Wood and Booth, 1983, used mild cases of mastitis five days milk loss (in addition to the sub clinical loss ) was assumed and for acute cases 5.9 –11% reduction in production was assumed. An approximate culling rate due to mastitis of 3% was found each year during a three years study by Whilesmith et al,1986. 1-10-1. Somatic cell count and milk reduction:Milk reduction represented the major impact of sub clinical mastitis. An inflammatory response is initiated when bacteria enter the mammary gland which is the body’s second line of defense. These bacteria multiply and produce toxins, enzymes and cell-wall compound which stimulate the production of numerous mediators of inflammation by inflammatory cells. The magnitude of the inflammatory response may be influenced by the causative agent, stage of lactation, age, immune status of the cow, genetics and 19 Literature Review nutritional status (Harmon, 1994). Polymorph-nuclear-leuckocyte. (PMN), and phagocytes move from bone marrow towards the invading bacteria and are attracted in large number by chemical messengers or chemotactic agent from damaged tissues. Masses of PMN may pass between milk producing cells into the lumen of the alveolus, thus increasing the somatic cell count as well as damaging secretory cells (Jones and Bailey (1998). The Dairy Herd Improvement Association (DHIA) has adopted an SCC scoring system that divides the SCC of compositive milk into 10 categories from 0 to 9 known as linear scores. The DHIA programs determine the SCC on each milk cow each month and report either the SCC or the linear score. Linear scores can be used to estimate production losses, but the average linear score for the lactation most accurately reflects reduced milk yield. Table (2): Milk loss related to CMT,WMT and somatic cell count Lactation Average linear SCC score CMT Score WMT(mm) 2 -ve -- 50,000 --- 3 -ve 2 100,000 3 4 Trace 5 200,000 6 Trace 8 300,000 7 Trace 10 400,000 8 Trace 12 500,000 9 +1 14-21 600,000-1,000,000 10 <2 24 1,200,000 >12 29 1,600,000 5 6 Somatic cell count cells/ml Milk loss% CMT= California Mastitis Test WMT= Wisconsin Mastitis Test Source: Dairy Herd improvement Association and Philpot (1984). 20 Literature Review According to . Tolle (1970) , the mean reduction in the milk yield in infected animals is as high as 17% . However a cell count of less than 500,000 per ml may be is regarded as acceptable for the average herd, even though it represents loss in yield of approximately 4%. Table (3): Reduction in milk yield in relation to SCC/ml SCC/ ml Reduction in milk yield % less than 250,000 - 250,000-500,000 3.9% 500,000-750,000 6.8% 750,000-1,000,000 15.4% More than 1,000,000 18% Source: Federal institute for Dairying , Kiel west Germany (1970). Jones and Bailey (1998) explained the relationship between somatic cell counts, milk production and intramammary infections as shown in table (4). Reduction of milk yield in relation to various factors Foremilk SCC Major pathogens Below 100,000 12,500 25,000 50,000 100,000 100-200,000 200-300,000 300-400,000 400-500,000 500-800,000 Over 800,000 5.9 First lactation 1b milk/day Older lactations 1b milk /day 50.8 50.4 49.7 49.3 48.6 48.2 48.0 47.5 47.1 43.8 64.2 62.9 61.6 60.3 59.2 58.3 57.6 57.2 55.6 51.8 11.7 17.3 18.8 23.5 25.2 19.5 Reichmuth et al , 1970 ; Philpot , 1967 and .King (1978) reported, the reduction in milk production in infected quarters in comparison 21 Literature Review to healthy quarter where indicated to the loss in milk yield in infected quarter from 8.8% -45% with increase in SCC as 10x1000 Anderson (1982) revealed the mean SCC over 10x1000 cell/ml related with loss in milk yield as 259 liter /cow/season. 1.11 The milk yield curve:Many workers was established the effect of mastitis in milk yield, on the other hand, other workers had been proved the milk yield and factors affected on it such as lactation length, dry period, lactation number and season. Wood defined the lactation curve is known to be the graphical representation of the relationship between milk yield and lactation length and also assumed to represent the total milk yield of a single lactation. On the other hand persistency is defined as the slope of the decreasing phase of the curve or it is extent to which peak yield is maintained (Wood, 1967). There fore , peak yield and persistency are the only variables of the lactation curves which are influenced by many factors , although the general shape of the curve remains substantially un changed El- Amin and Osman (1971). Also reported that month of calving did not influence persistency of Northern Sudan zebu cattle . Moon et. al, (1992) reported that winter calving cows had higher and later peak yield and lower persistency than cows calving in other seasons . Mudgal et al (1986) found significantly defferent peak yield between farms and among periods, genetic groups and parities. The importance of the curve lies in predicting the lactation yield by using both completed or part lactation length. Musa (2001) revealed that, the overall mean of milk yield per lactation was 1662.57±108.96 kg with coefficient of variation of 37.22% .The result also showed the effect of parity number on the 22 Literature Review milk yield that showed an increasing yield with advancing parity. The maximum milk yield per lactation was reached after the fifth parity. The milk yield after the fifth parity was significantly (P<.0.05) higher than the yield after the first, second and the third parities while it was similar to the yield in the fourth lactation .Also for the daily milk yield, the maximum was reached in the fifth parities and the daily milk yield of the first lactation was significantly (P<0.05) lower than the other parties for Butana and cross breed, (Musa,2001, Yousif et al, 1998, Sid Ahmed, 1996 and Ali, etal 1988). 1-12. Factors Affecting production traits: 1-12-1.Morphology of the mammary gland: The mammary glands together form a voluminous mass Known as the udder. This weighs between 12-30 kg-and may hold up to 20kg. Of milk, although fused externally the mammae are independent and comprise four glands or quarters the right and left anterior, the right and left posterior. The two posterior quarters are more fully developed than the two anterior, the former secreting 120150% as compared with the latter (C.Craplet,1963). 1-12-2. Milk yield:The milk yield reflects the genetic potential of the herd and is greatly affected by climatic conditions, stage of lactation number, management and feeding. In Sudan khalifa and Shafei (1965) in their study on the milk yield of Sudanese cattle as affected by the age of the cow, reported that no definite trend could be detect for the influence of age at first calving on milk production in the successive lactation. However, they interesting by noted that there was a negative trend for the average life –time production with increase in age at first calving. However , El Amin (1969) mentioned, that 23 Literature Review the yield tended to increase as the age at first calving increased , but the effect of age at first calving on first lactation milk yield was found to be not statistically significant. He also concluded that the month of calving effect was not significant . Ishag(2000) found that the period of calving as well as the parity number had a significant effect on milk yield per lactation .The performance of Butana and kenana cattle was investigated by many researchers, Alim (1962), Fangaly (1980), Abdalla et al, (1990) and El-Habeeb (1991) in the studies on Butana and Kenana cattle at Atbara and Umbenein Research Station calculated the average total lactation milk yield for Butana cows. It was 1419± 8.1kg, 1527.06± 928kg, 1807±562kg and 1599.73±502.9 kg and for kenana cattle was 1511±18.7 kg 1358.91± 819.30kg 2136±168kg and 1423.58±551.7kg for the first four lactation period. Milk yield of the indigenous dairy cattle in tropical countries was studied by many authors. In India for first lactation records of 580 Sahiwal cows studied by Bhatnagar et al, (1983) an estimate of 2083± 33kg for 305-day milk yield was obtain. Taneja and Bhatnagar (1985) reported that the first lactation milk yield for 322 days among Friesian cross–bred cows showed different traits according to the foreign blood percentage and the average was between 4136-5733 (1bs) (Ali et al, 1988) . Tharparkar records were 2289.9kg. In Pakistan, Ahmed and Sivarajasingam (1998) computed the first parity average milk yielded as 1613kg for 909 Sahiwal cows. 1-12-3. Lactation length:Lactation duration is defined as the period between two consecutive calving during which cows are capable of producing 24 Literature Review milk. The length of lactation is one of the lactation curve components. Many researchers have concluded that lactation length is positively correlated with milk yield (El-Amin, 1969; Rao and Dommerholt , 1981; and Bhatnagar et al. (1983) . The common unit of measurement of milk yield in days is a period of 305-days. Mahaderan (1958) showed that the advantage of the 305-days over other measurement is that the former is more closely related to the reproductive cycle of the cow calving once a year. Lactation length for Sudanese indigenous breeds such as kenana and Butana was investigated by several researchers, and it can be concluded that it falls well below the standard period of 305days. For Butana cattle at Atbara Research Station and kenana cattle at Umbenein Experimental station Abdalla et al. (1990) reported lactation length average of 283.2±57 and 283±43 days respectively. Where the Friesian cross-bred cows showed the positive correlation between lactation length and daily milk yield for 50% and was not significant for 62.5% (Ali et al, 1988). 1-12-4. Dry period: The term refers to the period of milking which is necessary for replenishing the body with nutrients that were depleted during lactation, repairing and regenerating the alveolar epithelium and gaining new stimulation for lactation as a result of parturition following gestation. Thus an optimum dry period is essential for maximum production of milk in the subsequent lactation. The standard measurement of dry period in days is a period of 60 days. 25 Literature Review Dry period influences the total lactation yield through its effect on the lactation period. Folley et al. (1972) stated that the cows which were given a longer dry period usually produced 62-75% as much milk in the subsequent lactation as their twins given a rest of bodies between lactation. In Sudan many researchers reported that the Sudanese indigenous dairy cattle have a dry period that exceeds the 60 days recommended in the literature. Alim, 1960, khallafalla, 1977 calculated the mean dry period for the kenana herd at Gezira Research station, Umbenein was 164 ± 94,174±5.1 days respectively .For Butana cattle At Atbara research station Fengaly (1980) reported that the mean dry period was 119.56±102.57 which is an estimate of limited value because of the rather large standard error. Dry period was investigated by many authers in tropical countries .In India Bhatnagar et al (1983) gave an estimate of 139.7 days for mean dry period in first lactation records of 580 Sahiwal cows .Ahmed and Sivarajasingam (1998)in Pakistan stated that the dry period for 909 first parity Sahiwal cows averaged 190.4 days . 1 shag (2000) working on Sudanese data from crossbred cows (Friesian x kenana) concluded that the average dry period for first and overall calving were 90.02±89.18 and 96.31±70.16 days respectively. 26 2- Materials and Methods 2,1 Location of the study: This investigation was carried out at Atbara Livestock Research Station. The station is situated in the River Nile State to the north of Atbara town in Northern Sudan. It is located at 17/42° N Latitude and 33/58° E Longitude at an altitude of approximately 345 meter above sea level. At Atbara there are three seasons: winter (November –February) dry summer (March-June) and wet summer (July-October) (Hewiston,1945). The atmospheric temperature in this area varies from a maximum of 47.7°C recorded in April, to the lowest minimum of 4.5°C was registered in January .However .Atbara falls in atypical semi-desert ecological zone with an average annual precipitation of 70m.m. These are subtype of Northern Sudan zebu. This breed is named after it is home land , the Butana plains of central Sudan (between the River Nil ,Atbara River and the Blue Nile). 2,2. Herd Management:Cows in five farms were fed on irrigated forage crops and grasses together with locally available concentrate feeds .In Atbara dairy Farm animals are allowed to graze sorghum grass and legumes twice daily for five hours whereas in the other farms animals were fed the same grasses in yard. The milking cows are fed a concentrate mixture composed of 34.5% cotton seed cakes or peanut oil meal, 34.5% wheat bran and 30% sorghum grains where in Atbara dairy farm milking cows are fed 50% Molasses , 30% wheat bran, 10% sorghum grains ,5% peanut oil meal, 3% urea and 2% salt and vitamins. Cows depending on their milk yield and the condition of the cows. Some changes to the general pattern of feeding policy 27 Materials and Methods outlined have taken place over the years either as deliberate policy or due to circumstances prevailing at particular occasion . Only natural mating was practiced. Bulls run freely with the appropriate herd groups .However, breeding bulls were selected from the offspring of highest yielding dams in the herd. Calves in Atbara dairy farm are bucket fed on colostrum and fresh milk to an age of one month after which the amount is reduced gradually as the calf advances in age and substitution amount of grass and concentrates are given in small amounts. In the private sectors calves are reared by lactating the dam beside feeding the calf. All cows are milked is done by hand twice daily. Animals are vaccinated against the major infectious livestock disease in Sudan. 2,3. Data collection and manipulation:A) Retrospective study : The data used in this study were extracted from the station records, which have been collected from 1972 until 1993 . The number of cows studied was five hundred and their records extended from first to twelfth lactation. The parameters used in the study were the followings: (1) Milk yield per lactation (2) Daily milk yield (3) Lactation length (4) Lactation number (5) Cow birth date (6) Calving date (7) Dry period (8) Season (9) Mastitis test (10) Milk curve 28 Materials and Methods (11) Discarded milk and the cost (12) Treatment cost B) Prospective studies : 1- Milk yield per lactation . 2- Daily milk yield. 3- Lactation length. 4- Dry period. 5- Season. 6- Mastitis test. 2,4. Milk tests and sampling A total of 136 cows were tested for mastitis using California Mastitis Test from apparently healthy local and cross bred (Local X Friesian). The tested cows belonged to five herds kept in dairy farm around Atbara town. They were at different lactation seasons . 2,4,1. California Mastitis Test. (C.M.T.): C.M.T. was performed on milk samples collected from individual udder quarters to detect subclinical mastitis. Before collection of milk sample for bacteriological examination, 2ml of foremilk were squeezed from each quarter into the cup of the paddle where equal volume of California mastitis test reagent (Alvetera rapid mastitis test kit-Alvetera Gmbh-Germany) was added. The milk and reagent were mixed together and the reaction between them was interpreted according to Schalm and Noorlander (1957) as follows: Negative (-ve):- The mixture remained liquid, homogenous with no evidence of thickening. Trace (T):- There was a slight thickening that was seen best by tipping the paddle back and forth and observing the mixture as it 29 Materials and Methods flows over the bottom of the cup. Trace reactions tended to disappear with continued rotation of the paddle. Weak positive (+1):- A distinct thickening of the liquid formed, but there was no tendency toward a gel formation. Distinct positive (+2):- The mixture thickened immediately, and a gel formation was suggested. Strong positive (+3):- A gel was formed, which caused the surface of the mixture to become elevated like a partially fried egg. (Duane, 1997). The C.M.T. reaction scores. C.M.T. score Average somatic count ( cells/ml) Negative 100,000 Trace 300,000 +1 900,000 +2 2,700,000 +3 8,100,000 2,4,2. The Direct Microscopic somatic cell count (Breed and Prescott test):From the milk 0.01 ml was spread on a fixed area of slide and stained by methylene blue (dissolved in tetra chloroethan and ethanol.) WBC which stained by the blue colour cells were counted under oil immersion lens. 2,4,3. pH test:Of Bromcresol purple was prepared by adding 0.9 g to Bromcresol–purle powder to 100ml distilled water. Of this 1.0 ml was added to 6.o ml of suspected milk. In mastitic milk the colour changed to purple. Bromcresol purple has a pH range of 5.2 to 6.8 30 Materials and Methods changing from yellow to purple. When the solution is added to normal milk the colour remains yellow and when added to mastitis milk the colour change to purple color. (Hall, 1981). 2,5. Sampling procedure:Before collection of milk samples from the tested cows, the teats were thoroughly cleaned using pieces of cotton soaked in 70% alcohol .The first streams were discarded and 5 ml of milk were then drawn into sterile bottles. The collected samples were then transferred in ice to Atbara veterinary Research laboratory where they were tested. 2,6. Laboratory examination of milk:2,6,1. Sterilization methods:Glassware used (bottles , petridishes , cotton–plugged test tubes , flasks and Pasteur’s pipettes )were sterilized at 170°C for one hour using hot air oven (Merchant and Packer,1967). Different laboratory media ,solutions, plastic –stoppers test tubes and screw capped bottles were sterilized by autoclaving at 115120°C (15Ib- in²) for 10-20 minutes (Cowan, 1985). 2,6,2. Preparation of culture media:Different culture media were prepared according to instructions of the manufacturer or as recommended by Cowan and steel (1985). The pH of different media prepared was adjusted using 10% NaoH and 1N-Hcl. Every step of media preparation was conducted under aseptic condition (either under flame or using sterile utensils). All prepared culture media were incubated overnight at 37°C to check their sterility. Unless otherwise stated all inoculated cultured media were incubated at 37°C. 31 Materials and Methods 2,6,2,1. Blood agar media :Twenty three grams of lab-lemco agar (Oxoid, CM.17) were suspended in one litre of distilled water .It was heated to dissolve completely using electric boiler and then sterilized . The media was then cooled to 50°C in a water bath and 5% of sterile defibrinated sheep blood was added and mixed well .The mixture was distributed into sterile petri dishes and left to solidify. 2,6,2,2. Edward’s medium: Forty one grams of Edward’s medium (Oxoid ,CM.27) were added to one liter of distilled water. The medium was then heated to dissolve , sterilized in the autoclave and left to cool to 50°C in a waterbath, 7% of sterile defibrinated sheep blood was added to the medium, mixed well, distributed in sterile petridishes and left to solidify . 2,6,2,3. MacConkey;s agar medium: Fifty grams of MacConkey agar (Merk,5465) were added to one liter of distilled water . It was then dissolved by heating , sterilized , distributed in to sterile petridishes and left to solidify . 2,6,2,4. Chapman stone medium: Of chapman medium (DIFCO, 00313:01) 20.2g were suspended in 100ml. of cold distilled water. It was then heated to dissolve, sterilized, dispensed in sterile petridishes and left to solidify. 2,6,3. Media for biochemical tests:2,6,3,1. 40% bile agar: Forty g of bile salt were added to 1 liter of melted nutrient agar .They were mixed , sterilized and cooled to 55°C .Fifty ml. of sterile equine serum was then added ,mixed well and distributed in sterile petridishes . 32 Materials and Methods 2,6,3,2. Hugh and Leif son's (O, F) medium: Two g of peptone, 5 g of sodium chloride, 0.03 g K2Hpo4 and 3 g of agar were mixed in one liter of distilled water. The medium was dissolved by heating, adjusted to pH7.1 and filtered. 1.5ml of bromo thymol blue was added and sterilized. One percent of appropriate carbohydrate was added, mixed and distributed in sterile tubes. These tubes were then steamed in the autoclave for 30 min. 2,6,3,3. Motility medium:Eighty g of gelatin were soaked in one liter of distilled water for half –an – hour. The 10g of peptone, 3g beef extract, 5g sodium chloride and 4 g of agar were added to the soaked gelatin. It was heated to dissolve, distributed in tubes and sterilized. 2,6,3,4. Aesculin broth:0.5 g of ferric citrate and 5g of aesculin were mixed with 1 litre of peptone water, distributed into Bijuo bottles and sterilized. 2,6,3,5. Arginine broth:Five g of peptone, 5g yeast extract, 2g k2Hpo4 , 0.5 g glucose and 3 g of arginine monohydrochloride were suspended in 1 liter of distilled water. They were then heated to dissolve adjusted to pH 7.0 and filtered. The medium was distributed in Bijuo bottles and sterilized 2,6,3,6. MacConkey’s broth purple:One tablet of MacConkey broth purple (Oxoid,CM.ba) was added to 10 ml of distilled water in a tube containing inverted Durham’s tubes and sterilized. 2,6,3,7. Bromo thymo1 blue: 0.2 g of Bromo thymo1 blue was dissolved in 100ml. Of distilled water. 33 Materials and Methods 2,6,4. Reagents : 2,6,4,1. Hydrogen peroxide 3% (volume 10): This was obtained from local pharmacies. 2,6,4,2. Oxidase test reagent: One gram of tetraethyl p-phenylene diamine dihydrochloride was dissolved in 100ml. of distilled water. 2,6,4,3. Nessler’s reagent: (Philps and Haris –Cat.No.5 59035/0). This was kindly donated by Microbiology Department (Central veterinary Research Laboratory) of soba/ Khartoum. 2,6,5. Culture methods : 2,6,5,1. Primary isolation: Blood agar and Edward’s media were inoculated by transferring a loop full of milk sample onto the surface of each plate and streaked to obtain discrete colonies. Inoculated plates were then incubated under aerobic condition for 3 days 2,6,5,2. Examination of inoculated culture media : Culture media were examined for the growth of the organism's, colony morphology, haemolysis, change in colour and consistency. 2,6,5,3. Purification: Recovered bacteria were purified by picking a single colony that was then streaked onto blood agar plates. 2,6,6. Primary identification : A drop of distilled water was placed on a clean slide . A single colony was taken, emulsified in the drop of water and then spread. The smear was dried, fixed by heating and stained by Gram’s or Ziehl – Nelsen stains (Cowan, 1985). 34 Materials and Methods 2,6,7. Identification of bacterial genera:The genera of the recovered bacteria were identified after Cowan (1985) through primary tests that include: a. Gram’s stain b. Acid-fastness c. Aerobic growth d. Motility Motility media was inoculated by staping the isolated bacteria with straight wire loop. The media was then incubated for up to 3days together with uninoculated media as a control. The growth of non- motile organism was confined to the stab, while the motile one was distributed out the stab. e. Catalase activity: On a clean slide a drop of 3% H2O2 was placed. A single colony from the suspected bacteria on nutrient agar was emulsified and added to the drop of H2O2. Positive reaction was indicated by evolution of gas. f. Oxidase test: A colony of the tested organism was smeared with glass rod across a filter paper saturated with 1% Tetramethyl-pphenylene diamine dihydrochloride. A positive result was shown by the development of dark purple color . g. Oxidation fermentation (O.F) test: Two tubes of O.F. medium were stabbed by the organism under test with straight loop. To one of the tubes, a layer of paraffin oil was added. The two tubes were incubated for 48hrs, with un inoculated tubes as a control. Development of yellowish color in the 2 inoculated tubes indicated fermentation. Whereas oxidation 35 Materials and Methods reaction was indicated by the development of yellow colour in the open tube only. 2,6,8. Identification of bacteria species: 2,6,8,1.Arginine hydrolysis: Arginine broth medium was inoculated with the tested organism and incubated over night with an un inoculated arginine medium as a control. Apositive reaction was indicated by change of colour to brown after addition of 0.25 ml of Nessler’s reagent. 2,6,8,2. Aesculin hydrolysis: Aesculin broth was inoculated with the test organism, incubated with an un inoculated aesculin broth as a control and observed for 7 days for blackening of the medium. 2,6,8,3. Eijkhman test: MacConkey's broth purple medium was inoculated with the tested organism and incubated at 44°C in waterbath for 48 hrs. With an un inoculated medium as a control. Development of a yellow colour in the medium and presence of gas in the inverted Durham’s tube indicated a positive result. 2-7. Statistical analysis:Data obtained were subjected to statistical analysis using Analysis of Variance (ANOVA). 36 3- Results 3,1. Recorded number of cases with subclinical mastitis and dead quarters in dairy farms :The total number of cows in five dairy farms investigated was 408 of which 175 (42.9%) were adult producing cows. Of the adult productive cows 39(22.3%) were dry and 136 (77.7%) were milking. The number f cows with dead quarter are shown in table (5). Ten 10 (7.4%) out of 136 milking cows had one dead quarter. The percentage of cows with one dead quarter varied from 0% (farm No.5) to 40% (farm No.1). One hundred nineteen 119 (87.5) out of 136 were positive reactors for California Mastitis Test (C.M.T) . The percentage of positive reactors varied from 9.2% (farm No.5) to 34.5% (farm No.1). 3,2. The number of cows with positive quarters examined by California Mastitis Test:Table (6) showed the number of quarters examined by C.M.T. Cows that had one positive quarter represented the highest percentage 42.9%.Those with three quarters represented the least percentage (10.9%). 3,3. The number of CMT performed in farm No .1 and the number of infected quarters:On farm No.1, lactating cows had been tested nine times and the positive cases varied from 90.9% as the highest percentage to 23.5 % as the least one. Table (7) shows the percentage of infected quarters within the positive cases 65% was the highest and 25% was the least percentage. 3,4. The allocation of infected quarters:The right posterior quarter represented the highest percentage (27.1%) within the infected quarters where the right anterior quarter represented the least percentage of infected quarter's table (8). 37 Results 3,5. The grading of infected quarters:Based on the CMT , strong positive result (+3) represented the highest percentage (50%) followed by the weak positive (+1) (27.6%) whereas the distinct positive (+2) result represented the least percentage (22.4%)(table9). 3,6. The relationship between CMT result and Microbial isolation:Table (10) show all samples with positive CMT were been positive for bacterial isolation. 3,7. The microorganisms isolated from milk samples:The bacteria isolated from milk samples included Gram-positive cocci, Gram-negative rods and Gram-positive poleomorphic bacilli. The bacteria isolated were staphylococci, stryptococci, micrococci enterobacteria. Fungi were also isolated from the samples (Table 11). 3,8. Somatic cell counts as they relate to estimated milk losses:Indirect estimation of somatic cell counts by CMT is shown in table (12) indirect SCCs varied between 100,000 to 1,200,000 cells. Whereas the direct SCCs varied between 10,000 to 1,980,000 cells/ml of milk. The estimation of milk reduction according to the indirect and direct SCCs ranged from 3% to more Resultsthan 12%. The results also showed that the reduction percentage according the number of infected quarters varied from 10% for one infected quarter to 48% for the four infected quarters (Table12). 3,9. The reduction percentage in milk yield (comparison between infected and healthy quarter yield):In Atbara dairy farm where the artificial rearing is adopted, the titre of milk yield for infected and healthy quarters were monitored for several days and be consistent many times and compared with them. In the other farms, the titre of milk yield was monitored at the same time of testing of 38 Results mastitis. The result revealed the average percentage of milk reduction was 12.5% (Table 13) 3,10. The percentage of infected cows in relation to the milk yield Table (14) showed the percentage of infected cows in Atbara dairy farm where the testing for mastitis was repeated many times. A high percentage of infected cows appeared in cows in lactation No.9,10 (85.7%) and lactation No. 11,12 (77.8%) whereas the least percentage was lactation No.8.The average milk yield/cow in relation to lactation number appeared high in lactation No.1 followed by lactation No.6, however, the least yield was in lactation No.10,11,12 followed by lactation No.3. The average milk yield /cow/day was highest No.1 is followed by lactation No.6 where the lactation No. (10, 11, 12.) gave the least average day milk yield. 3,11. The evaluation of the average daily milk yield:A total of 12 milking cows had been studied for their daily yield from calving to seven months. The average milk yield per month had been adopted .The result appeared irregularity in the average milk yield from the first to the seventh month (table 15). 3,12. Comparison of the milk yield of mastitic and non mastitic cows:The table (16, A,B) represented the mean reduction in milk yield per cow. Comparison between the mastitic and non mastitic cows is made. Taking into consideration the lactation number, total milk yield, of cows in one lactation number in both summer and winter season. 3,13. The effect of mastitis in milk yield:The mean effect of mastitis on total milk yield demonstrated that, there was no significant effect (presented in table 17). While the mean of effect of season showed that there was significant effect of season in winter than the summer. 39 Results Analysis of variance showed that, interplay between season and mastitis; mastitis, parity and season had significant effect (P<0.03), (P<0.05) respectively on total milk yield. Where the mastitis and season alone had no significant effect (P> 0.05). On the other hand, the parity had highly significant effect (P< 0.002) on total milk yield (table 18). The mean effect of mastitis on daily milk yield is presented in table (19). The mean showed that there was no significant effect of mastitis while the mean effect of season revealed that there was significant effect in winter. The analysis of variance for daily milk yield demonstrated that there was no significant effect of mastitis alone or as interaction with other factors (table20). 3,14. The cost of Treatment and discarded milk : From the records, 54 mastitis cows had been chosen to calculate the treatment cost and the milk dumped after antibiotic usage with the treatment period. The result revealed, the average period of treatment was 10 days and the period variability extended from 2days to 42 days . The average yield of the milk dumped after antibiotic usage was 50.4kg and the cost of discarded milk as average with updating the prices cost was 5670 SD/cow. Where the treatment cost denoted as average per cow to 5167 SD (table No.21). 3,15. The assessment of reduction in milk yield in relation to the methods usage: Table (22) showed the different methods used in the assessment of the reduction percentage in milk yield of infected quarter. The average reduction in the milk yield was 14.2% of the total milk yield per cow/season. 40 Results 3,16. The evaluation of economical loss : The economical losses were assessed according to the cost of reduction in milk yield in case of subclinical mastitic (one infected quarter ) that had been evaluated by 27325 SD represented as percentage by 67.2% followed by discarded milk (5670SD)(14.5%), treatment 5167(13.2%) and veterinary service 2000 (5.1%). The other costs (table 24) included the clinical cases and the consequence of the disease that had been probabilities between the kind of infection (peracute, acute and subacute or chronic form), the milk reduced due to the infection, the sort of treatment according to the infection, the dead quarter and finally, the culling, pre mature culling and mortality. 3,17. Milk yield carve: Fig(1)(2) showed the comparison between the non – mastitic curve of the average milk yield per cow and the curve of mastitic cow in summer and winter season .Also Fig (3)(4) showed the different shape of curve for non- mastitic and mastitic cows for average daily milk yield in summer and winter season. 41 Table (5) Subclinical mastitis and dead quarters in the dairy farms Farm No. Herd number 1 2 3 4 5 Total 90 150 120 25 23 408 Number of cows Dry cows 44 14 55 18 41 7 17 4 18 3 175 46 (43.9%) (26.3%) Lactating cows Number of cows with dead quarters 30 37 41 13 15 136 (77.7%) 4(40%) 3(30%) 2(20%) 1(10%) -(0%) 10 (7.4%) Number of cases with subclinical mastitis 41(34.5%) 28(23.5%) 26(21%) 13(10.9%) 11(9.2%) 119 (87.5%) Table (6) Results of the California Mastitis test Number of cows tested Negative test 44 37 41 13 15 150 Total 3 9 15 4 31 (20.7%) Number of cows with positive quarters One Tow Three Four quarter quarters quarters quarters 19 13 6 3 12 6 3 7 10 7 3 6 5 6 2 5 3 1 2 51 35 13 20 (42.9%) (29.4%) (10.9%) (16.8%) 42 Table (7) Results of California mastitis test in farm no. (1) Test No. Test No.1 Test No.2 Test No.3 Test No.4 Test No.5 Test No.6 Test No.7 Test No.8 Test No.9 Number of the tested cows 30 17 18 24 22 20 22 12 15 Number of negative cases 15 13 7 10 2 5 11 5 6 Number of positive cases 15(50%) 4(23.5%) 11(61.1%) 14(58.3%) 20(90.9%) 15(75%) 11(50%) 7(58.3%) 9(60%) Number of infected quarter 27(45%) 4(25%) 22(50%) 25(44.6%) 46(57.5%) 39(65%) 27(61.4%) 13(46.4%) 19(52.8%) Table (8) Allocation of infected quarters Farm No. 1 2 3 4 5 T Right anterior quarter 19 25.8% 11 18.3% 11 20% 8 32% 4 18.2% 53 22.5% Left anterior quarter 18 24.3% 13 21.7% 17 30.9% 3 12% 6 27.2% 57 24.1% 43 Right Posterior quarter 18 24.3% 16 26.7 13 23.6% 9 36% 8 36.4% 64 27.1% Left posterior quarter 19 25.8% 20 33.3 14 25.5% 5 20% 4 18.2% 62 26.3% Table (9) The grading of infected quarters positive to the CMT. Farm No. 1 2 3 4 5 Total +1 45 9 8 2 64 (27.6%) +2 9 14 11 12 6 52 (22.4%) +3 16 37 36 13 14 116 (50%) Table (10) Relationship between C.M.T result and microbial isolation Farm Number of lactating cows Number of sample Number positive for C.M.T result 1 2 3 4 5 44 37 35 13 15 10 13 10 5 11 10 13 10 5 11 44 Number positive for Bacterial isolation 6 7 4 5 7 Number positive for Fungal isolation 4 6 6 0 4 Table (11) Identification of bacteria isolated Test Farm(1) Number of samples Growth on blood agar Growth on MacConkey agar Growth on Edward mdium Catalase test Bromocresol purple Shape of bacteria 10 13 10 5 Farm(5)] 11 +ve +ve +ve +ve +ve 4+ve 6+ve 3+ve 3+ve 6+ve 4+ve 4+ve 3+ve 2+ve 6+ve 6+ve +ve 7+ve +ve 7+ve +ve 2+ve +ve 5+ve +ve G+ve cocci and pleomormphic bacilli +ve G+ve cocci and Gve rods +ve G+ve cocci -ve -ve 3+ve -ve -ve F.O 3+ve -ve -ve F.O 3+ve -ve -ve F.O 1+ve -ve -ve F.O 2+ve 3+ve 3+ve 3+ve 1+ve 2+ve 4+ve 3+ve 4+ve 3+ve 3+ve 3+ve 3+ve 1+ve 6+ve -ve -ve 4+ve 2+ve 4+ve -ve 3+ve 1+ve 3+ve 6+ve. Growth aerobically Oxidase test Motility test O.F test Growth on Chapman media Arginine hydrolysis Aesculin test Growth on 10% NaCl Eijkhman test Growth on 40% bile agar F.O Farm(2) Farm(3) Farm(4) 45 +ve G+ve G+ve cocci cocci and G+ve and G- pleomorphic ve rods +ve +ve Table No (12) Somatic cell counts as they relate to estimated milk losses Cow DCMT ISCe Red% DISCC Red % 3/2 7/2 20/2 21/2 30/2 5/5 6/5 7/5 8/2 32/2 31/2 12/5 14/5 15/5 29/2 1/5 11/5 2/2 26/2 33/2 3/5 12/2 17/2 34/2 4/5 1 q+2 1 q +2 1 q +2 1 q +2 2 q +2 1 q +2 1 q +2 2 q +2 1 q +3 1 q +3 1 q +3 1 q +3 1 q +3 1 q +3 2 q +3 2 q +3 2 q +3 3 q +3 3 q +3 3 q +3 3 q +3 4 q +3 4 q +3 4 q +3 4 q +3 12 х104 12 х104 12 х104 12 х104 24 х104 12 х104 12 х104 24 х104 16 х104 16 х104 16 х104 16 х104 16 х104 16 х104 32 х104 32 х104 32 х104 48 х104 48 х104 48 х104 48 х104 64 х104 64 х104 64 х104 64 х104 12 12 12 12 24 12 12 24 12 12 12 12 12 12 24 24 24 36 36 36 36 48 48 48 48 22х104 85 х104 72 х104 35 х104 21 х104 103 х104 98 х104 113 х104 36 х104 28 х104 26 х104 198 х104 141 х104 117 х104 43 х104 87 х104 135 х104 76 х104 82 х104 50 х104 107 х104 31 х104 10 х104 145 х104 94 х104 6 11 10 7 11 11 6 12 7 6 6 >12 12 12 8 11 12 10 11 9 12 8 3 12 11 DCMT= Degree of the California mastitis test DISCC= Direct somatic cell count Red %= Reduction percentage ISCC= Indirect somatic cell estimate q = quarter 46 Table (13) Reduction percentage in milk yield (comparison between infected and healthy quarters) Allocation of inf. quarters 27 1(ra) 7 2(la) 11 3(rp) 7 3(rp) 5 4(lp) 6 1-2(ra,la) 6 1-2(ra,la) 6 3-4(rp,lp) 7 3-4(rp,lp) 7 3-4(rp,lp) The total average reduction / quarter = Cow Numb. The C.M.T score +2 +1 +2 +3 +1 +1-+3 +3 +2 +3 +3-+2 % of milk Reduction 15.8% 17.55% 18.9% 17.9% 5.3% 35.8% 12.05% 40.2% 2.175% 8.1% 12.5% 1= right anterior 2= left anterior 3= right posterior 4= left posterior Table (14) The percentage of infected cows in relation to the milk yield L.N T.M.Y 1 2 3 4 5 6 7 8 9 10,11,12 46098.9 39123.4 16810.3 13299.1 10916 12248.3 19231.1 11703 10793.5 11382 N.of cow 22 24 13 10 7 6 10 8 7 9 116 Av.per Av.LL Av.D.M Inf.cows % cow (54.5) 12 7.1 297 2095.4 (45.8) 11 5.8 283 1630.1 (61.5) 8 5.2 247 1293.1 (50) 5 5.6 237 1329.9 (28.5) 2 5.1 306 1559.4 (66.6) 4 6.5 316 2041.4 (70) 7 5.5 352 1923.1 (25) 2 5.3 275 1462.9 (85.7) 6 5.5 278 1541.9 (77.8) 7 4.2 296 1264.7 16141.9 The average percentage of infected cows= 56.54% The average percentage of total milk yield Per cow/ season = 1614.19 kg TMY = Total milk yield LN = Lactation number Av. LL = Average of lactation length Av. DM = Average daily milk yield 47 Cow No 13 15 17 19 21 22 30 35 36 45 46 61 Table (15 ) Evaluation of the average daily milk yield MTR 1st 2nd CD L. N MTD Month Month 20.01.04 9 13.4.04 4 q +3 4.2 4.6 10.7.04 4 q +3 26.11.03 9 22.1.04 (AL,PL)+3 7.2 6.5 13.4.04 4 q +3 28.12.03 7 13.4.04 Pr+3 7.9 7.6 11.02.04 5 13.4.04 Ar+3 6.9 7.6 26.04.04 5 13.4.04 Pl+1 10.4 11.4 06.04.04 6 13.4.04 Ar+3 6.7 6.8 19.02.04 2 13.4.04 Al+3 7 6.9 10.04.04 3 13.4.04 3 q +2 4.8 5.4 22.11.03 3 13.4.04 3q 4.8 3.7 +1,+2+3 09.03.04 3 13.4.04 2 q +2 5.3 8.1 29.11.04 3 13.4.04 4 q +1 6.9 7.3 21.2.04 2 13.4 3 q +2 7.7 6.4 3rd month 5.4 4th month 6.2 5th month 6 6th month 5.3 7th month 3.6 5 5.7 4.9 2.6 2.4 8.1 6.7 9.4 7.2 6.7 4.8 3.4 8.3 5.9 7.9 9.2 6.8 4.5 3.8 8.3 7.5 8 6.2 6.5 4.4 4 8.2 5.9 8.7 5.9 5.7 4.3 4.4 7.3 6.3 7 5.4 5.8 3.3 4.1 5.7 7 7 6.4 6.9 7 6.2 6.1 6.4 6.2 6.2 6.5 5.8 6 6.8 CD=calving date LN=lactation number MTR = mastitis test result MTD= mastitis test date q = quarter 48 Table (16) Comparison between Mastitic and non- mastitic cows milk yield in both summer and winter . (A) (Non mastitic cows) Lact .no 1 2 3 4 5 6 7 8 Summer season Cow 9 6 2 3 4 1 3 3 number T.M.y 15057.7 6813.2 2832 2974.7 6132.2 2809.5 4714.6 4158.1 Winter season Cow 1 7 3 2 1 1 3 number T.M.y 1053 12279.7 3370.2 3338.6 2150.3 3855 4455 Table (16) B Comparison between Mastitic and non- mastitic cows in milk yield in both summer and winter(Mastitic cows) Lact .no 1 2 3 4 5 6 7 8 Summer season Cow 11 6 3 4 2 1 3 1 number T.M.y 1254.5 8711.6 4224.7 5673.4 2633.5 1320 7082.9 1790.3 Winter season Cow 1 5 5 1 3 4 1 number T.M.y 540 4791.3 5943.1 1312.1 4263.8 7451.6 1299.6 AV./M.Y.d =The average of milk yield per day AV./D.P = The average of dry period 49 9 3 5151.7 9 6 7695.8 7 9328 Table (17): The mean effect of mastitis and season on total milk yield. Source 1- mastitis Mean Standard error (+ve) (-ve) 2- Season Summer Winter 1167.4(A) 1252.7(A) 52.5 53.5 1212.5 1209.3(A) 49.4 57.1 Means with same letter are not significantly deferent (P>0.05) Table (18): Analysis of variance for total milk yield. Source Season Mastitis Parity Season &mastitis Season & Parity Mastitis & Parity & Season Mastitis & Parity Error Total Df 1 1 11 1 10 11 9 423 498 ** = significant at P <0.01. * = significant at P< 0.05. N.S. = Not significant. Df= degree of freedom MS= Mean square F = value of significance 50 MS 135976.8 1129008.1 1011210.2 1614855.9 615880.6 659621.7 546596.4 365861.8 F 0.73 N.S. 3.09 N.S. 2.76 ** 4.41 * 1.68 N.S 1.80 * 1.49 N.S. Table (19): The mean effect of mastitis and season on daily milk yield: Source 1- Mastitis (+ve) (-ve) 2- season Summer Winter Mean Standard error 4.3 (a) 4.4 (a) 0.15 0.15 4.5 4.2 (a) 0.14 0.17 Means with same letter are not significantly different P>0.05. Table (20): The analysis of variance for daily milk yield: Source Season Mastitis Parity Season & mastitis Season & Parity Mastitis, Parity & season Mastitis & Parity Error Total df 1 1 11 1 10 11 9 452 497 MS 8.46 2.11 8.24 6.25 3.59 4.42 3.54 3.09 ** = significant at P <0.01. N.S. = Not significant Df= degree of freedom MS= Mean square F = value of significance 51 F 2.74 0.68 2.76 2.02 1.16 1.43 1.15 N.S. N.S. ** N.S. N.S. N.S. N.S. Table (21) The discarded milk and treatment cost due to the mastitis treatment No of cows 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Treatment period Discard milk/kg 112 90 66 92 27 18 90 38 19 44 36 74 14 30 14 30 29 38 33 17.6 29 46 50 120 135 39 125 85 42 18 day 21 10 9 3 13 12 4 2 20 5 10 2 4 2 4 15 16 8 4 7 5 8 12 42 7 30 15 6 52 Cost of treatment /SD 50366 3490 2920 3430 1590 2380 4750 1120 510 1840 6000 1328 560 1000 1920 500 2150 2360 3960 2060 4760 140 1500 4240 15750 3900 4080 13000 6640 Continuous Cont. table (21) The discarded milk and treatment cost due to the mastitis treatment No of cows 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 AV. Discard milk/kg 60 79 3 4 6 104 21 17 84 57 120 9 60 140 25 30 40 13 31 40 52 14 42 18 80 50.4kg Treatment period 9 27 2 3 4 15 3 3 11 6 15 2 13 30 4 7 8 2 9 7 14 3 13 16 20 10days 53 Cost of treatment /SD 6430 7390 360 560 2140 2530 3705 1590 8800 5000 11350 1000 2300 16850 840 3370 2500 1000 13500 16140 6000 1250 2500 4840 8640 5167 Table No.(22) Reduction in milk yield as calculated by different methods used. Methods Reduction % 1- Through the somatic cell count a- Per infected one quarter 12% b-Per infected two quarters 24% c- Per infected three quarters 36% d- Per infected four quarters 48% e- Through Tolle (1970) procedure 18% 2- Through the comparison between infected and healthy quarter. a-The average of reduction in case of one infected quarter 12.5% b-The average of reduction in case of two infected 25% quarters c-The average of reduction in case of three infected quarters d-The average of reduction in case of four infected quarters The average of reduction percentage in average of total milk yield /cow/season 54 37.5% 50% 14.2 234 kg Table (23) the economical loss evaluation /cow/season Source of loss quantity 1-Reduction in milk yield/cow for one infected quarter 14.2%=234kg 2-Discaded milk = 50.4kg 3- Treatment cost = 5167.25SD 4- Other costs had been included as a- Veterinary services b- Culling and death cost (i) culling with replacement (ii) culling with out replacement (iii) death c- dead quarter (RA,LA,RP,LP) (i) In case of Anterior quarter =18.75 (ii) In case of posterior quarter = 31.25 55 cost 27325 SD 5670 SD 5167.25 SD 2000 SD 50.000 SD 111.596 SD 181594 SD 34049 SD 56749 SD Table No( 24) The expected cost of treatment and discarded milk (prospective) parity Average of milk yield No Sort of case 1 a b c d Subclinicl case One infected anterior quarter 2 infected ant.quarter one infected posterior quarter 2infected posterior qoarter 6.10 2 a b c d clinical case one infected anterior quarter 2 infecred ant. quarters one infected posterior quarter 2 infected post. quarters 6.10 Discarded milk Discarded milk cost 1.14kg 2.28kg 2kg 4kg 128.25 256.5 225 450 1800SD 3600SD 1800SD 3600SD 128.25 256.5 225 450 acute 4450 7450 4450 7450 1.14kg 2.28kg 2kg 4kg 56 Treat ment cost chronnic 1800 3600 1800 3600 Figure (1) Mastatic & non- mastatic- Average daily milk yeild ( winter season) 8 7.4 7 6.5 6.3 6.1 6 5.7 5.3 Av. D. M.Y. 5 5.1 4.9 4.8 4.4 4.3 4 4.1 4 3.9 3 2.6 2 1 0 9 8 7 6 5 4 Parity 57 3 2 1 Non- mastatic Mastatic Figure (2) Average daily Milk Yeild (Mastatic &non-mastatic) Summer season 7.6 8 5.5 5.3 4.9 5.7 5.8 5.2 4.4 4 4.1 4 5.3 4.2 3.8 3 2 1 0 Parity Av. D. M.y. 5 5.3 6 5.8 6.3 6 5.8 7 Parity 58 mastatic non mastatic Figure (3) (Mastatic &non-mastatic) Total milk yeild/ cow ( Summer season) 2809.5 3000 2500 2361 991.6 1673.1 1451.9 958.3 1500 1140.5 1000 500 9 9 8 8 7 7 6 6 5 5 4 4 Parity 59 3 3 2 2 1 1 0 M.Y/cow 1418.3 1316.7 1135.5 1320 1416 1571.5 1386 1282.6 1533 1717.2 2000 1790.3 Series1 Series2 Series3 Figure (4) (mastatic & non mastatic) Total milk yeild /cow ( Winter season) 4500 4000 3855 3500 Milk yeald 3000 2500 non mastatic mastatic 2150.3 2000 1862.9 1754.2 1669.3 1500 1485 1299.6 1332.6 1421.3 1312.1 1188.6 1123.4 1000 958.3 540 500 10.53 0 9 8 7 6 5 4 Parity 60 3 2 1 4-Discussion Bovine mastitis can be defined as the inflammation of the udder resulting from infection and is generally thought to be the most economically important disease in dairy cows. Losses in the U.S.A dairy industry alone amount to well over $2 billion/year. Prevalence of the disease in cows approaches or exceeds 25% of quarters at any time (Hurely and morine, 1996). Mastitis results in reduced quantity and quality of milk and milk products. Costs can be divided into those where, there is a decrease in revenue and those where there is an increase in outlay. Decreased milk production, discarded milk and lost value of cull cows are components of the former. While increased costs of replacement due to early culling, increased drug usage and increased veterinary and labour costs are examples of the later (Melnerney and Turner, 1989). In this investigation, the California mastitis test had been conducted as the main tool to verify the mastitis problem and its economical implication. Confirmatory tests such as bacterial isolation, somatic cell count and bromocresol purple had been performed .The study showed that, 10(7.4) out of 136 milkers had one dead quarter. These finding are inline with those reported by Zingeser et al. (1991) about health condition of udders. The dead quarter may be due to a consequence of tick infestation on the udder (Idris et al., 1991) or as the result of persistence of mastitis (El Tayeb and Habiballa, 1978). The apparently healthy cow can harbor subclinical mastitis, which creates tremendous loss in milk production. The present infection creates an elevation of somatic cell count in milk. The unseen infections are detected by several methods, including the direct microscopic somatic cell count (DMSCC), the Wisconsin Mastitis test (WMT), and the California Mastitis test (CMT). The DMSCC and WMT are laboratory tests however the CMT is a valuable tool yielding rapid results (Daune, 2000). Several studies had been published proofing positive correlation between CMT and somatic cell count of milk (Rao and Rao, 1986) and between CMT and the 61 Discussion presence of pathogen in both individual udder quarter and composite milk sample (Edmondson and Marshal, 1962; Ansley and Boul, 1965; and Brook Banks, 1966). In the present study, the number of cases with subclinical mastitis were represented by 119 (87.5%) out of lactating cows, while from data collected from health records of dairy, they were represented by (43.3%) Michel, (2000) reported, that the subclinical form are the vast majority of mastitis cases. How ever, for every one clinical mastitis, there were 20 to 40 subclinical cases. Subclinical mastitis in study involving one infected quarter represented the highest percentage followed by infection in two quarters. Infection of three quarters was least. infection of the posterior quarters was more frequent (27.1%, 26.3%) than that of the anterior quarters (22.5%, 24.1%). Similar result were obtained by Herms (2001) who reported that the two posterior quarters were most frequently infected than the two anterior ones. Carplet (1963) reported that the two posterior glands were more fully developed than the anterior ones and that the former glands secreted 120-150% milk as compared with latter. In this investigation, the microorganisms isolated from infected quarters were staphylococcus, streptococcus coliform, micrococcus and fungi. Many workers had reported that the staphylococci were found to be the most frequent causative agent of mastitis among cattle (Cargil and Bootas, 1970, Kapur and Singh, 1978, Shalalli et al, 1992 Aydin etal., 1995). The commonest bacteria isolated from subclinical mastitis were streptococus species (Hashim et al., 1991 keskintepe et al., 1992) and micrococcus spp. (Shalalli, et al., 1982 and Costa, et al., 1987). Coliforms causing mastitis included Escherichia coli, klebsiella pneumoniae and Enterobacter aerogenes. These bacteria are inhabitant of water and soil and are transmitted by flies contaminated water and feed (Merchant and Packer, 1967). Escherichia coli was considered as the most common Gram negative bacillus associated with clinical and subclinical mastitis (Elliot etal., 62 Discussion 1971 Jha et al.,1994) .Also Candida albicans was established as a causative agent of subclinical mastitis (Nizaml et al., 1989 Ognean et al., 1992). Somatic cell counts is one of the confirmatory methods to monitor udder health and thus milk quality. In the present study, somatic cells were counted directly microscopically or indirectly by CMT. The results showed the direct SCC ranged from 10x104 to 19.8x105. This reflects an elevation in SCC above the normal level confirming the evidence of mastitis. This finding is in accord with Jones and Bailey (2000) who reported, that many of the cows with SCC over 20x104 cells/ml had subclinical mastitis. Schroeder (1997) stated that normal milk should have less than 200,000 cells/ml. On the other hand, the estimation of somatic cell counts had been made using grads of CMT. (Dairy Herd Improvement Association) (DHIA)( 1984) and National Mastitis council (1996). This procedure had been adopted in this investigation. The results showed that, the somatic cell counts ranged from 60x104 and over 10x105 cells/ml of milk. Reduction in milk yield had been estimated through several methods. Based and SCC. Dairy Herd Improvement Association (DHIA 1984), National Mastitis Council (1996) and Schroeder (1997) estimated of reduction 10% and > 12% from the total milk yield/cow, the somatic cell counts was between 60x104 and 1.2x105 cell/ml. The Federal Institute for Dairying (Tolle 1970) estimated the reduction percentage in the total milk yield as 3.9% to 18% when SCC, was 25x104 and more than 10x105 cells/ml. In this investigation direct SCCs ranged from 10x104 to 19.8x105 and the reduction in milk yield ranged from 3% to 12% of milk yield for one infected quarter. Whereas indirectly (CMT), the estimated SCCs ranged from 10x104 to10x105 and the reduction in milk yield ranged from 10% to >12% of milk yield for one infected quarter. Comparison of milk yield of infected and healthy quarters showed a reduction of 12.5 % of milk yield for one infected quarter. This represented 67.2% of the total financial loss due to mastitis. Estimated financial loss due to reduction in milk yield was found to be about 27,325 Sudanese Dinnars. 63 Discussion Many workers collaborated in assessment of reduction percentage due to mastitis. Fustes, et al. (1986) reported that total loss for one infected quarter was about 10.36%. Wagner and Stull (1978) concluded that, mastitis had reduced the milk yield. This reduction amounted to per quarter 13.2% in mastitic cow when compared with a healthy parallel quarter. More over, Fetrow (1986) reported that the greatest loss of milk production was attributed to subclinical mastitis and represented by 75% of the total loss. Whereas Ahlner (2003) found it to cause 70% of the losses in milk production .De Graves and Fet row, (1993) found that the production loss from udder quarters with subclinical mastitis was from 1026%. This was also confirmed by Oleary and Leavitt (1983) who studied the effect of infusion of E.coli in quarters. The reduction in milk yield was 14.5%. While Meyer (1980) found that, milk yield from udder quarters with subclinical mastitis was 20.8% lower than from parallel healthy quarters. Moreover, he could estimated a total reduction in milk yield of about 13.2% in mastitic cows. The other financial losses found in this investigation were due to treatment cost and veterinary services. The percentage of milk dumped after treatment represented 14.5%, of total cost followed by treatment cost (13.2%) and veterinary service (5.1%). Many workers had obtained similar result with some simple variables, which may be due to the status of cow productivity. Fustes et al (1986) estimated the discarded milk to be 6.8% of the total financial loss. Where the drug cost was 1.93%, the heifer replacement 1.52% and veterinary service 0.36% to. Where National Mastitis council (1996) by Shroeder (1997) reported that the discarded milk percentage about 5.7%, replacement cost 22.6%, extra labor 1.14%, Drug cost 4.1% and veterinary services about 1.5%. Blosser (1979) reported about 70% of the loss was due to reduced milk yield, 11% to nonutilizable milk and the cost arising from treatment amounted to 1.7%. The simple variabilities between the results may be due to the status of the cow, mistreatment, drug cost variability. In the present study, the amount of reduced milk as averaged about 234 kg./one infected quarter/ season. Miller et al. (1983) 64 Discussion estimated the milk losses throughout lactation as 180 and 129 kg for Swedish Friesian and Swedish -Red cows respectively. Where Heeschen (1984) revealed that the losses in milk yield due to mastitis were 386kg/cow/year. Also Schroeder (1997) reported that the average production loss per lactation for one infected quarter was about 1600 pound "weight". Further more, Raubertas and Shook (1982) reported that, the yield loss for each increase in log cell count, was associated with a decrease of 135kg. This variability in amount of milk loss may be attributed to the stage of lactation. Ibtisam (1995) found there was significant effect of the week of lactation on which infection had occurred on milk yield ( p<0.05). Also, Lucey and Rowlands (1984) observed a reduction between peak of production and 10 weeks after peak. In the present study loss the reduced milk yield for one quarter infected with subclinical mastitis as an averaged of 14.2% (234kg) which cost 27325 Sudanese Diners. Whereas the average of discarded milk due to the antibiotic usage was 14.5% of the total loss which cost 5670SD. Treatment was (13.2%) cost 5167SD of the total and the veterinary service (5.1%) by 2000SD. The average total loss value of mastitis impact about 30252 SD for one infected quarter/ season. This was in agreement with the finding of Schroeder (1997) who estimated the annul loss due to mastitis were U.S.$184.00 . Also Dijkuizen and Stelwagen (1982) found the losses due to mastitis were Franc 125/cow. Websid (1989) found mastitis in about 40% of cows with at least two quarters affected causing loss of about U.S.$182.00/cow /year . While Jones and Baily (2000) explained that, the total cost of mastitis in average herd enrolled in DHIA was approximately U.S.$171 per cow. To verify effect of mastitis on milk yield, the study adopted the average daily milk yield for the mastitic cows for seven months. Also the average of total milk yield for mastitic and healthy cows with the same parity and season. The result demonstrated an irregulatory of average daily milk yield .and average of 65 Discussion total milk yield /cow. This may be due to many factors. The dominant of them may be the mastitis factor .Many workers had evaluated the milk yield per cow and reported that, an increasing milk yield related with advancing of parity number. Also revealed that, the maximum milk yield per lactation was reached after the fifth parity. Moreover, the milk yield after the fifth season was significantly (p<0.05) higher than the yield after the first, second and third parities, while it was similar to the yield in the fourth lactation. For the daily milk yield, also the maximum yield was reached in the fifth parities and the daily milk yield of the first lactation was significantly (p<0.05) lower than the other parities. This was in agreement with Sid Ahmed,(1986); Ali et al.,(1988),Yousif et al.,(1998) and Musa, (2001). Figure (1), (2), (3) and (4), illustrates of average daily milk yield and average of total milk yield. Regarding the productive traits, the mean effect of mastitis on total milk yield had no significant effect, while the season had significant effect in winter. The analysis of variance for total milk yield revealed that, there was no significant effect of mastitis alone. This may be due to the fact the effect of mastitis had cleared. In addition the allocation and kind of infection may be play a role in the effect of mastitis on total milk yield. Moreover, the other factors such as parity, dry period and lactation length had more significance. While the analysis of variance revealed that, there was significant effect of mastitis and season; mastitis, parity and season as display. This in line with Miller et al. (1983) who estimated the milk losses throughout lactation based on single test / day. Also Bauman, (1994) reported that, there are several factors predispose the dairy cow to mastitis such as season, parity, herd, stage of lactation and environment. They reported that, there is a positive relation between milk yield and incidence of mastitis. Musa, (2000); Yousif et al. (1998) and Ali, et al.,(1988) reported that, there is a significant effect of parity number and period on milk yield. The analysis of variance for the daily milk yield revealed that, there was no significant effect of 66 Discussion mastitis alone or as interaction with other factors. While parity had highly significant effect on daily milk yield. This may be due, to the fact that the daily milk yield was obtained from the total milk yield with lactation period as average. 67 Recommendations Veterinarian must be adopt this recommendation to protect the owner from mastitis infection through. 1- Milking parlour must be constructed for any dairy farm and this is must be supported by low and conductor. 2- Dairy farmers must be aware of three major factors that lead to mastitis the microorganisms as the causative agent of the disease, the cow as the host and the environment which can influence both the cow and the microorganisms. 3- Mastitis consequence must be understood by the owners who must recognize with the economical loss. 4- Mastitis management programs must be adopted in any dairy farm such as • Keep the cow's environment clean and stress –free. • Isolate the mastitic cows at milking time. • Monitor Somatic cell count in bulk tank, causative agent and sensitivity test. 5- Use proper milking procedure as:• Wash only teats and lower udder surface using water and disinfectant. • Dry each teat thoroughly with individual paper towels • Check quarters for abnormalities each milking by stripping. • Dip or spray teats, especially post. 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