ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ Identification of Aerobic Bacteria Isolated From Vagina of Cross-Bred Dairy Cows during Early Postpartum In University Farm, Khartoum North, Sudan M.Sc. Microbiology by Research By Nahid Bashir Mohmoud Sadig B .V.Sc, University of Khartoum (1998) Supervisor Dr. Suliman Mohamed Elhassan A thesis submitted in fulfillment of the requirements of the Master degree in veterinary Medicine Department of Microbiology Faculty of veterinary Medicine University of Khartoum 2010 ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ Dedication To my dear Mother To my dear Father To my Brothers and Sisters To my family, Husband , Sons and Daughter To my Colleagues and Friends With love Nahid i Acknowledgements First of all my thanks and praise is due to almighty Allah, the Merciful, for giving me health and strength to accomplish this work. Then I am most grateful for superb assistance, continuous guidance, encouragement, and meticulous attention, patience offered by my supervisor Dr. Suliman Mohamed El Hassan. My deep appreciation and thanks are extending to my father, brothers and sisters for their unlimited moral support and encouragement during the period of this study. My very best thanks are expressed to my husband Hassan Hussein for his moral support and efforts exerted by him. My thanks are also extended to my friend Dr. Faisal Alzubair department of obstetrics and gynecology Faculty of Veterinary Medicine ,University of Khartoum. and technicians and assistants in the Department of Microbiology mainly Fawzia, Mona and A/Azeem. ii Abstract A study was carried out to identify the bacteria which are found in the reproductive tract of dairy cows during parturient and to find the appropriate treatment. The area of study was Khartoum University Farm at Shambat, Khartoum North, Khartoum State. The cows examined were 4 – 8 years old cross-bred dairy cows (Friesian × Kenana). A total of 80 vaginal swabs were collected from 55 postparturient cows. These swabs were collected within 3 days after delivery. Thirty vaginal swabs were collected 1 – 3 days after parturition. Twenty – five vaginal swabs were collected from cows 1-3 days after parturition before treatment with antiseptic 1% Lugol's iodine and then vaginal swabs were collected from the same twenty – five cows 24 hours after treatment with the antiseptic 1% Lugol's iodine. The vagina of postparturient cow was sampled by sterile cotton wool. The collected swabs were inoculated on blood agar and MacConkey's agar. The isolated bacteria were identified by their cellular morphology, arrangement, staining reaction and biochemical properties. The antibacterial sensitivity of the isolated bacteria was examined to Ampicillin, Gentamysin, Chloramphincol, Cloxacillin, Erythromycin, Pencillin, Tetracycline, Streptomycin, Naladixic acid, Nitrofurantol, Cloistin and Cotrimoxazole and 1% Lugol's iodine. The indentified bacteria were Staphylococcus, Streptococcus, Corynobacterium, Bacillus, Escherichia, Kelbsielle, Aerococcus, Micrococcus and Proteus. The Gram positive bacteria isolated from 30 postpartum vaginal swabs within 1-3 days after parturition were Staphylococcus spp, Streptococcus spp, Bacillus spp, Micrococcus spp, Aerococcus spp, Lactobacillus spp and Corynobacterium spp. The Gram- positive bacteria isolated from 25 postpartum vaginal swabs before treatment with 1% Lugol’s Iodine were Staphylococcus spp, Streptococcus spp, Bacillus spp, Micrococcus spp and Corynobacterium spp. The Gram-positive bacteria isolated from 25 postpartum vaginal swabs after 1-3 days after treatment with 1% Lugol’s Iodine were Staphylococcus spp, Streptococcus spp, Bacillus spp, and Corynobacterium spp. The Gram- negative bacteria isolated from 30 postpartum vaginal swabs within 1 – 3 days after parturition were E. coli, Klebsiella spp, Protues spp, Enterobacter spp. The Gram- negative bacteria isolated from 25 postpartum vaginal swabs before treatment with 1% Lugol’s Iodine were E.coli, Klebsiella spp and Protues spp. The Gram- negative bacteria iii isolated from 25 postpartum vaginal swabs after treatment with 1% Lugol’s Iodine were E.coli, Klebsiella spp and Protues spp. The sensitivity of the isolates to antibacterial agents was variable. They were less sensitive to the commonly used drugs like Tetracycline and Cloxacillin. This may be due to misuse of these drugs, while they were highly sensitive to others like Gentamysin and Ampicillin which are less commonly used. The sensitivity of the isolates to 1% Lugol's iodine was variable. Some isolates were resistant and a few were sensitive. iv ﺍﻟﻤﺴﺘﺨﻠﺹ ﺃﺠﺭﻴﺕ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻤﻌﺭﻓﺔ ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺍﻟﺘﻲ ﺘﻭﺠﺩ ﻓﻲ ﻤﻬﺒل ﺃﺒﻘﺎﺭﺍﻟﺤﻠﻴﺏ ﺃﺜﻨﺎﺀ ﻓﺘﺭﺓ ﺍﻟﻨﻔﺎﺱ ﻭﻹﻴﺠﺎﺩ ﺍﻟﻌﻼﺝ ﺍﻟﻤﻨﺎﺴﺏ ،ﻭﺫﻟﻙ ﻓﻲ ﻤﺯﺭﻋﺔ ﺠﺎﻤﻌﺔ ﺍﻟﺨﺭﻁﻭﻡ ﺒﺸﻤﺒﺎﺕ ،ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻱ، ﻭﻻﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ.ﻓﺤﺼﺕ ﺃﺒﻘﺎﺭﺘﺭﺍﻭﺡ ﻋﻤﺭﻫﺎ ﺒﻴﻥ 4ﻭ8ﺴﻨﻭﺍﺕ ﻤﻥ ﺴﻼﻟﺔ ﻫﺠﻴﻥ ﻤﻥ ﺍﻟﻔﺭﻴﺯﻴﺎﻥ ﻤﻊ ﺍﻟﻜﻨﺎﻨﺔ .ﺠﻤﻌﺕ 80ﻋﻴﻨﺔ ﻤﻬﺒﻠﻴﺔ ﻤﻥ 55ﺒﻘﺭﺓ ﻓﻲ ﻓﺘﺭﺓ ﺍﻟﻨﻔﺎﺱ ،ﺨﻼل ﺜﻼﺜﺔ ﺃﻴﺎﻡ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ30.ﻤﻨﻬﺎ ﺠﻤﻌﺕ ﻤﺎ ﺒﻴﻥ ﺍﻟﻴﻭﻡ ﺍﻷﻭل ﻭﺍﻟﻴﻭﻡ ﺍﻟﺜﺎﻟﺙ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ25ﻋﻴﻨﺔ ﺠﻤﻌﺕ ﻤﻥ ﺃﺒﻘﺎﺭ ﺃﺜﻨﺎﺀ ﺍﻟﻴﻭﻡ ﺍﻷﻭل ﺇﻟﻲ ﺍﻟﺜﺎﻟﺙ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻭﻗﺒل ﺍﻟﻤﻌﺎﻟﺠﺔ ﺒﺎﻟﻤﻁﻬﺭ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل ﺜﻡ ﺠﻤﻌﺕ ﻋﻴﻨﺎﺕ ﻤﻥ ﻨﻔﺱ ﺍﻟﺨﻤﺱ ﻭﻋﺸﺭﻴﻥ ﺒﻘﺭﺓ ﺒﻌﺩ 24ﺴﺎﻋﺔ ﻤﻥ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺒﺎﻟﻤﻁﻬﺭ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل. ﺃﺨﺫﺕ ﺍﻟﻌﻴﻨﺔ ﻤﻥ ﺍﻟﻤﻬﺒل ﺒﻭﺍﺴﻁﺔ ﻤﻤﺴﺤﺔ ﻗﻁﻥ ﻤﻌﻘﻤﺔ .ﺯﺭﻋﺕ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺠﻤﻭﻋﺔ ﻓﻲ ﻭﺴﻁ ﺃﺠﺎﺭ ﺍﻟﺩﻡ ﻭﻤﺎﻜﻭﻨﻜﻲ.ﻋﺭﻓﺕ ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺍﻟﻤﻌﺯﻭﻟﺔ ﺒﻨﺎﺀ ﻋﻠﻰ ﺤﺴﺏ ﺸﻜل ﺍﻟﺨﻠﻴﺔ ﻭ ﺘﻔﺎﻋﻼﺘﻬﺎ ﺍﻟﺼﺒﻐﻴﺔ ،ﻭﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﺯﺭﻋﻴﺔ ﻭﺍﻟﺼﻔﺎﺕ ﺍﻟﻜﻴﻤﻭﺤﻴﻭﻴﺔ.ﺍﺨﺘﺒﺭﺕ ﺍﻟﺤﺴﺎﺴﻴﺔ ﻟﻠﻤﻀﺎﺩﺍﺕ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ: ﺃﻤﺒﺴﻠﻴﻥ ،ﺠﻨﺘﺎﻤﺎﻴﺴﻴﻥ ،ﻜﻠﻭﺭﺃﻤﻔﻴﻨﻴﻜﻭل ،ﻜﻠﻭﻜﺴﺎﺴﻴﻠﻴﻥ ،ﺇﺭﻴﺜﺭﻭﻤﺎﻴﺴﻴﻥ ،ﺒﻨﺴﻠﻴﻥ ،ﺘﺘﺭﺍﺴﺎﻴﻜﻠﻴﻥ، ﺇﺴﺘﺭﺒﺘﻭﻤﺎﻴﺴﻴﻥ ،ﻨﺎﻟﻴﺩﻴﻜﺴﻴﻙ ﺃﺴﻴﺩ ،ﻨﻴﺘﺭﻭﻓﻴﻨﻴﺘﻭل ،ﻜﻭﻟﻴﺴﺘﻴﻥ ،ﻜﻭﺘﺭﻴﻤﻴﻜﺴﻭل ﻭﺍﻴﻀﹰﺎ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل .ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺍﻟﺘﻰ ﺘﻡ ﺍﻟﺘﻌﺭﻑ ﻋﻠﻴﻬﺎ ﻜﺎﻨﺕ ﻋﻨﻘﻭﺩﻴﺔ ،ﻋﻘﺩﻴﺔ ،ﻭﺘﺩﻴﺔ ،ﻋﺼﻭﻴﺔ ،ﺇﺸﺭﻴﻜﻴﺔ ﻗﻭﻟﻭﻨﻴﺔ ،ﺍﻟﻜﻠﺒﺴﻴﻠﺔ ،ﺍﻹﻴﺭﻭﻜﻭﻜﺱ ،ﺍﻟﻤﻜﻭﺭﺓ ﺍﻟﺩﻗﻴﻘﺔ ﺍﻟﻤﺼﻐﺭﺓ ﻭﺍﻟﻤﺘﻘﻠﺒﺔ .ﺃﻤﺎ ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﻤﻭﺠﺒﺔ ﺍﻟﺠﺭﺍﻡ ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ 30ﻋﻴﻨﺔ ﻤﻥ ﻤﻬﺒل ﺍﻷﺒﻘﺎﺭ ﺨﻼل 3 –1ﺃﻴﺎﻡ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻓﻜﺎﻨﺕ ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﻨﻘﻭﺩﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﻘﺩﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﺼﻭﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻤﻜﻭﺭﺓ ﺍﻟﺩﻗﻴﻘﺔ ﺍﻟﻤﺼﻐﺭﺓ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﺍﻹﻴﺭﻭﻜﻭﻜﺱ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﺼﻭﻴﺔ ﺍﻟﻠﺒﻨﻴﺔ ،ﻭﺒﺎﻜﺘﻴﺭﻴﺎ ﻭﺘﺩﻴﺔ .ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﻤﻭﺠﺒﺔ ﺍﻟﺠﺭﺍﻡ ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ 25ﻋﻴﻨﺔ ﻤﻬﺒﻠﻴﺔ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻗﺒل ﺍﻟﻤﻌﺎﻟﺠﺔ ﺒـ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل ﻜﺎﻨﺕ :ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﻨﻘﻭﺩﻴﺔ، ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﻘﺩﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﺼﻭﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻤﻜﻭﺭﺓ ﺍﻟﺩﻗﻴﻘﺔ ﺍﻟﻤﺼﻐﺭﺓ .ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﻤﻭﺠﺒﺔ ﺍﻟﺠﺭﺍﻡ ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ 25ﻋﻴﻨﺔ ﻤﻬﺒﻠﻴﺔ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻭﺒﻌﺩ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺏ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل ﻜﺎﻨﺕ: ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﻨﻘﻭﺩﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﻘﺩﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻋﺼﻭﻴﺔ ،ﺒﺎﻜﺘﻴﺭﻴﺎ ﻭﺘﺩﻴﺔ ،ﻭ ﺒﺎﻜﺘﻴﺭﻴﺎ ﻤﻜﻭﺭﺓ ﺍﻟﺩﻗﻴﻘﺔ ﺍﻟﻤﺼﻐﺭﺓ .ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺴﺎﻟﺒﺔ ﺍﻟﺠﺭﺍﻡ ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ 30ﻋﻴﻨﺔ ﻤﻥ ﻤﻬﺒل ﺍﻷﺒﻘﺎﺭ ﺨﻼل3–1ﺃﻴﺎﻡ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻜﺎﻨﺕ :ﺍﻹﺸﺭﻴﻜﻴﺔ ﺍﻟﻘﻭﻟﻭﻨﻴﺔ ،ﺍﻟﻜﻠﺒﺴﻴﻠﺔ ،ﺍﻟﻤﺘﻘﻠﺒﺔ ﻭﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺍﻟﻤﻌﺩﻴﺔ .ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺴﺎﻟﺒﺔ ﺍﻟﺠﺭﺍﻡ ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ 25ﻋﻴﻨﺔ ﻤﻬﺒﻠﻴﺔ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻗﺒل ﺍﻟﻤﻌﺎﻟﺠﺔ ﺒـ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل ﻜﺎﻨﺕ :ﺍﻹﺸﺭﻴﻜﻴﺔ ﺍﻟﻘﻭﻟﻭﻨﻴﺔ ،ﺍﻟﻜﻠﺒﺴﻴﻠﺔ ،ﺍﻟﻤﺘﻘﻠﺒﺔ .ﺍﻟﺒﺎﻜﺘﻴﺭﻴﺎ ﺴﺎﻟﺒﺔ ﺍﻟﺠﺭﺍﻡ ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ 25ﻋﻴﻨﺔ ﻤﻬﺒﻠﻴﺔ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻭﺒﻌﺩ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺒـ %1ﺍﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل ﻜﺎﻨﺕ :ﺍﻹﺸﺭﻴﻜﻴﺔ ﺍﻟﻘﻭﻟﻭﻨﻴﺔ ،ﺍﻟﻜﻠﺒﺴﻴﻠﺔ ،ﻭﺍﻟﻤﺘﻘﻠﺒﺔ .ﻜﺎﻨﺕ ﺍﻟﺤﺴﺎﺴﻴﺔ ﻟﻠﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﺒﺎﻟﻨﺴﺒﺔ ﻟﻠﻌﻴﻨﺎﺕ v ﺍﻟﺘﻲ ﺘﻡ ﻋﺯﻟﻬﺎ ﻓﻲ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻤﺨﺘﻠﻔﺔ ،ﻓﻘﺩ ﺃﻅﻬﺭﺕ ﺒﻌﺽ ﺍﻟﻌﻴﻨﺎﺕ ﺇﺴﺘﺠﺎﺒﺔ ﻀﻌﻴﻔﺔ ﻟﺒﻌﺽ ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴﺔ ﺍﻟﺸﺎﺌﻌﺔ ﺍﻹﺴﺘﻌﻤﺎل ﻤﺜل ﺍﻟﺘﺘﺭﺍﺴﺎﻴﻜﻠﻴﻥ ﻭﺍﻟﻜﻠﻭﻜﺴﺎﺴﻴﻠﻴﻥ ﻭﺫﻟﻙ ﺭﺒﻤﺎ ﻟﺴﻭﺀ ﺍﻹﺴﺘﻌﻤﺎل ،ﺒﻴﻨﻤﺎ ﻜﺎﻨﺕ ﺍﻹﺴﺘﺠﺎﺒﺔ ﻋﺎﻟﻴﺔ ﻟﻠﻤﻀﺎﺩﺍﺕ ﺍﻷﺨﺭﻱ ﻜﺎﻟﺠﻴﻨﺘﺎﻤﺎﻴﺴﻴﻥ ﻭﺍﻷﻤﺒﺴﻠﻴﻥ ﺍﻟﺘﻲ ﻗﻠﻤﺎ ﺘﺴﺘﻌﻤل ﻋﺎﺩ ﹰﺓ .ﺤﺴﺎﺴﻴﺔ ﺍﻟﻌﻴﻨﺎﺕ ﻟﻤﻌﺯﻭﻟﺔ ﻓﻲ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻠﻤﻁﻬﺭ %1ﺃﻴﻭﺩﻴﻥ ﻟﻭﻗﺎل ﻜﺎﻨﺕ ﻤﺨﺘﻠﻔﺔ ،ﺒﻌﺽ ﺍﻟﻌﻴﻨﺎﺕ ﻜﺎﻨﺕ ﻤﻘﺎﻭﻤﺔ ﻭﺍﻟﻘﻠﻴل ﻤﻨﻬﺎ ﺃﻅﻬﺭ ﺤﺴﺎﺴﻴﺔ. vi Table of Contents Dedication ………………………………………………………………… i Acknowledgment....................................................................................... ii Abstract …………………………………………………………………… iii Arabic abstract …………………………………………………………… v Table of contents......................................................................................... vii List of tables............................................................................................... xii List of figures.............................................................................................. xiii List Of abbrevitioin.............................................................................................. xiv introdution.............................................................................................. xv CHAPTER ONE: Literature Review 1 1. 1 Postpartum period (postpartum) in dairy cows 1 1. 1. 1 Definition 1 1.1.2 Endocrinology of the postpartum period 2 1.1. 2.1 Luteinising hormone 2 1.1. 2. 2 Oestradiol 2 1.1. 2. 3 Progesterone 2 1.1. 2. 4 FSH 3 1.1. 2. 5 Oestrogens 3 1.1. 2. 6 Oxytocin 3 1.1. 2. 7 Prostaglandin 3 1.1. 2. 8 Prolactin 4 1.1. 2. 9 Insulin 4 1.1. 3 Iodine 4 1.1. 4 Suckling 4 1.1. 5 Milk yield 5 1.1. 6 Mastitis 5 1.1. 7 Season 5 1.1. 8 Nutrition 5 1.1. 9 Body condition score (BCS) 6 vii 1.1. 10 Parity 6 1.1. 11 Age 6 1.1. 12 Management 6 1.1. 13 Heat stress 7 1.1. 14 Uterine infection 7 1.2. Infertility in cows 9 1. 2. 1. Bacterial and protozoan infections 9 1. 2. 1. 1. Brucellosis 9 1. 2. 1. 2 Mycoplasmas 11 1. 2. 1. 3 Campylobacteriosis 12 1. 2. 1. 4 Leptospirosis 13 1. 2. 1. 5 Salmonellosis 14 1. 2. 1. 6 Mycobacteria 14 1.2.1.7 Non-specific bacterial infections 15 1. 2. 2 Viral infections 16 1. 2. 2. 1 Infectious bovine rhinotracheitis 16 1. 2. 2. 2 Bovine virus diarrhea 16 1. 2. 2. 3 Infectious bovine epididymitis and vaginitis complex 17 1.2.3 Parasitic infection 17 1. 2.3.1 Trichomoniasis 17 1.2.3.2 Neospora protozoa 18 1.2.3.3 Sarcocystis cruzi 18 1.2.3.4 Helminth parasitic infections 18 1.2.4 Mycotic infections 19 1. 3 Endometritis, metritis and pyometra, and the use of 19 prostaglandins in postpartum uterine pathology 1. 3. 1 Puerperal metritis 21 1. 3. 1. 1 Clinical pathology of puerperal metritis 21 1. 3. 1. 1.1 Clinical signs: diagnosis 23 1. 3. 2 Clinical endometritis and pyometra 23 1. 3. 2. 1 Signs and diagnosis of clinical endometritis and pyometra 25 viii 1. 3. 2. 2 Subclinical endometritis 25 CHAPTER TWO: Materials and Methods 2. 1 Study area 27 2. 2 Early postpartum uterine swabs collection and bacteriology 27 2. 3 Sterilization 27 2. 3. 1 Flaming 27 2.3. 2 Red heat 27 2. 3. 3 Sterilization of equipments (hot air oven). 27 2.3.4 Irradiation 28 2. 3.5 Sterilization of culture media and solutions 28 2. 4 Reagents and indicators 28 2. 4. 1 Reagents 28 2. 4. 1. 1 Tetramethyl-piphenylene diamine dihydrochloride 28 2. 4. 1. 2 Hydrogen peroxide 28 2. 4. 1. 3 Methyl red 28 2. 4. 1. 4 Alpha-naphthol solution 28 2. 4. 1. 5 Potassium hydroxide 28 2. 4. 1. 6 Nitrate reagent 28 2. 4. 1. 7 Kovac´s reagent 29 2.4.1.8 Lead acetate 29 2.4.1.9 Gram stain regents 29 2.4.1.9.1 Crysal violet 29 2.4.1.9.2 Lugols iodine 29 2.4.1.9.3 Decolorize ( acetone – alcohol) 30 2.4.1.9.4 Strong carbol fuchsin 30 2. 4. 2 Indicators 30 2. 4. 2. 1 Andrade´s indicators 30 2. 4. 2. 2 Bromothymol blue 30 2. 4. 2. 3 Phenol red 30 2. 5 Collection of blood for enriched media 31 2. 6 Preparation of media 31 2. 6. 1 Nutrient broth 31 2. 6. 2 Peptone water 31 ix 2. 6. 3 Peptone water sugars 31 2. 6. 4 Nitrate broth 31 2. 6. 5 Glucose phosphate medium (MR-VP test medium) 31 2. 6. 6 Nutrient agar 32 2. 6. 7 Blood agar 32 2. 6. 8 MacConkey agar 32 2. 6. 9 Motility medium – Cragie tube medium 32 2. 6. 10 Hugh and leifson´s (O/F) medium 32 2. 6. 11 Diagnostic sensitivity test agar 33 2. 6. 12 Urea agar 33 2. 6. 13 Simmon´s citrate agar 33 2. 6. 14 Dorset egg medium 34 2. 7 Culture of specimens 34 2. 8 Purification of culture 34 2. 9 Microscopic examination 35 2. 10 Identification of bacteria 35 2. 11 Biochemical methods 35 2. 11. 1 Oxidase test 35 2. 11. 2 Catalase test 35 2. 11. 3 Oxidation – Fermentation (O/F) test 35 2. 11. 4 Sugar fermentation test 36 2. 11. 5 Voges – Proskaur test 36 2. 11. 6 Nitrate reduction 36 2. 11. 7 Coagulase test 36 2. 11. 8 Indole production test 37 2. 11. 9 Methyl red (MR) test 37 2. 11. 10 Urease test 37 2. 11. 11 Citrate utilization 37 2. 11. 12 Motility test 37 2. 11. 13 Antibiotic sensitivity 37 CHAPTER THREE: Results 39 3. 1 The aerobic bacteria isolates 39 3.1.1 Gram positive bacteria 39 3.1.2 Gram negative bacteria 41 x 3.1.3 Antibiotics sensitivity test 42 CHAPTER FOUR: Discussion 65 Conclusions and recommendations 70 conlusions 70 71 reommendetions 72 references xi List of Tables Table Page 1 Gram positive bacteria isolated from vaginal swabs of postpartum 48 cows within 1 – 3 days after parturation. 2 Gram positive bacteria isolated from vaginal swabs collected from 49 postpartum cows within 1 – 3 days after parturition. 3 Gram negative bacteria isolated from vaginal swabs collected from 50 postpartum cows within 1 – 3 days after parturition. 4 Bacteria species isolated from vaginal swabs collected from same 51 cows within 1 – 3 days postpartum and then 24 hours after treatment with antiseptic 1 % Lugol’s iodine. 5 Characters and biochemical reactions of Staphylococcus species 52 isolated from vaginal swabs collected from postpartum cows 6 Characters and biochemical reactions of Streptococcus species 53 isolated from vaginal swabs collected from postpartum cows 7 Characters and biochemical reactions of Bacillus species isolated 54 from vaginal swabs collected from postpartum cows 8 Characters and biochemical reactions of Micrococcus species 55 isolated from vaginal swabs collected from postpartum cows. 9 Characters and biochemical reactions of Aerococcus species isolated 56 from vaginal swabs collected from postpartum cows. 10 Characters and biochemical reactions of Corynobacterium striatum 57 isolated from vaginal swabs collected from postpartum cows. 11 Characters and biochemical reactions of Gram negative bacteria 12 Antimicrobial sensitivity of Gram positive bacteria isolated from 60 vaginal swab samples collected from postpartum cows. 13 Antimicrobial sensitivity of Gram negative bacteria isolated from 61 vaginal swab samples collected from postpartum cows. xii 58 List of figures figure 3.1 page Antimicrobial sensitivity of Gram positive bacteria isolated from vaginal swab samples collected from postpartum cows. 3.2 62 Antimicrobial sensitivity of Gram negative bacteria isolated from vaginal swab samples collected from postpartum cows. 63 3.3 Pink colored small lactose fermenting Escherichia coli colonies on 64 MacConkey agar media. 3.4 Antibiotic sensitivity of Escherichia coli on DST agar. The isolates 64 were sensitive to Ampicillin, Colistin sulphate, Cotrimoxazole, Gentamicin, Nalidixic acid, Nitrofurantol and Tetracycline. xiii List of Abbreviations BCS Body condition score CL Calving interval DF Dominant follicle DO Day open FSH Follicle stimulating hormone GN Gram negative GnRH Gonadotrophin relasing hormone LH Luteinasing hormone PP Postpartum PPP Postpartum UI Uterine involution xiv INTRODUCTION A long postpartum interval is a major problem that limits the improvement of reproductive efficiency of dairy cows under tropical conditions (Shart et al., 1990; Williams, 1990). Cross-bred dairy cows in Sudan are known to have a long post partum interval to first oestrus (Elhaj, 2003) which affects their reproductive efficiency. Generally, uterine infection reduces the reproductive efficiency of dairy cows and 40% of postpartum cows had uterine infections (Barlt et al., 1986). More of the cows that show uterine infections usually increase herd health costs, reduce feed consumption and cause an appreciable reduction in milk yield. However, during early postpartum, it has long been shown that, uterine infections delay commencement of folliculogenesis and suppresses the rate of follicular growth in dairy cows by inhibiting FSH release ovarian activity (Peter and Basu, 1988). They also have presented that during the early postpartum excreted an important influence on the ability of the uterus to resist or eliminate bacterial infections. Arthar et al. (1998) reported that delayed commonly accompanied postpartum uterine infection. Unlikely bacterial infections of the genital tract have been recognized as important cause of infertility in the dairy cows (Nakoa et al., 1997). Infertility is caused by many factors such as anatomical abnormalities, functional disorders, managemental problems and infectious disease of the reproductive tract. These infectious micro- organisms include viruses, rickettsiae Chlamydia, mycoplasma, bacteria and fungi. It has been believed that man playes an important role in introducing these organisms. Man has interfered with natural breeding program by cross- breeding local breeds with temperate foreign breeds aiming to increase milk production. Cross- bred cows have been brought up in an environment which is incompatible with some of their inherited traits- such stresses may comprise predisposing factors that would lower the immunity of the xv animal and may thus make it more vulnerable and more susceptible to the infectious agents. Management practices of feeding and housing dairy cattle are sometimes adverse to animal health. In most cases they are the cause of appearance of certain diseases: metabolic disturbances pos- parturient paresis, high incidence of postpartum infection and mastitis among others. These diseases are, mostly the cause of infertility and create extra costs of veterinary assistance and drugs. The present work was carried out to : - Isolate and identify bacterial flora in the genital tract of cross-breed postparturient cows. - Examine the effect of antiseptic Lugol's Iodin on the bacterial flora of the vagina of postparturient cows. - Examine the sensitivity of isolated bacteria to antibacterial drugs. xvi CHAPTER ONE LITERATURE REVIEW 1.1 Postpartum period (postpartum) in dairy cows 1. 1. 1 Definition Postpartum is the time spans from parturition to complete involution of the uterus and it could be as short as 15 days or longer than 100 days (Malven, 1984). The postpartum period is divided into three periods, the puerperal period which is the period from parturition until the pituitary becomes responsive to Gonadotrophin release hormone (GnRH) (7 to 14 days), the intermediate period which begins after the increment of pituitary sense to GnRH and ends after the first postpartum ovulation (14 to 20 days) and the post-ovulatory period which is the time from the first postpartum ovulation until uterine involution is completed (Macmillan and Thatcher, 1991). The postpartum period depends on the level of nutrition, body condition score, suckling, milk yield, age, season, breed, mastitis, heat stress, management and uterine bacterial infection (Short et al., 1990; Stagger et al., 1995). The recrudescencene of follicular growth occurs soon after calving (Savio et al., 1990). The postpartum first ovulation in dairy cows occurs 3 to 5 weeks and it is related to energy balance during the first 3 weeks (Grummer and Corroll, 1991). The ovaries rebound within 30 to 102 days postpartum in dairy cows (Beam and Butler., 1997: Toribio et al, 1995). Nearly about 90% of dairy cows start oestrous cycles by 60 days postpartum, but only about 60% were detected correctly in oestrus at that time (Yanquist, 1988). 1 1. 1. 2 Endocrinology of the postpartum period The interval between calving and conception depends on the reestablishment of normal ovarian cycles after calving, the occurrence of oestrous behaviour at the appropriate time in the cycle, and the pregnancy rate following service (Peters, 1984). The interval between parturition and ovulation is characterised by sexual quiescence (postpartum anoestrus). The duration of this interval varies with breed, milk yield level, animal age, suckling or lactating status, nutritional level before and after calving, season and associated photoperiodism, climate, health status and calving difficulty. Of these factors, nutrition and suckling appear to be very important (Peters et al, 1984). 1. 1. 2. 1 Luteinising hormone (LH) Peters and Lamming (1983) estimated that a pulsatile pattern of LH secretion with a frequency of 0.25 to 1 per hour appears to be a prerequisite for the first ovulation postpartum. This results in gradually increasing LH concentration before the first LH surge. 1. 1. 2. 2 Oestradiol Early postpartum cows are sensitive to GnRH-induced gonadotrophin release and responds by secreting oestradiol (Peters and Lamming, 1986). 1. 1. 2.3 Progesterone Two types of luteal activity have been observed in the postpartum cow. Fifty to 80% of milked or suckled dairy cows exhibit an initial luteal phase in which increases in plasma progesterone concentration are of shorter duration and progesterone concentrations are lower than in the normal cycle (Donaldson et al, 1970; Schams et al, 1978; Odde et al, 1980; Peters and Riley, 1982). This is referred to as the short luteal phase and lasts 6 to 12 days. The second luteal phase lasts about 14 days and tends to have lower than normal progesterone levels. This dual progesterone pattern 2 occurs even after GnRH challenge (Fonseca et al, 1980), early weaning (Odde et al, 1980) or limited suckling with (Dunn et al, 1985) or without GnRH treatment (Flood et al, 1979). 1.1.2.4 Follicle stimulating hormone (FSH) The level of FSH is reported to increases from low concentration in prepartum to higher levels within the first 3 to 5 days postpartum (Lamming et al., 1981; Peters and Horst, 1981). The high FSH level is essential for initiation of ovarian activity during the postpartum (Peters and Lamming, 1994). 1.1.2.5 Oestrogens The level of oestrogen increases from day 26 before prepartum and then decreases during the first week postpartum (Smith et al., 2003: Bo et al., 1995: Haughian et al., 2002). The oestrogen increment triggers a positive feed back mechanism on the hypothalamus and anterior pituitary gland that enhances preovulatory LH surge (Arthur et al., 1998). 1.1.2.6 Oxytocin The level of oxytocin reaches its maximum level at the expulsive stage of parturition and falls to the minimum level at time of oestrus (Hafez, 1993). The oxytocin level during last pregnancy and early stages of parturition remains low and increases to reach the peak values when the fetal head emerges from the vulva. So it plays only major role in initiation of uterine contractions (Arthur et al., 1998). 1.1.2.7 Prostaglandin During early postpartum the uterus produces and metabolizes large amount of prostaglandin PGF2 (Guilbau1t et al., 1984). In dairy cows, the PGF2α increases after the end of parturition to reach peak values 3 days postpartum and does not return to basal levels until 15 day (Edquvist et al., 1980). Administration of PGF2α during early postpartum reduced the time taken for uterine involution, accelerated recrudescence of the first 3 postpartum oestrus, reduced the day open calving intervals and improved conception rate in cross-bred dairy cows (Michiel et al., 1999; Schofield et al., 1999; Elsheikh and Ahmed, 2004). 1.1.2.8 Prolactin Prolactin increases 2 to 4 days before parturition and continues to increase after parturition in both milking and suckling dairy cows (Hafez, 1993). 1.1.2.9 Insulin Insulin has gonadotropic effects on follicular development (Butler and Smith, 1989). Insulin concentration rather than glucose may directly affect LH secretion (Spicer, 1993). Moreover, insulin is largely responsible for regulation of LH pulse patterns that seem to be crucial for the onset and timing of postpartum ovarian function. High insulin concentration increased the proportion of dairy cows that ovulate 50 days postpartum and reduced.the intervals from calving to first ovulation (Gong et al., 2002). This because insulin is a potent stimulator of both proliferation and function of follicular cells (Spicer et al., 1993). 1.1.3 Iodine Iodine is an essential nutrient for optimal thyroid function in dairy cattle (Sarkar, 2006). Reproduction in dairy cows is influenced through iodine's action on the thyroid gland. This improves reproduction efficiency, ovarian activity and improves conception rate in dairy cattle (Bailey et al, 1995). 1.1.4 Suckling Suckling is the major factor that affects duration of the postpartum ovarian activity in dairy cows (Xu et al., 2000). Continuous suckling extends the interval from parturition to the first oestrous and ovulation (Mukasa et al., 1991). Suckling interferes with hypothalamic release of 4 GnRH and provokes a marked suppression on pulstile LH release which extends postpartum anoestrous in dairy cows (Short et al., 1990; Montiel and Ahuja, 2005). Contrarly, restriction of suckling to once-daily or complete isolation of calf from suckling dairy cows promotes an earlier ovulation through improvement of nutritional status compared to continuous suckling in dairy cows (Patton et al., 2006). Thus, temporary calf removal is effective for reduction of postpartum to first ovulation in dairy cows (Stagger et al., 1998; Mackey et al., 2000; Rhodes et al., 2003). 1.1.5 Milk yield High milk yield is known to increase the length of postpartum interval to first oestrous and calving to conception (Gutievres et al., 1999; Gong et al., 2002). This is definitely due to the high metabolic demand during high lactation (Sejresan and Neimman, 1982). Therefore, the influences of high milk yield on reproductive performance during early postpartum could not be separated from nutrition (King, 1968). 1.1.6 Mastitis Mastitis is an inflammation of the mammary gland (Fetrow et al., 1991) Occurrence of mastitis during early postpartum in dairy cows, increases the day-open and services per conception as compared to uninfected cows (Santos et al., 2004). Dairy cows suffering from mastitis during the first month postpartum show a delay in the onset of ovarian activity (Huszenicza et al., 2005). 1.1.7 Season The season influences the duration of the postpartum in both suckled and non suckled dairy cattle (Lamming et al., 1981). In temperate climates, animals that calfed before the summer has a longer interval to first ovulation than that calfed afterwards (Fonesca et al., 1983; Opsomer et al., 2000). In subtropical environments, reproductive performance decreases during the warm season, as compared to cool season, where the interval to 5 first ovulation is being longer in summer as compared to winter calving cows (Jonsson et al., 1997). 1.1.8 Nutrition Nutrition plays a major role on the reproductive efficiency in dairy cows (Fergusson 1996; Butler 2000; Short et al., 1990).Nutrition is known to influence the metabolic and hormonal profiles which in turn affect follicular development (Braum et al., 1986). Nevertheless, overfeeding during early postpartum reduces fertility (Henrichs, 1989), and excessive intake of protein during early postpartum delays the ovarian activity in dairy cows (Fergusson and Chaluba, 1989; Lucy et al., 1991). Malnutrition during postpartum suppresses LH releases and delays ovulation (Sinclair et al., 2002). 1.1.9 Body condition score (BCS) Body condition score (BCS) indicates the body reserves of the cow and it reflects the level of nutrition (Braun et al., 1986). The BCS has been developed to enable an accurate judgment of the body condition (Paul, 1995). The BCS also helps to predict the duration of postpartum anoestrous and conception rates (Folman et al., 1990). For each reduction in BCS the duration of postpartum anoestrous was found to increase (Schillo, 1992). Conception rates were also influenced positively by BCS during early postpartum (Moreira et al., 2000). 1.1.10 Parity Parity is the number of times a cow had maintained a foetus beyond 260 days of gestation (Williams and Griffith, 1995; Sharma et al., 1996; Stahl et al., 2006). Parity influences postpartum and total number of follicular waves (Lucy et al., 1992). Dairy cows with second parity have an increased fertility as compared with third parity dairy cows (Walter et al., 2002). 6 1.1.11 Age Postpartum of old cows tend to be longer as compared to heifers and middle age cows (Marion and Waltersetal, 2002). The same authors reported that primiparous dairy cows had longer postpartum than the multiparous. Cows that reached their seventh lactation had poor fertility (Esslemont and Kossaibati, 2000). 1.1.12 Management Most reproductive failures in dairy cattle herds are due to deficiencies in management stratiegies (Rhodes et al., 2003). Poor standard of herd management will lead to poor detection rates of heat (Arthur et al., 1998). Miss-management like over crowdness and muddy floors are known to reduce the accuracy of heat detection in dairy cows (Arthur et al., 1998). 1.1.13 Heat stress In heat stressed dairy cows appetite and dry matter intake are both reduced. This prolongs the postpartum period of negative energy balance, increases the calving interval and negatively influences the plasma concentrations of insulin, IGF-1 and glucose (Dobson and Smith, 1998). Heat stress stimulates the hypothalamic center that regulates the GnRH and LH release during follicular phase (Dobson et al., 2003). Similary, heat stress during early postpartum, affects the endocrine system especially the thyroid gland, which releases less thyroxine under stress (Buffington et al., 1978). Heat stress is reported to alter follicular development by reducing steroid production (Wolfenson et al., 1997; Wilson et al., 1998). Heat stress is associated with a delayed initiation of postpartum ovarian activity (Putney et al., 1998; Alkatanani et al., 1999). 1.1.14 Uterine infection Bacterial contamination of the uterus occurs in up to 90% of dairy cows during the first week postpartum (Shedon et al., 2002).The percentage of pathogenic bacteria in uterus during this critical period 7 causes endomeritis, pathological lesions on the endomtrium, delays uterine involution and pertubs featus survival (Sheldon et al., 2003; Sheldon et al., 2006; Foldi et al., 2006). The outcome of uterine bacterial contamination depends on the number and virulence of the organism present as well as the condition of the uterus and its inherent defense mechanisms (Huszenica et al., 2005). During early postpartum, uterine bacterial infection, bacterial products or the associated inflammation reduces FSH pituitary concentrations, suppress LH release and consequently perturbs postpartum ovarian follicular growth and function, which impairs ovulation in dairy cattle (Opsomer et al., 2000). The in-balance between uterine infection and the intra-uterine antimicrobial self-defence mechanisms, often results in complications, such as subclinical endometritis, puerperal metritis, clinical endometritis and false pyometra (Sheldon et al., 2002). Fourty percent of dairy cows were diagnosed with and treated for postpartum uterine infection. This postpartum uterine bacterial infection was associated with prolonged day open, lowered rate of service per conception lowered breeding and increased calving interval (Bartlett et al., 1986; Huszenicza et al., 1999). In particular, resistance of the uterus to bacterial infection and its ability to eliminate bacterial populations during early postpartum is influenced by ovarian activity (Foldi et al., 2006). The exact causes of uterine infection are unknown but are associated with several factors. Cows with dystocia, retained placenta, twins or still births and various metabolic disorders are more likely to develop metritis than other cows (Bell and Robert, 2007). Aberrant immune function before and after calving seems to predispose cows to severe uterine infection, which delays uterine involution. A few cows die from severe uterine bacterial infection, but dairy cows with mild uterine bacterial infection are more likely to be culled for poor reproductive performance (Fredriksson et al., 1988; Gregory, 8 1997). The frequent cultured pathogenic bacteria that cause postpartum uterine infections in dairy cows are, E. coli, Eubacterium spp, Arcanobacterium pyogenes, Pasteurella multocida, Staphylococcus spp and Streptococcus spp, but sometimes other non-specific pathogens are found such as Campylobacter spp and Haemophylus spp (Stevenson and Call, 1998). It has been reported that 75% of bovine uteri are infected up to day 15, 78% up to day 30, 50% up to day 45 and 9% up to day 60 postpartum (Leslie, 1983). Fusobacterium necrophorum and Arcanobacterium pyogenes are isolated for 3 to 5 weeks postpartum as well as E. coli, Pasteurella spp and Proteus are isolated for 2 to 3 weeks postpartum (Konigsson, 2001). Dairy cows having pyogenes in their uterine fluid suffer from severe endometritis and are infertile at the first service. The duration of infertility associated with uterine bacterial infection depends on the severity and duration of inflammation (Sheldon et al., 2006). Resolution of inflammation occurs with time (Opsomer et al., 2000) and the fertility is restored in normal cows by 40 to 50 days in milk. Ninety two percent of cows had bacterial infection during early PP.Cows that suffer from severe PP uterine bacterial infection have a longer time for uterine involution (UI) than dairy cows that suffer from mild PP utrine bacterial infection. This can be attributed to severe endomtritis that occurrs during early PPP, which leads to damage of the endometrium and suppresses PGF2α which is essential in this critical period for UI (Elsheikh and Ahmed, 2004). 1.2 Infertility in cows Cattle are deemed infertile when they are neither normally fertile nor completely sterile. Interest in bovine infertility increased with the introduction of artificial insemination in the 1950s and as the factors 9 involved became known to farmers, herdsmen physiologists and other workers (Roberts, 1956). The causes of infertility are many and can be complex (Arthur, 1982). They relate to Graafian follicle development and maturation, oestrus onset, successful coitus, ovulation, fertilization, implantation, and the development and delivery of the foetus and its membranes. Anything interfering with these routines, such as diseases, poor nutrition, inadequate herd management, hereditary and congenital factors, hormonal disturbances or environmental changes, makes the animal infertile, if only temporarily (Osmanu, 1979). 1. 2.1 Bacterial and protozoan infections Pathological lesions were observed in the reproductive tracts of 55.14% of 700 cows examined at post-mortem on two ranches in Shaba, Zaire (Binemo-Madi and Mposhy, 1982). Most of these were on the ovary and salphinx. They may have been caused by abnormal rectal manipulations or bacterial infections of the uterus, vagina and vestibulae. About 45% of the cows were still capable of breeding, indicating that pathological conditions do not necessarily render cows permanently sterile. Their seriousness depends on the location of the infection. 1.2.1.1 Brucellosis Importance and incidence: Brucellosis is found world-wide. It affects humans, domestic animals and wildlife. In cattle it is caused by Brucella abortus, but it maybe caused by B. melitensis and B. suis. De et al., (1982), in a study of 989 cows in West Bengal, attributed abnormal termination of pregnancy, cervicitis, endometritis, repeat breeding and anoestrus to brucellosis, campylobacteriosis, leptospirosis and trichomoniasis. Rao et al., (1977), Hussain and Muniraju (1984) and 10 Kaikini et al. (1983) noted that these, and other infections, can lead to inflammation with bursar and uterine adhesions, periglandular fibrosis, hydro- and pyo-salphinx, hydrometra, pyometra, endometritis, vaginitis, and metritis. Other effects include early embryonic loss repeat breeding, abortion, dystocia, retained membranes, stillbirth, and prolapse in late gestation. Brucellosis has been extensively studied, partly because it causes widespread economic losses due to abortion and extended calving intervals and because it affects humans. Many countries in the temperate regions have substantially reduced the incidence of brucellosis. Some, such as Denmark, UK, The Netherlands and Romania, have eradicated the disease, mainly through rigorous test and slaughter policies. The diagnosis of brucellosis is now well standardised and, in general, highly efficient. However, the disease is still prevalent in many countries, especially in the tropics (Hussain and Muniraju, 1984). The reported incidence of brucellosis in Africa varies from zero to 100% (Chukwu, 1985; 1987). Brucellosis was reported in Ghana (Oppong, 1966), in western Nigeria (Esoruoso, 1974), in Ethiopia (Meyer, 1980), in Malawi by Klastrup and Halliwell (1977), in Sudan (Dafalla, 1962; Mustafa and Nur, 1968; and Mustafa et al., 1976), in Kenya (Kagumba and Nandhoka, 1978), in Uganda, Kenya and Tanzania (Marinov and Boehnel, 1976) and in Somalia (Wernery et al., 1979) Brucellosis is normally acquired by cattle by ingesting the bacteria. Infection may also occur through the mucosa of the eye, nose and teat, and through the endometrium if the cow is artificially inseminated with infected semen. The multi-layered mucosa of the vagina seems to protect against infection following natural service. The disease is most serious in cows infected during pregnancy. The bacteria show a preference for the pregnant uterus, foetus and the lymph 11 glands of the udder. Both the membranes and foetus respond to Brucella infection by increasing their production of erythritol, a simple carbohydrate, which increases the growth rate of the bacteria. This usually results in abortion at about 6 to 8 months of gestation. The organism may also produce toxins and allergens, cause vascular thrombosis, increase uterine motility, and disturbance production of sex steroids and prostaglandins, contributing to abortion (Vandeplassche, 1982). In some cases the dead foetus is not aborted, but is retained in a mummified or macerated form. If a calf is born alive it is likely to be weak and many die soon after delivery. Aborting infected cows or heifers are a major source of infection for other cattle and people handling them. Aborted material and vaginal discharges from infected females are heavily infected with Brucella, and these contaminate pastures, pens and buildings. Organisms are also present in the milk of infected cows. Brucellosis is a professional hazard for cattle keepers and veterinarians. Foetal membranes are commonly retained because of uterine inertia, placentitis or both. Retained membranes must be handled with great care. Puerperal metritis may develop and cows may remain infertile for some time. The bacteria are rarely cultured, partly because diagnostic material, particularly foetuses, usually reaches the laboratory in a condition unsuitable for proper examination. Serological tests are therefore, commonly employed. However, the various tests used differ in convenience and accuracy. A good serological test would establish early diagnosis, identify chronic infections, and distinguish between the antibodies of vaccination and infection (Fensterbank, 1986). 12 1. 2.1.2 Mycoplasmas Several species of Mycoplasma cause disease in cattle. They have been associated with infertility, but their exact aetiological role is difficult to certain because they are present in the genital tracts of healthy animals. In a study of cattle in South Africa, Mycoplasma bovigenitalium was found in 43% of males, 47% of females, 25% of faeti and 11% of placentas (Trichard and Jacobsz, 1985). Mycoplasma arginini, M. alkalescens, M. laidawii, M. bovis, M. bovirhinis, M. verecundum and M. conjunctivae were also isolated. Mycoplasmas can be transmitted by discharges from the respiratory and reproductive tracts, and by milk, of infected animals. Infected cattle develop antibodies, but these do not give complete protection. 1.2.1.3 Campylobacteriosis Abortion in cattle and sheep was first associated with vibrionic organisms in 1918 by McFadyean and Stockman (Laing, 1963). The organisms were first named Vibrio fetus by Smith (1918), and later Campylobacter species. The species most commonly encountered in the bovine genital tract are Campylobacter fetus subsp. venerealis serotype A, C. fetus subsp. fetus serotype A and C. fetus subsp. fetus serotype B. Of these, infertility in cattle is most often associated with C. fetus venerealis A (90%) and C. fetus fetus B (10%) and only rarely C. fetus fetus A and B (Vandeplassche, 1982). The incidence of campylobacteriosis in the tropics varies. De et al (1982) found an incidence of 6.1% in West Bengal and India and none in Malawi, the incidence reported by Klastrup and Halliwell (1977) was 11.5% of the exotic bulls tested serologically. In Zimbabwe and 33% of the cows sampled were found to be positive (Terblanche, 1979). Garcia et al (1983) 13 stated that transmission is purely venereal or via artificial insemination, and that attempts to infect cattle by vulval contamination failed. Breeding records often give the first indication, with many cows returning to oestrus after repeated service: the repeat breeder syndrome. Oestrous cycles are longer than normal, usually more than a month (Clark, 1971), indicating early embryonic death. After variable periods of infertility many cows recover and regain normal fertility. Thus, as with trichomoniasis, infected females should be sexually rested. Breeding records showing low non-return rates, many repeat breeders and a high incidence of abortion at mid-term suggest campylobacteriosis. Isolation of C. fetus from the bull or cows will confirm the diagnosis (Clark, 1971). Treatment should aim to cure the infected bull. Fat-free cream containing 1% neomycin or polymixin can be applied. 1.2.1.4 Leptospirosis Bovine leptospirosis is a systemic disease characterised by fever and, sometimes, mastitis and abortion. Leptospirosis should be suspected when abortion occurs in cows showing other symptoms such as icterus and haemoglobinuria (Carroll, 1973). It is one of the most widespread zoonoses. Leptospirosis has not been extensively researched in Africa. Moch et al (1975), working in Ethiopia, found incidences of 91.2% in horses, 70.7% in cows, 57.1% in pigs, 47.3% in goats, 43.4% in sheep, 15.4% in camels and 8.3% in dogs. Ball (1966) reported incidences of 34% in cattle and 9.3% in goats sampled in Kenya and Uganda. The disease is caused by Leptospirae. These are spirochaetes. Some 120 Leptospira serotypes have been identified by the World Health Organization (Moch et al, 1975). Serotypes associated with bovine abortion are Leptospira pomona, L. canicola, L. australia, L. icterohaemorrhagica 14 and L. grippotyphosa. They are all sensitive to antiseptics and desiccation and their virulence and pathogenicity are highly variable. Lawson (1963) indicated that L. pomona is the serotype most commonly implicated in bovine abortion. Animals infected with Leptospira excrete the bacteria in their urine. Direct or indirect contact with the urine of infected animals is the major route of infection in both animals and man. The usual route of infection is via the digestive tract, but the disease may be contracted via the respiratory and reproductive tracts, eyes or skin. In cows, infection is often followed by pyrexia and reduced milk production (Stoenner, 1968). In the pregnant cow the organisms show an attraction for the uterus and attack the foetus or endometrial capillaries. This may result in abortion during the last trimester or the birth of a weak or dead calf. Aborted foetuses show no characteristic lesions other than subcutaneous oedema and fluid-filled cavities. Foetal membranes may be retained, sometimes causing metritis and infertility. The organisms also settle in the kidneys and in 2 or 3 weeks leptospiruria starts. If the bacteria invade the udder they may cause mastitis or agalactia. The udder is flaccid and milk becomes thick, yellow and clotted (Stoenner, 1968). 1.2.1.5 Salmonellosis Salmonellosis is an important cause of abortion in cattle. It is also a zoonosis. Salmonella dublin and S. typhimurium are the most common causes of salmonellosis in dairy cattle. Infected animals excrete the organisms in their faeces, contaminating pastures, water supplies and housing. From these, the bacteria infect healthy animals. Typical symptoms of salmonellosis include septicaemia, pyrexia and dysentery. Pneumonia may also occur. The bacteria are attracted to the uterus and together with severe enteritis, caused by endotoxins, and painful arthritis, cause abortion (Vandeplassche, 1982). 15 The primary cause of abortion is the release of prostaglandin (PGF2 a) induced by salmonella endotoxin. Following abortion, the uterus may become severely inflamed, resulting in the death of the cow. Cows that recover may continue to excrete the bacteria for years. Salmonellae can be passed to humans. Aborted material should therefore be handled with extreme care. 2.1.6 Mycobacteria Tuberclosis caused by Mycobacterium bovis is present world wide in cattle and is of major importance in diary cattle that are closely housed or pastured in limited area (Stephen, 1982). Tuberclosis cuses infertility either through general reduction in health caused by any infection whether genital tract is involved or not or directly by infecting the genital tract organs that ova and spermatozoa production will be impaired and thus preventing conception or causing abortion (Laing, 1955). Infection is transmitted from infected animals via sputum, faeces, milk, urine, semen and genital tract discharges (Stephen, 1982). 2.1.7 Non-specific bacterial infections Many species of bacteria inhabit the vagina, uterus, and cervix of cows. Some are symbionts that become pathogenic when the animal is stressed; others are immediately pathogenic. The normal microbial flora of the bovine genital tract is made up of a dynamic mixure of aerobic, facultative anaerobic and strict anaerobic microorganisms . Under natural conditions this environment is stable, protecting the host againist pathogenic or potentially pathogenic saprophytic microorganisms. The normal microbial flora in this tract is composed of bacteria of the genera Staphylococcus, Streptococcus, and coliform group (Hafez, 1993). Enterobacteriaceae specially Escherichia coli have been isolated from the urogenital tract of cattle in low numbers (Torres et al, 1994; Otero et al 16 2000). The microbial population of vagina in healthy cows has been previously described with Lactobacilli been normal constituents of these microbial flora (Otero et al 1999; Otero et al 2000). Namboothripad and Raja (1976), Eduvie et al (1984) and El-Azab et al (1988) isolated Staphylococcus aureus, Escherichia coli, Pseudomonas pyocyanea, Corynebacterium pyogenes, Proteus mirabilis, Streptococcus spp, Pasteurella multocida, Proteus vulgaris, Klebsiella spp and several anaerobic microorganisms from the uteri of cows with a history of repeat breeding, retained placenta and metritis, as well as from the uteri of normal suckling cows. Mycobacterium tuberculosis was isolated by Mohanty et al., (1980) from a Haryana heifer that was a chronic repeat breeder. Listeria monocytogenes may also cause abortion in cattle. When the organism infects a pregnant cow, it invades the foetal nervous system and forms necrotic foci on the liver, lungs and spleen (Watson, 1979), killing the foetus. Vandeplassche (1982) indicated that, although the organisms are easily eliminated from the uterus, they may persist in the mammary system. Antibodies to Listeria are short-lived, and immunity is thus only temporary and cows can be re-infected. Treatment is often futile, even with antibiotics. Ellaithi, (1983) isolated Listeria grai from the uterus of normal breeders and the cervices of both repeat and normal breeders. Listeria monocytogens was isolated by the same author from the uteries of repeat and normal breeders and was isolated from the cervices of repeat breeders only . 1.2.2 Viral infections Several viral diseases, including infectious bovine rhinotracheitis (IBR), bovine virus diarrhoea/mucosal disease (BVD/MD), para-influenza3, infectious bovine epididymitis and vaginitis complex (Epivag), transmissible fibropapilloma and epizootic bovine abortion have been 17 associated with cattle infertility (Florent, 1963; Gledhill, 1968; McKercher, 1969). The major infections are described below. 1.2.2.1 Infectious bovine rhinotracheitis Infectious bovine rhinotracheitis (IBR) is caused by a herpes virus. Its incidence in Africa is not widely reported but a recent field study by ILCA on two populations of indigenous Ethiopian zebu cattle indicated a prevalence of over 50% (Tekelye Bekele et al. 1989). IBR can affect the respiratory, reproductive, nervous and digestive systems of cattle. The disease can be transmitted sexually or by droplet inhalation; in its venereal form it has been associated with infertility. Cows and heifers served by an infected bull develop a pustular vulvo-vaginitis 2 to 3 days later. This may clear up after 2 weeks. Some develop superficial ulcers on the mucosa of the vestibulum and may discharge yellowish pus. Infection rarely extends directly to the cervix or uterus, and therefore pregnancy is rarely terminated. However, if cows are artificially inseminated with semen from an infected bull, the virus is deposited directly into the uterus and induces endometritis. Infection of the uterus disrupts the cow's oestrous cycles and reduces its fertility. The virus may also invade the uterus systemically in cows suffering from the respiratory form of the disease (Polydorou, 1984). 1.2.2.2 Bovine virus diarrhea Bovine virus diarrhoea/mucosal disease (BVD/MD) is widespread. It is caused by a Toga-virus. Infection may be acute, mild or chronic. When the virus infects a pregnant cow it may also infect the foetus and kill it. Calves born alive may be stunted, with cerebellar hypoplasia, brain cavitation and mucosal ulceration (Polydorou, 1984). 18 1.2.2.3 Infectious (contagious) bovine epididymitis and vaginitis complex Infectious bovine epididymitis and vaginitis complex (Epivag) appears to be confined to East and southern Africa. It was first described in Kenya and subsequently in South Africa (Arthur, 1964). Epivag is a venereal disease. In cows the main symptom of Epivag is muco-purulent discharges from the vagina .Epivag can cause permanent lesions on the Fallopian tubes. In the bull, it causes the epididymitis to swell, sometimes to the size of a golf ball. The disease can be controlled by slaughtering infected bulls and by using artificial insemination. 1.2.3 Parasitic infection 1.2.3.1 Trichomoniasis About 40 years ago, trichomoniasis was an important cause of infertility in cattle in many countries. The disease causes endometritis, pyometra, abortion and sterility. It is a venereal disease which spreads at service or by artificial insemination with improperly treated or handled semen. It is sometimes called Bovine Venereal Trichomoniasis. Its incidence has been greatly reduced where artificial insemination is widely used, as in the UK, The Netherlands, France and Cyprus. It nonetheless remains a problem in other countries, especially in dairy cattle, poorly managed herds and herds which use communal bulls (Akinboade et al., 1980). The incidence of trichomoniasis in Africa and the tropics is not widely reported, partly because diagnosis is complex and time-consuming. Consequently, it is not clear if the disease is widespread. However, in Egypt, Gawade et al., (1981) found an incidence of 4.6% among Holstein bulls, and in Nigeria Akinboade (1980) found an incidence of 14.9% among slaughter animals. 19 Trichomoniasis is caused by Trichomonas fetus, a protozoan about 15 m long with an undulating membrane. In bulls, the trichomonads normally colonise the crypts of the external mucous membrane of the penis and prepuce. Since these crypts are deeper in older bulls, the prevalence of the disease tends to increase with bull age. Infection does not induce any local antibodies or specific agglutinins in the blood of bulls. Bulls carry the disease for a long time without showing symptoms (Akinboade et al., 1980). Cows and heifers that have never been exposed to the disease become infected following either natural service by a carrier bull or artificial inseminations with contaminated semen. Following natural service, the protozoa first multiply in the vagina and cervix for about 3 weeks. In about a quarter of the cows, the organisms do not migrate to the uterus. With intrauterine artificial insemination, the uterus is directly infected (Kodagali, 1974). Trichomoniasis causes infertility, repeat breeding, delayed return to oestrus after mating, early embryonic death and, sometimes, abortion. It may directly cause the death of the embryo or may do so via uterine endometritis and marked leucocytic diapedesis into the endometrium (Vandeplassche, 1982). The affected cow returns to oestrus or may abort anytime from 2 to 7 months after conception. 1.2.3.2 Neospora protozoa Bovine protozoal abortion a newly recognized bovine abortion disease. The etiological agent is termed Neospora protozoa. The only clinical sing is abortion which occurrs between 4 and 6 month`s gestation. Repeated transplacental infection has been documented in cows following Neospora abortions. The mode of transmission is suggested to occur from 20 eating food contaminated with feaces containing coccidial oocysts (Bradd and Mark, 1993). 1.2.3.3 Sarcocystis cruzi Sarcocystis cause sporadic and inferquent bovine abortions, this is associated with Sarcocystis cruzi. The organism was idenifeid in 5.1% of 265 bovine abortions (Jerret et al, 1984). 1.2.3.4 Helminth parasitic infections Helminth parasitic infections are commonly known to affect the general health of animals (Kumar et al, 1991) Suggested that fasciolosis reduces the potential breeding, impairs the growth rate of young stock and prolongs oesrtus in mature animals. 1.2.4 Mycotoc infections The rate of bovine mycotic infections diagnosed vary from less than 1% to 10.4% depending on geographic location (Bradd and Mark, 1993). Patgiri and Uppal (1993) examined 84 cows and 46 buffalos which failed to conceive. Candida, Aspergillus, Absidia, Rhizopus, Cladosporium, Scopule riopsis and Fusarium were isolated from uterine biopsies taken at oestrus. Tainturier (1995) showed that calving to conception interval decreased by 75 days in a group of cows desensitized ageanst Candida by intradermal route during gestation. Al sayed (1994) reported isolation of Asperigllus fumigatus, Penicillium spp and Candida albicans from repeat breeder Fricsian heifers and cows, but other bacterial organisms were isolated from the same groups. He had shown that cows and heifers with fungal infection needed more services preconception than those with bacterial infection .Thus fungal infection played a role in the incidence of repeat breeding and impaired the breeding performance. In the same study, Aspergillus fumigutus, Aspergillus falvus, Aspergillus niger cladosporium, Mucor, Candida albicans were isolated from uteri of aborted cows. 21 Rhizopus and Aspergillus niger were isolated from retained placente. A. fumigatus was isolated together with S. aureus from retained placente were increased days from calving to first service (S.P.C), days open and thus calving interval . 1.3 Endometritis, metritis and pyometra, and the use of prostaglandins in postpartum uterine pathology There is little information on the impact of metritis, endometritis or pyometra on the fertility of zebu cattle. Among Bos taurus cattle, Tennant and Peddicord (1968) found that endometritis, as indicated by pus in the vagina, significantly reduced fertility. Cows with endometritis required significantly more services per conception, had lower conception to first service rate and longer calving interval, and more animals were culled for infertility. Infections of the reproductive tract are usually contracted at parturition. Non-specific infections of the uterus are more common where the placenta is retained, in cows that need assistance with calving and in cows with milk fever. Metritis is often also associated with uterine atony or inertia. Acute metritis causes fever and depression within a week of infection, and is commonly followed by chronic metritis, with persistent purulent vaginal discharge. Specific venereal infections, such as trichomoniasis, campylobacteriosis and brucellosis, may also lead to metritis (Kennedy and Miller, 1993). Pyometra is the accumulation of pus in the uterus. It is a common cause of anoestrus and cows with pyometra should be treated promptly. Postpartum metritis, endometritis and pyometra may be common where cows and heifers are confined at delivery time in a building or area in which others have recently calved (BonDurant, 1999). The uterus can resist or eliminate bacteria infection. However, this ability is related to ovarian activity (Paisley et al., 1986). The uterus is 22 highly resistant to infection during the oestrogenic phase, but very susceptible during the period of progesterone dominance. Cows with chronic metritis are anoestrous and may have a retained (persistent) corpus luteum. Where a corpus luteum is present, initial treatment should aim at removing it. This is best achieved by an intramuscular injection of prostaglandin. If there is no corpus luteum, endometritis can be treated by infusing antibiotic or sulphonamides into the uterus. Application should be repeated every 2 days for a week. Alternatively, about 100 ml of 2% iodine can be infused into the uterus. The iodine solution is an irritant and stimulates new endometrial growth. Where the oviduct, deeper layers of the uterus or the cervix or vagina is infected, antibiotic should be given intra-muscularly (Sheldon, 2004). Several other postpartum conditions can reduce fertility. Cervicitis and vaginitis often follow a delayed or complicated delivery. Metritis may cause abscesses in the uterus; if it spreads to the Fallopian tubes it may lead to salpingitis. Scars in the uterus and adhesions between parts of the reproductive tract can result in infertility or sterility. Routine examination of cows 1 or 2 months after delivery can diagnose such conditions early (Kennedy, 1993). 1.3.1 Puerperal metritis Puerperal metritis is the bacterial complication of the early puerperium, which occurs during the first 2 weeks after calving (typically on 4–10 days postpartum), and is characterized by (i) a large amount of foul smelling, reddish-brown, watery exudate with some necrotic debris in the uterus and a thin uterine wall in the first half of this period, or (ii) a limited amount of malodorous, purulent uterine exudate and a thick (oedematous) uterine wall some days later. Systemic signs of disease (dullness, prostration) including pyrexia may or may not be present 23 (Sheldon and Dobson, 2004; Sheldon et al., 2006). Incidence of puerperal metritis varies from 2.2% to 37.3% (Kelton et al., 1998). 1.3.1.1 Clinical pathology of puerperal metritis From pathological point of view, puerperal metritis is an acute putrid inflammatory disease due to the massive bacterial infection of the uterus. The degenerative and infiltrative processes lead to an excessive damage of luminal and glandular epithelium, and may extend to the entire thickness of the uterine wall, sometimes also to the serosa (perimetritis) and the suspensory ligaments (parametritis) (Paisley et al., 1986; Kennedy and Miller, 1993; BonDurant, 1999; Sheldon, 2004; Sheldon and Dobson, 2004). Although in the first 10 – 14 days after calving there may be a lot of other bacteria present in the uterus, A. pyogenes and E. coli combined with certain Gram negative (GN) anaerobic bacteria (Fusobacterium necrophorum, Bacteroides spp and Prevotella spp) are considered the main responsible pathogens in this complication (Sheldon and Dobson, 2004). A pyogenes, Gram-negative GN anaerobic bacteria and coliforms were isolated in 33–83%, 49–67% and 67–85% of cows with puerperal metritis, respectively (Huszenicza et al., 1999; Dohmen et al., 2000; Mateus et al., 2002; Sheldon et al., 2004). The bacterial growth densities for A. pyogenes, E. coli, non-hemolytic Streptococci and Mannheimia haemolytica were associated with a fetid mucus odour (Williams et al., 2005). An intensive invasion by pathogens may occur if the migration and functional (phagocytic, cell-killing, oxidative burst) capacity of neutrophils are reduced (Paisley et al., 1986; Cai et al., 1994; Mateus et al., 2002; Sheldon, 2004). A. pyogenes and GN anaerobes invade not only the endometrium, but usually also the submucosa, and occasionally the deeper layers of the uterine wall (Paisley et al., 1986; BonDurant, 1999). 24 Stagnating lochia in the uterine cavity provides an excellent medium for multiplication of E. coli and/or other coliforms, resulting in extensive liberation of a lipopolysaccharide like cell wall component (endotoxin) of these microbes (Peter et al., 1987, 1990; Dohmen et al., 2000; Mateus et al., 2003). The enormous quantity of endotoxin in lochia of cows with dystocia and/or RFM shortly after parturition enhances the development of uterine infection by A. pyogenes and GN anaerobes thereafter in the later postpartum period (Dohmen et al., 2000). However, despite its high local level endotoxin remains undetectable in the peripheral blood in most of the cows (Dohmen et al., 2000), or may be detected only in the most severe cases (Mateus et al., 2003). Endotoxin in the lochia provides a positive chemotactic signal for leucocytes, which activates them, and enhances their migration from the blood into the uterus. Furthermore, in the stimulated immune cells (mainly macrophages) endotoxin induces the release of histamine and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF α) and interleukins (IL1 and IL6), and activates the phospholipase A2, cyclooxygenase-2 and 5-lipoxygenase enzyme systems producing various eicosanoids (PGF2 α, PGE2 and also prostacyclines and thromboxanes) (Madej et al., 1984; Peter et al., 1987; Kindahl et al., 1992, 1996; BonDurant, 1999; Mateus et al., 2003; Sheldon, 2004; Sheldon et al., 2004b). One of these eicosanoids is the PGF2 α, with known luteolytic and immunostimulative properties, which also increases the myometrial contractility in cattle (Hirsbrunner et al., 2003; Lewis, 2004). Most recently Herath et al. (2006) reported that PGE2 is produced by endometrial stroma cells as a response to endotoxin, which may be the explanation of elevated PGE2 levels in severe metritis. 1.3.1.1 Clinical signs: diagnosis In puerperal metritis the viscous consistency of normal lochia changes to foul smelling, watery and reddish-brown exudate, its amount 25 may be much increased accompanying a thin uterine wall, because the atonic uterus is not able to expel its content. In some cases, however, uterine wall may be rather thick because of oedema, due to inflammatory process, with somewhat less amount of abnormal putrid content. Mostly all layers of the uterine wall show acute inflammatory signs associated with degeneration of endometrial and myometrial tissue. The most important sign, however, regardless of the amount of discharge, is the foul smell of the accumulated uterine content. In more severe cases, local symptoms are accompanied by general signs, like fever (>39.5 ◦C), loss of appetite, dullness, reduced milk production or diarrhoea with dehydration as a consequence (Paisley et al., 1986; Hussain, 1989; Hussain and Daniel, 1991; Lewis, 1997; Dohmen et al., 2000; Sheldon and Dobson, 2004; Sheldon et al., 2006). General signs occur in minority of cows with puerperal metritis and these symptoms are associated with the excessive release of inflammatory mediators, as well as of endotoxins and other bacterial products. Therefore, toxic puerperal metritis is considered as a synonym of the most severe form (Drillich et al., 2001; Sheldon and Dobson, 2004; Sheldon et al., 2006). Diagnosis of puerperal metritis is quite straight forward on the basis of clinical signs. Time elapsed from parturition, malodorous, watery, reddish-brown uterine discharge, with or without systemic signs are enough for the diagnosis (Sheldon and Dobson, 2004; Sheldon et al., 2006). The character and odour of vaginal mucus reflect the number of bacteria in the uterus (Williams et al., 2005). 1.3.2 Clinical endometritis and pyometra Clinical endometritis occurs from the third postpartum week (after postpartum day 14) onwards, characterized by presence of abnormal (mucopurulent or purulent) content in the uterine cavity, and the same 26 quality of vaginal discharge expelled through the cervix, which is still open. Bovine pyometra is a closely related inflammatory disease, which develops after the first ovulation in presence of an active (sometimes persistent) luteal tissue, usually from about the 20th–21st day onwards. Due to the luteal progesterone production the cervix is closed i.e. not permeable, consequently the mucopurulent or purulent exudate accumulates in the uterine cavity (BonDurant, 1999; Sheldon and Dobson, 2004; Sheldon et al., 2006). The reported lactational incidence of “endometritis” varies from 7.5% to 61.6% (Curtis et al., 1985; Markusfeld, 1987; Gilbert et al., 2005). During the puerperal period the uterus of almost all of the cows is contaminated, and perhaps in 90% of them a mild, nonpathological form of endometritis develops. In majority of cows the local antimicrobial defence mechanisms can eliminate the pathogens, and this mild non-pathological form of endometritis resolves within some days (Sheldon, 2004). However, when following incomplete recovery of puerperal metritis the uterus remains infected with A. pyogenes and gram negative GN obligate anaerobes (F. necrophorum, Prevotella and Bacteroides spp.), or it may become re-infected with these pathogens from the environment causing clinical endometritis. Unlike other uterine pathogens, the incidence and importance of E. coli gradually decreases with time: the involvement of coliforms in pathogenesis of clinical endometritis is only secondary (Paisley et al., 1986; Hussain, 1989; Hussain and Daniel, 1991). It has been supposed for a long time that in ruminants the postpartum uterus is less sensitive to invasion by pathogens or experimental bacterial challenge until the first ovulation, and during the follicular phase of the subsequent cycles. However, the susceptibility to bacterial invasion increases when functional corpora lutea (CL) is present (Paisley et al., 1986; Hussain, 1989; Hussain and Daniel, 1991a). In a model study, 27 progesterone treatment with progesterone for a few days, increased the susceptibility of animals to intrauterine inoculation with A. pyogenes and E. coli, reduced circulating PGF2 α, and elevated plasma PGE2 (Lwis,2003). Ovariectomy increased and progesterone treatment decreased the cellular immune response as measured by concanavalin A-stimulated lymphocyte proliferation. It was concluded that progesterone made the postpartum uterus susceptible to infection, ovariectomy allowed the dam to remain resistant, and uterine prostaglandins mediated the process (Lewis, 2003, 2004). Intrauterine inoculation of E. coli shortened the luteal life span in cattle (Gilbert et al., 1990). This finding is associated with intrauterine liberation of endotoxin triggering a subsequent endometrial PGF2α release directly (Kindahl et al., 1996), and/or through the activation of local TNF α -related PGF2 α producing paths (Okuda et al., 2002). 1.3.2.1 Signs and diagnosis of clinical endometritis and pyometra Diagnosis of clinical endometritis seems not difficult, but considering that uterus size and quality of content may show a large variation between individuals and strongly depend on the time from parturition, clinical diagnosis may be quite subjective. At the assessment, the actual stage of uterine involution should always be considered: 3 – 5 weeks after calving the uterus is still enlarged and has not been fully involuted. Later, however, it seems to be often normal at rectal palpation. Before the first ovulation and formation of the first CL the cervix is open. So, in endometritis at least a small amount of (muco) purulent cervical discharge exists, although this may not always be noticed in standing animals. No systemic signs occur, in most cases also the milk production is normal (BonDurant, 1999; Sheldon, 2004; Sheldon and Dobson, 2004; Sheldon et al., 2006). 28 When bacteria in swabs collected from the uterine lumen of dairy cows 21 and 28 days after calving were categorized by their expected pathogenic potential in the uterus, purulent vaginal mucus was associated with the growth density of pathogenic bacteria but not opportunist contaminants: the growth of A. pyogenes, F. necrophorum, Prevotella ssp. and Bacteroides ssp was associated with mucopurulent or purulent vaginal mucus (Dohmen et al., 1995; Williams et al., 2005). 1.3.2.2 Subclinical endometritis The recently defined subclinical endometritis occurs any time after the histological completion of the uterine involution (i.e. on and after week 8), and is characterized by endometrium extensively infiltrated with neutrophil granulocytes, which can be recognized only by cytological examination of endometrium. There is no or only a minimum of exudate accumulated in the uterus, resulting in the complete lack of cervical discharge with pathogenomic property (Kasimanickam et al., 2004; Gilbert et al., 2005; Sheldon et al., 2006). Subclinical endometritis is a chronic, unapparent inflammatory process of endometrium with a relatively high proportion of PMN leukocytes in the uterus, which suppresses the fertility of affected cows. Proportion of PMN cells considered “relatively high” depends on sampling technique as well as on the time from parturition. 29 CHAPTER TWO MATERIAL AND METHODS 2. Study area This study was carried out at Khartoum University Dairy Farm. The location of this farm is about 4 km North to the Faculty of Veterinary Medicine at Shambat, Khartoum North. The average rainfall per year is 176 mm. The maximum rainfall is between July and September. Temperature is very high and it exceeds 45oC during the days of summer months. 2.2 Early postpartum vaginal swabs collection A total of 80 vaginal swabs were collected randamly from cows of different age at postpartum period within 1 – 3 days after parturation. These samples were collected between January 2006 to November 2009.Thirty swabs were collected from postpartum cow's vagina within 1 – 3 days after parturition, 25 vaginal swabs were collected from postpartum cows within 1 – 3 days after parturition before treatment with 1% Lugol’s Iodine and 25 vaginal swabs were collected from same postpartum cows 24h after treatment with 1% Lugol’s Iodine. Some cows showed abnormal vaginal discharge,on cow had aretaind placenta. The swabs were transferred on ice to Department of Microbiology and were cultured within 2 hours of collection. The swabs were cultured on blood agar and on MacConkey's agar and incubated aerobically at 37°C for 24-48 hours. 2. 3 Sterilization 2.3.1 Flaming Flaming was used to sterilize slides, cover slips and glass rods. 2.3.2 Red heat It was used to streilizse wire loop, needles and spatulas by holding them over Bunsen burner flame until they became red. 30 2.3.3 Sterilization of equipment (hot air oven) Petri dishes, test tubes, forceps, flasks, Pasteur pipettes and graduated pipettes were sterilized in a hot air oven at 160° C for 1h. 2.3.4 Irradiation Ultraviolet irradiation for 20 minutes was used to sterilize media pouring room. 2.3.5Disinfection Alchol (70%) was used for disinfecting working places in the media preparation room and in the laboratory. 2.3.6 Sterilization of culture media and solutions Media and solutions were sterilized by autoclaving at 121° (15 Ib/sq. inch) for 15 minutes, but carbohydrates containing media were sterilized by autoclaving at 115° C (10 Ib/sq. inch) for 10 minutes. 2.4 Reagents and indicators 2.4.1 Reagents 2.4.1.1 Tetramethyl-diphenylene diamine dihydrochloride This reagents was obtained from British Drug House, London (BHD), Ltd. It was prepared as 3% aqueous solution. It was used for oxidase test. 2.4.1.2 Hydrogen peroxide (H2o2) This reagent was obtained from Agropharm limited, Buckinham. It was prepared as 3% aqueous solution and it was used for catalase test. 2.4.1.3 Methyl red solution This reagent was prepared by dissolving 0.04 g of methyl red in 40 ml ethanol. The volume was made to 100 ml with distilled water. It was used for methyl red (MR) test. 2.4.1.4 Alpha-naphthol solution Alpha-naphthol is a product of British Drug House (BDH) London. It was prepared as 5% aqueous solution for Voges-Proskauer (VP) test. 31 2.4.1.5 Potassium hydroxide This reagent was prepared as 4% aqueous solution. It was used for Voges-Proskaur test. 2.4.1.6 Nitrate reagent Nitrate test reagent was consisting of two solutions and they were prepared according to Barrow and Feltham (1993). Solution A was composed of 0.33% sulphanilic acid dissolved by gentle heating in 5Nacetic acid. Solution B was composed of 0.6% dimethyleamine-alphnephthylamine dissolved by gentle heating in 5N-acetic acid. 2.4.1.7 Kovac´s reagent This reagent contained 5g of para-dimethyl aminobenzaldehyde, 75 ml amyl alcohol and 25 ml concentrated hydrochloric acid. It was prepared as described by Barrow and Feltham (1993) by dissolving the aldehyde in the alcohol by heating in a water bath. It was then cooled and the acid was added. The reagent was stored at 4°C for later use in indole test. 2.4.1.8 Lead acetate Filter paper strips 4-5 mm wide and 50-60 mm long were impregnated in lead acetate saturated solution and then dried . it was used for hydrogen sulphide test. 2.4.1.9 Gram stain regents 2.4.1.9.1 Crysal violet Ingredients g/Iiter Crystal violet 20.0 g Ammonium oxalate 9.0 g Ethanol 95 ml Alcohol was added to the crystal violet and mixed well until the dye was completely dissolved. Ammonium oxalate was dissolved in about 200 ml of distilled water and it was added to the stain . Then made up to I litre 32 with distilled water and mixed well. The prepared solution was stored at room temperature. 2.4.1.9.2 Lugols iodine Ingredients g/Iiter Potassium iodide 20 g Iodine 10 g Distilled water 1L Potassium iodide was dissolved in about a quarter of water . Iodine was added to potassium iodide solution and mixed well. The solution was made up to 1liter with disstilled water, mixed well and then stored in dark place at room temperature. 2.4.1.9.3 Decolorize (acetone – alcohol) Ingredients ml/1iter Acetone 500 ml Ethanol or methanol, absolute 475 ml Disstilled water 25 ml Disstilled water was mixed well with alcohol; acetone was measured and added immediately to alcohol. The solution was mixed well and stored at room tempreature. 2.4.1.9.4 Strong carbol fuchsin Strong carbol fuchsin consisted of two solutions: 1- Solution A : 10g of basic fuchsin mixed with 10 ml of ethanol (95%) dissolved in stopper bottle and kept at 37°C overnight. 2- Solution B: 5 g of phenol were mixed with 100 ml of disstilled water and shaked to dissolved. Strong carbol fuchsin was prepared by pouring 10 ml of solution A into 100 ml of solution B 33 2.4.2 Indicators 2.4.2.1 Andrade´s indicators This indicator composed of 5 g acid fuchsin, I litre distilled water and 150 ml N-NaOH. The acid fuchsin was added to I litre distilled water and 150 ml N-NaOH, mixed and was allowed to stand at room temperature for 24 h with frequent shaking until the color changed red brown. 2.4.2.2 Bromothymol blue Bromothymol blue indicator was obtained from British Drug House, (BDH) Ltd. London . The solution was prepared by dissolving 0.2g of the bromothymol blue powder in 100 ml distilled water. 2.4.2.3 Phenol red This reagent was obtained from Hopkins and William Ltd, London. It was prepared as 0.2% solution in distilled water. 2.5 Collection of blood for enriched media Defibrinated sheep blood was used in preparing blood agar medium. The blood was collected aspetially from the jugular vein of a healthy sheep, in asterile flask containing glass beads and mixed gently after collection. The blood was distributed in 10 ml amounts in sterile screw capped bottles and stored in arefrigerator at 4°C. 2.6 Preparation of media 2.6.1 Nutrient broth Thirteen grams of nutrient broth (Oxoid, CM1) were added to 1 litre of distilled water, mixed well and distributed in 3 ml amounts into clean test tubes. Then sterlized by autoclaving at 121°C for 15 minutes. 2.6.2 Peptone water Fifty grams of peptone water powder (Oxoid, CM9 - CM10) were added to 1 litre of distilled water, mixed well, distributed in 3 ml amount into clean test tubes and sterlized by autoclaving at 121°C for 15 minutes. 34 2.6.3 Peptone water sugars This medium contained 15 g peptone, 10 ml Andrade's indicator, 10g sugar and 100 ml distilled water. The pH of peptone water sugar was adjusted to 7.2 before the addition of Andrade's indicator. After mixing; the complete medium was distributed into portions of 2 ml into clean test tubes containing Durham's tubes and sterilized by autocalving at 110°C (10 ib/inch²) for 10 minutes. 2.6.4 Nitrate broth The medium was prepaerd as described by Barrow and Feltham (1993). One gram of potassium nitrate was dissolved to 1 litre of nutrient broth, and distributed in 5ml amounts into clean test tubes. Then sterilized by autoclaving at 115°C for 20 minutes. 2.6.5 Glucose phosphate medium (MR-VP test medium) Five grams of peptone powder and 5 g phospahate buffer (K2HPO4) were added to 1 litre of distilled water, and dissolved by steaming. The pH was adjusted to 7.5. Five grams of glucose were added and mixed well. The complete medium was distributed into clean test tubes and sterilized by autoclaving at 110°C for 15 minutes. 2.6.6 Nutrient agar To one litre of nutrient broth (Oxoid, CM3) 15g of agar were added, dissolved by boiling and sterilized by autoclaving at 121°C for 15 minutes. Then cooled to about 50°C and distributed in 15 ml amounts per plate. The poured plates were left to solidify at room temperature on a leveled surface. 2.6.7 Blood agar Forty grams of blood agar base NO.2 (Oxoid, CM271) were suspended in 1 litre of distilled water, dissolved by boiling, mixed and sterilized by autoclaving at 121°C for 15 minutes. Then cooled to about 50°C and sterile defibrinated sheep blood was added aseptically to give final concentration of 10% and mixed gently. Fifteen ml of complete 35 medium was poured into each sterile Petri dish. The poured plates were allowed to solidity at room temperature on a leveled surface. 2.6.8 MacConkey's agar Fifty two grams of MacConkey's agar (Oxoid,CM69) were suspended in 1Liter of distilled water, and brought to boil to dissolve the ingredients copletely. Then sterilized by autoclaving at 121°C for 15 minutes and poured into sterile Petri-dishes in 15 ml amount. The poured plates were left to solidify at room temperature on a leveled surface. 2.6.9 Motility medium – Cragie tube medium Thirteen grams of dehydrated nutrient broth (Oxoid,CM1) were added to 5 g of Oxoid agar No.1 and dissolved in 1 litre of distilled water. The pH was adjusted to 7.4. This medium was dispended in volumes of 5 ml into 20 ml test tubes containing Cragie tubes, and then sterilized by autoclaving at 121°C for 15 minutes. 2.6.10 Hugh and leifson´s (O/F) medium This medium was prepared as described by Barrow and Feltham (1993). Two grams of peptone powder, 5 g of sodium chloride, 0.3 g of potassium hypophosphate and 3g of agar were added to 1 litre of distilled water. The mixture was heated in water bath at 55°C to dissolve the solids. The pH was adjusted to 7.1 and filtered. The indicator bromothymol blue (0.2% aqueous solution) was added and the mixture was sterilized by autoclaving at 110°C for 10 minutes. Then filtered sterile glucose solution was added aseptically to give a final concentration of 1%. The complete medium was mixed and distributed, asptically in 10 ml amounts into sterile test tubes of not more than 16 mm diameter. 2.6.11 Diagnostic sensitivity test agar (DST Agar) This medium was supplied by (Oxoid, CM261). It consists of protease peptone, veal infusion solids, dextrose, sodium chloride, disodium 36 phosphate, sodium acetate, adenine sulphate, guanine hydrochloride, uracil, xanthine and ion agar No.2. Forty grams of medium were suspended in 1 litre of distilled water then brought to boil to dissolve completely and sterilized by autoclaving at 121°C for 15 minutes. The sterilized medium was dispended into sterile Petri dishes in portions of 15 ml each. The poured plates were left to solidify at room temperature on a leveled surface. 2.6.12 Urea agar This medium composed of peptone, dextrose, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, phenol red and agar. It was obtained in dehydrated form Oxoid ( CM53). The medium was prepared according to manufacture´s instructions. Powder (2.4g) was dissolved in 95 ml distilled water by boiling and the pH was adjusted to 6.8. After sterilization by autoclaving at 110°C for 20 minutes, the basal medium was cooled to 50°C and aseptically, 5 ml of sterile 40% urea solution were added and distributed in 10 ml aliquots into sterile screw capped bottles, which were allowed to solidify in inclined position. 2.6.13 Simmon´s citrate agar The dehydrated medium of Oxoid ( CM155) cosisted of sodium chloride, magnesium sulphate, ammonium dihydrogen phosphate, sodium ammonium sulphate, sodium citrate, bromothymol blue and agar. Twenty-three grams of the dehydrated medium were dissolved in 1 liter distilled water by steaming. The pH was adjusted to 7.0. The medium was sterilized by autoclaving at 121°C for 15 minutes, distributed into sterile MacCarteny bottles, and allowed to set in slope and butt. 2.6.14 Dorset egg medium Nutrient broth Whole fresh eggs The nutrient broth was prepared and sterilized as described before. 37 The eggs were washed by scrubbing them carefully with soap and water followed by rinising in clean running water. The eggs were immersed in 70% v/v ethanol for 10 minutes. The eggs were breaked into a sterilized flask (permarked to hold 80 ml), which contained a few sterile beads. Then mixed well until the egg yolks and whites are completely homogenized. The egg mixture was strained through sterilized gauze or muslin into a sterilized bottle. When the nutrient broth cooled to 45 – 50°C, it was added aseptically to the egg mixture and mixed well. The medium was dispensed aseptically in 3 ml amounts in sterilized bijou bottles or screw-caped bottle. The medium was ispissated at 75 – 80°C with the bottles in an inchned position until the medium solidified. The bottles were tightly screwed and stored in a cooled place or at 2 – 8°C. 2.7 Culture of specimens The collected swabs were inoculated onto blood agar and MacConkey agar. The inoculated plates were then incubated for 24 – 48 h at 37°C. 2.8 Purification of culture All isolates were purified by several subculturing from well separated colonies of each type on primary culture. The purification was carried out on nutrient agar or blood agar. The purity was checked by examining Gram stained smear. The pure culture was then used for studying cultural and biochemical charactristics and sensitivity of isolates to antibactrial agents. The purified isolates were inculated onto dorset egg medium, incubated at 37ْC for 24 h, and then preserved at 4ْC for further examination.The pure culture was then used for studying cultural and biochemical charactristis and sensitivity of isolates to antibacterial agents. 38 2.9 Microscopic examination Smears were made from each type of colonies on primary culture and from purified colonies, fixed by heating and stained by Gram method (Barrow and Feltham, 1993). Then examined microscopically under oil immersion objective lens. The smear was examined for cell morphology, arrangement and staining reaction. 2.10 Identification of bacteria The purified isolates were identified according to the criteria described by Barrow and Feltham (1993). This included staining reaction, organism morphology, growth condition, the colonies characteristics on different media, haemolysis on blood agar, motility and biochemical characteristics. 2.11 Biochemical methods 2.11.1 Oxidase test Strip of filter paper was soaked in 1% solution of tetramethylphenylenediamine dihydrochloride, dried in hot-air oven and placed on clean glass slide by sterile forceps. A fresh young test culture on nutrient agar was picked off with sterile glass rod and rubbed on the paper strip. If a puple color developed within 5 – 10 seconds, the reaction was considered positive. 2.11.2 Catalase test A drop of 3% H2O2 was placed on clean slide and then a colony of test culture on nutrient agar was picked with a glass rod and added to the drop of 3% H2O2. A positive reaction was indicated by air bubbles. 2.11.3 Oxidation – Fermentation (O/F) test The test organism was inoculated with straight wire into duplication of test tubes of Hugh and Leifeson´s medium. To one of the test tubes a layer of sterile melted soft paraffin oil was added to the medium to seal it 39 from air. The inoculated tubes were incubated at 37°C and examined daily for 14 days. Yellow color in the open tube only indicated oxidation of glucose. Yellow color in both tube showed fermentation reaction and blue or green color in open tube and yellow color in the sealed tube indicated production of alkali. 2.11.4 Sugar fermentation test Peptone water sugar was inoculated with the organism under the test, incubated at 37°C and examined daily for several days. Acid production was indicated by appearance of reddish color, while gas production was indicated by appearance of empty space in the inverted Durham´s tubes. 2.10.5 Voges – Proskauer test The test culture was inoculated into glucose phosphate medium (MR – VP medium) and incubated at 37°C for 48 h. Three milliliter of 5% alpha-naphthol solution and 1ml of 40% potassium hydroxide were added. When bright pink color developed within 30 minutes, the raction was regarded as positive. 2.11.6 Nitrate reduction The test culture was lightly inoculated into nitrate broth and incubated at 37°C for 2 days. Then 1 ml of solution (A) followed by 1 ml of solution (B) of nitrate test reagents were added. Red color indicated positive reaction that showed nitrate had been reduced. If red color did not develop, powder zinc was added to see whether there was residual nitrate or not. Red color development indicated that nitrate in medium had been reduced to nitrite by zinc but not by organism, whereas unchanged color indicated nitrate in original medium had been reduced completely and nitrite was further broken down by the organism. 40 2.11.7 Coagulase test To 0.5 ml of 1: 10 dilution of human plasma in saline, 0.1 ml of 18 – 24 h old culture of the test organism was added, then incubated at 37°C and examined after 6 – 24 h for coagulation. Definite clot formation indicated positive result. The test was also performed on slide. Two colonies of test culture were placed on a clean slide, emulsified in drop of normal saline and a loopfull of human plasma was added to the drop of bacterial suspension. Appearance of coarse visible clump was recorded as positive result. 2.11.8 Indole production test The test organism was inoculated into peptone water and incubated at 37°C for 48 h. One milliliter of Kovac's reagent was run down along side of the test tube. Appearance of pink color in the reagent layer within a minute indicated positive reaction. 2.11.9 Methyl red (MR) test The test organism was inoculated into glucose phosphate medium (MR – VP medium), and incubated at 37°C for 48 h. Two drops of methyl red reagent were added, shaken well and examined. Appearance of red color indicated positive reaction, whereas orange or yellow color indicated negative reaction. 2.11.10 Urease test A slope of urea agar medium was inoculated with the test organism and incubated at 37°C. Change in color to red indcated positive reaction. 2.11.11 Citrate utilization Simmon´s citrate medium was inoculated with test organism and incubated at 37°C for up to 7 days and was examined daily for growth and color change. Blue color and streak of growth indicated positive citrate utilization. 41 2.11.12 Motility test The Cragie tube in semi-solid nutrient agar prepared as described by Cruckshank et al. (1975) was inoculated by straight wire. A small piece of colony of the bacterium under test was picked by the end of the straight wire and stabbed in the center of semi-solid agar in the Craigie tube and then incubated at 37°C overnight. The organism was considered motile if it produced turbidity in the medium in and outside the Craigie tube. 2.11.13 Antibiotic sensitivity Sensitivity of isolate, to a number of antibiotics was determined by disc diffusion technique (Cruckshank et al., 1975). The isolate was grown in peptone water and incubated at 37°C for 2 h. About 2 ml of culture was poured on a Petri dish containing diagnostic sensitivity test (DST) agar medium and the inoculum was evenly distributed by rotation. Excess fluid was withdrawn using asterile Pasteur pipette and plate was left to dry at room temperature for 15 minutes.Commercially prepared discs of Plamatic Laboratory (England) were placed on the surface of the medium with sterile forceps, pressed gently to ensure full contacts with the surface of the culture medium. The plates were then incubated at 37°C for 24 h and up to 48 h. Zones of no growth around discs indicated inhibition of growth of test organism by the antibiotic of that disc. The antibiotics used were Ampicilin, Gentamicin, Chloramphincol, Cloxacillin, Erythromycin, Pencillin, Tetracycline, Streptomycin, Naldixic acid, Nitrofurantol, Colistin and Cotirmoxazole. 42 CHAPTER THREE RESULTS Eighty samples examined in this study gave 404 isolates of bacteria; two cows didn't show bacterial growth despite of clinical symptoms (vaginal discharges). One cow had retained placenta. 3.1The aerobic bacteria isolates: 3.1.1 Gram positive bacteria The Gram positive bacteria isolated from 30 postpartum vaginal swabs collected within 1-3 days after parturition were 95 (52%) Staphylococcus spp, 52 (28%) Streptococcus spp , 18 (9%) Bacillus spp, 10 (5%) Micrococcus spp, 5 (2%) Aerococcus spp, 2 (1%) Lactobacillus spp and 1 (0.6%) Corynobacterium spp (Table 1). The Gram positive bacteria isolated from 25 postpartum vaginal swabs collected within 1-3 day after parturition and before treatment with 1% lugol iodine were 25 (100%) Staphylococcus spp, 17 (68%) Streptococcus spp, 9 (36%) Bacillus spp, 3 (12%) Micrococcus spp and 3 (12%) Corynobacterium spp. (Table 4). The Gram positive bacteria isolated from 25 postpartum vaginal swabsfrom the same above 25 cows 24 hrs after treatment with 1% lugol's iodine 12 (48%) Staphylococcus spp, 6 (24%) Streptococcus spp, 4 (16%) Bacillus spp, 2 (8%) and 2 (8%) Corynobacterium spp. These results are shown in Table (4). Staphylococcus spp isolates Ninety five (52%) isolates of Staphylococcus spp were isolated from vaginal samples collected within 1-3 days after parturition. Twenty five isolates from vaginal samples within 1-3 days after parturition 43 collected before treatment with potassium iodide. And 12 isolates after treatment with 1% lugol's iodine. They were Gram positive aerobic cocci. On blood agar smooth white or grey colonies were observed. Characters and biochemical reactions of staphylococcus species are shown in Table 5. Streptococci spp isolates: Fifty two (28%) isolates of Streptococcus spp were isolated from vaginal samples collected within 1-3 days after parturition, 17 isolates from vaginal samples collected within 1-3 days after parturition before treatment with antiseptic and 6 isolates after treatment with antiseptic. They were Gram positive aerobic, cocci. On blood agar smooth white or grey colonies were observed. Characters and biochemical reactions of Streptococcus species are shown in Table 6. Bacillus spp isolates: Eighteen (9%) isolates of Bacillus spp were isolated from vaginal samples collected within 1-3 days after parturition, 9 (36%) isolates from vaginal samples collected within 1-3 days after parturition before treatment of 1% lugol's iodine and 4 (16%) isolates after treatment with 1% lugol's iodine. They were Gram – positive rods on blood agar rough, flat, gray colonies were observed. Characters and biochemical reactions of Bacillus species are shown in Table 7. Micrococcus spp isolates: Ten isolates of Micrococcus spp were isolated from vaginal samples collected within 1-3 days after parturition, 3 isolates from vaginal samples collected within 1-3 days after parturition before treatment with 1% lugol's iodine and 2 isolates after treatment. 44 They were Gram positive aerobic cocci on blood agar red, yellow and orange colonies were observed. Characters and biochemical reactions of Micrococcus species are shown in Table 8. Aerococcus spp isolates: Five isolates of Aerococcus spp were isolated from vaginal samples collected within 1-3 days after parturition, none from vaginal samples collected within 1-3 days after parturition before and after treatment of 1% lugol's iodine. They were aerobic, microaerophilic Gram positive cocci on blood agar green zone around grey colonies observed. Characters and biochemical reactions of Aerococcus species are shown in Table 9. Corynobacterium spp isolates One isolate of Corynobacterium spp was isolated from vaginal samples collected within 1-3 days after parturition. 3 isolates from vaginal samples collected within 1-3 days after parturition before treatment of 1% lugol's iodine and 2 isolates after treatment with 1% lugol's iodine. They were aerobic and facultatively anaerobic Gram positive rods on blood agar white, smooth colonies observed. Characters and biochemical reactions of Corynobacterium. Species are shown in Table 10. 3.1.2 Gram negative bacteria: Gram negative bacteria isolated from 30 postpartum vaginal swabs within 1 – 3 days after parturition were 23 (76.7%) E. coli, 18 (60%) Klebsiella spp, 10 (33.3%) Protues spp and 4 (13.1%) Enterobacter spp (Table 3). The Gram negative bacteria were isolated from 25 postpartum vaginal swabs before treatement with 1% lugos iodide. 22 (88%) E. coli, 12 (48%) Klebsiella spp, 13 (52%) Protues spp. 45 The Gram negative bacteria isolated from 25 postpartum vaginal swabs after treatement with 1% lugol's iodine were 25 (100%) E. coli, 7 (28%) Klebsiella spp, 4 (16%) Protues spp. These results are shown in Table 4. E-coli isolates: Twenty three isolates of E-coli were isolated from vaginal samples within 1-3 days after parturition, 22 isolates from vaginal samples within 13 days after parturition before treatment with 1% lugol's iodine and 25 isolates after treatment. They were Gram-negative rods, on MacConky's agar medium, largepink, circular and smooth colonies were observed indicating lactose fermentation. Characters and biochemical reactions of E. coli isolates are shown in Table 11. Klebsiella spp isolates: Eighteen isolates of Klebsiella spp were isolated from vaginal samples within 1-3 days after parturition, 12 isolates from vaginal samples within 1-3 days after parturition before treatment with 1% lugol's iodine and 7 isolates after treatment. On MacConky's agar medium, colonies of Klebsiella spp appear large, mucoid and pink color, indicating lactose fermentation and capsule formation. Characters and biochemical reactions of Klebsiella spp isolates are shown in Table 11. Proteus spp isolates: Ten isolates of Proteus spp were isolated from vaginal samples within 1-3 days after parturition, 13 isolates from vaginal samples within 13 days after parturition before treatment with 1% lugol's iodine and 4 isolates after treatment. 46 3.1.3 Antibiotics sensitivity test: Antimicrobial sensitivity of Gram positive bacteria isolated from vaginal swabs after parturition is shown in Table 12. Antimicrobial sensitivity of Gram negative bacteria isolated from vaginal swabs after parturition is shown in Table 13. 47 Table 1 Gram positive bacteria isolated from vaginal swabs of postpartum cows within 1 – 3 days after parturation. Bacterial species No. of Number samples/ of examined isolates Frequency Frequency of of isolation isolation from % total number (404) Staphylococcus spp Streptococcus spp Bacillus spp Micrococcus spp Aerococcus spp Lactobacillus spp Corynobacterium spp TOTAL 30 30 30 30 30 30 30 95 52 18 10 5 2 1 52% 28% 9% 5% 2% 1% 0.6% 32% 18% 7% 3% 1% 1% 0.5% 30 182 97.6% 62.5% 48 Table 2 Gram positive bacteria isolated from vaginal swabs collected from postpartum cows within 1 – 3 days after parturation. Bacterial species Staphylococcus chromogenes Staphylococcus sacharolyticus Staphylococcus aureus Staphylococcus cascolyties Staphylococcus intermedius Staphylococcus hyicus Staphylococcus auricularis Staphylococcus epidermidis Streptococcus pocinus Streptococcus bovies1 Streptococcus aginosus Streptococcus pyogenes Streptococcus dysaglactia Streptococcus faecalis Bacillus mycoides Bacillus laterosporus Bacillus firmis Bacillus coagulaus Bacillus cereus Micrococcus nishinomigaensis Micrococcus luteus Micrococcus varaians Micrococcus agilis Aerococcus viridans Aerococcus pedicoccus Lactobacillus thermobacter Crynobacterium strataum TOTAL Total No. Number of samples/ of isolates examined 30 9 30 14 30 28 30 13 30 15 30 12 30 1 30 3 30 2 30 25 30 1 30 16 30 5 30 3 30 5 30 1 30 3 30 4 30 5 30 2 30 5 30 1 30 2 30 3 30 2 30 2 30 1 30 182 49 Frequency of isolation % 30 % 46.7% 93.3% 43.3% 50% 40% 3.33% 10% 6.7% 83.3% 3.3% 53.3% 16.7% 10% 16.7% 3.3% 10% 13.3% 16.7% 6.7% 16.7% 3.3% 6.7% 10% 6.7% 6.7% 3.3% 609.97 Table 3. Gram negative bacteria isolated from vaginal swabs collected from postpartum cows within 1 – 3 days after parturation. Bacterial species No. of Number Frequency Frequency samples/ of isolates of isolation of % isolation examined from total number (404) Escherichia coli Kelbsiella oxytoca Enterobacter cloacae Enterobacter sakazaki Proteus mirabilis TOTAL 30 30 30 30 30 30 23 18 2 2 10 55 50 76.7% 60% 6.7% 6.7% 33.3% 183.4% 17% 9% 0.5% 0.5% 6% 33% Table 4: Bacteria species isolated from vaginal swabs collected from same cows within 1 – 3 days postpartum and then 24 hours after treatment with antiseptic 1 % lugol's iodine Bacterial species Escherichia coli Staphylococcus spp Streptococcus spp Bacillus spp. Corynobacterium spp Proteus spp Micrococcus spp Kelbsella spp TOTAL No. of cows examined 25 25 25 25 25 25 25 25 25 No. (percentage) isolated before treatment 22 (88 %) 25 (100%) 17 (68%) 9 (36%) 3 (12%) 13 (52%) 3 (12%) 12 (48%) 104 (416%) 51 No. (percentage) isolated after treatment 25 (100 %) 12 (48 %) 6 (24 %) 4 (16 %) 2 (8%) 4 (16 %) 2 (8 %) 7 (28 %) 62 (248%) Table 5 Characters and biochemical reactions of Staphylococcus species isolated from vaginal swabs collected from postpartum cows :Bacterial species Characters Staph. Staph. Staph. Staph. Staph. Staph. Staph. Staph. aureus intermedius hyicus chromogenes epidermids auricularis sachrolyticus cascolytics Gram stain + ve + ve + ve + ve + ve + ve + ve + ve Shape Cocci Cocci Cocci Cocci Cocci Cocci Cocci Cocci Spores - ve - ve - ve - ve - ve - ve - ve - ve Motility - ve - ve - ve - ve - ve - ve - ve - ve Catalase + ve + ve + ve + ve + ve + ve + ve + ve Oxidase - ve - ve - ve - ve - ve - ve - ve - ve O/F test F F F F F F F F VP test + ve + ve - ve - ve + ve - ve ND - ve Nitrate utilization + ve + ve + ve + ve + ve - ve + ve - ve Urea hydrolysis + ve + ve + ve + ve + ve - ve + ve - ve Coagulase + ve + ve + ve - ve - ve - ve - ve `- ve Novoboicin S S S S S S S S Acid from :Lactose Maltose Mannitol Fructose Sucrose Trehalose Xylose Raffinose Mannose + ve + ve + ve + ve + ve + ve - ve - ve + ve ND/ not done S/ Sensitive + ve - ve + ve + ve + ve + ve - ve - ve + ve + ve - ve - ve + ve + ve + ve - ve - ve + ve + ve + ve + ve + ve + ve + ve - ve - ve + ve 1 + ve + ve - ve + ve + ve - ve - ve - ve + ve - ve + ve - ve + ve - ve + ve - ve - ve - ve - ve - ve - ve + ve - ve - ve - ve - ve + ve + ve + ve - ve + ve - ve - ve - ve + ve -ve Table 6 Characters and biochemical reactions of Streptococcus species isolated from vaginal swabs collected from postpartum cows. Bacterial species Strepto. Strepto. Strepto. Strepto. Strepto. pyogenes dysgalatiae aginosus procinus faecalis Gram stain + ve + ve + ve + ve + ve Shape Cocci Cocci Cocci Cocci Cocci Spores - ve - ve - ve - ve - ve Motility - ve - ve - ve - ve - ve Catalase - ve - ve - ve - ve - ve Oxidase - ve - ve - ve - ve - ve O/F test F F F F F Haemolysis β α Β Β Β VP test - ve - ve + ve + ve + ve H2O2 production - ve - ve - ve - ve - ve Yellow pigment - ve - ve - ve - ve - ve Characters Mannitol Sucrose Lactose Trehalose Raffinose Starch Glucose - ve + ve + ve + ve - ve + ve + ve - ve + ve + ve + ve - ve + ve + ve - ve + ve - ve + ve - ve + ve + ve + ve + ve - ve + ve - ve - ve + ve S/ sensitive W/ week ND/ not done 2 + ve + ve + ve + ve - ve + ve + ve Strepto. Bovis1 + ve Cocci - ve - ve - ve - ve F α + ve - ve - ve Strepto. durans + ve Cocci - ve - ve - ve - ve F β + ve - ve - ve - ve + ve + ve + ve + ve + ve + ve Acid from :- ve + ve + ve + ve - ve -ve + ve Table 7 Characters and biochemical reactions of Bacillus species isolated from vaginal swabs collected from postpartum cows. Bacterial species Characters Bacillus cereus Bacillus Bacillus Bacillus Bacillus Lactobacillus mycoides firmus coagoulus laterosporus therobacteria Gram stain + ve + ve + ve + ve + ve + ve Shape Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Motility +ve - ve + ve + ve + ve - ve Spores position and shape X V X V V - ve Utilization of citrate - ve - ve - ve - ve - ve ND Urease hydrolysis - ve - ve - ve - ve - ve ND Indole - ve - ve - ve - ve - ve ND VP + ve + ve - ve - ve + ve - ve Nitrate reduction + ve + ve + ve + ve + ve - ve Starch hydrolysis + ve + ve + ve + ve -ve ND Oxidase - ve - ve - ve - ve - ve - ve O/ F test F ND F F F F Acid from :Glucose + ve + ve + ve + ve + ve + ve gas Galactose - ve - ve - ve - ve - ve - ve Mannose - ve - ve - ve + ve - ve + ve Raffinose - ve - ve - ve + ve - ve ND Salcin + ve -ve - ve - ve + ve ND Xylose - ve - ve - ve - ve - ve - ve X Spore oval elliposoidal V Spore central subterminal 3 Table 8 Characters and biochemical reactions of Micrococcus species isolated from vaginal swabs collected from postpartum cows. Characters Gram stain Shape Spores Motility Growth in air Catalase Oxidase Nitrate reduced VP Pigmentation O/ F test Acid from:Glucose Fructose Sucrose Micrococcus lutcus + ve Cocci - ve - ve + ve + ve + ve - ve - ve Yellow O - ve - ve - ve Bacterial species Micrococcus Micrococcus varaians nishinomlyaensis + ve + ve Cocci Cocci - ve - ve - ve - ve + ve + ve + ve + ve + ve + ve + ve + ve - ve - ve Yellow Orange O O + ve + ve - ve - ve - ve - ve 4 Micrococcus agilis + ve Cocci - ve + ve + ve + ve + ve - ve - ve Red O - ve - ve - ve 5 vaginal swabs collected from postpartum cows. Characters Gram stain Shape Spores Motility Catalase Oxidase Glucose (acid) O/F Haemolysis VP H2O2 production Yellow pigment Mannitol Sucrose Lactose Trehalose Raffinose Starch Bacterial species Aerococcus pedicoccus Aerococcus viridians + ve + ve Cocci Cocci - ve - ve - ve W - ve - ve + ve + ve F F α Α - ve - ve + ve - ve - ve - ve - ve + ve - ve + ve - ve - ve 1 Fermentation of :- ve + ve + ve + ve - ve + ve Table 10 Characters and biochemical reactions of Corynobacterium striatum species isolated from vaginal swabs collected from postpartum cows. Bacterial species Corynobacterium striatum + ve Bacilli -ve - ve + ve + ve -ve -ve -ve + ve -ve F Characters Gram staian Shape Spores Motility Oxidase Catalase Nitrate reduction Urease hydrolysis Haemolysis Growth improved by blood serum VP O/F Acid from:Glucose Lactose Maltose Mannitol Salcin Starch Sucrose Trahalose Xylose + ve + ve + ve + ve -ve + ve - ve + ve - ve 2 Table 11: Characters and biochemical reactions of Gram negative bacteria isolated from vaginal swabs collected from postpartum cows. Characters Gram staian Shape Motility Yellow pigment Red pigment MacConkey growth Citrate utilization Urease hydrolysis Catalase Oxidase O/F Nitrate reduction Pigment production Haemolysis Requirement for blood or serum Growth on nutrient agar Gas from glucose Escherichia coli - ve Cocci + ve - ve - ve + ve - ve - ve + ve - ve F ND ND -ve ND Kellbsiella oxytoca - ve Cocci - ve - ve - ve + ve + ve + ve ND ND F ND ND ND ND ND + ve ND + ve ND + ve 3 Bacterial species Proteus Enterobacter mirabilis cloace - ve - ve Cocci Cocci + ve + ve - ve - ve - ve - ve + ve + ve -ve + ve + ve -ve ND ND ND ND F F ND ND ND ND ND ND ND ND ND + ve Enterobacter sakazaki - ve Cocci + ve + ve - ve + ve + ve - ve ND ND F ND ND ND ND ND + ve Table 11:Continued Bacterial species Escherichia Kellbsiella Proteus Enterobacter Enterobacter coli oxytoca mirabilis cloace sakazaki MR test + ve - ve - ve - ve - ve VP test - ve - ve - ve - ve - ve Indole + ve + ve - ve - ve - ve ND ND ND ND ND Acid from Glucose ND ND ND ND ND Adinotole -ve + ve -ve + ve + ve Arabinose + ve + ve -ve ND ND Lactose - ve + ve -ve -ve + ve Maltose + ve + ve -ve -ve + ve Mannitole + ve + ve -ve + ve + ve raffinose -ve + ve -ve -ve -ve Salcin - ve + ve -ve + ve + ve Sucrose + ve + ve -ve + ve + ve Sorbitol + ve + ve -ve + ve -ve Glycerol + ve + ve + ve -ve -ve Trehalose + ve + ve + ve + ve + ve Xylose +ve + ve + ve + ve + ve Starch - ve - ve - ve - ve - ve Gluconate - ve + ve + ve + ve + ve Malonate - ve + ve - ve + ve - ve Tabe 12 : Antimicrobial sensitivity of Gram positive bacteria isolated from vaginal swab samples collected from postpartum cows Characters species No. of isolates examined Percentage of isolates sensitive to Amp. Percentage of isolates sensitive to Erythro. Percentage of isolates sensitive to Gent. Percentage of isolates sensitive to Chlor. Percentage of isolates sensitive to Cloxa Percentage of isolates sensitive to Strepto. Percentage of isolates sensitive to Tetra Percentage of isolates sensitive to Penic. occus 132 100 % 50 % 100 % 100 % 50 % 0.0 % 50 % 100 % cus 75 100 % 32 % 100 % 70 % 32 % 46 % 30 % 90 % 4 pp. 31 cterium 6 100 % 73 % 100 % 98 % 0.0 % 9% 100 % 50 % 100 % 82% 100 % 0.0 % 22 % 100 % 60 % 100 % us spp 15 100 % 32% 100 % 15 % 0.0 % 73 % 100 % 41 % s spp 5 100 % 0.0 100 % 30 % 50 % 50 % 50 % 30 % 264 100 % 44.83% 100 % 52.16% 46.33% 65% 68.5% 24.16% Amp = Ampicillin ; Erythro = Erythromycin ; Gent = Gentamicin ; Chlor = Chloramphenicol ; Cloxa = Cloxacillin ; Strepto = Streptomycin ; Tetra = Tetracycline ; Penic = Penicillin ; Antiseptic = 1% Lugol’s Iodine 5 Table 9 Characters and biochemical reactions of Aerococcus species isolated from 13: Antimicrobial sensitivity of Gram negative bacteria isolated from vaginal swab samples collected from postpartum cows No. of Percentage Percentage isolates of isolates of isolates examined sensitive sensitive to Amp. to Gent. Percentage of isolates sensitive to Chlor. Percentage of isolates sensitive to Tetra Percentage of isolates sensitive to Strepto. Percentage of isolates sensitive to Nal Percentage of isolates sensitive to Nitro. Percentage of isolates sensitive to Col Percentage of isolates sensitive to Cot 70 100 % 100 % 12 % 50 % 50 % 82 % 50 % 11 % 62 % 27 100 % 100 % 32 % 33 % 92 % 70 % 50 % 88 % 90 % 37 100 % 100 % 50 % 63 % 88 % 62 % 23 % 70 % 100 % 134 100 % 100 % 31.33% 48.66% 76.66% 71.33% 41% 56.33% 84% Amp = Ampicillin ; Gent = Gentamycin ; Chlor = Chloramphenicol ; Tetra = Tetracycline ; Strepto = Streptomycin ; Nal = Nalidixic acid ; Nitro = Nitrofurantol ; Col = Colictin ; Cot = Cotrimoxazole ; Antiseptic = 1% Lugol’s Iodine 100 80 60 40 20 Sensitive Sensitive resistant Amp. 100 0 Erythro 44.83 55.17 Gent. 100 0 Chlor. 52.16 47.84 Cloxa 46.33 53.67 c. An t is ep tic resistant Pe ni Te tra o. St er pt ox a Cl r. lo Ch Ge nt . o th r Er y Am p. 0 Sterpto. 65 35 Tetra 68.5 31.5 Penic. 24.16 75.84 Fig1:Antimirobial sensitivity of (Gram positive)bacteria isolated from vaginal swab samples colleted from dairy cows in Khartoum state within1-3 days after parturition. Antiseptic 22.33 77.67 100 80 60 Sensitive 40 resistant 20 Sensitive resistant Amp. 100 0 Gent. 100 0 Chlor. 31.33 68.67 Tetra 48.66 51.34 Strepto 76.66 23.34 Go t An t is ep tic l Co Ni t ro Na l o St re pt Te tra Am p. Ge nt . Ch lo r. 0 Nal 71.33 28.67 Nitro 41 59 Col 56.33 43.67 Got Fig1:Antimirobial sensitivity of (Gram negative)bacteria isolated from vaginal swab samples colleted from dairy cows in Khartoum state within1-3 days after parturition. 84 16 Antiseptic 45.33 54.67 CHAPTER FOUR DISCUSSION Postpartum (PP) genital tract bacterial infection causes infertility in dairy cows (Nokoao et al., 1997). The genital tract is naturally protected against the entrance of micro-organisms by the vulval, cervical sphincters and by the natural defense mechanism which is influenced by endocrine system. Vaginal bacterial infection during early PP occurs due to improper interference, retention of the faetal membranes or ascending infection (Arthure et al., 1998; Crter, 1985). This study was carried out to isolate and identify bacteria which infect reproductive system of cross-bred cows during early PP period. Eighty samples were collected, 2.5% of the cows were noninfected despite of clinical symptoms abnormal vaginal discharge, this may be due to viral infection or fungal infection. Bardd and Mark (1993) who reported that the rate of bovine mycotic infections varies from less than 1% to 10.4% depending on geographic location. Alsayed (1994) also reported isolation of Aspergillus fumigatus, Penicillum spp and Candida albicans from genital tract of repeat breeder dairy cows. This study also confirms that, 1.25% of the examend cows had retained placenta which gave mixure bacterial infections Staphylococcus spp, E. coli and Kelbsiella this agree with Jooston et al. (1988) reported that retined placentae led to mixed bacterial infections in genital tract and caused metritis. Consequently cystic ovaries in more than 50%. The exsamed cows 98,7% showed mixture of vaginal bacterial infection 404 isolates. The identified bacteria were Staphylococcus, Streptococcus, Corynebacterium, Bacillus, E. coli, Micrococcus, Aerococcus, Proteus, Klebsiella and Lactobacillus. These mixture bacteria in some cases without clinical symptoms that means these bacteria were normal flora in genital tract in stable environment. This is consistent with Hafez (1993) who found that the normal microbial flora of genital tract is composed by bacteria of genera Staphylococcus, Streptococcus and Coliform group. This study also confirm that 32% of total isolates were Staphylococcus spp, 28% of these Staphylococcus spp were Staphylococcus aureus, 18% of total isolate were Streptococcus spp ,43% of these Streptococcus spp were Streptococcus bovis, Bacillus, 1% Corynobacterium, 3% Micrococcus, 1% Aerococcus, 17% E.coli, 6% Proteus, 9% Kelbsiella spp and 2% isolate were Lactobacillus. These bacteria are opportunistic pathogens that normally inhabit the environment of the cow and are present on the integument and in the faeces this agrees with Arthur et al. (1984), and Gibbons et al.(1954) who found Sterptococci Group C, Staphylococci, Corynebacterium pyogenes, E.coli, Micrococcus, Diphtherioids and Proteus. These organisms require predisposing factors to effect individual cows fertility. At parturition massive contamination with these opportunistic organisms may lead to infertility (Arthur et al 1998). The most commonly isolated organisms was E.coli (59%) fllowed by Staphylococcus aureus 57.4% and Staphylococcus epidermidis (Kamieniecki, 1991). The pathogens found in this study are similar to the pathogens reported in several study that were reviewed elsewhere (Nomboothripad and Raja, 1976; Eduvic et al., 1984; Elazab et al., 1988) they isolated Staphylococcus aureus, E.coli, Klebseilla spp, Psuedomonus pyocyanca, Corynebacterium pyogenes, Proteus vulgaris and several anaerobic microorganisms from reproductive tract of normal suckling cows during PP and also found these bacteria causes of repeat breeding, retained placenta and metritis. E.coli was represent 17% of total isolates. It is a well known to cause of endometritis and infertility in cattle (Sheldon et al., 2002). It is accepted that they come from environment most probably from the cattle's feaces (Yerham et al., 2006). Isolation of E.coli from genital tract agrees with William et al. (2005) who reported that aerobic bacteria E.coli and non-hcamolytic Streptococci were isolated from PP cows and from puerperal metritis. Paisely et al. (1986), Knnedy and Miller (1993), Lewis (1997) Bondurant (1999); Leblanc et al. 2002) Kasimancllen et al. (2004) and Gilbert et al. (2005) found E.coli non and haemolatic Streptococci caused puerperal metritis, pyometra and subclinical endometritis. Ahmed and Elshakh (2004) found that the major pathogenic bacteria isolated from genital tract of the PP dairy cows were Staphylococcus spp, Streptococcus spp, Pasteurella multocida and E.coli. The genital bacterial infection during early PP in diary cows is presumply due to poor sanitation and hygienc during normal delivery, dystocia or retention of the foetal membranes. In addition, contamination of the genital tract of the cow could be via the descending bacterial infection, uterine bacterial infection, or bacterial products, all these findings agree with the findings of this study. Isolated lactobacillus from genital tract at early PP agrees with Otero et al. (1999) and Otero (2000) who found the lactobacillus were isolated from posterior vagina of thirty healthy cows. Antimicrobial resistance has been recognized as important problem in human and veterinary medicine, and antimicrobial agent misuse is considered the most important factor for the emergence, selection and dissemination of antimicrobial agent-resistant bacteria (Sayah et al., 2005). Sensitivity testing of bacterial isolates was in this study showed resistance to many antibiotics commonly used for treatment of disease in animals. E.coli isolates showed medium and lower sensitivities to Tetracycline (50%) Streptomycin (50%) this agrees with Ellaithi (2004) who studied susceptibility of E.coli strains isolated from diarrhoaic calves and man. She found E.coli had medium and lower sensitivities to Tetracycline (45%) . E.coli isolated in this study was found sensitive to Gentamycin (100%). Which agrees with Orden (2001) who found that E.coli strains isolated form dairy calves affected by neonatal diarrhea were very susceptible (89-95%) to Gentamycin. All Streptococcus spp strains were sensitive to Penicillin and Ampicillin (90%-100%) these results agree with Geo et al. (2000) who reported that all beta hemolytic Streptococci were sensitive to Penicillin. While most Streptococcus spp isolates in this study were less sensitive to Erythromycin (32%) and Streptomycin (46%). Lowbury et al. (1959) reported that Streptococcus pyogenes isolates isolated from clinical sources were resistant to Erythromycin and this study confirms these findings. The strains of Streptococcus spp isolated in this study were less sensitive to Erythromycin (32%). Cloxacillin (32%) and Tetracycline (30%). These results agree with Geo et al. (2001) who reported that pneumococci was sensitive to Penicillin and resistant to Tetracycline and Erythromycin. The strains of Staphylococcus spp isolated in the present investigation were sensitive to Ampicilin (100%) Gentamycin (100%) Chloramphinol (100%) Penicillin (100%) and Cloxacillin (50%). This disagrees with Geo et al. (2001), who found Staphylococcus aureus resistant to Penicillin but in this study Staphylococcus was resistant to Streptomycin. The sensitivity results in Gram positive bacteria illustrate that Ampicillin and Gentamycin could be considered the drug of choice for treatment of vaginal postpartum bacterial infection as most of isolated bacteria (100%) were sensitive to both of them and Penicillin is the third drug of choice as many of the isolated bacteria (86.09%) were sensitive to it. For Gram negative bacteria Ampicillin and Gentamycin were also two drugs of choice (100%) and of the isolated bacteria 79.1% were sensitive to Cotrimoxazole The sensitivity of isolated bacteria was variable to antiseptic 1% Lugol’s Iodine examined in this study , Corynobacterium spp and Micrococcus spp were sensitive to 1% Lugol’s Iodine (50%). Staphylococcus (5%), Areococcus spp, Kelbseilla spp and Proteus spp were sensitive to 1% Lugol’s Iodine (50%). Streptococcus spp, Bacillus spp and E.coli were resistant to 1% Lugol’s Iodine. These results means that uterine wash is not good treatment for postparturient cows in case of retained placenta or normal delivery. Some cows gave more isolates after treatment with antiseptic 1% Lugol’s Iodine. While other cows had no bacterial infection before infusion of 1% Lugol’s Iodine, but bacteria were isolated after infusion of 1% Lugol’s Iodine this might be due to inhibition of normal cow’s vaginal flora or new infection due to contaminated material, water and non hygienic measures during PP of uterine wash. CONCLUSIONS From this study it can be concluded that:- The most common bacterium isolated from postpartum cows vaginal swabs were Staphylococcus spp, Sterptococcus spp and E. coli. - Staphylococcus aureus is the most common isolate of Staphylococcus spp and causes postpartum infection. - Ampicillin and Gentamycin are the drugs of choices for treatment of postpartum bacterial infection. - The antiseptic which was used in treat of postpartum cows may caused more bacterial contamination and some bacterial isolates were resistant to this antiseptic. 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