full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1123-1129 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1124by the passage of an infected placenta and orfoetal tissues, the main concern of transmissionof the infection to susceptible ewes in the periparturientperiod when the lambing environmentmay become contaminated with diseased tissues(18). Chlamydia-induced abortion in sheep wasrecognized to stimulate an immune response thatprotected against subsequent abortion (9). Ewesthat experienced abortion as a result ofexperimental infection with C. abortusmaintained an elevated systemic antibodyresponse to the organism and this was associatedwith a chronic reproductive tract infection (13)Since future fertility was apparently notcompromised, ewes that aborted are usuallyretained and reintroduced to the flock.Identification of those carrier ewes could aid thedevelopment of a management strategy toeliminate continued disease outbreaks in flocks.The purpose of the present study was to establishwhether or not C. abortus is shed through thereproductive tract following infection under naturalconditions.Material and MethodsExperimental design : Two sheep farms whereenzootic abortion was endemic were selected tostudy the carrier state of Chlamydophilaabortus. Fourteen ewes confirmed to have hadaborted due to the natural infection of C. abortussix months earlier were randomly selected forthe study and was investigated by sequentialcollection of vaginal secretions, faeces and serumsamples.Synchronization of oestrus cycle wascarried out in all the fourteen ewes by insertingvaginal progesterone impregnated sponges andby parenteral administration of pregnant mareserum gonadotrophin (PMSG). Blood and faecalsamples were collected on the day of spongeinsertion. Thirteen days later the sponge wasremoved before injecting each ewe with PMSG.Immediately Coagulated blood, faeces and vaginalsamples were collected. Again 2 and 4 dayslater vaginal swabs were collected. The eweswere euthanized 33 days later and the fallopiantube, mid uterus, and vagina were used forchlamydial isolation. All samples were stored at–80 o C until further use.Collection of samplesFaecal samples: Fresh rectal faeces collected(0.1 to 25 g) were placed in transport mediummixed with a rotomixer and centrifuged at 1000xg for 10 min. Twenty µl of the supernatant werethen used to inoculate a monolayer of McCoytogether with culture medium containing 200 µg/ml of gentamycin (15).Reproductive tract samples: The sections ofthe vagina distal, the mid region of the uterus andthe oviduct were obtained under aseptic conditionsand immersed into transport medium. Thesamples were cut up into smaller pieces withscissors and broken further with mortar andpestle. After centrifugation at 1000 g for 10 min,100 µl of the supernatant were used to inoculateMcCoy cells (12)Vaginal swabs: Vaginal swabs for chlamydialisolation and PCR were collected after gentlyinserting sterile swabs into the vagina and rotatingthem in both directions. Immediately aftersampling, the tip of the vaginal swab wasimmersed in transport medium, mixed withrotomixer and centrifuged at 1000 x g for 10 min.The supernatants were then used to inoculateMcCoy cell monolayers and for DNA extraction.The samples were kept at –80 o C until used.Bacterial identification and isolationMcCoy cells, grown in 24-well platemonolayer were used for the isolation. Inoculatedmonolayers were examined for the presence ofinclusion bodies after staining cover slips with DiffQuick stain at 48-72 hours post inoculation. SomeRabia Abadía Elzlitne et al