full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
- No tags were found...
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1123-1129 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1124by the passage <strong>of</strong> an infected placenta <strong>and</strong> orfoetal t<strong>issue</strong>s, the main concern <strong>of</strong> transmission<strong>of</strong> the infection to susceptible ewes in the periparturientperiod when the lambing environmentmay become contaminated with diseased t<strong>issue</strong>s(18). Chlamydia-induced abortion in sheep wasrecognized to stimulate an immune response thatprotected against subsequent abortion (9). Ewesthat experienced abortion as a result <strong>of</strong>experimental infection with C. abortusmaintained an elevated systemic antibodyresponse to the organism <strong>and</strong> this was associatedwith a chronic reproductive tract infection (13)Since future fertility was apparently notcompromised, ewes that aborted are usuallyretained <strong>and</strong> reintroduced to the flock.Identification <strong>of</strong> those carrier ewes could aid thedevelopment <strong>of</strong> a management strategy toeliminate continued disease outbreaks in flocks.The purpose <strong>of</strong> the present study was to establishwhether or not C. abortus is shed through thereproductive tract following infection under naturalconditions.Material <strong>and</strong> MethodsExperimental design : Two sheep farms whereenzootic abortion was endemic were selected tostudy the carrier state <strong>of</strong> Chlamydophilaabortus. Fourteen ewes confirmed to have hadaborted due to the natural infection <strong>of</strong> C. abortussix months earlier were r<strong>and</strong>omly selected forthe study <strong>and</strong> was investigated by sequentialcollection <strong>of</strong> vaginal secretions, faeces <strong>and</strong> serumsamples.Synchronization <strong>of</strong> oestrus cycle wascarried out in all the fourteen ewes by insertingvaginal progesterone impregnated sponges <strong>and</strong>by parenteral administration <strong>of</strong> pregnant mareserum gonadotrophin (PMSG). Blood <strong>and</strong> faecalsamples were collected on the day <strong>of</strong> spongeinsertion. Thirteen days later the sponge wasremoved before injecting each ewe with PMSG.Immediately Coagulated blood, faeces <strong>and</strong> vaginalsamples were collected. Again 2 <strong>and</strong> 4 dayslater vaginal swabs were collected. The eweswere euthanized 33 days later <strong>and</strong> the fallopiantube, mid uterus, <strong>and</strong> vagina were used forchlamydial isolation. All samples were stored at–80 o C until further use.Collection <strong>of</strong> samplesFaecal samples: Fresh rectal faeces collected(0.1 to 25 g) were placed in transport mediummixed with a rotomixer <strong>and</strong> centrifuged at 1000xg for 10 min. Twenty µl <strong>of</strong> the supernatant werethen used to inoculate a monolayer <strong>of</strong> McCoytogether with culture medium containing 200 µg/ml <strong>of</strong> gentamycin (15).Reproductive tract samples: The sections <strong>of</strong>the vagina distal, the mid region <strong>of</strong> the uterus <strong>and</strong>the oviduct were obtained under aseptic conditions<strong>and</strong> immersed into transport medium. Thesamples were cut up into smaller pieces withscissors <strong>and</strong> broken further with mortar <strong>and</strong>pestle. After centrifugation at 1000 g for 10 min,100 µl <strong>of</strong> the supernatant were used to inoculateMcCoy cells (12)Vaginal swabs: Vaginal swabs for chlamydialisolation <strong>and</strong> PCR were collected after gentlyinserting sterile swabs into the vagina <strong>and</strong> rotatingthem in both directions. Immediately aftersampling, the tip <strong>of</strong> the vaginal swab wasimmersed in transport medium, mixed withrotomixer <strong>and</strong> centrifuged at 1000 x g for 10 min.The supernatants were then used to inoculateMcCoy cell monolayers <strong>and</strong> for DNA extraction.The samples were kept at –80 o C until used.Bacterial identification <strong>and</strong> isolationMcCoy cells, grown in 24-well platemonolayer were used for the isolation. Inoculatedmonolayers were examined for the presence <strong>of</strong>inclusion bodies after staining cover slips with DiffQuick stain at 48-72 hours post inoculation. SomeRabia Abadía Elzlitne et al