2012 Jiva April Cover 220 GSM Art Card Glossy ... - Jivaonline.net

POLYMERASE CHAIN REACTION BASEDDETECTION OF Mycoplasma gallisepticum IN CHICKEN*RESEARCH ARTICLESurya Sankar, G. Krishnan Nair, M. Mini and Hiron M. HarshanDepartment of Veterinary Microbiology, College of Veterinary and Animal Sciences, Mannuthy*Part of PhD. thesis submitted by the first author to Kerala Agricultural University, ThrissurINTRODUCTIONAmong the prevailing diseases of poultry,mycoplasmosis has become one of the most seriousand frequently reported diseases throughout theworld, causing considerable economic losses.Among the mycoplasma affecting poultry,Mycoplasma gallisepticum is the most pathogenicand responsible for chronic respiratory disease(CRD) in chicken and infectious sinusitis in turkey.Although the mortality among birds affected by thedisease is not very high, the disease causessubstantial losses as a result of downgrading andcondemnation of carcasses, decreased eggproduction, poor hatchability, reduced feedconversion and retarded growth, as well asaggravation of various other disease conditions andalso the high cost incurred during control programme(Kleven, 1998). The severity of the disease is greatlyinfluenced by the degree of secondary infection withNewcastle disease and Infectious Bronchitis virusesand / or bacteria such as Escherichia coli.MATERIALS AND METHODSSource of samplesA total of 50 samples were obtained fromsick/apparently healthy birds of different age groupsfrom University Poultry Farm, Mannuthy; poultryfarms in different parts of Kerala; birds brought fordisease diagnosis to the Department of VeterinaryMicrobiology; birds necropsied in Centre ofExcellence in Pathology and College of Veterinaryand Animal Sciences, Mannuthy.Processing of SamplesFifty swab samples collected in Mycoplasmabroth were removed after four hour of incubation andone ml of the broth was transferred aseptically intosterile eppendorf tubes in a laminar airflow cabinetfor the preparation of template DNA for Mycoplasmagenus-specific PCR. The remaining broth mediawere further incubated till an appreciable colourchange of the broth to orange or yellow wasevidenced or up to 21 days, whichever was earlier.Twenty five samples were directly plated on to theMycoplasma agar plates also.Extraction of DNA from clinical samplesThe extraction of DNA was performedaccording to a previously described procedure (Liuet al., 2001).Extraction of DNA from other bacterial strainsPure colonies of Eschericia coli,Staphylococcus aureus and Pasturella multocidawere inoculated separately in to five ml of Brain0heart infusion (BHI) broth and incubated at 37 C for18 h. From this broth culture, 2 ml was transferred toan eppendorf tube and centrifuged at 3000 × g for 10min. The supernatant was discarded and the pelletwas washed twice with sterile PBS. The final pelletwas re-suspended in 50l of triple distilled water.The mixture was boiled for 10 min and immediatelychilled on ice for 30 min. The samples were thawedand centrifuged at 3000× g for 5 min and supernatant0was stored at -20 C for further use as template forPCR reactions.JIVA Vol. 10 Issue 1 April 20125