Houssam Shaib: “PCR use in epidemiological study of avian mycoplasmosis and control of Gumboro by herbal extracts”, 2004.

Abstract:

The first study determined the immunopotentiation activities of Calendula officinalis and Centaurea ainetensis against IBD (Gumboro) virus in broilers. Day-old broilers were divided into 5 groups, of 11 birds each. Group 1 and 2 did not receive any herbal extracts while group 3, 4, and 5 received daily known amounts of Calendula officinalis, or Centaurea ainetensis or a mixture of both extracts respectively, between 16 to 21 days of age. All groups, except for group 1, were challenged with IBDV live vaccine viruses of 20x log10EID50 per bird at 17 days of age. Group 1 deprived of extracts, group 3 receiving Calendula sp. extracts and group 5 receiving a mixture of Calendula sp. and Centaurea sp. showed the highest bursal indices (0.2755, 0.2382 and 0.2582 respectively, p<0.05), the lowest respective lesion scores (2 birds out of 11, 4/11 and 4/11, P<0.05), and the highest heterophil counts (4.41, 5.00 and 5.64 heterophils/2 fields/bursa, p<0.05). Group 3, 4, and 5 that received herbal extracts, showed the lowest respective IBDV viral antigen counts in the bursa of Fabricious namely, 8.18, 9.1 and 8.27 /3 fields/bursa. C. officinalis water extracts helped in the clearance of IBDV. The second study involved standardization of the RAPD-PCR method for MG subtyping in Lebanon, evaluation of the sensitivity, specificity, and reproducibility of the method, and identification of a gene by sequencing, and reproducibility of re-amplification of major amplicons. The standardized RAPD-PCR revealed maximum clear bands of 912, 416, 854, and 480 bp at 2 mM MgCl2, 0.8 mM of each dNTP, 0.025 nmole of each primer and 750 ng of MG DNA. It showed also high sensitivity resulting in amplification at low MG-DNA levels between 50 and 100 ng of DNA. The specificity of the method in differentiating MG from Salmonella Enteritidis (SE) and E. coli was 100% resulting in amplicons of 912, 854, 416 bp for MG; 353, 680, 769, and 935 bp for SE and 303, 384, 465, 517, 914, 1084 bp for E. coli. The reproducibility of the following MG amplicons: 912, 854, 416 bp; 912, 854, 729, 416 bp; 1158, 912, 854, 729, 416 bp were 87.5, 44, and 31% respectively. This is the first report enabling to identify a target cdsA gene encoding for CDP-diglyceride synthetase based on sequencing of a 916 bp amplicon resulting from the RAPD-PCR. The re-amplification of major MG amplicons of 1153 and 726 bp bands was 100% reproducible with each primer mix. The results produced by re-amplification will be used in future epidemiologic investigations as new genetic markers to trace and control the etiologic agents of MG infection.