الفهرس 
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Abstract
Mannheimia haemolytica is the most common bacterial isolates that cause respiratory tract infections in ruminants and is often a commensal organism in the upper respiratory tract of healthy cattle, stress provokes the bacteria in the nasal/tonsillar mucosa allowing them to be inhaled onto the lungs. M. haemolytica possesses multiple virulence factors which promote its adhesion, colonization and proliferation within the respiratory tract. This study was planned to assess the genetic diversity among M. haemolytica isolates which isolated from ruminants (cattle, buffaloes, sheep and goats) suffering from respiratory manifestations by partial genome sequencing of virulence related outer membrane lipoprotein (gs60) gene and antimicrobial resistance (blaROB-1) gene. To acheive these goals, collection of total number of 315 samples from different farms and freshly slaughtered animals at abattoirs in El-Bohaira and Alexandria governorate was performed for conventional culture isolation, microscopic, biochemical and molecular identification of the isolated M. haemolytica by PCR using specific primer, then detection of virulence releated genes of the positive isolated M. haemolytica by PCR. The screened virulence genes are gs60 and gcp genes, also occur detection of antibiotic resistance gene (blaROB-1). Total numbers of collected 315 samples were consisted of 200 nasal swabs (25, 65, 40 and 70) for cattle, sheep, buffaloes and goats respectively and115 clinically pneumonic lung tissue of freshly slaughtered animals (50, 25 and 40 from cattle, sheep and buffaloes respectively) Isolation rate of M. haemolytica from different collected samples (75 cattle, 80 buffaloes, 90 sheep, and 70 goats), recovered 4 (5%), 3 (3.33%) biochemically typical M. haemolytica from cattle and sheep respectively. No isolation from goat and buffaloes. from totally examined 315 samples from different tested animal species, 7 (2.2%) biochemically typical Mannheimia haemolytica were yielded. Identification of seven M. haemolytica isolates was confirmed by PCR technique based upon the use of specific (PHSSA) primers derived from serotype specific antigen (ssa) gene for detection of M. haemolytica. All M. haemolytica isolates that were positive by biochemical reactions were also detected to be positive by PCR. Screening of virulence related O-sialoglycoprotease (gcp) and outer membrane lipoprotein (gs60) genes in DNA extracted from seven PCR positive M. haemolytica, all isolates showed positive PCR bands of virulence-related gcp and gs60 genes at 227bp and 330bp respectively. After that, the seven isolates that were previously positive for gcp and gs60 genes were screened for antibiotic resistance gene (blaROB-1). In this study blaROB-1 gene was detected in only 2 isolates (one from pneumonic lung of cattle and other one from sheep pneumonic lung) of the total seven tested M. haemolytica isolates showing positive PCR bands at 685bp. The partial coding of gene sequencing of virulence-related gs60 gene in the seven yielded PCR-positive Mannheimia haemolytica strains done in MACROGEN, KOREA and deposited in GenBank under the following accession numbers KT074854 Mannheimia haemolytica MMD/Egypt-1, KT074855 Mannheimia haemolytica MMD/Egypt-2, KT074856 Mannheimia haemolytica MMD/Egypt-3, KT074857 Mannheimia haemolytica MMD/Egypt-4, KT074858 Mannheimia haemolytica MMD/Egypt-5, KT074859 Mannheimia haemolytica MMD/Egypt-6, KT074860 Mannheimia haemolytica MMD/Egypt-7, which revealed unique nucleotide substitutions (C59G, G175C and T266G) for KT074854, (A85T) for KT074857 and KT074858 and (C59A and T120A) for KT074860, while KT074855 Mannheimia haemolytica MMD/Egypt-2; KT074856 Mannheimia haemolytica MMD/Egypt-3 and KT074859 Mannheimia haemolytica MMD/Egypt-6 have no mutation. Nucleotide identity percentage of our seven strains was ranged between 99%and 100% in relation to genetically related circulating strains. 3D for the obtained protein sequences was carried out to predict the epitope mapping using I-Tasser software. Our findings revealed that KT074854 Mannheimia haemolytica MMD/Egypt-1, KT074857 Mannheimia haemolytica MMD/Egypt-4 and KT074858 Mannheimia haemolytica MMD/Egypt-5and KT074860 Mannheimia haemolytica MMD/Egypt-7 have a unique 3D protein form different from other genetically strains while KT074855 Mannheimia haemolytica MMD/Egypt-2; KT074856 Mannheimia haemolytica MMD/Egypt-3 and KT074859 Mannheimia haemolytica MMD/Egypt-6 have similar 3D protein form like the commonly circulating strains. The partial coding of gene sequencing of bla gene in the two yielded PCR-positive M. haemolytica isolates done and genbank accession numbers KT167392 and KT167393 were received. Multiple nucleotide alignment was conducted for detection nucleotide substitution sites which revealed unique nucleotide substitutions (T45A, A176C and C244A) for KT167392 P. haemolytica MMD/Egypt-1 Bla gene encoding beta-Lactamase, (T45A and C244A) for KT167393 P. haemolytica MMD/Egypt-2 Bla gene encoding beta-Lactamase. Nucleotide identity percentage of the two isolates was 100% with each other and ranged between 96- 99% in relation to genetically related M. haemolytica circulating strains in NCBI. 3D for the obtained protein sequences was carried out for bla gene. Our results revealed difference even between our two M. haemolytica isolates in comparison with reference M. haemolytica strain on Genbank in spite of maintaining the resistance to Ampicillin/Penicillin antibiotic.