الفهرس 
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Abstract
Mannheimia haemolytica is the most common bacterial isolates that cause pulmonary diseases in ruminants. This study examined the molecular characteristics and genome sequencing of virulence-related O-sialoglycoprotease (gcp) gene of Mannheimia haemolytica isolated from cattle, buffaloes, sheep and goats suffered from respiratory manifestations. A total number of 148 samples from different farms, freshly dead and freshly slaughtered at abattoirs in El-Bohaira and Alexandria Governorate for conventional culture isolation, microscopically, biochemically and molecular characterization of Mannheimia haemolytica in ruminants by PCR and genome sequencing technique of virulence-related O-sialoglycoprotease (gcp) gene.Total numbers of collected 148 samples were consisted of 56 nasal swabs collected from 18 cattle, 24 sheep and 14 goats, and 92 lung tissues collected from 36 cattle, 25 buffaloes, 18 sheep and 13 goats. Bacteriological examination of the collected 148 samples onto TSBA (Trypton soya blood agar) medium with 5% sheep blood revealed that 128 (86.5%) isolates have been yielded, of them 26 isolates showed α haemolysis with a percentage of 17.6%, while 85 isolates (gray and pin point) showed B haemolysis have been recovered with a percentage of 57.4%, and 17 isolates obtained with no haemolysis with a percentage of 11.5% but the rest 20 (13.5%) samples showed no growth. Gram staining of the yielded 85 B haemolytic isolates revealed that 70 isolates (82.4%) were Gram negative while the other 15 isolates (17.6%) were Gram positive. The cultivation of 70 yielded Gram negative isolates onto MacConkey’s agar revealed that 51 (72.9%) isolates were lactose fermenter, while 9 (12.9%) isolates were non lactose fermenter, but the rest 10 (14.3%) isolates not yielded again and showed negative growth. Biochemical identification of the obtained 51 isolates that showed a microscopically typical appearance of Mannheimia haemolytica (gram –ve bacilli) and that were with gray and pinpoint morphology, showed B haemolysis and lactose fermenter were biochemically identified by performing Oxidase, Catalase, Indole, MR, Citrate, TSI and Urease tests which reflected that 20 (39.2%) isolates were biochemically typical Mannheimia haemolytica that showed Oxidase +ve, Catalase +ve, Indole –ve, MR –ve, Citrate –ve, Urease –ve and in TSI it made yellow slant / yellow butt without H2S production while other 31 (60.8%) isolates were atypical. The incidence of biochemically typical Mannheimia haemolytica isolation in different collected samples from 54 cattle, 25 buffaloes, 42 sheep, and 27 goats, recovered 7 (13%), 2 (8%), 6 (14.3%) and 5 (18.5%) biochemically typical Mannheimia haemolytica, respectively. from totally examined 148 samples from different tested animal species, 20 (13.5%) biochemically typical Mannheimia haemolytica were obtained. PCR technique were applied used for detection of virulence related O-sialoglycoprotease (gcp) gene in DNA extracted from yielded biochemically typical 20 isolates of Mannheimia haemolytica, out of them, 2 isolates (10%) showed positive PCR bands of virulence-related gcp gene at 227bp; one of them was isolated from nasal swab of adult cattle and the other positive one isolated from lung tissue of sheep, while other 18 isolates (90%) showed negative PCR result. The partial coding of gene sequencing of virulence-related O-sialoglycoprotease (gcp) gene in the two yielded PCR-positive Mannheimia haemolytica strains done in MACROGEN, KOREA; one of them is Mannheimia haemolytic serotype A1 which isolated from cattle nasal swab, and the other is Mannheimia haemolytic serotype A2 which isolated from sheep lung tissue and deposited in GenBank under the following accession numbers: BankIt1789308 Seq1 KP411385 for Mannheimia haemolytica MS/Alex-1 sialoglycoprotease (gcp) gene, partial cds and BankIt1789312 Seq1 KP411386 for Mannheimia haemolytica MS/Alex-2 sialoglycoprotease (gcp) gene, partial cds .The percentages of identity of partial sequencing of virulence-related O- sialoglycoprotease (gcp) gene of recent Egyptian Mannheimia haemolytica strains clarified that both isolated strains of Egyptian Mannheimia haemolytica MS/Alex 1 and MS/Alex 2 had an identity percentage of 91% with each other while identity percentages of 40% with all of Mannheimia haemolytica PH196, Mannheimia haemolytica PH494, Mannheimia haemolytica PH2 and Pasteurella haemolytica lipopolysaccharide biosynthesis, for both types of isolated Egyptian Mannheimia haemolytica. from the previously mentioned data it could be suggested that Mannheimia Haemolytica is most commonly associated with respiratory manifestation in ruminant, PCR assay for detection of virulence related gene O- sialoglycoprotease (gcp) gene could suggested its pathogenicity, rapid, sensitive, specific, facilitate identification and does not require laboratory animal, genome sequencing for gcp gene confirmed that Mannheimia haemolytica serotype A1 is most prevalent in cattle while serotype A2 most common in sheep suffering from respiratory manifestation. These could help in determining epidemiology, bacterial factor and genetic basis of virulence and pathogenesis, control in outbreaks and development of the bovine respiratory disease and could also provide potential candidates for serotype-specific vaccine development against Mannheimia haemolytica. Genome sequencing can look at millions of transcripts in a single run and determine relative expression levels of individual genes. Those understanding of its virulence and pathogenesis will improve the genetic tools and techniques that have been developed for M. haemolytica will be useful in post sequencing genetic analysis of the organism bearing in mind the great economic importance of this bacterium. Mannheimia haemolytica continues to be an enigmatic organism. With the completion of the genome sequencing, our understanding of its virulence and pathogenesis will be improved.