الفهرس 
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Abstract
Bovine respiratory disease (BRD) is the most common and costly disease affecting bovine calves in the world causing great economic losses to farmers and animals owners by reducing average daily gain, feed efficiency, overall performance of beef calves and mortality .
The present study was designed to perform bacteriological and immunological studies on calves affected by respiratory manifestations with special reference to P. multocida and M. haemolytica as important causes of BRD. This study was carried out in different Governorates of Upper Egypt; including Giza, EL-Fayoum, Beni-Suef, Assiut and Sohag, during the period from January-December, 2017. A total number of 406 deep nasal swabs collected from calves affected with respiratory manifestation from different Governorates were bacteriologically examined for isolation of P. multocida and M. haemolytica and other bacterial pathogens.
The results of bacteriological examination and molecular identification revealed, that 108 isolates (26.6%) were identified as Pasteurella or Mannheimia species of which 74 P. multocida (18.2%) and 34 M. haemolytica (8.4%).
Regarding to the Governorates, the results revealed that EL-Fayoum Governorate showed the highest overall prevalence as 30.2% meanwhile, Beni-Suef Governorate showed the lowest as 19.3%. The highest prevalences of P. multocida and M. haemolytica infection were in EL-Fayoum as 20.8% and 9.4, respectively, while lowest prevalences were in Beni-Suef as 13.3% and 6.0%, respectively.
Regarding the prevalences of single and mixed P. multocida and M. haemolytica infections in different Governorates, P. multocida and M. haemolytica were singly isolated from 4.9% and 1.7% of cases, respectively. Regarding mixed infections, mixed P. multocida and M. haemolytica isolates with S. aureus was the most Prevalent as 4% and 2.7%, respectively. The current study concluded that total bacterial infection of P. multocida was most common in a high percentage in El-Fayoum and Sohag Governorates 20.8%, 19.7% respectively. Also, the total infections of M. haemolytica were most common in El-Fayoum and Assiut Governorates with percentages of 9.4% and 9.2%, respectively.
The in-vitro antimicrobial susceptibility of P. multocida and M. haemolytica isolates were tested. Generally, the results of the in in-vitro sensitivity testing of both P. multocida and M. haemolytica isolates showed high susceptibility to fluoroquinolones and cephalosporins. On the other hand, high resistances were obtained against tetracyclines, penicillins and aminoglycosides.
Other bacterial isolates from diseased calves in different Governorates were recovered and identified. The results revealed that, 56.2% of samples showed bacterial isolation other than P. multocida and M. haemolytica while 17.2% showed no bacterial isolation. The results of bacterial isolation and identification revealed that S. aureus, mixed S. aureus+ Streptococci isolates and Streptococcus species were the most prevalent as 12.6%, 11.3% and 10%, respectively. Then, mixed S. aureus + E. coli, E. coli, mixed S. aureus+ Streptococcus species+ E. coli, mixed Streptococcus species +E. coli, P. aeruginosa, Proteus species and Corynebacterium species as 5.2%, 4.2%, 4.2%, 3.2%, 2.7%, 1.5% and 1.2%, respectively. The highest prevalences of bacterial isolation were recorded in Beni-Suef (63.9%) and Assiut (60.9 %) while the lowest prevalence was recorded in Sohag (44.3%).
The collective prevalences of different bacterial isolates recovered from 406 diseased calves in different Governorates revealed that, 336 calves (82.8%) had bacterial isolation. Meanwhile 70 calves (17.2%) had no bacterial isolation.
On the immunological level, the data of the existing study showed that all respiratory affected calves recorded significant elevation of nitric oxide level when compared with normal control calves. However, all infected calves elucidated significant reduction of lysozyme activity. On the similar ground, infected calves display significant increase in serum interlukine-6.
Polymerase chain reaction (PCR) assay was applied for detection of virulence genes in P. multocida and M. haemolytica isolates from calves with respiratory disorders. PCR was applied on 5 P. multocida isolates; to determine 3 virulence genes (nanB, omp87 and toxA), and applied also on 5 M. haemolytica isolates to determine 3 virulence genes (ssa, gcp and lktC).
The results showed that all the tested P. multocida isolates (100%) represented all the virulence genes (nanB, omp87 and toxA). Meanwhile, only 3 tested M. haemolytica isolates (60%) represented each of the virulence genes (ssa, gcp and lktC).
Sequencing of P. multocida toxA gene and M. haemolytica lktC gene were applied.
Regarding P. multocida toxA gene, amino acid alignment report of the sequenced 276 amino acids of P. multocida toxA showed great homology between the Egyptian strain and the different P. multocida strains uploaded from genebank. Also, nucleotide alignment report of the sequenced 828bp nucleotides of P. multocida toxA showed great homology between the Egyptian strain and the different P. multocida strains uploaded from genebank.
Phylogenetic tree showed clear clustering of the isolated Egyptian strain with different P. multocida strains uploaded from genebank
Sequence distance of P. multocida toxA was created. Generally, the sequence identities between the isolated Egyptian strain and different P. multocida strains uploaded from genebank revealed 99.4-100% homology.
Concerning M. haemolytica lktC gene, amino acid alignment report of the sequenced 149 amino acids of M. haemolytica lktC showed great homology between the Egyptian strain and the different M. haemolytica strains uploaded from genebank. Also, nucleotide alignment report of the sequenced 447bp nucleotides of M. haemolytica lktC revealed great homology between the Egyptian strain and the different M. haemolytica strains uploaded from genebank.
Phylogenetic tree revealed clear clustering of the isolated Egyptian strain with different M. haemolytica strains uploaded from genebank.
Sequence distance of M. haemolytica lktC was created. Sequence identities between the isolated Egyptian strain and different M. haemolytica strains uploaded from genebank revealed 99.6-100% homology.