الفهرس 
ACKNOWLEDGMENT
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Abstract
A total of 300 samples were taken from different layer farms aged 33-75 weeks in Egypt (El-Sharqia, Elmina, Assiut and Sohag) showing remarkable signs for fowl cholera and examined for detection of P. multocida. Isolation of P. multocida was from liver, lung and trachea and identified biochemically and biologically. Suspected cultured were small mucoid dew drops like colonies that observed on on trpticase soy agar and non-haemolytic on blood agar after 18 hrs incubation at 37oC. Moreover, there was no growth on MacConkey agar revealing 16 suspected isolates to be P.mulocida .Typical Gram-negative, bipolar coccobacilli microorganism was revealed by Gram and Methylene blue stains from recent cultures, blood films and tissue smear prepared from positive sample respectively.
Biochemical reactions were performed using VITEK 2 system resulted in 8 isolates of P.multocida were identified. Isolates of P. multocida were found to be virulent with death times recorded at 24 h and 48 h. Heart blood smear, liver and spleen impression smears revealed characteristic bipolar organisms using Giemsa staining. The isolates showed typical culture for P. multocida Molecular identification using muliplex PCR isolates gave bands at 460 bp which are characteristic to Pasteurella subspecies multocida with an incidence of (2.6%) which in turn belong to capsular type A (1044 bp).
Phenotypic pattern of sensitivity and resistance of isolates was assessed using antibacterial agents and determined by two methods:-
A- The disc diffusion technique: according to inhibition zone the most effective antibiotics were amoxicillin, doxycycline, Flumequine. Strains showed an intermediate sensitivity toward gentamicin and sulfamethoxazole and Trimethoprim, while there were high resistant to Oxytetracycline streptomycin, chloramphenicol, ampicillin, erythromycin, Spectinomycin and Cephradine.
B- Determination of minimum inhibitory concentration (MICs): isolates were (100%) resistant to 4 antimicrobials (Lincomycin, erythromycin, sulphaquinxaline, Spectinomycin. All isolates were (87.5%) resistant to 3antimicrobials (streptomycin, Cephradine, florphenicol).All isolates were (100%) sensitive to amoxicillin). All isolates were (87.5%) sensitive to doxycycline .All isolates were (25%) sensitive to gentamicin. Different variations of MICs were detected for resistant strains, whereas the MICs of streptomycin and erythromycin were distinctly higher revealed128 μg/ml, while doxycycline was16μg/ml with50% and was (8 μg/ml) with 37.5% for all isolates.
Antimicrobial resistance phenotypes observed with PCR-positive results revealed 5 isolates harboring multiple resistance genes with 62.5%.A progressive increase of resistance with 75% to erythromycin, streptomycin and 87.5% for amoxicillin while the percentage of resistance to tetracycline was 37.5 %were detected among the P. multocida strains with amplified fragments of 488pb, 685, 1076 and 489pb corresponding to ermX, BlaROB1, tetH and aadA genes respectively.
Experimental infection of P. multocida type A in chickens (group1,sub-group A) produced characteristic mild clinical picture for respiratory system affection consisted of coughing, sneezing and frothy eyes within 48 h and persisted for 9dpi with 8% mortality. Mild septicemic lesions including: white necrotic foci and pinpoint hemorrhages in heart, liver and sever inflammation in pancreas were observed , Heart blood and liver impressions represented characteristic bipolarity of p. multocida stained with Giemsa and typical growth of dew drop, mucoid, non‐haemolytic colonies in sheep blood agar resulted from re-isolation, on the other hand, the treated group 1subgroup B, with PHENOXIPEN®,P. multocida was isolated in decreasing pattern within 3dpt with a lower frequency of clinical and macroscopic lesions. There were neither signs nor gross pathological lesions in the control group.