الفهرس 
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Abstract
This study was carried out mainly in the Department of Agricultural Microbiology, Faculty of Agric. Zagazig Univ., Egypt and Central Laboratory for Aquaculture Research, Abbassa, Abu-Hammad, El-Sharkia, Egypt during October 2018 and May 2019 in order to study the prevalence of Aeromonas spp. Phages in irrigation water and fish in Sharika Governorate. Also this study concentrated on the isolating and characterizing of different Aeromonas phages to take an overview about the prevalence of some phages in irrigation water and fish samples. In addition, four Aeromonas hydrophila strain (ATCC 13037) phages were isolated from different irrigation water and fish samples and characterized with electron microscope (TEM). Some factors affecting phages were performed also. Molecular characterization and in vitro phage cocktail application as well as phage therapy against pathogenic Aeromonas hydrophila strain (ATCC 13037) were also studied and the results could be summarized as follows :
1- One reference strain namely Aeromonas hydrophila strain (ATCC 13037) was kindly obtained from MIRCEN, Faculty of Agriculture, Ain Shams University, and was used in all experimentations as a host.
2- Aeromonas hydrophila strain (ATCC 13037) was sensitive to Amoxicillin, Chloramphenicol, Ciprofloxacin, Erythromycin, Levofloxcin, Neomycin, Norfloxacin, Ofloxacin, Penicillin G and Vancomycin.
3- Multiple antibiotic resistant was observed with A. hydrophila strain (ATCC 13037) which was resistant to 18 antibiotics. Also this strain was sensitive to 10 different antibiotics, while it was intermediate only to 4 antibiotics.
4- Many phages were isolated from different sources of irrigation water and fish using Aeromonas hydrophila strain (ATCC 13037) as a host for all of them. Four phages were chosen namely Ah01, Ah02, Ah03, and Ah04 for achieving this study.
5- Nine different Aeromonas strains (G-) were used to find out their susceptibility to the four Aeromonas phages isolated from fish and water.
6- Phages Ah01 and Ah02, exhibited the highest lytic activity against Aeromonas strains tested in this study with a proportion of i.e., 66.6%.
7- Four phages (Ah01, Ah02, Ah03, and Ah04) were infectious to two species of genus Aeromonas (i.e. Aeromonas hydrophila strain (DSM 30187) and Aeromonas hydrophila strain (ATCC 13037). Moreover, Aeromonas molluscorum strain (LMG 22214) was susceptible to the all phages examined.
8- A medium clear plaques (2.0 – 3.0 mm) with a sharp edge were obtained using phage Ah03 on Aeromonas hydrophila strain (ATCC 13037) while phage Ah02 gave a large clear plaques with a sharp edge (3.5 – 0.4 mm).
9- The heads of the four phages were appeared to be separated from the sheath by the necks. These necks were measured 15.1, 22.4, 25.5, and 14.3 nm in lengths and 16.3, 23.6, 22.4, and 15.4 in width for Ah01, Ah02, Ah03, and Ah04, respectively.
10- The adsorption rate constant (k) in ml/min values were 2.4x10-9, 3.4x 10-9, 2.8x10-9, and 3.4x10-9 ml/min for phages Ah01, Ah02, Ah03, and Ah04, respectively.
11- The latent period was 15 min for either of phages Ah01or Ah02, and the burst sizes for both phages were 102 and 209 PFU/infected cell, respectively. While the latent period was 10 min for either of phages Ah03 or Ah04, and the burst sizes were 94 and 103 PFU/infected cell, respectively.
12- The four phages Ah01, Ah02, Ah03 and Ah04 were stable to storage at 4oC until 6 months while they lost their infectivity after 8 months. The four phages were more stable in maintenance at refrigerator as well as freezer conditions than at as ambient temperature. However, it was more sensitive to the maintenance at below 0oC than at 4oC.
13- Thermal inactivation points (TIP) for these phages were 84oC hence the reduction of virus particles reached 100% after 10 min. of exposure to this degree.
14- Two phages (Ah03 and Ah04) were tolerant to a broad range of pH from 3 to 11 than the others (Ah01, and Ah02) and the latter lost their activities at pH 5 and pH 3, respectively.
15- The DNA fingerprint of the virulent phages Ah01, Ah02, Ah03, and Ah04 were assessed using the RAPD-PCR technique.
16- Six primers only succeeded to produce polymorphic DNA products varied in their sizes. with each primers were found in this study for the differentiation of the four phages Ah01, Ah02, Ah03, and Ah04 and 128 fragments were produced. The sizes of these fragments were between 251-6107 base pairs in gel electrophoresis.
17- Phage Ah03 had 6 structural proteins with molecular weights ranged from 110- 26 KDa: in which two of them were mutual with 2 protein bands appeared with phage Ah04 namely 36 and 27 KDa. On the other hand, phage Ah04 had 5 structural proteins with molecular weights of 181, 119, 36, 27 and 25 KDa.
18- Using cocktails contained two, three or four phages was employed in order to investigate their ability in controlling Aeromonas hydrophila strain cells at MOI 0.01 of a favorite one. No big differences was observed between treated bacterial cells with the cocktails containing (2) or (3) or (4) phages since the bacterial cell densities were 0.408 (OD600) while the bacterial densities with the cocktails of 2 phages were 0.121-0.132 (OD600) and the cocktails of 3, or 4 phages were between 0.115 and 0.136 (OD600) after 12h of incubation.
19- The efficacy of four different phages in comparison with ciprofloxacin antibiotic against Aeromonas hydrophila infection in the Nile tilapia; Oreochromis niloticus and the behavior and the external appearance of the infected fish were recorded daily. The infected fish was shown loss their appetite and equilibrium, dullness, sluggish swimming at the water surface. Some had a hemorrhage with enlarged abdomen and ascites, fin and tail rot, detachment of scales, skin erosion and ulcer. Others had exophthalmia or opaqueness of the eye, congested or pale gills, anemic and covered with excessive mucus. The mortality in the infected fish with Aeromonas hydrophila strain reached 83.34% at the end of the experiment (15 days), while in fish treated with ciprofloxacin, the mortality was decreased to 33.34%. Moreover, no mortality was detected when the phages were injected with the bacteria used.
20- Fish infected with A. hydrophila strain were found to have the highest values of neutrophils and lymphocytes when compared with other treatments.
21- A. hydrophila strain caused significant increase in total plasma bilirubin as well as direct and indirect bilirubin (G2) of O. niloticus. In addition, alanine and aspartate transferase enzymes (ALT & AST) elevated significantly in the same group (G2) in comparison with those in the control group (G1). These biochemical indicators showed less increase in the groups treated with ciprofloxacin (G3) and the phages. The effect of the phages against Aeromonas hydrophila strain was nearly the same as in the antibiotic ciprofloxacin.
22- Total protein was highly significantly decreased on the 3rd day and more highly significantly reduced (P<0.05) on the 15th day of infected fish group. The decrease in total protein was enhanced in the other groups treated with the antibiotic and phages but still lower than in the control. The same trend was showed with albumin, globulin and A/G ratio.
23- Finally, we concluded that phages help in treatment Aeromonas hydrophila infection and induced improvement in hematological and biochemical parameters in Nile tilapia. It is recommended to use phages instead of the antibiotics to protect fish production farms and the aquatic environment against antibiotic-resistance bacterial strains and consequently protect ourselves against zoonotic diseases.