الفهرس 
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Abstract
Vibriosis is was among the bacterial fish diseases that adversely affect a wide range of farmed marine fish worldwide. It causes significant economic losses and mass mortalities, negatively impacting the cage aquaculture industry in several countries, including Egypt. This study was focused on two major objectives: a) isolation and identification of Vibrio species affecting cultured marine fish in Egypt, and b) describing the relationship between environmental stressors and the incidence of this bacterial disease among marine fish stocks.
1. One hundred cage-cultured European seabass were collected from the private marine fish farm at Borg-El Arab, Alexandria province. Moribund fish samples were primarily subjected to clinical investigation to record the clinical signs and postmortem lesions on the diseased fish. Bacteriological examination was performed before the biochemical and molecular assays. In parallel, water samples were collected during fish sampling for successive seven-month periods to elaborate a close relationship between the water quality properties and disease occurrence. The results revealed that the unionized ammonia, nitrite, and water temperature were higher and over the permissible limits, especially during the summer months. Concurrently with the lower dissolved oxygen levels in water samples collected in the summer season. Thus, this is strongly related to the increasing number of fish mortalities during these months.
2. Clinical signs and postmortem lesions of diseased cage-cultured European seabass showed typical signs of septicemia and were characterized by hemorrhagic patches on the body surface, especially at the ventral of the abdomen, the base of the fins, and around the vent. In addition to the other samples showing scales detachment, skin darkening with ulcers, and eroded fins. As well, severe abdominal dropsy and anal prolapse were also detected. At the same time, PM examinations showed different degrees of liver congestion and hemorrhages. Also, the accumulation of hemorrhagic ascitic fluid in the abdomen with severe adhesion of the internal organs and the intestine were identified. Finally, Marked marked congestion of the gills was demonstrated in some fish samples.
3. After culturing on ordinary and selective media, V. alginolyticus appeared as large convex, yellow-colored colonies on TCBS media, swarmed creamy colonies on TSA media supplemented with 3% NaCl and non-hemolytic on blood agar media. Moreover, V. parahaemolyticus has appeared as convex, green-colored colonies with a dark blue center, giving alpha hemolysis on blood agar media. Besides, V. parahaemolyticus showed alpha hemolysis on Wagatsuma blood agar, so this strain is was negative for the Kanagawa phenomenon. Also, both Vibrio strains could grow at various levels of salt concentrations representing 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0%.
4. The incidence of bacterial isolates in the internal organs of all naturally examined fish species (liver, kidney, and spleen) has been studied, and the results revealed that the total number of bacterial isolates from the liver was (30 isolates), (10 isolates) from the kidney, and (20 isolates) from the spleen and which means that the highest percentage of isolates were from the liver (about 50 %) from all bacterial isolates. Moreover, it was found that V. alginolyticus had isolated the highest percentage from the liver, then spleen, and kidney. Conversely, the isolation of V. parahaemolyticus was mainly from the liver, followed by the kidney and spleen.
5. Conventional and VITEK biochemical analysis confirmed the suspected bacterial isolates.
6. PCR method used for confirming the Vibrio isolates were positively reacted to the 16S rRNA (663 bp) product. And they were also detecting the different virulence genes in both bacterial isolates. V. alginolyticus strain was positively reacted to the trh gene primer of expected size 250-bp. Nevertheless, this strain was negatively reacted to the tdh gene primer with 373 bp size band length. Moreover, V. parahaemolyticus strain was positively reacted to the toxR and trh gene primer at their specific segment sizes of 368 and 250-bp, respectively. Nevertheless, this strain was negatively reacted to the tdh gene with 373 bp size band length.
7. Sequencing and phylogenetic analysis displayed revealed that the sequence of V. alginolyticus (MM2) and V. parahaemolyticus (MM1) strains were deposited in the GenBank database under the accession number OL755952, and OL755953, respectively. The maximum-likelihood phylogenetic trees based on the 16S rRNA sequence cluster placed our bacterial isolates close to the reference strains of V. alginolyticus and V. parahaemolyticus.
8. Antibiogram assay clarified that both retrieved bacterial isolates were highly sensitive to several antimicrobial drugs such as ciprofloxacin, azithromycin, nitrofurantoin, gentamycin, co-trimoxazole, colistin, amikacin, and doxycycline. In contrast, these strains showed ascending degree degrees of resistance patterns to Amoxycillin amoxicillin and amoxiclav.
9. Experimental infection: - A total of 100 European seabass (average body weight of 30.0±5.0 g) were obtained from the marine fish hatchery at Kilo 21 region - Agami, Alexandria province. Fish were maintained in 1 m3 reinforced polyethylene ponds for acclimation to the new culture conditions for 2 weeks before the experiment. Fish were divided into 2 groups with 50 individuals in each. Before starting the experimental infections, each fish group was subdivided into 5 subgroups with 10 fish in each and kept separately in plastic aquaria at 22 ± 1 °C. Four subgroups were designated as experimental ones, while the 5th as control ones. Subgroups were inoculated intraperitoneally (IP) with the identified isolates of V. alginolyticus and V. parahaemolyticus using 0.2 ml of serial bacterial dilutions containing 3× 10 8 to 3× 105 CFU/ml. Control was inoculated with 0.2 ml sterile saline (0.9%). Then mortalities, clinical signs, and PM lesions were recorded for 14 days after the injection. Then samples for histopathological examination were taken. Reisolating of the injected bacteria was determined for verification of the cause of death. Results revealed that: -
a) Clinical signs appear as typical vibriosis and are were characterized by hemorrhagic eyes, exophthalmia, hemorrhagic ulcerations all over the body surface, and hemorrhages at the ventral abdominal surface.
b. PM lesions appeared as severe congestion in the liver, spleen, and intestine, while the most characteristic sign was bloody stained ascitic fluids with hemorrhagic enteritis.
c. Total mortalities were recorded caused by injecting 4 different bacterial doses of both Vibrio strains. Moreover, these results showed a strong relationship between increasing bacterial doses and an exaggerated number of fish mortalities. V. alginolyticus doses were 3× 10 8, 3× 107, 3× 10 6, and 3× 105 (CFU/mL), resulting in mortality rates of 70, 60, 50, and 30%, respectively. Furthermore, V. parahaemolyticus doses were 3× 10 8, 3× 107, 3× 10 6, and 3× 105 (CFU/mL), resulting in mortality rates of 60, 40, 30, and 20%, respectively.
d. The re-isolation of the bacterial isolates causing these signs and PM lesions was found to have the same culture and biochemical characteristics of as the injected bacterial strains. This confirms the cause of mortalities, signs, and PM lesions.
e. Histopathological results showed that there were severe hemorrhages, congestion of blood vessels, widespread necrosis, and vacuolar degeneration in the hepato-pancreatic tissues of experimentally infected European seabass. Moreover, marked activation of melanomacrophage centers, severe congestion of blood vessels, focal lymphoid depletion, and lymphoid cell necrosis were inspected in the splenic tissues.