الفهرس 
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Abstract
Columnaris, saddleback disease, cotton-wool disease, cotton mouth disease, and fin rot is caused by Flavobacterium columnare it belongs to the family Flavobacteriaceae and causes severe mortality among fish fresh or marine fishes. So, this study was carried-out to throw the light on the incidences of flavobacteriu columnaris in marine and fresh water fishes its signs, PM lesions and its losses and role of PCR in detection and diagnosis of flavobacterium columnaris diseases. In our investigation, a total number of 100 of both cultured fresh water and marine water fishes (50 each of Oreochromis.niloticus and sea bream Sparus aurata L.) of different body weight ranges (100-150 g) were collected showing clinical signs from private fish farms. The O.niloticus was obtained from Kafr-El sheikh governorate, while the sea bream (Sparus aurata L.) were obtained from Wadi-Mariut region at west Alexandria governorate, Egypt. Marine water Fishes were transferred to laboratory in the same farm as soon as possible . While the fresh water fishes transferred to private lab in Alexandria governorate . The freshly dead and moribund fishes specimens were subjected to full clinical, postmortem (PM), parasitic and mycological as well as bacteriological examinations. The fish were subjected to clinical and post-mortem examination, bacteriological, biochemical examination, antibiotic sensitivity test, PCR examination for detection of the characters of flavobacterium columnaris. Our results concluded that : 1) Flavobacteriosis, caused by pathogenic members of the genus Flavobacterium, considered as serious fish disease that severely impact freshwater aquaculture worldwide while, Tenacibaculum maritimum (formerly known as Flavobacterium maritimus) is the etiological agent of Tenacibaculosis and it has a specific tropism to certain parts of fish body especially skin, mouth, fins and tail of fish, causing eroded mouth, frayed fin, and tail rots and sometime necrosis on the gills and eye with severe necrotic and ulcerative lesions on the body surface with systemic signs. 2) Clinical signs and Postmortem (PM) lesions showed upon examination naturally infected examined O. niloticus advanced lesions may be round or oval in shape, yellowish-brown in color with an ulcer in the center when the infection progress, a yellowish-brown ulcer often is found in the center of the saddle additionally, others show opaqueness of the eyes Moreover showing partial brown to yellowish-brown lesions (sores) on their gills, The gill filaments were congested , swollen and covered by profuse mucus. Such lesions were the main cause of large mortalities among infected fishes . The previous findings could be attributed to the adherence and irritation of the bacteria with gill structure skin and/or fins the infection may lead to frayed fins others show accumulation of fluids in abdominal cavity while the others show congestion of the liver of examined fish ,protrusion of hemorrhagic anal opening and fin destruction. 3) Concerning to the most important Clinical signs and Postmortem (PM) lesions showed upon examination naturally infected examined Seabream (Sparus aurata L.) showed yellowish coloration of the head may spread on the body severe erosions and ulcers may form on the skin and penetrate to the underlying musculature and erythematic hemorrhages on the operculum may be present others showing swelling in the abdominal cavity (ascitis) other naturally examined Sea bream(Sparus aurata .L) showing opaqueness of the eyes and exophthalmia and other showing loss of eyes. 4) Culture, morphological and biochemical characteristics of the bacterial isolates both types of Bacterial cultures produced are long, thin Gram-negative rods that are motile on agar media by a creeping or flexing motion on Anchor and Ordal media. The columnaris bacteria can be differentiated from other bacteria by their size and shape (long, thin rods, 7 to 10 millimeters and approximately 10 to 20 times longer than wide) and the formation of haystacks By the use of selective media such as selective Cytophaga Agar (SCA) these media take advantage of the ability of columnaris bacteria to grow in the presence of neomycin (5 mg/l) and polymyxin B (200 units/ml), tenacibaculum maritimum can grow on this selective media by adding 3%Nacl both types of bacteria were biochemically tested and gave these results Gram-ve by Gram staining, motility +ve, oxidase activity, catalase production, flexirubin pigment, Congo red absorption, nitrate reduction and H2S production. 5) It is evident that the columnaris is primarily an epithelial disease of freshwater fish. It causes erosive / necrotic skin and gill lesions that may become systemic. It often presents as large erosions with loss of scales and red-grayish patches that may have a red margin on the head, back (saddleback lesion), and / or fins (fin rot) especially the caudal fin. Fragments of the fin rays may remain after the epithelium has sloughed, leaving a ragged appearance. Ulcerations with yellow or orange color were deeply seen in underlying tissue due to masses of pigmented bacteria growth. 6) The histopathological findings includes, the skin and gills, primary target organs, showed necrosis with intense neutrophilic response. Erosions and ulcerations were seen as sloughing the necrotic tissue. Such reaction was deeply penetrated the underlying tissue as dermis and muscle (myositis and perimyositis) in skin and gill-arch. The dermal lesions besides those of the epidermis are probably the reasons for easily detached scales. The a fore mentioned lesions were milder or completely absent with probiotic treated fish with numerous melanomacrophages infiltrations in these organs. The melanin pigment inside these cells is supposed to share in the defense mechanism as it is a response to probiotics. Melanin pigments are also found in the melanomacrophages within lysosomes; such pigments are considered to have a bactericidal effect through the production of free radicals. 7) The antibiotic sensitivity test cleared that, both F. columnare and T. maritimum more sensitive to ciprofloxacin and Flumequine. Also, the results indicated that, the T. maritimum more sensitive to ciprofloxacin, Flumequine, Doxycycline and Oxytetracycline. The results also cleared that, the both F. columnare and T. maritimum inter mediate sensitive to Gentamycin and Enrofloxacin. But, the F. columnare resist to Sulphamethoxazole and Trimethoprim, Oxytetracycline, Amoxycillin and Erythromycin. While, T.maritimum resist to Amoxycillin and Erythromycin. 8) A variety of media has been used to selectively isolate members of the genus Flavobacterium. Comparisons of the efficacy of the various selective mediums have, however, not been made. The isolation of Flavobacterium columnare in common medium is very difficult since the growth of the bacteria may be suppressed by other faster growing bacteria, by other bacteria which have extracellular products with cellulolytic or other antagonistic effects or by commensal bacteria. The composition of the medium should give sufficiently good growth conditions for the strains to be isolated, but an ideal medium for Flavobacterium disease did not exist. 9) The experimental design of LD50 of (Flavobacterium columnare ”F. columnare” and Tenacibaculum maritimum ”T. maritimum”) in both of seabream and O.niloticus were studied and one of the difficulties associated with assessing F. columnare virulence is ensuring accurate cell counts in cultures used for infection studies. The LD50 in O. niloticus was 108.5 and in seabream fish was 108.5. 10) F. columnare-specific PCR : a- PCR products from the Flavobacterium columnare gene in fish samples positive for Flavobacterium columnare Lane 1 , Lane M molecular weight markers, 1000 -bp ladder (BIOLINE); lanes 2,3,4,5,6,7,8 and 9, strains number 11, 17, 18, 20, 22, 24, 27 and 33, respectively; lane 10 and 11 , negative control(saline). b- PCR products from the Tenacibaculum maritimum gene in fish samples positive for Tenacibaculum maritimum Lane 1 ,Lane M molecular weight markers, 1000 -bp ladder (BIOLINE); lanes 2 to 5, strains number 6, 13, 26, and 41, respectively; lane 6 and 7, negative control. 11) The Transmission electron micrographs of thin sections of cells (X 1000000) F. columnare isolated from diseased O. niloticus . indicates a capsular structure strongly stained with ruthenium red. reason for the total fading of the PCR signal from the freeze-stored samples. Freezing caused severe damage to the cells of F. columnare, and in frozen samples the cell walls were completely disintegrated . However, freezing had no effect on E. coli cell morphology (data not shown).