الفهرس 
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Abstract
The current study was carried out to investigate the most common bacterial pathogens affecting freshwater fish species, particularly, African sharp teeth catfish, Clarias gariepinus Burshell 1822, raised in earthen pond aquaculture system with special concern with fish, which showed clinically diseased patterns in Fayoum Governorate, Egypt. The study was carried out during the period starting from October 2016 to October 2019 by available techniques to detect these pathogens. The study was designed to isolate and identify the bacterial pathogens incriminated in disease outbreaks. In parallel, samples of organic fertilizer (poultry droplets), poultry carcasses and chickens slaughter house byproduct (used as fish feeds) were also collected to investigate their role (s) in fish diseases outbreaks. Conventional phenotypic identification and genotypic detection of AMR determiners genes of the selected pathogens were also investigated. In parallel, clinical picture manner and prevalence rates were also investigated. Antibiogram assays were carried out to investigate the AMR among the revealed pathogens. Moreover, experimental trials were performed for replacement of antibiotics by alternative patent antimicrobial agents of herbal origin namely Trivir® (10% carvacrol).
The presented study showed that a total number of 250 cultured C. gariepinus (150 ± 10gms) were collected alive from Etsa and Senoris (cultured) at Fayoum Governorate, Egypt. The collected fish samples showed variety of clinical signs characteristic to septicemia. Externally, fish samples showed irregular hemorrhagic spots on the body surface, exophthalmia, hemorrhages, abdominal distention, ulceration of the head, peduncle region and dorsal musculature and protruded congested vent. Internally, the pathognomonic pictures include pale or congested liver, distended gall bladder, congested and enlarged spleen, congestion and enlargement of kidneys. Mostly, the parenchymatic organs friable and soft. Some cases showed yellowish exudates bloody tinged within the body cavity.
For isolation of causative agent (s), several microbiological media were used including both ordinary and selective ones. For example, but not limited, BHI, NA and TSA as ordinary enriched media, while Aeromonas selective agar base medium with supplement were used. For the isolation purpose, samples from kidneys, skin lesion and liver of the diseased fish were streaked on aforementioned prepared plates. On the basis of conventional biochemical tests and confirmed by API identification system (API 20E, API 20 NE, API 20 Strep) the obtained bacterial pathogens were identified.
The generated results showed a total of 116 bacterial isolates were recovered during the presented investigation (99 bacterial isolates were collected from C. gariepinus) including 83 isolates (83.8%) Gram negative and 16 isolates (16.2%) Gram positive. At the same manner, collected bacteria from the poultry were 17 isolates (14.6% of the total) representing 13 (76.5) Gram negative and 4 (23.5) were Gram positive ones. The isolated Gram negative bacteria were members of Aeromonads, Vibrios, Pseudomonads and Sh. Putrefaciens. Collected bacteria from the poultry were 17 isolates (14.6% of the total) representing 13 (76.5) Gram negative and 4 (23.5) were Gram positive ones. On the other hand, the isolated Gram positive isolated bacteria were the Enterococci and Staphylococci. The most prevalent bacterial isolates isolated from fish were belonged to Aeromonads species 28 (35.7%), particularly A.hydrophila 17 (14.6%) followed by A .salmonicida 11 (9.5%). followed by Vibrio species 17 (14.6 %) and Pseudomonads species 12 (10.3 %). On the other hand, the Gram positive isolates included E. faecalis (catfish, poultry) and S. aureus, with percentages of 9.4, 7.8% respectively. The bacterial isolates were mainly isolated from kidney showed the highest levels (37.9%) followed by skin ulcer (31.8%) and liver (30.1%). The higher incidence of bacterial isolation mainly recorded during summer (49.1%), followed by autumn (31.0 %) and winter (19.8 %). On the other hand, bacteria isolated from poultry and fish were mainly Gram positive representing E. feacalis as the main species, whereas Salmonella and E. coli species were the only ones of poultry origin. Although no septicemic fish bacterial pathogens were isolated from poultry origin, the emergence of antimicrobial resistance (AMR) were predictably driven them to acquire resistance as a result of using untreated poultry slaughter house byproducts as feeding staff for catfish and/or poultry droplets as organic fertilizers. As a fact, it is believed that the bacterial load of fish ponds including its sediment is associated with incorporation of organic fertilizer (manure and/or poultry droplets).
The pathogenicity test was carried out on cultured C. gariepinus by I/P injection of A. hydrophila BNS 0119 at dose 0.1 ml containing 3×108, 1.5×108, 1.5 x 107, 1.5 x 106, and 1.5 x 105 CFU/ml, showed cumulative mortality rates 50% with LD50 of 1.5x 107.
Conventional Antibiogram assays showed variable resistance levels of the tested pathogens to antibiotics. In general, the results revealed that the majority pathogenic bacterial isolates were found to be highly resistant against most of used antibiotics. Meanwhile, A. hydrophila experiences moderate resistances to Oxytetracycline, Erythromycin, Doxycycline and full resistances to Oxolinic acid, Nalidixic acid, Flumequine, Colistine sulphate, Ampicillin and Penicillin. On the other hand, Sh.putrifaciens showed high resistant to Oxytetracycline, Erythromycin, Doxycycline, Oxolinic acid, Nalidixic acid, Flumequine, Colistine sulphate, Ampicillin and Penicillin. Concerning results of antibiogram of E. faecalis, it showed sensitivity to Oxytetracycline while intermediate resistance was found to Erythromycin, Ampicillin, Penicillin and Doxycycline, while showed resistance to Oxolinic acid, Nalidixic acid, Flumequine and Colistin Sulphate. As a result, P. aeruginosa showed sensitivity to Oxytetracycline, Colistin Sulphate while intermediate resistance was found to Flumequine and Doxycycline while showed resistance to Oxolinic acid, Nalidixic acid, Erythromycin, Ampicillin and Penicillin. Finally, V. parahymoliticus showed sensitivity to Oxolinic acid, Nalidixic acid, Flumequine, Oxytetracycline, Colistin Sulphate and Doxycycline, While moderate to Erythromycin, Ampicillin and Penicillin.
On the level of antibacterial resistance genes detection by PCR, A. hydrophila BNS 0119 showed acquisition of resistance tetracycline genes and experienced resistant determinants specific for tet (A), tet (C), tet (B) and dhfr1 with production of amplicon sizes of 211, 897, 391 and 600 recpectively.
Production and control of fish health safely together with living in harmony with the nature, natural substances considered as an important area for future developments in aquaculture. In this respect, the current study was aimed to investigate the antibacterial activities of Trivir® (10% carvacrol), one of the major essential oils of oregano, against some fish pathogenic bacteria isolated from diseased catfish, C. gariepinus. Trivir® solution was designated to be used as broad-spectrum disinfectant for aquaculture according to the manufacture instructions. The antibacterial effects of the Trivir® were tested on clinical isolates of A. hydrophila BNS 0119, E. faecalis and Sh. putrificans. The minimum inhibitory concentrations (MICs) of Trivir® against A. hydrophila were estimated as 62 μg/ ml, 125μg/ml for Sh. putrificans and 250μg/ml for E. faecalis. Despite of its bacteriostatic activities, Trivir® at concentrations of 1000, 500,250, 125, 63, 32, 16, 8, 4 and 2μl/ml could not kill the tested bacterial isolates through using microplate assays. Additionally, the tested bacterial strains re-grow when they were plated on fresh BHI plates. Moreover, C. gariepinus exposed to 32, 62 and 125 μg/ml of Trivir® showed survival rates of 100% at all exposure times tested. On contrast, Trivir® showed its toxic effects at concentrations of 1000, 500, 250 μg/ml after exposure times of 15, 30 and 60 min causing 100% mortalities in catfish. In conclusion, Trivir® at high doses of 1000, 500, 250 μg/ml could be used as antimicrobial disinfectant for utensils used in fish farms, however, low doses (125, 64, 32μg/ml) could be used in vivo (fish) as a bacteriostatic antiseptic agent, particularly, during farm operational processes. Trivir® can be considered as an effective alternative for antibiotics usage in aquaculture and with an ultimate competent health and safe environment.