Skin histopathological responses of Oreochromis niloticus (Nile tilapia) to transportation in water with and without salt.

Oreochromis niloticus (Nile tilapia) is a well-known economic fish species that can thrive under the right environmental circumstances. The transport of live fish, either for food or as companion animals, presents a big issue for animal welfare at the same time it is considered one of stressful conditions. Hence, the present study investigated the skin histopathological responses of O. niloticus that were attributed to stress and salt addition during transportation. Three experimental groups of O. niloticus the 1st is the control non-transported group (CG), the 2nd is transport in water without salt (PT-S) and the 3rd is transport in water containing 5gL- 1salt (PT + S), the last 2 groups were transported in 5 h transport model. Results indicate that the skin of PT-S fish showed a marked decrease in epidermal thickness, decreased number of goblet cells, and an increase in the sub-epidermal and dermal pigments with the presence of large edematous vacuoles. Fish skin from PT + S demonstrated mild hydropic swelling in epidermal cells with normal goblet (mucous) cells density, and more or less normal melanin pigment distribution in sub epidermis and on the dermis layers, however, dermis showed mild edematous spaces. Scanning microscopy of PT-S skin tissue showed few scratched white patches among normal regions that may represent a thickened surface with the decreased number of goblets cell opening, while the PT + S group showed moderate preservation of surface skin architectures with the presence of goblet (mucous) cells opening in spite of presence of slight thickened white patches. The estimated total lesion changes present in PT-S group showed a significant increase (P < 0.001) compared with the control (CG) group. On the other hand, PT + S showed significant (P < 0.001) improvement in the overall previously recorded changes compared with the PT-S group, and a non- significant change in the histological architectures compared with the control group. Our findings underlined the importance of skin and its mucous cover health during transportation. The use 5 gL- 1salt during O. niloticus transportation appears to preserve the surface skin features, and keep the goblet (mucous) cells open to the external surface, and may act as a deterrent for the release of mucus from goblet (mucous) cells in response to stress and lessen the stress of transportation.

Impact of transportation in freshwater and brackish water on Nile tilapia (Oreochromis niloticus) resistance.

BACKGROUND: Oreochromis niloticus has great economic value and potential for farming and development. Transportation of fish was done for breeding or trading purpose and it is a challenging aspect of aquaculture. This study aimed to investigate the effect of transportation in freshwater and brackish water on the resistance of O. niloticus as well as transportation stress mitigation effect of NaCl. Four equal groups were used; each of 50 fish, the 1st group served as the control (P 1), while the 2nd group (PT 2) was transported in water without salt, the 3rd (PT 3) and 4th (PT 4) groups were transported in water containing 5 gL- 1 and 10 gL- 1 salt respectively. PT 2, PT 3 and PT 4 were transported for 5 h without any rest or sedative drugs. RESULTS: The serum cortisol of O. niloticus significantly increased at 0 h and then decreased at 12 and 24 h post transportation in the PT 2 group and non-significantly increased at all point times in the PT 3 and PT 4 groups comparing to P 1 group. Mucin2 gene (MUC2) expression was non-significantly up regulated in the PT 2 group and down regulated in the PT 3 and PT 4 groups at 0 h comparing with P 1 group, but at 12 and 24 h it was significantly up regulated in the PT 2, PT 3 and PT 4 groups. The ß Defensin-1 (ß D1) and 2 (ß D2) genes expression was non-significantly down-regulated in the PT 2 group and significantly up regulated in the PT 3 and PT 4 groups at 0 h., while at 12 and 24 h was significantly down regulated in the PT 2 group and non-significantly down regulated in the PT 3 and PT 4 groups, it significantly down regulated in the PT 2 and PT 3 group and non-significantly down regulated in the PT 4 group at 24 h. Non-significant up regulation in interleukin - 1ß (IL-1ß) gene expression was reported in the PT 2 group and non-significant down regulation in the PT 3 and PT 4 groups at 0 h. However, significant up regulation was recorded in the PT 2, PT 3 and PT 4 groups at 12 and 24 h. The Tumor necrosis factor-alpha (TNF-α) gene expression was non-significantly up regulated in the PT 2 group and non-significantly down regulated in the PT 3 and PT 4 groups at 0 h. However, it was significantly up regulated in the PT 2, PT 3 and PT 4 groups at 12 and 24 h. CONCLUSION: The results of this study confirmed the stressful effect of transportation on O. niloticus as well as the transportation stress mitigation effect of NaCl.

Metal Accumulation and DNA Damage in Oreochromis niloticus and Clarias gariepinus After Chronic Exposure to Discharges of the Batts Drain: Potential Risk to Human Health.

The present work showed the impact of long-term exposure to the Batts drain's discharges on O. niloticus and C. gariepinus fish species. The accumulation level of Cu, Zn, Fe, Cd, Pb and Al in five vital tissues was markedly elevated near the Batts drain (site2) compared to the samples from the reference site (site 1). At the same site, C. gariepinus displayed the highest accumulation capacities when compared to O. niloticus. Based on the metal pollution index, livers and kidneys, followed by gills, showed the maximum overall metal load. The degree of DNA damage (assessed by comet and diphenylamine assays) was relative to the accumulated metals in tissues with species and site specification. The values of the hazard index for human consumption showed that the studied metals were within safe values at normal consumption rates. While harmful health consequences were observed at the habitual consumption level at site 2.

Evaluation of Nephrotoxicity in Oreochromis niloticus After Exposure to Aluminum Oxide Nanoparticles: Exposure and Recovery Study.

Nephrotoxic impacts of Al2O3 nanoparticles (NPs) were studied in Oreochromis niloticus after seven days of exposure and fifteen days of recovery periods. Fish were classified as group I (dechlorinated water); group II (2 mg/L Al2O3NPs); group III (4 mg/L Al2O3NPs); group IV (8 mg/L Al2O3NPs). Blood creatinine and uric acid levels showed marked increases in groups III and IV. A dose-dependent disturbance in renal antioxidant components was recorded as indicated by elevated catalase, superoxide dismutase, thiobarbituric acid reactive substances levels, and decreased glutathione reduced concentration. Renal histopathology was recorded with the highest % of appearance in group IV. A reduction in renal Al content, kidney function biomarkers (excepting group IV), and enhanced antioxidant status were observed after applying a recovery strategy. Several structural damages were identified following the recovery period, but the alteration frequencies indicated regressive histopathological changes. The Al2O3NPs-induced nephrotoxicity can be reduced after applying a suitable recovery period.

Astragalus membranaceus nanoparticles markedly improve immune and anti-oxidative responses; and protection against Aeromonas veronii in Nile tilapia Oreochromis niloticus.

The effects of dietary administration of Astragalus membranaceus nanoparticles (ANP) on immune and anti-oxidative responses, growth performance and disease resistance of Oreochromis niloticus were evaluated in the present study. Fish were divided into three groups and received the ANP at rates of 0 (control), 1, and 2%/kg diet for four weeks. After the four-week feeding trial, three fish from each replicate were sampled for immune and anti-oxidative responses evaluation, ten fish from each group were challenged with A. veronii, and nine fish from each group were subjected to cold and hypoxia challenges. It was obvious from the results that ANP significantly enhanced lysozyme activity and nitrous oxide (NO) activities, as well as improved superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities. Also, aspartate aminotransferase, alanine transaminase, glucose, and cortisol measurements showed significantly lower levels in incorporated groups compared to the control. Growth performance; and amylase and lipase digestive enzymes activities also showed markedly improved results. Expression of heat shock protein 70 (HSP70) and interleukin 1, beta (IL-1ß) genes were significantly upregulated throughout the entire experimental period. When challenged with A. veronii, the mortality of treated groups was significantly (P < 0.05) lower than the control. Current results proofs that dietary ANP had a synergistic effect on immune and anti-oxidative responses, growth performance and disease resistance of Oreochromis niloticus.

Haemato-immunological responses and effectiveness of feed-based bivalent vaccine against Streptococcus iniae and Aeromonas hydrophila infections in hybrid red tilapia (Oreochromis mossambicus × O. niloticus).

BACKGROUND: Streptococcosis and Motile Aeromonad Septicemia (MAS) are important diseases of tilapia, Oreochromis spp. and causes huge economic losses in aquaculture globally. The feed-based vaccination may be an alternative to minimize major infectious diseases in tilapia. Thus, this study aims to evaluate the haemato-immunological responses and effectiveness of a newly developed feed-based killed bivalent vaccine against Streptococcus iniae and Aeromonas hydrophila in hybrid red tilapia. A total of 495 hybrid red tilapia of 61.23 ± 4.95 g were distributed into 5 groups (each with triplicate). The fish were immunized orally through bivalent (combined S. iniae and A. hydrophila) spray vaccine (BS group), bivalent formulate vaccine (BF group), monovalent S. iniae vaccine (MS group), monovalent A. hydrophila vaccine (MA group) and unvaccinated as a control group. The vaccine was orally administered on days 0, 14 and 42 applied feed-based bacterin at 5% body weight. The blood and spleen samples were collected from all groups on 7, 21 and 49 days post-vaccination, and also 96 h post-infection to assess their haemato-immune responses. RESULTS: Compared with the unvaccinated group, leukocyte, lymphocytes, monocytes, granulocytes counts in vaccinated groups were significantly (P < 0.05) increased on 21, 49 days post-vaccination and also 96 h post-infection, while erythrocytes, haemoglobin and haematocrit in vaccinated groups were significantly (P < 0.05) enhanced only 96 h post-infection. Additionally, the lysozyme and phagocytic activity and, serum antibody (IgM) were significantly higher (P < 0.05) against S. iniae and A. hydrophila in vaccinated groups compared to the unvaccinated group in the pre- and post-infection. Results from the challenge through co-infection with S. iniae and A. hydrophila showed the relative percent survival (RPS) in BF group was 76.67 ± 4.71%, which had the capacity to induce significant protection (P < 0.05) compared to others groups. CONCLUSIONS: This study demonstrates the bivalent formulate (BF) group could elicit significant non-specific and specific immunological responses with higher protection in hybrid red tilapia. In addition, this newly developed feed-based bivalent vaccination can be a promising technique for effective and large scale fish immunization in the aquaculture industry.

Influence of dietary fermented Saccharomyces cerevisiae on growth performance, oxidative stress parameters, and immune response of cultured Oreochromis niloticus.

Our trial was performed to investigate the effect of fully fermented yeast Sacharomyces cerevisiae (Hilyses, ICC Company, Brazil) on the growth performance and immune response of Oreochromis niloticus. In this study, a total of 270 O. niloticus (50.7 ± 0.8 g) were randomly divided into 3 groups in triplicates. The control group was fed on the basal diet while the other two groups were fed on a basal diet supplemented with 0.2% and 0.4% of Hilyses. The trial period extended for 2 months. At the end of the feeding trial, oxidant and antioxidant parameters (MDA, catalase, and glutathione reductase), some innate immunological parameters and immune-related gene expression were measured. Histological examination of liver, spleen, kidney, and intestine was performed. Further, fish groups were challenged against Gram-negative and Gram-positive bacteria; A. hydrophila and L. garvieae. The results revealed significant improvement (p < 0.05) in growth performance and feed utilization in Hilyses-treated groups versus the control group. Blood parameters and liver and kidney functions of Hilyses-supplemented groups were similar to those of the control group. The histological findings of treated groups showed normal tissue structure with multiple focal lymphoid aggregations in the spleen, kidney, and intestine. Both levels of Hilyses successfully enhanced phagocytic activity/index, lysozyme activity, and gene expression of TNF-α, and IL-1ß. Fish group fed on 0.4% Hilyses exhibited the highest expression of IL-1ß and the least mortality percentages post challenges. Thus, dietary supplementation of Hilyses could promote the growth performance and immunity and increase the resistance of O. niloticus against diseases.

Influences of Chlorella vulgaris dietary supplementation on growth performance, hematology, immune response and disease resistance in Oreochromis niloticus exposed to sub-lethal concentrations of penoxsulam herbicide.

Little is known regarding the impact of penoxsulam, a fluorinated benzenesulfonamid rice herbicide, on Oreochromis niloticus (O. niloticus). Therefore, the current study was undertaken to highlight the effects of penoxsulam exposure on O. niloticus and to evaluate the advantages of Chlorella vulgaris (CV) dietary supplementation against the induced effects. The 96-h lethal concentration 50 (LC50) penoxsulam value for O. niloticus was estimated at 8.948 mg/L by probit analysis in a static bioassay experiment. Next, 360 healthy fish were randomly allocated into 6 treatment groups. The T1 group served as the negative control and was fed a basal diet. The T2 group served as the positive control and was fed a basal diet supplemented with 10% CV. The fish in the T3 and T4 groups were exposed to 1/10 the 96-h LC50 of penoxsulam (0.8948 mg/L) and were fed the basal diet alone or the basal diet supplemented with 10% CV, respectively. The fish in the T5 and T6 groups were exposed to 1/5 the 96-h LC50 of penoxsulam (1.7896 mg/L) and fed the basal diet alone or the basal diet supplemented with 10% CV, respectively. Sub-acute penoxsulam exposure significantly altered hematological indices, as well as compromised the fish's immune defense mechanisms, including the phagocytic percentage, phagocytic index, nitric oxide production, immunoglobulin M levels and lysozyme, anti-trypsin and bactericidal activities subsequently decreasing O. niloticus's resistance to the Aeromonus sobria challenge and increasing disease symptoms and the mortality rate. Furthermore, sub-chronic penoxsulam exposure markedly altered growth performance, oxidant/antioxidant status and liver status and down-regulated the expression of interleukin-1ß (IL-1ß) and tumor necrosis-α (TNF-α). Interestingly, incorporating 10% CV into the diet protects fish against sub-acute penoxsulam-induced immunotoxicity via improvement of immune responses that increases the resistance against bacterial infection. Further, it improved the growth performance, oxidant/antioxidant status, liver status and markedly up-regulated immune-related gene expression, IL-1ß and TNF-α, in the spleens of fish sub-chronically exposed to penoxsulam. These outcomes showed that dietary CV supplementation can protect the commercially valuable freshwater fish O. niloticus against penoxsulam toxicity and may be a potential feed supplement for Nile tilapia in aquaculture.

Glucose enhances tilapia against Edwardsiella tarda infection through metabolome reprogramming.

We have recently reported that the survival of tilapia, Oreochromis niloticus, during Edwardsiella tarda infection is tightly associated with their metabolome, where the survived O. niloticus has distinct metabolomic profile to dying O. niloticus. Glucose is the key metabolite to distinguish the survival- and dying-metabolome. More importantly, exogenous administration of glucose to the fish greatly enhances their survival for the infection, indicating the functional roles of glucose in metabolome repurposing, known as reprogramming metabolomics. However, the underlying information for the reprogramming is not yet available. Here, GC/MS based metabolomics is used to understand the mechanisms by which how exogenous glucose elevates O. niloticus, anti-infectious ability to E. tarda. Results showed that exogenous glucose promotes stearic acid and palmitic acid biosynthesis but attenuates TCA cycle to potentiate O. niloticus against bacterial infection, which is confirmed by the fact that exogenous stearic acid increases immune protection in O. niloticus against E. tarda infection in a manner of Mx protein. These results indicate that exogenous glucose reprograms O. niloticus anti-infective metabolome that characterizes elevation of stearic acid and palmitic acid and attenuation of the TCA cycle. Therefore, our results proposed a novel mechanism that glucose promotes unsaturated fatty acid biosynthesis to cope with infection, thereby highlighting a potential way of enhancing fish immunity in aquaculture.

Marine Collagen Peptides from the Skin of Nile Tilapia (Oreochromis niloticus): Characterization and Wound Healing Evaluation.

Burns can cause tremendous economic problems associated with irreparable harm to patients and their families. To characterize marine collagen peptides (MCPs) from the skin of Nile tilapia (Oreochromis niloticus), molecular weight distribution and amino acid composition of MCPs were determined, and Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical structure. Meanwhile, to evaluate the wound healing activity, in vitro and in vivo experiments were carried out. The results showed that MCPs prepared from the skin of Nile tilapia by composite enzymatic hydrolysis were composed of polypeptides with different molecular weights and the contents of polypeptides with molecular weights of less than 5 kDa accounted for 99.14%. From the amino acid composition, the majority of residues, accounting for over 58% of the total residues in MCPs, were hydrophilic. FTIR indicated that the main molecular conformations inside MCPs were random coil. In vitro scratch assay showed that there were significant effects on the scratch closure by the treatment of MCPs with the concentration of 50.0 µg/mL. In the experiments of deep partial-thickness scald wound in rabbits, MCPs could enhance the process of wound healing. Therefore, MCPs from the skin of Nile tilapia (O. niloticus) have promising applications in wound care.

Occurrence of microplastics in commercial fish species from the Ethiopian rift valley's Lake Hawassa, Ethiopia.

Microplastics (MPs) have recently been detected as emergent pollutants in the Ethiopian rift valley lakes located close to rapidly expanding towns. We provide the first study of MPs ingestion of commercial fish species from Lake Hawassa, Ethiopia: Catfish (Clarias gariepinus) and Nile Tilapia (Oreochromis niloticus). A total of 60 individual fish species was collected from three sampling sites of lake Hawassa in October 2020. Across all sampling sites, there was a significant difference in ingested MPs between benthic omnivore catfish and pelagic phytoplanktivorous Tilapia (χ2 = 15.864, p < 0.001). The most common size of ingested MPs (84.6%) was 0.5-1 mm, with fragments (59.5%) dominating, followed by fibers (25.4%). On average, 4.03 ± 1.33 MPs with sizes ranging between 60 µm and 10.53 mm were detected per individual. White and yellow MPs were particularly numerous, accounting for 36.8% and 26.4% of the total, respectively. Because Lake Hawassa's fishery is so significant, the potential impact of MP pollution on the lake biota in general, and economically valuable fish species in particular, deserves attention, additional research, and, if possible, early mitigation.

Vitamin E supplementation in diet ameliorates growth of Nile tilapia by upgrading muscle health.

Vitamin E (VE), an important lipid-soluble antioxidant, has great influence on growth and maintenance in animal. The effects of VE supplemented diet on growth and feed usage in Nile tilapia (Oreochromis niloticus) was investigated in this study. Three formulated diets containing VE (0, 50 and 100 mg/kg) were fed to Nile tilapia (3.56 ± 0.16 g) in glass aquaria maintaining three replicate groups for 56 days (8 weeks). Survival, growth performance including weight gain, percent weight gain, and specific growth rate (WG, % WG, and SGR), and feed utilization comprising protein efficiency ratio and feed conversion ratio (PER and FCR) were calculated. Hemato-biochemical indices including hemoglobin level (Hb), white blood cell (WBC), red blood cell (RBC) and glucose level were analyzed. In addition, muscle morphology was examined after completion of the experiment. At the end of the trial, WG, %WG, SGR, FCR and PER increased significantly which had dietary VE supplimentation. However, no distinct changes were observed in Hb level, RBC count, WBC count and glucose level among these different dietary groups. Dietary VE treatments significantly upgraded the muscle fiber diameter and lowered the intra-muscle gap. Moreover, quantity of hyperplastic muscle fiber as well as nucleus also significantly enhanced by VE. Morphological structure of muscle characterized by a huge proportion of hyperplastic muscle that may be supposed to contribute the enhanced growth of Nile tilapia receiving VE supplemented diet. Therefore these results suggested that VE incorporation into the feed can be effective to improve the feed efficiency and maximize the growth of O. niloticus.

Evaluation and Association of Heavy Metals in Commonly Used Fish Feed with Metals Concentration in Some Tissues of O. niloticus Cultured in Biofloc Technology and Earthen Pond System.

For successful aquaculture, the primary need is the quality of fish feed, which determines fish flesh quality. The current study was conducted to evaluate the number and concentration of heavy metals in commonly used fish feeds and fish gills, liver, and muscle of biofloc technology and earthen pond systems. Besides this, the correlation between heavy metals in fish feeds with detected metals in the gills, liver, and muscle of fish was also determined. Results revealed that heavy metals concentration, including Cu and Cd, in feed B was significantly greater than in feed A, but the Zn level in feed A was significantly higher than in feed B. Furthermore, the concentration of heavy metals in fish of both aquaculture systems was significantly higher in the liver than in the gills and muscle. The metal concentration in fish feeds and fish edible parts (muscle) was lower than the WHO standard level; however, the amount of Pb was higher in the fish muscle, which is harmful for human consumption. Though the correlation test revealed that all of the metals from the feeds were positively correlated to the metals detected in the fish, but most of the estimated correlation was insignificant. From the current study, it can be concluded that the fish feed producers need to measure feed quality adequately to avoid hazardous contamination by heavy metals in the feed. The ultimate consumer, fish and humans, may, otherwise, be predisposed to assimilate and accumulate these heavy metals.

Ameliorative effect of vitamins (E and C) on biochemical alterations induced by sublethal concentrations of zinc oxide bulk and nanoparticles in Oreochromis niloticus.

The comparison between bulk and nano ZnO particles were antecedently studied but describing the dose-dependent toxicity and the ameliorative effect of vitamins (E and C) in Oreochromis niloticus, have not been previously documented. Therefore, the present study was designed to investigate the ameliorative effect of vitamins (E and C) against oxidative stress and biochemical alterations induced by sublethal concentrations of zinc oxide bulk particles (ZnOBPs) and zinc oxide nanoparticles (ZnONPs). Toxicity tests were carried out on O. niloticus and showed that 96 h LC50 values of ZnOBPs and ZnONPs were 84 mg/l and 5.6 mg/l respectively. Exposure of the studied fish to these sublethel concentrations for 7, 14, 21 & 28 days showed a significant increase (p < 0.05) in serum glucose, AST, ALT, creatinine, urea and uric acid compared to control groups while, fish groups exposed to ZnOBPs or ZnONPs and supplemented with vitamins E and C, their serum enzyme concentrations were decreased compared to the groups without supplementation after 7, 14, 21 and 28 day. On the other hand, antioxidant defense enzymes (SOD, CAT and GST) activity in O.niloticus fish were increased significantly (p < 0.05) when exposed to sublethal concentrations of ZnOBPs or ZnONPs compared to the control value. However, fish groups supplemented with vitamins (E and C) have a decrease in SOD, CAT and GST enzymes activity when compared to unsupplemented groups and the values returned to similar levels established in the control at low concentrations but still higher than control at the high concentrations.

Water assessment of the Itapemirim River/Espírito Santo (Brazil): abiotic and toxicogenetic aspects.

The Itapemirim River is considered one of the most important water resources in the state of Espírito Santo, Brazil. However, environmental problems due to continuous anthropogenic contamination are threatening its potential use. This study assessed water quality by analyzing abiotic and toxicogenetic aspects of the water from four stations along the river. Samples were collected in both dry and rainy seasons. Most of the abiotic variables were below the threshold established by CONAMA Resolution No. 357/2005, and so were most of the metals. However, Al and Cu contents were above those allowed by legislation, ranging from 0.2 to 0.9 mg/L. Regarding toxicogenetic aspects, genotoxic effects were observed in meristematic cells of Allium cepa, in micronucleus test and comet assay of Oreochromis niloticus, and CHO-K1 cells. Mutagenic effects were significant at RI 02 (0.34), RI 03 (0.46), and RI 04 (0.12) stations on the first campaign in A. cepa F1 cells, compared to the negative control (0.0). The second campaign revealed the same results, but with the addition of samples from RI 01 (0.17) and RI 03 (0.18) showing mutagenicity in the micronucleus test with fish erythrocytes when compared to the negative control (0.3). Essentially, all the samples evaluated in both campaigns showed damage in A. cepa, O. niloticus, and CHO-K1 cells, thus demonstrating that the water quality of the Itapemirim River is compromised and requires action plans for its recovery.

Molecular Typing, Antibiogram and PCR-RFLP Based Detection of Aeromonas hydrophila Complex Isolated from Oreochromis niloticus.

Motile Aeromonas septicemia is a common bacterial disease that affects Oreochromis niloticus and causes tremendous economic losses globally. In order to investigate the prevalence, molecular typing, antibiogram and the biodiversity of Aeromonas hydrophila complex, a total of 250 tilapia (Oreochromis niloticus) were collected randomly from 10 private tilapia farms (25 fish/farm) at El-Sharkia Governorate, Egypt. The collected fish were subjected to clinical and bacteriological examinations. The majority of infected fish displayed ulcerative necrosis, exophthalmia, and internal signs of hemorrhagic septicemia. The prevalence of A. hydrophia complex was 13.2%, where the liver was the most predominant affected organ (54.1%). Polymerase chain reaction (PCR) was used to verify the identification of A. hydrophila complex using one set of primers targeting gyrB as well as the detection of virulent genes (aerA, alt, and ahp). All isolates were positive for the gyrB-conserved gene and harbored aerA and alt virulence genes. However, none of those isolates were positive for the ahp gene. The antimicrobial sensitivity was carried out, where the recovered strains were completely sensitive to ciprofloxacin and highly resistant to amoxicillin. All retrieved strains showed the same phenotypic characteristics and were identical based on the restriction fragment length polymorphism (RFLP). Experimentally challenged fish presented a high mortality rate (76.67%) and showed typical signs as in naturally infected ones. In conclusion, the synergism of phenotypic and genotypic characterization is a valuable epidemiological tool for the diagnosis of A. hydrophila complex. RFLP is a fundamental tool for monitoring the biodiversity among all retrieved strains of A. hydrophia.