الفهرس 
CONTENT
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Abstract
Tilapia is the first Egyptian farmed fish. The environmental conditions as osmotic strength, oxygen levels, pH, salinity and temperature and sometimes poor management practices (inadequate nutrition, overcrowding and overfeeding) can cause stress to the cultured fish and thus make them more susceptible to diseases. Among the bacterial pathogens, Aeromonas species and Streptococcus species are the most common pathogens in tilapia that cause huge economic losses in its production. Various types of antibiotics are used to treat such infections. Misuse of antimicrobial agents in aquaculture provides a selective pressure creating reservoirs of drug-resistant bacteria and transferable resistance genes in fish pathogens and other bacteria in the aquatic environment. These resistance genes may disseminate and reach human pathogens, or the drug-resistant pathogens may reach humans directly. To overcome these situations an urgent need for new therapeutic agents are required. Probiotic bacteria mainly lactic acid bacteria and antimicrobial peptides are most important alternatives for control or treatment of fish diseases.
This study was conducted in two sections. Firstly, the prevalence and antibiotic resistance of Aeromonas hydrophila, Streptococcus agalactiae and Streptococcus iniae in tilapia fish and aquaculture water was investigated. Also, the water quality parameters were determined and their correlation with the isolated pathogens was assayed.
Secondly, alternative probiotic lactic acid bacteria and antimicrobial peptides were obtained from tilapia aquaculture.
I- Pathogen isolation
Seventy-nine tilapia fish and seventy water samples were collected in four visits from three farms in Assiut and Minia Governorates.
The results obtained in this study revealed that:
1. Aeromonas hydrophila prevalence, molecular identification and antibiotic sensitivity in tilapia aquaculture
Aeromonas hydrophila was isolated from tilapia five organs including the liver, kidney, intestine, spleen and gills with a percentage of 7.1%, 6.5%, 9.1%, 5.1%, 11.7%, receptively, and from water samples with 59.7%.
The isolates were molecularly confirmed as Aeromonas hydrophila in 70.9% and 82.4% of the tested isolates using 16S RNA and gyr-β primers, respectively. Aeromonas hydrophila isolates revealed a marked resistance for the tested antibiotics; amoxicillin and novobiocin (100%), doxycycline and trimethoprim/sulphamethoxazole (50%), colistin (43%), streptomycin (71.4 %), chloramphenicol (57%), ciprofloxacin and norfloxacin (14%). The isolates showed multiple antibiotic resistance indexes ranging from 0.3 to 1.
2. Streptococcus agalactiae prevalence, molecular identification and antibiotic sensitivity in tilapia aquaculture
Streptococcus agalactiae was isolated from tilapia five organs including the liver, kidney, intestine, spleen and gills with a percentage of 14.3%, 7.1%, 19.4%, 7.1%, 16.7%, receptively, and from water samples with 35.7%.
Two isolates were molecularly confirmed as Streptococcus agalactiae using the species specific 16-23S intergenic space primers. Streptococcus agalactiae isolates exhibited resistance against most tested antibiotics with multiple antibiotic resistance indexes ranging from 0.9 to 1.
3. Streptococcus iniae prevalence, molecular identification in tilapia aquaculture
Streptococcus iniae was isolated from tilapia five organs including the liver, kidney, intestine, spleen and gills with a percentage of 13.5%, 22%, 18.6%, 5%, 13.5%, receptively, and from water samples with 27.1%. However, no isolate was confirmed molecularly using the species specific lactate oxidase (lctO) gene primer
4. Water quality parameters
The water temperatures were 14.7, 13.4, 16.8, 28oC, respectively. The measured water pH among the examined ponds were 7.7, 8.7, 9 and 9.3 The dissolved oxygen values were 5.7, 4.3, 4.9 and 2.7 mg /L, the mean electrical conductivity values were 317.2, 527.6, 1513, and 1696.5 µS/ cm2. Furthermore, the total dissolved solids values were 138.4, 260.6, 757.2 848.2 in visit I II, III, and IV, respectively.
5. Correlation analyses
A- Between water parameters
The electrical conductivity and total dissolved solids showed a strong significant correlation. At the same time, each of them displayed a positive correlation with water temperature and pH. While, each of them displayed a negative correlation with the water dissolved oxygen. Moreover, there is a positive relationship between water temperature and pH. In contrast, the water dissolved oxygen displayed a negative correlation with other parameters.
B- Water parameters correlation with the isolated pathogens
Analyses of the correlation between water Aeromonas species isolates and different physicochemical parameters showed that, the water temperature, electrical conductivity and total dissolved solids showed significant positive correlation with the number of isolated Aeromonas species. Although, the water dissolved oxygen displayed negative linkage with number of isolated Aeromonas species isolates from the aquaculture water.
Analyses of the correlation between water Streptococcus isolates and the water physicochemical parameters showed that all parameters had significant positive correlation with the isolated pathogen except for water pH, the linkage was negative.
II- Antibiotic alternatives.
A- Probiotic lactic acid bacteria
The mean probiotic lactic acid count of tilapia gut was 5.67, 4.83, 5.43, and 5.78 log10 CFU count / ml in visit I II, III, and IV, respectively. Seventy-six lactic acid bacteria were selected for further characterization based on their phenotypic characters. The selected strains were tested for their safety and only twenty strains proved their safety. Furthermore, these 20 lactic acid bacteria isolates exerted antibacterial activity against multi-antibiotic resistant Aeromonas hydrophila and Streptococcus agalactiae isolates.
The probiotic bacteria exhibited different degrees of adaptability to pH and bile stress, and secreted different enzymes including lipase, protease, and amylase.
Four strains were selected for molecular identification and all were positive for PCR amplification. Two strains with the highest activity were further subjected to sequence analysis and one of them showed 98 % homology to Enterococcus faecium and the other showed 99% homology to Enterococcus faecalis.
B- Antimicrobial peptide
Tilapia hydrolysate was prepared under the optimum conditions of the hydrolyzing enzyme and the most active hydrolysate was further characterized for amino acid composition analysis. The extracted peptides exerted inhibitory activity presented in a halo zone around the tested pathogens; ranged from 10- 30 mm against Streptococcus agalactiae, and 10-18 mm against Aeromonas hydrophila.
The peptides showed different degrees of hydrolysis ranged from 34.5 to 41.9%. The most active peptide was investigated for amino acid composition and it was found that the peptide contains methionine, phenyl alanine, leucine, therionine and lysine