الفهرس 
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Abstract
River Nile is considered the main source of water in Egypt. In the last decades, the River Nile was subjected to different types of pollution. The most important pollution sources were agriculture run-off, domestic and industrial effluents. Therefore, the following study was conducted.
In the present study, some aquatic insects and fish were used as bioindicators of some zoonotic bacteriological pollutants (Aeromonads group; total vibrios and Salmonellae group) in the River Nile during one year (February 2009- January 2010) at different pollution sites representing: The main stream of River Nile (reference point), the mixed point of agriculture drain and the mixed point of wastewater.
1. Sample collection and identification:
Aquatic insects and fish samples were collected from the River Nile at different pollution sites and then identified as follows:
In the main stream of River Nile (reference point); the collected aquatic insects were Appasus urinator, Belostomatidae, Hemiptera and Enallagma vansomereni, Coenagriidae, Odonata. Fish samples were Oreochromis niloticus, Cichlidae, Perciformes. In the mixed point of agriculture drain; the collected aquatic insects were Appasus urinator, Belostomatidae, Hemiptera and Hydaticus leander, Dytiscidae, Coleoptera. Fish samples were Oreochromis niloticus, Cichlidae, Perciformes. In the mixed point of wastewater; the collected aquatic insects were Eristalis sp., Syrphidae, Diptera. Fish samples were Silurus triostegus, Siluridae, Siluriformes.
2. Determination of physicochemical parameters:
In the following study; physicochemical parameters were measured in water collected from the River Nile at different pollution sites. In case of the main stream of River Nile (reference point): The temperature and pH average measurements were: 23.5.C and 8.2, respectively. The average levels of DO, COD and EC were 7.4 mg O2/l, 20.1 mg O2/l and 390 ƒÊs, respectively. Chloride, TDS, hardness and alkalinity average levels were20.3, 226.6, 132.9 and 184 mg/l, respectively. The average level of ammonia was 0.15 mg/l while Nitrite was detected only in two months during the year of study (February 2009- January 2010) in March and November (0.01 mg/l). In case of water collected from the mixed point of agriculture drain; physicochemical parameters were detected. The average levels of temperature and pH were: 23.5.C and 8.3, respectively. The average levels were: 6.8 mg O2/l, 103.2 mg O2/l and 1656.6 ƒÊs for DO, COD and EC during the period of study. Chloride, TDS, alkalinity and hardness average levels were 153, 1012.5, 326.4 and 432.8, respectively. The average levels of Ammonia and Nitrite were 0.8 and
0.1 mgN/l, respectively. In case of water collected from the mixed point of wastewater: The average levels during one year were 23.5 .C and 8 for temperature and pH, respectively. The average levels were: 2.5 mgO2/l, 62.3 mgO2/l and 1749.9 ƒÊs for DO, COD and EC, respectively. Chloride, TDS, alkalinity and hardness average levels were 259.2, 1033.3, 242.3 and 294.4 mg/l, respectively. Also, the average levels were 5.1 and 0.4 for ammonia and nitrate respectively.
3. Bacteriological examination:
3.1. Determination of total vibrios:
3.1.1. Determination of total vibrios by MPN-TCBS method:
Total vibrios were determined in water collected from different pollution sites. Total vibrios were not detected in water collected from the main stream of River Nile before branches (control site). While the average values of total vibrios (MPN/100ml) recorded in water was 1.0 x 104 and 2.7 x 103 in the mixed point of agriculture drain and the mixed point of wastewater, respectively. Total vibrios were determined by MPN/gm in aquatic insects collected from River Nile at different pollution sites during one year (February 2009- January 2010). The average MPN/gm of total vibrios recorded in aquatic insects was 1.4 x 103, 1.7 x 105 and 1.3 x 105 in the main stream of River Nile before branches, the mixed point of agriculture drain and the mixed point of wastewater respectively.
Also, total vibrios were determined in fish collected from River Nile from different pollution sites in 3 organs; muscle, liver and gills. Muscle did not contain any Vibrio spp in the different sites. In the main stream of River Nile before branches, liver did not contain any Vibrio spp. The average MPN/gm values of total vibrios in liver isolated from fish collected from the mixed point of agriculture drain and the mixed point of wastewater were: 2.9 x 104 and 6.2 x 103, respectively. The average MPN/gm values of total vibrios in gills isolated from fish collected from the main stream of River Nile (reference point), the mixed point of agriculture drain and the mixed point of wastewater were: 3.8 x 103, 1.0 x 104 and 2.5 x 104 and respectively.
3.1.2. Determination of Vibrio spp. by PCR and multiplex PCR:
It was found that 425 total vibrios were isolated from aquatic insects, fish and water collected from River Nile at different pollution sites from which only 202 isolates were +ve Vibrio spp. when tested by PCR.
3.2. Determination of Aeromonads group:
3.2.1. Determination of Aeromonads group by surface plate technique:
Aeromonads group was determined by surface plate technique in water, aquatic insects and fish collected from River Nile at different pollution sites during one year (February 2009- January 2010). The average CFU/100ml of Aeromonads recorded in main stream of River Nile water was 3.3 x 104 for A. hyDROPhila and 3.2 x 104 for other Aeromonas spp. While the average CFU/100ml of Aeromonads recorded in mixed point of agriculture drain water was 4.4 x 105 for A. hyDROPhila and 3.2 x 105 for other Aeromonas spp. And in case of the mixed point of wastewater the average counts of Aeromonads recorded were 3.1 x 108 CFU/100ml for A. hyDROPhila and 2.5 x 108 CFU/100ml for other Aeromonas spp.
Aeromonads were determined by CFU/gm in aquatic insects collected from River Nile at different pollution sites. The average CFU/gm of Aeromonads recorded in aquatic insects collected from the main stream of River Nile (reference point) was 2.0 x 106 for A. hyDROPhila and 1.2 x 106 for other Aeromonas spp. In the mixed point of agriculture drain the average CFU/gm counts of Aeromonads recorded in aquatic insects collected from mixed point of agriculture drain were 1.1 x 106 for A. hyDROPhila and 2.5 x 106 for other Aeromonas spp. While in the mixed point of wastewater the average CFU/gm counts of Aeromonads recorded in aquatic insects were 1.0 x 106 for A. hyDROPhila and 8.3 x 105 for other Aeromonas spp.
Aeromonads were determined in tilapia fish (muscle, liver and gills) collected from the main stream of River Nile. The average A. hyDROPhila values of CFU/gm were: 1.8 x 103, 5.5 x 104 and 2.7 x 104 in muscle, liver and gills, respectively. Also the average values of others Aeromonas spp. were: 4.0 x 103, 9.1 x 103 and 1.1 x 104 CFU/gm in muscle, liver and gills, respectively. Also total Aeromonads were determined in tilapia fish collected from mixed point of agriculture drain. The average values of A. hyDROPhila were: 8.7 x 104, 7.2 x 104 and 1.2 x 106 CFU/gm in muscle, liver and gills, respectively. While the average values of other Aeromonas spp were: 1.4 x 105, 2.5 x 105 and 1.0 x 106 CFU/gm in muscle, liver and gills, respectively. Total Aeromonads were detected in catfish collected from the mixed point of wastewater. the average values of A. hyDROPhila were: 3.2 x 105, 1.5 x 107 and 1.4 x 107 CFU/gm in muscle, liver and gills, respectively. On the other hand, the average values of others Aeromonas spp. were: 2.0 x 105, 7.1 x 106 and 2.9 x 107 CFU/gm in muscle, liver and gills, respectively.
3.2.2. Determination of Aeromonas spp. and Aeromonas hyDROPhila by PCR:
Results indicated that 750 isolates of total Aeromonads were isolated from water, aquatic insects and fish collected from River Nile at different pollution sites. After confirmation by PCR; it was found that 672 isolates were Aeromonas spp. +ve from which only 309 isolates were A. hyDROPhila +ve.
3.3. Determination of Salmonellae group:
3.3.1. Determination of Salmonellae group by MPN method:
Salmonellae group were determined by MPN in water, aquatic insects and fish collected from River Nile at different pollution sites during one year (February 2009- January 2010). The average MPN/100 ml of Salmonellae recorded in water was 4.3 x 102, 1.3 x 104 and 1.3 x 105 in the main stream of River Nile before branches (reference point), the mixed point of agriculture drain and the mixed point of wastewater, respectively. Determination of Salmonellae by MPN/gm in aquatic insects collected from River Nile at different pollution sites during one year (February 2009- January 2010) was represented. The average MPN/gm of Salmonellae recorded in aquatic insects was 1.5 x 103, 1.9 x 105 and 1.6 x 104 in the main stream of River Nile before branches (reference point), the mixed point of agriculture drain and the mixed point of wastewater, respectively. Also Salmonellae were determined in tilapia fish collected from mixed point of agriculture drain and the main stream of River Nile and catfish in 3 organs; Muscle, liver and gills. In the main stream of River Nile before branches; the average values of MPN/gm were: 6.0 x 10, 1.5x 103 and 1.4 x 103 in muscle, liver and gills, respectively. While in fish collected from the mixed point of agriculture drain the average MPN/gm values were: 1.3 x 104, 1.8 x 104 and 3.0 x 105 in muscle, liver and gills, respectively. In the mixed point of wastewater the average values were: 1.9 x 104, 9.4 x 105 and 1.6 x 105 MPN/gm in muscle, liver and gills, respectively.
3.3.2. Determination of Salmonellae group by PCR and nested PCR:
The incidence of Salmonellae was studied in water, aquatic insects and fish collected from River Nile at different pollution sites. Samples were detected by MPN technique and confirmed by PCR techniques. Seven hundred and ten random isolates of typical colonies of salmonellae were determined by MPN method and then confirmed by PCR and nested PCR. Five hundred and fourteen isolates were Salmonella spp. +ve when tested by PCR from which only 144 isolates were +ve when tested by the nested PCR assay.
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4. Relationship between pathogenic bacteria and physicochemical parameters:
In this study correlation between pathogenic bacteria and physiochemical parameters in River Nile water at different pollution sites was determined using the two tailed Pearsones correlation coefficient. In water samples collected from the main stream of River Nile (reference point) it is clear that that total vibrios not detected in all tested samples during the period of study. While, total aeromonads showed positive relation without significance with temperature, pH, alkalinity, COD, ammonia and chloride (r = 0.46, 0.32, 0.26, 0.34, 0.33 and 0.30, respectively). The correlation of total aeromonads with DO, nitrite, EC, TDS and hardness revealed low and negative relation without significance (r = -0.53, -0.34, -0.15, -0.16, -0.11, respectively). While salmonellae group recorded highly significant negative correlation with ammonia (r = -0.88). Negative relation without significance with temperature, pH, alkalinity, COD and hardness (r = -0.49, -0.12, -0.11,-0.16 and -0.02, respectively). where there was a positive relation without significance with DO, nitrite, EC, TDS and chloride (r = 0.52, 0.08, 0.09, 0.01 and 0.17, respectively).
In the mixed point of agriculture drain samples, total vibrios showed positive relation without significance with all physiochemical parameters except with temperature, pH, COD and chloride (r = - 0.28, - 0.10, - 0.26 and - 0.21, respectively). On the other hand, no correlation were found between total aeromonads and EC (r = 0.00). Total aeromonads recorded positive relation without significance with temperature, pH, alkalinity, nitrite, TDS, chloride and hardness (r = 0.03, 0.07, 0.08, 0.03, 0.12, 0.04 and 0.15). And negative relation without significance with DO, COD and ammonia (r = -0.40, -0.31 and -0.15). whereas salmonellae showed poor positive correlation with pH, alkalinity, ammonia, nitrite, TDS, chloride and hardness (r =0.15, 0.32, 0.30, 0.35, 0.06, 0.11 and 0.10). Negative correlation with temperature, DO, COD and EC (r = -0.78, -0.27, -0.03 and -0.25).
In the mixed point of wastewater samples, total vibrios recorded negative relation without significance with all physiochemical parameters except temperature, DO, COD, nitrite and TDS (r = 0.23, 0.01, 0.56, 0.01 and 0.07, respectively). where total aeromonads recorded a highly significant correlation with chloride (r =0.76). Also, positive significant correlation with nitrite (r =0.69) and negative significant correlation with temperature (r = - 0.69) and no significant correlation with other physiochemical parameters. There was significant negative correlation between salmonellae and pH (r = -0.68) and negative relation without significance with other physiochemical parameters except COD, nitrite and hardness r= (0.47, 0.25 and 0.25).
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5. Single cell gel electrophoresis (comet assay):
The results indicated higher tail moment in fish and aquatic insects collected from the mixed point of wastewater than the mixed point of agriculture drain and the main stream of River Nile before branches. The highest percentage of comet cells were found in fish and aquatic insects collected from the mixed point of wastewater followed by the mixed point of agriculture drain. The main stream of River Nile before branches has the lowest comet percentage. The statistical analysis of DNA damage using one way ANOVA for tail moment of fish and aquatic insects collected from different aquatic sites showed insignificant differences between samples collected from control site (P= 0.08). While when analyzing other comet parameters (comet% and tail length) samples were significantly different (P= 0.04).