Morphometric parameters and level of Salmonella and Escherichia coli contamination of Tilapia guineensis and Sarotherodon melanotheron in the waterway of Southern Benin

Aim: The present study aims to evaluate the morphometric parameters and level of Salmonella and Escherichia coli contamination of Tilapia guineensis and Sarotherodon melanotheron in the waterway of Southern Benin. Materials and Methods: 183 T. guineensis and 195 S. melanotheron were collected from June to July 2014 in four waterways: Lake Ahémé, Nokoué Lake, coastal lagoon, and lagoon of Porto-Novo. Weight, total length, and standard length of these fish were evaluated. E. coli and Salmonella sp. were sought in fresh fish. Results: The results obtained in this study indicate that S. melanotheron presented high-performance of length and weight more than T. guineensis in Ahémé Lake and lagoon of Porto-Novo. However, in Nokoué Lake and coastal lagoon, no difference was observed between the two species of fish. As for bacteriological analysis, the population of T. guineensis was more contaminated with E. coli with respective values of 60% and 59.52% in the Nokoué Lake and coastal lagoon than in the two other streams. Regarding the population of S. melanotheron, she was most contaminated in the coastal lagoon with a percentage of 66.66% in Ahémé Lake, Nokoué Lake, and the lagoon of Porto-Novo. However, no Salmonella germ was detected in fish analyzed in this study. Conclusion: It appears that the morphometric parameters and weight of T. guineensis are lower than those of S. melanotheron. The evaluation of the microbiological quality revealed that T. guineensis is more contaminated with E. coli that S. melanotheron.


Introduction
Food security is today a challenge for the authorities.People are facing an animal protein deficit.Fishing is one of the very important activities to overcome this problem.Furthermore, fishing is a veritable source of animal protein for the majority of the world population.
In Benin, the national fish production is estimated at 40,363.63 tons in 2012 [1].Despite this contribution, the supply of fish products is below market needs that are around 8,657,033 tons [1].In view of the gap between supply and demand plus the decline in fisheries production [2], Benin is dependent on foreign countries for fishery products.This situation resulted in massive imports of frozen fish from neighboring countries and Europe.Fish products are preferred food because they are inexpensive, acceptable, and accessible to the consumers.However, the skin, shell, mucus, gills and gastrointestinal tract of these animals contain a significant number of microbial floras [3].In the United States, Olsen et al. [4] reported that fish was an accessory to 25% of the recorded food poisoning.
The sanitary degradation of the aquatic environment due to the increase in the lacustrine population of Benin affects the aquatic fauna of the Lake [5].Therefore, it is important to evaluate the morphometric parameters of fish species in some waterways in South Benin.It should also be taken into account the presence of zoonotic gastrointestinal bacteria such as Salmonella spp.and Escherichia coli in their bodies.Tilapia guineensis and S. melanotheron are two Indigenous species of cichlid of the Benin water which are strongly consumed.The present study evaluates the morphometric parameters and the presence of Salmonella and E. coli in two species of cichlid in some streams in Southern Benin.

Collection of fish
T. guineensis and S. melanotheron were collected in four wild waterways in southern Benin: Ahémé Lake, Nokoué Lake, coastal lagoon, and the lagoon of Porto-Novo.The age of the fish was not established.Collected fish were transported in 1 or 2 h maximum from the site to the Veterinary Clinical Laboratory and Pharmacy Complex of Animal Health Production of the University of Abomey-Calavi for morphometrical and microbiological analyzes.A temperature of 4°C was maintained during transport to avoid deterioration of the specimen.

Total length, standard length, and weight measurements
Once at the laboratory, fishes were immediately weighed with a digital scale (KERN ® : max 220 g, d=0.1 mg). Figure -1 shows the total length and the standard length of collected fish were taken with an ichtyometer.

Evaluation of the relationship weight-lengt
The type of growth presented by the fish was evaluated through the weight-length relationship.This exponential relationship between weight and length is computed based on Le Cren, ( 1951 The coefficient b varies between 2 and 4. It expresses the relative shape of the body of a fish.When it is equal to 3, fish growth is isometric; below 3, it's allometric, but when coefficient b, is above 3, it indicates better growth in weight and length and viz.[6].

Detection of E. coli
After morphometric analyzes, each fish sample was aseptically dissected to expose the abdominal region.The intestine was removed and cut into small pieces.
A sample of 25 g of intestine was removed.To this sample, 225 ml of buffered peptone water (Oxoid CM0509 lot 1390836, England) was added to obtain a stock solution.Successive dilutions were performed until 10 −4 from the stock solution.1 ml of each dilution was inoculated by incorporating on the medium RAPID E. coli 2. After incubation at 44°C for 24±2 h, the reading was taken.The colonies of E. coli are purple, and those of thermotolerant coliforms are blue.

Detection of Salmonella sp.
For detection of Salmonella sp., 225 ml of buffer peptone water (Oxoid CM0509 lot 1390836, England) was added to 25 g of sample intestine.After incubation at 37°C for 18 h, 0.1 ml of this culture was inoculated in 10 ml Rappaport-Vassiliadis broth (Bio-Rad lot 098282, France) and 2 ml in 20 ml of broth Mueller Kaufman.The incubation was performed at 37°C for 24±4 h.The isolation of Salmonella was done by seeding xylose lysine decarboxylase media (Oxoid CM0469 lot 1399006, United Kingdom) and Hektoen (Oxoid CM0419 lot 1423494, England).Selecting Salmonella characteristic colonies were made after incubation at 37°C for 24±4 h.The discrimination test was performed by inoculating the urea indole medium.Salmonella is devoid of urease [7].The API 20E gallery was finally planted.

Statistics analysis
Morphometric parameters measured were encoded in Excel and statistically analyzed using SAS [8].Proc GLM procedure was used for the analysis of variance.The F-test was used to determine the significance of the effect species.Means were compared pairwise by the Student's t-test.The Chi-square test was used to determine the significance of the frequencies of bacterial contamination levels by species and waterways.The test bilateral of Z was used to compare pairwise the frequency.

Total length, standard length, and weight measurements
In Ahémé Lake, the average values of morphometric parameters were, respectively, 133.58±1.98 and 102.56±1.53mm for the average total length and the average standard length for S. melanotheron.On the other hand, in T. guineensis, the average total length was 116.22±2.19mm with an average standard length of 88.24±1.70 mm.
The average body weight was 48.1±1.62 g in S. melanotheron against 32.44±1.79g in T. guineensis.These parameters varied significantly between the two species in the rivers (p<0.001)(Figure -2).
In the coastal lagoon, the average total length and average standard length for S. melanotheron were, respectively, 131.6±2.96 and 98.87±2.29 mm.In T. guineensis, the average total length was 128.31±3.06mm and the average standard length was 96.64±2.37 mm.As regards the body weight, S. melanotheron weighed 52.44±3.13against 44.93±3.24g for T. guineensis (Figure -4).These parameters have not varied significantly between the two species in the lagoon (p>0.05).
However, in the lagoon of Porto-Novo, the total length of S. melanotheron and T. guineensis was, respectively, 176.28±3.73 and 161.28±3.73mm, with respective average standard lengths of 135.54±2.91 and 123.88±2.91 mm.In this stream, the average body weight of S. melanotheron was 113±6.66 against 89.70±6.66g in T. guineensis.The statistical analysis showed that there is a significant difference between these parameters in these two species (p<0.01)(Figure -5).
As regards the weight of S. melanotheron, it was 75.46±2.87against 64.11±2.96g in T. guineensis.For these three parameters, the values were significantly different between the two species (p<0.01).
Table-1 shows the morphometric parameters of T. guineensis in different streams.The body weight of T. guineensis in Nokoué Lake (80.60±4.20 g) and in the lagoon of Porto-Novo (89.70±4.20 g) did not differ significantly (p>0.05).However, the body weight of this species in these two waterways is higher than the coastal lagoon and Ahémé Lake where the respective values of this parameter were 44.93±4.58 and 32.44±4.64g (p<0.05).As for the average total length, T. guineensis from Nokoué Lake and Porto-Novo lagoon showed the highest values (156.34±2.66 and 161.28±2.66mm, respectively) compared to those living in the Ahémé Lake and coastal lagoon.Furthermore, T. guineensis had a higher total length in the coastal lagoon than Lake Ahémé with respective values of 128.31±2.91 and 116.22±2.94mm (p<0.05).Regarding the standard length, it was higher in T. guineensis living in Nokoué Lake and Porto-Novo lagoon than the ones living in the coastal lagoon (p<0.05).
In addition, T. guineensis of the coastal lagoon was longer than the ones of Ahémé Lake.In sum, T. guineensis of Nokoué Lake and Porto-Novo lagoon had the highest morphometric parameters (p<0.001).

Morphometric parameters in S. melanotheron compared in different waterways
In the lagoon of Porto-Novo, the body weight, the total length, and standard length were, respectively, 113.00±4.65 g, 176.28±3.15,and 135.54±2.4mm for S. melanotheron.The values of these parameters were lower for those living in the Nokoué Lake with respective values of 86±4.65 g, 161.64±3.15,and 123.16±2.4mm (p<0.05).Furthermore, the values for these parameters in the Nokoué Lake were higher than those obtained in the Ahémé Lake and in the coastal lagoon (p<0.05)(Table-2).

Body weight and total length relationship in T. guineensis and in S. melanotheron
In T. guineensis, the values of growth rate ranged from 2.55 to 3.15 in Ahémé Lake, Nokoué Lake, the coastal lagoon, and the lagoon of Porto-Novo.In Ahémé Lake and coastal lagoon, the growth of T. guineensis was negative allometric (b<3) while in the lagoon of Porto-Novo, this species was positively allometric growth (b>3).By cons, T. guineensis of Nokoué Lake presented an isometric growth (b=2.92)(Table -3).Regarding S. melanotheron, the values of growth coefficient in the Ahémé Lake, coastal lagoon, and Porto-Novo lagoon were, respectively, 2.07, 2.76, and 2.53.In these waterways, S. melanotheron presented a negative allometric growth.By cons, in Nokoué Lake the growth of these fish was isometric (b=2.92)(Table -3).The body weight and total length relationship of T. guineensis and S. melanotheron were highly significant in Ahémé Lake, coastal lagoon, and lagoon of Porto-Novo (p<0.001).

Evaluation of the level of contamination by Salmonella and E. coli in T. guineensis and S. melanotheron taken from Lakes Ahémé, Nokoué; coastal lagoon, and the lagoon of Porto-Novo
The level of contamination of Salmonella and E. coli in T. guineensis and S. melanotheron taken from different waterways were presented in Table-4.In the coastal lagoon, 66.66% of the population of S. melanotheron were contaminated with E. coli against 59.52% of the population of T. guineensis (p<0.05).In Nokoué Lake, populations of T. guineensis were more contaminated with 60% against 20% in S. melanotheron.In the lagoon of Porto-Novo, we note the same registration with 30% in populations of T. guineensis and 10% in S. melanotheron (p<0.001).About the level of contamination of E. coli in T. guineensis and S. melanotheron in Ahémé Lake, no significant difference was observed.However, microbiological analyzes revealed no sample contamination by Salmonella sp.

Morphometric parameters of T. guineensis and S. melanotheron
The results obtained in this study are in accordance with those obtained by Fousséni [9] in these same waterways in Southern Benin.In Nigeria, it reports that the morphometric parameters of S. melanotheron are higher than those of T. guineensis [10].Other authors report that the quality and quantity of food, as well as environmental variables, affect fish growth [11].This can explain the difference in morphometric parameters and body weight between S. melanotheron and T. guineensis.S. melanotheron is particularly suitable in estuaries and brackish water.It tolerates a very wide range of salinity and low dissolved oxygen levels.It is an omnivorous species whose food resources vary in accordance with the environment [12].According to Halvorsen and Svenning [13], a temperature change causes a difference in growth.In the lagoon of Porto-Novo and Nokoué Lake, the temperature is lower than in the Lake Ahémé and coastal lagoon [9].This also explains the low morphometric parameter of T. guineensis in these two rivers.The same authors relate that T. guineensis presents low morphometric parameters and a relatively low weight gain when the salinity and dissolved oxygen levels are high.In their natural environment, the average total weight obtained in T. guineensis (64.11 g) is higher than that obtained in fish breeding (44.34 g) by Toko-Imorou et al. [6].In the same study, the population of S. melanotheron with highest morphometric parameters in the natural environment is found in Porto-Novo lagoon with an average weight of 113 g and a total length of 176.28 mm.It showed that S. melanotheron is less efficient growth when it is bread in cages than when it is bread Acadja in lagoon environment [14].Similarly, morphometric parameters, growth indicators, are influenced by the waterway effect in T. guineensis [15].

Weight-length relationship
In this study, the results obtained in T. guineensis in Ahémé Lake, coastal lagoon, and the lagoon of Porto-Novo are in accordance with those of Lalèyè et al. [16] at the Ahémé Lake.On the other hand, the research conducted by Fontaine et al. [15] in Ahémé Lake, the lagoon of Porto-Novo and Nokoué Lake from August to October 2013, reveal isometric growth in the first two waterways and allometric growth in the latter.These types of variations are also observed in Labeo barbus batesii during its growth [17].These changes can be explained by the fact that the growth factor is related to biotic factors, abiotic and availability of food as well as to habitat type [18].
In this work, regarding S. melanotheron, allometric coefficients obtained, respectively, in the Ahémé Lake, the coastal lagoon, and the lagoon of Porto-Novo are in accordance with those obtained by Fagnon [19] who reveals an allometric coefficient of 2.75 in the coastal lagoon and 2.73 in the lagoon of Porto-Novo.

Bacteriological analyzes in T. guineensis and S. melanotheron
The presence of E. coli in the intestine of T. guineensis and S. melanotheron is linked to unhealthy waterways [20].The results obtained in this study are in accordance with those obtained by Sèdogbo [21] and Dègnon et al. [20] on shrimp in the Nokoué Lake and the Ahémé Lake.The poor quality of the captured fish species is due to the high level of bacterial contamination of streams [22,23].Similarly, Dègnon et al. [24] reported high bacterial contamination of Trachurus trachurus by coliforms in Benin.The absence of Salmonella in samples analyzed may be explained by his absence in waterways or the influence of resistance of fish in relation to the bacteria.These results are consistent with those obtained by N'diaye [25] on fishery products in 1998 in Dakar.

Conclusion
This study allows understanding the morphometric parameters and weight of S. melanotheron and T. guineensis in some stream in Southern Benin.It appears that the morphometric parameters and weight of T. guineensis are lower than those of S. melanotheron.The evaluation of the microbiological quality revealed that T. guineensis is more contaminated with E. coli that S. melanotheron.Since these two species are very important in the commercial market, particular attention should be paid to different cooking techniques to prevent bacterial infections that can result from the consumption of these fish species.It would be also interesting to perform the analysis of bacterial contamination of edible parts of the studied fish since intestines are probably not consumed.In addition to state that there is a risk for the final consumer of those fish a quantification of the amount of the colony-forming unit of E. coli should be performed.
); the formula is as follows: W = a TL b Where: W = Fish weight (g); TL = Total length of the fish (cm); a = Coefficient related to the environment of fish (y-intercept of the regression line); b = The coefficient of relative growth and (b) as the relative growth rate of the mass (slope of the regression line).

Table - 1
: Morphometric parameters of Tilapia guineensis in different streams.

Table - 3
: Body weight and total length relationship in Tilapia guineensis and Sarotherodon melanotheron in streams in southern Benin.The sign (*) indicates that the value is significantly different from 3; a is ordered to the origin of the regression line; b or regression coefficient is the gradient of the regression line; r² is the coefficient of determination Table-4: Contamination level of salmonella and E. coli in Tilapia guineensis and Sarotherodon melanotheron.