CHAPTER The result obtained in this study was similar



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5.1       Discussion

bacterial load found in the skin, gill and intestine of Clarias gariepinus sampled from different farm locations in Ojo and
Badagry Local Governments of Lagos State was analysed in this study. The total
bacteria count, TBC (x 106CFU/mL) of the different parts ranged from
0.4 to 3.72. However, the result showed that the highest TBC was analyzed from
the intestine to be 3.72±0.81 and the lowest analyzed from the skin
(0.40±0.08). The result obtained in this study was similar in pattern to the
result observed by Abu and Uwadirioha (2016). The higher microbial load in the
gill and intestine of experimental fish may be due to increased bacteria that
were trapped in the gills and intestine during feeding and ingestion of food.
This was also observed by Akani and Obire (2014) in their study.

total bacterial count in this study was higher than the set standard for the
acceptable level of bacterial count/microbial safety of foods which is 1 to 100
cfu/mL (Ayinla et al., 1994). In
summary, the microbial load of the different parts of the experimental fish
collected from selected locations according to this study showed that fish
under culture is a reservoir of bacteria diversities. It is therefore noteworthy that
sanitary condition under which fishes are handled, fed and cultured be improved
upon to reflect standard or good management practices.

The biochemical characteristics of the isolated
organisms in the present study showed that there are both Gram positive and
Gram negative isolates, most of which are Gram negative.  Most of the isolates are Bacillus (rod-like
shape) while only three (3) of the isolates are cocci (round shape). Most of
them are also motile while only two (2) are non-motile. Moreover, all the
isolates showed positive reaction to glucose fermentation and catalase tests.
They showed varied reactions to lactose, mannitol, maltose fermentation, urea
and oxidase tests. All the organisms are negative to indole test except one.
Also, the isolates showed varied reactions to methyl red, hydrogen sulphide (H2S)
and Voges proskaeur. Four isolates were negative and only one was positive to
coagulase test. Most of the organisms isolated were positive to sucrose
fermentation test and citrate reaction. Only one isolate was positive while
most were negative to indole reaction.

The result of biochemical characteristics identified
the presence of a total number of 16 bacteria species. Based on the analysis
done using standard microbiological methods, this study revealed the presence
of  Staphylococcus
aureus S. saprophyticus, Escherichia coli, Bacillus subtilis, B. mycoides, B.
megaterium, Micrococcus luteus, Proteus mirabilis, P. vulgaris, Pseudomonas
fluorescens, P. alcaligens, P. putida, P. aeruginosa, Aeromonas hydrophila,
Klebsiella specie and Citrobacter freudii, in
the gills, skins and intestines of the sampled fish.

occurrence of many Gram negative bacteria (E.
coli, Proteus mirabilis, Pseudomonas fluorescens, Aeromonas hydrophila, P. alcaligens, P. vulgaris, P. putida, Klebsiella spp., Citrobacter  freudii and P. aeruginosa) and some Gram positive (Staphylococcus aureus, S. saprophyticus, Micrococcus luteus, Bacillus
subtilis, B. mycoides and B. megaterium) in the samples is in
concurrent with the work of Ibrahim and Adetuyi (2013). The isolation of Klebsiella spp, Proteus
spp. and E. coli from the fish
sampled indicated environmental contaminant and this supports the findings of
Yagoub and Ahmed (2004) and Naijah et al.
(2008). The occurrence of pathogenic bacteria especially E. coli in C. gariepinus may
jeopardize the fish health by causing infections in the fish under culture and
also induce diseases to human when in contact with them. Pseudomonas spp. and Proteus spp.
have been identified as responsible for some fatal diseases in fish as well as
zoonotic to man, Staphylococcus spp.
are known to cause severe diseases in man (Raghavan, 2003; Babu, 2000). Bacillus spp. and Staphylococcus spp. are major pathogens causing food borne diseases
(Bennett et al., 2013).

Although, pathogenicity of bacteria
isolates was not part of the focus, most of the bacteria species encountered in
this study are no doubt potentially pathogenic to different fish species under
culture especially Clarias gariepinus, as
also reported by Sowunmi et al (2008).

Length-weight relationship gives the
condition and growth patterns of fish. It provides important information
concerning the structure and function of fish populations (Hirpo, 2013). The
length and weight of experimental fish sampled from the different locations was
analysed. Result showed that the mean length (cm) ranged from 27.47±1.82 to
41.195±1.55 while the mean weight ranged from 155.26±25.44 to 483.62±41.21.
Length-weight relationship of the fish showed both linear and non-linear growth
pattern. However, the result showed that all the sampled fish had allometric
growth (b<3). Fafioye and Oluajo (2005) reported similar result of condition factor for C. gariepinus in Epe Lagoon as 0.79±0.15 and b value as 2.790. Bagenal and Tesch (1978) also reported 'b' values (2.9±4.8). The condition factor (K)  in this study  is less than 1 (K ranged from 0.69 to 0.89), the finding of Ayo-Olalusi (2014) also reported the K value to be low when they studied the length-weight relationship, condition factor and sex ratio of C. gariepinus reared in flow-through tank systems.  However, low K value may be due to reduced availability of food and or feed item. Natural products of plant origin remain the most important source of new drugs that synthesis into commercially available drugs for both therapeutic and prophylactic use in aquaculture. The majority of bioactive compounds present in these natural products include terpenoids, steroids, alkaloids, glycosides, phenolic compounds and derivatives, etc which are known to exhibit antibiotic, antiviral, antioxidant, insecticidal, nematocidal, etc effects (Nwokonkwo and Okeke, 2014). This present study analysed the presence of these bioactive compounds in the leaf, root, husk and bark extracts of Theobroma cacao. It was found out that Alkaloid was present in high concentration in the root and leaf extracts and low concentration in the bark and the husk of T. cacao. Flavonoid was present in high concentration in the leaf, low concentration in the root and not present in the bark and husk. Tannin, however, was present in high concentration in the husk and the leaf, and low in the root and bark. Steroid was analysed to be high in the bark and the husk, low in the root and not present in the leaf extracts. Moreover, combined anthraquinone and phlobatannin were only present in low concentration in the root and the bark and not found in the husk and the leaf. Saponnin was present in the root, bark and husk at low concentration but not found in the leaf. Terpenoid was found in all the parts but in low concentration. On the other hand, cardiacglycoside was not found in all the parts. Phenol was not present in the husk extract but was in the root, bark and leaf extracts in low concentrations. The phytochemical characteristic of bark extracts of T. cacao according to this study was similar to the result obtained by Nwokonkwo and Okeke (2014) when they studied the phytochemical constituents of the stem bark of the same plant. It has also been reported that the extracts of the leaves, root and husk of the some medicinal plants are rich in alkaloids, tannins, terpenoids, saponin and phenols; this was also observed in this study. Although, flavonoid was present in low quantity in some parts, the results of phytochemical screening of T. cacao parts extracts confirmed the possible use of this plant as a source of antibacterial drug against fish pathogens. The potentials of ten different synthetic drugs (Cefuroxime, Gentamycin, Amoxycillin, Penicillin, Streptomycin, Ciprofloxacin, Ofloxacin, Tetracycline, Ampicillin and Cotrimoxazole) on the identified bacteria (S. aureus, S. saprophyticus, E. coli, B. subtilis, B. megaterium, M. luteus, P. mirabilis, P. fluorescens, A. hydrophila, P. alcaligens, P. vulgaris, B. mycoides, P. putida, Klebsiella spp., C. freudii and P. aeruginosa) showed varying antibacterial efficacies based on the inhibition diameters recorded. The result of this study showed that Ofloxacin, Gentamycin, Ciprofloxacin, Tetracycline, Streptomycin and Cefuroxime have significant efficacies (p<0.05) on most of the bacterial pathogens. However, some Gram negative bacteria such as P. aeruginosa, E. coli and A. hydrophila  and some Gram positive bacteria such as S. aureus, Bacillus spp. and M. luteus produced a significant level (p<0.05) of resistance to Penicillin, Ampicillin and most especially Cotrimoxazole according to (CLSI, 2007). The resistivity of E. coli, P. aeruginosa, A. hydrophila and S. aureus is a significant threat to C. gariepinus under culture because they have been known to be important pathogens responsible for serious infections and mortalities in aquaculture. According to Munita and Arias (2016), there are a number of ways antibiotic resistances are exhibited in bacterial pathogens, such include the modifications of the antimicrobial molecule, the prevention by the pathogen to reach the antibiotic target (by decreasing penetration or actively extruding the antimicrobial compound); changes and/or bypass of target sites and resistance due to global cell adaptive processes. However in practice, it is important to note that irrational and inappropriate use of antibiotics is responsible for the development of resistance of most pathogenic bacteria especially Pseudomonas spp (Goswami et al., 2011). The results of the sensitivity assays of extracts of T. cacao against the isolated organisms showed that 100% of the extracts were most effective in the control of the isolated organisms than other concentrations. The leaf of T. cacao produced a good level of efficacy on P. putida (Inhibition diameter, 16.75±2.19), S. aureus inhibition diameter, 15.60±5.82), C. freudii (14.20±5.16) and B. megaterium (Inhibition diameter, 10.89±2.32). The husk of test plant produced a level of efficacy on P. aeruginosa (inhibition diameter, 11.63±2.00) and E. coli (inhibition diameter, 9.80±4.66). The result however, showed that root and bark extracts were least effective on the test isolates. Furthermore, leaf, husk, root and bark were resisted by the test organisms at the least concentration (25%). The in-vitro susceptibility testing of the leaf and husk extracts of T. cacao according to this study showed a promising antibacterial/antimicrobial potential in the control of pathogenic bacteria of C. gariepinus. The antibacterial efficacy of the leaf and husk may be influenced by the concentrations of medically active antibiotic chemical constituents especially good levels of tannin, flavonoids and phenols in them. Moreover, ethanolic extracts of many medicinal plants were reported especially to exhibit effective antibacterial and antioxidant on fish pathogens according to authors such as Sofowora (2008) and Alo, et al. (2012). The MICs of the synthetic antibiotics and T. cacao extracts on identified organisms were performed in order to obtain the minimum concentrations of the antimicrobial that will inhibit the growth of the sixteen (16) identified organisms. The results of the MIC performed showed varied efficacies among synthetic drugs. The result of MICs revealed that Gentamycine, Streptomycin, Ciprofloxacine and Ofloxacine had the most effective MICs, while Penicillin, Ampicillin and Cotrimoxazole had the least MICs on the test organisms. The results of the MIC of T. cacao parts extracts against the identified organisms showed varied efficacies. The results revealed that the husk MIC range (12.5 to 25%) and the leaf extract had the most effective MIC ranging from 12.5 to 50% while the bark extract (6.25 to 100%)  and root  (25 to 100%) had the least effective range on the test organisms. The MBCs of the synthetic antibiotics and T. cacao parts extracts on identified organisms were performed in order to obtain the minimum concentrations of each synthetic antibiotic and the T. cacao parts extracts that will kill about 99.5% of each of the sixteen (16) identified organisms. The results of the MBC performed followed similar trend as the MIC results. The bacteriostatic and bactericidal efficacy of the synthetic antibiotics on the 16 bacterial pathogens revealed the potential efficacies of the synthetic drugs, Gentamycine, Streptomycin, Ciprofloxacine and Ofloxacine and T. cacao part extracts, leaf and husk as a remedy for diseases caused by a host of pathogens identified in this study. The MICs and MBCs of leaf and husk extracts showed a promising efficacy than some synthetic drugs especially Penicillin and Cotrimoxazole if well purified.  Also, medicinal plants provide inestimable projections for new drug discoveries because of the matchless availability of chemical range (Alo et al., 2012).   5.2       Conclusion and Recommendations Herbal compounds have the ability to inhibit the generation of oxygen anions and scavenge free radical, hence reducing stress effects. It will be best if we use herbal plant extract instead of chemicals. Using of chemicals to control diseases leads to effluent remittance in the fish muscle which may cause side effects to the consumer. As this biological and eco-friendly approach is known to many of the farmers, awareness programmers should be done to popularize these herbal medicines. It is better to conduct result demonstration to disseminate this herbal approach rapidly among the farmers to control the diseases. Chemicals such as hydrogen peroxides, formalin, malachite green and others may offer a short term solution against parasitic problems. In sea cages, bathing fish is a labour intensive and costly operation. Although it usually brings temporary relief from parasites, the treatment also causes stress to the fish and increases the chances of future infections. Many of these chemicals are not environmentally sustainable and most of them are prohibited for use in aquaculture systems and banned in many countries. Authorities should review the current legislation regarding the use of herbal and natural remedies in aquaculture taking the above issues into consideration and allowing more flexibility in the use of herbal medicine in aquaculture.   The microbial analysis of the  Clarias gariepinus sampled from 10 selected farms in Abaranje (Ojo) and Ajara (Badagry) local government areas identified a number of 16 different pathogens (Staphylococcus aureus, Staphylococcus saprophyticus, E. coli, Bacillus subtilis, Bacillus megalopleural, Micrococcus luteus, Proteus mirabilis, Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas alcaligens, Proteus vulgaris, Bacillus mycoides, Pseudomonas putida, Klebsiella spp., Citrobacter cloaca and Pseudomonas aeruginosa) according to the biochemical tests carried in this study. The bacterial counts in the gills, intestine and skin specimen was above the permissible limit of WHO in fresh fish. This study showed that the fish samples contained a well load of pathogens which could be sourced from the fish's culture environment such as the feed, water, and human contact used in the culture practice.  The results of phytochemical screening of T. cacao parts especially the leaf and husk extracts showed that the plant has some bioactive compounds such as Alkaloid, Flavonoid, Tannin, Steroid, cardiac glycoside, Combined Anthraquinone, Saponin, Terpenoid, Phenol and Phlobatanin. This gave a promising potential use of this plant as a source of antibacterial agent against fish pathogens. Fish farmers can take advantage of this over the costlier synthetic drugs in their aquacultural practices, although the susceptibility of identified bacteria to synthetic drugs was higher compared to that of the T. cacao part extracts. The results of the MICs and MBs of T. cacao parts extracts against the identified organisms showed that the husk and the leaf extracts had better bacteriastatic and bacteriacidal effects than the root and bark. The bacteriostatic and bactericidal effects of synthetic and natural antimicrobials on these pathogens revealed the potential efficacies of Gentamycine, Streptomycin, Ciprofloxacine and Ofloxacine and T. cacao leaf and husk extracts as a remedy for diseases caused by a host of pathogens identified in this study. The MICs and MBCs of leaf and husk extracts showed a promising efficacy than some synthetic drugs especially Penicillin and Cotrimoxazole and can be more effective if well purified. It is however recommended that threobromine (not studied), which is the major constituent of cocoa (especially the cocoa beans) should be further investigated in order to actually get the accurate content and its efficacy in the treatment of some diseases of the African mudcatfish, C. gariepinus and therefore enhance profit maximization of the aquaculture industry. Furthermore, it is also recommended that further research on the in vivo experimentation of this study should be carried out to determine the actual efficacies of these synthetic drugs and T. cacao extracts on diseased or infected fish.