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Antibacterial Activities of Mango Leaf (Mangifera indica) Extracts on Catfish Clarias gariepinus (Burchell, 1822) Infected with Pseudomonas aeruginosa



F.A. Awe, A.M. Hammed, A.A. Akinyemi, O.O. Whenu and O.A. Olanloye
 
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ABSTRACT

Background and Objective: The side effects of antibiotics on fish and the consequences on the environment can be mitigated using plant extracts. This study was conducted to evaluate the efficacies of mango leaf extracts on pathogens isolated from African mud catfish Clarias gariepinus. Materials and Methods: Sub adult catfish collected from private fish farms in Odogbolu, Ogun state, were used for microbiological examination, biochemical tests, DNA extraction and molecular characterization of bacteria. The fish were infected with Pseudomonas aeruginosa with visible lesions on flesh and subsequent treatment with Mangifera indica leaf extracts. Qualitative and quantitative phytochemicals were later determined. Haematology and histological studies carried out and Complete Randomized Design experiment was used for the study. Dissolved oxygen, pH and temperature were monitored weekly on the experimental farm. Data were analyzed using Analysis of Variance. Results: Haematological profile of WBC (White Blood Cells), HGB (Haemoglobin), RBC (Red Blood Cells) and HCT (Haematocrit) after treatment with extracts improved significantly after post infection at 10 mg mL–1 with liver normalizes after 7 days of treatment with mango leaf extracts at 10.0 mg mL–1 concentration. There was also, atrophy or proliferation of the epithelial cells of secondary lamellae with fusion 7 days after treatment at 10 mg mL–1. Phytochemicals present were alkaloids, flavonoids, saponin, tannin, cardiac glycoside, phenol, anthraquinone, terpenoid, phylobatanin and steroid. Quantitatively alkaloids were 10.49±1.00a, flavonoids 2.93±1.00ab, saponin 3.52±1.00ab, tannin 3.52±1.00bcd and phenol 4.10±1.00abc. Conclusion: This study revealed that mango leaves extracts at 10 mg mL–1 contained sufficient phytochemicals that exerted antibacterial properties on the identified fish pathogens and reduce reliance on synthetic antibiotics. It is therefore suffice to say that plant extracts are a good source of novel products and can serve as alternative to antibiotics.

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  How to cite this article:

F.A. Awe, A.M. Hammed, A.A. Akinyemi, O.O. Whenu and O.A. Olanloye, 2019. Antibacterial Activities of Mango Leaf (Mangifera indica) Extracts on Catfish Clarias gariepinus (Burchell, 1822) Infected with Pseudomonas aeruginosa. Asian Journal of Agricultural Research, 13: 28-36.

DOI: 10.3923/ajar.2019.28.36

URL: https://scialert.net/abstract/?doi=ajar.2019.28.36
 
Received: December 04, 2018; Accepted: February 01, 2019; Published: April 24, 2019



INTRODUCTION

There was a remarkable increase in aquaculture productivity in the last decade with Africa recording the highest annual increase in the number of people engaged in fish farming followed by Asia, Latin America and the Caribbean respectively1. In Africa, culturing of catfish has surpassed tilapia from 2004 with Nigeria being on top of other nations1. The progressive dominance of this species in Nigeria and many African countries is due to its fast growth rate, high resistance to diseases and tolerance to environmental extremes and high consumer preference. Clarias gariepinus, Clarias anguillaris and Clarias buthupogon are cultured more than other species. Clarias sp., are generally strong fishes. Catfish accessory air breathing organs made them to adapt to different aquatic situation than other fish species2.

However, based on intensive fish culture, world aquaculture production is highly predisposed to disease occurrence that might affect part or outright loss of fish and farmers income3. The weakening of fish to disease outbreak and infection may be caused by overcrowding, periodic handling, high or sudden changes in temperature, poor water quality and poor nutritional status. Poor sanitation in an intensive aquaculture may be a source of introduction of pathogens resulting in fish death4,5.

In order to avoid economic losses related to disease outbreak, several veterinary drugs are commonly used in aquaculture to prevent or treat disease outbreaks. Antimicrobials and other veterinary drugs are administered regularly as additives in fish food or sometimes in baths and injections and are used as prophylactics (prevent diseases before they occur), therapeutics (treat sick animals) or growth promoters6. Nevertheless, due to awareness and various side effects of veterinary drugs on the environment, fish and man is a source of health safety challenge. For example, massive use of antibiotics have resulted in the development of resistant bacteria strains7,8 or the presence of residual antibiotics in the muscle of commercialized fish and thus has potential consequences on human health4,9.

Use of vaccines for treatment of fish against pathogenic infection occurrence is still in use at large for intensive aquaculture. However, commercial vaccines are too expensive for widespread use by fish producers and they have the downside that a single vaccine is effective against only one type of pathogens10,11. Disease management that is harmless and preventive is better than veterinary drug use because of their side effects on environment, man and fish coupled with low efficiency in terms of its working.

The alternative answer to reduce the outbreak of disease should be such that fish immunity and fitness be at optimum to avoid and face micro-organism invasion and block opportunistic bacteria from using a susceptible immune low fish as an avenue to invade12-14. Some of the proposed solutions are the use of natural products (plant extracts) or probiotics (beneficial microbial strains) in the culture of fish and shrimp15,16.

More so, there is an increasing interest in consuming organic and environmentally friendly food. Thereafter, use of natural and novel products in treatments could boost the consumption of aquaculture products due to the restriction on chemical products in aquaculture. Therefore, Mango plant Mangifera indica was used in the treatment of infected fish. In addition, in an in vitro agar diffusion method, mangiferin exhibited activities against 7 bacterial species, Bacillus pumilus, B. cereus, Staphylococcus aureus, S. citreus, Escherichia coli, Salmonella agona, Klebsiella pneumoniae, 1 yeast (Saccharomyces cerevisiae) and 4 fungi (Thermoascus aurantiacus, Trichoderma reesei, Aspergillus flavus and A. fumigatus)17.

MATERIALS AND METHODS

Experimental site: Catfish were raised from fingerlings to sub-adult and were inoculated with bacterium organism, Pseudomonas aeruginosa this were done at Lagos state University, Ojo, Lagos, Fish Ponds and hatchery farms.

Collection of plant parts: They were collected within Lagos State University, Ojo campus and taken to the Chemistry laboratory, Lagos State University for extraction while concentrated extracts for phytochemical screening was taken to Nigerian Institute of Science Laboratory Technology, Ibadan, Oyo state (NISLT) for analyses.

Plant part extraction: The plant parts such as the leaves and the barks were extracted using ethanol as solvent18.

Ethanolic extraction: Forty grams of the plant part were weighed using electronic sensitive analytical balance and was extracted by using Soxhlet extractor, 40 g of the part was placed inside the thimble and 150 mL of 95% ethanol was placed inside the round bottom flask and heat was applied at 60°C for 4 h. The ethanol inside the flask evaporated leaving the extract behind. Thereafter the extracts were air dried with vacuum pressure machine to reduce the volume to concentrated form. This was repeated for all the other plants. The extracts from the plants were now used for sensitivity tests on isolated and identified fish bacteria and for phytochemical screening.

Phytochemical screening: Qualitative19 and quantitative20 analyses of the plant parts were carried out at the NISLT, Ibadan, Oyo state, to determine the presence or absence of the different organic constituents in the Ethanolic extracts.

Qualitative and quantitative screening methods: Alkaloids, flavonoids, steroids, saponins, tannins, glycosides and cardiac glycosides were qualitatively determined following the process and procedure of Oloyede19, while Alkaloids, flavonoids, steroids and saponins were quantitatively analyzed using the procedure of Harborne20. Tannins and phytate were determined following the procedure of Pearson21, Oxalate22 and Cyanide23.

Identification of micro-organism: About 2 g of Flesh, Gills and Intestine were cut from each fish sample and were aseptically weighed and dropped in 10 mL sterile distilled water to release the bacteria organisms into it for serial dilutions. Thereafter, the isolated organisms on the nutrient agar slants were sub-cultured on nutrient agar plates to activate the pure culture. Characterization of the organism was based on colonial, morphological characteristic, biochemical tests and Molecular tests24-26.

In vivo experiment: This was carried out at LASU Fisheries Laboratory, healthy fish, numbering 40 were selected and acclimated to experimental plastic bowls (25 L capacity) at a stocking density of 10 fish per tank for the period of experimentation and done in triplicate. Each plastic tank was filled to 2/3 (15 L) of its volume with water supplied from the hatchery storage tanks. During acclimation period fishes were fed ad libitum with commercial diet twice daily. A completely randomized design was adopted for the experiment. The water in each tank was exchanged with fresh water daily, in the morning (07:30-08:00 h). Uneaten feed and faecal droppings were also removed daily by siphoning with minimal disturbance to the fishes. Fish mortality was monitored in each tank.

Bacterial culture: The bacterial culture was centrifuged at 1000 revolution speed for 10 min at 4°C. The supernatant was discarded and the isolated bacterial pellets were washed thrice and resuspended in distilled water at a pH of 7.4. The optical density of the solution was adjusted to 0.5 McFarland standards at 456 nm which corresponded to 3×108 CFU mL–1. About 10 mL each from the bacterial solution were taken using syringe and needle and dissolved directly into the water in the plastic bowls containing the experimental fish 10 in number and done in duplicate. The fish were carefully observed for any behavioural changes or mortality once they had been challenged.

Blood collection: The needle was gently pushed through the skin near the base of the caudal peduncle. After contact is made with the vertebral column, which is felt as firm resistance, the needle is directed slightly ventrally and lateral to the vertebral column, while syringe gently aspirates. It may be necessary to slowly rotate the needle before blood can be drawn. When one of the caudal vessels is entered artery or veins they run closely to one together, blood is aspirated and collected in vacutainer containing an anti-coagulant (EDTA). Same Fish where blood was aspirated were also sacrificed to obtain the gills and liver for histological analysis.

Estimation of blood parameters: The blood samples collected in vacutainer tubes were stored in the refrigerator before taken to Haematology Unit, University of Lagos Teaching Hospital, Idi-Araba, Lagos for haematological analyses using Auto Haematology Analyser, BC-3200, Shenzhen Mindray, Bio-Medical electronics CO., Ltd. The analyser was used for the quantitative determination of the following parameters. White blood cell or leukocyte (WBC), red blood cell or erythrocyte (RBC), haemoglobin concentration (HGB), mean corpuscular (erythrocyte) volume (MCV), mean cell (erythrocyte) heamoglobin (MCH), mean cell (erythrocyte) heamoglobin concentration (MCHC), red blood cell (erythrocyte) distribution width (RDW-CV), coefficient of variation (Red blood cell (erythrocyte) distribution width (RDW-SD), standard deviation hematocrit (HCT), platelet (PLT), Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), plateletcrit (PCT).

Histological examination: The liver and the gills were collected in the sample bottles and preserved with formalin and taken to Histology Unit at Department of Veterinary Microbiology, Federal University of Agriculture, Abeokuta for examination and reading.

Table 1:
Haematological parameters of C. gariepinus , control values, 7 days post infection and 7 days after treatment with antibiotics and Mango leaf extracts at different concentration against sub-adult C. gariepinus infected with Pseudomonas aeruginosa
Means of different superscript are significantly different at p<0.05 along the columns, ML: Mango leaf, GRP: Group, CTR: Control, PI: Post infection, T1: 25% concentration, T2: 50% concentration, T3: 75% concentration, T4: 100% concentration, ANT: Antibiotic 100% concentration, WBC: White blood cell or leukocyte, RBC: Red blood cell or erythrocyte, HGB: Haemoglobin concentration, MCV: Mean corpuscular (erythrocyte) volume, MCH: Mean cell (erythrocyte) haemoglobin, MCHC: Mean cell (erythrocyte) haemoglobin concentration, RBC: Red blood cell, RDW-CV: (Erythrocyte) distribution width coefficient of variation, RDW-SD: Red blood cell (erythrocyte) distribution width, HCT: Standard deviation hematocrit, PLT: Platelet, MPV: Mean platelet volume, PDW: Platelet distribution width, PCT: Plateletcrit

Table 2: Qualitative phytochemical analysis of the plant parts

RESULTS

The white blood cells, haemoglobin, red blood cells and haematocrit improved after treatment with mango plant extracts which were higher than control and post infection. Treatment at 10 mg mL–1 was higher than others and competes favourably with aquacryl antibiotics (Table 1). The phytochemicals present in mango extracts were alkaloids, flavonoids, saponin, tannin, cardiac glycoside, phenol, anthraquinone, terpenoid, phylobatanin and steroid (Table 2).

The white blood cells differentials indicated improvement from control, post infection and after treatment with mango extract and this were exhibited by the values of heterophils and lymphocytes especially at 10 mg mL–1 which also compared favourably with the antibiotic (Table 3). The quantitative values of phytochemicals indicated that alkaloid had a value of 10.49±1.00, while flavonoids was 2.93±1.00, saponin 3.52±1.00, tannin 3.52±1.00 and phenol was 4.10±1.00 (Table 4). Plate 1 and 2 revealed the histological examination of the liver and the gills. The liver was normal at 10 mg mL–1 after treatment with mango leaf extract while the gills showed atrophy of the secondary lamellae after treatment with extracts (Plate 1 and 2).

DISCUSSION

Mango leaf extracts competed very well with aquacryl antibiotic at 10 mg mL–1 on Pseudomonas aeruginosa. At this, the concentrations showed improved performance on the White Blood Cells (WBC), Haemoglobin (HGB), Red Blood Cells (RBC), Haematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin concentration (MCHC) when compared to post infection (Table 1). There was significant difference in their values and it is higher than control and post infection at (p<0.05) (Table 1). This result contradicts the findings of Awe et al.24 who reported a reduction in t he PCV of Nile Tilapia naturally infected with Aeromonas hydrophila. The WBC showed improvement after post infection over control and significant increase after treatment with plant extract and antibiotic, this could be due to ability of WBC to fight any foreign organism invading the fish system. This is in support of the findings of Hammed et al.25 and Sebastiao et al.27 who reported increased in the number of WBC in fish infected with different bacteria. George et al.28 also corroborated that White Blood Cells (WBC), neutrophils, monocytes, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentrations (MCHC) in fish exposed to the pesticides were significantly (p<0.05) higher than that of the control.

Plate1(a-g):
Histological section of liver cells to different concentration of mango leaf extracts on catfish inoculated with Pseudomonas aeruginosa, (a) Normal liver from catfish used as control experiment, (b) Diffuse vacuolar degeneration of liver post infection with Pseudomonas aeruginosa, (c) Mild vacuolar degeneration of liver 7 days after treatment with mango leaf at 2.5 mg mL–1 concentration against catfish inoculated with Pseudomonas aeruginosa, (d) Mild vacuolar degeneration of liver 7 days after treatment with mango leaf at 5.0 mg mL–1 concentration, (e) Mild vacuolar degeneration of liver 7 days after treatment with mango leaf at 7.5 mg mL–1 concentration, (f) Normal liver 7 days after treatment with mango leaf extracts at 10.0 mg mL–1 concentration against catfish inoculated with Pseudomonas aeruginosa and (g) Normal liver 7 days after treatment with aquacryl antibiotic at 10.0 mg mL–1 concentration against catfish inoculated with Pseudomonas aeruginosa, Haematoxylin and Eosin, x400

Table 3:
Total white blood cell count and differentials of C. gariepinus, control values, 7 days post infection and 7 days after treatment with antibiotics and Mango leaf extracts at different concentration against sub-adult C. gariepinus infected with Pseudomonas aeruginosa

Table 4: Quantitative phytochemical presence in plant parts

Plate 2(a-d):
Histological section of gills to different concentration of mango leaf extracts on catfish inoculated with Pseudomonas aeruginosa, (a) Normal gills of catfish used as control experiment, P: Primary lamellae, S: Secondary lamellae, (b) Proliferation of the epithelial cells of secondary lamellae with fusion 7 days after treatment with mango leaf extracts at 2.5 mg mL–1 concentration against catfish inoculated with Pseudomonas aeruginosa, (H and E, x 400), (c) Proliferation of the epithelial cells of secondary lamellae with fusion 7 days after treatment with mango leaf extracts at 10.0 mg mL–1 concentration (H and E, x 400), (d) Atrophy and fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with aquacryl antibiotic at 10 mg mL–1 concentration against catfish inoculated with Pseudomonas aeruginosa, Haematoxylin and Eosin, x 400

Findings from this work differ from what was reported by Rizkalla et al.29 who reported an insignificant reduction in the WBCs of infected Clarias gariepinus from the 1st week to the 3rd week. The histopathology of the liver was normal (Plate 1). The gills had atrophy and fusion and this could be due to inadequate dissolved oxygen content in the plastic bowl (Plate 2). Oxygen deficiency as a result of gill degeneration is the most common cause of the cellular degeneration in the liver. El-Murr et al.30 reported that fish exposed to fipronil showed pale gills and nervous manifestations beside congestion and hemorrhages of different internal organs. In addition, Khalesi et al.31 reported that the exposed fish showed fusion of gill lamellae, vessel dilatation, hyperaemia and hyperplasia of gill epithelial cells whereas muscle histology remained unchanged on common carp (Cyprinus carpio) exposure to sublethal concentrations of two non-essential heavy metals: cadmium (Cd: 8.4 mg L–1) and lead (Pb: 6.2 mg L–1) for 15 days to evaluate occurring biochemical and haematological effects. This finding is in line with findings of Eder and Gedigk32 on fish anatomy.

The gills (Plate 2), which participate in many important functions in the fish, such as respiration, osmoregulation and excretion, remain in close contact with the external environment and particularly sensitive to changes in the quality of the water are considered the primary target of the bacteria species. This is in agreement with the findings of Camargo and Martinez33 that studied Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream and Thophon et al.34 that examined the histopathological alterations of white seabass, pollutants on freshwater fish Lates calcarifer in acute and subchronic cadmium exposure. These pathological changes may be a reaction to bacteria inoculation and after treatment signs may be due to an adaptive response to prevent the entry of any harmful materials through the gill surface. The observed alterations like proliferation of the epithelial cells, partial fusion of some secondary lamellae and epithelial lifting are defense mechanisms, since, in general, these result in the increase of the distance between the external environment and the blood and thus serve as a barrier to the entrance of further materials. The findings of this work is in line with observation of Fernandes and Mazon35 on Environmental pollution and fish gill morphology, fish adaptation and Poleksic and Mitrovic-Tutundzic36 on sublethal and chronic effects of pollutants on freshwater fishes, fish gills as a monitor of sublethal and chronic effects of pollution.

The organ most associated with the detoxification, blood supply and biotransformation process is the liver (Plate 1) and it is one of the organs most affected by inoculated bacteria, plant extracts and antibiotics in the water. This is in line with findings of Camargo and Martinez33. The liver of some fish were normal others showed vacuolar degeneration in the hepatocytes, focal areas of necrosis, The vacuolization of hepatocytes might indicate an imbalance between the rate of synthesis of substances in the parenchyma cells and the rate of their release into the circulation system. This was also corroborated by Gingerich37 on Hepatic Toxicology of Fishes. However, Soufy et al.38 in their work biochemical and pathological investigations on monosex Tilapia following chronic exposure to carbofuran pesticides asserted that these changes may be attributed to direct toxic effects of pollutants on hepatocytes, since the liver is the site of detoxification of all types of toxins and chemicals.

The mango leaf extracts provides an alternative to the use of antibiotics in the control and treatment of fish diseases. Mango leaf is easily available, cheap to source, the method of extraction is simple, the plants phytochemicals are easily biodegradable, it is natural and not synthetic, their use with appropriate trials are not harmful in terms of dosage. Its use had been long with man in human medicine as an alternative to orthodox medicine. But in Fisheries and Aquaculture, its use is being developed systematically and is gradually being exploited. Mango leaves provides an alternative in areas of the active ingredients and phytochemicals that actually supports the immune system of the organisms and inhibit the growth of bacteria that cause damage to the fish growth. The mango leaf has little or no side effects on the fish, man and the environment. These properties when compared with synthetic antibiotics that are associated with side effects necessitated the use of an alternative product that can perform the same function as the antibiotics. The novelty of this product is similar to other terrestrial and mangrove plants being developed. From literatures, terrestrial plants have been studied and utilized widely over 100 years for the effect of a given agent of their extracts. Algae are also considered to be a rich source of bioactive molecules but only current researches have started to find out their chemical composition and to assess their prospective bioactivities, essentially against human pathogens39-41. Nevertheless, they present an enormous potential to be used in aquaculture as immunostimulant and preventing disease outbreaks as they present a wide range of original bioactive molecules42-44. The leaf seem to possess multiple strong bioactivities such as antibacterial and antihelminthic45,46 and the utilization of local algae can avoid the introduction of exogenous molecules in the marine environment.

CONCLUSION

Mango leaf is a good potential control of fish bacteria and this should be fully utilized, at 10 mg mL–1 it was able to successfully control the bacteria and the fish recovered fully.

SIGNIFICANCE STATEMENT

The importance of this study could be seen in the area of exploiting natural plants coupled with better way of extraction, high quality phytochemicals and improving the immunity of the fish, thereby exerting action against diseases.

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