Abstract: Background and Objective: The aftermath of heavy usage of antibiotics could be harmful on the fish and aquatic environment by creating drug resistant bacteria and transferable resistance genes in fish pathogens. This study was conducted to evaluate the efficacies of whole lemon grass plant extracts on pathogens isolated from African mud catfish and examined the histological and haematological indices of sub-adult African mud catfish collected from private fish farms in Odogbolu, Ogun state, Nigeria. Microbial examinations, biochemical tests, DNA extraction and molecular characterization of bacteria were conducted. Materials and Methods: About 150 experimental farm catfish were infected with bacteria (Aeromonas veronii) with visible lesions on flesh and was subsequent treatment with lemon grass (Cymbopogon citratus) leaf extracts at concentrations of 2.5, 5.0, 7.5 and 10 mg g–1 and aquaceryl antibiotic 10 mg g–1. Cymbopogon citratus was extracted using ethanol, while qualitative and quantitative phytochemical screening were carried out after being concentrated. Fish samples were analyzed for haematological and histological profiles. Complete randomised design experiment was used for this study. Dissolved oxygen, pH and temperature were monitored weekly on the experimental farm. Results: Data were analyzed using analysis of variance. Phytochemicals revealed the presence of alkaloids, tannin, saponin, flavonoids and phenol with quantitative values of alkaloid at 6.78±1.00 mg g–1, flavonoids 3.22±1.00 mg g–1, saponin, tannin and phenol had 0.42±1.00, 1.68±1.00, 4.29±1.00 mg g–1, respectively. The haematological profile of fish post inoculation showed that White Blood Cells (WBC) increased significantly (p<0.05) compared with control. After treatment with plant extracts, the blood parameters including WBC, Haemoglobin (HGB), Red Blood Cells (RBC), Haematocrit (HCT), Mean Corpuscular Volume (MCV) and Mean plateletcrit Volume (MPV) increased significantly (p<0.05) at 2.5 mg g–1. The liver and the gills were normal after treatment with extract. Conclusion: This study showed that whole lemon grass plant improved the haematological and histological profile of inoculated fish.
INTRODUCTION
Histological and haematological techniques in fish culturing have been growing in relevance for toxicological research, environmental monitoring and fish health conditions. Many studies have been conducted on haematological changes of pesticides in the fish such as Das and Mukherjee1, Adebayo et al.2 and Patnaik and Patra3. Samprath et al.4 was of the opinion that there is a possibility that studies on fish blood might reveal conditions within the body of the fish long before there is any outward manifestation of disease. Railo et al.5 reported that the blood parameters of diagnostic importance are erythrocyte and leucocytes counts, heamoglobin, haematocrit and leucocyte differential counts would readily respond to incidental factor such as physical stress and environmental stress due to water contaminants.
Research works have revealed that when the water quality is affected by toxicants, any physiological changes would be revealed in the values of one or more of the haematological parameters6,7. Blood cells responses are important indicators of changes in the internal and/or external environment of animals. In fish, exposure to chemical pollutants can induce either increases or decreases in haematological levels. Their changes depend on fish species, age, the cycle of the sexual maturity of spawners and diseases8,9.
Fish live in very intimate contact with their environment and are therefore very susceptible to physical and chemical changes which may be reflected in their blood components10. Blood tissues essentially reflect physical and chemical changes occurring in organism; therefore, detailed information can be obtained on general metabolism and physiological status of fish in different groups of age and habitat. Early diagnosis is also possible when evaluating haematological data, particularly blood parameters8,9. Furthermore, it should be noted that haematological indices are of different sensitivity to various environmental factors and chemicals11. Previous haematological study of nutritional effects12, infectious diseases13 and pollutants14 brought knowledge that erythrocytes are a major and reliable indicator of various sources of stress15,16.
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 immuned low fish as an avenue to invade17-19. Some of the proposed solutions are the use of natural products (plant extracts) or probiotics (beneficial microbial strains) in the culture of fish and shrimp20,21.
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, whole lemon grass plant Cymbopogon citratus was used in the treatment of infected fish of bacteria Aeromonas veronii.
MATERIALS AND METHODS
Experimental site: Catfish were raised from fingerlings to sub-adult and were inoculated with bacterium organism, Aeromonas veronii this were done at Lagos State University, Ojo, Lagos, Nigeria Fish Ponds and hatchery farms.
Collection of plant parts: Whole lemon grass plant was collected from 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 solvent22.
Ethanolic extraction: Forty gram 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. The extracts from the plants were now used for sensitivity tests on isolated and identified fish bacteria and for phytochemical screening.
Phytochemical screening: Qualitative23 and quantitative24 analyses of the plant parts were carried out to determine the presence or absence of the different organic constituents in the ethanolic extracts was out at the NISLT, Ibadan, Oyo state.
Qualitative and quantitative screening methods: Alkaloids, flavonoids, steroids, saponin, tannins, glycosides and cardiac glycosides were qualitatively determined following the process and procedure of Oloyede23, while Alkaloids, flavonoids, steroids and saponins were quantitatively analyzed using the procedure of Harborne 24. Tannins and phytate were determined following the procedure of Pearson25, Oxalate26 and Cyanide27.
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 tests.
In vivo experiment: This was carried out at LASU Fisheries Laboratory, healthy fish, numbering 150 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 standard 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 triplicate. 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 analyses.
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 analyzer 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.
RESULTS
Table 1 indicates that the highest value of 6.78 was the quantitative value found in alkaloids while the least value of 0.42 was found in saponin. Phenol values closely followed alkaloid and next to it is flavonoids and tannin.
| Table 1: | Quantitative phytochemical presence in plant parts |
| Table 2: | Qualitative phytochemical analysis of the plant parts |
| +: Presence of the phytochemical, -: Absence of phytochemical | |
| Table 3: | Haematological parameters of C. gariepinus, control values, 7 days post infection and 7 days after treatment with anti-biotics and whole lemon plant extracts at different concentrations against sub-adult
C. gariepinus infected with Aeromonas veronii |
Mean values with different superscript along the column are significantly different at p<0.05, 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 |
|
The plant extracts indicated the absence of cardiac glycoside, anthraquinone, terpenoid, phylobatanin and steroid. However, the plant extracts indicated the presence of alkaloid, flavonoids, saponin, tannin and phenol (Table 2).
Table 2 shows the haematological parameters of C. gariepinus, control values, 7 days post infection and 7 days after treatment with antibiotics and whole lemon plant extracts at different concentrations against sub-adult C. gariepinus infected with Aeromonas veronii showed that at T1: 2.5 mg mL–1 concentration, the blood profile such as WBC: White blood cell or leukocyte, HGB: Haemoglobin concentration, RBC: Red blood cell or erythrocyte, HGB: Haemoglobin concentration, HCT: Hematocrit increased significantly followed by T3 at 7.5 mg mL–1 concentration, with the least at T2 which is 5.0 mg mL–1 concentration of the extract (Table 3). The white blood cells differentials also improved significantly from post infection and after treatment with plant extract.
DISCUSSION
Table 1 revealed the quantitative phytochemical of whole lemon grass plant with different concentration in values of alkaloids, flavonoids, saponin, tannin and phenol. With quantitative values of alkaloid at 6.78±1.00 mg g–1, flavonoids 3.22±1.00 mg g–1, saponin, tannin and phenol had 0.42±1.00, 1.68±1.00 and 4.29±1.00 mg g–1, respectively.
Table 2 shows the qualitative phytochemical which indicated the presence of alkaloids, flavonoids, saponin, tannin and phenol while cardiac glycoside, anthraquinone, terpenoids, phylobatanin and steroid were absent. Which indicated flavonoids, carbohydrates, tannins, alkaloids, steroids and phytosteriods tested were all detected, except glycosides and phenols that were absent in the acetone and chloroform leaf extracts. However, my result indicated presence of phenol as compared to his work which reported absence, also steroid was absent in this work but present in his study.
Table 3 shows haematological parameters of C. gariepinus, control values, 7 days post infection and 7 days after treatment with antibiotics and whole lemon plant extracts at different concentrations against sub-adult C. gariepinus infected with Aeromonas veronii. The Haemoglobin level decreased from 8.7 g dL–1 in the control to 8.2 g dL–1 in post infection PI. However, it decreases in treatment ANT to 8.0 and at T4 is 8.7 and further increased to 10.7 g dL–1 in the treatment containing the concentrations of T1 and T3, respectively.
The PCV or Haematocrit HCT of C. gariepinus showed a significant increase from 26% in the control and post infection to 38.70% in the fish treated to the highest concentration (treatment 4). Also, it increased from 35-37% in treatments 3 and 1, respectively and also increased from 27% in ANT antibiotic to 30.20 in treatment 2.
This increase could be as a result of immunological status of the fish used as this could vary among species of the same strain, age and environmental condition. The significant increases in PCV levels were in variance with observation of Fafioye28. He noted significant decreases in the blood cell parameter between the control and experimental specimens of both C. gariepinus and O. niloticus on exposure to sub-lethal concentrations of the aqueous and ethanolic extracts of Rafia vinifera and Parkia biglobosa. The decreased value in PCV may be due to anaemia and haemodilution or haemolysis of RBC as was also reported by Fafioye28. The Haemoglobin level decreased from 8.7 g dL–1 in the control to 8.2 g dL–1 in post infection PI. However, it decreases in treatment ANT to 8.0 and at T4 is 8.7 and further increased to 10.7 g dL–1 in the treatment containing the concentrations of T1 and T3, respectively. This was in line with observation of Fafioye28. He noted significant decreases in these blood cell parameters. Reduced RBC level may also be due to swelling of erythrocyte or haemolysis which may be due to increased protein-carbon IV oxide in the blood. It may also be as a result of anaemia which is possibly due to haemodilution resulting from impaired osmoregulation across the gill epithelium as reported by Wedemeyer et al.29 and Svoboda et al.30.
The white blood cell increased significantly from 9.5 109 L–1 in the control experiment to 17.0 109 L–1 in the lowest concentration of T1 which is 2.5 mg mL–1. The RBC value of C. gariepinus increased significantly from 1.76 106 μL in the control to 2.7 106 μL in treatment T1 containing the lowest concentration and T3. There was an increase in values from treatment T2, T4 and ANT from 2.13 106 μL, 2.2 106 μL and 2.0 106 μL. There was a significant increase (p<0.05) in the mean corpuscular volume from 146 fl in the control to 175 fl in treatment T4 and significant decrease (p<0.05) in mean corpuscular heamoglobin from 51.8 pg in the control to 39.30 pg in treatment T3.
Though, the lymphocyte level increased significantly in the experiment so as to increase the resistance of the fish by boosting the immunological actions of the white blood cells to the effect of extract. This is in agreement with Agrawal and Srivastava31 that, increase in White blood cell count has been attributed to several factors such as increase in thrombocytes, lymphocyte or squeezing of leucocytes in peripheral blood. Also it is in consonance with the report of Santhakumar et al.32 that leucocytes are involved in the regulation of immunological function of body. An increase in white blood cell indices such as lymphocyte thus occurs as a protective response to stress33.
The significant increase in values of those parameters recorded after treatment with the extracts at different concentration could be attributed to the stress-mediated condition which prompts the release of new erythrocytes from the erythropoietic tissue to improve the oxygen carrying capacity of exposed fish blood with resultant higher values of erythrocyte count and heamoglobin concentration. This was in agreement with the report given by Fafioye28 and Saeed et al.34.
The summary of histological changes observed in the gills and liver of Clarias gariepinus subjected to different concentration of whole lemon grass plant extract as presented in Fig. 1 and 2. There were no significant changes observed in the test fish in the control experiment. The gills of fish in the control set-up consist of a well arranged primary and secondary lamellae or filaments with slender branching projection lined by highly vascularized simple epithelium. Whereas the gills of fish treated at different concentration varied from normal 7 days after treatment with whole lemon grass extract at 2.5 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii to partial fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with whole lemon grass extract at 5.0 mg mL–1 concentration. Atrophy with fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with whole lemon grass at 7.5 mg mL–1 concentration. Exfoliation of epithelial cells of primary filament with atrophy of epithelial cells of secondary lamellae 7 days after treatment with whole lemon grass extracts at 10.0 mg mL–1 concentration. Fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with aquaceryl anti-biotic at 10.0 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii. This agreed with report given by Adeogun35,36 that different toxicant produced remarkable lesions on gills of fish such as scattered gill filaments, thickened gill filament, inflammation of lamellae and necrosis in higher concentration.
The liver was normal to diffuse vacuolar degeneration of the liver cells post inoculation with Aeromonas veronii. Normal liver were seen 7 days after treatment with whole lemon grass plant at 2.5 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii.
| Fig. 1(a-g): | Histological section of liver cells to different concentration of whole lemon grass extracts on catfish inoculated with Aeromonas veronii, (a) Normal liver from catfish used as control experiment, (b) Diffuse vacuolar degeneration of the liver cells post inoculation with Aeromonas veronii, (c) Normal liver 7 days after treatment with whole lemon grass plant at 2.5 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii, (d) Normal liver 7 days after treatment with whole lemon grass plant at 5.0 mg mL–1 concentration, (e) Moderate diffuse vacuolar degeneration of cells 7 days after treatment with whole lemon grass plant at 7.5 mg mL–1 concentration, (f) Moderate diffuse vacuolar degeneration of cells 7 days after treatment with whole lemon grass plant at 10.0 mg mL–1 concentration and (g) Normal liver 7 days after treatment with aquaceryl anti-biotic at 10 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii, Haematoxylin and Eosin, x400 |
Normal liver 7 days after treatment with whole lemon grass plant at 5.0 mg mL–1 concentration. There was moderate diffuse vacuolar degeneration of cells 7 days after treatment with whole lemon grass plant at 7.5 mg mL–1 concentration. Moderate diffuse vacuolar degeneration of cells 7 days after treatment with whole lemon grass plant at 10.0 mg mL–1 concentration. Normal liver 7 days after treatment with aquaceryl anti-biotic at 10 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii. Vacuolation of some portions of the liver tissue observed probably resulted from the excessive work required by the fish to get rid of the bacteria and extract from its body during the process of detoxification by the liver. The inability of fish to regenerate new liver cells may also have led to it. The phytochemicals present in the plant exert good anti-bacterial properties in the healing of the fish diseases.
| Fig. 2(a-g): | Histological section of gills to different concentration of whole lemon grass extracts on catfish inoculated with Aeromonas veronii (Haematoxylin and Eosin), (a) Normal gill of catfish used as control experiment, P: Primary lamellae, S: Secondary lamellae, (b) Necrosis, atrophy and fusion of epithelial cells of secondary lamellae post inoculation with Aeromonas veronii, (c) Normal gill 7 days after treatment with whole lemon grass extract at 2.5 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii (H and E, x 400), (d) Partial fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with whole lemon grass extract at 5.0 mg mL–1 concentration, (e) Severe atrophy with fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with whole lemon grass at 7.5 mg mL–1 concentration, (f) Exfoliation of epithelial cells of primary filament with atrophy of epithelial cells of secondary lamellae 7 days after treatment with whole lemon grass extracts at 10.0 mg mL–1 concentration and (g) Fusion of epithelial cells of secondary lamellae of the gills 7 days after treatment with aquaceryl antibiotic at 10.0 mg mL–1 concentration on catfish Clarias gariepinus inoculated with Aeromonas veronii Haematoxylin and Eosin, x 400 |
CONCLUSION
About 2.5 mg mL–1 of whole lemon grass plant extracts performed the needed healing properties required for the fish to be normal. This healing property could be found in the phytochemical properties of the plant which exerts its anti-bacterial potential on the infected disease thereby increasing the fish immunity.
From this study, the use of whole lemon grass plant extract could be used as an alternative to aquaceryl anti-biotics based on its performances which reflected in the improved haematological profile and histological profile of catfish.
SIGNIFICANCE STATEMENT
Lemon grass (Cymbopogon citratus) whole plant extracts provides an alternative to the use of anti-biotics in the control and treatment of fish diseases. Available everywhere, cheap to source with simple method of extraction, 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. Lemon grass (Cymbopogon citratus) is a tufted perennial C4 grasses with numerous stiff stems arising from a short, rhizomatous rootstock as with citrus flavour and can be dried and powdered or used fresh. 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 fish diseases.