Antibiotics resistance of Vibrio spp. isolated from diseased seabass and tilapia in cage culture

Vibriosis has become one of the most important bacterial diseases in marine cultured organisms in recent years. This study was focusing on isolation and identification of Vibrio spp. isolated from diseased seabass (Lates calcarifer), tilapia (Oreochromis niloticus) and seawater from Sri Tujuh lagoon in East Coast of Malaysia; also determination of antibiotic resistance patterns among Vibrio spp. Vibrio species isolated from diseased seabass in Pantai Sri Tujuh, Tumpat, Kelantan were screened for their antibiotic sensitivity patterns by Kirby-Bauer method. A total of 47 isolates belonging to three different species were identified which are V. parahaemolyticus, V. vulnificus, and V. alginolyticus. In this study, high incidence of erythromycin, chloramphenicol and sulfamethoxazole resistance was observed among the Vibrio isolates, whereas all isolates were susceptible to oxytetracycline. Vibrio isolates were 96% resistant to one or more different classes of antibiotic, and 17 different resistance patterns were identified. The MAR index of 0.4 indicating the Vibrio spp. in these farmed fish might have been indiscriminately and continuously exposed to those antibiotics during culturing stages of the fish. This study showed that multidrug-resistant Vibrio spp. were common in diseased seabass and tilapia cultured at Sri Tujuh lagoon. These essential findings suggested involvement of seafood in transmission of these pathogen to human. In addition, oxytetracycline can be used as a treatment to combat vibriosis in diseased seabass and tilapia

The effects of Vitex trifolia, Strobilanthes crispus and Aloe vera herbal-mixed dietary supplementation on growth performance and disease resistance in red hybrid tilapia (Oreochromis sp.)

Herbs and herb mixtures have been used as a growth promotant in livestock and aquaculture production. The objective of the study was to evaluate the effect of a dietary herbal mix comprised of Vitex trifolia (VTE), Strobilanthes crispus (SCE) and Aloe vera (AVE) on the growth performance, disease resistance and histology of Oreochromis sp. for 60 days. The fishes were divided into i) control, infected fish, fed with normal diet and infected fishes treated with different herbal-mixed supplementation diets of ii) VTE and SCE iii) SCE and AVE iv) AVE and VTE. All experimental groups were challenged using with Streptococcus agalactiae (1 × 107 cfu/mL) via intraperitoneal route on day 46. On day 46th (pre-challenge) and 60th (post-challenge), five fish were randomly chosen from each tank for each experimental and control groups to blood collection. The cumulative mortality and survival rate were assessed every day. Tissues from kidney, liver and spleen were examined. The fish supplemented with herbal-mix with the combination of VTE and SCE and AVE and VTE showed improved growth performance. For haematological assays, RBC, Hb, and WBC were higher (P<0.05) in fish supplemented with these herb mix, while the alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were significantly affected by mixed-herbal supplementation. Histopathological examination of the organs revealed no negative effects in tissues. In conclusion, this study suggested that methanolic extracts of herbal mix composed of V. trifolia, S. crispus and A. vera extracts were effective as growth promoters and bacterial disease treatment in Oreochromis sp. when supplemented in daily diet

The effects of dietary supplementation of methanolic extracts of herbal medicine on haematological variable of red hybrid tilapia (Oreochromis sp.)

The most common strategy to treat in aquaculture disease is the use of antibiotics, however, such utilization has been accounted to have antagonistic impacts like accumulation of drugs in tissues, development of drug resistance and immunosuppression. One of the most promising methods of controlling diseases in aquaculture is strengthening the defence mechanisms through therapeutic administration. Vitex trifolia, Strobilanthes crispus, and Aloe vera have been reported to have better antimicrobial activity in vitro against Streptococcus agalactiae. However, there is no report on the application of the extracts on the treatment of Oreochromis sp. The objective of the study was to assess the effectiveness of diet supplementation of selected plant extract for 14 days as disease treatment. In red blood cell (RBC), haemoglobin (Hb), mean corpuscular volume (MCV), white blood cell (WBC), alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) showed significant differences between treatment and control. RBC, Hb and WBC levels of the infected group were significantly higher (P<0.05) than those of the control group indicating improve defence system in the fish fed with V. trifolia, S. crispus, and A. vera. These results suggested that of methanolic mixed herbal to applying S. agalactiae infected Oreochromis sp had a synergistic restorative effect on the haematological variables

Studies on pathogenicity effect of Aeromonas hydrophila infection in juvenile red hybrid tilapia Oreochromis sp.

This study was conducted to evaluate the ability of motile Aeromonas species (MAS) to induce clinical symptoms and some pathological changes in juvenile red hybrid tilapia. A standard dose of infection was selected based on predetermined LD50. Infected fish were observed for any clinical sign and symptom for 96 hours. Samples of liver, spleen and kidney were collected for histopatological changes due to Aeromonas hydrophila infection. Clinical signs of fish included abnormal swimming behaviour and loss of balance. Most of infected fish suffered from haemorrhagic eyes and lesions on the body surface. Some of the them also developed bilateral exophthalmia. Histopathological changes were observed in kidney, liver and spleen. The infected liver showed severe congestion of hepatic veins and vacuoles formation while anterior kidney manifested degeneration of excretory tubules and glomeruli followed by severe haemorrhages and hyaline droplets degeneration. Spleen on the other hand showed tissues degeneration and vacuoles formation. All targated tissues also showed deposition of haemosiderin pigments followed by proliferation of melanomacrphage centres. Development of such symptoms was associated with the infection with Aeromonas hydrophila. The current study has shown that MAS could serve as the primary cause of severe infection not only in red hybrid tilapia but may also infected other freshwater fish species.The results of histological analysis of various tissues indicates a direct correlation between disease infection and histopathological disorders observed in the tissues

Histopathological evaluation and molecular detection of natural iridovirus infection in cultured grouper fish in Malaysia

Iridovirus is the causative agent of grouper iridovirus infection (GIV), which causes severe epizootics resulting in large-scale mortalities and huge economic losses in cultured and marine fishes worldwide. The current study evaluated the gross and histopathological lesions, and molecular detection of GIV in naturally infected grouper farms in Malaysia. A total of 150 moribund fish showing different clinical signs, presented to the Aquatic unit were used for this study. The fish were necropsied and visceral organs (spleen, kidney, heart, liver, intestine, brain, eyes, gills, and some parts of skin with ulcer) were collected and fixed in 10 % buffered formalin for histopathological processing and evaluation. Molecular detection of the virus was done by polymerase chain reaction (PCR). Grossly, the groupers had ulceration on the operculum and close to the caudal fin with hemorrhages on the margin of the caudal fin. In some fish, there was necrosis of the caudal fin, sloughing of the epidermis, dermal ulceration and popeyes Histological sections showed large basophilic cytoplasmic inclusions and vacuolations in the cytoplasm of hepatocytes. Basophilic or eosinophilic enlarged cells with the presence of mononuclear cellular infiltrations were seen in the kidney, liver, eye and gills, which is the distinctive feature of this disease. PCR detection showed positive amplification from 27 groupers. Based on the gross, histopathological and molecular detection GIV infection was established

Assessment of therapeutic and antimocrobial effects of local plants on bacterial diseases in red hybrid tilapia (Oreochromis sp.)

Currently, aquaculture is regarded as one of the potential strategic industries for Malaysia. The percentage on its production increases year by year. The growth are obvious due to obstacle in continuous dependent on finite stocks of protein source from wild-caught fish which is shown to be over-exploited or near to be depletion. One of main problem in aquaculture is outbreaks of infectious diseases which has incurred big losses to the industry. Herbal medicine has been used in most part of the world as an alternative medicine for human being and domestic animals. The plant compounds have been shown to be beneficial and rich in a wide variety of phytonutrients, and thus used as chemotherapeutics and feed additives. Hence, the general objectives of this study were to determine the effect of herbal supplementation on the growth performance, disease resistant, disease treatment in Red hybrid tilapia (Oreochromis sp.). Six extracts of Vitex trifolia, Aloe vera, Strobilanthes crispus, Clinacanthus nutans, Pereskia grandifolia and Peperomia pellucida were determined for phytochemical properties and their antibacterial activities against common freshwater pathogens i.e. Streptococcus agalactiae, Aeromonas hydrophila and Enterobacter cloacae. Qualitatively screening of phytochemical properties in herbs were determined for flavonoids, tannins, saponin, alkaloids steroid and glycoside. The results showed presence of flavonoid in all plant extracts. For the antimicrobial activity, the aqueous and methanolic extracts were tested by using disk diffusion method. Antimicrobial assay of methanolic extracts (25 to 100 mg/mL) showed effectiveness against the pathogenic bacteria. Comparatively, all aqueous extracts did not show any antimicrobial activity. Moderate antibacterial activity was shown by the methanolic extracts of V. trifolia, A. vera and S. crispus while weak antimicrobial activity was shown by C. nutans, P. grandifolia and P. pellucida. From the antimicrobial result, three potential plant extracts namely V. trifolia (VTE), S. crispus (SCE) and A. vera (AVE) were test for toxicity and feeding experiment. The cytotoxicity test using Artemia salina (45.9 - 66.7 μg/mL) and BF-2 fish cell line (85.0 - 120.50 μg/mL). The median lethal dose (LD50) value in Oreochromis sp. injected intraperitoneal were equivalent to 2.67 X 107 CFU/mL (S. agalactiae), 3.10 X 106 CFU/mL (E. cloacae) and 3.75 X 105 CFU/mL (A. hydrophila). Oreochromis sp. were fed with diet supplemented with VTE, SCE and AVE at 2g/kg, 5g/kg, 7g/kg and 9g/kg for 45 days, followed by challenged with 0.1 ml of 107 CFU/ml S. agalactiae. The results showed Oreochromis sp. supplemented with VTE-7 was the best growth promoter among treatments. It showed 57.3% in body weight gain with 100% survivability. For haematological the value of Hb after challenged with S. agalactiae showed significant different (P<0.05) compared to control except in SCE-2 and SCE-7. While, in WBC value showed significant different (P<0.05) except in AVE-5 and AVE-7. In MCV value showed significant different except in SCE-5, AVE-9 and in MCHC value showed significant except in VTE-2 and SCE-5. In biochemical parameters of blood, there was significant different in ALT value observed in fish fed with VTE-2, VTE-5, VTE- 7, SCE-2, SCE-5, SCE-7, AVE-5 and AVE-7 compared to control (infected). In AST value showed significant different (P<0.05) in VTE-5, VTE-7, SCE-9, AVE- 2 and AVE-7 compared to control (infected). Fish fed with SCE-5 was the best for the disease resistant showed the highest level of WBC value indicated increase immune responses and the ability of the fish to fight S. agalactiae. In histology examination of different selected herbal extracts showed mild degeneration in liver and kidney tissues. To determine the growth performance and disease resistant of Oreochromis sp. supplemented with herbal-mixed of the selected herbal extracts, the fish were fed with diet containing mixtures of AVE&VTE, VTE&SCE, SCE&AVE. Herbal mixed of AVE&VTE showed the most significant increased as compared with others. For SGR, only AVE&VTE showed promising result with significant higher (P< 0.05) than control. Fish survival after the disease challenge showed significant difference (P< 0.05) compared to control. In haematological assays, the post-challenged results showed the significant increased (P<0.05) of RBC, Hb and PCV values. The WBC value in pre- and post-challenged showed no significant different (P>0.05) of all treatments compared to control. As for MCHC value reading with the mixed-herbals, AVE&SCE was the only treatment which revealed no significant difference (P>0.05) in pre-challenged and showed decreased significant difference (P<0.05) in post-challenged in all treatments. On the other hand, MCV and plasma protein values did not show any significant difference (P>0.05) as compared to control. The biochemical parameters of ALT value and AST value were significantly affected by mixed-herbal supplementation. However in the post-challenged, the ALP value showed no significant difference compared to control. Histopathological changes, which included necrosis and haemorrhage in the kidney, liver and hepatopancreas tissues could be due to the cytotoxin produced by the bacteria. Also, a study was carried out to evaluate the effects of dietary supplementation of methanol extracts of V. trifolia, S. crispus and A. vera Oreochromis sp. as disease treatment. Results showed that RBC levels were significantly increased (P<0.05) in VTE-2, SCE-2, SCE-5, SCE-9, AVE-5, AVE-7 and control (OTC) as compared to control (infected). While, Hb value showed significant increased in VTE-2, SCE-5, SCE-9, in contrast to significant decreased in AVE-7 and AVE- 9 as compared to control. For PCV value showed significant (P<0.05) increased in all treatments included control (OTC) as compared to control (infected). On the other hand, in MCHC value no significant different for SCE-5. Plasma protein value showed significant increased (P<0.05) after supplementation of VTE-2, VTE-5, SCE-2, SCE-5, SCE-9, AVE-2, AVE-5, AVE-9. For ALT and AST values, they showed significant decrease in all treatments including control (uninfected) and control (OTC) (P<0.05). Also in ALP value, it showed significant decreased in all treatments including the control (OTC). The survivability of fish showed increase after fed with the herbal supplementation (P< 0.05) in VTE-5, VTE-7, AVE-2, AVE-5 and AVE-9 as compared to control (infected). All the haematological measurements in Oreochromis sp. that were infected with S. agalactiae with supplemented with VTE, SCE and AVE showed similar values compared with the control and the treatment with antibiotic. Its indicated the fish that were infected with S. agalactiae able to restore their haematological indices to near normal values. The results were agreeable with literatures which reported the numerous efficacies of the use of herbal in aquaculture to control various bacterial pathogens. Also from current research findings, suggested that mixed herbal of methanolic-extract given to S. agalactiae infected Oreochromis sp. had a synergistic restorative effect on the hematological variables. In conclusion, current study suggested that V. trifolia, S. crispus and A. vera extracts and mixed herbal were effective as growth promoters and bacterial disease treatment in Oreochromis sp. It also proved the potential of the selected herbal plant as aquaculture therapeutant as they were environmental friendly and it showed negligible side effect in the intended fish

Screening of phytochemical properties and antimicrobial activity of Malaysian medicinal plants against aquatic bacteria

Aims: Quantitative screening of phytochemical properties and antimicrobial activities were done on some plants of importance in human medicine as traditional herbs to treat fish diseases in Malaysia. Six extracts of Vitex trifolia, Aloe vera, Strobilanthes crispus, Clinacanthus nutans, Pereskia grandifolia and Peperomia pellucida were determined for phytochemical properties and their antibacterial activities against common freshwater pathogens i.e. Streptococcus agalactiae, Aeromonas hydrophila and Enterobacter cloacae. Methodology and results: Qualitative screening of phytochemical properties in herbs were determined using conversional method for flavonoids, tannins, saponin, alkaloids steroid and glycoside The results showed flavonoid was presence in all plant extracts. For the antimicrobial activity, the aqueous and methanolic extracts were tested by using disk diffusion method. Antimicrobial assay of methanolic crude extracts (25 to 100 mg/mL) showed effectiveness against the pathogenic bacteria. Comparatively, all aqueous extracts did not show any antimicrobial activity. Strong antibacterial activity was shown by the methanolic extracts of V. trifolia, A. vera and S. crispus while moderate antimicrobial activity was shown by C. nutans, P. grandifolia and P. pellucida. Conclusion, significance and impact study: The current results indicated that the studied plants might indeed be potential sources of natural antimicrobial agents to control fish diseases