An in-vivo experimental evaluation of the efficacy of fish-derived antimicrobial peptides against multidrug-resistant Pseudomonas aeruginosa.

Introduction: due to the fact that antimicrobial peptides antimicrobial peptides (AMPs) from climbing perch have not been fully explored for their antimicrobial potency, this investigation was undertaken to explore that possibility. Methods: antimicrobial peptides (AMPs) from the mucous secretion of climbing perch were obtained and an in-vivo analysis was conducted using mice. Results: the results showed inhibitory effects on multidrug-resistant multidrug-resistant Pseudomonas aeruginosa with reduced mortality from 100% among the non-treated group to 25%. Similarly, the level of serum transaminase enzymes (AST and ALT), creatinine levels, and pro-inflammatory cytokines (TNF-α and IL-6) were all found to be higher in the non-treatment group compared to the AMP-treatment group. Also, extensive tissue damage in the lung, liver, and spleen of the non-treated control group mice was observed based on the histopathological lesions recorded. As expected, AMPs from climbing perch significantly alleviated multidrug-resistant P. aeruginosa infection in-vivo and produced enhanced therapeutic efficacy superior to the ciprofloxacin treatment. Conclusion: this study provides insight into the potential antimicrobial activity of fish innate immune system-derived peptides that could serve as a candidate for the substitute of antibiotics.

On-Farm Practices Associated with Multi-Drug-Resistant Escherichia coli and Vibrio parahaemolyticus Derived from Cultured Fish.

Aquaculture activities have been implicated as responsible for the emergence of antimicrobial resistance (AMR), leading to broad dissemination and transference of antibiotic resistance to pathogens that affect humans and animals. The current study investigates the on-farm practices and environmental risk factors that can potentially drive the development and emergence of multi-drug-resistant (MDR) Escherichia coli and Vibrio parahaemolyticus in the aquaculture system. A cross-sectional study was conducted on 19 red hybrid tilapia (Oreochromis spp.) and 13 Asian seabass (Lates calcarifer, Bloch 1970) farms on the west coast of peninsular Malaysia. Data were collected using a structured questionnaire pertaining to farm demography, on-farm management practices and environmental characteristics. Multi-drug-resistant E. coli (n = 249) and V. parahaemolyticus (n = 162) isolates were analyzed using multi-level binary logistic regression to identify important drivers for the occurrence and proliferation of the MDR bacteria. On-farm practices such as manuring the pond (OR = 4.5; 95% CI = 1.21-16.57) were significantly associated with the occurrence of MDR E. coli, while earthen ponds (OR = 8.2; 95% CI = 1.47-45.2) and human activity adjacent to the farm (OR = 4.6; 95% CI = 0.75-27.98) were associated with an increased likelihood of MDR V. parahaemolyticus. Considering the paucity of information on the drivers of AMR in the aquaculture production in this region, these findings indicate the targeted interventions implementable at aquaculture farms to efficiently abate the risk of MDR amongst bacteria that affect fish that are of public health importance.

Prevalence and Antimicrobial Resistance of Escherichia coli, Salmonella and Vibrio Derived from Farm-Raised Red Hybrid Tilapia (Oreochromis spp.) and Asian Sea Bass (Lates calcarifer, Bloch 1970) on the West Coast of Peninsular Malaysia.

Antibiotics are widely used in intensive fish farming, which in turn increases the emergence of antimicrobial-resistant (AMR) bacteria in the aquatic environment. The current study investigates the prevalence and determines the antimicrobial susceptibility of E. coli, Salmonella, and Vibrio in farmed fishes on the west coast of Peninsular Malaysia. Over a period of 12 months, 32 aquaculture farms from the Malaysian states of Selangor, Negeri Sembilan, Melaka, and Perak were sampled. Both E. coli and Salmonella were highly resistant to erythromycin, ampicillin, tetracycline, and trimethoprim, while Vibrio was highly resistant to ampicillin and streptomycin. Resistance to the antibiotics listed as the highest priority and critically important for human therapy, such as colistin in E. coli (18.1%) and Salmonella (20%) in fish, is a growing public health concern. The multi-drug resistance (MDR) levels of E. coli and Salmonella in tilapia were 46.5% and 77.8%, respectively. Meanwhile, the MDR levels of E. coli, Salmonella, V. parahaemolyticus, V. vulnificus and V. cholerae in Asian seabass were 34%, 100%, 21.6%, 8.3% and 16.7%, respectively. Our findings provide much-needed information on AMR in aquaculture settings that can be used to tailor better strategies for the use of antibiotics in aquaculture production at the local and regional levels.

Estimating the Economic Loss Due to Vibriosis in Net-Cage Cultured Asian Seabass (Lates calcarifer): Evidence From the East Coast of Peninsular Malaysia.

This study aims to estimate the economic loss due to vibriosis in the production of Asian seabass in floating net-cages on the east coast of Peninsular Malaysia. Asian seabass has contributed significantly to Malaysia's economic activities and food security. However, its production can be hindered by the occurrence of diseases, such as vibriosis, causing severe economic losses to farmers. A questionnaire-based survey was conducted on 14 small-scale monoculture Asian seabass net-cage farms. Using a stochastic bioeconomic model and inputs from the survey, existing literature, and expert opinion, the economic losses were determined. Moreover, this model considered the prevalence of Vibrio spp. at a farm on the east coast and the risk posed by its infection from hatcheries. The results showed that 71.09% of Asian seabass simulated in the stochastic model survived. The mortality rate due to vibriosis and other causes was at 16.23 and 12.68%, respectively. The risk posed by Vibrio spp. infection from hatcheries contributed to 2.77% of the increase in Asian seabass mortality. The stochastic model estimated that the total cost of producing a tail of Asian seabass was €2.69 per kilogram. The economic loss of vibriosis was estimated at €0.19 per tail per kilogram, which represents 7.06% of the total production cost of Asian seabass per kilogram. An increase in the prevalence of clinical vibriosis and vibriosis case fatality rate at 42 and 100%, respectively, will lead to an increase in the cost of grow-out Asian seabass by €0.29 per tail from the default value. An increase in pellet price per kilogram by €1.38 and feed conversion ratio pellet by 0.96 will consequently increase the cost of grow-out Asian seabass by €2.29 per tail and €0.82 per tail, respectively. We find that the occurrence of Vibrio spp. infection at the hatchery level can contribute to an increased risk in the mortality of Asian seabass during the grow-out phase. Hence, we also need to focus on the control and prevention of vibriosis infection from hatcheries.

An update on the ornamental fish industry in Malaysia: Aeromonas hydrophila-associated disease and its treatment control.

Malaysia is the world's major producer and exporter of ornamental fish, contributing 9% to the global trade and taking the second position after Singapore. Because of their artistic appeal and tremendous commercial value for international trade, ornamental fish recently gain rapid importance for foreign exchange and as a source of employment. While ornamental fish production is growing, there is an increase in infectious diseases, resulting in high fish mortality with significant economic loss. Bacterial disease is a serious problem for ornamental fish industry. Bacterial species surveillance in diseased freshwater ornamental fish from an aquarium shop reveals that Aeromonas hydrophila is the most dominant bacteria isolated. Consequently, Malaysia is stepping up its efforts by implementing the Economic Transformation Program and other biosecurity steps to address the aquaculture issues and encourage the regrowth of the ornamental fish market. Chemotherapeutic medications, phytobiotics, probiotics, yeast extracts, vaccines, and disinfectants can be used in controlling bacteria. Further studies should be done to find new antibacterial agents from natural sources to combat bacterial fish diseases and reduce fish mortality rate in sustainable aquaculture farms. This review summarizes the literature on ornamental fish industries and aquaculture production in relation to A. hydrophila-associated diseases and ornamental fish health management in Malaysia.

Glyphosate Herbicide Induces Changes in the Growth Pattern and Somatic Indices of Crossbred Red Tilapia (O. niloticus × O. mossambicus).

The development of glyphosate-resistant genetically modified organisms (GMO) has increased the use of herbicide glyphosate by several magnitudes in recent years. It is now the most commonly used pesticide globally that affects aquatic habitats, especially fish. This study aims to add new knowledge on the effect of technical grade glyphosate on several toxicity parameters and to identify the most effective parameter in predicting technical grade glyphosate chronic toxicity (seven weeks) to fish, especially Malaysia's heavily farmed red tilapia. The results show that a relatively high concentration of technical grade glyphosate is needed to induce significant changes in all tested parameters. However, the results also indicate that the bodyweight index is the most sensitive toxicity parameter in that a reduction in body weight was observed at 25 mg/L of glyphosate. Negative correlations between the glyphosate concentration and toxicity parameters such as specific growth rate (SGR), hepato-somatic index (HIS), and gonado-somatic index (GSI) were observed. The fish condition factor and feed conversion ratio were found not to be affected at the highest glyphosate concentration tested (150 mg/L). To conclude, crossbred red tilapia (O. niloticus × O. mossambicus) is one potential species for evaluating the toxic effects of technical grade glyphosate on fish.

Short- and long-term probiotic effects of Enterococcus hirae isolated from fermented vegetable wastes on the growth, immune responses, and disease resistance of hybrid catfish (Clarias gariepinus × Clarias macrocephalus).

This study evaluated the short- and long-term effects of dietary supplementation with Enterococcus hirae strain UPM02 on the growth performance, immunity, and disease resistance of hybrid catfish (Clarias gariepinus × Clarias macrocephalus) against Aeromonas hydrophila infection. In the long-term trial, fingerling fish were fed diets containing 0 (control), 2 × 105, or 2 × 107 CFU/g E. hirae UPM02 for 120 days. Administration of E. hirae UPM02 had significant effects on the specific growth rate (SGR), feed utilization efficiency, body indices (P < 0.05), and gut villus physiology of the catfish. E. hirae UPM02 application also significantly increased the complete blood cell counts, phagocytic activity, respiratory burst, lysozyme activity, and alternative complement pathway hemolytic (ACH50) activity in tested catfish throughout the experimental periods (P < 0.05). Dietary E. hirae UPM02 at both concentrations significantly increased the expression levels of the alpha-2-macroglobulin (α2M), CC chemokines, CXC chemokines, lysozyme c (LYZC), myeloperoxidase (MYE), NF-kappa-B1 p105 subunit (NF-K), and bactericidal permeability-increasing protein (BPIP) genes in the head kidney, liver, and spleen (P < 0.05) at days 80, 100 and 120 after application. However, heat shock protein 70 (HSP70) gene expression was slightly downregulated in these organs. Interestingly, fish fed the diets containing 2 × 105 and 2 × 107 CFU/g E. hirae UPM02 exhibited a significantly lower (P < 0.05) postchallenge mortality rates (32% and 30%, respectively) after 14 days of A. hydrophila challenge than the control fish (58%). In short-term (28 days) application to juvenile catfish, the two concentrations of E. hirae did not affect all growth parameters. Nevertheless, these concentrations markedly elevated all tested immune parameters, similarly to long-term application. Immune-related gene expression was significantly upregulated at day 28 in the head kidney, at day 14 in the liver, and at day 7 in the spleen in fish treated with the two concentrations of the probiotics (P < 0.05). Mortality at 14 days after challenge with A. hydrophila in the groups receiving the two concentrations of the probiotic was significantly lower than that in the control group, at 28, 24, and 48%, respectively (P < 0.05). These results collectively suggest that dietary supplementation with E. hirae UPM02 at 2 × 105 and 2 × 107 CFU/g effectively influenced immune responses, enhanced disease protection, and stimulated immunity-related gene expression in hybrid catfish under both short- and long-term application. However, growth enhancement was significantly evidenced with long-term application only.

Mucosal responses of brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) following intraperitoneal infection with Vibrio harveyi.

Groupers are popular aquaculture species in South-East Asia, but their cultivation is affected by infectious disease outbreaks. Mucosa-associated lymphoid tissues provide a first-line defence against pathogens; however, few studies are available relating to cellular or proteomic responses of mucosal immunity in grouper. Skin, gill and intestine were sampled from brown-marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) at 4 and 96 hr post-infection (hpi) and 7 days post-infection (dpi) following intraperitoneal infection with Vibrio harveyi, and stained with haematoxylin/eosin and Alcian Blue/periodic acid-Schiff. Skin mucus was analysed by 2D-gel electrophoresis, and proteins modulated by the bacterial infection identified. In the infected fish, significant increases in sacciform cells in skin and increased levels of nucleoside diphosphate kinase in mucus were detected at 4 hpi. At 96 hpi, goblet cells containing acidic mucins significantly increased in the intestine, while those containing mixed mucins increased in skin and gills of infected fish. Proteasome subunit alpha type-I and extracellular Cu/Zn superoxide dismutase levels also increased in mucus. Rodlet and mast cells did not appear to respond to the infection. Mucosal tissues of grouper appeared actively involved in response to Vibrio infection. This information may help future research on improving grouper health, production and vaccine development.

In vivo and in vitro effects on cholinesterase of blood of Oreochromis mossambicus by copper.

The present study is aimed to evaluate the effects of sub-acute toxicity testing of copper sulphate (CuSO4), on behavioural, histological and biochemical changes of the Oreochromis mossambicus (black tilapia) blood tissues. The effects were assessed according to the previous results on sub-acute toxicity test after exposing fish to several concentrations (0.0, 2.5, 5.0, and 10.0 mg/L). The observations of scanning electron microscope, and transmission electron microscope studies revealed severe histopathological changes on the surface and the cellular changes in blood tissues, respectively. The morphological alterations in blood involved irregular structure of red blood cell and blood clot formation. CuSO4 affected the biochemical alteration of the blood cholinesterase also known as serum cholinesterase (ChE). Blood ChE inhibited up to 80% of activity when exposed to 10.0 mg/L CuSO4. The findings from this study can further improve the quality standards of aquaculture industry and the fundamental basis in selecting suitable strains among freshwater fish species to be used as bioindicator.

Quorum sensing is required for full virulence of Vibrio campbellii towards tiger grouper (Epinephelus fuscoguttatus) larvae.

The link between quorum sensing in Vibrio campbellii and its virulence towards tiger grouper (Epinephelus fuscoguttatus) was investigated using V. campbellii wild type and quorum-sensing mutants with inactive quorum sensing or constitutively maximal quorum-sensing activity, and signal molecule synthase mutants. The results showed that wild-type V. campbellii is pathogenic to grouper larvae, causing more than 50% mortality after 4 days of challenge. Furthermore, the mortality of larvae challenged with the mutant with maximally active quorum sensing was significantly higher than that of larvae challenged with the wild type, whereas a higher survival was observed in the larvae challenged to the mutant with a completely inactive quorum-sensing system. Grouper larvae challenged with either the signal molecule synthase triple mutant, the harveyi autoinducer-1 (HAI-1) synthase mutant and the autoinducer-2 (AI-2) synthase mutant showed higher survival than larvae challenged with the wild type. In contrast, larvae challenged with the cholerae autoinducer-1 (CAI-1) synthase mutant showed high mortality. This indicates that HAI-1 and AI-2, but not CAI-1, are required for full virulence of V. campbellii towards grouper larvae. Our data suggest that quorum-sensing inhibition could be an effective strategy to control V. campbellii infections in tiger grouper.

Live vaccines against bacterial fish diseases: A review.

Fish diseases are often caused either by bacteria, viruses, fungi, parasites, or a combination of these pathogens. Of these, bacterial fish diseases are considered to be a major problem in the aquaculture industry. Hence, the prevention of such diseases by proper vaccination is one of the integral strategies in fish health management, aimed at reducing the fish mortality rate in the aquaculture farms. Vaccination offers an effective yet low-cost solution to combat the risk of disease in fish farming. An appropriate vaccination regime to prevent bacterial diseases offers a solution against the harmful effects of antibiotic applications. This review discusses the role of live-attenuated vaccine in controlling bacterial diseases and the development of such vaccines and their vaccination strategy. The current achievements and potential applications of live-attenuated and combined vaccines are also highlighted. Vaccine development is concluded to be a demanding process, as it must satisfy the requirements of the aquaculture industry.

Ultrastructural analysis of Apicomplexa-Like parasites in two conch species Laevistrombus canarium and canarium urceus from Johor Straits, Malaysia.

The tropical conch, Laevistrombus canarium (Linnaeus, 1758) and Canarium urceus (Linneaus, 1758) are ecologically and economically important shellfish species in Malaysia and neighboring region. Their populations, however are currently declining and this histopathological study investigates the aspect of parasitism and diseases that may affect their well-being. Conch samples were randomly collected from their natural habitat and histological sections (4-5 µm) of various organs and tissues were examined under light microscope. This was followed by ultrastructure analysis on infected tissues using transmission electron microscope (TEM). Based on the histological analysis, large numbers of gamonts, sporocysts and trophozoites of Apicomplexa-like parasites were observed in the vacuolated cells and pyramidal crypt cells of the digestive tubules, and in the digestive ducts. Furthermore, coccidian and oocysts-like Pseudoklossia sp. stages were also observed in the cells of the kidney. Apart from that, spores with cyst-like structure were observed in the digestive gland and kidney. Although the parasites were present in most of the organs analyzed, there was no obvious symptom, inflammatory response or mortality incurred on both species, which implies the possibility of a non-virulent relationship like commensalisms or mutualism. However, more investigations, including molecular studies, are needed to confirm the parasite identification and dynamics, and to further evaluate the nature of relationship between Apicomplexa parasites and their host.

Toxic effects of copper on liver and cholinesterase of Clarias gariepinus.

The release of pollutants, especially heavy metals, into the aquatic environment is known to have detrimental effects on such an environment and on living organisms including humans when those pollutants are allowed to enter the food chain. The aim of this study is to analyse the damage to Clarias gariepinus' liver caused by exposure to different concentrations of copper. In the present study, samples of C. gariepinus were exposed to sub-lethal copper sulphate (CuSO4) concentrations (from 0.2 to 20.0 mg/L) for 96 h. Physiological and behavioural alterations were observed with respect to their swimming pattern, mucus secretion and skin colour. Mortality was also observed at high concentrations of copper. Histopathological alterations of the liver were analysed under light, transmission and scanning electron microscopies. The liver of the untreated group showed normal tissue structures, while histopathological abnormalities were observed in the treated fish under light and electron microscopes with increased copper concentrations. Histopathological abnormalities include necrosis, melanomacrophage, hepatic fibrosis and congested blood vessels. In addition, the enzyme activity of liver cholinesterase (ChE) was also found to be affected by copper sulphate, as 100% of cholinesterase activity was inhibited at 20.0 mg/L. Thus, liver enzyme activity and histopathological changes are proven to be alternative sources for biomarkers of metal toxicity.

Experimental infection of Aphanomyces invadans and susceptibility in seven species of tropical fish.

AIM: Epizootic ulcerative syndrome (EUS) causes by aquatic oomycete fungus, Aphanomyces invadans is a dangerous fish disease of a wide range of fresh and brackish water, wild and farmed fish throughout the world. The objective of the present study was to determine the susceptibility of a number of tropical fish species to the EUS and compare the severity of infection between experimental groups. MATERIALS AND METHODS: Snakehead, Channa striata (Bloch, 1793); snakeskin gourami, Trichopodus pectoralis (Regan, 1910); koi carp, Cyprinus carpio (Linnaeus, 1758); broadhead catfish, Clarias macrocephalus (Günther, 1864); goldfish, Carassius auratus (Linnaeus, 1758); climbing perch, Anabas testudineus (Bloch, 1792); and Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) were challenged by intramuscular injection using zoospores of Aphanomyces invadans (NJM9701). The infected fish skins and muscles were examined for EUS histopathological characteristics, and the results on the severity of lesions and mortality were analyzed using SPSS program. RESULTS: All zoospore-injected fish were shown to be susceptible to the EUS infection except Nile tilapia. Although, the general histopathological pattern was similar in the zoospore-injected group, but there were some variation in granulomatous reaction, that is the presence or absence of giant cells, and time of mortality were detected. The result of statistical analysis showed that there was a significant difference between species, (c (2)=145.11 and p<0.01). CONCLUSION: Gourami, koi carp, and catfish were demonstrated to be highly susceptible while goldfish and climbing perch were found to be moderately susceptible to the EUS infection. These findings suggested that the cellular response of fish to mycotic infection and granulomatous reaction varied in different fish species, which could not be an indicator of susceptibility or resistant to the EUS itself, although it was shown that the granulation rate and the level of maturity or solidification (consolidation of granulomas) were higher in resistant fish.

Vacuolating encephalopathy and retinopathy associated with a nodavirus-like agent: a probable cause of mass mortality of wild Golden grey mullet (Liza aurata) and Sharpnose grey mullet (Liza saliens) in Iranian waters of the Caspian Sea.

Mullets are dominant fishes in the catch composition in the southern coasts of the Caspian Sea and after (Rutilus frisii kutum Kamensky, 1901) have a worthy role in production of marine proteins and incomings of north provinces of Iran. Mullets stocks decreased dramatically in recent decades in the Caspian Sea and catch amount reached from 6446 MT on 2002 to 2151 MT in 2012. Mysterious mortalities occurred in wild mullet (Liza auratu) and (Liza saliens) in Iranian waters of Caspian Sea in recent years. Regarding to investigation of causative agent of mentioned outbreak about 322 suspected samples were collected from coastal capture sites of Iranian north provinces in 2008 till 2011. Moribund fish revealed skin darkening, erratic swimming, belly-up at rest and high distension of swim bladder. Target tissues such as brain and eye were removed and then fixed for histopathology and TEM assay. Widespread and massive vacuolation were observed in brain, spinal cord, retina and optical nerve and intracytoplasmic vacuoles and virus particles in retina. So concerning to clinical signs, histopathological and TEM findings, it could be concluded that nodavirus-like agent could be probable cause of mass mortality of wild mullet in Iranian waters of the Caspian Sea.

Histological features of the gastrointestinal tract of wild Indonesian shortfin eel, Anguilla bicolor bicolor (McClelland, 1844), captured in Peninsular Malaysia.

This study was conducted to record the histological features of the gastrointestinal tract of wild Indonesian shortfin eel, Anguilla bicolor bicolor (McClelland, 1844), captured in Peninsular Malaysia. The gastrointestinal tract was segmented into the oesophagus, stomach, and intestine. Then, the oesophagus was divided into five (first to fifth), the stomach into two (cardiac and pyloric), and the intestine into four segments (anterior, intermediate, posterior, and rectum) for histological examinations. The stomach had significantly taller villi and thicker inner circular muscles compared to the intestine and oesophagus. The lamina propria was thickest in stomach, significantly when compared with oesophagus, but not with the intestine. However, the intestine showed significantly thicker outer longitudinal muscle while gastric glands were observed only in the stomach. The histological features were closely associated with the functions of the different segments of the gastrointestinal tract. In conclusion, the histological features of the gastrointestinal tract of A. b. bicolor are consistent with the feeding habit of a carnivorous fish.

White mulberry (Morus alba) foliage methanolic extract can alleviate Aeromonas hydrophila infection in African catfish (Clarias gariepinus).

Two experiments were simultaneously conducted with Morus alba (white mulberry) foliage extract (MFE) as a growth promoter and treatment of Aeromonas hydrophila infection in separate 60 and 30 days trail (Experiments 1 and 2, resp.) in African catfish (Clarias gariepinus). In Experiment 1, four diets, control and control supplemented with 2, 5, or 7 g MFE/kg dry matter (DM) of diet, were used. In Experiment 2, fish were intraperitoneally infected with Aeromonas hydrophila and fed the same diets as experiment 1 plus additional two diets with or without antibiotic. Results of experiment 1 showed that growth was unaffected by dietary levels of MFE. Treatments with the inclusion of MFE at the levels of 5 and 7 g/Kg DM had no mortality. Red blood cells (RBC), albumin, and total protein were all higher for the treatments fed MFE (5 and 7 g/Kg DM). Results of experiment 2 showed RBC, hemoglobin, hematocrit, globulin, albumin, and total protein improved with the increase in MFE in the infected fish. The dietary MFE at the level of 7 g/kg DM reduced mortality rate. In conclusion, MFE at the level of 7 g/kg DM could be a valuable dietary supplement to cure the infected fish.

Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian Macrobrachium rosenbergii de Man.

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.

Development and partial characterization of new marine cell line from brain of Asian sea bass Lates calcarifer for virus isolation.

A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses.

Sharing Malaysian experience with the development of biotechnology-derived food crops.

Biotechnology-derived food crops are currently being developed in Malaysia mainly for disease resistance and improved post harvest quality. The modern biotechnology approach is adopted because of its potential to overcome constraints faced by conventional breeding techniques. Research on the development of biotechnology-derived papaya, pineapple, chili, passion fruit, and citrus is currently under way. Biotechnology-derived papaya developed for resistance to papaya ringspot virus (PRSV) and improved postharvest qualities is at the field evaluation stage. Pineapple developed for resistance to fruit black heart disorder is also being evaluated for proof-of-concept. Other biotechnology-derived food crops are at early stages of gene cloning and transformation. Activities and products involving biotechnology-derived crops will be fully regulated in the near future under the Malaysian Biosafety Law. At present they are governed only by guidelines formulated by the Genetic Modification Advisory Committee (GMAC), Malaysia. Commercialization of biotechnology-derived crops involves steps that require GMAC approval for all field evaluations and food-safety assessments before the products are placed on the market. Public acceptance of the biotechnology product is another important factor for successful commercialization. Understanding of biotechnology is generally low among Malaysians, which may lead to low acceptance of biotechnology-derived products. Initiatives are being taken by local organizations to improve public awareness and acceptance of biotechnology. Future research on plant biotechnology will focus on the development of nutritionally enhanced biotechnology-derived food crops that can provide more benefits to consumers.