The Aquatic Animal Health Research Institute

Table of Contents

STUDY ON WHITE ULCERATIVE DISEASE IN GUPPY (Poecilia reticulata)

FIRST REPORT OF SYSTEMIC AMOEBOSIS IN OSCAR, Astronotus ocellatus

CHARACTERISTIC AND PATHOGENICITY OF Flexibacter maritimus FROM DISEASED MARINE SEABASS, Lates calcarifer

ACUTE TOXICITY OF SOME CHEMICALS ON 15 DAYS OLD FRY AND 55 DAYS OLD FINGERLING OF GIANT GOURAMY, Osphronemus goramy (Lacepede)

PHARMACOKINETIC STUDY OF NORFLOXACIN IN SEABASS (Lates calcarifer)

EFFECT OF OXYTETRACYCLINE ON GROWTH AND RESIDUE IN Penaeus monodon

RESISTANCE OF Acinetobacter spp. IN FISH INTEGRATED FARMS

EVALUATION OF CEFTIOFUR AS A CHEMOTHERAPEUTIC AGENT IN GRASS CARP, Ctenopharyngodon idella

DEVELOPMENT OF MONOCLONAL ANTIBODIES TO IMMUNOGLOBULIN M (IGM) OF SNAKEHEAD (Channa striata)

THE PRESENCE OF MYCOBACTERIUM SP. IN THE BLOOD AND TISSUES OF SNAKEHEAD (Channa striata) DURING ARTIFICIAL INFECTION, DETECTED USING POLYMERASE CHAIN REACTION (PCR) AND IN SITU HYBRIDIZATION (ISH)

ESTABLISHMENT OF A CELL LINE DERIVED FROM CHINESE SOFT-SHELLED TURTLE, Trionyx sinensis Wiegmann

COMPLILATION OF SMALL-SCALE FRESHWATER AQUACULTURE IN THAILAND

SURVEY ON AQUACULTURE ACTIVITIES OF EXTENSION OFFICER

All abstract in this issue are from papers presented at The Seminar on Fisheries 2001 held at Department of Fisheries, Ministry of Agriculture & Cooperative, Bangkok, Thailand during 18 - 20 September 2001. Subject to review, these paper will be published in Proceeding of The Seminar on Fisheries 2001.

Thitiporn Laoprasert, Chalearmpol Khaisri and Supranee Chinabut
Aquatic Animal Health Research Institute, Bangkok, Thailand

Study on white ulcerative disease, which is the serious problem particularly in tropical aquarium fish was conducted in guppy (Poecilia reticulata). Infected fishes were collected from guppy farms in Bangkok and the other areas nearby. The clinical sign of infected fish is whitish patch lesions on the body due to massive of ciliated protozoa in the scale sockets. Fish scales are bristled as the outer skin is lost and the muscular tissues swollen. These ciliated protozoa can pervade through skin lesions, into muscle and also other internal organs. Histopathological changes were severe extensive necrosis occur in subdermal and muscular tissues. Inflammatory reaction was rarely observed. The infection results in mortality. It was possible to conclude that ciliated protozoan, Tetrahymena sp. is the main causative agent of white ulcerative disease in guppy. It disintegrates host tissue and feeds on cell debris. The endemic of this disease may relate to the importation of ornamental fish into the country without any quarantine system. The proper health management in the farms is recommended for preventing of this disease.

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Thitiporn Laoprasert, Somkiat Kanchanakhan and Supranee Chinabut
Aquatic Animal Health Research Institute, Bangkok, Thailand

Disease diagnosis was performed by using conventional method of sudden death oscar (Astronotus ocellatus). Diseased fish showed , lose of appetite, emaciation, skin darkening, small ulcers on the bodies, aggregation on the bottom of the tanks. Mortality rate at 80 to 100 percent was found within 3 to 5 days after disease on set. Typical trichodinid and monogenean parasites were found on gill and skin of diseased fish. Application of formalin at the concentrations of 40 to 45 ppm was effectively killed both parasites. However, mortality still continued. Neither virus nor bacteria was isolated. Histopathological studies showed numerous of amoebic-like organisms in gill, kidney and muscle tissues of infected fish. The organism was isolated and maintained in axenic culture. Morphological studies by inverted microscope was conducted and found two distinctive forms of trophozoites that have spiny-liked pseudopod and polygonal shape cysts. Application of Polymerase Chain Reaction technique with specific primer indicated that this organism is an amoebic protozoan belonging to the genus Acanthamoeba, which should be the causative agent of this disease.
Keyword: Amoebosis, oscar(Astronotus ocellatus), Acanthamoeba

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Yaowanit Danayadol and Jeeranun Uraiprasit
National Institute of Coastal Aquaculture, Songkhla, Thailand

Diseased seabass (Lates calcarifer) showed the lesions of body, fins and head were studied. Gliding bacteria with seawater requirement were isolated. Biochemical characterization indicated that the gliding bacteria belonged to the species Flexibacter maritimus. Pathogenicity test was succeeded by wounded the skin before bath challange. The bacterial isolates were resisted to sulfamethoxazole and nalidixic acid, but were sensitive to several antibiotics such as penicilin, norfloxacin, tetracycline and ampicilin. From these results it could be concluded that Flexibacter maritimus is the pathogenic bacteria in seabass.

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Bungong Jamnongsittathum
Uthai Thani Inland Fisheries Station, Uthai Thani, Thailand

The acute toxicity of dipterex, formalin and malachite green to 15 and 55 days. Osphronemus goramy (Lacepede) were determined. The experiment was carried out during July to October 2000 at Uthai Thani Inland Fisheries Station at 28.0^o – 29.5^oC of water temperature.
The results showed that the 96-hour LC₅₀ of dipterex, formalin and malachite green on Osphronemus fry and fingerling were 0.665 and 0.682 ppm, 179.99 and 196.79 ppm and 0.146 and 0.668 ppm, respectively. The safety concentration of dipterex, formalin and malachite green for indefinite treatment were 0.07 and 0.07 ppm, 17.99 and 19.68 ppm and 0.015 and 0.067 ppm, respectively. The results indicated that all tested chemicals were more toxic to Osphronemus fingerling than fry. However, the toxicity of each chemical to fry was most obvious in the first 12-24 hours than to fingerling.
Dipterex showed no effect on water pH, alkalinity and hardness during the exposure except lowering the dissolved oxygen in the toxicity test on the 55 days old fingerling. While formalin and malachite green significantly reduced water pH and dissolved oxygen for 96 and 72 hours, respectively, during the exposure time. Conclusively, the reduction of water pH and dissolved oxygen were correlated with the concentration of formalin and malachite green.

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Walai Kleechaya
Samutsakorn Coastal Aquaculture Development Center, Samutsakorn, Thailand

The pharmacokinetics and bioavailability of norfloxacin (NFX) was estimated after drug administered to seabass (Lates calcarifer) at 10 mg/kg body weight for intravascular administration (iv.) and 30 mg/kg body weight for oral administration (po.). The levels of NFX in seabass serum at time interval were determined by using HPLC. The kinetics of NFX was described by two compartment model for iv. Data. NFX has slowly distribution half-life (T_1/2 = 2.56 h) and prolong elimination half-life (T_1/2 = 38.5 h). For po. data, the kinetic after dosing was fitted to one compartment model. NFX was rapidly assimilated (T_1/2a = 2.21 h) and also rapidly eliminated(T_1/2 = 17.7 h). Bioavailability (F) refers to the rate and extent of drug absorption by calculating from AUC (Area under the concentration-time curve from zero to infinity) after iv. and po. administration. The bioavailability (F = 6.90 %) was quite low, while A peak serum concentration (C _max) from po. was 0.81 g/ml and reached (T _max) at 9 h. The dosage regimen for multiple oral dosing by refer to the minimum effective concentration (MEC) for causative agent for vibriosis (2 times of MIC; MEC = 0.4 g/ml for Vibrio anguillarum) was 30 mg/kg under the experimental condition.

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Wannapa Somboonsumran, Supis Thongrod, Supapum Brillanstes and U-ma Chukaew
Feed Quality Control and Development Division, Bangkok, Thailand.

Shrimp fed with diet containing oxytetracycline (OTC) at 0, 0.5, 5 and 10 grams/kg feed for 7 days and 5 grams/kg for 14 days, showed that both amount of OTC accumulating in tissue and duration of residue depend on the concentrations of OTC in feed and duration of feeding. In this experiment, shrimp fed with OTC at 10 grams/kg feed has the highest concentration of residue at 1.66 ppm, and the longest deposition, 9 days, while shrimp fed 0.5 and 5 grams/kg feed, 0.065 and 1.5 ppm, respectively, with the deposition at 6 days. Deposition of OTC residue accumulated in shrimp fed with diet containing OTC at 5 grams/ kg feed for 14 days and 7 days were 9 and 6 days, respectively. The result of this experiment indicated that shrimp fed with higher concentration of OTC showed higher amount of OTC accumulation in tissue and longer duration of residue. The longer feeding period gave longer deposition of OTC.
After 12 weeks of the experiment on the effect of OTC on growth, average body weight of shrimp fed with 0, 0.05, 5 and 10 grams/kg feed were 6.14 0.65, 4.66 0.10, 4.65 0.11 and 5.07 0.33 grams, respectively. Shrimp fed with no oxytetracycline (control group) showed significantly higher average body weight than the others (P < 0.05), while the body weight of shrimp fed with diet containing OTC at different concentrations were not significantly different (P > 0.05). Survival rates of shrimp from this experiment were not significantly different (P > 0.05) and varied between 55.0 – 77.5 %.

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Kantimanee Phanwichin¹, Temdoung Somsiri², Supranee Chinabut², Tawatchai Kaewmak² and Andrease Petersen³
1 : Department of Zoology, Kasetsart University, Bangkok, Thailand
2 : Aquatic Animal Research Institute, Bangkok, Thailand
3 : Royal Veterinary and Agriculture University, Denmark

A number of antibiotic substances have been applied into fish integrated farm as growth promoters and for bacterial diseases treatment and prevention, which has led to increasing frequencies of resistant bacteria. In present study, Acinetobacter spp. is chosen as such an indicator organism due to their ubiquitous distribution in the aquatic environment to determine the association between use of antibiotic in the integrated fish/poultry, fish/pig farms and fish farm and the bacterial antibiotic susceptibility patterns. The 979 strains of Acinetobacter spp. were isolated from chicken/pig/duck manure, water from fish pond and gill tissues of fish. They were identified as Acinetobacter spp. by colony hybridization. Antimicrobial susceptibility testing on amoxicillin, chloramphinical, norfloxacin, gentamycin, oxytetracycline and sulfamethoxazone against these organisms by agar disk diffusion method had been done. The bacteria exhibited resist to oxytetracycline 49%, sulfamethoxazone 38%, chloramphinical 25% and amoxicillin 11%, respectively. The isolates showed 100% sensitive to gentamycin and 95% sensitive to norfloxacin. Triple resistance on 3 antibiotic including norfloxacin, oxytetracycline and sulfamethoxazone has been presented.
The 60 isolates, sampling from bacteria that show wide diameter of clean zone, had been tested on minimal inhibitory concentration (MIC) against norfloxacin, oxytetracycline and sulfamethoxazone by agar plate dilution MIC values of norfloxacin, oxytetracycline and sulfamethoxazone ranged respectively from 0.125-64.0, 0.3125-160.0 and 1.0-1024.0 microgram/milliliter. There are relationship between use of antibiotic in integrated farms and bacterial resistance

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Amornchai Somjetlertcharoen
Aquatic Animal Research Institute, Bangkok, Thailand

Ceftiofur sodium, a third generation of cephalosporins, was studied to determine the potential of this drug as an alternative bacterial therapeutic agent for aquaculture industry. Pharmacokinetics and histopathological observations were investigated in grass carp (Ctenopharyngodon idella). Ceftiofur sodium was administered a single time to grass carp by four different routes : intracardiac (IC), intraperitoneal (IP), intramuscular (IM) and oral (PO) at a dosage of 8 mg/kg body weight. Oral absorption was not observed in this species. Following, IC, IP and IM ceftiofur sodium administration, the final elimination half-lives, maximum plasma concentration, time to reach maximum concentration, volume of distribution and plasma clearance were 0.38, 0.45 and 13.86 hours ; 157.09, 31.54 and 8.86 g/ml ; 0, 0.25 and 0.5 hours ; 0.09, 0.17 and 0.53 l/kg ; and 0.21, 0.26 and 0.26 ml/min.kg, respectively. For histopathological studies, decreased number of renal tubules, increased tubular necrosis and infiltration of inflammatory cells were seen in the posterior kidney at the dosage of 80 mg/kg. In summary, ceftiofur sodium is probably not appropriate for production food fish because of its lack of oral absorption but has potential as an efficacious chemotherapeutic agent for controlling bacterial infection in brood stock and ornamental fish.

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Suppalak Puttinaowarat¹, Kim D. Thompson² and Alexandra Adams²
1 Aquatic Animal Health Research Institute, Department of Fisheries, Bangkok, Thailand
2 Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK

Monoclonal antibodies have proven to be a very powerful tool to investigate the immune response of fish. Monoclonal antibodies were developed here to snakehead (Channa striata) IgM to use as probes for monitoring the immune response of snakehead, especially during vaccine development for this species. Two monoclonal antibodies (7D2 and 1C3) were produced. Mab 7D2 was characterised as an IgG_2a subclass with a light chain, while Mab 1C3 was of an IgG1 subclass, also with a light chain. Mab 7D2 and 1C3 were highly reactive to snakehead IgM in ELISA. They both reacted with the heavy chains and light chains of IgM in Western blot analysis, where the molecule bands were located at 68 and 27 kDa respectively. When tested with the IgM of another nine fish species, both Mab 7D2 and 1C3 cross-reacted with IgM of giant snakehead (Channa micropeltes), Nile tilapia (Oreochromis niloticus) and Silver balb (Babodes gonionotus).

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Suppalak Puttinaowarat, Jaree Polchana & Pojanee Saengthong
Aquatic Animal Health Research Institute, Department of Fisheries, Bangkok, Thailand

Mycobacteriosis caused by Mycobacterium spp. is a common disease in a wide range of ornamental and food fishes. The pathogenesis of the disease has been extensively studied however there is a lack of information about the presence of the bacterium circulating in the blood of fish during infection. Therefore, the aim of this study was to investigate the presence of the pathogen in tissues and blood after artificial infection with a Mycobacterium sp.
Forty snakehead (Channa striata) were intraperitoneally injected with Mycobacterium marinum (S268) at 1 x 10⁸ cells/ml. Five fish was sampled every week throughout the experiment. Spleen, liver and kidney were collected and analysed by PCR and ISH, while blood was tested by PCR only.
No significant clinical signs were observed during the first week of injection, although minor pathological changes were observed. Bacteria were detected in the tissues of infected fish by PCR, but no bacteria were present in the blood using this method of detection. By comparison bacteria were only detected in the kidney and spleen of infected fish by ISH. During the second week of the experiment, some fish became lethargic and appeared pale, and bacteria were detectable both in tissues and blood samples tested by PCR and ISH. The level of the pathogen appeared to increase within the blood and tissues during the infection period, although the clinical signs of the disease were not particularly obvious.

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Somkiat Kanchanakhan and U-malai saduakdee
Aquatic Animal Research Institute, Bangkok, Thailand

A new cell line was derived from Chinese soft-shelled turtle embryo, Trionyx sinensis Wiegmann. The cells grew well in L-15 Leibovitz medium containing 5-15% fetal bovine serum with optimum temperature ranging from 20 to 30^oC but no growth at 15^oC. They have been subcultured for more than 130 passages. This cell line was epithelium-like and designated as STE. STE had high efficiency of plating up to 74.7% and its optimum ratio of subculture was 1:6. The cells were capable to remain monolayer in tissue culture flask for at lease 30 days at 25-30^oC without replacing the medium. No adventitious viruses, bacteria, fungi or mycoplasmas were detected in STE line. The STE could be stored safely in liquid nitrogen tank. STE line was susceptible to many viruses, infectious pancreatic necrosis virus (IPNV), sand goby virus (SGV), snakehead rhabdovirus (SHRV), ulcerative disease rhbdovirus (20E), and other rhabdoviruses that isolated from epizootic ulcerative syndrome-effected fishes.

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Temdoung Somsiri¹, Supranee Chinabut¹, Yolprapa Dumrongphol¹ , M. Crumlish², K. Thompson²
1 Aquatic Animal Research Institute, Bangkok, Thailand
2 Institute of Aquaculture, University of Stiring, Stirling FK9 4LA, Scotland

A small scale freshwater fish farm survey in 2000 has been conducted to assess problems associated with husbandry and fish disease. Three hundred and four households were surveyed in 26 provinces, and were randomly selected using a devised survey sample frame, which incorporated information from the Department of Fisheries database (1999). Relative risk analyses were performed using Epi-Info version 6 and SPSS version 9.01, and identified potential risk factors from different exposure variables associated with disease as the outcome variables from data collected from the fish farms.
There were more households practised polyculture compared with any other type of aquaculture system. Only 69 households claimed that fish farming was their main source of income and 39 had experienced disease problems on their farms. There were 288 households with ponds (others had small or large cages and pens) and most had one or two ponds in total and in use. The most common pond size was found to be 400 m² and cage size was found to be 9 m². More households cultured tilapia compared with any other species. Most families harvested their fish for family consumption as well as for the local market purposes and the individual weight ranged between 0.11-0.3 kilograms. The majority of households fed their fish with pellets but a high proportion of them used rice bran and vegetables. The highest percentage households with water quality problems were found in Mudaharn, Nakhonpathom, Nongboulumpoo and Ubonratchathani and dead or sick fish were observed during this time. Less than 40% of farmers had encountered disease problems which mostly occurred during November to February but the highest number reported diseases occuring in December. A statistically significant association was found between not preparing the farm before every crop and having experienced disease problems.

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Temdoung Somsiri¹, Supranee Chinabut¹, Yolprapa Dumrongphol¹ , M. Crumlish²,
K. Thompson²
1 Aquatic Animal Research Institute, Bangkok, Thailand
2 Institute of Aquaculture, University of Stiring, Stirling FK9 4LA, Scotland

The aquaculture activities of extension services in 26 provinces were evaluated using a structured survey questionnaire, Epi-Info version 6 was used as a database and frequency analysis performed. The questionnaire was sent to extension officers in 26 provinces, but only 18 provinces responsed. The main work of extension officer was to provide advice on variousaspects of aquaculture to fish farmers and stimulate people in their area to participate in aquaculture activities. Approximately 40% of the officers included in the survey had been trained on aquaculture from the Department of Fisheries. Most of officers were responsible for more than 20 fish farms in total and 60% of these officers had visited the fish farms once a month. Most farmers asked advice on disease prevention and treatments. Fifty-five percent of officers recorded information on the farms visited, which included disease problems and farm management strategies. More than 90% of officers have ever gave advice on disease prevention and treatment. A higher number of farmers in Nongkhai, Pattalung and Ubonratchathani had contacted the extension officers when they had encountered disease problem compared with other provinces. More than 80% of extension officers required additional information and training on the application of chemotherapeutic agents in aquaculture and information regarding fish health management.

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