RESEARCH ON THE NOCARDIOSIS AND PATHOGEN IN REARED SNAKEHEAD,OPHIOCEPHALUS ARGUS CANTOR

Epizootics of nocardiosis in reared snakehead,Ophiocephalus argus Cantor,occurred in the north part of Zhejiang Province,China,in recent years.It is a chronic systematic disease which bring significant loss in aquiculture.And an epizootic disease of snakehead,cultured in brackish ponds was investigated by our workgroup in Xiaoshan,in Zhejiang Province,China,from June to July 2006.The cumulative mortality within 30d was 35%(180000 out of 500000) and the mortality was as high as 100%.The clinical signs,pathogenic morphology,virulence,biochemical characterization,ultrapathology and 16S rRNA gene were observed and analyzed.This is the first study on Nocardia sp.-infected snakehead in aquaculture.We try to make it the reference of prevention and cure on nocardiosis in snakehead and also provide new contents about diseases in snakehead.The diseased fish were 18 months old with individual length from 30 to 35 cm,body weight from 250 to 400 g.Multiple,ivory-white nodules,1-5 mm in diameter,were scattered on several internal organs,especially the liver,spleen and kidney.Gram-positive rods,often seen as branched fragmented filaments,were detected in Gram-stained smears of scrapings of the organ lesions.Bacteria were isolated from the liver and kidney of diseased fish and numbered W060622.Experimentally induced nocardiosis in snakeheads challenged with the bacterium isolated from a natural outbreak produced the same pathological changes as observed in naturally infected fish.In addition,the bacterium was re-isolated in pure culture from challenged animals only.These observations confirm the bacterium as the aetiological agent of this disease in cultured snakeheads.The morphology of isolated bacteria from Tryptic soy agar(TSA) and Ogawa's medium(OM) were bead-like or long,slender,filamentous rods both under microscope and electron microscope.Batches of bacteria were found in kidney tissue and cells,whereas,no virus and other prokaryotes were found under electron microscope.Biochemical characterization contains enzyme production,decomposition,growth on sole carbon sources were tested and draw similarities to Nocardia.Almost complete sequence of the 16S rRNA gene of the organism was sequenced to be 1430 bp(GenBank Accession No.EF192033) and compared with the correlative sequences in Genbank database.It was found to be highly similar with that of the type strain of Nocardia seriolae JCM 3360T(Z36925) and the similarity reached 99.9%.Based on the results of the experimental,morphological feature,physiological,biological properties and 16S rRNA sequence,the pathogenic organism W060622 was identified as Nocardia seriolae.In the experimental infection,we found that healthy snakehead could be infected by intraperitoneal(IP) injection,intragastric(IG) infection and also by immersion challenge.This indicated that the bacterium colonizes in the fish via manifold routes,such as injured skin,gill or digestive tract.Lots of nodules were found on the liver,spleen and especially kidney of challenged fish which probably reflected that the pathogen multiplying related to blood circulation.Epidemiology and environmental conditions showed the outbreak might associate with depressed germplasm resource by inbreeding,water contaminated and unpasteurized diets.The high density at which fish were reared and eutrophication water acted as external stressors might decrease the fish's resistance to bacterial infection.These observations suggested an opportunistic role of Nocardia in infected snakeheads.Because of the great harm of this disease,no effectual approaches were found in early diagnosis and prevention and cure.So the pathology,immunology,detection and medication of nocardiosis in snakehead warrants further investigation.