Studies on Identification, Pathogenicity and Rapid Diagnostic Method of Nocardia seriolae from Sea Bass (Lateolabrax japonicus)

• Main Authors: chin-chu lai, 賴金鞠
• Other Authors: shin-chu chen
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/33527558075671237242

碩士 === 國立屏東科技大學 === 獸醫學系 === 89 === An investigation on an epizootic of Nocardia seriolae (507, 336, 300 and 531) infected of sea bass (Lateolabrax juponicus), strip bass (Moronme saxatilis) and largemouth bass (Micropterous salmonids) occurred at Yulin in Taiwan, respectively. The growth character, morphological and biochemical properties of four isolates, Nocardia spp. from diseased fish of the fresh water pound were similar to those of N. seriolae. The 16S rDNA sequence for 1500bp PCR amplicon from fish isolates (507, 300, 336 and 531) was determined and deposited in the GenBank data base under accession number AF251566、AF254418、AF254417 and AY017474. Those sequences showed 99.9~100% identity with N. seriolae (GenBank accession number Z36925). The pathogenicity experiments were tested by intraperitoneal injection (IP), intramuscular injection (IM), gastric intubation, bathing in juvenile sea bass (35~40g). After 24 days, half lethal dose (LD50) of the experimental intraperitoneal injection were 1.5 ×103 CFU/ml. There were no death terminated during the two months in both bathing group (water contained N. seriolae 1.2 ×109 CFU/ml) and gastric intubation, without showed clinical signs and significant lesion. Fish were received 0.5ml of 1.2×109 CFU/ml of bacterial suspension for IP and IM showed 100% mortality after 9 days, respectively. Pure cultures of bacteria were reisolated from the kidney and peritoneum. In the gross finding, the lesion were severe peritoneal granulomas in IP group. The IM group showed nodules in the heart, liver, spleen, kidney and gill were similar to those naturally infected fish were seen. In naturally infected fish and experimental fish, in the necrotic area and/or the center of granulomas showed bead-like, filamentous and branching bacteria by Ziehl-Neelsen stain, and by immunocytochemistry method. The isolated bacteria used in two infecting trial in which tilapia received intraperitoneal and intramuscular injection of the same concentration. Mortalities of IP and IM group were 100% and 50% after 6 days and 10 days, respectively. The histopathologicl changes in experimentally infected tilapia were similar those naturally and experimentally infected sea bass and reisolated bacteria from the nodules organs of infected tilapia. It also accredited that tilapia were susceptible to N. seriolae infection. Altogether the results constitute the first confirmation that N. seriolae can cause disease outbreaks in the sea bass, Lateoabrax japonicus. Genus-specific sequence of 16S rDNA of Nocardia were amplified by PCR using the NG1 and NG2 primer. A 596 bp amplicom was observed in all the nodules organ and bacterial suspension. The results demonstrated that all PCR amplicon exhibited one Mnl I recognition site, producing two DNA fragments of 278 and 318 bp. The technique is conceived to enable the rapid identification of the Genus Nocardia in fish.