高層培地法および凍結法によるＰａｓｔｅｕｒｅｌｌａ ｐｉｓｃｉｃｉｄａの保存

Abstract

Long-term preservation of Pasteurella piscicida is necessary for producing highly effective bacterins for the control of pseudotuberculosis in cultured yellowtail Seriola quinqueradiata. This report describes the survival and pathogenicity of P. piscicida in stab culture and frozen storage. Strain 5866 virulence was maintained by fish passage. And then both preservation methods were investigated. With stab culture in BHIA, strain 5866 was isolated from every fish passage and stocked at 15°C. For frozen storage, a culture medium composed of polypeptone, yeast extract and NaCl was used. Cultures of strain 5866 were stored in either glycerol (10, 30 and 50% V/V), defibrinated horse blood (10, 30 and 50% V/V), 20% skim milk (10, 30 and 50% V/V), calf or horse serum (50% V/V) or dimethyl sulfoxide (10% V/V) and dispensed in sterile screw-cap vials. The suspensions were stored frozen at -20 and -80°C.After 3 months, strain 5866 lost virulence when stored in BHIA stab cultures. After 6 months in frozen storage, the relationship between viability and the use of protective additives was not clear. All cultures stored at -20°C showed a gradual decrease in numbers of surviving cells and viability was lost between 1 and 6 months. Cultures stored at -80°C gave a high recovery of viable cells showing the lower temperature was preferable for prolonged storage. Strain 5866 stored at -80°C in skim milk maintained virulence and pathogenicity after 6 months.