ture of 37C is in excess of the normal growth temperature of many fish species

Ture of 37c is in excess of the normal growth

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ture of 37°C is in excess of the normal growth temperature of many fish species, notably salmonids. This indicates that the organisms may well have been derived from warm-blooded animals, and may, therefore, constitute a pubHc health risk. Heart infusion agar supplemented with thallium acetate and oxolinic acid or with colistin sulphate and oxolinic acid was evaluated for the selective recovery of Str. iniae from Japanese flounder and the fish farm environment. Defibrinated horse blood was also added to determine haemolysin pattern. The result was recovery of Str. iniae from brain, intestine and kidney of diseased fish (Nguyen and Kanai, 1999). AEROBIC GRAM-POSITIVE RODS AND COCCI Renibacterium salmoninarum Cultivation of the causal agent of BKD in vitro from chinook salmon was not achieved until Earp (1950) used a nutrient-rich medium, containing fish extract, glucose, yeast extract and bovine serum/meat infusion, with incubation at 15 or 20°C. An improvement resulted from use of minced chick embryos in 1% (w/v) agar or on Dorset egg medium (Earp et al, 1953). Nevertheless, growth was generally poor, even after prolonged incubation periods of >14 days. To prove that the growth was of the pathogen, Earp successfully inoculated the bacterial culture into healthy chinook salmon, and eventually recovered it again from the resultant kidney lesion. Continuing this pioneering work, Ordal and Earp (1956) supplemented Dorset egg medium with 0.05-1.0% (w/v) L-cysteine, tryptone and yeast extract, and succeeded in isolating the pathogen in 3-4 weeks following incubation at 17°C. The outcome of this work was the formulation of cysteine blood agar with which Koch's postulates were fulfilled (Appendix 5.1; Ordal and Earp, 1956). Foetal calf serum was substi- tuted for human blood in a modification proposed by Evelyn et al (1973). This was developed further by removing sodium chloride and substituting peptone for tryp- tone and beef extract (KDM2; Appendix 5.1; Evelyn, 1977); this medium is now used commonly for growth of the BKD organism. In a parallel development. Wolf and
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Isolation/Detection 157 Dunbar (1959) used Mueller-Hinton agar supplemented with 0.1% (w/v) L-cysteine hydrochloride (MHC) to culture the pathogen, although success with this medium did not occur with Smith (1964). However, Bullock et al. (1974) confirmed the value of MHC, although this has been subsequently contended by Evelyn (1977). Serum- rich KDM2 was considered to be superior to serum-deficient MHC, indicating the benefit of serum for the cultivation of the BKD organism. This was further supported by Paterson et al. (1979), who supplemented MHC with 10% (v/v) foetal calf serum, and successfully used the medium for isolating the pathogen from Atlantic salmon. Daly and Stevenson (1985a) proposed replacing serum with charcoal, which serves as a detoxicant (Appendix 5.1—charcoal agar). However, these media, being extremely rich in composition, are generally suitable for the growth of many aerobic, hetero- trophic bacteria. Moreover, fast-growing organisms may rapidly outcompete and overgrow the slower-growing BKD organism. A solution was proposed by Evelyn
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