Temperature, pH and bile dependent in vitro cultivation of Hexamita salmonis from rainbow trout Oncorhynchus mykiss intestine

Abstract

The intestinal diplomonadid flagellate Hexamita salmonis was isolated from the intestinal contents of freshwater reared ra~nbow trout Oncorhynchus mykiss in Denmark and maintained in axenic cultures in the laboratory. A cell culture medium (MEM) or MEM supplemented with a bile solution (Keister's modified TYI-S-33) and penicillin, gentamicin and nystatin (mycostatin) was used as culture substrate. Temperature, pH and bile requirements for laboratory maintenance were investigated. Optimum temperature for H. salmonis population increase was determined to be 10°C. Satisfactory although decreased growth was found at 5OC. At 15°C and 20°C flagellate cells survived but did not exhibit significant population growth. In the pH range from 4 to 10 parasite survival was seen from 5.5 to 9.0, although optimum pH conditions for population Increase were pH 7.5 to 8.0. Bile In low concentrations (30 to 960 mg ]-') enhanced flagellate propagation sl~ghtly. This study on the basic requirements explains the occurrence of the flagellate in rainbow trout in less warm seasons and biotopes as well as particular microhabitats in the host.

In vitro cultivation and temperature-dependent growth of two strains of Spironucleus barkhanus (Diplomonadida:Hexamitidae) from Atlantic salmon Salmo salar and grayling Thymallus thymallus

“…Spironucleus barkhanus seems to be able to live and multiply at high densities. A cell density of more than 10' cells ml-' in culture is 5 times higher than reported for Giardia lamblia (Visvesvara 1980, Keister 1983) and 40 times higher than reported for Hexamita salmonis (Uzmann & Hayduk 1963, Buchmann & Uldal 1996. This could reflect the suitability of the medium for S. barkhanus, but a potential for high reproductive activity was also seen in vivo in the salmon suffering from systemic spironucleosis, where large muscle abscesses were found to contain a high density of trophozoites in pure culture (Poppe et al 1992).…”

“…It is believed that in vitro cultivation will be a valuable tool for obtaining flagellates for future experimental studies of these mechanisms. This was suggested by Buchmann & Uldal (1996) and has already been shown for the related genus Giardia. Trypticase yeast extract iron serum medium (TYI-S-33) (Diamond et al 1978), as modified by Keister (1983), is now widely used for routine cultivation of Giardia duodenalis for experimental studies.…”

“…According to Poynton et al (1995). However, Keister's medium failed to support growth of Hexamita salmonis (Buchmann & Uldal 1996), which is more closely related to the genus Spironucleus than Giardia spp. are.…”

Spironucleus vortens, a possible cause of hole-in-the-head disease in cichlids

“…The in vitro method described by Buchmann & Uldal (1996) for Hexamita salmonis was used here for screening tropical fish for hexamitids. In the present study bile salts (bovine Sigma B-8381) added at a concentration of 120 to 150 mg l -1 substituted for Keister's TYI-S-33 medium.…”

“…A modification of Buchmann & Uldal's (1996) in vitro culture technique for Hexamita salmonis from rainbow trout Oncorhynchus mykiss was used to screen the fish for the presence of flagellates.…”