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

Abstract

Spironucleus barkhanus from muscle abscesses of farmed Atlantic salmon Salmo salar L., and from the gall bladder of grayling Thymallus thymallus (L.) was cultivated axenically in a medium routinely used for cultivation of the human pathogen Giardia duodenaljs. Trophozoites multiplied by binary division, but nlultinucleated cells (< 10 nuclei) were frequently observed Complete cell divisions were never observed in these cells. Both strains grew at all temperatures tested (5, 10, 15 and 20°C). However, continuous growth of the salmon strain at 20°C was not possible. Growth potential, calculated as minimum generation time, showed relatively small differences both between strains and among temperatures, but the results indicate that the grayling strain had both a slightly higher optimum temperature and a higher upper temperature limit than the salmon strain.

In vitro studies on optimal requirements for the growth of Spironucleus vortens, an intestinal parasite of the freshwater angelfish

“…The parasites are normally found in the intestine (O'Brien et al 1993, Poynton et al 1995. but may be found in other organs including the gall bladder (Molnar 1974, Sterud 1998b. However, the results of this study indicate that S. vortens is less tolerant to bile, therefore suggesting that it may only accidentally enter the gall bladder during heavy intestinal or systemic infections.…”

“…The optimal temperature and pH for Hexamita salmonis isolated from rainbow trout were 10°C and pH 7.5 to 8.0 (Buchmann & Uldal 1996). Spironucleus barkhanus isolated from grayling and salmon have a suitable temperature range for growth from 5 to 20°C (Sterud 1998b). Both of these organisms are parasites of coldwater fish.…”

“…are still unknown. In addition, most of the in vitro cultivation of diplomonads, including fish hexamitids, have been done in culture medium supplemented with bovine bile (Keister 1983, Buchmann & Uldal 1996, Sterud 1998b. To date, none of the fish hexarnitids have been cultivated in medium supplemented with fish bile.…”

“…The organism can be maintained and propagated in artificial medium (Poynton et al 1995). A few studies have suggested the in vitro growth requirements of fish hexamitids (Poynton et al 1995, Sterud 1998b), but none have examined the specific optimal requirements for growth of S. vortens. Although S. barkhanus cultivated in bile-supplemented medium have shown a slight difference in optimal temperatures between strains isolated from salmon and grayling (Sterud 1998b), the optimal pH and bile requirement for Spironucleus spp.…”

Detection and quantification of Spironucleus barkhanus in experimentally infected Atlantic salmon Salmo salar

“…The ELISA was performed in flat bottom microplates (96 well, high binding polystyrene, Costar). Each well in the plate was coated with 50 µl of coating buffer (0.429 g Na 2 CO 3 × 10 H 2 O, 0.293 g NaHCO 3 in 100 ml double distilled water, pH 9.6) containing 50 µg ml -1 of sonicated Spironucleus barkhanus antigen (from axenic cultures of S. barkhanus in a modified Diamond's TYI-S-33 medium) (Diamond et al 1978, Sterud 1998. Total protein content was measured using the Bio-Rad Protein Assay and incubated at 37°C for 60 min.…”