Activation of an Aquareovirus, Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis.

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal-lysosomal system and released in a more infectious form. When allowed to swim in CSV-infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.

Some but not All Tetrahymena Species Destroy Monolayer Cultures of Cells from a Wide Range of Tissues and Species.

The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.

Delineating cellular interactions between ciliates and fish by co-culturing Tetrahymena thermophila with fish cells.

Although several species of Tetrahymena are often described as histophagous and opportunistic pathogens of fish, little is known about ciliate/fish cell interactions, but one approach for studying these is in vitro with cell lines. In this study, T. thermophila, B1975 (wild type) and NP1 (temperature sensitive mutant for phagocytosis) were cultured on monolayers of 3 fish epithelial cell lines, CHSE-214, RTgill-W1, and ZEB2J, and the rabbit kidney epithelial cell line, RK-13. Generally the ciliates flourished, whereas the monolayers died, being completely consumed over several days. The destruction of monolayers required that the ciliates could make contact with the animal cells through swimming, which appeared to dislodge or loosen cells so that they could be phagocytosed. The ciliates internalized into food vacuoles ZEB2J from cell monolayers as well as from cell suspensions. Phagocytosis was essential for monolayer destruction as monolayers remained intact under conditions where phagocytosis was impeded, such as 37°C for NP1 and 4°C for B1975. Monolayers of fish cells supported the proliferation of ciliates. Thus T. thermophila can 'eat' animal cells or be histophagous in vitro, with the potential to be histophagous in vivo.

Survival and growth of Tetrahymena thermophila in media that are conventionally used for piscine and mammalian cells.

The transfer of Tetrahymena thermophila from normosmotic solutions (~20-80 mOsm/kg H(2)O) to hyperosmotic solutions (> 290 mOsm/kg H(2)O) was investigated. During the first 24 h of transfer from proteose peptone yeast extract (PPYE) to either 10 mM HEPES or PPYE with added NaCl to give ~300 mOsm/kg H(2)O, most ciliates died in HEPES but survived in PPYE. Supplementing hyperosmotic HEPES or PPYE with fetal bovine serum (FBS) enhanced survival. When ciliates were transferred from PPYE to a basal medium for vertebrate cells, L-15 (~320 mOsm/kg H(2)O), only a few survived the first 24 h but many survived when the starting cell density at transfer was high (100,000 cells/ml) or FBS was present. These results suggest that nutrients and/or osmolytes in either PPYE or FBS helped ciliates survive the switch to hyperosmotic solutions. FBS also stimulated T. thermophila growth in normosmotic HEPES and PPYE and in hyperosmotic L-15. In L-15 with 10% FBS, the ciliates proliferated for several months and could undergo phagocytosis and bacterivory. These cell culture systems and results can be used to explore how some Tetrahymena species function in hyperosmotic hosts and act as opportunistic pathogens of vertebrates.

Use of Tetrahymena thermophila to study the role of protozoa in inactivation of viruses in water.

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4 degrees C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.