Interactions of ciliates with cells and viruses of fish
Pinheiro, Marcel D.O.
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This thesis develops and utilizes in vitro approaches to study ciliate/fish interactions. The thesis is divided into six chapters. Chapter one reviews the literature on culturing ciliates and fish cells. Chapter two develops methods for culturing the ciliate Tetrahymena thermophila in media developed and used for mammalian and piscine cells. Chapter three explores the interactions of T. thermophila with monolayers of epithelial cells from fish and mammals. Chapter four studies the interactions of T. corlissi, T. thermophila, and T. canadensis with monolayers of epithelial and fibroblasts from a wide range of animals. The interactions of T. thermophila with the fish viruses are described for the rhabdovirus, viral hemorrhagic septicemia virus (VHSV), in chapter five and for the aquareovirus, Chum salmon reovirus (CSV) in chapter six. The summaries for these six chapters are presented in the following six paragraphs. How the ciliates of fish can be cultured and used to study ciliate/fish interactions are reviewed. The culturing of ciliates is done in media based on either freshwater, seawater, or vertebrate bodily fluids together either with bacteria, fish cells, or organic matter, which can be undefined, such as proteose peptone, or defined. Some ciliates can be pathogenic but with a variable dependency on the fish host. The most dependent and difficult to culture has been Ichthyophthirius multifiliis. Cryptocaryon irritans has been maintained successfully in co-cultures with fish cells. Pathogenic scuticociliates and tetrahymenas can be cultured axenically. Established cultures have been used to screen drugs for their potential chemotherapeutic value and to study pathogenic mechanisms. As well as being pathogens, ciliates interact with fish in other ways. Free-living forms can modulate the activities of other fish microbial pathogens and be food for fish larvae. Tetrahymena spp. have been shown in culture to phagocytose pathogenic bacteria and microsporidia spores. Large-scale cultures of both freshwater and marine ciliates have been achieved and could be a source of feed for fish larvae. In the future cell cultures should be invaluable in studying these and other possible relationships between fish and ciliates. The transfer of Tetrahymena thermophila from normosmotic solutions (~20 to 80 mOsm/kg H2O) to hyperosmotic solutions (> 290 mOsm/kg H2O) 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 H2O, 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 H2O), 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. 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 three fish epithelial cell lines, CHSE-214, RTgill-W1, and ZEB2J, and of 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 be able to make contact with the animal cells through swimming, which appeared to dislodge or loosen cells so that they could concurrently 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 proliferation of ciliates. These results show for the first time that T. thermophila can ‘eat’ animal cells or be histophagous in vitro, with the potential to be histophagous in vivo. The activities of T. corlissi, T. thermophila, and T. canadensis were studied in co-culture 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 undergo phagocytosis and did not destroy monolayers, whereas T. canadensis was able to undergo phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. Additionally 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. Incubating the fish pathogen VHSV with the ciliate T. thermophila, inactivated the virus, depending on the incubation temperature. Without the ciliates, the VHSV titre declined significantly over 72 h at 30 °C, but remained unchanged at 22 °C and 14 °C. At 30 °C, the ciliates only slightly enhanced the heat inactivation of VHSV. At 22 °C, the ciliates inactivated a substantial proportion of the VHSV by 24 h but no inactivation had occurred by 72 h at 14 °C. The ciliates vigorously phagocytosed fluorescent beads at 22 °C but not at 14 °C. When VHSV were labeled with the nucleic acid stain SYBR Gold, internalization of the virus into food vacuoles was seen at 22 °C. Thus phagocytosis was one possible mechanism for VHSV inactivation by ciliates. However, another VHSV/ciliate interaction was revealed by immunofluorescent staining and might contribute to inactivation. After being incubated for 24 h with VHSV, washed, and stained at various times afterwards for VHSV G protein, the ciliates stained transitorily. The strongest staining was seen at approximately 30 minutes after washing and was confined largely to the cilia but after 60 minutes this staining was lost. Tetrahymena thermophila strains B1975, wild type, and NP1, a temperature sensitive mutant, activated the fish aquareovirus CSV, depending on the temperature. CSV caused fish cells to form syncytia. This cytopathic effect (CPE) was used to titre CSV in the fish cell line, CHSE-214. The CSV titre remained stable during incubations of up to 96 h in Leibovitz’s L-15 with FBS at 4, 14, 22 and 30 °C. When CSV was incubated with B1975 or NP1 at 22 °C in the same medium for between 24 and 96 h, the virus titre increased approximately 3 log. At 4 °C, the titre was unchanged by ciliates and T. thermophila was unable to phagocytose beads. At 30 °C, B1975 enhanced CSV infectivity and underwent phagocytosis, whereas NP1 did neither. When CSV were labeled with the nucleic acid stain SYBR Gold, internalization of the virus into B1975 food vacuoles was seen. Therefore the viral activation pathway likely involved phagocytosis. Tetrahymena canadensis were incubated with CSV-infected CHSE-214, washed by centrifugation through a step gradient of polysucrose, and transferred to new CHSE-214 cultures, which developed the characteristic CSV CPE. Thus as well as activating CSV, ciliates could transport CSV.