Abstract

A cell line was established from the bulbous arteriosus of Tilapi mossambica. These cells, designated TmB, have been passaged 85 times during the last two years. The mean and modal chromosomal number of the cells at passages 1 and 84 were 43.2 ± 0.4 and 46, respectively. The cells grow well in Eagles MEM containing 10 percent fetal bovine serum at a temperature of 25ºC. Cytopathic effects, which included giant cell formation and cell lysis, were observed 5 to 8 days after exposure of cells to channel catfish virus (CCV), two eel virus isolates (EVEX, FVA), golden shiner virus (G5V), infectious hematopoietic necrosis virus (IHN), infectious pancreatic necrosis (IPN), lymphocystis virus (LC), and Rhabdovirus carpio.

Introduction

In a review of the available piscine cell lines and viral agents, Wolf and Mann listed 61 cell lines representing 17 families and 36 species of fish (1). Although 32 viruses of piscine origin were listed, none were reported from Tilapia. Tilapia cell lines were not among those listed in the review, and a subsequent search of the literature failed to reveal descriptions of tilapia cell culture systems.

Tilapia are important food fish in many areas of the world and, like other intensively maintained fish species, are probably subject to virus infections. The absence of tilapia cell culture systems would however, preclude detection of tilapia host specific viral agents. Efforts were thus directed toward developing a tilapia call culture system for use in virus screening of tilapia.

Materials and Methods

Three 250-300 g female Tilapia mossambicawere anesthetized in an ice-water mixture 10 min. The fish were then soaked 5 min in a cold (2-5ºC) benzalkoniurn chloride solution and washed with cold sterile distilled water. The fish were dissected and fragments of bulbous arteriosus minced in cold Hank's balanced salt solution (HBSS), washed two times, resuspended in 100 ml HBSS containing 0.25% trypsin and 0.02% versine, pH 7.9 (TV solution). After two hours trypsinization with stirring at room temperature, the preparation was filtered through gauze and the cells washed three-times in Eagle's minimum essential medium (MEM). Cells were dispensed (2 x 10⁶ cells/ml) in MEM containing 10% fetal bovine serum, 5 uM 1-glutamine, chloramphenicol 50 ug/ml, and gentamicin 50 ug/ml (MEM-10) into 25 cm² polystyrene flasks (Corning Glass Works, Corning, NY, 14850), 5 ml cell suspension/ flask. For subcultivation, the cells were dispersed by removing culture medium, washing the monolayer with MEM, and covering it with 0.2 ml TV solution/flask. After 1-2 min digestion cells were harvested, resuspended in MEM-10, and incubated at 22ºC. The cell suspension from each flask was dispensed into two flasks. During early subcultivations, the cultivation medium consisted of equal parts fresh medium and conditioned medium. After 15 passages, the cells were maintained in fresh medium.

Rabbit antiserum was prepared against tilapia bulbous arteriosus tissue, arid brown bullhead catfish cells (BB) for use in identifying cells by the cytotoxic dye exclusion test described by Greene et at. (2). Bulbous arteriosus tissues were dissected from three 250-300 g T. mossambica, minced, and the preparation filtered through gauze. Three day cultures of TmB and BB were trypsinized, washed three times in HBSS, and adjusted to 2 x 10⁵ cells/ml. The tissue and cell culture suspensions were sonified 30 seconds (20 kHz at 60W) with a Branson sonifier (Model W140) and the resulting suspension used as antigen.

Rabbits received 0.5 ml of the cell suspension intravenously on days 1 and 5. The animals were then inoculated intramuscularly with 1.0 ml cell suspension, Freund's complete adjuvant mixture on days 8 and 16, and bled 2 weeks later.

Cytotoxic dye exclusion tests were performed on 3-day cultures of TmB and BB cells. The cells were trypsin dispersed and washed three times in HBSS. Cell concentration was adjusted to ca 2 x 10⁵ cells/ml. Prior to use, rabbit antiserums were complement inactivated by incubation at 56ºC for 1 hr. A preliminary viable cell count of the cell culture suspension was conducted with Trypan Blue. One milliliter of cells in HBSS was incubated with 0.1 ml of the appropriate antiserum for 20 min at 25ºC.

Following incubation, 0.1 ml guinea pig complement (Difco Laboratories, Detroit, MI) was added to the mixture and the mixture incubated 2 hr at 37 C. Controls consisted of cells and antiserum, but no complement. Cell viability was assessed by Trypan Blue dye exclusion.

Chromosome preparations of cells (passages 1 and 84) were made from Leighton tube cover-slip cultures using 10^-6 M colchimine. Fifty well spread chromosome preparations were counted for the cell passages.

Lactic dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PD) isoenzyme profiles of the TmB cells were compared with those of hamster kidney, BB, and mosquito cell lines. For this purpose, approximately 10 3-day cells were obtained by trypsinization and washed three times in MEM. After the last wash, supernatants were removed and cell pellets frozen at -20ºC. The frozen cells were lysed in distilled water and the lysate subjected to electrophoresis on cellulose acetate (Titan III, Helena Laboratories, Beaumont TX) (3) for 30 min at 200 V uising glycine-borate pH 8.4, µ = 0.02.

For sterility testing, TmB cells were grown in MEM-10 without antibiotics for five successive passages. The cells were examined for mycoplasmas (4), bacteria, and fungi (5).

Susceptibility of TmB cells to channel catfish virus (CCV), golden shiner virus (GSV), infectious hematopoietic necrosis virus (IHN), Rhabdovirus carpio, infectious pancreatic necrosis virus (IPN), lymphocystis virus, infectious bursal disease virus (IBD) of avian origin and bovine enterovirus -- serotype 2 (BEV-2) -- was determined. Each virus was propagated in appropriate cell culture systems and the fluids diluted 10³ prior to exposure to TmB cells. Leighton tube cultures of TmB cells were drained, 0.2 ml virus inoculum added, and incubated 1 hr at 25ºC. The medium was replaced and the culture incubated at 25ºC for 14 days. Coverslips were removed from 3 tubes daily for 14 days and stained with giemsa to reveal cytopathic effects.

Results

The cytotoxic dye exclusion test revealed antigenic similarity between the TmB cell line and tilapia bulbous arteriosus tissue and non-relatedness of TMB to the BB cell line. When treated with complement and heat inactivated rabbit anti-tilapia bulbous arteriosus tissue serum or anti-serum, 90-95% of the BB cells were viable, whereas viability was demonstrable in less than 2% of the treated TmB cells. In the absence of complement, the number of viable cells following treatment was essentially the same in all cases.

At passages 1 and 84, TmB cells revealed a mean and modal chromosome number of 43.2 + 0.4 and 46, respectively.

LDH and G6PD isoenzyme profiles of hamster kidney, mosquito, BB, and TmB cell lines provided a basis for clearly distinguishing the cell lines examined.

Neither bacteria, fungi, nor mycoplasma were demonstrable in the cells.

Eight of the ten viruses tested caused cytopathic effects 5-8 days after initial exposure of the cells to the viral agents. Cytopathic effects were not detected in TMB cells exposed to BEV-2 and IBD.

Discussion

The development of TmB cells provides a non-fastidious cell culture system with an apparent broad range of fish virus susceptibility. It is one of the few non-ictaturid cell lines susceptible to channel catfish virus. The two non-piscine viral agents (BEV-20 and IBD) were included in the study to assess the potential susceptibility of tilapia to agents sometimes encountered in the environments exposed to animal manures and because the agents induce cytopathic effects in a wide variety of homeothermic animal cells. Neither of the agents produced cytopathic effects in TmB during the 14 day incubation period.

As an homologous system, the TmB cell line has a potential for applications that will contribute to knowledge of diseases of tilapia, a major cultured species in many areas of the world.

Acknowledgement

Funds for the project upon which this paper is based were made available through the U.S.-Israel Binational Agricultural Research and Development Fund (Project No. US-313-801). The authors gratefully acknowledge the technical assistance of Mrs. N.M. Charanza.

References

1. Wolf, K. and Mann, J.A. Poikilothermic verterbrae cell lines and viruses: A current listing for fishes. In Vitro 16: 168-179 (1980).
2. Green, A.E.; Coriell, L.L.; and Charney, J.A. A rapid cytotoxic antibody test to determine species of cell cultures. J. Natl. Cancer Inst. 32: 779-786 (1964).
3. Harris, H. and Hopkinson, D. Handbook of enzyme electrophoresis and human genetics (loose-leaf). North Holland Publishing Co., Amsterdam, 1976.
4. Macy, M.L. Tests for mycoplasmal contamination of cultured cells as applied at the ATCC. Procedure 75305, TCA Manual 4: 1151-1155 (1979).
5. Cour, I.; Maxwell, G.; and Hay, R.J. Tests for bacterial and fungal contaminants in cell cultures as applied at the ATCC. Procedure 75494, TCA Manual 4: 1157-1160 (1979).