Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 27, Num. 1, 2009, pp. 35-39

Indian Journal of Medical Microbiology, Vol. 27, No. 1, January-March, 2009, pp. 35-39

Brief Communication

In vitro culture of various species of microsporidia causing keratitis: Evaluation of three immortalized cell lines

Joseph J, Sharma S

Ocular Microbiology Service, L. V. Prasad Eye Institute, Bhubaneswar
Correspondence Address:Ocular Microbiology Service, L. V. Prasad Eye Institute, Bhubaneswar, savitri@lvpei.org

Date of Submission: 01-Jan-2008
Date of Acceptance: 19-Feb-2008

Code Number: mb09008

Abstract

Keywords: Cell culture, keratitis, microsporidia

Microsporidia are obligate intracellular protozoan parasites that infect both invertebrates and vertebrates. ^[1] Though not commonly reported, microsporidial keratitis may in fact occur more commonly than is believed. We have earlier reported the largest case series in the world for both microsporidial stromal keratitis ^[2] and microsporidial keratoconjunctivitis ^[3] but we have just scratched the surface of this emerging ocular pathogen, and a concerted approach is required to define the true epidemiological extent of microsporidial keratitis. Routine diagnosis of microsporidia in clinical laboratories has relied mostly on special staining and microscopic techniques ^[4] ; however, it is possible that microsporidial organisms may be present in very small numbers and they can be easily missed during histologic examination. ^[1],[5] Some microsporidia such as Encephalitozoon species and Brachiola , even when they are present in small numbers, have the potential to become established in cell cultures, thus facilitating their easy identification at a later time. ^[5] Microsporidia cannot be cultivated axenically because of their obligate intracellular development, but they have been successfully cultivated in a number of mammalian cell lines. Microsporidia have been isolated from various biological specimens (conjunctival scrapings, urine, faeces, bronchoalveolar lavage) by In vitro cultivation on different established cell lines or primary cell culture, ^[6],[7] including monkey and rabbit kidney cells (vero and RK13), human foetal lung fibroblasts (MRC-5), MDCK cells, and several other cell lines. ^[5] Species that have been cultivated In vitro from a variety of human specimens include E. hellem , E. cuniculi , E. intestinalis , Vittaforma corneae , and T. hominis . ^[5] With an appropriate In vitro model, the same cell monolayer can be maintained for long periods of time by weekly replacement of the medium, permitting harvest of a large number of spores. However, further evidence is needed to prove this hypothesis. Hence we decided to make an In vitro growth comparison of four microsporidial species causing keratitis in three different cell lines available in the laboratory to assess the most suitable host-parasite In vitro system.

Materials and Methods

Reference samples

Standard strains of Encephalitozoon hellem (ATCC 50504), Encephalitozoon cuniculi (ATCC 50789), Encephalitozoon intestinalis (ATCC 50651), and Vittaforma corneae (ATCC 50505) were obtained from the American Type Culture Collection, (Manassas, VA, USA). The standard strains were obtained in a frozen state grown in ATCC CCL-26 (African green monkey) cell line and maintained in ATCC 30-2003 (Eagle′s minimum essential medium with 2 mM L-glutamine and Earle′s BSS adjusted to contain 1.2 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate).

The continuous cell lines used for this study were SIRC (rabbit corneal epithelium), vero cell line from monkey kidney, and HeLa cell culture (National Facility for Animal Tissue and Cell Cultures, Pune, Maharashtra, India).

Maintenance of ATCC strains

To initiate cultures, the reference strains were immediately thawed in a water bath set at 35°C for two to three minutes and inoculated into a monkey kidney (vero) cell culture that was maintained on Eagle′s minimal essential medium (EMEM) fortified with 2 mM glutamine and 10% foetal bovine serum (FBS). The medium also contained 50 µg of gentamicin, 1,000 units of penicillin, and 5 µg/mL of amphotericin B to prevent bacterial and fungal overgrowth. The culture medium from each flask (Nunc, 25 cm ² ) was removed daily for the first week and twice weekly thereafter and replaced with fresh medium containing the antibiotics. After centrifugation, spent medium was removed from the spore pellets by aspiration. Spores were re-introduced into the original cell culture flasks and re-incubated as described above. The cultures were incubated at 37°C. After four to six weeks of such manipulations, many spores were seen in the culture supernatants and many cells had become enlarged and distended with developing stages and spores [Figure - 1].

Preparation of spores

Spores were harvested from the cell monolayers and pelleted. Cell debris was removed by centrifugation with an isopycnic gradient of 80% Percoll (Amersham Pharmacia Biotech, Piscataway, N.J.). Tubes containing the spore Percoll mixture were centrifuged at room temperature at 2,300 g for 30 minutes. The pellet containing purified spores was washed with sterile phosphate buffered saline (PBS), centrifuged again at 1,300 g , and resuspended in sterile PBS. A haemocytometer (Neubauer-ruled Bright Line counting chambers; Hausser Scientific, Horsham Pa. USA) was used to count the spores; the spore concentration was adjusted to 1 x 10 ⁸ spores/mL of PBS, and the standard suspension was inoculated into monolayers of vero/SIRC/HeLa cells growing on glass cover slips placed in the wells of 24-well Nunc plates.

In vitro microwell plate assay

Cell culture conditions that were optimized in the laboratory were used to determine rate of growth in various cell lines. Vero, SIRC, and HeLa cells were grown by using a 24-well plate format. Cells were removed from cell culture flasks with trypsin-EDTA (Sigma Chemical Co.) at 35°C. Culture medium containing FBS was then added to the flasks to stop the trypsin effect and dilute the cells for transfer to multiwell plates. The multiwell plates were seeded by placing 1 mL of cell suspensions mixed with culture medium onto 15-mm ² coverslips, in the wells in the 24-well Nunc plates. The plates were incubated at 35°C in the presence of 5% CO ₂ with humidity for two days, a time period predetermined to produce almost confluent monolayers. The spores at a target concentration of 1 x 10 ⁸ spores/mL were diluted tenfold in 0.05 M phosphate buffer, to prepare various suspensions of spores ranging from 1 x 10 ⁴ /mL to 1 x 10 ⁸ /mL. One millilitre of each spore suspension was inoculated onto vero/SIRC and HeLa cell monolayers, grown on coverslips as described above, and replenished immediately before inoculation of spores along with 1 mL of minimal essential medium (Sigma) containing 10% foetal bovine serum.

Four wells were inoculated for each spore suspension (5 x 4 wells) for growth assessment at different time points at weekly intervals. In the remaining four wells, no spores were inoculated, to be used as controls for our study. Immediately after inoculation, the 24-well plates were incubated at 35°C in the presence of 5% CO ₂ with humidity for a total period of four weeks. Media were replaced every three to four days, and care was taken not to remove organisms from the bottoms of the wells. After a few days of culture manipulations, spores of the isolate appeared in large numbers in the culture medium, indicating successful establishment of the cultures. As development proceeded, these foci of infection increased in number; and within a few weeks, about 70% to 80% of the monolayers were infected and the host cells were completely filled with spores. At weekly intervals, the coverslips were removed and 10% sodium dodecyl sulphate was added to the wells to release organisms from host cells. The spent culture medium, along with the cells containing distended spores, was centrifuged and the pellet was used for spore-counting in a haemocytometer. Each experiment was performed in triplicate and the average count was used for the analysis. The presence of spores in smears made from centrifuged sediments from cultures was confirmed by staining with either the 1% acid-fast stain or KOH/Calcofluor white stain.

Results

V. corneae, E. hellem, E. cuniculi, and E. intestinalis spores at various concentrations (1 x 10 ⁸ to 1 x 10 ⁴ spores/mL) were added to confluent monolayers of vero, SIRC, and HeLa cells on day 0, and the total number of organisms were measured at weekly intervals. Comparative growth curves are illustrated in [Figure - 2] for the different species grown in all the three cells after four weeks. Calcofluor white staining indicated that spores were plentiful in the supernatant fluid when the cytopathic effect (CPE) was well advanced. Microsporidia were not detected in uninfected control cells of all the three cell lines. The initial spore production showed a decrease in the number of spores after the first week of culturing for all species studied, irrespective of the cell line supporting their growth. All Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, whereas they did not show any significant differences in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. However, in vero cells too it showed a considerable increase in growth compared to the initial inoculum. HeLa cells showed the least amount of growth for all the species included in this study.

In vero cells, the weekly output of spores exceeded the initial inoculum at fourth and fifth week in all species, whereas it remained below the initial inoculum in case of HeLa cells for E. cuniculi and E. intestinalis. Spore output in vero cells was well above that in HeLa cells. All Encephalitozoon species cultivated in vero cells produced twice as many spores compared to SIRC and thrice as many spores compared to HeLa cells.

Comparison of the cell lines to determine the lowest number of spores that supported growth [Figure - 3] showed the initial inoculum as 1 x 10 ⁴ spores/mL for vero cells for all the species studied.

In comparison, V. corneae and E. hellem required an initial inoculum of 1 x 10 ⁴ for growth in SIRC cells. The initial inoculum that showed good growth in SIRC cells for E. cuniculi and E. intestinalis was 1 x 10 ⁵ spores/mL. In contrast, HeLa cells could support growth of microsporidia only with initial inoculum above 1 x 10 ⁷ spores/mL for E. intestinalis and 1 x 10 ⁶ for E. cuniculi and 1 x 10 ⁵ for V. corneae and E. hellem. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris.

Discussion

During the In vitro replication cycle, Encephalitozoon spp. typically form cytoplasmic parasitophorous vacuoles filled with large numbers of mature spores; these vacuoles eventually rupture and release spores into the culture medium. ^[5] Few attempts have been made to optimize culture conditions and spore enumeration methods so that standardized quantitative assays may be used to monitor changes in the In vitro growth of microsporidia. ^[8] Most, but not all, of the human-infecting microsporidia grow well in a variety of cell lines, which permits workers to use an In vitro model to evaluate organism infectivity and growth (Visvesvara, 2002). In this study, an assay performed with vero, SIRC, and HeLa cell culture systems for growth of various species of microsporidia known to cause ocular infections proved to be a reproducible standardized assay for determining the most suitable cell line in a tissue culture laboratory for the In vitro growth of microsporidia. Using 24-well plates facilitated replicate testing at several concentrations and for several time periods. The selection of In vitro parameters was based on previous studies (Didier et al., 1991; Visvesvara et al., 1995; Didier et al., 1996) and ATCC recommendations. These results show vero cells to be the most suitable for growth of all species known to cause ocular infections.

The initial trend of spore production observed in all host cell-parasite systems (decrease in the number of spores collected after first week of culturing) has already been reported by other authors ^[9],[10] and may indicate that spores which were unable to invade the host cells were also harvested. Also, after the first days of culturing, some parasites lose their infectivity and only those which are able to develop in the host cell can propagate. V. corneae grew better compared to the other isolates in all cell lines and underwent a shorter lag phase and rapidly entered logarithmic phase. On the other hand, the lag phase for E. intestinalis was twice as longer and the output of spores produced was also less compared to the other species included in the study. These results confirm that microsporidial parasites vary among species in terms of their capacity for In vitro propagation. ^[8] Although testing with this system is labour intensive, it is less demanding than testing with spores harvested from infected flask-based systems. Many variables affect the results of an In vitro assay. In this study, tests were previously performed in an attempt to optimize centrifugation, the age of cells, the cell density, and the method used for spore purification and enumeration. Other key variables, such as the source of foetal bovine serum, the pH of the medium, also affect the final viability and spore count. The multiwell vero cell assay has potential uses in a variety of situations. The analytical sensitivity was found to be 1 x 10 ⁴ spores/mL for vero cell line, compared to 1 x 10 ⁵ spores/mL for SIRC cell line and 1 x 10 ⁷ spores/mL for HeLa cell line. Because vero cells are easily cultivated and rapid growth of Encephalitozoon sp. occurs with these cells, the system can facilitate assessment of quantitative effects of cell culture variables on the growth of microsporidia. This preliminary study has shown the superiority of vero cells over SIRC and HeLa in the isolation of microsporidia for the laboratory diagnosis of microsporidial keratitis.

In a study comparing vero, MDCK, and RK13 cell lines for growth of all three Encephalitozoon spp, ^[8] the vero cell line was reported as the most suitable model for large-scale spore production for the microsporidial species tested. The observations of this study agreed with those of the Ludovisi study in finding vero cell line as the most suitable for cultivation of microsporidia compared to SIRC and HeLa. However, the results should be interpreted with caution since clinical isolates were not used, wherein presence of inhibitors may also affect the growth of these organisms. Awareness of these conditions may eventually facilitate In vitro growth of the microsporidian species that currently cannot be cultivated by traditional methods.

However, for making a confirmatory diagnosis of microsporidiosis, Gram or Calcofluor white stains were necessary for visualization of spores, as these could be mistaken for artefacts. Hence, considering that the analytical sensitivity is 1 x 10 ⁴ spores/mL and time required for visualization of spores in culture supernatant is four weeks, it is concluded that In vitro culture of microsporidia may have limited application in the diagnosis of microsporidial keratitis. Further studies are warranted using samples from patients, in a statistically adequate sample size, to define their complete clinical utility. To conclude, based on a "MEDLINE" search, it is believed that this is the first report of the comparison of vero, SIRC, and HeLa for the growth of microsporidia causing ocular infections. Cultivation of microsporidia using cell lines may have limited applicability as a diagnostic tool in clinical laboratories. Nevertheless, inclusion of vero cell line, when available, in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield; and this information is a fundamental step in developing molecular and immunological diagnostic methods for the study of microsporidia infecting humans.

Acknowledgement

References

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