In vitro Model for Cryptosporidium parvum Infection

INTRODUCTION

Cryptosporidium is a genus of protozoan parasites which is a part of the coccidia group, from the phylum Apicomplexa, which also includes Isospora and Cyclospora.¹ until the mid-1990s, several species of Cryptosporidium were described depending on morphology and host specificity. C. parvum, C. muris, C. felis and C. wrairi were identified as species that infect mammals. C. parvum was the primary species that have been isolated from infected humans^2,3 and cross-transmission studies indicated that C. parvum isolates can be transmitted from humans to animals and between different animals.⁴

C. parvum is an intracellular parasite that infects the microvillous region of epithelial cells in the digestive tract of vertebrates, resulting in watery diarrhea.

 In immunosuppressed individuals, the symptoms are particularly severe and can be fatal.^5,6 The parasite is primarily spread through the fecal-oral route, often through contaminated water.^7,8       The life cycle of C-parvum begins with the ingestion of oocysts by the host. In the intestine, four sporozoites are excysted, which then infect epithelial cells and initiate asexual development. They become internalized and undergo merogony, in which eight merozoites are produced. Merozoites from this type I meront can either produce another type I or may develop as a type II meront with four merozoites. The four merozoites released from the type II merogony give rise to the sexual developmental stages, the micro and macrogamonts. The release of microgametes, and their union with macrogametes, gives rise to the zygote, which, after two asexual divisions, forms the environmentally resistant oocyst containing four sporozoites while still within the host cell.⁹        Environmentally robust oocysts are shed by infected hosts into the environment. These oocysts can survive the adverse conditions on the environment for months until it is ingested by a new suitable host. In the new host, the life cycle starts again, and multiplication occurs, using resources of the host.⁹

Methods for the in vitro cultivation of the parasite were advanced significantly in the recent years. The parasite can develop in a variety of epithelial cell lines including human enterocyte lines (e.g. Caco 2, HT-29)^10,11 and a similar murine cell line (CMT-93)¹² Parasite development is limited, but in vitro, growth is enough to support a variety of research studies. Routine methods for parasite preparation and cell culture infection assays for parasite development have been established, however. C. parvum oocysts can be generated in rodent animal models¹³ but large-scale production is generally accomplished in neonatal livestock, especially bovine calves and goat kids.¹⁴ Several methods have been described for purification, concentration and surface sterilization of oocysts.¹⁵ Oocysts generated in animals have limited durability, with infectivity markedly diminished within 6-8 weeks for in vitro and in vivo studies. Moreover, in vitro models enable the amplification of parasitic material for further immunological, biochemical and molecular studies.^{15, 16} Parasite development in cell culture can be assessed microscopically with a variety of histological stains or immunofluorescence¹⁷ and by enzyme immunoassay¹⁸ or PCR assays.¹⁹

The study aimes to evaluate and validate in vitro models of C. parvum and to establish an in vitro culture system for the parasite for further research studies.

MATERIALS AND METHODS

All plastic consumables (e.g. flasks, pipettes and tissue culture plates) were purchased from Vacsera Vaccination Centers, Cairo, Egypt.

        HT-29: Human Colorectal Adenocarcinoma cell line was obtained from Cell Culture Lap, Faculty of Pharmacy, Al-Azhar University, Cairo. Dulbecco’s modified Eagle medium (DMEM), heat-inactivated fetal bovine serum,  penicillin-streptomycin, glutamine, nonessential amino acids, phosphate-buffered saline (PBS), trypsin and all other chemicals were obtained from Vacsera Vaccination Centers.

         HT-29: Human Colorectal Adenocarcinoma cell lines were grown in DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 4 mM glutamine, 100 U/ml penicillin, 100mg/ml streptomycin and 1% non-essential amino acids in a 5% CO2 and incubated at 37oC. For experiments, trypsinized cells were seeded into 24- well plastic tissue culture plates with 13mm diameter glass coverslips and grow to confluence over 5 days. All experiments were done in triplicate.²⁰

         C. parvum oocysts were obtained from Theodor Bilharz Research Institute (TBRI) Cairo, Egypt. Before use, oocysts will surface sterilized by suspension in 10% commercial bleach solution (0.55% sodium hypochlorite) for 10 minutes and subsequently washed 3 times in DMEM before enumeration in a Neubauer hemocytometer.²¹

Infection of cell lines with oocysts.

          Confluent cell monolayers were infected with 4X105 viable C. parvum oocysts suspended in 200 ml cell culture medium. Cells were then incubated for 2 hours at 37°C to allow excystation and host cell invasion to occur. Following this period, unattached parasites and oocyst debris were removed by washing twice with the medium.

Wells were subsequently filled with fresh medium and incubated at 37°C for the stated time.²¹        

Cell monolayers that grown on 13 mm glass coverslips were washed twice with PBS to remove any traces of medium and fixed in methanol for 1 min, stained with 10% Giemsa (diluted in PBS, pH 7.2) for 2 h, washed in de-ionized water and then mounted on glass slides. Using a Zeiss Axioplan microscope at X1000 magnification with oil-immersion the parasites were counted in 20 fields across the diameter of the coverslip starting from the periphery. ²¹  

      The obtained data were expressed as means ± SD. Significance analysis was carried out using a two-tailed Student’s t-test for unpaired means.

RESULTS

In vitro development of c. parvum in intestinal epithelial cells showed that; confluent monolayers of HT-29 to which C. parvum oocysts were added, fixed and stained with Giemsa at different periods (6, 24, 48 and 72 hours) after incubation at 37oC. The tested cell line (HT-29) was found to support C. parvum growth in vitro. (Figure 1)

Infection of cell monolayers by different concentrations of oocysts

The maximal number of parasites was obtained by using 4x10⁵ oocysts with no significant difference in infection observed between 4x10⁵ and 1x10⁵oocysts (p>0.05). Fewer parasites were obtained when used 2x10⁴ compared with 1x10⁵(p<0.02) and 4x10⁵ (p<0.008) compared with 24h.  (Figure 4)

Fig. 4: C. parvum infection in HT-29 cells using variant numbers of viable oocysts. Infection of cell monolayer by using 4x10⁵ oocysts produced the highest numbers of parasites with no significant difference in infection observed between 4x10⁵ and 1x10⁵oocysts (p >0.05). Fewer parasites were obtained when used 2x10⁴ compared with 1x10⁵ (p <0.02) and 4x10⁵ (p <0.008).

DISCUSSION

A key goal of this study was to confirm the development of C. parvum in the intestinal epithelial cell line. The present study was shown that C. parvum can invade and develop within HT-29. However, only asexual development was predominantly observed with gametes being observed only occasionally. Complete development of C. parvum in vitro has been previously described ^{1, 5} and parasite growth was maintained for up to 25 days by multiple sub-culturing. It appears that adjustment of pH (7.2 -7.6) by the regular change of culturing media at 2-3 days intervals and adding HEPES buffer may be the key to successful long-term in vitro cultivation of C. parvum.¹⁵Also, thin-walled oocysts are required to maintain parasite growth by releasing sporozoites that infect new cells (autoinfection) and it was reported that thin-walled oocysts occurred in vitro after the passage of parasite for 12 days.¹⁵Lack of thick-walled oocysts may reflect the absence of certain enzymes and hormones that are present in vivo or a lack of effective host immune response that may induce the parasite to produce this type of oocysts.^{15, 22} For my thesis research, this long term cultivation was not essential.

        In the present study, the maximal number of parasites was obtained at 6-24h and development had decreased by 48-72h of infection. Only immature stages were showed after 6h of infection, but many immature and asexual mature stages were observed in the cells 24 and 48h post-infection as shown in fig. (3a- c). C. parvum developmental stages were easily identified by Giemsa staining in HT-29 cells. Interestingly, infection of cell monolayers by different concentrations of C. parvum oocyst reveals that the maximal number of parasites was obtained by using 4x10⁵ oocysts with no significant difference in infection observed between 4x10⁵ and 1x10⁵oocysts. Taken together, these results recommend the use of HT-29 cells to support the study of C. parvum development in vitro.

        Having demonstrated that C. parvum grows in the HT-29 cell line, further investigation of parasite interactions with host cells was possible using these in vitro models of infections.

CONCLUSION

It was concluded that the HT-29 cell line supports the in vitro cultivation of C. parvum protozoan parasite.