Aged HCT-8 cell monolayers support Cryptosporidium parvum infection

Abstract

Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

FIG. 1.

Average (±standard deviation [SD]) clusters of infection for experiment 1 with fresh and aged monolayers (n, number of replicate monolayers): 2 day old, n = 10; 8 day old, n = 16; 32 day old, n = 16; 38 day old, n = 8; 67 day old, n = 8.

FIG. 2.

Average (±standard deviation [SD]) clusters of infection for experiment 2 with fresh and aged monolayers (n, number of replicate monolayers): 2 day old, n = 15; 8 day old, n = 16; 11 day old, n = 13; 15 day old, n = 16.

FIG. 3.

Average (±standard deviation [SD]) clusters of infection for experiment 3 with fresh and aged monolayers (n, number of replicate monolayers): 2 day old, n = 8; 8 day old, n = 8; 16 day old, n = 8; 22 day old, n = 8.