Oocysts of Cryptosporidium species are frequently found in surface water.
Detection methods, such as US Environmental Protection Agency Method 1623, are based on immunofluorescence and
therefore do not discriminate between infectious and dead oocysts. Consequently, testing
does not provide information on the risk of waterborne oocysts to the consumers. The
infection of cell monolayers with C. parvum and C. hominis, the two Cryptosporidium
species responsible for most human infections, are commonly used as alternatives to
animal models to determine the infectivity of oocysts (Rochelle et al., 2002). To a
limited extent, cell culture mimics the interaction between host cells and parasites in vivo
and is the only known method which can discriminate between infectious and dead
oocysts without relying on laboratory animals. An advantage of cell culture over the
mouse model, is that infectious oocysts of both species, C. parvum and C. hominis, can
be cultured, whereas C. hominis is not infectious to mice. The limitation of the culture
method is that Cryptosporidium development is transient and continuous propagation is
not commonly achieved (Hijjawi et al., 2001). The reason for the temporally limited
proliferation of the parasite in cell monolayers is unknown, but the lack of sexual
differentiation and the death of infected cells are some of the proposed explanations. The
practical consequence of the lack of parasite proliferation, is that small numbers of
infectious oocysts present in a sample are difficult to detect.
We tested different modifications of a standard culture method in human
epithelial cell monolayer for their effect on growth of C. parvum. Whereas none of these
methods supported a sustained increase in parasite numbers, we found that not all cells in
a monolayer become infected. This heterogeneity might be related to the mitotic cell cycle. A large proportion of monolayer cells remain uninfected. To assess the proportion of
infected cells in a monolayer, parallel monolayers were infected with oocysts at a 1:1 host
cell-to-oocyst ratio, or mock infected with the same number of heat inactivated oocysts.
Since each oocyst contains four sporozoites, this dose theoretically corresponds to a fourfold
excess of parasites over cells. Because the proportion of oocysts which do not
release sporozoites is unknown, the exact cell-to-sporozoite ratio is also unknown. Cell
suspensions double labelled with an anti-Cryptosporidium polyclonal antibody (Alexa
488) and propidium iodide (PI) were analyzed by flow cytometry and infected cells
identified by flow cytometry on a FL1 vs FL3 bivariant plot. Each infected monolayer
was then compared with its mock infected control and the infected cell population
delineated by comparing the distribution of FL1 signals in the corresponding plots and
FL1 histograms. The average proportion of infected cells was 11.9% (n=5; SD =4.6). At 48 hr post-infection 38.1% of the cells were infected in one experiment. The correlation
between oocyst age and prevalence of infected cells was not significant, indicating that
oocyst age did not influence the results.
To investigate the effect of increasing oocyst doses on the infection of HCT-8
monolayers, parallel monolayers were infected with 10-fold and 100-fold incremented
oocyst doses and analyzed at four and seven days post-infection. The experiment was
designed to test the relationship between infectious dose and parasite density in the
monolayer and specifically to determine whether infections with incremented oocyst
doses would result in proportionally higher parasite densities. The lowest dose was
equivalent to 1 oocyst per 10,000 cells, and was chosen intentionally low to enable
parasite growth over the 4- and 7-day experimental period.
Increasing oocyst doses resulted in higher parasite densities, however the increment was
not proportional to the dose, even when low oocyst-to-cell ratios of 10-4 or 10-3 were used. Includes 12 references, table, figu
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| Published : | 11/01/2005 |
