AWWA WQTC62401

Oocysts of Cryptosporidium species are frequently found in surface water.Detection methods, such as US Environmental Protection Agency Method 1623, are based on immunofluorescence andtherefore do not discriminate between infectious and dead oocysts. Consequently, testingdoes not provide information on the risk of waterborne oocysts to the consumers. Theinfection of cell monolayers with C. parvum and C. hominis, the two Cryptosporidiumspecies responsible for most human infections, are commonly used as alternatives toanimal models to determine the infectivity of oocysts (Rochelle et al., 2002). To alimited extent, cell culture mimics the interaction between host cells and parasites in vivoand is the only known method which can discriminate between infectious and deadoocysts without relying on laboratory animals. An advantage of cell culture over themouse model, is that infectious oocysts of both species, C. parvum and C. hominis, canbe cultured, whereas C. hominis is not infectious to mice. The limitation of the culturemethod is that Cryptosporidium development is transient and continuous propagation isnot commonly achieved (Hijjawi et al., 2001). The reason for the temporally limitedproliferation of the parasite in cell monolayers is unknown, but the lack of sexualdifferentiation and the death of infected cells are some of the proposed explanations. Thepractical consequence of the lack of parasite proliferation, is that small numbers ofinfectious oocysts present in a sample are difficult to detect.We tested different modifications of a standard culture method in humanepithelial cell monolayer for their effect on growth of C. parvum. Whereas none of thesemethods supported a sustained increase in parasite numbers, we found that not all cells ina 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 ofinfected cells in a monolayer, parallel monolayers were infected with oocysts at a 1:1 hostcell-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 fourfoldexcess of parasites over cells. Because the proportion of oocysts which do notrelease sporozoites is unknown, the exact cell-to-sporozoite ratio is also unknown. Cellsuspensions double labelled with an anti-Cryptosporidium polyclonal antibody (Alexa488) and propidium iodide (PI) were analyzed by flow cytometry and infected cellsidentified by flow cytometry on a FL1 vs FL3 bivariant plot. Each infected monolayerwas then compared with its mock infected control and the infected cell populationdelineated by comparing the distribution of FL1 signals in the corresponding plots andFL1 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 correlationbetween oocyst age and prevalence of infected cells was not significant, indicating thatoocyst age did not influence the results.To investigate the effect of increasing oocyst doses on the infection of HCT-8monolayers, parallel monolayers were infected with 10-fold and 100-fold incrementedoocyst doses and analyzed at four and seven days post-infection. The experiment wasdesigned to test the relationship between infectious dose and parasite density in themonolayer and specifically to determine whether infections with incremented oocystdoses would result in proportionally higher parasite densities. The lowest dose wasequivalent to 1 oocyst per 10,000 cells, and was chosen intentionally low to enableparasite growth over the 4- and 7-day experimental period. Increasing oocyst doses resulted in higher parasite densities, however the increment wasnot proportional to the dose, even when low oocyst-to-cell ratios of 10-4 or 10-3 were used. Includes 12 references, table, figu

Product Details

Edition:

Vol. - No.

Published:

11/01/2005

Number of Pages:

6

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1 file , 680 KB