A NUDE-MOUSE MODEL AS AN INVIVO INFECTIVITY ASSAY FOR CRYPTOSPORIDIOSIS

Cryptosporidium parvum is an important pathogen of diarrheal disease which has been implicated in several outbreaks associated with contamination of surface waters. In monitoring for C. parvum in drinking water sources, it is important to ascertain the viability, and more importantly, the infectivity of low numbers of recovered oocysts. Groups of 10 Balb/C nude (nu/nu) mice, 4-8 weeks old at time of inoculation, were infected with C. parvum oocysts from naturally infected calves and purified using Sheather's sucrose gradients. Oocysts were counted using the Merifluor IFA kit (Meridian). Each group of 1 0 mice were infected with 1, 10, 100 and 1000 oocysts respectively. Numbers of oocysts per inoculation were determined by limiting dilution, and parallel inocula were counted microscopically to ascertain the accuracy of the dilutions. Two uninfected nude mice were kept in each cage to serve as controls. Mouse stools were collected every 4 days, concentrated using the Fekal Kontrate Concentration Kit (Meridian) and oocysts were counted with a UV microscope using the Merifluor IFA Kit (Meridian). Oocyst counts were expressed in terms of number of oocyst/g feces. Mice inoculated with 1000 oocysts began to shed oocysts on day 32, mice inoculated with 100 oocysts began to shed on days 44-48, mice inoculated with 10 oocysts began to shed on days 56-60, and mice inoculated with 1 oocyst shed on days 68-88. All infected mice continued to shed oocysts intermittently and with variable oocyst counts until day 180 when the experiment was terminated. This study established that it is possible to infect nude mice with very low numbers, down to a single oocyst. We are currently in the process of correlating the nude mouse assay with other viability assays.