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

Cryptosporidium parvum is an important pathogen of diarrheal disease w hich has been implicated in several outbreaks associated with contamin ation of surface waters. In monitoring for C. parvum in drinking water sources, it is important to ascertain the viability, and more importa ntly, the infectivity of low numbers of recovered oocysts. Groups of 1 0 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 pu rified 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 inocul a were counted microscopically to ascertain the accuracy of the diluti ons. Two uninfected nude mice were kept in each cage to serve as contr ols. Mouse stools were collected every 4 days, concentrated using the Fekal Kontrate Concentration Kit (Meridian) and oocysts were counted w ith a UV microscope using the Merifluor IFA Kit (Meridian). Oocyst cou nts were expressed in terms of number of oocyst/g feces. Mice inoculat ed 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 inter mittently and with variable oocyst counts until day 180 when the exper iment was terminated. This study established that it is possible to in fect nude mice with very low numbers, down to a single oocyst. We are currently in the process of correlating the nude mouse assay with othe r viability assays.