The mock-immunized group that received an AJ challenge were reduced to two mice in the group because of a technical error during challenge. The resulting blood-stage infections were followed by microscopic examination of Giemsa’s solution-stained thin blood smears taken daily using venous blood from the tail. In order to determine the day at which parasites first became detectable in the blood, at least 10 000 red blood cells were examined per smear. For the generation of sporozoites, Anopheles stephensi mosquitoes were allowed to feed on anaesthetized mice that had been inoculated with 1 × 106 iRBCs IP 6 days

previously. Prior to feeding, mouse blood was checked for this website the presence of gametocytes, and their viability assessed by the observation of exflagellation of microgametocytes in fresh blood CP-690550 mw preparations. Seven to 10 days post-feed, mosquito mid-guts

were dissected and the presence of oocysts confirmed. Sixteen days post-feed, mosquito salivary glands were dissected into a 50 : 50 solution of FCS and Ringer’s solution, crushed in a glass and Teflon tissue homogeniser, and the numbers of sporozoites in the homogenate assessed by counting with a haemocytometer. In order to assess sporozoite viability, only those sporozoites displaying circular gliding motility were considered viable. There were no discernable differences in the viability of CB and AJ sporozoites, and sporozoites of both strains were handled in exactly

the same manner prior to immunization and challenge inoculation. All mice were kept on 0·05% para-aminobenzoic acid (PABA)-supplemented water ad libitum and were housed at 21°C on a 12 h-light–dark cycle. Anopheles stephensi mosquitoes were fed with 0·05% PABA-supplemented 10% glucose solution and were housed at 27°C and 70% humidity on a 12-h light–dark cycle. We used R version 2·7·0; The R Foundation for Statistical Computing; http://www.R-project.org) for data analysis. To analyse patterns of parasitaemia during infections, we used mixed effects models because, by treating each infection as a ‘random’ effect, we can account for repeated measures from each infection and overcome pseudoreplication problems associated with such data. These 4-Aminobutyrate aminotransferase models were fitted with Poisson error distributions and minimized following stepwise deletion of the least significant term, using log-likelihood ratio tests to evaluate the change in model deviance, until only significant terms remained. We present F-ratios for fixed effects remaining in minimal models. Mann–Whitney tests were used to compare patency data. Cumulative, summary data were analysed with linear models, using anova (F ratios) to evaluate significance of terms. The days on which parasites became detectable by microscopy (patent) in the blood of mice subjected to various immunization and challenge regimens are shown in Table 1.