Of Cryptosporidium DNA in ileo-caecal tissues, 17 mice were selected. The standard

Of Cryptosporidium DNA in ileo-caecal tissues, 17 mice were selected. The standard curves generated for both Cryptosporidium and beta-actin showed a reproducible linear relationship between the Ct value and the log transformed number of copy over almost five orders of magnitude of DNA dilution. Correlation coefficient obtained by linear regression analysis of three independent experiments was R2 = 0.99 for both Cryptosporidium and mouse plasmids. DNA amplification efficiencies were 99 for Cryptosporidium and 89 for mouse. Standard curves were also performed with Cryptosporidium genomic DNA as well as mouse genomic DNA. For both, plotting of the delta Ct values (Ct plasmid DNA – Ct genomic DNA) against the logarithm of the dilution factors resulted in a curve slope lower than 0.1 (data not shown), demonstrating that plasmid DNA could be used to quantify genomic DNA. The number of Cryptosporidium and the amount of murine DNA present in each sample were quantified by interpolation of the corresponding Ct values in the standard curves for Cryptosporidium DNA and for the 15481974 murine beta-actin gene. Levels of parasite DNA in Anlotinib tissues of studied animals are shown in Table S1. In total, 14 out of 15 studied inoculated mice were colonized with Cryptosporidium. The parasite load in tissues of mice inoculated with get Solvent Yellow 14 higher inoculum (105) was higher when compared to mice inoculated with low doses (#100 oocysts) (p,0.001). However, when comparing tissue loads at the same time P.I. (45 days) between the lowest and the maximal inoculum, mouse Nu12, inoculated with 100,000 times more oocysts than mouse Nu1, had only 3.6 fold more parasite loads. No Cryptosporidium DNA was found in one mouse (Nu7) inoculated with 10 oocysts (Table S1). This mouse developed neither infection nor neoplastic lesions, as confirmed also by IMS-DFA. For 6 samples, the Cryptosporidium qPCR was not positive for all 3 replicates assuming a Poisson distribution of template when detecting very low copy numbers of the target. For such samples, the obtained Ct values were between 39 and 40 signifying that the PCR reaction contained theoretically 1 genome copy. In fact, the detection limit of the assay was reached and we could not attempt quantification with an acceptable level of accuracy and reliability. Runs of Cryptosporidium qPCR were tested with samples at a lower dilution point in order to obtain lower Ct values. Unlikely, they were not validated due to a negative PCR result (Ct absence) or because the average shifts in Ct did not produce the expected change (respecting the 10-fold dilution). Histological lesions were always associated with the presence of parasites as it was observed by microscopy (Figures 2B, 2D) and qPCR (Table S1). The DNA detection of parasites through qPCR corroborates that even mice with lowest parasites loads in tissues had neoplastic lesions. Neither parasites nor lesions were detectedAdenocarcinoma Induced by Low Doses of C. parvumFigure 1. Pattern of oocyst shedding (oocyst/g feces) of Dex-treated SCID mice. Experimental groups were inoculated with intended doses of 1, 10, 100 and 105 oocysts. Each point represents one mouse, the lines being the geometric means of oocyst shedding per group. Only animals with oocyst shedding at a precise moment of the day are represented. None of mice infected with one oocyst released parasites until day 15 (see material and methods for oocyst shedding assessment). doi:10.1371/journal.pone.0051232.gin negative control gro.Of Cryptosporidium DNA in ileo-caecal tissues, 17 mice were selected. The standard curves generated for both Cryptosporidium and beta-actin showed a reproducible linear relationship between the Ct value and the log transformed number of copy over almost five orders of magnitude of DNA dilution. Correlation coefficient obtained by linear regression analysis of three independent experiments was R2 = 0.99 for both Cryptosporidium and mouse plasmids. DNA amplification efficiencies were 99 for Cryptosporidium and 89 for mouse. Standard curves were also performed with Cryptosporidium genomic DNA as well as mouse genomic DNA. For both, plotting of the delta Ct values (Ct plasmid DNA – Ct genomic DNA) against the logarithm of the dilution factors resulted in a curve slope lower than 0.1 (data not shown), demonstrating that plasmid DNA could be used to quantify genomic DNA. The number of Cryptosporidium and the amount of murine DNA present in each sample were quantified by interpolation of the corresponding Ct values in the standard curves for Cryptosporidium DNA and for the 15481974 murine beta-actin gene. Levels of parasite DNA in tissues of studied animals are shown in Table S1. In total, 14 out of 15 studied inoculated mice were colonized with Cryptosporidium. The parasite load in tissues of mice inoculated with higher inoculum (105) was higher when compared to mice inoculated with low doses (#100 oocysts) (p,0.001). However, when comparing tissue loads at the same time P.I. (45 days) between the lowest and the maximal inoculum, mouse Nu12, inoculated with 100,000 times more oocysts than mouse Nu1, had only 3.6 fold more parasite loads. No Cryptosporidium DNA was found in one mouse (Nu7) inoculated with 10 oocysts (Table S1). This mouse developed neither infection nor neoplastic lesions, as confirmed also by IMS-DFA. For 6 samples, the Cryptosporidium qPCR was not positive for all 3 replicates assuming a Poisson distribution of template when detecting very low copy numbers of the target. For such samples, the obtained Ct values were between 39 and 40 signifying that the PCR reaction contained theoretically 1 genome copy. In fact, the detection limit of the assay was reached and we could not attempt quantification with an acceptable level of accuracy and reliability. Runs of Cryptosporidium qPCR were tested with samples at a lower dilution point in order to obtain lower Ct values. Unlikely, they were not validated due to a negative PCR result (Ct absence) or because the average shifts in Ct did not produce the expected change (respecting the 10-fold dilution). Histological lesions were always associated with the presence of parasites as it was observed by microscopy (Figures 2B, 2D) and qPCR (Table S1). The DNA detection of parasites through qPCR corroborates that even mice with lowest parasites loads in tissues had neoplastic lesions. Neither parasites nor lesions were detectedAdenocarcinoma Induced by Low Doses of C. parvumFigure 1. Pattern of oocyst shedding (oocyst/g feces) of Dex-treated SCID mice. Experimental groups were inoculated with intended doses of 1, 10, 100 and 105 oocysts. Each point represents one mouse, the lines being the geometric means of oocyst shedding per group. Only animals with oocyst shedding at a precise moment of the day are represented. None of mice infected with one oocyst released parasites until day 15 (see material and methods for oocyst shedding assessment). doi:10.1371/journal.pone.0051232.gin negative control gro.