Th slight modifications [8,10]. Briefly: 0, no lesion; 1, inflammation and/or regenerative changes; 2, low grade intraepAdenocarcinoma Induced by Low Doses of C. parvumTable 1. Experimental cryptosporidiosis in SCID mice: influence of inoculum size on infectivity and histopathological findings.Group of miceIntended infective oocyst doseMean of infective oocysts after verification of prepared doses (SD)Number of infected mice/total mice per groupe ( )Histopathological findings: score of severitya Stomach Ileo-caecal area 2 to 3 2 to 3 3 3 to 4 01 2 3 4 5bc1 10 100 100,000 03.6+1.8 11.6+3.6 47.2+25 ND ND ND2/7 (28.5) 6/8 (75)d2 to 5 2 to 4 3 to 5 3to 5 07/7 (100)e 4/4 (100) 0/3 (0)f 0/3 (0)fEuthanasia was done 45 to 100 days P.I. a 0, no lesion; 1, inflammation and/or regenerative changes; 2, low grade intraepithelial neoplasia (LGIEN); 3, high grade intraepithelial neoplasia (HGIEN), carcinoma in situ (limited to the epithelium) or intramucosal adenocarcinoma (invasion into the lamina propria through the basement membrane of glands). 4, submucosal adenocarcinoma when glands penetrate through the muscularis mucosa; 5, invasive adenocarcinoma with the invasion through the muscularis into the RE 640 subserosa. b Inoculation with PBS. c Inoculation with 105 heat-inactivated oocysts. d One mouse found dead on day 2 P.I. e One mouse found dead on day 5 P.I. f One mouse found dead on day 1 P.I. ND: Note done. doi:10.1371/journal.pone.0051232.tithelial neoplasia (LGIEN); 3, high grade intraepithelial neoplasia (HGIEN), carcinoma in situ (limited to the epithelium) or intramucosal adenocarcinoma (invasion into the lamina propria through the basement membrane of glands). 4, submucosal adenocarcinoma when glands penetrate through the muscularis mucosa; 5, invasive adenocarcinoma with the invasion through the muscularis into the subserosa. The following histochemical and immunohistochemical analyses were performed using the BenchMark XT staining module (Ventana medical system, Meylan, France). The Volgens-Gomori stain (Reticulin) [13] was employed for assessment of basement membrane integrity. A mouse monoclonal antibody to cytokeratin (Arg8-vasopressin undiluted) (AM071-5 M; Biogenex, Netherlands) was used to mark epithelial cells. Muscle fibers were stained using an anti-alpha smooth muscle actin monoclonal antibody (dilution 1:100) (M0851; Dako, Denmark). Sections were examined using a Leica DMRB microscope equipped with a Leica digital camera connected to an Imaging Research MCID analysis system (MCID software, Cambridge, United Kingdom).Quantification of parasites in mouse tissueDNA extraction from formalin-fixed paraffin-embedded tissue samples. Paraffin embeded tissues from ileo-caecalregion from 17 mice were available for molecular analysis. DNA was extracted from a mixture of 2 sections of 25 mm of each tissue block. Histologic sections were processed by using xylene and ethanol for paraffin removal and were then rehydrated. To disrupt the oocysts, the samples were frozen (280uC, 5 min) and thawed (99uC, 4 min) six times and were at last sonicated during 1 min. DNA was then extracted using the NucleoSpin tissue (Machery Nagel, Duren, Germany) following the manufacturer instructions ?except that the proteinase K digestion was performed overnight. Real time quantitative PCR (qPCR) assays. Two TaqMan systems were developed: the Cryptosporidium Taqman assay and the in-house mouse Taqman assay. The primers and TaqMan probe used for the Cryptosporidium qPCR assay were t.Th slight modifications [8,10]. Briefly: 0, no lesion; 1, inflammation and/or regenerative changes; 2, low grade intraepAdenocarcinoma Induced by Low Doses of C. parvumTable 1. Experimental cryptosporidiosis in SCID mice: influence of inoculum size on infectivity and histopathological findings.Group of miceIntended infective oocyst doseMean of infective oocysts after verification of prepared doses (SD)Number of infected mice/total mice per groupe ( )Histopathological findings: score of severitya Stomach Ileo-caecal area 2 to 3 2 to 3 3 3 to 4 01 2 3 4 5bc1 10 100 100,000 03.6+1.8 11.6+3.6 47.2+25 ND ND ND2/7 (28.5) 6/8 (75)d2 to 5 2 to 4 3 to 5 3to 5 07/7 (100)e 4/4 (100) 0/3 (0)f 0/3 (0)fEuthanasia was done 45 to 100 days P.I. a 0, no lesion; 1, inflammation and/or regenerative changes; 2, low grade intraepithelial neoplasia (LGIEN); 3, high grade intraepithelial neoplasia (HGIEN), carcinoma in situ (limited to the epithelium) or intramucosal adenocarcinoma (invasion into the lamina propria through the basement membrane of glands). 4, submucosal adenocarcinoma when glands penetrate through the muscularis mucosa; 5, invasive adenocarcinoma with the invasion through the muscularis into the subserosa. b Inoculation with PBS. c Inoculation with 105 heat-inactivated oocysts. d One mouse found dead on day 2 P.I. e One mouse found dead on day 5 P.I. f One mouse found dead on day 1 P.I. ND: Note done. doi:10.1371/journal.pone.0051232.tithelial neoplasia (LGIEN); 3, high grade intraepithelial neoplasia (HGIEN), carcinoma in situ (limited to the epithelium) or intramucosal adenocarcinoma (invasion into the lamina propria through the basement membrane of glands). 4, submucosal adenocarcinoma when glands penetrate through the muscularis mucosa; 5, invasive adenocarcinoma with the invasion through the muscularis into the subserosa. The following histochemical and immunohistochemical analyses were performed using the BenchMark XT staining module (Ventana medical system, Meylan, France). The Volgens-Gomori stain (Reticulin) [13] was employed for assessment of basement membrane integrity. A mouse monoclonal antibody to cytokeratin (undiluted) (AM071-5 M; Biogenex, Netherlands) was used to mark epithelial cells. Muscle fibers were stained using an anti-alpha smooth muscle actin monoclonal antibody (dilution 1:100) (M0851; Dako, Denmark). Sections were examined using a Leica DMRB microscope equipped with a Leica digital camera connected to an Imaging Research MCID analysis system (MCID software, Cambridge, United Kingdom).Quantification of parasites in mouse tissueDNA extraction from formalin-fixed paraffin-embedded tissue samples. Paraffin embeded tissues from ileo-caecalregion from 17 mice were available for molecular analysis. DNA was extracted from a mixture of 2 sections of 25 mm of each tissue block. Histologic sections were processed by using xylene and ethanol for paraffin removal and were then rehydrated. To disrupt the oocysts, the samples were frozen (280uC, 5 min) and thawed (99uC, 4 min) six times and were at last sonicated during 1 min. DNA was then extracted using the NucleoSpin tissue (Machery Nagel, Duren, Germany) following the manufacturer instructions ?except that the proteinase K digestion was performed overnight. Real time quantitative PCR (qPCR) assays. Two TaqMan systems were developed: the Cryptosporidium Taqman assay and the in-house mouse Taqman assay. The primers and TaqMan probe used for the Cryptosporidium qPCR assay were t.
