gCryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidiumpositive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

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Ninety-two Cryptosporidium sp.-positive fecal samples of dairy diarrheic or non-diarrheic calves from 30 cattle herds in Normandy (France) were selected. Here, the aim was to investigate the species of Cryptosporidium excreted as well as the subtypes of Cryptosporidium parvum found in 7-17-day-old dairy calves. Excretion levels were comprised between 2 × 10(4) and 4 × 10(7) oocysts per gram of feces. Here, a nested 18S SSU rRNA PCR associated with sequencing was performed for identification of Cryptosporidium species and revealed the presence of C. parvum in most cases (80/82), except for two animals which were infected with Cryptosporidium bovis. Then, C. parvum samples were submitted to gp60 PCR. For 39 samples from 24 different herds, a multilocus analysis based on four mini-microsatellites loci (MM19, MM5, MSF, and MS9-Mallon) were conducted. These results were combined with sequence analysis of the gp60 to obtain multilocus types (MLTs). Here, C. parvum gp60 genotyping identified three subtypes in the IIa zoonotic allele family: IIaA15G2R1 (88%), IIaA16G3R1 (10%), and IIaA19G2R1 (2%), and we identified 12 MLTs. The MS9-Mallon locus was reported as the most polymorphic (five alleles). The most common MLT was MLT 1 with 15 samples in 10 farms: (MS9-M: 298, MSF: 165, MM5: 264, MM19: 462, and gp60 subtype: IIaA15G2R1). When comparing diarrheic and non-diarrheic fecal samples, no difference was seen for distribution of Cryptosporidium species, C. parvum gp60 subtypes, and MLTs. Here, in a range of oocyst excretion of 10(4)-10(7) opg, both in diarrheic and non-diarrheic calves, infection was mainly due to C. parvum and to the zoonotic subtype: IIaA15G2R1.

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The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.