Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (
Feces from 142 animals were collected on 15 farms in the region of Brittany, France. Each sample was directly collected from the rectum of the animal and identified with the ear tag number. Animals were sampled three times, at 5, 15 and 22 weeks of age. After DNA extraction from stool samples, nested PCR was performed to amplify partial 18S-rDNA and 60 kDa glycoprotein genes of Cryptosporidium. The parasite was detected on all farms. One hundred out of 142 calves (70.4%) were found to be parasitized by Cryptosporidium. Amplified fragments were sequenced for Cryptosporidium species identification and revealed the presence of C. parvum (43.8%), C. ryanae (28.5%), and C. bovis (27%). One animal was infected with Cryptosporidium ubiquitum. The prevalence of these species was related to the age of the animal. C. parvum caused 86.7% of Cryptosporidium infections in 5-week-old calves but only 1.7% in 15-week-old animals. The analysis of the results showed that animals could be infected successively by C. parvum, C. ryanae, and C. bovis for the study period. C. parvum gp60 genotyping identifies 6 IIa subtypes of which 74.5% were represented by IIaA15G2R1. This work confirms previous studies in other countries showing that zoonotic C. parvum is the dominant species seen in young calves.
Genomic DNAs from humanCryptosporidium spp. are Apicomplexan parasites that infect the gastrointestinal or respiratory tract of humans and animals. In immunocompetent hosts, the infection is typically acute and self limiting, whereas in immunocompromised individuals, such as persons receiving immunosuppressive drugs and AIDS patients, cryptosporidiosis is often a chronic disease. Since drug therapy to control or eliminate these organisms is not yet available, persistent infections in these patients are therefore especially severe and can be life threatening. The potential of Cryptosporidium as an opportunistic parasite and the recent reports of major outbreaks of cryptosporidiosis in the United States, United Kingdom, and Australia due to contamination of drinkable water supplies indicate that Cryptosporidium should be considered a major public health problem (12,25).To date, eight Cryptosporidium species have been regarded as valid on the basis of host specificity, pathogenesis, and oocyst morphology (13). These included Cryptosporidium parvum in mammals, Cryptosporidium muris in rodents and ruminants, Cryptosporidium felis in domestic cats, Cryptosporidium wrairi in guinea pigs, Cryptosporidium baileyi and Cryptosporidium meleagridis in birds, Cryptosporidium serpentis in reptiles, and Cryptosporidium nasorum in fishes. According to this classification, the causative agent of cryptosporidiosis in humans and a range of mammalian species is the species C. parvum. Numerous PCR-based assays have been described for detection of Cryptosporidium parasites. The primers of these PCR assays are based on either undefined genomic sequences (2, 3, 22, 27, 46) or specific genes (4, 27-29, 35, 40, 41, 43, 45, 48, 49). Most PCR assays have led to the confirmation of C. parvum as the major cause of cryptosporidiosis in humans and to the identification of two genotypes within this species: the "human" genotype (genotype 1), which has so far been found exclusively in humans, with the exception of a single nonhuman primate (39) and a dugong (33), and the "bovine" genotype (genotype 2), found in domestic livestock such as cattle, sheep, and goats, etc., which can also infect humans. Additional genotypes have then been distinguished in C. parvum (32). However, most of the available genotyping tools were designed to analyze clinical specimens, and their specificities for other C. parvum genotypes or other Cryptosporidium species were not always established.Laxer et al. were among the first authors to publish primers for detection of Cryptosporidium (22). These primers, which were not known to target a specific gene but to amplify a 452-bp fragment of an unidentified region, as well as the reported 452-bp sequence, have been widely used (1, 6-11, 14, 15, 17, 18, 20, 21, 23, 37, 38). The purpose of the present study was to investigate the extent of sequence heterogeneity for this diagnostic DNA fragment among human isolates of Cryptosporidium.Sample analysis. Fecal samples used in this study were obtained from infected bovine (named...
Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived Pneumocystis. Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.
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