RESUMEN
A polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of a 587-bp region of the Cryptosporidium parvum 70-kDa heat shock protein (HSP70) gene was developed for the detection and discrimination of the two major genotypes of C. parvum, genotype 1 and genotype 2. Ten Cryptosporidium isolates from non-immunocompromised people were identified as genotypes 1 and 2 (five each) by DNA sequencing of the 587-bp PCR product. This distinction was also achieved with the combination of two endonucleases, HinfI and ScaI, which generated a specific pattern for each genotype. A thorough screening of published sequences showed that this combination of enzymes could also be used for the discrimination of other species/genotypes of Cryptosporidium, especially Cryptosporidium meleagridis and the 'dog' genotype of C. parvum, both of which are infectious in humans. The PCR, conducted on genotypes 1 and 2 of C. parvum, could detect one oocyst per reaction. This new and sensitive genotyping procedure should be of particular interest when applied to the monitoring of water resources in which low concentrations of parasites usually occur.
Asunto(s)
Cryptosporidium parvum/clasificación , Cryptosporidium parvum/genética , Genes Protozoarios/genética , Proteínas HSP70 de Choque Térmico/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Criptosporidiosis/parasitología , Cartilla de ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Genotipo , Proteínas HSP70 de Choque Térmico/clasificación , Humanos , Mapeo RestrictivoRESUMEN
Two mRNA extraction methods were compared in this study to clarify the discrepancies found between authors regarding the presence of mRNA in inactivated Cryptosporidium parvum oocysts. Cryptosporidium parvum heat shock protein 70 (hsp70) mRNA extraction was performed by using oligo(dT)20-labeled magnetic beads or by incubating oocyst lysates with DNase I. Significant differences in mRNA recovery rates between these 2 techniques were observed when working on inactivated oocysts. We consistently detected hsp70 mRNA in oocysts heated at 60 C for 30 min and oocysts incubated in 10% formalin for 2 hr when using DNase I in the mRNA extraction procedure. In contrast, no mRNA was detected in such oocysts when magnetic beads were used for the mRNA extraction. The selective capture of long poly-A tail mRNA, when using oligo(dT)20-labeled magnetic beads, is proposed in this paper for explaining the discrepancies observed between the two mRNA extraction methods compared in this study. DNA decay in inactivated and aging oocysts makes quantitative polymerase chain reaction a potential alternative technique for assessing C. parvum oocyst viability status in environmental samples.
Asunto(s)
Criptosporidiosis/parasitología , Cryptosporidium parvum/crecimiento & desarrollo , Cryptosporidium parvum/genética , Proteínas HSP70 de Choque Térmico/genética , ARN Mensajero/genética , Animales , Niño , Proteínas HSP70 de Choque Térmico/metabolismo , Calor , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of the Cryptosporidium genome were evaluated for the detection of C. parvum, the agent of human cryptosporidiosis, and C. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridis couldn't be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.
Asunto(s)
Cryptosporidium parvum/genética , Cryptosporidium parvum/aislamiento & purificación , Cryptosporidium/genética , Cryptosporidium/aislamiento & purificación , ADN Protozoario/genética , Reacción en Cadena de la Polimerasa/métodos , Animales , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Cryptosporidium/clasificación , Cryptosporidium parvum/patogenicidad , Cartilla de ADN/genética , Genes Protozoarios , Humanos , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y Especificidad , Especificidad de la Especie , Microbiología del AguaRESUMEN
We developed a PCR-based method that can be used to identify Cryptosporidium parvum in human feces. Fecal oocysts were concentrated by centrifugation on a sodium chloride gradient and filtration on a nitrocellulose filter prior to DNA extraction and PCR amplification of a 452-bp C. parvum-specific DNA sequence with a protocol including dUTP and uracil-N-glycosylase. All samples obtained from naturally infected humans (n = 10), calves (n = 4), and goats (n = 2) were positive. A 100% detection rate was achieved with both formed and solid stools (n = 10) seeded with 1,000 C. parvum oocysts per g. Procedures based on stool concentration by a modified Ritchie method and subsequent oocyst identification by immunofluorescent labeling or acid-fast staining require concentrations of 50,000 to 500,000 oocysts per g to achieve a 100% detection rate with formed stools. The described PCR-based assay thus has a 50- to 500-fold increase in sensitivity compared to those of the methods commonly used to analyze formed feces.
Asunto(s)
Cryptosporidium parvum/aislamiento & purificación , ADN Glicosilasas , Heces/parasitología , Reacción en Cadena de la Polimerasa/métodos , Animales , Humanos , N-Glicosil Hidrolasas , Uracil-ADN GlicosidasaRESUMEN
In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.