RESUMO
The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.
Assuntos
Criptosporidiose/parasitologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Água Doce/parasitologia , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Cryptosporidium/classificação , DNA de Protozoário/genética , DNA Ribossômico/genética , Genótipo , Proteínas de Choque Térmico HSP90/genética , Humanos , Proteínas de Protozoários/genética , RNA Ribossômico 18S/genéticaRESUMO
The biological basis for the specificity of host infectivity patterns of Cryptosporidium spp., in particular C. hominis and C. parvum, has yet to be fully elucidated. Comparison of the C. parvum and C. hominis P23 and GP900 predicted amino acid sequences revealed 3 differences in P23 and 4 and 17 differences in GP900 domains 1 and 5, respectively. Using monoclonal antibodies developed against the surface (glyco)proteins P23 and GP900 of the C. parvum Iowa isolate, solubilized glycoprotein from three C. hominis isolates was screened for reactivity using Western immunoblots. One of ten P23 MAbs and three of 21 GP900 MAbs were not reactive with any of the three C. hominis isolates. The non-reactive P23 MAb binds to a peptide epitope, while the non-reactive GP900 MAbs bind to either carbohydrate/carbohydrate-dependent or peptide epitopes of C. parvum. These results demonstrate phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/imunologia , Cryptosporidium/imunologia , Proteínas de Protozoários/imunologia , Substituição de Aminoácidos/genética , Animais , Western Blotting , Carboidratos/imunologia , DNA de Protozoário/química , DNA de Protozoário/genética , Epitopos/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Ligação Proteica , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosAssuntos
Cryptosporidium parvum/genética , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cryptosporidium parvum/isolamento & purificação , Genótipo , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Alinhamento de SequênciaRESUMO
Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.
Assuntos
Cryptosporidium parvum/isolamento & purificação , DNA de Protozoário/análise , Separação Imunomagnética/métodos , Animais , Imunofluorescência , Reação em Cadeia da PolimeraseRESUMO
Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.
