Intraspecies Polymorphism of Cryptosporidium parvum Revealed by PCR-Restriction Fragment Length Polymorphism (RFLP) and RFLP-Single-Strand Conformational Polymorphism Analyses
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ABSTRACT A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C . parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C . parvum .Keywords:
Cryptosporidium parvum
Single-strand conformation polymorphism
The single-strand conformation polymorphism(PCR-SSCP) method was used to analyze for polymorphism at the 5'-flanking region of the HSPA1A gene.The PCR-SSCP products of primer showed polymorphisms and could be divided into four genotypes: AA,AB,AC,CC among 70 Jersey and Holstein cows.The ac genotypic cows showed higher heat tolerant capacity than those of AA,AB,and BB genotype.These mutation sites can be used as molecular genetic markers to assist the selection for anti-heat stress cows.
Single-strand conformation polymorphism
Primer (cosmetics)
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PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts.
Cryptosporidium parvum
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ABSTRACT Healthy adults are susceptible to infection with small numbers of Cryptosporidium parvum oocysts, resulting in self-limited infection. We investigated if infection of humans with C. parvum is protective 1 year after primary exposure. At 1 year after a primary challenge with 30 to 10 6 oocysts, 19 healthy immunocompetent adults were rechallenged with 500 oocysts and monitored for the development of infection and/or illness. Oocyst excretion was quantitated by direct immunofluorescence with a C. parvum -specific monoclonal antibody, and anti- C. parvum antibodies in serum were detected by an enzyme-linked immunosorbent assay. Fewer subjects shed oocysts after the second exposure (3 of 19; 16%) than after the first exposure (12 of 19; 63%) ( P < 0.005). Although the rates of diarrhea were comparable after each of the two exposures, the clinical severity as determined by the mean number of unformed stools passed was lower after reexposure (11.25 versus 8.62; P < 0.05). The number of anti- Cryptosporidium immunoglobulin G and A seroconversions increased after secondary exposure. However, the C. parvum serum antibody response did not correlate with the presence or absence of infection.
Cryptosporidium parvum
Immunoglobulin M
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Cryptosporidium parvum
Infectivity
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Abstract Cryptosporidium parvum is an apicomplexan parasite that can cause serious watery diarrhea, cryptosporidiosis, in human and other mammals. C. parvum invades gastrointestinal epithelial cells, which have abundant glycosaminoglycans on their cell surface. However, little is known about the interaction between C. parvum and glycosaminoglycans. In this study, we assessed the inhibitory effect of sulfated polysaccharides on C. parvum invasion of host cells and identified the parasite ligands that interact with sulfated polysaccharides. Among five sulfated polysaccharides tested, heparin had the highest, dose-dependent inhibitory effect on parasite invasion. Heparan sulfate-deficient cells were less susceptible to C. parvum infection. We further identified 31 parasite proteins that potentially interact with heparin. Of these, we confirmed that C. parvum elongation factor 1α (CpEF1α), which plays a role in C. parvum invasion, binds to heparin and to the surface of HCT-8 cells. Our results further our understanding of the molecular basis of C. parvum infection and will facilitate the development of anti-cryptosporidial agents.
Cryptosporidium parvum
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Single-strand conformation polymorphism
Amplicon
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Although polymerase chain reaction (PCR) can sensitively detect parasitic or other infections, its use with fecal samples is extremely limited, primarily because of the presence of substances that inhibit DNA extension. Here an improved protocol is reported for directly isolating DNA from aged or fresh formalin-fixed stools, which can then be used to detect Cryptosporidium parvum by nested PCR. This method is highly reproducible, sensitive, and specific. It detects <1 pg of C. parvum DNA in human stool, and there are no cross-reactions with other parasites commonly found there.
Cryptosporidium parvum
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Removal of Cryptosporidium parvum oocysts and Cryptosporidium-sized microspheres was evaluated in full-scale swimming pools via high-rate sand filtration (31–34 m/h) with coagulation. Results showed that at least 90% of C. parvum oocysts and microspheres were removed by filtration with an initial dosage of coagulant B (1.56 mg/L), D (1.9 mg/L or 305 g/m2), or F (1.56 mg/L) from each swimming pool. Filtration with an initial dosage of coagulant E (0.1 mg·Al/L) achieved 82% C. parvum oocyst removal and 97% microsphere removal. Coagulants B and F had a tendency to overdose over time with continuous feeding (based on corresponding pilot-scale experiments) and did not consistently achieve removals greater than 90% in the full-scale trials. As high as 99% of C. parvum oocysts and 98% of microspheres were removed with a continuous dosage of coagulant D. Up to 98% (1.7 log) of C. parvum oocysts and 93% (1.1 log) of microspheres were removed by continuous dosing of coagulant E at 27 m/h. Consistent oocyst and microsphere removal by aluminum-based coagulants (D and E) was achieved under the tested swimming pool conditions.
Cryptosporidium parvum
Filtration (mathematics)
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Excystation ofCryptosporidium parvumat temperatures that are reached during solar water disinfection
SUMMARY Species belonging to the genera Cryptosporidium are recognized as waterborne pathogens. Solar water disinfection (SODIS) is a simple method that involves the use of solar radiation to destroy pathogenic microorganisms that cause waterborne diseases. A notable increase in water temperature and the existence of a large number of empty or partially excysted (i.e. unviable) oocysts have been observed in previous SODIS studies with water experimentally contaminated with Cryptosporidium parvum oocysts under field conditions. The aim of the present study was to evaluate the effect of the temperatures that can be reached during exposure of water samples to natural sunlight (37–50°C), on the excystation of C. parvum in the absence of other stimuli. In samples exposed to 40–48°C, a gradual increase in the percentage of excystation was observed as the time of exposure increased and a maximum of 53·81% of excystation was obtained on exposure of the water to a temperature of 46°C for 12 h ( versus 8·80% initial isolate). Under such conditions, the oocyst infectivity evaluated in a neonatal murine model decreased statistically with respect to the initial isolate (19·38% versus 100%). The results demonstrate the important effect of the temperature on the excystation of C. parvum and therefore on its viability and infectivity.
Cryptosporidium parvum
Infectivity
Contaminated water
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Cryptosporidium parvum
Infectivity
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