Giardia and Cryptosporidium are intestinal protozoan parasites that infect a wide range of host species, including humans. DNA sequencing of Giardia and Cryptosporidium isolates from human and animal sources has identified numerous species and genotypes, and has demonstrated that a number of Giardia and Cryptosporidium genotypes are shared between animals and humans. Therefore, livestock may act as a source of contamination of the food and water supply. The goal of this study was to optimize methods for the detection and molecular characterization of Giardia and Cryptosporidium. Achieving this objective involved the incorporation of immunomagnetic separation, as well as the evaluation of different methods, including microscopy, flow cytometry and polymerase chain reaction. A number of faecal samples from adult cattle and calves were collected from farms in Ontario and Prince Edward Island (PEI). Following DNA extractions from stool samples, a nested-PCR was used for Giardia to amplify a fragment of the 18S rRNA gene generating a 292-bp product. Nested-PCR protocol was also used for Cryptosporidium to amplify fragments of the heat-shock protein 70 (HSP-70) gene (ca. 325 bp). For Giardia, of 143 cattle samples analyzed by PCR, 32 (22.4 %) were positive. When IMS was incorporated into the methodology, 64 out of 143 (44.8 %) samples analyzed were positive for Giardia. For Cryptosporidum, out of 143 cattle faecal samples analyzed by the PCR method using the HSP-70 gene, 58 were positive (40.6 %), while using IMS, plus PCR, 60 samples were positive (42 %). Results from this study indicated that incorporation of IMS significantly improve the sensitivity of PCR for the detection of both Giardia (p<0.01) and Cryptosporidium (p=0.02). Among the other genes that were targeted, including the beta-giardin gene and glutamate dehydrogenase (GDH) for Giardia, and the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA for Cryptosporidium, the 18S rRNA for Giardia , and HSP-70 for Cryptosporidium were found to be the "best genes". When different methods, including microscopy, flow cytometry and PCR-IMS were compared, the PCR method showed the highest sensitivity in detecting both parasites. Genotyping done by DNA sequencing showed that there was a high prevalence of zoonotic genotypes (Assemblage A for Giardia , and C. parvum bovine genotype for Cryptosporidium ) among the samples from both PEI and Ontario. In addition, a temporal study was done on calf samples from Ontario and showed that over time there was a decrease in Cryptosporidium infections, concomitant with an increase in Giardia infections.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/27823 |
| Date | January 2007 |
| Creators | Coklin, Tatjana |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 144 p. |
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