Una Ryan

In this study, genetic variation in two medically important protozoan parasites Cryptosporidium and Giardia, was investigated using the novel PCR-based technique of RAPD (Random Amplified Polymorphic DNA) analysis. This was undertaken in order to obtain fundamental knowledge on the population structure and zoonotic potential of Giardia and Cryptosporidium, both of which are important to fully understand the epidemiology of these parasites. The RAPD technique was also applied to the development of diagnostic PCR primers for Giardia and Cryptosporidium in view of the limitations of currently available diagnostic procedures. Previous studies on Cryptosporidium have contributed little to our understanding of its population structure and zoonotic potential due to the lack of adequate in vitro cultivation techniques for laboratory cultivation and amplification, and the difficulties in obtaining sufficient quantities of the parasite from conventional analyses. A significant proportion of Giardia isolates are refractory to in vitro establishment and amplification and there is concern that the isolates of Giardia we study may be the result of laboratory-induced artificial selection. In addition, current detection and diagnosis of these parasites are time consuming and lack sensitivity. Therefore, the major aims of this work, were to: 1. evaluate the suitability of RAPDs as a technique for analysing genetic variation in Cryptosporidium and Giardia; 2. compare the level of variation generated by RAPD analysis and isoenzyme electrophoresis; 3. use the RAPD technique for the development of diagnostic PCR primers for both parasitic infections. Application of the RAPD technique to Cryptosporidium over a wide range of conditions showed the technique to be both reproducible and reliable and a suitable method for analysing variation in this parasite. RAPD analysis was applied to 37 isolates of Cryptosporidium from both human and animal hosts. This is the largest and most comprehensive study of genetic variability in Cryptosporidium to date. The analysis produced one major group within the animal isolates, while human isolates formed two major groups with the exception of a number of isolates including two human isolates which were grouped with the domestic animal isolates. The observed variation between human and animal isolates may suggest that distinct animal and human strains of C. parvum exist and that zoonotic transmission does not occur with all isolates of the parasite. Other possible explanations for the observed differences between human and animal isolates include chance selection of the relatively small number of isolates examined; host-adapted strains or sub-species which may normally circulate among livestock and humans but are, nonetheless, able to cross-infect other hosts; or some form of host determined clonal selection. The occurrence of RAPD genotypes found in distant localities and at different times tends to suggest that Cryptosporidium may have a clonal population structure. More extensive studies in localised endemic areas are required RAPD analysis on isolates of Giardia correlated well with isoenzyme analysis of the same isolates, indicating that RAPD is a useful technique for population genetics and molecular epidemiological studies on Giardia and giardiasis. It is particularly useful when starting material is limited, as RAPDs require considerably less material than isoenzyme electrophoresis. The correlation between the RAPD and isoenzyme techniques also supports the concept of a clonal population structure for Giardia which has been put forward by other researchers. In addition, some human and animals isolates of Giardia were grouped into very similar rapdemes, which supports the occurrence of zoonotic transmission. However, for RAPD analysis to be of use in routinely examining variation in isolates of Giardia that are refractory to in vitro amplification a more effective method of purifying Giardia cysts needs to be developed. RAPD analysis was successfully used to develop diagnostic primers for Cryptosporidium. The diagnostic primers have been shown to be both specific and sensitive, with detection limits as high as one oocyst and could also amplify Cryptosporidium directly from boiled faeces. A total of 38 different isolates of C. parvum isolates of both human and bovine origin from diverse geographic locations were screened using these primers and all amplified the desired 680 bp band, indicating that the sequence defined by the primers is conserved amongst isolates. RAPD analysis was also used to develop diagnostic PCR primers for Giardia. The primers developed were specific for Giardia but due to the extent of genetic heterogeneity amongst isolates of Giardia the primers were unable to identify a number of the genetically more distinct isolates. The greater the extent of genetic diversity within species, the more difficult it becomes to develop diagnostic PCR primers that will recognise all isolates within that species. The level of genetic heterogeneity exhibited by Giardia isolates made it more difficult to develop diagnostic PCR primers that would recognise all Giardia isolates. The results of this study show that RAPD analysis is a useful technique for analysing genetic variation, particularly in organisms that can only be obtained in small quantities and for which little sequence information is available. Isolates of C. parvum and particularly G. duodenalis were shown to be genetically diverse and tentative evidence for zoonotic transmission was shown by the fact that some animal and human isolates of both parasites were grouped similarly. The source of the genetic diversity exhibited by these parasites is consistent with a clonal population structure for Giardia and perhaps also for Cryptosporidium, however further studies are required in order to confirm this. The RAPD technique was also shown to be very successful in the development of diagnostic PCR primers for both parasites.