[Occurrence of Giardia species and genotypes in humans and animals in Wielkopolska region, Poland].

Giardia is the most common intestinal protozoan parasite found in humans and animals worldwide. Although it has been known for three hundred years, the nomenclature, taxonomy, host specificity, and pathogenicity of Giardia still arouse numerous controversies and ambiguities. Giardia is classified into six species, that are characterised by various ranges of hosts. The most dubious species is G. intestinalis, which includes a dozen or so genotypes, and only two of them (genotype A and B) have wide ranges of hosts, including humans. Moreover, in some genotype assemblages of G. intestinalis certain subgenotypes were distinguished and it was proven that in the same host species various subgenotypes of this parasite may occur. Bearing in mind the significant genetic heterogeneity of G. intestinalis and the fact that various genotypes and subgenotypes of this parasite are characterised by the broad or narrow host specificity, the data concerning the frequency of giardiosis occurrence are insufficient. It is necessary to use molecular biology techniques in order to define the genotype and/or the subgenotype of G. intestinalis that are found in humans and in certain animal species. Furthermore, since more and more pieces of evidence connected with a possibility of the sexual recombination of Giardia are gathered, it is unknown if genotypes and subgenotypes of this parasite are stable in time. The aim of this thesis was to define the frequency of Giardia occurrence in humans and animals in Wielkopolska region, to identify species and genotypes of Giardia that occur in humans and animals, as well as to obtain an axenic culture of the chosen isolates of Giardia from animals and to compare the sequence of the beta-giardin gene fragment obtained from the DNA isolated from cysts and trophozoites in order to check if the axenisation of G. intestinalis leads to the selection of genotypes or if Giardia genotypes are stable in time. Altogether, 2183 faecal samples were examined for the presence of Giardia cysts; 447 faecal samples were taken from 232 persons coming from 5 cities situated in Wielkopolska, and 1736 faecal samples were obtained from 123 animal species, including: 266 faecal samples from 113 species of animals kept in the Zoological Garden in Poznań, 1286 samples from 4 species of breeding animals, 118 samples from dogs, and 66 samples from 5 species of wild animals. Faecal samples were taken from animals coming from 25 places in Wielkopolska. Moreover, seven isolates of G. intestinalis were used in the studies, which were obtained from various species of hosts and kept in an axenic in vitro culture. Microscopic, molecular and bio-informative methods were used in the studies. From each faecal sample fresh smears were made in a 0.6% solution of physiological salt and in Lugol's solution, as well as a permanent smear stained with trichrome was made. Moreover, the following molecular techniques were implemented in the studies: DNA extraction and purification, the PCR technique (two molecular markers), electrophoresis and visualisation of PCR products, and sequencing. A fragment of the beta-giardin gene was used as a molecular marker in order to define the genotype and subgenotype of Giardia. Only in the case of genotyping of two Giardia isolates obtained from Peromyscus eremicus another molecular marker (SSU rRNA)was additionally used. Some widely available computer programmes (Chromas, CAP 3, BioEdit, BLASTn, MEGA version 4.0) were utilised in the analysis of the sequence of the beta-giardin gene fragment and in the phylogenetic analysis. The culture of Giardia trophozoites was established to compare the sequence of the partial beta-giardin gene from cysts and trophozoites. Concentration and purification of Giardia cysts in the saccharose gradient, and the excystation technique were applied in the studies to obtaining an axenic in vitro culture. In this study, Giardia cysts were found in 12 faecal samples obtained from 3 persons and 5 animal species. Giardia cysts were found only in faecal samples from humans living in Poznań and the samples obtained from animals coming from Poznań and around Puszczykowo. The highest frequency of infection was stated in domestic animals (2.5%) and in animals kept in the Zoological Garden (2.0%), whereas a slightly lower frequency was noticed in wild animals (1.5%) and in humans (1.3%). No Giardia cysts were found in the faecal samples collected from breeding animals. Two new species of Giardia hosts were identified, namely Rhinella marina and Peromyscus eremicus; however, due to a minimal amount of faecal samples supplied for the study it was impossible to define the species and genotype of this parasite. PCR products (the partial of beta-giardin gene) were obtained in seven faecal samples out of the ten studied, including three samples from people and four faecal samples derived from three animal species (i.e. dog, tamandua, red deer). Moreover, molecular characterization of seven Giardia isolates from three persons and four animal species (red-bellied monkey, silver marmoset, Thomson's gazelle, and sheep) kept in an axenic in vitro culture was performed. Based on the beta-giardin sequence fragment analysis, four assemblages of G. intestinalis genotypes were identified (A, B, C and D). In humans, A and B G. intestinalis genotypes and three subgenotypes, including a cosmopolitan subgenotype A2 and two new subgenotypes A and B were detected. Furthermore, four G. intestinalis genotypes were found in animals, including three genotypes which are non-infectious to humans, namely: genotypes C and D in dogs and a cervids-specific genotype A in red deer (Cervus elaphus), which indicate that these animals do not constitute the source of infection to humans. On the other hand, in a tamandua from the Zoological Garden in Poznań a new subgenotype B of G. intestinalis was identified, which due to a close relationship with Giardia isolates obtained from humans is potentially infectious to man. In none of the studied faecal samples a mixed infection of Giardia was found. To date, nine sequences of the partial beta-giardin gene have been deposited in the National Center for Biotechnology Information (NCBI), including two sequences of Giardia isolates obtained from humans (GenBank accession numbers FJ009207, FJ009208), three sequences of isolate obtained from red deer (GenBank accession numbers EU621373, EU626198, EU216429), two sequences of both Giardia isolates obtained from dogs (GenBank accession numbers FJ009205, FJ009206), and the single sequences obtained from tamandua (GenBank accession number FJ009209) and from Thomson's gazelle (GenBank accession number EU626199). According to the literature, an axenic in vitro culture of G. intestinalis was obtained from a red deer for the first time. Based on the analysis of the sequence of the beta-giardin gene fragment obtained from the DNA isolated from cysts and trophozoites it was proven that the red deer was infected with a single population of Giardia and that during the axenisation of the culture no mutation in the DNA of the parasite's trophozoites took place. Probably the time distance that the DNA was isolated from the trophozoites kept in the culture was too short to cause the mutation. This suggestion is confirmed by the results of the genotyping of seven G. intestinalis isolates obtained from various host species and kept in an axenic in vitro culture for at least a number of years. Based on the molecular characteristics it was stated that all the studied isolates from the axenic culture were identical and belonged to the same assemblage B. The comparision with the sequences from GenBank database revealed that all mentioned isolates were 99% similar to the sequence of Giardia Nij5 isolate obtained from a person from the Netherlands and characterised as genotype B1. Due to the sameness of the molecular marker sequences it seems improbable that the identical G. intestinalis genotype occurred in various time periods (the largest difference was 14 years) in humans and in a number of animal species in diverse areas of Wielkopolska region. Quite opposite, the long-term keeping of these isolates in the homogenous conditions of an axenic in vitro culture leads to the selection of a genotype or proves the instability of genotypes of this parasite. Long-term studies need to be conducted in order to verify these hypothesis. Their results will have a key meaning in explaining the genetic structure of the Giardia population and in understanding the molecular epidemiology of giardiosis.