| Objective:The general purpose of this doctoral dissertation is to carry out a extending study on Ascaris population genetics from humans and pigs in China and to shed light on molecular genetics, physiology and epidemiology of Ascaris and ascariasis, as well as to lay a theoretical foundation for the control strategy of ascariasis in humans in China. The concrete objective for respective chapters after the literature review is as follows:1. For chapter 2:It is to obtain further understanding of genetic structure and evolutionary relationship of Ascaris from humans and pigs.2. For chapter 3:It is to further reveal the genetic variation of Ascaris individuals with genotype G2, and to make up for the deficiency of ITS genetic marker not able to discriminate this shared genotype.3. For chapter 4:It is to provide the basic pattern of frequency distribution of cross infection and hybrid individual between Ascaris from human and pig in its area, host and genotype in China.4. For chapter 5:It is to determine whether single Ascaris suum female could mate with multiple males.Methods:1. Phylogeography study on twelve populations from six endemic regions in China was conducted using mitochondrial DNA markers (COX1 and NAD1) and the software programs of DnaSP 5.0, Arlequin 3.0, MEGA4.0 and NETWORK 4.5.1.6.2. Twenty autosomal microsatellite markers were employed and capillary electrophoresis was used to determine the allele size. The software CERVUS V2.0, ARLEQUIN V3.11, FSTAT V2.9.3 and GENETIX V4.05 were used to analyze the genetic diversity of 69 Ascaris worms from humans and pigs with the shared genotype G2.3. Twenty microsatellite markers rich in polymorphism were selected to screen 258 Ascaris worms from humans and pigs from six endemic regions in China. The software STRUCTURE, BAPS and NEWHYBRIDS were employed to determine the case of cross-infection of human with pig-derived Ascaris or pig infected with human-derived Ascaris, and hybrids of human and pig Ascaris.4. Seven sex-linked microsatellite markers were employed and paternal genetic analyses were conducted. Totally 62 offspring individuals from 3 single females were screened and the numbers of fathers in each family were determined using allele counting methods and the program GERUD (version 2.0).Results:1. The phylogeographical analysis showed a) genetic diversity of Ascaris varied with hosts and locations, but no distinct geographical distribution pattern was found, b) higher level of genetic diversity and differentiation was found in pig-derived populations in contrast to human-derived ones, and in populations of human-derived Ascaris from the southern regions in comparison to that from the middle and northern locations, but similar geographical difference was not observed within pig-derived populations, c) historical population expanding was detected from a large part of human-derived Ascaris populations but not in pig-derived Ascaris, d) high level of gene flow was detected between human- and pig-derived Ascaris, and also among human-derived populations, e) network analysis from haplotype of COX1 indicated an ancestral haplotype from human-derived Ascaris.2. The screening on the 69 genotype G2 Ascaris from humans and pigs showed that a total of 378 different alleles were detected in Ascaris from humans, ranging from 3 to 31, with an average of 18.9 per locus; a total of 229 different alleles were detected in the Ascaris from pigs, ranging from 5 to 19, with an average of 11.45 per locus. The allele frequency of Ascaris from human was found in a range of 0 to 0.821; in contrast to the range from 0 to 0.833 of Ascaris from pigs. There are 14 sites out of the 20 genetic loci had the same advantage alleles in Ascaris from both hosts but 6 gene loci (locus 1,6,10,12,16 and 17) was inconsistent. There was no specific allele in the sites 13 for Ascaris from pigs and in the sites 15 for Ascaris from human, but at the other sites there were their respective special alleles. Ascaris from humans had 204 special alleles in contrast to 55 special alleles for the Ascaris from pigs. Among Ascaris from humans and pigs, the average expected heterozygosity (HE) was 0.802 and 0.777, and the polymorphism information content (PIC) 0.779 and 0.737, respectively. The analysis of molecular variation (AMOVA) showed 2.6% genetic variation between population and 68.6% within individuals. The FST estimates and gene flow (Nm) between two populations were 0.0268 and 9.08 respectively.3. Cross infection was detected in either human or pig host but mostly in humans. Of the total 20 cross infection cases,19 were indentified as pure pig breed Ascaris parasitizing in human body in contrast to only one pure human breed Ascaris parasitizing in pig host. The distribution of cross infection also showed obvious variation in genotypes, in that genotype G1 (human type), G2 (share type) and G3 (pig type) were account for 45%,45% and 10% respectively. Similar to the findings in the case of cross infection, hybrids Ascaris was also detected either in human and pig host with the far most in humans. Of the 20 worms indentifies as hubris,19 were obtained from human bodies with only one from a pig body. The distribution of hybrids also showed significant difference between south, and north region, because more than half hybrids (11 out of 20) were from the 2 southern provinces and the other 9 hybrids were from other 4 northern provinces. The frequency distribution of the hybrids also genotype-related because the genotype G1 (human type), G2 (share type) and G3 (pig type) were count for 55%,30% and 15% respectively.4. The 7 sex-linked microsatellite loci showed high polymorphism and revealed that one out of three families (allele counts) and two out of three families (GERUD) of the sampled families had at least 2 sires (2～6).Conclusions:1. The present study revealed new information of Ascaris on the aspects of genetic diversity, population differentiation and historical demographic patterns, gene flow, phylogenesis reconstruction and haplotype network, discussed the results with historical demographic migration of humans and domestication of wild boar in China, raised a different assumption about the evolutionary relationship of the two roundworms that the ancestor of Ascaris of pigs may originate from human Ascaris. This study should have certain enlightenment for the epidemiology and the evolutionary and taxonomy relationship of Ascaris from humans and pigs.2. High genetic diversity was found in the genotype G2 Ascaris from humans and pigs. No obvious genetic differentiation between the two populations was revealed but gene flow between them was detected. However, the differences in specific alleles and the advantage alleles suggested an important genetic difference, which might be partly explained their different host parasitic specificity.3. The frequency of cross infection and hybrids suggests an obvious host and genotype preference. Furthermore, the frequency of hybrids showed geographical variance in terms of north and south. These results strongly suggest that pig Ascaris have become important source of infection to humans in endemic area where human and pig Ascaris both exist. It is also a new phenomenon in epidemiology of ascariasis. In consideration of current control strategy for ascariasis in humans in China in which pig Ascaris has not being considered as an infection source for humans, this research provided the theoretical guidance and evidence supports for a necessary revise of the current control measures for human ascariasis in China. The knowledge on cross-transmission and hybridization between Ascaris lumbricoides and Ascaris suum has also important meaning not only in public health, but also in further studies in the genetic evolution, taxonomy and molecular epidemiology of Ascaris.4. It is the first molecular evidence that females of A. suum can mate with multiple males, adding new knowledge to reproduction physiology of Ascaris. These findings have implications for the genetic structure of Ascaris populations, the probability of adaptation to other host species, and the rate of spread of drug resistance, even for other parasites. |
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