Genotyping Ascaris nematodes from diverse geographic locations translation missing: es.hyrax.visibility.files_restricted.text

Richins, Travis (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/j6731468r?locale=es
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Abstract

Introduction:

There is some debate as to whether the pig intestinal giant roundworm, Ascaris suum, is a distinct species from Ascaris lumbricoides, the human intestinal giant roundworm. Over 800 million people world-wide are infected with this disease. The large amounts of morbidity and economic loss that this disease causes makes it important to continue to add evidence in respect to the speciation debate, as it will influence future public health interventions. The goal of this study is to identify the relationship between the ascaris genetic material (mitochondrial and nuclear) with the location or the host of infection (humans or pigs) in global ascaris populations.

Methods:

142 unique samples used in this survey of Ascaris spp. were collected by various programs and researchers. The samples had four main origins: Guatemala, Solomon Islands, Slovakia, and the state of Georgia. All of the samples were fecal specimens preserved in ethanol with the exception of the positive control, which was a whole worm preserved in ethanol. DNA extraction was used to isolate the genetic material, while a novel PCR assay developed after identifying descriptive loci in the genome of Ascaris spp. worms was used to amplify the selected targets. These targets include COXI 6A, COXI 7A, and TR3. Geneious was utilized to analyze the sequenced amplicons.

Results:

The dendrograms for the amplified targets CoxI 6A (76 sequences), CoxI 7A (23 sequences), and TR3 (18 sequences) were generated to show the clusters of samples of all geographic origins. In all three loci, different geographic areas seem to cluster more closely together than samples from different geographic areas. For example, when utilizing the COXI 6A locus all ten detected Solomon Island samples clustered closely together, as did the four detected Slovakian samples. In the COXI 7A locus, the Slovakian samples clustered together. While in the TR3 locus the Slovakian samples clustered together as did the Solomon Island samples. The samples from Georgia and Guatemala frequently were clustered together. In each loci, human and pig ascaris clustered closely together. For example, in the COXI 6A locus, a Georgian pig sample (P2) clustered with a large group of Solomon Island human samples. When utilizing the COXI 7A locus, one Guatemala (G66) human sample clustered with one Georgian pig sample (P3) as well as the pig positive control. In the analysis of the TR3 locus, it was found that a Georgian (P2) pig sample clustered with three Guatemala (G31, G38, G46) human samples as well as the pig positive control.

Discussion:

The results of this study suggest that this method can genotype soil-transmitted helminth infections. It also suggests that it can group them in accordance with their geography and by host, whether human or pig. While the clusters were helpful in illustrating the origin of the infection, they also give evidence to the thought that there may be exchange of genetic information between certain populations. This is shown in the clustering of samples from different geographic locations together, as well as the clustering of samples from different hosts together.

Table of Contents

Table of Contents

Literature Review1

Introduction1

Ascaris life cycle1

Ascariasis3

Diagnosis4

Methods for Assessing Population Genetic Structure13

Potential Public Health Impact of an Effective Genotyping Tool 14

Study Goals and Aims15

Significance16

Methods and Materials17

Ethics Statement17

Samples17

PCR Primer Design18

Isolation of Ascaris DNA20

PCR Annealing Temperature Optimization20

Sequencing Amplified Targets22

Results24

CoxI 6A amplicon genetic analysis25

CoxI 7A amplicon genetic analysis26

TR3 amplicon genetic analysis28

Discussion30

Public Health Implications33

References34

Appendix A37

Emory IRB determination37

Guatemala IRB determination38

Solomon Island IRB determination39

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