Author ORCID Identifier
Date Available
7-23-2022
Year of Publication
2022
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Agriculture, Food and Environment
Department/School/Program
Veterinary Science
First Advisor
Dr. Martin K. Nielsen
Abstract
Parasitic nematodes, including the large roundworms colloquially known as ascarids, affect the health and well-being of livestock animals worldwide. The equine ascarid, Parascaris spp., was the first ascarid parasite to develop wide-spread anthelmintic drug resistance, with other species slowly following suit. There are no new classes of anthelmintics currently in development, and a solution to the ever-increasing prevalence of resistance is desperately needed. The microbiome has been shown to be an important factor in the fitness and health of many organisms and changes to microbiome composition have been associated with a plethora of diseases. The microbiome is also important to the health of parasitic nematodes, and the endosymbiotic bacterium Wolbachia, whose presence is essential for the viability of filarial nematodes, has been exploited for treatment of filariasis in humans by using both broad-range and, more recently, specific anti-Wolbachial antimicrobial treatments. Despite this success, parasite microbiomes are understudied. The overarching goal of this dissertation was to characterize the microbiome of Parascaris spp. by identifying a common core microbiota, by comparing microbiota diversity metrics for the whole worm at different life stages and in individual organs in male and female parasites, and by assessing the female gonad microbiota in greater detail.
Worms, along with jejunal content samples, were collected from foals at necropsy and used for both the whole worm study, which utilized a total of 27 parasites (9 male, 9 female, 9 immature), and in the organ study, which utilized a total of 46 adult parasites (24 male, 22 female). DNA extracted from these samples was used to produce a library using a 16S rRNA metagenomic sequencing protocol, and this library was sequenced using the Illumina MiSeq platform. A bioinformatics pipeline was developed to identify taxa and their relative abundance in the samples, and subsequent data analysis was carried out using R packages including Vegan, DESeq2, corncob, metagenomeSeq, and ANCOM.BC. The 22 female gonad samples were further analyzed using next generation metagenomic sequencing following the same protocol as the other two studies, and then using a kit that targeted to multiple regions and that allowing consensus sequences to be assembled. Additionally, another female worm was also collected, immediately fixed, dissected, and submitted for sectioning and examination by transmission electron microscopy.
A common core microbiota consisting of eleven genera was established for Parascaris spp. and consisted of: Acinetobacter, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (ANPR), Clostridium senso stricto 1, Gemella, Janthinobacterium, Lactobacillus, Reyranella, Sarcina, Sphingomonas, Streptococcus, and Veillonella. When comparing organs, Veillonella was differentially abundant when using DESeq2 and ANCOM-BC (p < 0.0001), corncob (p = 0.0008), and metagenomeSeq (p = 0.0118) and Sarcina was differentially abundant across all four analytical methods (p < 0.0001).
Alpha and beta diversity for the whole worm microbiota was similar across groups for all three taxonomic levels. Alpha diversity for the organ microbiota was significantly different based upon both sex and location at all three taxonomic levels. Simpson alpha diversity was significantly different between the female intestine (FI) and male gonad (MG) at the phylum (p < 0.0001), family (p = 0.0058) and genus (p = 0.0018) levels, and between both the female gonad (FG) and FI (p < 0.0001) and the FI and male intestine (MI; p = 0.0072) at the phylum level. Shannon alpha diversity was significantly different between the FI and the FG (p < 0.0001), the horse jejunum (HJ; p = 0.0483), the MG (p < 0.0001) and the MI (p = 0.0007) at the phylum level, between the FI and MG (p = 0.0003) at the family level and between the FG and MG (p = 0.0130), the FI and HJ (p = 0.0383) and the FI and MG (p = 0.0001) at the genus level. Beta diversity was significantly different between FI and FG (p = 0.0377) at the phylum level, MG and FG (p = 0.0010), FI (p = 0.0174), and HJ (p = 0430), and FG and MI (p = 0.0061) at the family level, and MG and FG (p = 0.0006), MI and FG (p = 0.0093), and MG and FI (p = 0.0041) at the genus level.
Twelve species were identified in the female gonad, and phylogenetic trees were created for the genera Aminobacter, Reyranella, Limosilactobacillus and Ligilactobacillus. Cladograms indicated that consensus sequences from members of these genera were related to species found in soil and water, and to those that had previously been found in horses, and thus the presence of related bacteria in parasites makes biological sense. Finally, morphological structures identified as candidate bacteria were found in the cells of Parascaris spp. female gonad sections, indicating that there are also possibly endosymbionts associated with these parasites.
In summary, the overarching goal of this research was met. A common core microbiota was established for Parascaris spp., diversity metrics were compared for different life stages and organs, and the female gonad was explored in more detail. This research lays the groundwork for future studies involving the Parascaris spp. microbiome and provides more data to the effort to understand parasite microbiomes.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2022.254
Funding Information
This work was funded by the National Center for Veterinary Parasitology from 2018-2020 and Zoetis from 2021-2022.
Recommended Citation
Cain, Jennifer Lynn, "The microbiome of the equine roundworm, Parascaris spp." (2022). Theses and Dissertations--Veterinary Science. 57.
https://uknowledge.uky.edu/gluck_etds/57
