Genomic studies on Strongyloides stercoralis in northern and western Thailand

Abstract

Background: Strongyloidiasis is a soil borne helminthiasis, which in most cases is caused by Strongyloides stercoralis. Human infections with S. fuelleborni fuelleborni and S. fuelleborni kellyi also occur. Although up to 370 million people are currently estimated to be infected with S. stercoralis, this parasite is frequently overlooked. Strongyloides stercoralis is prevalent among humans in Thailand; however, S. fuelleborni fuelleborni has also been reported. Three recent genomic studies of individual S. stercoralis worms found genetically diverse populations of S. stercoralis, with comparably low heterozygosity in Cambodia and Myanmar, and less diverse populations with high heterozygosity in Japan and southern China that presumably reproduce asexually.

Methods: We isolated individual Strongyloides spp. from different localities in northern and western Thailand and determined their nuclear small ribosomal subunit rDNA (18S rDNA, SSU), in particular the hypervariable regions I and IV (HVR-I and HVR-IV), mitochondrial cytochrome c oxidase subunit 1 (cox1) and for a subset whole genome sequences. These sequences were then compared with each other and with published sequences from different geographical locations.

Results: All 237 worms isolated from 16 different human hosts were S. stercoralis, no S. fuelleborni was found. All worms had the common S. stercoralis SSU HVR IV haplotype A. Two different SSU HVR I haplotypes (I and II), both previously described in S. stercoralis, were found. No animal heterozygous for the two haplotypes was identified. Among the twelve cox1 haplotypes found, five had not been previously described. Based upon the mitochondrial cox1 and the nuclear whole genome sequences, S. stercoralis in Thailand was phylogenetically intermixed with the samples from other Southeast Asian countries and did not form its own branch. The genomic heterozygosity was even slightly lower than in the samples from the neighboring countries.

Conclusions: In our sample from humans, all Strongyloides spp. were S. stercoralis. The S. stercoralis from northern and western Thailand appear to be part of a diverse, intermixing continental Southeast Asian population. No obvious indication for genetic sub-structuring of S. stercoralis within Thailand or within the Southeast Asian peninsula was detected.

Keywords: Neglected tropical disease; Phylogeny; SSU; Strongyloides stercoralis; Strongyloidiasis; cox1.

Fig. 1

cox1 gene neighbor-joining tree of different S. stercoralis isolates based on 552-bp partial sequences. Shown are the sequences found in this study (green box) and selected published S. stercoralis haplotypes representing the major phylogenetic groups described in recent S. stercoralis cox1 phylogenies. The scale-bar represents 0.02 substitutions per site. The bootstrap values represent 1000 bootstrapping repetitions. Bootstrap values for neighbor-joining and maximum likelihood analysis are shown above or near the branches. The labels are composed as follows: author of the reference; haplotypes names according to this reference; host the isolate was derived from; country the isolate was isolated from; GenBank accession number. The host a particular sequence was found in is further highlighted with a red filled circle for “human” and a blue filled square for “dog”. The two columns on the right indicate the SSU HVR-I and HVR-IV haplotypes found among the worm individuals of the respective cox1 haplotype according to the respective authors, if known. Published sequences can be found under the following references [–20]

Fig. 2

Phylogenetic tree based on whole genome sequences. Sequences newly determined for this study are highlighted in color, followed by the cox1 haplotype number and the SSU HVR-I and HVR-IV haplotypes in parentheses. The colors indicate the provinces: red, Chiang Mai; blue, Lamphun; green, Lampang; grey, Mae Hong Son; yellow, Tak. For comparison, selected published S. stercoralis whole genome sequences described in recent whole genome-based S. stercoralis phylogenies are shown (not highlighted). Samples ending with “cam” are from Cambodia [16]. Samples starting with “My” and “Rk” are from Myanmar and Japan, respectively [23]. Sample cn-379 is from southern China [20]. sste_f1_ERR422406 is a free-living female of the S. stercoralis reference isolate [42]. If listed in the corresponding reference, the SSU HVR-I and HVR-IV haplotypes are indicated in parentheses (note that HVR-IV haplotype C in [20] is the same as HVR-IV haplotype E in [12]; this was noticed by Barratt et al. [22] and occurred because the two publications appeared almost simultaneously). Key for comparing samples from Cambodia with figure 4 of [20]: sste3_cam to sste26_cam correspond to Kh-3 to Kh-26; sste_DC51FLfemale1_cam = Kh-27; sste_DC51FLfemale2_cam = Kh-28; sste_DC51FLmale1_cam = Kh-29; sste_DC51FLmale3_cam = Kh-30; sste_DC69FLmale1_cam = Kh-31; sste_DC69FLmale3_cam = Kh-32; sste_DC69FLmale4_cam = Kh-33

Fig. 3

Heterozygosity of individual S. stercoralis. The heterozygosity on the autosomes is plotted against the heterozygosity on the X chromosome. For comparison, published data from S. stercoralis individuals from different geographical locations are included. Cn, China [20]; Kh, Cambodia [16]; My, Myanmar; Rk, Japan (both [23]); Thai, Thailand (present study); US-Ref, USA-derived reference laboratory strain PV001 [42]. For Cn, Kh, My, Rk and the USA reference, the same samples as in Fig. 5 of [20] are included. For Thailand, all samples in Fig. 2 that fulfilled the read coverage criteria (see Methods) were included in this analysis