Different but overlapping populations of Strongyloides stercoralis in dogs and humans-Dogs as a possible source for zoonotic strongyloidiasis

Strongyloidiasis is a much-neglected soil born helminthiasis caused by the nematode Strongyloides stercoralis. Human derived S. stercoralis can be maintained in dogs in the laboratory and this parasite has been reported to also occur in dogs in the wild. Some authors have considered strongyloidiasis a zoonotic disease while others have argued that the two hosts carry host specialized populations of S. stercoralis and that dogs play a minor role, if any, as a reservoir for zoonotic S. stercoralis infections of humans. We isolated S. stercoralis from humans and their dogs in rural villages in northern Cambodia, a region with a high incidence of strongyloidiasis, and compared the worms derived from these two host species using nuclear and mitochondrial DNA sequence polymorphisms. We found that in dogs there exist two populations of S. stercoralis, which are clearly separated from each other genetically based on the nuclear 18S rDNA, the mitochondrial cox1 locus and whole genome sequence. One population, to which the majority of the worms belong, appears to be restricted to dogs. The other population is indistinguishable from the population of S. stercoralis isolated from humans. Consistent with earlier studies, we found multiple sequence variants of the hypervariable region I of the 18 S rDNA in S. stercoralis from humans. However, comparison of mitochondrial sequences and whole genome analysis suggest that these different 18S variants do not represent multiple genetically isolated subpopulations among the worms isolated from humans. We also investigated the mode of reproduction of the free-living generations of laboratory and wild isolates of S. stercoralis. Contrary to earlier literature on S. stercoralis but similar to other species of Strongyloides, we found clear evidence of sexual reproduction. Overall, our results show that dogs carry two populations, possibly different species of Strongyloides. One population appears to be dog specific but the other one is shared with humans. This argues for the strong potential of dogs as reservoirs for zoonotic transmission of S. stercoralis to humans and suggests that in order to reduce the exposure of humans to infective S. stercoralis larvae, dogs should be treated for the infection along with their owners

Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy

Sample relatedness analysis on whole genome data.

<p>A) Neighbor joining tree based on a genomic identity-by-state relationship matrix in cooperating 1326 SNPs (thresholds: LD = 0.05, MAF = 0.05). B) Neighbor joining tree based on pairwise similarities of the 23 individual genomes estimated using kWIP. The same samples in the two trees are connected with dotted lines colored in red for human derived worms and in blue for dog derived worms. ERX044031 indicates the reference genome short read data set [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005752#pntd.0005752.ref057" target="_blank">57</a>], which has HVR I haplotype I and HVR IV haplotype A. The labels contain: [identifier of the worm] | [identifier of the host individual] | [HVR I haplotype, HVR IV haplotype], with each attribute separated by vertical lines.</p

Genotypes of free-living mothers and their progeny.

<p>Genotypes of free-living mothers and their progeny.</p

The life cycle of <i>S</i>. <i>stercoralis</i>.

<p>The life cycle of <i>Strongyloides stercoralis</i>. The numbers refer to the numbers of the developmental options in the description of the life cycle in the text.</p

Gene tree of the mitochondrial gene <i>cox</i>1.

<p>Maximum likelihood tree of the 17 different <i>cox</i>1 sequences we found and representative previously published sequences. The numbers are bootstrap values based on 1000 bootstraps. For haplotypes isolated in this study the labels have the following format: Haplotype number (accession number) host country (number of individuals this haplotype was found in). ¹For sequences previously published by Hasegawa and colleagues [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005752#pntd.0005752.ref036" target="_blank">36</a>] the label starts with Hasegawa. ²For sequences previously published by Laymanifong and colleagues [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005752#pntd.0005752.ref006" target="_blank">6</a>] the label starts with the <i>cox</i>1 clade this reference assigned the particular sequence to. This is followed by: (accession number) host country (CAR = Central African Republic). The hosts are also highlighted by red circles (human) and blue squares (dog). Entries to the right of the tree indicate for each <i>cox</i>1 haplotype the <i>SSU</i> haplotypes it was found together in the same individual. If a given <i>cox</i>1 haplotype existed in the context of multiple <i>SSU</i> haplotypes, the number of worms with this particular combination is given in parentheses. Note: the <i>cox</i>1 haplotypes 2 and 3 were found in both hosts and are included twice in this tree. For the sequencing results for both <i>SSU</i> HVR and <i>cox</i>1 for each individual worm see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005752#pntd.0005752.s002" target="_blank">S1 Table</a>.</p

Primers and PCR conditions.

<p>Primers and PCR conditions.</p

<i>SSU</i> HVR I haplotypes of <i>S</i>. <i>stercoralis</i> with HVR IV haplotype A isolated from humans and dogs, 2013 and 2016.

<p><i>SSU</i> HVR I haplotypes of <i>S</i>. <i>stercoralis</i> with HVR IV haplotype A isolated from humans and dogs, 2013 and 2016.</p

Genotypes of free-living parents and their progeny at <i>ytP274</i>.

<p>Genotypes of free-living parents and their progeny at <i>ytP274</i>.</p