Discussion

The ability to integrate large sequence datasets from multiple studies composed of specimens with varying combinations of MLST markers amplified, and specimens that are heterozygous, represents an important advantage of the ML procedure employed here over traditional methods utilized in the field of population genetics. Furthermore, the way in which haplotypes are defined using our ML procedure avoids problems associated with the treatment of indels that are inherent in many phylogenetic methods; indels (alignment gaps) are often treated as missing data in phylogenetic analyses and are excluded altogether (Truszkowski and Goldman, Reference Truszkowski and Goldman2016; Donath and Stadler, Reference Donath and Stadler2018). This exclusion of alignment gaps is problematic for the analysis of Strongyloides HVRs because these loci contain informative indels that differentiate haplotypes. Indels do not represent a problem for the ML approach used here which represents another advantage of this approach over phylogenetic methods. This method facilitated the inclusion of hundreds of genotyped S. stercoralis specimens from multiple studies in a single analysis, despite that many published genotyping studies examined different, yet overlapping, combinations of markers. Therefore, this analysis provides a broader view of potential host-associations and geographic patterns that exist in populations of these worms than could be provided in previous studies given the data analysis challenges mentioned above. The large number of specimens retained for analysis here meant that any observable trends are supported with greater statistical power than could be achieved within the smaller genotyping studies. Consequently, this ML procedure represents a powerful alternative to traditional phylogenetic methods. Using this approach, we provide evidence supporting that S. stercoralis represents a species complex and that African and s.e. Asian S. fuelleborni are distinct. These results are supported by a synthesis of scientific literature, focusing on observed patterns relating to host preference and geography reported on the basis of biological and experimental evidence published by pioneering investigators who were predominantly active in the early to middle 20th century. This synthesis highlights the agreement between our current analysis and the observations of these classic parasitologists who lacked the molecular tools required to help resolve these important taxonomic questions.

Since Fuelleborn's initial discovery of a Strongyloides infection in a Chinese dog, the status of dog-derived isolates as a valid species or as a variant of human S. stercoralis, and thus its zoonotic potential, has remained a subject of debate (Fuelleborn, Reference Fuelleborn1914). While many authors regarded canine and human isolates as more or less morphologically identical strains with differing host specificity within the same species S. stercoralis (Hung and Höppli, Reference Hung and Höppli1923; Kreis, Reference Kreis1932), others recognized the potential existence of ‘geographic races’ or even suggested the separation of canine Strongyloides on experimental and epidemiological grounds in spite of negligible morphological differences (Brumpt, Reference Brumpt1922; Augustine and Davey, Reference Augustine and Davey1939). Brumpt (Reference Brumpt1922) argued for the establishment of a new species Strongyloides canis on the basis of geographic disparities in prevalence, barriers in experimental cross-transmission, and reported developmental differences in vitro. As most early 20th century authors were averse to applying for specific status without important morphological differences (Chandler, Reference Chandler1925; Sandground, Reference Sandground1925; Goodey, Reference Goodey1926; Kreis, Reference Kreis1932), S. canis was never recognized as a valid name. However, the modern availability of genetic data – including our results – and the resulting shift in taxonomic dogmas allows this question to be revisited.

The body of experimental evidence demonstrates varying abilities of human-derived S. stercoralis from different geographic regions to establish infections in dogs. This could correspond with the ‘spectrum’ between human- and apparently canine-restricted genetic lineages presented here (Fig. 2). In Fuelleborn's investigations (Fuelleborn, Reference Fuelleborn1914, Reference Fuelleborn1927), dogs developed only short duration infections (2–3 weeks) with human-derived Chinese S. stercoralis and were refractory to infection with East African S. stercoralis. Sandground successfully infected dogs with a USA human-derived strain, though the majority of dogs eventually self-cured after about 3–10 weeks and were refractory towards repeated exposures (Sandground, Reference Sandground1928; Galliard, Reference Galliard1939, Reference Galliard1951b). Galliard reported ease in infecting dogs with human Strongyloides from Vietnam (Galliard, Reference Galliard1939), but that dogs were more or less refractory to isolates from the West Indies and North Africa (Galliard, Reference Galliard1951a). Also, in his experiments, dogs that were not successfully infected with the African strain developed severe infections after exposure to the Vietnamese strain.

Sandosham (Reference Sandosham1952) reported short-term, unstable infection in a dog repeatedly inoculated with many larvae derived from former prisoners of war in Thailand, concluding that dogs could not have been the reservoir host for S. stercoralis in that setting despite their supposed abundant presence in the prison camps (Sandosham, Reference Sandosham1952). Many years later, attempts to infect laboratory dogs with S. stercoralis derived from a human patient infected in Southeast Asia yielded mixed results; in one dog, a chronic infection lasting at least 15 months was established, but in four others, fecal larval counts peaked at 3 weeks and dropped off drastically after that (Grove and Northern, Reference Grove and Northern1982). A similar pattern of unstable, transient patent infections has been noted previously in human volunteers infected with other animal Strongyloides species, such as Strongyloides procyonis (raccoons) and Strongyloides ransomi (swine), further suggesting that host adaptation strongly influences the duration of infection and larval output (Freedman, Reference Freedman1991). Reciprocally, Augustine and Davey (Reference Augustine and Davey1939) failed to infect a human volunteer, guinea pigs and cats with filariform larvae reared from a naturally-infected dog in Massachusetts but readily infected both young and aged dogs, which developed long-lasting patent infections.

Clearly, historical workers had differing levels of success infecting dogs, and one varying factor was the geographic origin of isolates. Though fragmentary, it is interesting to compare the geographic origins of parasites used in dog infection trials to the emerging picture of the S. stercoralis global population structure shown here. For example, dogs were found to be refractory or developing very short-lived infections when exposed to human-derived isolates of S. stercoralis from China, East Africa, North Africa and the West Indies (Fuelleborn, Reference Fuelleborn1914; Galliard, Reference Galliard1950; Galliard and Berdonneau, Reference Galliard and Berdonneau1953). Notably, the lineages of S. stercoralis, represented by clusters 1, 2, 3 and 6 were almost all obtained from human samples and known to occur in these locations (Fig. 2). Intermediate results (e.g. short-lived infections, low larval outputs, and/or resistance to reinfection in most dogs, with occasional chronic infections) were seen with North American and some Southeast Asian S. stercoralis strains (Sandground, Reference Sandground1925; Faust, Reference Faust1933; Grove and Northern, Reference Grove and Northern1982; Genta, Reference Genta1989); these locations are represented by lineages of S. stercoralis assigned to genetic clusters 4 and 7, where we observe genotypes that are seemingly capable of infecting both hosts, yet with the majority of specimens assigned to these clusters collected from dogs and humans, respectively (Fig. 2, Table 1). The sole trial where dogs were readily infected with no immunity to reinfection used a human-derived S. stercoralis from Vietnam (Galliard, Reference Galliard1939), which is in close geographic proximity to the origin of roughly half of all specimens assigned to cluster 7 (n = 20, Myanmar and Thailand). In summary, the observed historical variations show some overlap with both the locations and host frequencies in our observed S. stercoralis clusters.

Our analysis suggests a disparity between where human and canine S. stercoralis infections are occurring; infections caused by S. stercoralis assigned to cluster 5 are almost exclusive to Southeast Asia (98%). Our analysis also indicates that different genotypes of S. stercoralis are more frequently reported in specific hosts (dogs are over-represented hosts in clusters 4 and 5), based on a χ ² analysis (Table 1). These observations were also extensively discussed by historical authors who commented on the marked disparity in the occurrence of canine and human S. stercoralis infections i.e. in places where dog S. stercoralis infections occur commonly, human infections are rare, and vice versa. Outside of East and Southeast Asia, natural infections in dogs appeared to only occur in North America, and reports of dog infections were exceedingly rare to absent in areas where prevalence was high in humans (Brumpt, Reference Brumpt1922; Faust, Reference Faust1933; Augustine, Reference Augustine1940; Galliard, Reference Galliard1950; Sandosham, Reference Sandosham1952; Genta and Grove, Reference Genta, Grove and Grove1989). For example, not a single infection was detected in 528 dog faecal specimens during a survey in a highly endemic region of Colombia, where at least 14% of humans were S. stercoralis-positive (Faust and Giraldo, Reference Faust and Giraldo1960); even if some infections were missed due to the use of flotation techniques, infection would still be very rare in dogs vs humans. Recent case reports and molecular evidence show that natural canine infections also occur in Europe and Australia (Paradies et al., Reference Paradies, Iarussi, Sasanelli, Capogna, Lia, Zucca, Greco, Cantacessi and Otranto2017; Basso et al., Reference Basso, Grandt, Magnenat, Gottstein and Campos2019; Beknazarova et al., Reference Beknazarova, Barratt, Bradbury, Lane, Whiley and Ross2019; Barratt et al., Reference Barratt, Lane, Talundzic, Richins, Robertson, Formenti, Pritt, Verocai, Nascimento de Souza, Mato Soares, Traub, Buonfrate and and Bradbury2019b). However, an important caveat is that results based solely on microscopy of feces must be interpreted with caution as free-living nematode larvae, common contaminants of specimens collected from the ground can easily be confused with Strongyloides spp. (Speare, Reference Speare and Grove1989). Direct epidemiologic evidence for cross-transmissibility is limited, in part due to the apparent variability in host adaptation and geographic occurrence of these strains. In one instance, a kennel worker in the USA with no foreign travel acquired Strongyloides from dogs under his care; this strain was successfully used to infect laboratory dogs (Georgi and Sprinkle, Reference Georgi and Sprinkle1974). A small study in an area of Japan considered endemic found no association in S. stercoralis infection status among owners and dogs (Takano et al., Reference Takano, Minakami, Kodama, Matsuo and Satozono2009). Overall, there are still many knowledge gaps in our understanding of canine strongyloidiasis as a zoonosis and its epidemiology, and interpretation will require further characterization of strains across a diverse geographic range.

While establishing the specific identity of canine Strongyloides will require further investigation, based on historical reports and our novel analysis, it appears that different S. stercoralis lineages display differences in infection frequency for canine and human hosts – which may exclude or drastically limit cross-transmission potential in some cases (Table 1). Given the epidemiological implications of this genetic variability and the now large body of evidence across many independent studies, it now seems appropriate to treat S. stercoralis as a species complex. For example, ‘S. stercoralis sensu lato’ could be useful for referring to all isolates, and ‘S. stercoralis sensu stricto’ could be considered for the human-genotypes represented in 6 of the 7 clusters associated with HVR-IV haplotype A (Fig. 2, excluding cluster 5). This would also include specimens assigned to genetic cluster 4 which seems associated with the 18S rDNA genotype VI + A, and likely represents a canine adapted lineage of S. stercoralis sensu stricto. Subspecific ranks (e.g. S. stercoralis canis) were suggested nearly a century ago by Chandler for S. stercoralis of different animal hosts (Chandler, Reference Chandler1925). This seems reasonable, given the exclusive finding in canine hosts (100% of cluster 5), the restricted geographic range of S. stercoralis assigned to cluster 5 (98% were from Southeast Asia), and the fact this lineage (associated with HVR-IV haplotype B) naturally clusters as an outgroup to all other S. stercoralis (Fig. 2).

An important outstanding question is whether the canine infections in certain human host-dominated populations represent transient, spurious, or incidental infections, or if there exists a substructure that is not clearly apparent due to under-sampling of dog-derived genotypes from specific locations. Based on our analysis, dog-derived genotypes assigned to clusters 1–3 and 6 likely represent transient infections with strains originating from humans; this is supported by the experiments of Fuelleborn, Sanground and Galliard who infected dogs with human-derived S. stercoralis which often resulted in transient infections (Fuelleborn, Reference Fuelleborn1914, Reference Fuelleborn1927; Sandground, Reference Sandground1928; Galliard, Reference Galliard1939, Reference Galliard1951a, Reference Galliard1951b). However, for cluster 7 it seems possible that some additional population substructure exists. Cluster 7 contains a small ‘sub-cluster’ composed of eight cox1 sequences derived exclusively from S. stercoralis from Japanese dogs. Sequencing of HVR-I and/or HVR-IV from these worms, and the sampling of worms from additional dogs in the same geographic area that these specimens were collected, would provide greater clarity as to what this smaller ‘sub-cluster’ of Cluster 7 might represent.

The identity of Strongyloides spp. infecting cats is a more complex question, and it appears multiple species are involved, including S. stercoralis. Chandler first reported S. stercoralis in cats in India (Chandler, Reference Chandler1925), and some attempts to infect cats with S. stercoralis from dogs and humans have been successful; though infections were usually short-lived, and it appears that cats are competent but abnormal hosts for S. stercoralis (Sandground, Reference Sandground1928; Desportes, Reference Desportes1944; Wulcan et al., Reference Wulcan, Dennis, Ketzis, Bevelock and Verocai2019). Strongyloides felis has been described from India and Australia, and is somewhat similar morphologically to S. stercoralis although no molecular data are available (Speare and Tinsley, Reference Speare and Tinsley1986). Strongyloides planiceps occasionally infects cats but is dissimilar in its life cycle and morphologic characteristics to S. stercoralis (Thamsborg et al., Reference Thamsborg, Ketzis, Horii and Matthews2017). A fourth species, Strongyloides tumefaciens, is of interest as it has been reported as causing colonic nodules, an unusual clinical presentation for Strongyloides (Thamsborg et al., Reference Thamsborg, Ketzis, Horii and Matthews2017). However, the original description was incomplete, and a recent study cast doubt on the validity of S. tumefaciens. Strongyloides sp. extracted from nodules of necropsied cats were found to have cox1 sequences that matched S. stercoralis. These sequences were assigned to genetic cluster 4 in this study (Fig. 2), suggesting that S. tumefaciens infections could simply be an unusual pathological presentation of S. stercoralis (Wulcan et al., Reference Wulcan, Dennis, Ketzis, Bevelock and Verocai2019). There is clearly a need for further investigation into Strongyloides spp. of domestic cats, especially in regard to their zoonotic potential. The analytic approach presented here, along with morphological and biological characterization, should prove beneficial in answering these questions.

The other topic of interest in our study was the identity and population structure of S. fuelleborni as it relates to human and other primate infections. The validity and number of species infecting primates have been long debated. Genetic analyses, including the approach used here, have proven valuable in reconciling some of these viewpoints, as morphological comparisons have confounded historical investigations of Strongyloides taxonomy. This challenge mainly owes to the variability of characters and their interpretation – importantly, how much relative weight was given to which characteristics for species designations. For example, both Chandler and Little felt that morphologic features of the parasitic female were most reliable (Chandler, Reference Chandler1925; Little, Reference Little1966a), whereas Looss and Goodey placed more importance on the free-living adult stages (Looss, Reference Looss1911; Goodey, Reference Goodey1926). These discrepancies, along with the broad geographic and host range from which Strongyloides specimens were derived in these investigations, lead to differing conclusions and opinions regarding the number of species from primates.

Formerly, two species names were used for Strongyloides derived from Old World primates – S. fuelleborni Von Linstow, Reference Von Linstow1905, described from Pan troglodytes and Papio cynocephalus from Africa, and S. simiae Hung and Höppli, Reference Hung and Höppli1923 from Asian Macaca sp. The latter species was established on the basis of a smaller oesophagus to total body length ratio in comparison to S. fuelleborni, a lack of prominent constriction behind the vulva of the free-living female, and a ‘finely-striated cuticle’ (the authors considered S. fuelleborni to have a smooth cuticle) (Hung and Höppli, Reference Hung and Höppli1923). However, other authors report that S. fuelleborni and many, if not all other Strongyloides spp., indeed has a striated cuticle though sometimes very difficult to observe (Sandground, Reference Sandground1925; Grove, Reference Grove1989). As such, the validity of S. simiae has been scrutinized by several authors, most of whom regarded the morphological evidence for specific status insufficient or too highly variable for unequivocal species discrimination (Sandground, Reference Sandground1925; Premvati, Reference Premvati1959; Little, Reference Little1966a). Staphylococcus simiae became a junior synonym of S. fuelleborni, which is the name currently applied to all egg-passing Strongyloides from Old World apes and monkeys (Grove, Reference Grove1989).

Some biological evidence suggests there may still be valid differences between ‘S. simiae’ from Macaca spp. and S. fuelleborni. Experimental work by Augustine revealed that crosses between strains derived from Macaca and strains derived from Pan failed to produce offspring – suggesting perhaps that S. simiae is indeed a separate species, but nearly impossible to distinguish morphologically from S. fuelleborni (Augustine, Reference Augustine1940). It was also observed that Cebus-derived strains (later designated S. cebus – a species currently considered valid) also failed to cross with the other primate strains, supporting S. cebus as a distinct species despite morphologic overlap with S. fuelleborni in several characters (Augustine, Reference Augustine1940; Premvati, Reference Premvati1959). Another potential point of interest is that Von Linstow's original description of S. fuelleborni was based on parasites from two African host species (Pan troglodytes and Papio cynocephalus); though no major morphological differences were observed, this could have created inherent variability and lead to assumptions on a lack of host specificity (Von Linstow, Reference Von Linstow1905). While it has been established that New World and Old World-primate-derived Strongyloides (i.e. S. cebus and S. fuelleborni) are not capable of cross-infection (Faust, Reference Faust1931), no experiments have been conducted to compare the host specificity within African and Asian S. fuelleborni from different primate hosts.

The distinction between S. fuelleborni lineages on the basis of geography is supported by our analysis, where specimens obtained from Africa (Fig. 3, clusters 1 and 2) are clearly distinct from Asian genotypes. The finer division of Asian S. fuelleborni into multiple lineages is also supported (Fig. 3). Strongyloides fuelleborni from Bornean monkeys (proboscis monkeys, long-tailed macaques, silvered leaf monkeys) and orangutans form an outgroup to other genotypes (Fig. 3, clusters 6 and 7), and are also distinct from an undescribed Strongyloides sp. collected from lorises as part of the same Bornean survey (Frias et al., Reference Frias, Stark, Lynn, Nathan, Goossens, Okamoto and MacIntosh2018). We also note that the host range of S. fuelleborni from Malaysian Borneo (Fig. 3, clusters 6 and 7) and mainland Southeast Asia (Fig. 3, cluster 3) overlap; both genotypes were found in long-tailed macaques, yet the genetic distinction between Bornean and mainland Southeast Asian genotypes is clear. Japanese macaques (Fig. 3, cluster 4) harbour a lineage of S. fuelleborni that shares an affinity with isolates from mainland Southeast Asia and Southern Asia (Fig. 3, cluster 3), but these two clusters may still represent distinct groups. We note, however, that the specimens from Japanese macaques were based on an inferred genotype so the analysis was performed a second time, excluding data from Japanese macaques. The structure of the resulting dendrograms remained virtually unchanged following exclusion of this data, indicating that the distinction between S. fuelleborni from Malaysian Borneo, Africa and mainland Southeast Asia/Southern Asia remains supported (Supplementary File S2, Appendix part D). Further sampling of primate Strongyloides across these areas of interest and from other primate species, will help to resolve the true picture of species diversity.

Similar to how the genetic differences observed in host varieties raised important questions on the most appropriate naming of S. stercoralis across hosts, the geographic differences we uncovered for S. fuelleborni may also prompt a possible nomenclatural revision. Prior to the availability of molecular analyses, Ashford and Barnish stated that ‘In the event of S. fuelleborni is as thus conceived including more than one species, the name S. simiae is available for the parasites of Asian primates’ (Ashford and Barnish, Reference Ashford, Barnish and Grove1989). In the ‘molecular era’, Hasegawa et al. (Reference Hasegawa, Hayashida, Ikeda and Sato2009, Reference Hasegawa, Sato, Fujita, Nguema, Nobusue, Miyagi, Kooriyama, Takenoshita, Noda, Sato, Morimoto, Ikeda and Nishida2010) also remarked on the considerable diversity of S. fuelleborni across Asian and African varieties, suggesting that subspecific designations might be indicated for varieties they investigated. Though not possible to definitively confirm the specific status and assign ‘S. simiae’ or suggest another name for Asian isolates from this work alone, this opens the possibility for such if further characterization continues to support that conclusion. It is also possible that Macaca spp. and other primates may harbour multiple, possibly cryptic, Strongyloides species. Importantly, genetic differences between and among African and Asian primate S. fuelleborni may lead to corresponding differences in zoonotic potential, which could explain the nearly exclusive restriction of human S. fuelleborni infections to sub-Saharan Africa. If this is the case, recognizing separate species would be useful if further investigation supports this. Presently, S. fuelleborni genotyping datasets are smaller and less complete compared to those available for S. stercoralis. Additional sequence data from morphologically characterized African and Asian S. fuelleborni would be required from a range of hosts before any taxonomic changes are formalised.

Continuing genetic analyses will aid in resolving a major outstanding question on whether the occurrence of S. stercoralis in animals and humans represents zoonotic spillover (animal to human transmission) or spillback (human to animal transmission). Apart from occasional S. stercoralis infections in great apes living in proximity to infected humans, bona fide S. stercoralis infections, or infections with similar species are very seldom detected in herbivores or the omnivorous primates (Grove, Reference Grove1989). Therefore, it seems that S. stercoralis would have originally evolved in canids or allied taxa (e.g. within the suborder Caniformia) rather than in hominids.

Finally, historic experimentation reveals that two Strongyloides species described from procyonids, S. procyonis and S. nasua, show interesting patterns analogous to observations of S. stercoralis of dogs and humans. The raccoon-associated species S. procyonis bears close morphological similarities to S. stercoralis (see Little, Reference Little1966a). In one trial, a short-lived, transient patent S. procyonis infection was successfully established in a human volunteer; one dog also developed a moderate-duration patent infection (3.5 months) (Little, Reference Little1966b). Raccoons may also be susceptible to human S. stercoralis – a young raccoon developed a patent infection of moderate duration (92 days) following exposure to filariform larvae collected from an infected human (Johnson, Reference Johnson1962). Strongyloides nasua of coati (Nasua spp.) – is also highly similar to S. stercoralis and some authors regard Strongyloides nasua as a synonym for S. stercoralis (Moraes et al., Reference Moraes, da Silva, Tebaldi and Lux Hoppe2019). Also, a patent infection was established in a white-nosed coati (Nasua narica) using human-derived S. stercoralis (Sandground, Reference Sandground1926). The observation of morphological overlap and cross-transmissibility among these species of S. stercoralis-like parasites (S. procyonis, S. nasua, S. stercoralis of dogs, and S. stercoralis of humans) supports the idea that the common ancestor of the S. stercoralis-like parasites originated in some ancestral Caniformia carnivore, that adapted to infecting humans sometime during the domestication of dogs. This idea was also suggested by Nagayasu et al. (Reference Nagayasu, Htwe, Hortiwakul, Hino, Tanaka, Higashiarakawa, Olia, Taniguchi, Win and Ohashi2017) on the basis of S. procyonis being the closest extant relative to S. stercoralis, which is also supported by phylogenies constructed from 18S rDNA sequences by Hino et al. (Reference Hino, Tanaka, Takaishi and Fuji2014). Further genetic characterization of the procyoid parasites (S. procyonis, S. nasua) and other currently undescribed Strongyloides of other caniforms, including both true canids and arctoid mammals (e.g. bears, mustelids), will be necessary to investigate this evolutionary hypothesis.

It follows from the evidence above that S. fuelleborni, rather than S. stercoralis, is probably best regarded as the true ‘human Strongyloides’ that has coevolved with our species, given the primate origin of the former, and that S. stercoralis probably represents an evolutionary spillover event. The inability to morphologically distinguish distinct genetic lineages of S. stercoralis, the relationship between S. stercoralis and other Strongyloides infecting mammals of the order Caniformia, in conjunction with the clustering of canine S. stercoralis (cluster 5) as an outgroup in this analysis, support the hypothesis of Nagayasu et al. (Reference Nagayasu, Htwe, Hortiwakul, Hino, Tanaka, Higashiarakawa, Olia, Taniguchi, Win and Ohashi2017) who proposed that the origin of human-infecting S. stercoralis was related to the domestication of dogs circa 14,000–6500 years before present (MacHugh et al., Reference MacHugh, Larson and Orlando2017). However, our data indicate that some genotypes have seemingly adapted to the human host to a point where they rarely infect dogs (Fig. 2, excluding clusters 4 & 5). Therefore, these rare cases of dog infection caused by clearly human-adapted genotypes (e.g. Fig. 2, clusters 1–3 & 6) possibly represent modern and ongoing zoonotic ‘spillback’. This highlights that generalizations regarding the role of dogs in human strongyloidiasis are difficult to make without understanding the population structure of this species complex.