Assessing the zoonotic potential of Ascaris suum and Trichuris suis: looking to the future from an analysis of the past

The two geohelminths, Ascaris lumbricoides and Trichuris trichiura, infect more than a billion people worldwide but are only reported sporadically in the developed part of the world. In contrast, the closely related species A. suum and T. suis in pigs have a truly global distribution, with infected pigs found in most production systems. In areas where pigs and humans live in close proximity or where pig manure is used as fertilizer on vegetables for human consumption, there is a potential risk of cross-infections. We therefore review this relationship between Ascaris and Trichuris in the human and pig host, with special focus on recent evidence concerning the zoonotic potential of these parasites, and identify some open questions for future research.     

Ascaris and ascariasis

Ascaris lumbricoides and Ascaris suum are widespread parasitic nematodes of humans and pigs respectively. Recent prevalence data suggests that approximately 1.2 billion people are infected. Adult worms exhibit an overdispersed frequency distribution in their hosts and individuals harbouring heavy burdens display associated morbidity. In this review, we describe the parasite, its distribution and measures undertaken to control infection.     

Some experimental approaches to the study of nutrition and parasitic infection

Two experimental models to examine the relationship between host nutrition and parasitic infection are considered. Moniliformis dubius, an acanthocephalan parasite of the laboratory rat, is dependent on the type and amount of host dietary carbohydrate for growth and reproduction. Starch, glucose, fructose, and mannose in host diets support parasite growth and development, but much lower dietary amounts of starch, fructose, and mannose are needed compared to free glucose. The pig-Ascaris suum relationship is considered as a model for human ascariasis. Experimental Ascaris infections in pigs resulted in reduced growth, reduced food intake, lowered biological value of dietary protein, and reduced intestinal lactase activity and lactose tolerance. These results are similar in some respects to reported effects of Ascaris infection in humans.     

Biochemical and immunological systematics of some ascaridoid nematodes: genetic divergence between congeners

Vertical starch gel electrophoresis and trefoil immunodiffusion were used to study the systematics of some ascaridoid nematodes. Within the Ascarididae, the time scale of divergence was too great for intergeneric electrophoretic comparisons. Congeneric electrophoretic comparisons of Baylisascaris procyonis (host--raccoon) versus Baylisascaris transfuga (host--black bear), and Toxocara canis (host--domestic dog) versus Toxocara cati (host--domestic cat) yielded Nei genetic distance coefficients of 1.21 and 1.55, respectively. Estimates of times of divergence made from 1 electrophoretic clock calibration suggest that the Baylisascaris species have not shared a common ancestor for 25 million years (Myr), and that the Toxocara species diverged 33 Myr ago. The Baylisascaris divergence estimate corresponds to host-family divergence estimates based on immunological and paleontological evidence, which suggests that cospeciation has occurred. In contrast to this, Ascaris suum (host--pig) and Ascaris lumbricoides (host--human) have a distance coefficient of 0.09. This indicates that these species diverged comparatively recently and may represent a case of host range expansion. Trefoil immunodiffusion comparisons of ascaridoid albumins yielded reactions of identity for A. suum, A. lumbricoides, Parascaris equorum, B. procyonis, B. transfuga, T. canis, and T. cati. This confirms that these taxa are members of a monophyletic group.     

The dangers of using single locus markers in parasite epidemiology: Ascaris as a case study

Molecular markers are used widely to discriminate between closely related species of parasites, and in many cases a single locus is used for this purpose. This article aims to show how molecular data derived from a single genetic marker or linkage group - in this case mitochondrial DNA - can lead to ambiguous conclusions and to illustrate how a multilocus approach has enhanced our understanding of the epidemiology of two closely related parasites, the nematodes Ascaris suum, which infects pigs, and Ascaris lumbicoides, which infects humans.     

Ascaris suum: are trypsin inhibitors involved in species specificity of Ascarid nematodes?

Inhibitors of porcine trypsin were prepared from aqueous extracts of the parasitic nematodes Ascaris suum (hogs) and Ascaris lumbricoides (human). In this study three experiments were performed. (1) Polyclonal antibodies were prepared against one isoform of trypsin inhibitor from each parasitic nematode. Each antibody reacted with all isoforms from itself as well as all isoforms from the other parasite. (2) Association equilibrium constants were measured by titrating host trypsins (porcine or human) with the isoforms of trypsin inhibitors from A. suum and A. lumbricoides. While three of the combinations formed tight complexes that can be precipitated, the fourth complex, A. suum trypsin inhibitor-human trypsin has a Ka that is a 300 to 1000 times weaker interaction than the three other titration pairs. (3) Live A. suum worms were incubated in isosmotic media that contained either porcine trypsin or human trypsin. A suum worms survived in porcine trypsin and in the controls but were killed and digested after exposure for 5 days in human trypsin. The first experiment suggests that the trypsin inhibitors from A. suum and A. lumbricoides have similar epitopes, while the second experiment suggests that there are differences near the reactive site of the inhibitors. The consequences of these differences are dramatically demonstrated by the third experiment in which live A. suum worms in the presence of human trypsin die and are digested but those in porcine trypsin survive. These experiments suggest that in order to parasitize a host, a nematode requires a complement of protease inhibitors that interact strongly with those host proteases that are in their environment.     

Scanning electron microscopy of the eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax.

Eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax were examined by scanning electron microscopy (SEM). All species under study exhibited pronounced surface ridges. The ridges formed distinctive patterns in T. canis and T. mystax. In the Ascaris species, the ridges are similar except that they are more pronounced in the eggs of A. suum. Operculumlike structures were observed only in Ascaris. Correlation of data from SEM with previously reported transmission electron microscopy suggests that the surface ridges seen in Ascaris eggs are formed by the chitinous layer of the shell.     

Pigs become infected after ingestion of livers and lungs from chickens infected with Ascaris of pig origin

An experimental infection with Ascaris of pig origin showed that Ascaris suum larvae can migrate extra-intestinally in chickens. Furthermore, after feeding piglets with Ascaris infected chicken liver and lungs, it was possible to recover larvae from their lungs. These observations suggest that the chicken could serve as a paratenic host for Ascaris. There is also the possibility for zoonotic transmission if raw chicken livers are consumed by humans.     

Toxocara infection in pigs. The use of indirect fluorescent antibody tests and an in vitro larval precipitate test for detecting specific antibodies.

An in vitro larval precipitate test using second-stage Toxocara canis larvae and an indirect fluorescent antibody (IFA) test employing cuticles of T. canis larvae as antigen were evaluated using antisera produced in pigs experimentally infected with T. canis, T. cati, Ascaris suum, Toxascaris leonina and Parascaris equorum. The former test was both specific and sensitive and is suggested as a reliable and simple method of detecting Toxocara antibodies in pigs. The latter test was considered unsuitable because of cross-reactions that occurred when sera from pigs infected with other ascarids were tested. An IFA test for Ascaris antibodies, employing cuticles of A. suum larvae as antigen, is described. The degree of specificity of this test suggests that it may be of value in the detection of antibodies to Ascaris in pigs under natural conditions.     

Interactions Between the Nematode Parasite of Pigs,Ascaris suum,and the Earthworm Aporrectodea longa

Pig faeces in which Ascaris suum eggs had been embryonating for 57 days were placed in buckets of soil containing either 30 or no earthworms (Aporrectodea longa). When present, earthworms consumed the faeces and transported the eggs down into the soil, without inflicting any visible damage on the eggs. In later experiments 10 earthworms from the above experiment were fed to each of ten pigs, and another 40 earthworms were dissected. None of the 10 pigs became infected with A. suum through consumption of earthworms, and none of the dissected earthworms were found to contain A. suum larvae. This experiment indicates that A. longa did not act as a paratenic host for A. suum but shows that earthworms are very efficient in transporting A. suum eggs from faeces deposited on the soil surface into the soil.     

The population genetics of the apple maggot fly,Rhagoletis pomonella and the snowberry maggot,R. zephyria: implications for models of sympatric speciation

True fruit flies belonging to the Rhagoletis pomonella (Diptera:Tephritidae) sibling species complex possess several attributes consistent with a history of sympatric divergence via host plant shifts. Here, we investigate whether hybridization and genetic introgression is occurring between two members of the group, Rhagoletis pomonella (Walsh), whose primary hosts are domestic apples (Malus pumila) and hawthorns (Crataegus spp., and R. zephyria (Snow) whose host is snowberries (Symphoricarpos spp.). These flies are important because they appear to be at a transition stage between taxa reproductively isolated solely on the basis of host plant-related adaptations and those that have evolved additional non-host dependent sterility and inviability. Observing extensive hybridization and introgression between R. pomonella and R. zephyria would have major repercussions for current models of sympatric speciation.In a survey of allozyme and mitochondrial DNA (mtDNA) variation for 1105 flies collected throughout the northern United States we found two results suggesting that low level hybridization occurs between R. pomonella and R. zephyria. (1) Two flies reared from snowberries and one fly reared from hawthoms had genotypes indicative of them being R. pomonella and R. zephyria, respectively. Rhagoletis pomonella and R. zephyria adults may therefore occasionally frequent each others host plant, providing the opportunity for hybridization. (2) Four flies collected from hawthorns and one from snowberries had genotypes that made them likely to be F1 hybrids.Likelihood analysis revealed the data were also consistent with an hypothesis of shared ancestral alleles (i.e., the pattern of genetic variation could also be explained by R. pomonella and R. zephyria sharing alleles/haplotypes whose origins date to a common ancestor). We estimated that, in the absence of interspecific mating, random assortment of genes within R. pomonella and R. zephyria populations would produce an average of 5.4 flies with genotypes suggesting they were F1 hybrids – a number equivalent to the 5 putative F1 hybrids observed in the study. Our results therefore underscore the difficulty in distinguishing between hypotheses of low level introgression and shared ancestral polymorphism. But even if hybridization is occurring, the data suggest that it is happening at a very low and probably evolutionarily insignificant level (perhaps 0.09% per generation), consistent with sympatric speciation theory. Future tests are discussed that could help resolve the hybridization issue for R. pomonella and R. zephyria.     

Proteinase inhibitors in Ascarida

Ascaris suum and A. lumbricoides are intestinal parasites that survive in a hostile hydrolytic environment They contain low-molecular-weight proteins that can inactivate most of the proteinases present in the worm's surroundings. Jeffrey Hawley, Mark Martzen and Robert Peanasky suggest that host specificity of these nematodes may be associated with the efficiency of their inhibitors.     

Influence of hydrogeographic history and hybridization on the distribution of genetic variation in the pupfishes Cyprinodon atrorus and C. bifasciatus

The evolutionary importance of hybridization in animals has been subject of much debate. In this study, we examined the influence of hydrogeographic history and hybridization on the present distribution of nuclear and mitochondrial DNA variation in two pupfish species, Cyprinodon atrorus and Cyprinodon bifasciatus. Results presented here indicate that there has been limited introgression of nuclear genes; however, mtDNA introgression has been substantial, with complete replacement of the C. bifasciatus mitochondrial genome by that of C. atrorus. Subsequent to this replacement, there has been diversification of mitochondrial haplotypes along major geographic regions in the basin. Evidence was also found that mitochondrial replacement follows a predictable, cyclical pattern in this system, with isolation and diversification followed by re-contact and replacement of C. bifasciatus mitochondrial haplotypes by those of C. atrorus. This pattern is best explained by a combination of a numeric bias towards C. atrorus and mating site selection rather than selection for C. atrorus mitochondrial genome. These results demonstrate the important role hybridization can play in evolution.     

Y‐chromosome evidence supports asymmetric dog introgression into eastern coyotes

Hybridization has played an important role in the evolutionary history of Canis species in eastern North America. Genetic evidence of coyote–dog hybridization based on mitochondrial DNA (mtDNA) is lacking compared to that based on autosomal markers. This discordance suggests dog introgression into coyotes has potentially been male biased, but this hypothesis has not been formally tested. Therefore, we investigated biparentally, maternally, and paternally inherited genetic markers in a sample of coyotes and dogs from southeastern Ontario to assess potential asymmetric dog introgression into coyotes. Analysis of autosomal microsatellite genotypes revealed minimal historical and contemporary admixture between coyotes and dogs. We observed only mutually exclusive mtDNA haplotypes in coyotes and dogs, but we observed Y‐chromosome haplotypes (Y‐haplotypes) in both historical and contemporary coyotes that were also common in dogs. Species‐specific Zfy intron sequences of Y‐haplotypes shared between coyotes and dogs confirmed their homology and indicated a putative origin from dogs. We compared Y‐haplotypes observed in coyotes, wolves, and dogs profiled in multiple studies, and observed that the Y‐haplotypes shared between coyotes and dogs were either absent or rare in North American wolves, present in eastern coyotes, but absent in western coyotes. We suggest the eastern coyote has experienced asymmetric genetic introgression from dogs, resulting from predominantly historical hybridization with male dogs and subsequent backcrossing of hybrid offspring with coyotes. We discuss the temporal and spatial dynamics of coyote–dog hybridization and the conditions that may have facilitated the introgression of dog Y‐chromosomes into coyotes. Our findings clarify the evolutionary history of the eastern coyote.     

Morphological changes of Ascaris spp. eggs during their development outside the host

Information on the infective stage of Ascaris lumbricoides and the pathology caused by the parasite is widely available in the literature. However, information about early embryonic development of A. lumbricoides and its life cycle outside the host is limited. The purpose of this study was to describe the morphological changes within the developing embryo during incubation in vitro at 28 C, as well as to explore differences in egg viability during incubation. Ascaris suum eggs (4,000 eggs/ml), used as a model for A. lumbricoides , were placed for incubation in 0.1N H(2)SO(4) at 28 C in the dark for 21 days. Every day, sub-samples of approximately 100 A. suum eggs were taken from the incubation solution for microscopic evaluation. Development, morphological changes, and viability of the first 40 eggs were observed and documented with photos. During this study, 12 stages were identified in the developing embryo by standard microscopy, 2 of which had not been previously reported. By the end of the first wk, most developing embryos observed were in the late-morula stage (72.5%). On day 14 of incubation, 90% had developed to larva-1 stage, and by day 21, 100% had developed to larva-2 stage. No significant differences were found in the viability recorded in a continuum from day 5 to day 21 of incubation (chi-square, P > 0.05). The result of this study complements and expands the stages of development of Ascaris spp. outside the host previously reported in the literature. It also suggests the potential use of early stages of development of the nematode to determine viability and safety of sewage sludge, wastewater, or compost after treatment recommended by USEPA.     

Phylogeographical Studies of Ascaris spp. Based on Ribosomal and Mitochondrial DNA Sequences

Background

The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the world''s most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples.

Methodology

151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions.

Principal Findings

No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum.

Conclusions/Significance

These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This conclusion has implications on the systematics, transmission and control programs relating to ascariasis.     

Chlorophyll-binding proteins revisited - a multigenic family of light-harvesting and stress proteins from a brown algal perspective

Background

Mitochondrial introgression may result in the mitochondrial genome of one species being replaced by that of another species without leaving any trace of past hybridization in its nuclear genome. Such introgression can confuse the species genealogy estimates and lead to absurd inferences of species history. We used a phylogenetic approach to explore the potential mitochondrial genome introgression event(s) between two closely related green pond frog species, Pelophylax nigromaculatus and P. plancyi.

Results

DNA sequence data of one mitochondrial and two nuclear genes from an extensive sampling of the two species were collected, and the genealogies of the three genes were constructed and compared. While the two nuclear genes congruently showed mutual reciprocal monophyly of both species, the mitochondrial phylogeny separated a Korean P. nigromaculatus clade, a paraphyletic central China P. plancyi assemblage, and a large well-supported introgression clade. Within the introgression clade, the mitochondrial haplotypes of the two species were mixed together. This reticulated pattern can be most parsimoniously explained by an ancient mitochondrial introgression event from P. plancyi to P. nigromaculatus that occurred at least 1.36 MYA, followed by multiple recent introgression events from P. nigromaculatus back to P. plancyi within the last 0.63 MY. The re-constitution of previously co-adapted genomes in P. plancyi may be responsible for the recent rampant introgression events. The Korean P. nigromaculatus clade likely represents the only surviving "true" mitochondrial lineage of P. nigromaculatus, and the central China P. plancyi assemblage likely represents the "original" P. plancyi mitochondrial lineage. Refugia in the Korean Peninsula and central China may have played a significant role in preserving these ancient lineages.

Conclusions

The majority of individuals in the two species have either introgressed (P. nigromaculatus) or reclaimed (P. plancyi) mitochondrial genomes while no trace of past hybridization in their nuclear genomes was detected. Asymmetrical reproductive ability of hybrids and continuous backcrossing are likely responsible for the observed mitochondrial introgression. This case is unique in that it includes an ancient "forward" introgression and many recent "backward" introgressions, which re-constitutes the original nuclear and mitochondrial genomes of P. plancyi. This hybrid system provides an excellent opportunity to study cyto-nuclear interaction and co-adaptation.     

Chromatin diminution in nematode development

Chromatin diminution in Parascaris and Ascaris represents the classical case of a developmentally programmed genome rearrangement. The process is very specific with respect to ontogenetic timing and chromosomal localization, and involves chromosomal breakage, new telomere formation and DNA degradation. Recent evidence from Ascaris lumbricoides var. suum suggests that chromatin diminution might have a function in gene regulation.     

Chronic ascariasis and malnutrition

Malnutrition is the most widespread ill of mankind. Its global distribution coincides indistinguishably with that of the common roundworm. Ascaris lumbricoides is common - infecting about one-quarter of the world's population. Acute clinical ascariasis sometimes requires hospital treatment, but chronic infections contribute to long-term malnutrition. This alone should justify greater expenditure on the treatment and control of Ascaris.     

Trichinella spiralis: a 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody

Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with Trichinella spiralis larvae and an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA.