Molecular Confirmation of Ascaris suum: Further Investigation into the Zoonotic Origin of Infection in an 8-Year-Old Boy with Loeffler Syndrome.

An 8-year-old male from south Louisiana was diagnosed with Loeffler syndrome of suspected Ascaris origin. Further investigation of the farm recovered larvated, infective Ascaris eggs from the soil in drains surrounding pens on the family's small hog farm. Molecular analysis of the recovered eggs, in conjunction with Ascaris-specific IgE, inadequate farm management and sanitation, and behavioral risk factors indicate the patient had an Ascaris suum soil-transmitted infection.

Molecular identification of Ascaris lumbricoides and Ascaris suum recovered from humans and pigs in Thailand, Lao PDR, and Myanmar.

Ascaris lumbricoides is the largest roundworm known from the human intestine while Ascaris suum is an internal parasite of pigs. Ascariasis, caused by Ascaris lumbricoides, has a worldwide distribution. Here, we have provided the first molecular identification of Ascaris eggs and adults recovered from humans and pigs in Thailand, Lao PDR, and Myanmar. We amplified and sequenced nuclear ribosomal DNA (ITS1 and ITS2 regions) and mitochondrial DNA (cox1 gene). Sequence chromatograms of PCR-amplified ITS1 region revealed a probable hybrid genotype from two human ascariasis cases from Chiang Mai Province, northern Thailand. All complete ITS2 sequences were identical and did not differ between the species. Phylogenetic trees and haplotype analysis of cox1 sequences showed three clusters with 99 haplotypes. Forty-seven samples from the present study represented 14 haplotypes, including 7 new haplotypes. To our knowledge, this is the first molecular confirmation of Ascaris species in Thailand, Lao PDR, and Myanmar. Zoonotic cross-transmission of Ascaris roundworm between pigs and humans probably occurs in these countries.

Phylogeography of Ascaris lumbricoides and A. suum from China.

In order to obtain further understanding of genetic structure and evolutionary relationship of Ascaris from humans and pigs, phylogeography study on 12 populations from six endemic regions in China was conducted using mitochondrial DNA markers (cytochrome c oxidase subunit 1 (COX1) and NAD1) and the software programs of DnaSP 5.0, Arlequin 3.0, MEGA 4.0, and NETWORK 4.5.1.6. Results showed that (a) genetic diversity of Ascaris varied with hosts and locations, but no distinct geographical distribution pattern was found, (b) a higher level of genetic diversity and differentiation was found in pig-derived populations in contrast to human-derived ones, and in populations of human-derived Ascaris from the southern regions in comparison to that from the middle and northern locations, but similar geographical difference was not observed within pig-derived populations, (c) historical population expanding was detected from a large part of human-derived Ascaris populations but not in pig-derived Ascaris, (d) a high level of gene flow was detected between human- and pig-derived Ascaris and also among human-derived populations, and (e) network analysis from haplotype of COX1 indicated an ancestral haplotype from human-derived Ascaris. In conclusion, the present study revealed new information on Ascaris on the aspects of genetic diversity, population differentiation and historical demographic patterns, gene flow, phylogenesis reconstruction, and haplotype network, discussed the results with historical demographic migration of humans and domestication of wild boar in China, and raised a different assumption about the evolutionary relationship of the two roundworms. This study should have certain enlightenment for the epidemiology and the evolutionary and taxonomy relationship of Ascaris from humans and pigs.

Ascariasis in Japan: is pig-derived Ascaris infecting humans?

Human ascariasis is caused by infection with the common roundworm Ascaris lumbricoides, although the pig roundworm Ascaris suum has also been reported to infect humans and develop into the adult stage. To elucidate whether pig-derived Ascaris infects humans in Japan, 9 Ascaris isolates obtained from Japanese patients and a further 9 Ascaris isolates of pig origin were analyzed to determine their internal transcribed spacer-1 sequences. Six of the 9 clinical isolates showed the Ascaris genotype which predominantly infects humans in endemic countries, while the other 3 clinical isolates and 9 pig-derived isolates showed the genotype predominant in pigs worldwide. These results suggest that at least some cases of human ascariasis in Japan are a result of infection with pig-derived Ascaris.

Some aspects of nematode infection in pigs from small herds.

Two traditionally maintained, small herds from southern Poland, with 8 and 12 sows, respectively, were surveyed coprologically during 2006-2007. In one of the herds, while deworming a group of sows with levamisole, faecal samples were collected on Day -7, Day 0 (the day of treatment) and Day 10, in order to assess the therapeutic effect of the drug. Coprological investigation was performed also in 26 fatteners originating from other small farms and slaughtered in a local abattoir, with their intestines washed through for the presence of roundworms. In both herds examined, Ascaris suum and Oesophagostomum spp. were prevalent, whilst Trichuris suis appeared only very rarely. Mainly fatteners, replacement gilts and young sows were highly infected with A. suum. The roundworm occurrence in 2- 3-week-old piglets, with the intensity of 300 eggs per gram of faeces (EPG), indicated the possibility of parasite transmission to offspring very early in age. The highest level of Oesophagostomum spp. infection was observed in sows, but weaners were also much affected. For the group of dewormed sows, the mean faecal egg count reduction (FECR) was estimated to be 77.1- 80.4%, suggesting the presence of resistant nodular worms. A very high false-positive A. suum egg counts found in slaughtered animals (240 to 320 EPG) testified to a high contamination level of the environment of small piggeries, as well. Since the reciprocal transmission of parasites between pigs and poultry might occur, it implies that the flocks should be raised separately.

PAS-1, a protein from Ascaris suum, modulates allergic inflammationvia IL-10 and IFN-c, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effectorphases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolatedfrom Ascaris suum worms, has an inhibitory effect on lung allergic inflammation due to its abilityto down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here,we investigated the role of IL-12, IFN-c and IL-10 in the PAS-1-induced inhibitory mechanism using amurine model of asthma. Wild type C57BL/6, IL-12 / , IFN-c / and IL-10 / mice were immunized withPAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-1was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophilperoxidase activity in OVA + PAS-1-immunized wild type mice. This effect well correlated with a significantreduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-1 was alsoobserved in IL-12 / mice, but not in IFN-c / and IL-10 / animals. These data show that IFN-c andIL-10, but not IL-12, play an important role in the PAS-1 modulatory effect.

[Comparative studies on the difference between Ascaris lumbricoides and Ascaris suum].

There has been continued controversy on the taxonomy of Ascaris lumbricoides Linnaeus, 1758 from humans and Ascaris suum Goeze, 1782 from pigs. This article reviews a range of comparative studies related to host susceptibility, morphology, karyotype, immunology and biochemistry, as well as molecular genetics in recent years.

MSP domain protein-1 from Ascaris suum and its possible role in the regulation of major sperm protein-based crawling motility.

Nematode sperm utilize a crawling motility based on a nematode sperm-specific cytoskeletal protein called the major sperm protein (MSP). Although MSP has no similarity to actin in sequence or structure, the motility mediated by these two proteins is nearly indistinguishable at a phenotypic level. As with the traditional actin cytoskeleton, the central component of MSP-based motility (MSP) interacts with accessory proteins that regulate polymerization and depolymerization and play a key role in cell motility. A bioinformatics approach has led to the identification of proteins with enhanced expression in the Ascaris suum male germ line, including five new proteins each containing an MSP domain. One of these MSP domain proteins (As-MDP-1) contains an MSP domain in the C-terminus and a N-terminal extension rich in prolines and alanines. The 15.6 kDa As-MDP-1 was shown to be >90% identical at the amino acid level to members of a small family of Ascaris proteins that have been shown to bind to the MSP cytoskeleton and to negatively regulate MSP fiber growth. Further, it was demonstrated that As-MDP-1 is the smallest member of the MFP1 triplet of negative regulators of MSP cytoskeleton formation. Antibodies were used to detect the presence of As-MDP-1 along the entire length of the MSP fibers suggesting that As-MDP-1 binds directly to the higher order forms of MSP. This protein has orthologues in other nematode species, is present in Ascaris in at least six allelic forms, and is likely to form multimers.

Identification of tissue-embedded ascarid larvae by ribosomal DNA sequencing.

Polymerase chain reaction (PCR) was applied to identify tissue-embedded ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), ITS1 and ITS2, of the ascarid parasites were amplified and compared with those of ascarid-nematodes registered in a DNA database (GenBank). The ITS sequences of the PCR products obtained from the ascarid parasite specimen in our laboratory were compatible with those of registered adult Ascaris and Toxocara parasites. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. Using this method, ascarid larvae embedded in the liver of a naturally infected turkey were identified as Toxocara canis. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions.

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.

Relationships among some ascaridoid nematodes based on ribosomal DNA sequence data.

The nuclear ribosomal DNA (rDNA) region spanning the first (ITS-1) and second (ITS-2) internal transcribed spacers was sequenced for 15 taxa of ascaridoid nematodes. The length of the ITS-1 and ITS-2 sequences in the 15 taxa ranged from 392-500 bp and 240 348 bp, respectively. While nucleotide variation of 0-2.9% in the ITS-1 and/or ITS-2 sequences was detected within taxa where multiple samples were sequenced, significantly higher level of nucleotide difference (9.4-66.6%) was detected between the taxa, except for Ascaris suum and A. lumbricoides whose taxonomic status remains uncertain. These interspecific differences were linked with the considerable size differences (0-108 bp) in the rDNA spacers. Phenograms based on the genetic differences among the 15 taxa showed some concordance with previous classification schemes derived from morphological data.

Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA-intercalating agent.

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold-supplemented gels. The latter was achieved because of differences (approximately 0.1-8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11-39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR-amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.