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.     

Species-specific proteins identified in Ascaris lumbricoides and Ascaris suum using two-dimensional electrophoresis

The protein profile of adult female Ascaris lumbricoides and Ascaris suum originating from humans and pigs, respectively, was studied using two-dimensional polyacrylamide gel electrophoresis. Six different major protein spots specific for A. lumbricoides were identified irrespective of their geographical origin and no major specific spot was encountered in A. suum. No major differences in the protein profiles between the extract by phosphate-buffered saline and urea were encountered for either Ascaris species. It is therefore possible to use 2D-PAGE as a tool for discriminating the closely related Ascaris species from humans and pigs.     

Homology and heterology between the secreted antigens of the parasitic larval stages of Ascaris lumbricoides and Ascaris suum.

The materials released in vitro by the tissue-parasitic larval stages of the large roundworm of man, Ascaris lumbricoides, were analysed by radio-iodination, immunoprecipitation, and SDS-PAGE. The antigens were found to be heterogeneous, ranging in molecular weight from 14 to 410 kD, and were found to alter radically during the parasites' migration to the lungs. The antigens secreted by the infective and lung-stage larvae of the pig homologue, Ascaris suum, were compared with those of the human worms. This revealed a remarkable degree of homology between the products of the two, at both the molecular and immunological levels. The two species could be discriminated, however, on the basis of the SDS-PAGE profiles of the antigens secreted by both developmental stages of the parasites examined. Finally, antiserum to the canine ascarid infective to man, Toxocara canis, was found to precipitate a significant proportion of Ascaris-secreted molecules. These studies, therefore, confirm the potent antigenicity of excretory/secretory materials, and their potential for use in immunodiagnosis, but predict serious difficulties for seroepidemiology and the specific detection of ascariasis in man.     

Antigenic relationships between the surface-exposed, secreted and somatic materials of the nematode parasites Ascaris lumbricoides, Ascaris suum, and Toxocara canis.

The cosmopolitan nematode parasites Ascaris lumbricoides, Ascaris suum, and Toxocara canis are closely related phylogenetically, and are all pathogenic to man. In the case of the latter, the antigens released by the tissue-invasive parasitic larvae in vitro ('excretory/secretory' or 'ES' antigens) are routinely used for serodiagnostic purposes. Here we have found, using radioimmunoprecipitation with defined rabbit antiserum, and SDS-PAGE, that there is a significant antigenic similarity between the secreted and somatic antigens of the three nematodes, and have characterized cross-reactive components. Among these is a 14 kD internal protein which has a homologue in all three parasites. This molecule is the subject of an IgG antibody response in Ascaris infection, but there is no measurable response to it in toxocariasis. Lastly, using quantitative immunofluorescence, the antigens exposed on the surface of intact, living, larvae were found to be cross-reactive or specific depending on the developmental stage of the parasites. This means that the surface of tissue-invasive Ascaris larvae bears stage-specific epitopes.     

The role of dogs in transmission of Ascaris lumbricoides for humans

The dog’s role as a definitive host for a number of zoonotic parasites has been widely studied and recognized as being a significant public health problem worldwide. This study aimed to report, for the first time, our investigation into the role of dogs as a biological transmitter for Ascaris lumbricoides, via necropsy of a sample of rural stray dogs in a developing community in Giza governorate, Egypt, where promiscuous defecation by human was common, and examination for A. lumbricoides worms as well as other ascaridiod nematodes of dogs. The recovered worms were identified in the laboratory after observing cephalic alae and egg morphology under a microscope, as well as scanning electron microscopy of their anterior ends. Of the 25 dogs examined, 14 were infected with Toxocara canis (56.0%), two with Toxascaris leonina (8.0%), and two with A. lumbricoides (8.0%). One dog was co-infected with T. canis and T. l eonina. A. lumbricoides eggs were shown to be viable and 75–80% of eggs embryonated following 3 weeks of incubation at 28°C. The present study suggested that dogs could act as reservoir hosts of A. lumbricoides and environmental contaminators that increase risk of infection in humans.     

N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum, and MHC-restricted IgE responses to it.

A protein allergen of the parasitic nematode Ascaris has been purified to homogeneity by immunoaffinity chromatography. It is the most abundant protein species in the parasite's body fluid and has been named ABA-1. The allergen's molecular weight (MW) has been previously estimated at 14,000, but this sizing is currently under re-evaluation. The immunological activity of the protein was intact after purification, as attested by immunoprecipitation and passive cutaneous anaphylaxis. The IgE response to ABA-1 was under major histocompatibility complex (MHC) restriction in the rat, in which only RT1u strains were found to respond following infection with the parasite. The tissue-invasive and intestinal stages of both Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) have an antigen of similar MW to ABA-1 in their secretions or among their somatic antigens. These are antigenically indistinguishable; they were found to have similar amino acid compositions, and their N-terminal amino acid sequences were identical to 41 residues. Finally, the apparent MW, amino acid composition and isoelectric point of ABA-1 all argue for close similarity to the previously described Allergen A of the parasite.     

Changes in locomotory behavior and cAMP produced in Ascaris suum by neuropeptides from Ascaris suum or Caenorhabditis elegans

Injection of Ascaris FMRFamide-like (AF) peptides and peptides encoded by genes in Caenorhabditis elegans were analyzed for effects on locomotion, body waveforms, and cAMP concentrations in adult female Ascaris suum. Injection of AF1 (KNEFIRFamide) or AF2 (KHEYLRFamide) inhibited the propagation of locomotory waves and reduced the number of waveforms, decreased the body length, and caused a large, long-lasting increase in cAMP. Muscle tissue was identified as a major source of the cAMP response induced by AF1. The AF1 analog AF1R6A did not affect cAMP levels by itself, but inhibited the cAMP response produced by AF1. AF8 (KSAYMRFamide) produced ventral coiling in the behavioral assay, and AF10 (GFGDEMSMPGVLRFamide) decreased the body length and increased the number of body waveforms. In dorsal muscle strips, AF10 produced a long-lasting contraction. Neither AF8 nor AF10 changed cAMP concentrations. AF17 (FDRDFMHFamide) increased body length and decreased cAMP. The neuropeptides encoded by C. elegans genes flp-4, flp-7, flp-9, and flp-13 produced paralysis and loss of waveforms, increased body length and, like AF17, decreased cAMP. Three new predicted peptides from C. elegans genome sequences were synthesized and tested. One produced ventral coiling but no change in cAMP; the other two gave no detectable responses. The fact that C. elegans neuropeptides produce behavioral and physiological effects in A. suum suggests that structurally related peptides may exist in A. suum. The profound changes in cAMP produced by some neuropeptides has important implications for understanding cAMP signaling and shows that neuropeptide-mediated signal transduction pathways are potential targets for anthelmintic drug development.     

The secreted and somatic antigens of the third stage larva of Anisakis simplex, and antigenic relationship with Ascaris suum, Ascaris lumbricoides, and Toxocara canis

The in vitro-released 'excretory/secretory' (ES) and somatic antigens of the third stage (infective) larva of Anisakis simplex were characterised by radioiodination, immunoprecipitation, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Oral infection with the parasite elicited a substantial IgG antibody response to ES in infected rabbits and humans, with a minimal response to somatic materials. Serial serum sampling in experimental infection showed that there was a sequential recognition of distinct ES components. In contrast to oral infection, intraperitoneal exposure of rats with living parasites induced a strong response to both ES and somatic antigen preparations. Sequential recognition of ES antigens, and differential responses to somatic components, might, therefore, have application in the estimation of the age and degree of penetration by the nematodes in human infection. Extensive antigenic relationships were found between A. simplex and three other species of ascaridoid nematodes, namely Ascaris lumbricoides, Ascaris suum, and Toxocara canis, but none with a panel of non-ascaridoid nematodes. Evidence is presented that a Mr 14,000 component of A. simplex has a homologue in all of the ascaridoids examined, but does not elicit an antibody response in anisakiasis. Finally, the ES of A. simplex is shown to contain two proteinase activities, of approximately Mr 23,400 and 46,100, as revealed by separation on gelatin substrate gels, although the antigenicity of the enzymes remains to be established.     

Immunohistochemical characterization of hepatic lesions associated with migrating larvae of Ascaris suum in pigs

This paper describes the histopathological features and the cellular distribution of T lymphocytes (CD3), B cells (CD79a), immunoglobulin (IgG, IgA, IgM)-bearing plasma cells, macrophages (Mac387 and alpha-1-antitrypsin), MHC class II antigen and S-100 protein in hepatic white spots associated with naturally occurring Ascaris suum parasitism in 35 pigs. Hepatic granulomas were observed in 10 pigs, whereas lymphoid proliferation with a diffuse or lymphonodular pattern was the main histopathological lesion in 14 other pigs, and portal fibrosis in a further 11 animals. In lymphonodular lesions, the distribution of immunoreactive cells with all antibodies tested was closely similar to that found in the cortex of lymph nodes. Thus, lymphoid follicles were composed mainly of CD79a(+)B cells and interfollicular tissue was composed mainly of CD3(+)T lymphocytes. The presence of follicular dendritic and interdigitating cells expressing S-100 protein and MHC class II antigen in lymphonodular lesions suggested that these are highly organized structures developed to enhance antigen presentation to B and T cells, and consequently the local immune response against the parasite. The humoral local response was represented mainly by IgG-secreting plasma cells. Copyright Harcourt Publishers Ltd.     

Molecular analysis of clinical isolates of Mycobacterium bovis recovered from humans in Italy

In order to achieve a better knowledge of Mycobacterium bovis epidemiology in Italy, 42 clinical isolates from humans were genotyped. Predominant molecular patterns were found in one cluster of 15 isolates sharing spoligotype (ST482), variable-number tandem repeat (VNTR), and IS6110-based restriction fragment length polymorphism (one 1.9-kb band) profiles and in two clusters of 6 and 3 Mycobacterium bovis BCG isolates differing by one VNTR character. The remaining 18 isolates yielded unique profiles. Our results confirm the potential utility of spoligotyping and VNTR typing as a major typing system of M. bovis isolates.     

Molecular epidemiological study of hepatitis B virus among migrant workers from Cambodia,Laos, and Myanmar to Thailand

Although hepatitis B virus (HBV) infection is endemic in Southeast Asia, molecular epidemiological data on HBV circulating in some countries are limited. The aims of this study were to evaluate the seroprevalence of HBV and its genetic variability among migrant workers from Cambodia, Laos, and Myanmar in Thailand. Sera collected from 1,119 Cambodian, 787 Laotian, and 1,103 Myanmarese workers were tested for HBsAg. HBV DNA was amplified and the pre‐S/S region was sequenced for genotyping and genetic mutation analysis. HBsAg was detected in 282 (9.4%). The prevalence of HBsAg among migrant workers from Cambodia, Laos, and Myanmar was 10.8%, 6.9%, and 9.7%, respectively. Of 224 subjects positive for HBV DNA, 86% were classified as genotype C (99% were sub‐genotype C1) and 11.6% were genotype B (30.8%, 34.6%, and 30.8% were sub‐genotypes B2, B3, and B4, respectively). Various point mutations in the “a” determinant region were detected in approximately 18% of these samples, of which Ile126Ser/Asn was the most frequent variant. Sequencing analysis showed that 19.1% of samples had pre‐S mutations, with pre‐S2 deletion as the most common mutant (7.7%) followed by pre‐S2 start codon mutation (3.8%) and both pre‐S2 deletion and start codon mutation (3.3%). High prevalence of HBV infection (approximately 7–11%) was found among migrant workers from Cambodia, Laos, and Myanmar, which may reflect the current seroprevalence in their respective countries. The data also demonstrated that HBV sub‐genotype C1 was the predominant strain and various mutations of HBV occurring naturally were not uncommon among these populations. J. Med. Virol. 82:1341–1349, 2010. © 2010 Wiley‐Liss, Inc.     

An investigation of the antigens of Ascaris lumbricoides using a radioimmunoassay and sera of naturally infected humans

Of 5 humans from an Ascaris lumbricoides(var. suum)-endemic area who were positive for Ascaris infection (past or current) when examined by a radioallergosorbent test (RAST) for serum immunoglobulin E, only 1 contained detectable levels of Ascaris specific serum IgG antibodies. These antibodies were not detectable when the radioimmunoassay was replaced with a precipitin assay. There was a wide ringe of molecular weights and isoelectric points of antigens in Ascaris body fluid (ABF) which reacted with antibodies in this infected human. However, unlike the IgE-binding ABF antigens, the IgG-binding ABF antigens were particularly evident in one area of molecular weight and isoelectric point. This human also had IgA and IgM to Ascaris antigens whereas the others had barely detectable amounts.     

Protective effect of an extract from Ascaris suum in experimental arthritis models

We investigated the effect of an extract from a helminth (Ascaris suum) in zymosan-induced arthritis (ZYA) or collagen-induced arthritis (CIA). Rats and mice, respectively, received 1 mg and 0.1 mg zymosan intra-articularly (i.a.). Test groups received an A. suum extract either per os (p.o.) or intraperitoneally (i.p.) 30 min prior to i.a. zymosan. Controls received saline. Hypernociception was measured using the articular incapacitation test. Cell influx, nitrite, and cytokine levels were assessed in joint exudates. The synovia and distal femoral extremities were used for histopathology. Cartilage damage was assessed through determining glycosaminoglycan (GAG) content. DBA/1J mice were subjected to CIA. The test group received A. suum extract i.p. 1 day after CIA became clinically detectable. Clinical severity and hypernociception were assessed daily. Neutrophil influx was determined using myeloperoxidase activity. The A. suum extract, either i.p. or p.o., significantly and dose-dependently inhibited cell influx and hypernociception in ZYA in addition to reducing GAG loss and ameliorating synovitis. The A. suum extract reduced i.a. levels of NO, interleukin-1beta (IL-1beta), and IL-10 but not tumor necrosis factor alpha (TNF-alpha) in rats subjected to ZYA while reducing i.a. IL-10, but not IL-1beta or TNF-alpha, levels in mice. Clinically, mice subjected to CIA treated with the A. suum extract had less severe arthritis. Hypernociception, myeloperoxidase activity, and synovitis severity were significantly reduced. These data show that a helminth extract given p.o. protects from arthritis severity in two classical arthritis models. This A. suum effect is species independent and functions orally and parenterally. The results show clinical and structural benefits when A. suum extract is given either prophylactically or therapeutically.     

Isolation and partial characterization of a fatty-acid-binding protein from Ascaris suum reproductive tissue

A 12-kDa fatty-acid-binding protein was purified to homogeneity from Ascaris suum reproductive tissue as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino-acid-sequence analysis of the protein revealed its identity with the ABA-1 allergen protein isolated from A. suum pseudocoelomic fluid. Fatty-acid binding by the protein from A. suum reproductive tissue was investigated using the Lipidex 1000 assay, which revealed the presence of a single class of fatty-acid-binding sites with an apparent dissociation constant for palmitate of about 0.8 μM. Received: 8 December 1996 / Accepted: 20 December 1996     

Pyruvate and citrate metabolism in the muscle tissue of Ascaris lumbricoides

Summary Aconitase and NAD linked isocitrate dehydrogenase were present in Ascaris lumbricoides muscle at only very low activities, whilst there were significant levels of citrate synthase, NADP linked isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase and succinic thiokinase. Pyruvate dehydrogenase was present in A. lumbricoides muscle at levels comparable with mammalian tissues and results suggest that it is modulated via a phosphotransferase/phosphatase system.The tricarboxylic acid cycle intermediates, citrate, isocitrate and 2-oxoglutarate were all detected in freeze clamped muscle, but their steady state levels were considerably lower than those found in mammalian tissues.     

Comparison of collagen gene sequences in Ascaris suum and Caenorhabditis elegans

The nucleotide sequences of a 3060-bp fragment containing almost the entire coding sequence of one Ascaris suum collagen gene, and a 3019-bp pair fragment containing the 3' end of another A. suum collagen gene have been determined. The polypeptides encoded by these genes show a striking similarity to two Caenorhabditis elegans cuticular collagens, both in the position of the triple-helical regions and in the position of cysteine residues. The results of Northern blot hybridisation experiments together with dot blot analysis of RNA isolated from different adult worm tissues suggest that one of these genes is expressed in the adult nematode and that it encodes a protein of approximately 30 kDa.