Genomics of the Parasitic Nematode Ascaris and Its Relatives.

Nematodes of the genus Ascaris are important parasites of humans and swine, and the phylogenetically related genera (Parascaris, Toxocara, and Baylisascaris) infect mammals of veterinary interest. Over the last decade, considerable genomic resources have been established for Ascaris, including complete germline and somatic genomes, comprehensive mRNA and small RNA transcriptomes, as well as genome-wide histone and chromatin data. These datasets provide a major resource for studies on the basic biology of these parasites and the host-parasite relationship. Ascaris and its relatives undergo programmed DNA elimination, a highly regulated process where chromosomes are fragmented and portions of the genome are lost in embryonic cells destined to adopt a somatic fate, whereas the genome remains intact in germ cells. Unlike many model organisms, Ascaris transcription drives early development beginning prior to pronuclear fusion. Studies on Ascaris demonstrated a complex small RNA network even in the absence of a piRNA pathway. Comparative genomics of these ascarids has provided perspectives on nematode sex chromosome evolution, programmed DNA elimination, and host-parasite coevolution. The genomic resources enable comparison of proteins across diverse species, revealing many new potential drug targets that could be used to control these parasitic nematodes.

The application of proteomic methods (MALDI-toff MS) for studying protein profiles of some nematodes (dirofilaria and ascaris) for differentiating species.

INTRODUCTION: Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-toff MS) is a reliable method for diagnosing a number of bacterial and fungal infections. It is also effective as a method of rapid diagnosis of several parasitic agents. We used MALDI-toff MS to study the protein profiles of four nematodes: Dirofilaria repens, Dirofilaria. immitis, Ascaris suum and Ascaris lumbricoides. METHODS: We studied the protein profiles of dirofilaria (five of each species: D. repens and D. immitis) and ascaris (five of each species: A. suum and A. lumbricoides), using a proteomic analysis based on MALDI-toff MS. RESULTS: Analysis of protein extracts of dirofilaria and ascaris showed spectra with high-intensity peaks in the range of 2-20 kDa. The quality of the spectra (clear graphical reflection of mass/charge to luminous intensity, consistent in repeated analyzes) and the intensity of the spectral peaks were consistent in all samples of the same species. The spectra profiles of D. repens and D. immitis differed in eight major peaks which makes it possible to differentiate species according to the protein profile. The spectra profiles obtained from A. suum and A. lumbricoides proteins differed slightly in 3 major peaks in both species and were discovered in m/z 13000; 13400 and 14400. The protein peaks in diapason 3000 kD-7300 kD specific for all genus ascaris are constant. CONCLUSIONS: MALDI-toff MS-based proteomic analysis can serve as an effective taxonomic tool for parasitological studies.

Long-Tailed Macaques (Macaca fascicularis) in Urban Landscapes: Gastrointestinal Parasitism and Barriers for Healthy Coexistence in Northeast Thailand.

Gastrointestinal parasites have diverse life cycles that can involve people, animals, and the environment (e.g., water and soil), demonstrating the utility of One Health frameworks in characterizing infection risk. Kosumpee Forest Park (Thailand) is home to a dense population of long-tailed macaques (Macaca fascicularis) that frequently interact with tourists and local residents. Our study investigated the presence of zoonotic parasites, and barriers to healthy coexistence by conducting stool analysis on macaques (N = 102) and people (N = 115), and by examining risk factors for infection with a household questionnaire (N = 95). Overall, 44% of macaques and 12% of people were infected with one or more gastrointestinal helminths, including Strongyloides spp., Ascaris spp., and Trichuris sp. An adults-only generalized linear mixed model identified three factors significantly associated with human infection: household size, occupational exposure, and contact with macaque feces at home. Participants identified both advantages and disadvantages to living in close contact with macaques, suggesting that interventions to improve human and animal health in Kosumpee Forest Park would be welcome.

Quantitative Polymerase Chain Reaction for Diagnosis of Soil-Transmitted Helminth Infections: A Comparison with a Flotation-Based Technique and an Investigation of Variability in DNA Detection.

Appropriate diagnostic techniques are crucial to global soil-transmitted helminth (STH) control efforts. The recommended Kato-Katz method has low sensitivity in low-transmission settings. Quantitative polymerase chain reaction (qPCR) is a highly sensitive alternative diagnostic option. However, little is known about the variability in qPCR results, and there are few published comparisons between qPCR and other microscopy-based techniques such as sodium nitrate flotation (SNF). Using 865 stool samples collected from 571 individuals, we compared SNF and qPCR in terms of diagnostic sensitivity and infection intensity measurements. In addition, we conducted repeated examinations on a single Necator americanus-positive stool sample over a 6-month period. Results showed good diagnostic agreement between SNF and qPCR for Ascaris spp. (κ = 0.69, P < 0.001), and moderate agreement for hookworm (κ = 0.55, P < 0.001) and Trichuris spp. (κ = 0.50, P < 0.001). Quantitative polymerase chain reaction demonstrated higher sensitivity than SNF for Ascaris spp. (94.1% versus 68.1%) and hookworm (75.7% versus 66.9%) but not for Trichuris spp. (53.1% versus 81.3%), which had very low prevalence. Sodium nitrate flotation and qPCR infection intensity measurements were strongly correlated for Ascaris spp. (ρ = 0.82, P < 0.001) and moderately correlated for hookworm (ρ = 0.58, P < 0.001). Repeated examinations using qPCR showed that N. americanus cycle threshold values decreased significantly at 1 month and remained stable thereafter. Results confirm the high diagnostic sensitivity of qPCR for Ascaris spp. and hookworm, particularly for light-intensity infections, which is ideal for settings approaching transmission elimination. Results support the potential for qPCR to be used as a quantitative assay for STH. Further research is needed in settings where Trichuris trichiura is endemic.

Diversity and prevalence of gastrointestinal parasites in two wild Galago species in Gabon.

In this study, we characterize the diversity and estimated infection levels of gastrointestinal parasites circulating in two galago species, Galago demidoff and G. thomasi in two sites situated in the Southeastern forests of Gabon. Our study reveals that eleven parasites including nine helminthes (Ascaris spp., Ankylostoma spp., Dicrocoelium spp., Gongylonema spp., Oesophagostomum spp., Lemuricola spp., Strongyloides spp. Trichostrongylus spp. and Trichuris spp.) and two protozoans (Balantidium spp. and Entamoeba spp.) may infect Galago spp. with high infection rates. The results show that: a very similar parasite spectrum is found in both host species; all the taxa identified were previously observed in other Primate species and/or Man. They also show that age, gender and forest type may influence infection rates and/or parasite diversity found in a particular host and/or geographic area.

Ancient Ascaris DNA Sequences of Cytochrome B, Cytochrome C Oxidase Subunit 1, NADH Dehydrogenase Subunit 1, and Internal Transcribed Spacer 1 Genes from Korean Joseon Mummy Feces.

We analyzed Ascaris ancient DNA of cytochrome b, cytochrome c oxidase subunit 1, NADH dehydrogenase subunit 1, and internal transcribed spacer 1 genes extracted from the feces or precipitates of 15- to 18th-century Korean mummies. After multiple Ascaris genes in ancient samples were successfully amplified by polymerase chain reaction (PCR), consensus sequences could be determined by the alignment of the sequences of cloned PCR products. The obtained sequences of each gene were highly similar to those of Ascaris spp. reported thus far but were genetically distinct from Baylisascaris, Parascaris, and Toxascaris spp. The current report establishes that the genetic characteristics of the Ascaris spp. infecting pre-modern Korean societies were not uniform but were diverse to some degree.

Ancient mitochondrial DNA analyses of ascaris eggs discovered in coprolites from joseon tomb.

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.

Characterization of ascaris from ecuador and zanzibar.

To shed light on the epidemiology of ascariasis in Ecuador and Zanzibar, 177 adult worms retrieved by chemo-expulsion from either people or pigs were collected, measured and subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ribosomal internal transcribed spacer (ITS) region. Upon double digestion with RsaI and HaeIII, PCR-RFLP analysis revealed the presence of A. lumbricoides in people and A. suum in pigs in Ecuador. In contrast, while there are no pigs on Zanzibar, of the 56 worms obtained from people, one was genotyped as A. suum. No additional genetic variation was detected upon further PCR-RFLP analysis with several other restriction enzymes. Upon measurement, worm mass and length differed by location and by species, A. suum being lighter and longer. While there is no evidence to suggest zoonotic transmission in Ecuador, an enduring historical signature of previous zoonotic transmission remains on Zanzibar.

DNA typing of ancient parasite eggs from environmental samples identifies human and animal worm infections in Viking-age settlement.

Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp., and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of: the human whipworm, Trichuris trichiura , using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07; and Fasciola hepatica using ITS1 sequence homology. The identification of T. trichiura eggs indicates that human fecal material is present and, hence, that the Ascaris sp. haplotype 07 was most likely a human variant in Viking-age Denmark. The location of the F. hepatica finding suggests that sheep or cattle are the most likely hosts. Further, we sequenced the Ascaris sp. 18S rRNA gene in recent isolates from humans and pigs of global distribution and show that this is not a suited marker for species-specific identification. Finally, we discuss ancient parasitism in Denmark and the implementation of aDNA analysis methods in paleoparasitological studies. We argue that when employing species-specific identification, soil samples offer excellent opportunities for studies of human parasite infections and of human and animal interactions of the past.

Ancient Mitochondrial DNA Analyses of Ascaris Eggs Discovered in Coprolites from Joseon Tomb

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.

Molecular paleoparasitological hybridization approach as effective tool for diagnosing human intestinal parasites from scarce archaeological remains.

Paleoparasitology is the science that uses parasitological techniques for diagnosing parasitic diseases in the past. Advances in molecular biology brought new insights into this field allowing the study of archaeological material. However, due to technical limitations a proper diagnosis and confirmation of the presence of parasites is not always possible, especially in scarce and degraded archaeological remains. In this study, we developed a Molecular Paleoparasitological Hybridization (MPH) approach using ancient DNA (aDNA) hybridization to confirm and complement paleoparasitological diagnosis. Eight molecular targets from four helminth parasites were included: Ascaris sp., Trichuris trichiura, Enterobius vermicularis, and Strongyloides stercoralis. The MPH analysis using 18th century human remains from Praça XV cemetery (CPXV), Rio de Janeiro, Brazil, revealed for the first time the presence E. vermicularis aDNA (50%) in archaeological sites of Brazil. Besides, the results confirmed T. trichiura and Ascaris sp. infections. The prevalence of infection by Ascaris sp. and E. vermicularis increased considerably when MPH was applied. However, a lower aDNA detection of T. trichiura (40%) was observed when compared to the diagnosis by paleoparasitological analysis (70%). Therefore, based on these data, we suggest a combination of Paleoparasitological and MPH approaches to verify the real panorama of intestinal parasite infection in human archeological samples.

Paleoparasitological surveys for detection of helminth eggs in archaeological sites of Jeolla-do and Jeju-do.

A paleoparasitological survey to detect helminth eggs was performed in archaeological sites of Jeolla-do and Jeju-do, the Republic of Korea. Total 593 soil samples were collected in 12 sites of Jeolla-do and 5 sites of Jeju-do from April to November 2011, and examined by the methods of Pike and coworkers. A total of 4 helminth eggs, 2 eggs each for Trichuris trichiura and Ascaris sp., were found in soil samples from 1 site, in Hyangyang-ri, Jangheung-eup, Jangheung-gun, Jeollanam-do. The egg-recovery layer was presumed to represent a 19th century farm, which fact suggested the use of human manures. This is the third archaeological discovery of parasite eggs in Jeolla-do. Additionally, no helminth eggs in archaeological sites of Jeju-do is an interesting problem to be solved in the further investigations.

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.

Paleoparasitological Surveys for Detection of Helminth Eggs in Archaeological Sites of Jeolla-do and Jeju-do

A paleoparasitological survey to detect helminth eggs was performed in archaeological sites of Jeolla-do and Jeju-do, the Republic of Korea. Total 593 soil samples were collected in 12 sites of Jeolla-do and 5 sites of Jeju-do from April to November 2011, and examined by the methods of Pike and coworkers. A total of 4 helminth eggs, 2 eggs each for Trichuris trichiura and Ascaris sp., were found in soil samples from 1 site, in Hyangyang-ri, Jangheung-eup, Jangheung-gun, Jeollanam-do. The egg-recovery layer was presumed to represent a 19th century farm, which fact suggested the use of human manures. This is the third archaeological discovery of parasite eggs in Jeolla-do. Additionally, no helminth eggs in archaeological sites of Jeju-do is an interesting problem to be solved in the further investigations.

Contamination of vegetables, fruits and soil with geohelmints eggs on organic farms in Poland.

The objective of this study was to evaluate the contamination of vegetables, fruits and soil with zoonotic parasite eggs on organic and conventional farms in south-eastern Poland. To evaluate the contamination with eggs of zoonotic parasites, examinations were conducted on 8 conventional and 11 organic farms in south-eastern Poland from May-October in 2008 and 2009. The following fruit and vegetables were selected for the experiment: strawberry, leek, onion, carrot, zucchini, beetroot, parsley, potatoes, celery, rhubarb, lettuce, cabbage, broccoli, pumpkin, young beetroot leaves, cauliflower, French beans, turnip, fennel and sorrel. A total of 187 samples of vegetables, fruits and soil were examined by means of a modified flotation method according to Quinn et al. (1980). Contamination with Ascaris, Trichuris and Toxocara eggs was found, with a higher number of positive samples revealed on conventional (34.7%), compared to organic farms (18.9%). The level of contamination in soil samples from conventional farms was higher (88.5% positive samples), than of those from organic farms (32.8%). Of the 15 geohelmints eggs, positive samples were found in vegetables: 9 Toxocara eggs, 4 Ascaris eggs and 2 Trichuris eggs. No geohelmints eggs were observed in the strawberry samples. The consumption of vegetables and fruits contaminated with the eggs of parasites may be the cause of parasitoses in humans. Stricter sanitary standards on farms of all types may limit the incidence of parasitic zoonoses.

Pig Ascaris: an important source of human ascariasis in China.

The aim of the present study is to detect the frequency and distribution of cross infection and hybridization of human and pig Ascaris in China. Twenty high polymorphic microsatellite loci were selected to screen 258 Ascaris worms from humans and pigs from six provinces in China. The software programs Structure, Baps and Newhybrids were used to determine the case of cross infection and hybridization of human and pig Ascaris. Results showed that cross infection was detected in all sampled locations and of the total 20 cross infection cases, 19 were indentified as human infections by pure-bred pig type Ascaris in contrast to only one case of pig infection by pure-bred human type Ascaris. Similar to the findings in cross infection, hybrid Ascaris was also detected in all locations and both host species and most of hybrids (95%) were detected from human host. The distribution of cross infection and hybrids showed significant difference between the two host species and among three categories of genotype in terms of G1, G2 and G3, and also between the south and north regions (for hybrids only). The results strongly suggest pig Ascaris as an important source of human ascariasis in endemic area where both human and pig Ascaris exist. In consideration of current control measures for human ascariasis targeting only infected people, it is urgently needed to revise current control measures by adding a simultaneous treatment to infected pigs in the sympatric endemics. The knowledge on cross transmission and hybridization between human and pig Ascaris is important not only for public health, but also for the understanding of genetic evolution, taxonomy and molecular epidemiology of Ascaris.

Ascaris: development of selected genotypes in mice.

Using nucleotide variation in the first internal transcribed spacer of nuclear ribosomal DNA, five different genotypes (designated G1-G5) have been identified and the preponderance of genotype G1 in humans and of genotype G3 in pigs led to the proposal that parasites bearing the two genotypes have an affinity for a particular host species. A subsequent study using eggs of genotype G1 from humans and G3 from pigs to infect pigs and mice indicated that there is a significant difference in the ability to infect and establish as larvae in mice and as adults in pigs between the two genotypes. Extending previous investigations, the present study investigated whether there are differences in development as designated by egg hatching, larvae migration and distribution in the mice between the Ascaris strains with known genotypes. Ascaris eggs of genotypes G1 (predominating in human-derived worms) and G3 (predominating in pig-derived worms) were used to infect C57BL/6 mice orally. Eggs/larvae were examined from the small and large intestines, thoracic and abdominal cavities, peripheral blood, livers and lungs at intervals of 2h until 12h post-infection, then periodically until 34 days of infection. Results showed distinct differences in egg hatching (the timing and location of hatching, and the numbers hatched), and in larvae migration and distribution (the means and constituent ratios, the time of peak recovery, and larvae reappearing in intestines) between the two strains. The results can explain the findings of significantly higher larval recovery of genotype G1 than G3 in the mice, and may shed some enlightenment to understand the difference in host affiliation of Ascaris of different genotypes.

Ascariasis in people and pigs: new inferences from DNA analysis of worm populations.

Ascaris is a large parasitic roundworm (nematode) of the small intestine of humans and pigs. These roundworms cause the socioeconomically important disease, ascariasis. For the past 20 years, molecular markers have been used in studies on Ascaris and ascariasis, and added valuable information to the understanding of these roundworms. Here, we provide a review of these studies on human and pig roundworms. We begin with a summary of studies using molecular phenotypic markers to compare Ascaris from humans and pigs, followed by a synopsis of comparisons using genetic markers. We then draw forth inferences in the aspects of host affiliation and infection success, transmission between and among humans and pigs, evolutionary history of Ascaris. We also highlight additional topics such as mating dynamics, diagnostics, and paleoparasitology where molecular epidemiological approaches have been utilized.

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.