End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3'-overhangs before the addition of neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible for nematode PDE.

Worldwide absence of canonical benzimidazole resistance-associated mutations within ß-tubulin genes from Ascaris.

BACKGROUND: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with ß-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the ß-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the ß-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. METHODS: To further determine the putative absence of key ß-tubulin polymorphisms, we screened two ß-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris ß-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. RESULTS: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of ß-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for ß-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of ß-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. CONCLUSIONS: Our work suggests an absence of canonical ß-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

A molecular and morphological study of Ascaris suum in a human-pig contact scenario in northeastern Brazil.

The aim of the present study was to assess morphologic and genetic data on ascariasis in swine (Sus scrofa domesticus) and humans in low-resource rural and periurban communities in the state of Piauí, Brazil. Our cross-sectional survey included 100 fecal samples obtained from swine and 682 samples from humans. Fifteen pigs were necropsied. Human and porcine fecal samples were examined to identify Ascaris eggs. Parasites obtained in the swine necropsies were studied using scanning electron microscopy (SEM), and the mitochondrial gene encoding the cytochrome oxidase 1 (cox1) enzyme was partially amplified and sequenced for molecular taxonomy and phylogenetic analyses. The overall prevalence of Ascaris eggs in the swine fecal samples was 16/100 (16%). No Ascaris eggs were identified in the human fecal samples. SEM of six worms recovered from pigs demonstrated morphological characteristics of A. suum. Cox1 sequences were compatible with A. suum reference sequences. Original and reference (GenBank) nucleotide sequences were organized into clusters that did not segregate the parasites by host species or and region. The largest haplogroups were dominated by haplotypes H01, H02 and H31. In the communities studied, there was no epidemiological evidence of the zoonotic transmission of ascariasis at the human-swine interface.

First molecular data on the human roundworm Ascaris lumbricoides species complex from the Bronze and Iron Age in Hallstatt, Austria.

Palaeoparasitological studies can provide valuable information on the emergence, distribution, and elimination of parasites during a particular time in the past. In the prehistoric salt mines of Hallstatt, located in the Austrian Alps, human faeces have been conserved in salt. The aim of this study was to recover ancient DNA of intestinal parasites from these coprolites. Altogether, 35 coprolites from the Hallstatt salt mines, dating back to the Bronze Age mining phase (1158-1063 BCE) and the Iron Age mining phase (750-662 BCE), respectively, were analysed by microscopy and molecular methods. In 91% of the coprolite samples, eggs of soil-transmitted helminths (STH), namely of Trichuris and/or Ascaris were detected by light microscopy. The Ascaris eggs were exceptionally well preserved. For further analysis, DNA was extracted from the palaeofaecal samples and species-specific primers targeting different genes were designed. While amplification of Trichuris DNA remained unsuccessful, sequence data of A. lumbricoides species complex were successfully obtained from 16 coprolites from three different genes, the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), the mitochondrial cytochrome B gene (cytB) and the mitochondrial NADH dehydrogenase subunit 1 gene (nadh1). Importantly, these included two Ascaris sequences from a coprolite from the Bronze Age, which to the best of our knowledge are the first molecular data of this genus from this period.

Programmed DNA elimination in the parasitic nematode Ascaris.

In most organisms, the whole genome is maintained throughout the life span. However, exceptions occur in some species where the genome is reduced during development through a process known as programmed DNA elimination (PDE). In the human and pig parasite Ascaris, PDE occurs during the 4 to 16 cell stages of embryogenesis, when germline chromosomes are fragmented and specific DNA sequences are reproducibly lost in all somatic cells. PDE was identified in Ascaris over 120 years ago, but little was known about its molecular details until recently. Genome sequencing revealed that approximately 1,000 germline-expressed genes are eliminated in Ascaris, suggesting PDE is a gene silencing mechanism. All germline chromosome ends are removed and remodeled during PDE. In addition, PDE increases the number of chromosomes in the somatic genome by splitting many germline chromosomes. Comparative genomics indicates that these germline chromosomes arose from fusion events. PDE separates these chromosomes at the fusion sites. These observations indicate that PDE plays a role in chromosome karyotype and evolution. Furthermore, comparative analysis of PDE in other parasitic and free-living nematodes illustrates conserved features of PDE, suggesting it has important biological significance. We summarize what is known about PDE in Ascaris and its relatives. We also discuss other potential functions, mechanisms, and the evolution of PDE in these parasites of humans and animals of veterinary importance.

Genetic diversity and identity of Ascaris worms from human and pig hosts in Thailand.

Ascaris roundworms are of public health and socio-economic importance worldwide. They are conventionally attributed to two taxa - A. lumbricoides infecting principally human and A. suum infecting principally pig. Phylogenomic analysis has revealed that Ascaris worms from both human and pig are represented in Clades A and B. A recent study indicates that the Ascaris worms from human and pig in Thailand belong to Clade A. We examined adult Ascaris worms from human and pig in Thailand by means of the partial sequences of three mitochondrial genes (cox1, cox2 and nad1) and concatenation of these genes. Phylogenomic analysis indicates that two isolates (H1,H2) of A. lumbricoides from human belonged to Clade B; one isolate (H3) belonged to Clade A (based on cox1, cox2 and concatenated sequences) or as an outlier to Clades A and B (based on nad1 sequences). All the eight isolates of A. suum from pig clustered in Clade A. The partial nad1 and the concatenated sequences revealed two lineages of A. suum isolates which were distinct from the two A. lumbricoides isolates of Clade B. It is evident that greater genetic diversity, and a more robust phylogeny, could be uncovered by the application of multiple genes. In sum, the present study reveals the presence in Thailand of A. lumbricoides from human in Clades A and B which necessitates appropriate treatment and control measures; Clades A and B have been reported to contain haplotypes of Ascaris worms from both human and pig in other parts of the world. A country wide study is needed to elucidate the identity, distribution, prevalence, cross transmission, genetic diversity and phylogeny of the Ascaris worms in Thailand.

Molecular epidemiology of Ascaris species recovered from humans and pigs in Cameroon.

BACKGROUND: In Cameroon, considerable research has been conducted on human ascariasis, but no studies have been undertaken to determine whether pigs contribute to the persistence of the infection in children or to unravel the evolutionary relationship between human-derived and pig-derived Ascaris. METHODS: DNA was extracted from adult Ascaris worms collected from humans and pigs. Segments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes were sequenced and analysed for 83 worms to dissect the local transmission dynamics of Ascaris in Cameroon. RESULTS: The data showed high genetic diversity and revealed demographically expanding populations in the human and pig Ascaris samples. A restricted gene flow between Ascaris lumbricoides and Ascaris suum populations correlating with the preference for humans and pigs, respectively, as hosts was evident. Phylogenetic analyses and haplotype networks split the haplotypes into two major clusters, A and B. However, support for cross-transmission between hosts and hybridization were revealed through shared haplotypes among worms from both hosts. CONCLUSIONS: This study provides useful baseline information for future studies of the genetics of Ascaris in Cameroon and suggests that effective and sustainable control of human ascariasis should target both human and pig hosts.

Small RNA pathways in the nematode Ascaris in the absence of piRNAs.

Small RNA pathways play key and diverse regulatory roles in C. elegans, but our understanding of their conservation and contributions in other nematodes is limited. We analyzed small RNA pathways in the divergent parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that associate with secondary 5'-triphosphate 22-24G-RNAs. These small RNAs target repetitive sequences or mature mRNAs and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Even in the absence of a piRNA pathway, Ascaris CSR-1 may still function to "license" as well as fine-tune or repress gene expression. Ascaris ALG-4 and its associated 26G-RNAs target and likely repress specific mRNAs during testis meiosis. Ascaris WAGO small RNAs demonstrate target plasticity changing their targets between repeats and mRNAs during development. We provide a unique and comprehensive view of mRNA and small RNA expression throughout spermatogenesis. Overall, our study illustrates the conservation, divergence, dynamics, and flexibility of small RNA pathways in nematodes.

Ascaris exposure and its association with lung function, asthma, and DNA methylation in Northern Europe.

BACKGROUND: Ascaris infections, with a worldwide prevalence above 10%, can cause respiratory pathology. However, long-term effects on lung function in humans are largely unknown. OBJECTIVE: We investigated the associations of Ascaris exposure with lung function, asthma, and DNA methylation. METHODS: Serum Ascaris IgG antibodies were measured in 671 adults aged 18 to 47 years (46% women) from Aarhus, Bergen, and Tartu RHINESSA study centers. Seropositivity was defined as IgG above the 90th percentile. Linear and logistic regressions were used to analyze Ascaris seropositivity as associated with lung function and asthma, adjusted for age, height, and smoking and clustered by center. DNA methylation in blood was profiled by a commercial methylation assay. RESULTS: Ascaris seropositivity was associated with lower FEV1 (-247 mL; 95% CI, -460, -34) and higher odds for asthma (adjusted odds ratio, 5.84; 95% CI, 1.67, 20.37) among men but not women, also after further adjusting for house dust mite sensitivity, consistent across study centers. At a genome-wide level, Ascaris exposure was associated with 23 differentially methylated sites in men and 3 in women. We identified hypermethylation of the MYBPC1 gene, which can regulate airway muscle contraction. We also identified genes linked to asthma pathogenesis such as CRHR1 and GRK1, as well as a differentially methylated region in the PRSS22 gene linked to nematode infection. CONCLUSION: Ascaris exposure was associated with substantially lower lung function and increased asthma risk among men. Seropositive participants had sex-specific differences in DNA methylation compared to the unexposed, thus suggesting that exposure may lead to sex-specific epigenetic changes associated with lung pathology.

Human-type and pig-type Ascaris hybrids found in pigs.

The discovery of hybrids between Ascaris lumbricoides and Ascaris suum has complicated our understanding of the relationship between the two species. We examined the same Ascaris specimens (48 from humans and 48 from pigs) using two methods: microsatellite markers combined with Bayesian clustering and PCR-RFLP of the nuclear internal transcribed spacer region. The results obtained by the two methods were inconsistent but showed that hybrid Ascaris identified through both approaches could infect pigs. The results of this study suggest that PCR-RFLP of ITS alone is not suitable for molecular identification of human-type and pig-type Ascaris hybrids. Use of multiple SSR markers combined with Bayesian analysis was the most reliable method in our study. Our results indicate that, in addition to host-specific Ascaris types, there may be some that do not show host specificity. Our results show for the first time that hybrid individuals can infect pigs as well as humans. This study has important theoretical and practical implications, including suggesting the need to re-evaluate long-term ascariasis control strategies.

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.

Microenvironment-Sensitive Fluorescent Ligand Binds Ascaris Telomere Antiparallel G-Quadruplex DNA with Blue-Shift and Enhanced Emission.

The development of small molecules that can selectively target G-quadruplex (G4) DNAs has drawn considerable attention due to their unique physiological and pathological functions. However, only a few molecules have been found to selectively bind a particular G4 DNA structure. We have developed a fluorescence ligand Q1, a molecular scaffold with a carbazole-pyridine core bridged by a phenylboronic acid side chain, that acts as a selective ascaris telomere antiparallel G4 DNA ASC20 ligand with about 18 nm blue-shifted and enhanced fluorescence intensity. Photophysical properties revealed that Q1 was sensitive to the microenvironment and gave the best selectivity to ASC20 with an equilibrium binding constant Ka =6.04×105  M-1 . Time-resolved fluorescence studies also demonstrated that Q1 showed a longer fluorescence lifetime in the presence of ASC20. The binding characteristics of Q1 with ASC20 were shown in detail in a fluorescent intercalator displacement (FID) assay, a 2-Ap titration experiment and by molecular docking. Ligand Q1 could adopt an appropriate pose at terminal G-quartets of ASC20 through multiple interactions including π-π stacking between aromatic rings; this led to strong fluorescence enhancement. In addition, a co-staining image showed that Q1 is mainly distributed in the cytoplasm. Accordingly, this work provides insights for the development of ligands that selectively targeting a specific G4 DNA structure.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs.

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

Risk factors for infection with soil-transmitted helminths during an integrated community level water, sanitation, and hygiene and deworming intervention in Timor-Leste.

Water, sanitation and hygiene interventions have been advocated as important complements to deworming programs to improve soil-transmitted helminth control. Evidence for the impact of water, sanitation and hygiene on soil-transmitted helminth infections is mixed, and based mainly on cross-sectional studies. In this study, we assessed associations between individual- and household-level water, sanitation and hygiene variables and soil-transmitted helminth infections, using data collected during the 2 year follow-up study period of the WASH for WORMS randomised controlled trial in Timor-Leste. Data were collected across four surveys, conducted at 6 monthly intervals in 23 communities. We analysed water, sanitation and hygiene and sociodemographic variables as risk factors for infection with Necator americanus, Ascaris spp., and undifferentiated soil-transmitted helminth infection, using generalised linear mixed models to account for clustering at community, household and participant levels. Water, sanitation and hygiene risk factors were examined both concurrently and with a 6 month lag period that coincided with the most recent deworming. The analysis included 2333 participants. Factors associated with N. americanus infection included age group, male sex (adjusted odds ratio (aOR) 3.1, 95% confidence interval (CI) 2.4-4.2), working as a farmer (aOR 1.7, 95% CI 1.2-2.4), and completing secondary school or higher (aOR 0.29, 95% CI 0.16-0.53). Risk factors for Ascaris spp. infection included age group, living in a dwelling with more than six people (aOR 1.6, 95% CI 1.1-2.3), having a tube well or borehole as the household water source (aOR 3.7, 95% CI 1.3-10.8), and using a latrine shared between households 6 months previously (aOR 2.3, 95% CI 1.2-4.3). Handwashing before eating was protective against infection with any soil-transmitted helminth (aOR 0.79, 95% CI 0.65-0.95). In the context of regular deworming, few water, sanitation and hygiene-related factors were associated with soil-transmitted helminth infections. Future research examining the role of water, sanitation and hygiene in soil-transmitted helminth transmission is required, particularly in low transmission settings after cessation of deworming. Identifying improved indicators for measuring water, sanitation and hygiene behaviours is also a key priority.

Molecular analysis of human- and pig-derived Ascaris in Honduras.

Ascaris sp. is a soil-transmitted helminth (STH) significantly affecting the health of human and swine populations. Health inequities and poverty, with resulting deficiencies in water, sanitation and hygiene, are directly associated with Ascaris lumbricoides prevalence in humans. Resource constraints also lead to small-scale livestock production under unsanitary conditions. Free-ranging pigs, for instance, are exposed to a number of infectious agents, among which Ascaris suum is one of the most common. Under these conditions, close proximity between people and pigs can result in cross-contamination; that is, pigs harbouring human Ascaris and vice versa. Moreover, the potential interbreeding between these two Ascaris species has been demonstrated. The present study analysed Ascaris worms obtained from children and pigs in Honduras. Adult worms were collected from stool samples of children after pharmacological treatment, and from pigs' intestines after slaughter for commercial purposes at a local abattoir. A nuclear ribosomal internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (PCR) and digested with a restriction enzyme in order to separate putative human- and pig-derived Ascaris isolates. PCR products were also sequenced, and cladograms were constructed. All parasites isolated from children showed the typical human-derived genotype of Ascaris, whereas 91% of parasites from pigs showed the expected pig-derived genotype. Cross-infections between hosts were not demonstrated in this study. Nine per cent of pig-derived worms showed a restriction band pattern highly suggestive of a hybrid human-pig Ascaris genotype. These results contribute to the understanding of ascariasis epidemiology and its zoonotic potential in a highly endemic region.

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.

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.

Genetic Diversity of Ascaris in China Assessed Using Simple Sequence Repeat Markers.

The giant roundworm Ascaris infects pigs and people worldwide and causes serious diseases. The taxonomic relationship between Ascaris suum and Ascaris lumbricoides is still unclear. The purpose of the present study was to investigate the genetic diversity and population genetic structure of 258 Ascaris specimens from humans and pigs from 6 sympatric regions in Ascaris-endemic regions of China using existing simple sequence repeat data. The microsatellite markers showed a high level of allelic richness and genetic diversity in the samples. Each of the populations demonstrated excess homozygosity (Ho0). According to a genetic differentiation index (Fst=0.0593), there was a high-level of gene flow in the Ascaris populations. A hierarchical analysis on molecular variance revealed remarkably high levels of variation within the populations. Moreover, a population structure analysis indicated that Ascaris populations fell into 3 main genetic clusters, interpreted as A. suum, A. lumbricoides, and a hybrid of the species. We speculated that humans can be infected with A. lumbricoides, A. suum, and the hybrid, but pigs were mainly infected with A. suum. This study provided new information on the genetic diversity and population structure of Ascaris from human and pigs in China, which can be used for designing Ascaris control strategies. It can also be beneficial to understand the introgression of host affiliation.

Respiratory disease associated with migrating Ascaris larvae in a beef calf.

A group of 4-month-old beef calves were examined for clinical respiratory disease with labored breathing, coughing, and fevers of over 104°F. Necropsy of one of the calves revealed lungs that were not collapsed but had red mottled appearance on cut surface. Assessment of lung tissue by bacterial culture and PCR did not reveal bovine bacterial or viral respiratory pathogens. Histopathology of affected tissues and lymph nodes revealed larval ascarid nematodes. In combination with phylogenetic analysis, amplification and sequencing of ITS1 was used to identify the larvae as Ascaris.

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.