Sensory neuroanatomy of a passively ingested nematode parasite, Haemonchus contortus: amphidial neurons of the third-stage larva

The sensory neuronal ultrastructure of the amphids of the infective larva (L3) of Haemonchus contortus was analyzed, compared, and contrasted with that of the first-stage larva (L1). As in L1, each amphid of the L3 is innervated by 12 neurons. Thirteen ciliated dendritic processes of 10 neurons, 3 with double processes, lie in each amphidial channel. The dendritic process of each finger cell neuron ends in a large number of digitiform projections or "fingers," many more than in the L1. Processes of another pair of specialized neurons, probable homologs of wing cells in Caenorhabditis elegans, extend into the extreme anterior tip of the larva; they are much longer than those in L1. In L3, the neurons exit through the posterior wall of the amphidial chamber individually rather than in a bundle, as in L1. Cell constancy between L1 and L3 was confirmed, and the neurons were individually identified. Significant neuron-specific variations, presumably related to functional differences between the 2 stages were observed. In contrast, species-specific differences are surprisingly small. Haemonchus contortus is closely related to hookworms and has amphidial structure nearly identical to that in hookworms and similar to that in C. elegans, to which it is also closely related.     

Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum

During its development from free-living infectious third-stage larvae to the adult worms in the large intestines of pigs, Oesophagostomum dentatum experiences several environmental changes. Differences in protein patterns can reflect such changes. Somatic and ES antigens and glycoproteins of pre-parasitic, histotropic and intestinal stages were compared by single-dimension SDS–PAGE and stage-specific proteins were defined. Furthermore, fourth-stage larvae derived from different sources—in-vitro cultivation and intestinal contents—were compared and also found to be different. It is hypothesised that O. dentatum reacts to environmental stimuli by differential expression of specific proteins as a possible mode of adaptation to the host.     

Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum

BackgroundThe porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.MethodsDifferent enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.ResultsWhereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered ‘male’, while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc_2–4PC_2–4 motifs not observed in our previous studies on other nematodes.ConclusionThe interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.General significanceUnusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.     

Oesophagostomum dentatum and Oesophagostomum quadrispinulatum: characterization of the complete mitochondrial genome sequences of the two pig nodule worms

In the present study, the complete mitochondrial DNA (mtDNA) sequences of the pig nodule worm Oesophagostomum quadrispinulatum were determined for the first time, and the mt genome of Oesophagostomum dentatum from China was also sequenced for comparative analysis of their gene contents and genome organizations. The mtDNA sequences of O. dentatum China isolate and O. quadrispinulatum were 13,752 and 13,681 bp in size, respectively. Each of the two mt genomes comprises 36 genes, including 12 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, but lacks the ATP synthetase subunit 8 gene. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T are 75.79% and 77.52% for the mt genomes of O. dentatum and O. quadrispinulatum, respectively. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), all revealed that O. dentatum and O. quadrispinulatum represent distinct but closely-related species. These data provide novel and useful markers for studying the systematics, population genetics and molecular diagnosis of the two pig nodule worms.     

Survival and viability of Ascaris suum and Oesophagostomum dentatum in ensiled swine faeces

The survival and viability of eggs from Ascaris suum and Oesophagostomum dentatum and of infective larvae (L3) from O. dentatum were determined in the ensiled solid fraction of swine faeces after 0, 7, 14, 28 and 56 days of ensiling. The experiment had two treatments, un-ensiled and ensiled manure, in a split-plot design. Each of 50 containers was inoculated with 40,000 eggs of both A. suum and O. dentatum, and another 50 containers were inoculated with 32,747 L3 of O. dentatum each. A. suum eggs were not destroyed by the ensiling process, although their viability was diminished. O. dentatum eggs and larvae were destroyed during the first 7-14 days of the ensiling process.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

ACTH-immunoreactive neurons and their projections in the cat forebrain

The organization of adrenocorticotropin (ACTH)-immunoreactive (IR) cell bodies and fibers in the cat forebrain is described. ACTH-IR cell bodies are found only in and around the arcuate nucleus of the hypothalamus (ARH). They are not detected elsewhere even after pretreatment with colchicine. ACTH-IR fibers are present in discrete areas of the hypothalamus, the septo-limbic areas and in the paraventricular thalamic nucleus. Complete electrolytic lesions of the ARH destroy ACTH-IR cell bodies as well as fibers in all parts of the brain. These results suggest that, in the cat forebrain, the ARH is the only source of ACTH-IR fibers.     

The neurons of class ALD mediate thermotaxis in the parasitic nematode, Strongyloides stercoralis

Strongyloides stercoralis, a skin-penetrating nematode parasite of homeotherms, migrates to warmth. In nematodes, the amphids, anteriorly positioned, paired sensilla, each contain a bundle of sensory neurons. In the amphids of the free-living nematode Caenorhabditis elegans, a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors, and have been named the finger cells (class AFD). A similar neuron pair in the amphids of the parasite Haemonchus contortus is also known to be thermosensory. Strongyloides stercoralis lacks finger cells but, in its amphids, it has a pair of neurons whose dendrites end in a multi-layered complex of lamellae, the so-called lamellar cells (class ALD). Consequently, it was hypothesised that these lamellar cells might mediate thermotaxis by the skin-penetrating infective larva of this species. To investigate this, first stage S. stercoralis larvae were anaesthetised and the paired ALD class neurons were ablated with a laser microbeam. The larvae were then cultured to the infective third stage (L3) and assayed for thermotaxis on a thermal gradient. L3 with ablated ALD class neuron pairs showed significantly reduced thermotaxis compared with control groups. The thermoreceptive function of the ALD class neurons (i) associates this neuron pair with the host-finding process of S. stercoralis and (ii) demonstrates a functional similarity with the neurons of class AFD in C. elegans. The structural and positional characteristics of the ALD neurons suggest that these neurons may, in fact, be homologous with one pair of flattened dendritic processes known as wing cells (AWC) in C. elegans, while their florid development and thermosensory function suggest homology with the finger cells (AFD) of that nematode.     

A Study on the Transmission of Oesophagostomum dentatum and Hyostrongylus rubidus among Outdoor Reared Pigs in Denmark

This study was carried out to obtain basic information on the transmission of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor reared pigs in Denmark. Eighteen 10 weeks old worm-free pigs were allocated into 3 groups of 6 pigs each. In May, all pigs were turned out on the same parasitologi-cally naive pasture, and after 2 weeks the pigs in groups 2 and 3 were experimentally infected with 10,800 O. dentatum and 8,700 H. rubidus infective larvae, respectively. Pigs in group 1 served as non-infected controls. All pigs were reared together on the experimental pasture for further 134 days until slaughter in October. Strongyle egg counts, differentiation of infective larvae at species level, serum pepsinogen, and herbage larval infectivity were monitored at regular intervals throughout. Both strongyle species established in the originally parasite-free pigs (group 1) and cross infections were established in group 2 and 3. The pigs were exposed to steadily increasing herbage infectivity of both species of strongyles. At the end of the experiment, geometric mean worm burdens of O. dentatum in groups 1,2 and 3 were 1202, 6136 and 1431 respectively, the burden in group 2 being significantly higher (p<0.05) than that of the 2 other groups. The geometric mean worm burdens of H. rubidus in groups 1, 2 and 3 were 4907, 3679 and 5246 respectively, showing no significant differences between groups.     

Carotenoids found in Littorina littorea and their relationship to parasitic infection by larva trematodes

Littorina littorea from Long Island Sound feed primarily on algae: Chlorophyceae (three species) and Rhodophyceae (two species). Carotenoids from the algae accumulate in tissues of the snail in either an unchanged or a metabolized state. β-Carotene, the major pigment of green and red algae, was isolated from the foot, hepatopancreas, and nephridium of these snails. Six oxygenated carotenoids, not completely identified, were isolated from the same tissues. The snails show a variation in foot color from white to brown to red. L. littorea is parasitized by trematode larvae of Cryptocotyle lingua and Cercaria parvicaudata from which β-carotene and one oxygenated carotenoid were isolated. Contrary to previous work, there is no relation between foot color of the snail and parasitic infection. Neither age nor sex appears to have any relation to foot color. Although carotenoid pigments are known to cause the variation in foot color, the reasons or factors for their accumulation in the snail tissue have not been established. Some hypothetical explanations are discussed.     

Exsheathment of the infective larva of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby Macropus eugenii.

The infective larva of L. eugenii is enveloped in two cuticles which are discarded when the larva exsheaths in the sacculated portion of the wallaby's stomach. In vitro larvae exsheathed in a 0·85% solution of sodium chloride at 37°C, buffered to pH 7 with bicarbonate ion and 40% carbon dioxide. Survival was enhanced if the liquid phase contained medium 199 and serum, and exsheathment was quicker if exposure to carbon dioxide was 1 h rather than 1 day or 7 days. As larvae exsheathed, contractions of the pharynx commenced, and medium was ingested, even when larvae were enveloped in both cuticles. The stimuli for exsheathment and the subsequent pattern of events are like those already recognised in some trichostrongyles.     

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3-5.2% for pcox1, 0-4.9% for pnad1, and 0-7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7-13.4% for pcox1, 11-14.6% for pnad1, and 14.9-18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.     

The effect of increasing levels of insoluble dietary fibre on the establishment and persistence of Oesophagostomum dentatum in pigs

This investigation compared the effect of diets with increasing content of insoluble dietary fibre (DF) on the establishment and persistence of Oesophagostomum dentatum in growing pigs. Twenty-eight worm-free pigs, from a specific pathogen-free farm were randomly divided to four groups of seven animals. The animals were assigned to the following diets: diet A, barley flour plus protein mixture (70%:30%); diet B, barley flour, oat husk meal plus protein mixture (65%:7%:28%); diet C, barley flour, oat husk meal plus protein mixture (60%:14%:26%) and diet D, barley flour, oat husk meal plus protein mixture (55%:21%:24%). The diets were formulated to provide increasing content of DF but constant levels of digestible protein per feeding unit for pigs. All pigs were experimentally inoculated with 6,000 infective O. dentatum larvae and followed coprologically for 11 weeks post infection, whereafter they were slaughtered. The experimental diets influenced the mean transit time and the metabolism in the large intestine significantly. Diets C and D, with highest content of insoluble DF, provided favourable conditions for establishment of O. dentatum, but diets A and B led to a significant lower worm numbers and fecundity.     

A reversible protein phosphorylation system is present at the surface of infective larvae of the parasitic nematode Trichinella spiralis

Trichinella spiralis infective larvae have externally oriented enzymes catalysing reversible protein phosphorylation on their surface. Incubation of larvae with exogenous ATP resulted in phosphorylation of surface bound and released proteins. Exposure of the parasites to bile, a treatment which renders them infective for intestinal epithelia, resulted in increased release of protein and an altered profile of phosphorylation. Both serine/threonine and tyrosine phosphorylation and dephosphorylation reactions took place at the parasite surface. Examination of the structural characteristics of the larvae following exposure to bile showed that the non-bilayer surface coat was not shed but was structurally reorganised.