Quantitative proteomics analysis of Angiostrongylus vasorum-induced alterations in dog serum sheds light on the pathogenesis of canine angiostrongylosis.

Blood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Infections with the blood-dwelling cardiopulmonary nematode Angiostrongylus vasorum in dogs manifest with a broad spectrum of clinical signs including respiratory distress, bleeding diathesis and neurological signs, and are associated with a perturbed blood protein profile in dogs. However, current knowledge does not completely explain the observed pathologies induced by A. vasorum infections, including bleeding disorders. Using sera from experimentally infected dogs, dog serum proteome was analysed by quantitative mass spectrometry methods over several time points before and after inoculation. Following computational analysis, we identified 139 up- and downregulated proteins after infection (log2 ratio cut-off ≥ 1.0; q-value ≤ 0.05). Among upregulated proteins were chitinase 3-like 1 and pulmonary surfactant-associated protein B (log2 fold-changes ≥ 5). Pathway enrichment revealed the complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of downregulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factor XIII-B. These results bring new elements towards understanding the underlying pathomechanisms of bleeding diatheses observed in some A. vasorum-infected dogs.

Combined Serological Detection of Circulating Angiostrongylus vasorum Antigen and Parasite-specific Antibodies in Dogs from Hungary.

The occurrence of the nematode Angiostrongylus vasorum, also known as the French heartworm, is increasingly being reported from various European countries. The adults of this parasite species live in the pulmonary arteries and right cardiac ventricle of wild canids and domestic dogs. Larval stages and eggs in the lungs induce inflammatory verminous pneumonia, causing severe respiratory disease in dogs. Furthermore, haematological and neurological signs and even death may occur. In Hungary, A. vasorum has been identified in red foxes, golden jackals and in two dogs and some slugs. In this first large-scale survey, 1247 sera from pet dogs were collected and tested by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA to detect specific antibodies against the parasite. A total of 1.36% (n = 17, 95 % confidence intervals, CI: 0.80 - 2.17 %) of the animals were positive in both ELISAs, while 1.76 % (n = 22, CI: 1.11 - 2.66 %) of the tested dogs were antigen-positive only and 2.73 % (n = 34, CI: 1.90 - 3.79 %) were positive for specific antibodies only. Regions with antigen- and antibody-positive animals overlapped and were distributed over nearly the whole sampled areas of the country. A considerable number of cases was observed in Budapest and also in the southern part of the country bordering Croatia, while in the most eastern part bordering Ukraine no positive samples were detected. These results confirm the endemic occurrence of A. vasorum in dogs originating from different parts of Hungary and the significant advantages of A. vasorum serology in epidemiological studies.

Insights into the immuno-molecular biology of Angiostrongylus vasorum through transcriptomics--prospects for new interventions.

Angiostrongylus vasorum is a metastrongyloid nematode of dogs and other canids of major clinical importance in many countries. In order to gain first insights into the molecular biology of this worm, we conducted the first large-scale exploration of its transcriptome, and predicted essential molecules linked to metabolic and biological processes as well as host immune responses. We also predicted and prioritized drug targets and drug candidates. Following Illumina sequencing (RNA-seq), 52.3 million sequence reads representing adult A. vasorum were assembled and annotated. The assembly yielded 20,033 contigs, which encoded proteins with 11,505 homologues in Caenorhabditis elegans, and additional 2252 homologues in various other parasitic helminths for which curated data sets were publicly available. Functional annotation was achieved for 11,752 (58.6%) proteins predicted for A. vasorum, including peptidases (4.5%) and peptidase inhibitors (1.6%), protein kinases (1.7%), G protein-coupled receptors (GPCRs) (1.5%) and phosphatases (1.2%). Contigs encoding excretory/secretory and immuno-modulatory proteins represented some of the most highly transcribed molecules, and encoded enzymes that digest haemoglobin were conserved between A. vasorum and other blood-feeding nematodes. Using an essentiality-based approach, drug targets, including neurotransmitter receptors, an important chemosensory ion channel and cysteine proteinase-3 were predicted in A. vasorum, as were associated small molecular inhibitors/activators. Future transcriptomic analyses of all developmental stages of A. vasorum should facilitate deep explorations of the molecular biology of this important parasitic nematode and support the sequencing of its genome. These advances will provide a foundation for exploring immuno-molecular aspects of angiostrongylosis and have the potential to underpin the discovery of new methods of intervention.

Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.

The spliced leader gene of Angiostrongylus cantonensis.

A 5' leader sequence has been identified on mRNAs of the parasitic nematode Angiostrongylus cantonensis. A 720-bp XhoI restriction fragment containing the gene encoding the leader sequence has been cloned and sequenced. It contains a 22-nt sequence identical to that of the leader sequence of Caenorhabditis elegans, a consensus splice site and a putative Sm antigen binding site. The gene is present as a tandem repeating unit of approximately 60 copies, and unlike C. elegans it is not associated with the 5S ribosomal RNA gene. The SL-RNA is 110 nt long and the sequence and primer extension studies suggest that it is transcribed from the tandemly repeating SL gene. It is precipitable from cell-free extracts of adult nematodes by anti-Sm anti-sera, and from RNA by anti-TMG anti-sera, thus suggesting its inclusion with small nuclear ribonucleoproteins in RNA splicing.

Application of 15N-nuclear magnetic resonance (N.M.R.) spectroscopy for the study of nitrogen metabolism of a parasite: transamination in Angiostrongylus cantonensis eggs.

In an attempt to study nitrogen metabolism in a parasite, we applied 15N-nuclear magnetic resonance (NMR) technology with a stable isotope, a 15N-labeled compound, for the study of the transamination system in A. cantonensis eggs, and demonstrated that 15N-aspartic acid can serve as an amino group donor for both the 2-oxoglutaric-glutamic acid and the pyruvic acid-alanine transamination systems in the eggs.

Lipids of Angiostrongylus cantonensis (Nematoda: Metastrongyloidea): a comparison between young adults and gravid worms.

1. All major classes of lipids were found in the young adults in brain (22 days post-infection) and gravid Angiostrongylus cantonensis in lung of rats (34 days post-infection) comprising approximately 60% of phospholipids, 30% of neutral lipids and the rest, glycolipids. 2. The relative composition of phospholipids were quite similar between worms from the two different habitats, with phosphatidylcholine predominating. The glycolipid profiles were also similar. 3. More neutral lipids in the worms from brain existed as cholesterol and cholesterol esters than those from the lung. More than 20% of the fatty acids in these lipids of the brain were found as C10-C14 acids while oleic acid was the main component in the lung worm.

1H- and 13C-NMR spectroscopic study of the metabolites in young adult Angiostrongylus cantonensis maintained in vitro.

1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy were used to study metabolites excreted by young adult Angiostrongylus cantonensis maintained aerobically in the presence of D-[13C6]glucose. End-products of glucose metabolism identified and quantitated by means of 1H-NMR were lactate, acetate and alanine, in the molar ratio of 1:0.13:0.05 for males and 1:0.07:0.04 for females. 13C-NMR analyses proved that all the three products originated from the glucose present in the medium.

Glycolytic enzymes in juvenile and adult Angiostrongylus cantonensis.

In extracts of adult Angiostrongylus cantonensis, the activities of enzymes including glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, triosepho sphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerokinase, phosphoglyceromutase, enolase, pyruvate kinase, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, glycerophosphate dehydrogenase and pyruvate dehydrogenase complex were demonstrated. The present of significant activity of glycerophosphate dehydrogenase and glucose 6-phosphate dehydrogenase may indicate the possibility of an operative of alpha-glycerophosphate and pentose phosphate pathway.

De novo purine ribonucleotide biosynthesis in adult Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

1. Radioactivity was found to be associated with purine ribonucleotides in extracts of gravid adult Angiostrongylus cantonensis from the lungs of rats when the parasite was incubated in a medium containing [14C]glycine. 2. The rate of incorporation was at least 0.2 pmole/min per mg of parasite and approximately equal amounts of adenine and guanine ribonucleotides were synthesized. 3. This is the first demonstration of the capacity for a mammalian parasitic helminth to synthesize purines de novo.