A case of asymptomatic infection of Ascaris suum identified by PCR-restriction fragment length polymorphism and DNA sequence analysis.

Human ascariasis is a soil-transmitted helminthiasis and remains a neglected tropical disease. Ascaris suum has the potential to cause cross-infections between humans and pigs. In this study, we present a rare case of a patient with asymptomatic infection by Ascaris suum. A 66-year-old male underwent colonoscopy, and a white linear worm body was found in the hepatic curvature. The worm was collected by aspiration and submitted to the laboratory for parasite identification. The patient had no symptoms related to parasitic infection. The worm was highly suspected to be of the genus Ascaris. Because of the difficulty of morphological classification, genetic analysis was performed. From PCR-restriction fragment length polymorphism results and sequence analysis of the internal transcribed spacer-1 region, it was determined to be A. suum. The experience with rapid differentiation of A. suum by performing genetic analysis will be useful for future examinations of parasitic infections.

A molecular and morphological study of Ascaris suum in a human-pig contact scenario in northeastern Brazil / Estudo molecular e morfológico de Ascaris suum em um cenário de contato entre suínos e humanos no nordeste brasileiro

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.(AU)

O presente estudo teve como objetivo acessar dados morfológicos e genéticos sobre a ascaridíase em suínos (Sus scrofa domesticus) e humanos, em comunidades rurais e periurbanas no estado do Piauí. O estudo transversal incluiu 100 amostras fecais de suínos e 682 amostras obtidas de humanos. Quinze suínos foram necropsiados. Amostras fecais suínas e humanas foram examinadas para detecção de ovos de Ascaris. Os parasitas adultos, obtidos nas necropsias, foram estudados através de microscopia eletrônica de varredura (MEV), e o gene mitocondrial codificante da enzima citocromo oxidase 1 (cox1) foi parcialmente amplificado e sequenciado para análises filogenéticas e de taxonomia molecular. A prevalência de Ascaris em amostras fecais de suínos foi 16/100 (16%), não sendo identificado nenhum caso de infecção por este parasita em humanos. A análise por MEV de parasitas recuperados de suínos demonstrou características morfológicas de Ascaris suum. As sequências nucleotídicas de cox1 foram compatíveis com A. suum. As sequências originais e de referência (obtidas no GeneBank) foram organizadas em clusters que não segregaram os parasitas por hospedeiro ou região geográfica. Os maiores haplogrupos foram dominados pelos haplótipos H01, H02 e H31. Nas comunidades estudadas, não foi evidenciada transmissão zoonótica de A. suum na interface suíno-humana.(AU)

Absence of mutations associated with resistance to benzimidazole in the beta-tubulin gene of Ascaris suum

Abstract INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.

Three independent techniques localize expression of transcript afp-11 and its bioactive peptide products to the paired AVK neurons in Ascaris suum: in situ hybridization, immunocytochemistry, and single cell mass spectrometry.

We utilized three independent techniques, immunocytochemistry (ICC), single cell mass spectrometry (MS), and in situ hybridization (ISH), to localize neuropeptides and their transcripts in the nervous system of the nematode Ascaris suum . AF11 (SDIGISEPNFLRFa) is an endogenous peptide with potent paralytic effects on A. suum locomotory behavior. A highly specific antibody to AF11 showed robust immunostaining for AF11 in the paired AVK neurons in the ventral ganglion. We traced the processes from the AVK neurons into the ventral nerve cord and identified them as ventral cord interneurons. MS and MS/MS of single dissected AVKs detected AF11, two previously characterized peptides (AF25 and AF26), seven novel sequence-related peptides, including several sharing a PNFLRFamide C-terminus, and peptide NY, a peptide with an unrelated sequence. Also present in a subset of AVKs was AF2, a peptide encoded by the afp-4 transcript. By sequencing the afp-11 transcript, we discovered that it encodes AF11, all the AF11-related peptides detected by MS in AVK, and peptide NY. ISH detected the afp-11 transcript in AVK neurons, consistent with other techniques. ISH did not detect afp-11 in the ALA neuron, although both ICC and MS found AF11 in ca. 30% of ALAs. All 10 AF11-related peptides reduced acetylcholine-induced muscle contraction, but they differed in their rate of reversal of inhibition after removal of the peptide.

Ascaris suum enolase is a potential vaccine candidate against ascariasis.

Ascariasis caused by Ascaris is the most common parasite problem in humans and pigs worldwide. No vaccines are available for the prevention of Ascaris infections. In the present study, the gene encoding Ascaris suum enolase (As-enol-1) was amplified, cloned and sequenced. Amino acid sequence alignment indicated that As-enol-1 was highly conserved between different nematodes and shared the highest identity (87%) with enolase from Anisakis simplex s.l. The recombinant pVAX-Enol was successfully expressed in Marc-145 cells. The ability of the pVAX-Enol for inducing immune protective responses against challenge infection with A. suum L3 was evaluated in Kunming mice. The immune response was evaluated by lymphoproliferative assay, cytokine and antibody measurements, and the reduction rate of recovery larvae. The results showed that the mice immunized with pVAX-Enol developed a high level of specific antibody responses against A. suum, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or blank controls, respectively. There was a 61.13% reduction (P<0.05) in larvae recovery compared with that in the blank control group. Our data indicated that A. suum enolase is a potential vaccine candidate against A. suum infection.

Changes in cyclic nucleotides, locomotory behavior, and body length produced by novel endogenous neuropeptides in the parasitic nematode Ascaris suum.

Recent technical advances have rapidly advanced the discovery of novel peptides, as well as the transcripts that encode them, in the parasitic nematode Ascaris suum. Here we report that many of these novel peptides produce profound and varied effects on locomotory behavior and levels of cyclic nucleotides in A. suum. We investigated the effects of 31 endogenous neuropeptides encoded by transcripts afp-1, afp-2, afp-4, afp-6, afp-7, and afp-9-14 (afp: Ascaris FMRFamide-like Precursor protein) on cyclic nucleotide levels, body length and locomotory behavior. Worms were induced to generate anteriorly propagating waveforms, peptides were injected into the pseudocoelomic cavity, and changes in the specific activity (nmol/mg protein) of second messengers cAMP (3'5' cyclic adenosine monophosphate) and cGMP (3'5' cyclic guanosine monophosphate) were determined. Many of these neuropeptides changed the levels of cAMP (both increases and decreases were found), whereas few neuropeptides changed the level of cGMP. A subset of the peptides that lowered cAMP was investigated for effects on the locomotory waveform and on body length. Injection of AF19, or AF34 (afp-13), AF9 (afp-14), AF26 or AF41 (afp-11) caused immediate paralysis and cessation of propagating body waveforms. These neuropeptides also significantly increased body length. In contrast, injection of AF15 (afp-9) reduced the body length, and decreased the amplitude of waves in the body waveform. AF30 (afp-10) produced worms with tight ventral coils. Although injection of neuropeptides encoded by afp-1 (AF3, AF4, AF10 or AF13) produced an increased number of exaggerated body waves, there were no effects on either cAMP or cGMP. By injecting peptides into behaving A. suum, we have provided an initial screen of the effects of novel peptides on several behavioral and biochemical parameters.

The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.

Evolution of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides in nematodes: putative rearrangement of disulfide bonding patterns.

ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides are defensin-like cysteine-rich peptides that are widely distributed in the phylum Nematoda. In known members of ASABF-type antimicrobial peptides, an array consisting of eight cysteine residues is completely conserved. In this study, we report a novel member ASABF-6Cys-alpha, which contains only six cysteine residues, in the pig round worm A. suum. The two cysteine residues deleted in ASABF-6Cys-alpha were not identical to a pair of half-cystine forming a disulfide bridge in ASABF-alpha, suggesting a rearrangement of disulfide bonding patterns. Gene organization and phylogenetic analyses suggested that ASABF-6Cys-alpha was generated from an ancestral ASABF gene after the divergence of Ascaridida from Rhabditida. ASABF-6Cys-alpha transcripts dramatically increased after bacterial injection, suggesting that ASABF-6Cys-alpha may contribute to immunity in nematodes.

Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme.

Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.

Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm.

The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A.suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A.suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m1A9). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of aminoacylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m1A9 is a key residue for the activity of T-armless tRNAs. Thus, m1A9 is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

Common structural properties specifically found in the CSalphabeta-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin?

The structural properties of the Ascaris suum antibacterial factor (ASABF)-type antimicrobial peptides, isolated from nematodes, were compared with the CSalphabeta-type antimicrobial peptides found in other organisms. The spacing of the half-cystine residues, cysteine pairings, and organization of the precursor were different from the 'classical' CSalphabeta-type antimicrobial peptides, such as drosomycin and plant defensins, and identical only to the MGD and myticin in mollusks. In addition, ABF-5, a member of the ASABF-type antimicrobial peptides in Caenorhabditis elegans, is predicted to contain a basic mature region and an acidic pro-region, similar to MGD and myticin. These results suggest that the ASABF-type antimicrobial peptides, MGD and myticin are similar in their structure.

Induction of ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides by bacterial injection: novel members of ASABF in the nematode Ascaris suum.

Recently, invertebrate models have been widely used for the study of innate immunity. Nematodes are novel potential candidates because of the experimental advantages of Caenorhabditis elegans. However, whether nematodes have active immune responses is still ambiguous. Previously, we reported ASABF (Ascaris suum antibacterial factor)-type antimicrobial peptides in the parasitic nematode Ascaris suum and the genetic model nematode C. elegans. Further screening of a cDNA library and an expressed-sequence-tag database search detected five novel members of ASABF (ASABF-beta, -gamma, -delta, - epsilon and -zeta) in A. suum. The transcripts for ASABF-alpha, -beta, -gamma, and -delta clearly increased in the body wall, and also in the intestine for ASABF-delta, 4 h after injection of heat-killed bacteria into the pseudocoelom (body cavity), suggesting that these peptides are inducible in the acute phase of immune response. These results also suggest that the nematodes can recognize bacteria in the pseudocoelomic fluid and evoke an active immune response.

Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase.

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.

Requirement of modified residue m1A9 for EF-Tu binding to nematode mitochondrial tRNA lacking the T arm.

Most of nematode mitochondrial (mt) tRNAs lacking the T arm have 1-methyladenosine (m1A) at position 9. To investigate the effect of m1A, we constructed a nematode Ascaris suum mt tRNA(Met) containing only m1A9 as the modified nucleoside by means of molecular surgery. Although the unmodified A. suum mt Met-tRNA(Met) did not bind to nematode mt EF-Tu, the m1A9-containing tRNA bound to the EF-Tu, suggesting that m1A at position 9 is necessary for binding of nematode mt tRNAs lacking the T arm to the EF-Tu, probably because of maintenance of the L-shape-like structure or interaction with the C-terminal amino acid residues of the EF-Tu.

Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

Cloning and characterisation of a peroxiredoxin from the swine roundworm Ascaris suum.

Antioxidant enzymes in parasites play an important role in protection against the oxygen radicals by generating during aerobic metabolism, as well as in defence against host immune cell assault. Here we report the cloning and characterisation of a cDNA encoding peroxiredoxin from Ascaris suum (AsPrx). AsPrx is 776bp long and contains the nematode 22bp splice leader sequence at the 5' end and polyadenylation signal followed by poly(A) tail at the 3' end. AsPrx codes a full-length protein with a predicted molecular mass of 22. 6kDa, and possesses two cysteine residues at amino acid 49 and 168 that are conserved among Prx proteins. GenBank() analysis showed that the deduced amino acid sequence had significant similarity to parasite and mammalian Prx at the amino acid level. DNA nicking revealed that Escherichia coli-expressed recombinant AsPrx (rAsPrx) is enzymatically inhibited to form oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse anti-rAsPrx serum reacted two major constituent protein spots in extracts of adult female worms, suggesting that the native AsPrx might be function as a major antioxidant enzyme in Ascaris suum.

The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties.

The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units.

Secretion of a novel class of iFABPs in nematodes: coordinate use of the Ascaris/Caenorhabditis model systems.

A novel fatty acid binding protein, As-p18, is secreted into both the perivitelline and perienteric fluids of the parasitic nematode, Ascaris suum, and at least eight potential homologues of As-p18 have been identified in the Caenorhabditis elegans genome. The products of the three most closely related homologues are fatty acid binding proteins (LBP-1, LBP-2 and LBP-3) which contain putative secretory signals. Phylogenetic analysis revealed that these secreted fatty acid binding proteins comprise a distinct gene class within the fatty acid binding protein family and are possibly unique to nematodes. To examine the potential sites of As-p18 secretion, the expression of the putative promoters of the C. elegans homologues was examined with GFP reporter constructs. The developmental expression of lbp-1 was identical to that of As-p18 and consistent with the secretion of LBP-1 from the hypodermis to the perivitelline fluid. The expression patterns of lbp-2 and lbp-3 were consistent with the secretion of LBP-2 and LBP-3 from muscle into the perienteric fluid later in development. These studies demonstrate that at least some perivitelline fluid proteins appear to be secreted from the hypodermis prior to the formation of the cuticle and, perhaps more importantly, that this coordinate C. elegans/A. suum approach may be potentially useful for examining a number of key physiological processes in parasitic nematodes.

Ligand-gated chloride channel subunits encoded by the Haemonchus contortus and Ascaris suum orthologues of the Caenorhabditis elegans gbr-2 (avr-14) gene.

The alternatively-spliced Caenorhabditis elegans gbr-2/avr-14 gene encodes two subunits of the nematode ligand-gated chloride channel family which forms an important molecular target for the avermectin and related anthelminthics. We used reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to isolate cDNAs encoding the products of the gbr-2/avr-14 orthologues from the parasitic nematodes Haemonchus contortus and Ascaris suum. The predicted polypeptides possess all the characteristics of subunits of the ligand-gated chloride channels, sharing greater than 80% amino-acid identity with their counterparts in C. elegans and with partial sequences from the filarial species Onchocerca volvulus and Dirofilaria immitis. The pattern of alternative splicing of the gbr-2/avr-14 gene observed in C. elegans is conserved in H. contortus but may not be in A. suum. Affinity-purified anti-GBR-2 antibodies were used to study the expression of these subunits in adult worms and they reacted specifically with the nerve ring, the ventral and dorsal nerve cords, the anterior portion of the dorsal sub-lateral cord and motor-neuron commissures in H. contortus. Specific immunofluorescence of the nerve cords was confirmed in A. suum; isolated muscle cells did not react with the antibody.