Ancylostoma Ceylanicum Research Articles

The hookworm Ancylostoma ceylanicum intestinal transcriptome provides a platform for selecting drug and vaccine candidates.

BackgroundThe intestine of hookworms contains enzymes and proteins involved in the blood-feeding process of the parasite and is therefore a promising source of possible vaccine antigens. One such antigen, the hemoglobin-digesting intestinal aspartic protease known as Na-APR-1 from the human hookworm Necator americanus, is currently a lead candidate antigen in clinical trials, as is Na-GST-1 a heme-detoxifying glutathione S-transferase.MethodsIn order to discover additional hookworm vaccine antigens, messenger RNA was obtained from the intestine of male hookworms, Ancylostoma ceylanicum, maintained in hamsters. RNA-seq was performed using Illumina high-throughput sequencing technology. The genes expressed in the hookworm intestine were compared with those expressed in the whole worm and those genes overexpressed in the parasite intestine transcriptome were further analyzed.ResultsAmong the lead transcripts identified were genes encoding for proteolytic enzymes including an A. ceylanicum APR-1, but the most common proteases were cysteine-, serine-, and metallo-proteases. Also in abundance were specific transporters of key breakdown metabolites, including amino acids, glucose, lipids, ions and water; detoxifying and heme-binding glutathione S-transferases; a family of cysteine-rich/antigen 5/pathogenesis-related 1 proteins (CAP) previously found in high abundance in parasitic nematodes; C-type lectins; and heat shock proteins. These candidates will be ranked for downstream antigen target selection based on key criteria including abundance, uniqueness in the parasite versus the vertebrate host, as well as solubility and yield of expression.ConclusionThe intestinal transcriptome of A. ceylanicum provides useful information for the identification of proteins involved in the blood-feeding process, representing a first step towards a reverse vaccinology approach to a human hookworm vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1795-8) contains supplementary material, which is available to authorized users.

Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: A new tool for anthelmintic research

The screening of candidate compounds and natural products for anthelmintic activity is important for discovering new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device (‘chip’) that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of the worm's pharynx. These electropharyngeograms (EPGs) are recorded simultaneously from multiple worms per chip, providing a medium-throughput readout of muscular and neural activity that is especially useful for compounds targeting neurotransmitter receptors and ion channels. Microfluidic technologies have transformed C. elegans research and the goal of the current study was to validate hookworm and Ascaris suum host-stage larvae in the microfluidic EPG platform. Ancylostoma ceylanicum and A. caninum infective L3s (iL3s) that had been activated in vitro generally produced erratic EPG activity under the conditions tested. In contrast, A. ceylanicum L4s recovered from hamsters exhibited robust, sustained EPG activity, consisting of three waveforms: (1) conventional pumps as seen in other nematodes; (2) rapid voltage deflections, associated with irregular contractions of the esophagus and openings of the esophogeal-intestinal valve (termed a ‘flutter’); and (3) hybrid waveforms, which we classified as pumps. For data analysis, pumps and flutters were combined and termed EPG ‘events.’ EPG waveform identification and analysis were performed semi-automatically using custom-designed software. The neuromodulator serotonin (5-hydroxytryptamine; 5HT) increased EPG event frequency in A. ceylanicum L4s at an optimal concentration of 0.5 mM. The anthelmintic drug ivermectin (IVM) inhibited EPG activity in a concentration-dependent manner. EPGs from A. suum L3s recovered from pig lungs exhibited robust pharyngeal pumping in 1 mM 5HT, which was inhibited by IVM. These experiments validate the use of A. ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for screening anthelmintic candidates or investigating parasitic nematode feeding behavior.

Phenolic Metabolites of Dalea ornata Affect Both Survival and Motility of the Human Pathogenic Hookworm Ancylostoma ceylanicum.

Hookworms are ubiquitous human parasites, infecting nearly one billion people worldwide, and are the leading cause of anemia and malnutrition in resource-limited countries. Current drug treatments rely on the benzimidazole derivatives albendazole and mebendazole, but there is emerging resistance to these drugs. As part of a larger screening effort, using a hamster-based ex vivo assay, anthelmintic activity toward Ancylostoma ceylanicum was observed in the crude extract of aerial parts of Dalea ornata. These studies have led to the isolation and characterization of phenolic metabolites 1-10. The structures were determined by 1D and 2D NMR spectroscopy, and the absolute configuration of 1 was assigned using electronic circular dichroism data. The new compound, (2S)-8-(3-methylbut-2-en-1-yl)-6,7,4'-trihydroxyflavanone (1), was weakly active at 7.3 μM, with 17% reduction in survival of the hookworms after 5 days. The rotenoids deguelin (9) and tephrosin (10), predictably perhaps, were the most active, with complete worm mortality observed by day 4 (or earlier) at 6.3 and 6.0 μM, respectively. The effects of 1-10 on hookworm motility and on toxicity to hamster splenocytes were also explored as important measures of treatment potential.

A Case of Ancylostoma ceylanicum Infection Occurring in an Australian Soldier Returned from Solomon Islands

A 26-year-old male member of the Australian Defense Force presented with a history of central abdominal pain of 4 weeks duration and peripheral eosinophilia consistent with eosinophilic enteritis. Acute hookworm disease was diagnosed as the cause. Adult worms recovered from feces after therapy with albendazole were morphologically consistent with Ancylostoma ceylanicum. As the patient had been deployed with the Regional Assistance Mission to Solomon Islands for 6 months prior to this presentation, it is very likely that the A. ceylanicum was acquired in Solomon Islands. Until now, it has been assumed that any Ancylostoma spp. recovered from humans in Solomon Islands is A. duodenale. However, this case demonstrates that human hookworm infection acquired in the Solomon Islands could be caused by A. ceylanicum.

Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo.

Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite’s resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck’s cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite’s CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN’s somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole.

Hamsters vaccinated with Ace-mep-7 DNA vaccine produced protective immunity against Ancylostoma ceylanicum infection

Hookworms are intestinal nematodes that infect up to 740 million people, mostly in tropical and subtropical regions. Adult worms suck blood from damaged vessels in the gut mucosa, digesting hemoglobin using aspartic-, cysteine- and metalloproteases. Targeting aspartic hemoglobinases using drugs or vaccines is therefore a promising approach to ancylostomiasis control. Based on homology to metalloproteases from other hookworm species, we cloned the Ancylostoma ceylanicum metalloprotease 7 cDNA (Ace-mep-7). The corresponding Ace-MEP-7 protein has a predicted molecular mass of 98.8 kDa. The homology to metallopeptidases from other hookworm species and its predicted transmembrane region support the hypothesis that Ace-MEP-7 may be involved in hemoglobin digestion in the hookworm gastrointestinal tract, especially that our analyses show expression of Ace-mep-7 in the adult stage of the parasite. Immunization of Syrian golden hamsters with Ace-mep-7 cDNA resulted in 50% (p < 0.01) intestinal worm burden reduction. Additionally 78% (p < 0.05) egg count reduction in both sexes was observed. These results suggest that immunization with Ace-mep-7 may contribute to reduction in egg count released into the environment during the A. ceylanicum infection.

Prevalence of gastrointestinal helminth parasites of zoonotic significance in dogs and cats in lower Northern Thailand.

Gastrointestinal zoonotic helminths of dogs and cats have a public health concern worldwide. We investigated the prevalence of gastrointestinal helminths of zoonotic significance in dogs and cats in lower Northern Thailand and utilized molecular tools for species identification of hookworms and Opisthorchis viverrini. Fecal samples of 197 dogs and 180 cats were collected. Overall prevalence of infection using microscopy was 40.1% in dogs and 33.9% in cats. Helminth infection found in both dogs and cats included hookworms, Spirometra spp., Taenia spp., Toxocara spp., O. viverrini, Strongyloides spp. and Trichuris spp. Hookworms were the most common helminth in dogs, while Spirometra spp. were the most prevalent in cats. Among hookworm infection in dogs and cats, Ancylostoma ceylanicum was the most prevalent hookworm, being 82.1% in hookworm infected dogs and 95.8% in hookworm infected cats. Mixed-infection due to hookworms and Spirometra spp. was the most dominant in both dogs and cats. Our finding showed that zoonotic helminth infection is highly prevalent in dogs and cats in the lower Northern area of Thailand.

Vaccination with a cocktail of Ancylostoma ceylanicum recombinant antigens leads to worm burden reduction in hamsters

Hookworms, a group to which Ancylostoma ceylanicum belongs, are gastrointestinal nematodes that infect more than 700 million people around the world. They are a leading cause of anemia in developing countries. In order to effectively prevent hookworm infections research is conducted to develop an effective vaccine using recombinant antigens of the parasite. The aim of this study was to examine the influence of the hosts' on protection against ancylostomiasis and the shaping of the humoral immune response among Syrian hamsters after immunization with a cocktail of five A. ceylanicum recombinant antigens. Ace-ASP-3, Ace-ASP-4, Ace-APR-1, Ace-MEP-6 and Ace-MEP-7 were obtained in the pET expression system. Immunization with a vaccine cocktail resulted in a 33.5% worm burden reduction. The immunogenicity of the recombinant proteins were determined using ELISA. Statistical analysis showed that vaccinated hamsters developed stronger humoral responses to four of five recombinant antigens (the exception being Ace-ASP-3) compared to hamsters from the control group.

Detection of Helminth Eggs and Identification of Hookworm Species in Stray Cats, Dogs and Soil from Klang Valley, Malaysia.

The present study was conducted to determine the prevalence of helminth eggs excreted in the faeces of stray cats, dogs and in soil samples. A total of 505 fresh samples of faeces (from 227 dogs and 152 cats) and soil were collected. The egg stage was detected via microscopy after the application of formalin–ether concentration technique. Genomic DNA was extracted from the samples containing hookworm eggs and used for further identification to the species level using real-time polymerase chain reaction coupled with high resolution melting analysis. Microscopic observation showed that the overall prevalence of helminth eggs among stray cats and dogs was 75.7% (95% CI = 71.2%–79.9%), in which 87.7% of dogs and 57.9% of cats were infected with at least one parasite genus. Five genera of heliminth eggs were detected in the faecal samples, including hookworms (46.4%), Toxocara (11.1%), Trichuris (8.4%), Spirometra (7.4%) and Ascaris (2.4%). The prevalence of helminth infections among stray dogs was significantly higher than that among stray cats (p < 0.001). Only three genera of helminths were detected in soil samples with the prevalence of 23% (95% CI = 15.1%–31%), consisting of hookworms (16.6%), Ascaris (4%) and Toxocara (2.4%). The molecular identification of hookworm species revealed that Ancylostoma ceylanicum was dominant in both faecal and soil samples. The dog hookworm, Ancylostoma caninum, was also detected among cats, which is the first such occurrence reported in Malaysia till date. This finding indicated that there was a cross-infection of A. caninum between stray cats and dogs because of their coexistent within human communities. Taken together, these data suggest the potential role of stray cats and dogs as being the main sources of environmental contamination as well as for human infections.

A novel isothermal microcalorimetry tool to assess drug effects on Ancylostoma ceylanicum and Necator americanus.

Soil-transmitted helminths, which affect the poorest communities, worldwide cause a range of symptoms and morbidity, yet few treatment options are available and drug resistance is a concern. To improve and accelerate anthelminthic drug discovery, novel drug screening tools such as isothermal microcalorimetry (IMC) have been tested with great potential. In this study, we used a novel microcalorimeter, the calScreener™, to study the viability on the hookworms Necator americanus and Ancylostoma ceylanicum as well as the whipworm Trichuris muris. Significant heat flow signals could be obtained with already one adult worm per channel for all three species. High-amplitude oscillations were observed for the hookworms; however, adult T. muris showed a twofold heat flow decrease during the first 24 h. Antinematodal effects of ivermectin and levamisole at 1, 10, and 100 μg/ml were evaluated on adult N. americanus and A. ceylanicum. Levamisole-treated hookworms showed a decline in heat flow and oscillation amplitude in a dose-response manner. Heat flow for ivermectin-treated hookworms increased proportionally with increased concentrations of ivermectin, though the wavelet analysis showed an opposite trend as observed by flatter wavelets. In conclusion, the calScreener™ is an excellent tool to study drug effects on intestinal hookworms at the adult worm stage as it offers a lower detection limit than other IMC devices and the possibility to monitor worm viability online.

Expression profile of heat shock response factors during hookworm larval activation and parasitic development

When organisms are exposed to an increase in temperature, they undergo a heat shock response (HSR) regulated by the transcription factor heat shock factor 1 (HSF-1). The heat shock response includes the rapid changes in gene expression initiated by binding of HSF-1 to response elements in the promoters of heat shock genes. Heat shock proteins function as molecular chaperones to protect proteins during periods of elevated temperature and other stress. During infection, hookworm infective third stage larvae (L3) undergo a temperature shift from ambient to host temperature. This increased temperature is required for the resumption of feeding and activation of L3, but whether this increase initiates a heat shock response is unknown. To investigate the role of the heat shock in hookworm L3 activation and parasitic development, we identified and characterized the expression profile of several components of the heat shock response in the hookworm Ancylostoma caninum. We cloned DNAs encoding an hsp70 family member (Aca-hsp-1) and an hsp90 family member (Aca-daf-21). Exposure to a heat shock of 42°C for one hour caused significant up-regulation of both genes, which slowly returned to near baseline levels following one hour attenuation at 22°C. Neither gene was up-regulated in response to host temperature (37°C). Conversely, levels of hsf-1 remained unchanged during heat shock, but increased in response to incubation at 37°C. During activation, both hsp-1 and daf-21 are down regulated early, although daf-21 levels increase significantly in non-activated control larvae after 12h, and slightly in activated larvae by 24h incubation. The heat shock response modulators celastrol and KNK437 were tested for their effects on gene expression during heat shock and activation. Pre-incubation with celastrol, an HSP90 inhibitor that promotes heat shock gene expression, slightly up-regulated expression of both hsp-1 and daf-21 during heat shock. KNK437, an inhibitor of heat shock protein expression, slightly down regulated both genes under similar conditions. Both modulators inhibited activation-associated feeding, but neither had an effect on hsp-1 levels in activated L3 at 16h. Both celastrol and KNK437 prevent the up-regulation of daf-21 and hsf-1 seen in non-activated control larvae during activation, and significantly down regulated expression of the HSF-1 negative regulator Aca-hsb-1 in activated larvae. Expression levels of heat shock response factors were examined in developing Ancylostoma ceylanicum larvae recovered from infected hosts and found to differ significantly from the expression profile of activated L3, suggesting that feeding during in vitro activation is regulated differently than parasitic development. Our results indicate that a classical heat shock response is not induced at host temperature and is suppressed during larval recovery and parasitic development in the host, but a partial heat shock response is induced after extended incubation at host temperature in the absence of a developmental signal, possibly to protect against heat stress.

Dalea emoryi revisited: Anthelmintic and new components of a previously-studied plant

Detailed chemical studies of Dalea emoryi A. Gray (syn: Psorothamnus emoryi (A. Gray) Rydb. (Fabaceae) were undertaken using taxonomic rationale. Other Dalea spp. have yielded anthelmintic components in our biological testing. D. emoryi was the subject of prior studies, and, interestingly, the crude extract was inactive in our bioassay. Nevertheless, new and active compounds 1 and 2, for example, were revealed upon further purification of the crude D. emoryi materials. These findings agree with prior observations regarding the importance of screening partially purified materials in addition to crude extracts. Anthelmintic activities were determined in an ex vivo assay with the hookworm Ancylostoma ceylanicum. Isolation of compounds was by classical open-column chromatographic methods, with characterization using extensive 1D and 2D NMR spectroscopic methods and by HRESIMS. Details of the bioassay methods, results, and the isolation and characterization of D. emoryi compounds will be presented.

Protection and Delivery of Anthelmintic Protein Cry5B to Nematodes Using Mesoporous Silicon Particles.

The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed in vitro and in vivo. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 μm size are noticeably ingested by both species within 2 h of introduction in vitro, whereas 5 ± 2 μm particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 μm display the highest level of in vitro toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. In vivo experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal.

The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families.

Hookworms infect over 400 million people, stunting and impoverishing them1–3. Sequencing hookworm genomes and finding which genes they express during infection should help in devising new drugs or vaccines against hookworms4,5. Unlike other hookworms, Ancylostoma ceylanicum infects both humans and other mammals, providing a laboratory model for hookworm disease6,7. We determined an A. ceylanicum genome sequence of 313 Mb, with transcriptomic data throughout infection showing expression of 30,738 genes. Approximately 900 genes were upregulated during early infection in vivo, including ASPRs, a cryptic subfamily of activation-associated secreted proteins (ASPs)8. Genes downregulated during early infection included ion channels and G protein–coupled receptors; this downregulation was observed in both parasitic and free-living nematodes. Later, at the onset of heavy blood feeding, C-lectin genes were upregulated along with genes for secreted clade V proteins (SCVPs), encoding a previously undescribed protein family. These findings provide new drug and vaccine targets and should help elucidate hookworm pathogenesis.

Identification of Ancylostoma ceylanicum in children from a tribal community in Tamil Nadu, India using a semi-nested PCR-RFLP tool.

It is generally assumed that hookworm infections in humans are caused by Necator americanus and Ancylostoma duodenale. However, previous studies have also reported the presence of the animal hookworm A. ceylanicum in human stools. We determined hookworm infections in children in a tribal community in Tamil Nadu, India, using a semi-nested PCR-RFLP approach. The results indicate that human species account for a majority of the hookworm infections (N. americanus 39/41 [95%]; A. duodenale 6/41 [15%]), whereas the animal hookworm A. ceylanicum only accounts for a minority of the infections (5%; 2/41). The results emphasize the need to consider zoonotic ancylostomiasis while developing strategies to control hookworm infections.

Molecular cloning and characterization of a nematode polyprotein antigen/allergen from the human and animal hookworm Ancylostoma ceylanicum

Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and retinol binding proteins that nematodes produce (fatty acid and retinol binding (FAR) and nematode polyprotein antigen/allergen (NPA)) are used to meet this need. A partial cDNA has been cloned corresponding to four subunits of a putative Ancylostoma ceylanicum NPA (AceNPA). The translated amino acid sequence of AceNPA shares sequence identity with similar proteins from Dictyocaulus viviparus, Ascaris suum, and Ostertagia ostertagi. Immunoblot experiments using a polyclonal anti-AceNPA IgG revealed proteins corresponding to the expected sizes of single, as well as two or three un-cleaved NPA subunits in adult excretory/secretory proteins and soluble adult worm extracts. Immunohistochemistry experiments localize AceNPA to the cuticle, pseudocoelomic space and testes suggesting a role in hookworm biology that is distinct from what has previously been defined for other hookworm lipid binding proteins. A single recombinant subunit of AceNPA (rAceNPAb) demonstrated binding in vitro to fluorescent fatty acids DAUDA, cis-parinaric acid, as well as retinol, at equilibrium dissociation constants in the low micromolar range. Further, in vitro data reveal that rAceNPAb binds fatty acids with chain lengths of C12–C22, with the greatest affinities for arachidonic, linoleic (C18), and eicosapentaenoic (C20) acids.

The crowding effect in Ancylostoma ceylanicum: density-dependent effects on an experimental model of infection.

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8% to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.

Evaluation of biochemical, hematological and parasitological parameters of protein-deficient hamsters infected with Ancylostoma ceylanicum.

BackgroundHookworms infect millions of people worldwide and can cause severe clinical symptoms in their hosts. Prospective cohort studies in Brazil show high rates of hookworm reinfection in malnourished children compared to well-nourished children, despite previous treatment. Additionally, soil-transmitted helminth (STH) infections can worsen the nutritional status of affected populations. Therefore, this study aims to clarify the effects of host malnutrition during Ancylostoma ceylanicum infection and how this infection affects host physiological parameters using a hamster model.Methodology/Principal FindingsHamsters were divided into four experimental groups: normal diet or low-protein diet (also referred to as “malnourished”) and A. ceylanicum infection or no infection. More severe pathogenesis was observed in the infected malnourished group, as demonstrated by significant decreases in the hemoglobin concentration, erythrocyte number and packed-cell volume compared to the non-infected malnourished group. Greater numbers of adult parasites and eggs were observed in the malnourished group compared to the control group; however, the oviposition rate was lower in the malnourished group. In general, greater values of total lipids were observed in malnourished animals compared to control animals, including lipids excreted in the stool.ConclusionsIn this work, we have demonstrated that animals fed an isocaloric low-protein diet presented more severe pathogenesis when infected with A. ceylanicum. The increased lipid concentration in the liver and blood is related to the conversion of the excess carbohydrate into fatty acids that increase the concentration of triglycerides in general. Triglycerides were excreted in the feces, indicating that infection associated with malnutrition caused a greater loss of these molecules for this group of animals and confirming the hypothesis that both nutrition and infection are responsible for the malabsorption syndrome. Taken together, the results found in this work confirm the hypothesis that the nutritional condition of the host greatly influences the course of the infection.

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.

The prevalence and distribution of gastrointestinal parasites of stray and refuge dogs in four locations in India

A gastrointestinal parasite survey of 411 stray and refuge dogs sampled from four geographical and climactically distinct locations in India revealed these animals to represent a significant source of environmental contamination for parasites that pose a zoonotic risk to the public. Hookworms were the most commonly identified parasite in dogs in Sikkim (71.3%), Mumbai (48.8%) and Delhi (39.1%). In Ladakh, which experiences harsh extremes in climate, a competitive advantage was observed for parasites such as Sarcocystis spp. (44.2%), Taenia hydatigena (30.3%) and Echinococcus granulosus (2.3%) that utilise intermediate hosts for the completion of their life cycle. PCR identified Ancylostoma ceylanicum and Ancylostoma caninum to occur sympatrically, either as single or mixed infections in Sikkim (Northeast) and Mumbai (West). In Delhi, A. caninum was the only species identified in dogs, probably owing to its ability to evade unfavourable climatic conditions by undergoing arrested development in host tissue. The expansion of the known distribution of A. ceylanicum to the west, as far as Mumbai, justifies the renewed interest in this emerging zoonosis and advocates for its surveillance in future human parasite surveys. Of interest was the absence of Trichuris vulpis in dogs, in support of previous canine surveys in India. This study advocates the continuation of birth control programmes in stray dogs that will undoubtedly have spill-over effects on reducing the levels of environmental contamination with parasite stages. In particular, owners of pet animals exposed to these environments must be extra vigilant in ensuring their animals are regularly dewormed and maintaining strict standards of household and personal hygiene.