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.

Knowledge attitudes and practices of grade three primary schoolchildren in relation to schistosomiasis, soil transmitted helminthiasis and malaria in Zimbabwe.

BACKGROUND: Helminth infection rates in grade three children are used as proxy indicators of community infection status and to guide treatment strategies in endemic areas. However knowledge, attitudes and practices (KAP) of this target age group (8-10 years) in relation to schistosomiasis, soil transmitted helminthiasis (STHs) and malaria is not known at a time when integrated plasmodium - helminth control strategies are being advocated. This study sought to assess KAP of grade 3 children in relation to schistosomiasis, STHs and malaria in order to establish an effective school based health education for disease transmission control. METHODS: Grade 3 children (n = 172) attending four randomly selected primary schools (one in rural and 3 in the commercial farming areas) in Zimbabwe were interviewed using a pre-tested interviewer administered questionnaire. The urine filtration technique was used to determine S. haematobium infection status. Infection with S. mansoni and STHs was determined using a combination of results from the Kato Katz and formol ether concentration techniques. P. falciparum was diagnosed by examination of Giemsa stained thick blood smears. RESULTS: It was observed that 32.0%, 19.2% and 4.1% of the respondents had correct knowledge about the causes of schistosomiasis, malaria and STHs, respectively, whilst 22.1%, 19.2% and 5.8% knew correct measures to control schistosomiasis, malaria and STHs. Sixty-two percent and 44.8% did not use soap to wash hands after toilet and before eating food respectively, whilst 33.1% never wore shoes. There were no functional water points and soap for hand washing after toilet at all schools. There was a high prevalence distribution of all parasites investigated in this study at Msapa primary school - S. haematobium (77.8%), S. mansoni (33.3%) hookworms (29.6%) and P. falciparum (48.1%). Reports that participant had suffered from schistosomiasis and malaria before were significant predictors of these diseases (p = 0.001 and p = 0.042, respectively). Report that participant had blood in urine on the day of examination was a significant predictor of schistosomiasis (p = 0.045). CONCLUSION: There is a critical need for targeting health messages through schools in order to reach the most susceptible schoolchildren. This will empower the schoolchildren with the basic knowledge and skills ultimately protecting them from acquiring schistosomiasis, STHs and malaria.

Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.

Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three beta-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum.

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.

The mucosal response of hamsters to a low-intensity superimposed secondary infection with the hookworm Ancylostoma ceylanicum.

An experiment was conducted to assess the mucosal response to low-dose superimposed challenge with Ancylostoma ceylanicum. Hamsters were assigned to five treatment groups (1-5 respectively): naïve controls; primary immunizing infection controls; challenge controls; immunized, anthelmintic-treated, challenged group; immunized, superimposed challenge group. Group 4 hamsters were resistant to challenge, whereas most of the challenge inoculum larvae established in Group 5. Villus height and crypt depth measurements were initially markedly divergent between these two groups but over time post-challenge (pc) values for both parameters drew nearer and by day 31 pc they were indistinguishable. The greatest change was experienced by Group 4 which showed increasing inflammation and gut pathology during the challenge infection. Mitotic activity in crypts and mast cell counts in the mucosa were highest in Group 5 on day 10 pc, but there was little to distinguish between Groups 4 and 5 by day 31 pc. Goblet cell, eosinophil and Paneth cell counts were very similar throughout in both groups but, in the case of Paneth cells, they were consistent with a possible role in protective immunity to challenge. Some adult worms survived throughout the period of intense inflammation, emphasizing their tremendous resilience and resistance to mucosal host protective responses.

Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae).

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.

Characterizing Ancylostoma caninum transcriptome and exploring nematode parasitic adaptation.

BACKGROUND: Hookworm infection is one of the most important neglected diseases in developing countries, with approximately 1 billion people infected worldwide. To better understand hookworm biology and nematode parasitism, the present study generated a near complete transcriptome of the canine hookworm Ancylostoma caninum to a very high coverage using high throughput technology, and compared it to those of the free-living nematode Caenorhabditis elegans and the parasite Brugia malayi. RESULTS: The generated transcripts from four developmental stages, infective L3, serum stimulated L3, adult male and adult female, covered 93% of the A. caninum transcriptome. The broad diversity among nematode transcriptomes was confirmed, and an impact of parasitic adaptation on transcriptome diversity was inferred. Intra-population analysis showed that A. caninum has higher coding sequence diversity than humans. Examining the developmental expression profiles of A. caninum revealed major transitions in gene expression from larval stages to adult. Adult males expressed the highest number of selectively expressed genes, but adult female expressed the highest number of selective parasitism-related genes. Genes related to parasitism adaptation and A. caninum specific genes exhibited more expression selectivity while those conserved in nematodes tend to be consistently expressed. Parasitism related genes were expressed more selectively in adult male and female worms. The comprehensive analysis of digital expression profiles along with transcriptome comparisons enabled identification of a set of parasitism genes encoding secretory proteins in animal parasitic nematode. CONCLUSIONS: This study validated the usage of deep sequencing for gene expression profiling. Parasitic adaptation of the canine hookworm is related to its diversity and developmental dynamics. This comprehensive comparative genomic and expression study substantially improves our understanding of the basic biology and parasitism of hookworms and, is expected, in the long run, to accelerate research toward development of vaccines and novel anthelmintics.

Identification and localization of hookworm platelet inhibitor in Ancylostoma ceylanicum.

Ancylostoma ceylanicum is a zoonotic hookworm, which mainly causes iron deficiency anemia (IDA) in humans and animals. Hookworm platelet inhibitor (HPI) has been isolated from adult Ancylostoma caninum and linked to the pathogenesis of hookworm associated intestinal hemorrhage and IDA. However, there is no available data about HPI from A. ceylanicum. To study the molecular characteristics of A. ceylanicum HPI (Ace-HPI), its corresponding cDNA was amplified from adult A. ceylanicum mRNA using the primers designed based on the Ac-HPI gene sequence, and its sequence homology and phylogenetic relationship were analyzed. The differential expression of Ace-hpi mRNA in the adult and third larval (L3) stages was compared using the quantitative real-time PCR. Ace-HPI reactivity and tissue localization were studied by Western blot and immunofluorescence, respectively. Platelet aggregation activity was monitored in a 96-well microplate reader. The results showed that the Ace-HPI encoding gene was 603 bp in length. Ace-HPI showed 91% homology to Ac-HPI, was closely related to Ac-ASP3, and belonged to the CAP superfamily. Ace-hpi transcripts were most abundant in the adult stage, followed by serum-stimulated infective larvae (ssL3), and finally in L3 stage, with a significant difference. Escherichia coli-expressed recombinant protein had good reactivity with the positive serum of A. ceylanicum-infected dogs. Immunolocalization indicated that Ace-HPI was located in the esophagus and cephalic glands of the adult. As well as, recombinant Ace-HPI inhibited the platelet aggregation in-vitro. HPI overexpression, anatomical location in adults, antigenicity and its in-vitro activity indicate its possible role in adult worm blood-feeding and as a valuable target for hookworm vaccine and drug development.

Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum.

Hookworm infection is still a public health problem in developing countries. Aspartic protease plays an important role in parasite invasion and migration in the host. Aspartic protease gene from Ancylostoma caninum has been reported (Biochem Biophys Res Commun 227:294-302, 1996), but the activity of Acasp in eggs and larvae stage has not been studied. This paper reported the cloning of Acasp, expression in Escherichia coli, and characterization in eggs and larval stage. The mRNA encoding Acasp was detected using reverse transcriptase polymerase chain reaction in L4 and adult worm. A polyclonal antiserum against E. coli expressed recombinant Acasp was produced and used to detect and localize the Acasp in various developmental stages of A. caninum. Results from Western blot and indirect fluorescent immunoassay showed that the Acasp was present in the embryo, larvae, as well as in the adult worms. The recombinant Acasp exhibited the protease activity by the gelatin gel digestion assay.

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm, Ancylostoma caninum.

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the ß-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous ß-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.

Of dogs and hookworms: man's best friend and his parasites as a model for translational biomedical research.

We present evidence that the dog hookworm (Ancylostoma caninum) is underutilised in the study of host-parasite interactions, particularly as a proxy for the human-hookworm relationship. The inability to passage hookworms through all life stages in vitro means that adult stage hookworms have to be harvested from the gut of their definitive hosts for ex vivo research. This makes study of the human-hookworm interface difficult for technical and ethical reasons. The historical association of humans, dogs and hookworms presents a unique triad of positive evolutionary pressure to drive the A. caninum-canine interaction to reflect that of the human-hookworm relationship. Here we discuss A. caninum as a proxy for human hookworm infection and situate this hookworm model within the current research agenda, including the various 'omics' applications and the search for next generation biologics to treat a plethora of human diseases. Historically, the dog hookworm has been well described on a physiological and biochemical level, with an increasing understanding of its role as a human zoonosis. With its similarity to human hookworm, the recent publications of hookworm genomes and other omics databases, as well as the ready availability of these parasites for ex vivo culture, the dog hookworm presents itself as a valuable tool for discovery and translational research.

A Critical Role for Thermosensation in Host Seeking by Skin-Penetrating Nematodes.

Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are a major source of neglected tropical disease [1-6]. Their life cycle includes an infective third-larval (iL3) stage that searches for hosts to infect in a poorly understood process that involves both thermal and olfactory cues. Here, we investigate the temperature-driven behaviors of skin-penetrating iL3s, including the human-parasitic threadworm Strongyloides stercoralis and the human-parasitic hookworm Ancylostoma ceylanicum. We show that human-parasitic iL3s respond robustly to thermal gradients. Like the free-living nematode Caenorhabditis elegans, human-parasitic iL3s show both positive and negative thermotaxis, and the switch between them is regulated by recent cultivation temperature [7]. When engaging in positive thermotaxis, iL3s migrate toward temperatures approximating mammalian body temperature. Exposing iL3s to a new cultivation temperature alters the thermal switch point between positive and negative thermotaxis within hours, similar to the timescale of thermal plasticity in C. elegans [7]. Thermal plasticity in iL3s may enable them to optimize host finding on a diurnal temperature cycle. We show that temperature-driven responses can be dominant in multisensory contexts such that, when thermal drive is strong, iL3s preferentially engage in temperature-driven behaviors despite the presence of an attractive host odorant. Finally, targeted mutagenesis of the S. stercoralis tax-4 homolog abolishes heat seeking, providing the first evidence that parasitic host-seeking behaviors are generated through an adaptation of sensory cascades that drive environmental navigation in C. elegans [7-10]. Together, our results provide insight into the behavioral strategies and molecular mechanisms that allow skin-penetrating nematodes to target humans.

Short report: an agar plate method for culturing hookworm larvae: analysis of growth kinetics and infectivity compared with standard coproculture techniques.

An agar plate (AP) method has been developed for culturing infectious larvae of the hookworm Ancylostoma ceylanicum. The third-stage larvae reared using the AP method displayed similar morphology to those cultured using Baermann or Harada-Mori coproculture techniques. The yield of viable larvae from the AP method (50%) was comparable to that of the Baermann (47%), and both were superior to Harada-Mori (2.1%). Third-stage larvae cultured by the AP method established patent infection in naturally permissive laboratory hosts, although the yield of adult worms was reduced compared with animals infected with L3 obtained by Baermann culture. The AP method is useful for defining growth requirements for hookworm development, as well as characterizing the effects of bacterially expressed compounds on hookworm larvae in vivo.

Impact of population structure on genetic diversity of a potential vaccine target in the canine hookworm (Ancylostoma caninum).

Ancylostoma caninum is a globally distributed canine parasitic nematode. To test whether positive selection, population structure, or both affect genetic variation at the candidate vaccine target Ancylostoma secreted protein 1 (asp-1), we have quantified the genetic variation in A. caninum at asp-1 and a mitochondrial gene, cytochrome oxidase subunit 1 (cox-1), using the statistical population analysis tools found in the SNAP Workbench. The mitochondrial gene cox-1 exhibits moderate diversity within 2 North American samples, comparable to the level of variation observed in other parasitic nematodes. The protein coding portion for the C-terminal half of asp-1 shows similar levels of genetic variation in a Wake County, North Carolina, sample as cox-1. Standard tests of neutrality provide little formal evidence for selection acting on this locus, but haplotype networks for 2 of the exon regions have significantly different topologies, consistent with different evolutionary forces shaping variation at either end of a 1.3-kilobase stretch of sequence. Evidence for gene flow among geographically distinct samples suggests that the mobility of hosts of A. caninum is an important contributing factor to the population structure of the parasite.

Ancylostoma ceylanicum infective third-stage larvae are activated by co-culture with HT-29-MTX intestinal epithelial cells.

BACKGROUND: Human hookworm larvae arrest development until they enter an appropriate host. This makes it difficult to access the larvae for studying larval development or host-parasite interactions. While there are in vivo and in vitro animal models of human hookworm infection, there is currently no human, in vitro model. While animal models have provided much insight into hookworm biology, there are limitations to how closely this can replicate human infection. Therefore, we have developed a human, in vitro model of the initial phase of hookworm infection using intestinal epithelial cell culture. RESULTS: Co-culture of the human hookworm Ancylostoma ceylanicum with the mucus-secreting, human intestinal epithelial cell line HT-29-MTX resulted in activation of infective third-stage larvae, as measured by resumption of feeding. Larvae were maximally activated by direct contact with fully differentiated HT-29-MTX intestinal epithelial cells. HT-29-MTX cells treated with A. ceylanicum larvae showed differential gene expression of several immunity-related genes. CONCLUSIONS: Co-culture with HT-29-MTX can be used to activate A. ceylanicum larvae. This provides an opportunity to study the interaction of activated larvae with the human intestinal epithelium.

Gene expression profiles associated with the transition to parasitism in Ancylostoma caninum larvae.

Ancylostoma caninum is a common canine parasite responsible for anemia and death in infected dogs. Gene expression profiling was used to investigate molecular differences between two different forms of the third larval stage (L3s): infective free-living larvae and in vitro serum-stimulated larvae that mimic the initial stages of parasitism of a host. We developed an A. caninum cDNA microarray consisting of 4191 EST clones, and used it to identify a set of 113 genes that are differentially regulated between infective and parasitic larval stages. Real-time RT-PCR was used to confirm the expression differences of a subset of the genes. Of the genes repressed upon serum stimulation, seven encode members of the 'Ancylostoma secreted protein' ASP family, while another transcript encoding a 24 kDa excretory protein with similarity to ASP was up-regulated in serum-stimulated L3s. This suggests that different members of a protein family that has important implications for the hookworm's parasitic lifestyle are regulated in a complementary manner in response to serum stimulation. Comparison of two strains of A. caninum from North Carolina and Maryland only identified a single gene, one of the members of the ASP family, that was differentially repressed upon serum stimulation.

Cloning and characterisation of an aspartyl protease inhibitor (API-1) from Ancylostoma hookworms.

Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878 bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme-inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine.

Giardia intestinalis and other zoonotic parasites: prevalence in adult dogs from the southern part of Mexico City.

The protozoan Giardia intestinalis is a mammalian-infecting parasite. It produces diarrhoea and malabsorption in its hosts. There is growing evidence that dogs could be reservoirs and play an important role in transmission. In Mexico, there are few data on the frequency of G. intestinalis. Therefore, we studied the small intestine of stray dogs, euthanazed at the "Culhuacan" Control Canine Centre, towards the end of 1997 and during the summer of 1998. We microscopically analysed intestinal contents and mucus samples taken every 3cm. During the cold season (winter), parasites were not found in 38/100 dogs, in contrast to 8/100 through the warm season. We found that 42/100 in winter and 51/100 in summer harboured G. intestinalis. To our knowledge, these G. intestinalis frequencies are the highest found in adult dogs worldwide. The results showed a rise in Ancylostoma spp. from 23/100 to 67/100 during the cold and warm seasons. Toxocara canis frequencies varied between 12/100 and 18/100, respectively. The data suggest that the probability of infection is higher during the hottest months compared to the coldest months of the year. Both puppies and adult dogs are highly infected. Dogs are reservoirs for zoonotic parasites; for this reason, it is imperative for humans to avoid fecal contamination in streets, public gardens and parks.

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.