The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
D I Pritchard - One of the best experts on this subject based on the ideXlab platform.
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the physicochemical fingerprint of Necator Americanus
PLOS Neglected Tropical Diseases, 2017Co-Authors: Veeren M Chauhan, David J Scurr, Thomas Christie, Gary Telford, Jonathan W Aylott, D I PritchardAbstract:Necator Americanus, a haematophagous hookworm parasite, infects ~10% of the world’s population and is considered to be a significant public health risk. Its lifecycle has distinct stages, permitting its successful transit from the skin via the lungs (L3) to the intestinal tract (L4 maturing to adult). It has been hypothesised that the L3 larval sheath, which is shed during percutaneous infection (exsheathment), diverts the immune system to allow successful infection and reinfection in endemic areas. However, the physicochemical properties of the L3 larval cuticle and sheath, which are in direct contact with the skin and its immune defences, are unknown. In the present study, we controlled exsheathment, to characterise the sheath and underlying cuticle surfaces in situ, using atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). AFM revealed previously unseen surface area enhancing nano-annuli exclusive to the sheath surface and confirmed greater adhesion forces exist between cationic surfaces and the sheath, when compared to the emergent L3 cuticle. Furthermore, ToF-SIMS elucidated different chemistries between the surfaces of the cuticle and sheath which could be of biological significance. For example, the phosphatidylglycerol rich cuticle surface may support the onward migration of a lubricated infective stage, while the anionic and potentially immunologically active heparan sulphate rich deposited sheath could result in the diversion of immune defences to an inanimate antigenic nidus. We propose that our initial studies into the surface analysis of this hookworm provides a timely insight into the physicochemical properties of a globally important human pathogen at its infective stage and anticipate that the development and application of this analytical methodology will support translation of these findings into a biological context.
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hookworm Necator Americanus larval enzymes disrupt human vascular endothelium
American Journal of Tropical Medicine and Hygiene, 2010Co-Authors: Nahed Souadkia, Alan Brown, Lopa Leach, D I PritchardAbstract:Knowledge of the molecular mechanisms used by Necator Americanus larvae to penetrate the human skin and the vasculature would aid the development of effective vaccines against this important pathogen. In this work, the impact of N. Americanus exsheathing fluid (EF) and excretory/secretory products (ES) on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Cellular responses were assessed by investigating molecular changes at cell–cell junctions and by determining levels of secreted IL-6, IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. It would appear that a repertoire of larval proteases caused a dose-related increase in endothelial permeability as characterized by a decrease in monolayer resistance with increased permeation of tracer-albumin. These barrier changes were associated with disruption of junctional vascular endothelial cadherin (VE-cadherin) and F-actin and an increase in endothelial secretion of IL-6 and IL-8. Our data suggest that larval proteases play an important role in negotiating the endothelium.
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dose ranging study for trials of therapeutic infection with Necator Americanus in humans
American Journal of Tropical Medicine and Hygiene, 2006Co-Authors: Kevin Mortimer, D I Pritchard, Alan Brown, Johanna Feary, Chris Jagger, Sarah Lewis, Marilyn Antoniak, John BrittonAbstract:Epidemiological studies suggest that a hookworm infection producing 50 eggs/gram of feces may protect against asthma. We conducted a dose-ranging study to identify the dose of hookworm larvae necessary to achieve 50 eggs/gram of feces for therapeutic trials of asthma. Ten healthy subjects without asthma or airway hyperresponsiveness to inhaled methacholine received 10, 25, 50, or 100 Necator Americanus larvae administered double blind to an area of skin on the arm. Subjects were seen weekly for 12 weeks and were then treated with mebendazole. Skin itching at the entry site and gastrointestinal symptoms were common at higher doses. Lung function did not change. Levels of blood eosinophils and IgE increased transiently, and levels of IgG increased progressively. All doses resulted in at least 50 eggs/gram of feces in the eight subjects who completed the study. Infection with 10 N. Americanus larvae is well tolerated, elicits a modest host eosinophil response, and is potentially suitable for use in preliminary clinical therapeutic trials.
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a proof of concept study establishing Necator Americanus in crohn s patients and reservoir donors
Gut, 2006Co-Authors: John Croese, D I Pritchard, J Masson, Sharon E. Cooke, Wayne Melrose, J Oneil, Rick SpeareAbstract:The emergence of autoimmunity, including Crohn’s disease (CD) where the immune relationship with commensal bacteria is corrupted, has been linked to hygiene.1,2 A gradual decline in endoparasites is but one argument that might explain this phenomenon.3 Weinstock and colleagues have successfully tested the pig whipworm, Trichuris suis , in patients with inflammatory bowel disease (IBD).4,5 However, repeated inoculation was required and concern has been raised that aberrant migration could occur.6 The haematophagous hookworm, Necator Americanus (NA), is proposed as an alternative. We have tested if CD patients tolerate hookworm infection, and the practical issues associated with establishing reservoir donors (RDs). Over 700 million people remain infected with hookworms. Infective larvae (L3i) are acquired through skin contact with contaminated soil.7 Auto-reinfection, direct person to person infection, aberrant migration, and hypobiosis do not occur. Adult worms live …
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identification of a collagen binding protein from Necator Americanus by using a cdna expression phage display library
Journal of Parasitology, 2001Co-Authors: A Viaene, D I Pritchard, Annick Crab, M Meiring, Hans DeckmynAbstract:A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator Americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TG1 cells yielded 3 × 108 pG6A, 1.9 × 108 pG6B, and 1 × 108 pG6C transfectants for N. Americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. Americanus libraries. PCR analysis revealed v...
Jerzy M Behnke - One of the best experts on this subject based on the ideXlab platform.
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hookworm infections among migrant workers in malaysia molecular identification of Necator Americanus and ancylostoma duodenale
Acta Tropica, 2017Co-Authors: Norhidayu Sahimin, Jerzy M Behnke, Benacer Douadi, Mohd Khairul Nizam Mohd Khalid, Johnjames Wilson, Siti Nursheena Mohd ZainAbstract:Ongoing urbanisation of the working population as well as cross-border migration of workers particularly into large cities has contributed to the development and growth of urban slums. These deprived areas are conducive for the transmission of intestinal pathogens including hookworm. The aim of this study was to determine both the prevalence and species identity of hookworm infections among the migrant worker community in Malaysia. A total of 388 faecal samples were collected from migrant workers between September 2014 and August 2015, representing workers from five employment sectors: construction, manufacturing, agriculture and plantations, food services and domestic services. Faecal samples were examined by microscopy and positive samples were subjected to molecular analysis. A total of 51 samples (13.1%) were positive by microscopy for hookworm infections. A two-step PCR based method amplifying a fragment of the 28S rRNA-ITS2 region was used to identify infections by Necator Americanus and Ancylostoma spp. PCR products positive for Ancylostoma spp. were sequenced bidirectionally, and sequences analysed through BLAST and phylogenetic analysis. Samples containing Ancylostoma duodenale were further characterized by amplification and sequencing a fragment of cytochrome c oxidase subunit 1 (cox1) gene. PCR amplicons were successfully obtained from 42 (82.4%) of 51 samples, with 81.0% (34 of 42) identified as Necator Americanus, 16.7% (7 of 42) as Ancylostoma spp. and 2.4% (1 of 42) as mixed infections of both species. All eight Ancylostoma spp. were confirmed to be Ancylostoma duodenale and this is the first time A. duodenale was reported in Malaysia. Samples containing A. duodenale from Nepalese and Indonesian workers shared high-similarity and were distinct compared to sequences from other countries. This study highlights the prevalence of hookworm infections among migrant workers living in Malaysia. Our findings underscore the necessity of screening migrant workers for hookworm infections, particularly those working in food-related services and industries.
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in vitro studies on the relative sensitivity to ivermectin of Necator Americanus and ancylostoma ceylanicum
International Journal for Parasitology, 1995Co-Authors: J C Richards, Jerzy M Behnke, Ian R DuceAbstract:Abstract Experiments were carried out to compare the sensitivity of Ancylostoma ceylanicum and Necator Americanus to ivermectin (IVM) and pyrantel in vitro . Loss of motility and inhibition of ingestion by IVM were compared and A. ceylanicum was found to be approximately 40–50 times more sensitive to IVM than N. Americanus . Both species showed a similar sensitivity to pyrantel. Uptake of [ 3 H]IVM across the cuticle was compared and shown to be unlikely to account for the differences in sensitivity to IVM between the two species.
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density dependent regulation of the growth of the hookworms Necator Americanus and ancylostoma ceylanicum
Parasitology, 1994Co-Authors: S M B Norozianamiri, Jerzy M BehnkeAbstract:Laboratory bred DSN hamsters were exposed to varying doses of infective larvae of Ancylostoma ceylanicum (orally) or Necator Americanus (percutaneously) and were autopsied at times which corresponded to a period immediately before cessation of growth of worms or soon afterwards. A total of 829 (404 male and 425 female) A. ceylanicum and 1582 (781 male and 801 female) N. Americanus were measured. At worm burdens of fewer than 100, the length of A. ceylanicum appeared to increase with infection intensity and no evidence was found that growth was retarded under crowded conditions. In an experiment comparing directly low (mean worm burden=22) and heavy infections (mean worm burden =180) significant negative associations between both weight and width, and worm burden were detected, but length again increased with form burden
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Resistance of the hookworms Ancylostoma ceylanicum and Necator Americanus to intestinal inflammatory responses induced by heterologous infection
International Journal for Parasitology, 1994Co-Authors: Jerzy M Behnke, Richard A Rose, Jennifer LittleAbstract:BEHNKE J. M., ROSE R. and LITTLE J. 1994. Resistance of the hookworms Ancylostoma ceylanicum and Necator Americanus to intestinal inflammatory responses induced by heterologous infection. International Journalfor Parasitology 24: 91-101. Experiments were carried out to ascertain whether the acute inflammatory phase of the intestinal response of hamsters to infection with Trichinella spiralis would adversely affect hookworms in concurrently infected animals. The survival and growth of hookworms were unaffected. However, the presence of hookworms reduced the establishment of T. spiralis, the initial growth of female worms and their fecundity. The expulsion of T. spiralis was also significantly slower in concurrently infected animals and there was significant depression of the serum IgG antibody response to muscle stage and adult worm antigens of T. spiralis in concurrently infected animals. These results are discussed in relation to the chronicity of human hookworm infections.
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sensitivity to ivermectin and pyrantel of ancylostoma ceylanicum and Necator Americanus
International Journal for Parasitology, 1993Co-Authors: Jerzy M Behnke, Richard A Rose, Paul GarsideAbstract:AbStrBCt-BEHNKE J.M., ROSE R. and GARSIDE P. 1993. Sensitivity to ivermectin and pyrantel of Ancylosioma ceylanicum and Necator Americanus. International Journal for Parasitology 23: 945-952. Experiments were carried out in the hamster to compare the relative susceptibility of Necator Americanus and Ancylostoma ceylunicum to treatment with ivermectin. A.ceylanicum was found to be 300 times more sensitive to the anthelmintic with a 50% effective dose (ED,,) of the order of lo-15pg kg-’ body weight whilst that for N.Americanus approximated to 3-5 mg kg-‘. Furthermore, whereas complete clearance of A.ceylunicum was observed with a dose of lOOfig kg- ‘, Namericunus was not totally removed after treatment with 25 mg kg-‘, the highest dose tested. Both parasites proved equally sensitive to pyrantel with an ED,, of 1-12 mg kg-’ for A.ceylanicum and 5-25 mg kg-’ for N.Americanus. Treatment with pyrantel at 1OOmg kg-’ completely eliminated worms of both species and doses of 25-50 mg kg-’ were > 90% effective. In addition to worm burdens, changes in host weight and PCV were also recorded and it was shown that both parameters could be used to evaluate the success/failure of treatment.
Sanjeev Kumar - One of the best experts on this subject based on the ideXlab platform.
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The response of adult and free-living stages of Necator Americanus, in vitro, to anthelmintics
Revista De Biologia Tropical, 2016Co-Authors: Sanjeev KumarAbstract:The in vitro response of adults (males and females) and free-living stages of Necator Americanus one of the human hookworms, to a wide variety of 20 broad and narrow spectrum anthelmintics was tested. Almost all the broad spectrum anthelmintics influenced males, females and free-living stages at different levels and showed good activity with EC 50 values varying from about 0.0002 and 0.0007 mg/l for pyrantel pamoate and tricofenol piperazine respectively to about 8.47 and 7.6 mg/l for morantel tartrate and amoscanate respectively. Certain drugs (emetine, praziquantel and suramin) exerted their effect either on male or female or free-living stages at 10.0 mg/l level. It is concluded that either sex or life-cycle stage alone may not be an effective criteria for screening of anthelmintics which have been employed at large; and females of nematodes (in particular those of N. Americanus ) should be taken into account for proposing EC 50 as they were found to require relatively highest EC 50 level in almost all the instances studied presently.
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New observations on proteases of the human hookworm Necator Americanus
Iubmb Life, 1993Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Two protease populations have been identified in the somatic products of adult Necator Americanus. One population (158, 138, 34 and 31 kDa) loses, whereas the other population (107, 74, 51 and 20 kDa) retains, its proteolytic activity after elution from substrate gels. The present study warns that hookworm proteases may oligomerise and/or lose their activity under the conditions necessary for their purification. We suggest that following assessment of protease activity on substrate gels, the protease of interest should be eluted from plain gels; their isolation from substrate gels may lead to a reduced or no yield.
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Secretion of metalloproteases by living infective larvae of Necator Americanus
Journal of Parasitology, 1992Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Fresh living third-stage larvae of Necator Americanus released a significant amount of label within 2 hr of their incubationonn 25I-labeled gelatin-coated polystyrene plastic plates. This protease activity was primarily susceptible to o-phenanthroline, which identifies the activity as predominantly metalloprotease. The third-stage (L3) larvae of Necator Americanus produce proteolytic secretions (Matthews, 1975) that are reputed to be associated with secretory granules present in the esophageal glands of the parasite (Smith, 1976). Matthews (1982) subsequently proposed a role for these proteolytic secretions in skin penetration, but little has been done since then to confirm these findings or to characterize and identify the type(s) of proteolytic enzymes present in these secretions. The present paper describes experiments that address these points and resurrects the suggestion (Thorson, 1956a, 1956b, 1956c) that proteolytic secretions of hookworms constitute a valid target for a host-protective immune response. Necator Americanus was maintained and passaged in hamsters (Sen and Seth, 1967; Behnke et al., 1986), and infective larvae (L3) were cultured from feces in Harada-Mori tubes (Harada and Mori, 1955). Feces collected from N. Americanus-infected hamsters were mixed with charcoal and fungizone (amphotericin B 250 ,g/ml, GIBCO, Grand Island, New York; 3% v/v approximately) and were made into a paste. The paste was then applied in the form of a thin layer on the upper half of 15 x 2.5-cm Whatman strips (folded along their lengths) and the strips placed in 15 x 2-cm glass tubes (2-4 strips per tube) containing 10 ml of distilled water. The tubes were sealed with parafilm and were incubated at 25 C for 10 days. The strips were taken out of the tubes and the larvae, which had by now moved down to the distilled water, were transferred to a measuring cylinder and were left overnight to settle. Labeling of gelatin with 1251 was performed using the water-insoluble chloroamide 1,3,4,6tetrachloro-3a,6a-diphenylglycoluril (lodo-gen, Pierce Chemical Co., Rockford, Illinois) as described by Fraker and Speck (1978). In brief, 100 ,gg of protein (gelatin or fibrinogen) in 200 ml of phosphate-buffered saline (PBS) was added per tube, followed by 10 gl of potassium iodide solution (11 gg/gl) and 5 megabequerel of 125I. The reaction was allowed to proceed for 10 min with gentle agitation of the tubes every 2 min. Separation of the labeled protein from the iodination reaction mixture, which includes still unreacted iodid , was accomplished by gel filtration using a Sephadex G-25 column. The radiolabeled mixture wa added and washed through with PBS, the fi st 10 fractions of 1.5 ml volume each being collected. Each fraction subsequently was analyzed for labeled protein by trichloroacetic acid (TCA) p ecipitation and the fractions containing the highest TCA-precipitable counts were retained. The fractions were dialyzed against PBS at 4 C for 24 hr, and TCA precipitations were performed once again. Protein radiolabeled in t is manner was stored at 4 C and used within
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Skin penetration by ensheathed third-stage infective larvae of Necator Americanus, and the host's immune response to larval antigens.
International Journal for Parasitology, 1992Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Abstract Kumar S. and Pritchard D. I. 1992. Skin penetration by ensheathed third-stage infective larvae of Necator Americanus , and the host's immune response to larval antigens. International Journal for Parasitology 22 : 573–579. In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator Americanus . The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator Americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.
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the partial characterization of proteases present in the excretory secretory products and exsheathing fluid of the infective l3 larva of Necator Americanus
International Journal for Parasitology, 1992Co-Authors: Sanjeev Kumar, D I PritchardAbstract:Abstract Kumar S. and Pritchard D. I. 1992. The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator Americanus . International Journal for Parasitology 22 : 563–572. Following the observation that live third-stage larvae (L3) could digest gelatin in vitro , gelatinolytic protease activity has been demonstrated at pH 8.5, in both exsheathing fluid (EF) and excretory/secretory (ES) products of infective L3 of Necator Americanus . EF resolved as a single band of proteolytic activity, with a mol. wt of 116 kDa, while L3 ES products exhibited multiple bands of proteolysis, at 219, 200, 195, 166, 137, 92, 72 and 62 kDa; weak bands were detectable at 92 and 72 kDa. The EF protease was characterized as cysteine, whereas ES apparently possessed one serine (195 kDa) and seven (219, 200, 166, 137, 92, 72 and 62 kDa) cysteine protease bands and a combination of metallo- and cysteine proteases of approximately the same mol. wts (62, 137 and 219 kDa). Though EF was not able to cleave immunoglobulins, ES was shown to cleave IgG, IgA and IgM, but not IgD or IgE. The activity appeared to be directed toward the Fc portion of the molecule, and was inhibited by PMSF, which is indicative of serine protease activity. The significance of the presence of such apparently diverse proteases in larval products is discussed.
David I. Pritchard - One of the best experts on this subject based on the ideXlab platform.
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A proof of concept study establishing Necator Americanus in Crohn’s patients and reservoir donors
Gut, 2006Co-Authors: John Croese, David I. Pritchard, J O’neil, J Masson, Sharon E. Cooke, Wayne Melrose, Rick SpeareAbstract:The emergence of autoimmunity, including Crohn’s disease (CD) where the immune relationship with commensal bacteria is corrupted, has been linked to hygiene.1,2 A gradual decline in endoparasites is but one argument that might explain this phenomenon.3 Weinstock and colleagues have successfully tested the pig whipworm, Trichuris suis , in patients with inflammatory bowel disease (IBD).4,5 However, repeated inoculation was required and concern has been raised that aberrant migration could occur.6 The haematophagous hookworm, Necator Americanus (NA), is proposed as an alternative. We have tested if CD patients tolerate hookworm infection, and the practical issues associated with establishing reservoir donors (RDs). Over 700 million people remain infected with hookworms. Infective larvae (L3i) are acquired through skin contact with contaminated soil.7 Auto-reinfection, direct person to person infection, aberrant migration, and hypobiosis do not occur. Adult worms live …
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New observations on proteases of the human hookworm Necator Americanus
Iubmb Life, 1993Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Two protease populations have been identified in the somatic products of adult Necator Americanus. One population (158, 138, 34 and 31 kDa) loses, whereas the other population (107, 74, 51 and 20 kDa) retains, its proteolytic activity after elution from substrate gels. The present study warns that hookworm proteases may oligomerise and/or lose their activity under the conditions necessary for their purification. We suggest that following assessment of protease activity on substrate gels, the protease of interest should be eluted from plain gels; their isolation from substrate gels may lead to a reduced or no yield.
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Secretion of metalloproteases by living infective larvae of Necator Americanus
Journal of Parasitology, 1992Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Fresh living third-stage larvae of Necator Americanus released a significant amount of label within 2 hr of their incubationonn 25I-labeled gelatin-coated polystyrene plastic plates. This protease activity was primarily susceptible to o-phenanthroline, which identifies the activity as predominantly metalloprotease. The third-stage (L3) larvae of Necator Americanus produce proteolytic secretions (Matthews, 1975) that are reputed to be associated with secretory granules present in the esophageal glands of the parasite (Smith, 1976). Matthews (1982) subsequently proposed a role for these proteolytic secretions in skin penetration, but little has been done since then to confirm these findings or to characterize and identify the type(s) of proteolytic enzymes present in these secretions. The present paper describes experiments that address these points and resurrects the suggestion (Thorson, 1956a, 1956b, 1956c) that proteolytic secretions of hookworms constitute a valid target for a host-protective immune response. Necator Americanus was maintained and passaged in hamsters (Sen and Seth, 1967; Behnke et al., 1986), and infective larvae (L3) were cultured from feces in Harada-Mori tubes (Harada and Mori, 1955). Feces collected from N. Americanus-infected hamsters were mixed with charcoal and fungizone (amphotericin B 250 ,g/ml, GIBCO, Grand Island, New York; 3% v/v approximately) and were made into a paste. The paste was then applied in the form of a thin layer on the upper half of 15 x 2.5-cm Whatman strips (folded along their lengths) and the strips placed in 15 x 2-cm glass tubes (2-4 strips per tube) containing 10 ml of distilled water. The tubes were sealed with parafilm and were incubated at 25 C for 10 days. The strips were taken out of the tubes and the larvae, which had by now moved down to the distilled water, were transferred to a measuring cylinder and were left overnight to settle. Labeling of gelatin with 1251 was performed using the water-insoluble chloroamide 1,3,4,6tetrachloro-3a,6a-diphenylglycoluril (lodo-gen, Pierce Chemical Co., Rockford, Illinois) as described by Fraker and Speck (1978). In brief, 100 ,gg of protein (gelatin or fibrinogen) in 200 ml of phosphate-buffered saline (PBS) was added per tube, followed by 10 gl of potassium iodide solution (11 gg/gl) and 5 megabequerel of 125I. The reaction was allowed to proceed for 10 min with gentle agitation of the tubes every 2 min. Separation of the labeled protein from the iodination reaction mixture, which includes still unreacted iodid , was accomplished by gel filtration using a Sephadex G-25 column. The radiolabeled mixture wa added and washed through with PBS, the fi st 10 fractions of 1.5 ml volume each being collected. Each fraction subsequently was analyzed for labeled protein by trichloroacetic acid (TCA) p ecipitation and the fractions containing the highest TCA-precipitable counts were retained. The fractions were dialyzed against PBS at 4 C for 24 hr, and TCA precipitations were performed once again. Protein radiolabeled in t is manner was stored at 4 C and used within
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Skin penetration by ensheathed third-stage infective larvae of Necator Americanus, and the host's immune response to larval antigens.
International Journal for Parasitology, 1992Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Abstract Kumar S. and Pritchard D. I. 1992. Skin penetration by ensheathed third-stage infective larvae of Necator Americanus , and the host's immune response to larval antigens. International Journal for Parasitology 22 : 573–579. In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator Americanus . The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator Americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.
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The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator Americanus
International Journal for Parasitology, 1992Co-Authors: Sanjeev Kumar, David I. PritchardAbstract:Abstract Kumar S. and Pritchard D. I. 1992. The partial characterization of proteases present in the excretory/secretory products and exsheathing fluid of the infective (L3) larva of Necator Americanus . International Journal for Parasitology 22 : 563–572. Following the observation that live third-stage larvae (L3) could digest gelatin in vitro , gelatinolytic protease activity has been demonstrated at pH 8.5, in both exsheathing fluid (EF) and excretory/secretory (ES) products of infective L3 of Necator Americanus . EF resolved as a single band of proteolytic activity, with a mol. wt of 116 kDa, while L3 ES products exhibited multiple bands of proteolysis, at 219, 200, 195, 166, 137, 92, 72 and 62 kDa; weak bands were detectable at 92 and 72 kDa. The EF protease was characterized as cysteine, whereas ES apparently possessed one serine (195 kDa) and seven (219, 200, 166, 137, 92, 72 and 62 kDa) cysteine protease bands and a combination of metallo- and cysteine proteases of approximately the same mol. wts (62, 137 and 219 kDa). Though EF was not able to cleave immunoglobulins, ES was shown to cleave IgG, IgA and IgM, but not IgD or IgE. The activity appeared to be directed toward the Fc portion of the molecule, and was inhibited by PMSF, which is indicative of serine protease activity. The significance of the presence of such apparently diverse proteases in larval products is discussed.
A M Polderman - One of the best experts on this subject based on the ideXlab platform.
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aflp fingerprinting for the analysis of genetic diversity within Necator Americanus
Molecular and Cellular Probes, 2006Co-Authors: Johanna M De Gruijter, A M Polderman, Lenie Dijkshoorn, Helen Roberts, Juventus B Ziem, Chhatra B Kunwar, Robin B GasserAbstract:Abstract In the present study, we utilised the method of AFLP to screen for genetic variation within and among individuals of the blood-feeding human hookworm Necator Americanus (Nematoda) from Africa, Asia and South America. A total of 45 adult worms (i.e. 20 from Ghana, 16 from Colombia and 9 from Nepal) were subjected to analysis using the restriction enzyme/primer combination Hind III+AG/ Bgl II+AC. Cluster analysis divided N. Americanus into multiple, genetically distinct groups, consistent with previous findings using ribosomal and mitochondrial DNA data sets. The results demonstrated the usefulness of AFLP fingerprinting for establishing genetic variation within N. Americanus and reinforce its applicability to other parasitic helminths of human and/or veterinary health importance.
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the role of pigs as transport hosts of the human helminths oesophagostomum bifurcum and Necator Americanus
Acta Tropica, 2000Co-Authors: N R Steenhard, P Nansen, P A Storey, L Yelifari, A M PoldermanAbstract:Abstract We conducted a study in an endemic area of both Oesophagostomum bifurcum and Necator Americanus in northern Ghana to examine the possibility of pigs acting as transport hosts for these two human helminth species, due to the commonly observed coprophagic habits of pigs. Under controlled conditions four parasite-free pigs consumed fresh faeces from people heavily infected with both helminths, and faeces were subsequently collected from the rectum of the pigs from 5 to 50 h post-feeding. Four to five per cent of the O. bifurcum and N. Americanus eggs fed to the pigs were viable and retrieved as third-stage larvae after coproculture of the pigs’ faeces. We discuss the possible impact of the coprophagic habits of pigs as potential parasite transport hosts during different seasons in this area of West Africa.
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Necator Americanus nematoda ancylostomatidae from africa and malaysia have different its 2 rdna sequences
International Journal for Parasitology, 1998Co-Authors: A Romstad, A M Polderman, Robin B Gasser, P Nansen, Neil B ChiltonAbstract:Abstract The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator Americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. Americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. Americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. Americanus , or that N. Americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.
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characterization of oesophagostomum bifurcum and Necator Americanus by pcr rflp of rdna
Journal of Parasitology, 1997Co-Authors: A Romstad, A M Polderman, Robin B Gasser, P Nansen, J R Monti, Neil B ChiltonAbstract:Esophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana, where Necator Americanus (human hookworm) also exists at high prevalence. Yet, very little is known about the transmission patterns of O. bifurcum, which is in part due to the difficulties in diagnosis and in differentiating some life-cycle stages of O. bifurcum from N. Americanus using morphological features. As a first step toward developing a molecular-diagnostic assay, it was evaluated whether ribosomal (r)DNA could provide genetic markers for the identification of O. bifurcum and N. Americanus to species. Internal transcribed spacer rDNA (plus flanking and intervening sequences) was analyzed by polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) using several restriction endonucleases. The analysis showed that there was no detectable intraspecific difference in the size of the PCR products among multiple samples, that there was a consistent size difference in the products (of 110 bp or 350 bp, depending on region amplified) between the species, and that there was no significant variation in restriction patterns within each species. These results indicate that the rDNA spanning the internal transcribed spacers provides useful genetic markers for the identification of O. bifurcum and N. Americanus to species, which has important implications for developing PCR-based tools to study the epidemiology and population biology of O. bifurcum.
