Studies on Necator americanus, Thai-Strain. The prevalence, distribution and faecal egg count of Necator Americanus in the human intestine.
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The anti-haemostatic strategies of the human hookworm Necator americanus.
The human hookworm Necator americanus appears to have evolved a number of complementary strategies to overcome the host's haemostatic processes. These include the inhibition of blood coagulation, platelet aggregation and mediator release, and the secretion of fibrinogenolytic enzymes. These strategies presumably allow the parasite to establish the chronic infections so often documented in human populations.
Animals↗
The molecular forms of acetylcholinesterase from Necator americanus (Nematoda), a hookworm parasite of the human intestine.
Necator americanus (Nematoda: Strongyloidea), a human hookworm parasite, is known to release considerable amounts of acetylcholinesterase (AChE) [Pritchard, D. I., Leggett K. V., Rogan, M. T., McKean, P. G. & Brown, A. (1991) Necator americanus secretory acetylcholinesterase and its purification from excretory/secretory products by affinity chromatography, Parasite Immunol. 13, 187-199]. The present study deals with AChE activity recovered in sequential somatic extracts, and excretory/secretory products, of the adult stage of the parasite. 97% of AChE was extractable in low-salt and high-salt detergent-free buffers, and only 3% was solubilised by a further extraction in the presence of Triton X-100. AChE in all three extracts was affected by the AChE inhibitors eserine, bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide and edrophonium chloride, but was resistant to the effects of tetramonoisopropylpyrophosphortetramide, a butyrylcholinesterase inhibitor. Sucrose density centrifugation revealed that AChE in all somatic extracts (low-salt, high-salt and detergent) resolved almost exclusively as a single peak between 6.9-7.5 S, while excretory/secretory products resolved at 8.2 S. These values are all compatible with dimers of catalytic subunits and no evidence was found for the presence of higher oligomers such as asymmetric forms. The only sample to show a shift in sedimentation following the inclusion of detergent (Triton X-100, Brij 96) in the gradient was a component of the detergent-soluble extract, indicating the existence of a minor amphiphilic form. In low-salt-soluble and high-salt-soluble extracts, AChE was solubilised as a hydrophilic globular form, probably a dimeric G2. The analysis of diisopropylfluorophosphate-labelled extracts by SDS/PAGE, and unlabelled extracts by immunoblotting using a polyvalent antiserum to N. americanus AChE, indicated that the AChE isolated in each extract was biochemically and immunologically similar. The banding patterns obtained were comparable to that seen when purified AChE was analysed by SDS/PAGE and immunoblotted. This suggests that the basic catalytic subunit has a mass of 66-70 kDa with the active site being located in a 30-kDa domain. All experimental data indicate the existence of only one AChE class in Necator homologous to AChE of class B from Caenorhabditis elegans. The solubility characteristics and globular nature of this hookworm AChE suggest that its major function is as an excretory or secretory product. This again raises the question of the true biological function of this 'non-cholinergenic' nematode secretion.
Acetylcholinesterase↗
Further Observations On The Prevalence Of Ancylostoma Duodenale And Necator Americanus In The Taegu Area.
In order to determine the prevalence of Necator americanus, 182 fecal samples were collected from school children in the Taegu vicinity. These sample were subsequently cultured by the Harada-Mori technique. Necator americanus was differentiated from Ancylostoma duodenale using the bases of morphological characteristics of filariform larvae. Necator americanus was not found in the vicinity of Taegu.
Journal Article↗
Is Necator americanus approaching a mutualistic symbiotic relationship with humans?
The hookworm Necator americanus establishes infections of impressive longevity in the immunologically hostile environment of its human host. In the process, it promotes pronounced T-helper 2 (Th2) cell activity, which in turn seemingly affords the host at least a degree of protection. Given the relatively asymptomatic nature of infection, we argue here that Necator americanus might be approaching a mutualistic symbiotic relationship with humans. In our view, infection is controlled by the immune system while being supported by a subtle immune-evasion strategy that is tolerated and possibly beneficial to the host in certain immunological circumstances, such as in counterbalancing potentially damaging Th1 responses.
Amino Acid Sequence↗
Construction and analysis of cDNA library of Necator americanus third stage larvae.
OBJECTIVE: To obtain the genetic information on Necator americanus and to search for the purpose genes. METHODS: mRNA was isolated from the third stage larvae of Necator americanus maintained in hamsters. Double strand cDNA was synthesized and ligated to lambda ZAPII vector to construct the cDNA library. Expressed sequence tages (ESTs) were obtained by single pass sequencing of randomly isolated cDNA clones from the established library. RESULTS: A cDNA library of N. americanus was successfully constructed with high recombinant efficiency. The titer of unamplified library was 1 x 10(7). The insert size was about 750-3,000 bp. Of 11 ESTs obtained from the library, 7 have a significant homology with certain functional genes. CONCLUSION: A high quality and high representative cDNA library of N. americanus was constructed at the first time and some functional genes were identified from the library by ESTs.
Animals↗
Skin penetration by Necator americanus larvae.
Skin penetration by Necator americanus larvae has been investigated in vitro. Larvae were able to penetrate completely human skin from both the epidermal and dermal directions; their passage through the epidermis was marked by cellular destruction. Removal of chloroform soluble skin lipids affected both the percentage of larvae invading and the percentage exsheathing. The larvae released an enzyme at about 37 degrees C, which showed peak activity against azocoll at 37 degrees C and pH 8. It is suggested that initial invasion is a mechanical process and that the enzyme is functional in passage through the germinal layers of the epidermis.
Animals↗
[Sequencing of cytochrome C oxidase 1 gene of Ancylostoma duodenale and Necator americanus].
AIM: To identify the genetic diversity between Ancylostoma duodenale and Necator americanus. METHODS: Mitochondrial cytochrome C oxidase subunit 1 (CO1) gene was amplified from genomic DNA of human hookworms collected from infected patients in Hejiang County, Sichuan Province, and the purified PCR products were directly sequenced by using Licor auto-sequencer. RESULTS: The PCR products were about 700 bp. Alignment of CO1 gene fragment sequences showed 89.7% similarity between Ancylostoma duodenale and Necator americanus, but still certain nucleotide variations (10.3%) existed. CONCLUSION: CO1 gene sequence can be used as a marker to identify the two species of human hookworms.
Amino Acid Sequence↗
Genes and genomes of Necator americanus and related hookworms.
The human hookworms (Necator americanus and Ancylostoma duodenale) infect over one billion people. The phylogenetic relationships of the human hookworms suggest independent acquisition of the human host. The hookworms probably have a haploid chromosome number n = 6, and an XX-XO sex determination mechanism is likely to be used. Genetic and molecular research on hookworms is in its infancy, but several important genes and gene products have already been identified. Of note are cathepsin genes, a family of secreted proteins known as Ancylostoma activation-associated proteins and a family of anticoagulants. The inception of an expressed sequence tag program on the human hookworm, N. americanus, promises to yield many new genes with novel functions in the biology of these important parasites.
Animals↗
Cultivation of the third-stage larvae of Necator americanus in vitro.
When filariform larvae of Necator americanus, enclosed within the second sheath, were cultured in various semi-defined media, the best larval development was achieved in Waymouth's MB 752/1 with 20% foetal calf serum (FCS). In this medium, most of the filariform larvae cast off the second sheath and developed into third-stage larvae with provisional buccal capsules and some larvae developed further; 26% were fourth stage after 49 days of culture. Of several supplements tested, only FCS supported such development and no further development occurred if the medium was further supplemented with human red blood cells, liver extract, intestinal extract or tryptose phosphate broth. Larval development stopped at or before early third stage in Eagle's Basal Medium, F-12, NCTC 135 and L-15, even when these media were supplemented with FCS.
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An initial characterization of the proteolytic enzymes secreted by the adult stage of the human hookworm Necator americanus.
The proteolytic activities present in adult Necator americanus excretory-secretory products have been assessed using biologically relevant, naturally occurring substrates (haemoglobin and fibrinogen) and a number of synthetic fluorogenic and chromogenic substrates. One broad peak of activity was observed against haemoglobin in the pH range 5 to 7, with maximum activity at pH 6.6, while fibrinogenolytic activity was shown to be greater at pH 3.5. Inhibition studies against haemoglobin, fibrinogen and synthetic substrates using a battery of appropriate protease inhibitors indicated the presence of a mixture of aspartyl, cysteinyl and serine proteases. Metal ion (Ca2+, Zn2+ and Fe2+) stimulation was demonstrated, with stimulation by Zn2+ being the most marked. These results are discussed in the context of recent developments in the field of parasite proteolytic enzymes, where they have been suggested as targets for immuno- and chemotherapy.
Amino Acid Sequence↗
The identification of a species-specific antigen from Necator americanus.
A 17 kD protein of Necator americanus was isolated by SDS-PAGE and used to raise monospecific antisera in rabbits. ELISA and Western blotting against a range of parasite extracts demonstrated the species specificity of this protein. It is expressed at all stages of the life-cycle, appears to be accumulated through the larval stages to adulthood, and can be localized in the oesophageal glands and cuticle of the adult parasite. The possible nature and diagnostic potential of this protein is discussed.
Animals↗
The growth and migration of Necator americanus following infection of neonatal hamsters.
Necator americanus was studied in neonatally infected hamsters in order to determine precisely the growth and migration of the parasite in this laboratory host. Most larvae stayed at the skin infection site for at least 48 hours following administration of larvae and the movement to the lungs commenced on day 3. There was no significant growth at the skin site or during the first two days in the lungs. 98% of the larvae were recovered from the lungs by day 6 and showed signs of some growth and development. Moulting larvae were seen in the lungs on days 7 and 8, but intestinal worms, which were first detected on day 7, were all L4 larvae. These worms were significantly longer than the lung stages and henceforth grew rapidly. Over 80% of the worms were recovered from the intestine on day 9, only small numbers of larvae persisting in the lungs until day 12. Moulting worms were observed in the intestine on days 17 to 21, after which growth continued and did not slow until about the fifth week. Small quantities of eggs were occasionally detected as early as day 34 and continuous egg production commenced in the seventh week of infection reaching a peak by about the 10th week.
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Secretion of metalloproteases by living infective larvae of Necator americanus.
Fresh living third-stage larvae of Necator americanus released a significant amount of label within 2 hr of their incubation on 125I-labeled gelatin-coated polystyrene plastic plates. This protease activity was primarily susceptible to o-phenanthroline, which identifies the activity as predominantly metalloprotease.
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Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variation.
Necator americanus is a blood-sucking, intestinal nematode of major human health importance in many tropical and subtropical regions of the world. The aim of the present study was to compare the complete mitochondrial genome sequence from one N. americanus individual from Togo with another from China, in order to estimate the magnitude of genetic variability for different mitochondrial genes and non-coding regions. For the 12 protein genes, this comparison revealed sequence differences at both the nucleotide (3-7%) and amino acid (1-7%) levels. The most conserved of these was the nad4L gene, whereas the nad1 gene was least conserved at both the nucleotide and amino acid levels. Nucleotide differences were also detected in 14 of the 22 transfer RNAs (trns) (1-13%), the AT-rich region ( approximately 8%), non-coding regions (8-25%) and in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rrn) ( approximately 1%). Comparison of the rrnL sequences among multiple individual worms revealed nine unequivocal nucleotide differences between N. americanus from the two countries. Consistent with previous studies, these findings provide evidence for substantial genetic variation within N. americanus, which may have implications for the transmission and control of hookworm disease.
Amino Acid Sequence↗
Genetic structure of populations of the human hookworm, Necator americanus, in China.
Twenty-one to 58 individual Necator americanus were sampled from each of four villages in south-western China. Each nematode was sequenced for 588 bp of the mitochondrial cytochrome oxidase I gene. Allelic and nucleotide diversity varied two-fold among villages. Overall FST among populations was approximately 0.28, but this large value resulted from one low-diversity population that had a large genetic distance to the other three populations (F(ST) = 0.10 without that population). There was no correlation between geographical and genetic distance among sites. Thus, the genetic structure of this species in China may be characterized by variable effective sizes and uneven movement among sites. We discuss the implications of this genetic structure for vaccine development and the spread of drug resistance in human hookworms, and compare the genetic structure of hookworms with that of other nematodes.
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[Scanning electron microscopic observations on the copulatory spicules of the male Necator americanus and Ancylostoma duodenale].
Scanning electron microscopic observations were made on the morphological structures of the two copulatory spicules of the male Necator americanus and Ancylostoma duodenale. In both species, one of the two copulatory spicules was in the shape of a concave groove and the other, in the shape of an oblate tube. Owing to the difference in the concavity of the groove shaped copulatory spicule between the two species as shown by the cross sections, Necator americanus usually appear to have only one copulatory spicule whereas Ancylostoma duodenale usually show two separate copulatory spicules in appearance (Figs 1-10).
Ancylostoma↗
Antigen expression during development of the human hookworm, Necator americanus (Nematoda).
The accumulated and de novo synthesized antigens expressed by L3, L4 and adult Necator americanus, recognized by both the natural host, man, and the experimental host, the hamster, were identified by immunoblotting and immunoprecipitation analysis. Following infection of neonatal hamsters serum samples were taken on days 17, 35 and 117. Only serum taken 117 days after infection showed significant reactivity in immunoblotting experiments, recognizing adult epitopes of 30,000, 33,000, 48,000 and 69,000 mol. wt thereby suggesting that few accumulated antigens are shared between developmental stages. By contrast, immunoprecipitation analysis of metabolically labelled proteins suggested that L3 and in particular L4 larvae synthesize some antigens which comigrate with those synthesized and accumulated by adult worms. In addition, L4 larvae synthesize a 41,000 mol. wt excretory/secretory (ES) stage specific antigen. Parallel experiments using serum samples from infected humans, demonstrated that hamsters and man recognize many antigens of identical molecular weight. Notable in this respect are accumulated adult antigens of 30,000, 33,000, 48,000 and 69,000 and de novo synthesized antigens of 30,000, 33,000, 44,000, 46,000 and 69,000 mol. wt. Some individual human sera mainly recognized L3 antigens of 47,000-69,000 mol. wt in immunoblotting experiments whilst others simultaneously recognized adult epitopes. This differential recognition of developmental stages by individual human sera suggests that genetic or epidemiological factors are operative and warrants further study. Overall, these studies confirm the pronounced immunogenicity of Necator americanus in both man and an animal model and pave the way for analysis of the relevance of these antigens to field situations.
Animals↗