Ancylostoma duodenale: maintenance through six generations in helminth-native pups.

An Indian strain of Ancylostoma duodenale with a known propensity for arrested development in man was passaged serially in 2-month-old, helminth-naive, male beagles. The sixth passage was initiated about 500 days after laboratory maintenance began. Experimental animals were infected by stomach tube with 1500 larvae each. These infections became patent in 22–31 days. In a line passaged in dogs without the use of a corticosteroid (prednisolone), adaptation, as judged by fecal egg count, increased through the first three generations and then declined sharply. This line appears destined for extinction. In another line passaged in prednisolone-treated pups, the strain's original viability is being maintained. Infections were long lived, frequently surviving for 90 days or more. Maximum egg output in terms of eggs per gram of feces (EPG) was highly variable between generations and between individuals within generations, but commonly exceeded 1500 EPG. In pups necropsied between 44 and 55 days, harboring fifth- and sixth-generation infections, adult worm burdens ranged from 47–146 and 34–128 in the nonprednisolone and prednisolone lines, respectively. Arrested larvae have been found in the same pups and apparently contribute to a turnover in the population of adult worms. Although A. duodenale has yet to be fully adapted to dogs in the sense that indefinite survival without prednisolone is assured, the present system yields a variety of life history stages in quantities suitable for many experimental purposes. Thus it provides a utilitarian laboratory model for the investigation of one of man's major hookworms.     

Molecular Detection of Ancylostoma duodenale,Ancylostoma ceylanicum,and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.     

Direct transfer of adult hookworms (Ancylostoma duodenale) from dog to child for therapeutic purposes

To induce chronic and continuous intestinal blood loss as therapy for congenital polycythemia in a 3 year old child, adult Ancylostoma duodenale were transferred directly from a dog to the patient via a nasoduodenal tube. By transferring adult worms, larval migration via the skin, blood and lungs--with possible attendant undesired side effects--was avoided. Furthermore, by eliminating larval migration with associated intimate tissue contact, immunogenicity was presumably reduced, and a known number of adult worms could be delivered directly to the final predilection site, the small intestine. An eosinophilic reaction of up to 23,000 cells/mm3 was observed, which may have adversely affected attempted superinfection. The relatively small numbers of parasites given on three separate occasions did not result in blood loss to a degree sufficient to eliminate the need for other forms of therapy.     

The mitochondrial genomes of the human hookworms, Ancylostoma duodenale and Necator americanus (Nematoda: Secernentea).

The complete mitochondrial genome sequences were determined for two species of human hookworms, Ancylostoma duodenale (13,721 bp) and Necator americanus (13,604 bp). The circular hookworm genomes are amongst the smallest reported to date for any metazoan organism. Their relatively small size relates mainly to a reduced length in the AT-rich region. Both hookworm genomes encode 12 protein, two ribosomal RNA and 22 transfer RNA genes, but lack the ATP synthetase subunit 8 gene, which is consistent with three other species of Secernentea studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T, but low in G and C. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. For both hookworm species, genes were arranged in the same order as for Caenorhabditis elegans, except for the presence of a non-coding region between genes nad3 and nad5. In A. duodenale, this non-coding region is predicted to form a stem-and-loop structure which is not present in N. americanus. The mitochondrial genome structure for both hookworms differs from Ascaris suum only in the location of the AT-rich region, whereas there are substantial differences when compared with Onchocerca volvulus, including four gene or gene-block translocations and the positions of some transfer RNA genes and the AT-rich region. Based on genome organisation and amino acid sequence identity, A. duodenale and N. americanus were more closely related to C. elegans than to A. suum or O. volvulus (all secernentean nematodes), consistent with a previous phylogenetic study using ribosomal DNA sequence data. Determination of the complete mitochondrial genome sequences for two human hookworms (the first members of the order Strongylida ever sequenced) provides a foundation for studying the systematics, population genetics and ecology of these and other nematodes of socio-economic importance.     

Necator americanus and Ancylostoma duodenale: life history parameters and epidemiological implications of two sympatric hookworms of humans

The life history data available for two species of hookworm that commonly infect humans, Necator americanus and Ancylostoma duodenale, were compiled. The data were then analyzed in the light of two ecological theories: r and K selection and the constraints of body size on reproduction. Field and laboratory experiments have shown that N. americanus is smaller than A. duodenale and produces fewer but larger eggs. The former is less virulent but the latter is hardier during its free-living stages and can infect orally as well as via the skin. Such species differences reflect the greater opportunism of A. duodenale and the greater degree of accommodation to the human host of N. americanus. A. duodenale optimizes the probability of finding a host and invading it, while N. americanus optimizes the chance of survival once within the host. On the basis of population density, longevity, and fecundity, N. americanus is the more K selected of the two species. However, the theory of r and K selection alone is insufficient to explain the different adaptations of closely related and sympatric parasites with complex life histories. N. americanus does not show greater competitive ability in its freeliving stage, and its mortality is not demonstrably more dependent on density than A. duodenale, as would be predicted by r and K selection theory as presently applied. Epidemiological differences between the two hookworm species are related to such life history parameters as lifespan, fecundity, age-specific survivorship, and density relationships.     

Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children

Background

Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi.

Methodology and Findings

A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16–5.33) and 9.04 (95%CI 2.52–32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18–11.20); 16.98 (95%CI 3.88–74.35) and 44.91 (95%CI 5.23–385.77) for low, moderate and high load respectively).

Conclusions/Significance

This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings.     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.