Cross-reactivity between Necator americanus and Schistosoma mansoni in mice.

, , and 1992. Cross-reactivity between Necator americanns and Schistosoma mansoni in mice. International Journal for Parasitology 22: 1143–1149. Poly-parasitism is common in endemic communities and reactivity of sera from hookworm-infected patients against schistosomular antigens has been reported. Protective cross-immunity between N. americanus and S. mansoni was investigated in NIH and BALB/c mice. Protective resistance to homologous challenge with both parasites was confirmed in this model, however, functional immunity to heterologous challenge was not demonstrated. Sera from animals which had received homologous challenge with N americanus and from hookworm-infected mice, which had previously been exposed to radiation-attenuated S. mansoni, exhibited an enhanced IgGAM response to infective stage N. americanus somatic antigens. The implications of these results with respect to serodiagnosis are discussed.     

The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus-Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30-46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development.     

X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection

Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages of the parasite, including a family of pathogenesis-related (PR) proteins known as the Ancylostoma-secreted proteins (ASPs). A novel crystal structure of Na-ASP-2, a PR-1 protein secreted by infective larvae of the human hookworm Necator americanus, has been solved to resolution limits of 1.68 A and to an R-factor of 17% using the recombinant protein expressed in and secreted by Pichia pastoris. The overall fold of Na-ASP-2 is a three-layer alphabetaalpha sandwich flanked by an N-terminal loop and a short, cysteine-rich C terminus. Our structure reveals a large central cavity that is flanked by His129 and Glu106, two residues that are well conserved in all parasitic nematode L3 ASPs. Na-ASP-2 has structural and charge similarities to chemokines, which suggests that Na-ASP-2 may be an extra-cellular ligand of an unknown receptor. Na-ASP-2 is a useful homology model for NIF, a natural antagonistic ligand of CR3 receptor. From these modeling studies, possible binding modes were predicted. In addition, this first structure of a PR-1 protein from parasitic helminths may shed light on the molecular basis of host-parasite interactions.     

Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5–10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.     

唐鱼养殖种群与广州附近4个野生种群的遗传关系

唐鱼(Tanichthys albonubes)是为数不多的几种原产中国的世界性观赏鱼类之一。自2003年以来, 多个唐鱼野生种群相继被发现, 其濒危状态和等级由野外灭绝降为极危。为研究唐鱼养殖种群与广州附近野生种群之间的遗传关系, 本文分析了唐鱼3个代表性养殖种群和4个野生种群, 共计186个样本的Cyt b基因、2个核基因(ENC1和RAG1)以及13个微卫星位点数据。基于K2P模型的遗传距离结果显示, 唐鱼野生种群间的遗传距离在0.005-0.015之间, 养殖种群间的遗传距离为0.001-0.009。系统发育分析表明, 唐鱼养殖种群包含4个单倍型谱系分支, 其中2个分别与广州附近2个野生种群聚在一起, 另外2个分别独立成支。单倍型网络亲缘关系分析显示, 清远种群只有1个单倍型且与芳村养殖种群共享, 芳村养殖种群拥有最多的单倍型。基于微卫星数据的STRUCTURE分析表明, 所有种群最佳分簇数为2, 清远种群与养殖种群聚为一簇, 良口和石门种群聚为另一簇。主成分分析结果显示, 养殖种群高度重叠并能与野生种群分开, 清远种群与养殖种群存在部分重叠。利用IMa3的基因流分析表明, 存在清远种群至芳村养殖种群的单向基因流。综合本文结果, 作者认为唐鱼养殖种群应起源于广州附近多个野生种群。清远种群来源于养殖种群中的芳村养殖种群。建议在未来唐鱼的保护策略中, 应禁止不规范的放流活动并且禁止将不同野生种群补充至养殖种群, 同时加强唐鱼养殖种群和野生种群的遗传资源管理和持续监测。     

Genetic structure of Lake Magadi tilapia populations

The control region of the mitochondrial DNA haplotype frequencies were significantly different among the two separate lagoon populations of Oreochromis alcalicus grahami in Lake Magadi and of O. a. alcalicus from lake Natron, and DNA fingerprint similarity indices were significantly higher for intra-population comparisons of the two Magadi lagoon populations and the Lake Natron population than the inter-population similarity indices among these populations. A modified F _st measure indicated population sub-division and the phylogeographic partitioning of the VNTR fragments observed were unique to specific populations further indicating substantial genetic differentiation. The lagoon populations within Lake Magadi demonstrated the same degree of genetic differentiation as either of these populations did to the outgroup (the Lake Natron population). There appears to be limited gene flow between Lake Magadi tilapia populations and this population structure has important implications for protecting locally adapted populations within this unique ecosystem.     

Effects of Pluronic F-68 on larval development of the hookworm,Necator americanus

Summary Eggs of the nematode,Necator americanus, were incubated at 28°C for up to 120h in medium (prepared from mouse faeces) supplemented with 0.4% (w/v) or 5% of either commercial grade or a silica-Amberlite purified fraction of the non-ionic surfactant, Pluronic F-68. Exposure to commercial grade or purified Pluronic at both concentrations delayed egg hatching. However, subsequent larval growth, as assessed by length of larva, buccal capsule, oesophagus and tail, was significantly (P<0.05) increased (maximally following exposure to 5.0% of the purified fraction). No further development occurred in larvae exposed to other Pluronic fractions tested after 48h, irrespective of concentration. These results suggest that Pluronic may be a valuable supplement in media used for the axenic culture of nematode larvae.     

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.     

Mitochondrial haplotypes in populations of the plant-infecting fungus Sclerotinia sclerotiorum: wide distribution in agriculture, local distribution in the wild

Analysis of mitochondrial DNA (mtDNA) haplotypes of Sclerotinia sclerotiorum points to a common origin of some genotypes from agricultural populations, especially when compared with two wild populations that are sharply distinguished from the agricultural sample and from each other. Five agricultural population samples from canola (Alberta, Canada and Norway), cabbage (North Carolina, USA), sunflower (Manitoba, Canada and Queensland, Australia) and two Norwegian populations from a wild plant, Ranunculus ficaria were compared. Haplotypes were determined by Southern hybridization of purified organelle DNA from S. sclerotiorum and Neurospora crassa to total genomic DNA of S. sclerotiorum. Each isolate had one haplotype. Haplotypes of S. sclerotiorum from R. ficaria were different between the two wild populations and also from all haplotypes observed in the agricultural populations. Among the wild isolates, DNA fingerprint, mtDNA haplotype and location in the sampling transect were all associated. Among the agricultural isolates, four haplotypes were observed in at least two agricultural populations and one haplotype was observed in all agricultural populations. In the Canadian canola sample some clones had one mtDNA haplotype, indicating association with DNA fingerprint, some clones had more than one haplotype, and some groups of clones shared haplotypes. Some of the haplotype diversity may be due to the presence of extra-chromosomal elements associated with the mitochondria of S. sclerotiorum.     

Genetic relationships among populations of wild resident, and wild and hatchery anadromous brook charr

To determine the genetic relationship of anadromous and resident life-history types within and among drainages, and compare several hatchery strains to their progenitor populations, brook charr Salvelinus fontinalis were examined for allozyme and mitochondrial DNA variation. Greater genetic similarity of sympatric anadromous and resident charr was found compared to similar life-history forms allopatrically, suggesting the two life-history types are not reproductively isolated. Low divergence among the mtDNA haplotypes suggests that the two life-history types are members of the same evolutionary lineage. Population differentiation from mtDNA data exceeded that from estimates based on allozymes. Genetic deviations from expectations suggest that the hatchery strains were derived from few individuals.     

Genetic differentiation among Danish brown trout populations, as detected RFLP analysis of PCR amplified mitochondrial DNA segments

An initial screening with 18 restriction endonucleases of brown trout mitochondrial DNA from three regions of Denmark revealed no variation in the D-loop while seven restriction endonucleases were found to detect variability in the NADH-dehydrogenase 1 and/or 5/6 regions. Thirteen different haplotypes were observed, fewer in the Lake Hald populations than in the other samples, probably a result of nonexisting gene flow due to an impassable dam. No correlation was found between geography and the genetic relationships among haplotypes. Significant genetic differentiation was observed among the three main regions. Genetic differentiation among populations was much more pronounced in the Lake Hald than in the Bornholm region. The genetic relationships among populations of the Lake Hald region suggested by mtDNA data were not in accordance with allozyme data and the known history of the populations, while estimates of genetic differentiation and gene flow inferred by the two methods were in agreement. (c) 1996 The Fisheries Society of the British Isles     

Genetic population structure of the catadromous Perciform: Macquaria novemaculeata (Percichthyidae)

Genetic population structure in the catadromous Australian bass Macquaria novemaculeata was investigated using samples from four locations spanning 600 km along the eastern Australian coastline. Both allozymes and mtDNA control region sequences were examined. Population subdivision estimates based on allozymes revealed low levels of population structuring ( G_st =0·043, P <0·05). However, mtDNA indicated moderate levels of geographic population structure ( G_st =0·146, P <0·0l). Phylogenetic analysis of mtDNA control region sequences (mean sequence divergence 1·9%) indicated little phylogeographic structuring. Results suggested that genotypic variation within each river population, while being affected primarily by genetic drift, was also prevented from more significant divergence by homogenizing levels of gene flow—synonymous with a one-dimensional stepping-stone model of population structure. The catadromous life history of Macquaria novemaculeata was considered to be influential on the pattern of population structure displayed. Results were compared to the few population genetic studies involving catadromous fishes, indicating that catadromy alone is unlikely to be a good predictor of population structure. A more comprehensive suite of biological characteristics than simple life-history traits must be considered fully to allow reliable predictive models of population structure to be formulated.     

Genetic differentiation of European grayling populations across the Main, Danube and Elbe drainages in Bavaria

Analysis of mitochondrial ( ND-1/3/4 genes) and nuclear ( GH-1 gene, four microsatellite loci) DNA markers of European grayling Thymallus thymallus populations identified strong genetic differentiation between the Rhine/Main, Elbe and Danube drainages in Bavaria, as a result of both present processes and past history. The Danube and the Main-Elbe group diverged about five times earlier than the populations of Main and Elbe. These data suggest that exchange and transfer of grayling stocking material between these drainages should be avoided, to maintain the genetic diversity and integrity of populations.     

Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined.     

Genetic structure and colonization processes in European populations of the common vole, Microtus arvalis

The level of genetic differentiation within and between evolutionary lineages of the common vole (Microtus arvalis) in Europe was examined by analyzing mitochondrial sequences from the control region (mtDNA) and 12 nuclear microsatellite loci (nucDNA) for 338 voles from 18 populations. The distribution of evolutionary lineages and the affinity of populations to lineages were determined with additional sequence data from the mitochondrial cytochrome b gene. Our analyses demonstrated very high levels of differentiation between populations (overall FST: mtDNA 70%; nucDNA 17%). The affinity of populations to evolutionary lineages was strongly reflected in mtDNA but not in nucDNA variation. Patterns of genetic structure for both markers visualized in synthetic genetic maps suggest a postglacial range expansion of the species into the Alps, as well as a potentially more ancient colonization from the northeast to the southwest of Europe. This expansion is supported by estimates for the divergence times between evolutionary lineages and within the western European lineage, which predate the last glacial maximum (LGM). Furthermore, all measures of genetic diversity within populations increased significantly with longitude and showed a trend toward increase with latitude. We conclude that the detected patterns are difficult to explain only by range expansions from separate LGM refugia close to the Mediterranean. This suggests that some M. arvalis populations persisted during the LGM in suitable habitat further north and that the gradients in genetic diversity may represent traces of a more ancient colonization of Europe by the species.     

Cu/Zn superoxide dismutase in excretory-secretory products of the human hookworm Necator americanus. An electron paramagnetic spectrometry study.

EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.