Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent "sphaerocrystals" located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.     

Survival of Ancylostoma caninum on bluegrass pasture.

The survival of infective larvae of Ancylostoma caninum on outdoor grass plots was studied in 40 experiments over 1 year. Weather data were collected over the period. Mean larval survival from August to early November was 24 days (range 1 to 49), from December through February was 0 days, and from March to mid-August was 6.6 days (range 0 to 21). Moderate to high temperatures and substantial rainfall favored larval survival; low temperatures and rainfall favored larval destruction.     

A pore-forming haemolysin from the hookworm, Ancylostoma caninum

Hookworms feed on blood, but the mechanism by which they lyse ingested erythrocytes is unknown. Here we show that Ancylostoma caninum, the common dog hookworm, expresses a detergent soluble, haemolytic factor. Activity was identified in both adult and larval stages, was heat-stable and unaffected by the addition of protease inhibitors, metal ions, chelators and reducing agents. Trypsin ablated lysis indicating that the haemolysin is a protein. A closely migrating doublet of hookworm proteins with apparent molecular weights of 60-65 kDa bound to the erythrocyte membrane after lysis of cells using both unlabeled and biotinylated detergent-solubilised hookworm extracts. In addition, separation of detergent-soluble parasite extracts using strong cation-exchange chromatography, resulted in purification of 60-65 kDa proteins with trypsin-sensitive haemolytic activity. Erythrocytes lysed with particulate, buffer-insoluble worm extracts were observed using scanning electron microscopy and appeared as red cell ghosts with approximately 100 nm diameter pores formed in the cell membranes. Red blood cell ghosts remained visible indicating that lysis was likely caused by pore formation and followed by osmotic disruption of the cell.     

Host-finding and host recognition of infective Ancylostoma caninum larvae

A. caninum larvae responded to environmental and host stimuli with four behavioral phases of host-finding. (1) Snake-like movement was stimulated by warmth and by defined vibrations of the substratum. (2) Waving behavior was a prerequisite for the passive change-over to dog hairs. It was stimulated by heat radiation and by the CO2 content, warmth, and humidity of an air stream. (3) Creeping direction: the larvae were attracted by heat in temperature gradients as weak as 0.04 degrees C mm-1 and by dog hydrophilic skin surface extracts, but not by skin lipids or serum. (4) Penetration into agar was stimulated by heat, dog hydrophilic skin fraction, and dog serum. The effective component of serum had a molecular weight of between 5000 and 30,000 and proved to be a protein, since it lost its effectiveness after digestion with proteinases. Dog saliva, urine, milk, and various pure dog serum components did not stimulate penetration. A. caninum larvae were able to penetrate mouse skin repeatedly, but they did not follow the tracks of previously penetrated larvae in agar.     

Strategies for the storage of Ancylostoma caninum third-stage larvae

Although cryopreservation protocols for storage of hookworm larvae have been described, the circumstances under which the technique is necessary to ensure larval survival are not well defined. The motility of infective-stage larvae (as judged by observation) and their ability to migrate through canine skin in vitro were measured over a 7-mo period in worms held at room temperature and worms that had been cryopreserved at the start of the experiment. Cryopreserved worms showed motility and migration proportions of 45.6-48.0% and 26.8- 34.0%, respectively, throughout the experiment, compared with percentages of 92.7 and 84.1%, respectively, in the original fresh worms. Larvae held at room temperature showed a gradual decrease in motility and migration ability over the experimental period. Motility and migratory ability of cryopreserved larvae was only significantly higher (P < 0.01) than room temperature-stored larvae from 4 and 5 mo onward, respectively.     

Generation of bioactive recombinant Ancylostoma caninum anticoagulant peptide c2

Thrombus formation is a crucial factor in the precipitation of unstable angina or myocardial infarction. Recently, several anticoagulant serine protease inhibitors have been identified from adult Ancylostoma caninum hookworms. One of them, A. caninum anticoagulant peptide c2 (AcAPc2), can inhibit the activity of factor VIIa/tissue factor complex to exert its antithrombotic effect. However, it is difficult to adopt traditional expression and purification systems to yield high-purity recombinant AcAPc2 (rAcAPc2). Here, we employed a simple method to produce high-yield and high-purity rAcAPc2. We obtained the full-length double-stranded cDNA encoding AcAPc2 by overlapping PCR and cloned it into an intein-based expression vector. The AcAPc2 cDNA was expressed in Escherichia coli and comprised 30% of the total bacterial proteins. The expressed rAcAPc2 was purified by cleaving the fused chitin-binding domain at pH 7.2. Finally, we produced a high yield of rAcAPc2 at a purity of greater than 98%. Importantly, the generated rAcAPc2 prolonged the prothrombin time (PT) and activated partial thromboplastin time (aPTT) of human plasma in vitro in a dose-dependent manner. Therefore, this method to generate the high-purity and bioactive rAcAPc2 may contribute to the scientific research on its biological function and the treatment of thrombotic diseases.