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.     

Serum-stimulated feeding in vitro by third-stage infective larvae of the canine hookworm Ancylostoma caninum

Developmentally arrested nonfeeding infective larvae of hookworms resume development after entry into the host, presumably in response to a signal encountered during invasion. Logically, an initial step in the resumption of development might be the resumption of feeding. An in vitro assay for feeding is described for the third-stage larvae of the canine hookworm Ancylostoma caninum. Populations of larvae incubated under hostlike conditions in the presence of 10% canine serum resume feeding within 6 hr, as evidenced by the uptake of fluorescein-labeled bovine serum albumin. Feeding is dependent on the presence of canine serum, and peaks by 24 hr incubation. Maximal feeding levels occur at temperatures above 34 C with a gas phase of 5% CO2/95% air, whereas culture medium and pH are unimportant for feeding. Serum concentrations between 0.1% and 1.0% (v/v) initiate feeding, and the response peaks at approximately 8.0% serum. Serum triggers feeding within 6 hr and is not required for feeding to continue once initiated. The saturation effect and the trigger phenomenon suggest that the initiation of feeding is a receptor-mediated response.     

A comparative in vitro study of antibody binding to different stages of the hookworm Ancylostoma caninum.

The Indirect Fluorescent Antibody Technique (IFAT) and the Indirect Immuno Peroxidase Technique (IIPT) have been applied to cryostat sections and intact stages of the hookworm species Ancylostoma caninum with sera from infected dogs. Especially the role of the body surface (= cuticle (cortex, matrix, basal layer) and hypodermis) in immunity was studied. Using cryostat sections and dead intact stages as the antigen, specific antibody binding was demonstrated round the ovum membrane and the cuticle of all stages of this hookworm species. Cryostat sections of adult worms showed, that it probably is not the cuticle itself that is antigenic, but that the specific reaction that is observed consisted of a layer, covering the cortex of the cuticle. Infective and parasitic living stages, however, showed no antibody binding in contrast to the free-living stages in which specific antibody binding was demonstrated.     

In vitro reactivation of Ancylostoma caninum tissue-arrested third-stage larvae by transforming growth factor-beta

Developmental arrest in Ancylostoma caninum is associated with preparasitic, free-living third-stage (L3) larvae, as well as anthelmintic-resilient hypobiotic L3 larvae within the tissues of an infected dog. With the tissue-arrested larvae, pregnancy and, more specifically, the hormonal effects of estrogen and prolactin mediate reactivation resulting in transmammary transmission of infection to nursing puppies. Estrogen and prolactin have been shown to be critically involved in upregulation of transforming growth factor (TGF)-beta2 during pregnancy, and studies on the soil nematode Caenorhabditis elegans further implicate TGF-beta and insulin-like signaling pathways with larval arrest and reactivation. In this report, an in vitro assay was used to show that neither estrogen, prolactin, nor insulin had a direct effect on the feeding/reactivation response of tissue-arrested larvae; however, TGF-beta isoforms 1 and 2 both had significant stimulatory effects that were comparable to the effects of dog serum. The stimulatory effects of serum could be blocked by preincubation with anti-TGF-beta antibodies. Taken together, the results support the hypothesis that during pregnancy, host-derived TGF-beta can signal a parasite-encoded receptor to trigger the reactivation of tissue-arrested larvae. TGF-beta had no effect on preparasitic larvae, suggesting that different signals may be involved in reactivation of the 2 different arrested forms of A. caninum L3 larvae.     

Effects of alcohol on natural resistance to Ancylostoma caninum in mice.

Experiments were performed to study the effect of alcohol on natural immunity of Swiss albino mice during Ancylostoma caninum infection. Natural resistance is lowered in alcoholic mice; 40% alcohol by mouth produced significant effect after 20 daily injections. The effect of the drug on natural immunity depended upon the concentration and the period of consumption. Animals retained maximum number of larvae during alcohol treatment.     

Albumin and a dialyzable serum factor stimulate feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum.

Previous studies demonstrated that third-stage, developmentally arrested larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to canine serum and hostlike temperature. Experiments to determine the identity of the serum stimulus are described. Serum from several nonhost species stimulated feeding, but to levels lower than canine serum. Heating the serum to 57 C had no effect on its stimulatory ability. Dialysis reduced serum stimulatory activity by 50%, and ultrafiltration through 10- and 30-kDa molecular weight cut-off membranes decreased activity in both the filtrates and retentates similarly. Recombination of the filtrates and retentates restored activity to whole serum control levels. Commercial canine and bovine albumin stimulated feeding to serum control levels at 10 and 50 mg/ml, respectively. These results suggest that albumin and an unidentified low molecular weight compound(s) are capable of inducing in vitro feeding by A. caninum L3.     

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.     

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.