Larval migration in PERL chambers as an in vitro model for percutaneous infection stimulates feeding in the canine hookworm Ancylostoma caninum

Abstract

BackgroundAncylostoma caninum third-stage larvae are the non-feeding infective stage of this parasite and are able to infect potential hosts via different infection routes. Since percutaneous infection is one of the most important routes and skin penetration is the first step into parasitic life, an existing in vitro model for percutaneous migration was modified and evaluated. The main parameter used to evaluate migration was the migration ratio (migrated larvae as a percentage of total number of larvae recovered). Additionally, the skin lag was calculated, expressing the percentage of larvae remaining in the skin and therefore not being recovered. Since initiation of feeding is proposed to be an important step in the transition from free-living to parasitic A. caninum larvae, feeding assays were performed with in vitro percutaneously migrated larvae. Additionally, infective larvae of A. caninum were activated via serum-stimulation and feeding behaviour was analysed and compared between percutaneously migrated and serum-stimulated larvae.ResultsMaximum skin migration levels of infective larvae were observed at temperatures above 32°C when larvae were placed on the epidermal side of skin for more than 12 hours. The medium beneath the skin had no effect on migration ratio, and no significant difference between the migration ratios through fresh and frozen/thawed skin was observed.Maximum feeding levels of 93.2% were observed for percutaneously migrated larvae after 48 h incubation, whereas serum-stimulated larvae reached the maximum of 91.0% feeding larvae after 24 h.ConclusionsThe PERL chamber system was optimised and standardised as an in vitro model for percutaneous migration. The larvae recovered after percutaneous migration showed characteristic signs of activation similar to that of serum-stimulated larvae. The observed difference in time course of resumption of feeding indicates that percutaneously migrated larvae are not identical to serum-stimulated larvae, which are currently representing the model for early parasitic stages.

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion

“…Excised native skin and simple hydrogels were generally used so far to study the penetration behavior of skin-invading parasitic helminths (Franke et al, 2011;Haas, 2003;McKerrow and Salter, 2002;Safer et al, 2007). Already 1993, Fusco et al (1993 studied the penetration success of S. mansoni into human skin equivalents derived from fibroblast and keratinocyte layers embedded in a collagen scaffold.…”

“…The infection of human full skin was performed in Franz type diffusion chambers (Gauer Glas, Puettlingen, Germany). This chamber system allows the application of a tissue with a higher thickness, compared to the skin equivalents (Franke et al, 2011). Human skin and ex vivo epidermis were prepared one day before the infection experiments.…”

“…Larval invasiveness was assessed by expressing the total number of larvae in the donor and the acceptor compartment as percentage of total number larvae applied to infect the model. The skin lag as a measure of larvae in the model was calculated according to a previously described and modified protocol (Franke et al, 2011):…”

“…Currently, larval invasion studies are based on animal models and excised animal skin (Franke et al, 2011;Stirewalt and Uy, 1969). In contrast to human skin samples, model organisms are characterized by a high availability.…”

Advances in the Molecular and Cellular Biology of Strongyloides spp.

“…Studies deploying in vitro models of skin penetration by iL3 of Strongyloides spp. and hookworms such as Ancylostoma spp., which may also invade the host percutaneously, have revealed that this event may trigger early events comprising reactivation of these developmentally quiescent infective larvae [72,73]. These model systems generally involve a glass apparatus, dubbed a PERL chamber that comprises upper and lower compartments filled with a physiological medium, the lower compartment being warmed to host body temperature, and separated by a layer of mammalian skin recovered from some laboratory animal at necropsy.…”