VASION MECHA SMS OF HOOKWORMS SKIN PENET ATION BY ANCYLOSTOMA TUBAEFORME - by BERNARD E. MATTHEWS B.Sc. (Reading), H.N.C. A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Science of the University of London Imperial College Field Station, Ashurst Lodge, Sunninghill, Ascot, Berkshire. February 1973 2 Abstract An in vitro system has been developed to study hookworm penetration. This system allows replication of results and has been-used in an extensive study of the process of penetration by the cat hookworm Ancylostoma tubaeforme. The process of invasion of the skin has been shown to be an active one in which proteolytic enzymes do not appear to play a part and a mechanical hypothesis for entry has been advanced. Further studies on the mechanism of penetration through the dermal tissues allowed this to be expanded and the data obtained have been explained in terms of mechanical penetration. As the penetration process had been shown to be an active one, the in vitro apparatus was used to assay the penetrability of A. tubaeforme. A study was undertaken to relate ageing with activity and penetration of the cat hookworm. A preliminary attempt was made to compare penetration in vitro by Necator americanus with that of A. tubaeforme, a number of differences were found and these are discussed in terms of their possible significance in causing human hookworm disease. 3 Acknowledgements My thanks are due primarily to my supervisor Dr. N.A. Croll, for his continual interest, encouragement and advice throughout this work, and to Professor T.R.E. Southwood in whose department the work was conducted. In addition I would like to thank Miss J. Fillery of the Botany Department at Imperial College for the use of the stereo- scan microscope; Dr. J.A. Clegg and Dr. J.R. Kusel of the National Institute for Medical Research for their assistance with the radio-assay technique; and Dr. P. Ball and Miss A. Bartlett of the Nuffield Institute of Comparative Medicine for the supply of Necator material. Finally I should like to thank Mrs. Catharine Gower for typing the manuscript and all those at Imperial College Field Station who have assisted in so many ways. The work was conducted during the tenure of a Medical Research Council Studentship. 4 Contents Page Abstract 2 Acknowledgements 3 Contents --, 4- Introduction 7 Section I - Invasion of the skin by Ancylostoma tubaeforme larvae 11 Introduction 12 Materials and Methods Development of the apparatus 13 Preparation of membranes 17 Procedures In vitro penetration tests 21 Effect of gravity 23 In vivo penetration tests 23 Preparation for Scanning Electron Microscopy 24 Results 24 The process of invasion 29 Discussion 44 Page Section II - Migration through the dermis and the mechanism of penetration 56 Introduction 57 Filmed sequences of larval migration 58 Dermal migration in vitro 63 Time course of penetration 69 Influence of pH and ionic changes on penetration 78 Assays for enzyme activity a)Dye release from azocoll 82 b)Release of radio-isotopes from labelled proteins 85 c)Haemolytic activity of larval secretions. 86 Discussion 89 Section III - Influence of ageing on activity and penetration of A. tubaeforme 105 Introduction 106 Materials and Methods 107 Results 108 Discussion 121 6 Page Section IV - Comparison of penetration by A. tubaeforme and Necator americanus 127 Introduction 128 Materials and Methods 128 - Re-Sults 129 Discussion 134 References 147 7 Introduction Allen (1888) observed that boys in Egypt who bathed in the rivers regularly, were more liable to schistosomiasis than girls. He postulated that the disease was transmitted by an unknown skin penetrating stage in the life cycle. In 1909 Fujinami & Nakamura working with Schistosoma Aaponicum sticeeded in demonstrating pene- tration of an invasive parasite through intact human skin. Looss (1911) in his classic monograph on the life history of Ancylostoma duodenale followed this up by showing active penetration by the infective stage of a nematode through mammalian skin. Since these early records the percutaneous route of entry has been demonstrated for many parasites. Stirewalt (1966) quotes over 50 species in 30 or more genera spread among, the arthropods, nematodes and platyhelminths that invade their vertebrate hosts through the skin. In addition to the parasites invading vertebrates, Tripius sciarae has been shown to penetrate the cuticle of the larvae and pupae of its fly host Bradysia paupera (Poinar & Doncaster, 1965), and this route has been suggested for other entomophilic nematodes (Keilin & Robinson, 1933; Welch, 1964). Miracidia of 8 trematode species normally enter their intermediate molluscan hosts by penetration through the skin (Wajdi, 1971; Kinoti, 1971; Wilson, Pullin & Denison, 1971 inter alia), and it seems probable that many further examples among invertebrates await description. Although such a common invasion route surprisingly little work has been reported on the mode of entry of actively penetrating parasite stages. A period of confirmation and amplification followed the original descriptions both for schistosomes (Meyagawa & Takamoto, 1921; Leiper, 1915, 1916, 1918; Cort, 1921; Koppisch, 1937), and nematodes (Goodey, 1922, 1925; Kosuge, 1924). Godpfey (1922) introduced the "floating raft" technique and subsequently (1925) established some of the factors governing penetration by A. caninum and Strongyloides spp. but no quantitative studies were attempted until Rogers (1939) used the technique to investigate the infectivity of the cat strain of A. caninum. Throughout the 1940s little work on penetration seems to have been attempted but the papers of Gordon & Griffiths (1952) and Standen (1953) seem to have re-awakened interest in this stage in the life cycle of schistosomes. Levine et al (1948) and Lewert & Lee (1954, 1956, 1957) attempted to approach the subject from an alternative direction 9 and to study the chemical factors that assist penetration. Of the species they investigated only Schistosoma mansoni and Strongyloides ratti were found to have any activity against the modified azo-collagen that they used for assay. Subsequent investigation by Milleman & Thonard (1959) has suggested that .azoco.11 activity does not necessarily reflect the presence of true collagenolytic activity. Stirewalt (1963) has reviewed fully the literature on larval helminth secretions and Gazzinelli & Pellegrino (1964) have subsequently demonstrated an elasto- lytic activity in extracts of S. mansoni. Attempts at in vitro studies of penetration were initiated by Goodey (1922) and used by Rogers (1939). Quantitative studies of penetration were not reported until Stirewalt and Uy (1969) made a detailed study of penetration stimuli resulting in optimal schistosomule harvest of S. mansoni, and Clegg- (1969) investigated the effect of skin products on penetration by Austrobilharzia terrigalensis. Most of the recent work on penetration has been conducted on schistosome cercariae and the present investigations have attempted to establish quantitative data for the penetration of hookworm larvae into mammalian skin and to determine the most important factors governing penetration. 10 For the sake of convenience the penetration process has been divided into two parts, firstly, the initial invasion of the skin and entry of the larvae into the stratum corneum and secondly, the migration through the dermis and epidermis. 11 SECTION I INVASION OF THE SKIN BY ANCYLOSTOMA TUBAEFORME LARVAE 12 SECTION I INTRODUCTION The in vitro study of penetration of infective nematode larvae into skin was initiated by Goodey (1922) with the intro- duction of the "floating raft" technique. Subsequently Rogers (1939) and Barrett (1969a) using the cat strain of Ancylostoma caninum and Strongyloides ratti respectively measured infectivity as the number of larvae that completely penetrated a stretched skin membrane. Stirewalt, Minnick and Fregeau (1966) used a modified Rose chamber (Rose, 1954) to investigate the change from cercaria to schistosomulum during penetration of Schistosoma mansoni through dried skin. This was further developed by Stirewalt and Uy (1969) in a detailed study of penetration stimuli resulting in optimal schistosomule harvest. Clegg (1969) used a further modification of the same raft principle in his study of the effect of skin products on the penetration behaviour of cercariae of the bird schistosome Austrobilharzia terrigalensis showing that skin lipids especially cholesterol stimulate penetration. 13 In each of these studies skin has presented a barrier between two fluids and little attempt has been made to investigate the course of penetration through it. The present investigations were aimed at providing an in vitro method of studying the pene- tration of hookworm larvae through skin that would give qualita- tive information about the course, route and mechanisms of pene- tration comparable to that obtained in vivo and to quantify larval invasion. This section deals with the development of the apparatus used, the initial invasion of the skin by Ancylostoma tubaeforme and entry into the epidermis. Section II covers the later migrations of the larvae through the dermal tissues. MATERIALS AND METHODS Development of the apparatus Goodey (1922) in his original floating raft experiments, used skin stretched across a half inch diameter hole cut in the centre of a sheet of cork and floated on warm saline. Penetration under optimal conditions was demonstrated by placing infective larvae on the surface and some of the