BERNARD E. MATTHEWS B.Sc. (Reading), H.N.C

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 stereoscan 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

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 introduction 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 penetration of hookworm larvae through skin that would give qualitative information about the course, route and mechanisms of penetration 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 factors affecting penetration by

A. caninum and Strongyloides spp. were established. Although the presence of larvae within the skin was demonstrated (Goodey, 1925)

FIGURE 1

PENETRATION CELLS

SKIN

Igo C') COLLECTING I CELLS 000 COVER SLIPS I .0 RUBBER GASKET

WATER JACKET

RUBBER GASKET

• 8 BASE PLATE

n vitro penetration apparatus. 15

no attempt was made to follow the course of penetration or to quantify the results.

The modified apparatus (Fig. 1) was designed to answer questions relating as much to the host skin as to the parasite.

The effective area of skin exposed to the larvae was 3 mm in diameter permitting serial sectioning of the entire area and subsequent investigation of the course of penetration. The size and shape were such that three penetration sites were established on a single piece of skin 5 cm by 1 cm which allowed replication while reducing to a minimum the effects of the inherent variations in skin structure.

The skin surface temperature of a cat was measured using a thermistor probe (Table 1). The average temperature was 31-32°C and there is thus a temperature difference of 10-15°C between laboratory temperature and that of the skin surface. This difference was simulated in vitro by adjusting the temperature of the water jacket so that the skin surface was 32°C (Fig. 2).

The skin was held under slight tension by pins passing through it and the top plate, finally being retained by the bolts clamping the top two plates. The collecting cells were filled with normal saline buffered with M/15 phosphate buffer to pH 6.8, care being taken to ensure that all air was excluded when the cover glasses were slid into place. The first gasket was then 16

20.

10 20 30 40 50 water tamp.41 C FIGURE 2. Effect of change in circulating water temperature on the temperature of the skin surface ( ) and fluid on the surface ( — — — ) in vitro.

10 20 30 40 50 60 70 80 90 time (mins.)

FIGURE 3. Equilibration curve for the in vitro penetration apparatus. Each line represents the skin surface temperature for a single penetration cell. 17

Table I

Surface temperatures of a cat under light Nembutal anaesthesia

Clipped cat skin 29-30°C Beneath fur 33 _Between foot pads 32

Surface of protruded tongue 30 Back of throat 33

fitted and the whole apparatus assembled. The flow rate through each cell of the water jacket was balanced to reduce temperature differences between the skin and penetration chambers. Water at

37°C was pumped through the water jacket for 30 min before each test to equilibrate the skin to 37°C (Fig. 3).

The self contained nature of the assembled apparatus meant that movement for observation under a microscope or inversion to study gravitational effects could be readily achieved.

Preparation of membranes

Both natural and artificial membranes have been used (Table 18

Table II

Penetration membranes tested against A. tubaeforme in vivo and in vitro

Membrane Penetration by Comments A. tubaeforme

Natural Membranes

Cat skin: Living

Freshly excised

Frozen

Rabbit skin: Living

Freshly excised

Frozen

Dried & scraped

Isolated epidermis

Hare skin: Frozen

Rat skin: Freshly excised

Dried & scraped MI6

Adult mouse skin: Freshly excised

Dried & scraped Penetration age Baby mouse skin: Freshly excised dependant ,Guinea pig skin: Freshly excised

Gerbil skin: Freshly excised

Frozen 19

Table II Contd.

Membrane Penetration by Comments A. tubaeforme

Human skin: Frozen

Chicken skin: Freshly excised el•

Dried & Plucked

Turkey skin: Freshly excised

Dried & plucked /NW

Frog skin: Freshly excised

Artificial Membranes

Filter paper: Whatman No. 1 Very limited penetration 4

540 Very limited penetration 541

Tanned gelatine

Filter paper/gelatine laminates Only partial penetration

The skin was either fresh, deep frozen prior to use or dried, plucked and sanded using the method proposed by Stirewalt et al

(1966). Hair was removed by careful clipping with fine scissors as shaving, however carefully performed, invariably disrupts the 20

outer layers of the stratum corneum and removes the rough debris naturally occurring there. (Head, 1970).

Membranes consisting of filter paper, gelatine sheets and laminates of filter paper with gelatine were used as artificial membranes in an attempt to reduce the inherent variability of whole-skin. Gelatine membranes were chrome tanned following the method described by Clegg (1969).

The grade of filter paper used is important. Hardened papers while having the advantage of greater wet strength, were too firm and only small numbers of larvae were able to penetrate.

The qualitative grade Whatman No. 4 proved suitable. The quoted mean pore size of this paper is 3.4 - 5.0jam and the mean larval diameter 27.0 4- 1.3iim so that activity is required for penetration.

Laminated membranes were prepared either by casting 10% (w/v) sheet gelatine (May and Baker laboratory grade) in distilled water in a 1 mm deep oblong mould 75 x 25 mm and applying No. 4 filter paper to the almost set top surface, or by placing filter paper in the mould and pouring gelatine over it. The gelatine was subsequently tanned using freshly prepared 0.006M chrome alum (CrK(SO4)2

12H 0. B.D.H., A.R.) in 0.2M sodium acetate/acetic acid buffer at 2 pH5.0 for 45 min (Clegg, 1969). Such membranes were washed and stored in cold water and used on the same day. 21

Soluble skin fractions were removed from closely clippped skin using redistilled chloroform on cotton wool pads handled with forceps (Clegg, 1969). Half the chloroform extract was applied to half the area from which it had been prepared and the chloroform allowed to evaporate, providing a membrane with the lipid-replaced. The remaining chloroform/lipid extract was added to No. 4 filterpaper by capillary action and the chloroform allowed to evaporate.

Procedures

In vitro penetration tests

Infective third stage A. tubaeforme larvae were cultured from infected cat faeces in 9 cm Petri dishes at 30°C (Croll, 1972a)and were used between 4 and 8 days of culturing. Separation of larvae from the cultures was either by Baermann funnel or by hand using a micro-pipette. Larvae, generally 50 or 100, per penetration cell, were counted into cells of microtitre agglutination.plates (Flow Labs) and allowed to settle. The volume of water was then reduced to 0.2 ml and the larvae transferred to the top surface of the penetration membrane and left for the duration of the experiment. In the longer experiments water was replaced as it evaporated from the skin surface. At the end of the experiment those larvae that had not entered the membrane were 22

removed, initially by pipette and subsequently by thorough washing

into a watch glass. The apparatus was then carefully dismantled,

the saline in the collecting cells removed and the under surface

of the membrane also washed thoroughly, the saline and washings being

collected. For histological examinations the exposed area of

skin_was removed, fixed in 7 ethanol for at least 18 h,

dehydrated in graded alcohols, cleared in cedarwood oil and after a

15 min wash in benzene embedded in Paraplast (Shandon). Routinely)

serial 10 jum sections were cut and stained with haematoxylin and

eosin.

For quantitative experiments it was essential to know not only how many larvae had completely penetrated the membrane, but also how many had entered it. The method used to remove the larvae depended on the membrane concerned. Skin membranes were cut into 1-2 mm squares and digested in a 1:1 mixture of 4% pepsin and 0.5% HC1 at 25°C until completely broken down. Larvae were not affected by this treatment and could be removed from the digest. Paper membranes were teased apart in water and larvae lodged among the fibres removed.

Croll (1972a) has shown that hookworm larvae from different cultures show measurable variation. To minimise such differences, experiments were conducted in pairs using skin and filter paper membranes as mutual controls. 23

Effect of Gravity

To investigate the effect of gravity on penetration a slight modification of the normal technique was used. The larvae were applied in water to the cold top surface of two separate pieces of penetration apparatus and left for 10 min to settle. The depth of fluid was then reduced from 3 mm to 1 mm with a micro- pipette, taking care not to remove the larvae. Cover slips with a small quantity of vaseline on the corners were applied to the top of the penetration chambers and one apparatus inverted. The cover slips prevented the skin surface drying out as it was impossible to replace evaporation losses from inverted cells.

The water supply was then connected and the experiment started.

This method avoided the problem of larvae having to migrate through 3 mm of water before reaching the membrane surface.

In vivo penetration tests

In vivo tests demonstrated the applicability of the in vitro results. The animal was anaesthetised using Nembutal (Abbott) and the abdominal hair removed using fine cutters on electric clippers and finally carefully trimmed with fine scissors. Strips of zinc oxide tape coated with vaseline on the non-adhesive surface and from which 6 mm diameter holes had been cut, were 24

stuck across the clipped area and drops of water containing infective larvae placed in the circle. After infection the animal was killed and the centre of each area marked with a fine pin.

The skin was then removed and processed for histological examination.

Preparation for Scanning Electron Microscopy

Skin exposed to larvae in vitro was dehydrated in graded ethanol, allowed to air dry, mounted on aluminium studs using

Silver Dag and coated with gold/palladium for examination in the

Cambridge Stereoscan Microscope.

RESULTS

Preliminary experiments were designed to establish a method of assaying the penetration process and a wide range of natural and artificial membranes were tested (Table II). The structure and thickness of skin varies widely from one part of an animal to another and between individuals. For quantitative use with

S. mansoni Stirewalt et al (1966) used dried, plucked and sanded rat skin, however larvae of A. tubaeforme were not able to penetrate either rat or adult mouse skin. Penetration did occur into cat, rabbit and guinea pig skin but the greater dermal thickness and more complex arrangement of hair follicles in these animals made it impracticable to prepare dried membranes of 25

sufficient constancy to give reproducible quantitative results.

A suitable artificial membrane with constant characteristics was thus desirable.

Gelatine membranes tanned to withstand the temperatures used in penetration tests were not penetrated by larvae but filter paper was. Of a number of different papers Whatman No. 4 proved the most suitable. Laminated membranes consisting of

No. 4 filter paper firstly on top of and secondly impregnated with, tanned gelatine demonstrated that the larvae could enter the paper but were not able to penetrate the gelatine (Plate 1).

Figure 4 gives regression lines for data extracted from pairs of experiments using both cat and rabbit skin and No. 4 filter paper, and shows a linear relationship between percentages entering the various membranes. There were absolute differences between batches of larvae and these differences were reflected in the results of penetrants recovered from both types of membrane.

The effect of changing the temperature of the circulating water on the percentage of larvae penetrating a filter paper membrane is plotted in Figure 5.

Inverting the apparatus so that larvae had to migrate through

3 mm of water before reaching the membrane caused a ten-fold decrease in the number of larvae entering the membrane in 2h,

54.8% entering with gravity and only 5.1% against it. By 26

PLATE 1.

Third stage A. tubaeforme larva that has penetrated No. 4

filter paper but has been stopped by tanned gelatine layer.

Haemotoxylin and eosin. X 300 +

28 FIGURE 4.

100 -

CAT SKIN

PER • / A P • / RABBIT SKIN 60 •

NG /. • RI R 450

TE / • N E 40 • • /It '909 • GE . • • TA N 20 PERCE

O 20 40 60 80 100 PERCENTAGE ENTERING SKIN

Relationship between entry of A. tubaeforme into cat ( and rabbit ( ) skin and No, If filter paper.

FIGURE 5.

20 40 50 water temp. °C

Effect of change in circulating water temperature on the percentage p....„Labattgmalt larvae entering a No. If filter paper membrane in 2 h in vitro. 29

modifying the technique so that both control and test larvae were in a 1 mm water layer resulted in only a slight reduction in the percentage of larvae entering the filter paper membrane

(Table III). This reduction was not significantly different (P >.05) from the control situation with the apparatus normally orientated. The addition of chloroform soluble skin products did not affect this result (Table III).

Table III

Mean percentage larvae entering filter paper membranes in 2 h with and against gravity Membranes Expt. 1 Expt. 2 Mean+S.E. Mean+S.E. No. 4 Filter paper 44.815.0 58.9+3.6 No. 4 filter paper (apparatus inverted) 38.8+2.8 51.6+5.6

No. 4 filter paper + cat skin lipids 52.3+6.0 60.1+3.2 No. 4 filter paper + cat skin lipids (apparatus inverted) 46.2+6.5 53.9±5.4

Removing lipids and replacing them in either cat skin or filter paper did not significantly affect the percentage of larvae entering these membranes (Figure 6).

The process of invasion Larvae have been observed during the process of invasion on FIGURE 6.

50 1M Z ICAT SKIN 0.4 FILTER 4 c::t o CONTROL PAPER o CONTROL 40 ~ LIPID fX'YVl PLUS SKI N ~ ~ LIPID 10&1 ~ EXTRACTED ~ fiAIO

to-~ .,.,Z u c: ~ 10 Z <.t 10&1 :E o hlYi1 'V)! 1 2 2 EXPERIMENT NUMBER

'Effect of chloroform soluble cat skin lipids on the entry of A. tubaeforme larvae into cat skin and No. 4 filter paper in vitro. Each column represents the mean of six determin~tions. The vertical lines indicate standard errors of the mean.

\J.Io 31

many occasions and the following description has been compiled from these observations together with histological and scanning electron microscopic evidence. The later stages of penetration are described in Section II.

When larval suspensions are placed on the skin surface the worms move rapidly and actively towards it, at least 90% being found on or very close to the skin surface within 10 min. The approach to the skin surface followed a very similar pattern for all membranes tested, regardless of whether penetration through the membrane was possible. The mechanical disturbance inherent in transferring the larvae by pipette, together with the. temperature difference, stimulate larvae that have been inactive in the agglutination plate cavities into activity. As the larvae are slightly more dense than water (Croll, 1972b) gravity may play a passive role in the approach to the membrane surface. Table III and Figure 6 illustrate that neither gravity nor the presence or absence of skin lipids significantly affected the percentage entering.

Initial contact with the skin is normally made with the worm roughly perpendicular to the surface. The larvae remain at a steep angle to the skin surface for as long as they remain active and as long as there is a sufficient depth of water. If the water level falls below the length of the larvae, about 650 ,Am, the angle decreases as the worms are pressed to the substratum. 32

PLATE 2.

Transverse section of ensheathed A. tubaeforme larva within

the stratum corneum of the cat.

Haematoxylin and eosin. X 1,500

The time spent searching for an entry site varies greatly. Larvae have been found in the epidermis within 5 min (Plate 2) but there is continuous invasion of the skin for at least 4 h if the surface is kept moist (Figure 7). Disappearance of the surface water film causes cessation of movement and thus penetration.

The surface of skin is not smooth, the outer layers of the stratum corneum are thrown into folds and rugosities and the surface is broken by the apertures of the hair follicles and by numerous micro-lesions and desquamations of the keratinized cells

(Plate 3). These provide the entry sites for the larvae.

Plate 4 shows a larva that has passed into the outermost layers of the stratum corneum and come back on to the surface. This site of entry is in an area of the skin well away from the bundles of hair follicles. Entry appears to be possible at any site on the surface at which a suitably sized fissure is located, and although hair follicles are also used (Plate 5), larvae are not restricted to this route. Once an entry site has been found the larvae orientate parallel to the skin surface (Plates 2, 6) following the line of least resistance into the skin. On no occasion has evidence been found for any destruction of the keratinized layers

by larval enzymic activity. Lee (1972) has recently described a very similar entry process for Nippostrongylus brasiliensis into mouse skin. 35

FIGURE 7.

100

• 80 Z. ag No.4 FILTER PAPER 60 0 • R•973 ac mg 40

CAT SKIN stlj • 20 -R -994 2 ir" V • • fAl A. 60 120 180 240 TIME (min.)

Effect of time on the percentage A. tubaeforme larvae entering cat skin (— — ) and No. 4 filter paper ( ) in vitro. Each point represents the mean of four determinations. 36

PLATE 3.

Scanning electron micrograph of cat skin showing the bases of

a number of hairs and the micro-lesions of the surface, of the

stratum corneum. X 540 C"- rt-\ 38

PLATE 4.

Scanning electron micrograph of an ensheathed A. tubaeforme

larva that has entered the stratum corneum at one of the surface

lesions but failed to penetrate further. X 400

PLATE 5.

Scanning electron micrograph of ensheathed larva entering hair follicle. X 1,250

142

PLATE 6.

Scanning electron micrograph of ensheathed larva laying

parallel to the skin surface after passage beneath the outer-

most layers of the stratum corneum. X 2,500.

Infective third-stage hookworm larvae are surrounded by the retained cuticle of the second-stage larvae after the second moult. This sheath is not necessarily lost before penetration and is clearly visible in both the scanning electron micrographs and light microscope sections.

DISCUSSION

The success of an in vitro penetration system that sets out to provide results comparable to the in vivo situation must rely on the fact that the skin used in vitro possesses characteristics that are not essentially different from living tissue. In this respect skin is a particularly useful tissue. In vitro culture of skin cells is readily achieved in suitable media (Trowell,

1959) full thickness explants remain viable for up to six weeks 14 (Summerlin, Charlton and Karasec, 1970) and C praline is incorporated into biopsy material for a number of hours after excision (Ditto, Lindy, Turto and Danielson, 1971). Deep freezing or freeze drying appear to have little effect on the viability of skin as shown by subsequent grafting experiments

(Billingham & Medawar, 1951) and the competance of the epidermis as shown by diffusion of water through it is retained for up to 45

nineteen days (Bettley & Donoghue, 1960). The stratum corneum

is composed of non-nucleated, flattened, hollow cells with the

peripheral cytoplasm keratinized and these are even less liable

to change than the living cells of the deeper skin tissues.

Although quantitative work on penetration has been conducted

in vitro, as this was more reproducible, control experiments have

been done in vivo and on each occasion the same picture of pene-

tration has emerged.

The'migration of larvae through a filter was shown by Looss

(1911) to occur when the temperature was sufficiently high to

permit larval mobility. This ability to penetrate blotting

paper has been explained in terms of positive thigmotaxy by Lane

(1933) who suggested that larvae attempt to retain their "anterior

faces" in contact with a solid surface. On irregular surfaces

such as paper (Plate 7) asymmetrical movement causes them to slip

from fibres into spaces. The normal undulatory movement then

carries the worm forward until the anterior is in contact with

another fibre when the process is repeated. Penetration through

the paper is made up of a series of such stages. This mechanism

may also explain the initial entry of hookworm larvae into the

stratum corneum, the fissures of which act in the same way as the

spaces between paper fibres. 1+6

PLATE 7.

Scanning electron micrograph of the surface of number 4

filter paper. X 550.

The direct relationship between entry into filter paper and entry into cat and rabbit skin (figure 4) suggests that entry may depend as much on the surface structure of the membrane as on its chemical composition. The different slopes for the two skin types probably reflect differences inherent in the physical characteristics of the outer layers of the two animals. Little pattern appeared from the skins into which A. tubaeforme larvae could enter. The failure to enter bird or amphibian skin suggests that the mammalian stratum corneum surface may be essential, Smirnov and Kamalov (1950) were also unable to illustrate penetration by either A. duodenale or N. americanus into frog skin. Larvae were incapable of entering the skin of adult rats or mice, both these animals have a relatively smooth epidermis (Plate 8) and the fissures in this may be too small for the larvae to obtain an initial point of entry. Alternatively the epidermal cells may be so firmly attached to the underlying layers that the larvae are unable to separate them to effect entry. Norris (1971) reported a very low percentage recovery of A. tubaeforme following cutaneous exposure of mice and rats, as he shaved the abdominal skin prior to infection it is possible that those larvae that did enter made use of artificially produced lesions.

The surface characteristics may also partly explain the inability of the larvae to enter tanned gelatine membranes as 1+9

PLATE 8.

Scanning electron micrograph of young adult mouse skin:

X 21+0

these have a smooth surface. The results from the laminated membranes however suggest that they are incapable of penetrating the gelatine even if provided with filter paper against which to obtain purchase (Plate 1).

According to Goodey (1925) Kosuga (1924), working with

Strongyloides larvae, showed that in the case of thin skinned animals the entry was by epidermal scales, whereas in thick skinned animals it was via the hair follicles. Although the skin types used were not specified it seems probable that cat and rabbit skin would be classed as thick in this context. The use of the scanning electron microscope, coupled with light microscopy, has shown that penetration of A. tubaeforme may be by either route but the former is the more usual. Vetter (1970) and Vetter and

Leegwater (1971) have briefly reported on penetration of A. ceylanicum, A. braziliense and A. caninum and suggested that entry is through the stratum corneum but that the hair follicles are often used as a migration route to the lower skin layers.

The question of exsheathment by infective larvae prior to penetration of the skin received some attention from earlier workers (Loose, 1911; Goodey, 1922) and it seems to have been tacitly accepted that it was essential in all cases before penetration could commence. Looss (1911) suggested that exsheathment occurred when friction between the substrate and the sheath 52

was so great that the sheath could no longer move but the enclosed larva was able to force an exit. Larvae of A. tubaeforme exsheath if allowed to dry on an agar plate under constant conditions as the sheath becomes held by the drying surface (Croll, 1972c).

The organized exsheathment shown by Lapage (1935) for trichostrongyle larvae does not appear to occur in hookworm larvae nor is any specific exsheathing stimulus apparently required. Rogers and

Sommerville (1963) considered that it was unlikely that penetration of exsheathed larvae could occur due to the structure of the sheath. They felt that the frictional properties of the host skin would be insufficient to allow mechanical exsheathment, and concluded that penetration probably involves secretion of proteases and hyalizronidases and that these may first help to break down the sheath. The sheath of A. tubaeforme is not necessarily lost on entry into the skin in vivo or in vitro and may be discarded at any stage in the penetration process. There thus seems to be less case to postulate the use of enzymes. The sheath includes a type of.collagen which is partly keratinized and is unique to the nematodes (Simmonds, 1958; Bird and Bird, 1969). It seems unlikely that larvae would be able to retain their sheaths during penetration if an enzyme sufficiently powerful to soften the keratinized layers of the stratum corneum were produced. The 53

direct relationship between entry into skin and non-living filter paper membranes also suggests that a mechanical rather than an enzymic mechanism may be operative. None of this evidence is conclusive and a more detailed study of this question is presented in Section II.

JGoodey (1925) reported finding large numbers of discarded

A. caninum sheaths on the skin surface after penetration, these have not been found with A. tubaeforme, perhaps because Goodey used young rat skin rather than that of the definitive host. Using

A. tubaeforme it has not been possible to demonstrate entry into rat skin, Norris (1971) however showed that A. caninum was better able to penetrate rat skin in vivo than A. tubaeforme. There is also a possibility that Goodey transferred sheaths from the cultures, the age of the larvae used was not given and exsheathment within old cultures is not unusual. Although apparently very closely related, the penetration method employed by A. tubaeforme and

A. caninum may differ to such an extent that exsheathment may play a more important part in the invasion process of the latter species.

The differences inherent in skin structure may also explain

Goodey's (1925) inability to demonstrate penetration against gravity or penetration from deep water drops. Qualitatively entry of larvae into cat skin has been demonstrated against gravity from 3 mm water drops; from 1 mm drops entry into filter paper 54

did not differ significantly with or against gravity (Table III).

Larvae were consistently found in sections taken from all parts of the exposed area demonstrating that the edge of the penetration chamber was not providing mechanical assistance.

The importance of skin lipids in the penetration of larval trematodes has recently been demonstrated. Clegg (1969) has shown that cholesterol, the largest fraction in chicken skin lipid, can stimulate the penetration of Austrobilharzia terrigalensis cercariae through tanned gelatine. The most polar fraction of rat skin lipid has been shown to be the most stimulatory to

Schistosoma mansoni cercariae (Austin, Stirewalt and Danziger,

1972). Wilson, Pullin and Denison (1971) have demonstrated that miracidia of Fasciola hepatica are stimulated to increase their time of attachment by host snail mucus and fatty acids of chain length C7_9. Lee (1972) has suggested that larvae of Nippostrongylus brasiliensis collect a layer of lipid around them that reduces evaporation and may eventually replace the water film and allow movement and penetration for longer periods. Skin lipids do not seem to play a part in the penetration process of A. tubaeforme as removal and replacement of the chloroform soluble fraction has no effect on the percentage of larvae entering skin or filter paper (Table III, Figure 6). ,The lipids appear to play no part in attracting larvae to the skin, in stimulating penetration behaviour or in assisting in the process of penetration. 55

Croll and Smith (1972) have demonstrated the very marked response of hookworm larvae to temperature changes and the evidence points to this factor being the most important in attracting larvae to the skin surface and initiating penetration behaviour.

Stirewalt (1971) and Austin et al (1972) have come to a similar conclusion with regard to schistosome cercariae, surface lipids acting as contact stimuli only. Figure 2 shows the effect of raising the temperature of the water circulating through the penetration apparatus. This has the effect of increasing both the steepness of the temperature gradient that the worm is in and also the actual temperature of the fluid above the membrane.

The effect of this on percentage penetration is indicated in

Figure 5, the curve is very similar to that given by Barrett

(1969b) for aronsyloides ratti activity and Al- Hadithi (1972) who looked at the percentage of A. tubaeforme larvae migrating towards the hot end of different temperature gradients. The similarity between the purely thermotactic response shown by

Barrett and Al-Hadithi and the penetration results in Figure 5 provides additional evidence that the initial entry into the skin may be mechanical. The temperature difference between the host skin and the environment results in an increase in activity which, if penetration is mechanical, is the time that it is most needed. Location of a suitable site and penetration before the surface moisture dries is of the utmost significance and depends on the worms being fully active. 56

SECT ION II

MIGRATION THROUGH THE DERMIS

AND

THE MECHANISM OF PENETRATION 57

SECTION II

INTRODUCTION

The great majority of investigations into the process of larval helminth penetration through the skin has been conducted on schistosome cercariae (Gordon & Griffiths, 1951; Standen, 1953;

Stirewalt, 1959, 1963, 1966; Clegg, 1969; Bruce et al, 1970; among many others). There seems to be little dispute amongst workers on schistosomes that cercarial penetration is assisted by enzymes produced by the worm, both pre- and post-acetabular gland contents are depleted during passage through the skin (Stirewalt &

Kruidenier, 1961).

Lytic activity has been demonstrated by a range of different methods; 1) by histochemical techniques (Lewert & Lee, 1954;

Stirewalt, 1956; Stirewalt & Kruidenier, 1961); 2) by the spread of dye following larval penetration (Kuntz, 1953; Lewert & Lee,

1954); 3) by swelling and disintegration of host tissues incubated with larval extracts (Gordon & Griffiths, 1951; Lewert

& Lee, 1954); and 4) by assay against specific substrates

(Lewert & Lee, 1956; Milleman & Thonard, 1959; Mandlowitz,

Dusanic & Lewert, 1960; Gazzinelli & Pellegrino, 1964). Of 58

these only Lewert & Lee (1954, 1956) and Mandlowitz et al (1960) included nematode larvae with the cercariae studied.

Looss (1911) described the movement of Ancylostoma duodenale larvae within the dermis with the words "the loose connective tissue does not appear to offer the least resistance to their advance". Because of the preponderance of work on cercarial penetration there seems to have been a tacit understanding that all penetrating helminths use enzymic secretions to effect entry.

As shown in Section I the initial entry into the stratum corneum can be explained in terms of a mechanical rather than an enzymic process. The purpose of the work in this section was to establish when, if at all, enzymic activity comes into play in the penetration of Ancylostoma tubaeforme

FILMED SEQUENCES OF LARVAL MIGRATION

MATERIALS AND METHODS

Two No. 0 cover glasses were cemented 5 mm apart on a glass microscope slide. Sections of skin both fresh and deep frozen, about 150 /um thick were cut by hand at right angles to the skin surface and mounted on the slide between the cover glasses. A third cover slip was applied to the top surface and light pressure 59

applied so that it rested on the two cemented glasses and was in intimate contact with the skin section. A suspension of A. tubaeforme in phosphate buffered isotonic saline was carefully introduced under the top cover glass and the larvae allowed to enter the skin. The process of movement within the skin was filmed under the microscope at 16 frames per second on K 11 type

A film using a Vinten 16 mm camera and phase contrast illumination.

The whole process was completed within 15 min of the death of the animal in the case of fresh skin, or within the same time of thawing in the case of deep frozen tissue, in order to reduce post-mortem changes to a minimum. After filming the skin was fixed in 70% ethanol and processed for histological examination.

RESULTS

Plate 9 presents a series of still frames taken at half second'intervals from the cine film. The anterior of the worm moved from inside the frame to the lower edge, a real distance of

77 iy m, in 3.5 seconds. This velocity of 22 io m/sec is about the mean for a number of analyses from various filmed sequences.

There was no visible evidence of enzymic activity, the dermis

"closed" after the passage of the worm and no evidence was found 6o

PLATE 9

Series of still frames taken at half second intervals from a

16 mm cine film showing an ensheathed A. tubaeforme larva

migrating through cat dermal tissue.

100/4n 7" 0 62

of any permanent tunnels or permanent damage to the tissue.

The dermis appeared as a soft gel-like material rather than the rigid fibrous lattice seen in fixed sections and was capable of flowing movements as the larvae moved through it. The undulatory locomotion of the larvae set up motion in the gel matrix which extended well over 100 iy m around the migrating worms. Eddies were set up around the worm and are visible in Plate 9. The larvae thus appeared to be moving smoothly through the dermis as if it were a highly viscous solution and their progress was not noticeably impeded by any structural fibre lattice.

The sheath was retained by some larvae migrating through the dermis and did not appear to cause any hindrance.

In some sequences it was possible to watch the larvae burrowing below the plane of focus, deeper into the skin and sections of the skin cut after filming showed worms throughout the tissues, confirming that the film was not an artefact of the larvae on the cut top skin surface.

Essentially the same picture emerged for A. tubaeforme in cat and rabbit skin although the latter was less viscous, and Necator americanus in human skin. 63

DERMAL MIGRATION IN VITRO

MATERIALS AND METHODS

As much as possible of the sub-dermal adipose tissue was dissected from a piece of clipped cat skin without damaging the dermis itself. This was inserted in the in vitro penetration apparatus that was set up as described in Section I (p. 15) but with the dermal surface uppermost and the epidermal surface in contact with the collecting cell fluid. On connecting the water circulation a temperature gradient was set up across the membrane but in the reverse direction from that normally obtaining.

One hundred third stage A. tubaeforme larvae were applied to the dermal surface and allowed to penetrate for times ranging from 30 min to 4 h. After the alotted time the skin was removed, fixed in

70% ethanol and processed for histological examination. 10 etc m serial sections were cut and each section divided by eye into five regions; 1) extending from the sub-dermal surface to a quarter of the way to the dermo-epidermal junction; 2) from a quarter to mid- way through the section; 3) from mid-way to three quarters;

4) from three quarters to the dermo-epidermal junction; and

5) the epidermis. The number of larval sections in each of the different regions in each section was counted.

Control tests using filter paper membranes were conducted concurrently to establish the normal behaviour of the penetrating larvae. Numerous tests conducted in vitro and in vivo with the 64

epidermis uppermost provided data on the normal course of pene-

tration through the li'ving layers of the epidermis and the dermal

tissue.

RESULTS

The percentage of the total number of larval sections found

in each of the regions of the skin at times up to 4 h is plotted

in Figure 8. After the first half hour all larvae were found in the surface quarter of the skin. As time progressed the pre-

ponderance of larvae at the surface became less pronounced and

there was a steady build up in the number of larvae in the deepest

region of the dermis (4) where they came to lie along the dermo- epidermal junction (Plate 10). On no occasion did any larvae

enter the epidermis or completely penetrate the skin in these

tests. The comparatively high and constant percentage of larvae

found in region 1 after half an hour is accounted for by the

retention of some of the sub-dermal adipose tissue. Once within

the lipid globules the larvae appeared to have difficulty escaping

from the cells.

Table IV summarises data on total penetrants collected from • a large number of in vitro tests on cat and rabbit skin with both FIGURE 8.

100

80 X z °X 060

0 on z1 40 V o. 20

0 2 REGION OF SKIN

0.5 1 2 4 TIME (HOURS)

Dermal migration of A. tubaeforme. Each block represents the percentage of sections of worms found in each of four equal thickness regions of the skin at times up to 4 h after exposing100 larvae to the dermal surface of cat skin in vitro. Region 1 is the dermal surface quarter and region 4 the quarter next to the dermo-epidermal junction. PLATE 10.

Section of exsheathed A. tubaeforme larva in the dermo-epidermal region of cat skin after migration from the dermal surface.

Haematoxylin and eosin. X 350

the epidermis and dermis uppermost. There is a ten-fold difference in the percentage of larvae totally penetrating the same type of membrane with reversed orientation. The actual numbers completely

penetrating with the dermis uppermost are so low that they may have

been caused by experimental errors from contaminated glassware.

Complete removal of the epidermis so that the membrane consisted of dermis only resulted in up to 40% of the larvae completely

penetrating the membrane in 2 h. The dermo-epidermal junction

thus appears to act as a barrier to the migration of the larvae. figure 9 gives frequency distributions for the numbers of larvae

totally penetrating cat and rabbit skin membranes with the

epidermis uppermost. 69

Table IV

Infective A. tubaeforme completely penetrating cat and rabbit skin

in vitro

Tissue No. of Total larvae No. totally % penetrants tests administered penetrating

Rabbit skin Epidermis up 40 3,550 148 4.20 Dermis up 8 950 6 0.63

Cat skin Epidermis up 67 4,200 75 1.80 Dermis up 22 2,500 5 0.20

TIME COURSE OF PENETRATION

As stated in section I and illustrated in Figure 7 invasion of the skin continues for at least 4 h. The analysis required to obtain these data involved digestion of the skin and the time course of penetration was not obtainable, this information is 70 FIG.

'61 30 RABBIT

10 12 14 16 18 20 22 24 total penetrants

s. I30 CAT

10 12 14 16 18 20 22 24 total penotants

Frequency distributions of totally penetrating A. tubaeforme larvae recovered from rabbit and cat skin in vitro. 71

available however from the sections cut after both in vivo and in vitro penetration tests.

The precise time course varies from skin to skin; for the cat$ ensheathed larvae have been found within the stratum corneum after 5 min. The living layers of the epidermis of the cat are thin,--usually only two or three layers of cells, and penetrating larvae have only rarely been found within them. Large numbers of sections have been examined and no evidence of epidermal cellular destruction has been found following passage of A. tubaeforme into the dermis. In young mice the germinal layers are thicker and larvae have been found among them. Plate 11 shows such a worm, the layers of cells have been displaced but there is no evidence of cellular destruction (c.f. Plate 14 ) and the worm appears to be passing between the layers of cells rather than

digesting them. The first larvae to penetrate through into the

dermis have been found after 60 min and some may penetrate the skin completely in this time.

No evidence of cellular reaction was found in in vivo tests of

up to 4 h duration.

Ensheathed larvae have regularly been found within the dermis

(Plates 12 and 13) and up to 5 of larvae that completely

penetrated the skin were still ensheathed. Exsheathment may occur

at any point in the penetration process and discarded sheaths 72

PLATE 11.

Section of A. tubaeforme exsheathed larva within the stratum

granulosum of day old mouse skin.

Haematoxylin and eosin. X 1,400.

7L4.

PLATE 12.

Section of ensheathed A. tubaeforme within the dermis of the

cat following penetration through the epidermis.

Haematoxyiin and eosin. X 2,500

PLATE 13.

Two sections of the same A. tubaeforme larva exsheathing within

the dermis of the cat following penetration through the epidermis.

Haematoxylin and eosin. X 1,500. 77 78

have been found on the surface, within the epidermis and within the dermis. Plate 13 shows two sections through the same worm exsheathing within cat dermis. The exsheathed portion of the worm is surrounded by a clear area whereas the tissues are closely apposed to the sheath with the worm loose within it. The measure- mentsPresented in Table V confirm that alcohol fixation causes more severe shrinkage to the worms than to the sheath.

Table V

Mean and standard deviation of the diameter of living and alcohol

fixed A. tubaeforme larvae

Living larvae Fixed larvae

Diameter of sheath 27.0 + 1.23 ft m 23.9 + 3.9 Acm

Diameter of worm 21.6 + 0.42 /m 13.7 + 2.7 ia m

INFLUENCE OF pH AND IONIC CHANGES ON PENETRATION

Lewert & Lee (1956) found that the addition of various cations to the incubation medium affected azocoll dye release with S. ratti 79

+++ and S. mansoni extracts and Clostridium welchii toxin. As ++ ++ Ca and Mg ions all activated the extract to some extent.

Chelation by the disodium salt of ethylene diamine tetraacetic acid

(E.D.T.A.) caused almost complete inhibition of the activity from

S. ratti extract and C. welchii toxin but did not affect S. mansoni

extract.

Lewert, Hopkins & Mandlowitz (1966) showed an increase in ++ ++ infectivity of S. mansoni when additional Ca and Mg ions were

present in the medium and a decrease in infectivity in the presence

of E.D.T.A. Lee (1972) has suggested that local ionic changes in

the immediate environment of migrating N. brasiliensis larvae may

be sufficient to ease the passage of the worms through the skin.

A series of experiments were thus conducted to investigate the

effect of various ions and changes in pH on the percentage of

A. tubaeforme larvae entering cat skin.

MATERIALS AND METHODS

Deionized distilled water was used to prepare solutions -3 ++ 3 ++ -3 -4 containing 10 M Ca , 10 M Mg , 10 M E.D.T.A., 5 x 10 M A.T.P.

and M/20 phosphate buffers of pH value 6.1, 7.2 and 8.6.

Infective, third stage A. tubaeforme larvae were isolated from

faecal cultures, washed thoroughly by centrifugation initially with 8o

water and finally with the relevant solution and suspended in fresh solution overnight. Aliquants containing 50 larvae were removed and applied to the top surface of clipped cat skin in the in vitro penetration apparatus. After 1 h the non-penetrants and total penetrants were removed and the exposed area of skin digested in acid-pepsin overnight to remove the larvae that had entered the skin. Each test was repeated at least eight times.

RESULTS

The mean percentage of larvae entering the skin after treatment with the various ions is listed in Table VI. There was no significant difference between the water controls and the various ionic solutions. There was some indication of a slight increase in the percentage penetrating at an alkaline pH over those at pH6.1 but this result was not significant at the 5% level. 81

Table VI

Mean and standard error of percentage A. tubaeforme larvae entering clipped cat skin in 1 h following treatment with various

solutions

Treatment Mean %

Water controls 26.7 + 3.6 -3 ++ lo m Ca 21.5 ± 3.9 10 3M Mg++ 22.9 + 4.3 10 3M E.D.T.A. 28.4 + 6.8 -4 5 x 10 m A.T.P. 28.0 + 4.6 M/20 phosphate buffer pH 6.1 10.2 + 1.7 7.2 10.0 + 2.1 8.6 17.3 ± 3.3 82

ASSAYS FOR ENZYME ACTIVITY

a) Dye release from azocoll

Azocoll (Wellcome) is an insoluble, denatured hide collagen to which an azo-dye has been coupled (Oakley, Warrack & van

Heyningen, 1946). The dye is released into solution when exposed to an enzyme capable of rupturing the link between it and the substrate. The release of the dye may be measured spectrophotometrically to give a quantitative assay of the activity of the enzyme concerned (Lewert & Lee, 1956).

MATERIALS AND MNAHODS

15 x 103 A. tubaeforme, 20 x 103 N. americanus and 12 x 103

Strongyloides fUlleborni infective larvae were isolated and washed thoroughly in three changes of distilled water and two changes of isotonic phosphate buffered saline at pH 7.2 before suspension in

8 ml ftesh saline. Each group was divided into two, one half was killed by raising the temperature to 100°C for a few seconds.

A control consisting of 10 mg/ml pepsin in phosphate buffered saline at pH 7.2 was included.

Each sample was made up to 5 ml with saline in a 10 ml centrifuge tube, 25 mg azocoll was added to each tube and they 83

were incubated in a 37°C water bath. At alotted time intervals the tubes were centrifuged and the supernatant removed and its absorbance measured spectrophotometrically. After measurement the supernatant was returned to the tube for further incubation.

Initially a spectral absorption curve was prepared for the azocoil dye released after incubation with pepsin using a Beckman spectrophotometer, this showed a peak absorbance at 540 nm

(Figure 10). Samples were measured on both the Beckman spectro-

photometer at 540 nm and a Unicam SP 1300 Series 2 colorimeter using an Ilford 625 filter with a transmission range of 510-590 nm.

No significant difference was found between the readings'for the same samples with the two instruments and the colorimeter was used

for subsequent assays. Fresh phosphate buffered saline was used as the control blank. At the end of the experiment larval viability

was checked by assessing activity.

RESULTS

The absorbance of the dye released by the various samples

during 4 h incubation at 37°C is plotted in Figure 11. The curves for different species of heat killed worms were virtually

identical and a single representative curve has been plotted for

3S0 400 450 SOO SSO 600 650 700 750 wave length nes FIGURE 10. .Spectral absorption curve for dye released from azocoll.

1.4

1.2

1.0 • c 0

•.6 .0 0 .4

1 3 4 time (hours)

FIGURE 11. Azocoll dye release from infective nematode larvae over 4 h. Circle 10 reml pepsin; inverted triPngles, 10 x 10 N. amorionnu5: squnres 3 6 x 10 S. fUlleborni; triangles 7.5 x 10 A. tubaeforme; diamonds, 7.5 x 105 heat killed A. tubaeforme. 85

clarity. There was no dye release from A. tubaeforme larvae but both S. fUlleborni and N. americanus larvae produced an azocoll positive secretion indicating that the method was sufficiently sensitive to detect biological levels of enzyme activity. No marked mortality was found among the larvae.

b) Release of radio-isotopes from labelled proteins

Smith (1972) in a study of the release of enzymes from schistosome eggs, developed a technique that is possibly the most sensitive of all assay systems for proteolytic enzymes. . The

leophilic sites of a protein substrate were externally labelled with 1251 (Hunter & Greenwood, 1962). Any proteolytic activity released labelled peptides which remained in solution when the protein was precipitated with trichloroacetic acid. Addition of laatalbumin hydrolysate provided a more complete release of the peptides as it competed with them for sites on the polypeptide chain of the precipitated protein.

MATERIALS AND METHODS

0.2 ml bovine serum albumin externally labelled with 1251 to an activity of 25 "Ci/100 jig was incubated at 37°C with 7.5 x 103 86

living A. tubaeforme larvae, 7.5x103 heat killed larvae and

10 mg/ml trypsin (A.R. Monk and Co.) each in 0.5 ml 0.1M tris/HC1

bUffer at pH 8.6. At each time interval 100 ,tl aliquots were removed, 1 ml 10% trichoroacetic acid containing 0.1% lactalbumin hydrolysate was added and 100 ,tl 1% cold bovine serum albumin to ensure precipitation of any remaining iodinated substrate. The

precipitated protein was spun down at 5,000 x g and 0.9 ml of the supernatant removed, filtered through a Whatman No. 1 filter paper and counted for 5 min in a Packard gamma spectrometer.

RESULTS

Figure 12 presents the counts per 5 min over the 3 h of the experiment. There was no difference between the living and dead larvae. The counts from the larval samples were so low that no evidence for enzymic activity was indicated.

c) Haemolytic activity of larval secretions

Since neither of the previous assay methods had shown any proteolytic activity emanating from infective A. tubaeforme larvae a method was devised to investigate the possibility that some other factor might be present that could affect cell walls without 87

'FIGURE 12.

500.d

400 ,

c 300

• 4- 200.

100 '

1 2 3 time (hours)

125 Release of labelled peptides from I bovine serum albumin. 3 Circles, tryrsin;, triangles 7.5 x 10 A. tubaeforme; squares, 7.5 x 103 heat killed A. tubaeforme. 88

causing gross histolysis. Such a factor, if present, might ease the process of penetration through the epidermal layers without necessarily leaving histological evidence of its presence.

MATERIALS AND METHODS

The in vitro penetration apparatus was set up with No. 4 filter paper membranes and buffered isotonic saline in the collecting cells. 7.5 x 103 A. tubaeforme, 10 x 103 N. americanus and 6 x 103 S. fUlleborni in 0.2 ml saline were placed on the top surface and allowed to penetrate for 5 h, the fluid level was maintained with distilled water as evaporation occurred. After the time the apparatus was dismantled and the larvae from above and below the membrane together with the saline and the membranes themselves were collected, made up to 0.5 ml with isotonic saline and centrifuged. The supernatant was removed and used for assay.

Fresh isotonic saline, 10 mg/ml pepsin and 0.1 mg/ml lysolecithin

(Koch Light) in isotonic saline were used as controls.

Rabbit red blood cells were thoroughly washed by centrifuging three times in isotonic phosphate buffered saline and suspended in fresh saline. The number of cells was counted using a dilution of 1 : 200 and a haemocytometer with Neubauer ruling following the method of Archer (1965). 0.4 ml of each of the test extracts and 89

0.4 ml of the blood suspension were mixed and the cell number counted. 0.1 ml of these suspensions was added to 0.1 ml of different saline concentrations and the cell number counted at each concentration. Each count was performed ten times, and the counts corrected to compensate for the dilution effect.

RESULTS

The mean red blood cell counts at different saline concentrations are plotted in Figure 13. Lysolecithin has a very powerful haemolytic activity but in low concentration (0.1 mg/ml) at which lysis did not occur in isotonic saline there was an effect on the cell walls that resulted in lysis when the concentration was reduced to 0.3 - 0.4 %. The cell counts were not significantly different (P = 0.05) for any of the samples at saline concentrations above 0.4%; below this value the lysolecithin values were significantly lower than the others which did not differ among themselves. The proteolytic activity of pepsin did not cause haemolysis and there was no evidence of any lytic activity from the larval extracts.

DISCUSSION

The barrier faced by skin penetrating larvae is a complex FIGURE 13.

M E

0 C C o

.4 .5 .6 .7 percentage saline

Effect of saline concentration on haemolysis of rabbit red blood cells incubated with supernatants from various infective larvae. Solid symbols: squares, SILwyloide ; circles, Ancylostoma tubaeforme; inverted triangles, Necator americanus; diamonds, 0.9,6 saline; triangles, 10 mg/ml pepsin. Open squares, 0.1 m ml lysolecithin. 91

one. The structure and composition of the outer layers of the stratum corneum and the process of entry into them by A. tubaeforme have been described in the previous section.

Beneath the stratum corneum lie the living layers of the epidermis. The thickness of these layers varies widely from species to species and in different regions of the same animal.

The epidermis of the abdominal skin of small laboratory animals is thin, usually no more than three layers of cells and about

20 Jim thick. These cells, unlike the stratum corneum, are metabolically and mitotically active and produce the keratinIzed cells that make up the stratum corneum (Jarrett & Spearman, 1964).

Desmosomal junctions link the membranes of apposing cells which are thus held closely at these points (Breathnach, 1971). The dermo-epidermal junction is marked by a basal lamella composed of a P.A.S. positive, polymucosaccharide containing protein. Lewert

& Lee (1954) in their study of the effect of helminth secretions on skin tissues, followed the suggestion of Gersh & Catchpole

(1949) and considered the basal lamella as being continuous with the acellular ground substance of the dermis. The excellent electron micrographs of Breathnach (1971) however, show hemi- desmosomal junctions between the basal layer of epidermal cells and the basal lamella and it now appears that it is epidermal rather than dermal in origin. 92

The dermis consists largely of acellular material. The generally accepted view is that the collagen which makes up

70% of the dry weight of the dermis, occurs in fibre bundles which are visible in both the light and electron microscope.

These fibres are thought to be accompanied by elastic fibres, the whole being embedded in an amorphous, acellular ground substance.

Jarrett (1958) found that the only fibres observable in fresh, unfixed, cryostat cut sections stained with an intra-vital fluorochrome dye, were removed by elastase. He concluded that these were the elastic fibres and that the collagen was in a relatively unpolymerised, non-fibrillar state. Formally' fixation resulted in gross changes which when followed by dehydration and wax impregnation gave preparations in which the dermis appeared to be composed of bundles of collagen fibres.

It is probably not a coincidence that "good" fixatives contain aldehyde groups as these are of importance in the formation of cross linkages between tropocollagen molecules. Two types of linkage are known, both are dependant on the conversion of a lysine residue to its aldehyde derivative, allysine.

amine R - (CH )- NH > R1 - (CH2) - CHO 1 2 2 oxidase

This unsaturated aldehyde can then react with an adjacent amine 93

group of another lysine residue in a second molecule to form a

Schiff base band.

R (CH ) - CHO + H N - (CH ) - R 3> R1(cH2)3 - CH = N 1 2 3 2 2 2 -(CH ) - R + H2O 2 4 2

The other linkage is formed by the condensation of two aldehyde

groupings in adjacent molecules to establish an aldol link

(Jarrett& Matthews, 1973).

R - (CH CH + CHO - (CH2) 3- R Ri - (CH2)2 1 2 )2 I 2 2 1 CHOHO

=CH(CH ) - R + H2O 2 3 2

The filmed sequences of larvae moving within fresh, unfixed

dermal tissue provided a dynamic demonstration of the in vivo

state of the dermis. No evidence of a rigid lattice of collagen

fibres•was found. The ease with which the larvae were able to

move through the tissue and the eddies set up around the worms

supported the idea that the collagen is in an unpolymerised state.

The "blocks" of collagen visible in Plates 12 & 13 thus appear to

be fixation artefacts. 94

The clear "tunnel" around the exsheathed larvae shown in

Plates 10, 11, 13 would also appear to be artefactual. Shrinkage

of the larvae is much greater than the sheath (Table V) and no

clear area is visible around the sheath of ensheathed larvae

(Plates 12, 13) although the worm has shrunk within it. In the

film sequences it was not possible to follow the route taken by

the migrating larvae after they had passed through and no permanent

damage to the tissue was observed. Stirewalt (1958, 1959)

reported that the path of migrating S. mansoni cercariae in mouse

skin was marked by a loss of continuity of the hOst tissue and

by the necrosis of cells but no such reaction was found to

A. tubaeforme and an alternative penetration mechanism appears

likely.

The ease and rapidity of passage through the dermis, coupled

with the retention of the sheath, the non-formation of permanent

penetration tunnels and the consistency of the dermis that emerged

from the film analysis all suggest that enzymic activity may play

little•or no part in dermal migration of A. tubaeforme. This is

further borne out by the fact that complete penetration of the skin

occurs in approximately the same time as migration through the

living layers of the epidermis. The dermal tissues thus apparently

offer little resistance to migrating larvae, as reported by Looss

(1911). 95

Lewert & Lee (1954) used a range of methods to compare the penetration of various helminths. They found histological and histochemical evidence of changes in the basal lamella and dermal ground substance after passage of schistosome cercariae, S. ratti,

S. simiae and A. caninum larvae. These changes were interpreted as indicating the production of an enzyme that acted to depoly-

merise the polysaccharide containing protein of the basal lamella and thus effect entry into the dermis. Penetration by N. muris larvae however, was effected without such changes, although there

was an increase in the solvent water in the dermal connective

tissue as shown by the Evans blue technique.

Lewert & Lee (1954) also showed azocoll positive activity

from living S. ratti, S. simiae, S. mansoni and A. caninum but

were unable to do so for Trichinella spiralis and N. muris. The

results for A. caninum were equivocal and in a subsequent quanti-

tative study (Lewert & Lee, 1956) they found no activity from

A. caninum larvae. Figure 11 illustrates similar results for

A. tubaeforme. Both N. americanus and S. ffilleborni larvae

showed an azocoll positive secretion which suggests that the method

used was sufficiently sensitive to detect enzymic activity that

was present. Azocoll is a much modified substrate and Milleman

& Thonard (1959) have presented evidence that activity against it

may not reflect true collagenolytic activity against native collagen. 96

They have stressed that the demonstration of positive proteolytic activity is dependant on the substrate used. A generalized protein, bovine serum albumin, was used for the radio-assay as it was considered that any proteolytic action was more likely to be demonstrated this way. In this method the actual site of cleavage is not important as it is only necessary to split the substrate protein into small enough sub-units to remain in solution after precipitation with trichloroacetic acid. Smith (personal communication) has shown enzyme activity from as few as 500 schistosome eggs using this method and the absence of any activity from 7,500 A. tubaeforme larvae provides strong evidence that there is no proteolytic activity from secretions from these worms.

There is a possibility that an enzyme is present but was not released under the experimental conditions. A temperature rise from ambient (17 - 20°C) to 37°C produced azocoll positive reactions from N. americanus and S. fillleborni and it is considered likely that these conditions would have released any enzyme from

A. tubaeforme.

If A. tubaeforme produced a powerful proteolytic enzyme to digest a route through the skin it might be anticipated that equal numbers of larvae would be able to penetrate from either the dermal or epidermal surface. This result was not found however

(Table IV ) and larvae come to rest along the dermo-epidermal junction (Figure 8, Plate 10). The results presented in Figure 8 97

are unfortunately not open to statistical analysis. It has been assumed that the number of worm sections is directly related to the actual number of worms present, an assumption that relies on random orientation of the larvae in each region. Regions 1 and 4

however have different characteristics from 2 and 3 and these may

have affected the larval orientation and consequently the number of sections into which they were cut. It is felt that the errors introduced by this change in orientation are unlikely to have altered the overall result, and that the general build up of larvae along the dermo-epidermal junction is the true one. The dermo-

epidermal junction thus appears to act as a barrier that• the larvae cannot pass when penetrating from the dermal surface. Duke

(1971) has suggested that Onchocerca microfilariae, which are able to migrate through the dermis, do not pass the epidermal basal lamella and are thus restricted to the dermis whence they are ingested by biting simuliids.

Lee (1972) has presented electron microscopic evidence that

N. braziliensis larvae enter rat and mouse skin by passage between the epidermal cells and, although he found evidence of collagen

fibre breakdown around the larvae in the dermis, no widespread cellular destruction was found. Indeed, dermal cells close to the migrating worm were apparently unaffected. The conclusion

drawn was that a change in pH or the ionic state in the immediate 98

environment of the worm may have been sufficient to cause such microscopic changes. Histological studies have provided no evidence that cellular destruction occurs in the epidermis during the passage of A. tubaeforme and it is thought that they are also able to pass between the epidermal cells. The haemolysis experiment provided no evidence that the larvae produced a factor with specific activity against cell membranes. The presence of such a factor acting on the desmosomal junctions of the epidermis would consider;.bly ease the problem of penetration and it is still possible that some undetected factor may emanate from migrating worms.

Cellular reactions to oral secretions of haematophagous arthropods are well known and appear rapidly, some skin reaction often appears within 10-15 min following the bite (Benjamini &

Feingold, 1970). Larrivee et al (1964) showed that there was a marked variation between guinea pigs exposed to bites of Cteno- cephalides fells fells but when a response did occur it was manifest within 20 min although development might continue for 24 h.

Immediate reaction of this type only occured after prior sensitization of the skin with the antigen. Although the cats used for in vivo tests had low residual patent A. tubaeforme infections or had just lost such an infection, these had been acquired by ingestion or sub-dermal injection of infective larvae and not 99

percutaneously. It is doubtful whether an intestinal helminth acquired via an alternative infection route, is able to sensitize the skin to subsequent penetration by a larval stage, and the absence of a cellular reaction following passage of A. tubaeforme into cats in vivo for up to 4 h is not surprising. Taliaferro &

Sarles (1939) found that cellular changes did not occur until 8 h after infection with N. brasiliensis and Lee (1972) with the same species found no response in his experimental exposures of up to 4 h.

A body of evidence has been built up against the presence of a proteolytic mechanism of penetration by A. tubaeforme and an alternative explanation of the penetration process is thus required.

This is summarised diagrammatically in Figure 14. Larvae placed on the epidermal surface of the skin enter the stratum corneum by undulatory activity (Section I), they orientate parallel to the skin surface and probably follow the weaker junction between the stratum corneum and the upper layer of the stratum granulosum.

The exact point at which the larvae penetrate through the epidermal layers has not been observed but entry via the hair follicles and their attendant sebaceous glands would appear to be unusual.

Plate 11 suggests that the larvae separate the epidermal cells and presumably this involves breaking the desmosomal junctions uniting the cell membranes. 100

FIGURE 14.

Diagrammatic summary of proposed penetration mechanism by A. tubaeforme. A. Uninfected epidermis with stratum corneum, stratum granulosum and basal lamella. B. Larva orientated parallel to the skin surface moving along the junction between the stratum corneum and stratum granulosum. C.)Larval penetration between the cells of the stratum granulosum, D.)separating the cells and placing the basal lamella under tension. E. Complete separation of the epidermal cells and passage into the dermis. F. Larva approaching the epidermis from the dermal surface faced by the smooth unbroken basal surface of the epidermis. 101

Little is known of the exact nature of the adhesive forces acting on cells in tissues. A number of hypotheses have been put forward (reviewed by Curtis, 1972). Szent-Gyorgyi (1969) presented two mechanisms of equal strength capable of holding cells together, one used a small number of strong bonds and the other a large number of weak ones. The former would result in a rigid whereas the latter would provide a supple tissue. If each of the bonds in a multiple bond junction is weak only a weak force is required to break it, providing the separation is initiated at one end uncovering successive bonds as rupture proceeds. The almost fluid deformation of the skin suggests that epidermal cells may be joined by this type of junction.

It has been suggested that a balance between electrostatic forces of repulsion and attraction arising from the London-van der

Weals dispersion forces between two surface membranes may play a part in cell adhesion (Curtis, 1972). One of the characteristics of this type of bond is that it is of low energy and easily broken.

Any change that occurs to influence the surface charge will affect the forCes of attraction and repulsion. If this type of bond occurs between the cells of the stratum granulosum it is possible that an influx of fluid with the migrating worm may be sufficient to upset the surface charge and thus initiate cellular separation.

Once started the process is progressive as each bond is exposed 102

to the disrupting medium. The movements of the worm would act to physically separate the cells. The basal lamella is joined to

the basal layer of the epidermis by hemidesmosomes, if the cells are pushed apart the basal lamella will be placed under tension and may rupture to allow the worm to pass through into

the dermis. The natural resiliency of the cell layers will tend

to close the gap through which the worm has passed and the bonds reform as the surface charge equilibrium is restored leaving no

histological evidence of passage.

The retention of the sheath may contribute to the transfer

of fluid into the skin. Attempts to improve the visibility of

larvae by vital staining with methylene blue were unsuccessful.

_Exsheathed larvae were not stained but the dye was able to

penetrate the sheath and the larvae appeared blue. On return to

water the dye migrated from the sheath leaving the larvae unstained.

If the epidermal cells are parted by specific ionic changes these

ions may be held in solution within the sheath to be expressed

during. penetration.

Changing the ionic concentration of the solution from which ++ ++ the worms penetrated with Ca and Mg ions did not significantly

affect the percentage of larvae entering cat skin (Table VI). ++ ++ Lewert et al (1966) found that both Ca and Mg ions increased

the penetration ability of S. mansoni cercariae, they correlated 103

this effect with the increase in enzyme activity found in vitro

(Lewert & Lee, 1956). Chelation of the metal ions with E.D.T.A. or A.T.P. which greatly reduced penetration by S. mansoni did not affect the penetrative ability of A. tubaeforme.

Stirewalt (1966) has pointed out that alkaline conditions cause- keratin to swell and soften. She has related the alkaline secretions from the post acetabular, mucus-producing glands of

S. mansoni cercariae with their ability to penetrate the horny layer. The slight increase in penetration found at pH 8.6 may have been due to an effect on the intercellular membranes but a direct effect on the keratin of the stratum corneum appears more likely. The fact that at both pH 6.1 and 7.2 the percentage penetrating was equal suggests that there was no effect on cell adhesion.

The epidermis is thin and the posterior of a penetrating worm remains in the stratum corneum when the anterior has passed into the dermis. In this way the keratin of the stratum corneum provides a rigid structure against which the worm can push to develop the forward motion needed to effect entry. When placed on the dermal surface of the skin the larvae migrate rapidly through the dermal tissues until they reach the dermo-epidermal region where they are faced by the smooth, unbroken surface of 104

the basal lamella and lower surface of the tightly inter-connected

basal layer of epidermal cells. The worms are in the semi-

fluid dermal tissue which is, itself, less dense in the dermo-

epidermal region and considerable slip accompanies undulatory

movement. Any fluid that may have entered the dermis with the

worms-initially is unlikely to have been transported across the

thickness of the dermis. With no solid substrate against which

to obtain purchase they are unable to break the epidermal layers

and penetrate. If the epidermis is removed the larvae are

.readily able to migrate and completely penetrate a wholly dermal

membrane. 105

SECTION III

INFLUENCE OF AGEING ON ACTIVITY AND PENETRATION

A. TUBAETORME 106

SECTION III

INTRODUCTION

The infectivity of animal parasitic nematodes has been

investigated by a number of in vivo and in vitro methods. Rogers

(1939) used the "floating raft" method of Goodey (1922) to establish

the percentage of infective larvae of the "cat strain" of

Ancylostoma caninum that would penetrate the skin of young mice

and Barrett (1969) used the same method with Strongyloides ratti.

In vivo tests have been used by Haley and Clifford (1960) for

Nippostrongylus brasiliensis, Rogers (1940) and Rose (1963) for

Haemonchus contortus and Elliott (1952) for Ascaridia galli. In

each of these infectivitywas assessed by counts of adult worms at

autopsy or by counting eggs per gram in faeces at known times after

a standard infection with aged eggs or larvae..

After hatching, the first and second stage hookworm larvae are

microbivorous and synthesise a lipid reserve that is used by the

infective larvae following the second moult (Croll, 1972b). This

lipid reserve is reduced with time at different rates under various

storage conditions (Croll, 1972a). Rogers (1939, 1940) and Rose

(1963) have suggested that infectivity and longevity are increased

after storage at reduced temperatures compared with storage at

room temperature, and this has been correlated with the decrease

in the lipid reserve of the larvae." Many authors have related 107

the loss of lipid with the loss of infectivity to such an extent that it has been assumed that the physiological age of larvae may be determined by assessment of the level of lipid reserve (Payne,

1923; Rogers, 1939, 1940; Wilson, 1965; Barrett, 1969; Clark,

1969; among others). Rogers (1939) and Barrett (1969) are among the few authors who have measured the activity of larvae at different ages. It has been stressed in Sections I and II that penetration

by A. tubaeforme is an active process with little if any, assistance from enzymic activity. A study was thus undertaken to assess

the effect of changes in lipid level and activity with those of

penetrability of A. tubaeforme.

MATERIALS AND METHODS

Larvae were cultured and separated after three days as

described earlier (Croll, 1972a). Several hundred larvae were

stored in 150 ml bottles, containing 100 ml of 1.5% saline in

M/20 phosphate buffer at pH 7 (isotonic, Croll, 1972a) or 100 ml

of 1.5% buffered saline plus 0.1 mg/ml neostigmine bromide at 10°C

and 26°C. Larvae were stimulated by bubbling air through the 108

solutions for either 15 min daily or for 2 h periods with 2 h rest periods.

Three measurements were made of larval activity: percentage moving, rate of movement and percentage penetrating into a filter paper membrane. As shown in Figure 4 (p. 28) penetration into filter paper is linearly related to entry into skin and a preliminary experiment showed no significant difference between results obtained using natural or artificial membranes. All subsequent tests were conducted using filter paper membranes.

Larvae were removed daily and transferred to a solid watch glass in the storage solutions in light at 30° + 2°C on a microscope stage. After 15 min the percentage active and rate of activity, in undulations/min, were measured. Infectivity was assayed by transferring 50 larvae in the storage solutions, to the top surface of a No. 4 filter paper membrane in the in vitro penetration apparatus and collecting the penetrants after 2 h.

A sample of freshly extracted larvae, prior to storage, was stained in Oil Red 0, for unbound neutral lipid (Croll 1972a).

Following each activity assay the larvae were similarly stained, care being taken to ensure that dead larvae were not included.

The possibility that larvae with a high lipid level would penetrate more rapidly was checked by measuring the lipid level of both penetrants and non-penetrants for various penetration times. 109

All samples were mounted in glycerol and measured at 517 nm, using a Vickers M85 scanning microdensitometer (Croll, 1972a).

RESULTS

The percentage active, rate of activity and percentage penetrants all decreased with time, from a maximum immediately after the second moult. The form of the decrease varied somewhat depending on the parameter measured. The percentage active dropped faster than the rate of activity (Figs. 15 & 16). The product of percentage active and rate of activity gave a measure of the overall "population activity", the rate of decrease of this parameter with time, measured as the slope of the fitted linear regression lines, did not differ significantly at the 5% level for any of the treatments (Fig. 17). The relationship between percentage active and activity rate was approximately linear

(Fig. 18). Percentage activity decreased linearly with time whereas activity rate decreased logarithmically. The portions of the curves over which the results were measured gave linear correlations significant at P = .01. The slopes of the three lines, which reflect the change in percentage activity with 110

FIGURE 15.

2 4 6 8 10 12 14 larval age (days)

Percentage activity at 30°C of A. tubaeforme larvae stored under various conditions. Squares, 1.5% saline; circles, 1.5% saline plus 0.1 meml neostigmine bromide; triangles, 1.5% saline bubbled intermittentlg. Solid symbols represent storage at 26°C, open symbols 10 C.

111 FIGURE 16.

y 40 C: O O "tz 20

larval age (days)

Activity rate in undulations/min at 30°C of A. tubaeforme larvae stored under various conditions. Squares, 1.5% saline; circles, 1.5% saline plus 0.1 mg/ml neostigmine bromide; triangles, 1.5% saline bubbled intermittently. Solid symbols represent storage at 26°C, open symbols 10°C. 112

FIGURE 17

ft 18 0

X 16 )10 00 woo tow 14 a 12 O 10

r 0.5 1.0 1.5 log time

"Population activity" of A. tubaeforme larvae stored under various conditions. Squares, 1.5 saline; circles, 1.5% saline plus 0.1 mg/ml neostigmine bromide; triangles, 1.5% saline bubbled intermittently. Solid symbols represent storage at 26°C, open symbols 10°C. Fitted linear regression lines have been plotted through the data. FIGURE 18.

ti 40 • /1'0.958 • "1".■ 0 a /1 0.938 '' 20 ca

10 20 30 40 50 60 70 80 90 100 percentage motile

Relationship between activity rate (undulations/min) and percentage activity of larvae stored under different conditions. Squares, 1.5% saline; circles 1.5% saline plus 0.1 mg/ml neostigmine bromide, triangles, 1.5% saline bubbled intermittently. Fitted regression lines y = 1.21 + 0.1325x, y = -10.48 + 0.3115x and y = -0.67 + 0.2364x respectively are plotted on the data and correlation coefficients added. 114

activity rate, do not differ significantly.

The percentage of larvae entering a filter paper membrane decreased logarithmically with time (Fig. 19). The presence of neostigmine bromide, which increased activity at any given age, also increased penetration. Figure 20 gives curves for the relationship between the percentage of larvae penetrating, their rate of activity and the percentage active. The direct relationship between the logarithms of these parameters confirms that activity is the major factor in larval penetration.

The storage conditions were selected so that at any given chronological age of a single batch of larvae a range of lipid levels was obtained (Fig. 21). Storage at 10°C resulted in a higher lipid level than storage at 26°C after about four days, o regardless of other conditions. At 10 C larvae were immobile, even in the presence of neostigmine bromide and after mechanical stimulation, however activity was restored on return to 26°C.

For any given lipid level the percentage active, activity rate and percentage penetrants were higher in the presence of neostigmine bromide, but the rate of decrease was the same as control larvae in isotonic saline (Figs. 22-24).

Figure 25 is typical of results obtained by staining both penetrant and non-penetrant larvae. There is no significant difference between the levels and no indication that larvae with higher lipid levels penetrate more rapidly. 115

FIGURE 19.

60- of

Cti 50 4.1

40 (7, C • •

1:4 30

a) 20 L • 42) ■ Ct. 0 10

4 4 8 lo 12 14 larval age (days)

Percentage of A. tubaeforme larvae penetrating a No. 4 filter paper.membrane after storage under various conditions. Squares, 1.5% saline; circles, 1.5% saline plus 0.1 mg/ml neostigmine bromide; triangles, 1.5% saline bubbled intermittently. Solid symbols represent storage at 26 C, open symbols 10°C.

FIGURE 20.

2.0

P 0.782 1.0-

1.0 2.0 1.0 2 . 0 log undulations/30 sec log percent motile

Relationship between log percentage penetrants and log activity rate and log percentage active. The plotted linear regression lines y = 0.25 + 0.9982x and y = -0.66 + 1.1248x respectively have been calculated from 34 separate results from the various experiments and correlation coefficients added.

117

FIGURE 21.

ep 9 cI3

7 2 4 6 8 10 12 14 larval age (days)

Lipid levels of P. tubaeforme larvae stored under various conditions. Squares, 1.5% saline; circles, 1.5% saline plus 0.1 mg/ml neostigmine bromide; triangles, 1.5% sant.% bubbled intermittently. Solid symbols represent storage at 26°C, open symbols 10°C. 118 FIGURE 22.

8 9 10 11 12 13 14 15 16 larval lipid Relationship between activity rate and larval lipid level of A. tubae- frome larvae stored in 1.5% saline, squares, and 1.5% saline plus 0.1 mg/ml neostigmine bromide, circles. Fitted linear regression lines y = -50.75 + 0.0495x and y = -23.052 + 0.0354x respectively are plotted and correlation coefficients added.

8 9 10 11 12 13 14 16 larval lipid Relationship between percentage activity and larval lipid level of A. tubaeforme larvae stored in 1.5% saline, squares, and 1.5% saline plus 0.1 mg/ma neostigmine bromide, circles. Fitted linear regression lines y = -136.84 + 0.1535x and y = -81.33 + 0.1297x respectively are plotted and correlation coefficients added. 119

FIGURE 24

40 e tag en

erc 20 • p

8 9 10 11 12 13 14 15 16

larval lipid

5 Relationship between percentage penetrant/and larval lipid level of A. tubaeforme larvae stored in 1.5% saline, squares, and 1.5% saline plus 0.1 mg/ml neostigmine bromide, circles. Fitted linear regression lines y = -32.5 + 0.017x and y = -63.5 + 0.002x respectively are plotted and correlation coefficients added. FIGURE 25.

2200

2100

2000

1900

1800

1700

16 00

1500

1400 ti 15 30 60 120 240 time (min.)

Lipid levels of A. tubaeforme larvae that penetrated, cross hatched, and did not penetrate, stipled, a filter paper membrane in vitro for various times. Each column represents the mean of at least 25 larvae. The vertical lines indicate standard errors of the means. 121

DISCUSSION

Most of the previous reports of nematode infectivity have indicated a more or less logarithmic decrease with time (Rogers,

1939, 1940; Haley and Clifford, 1960; Barrett, 1969; Herlich

& Ryan, 1970), and Olivier (1966) has shown a similar pattern for

S. mansoni cercariae. The present results confirm that A. tubaeforme also shows a similar decrease. Each of these studies has been conducted on a free living stage that relies to a large extent on activity to attain its definitive host. Even in the case of H. contortus and Trichostrongylus axei larvae that do not actively penetrate the integument of their host, migration from the faeces is important in increasing the chance of being ingested.

Elliott (1952) investigated the infectivity of Ascaridia galli where the infective stage is a fully embryonated egg rather than a free living larva. The shape of the curve that she obtained differed from that of other authors; there was a long plateau period.during which infectivity, never very high, remained fairly constant, followed by a rapid linear decrease. This difference in curve shape may reflect an evolutionary adaptation that has resulted in increased longevity of the infective stage. This could to some extent, compensate for the inability of the larvae to attain a position that will increase their probability of reaching their final host. 122

The longevity of larvae as indicated by laboratory experiments on infectivity, may be misleading as storage conditions play a large part in determining the final length of larval life. Rogers

(1940) showed that after storage at 26°C and 30°C the infectivity of H. contortus, measured as eggs per gram four weeks after feeding goats_1000 infective larvae, fell to zero after six and four weeks respectively whereas storage at 7oC resulted in a small residual infectivity continuing beyond seven weeks. Rose (1963) has subsequently shown that the maximum longevity of H. contortus varies from 35 weeks at 24-25°C to 87 weeks at 4-5°C, and that they remain infective to clean lambs for up to 8i months. The difference in storage conditions may explain the difference in time- scale obtained by Rogers (1939) for the "cat strain" of A. caninum at 37°C and A. tubaeforme in the present investigation. Biocca

(1954) and Burrows (1962) have presented evidence that A. caninum and A. tubaeforme are distinct species rather than physiological strains, and it seems probable that the parasite used by Rogers was in•fact A. tubaeforme. The results obtained by Rogers (1939) at 7°C are anomalous, infectivity was still about 60% after five weeks when activity had apparently fallen to a low level. Fig. 20 supports the idea that activity is the most important factor in penetration, and thus infectivity, in vitro and I am unable to resolve Rogers' result. 123

The logarithmic decrease in activity rate and "population activity" with time (Figs. 16, 17) confirm the results obtained by

Rogers (1939) and Barrett (1969). It is difficult to compare results quantitatively as the exact methods used to measure the parameters were different. Larvae were necessarily exposed to irregular thermal, photic and mechanical stimuli but these were standardised as far as possible. Both Rogers and Barrett measured the activity of 20 randomly selected larvae whereas in the present experiments 20 active larvae were selected. Although random selection has advantages, there is no way of distinguishing dead from non-motile larvae and this may introduce errors into any measurements. The approximately linear decrease in percentage larvae motile with time (Fig. 15) means that the combined curve

(Fig. 17) probably reflects most closely the total activity of the population.

Both Elliott (1952) and Barrett (1969) found that the lipid level fell rapidly initially and then more slowly. In the present series'of experiments when activity was present (26C in 1.5% saline) the lipid level and behavioural parameters decreased at a comparable rate (Figs. 15, 16, 17, 19). When stored at 10°C activity was not possible and the lipid level decreased more slowly, however when restored to conditions favouring activity, the larvae were no more active than controls that had been at 26°C throughout. The rate of decline in behavioural activity was very similar at 10°C or 26°C in 1.5% saline when bubbled for 15 min daily or intermittently 121+

for 50% of the time. Although lipid levels were not always appreciably reduced the metabolic mechanisms controlling behavioural activity had "aged" at a comparable rate to the controls. It is not clear whether the mechanisms involved are those governing the mobilization of energy or controlling behavioural activity itself.

A decrease in enzyme activity with age has been shown for a few nematodes. Erlanger & Gershon (1970) and Gershon & Gershon

(1972) working with the microbivorous Turbatrix aceti have illustrated that acetyl cholinesterase, ocamylase, malic dehydrogenase and isocitrate lyase all decrease in activity with time.• The latter enzyme was subjected to further investigation and it was shown that although the activity of the enzyme decreased there was an increase in protein precipitated by antibody formed against isocitrate lyase antigen. The conclusion reached was that ageing resulted in an increase in non-functional protein. Van Gundy et al (1967) reported that esterase and acid phosphatase, both of which function in the hydrolysis of lipids and phospholipids, had lower activity in aged than fresh larvae of the plant parasitic nematode Meloidogyne javanica. Wilson (1965) and Clark (1969) working with N. braziliensis and A. caninum larvae have shown that total protein levels are lower in old than young worms. In the light of these results it is tempting to speculate that the 125

decrease in behavioural activity and thus infectivity may be due to a reduction in specific enzyme activity in the mobilization of the lipid that is still present in the larvae. However in the absence of any precise data such an hypothesis must remain • tentative.

- -Cooper & Van Gundy (1971) have distinguished between senescence, quiescence and cryptobiosis in the ageing of nematodes.

Quiescence is an ill defined state in which a "metabolic slowdown"

(Cooper & Van Gundy, 1971) is experienced under adverse conditions.

It has been suggested that this prolongs the life of the nematode and thus increases its chance of survival until conditions improve.

Croll (1972a), has postulated that within a series of environmental parameters there is a "sphere" for larval activity outside of which quiescent survival may occur. There is no evidence of

A. tubaeforme having entered a cryptobiotic state nor a total state of quiescence. Although activity was not possible at 10°C lipid was still lost, albeit rather more slowly and the ageing process continued unaffected. It is possible that the conditions induced a state of partial quiescence so that a "slowdown" occurred in only some of the metabolic processes allowing others to continue at the normal rate. More extreme conditions might have induced a'truly cryptobiotic state. This appears unlikely however, as it has been shown that at 4°C and under osmotic stress at 26°C 126

ageing was unaffected by storage conditions that eliminated activity and reduced lipid loss (Croll & Matthews, 1973). To be of any significance in improving survival the ageing process would need to be the first system to be curtailed in any hierarchy and activity, which might provide a means of escape to more favourable conditions, the last. Exactly the reverse has been found and it would appear that the cat hookworm does not have any specialised system to increase longevity if environmental conditions should deteriorate.

Neostigmine bromide had the effect of increasing the percentage active and the percentage penetrating over the untreated controls.

Croll & Al Hadithi (1972) have suggested that neostigmine bromide lowered the post-synaptic threshold of the muscles through inhibition of acetylcholine esterase, and the present results support this idea.

Neostigmine bromide had a greater effect on the percentage larvae active (Fig. 15) than on the rate of activity (Fig. 16). This further supports the notion that it lowers the threshold for activity, rather than increasing its rate. The temperature range over which larvae were active was not affected by the presence of neostigmine. At any given lipid level there was an increase in larval activity in neostigmine, but the rate of decline in activity with age did not differ significantly from the controls (Figs. 22,

23, 24). 127

SECT ION IV

COMPARISON OF PENETRATION BY

ANCYLOSTOMA TUBAEFORME AND NECATOR AMER ICANUS 128

INTRODUCTION

The results presented in sections I and II suggested that for

A. tubaeforme penetration is a mechanical process. No evidence was found of enzymic activity and it was concluded that for this species proteolytic enzymes were not involved in penetration.

These results are rather contrary to the generally accepted view of hookworm penetration and it was considered of value to utilize some of the techniques developed for study of the cat hookworm on other species, to establish the applicability of the A. tubaeforme results. A limited supply of N. americanus larvae was made available for selected experiments through the generosity of

Dr. P. Ball and Miss A. Bartlett of the Nuffield Institute of

Comparative Medicine.

As the supply of material was limited it was not possible to plan all the experiments with adequate replicates or to follow up some of the results and, although of intrinsic interest, they must remain somewhat tentative.

MATERIALS AND METHODS

Yoshida and Kutome (1967) have shown that rabbits can be infected percutaneously with N. americanus and, in the absence of 129

a plentiful supply of human skin, rabbit skin was used for

comparative tests. In vitro experiments were conducted using No.

4 filter paper and clipped rabbit skin. The methods used were

the same as described earlier. Natural membranes were either

processed for histological examination or digested to determine

the percentage of larvae entering. Filter paper membranes were

macerated in water and the larvae removed.

N. americanus larvae were included in both the azocoll and

haemolysis assays for enzyme activity described earlier. A

short film sequence was prepared of larval movement within human

dermis.

RESULTS

The relationship between entry into filter paper and rabbit skin was linear, P = .001. (Fig. 26). The percentage entering

skin was greater than that entering paper. The time relationship showed that larval entry continued for up to 4 h but in place of

the linear relationship obtained with A. tubaeforme an approximately

hyperbolic relationship was fd4nd (Fig. 27).

The film sequence showed that ensheathed larvae were able to

130 FIGURE 26.

100

90 1 80 * 70

160 • g 50 2 40 • • • 30

10 20 30 40 SD 60 70 80 90 100 percentage entering skin Relationship between entry of N. americanus into rabbit skin and No. 4 filter paper.

FIGURE 27. 100

1 80•

• 60-

40 • 0.

0.5 1 2 4 time ( hours) Effect of time on the percentage of N. americanus larvae enteritg rabbit skin (.. ) and No. 4 filt;Tpaper7= - ) . 131

move within dermal tissues but, on no occasion, were ensheathed larvae found within the skin following exposure to either the dermal, or epidermal surface in in vitro penetration tests.

Substantial numbers of discarded sheaths were recovered with the larvae that had not penetrated.

the process of entry appeared to be the same for N. americanus as for A. tubaeforme but penetration through the stratum granulosum presented a different picture. Although only a limited number of tests was performed, more N. americanus larvae were found in the stratum granulosum than in all the tests with the cat hookworm, suggesting that passage through the epidermis is a longer process.

Epidermal cellular destruction was found during larval penetration

(Plate 14) and a true penetration tunnel through the epidermis was observed in one instance. Tunnels were not found in the dermis and the film showed that the dermis "closed" behind migrating larvae.

10% of larvae placed on the epidermal surface of rabbit skin and 12% of those placed on the dermal surface completely penetrated within 4 h. None of the total penetrants retained their sheath.

The results of the azocoll and haemolysis assays are presented in Figures 11 and 13 respectively and curves for N. americanus are included in these. There was no evidence of any haemolytic 13

PLATE 14.

Section of exsheathed N. americanus larva between the epidermis

and dermis of rabbit skin.

Haematoxylin and eosin. -X 800.

134

activity but a powerful azocoll positive enzyme was indicated. 3 This enzyme from 10 x 10 larvae gave approximately the same dye release as a 10 mg/ml pepsin solution.

DISCUSSION

The initial stage of penetration by N. americanus is again an active process and entry into filter paper and rabbit skin are directly related (Fig. 26). There is however, a difference in intercept between the results for A. tubaeforme and N. americanus.

This reflects a difference in the ease with which the larvae enter the respective membranes. At low percentag6s (short times)

N. americanus larvae more readily achieved entry into skin than into paper, for A. tubaeforme in rabbit skin, the rates of entry were more equal. This more rapid entry into skin is also shown by Figure 27. The percentage A. tubaeforme entering skin with time was always less than that entering paper (Fig. 7) whereas the converse was true for N. americanus.

The shape of the time/penetration curves may be significant.

For A. tubaeforme these curves were linear (Fig. 7) whereas for

N. americanus they were hyperbolic. It is not clear whether these differences were due to a different pattern of activity for the 135

two species or whether they reflect a different penetration mechanism. Croll (personal communication) has found that the decay rate for activity of N. americanus larvae following a mechanical stimulus was similar to that reported for A. tubaeforme

(Croll & Matthews, 1973). An azocoll positive secretion was found emanating from N. americanus larvae incubated at 37°C, and it is possible that the initial mechanical and thermal stimulation inherent in transfer to the penetration membrane caused a change in activity and, the release of a penetration enzyme. An alternative explanation is that N. americanus larvae show a less marked response to a thermal gradient than A. tubaeforme so that an equilibrium is set up between larvae entering and leaving the membranes. N. americanus larvae were able to completely penetrate skin membranes from the dermal surface so that migration from the epidermis appears more possible than for A. tubaeforme. This mechanism does not however, explain why entry into non-living filter paper membranes paralleled so closely that of entry into skin.

More larvae were found in the stratum granulosum in the few tests conducted on Necator than in all the A. tubaeforme experiment. The stratum granulosum appeared to present a greater barrier to penetrating larvae and they spent a longer time passing from the stratum corneum to the dermis. 136

The presence of an azocoll positive secretion, the destruction of epidermal cells (Plate 14) and the formation of true penetration tunnels after passage of N. americanus through the the epidermis all argue that enzymes play a part in the penetration process. Additional evidence that Necator penetration differs from that of A. tubaeforme and is accompanied by enzymic activity is provided by the fact that larvae penetrating from the dermal surface were able to completely penetrate skin and, that no ensheathed larvae were ever found within the skin. Ensheathed larvae were able to move within dermal tissues without any apparent enzymic activity as shown by the filmed sequences but, as the film was taken without any attendant thermal stimulus enzyme release may not have been stimulated. 137

Hookworms may cause two distinct types of disease. In susceptible hosts successful skin penetration is followed by migration via the blood or lymphatic systems to the heart and lungs. The larvae ascend the bronchi to the oesophagus and pass to the intestine. During this migration the third moult occurs and, after reaching the intestine the final one takes place, the adults remaining in the intestine. Hookworm larvae that enter hosts in which further development cannot or does not occur may remain in the skin and give rise to the condition known as creeping eruption or cutaneous larval migrans. Creeping eruption has not been reported from laboratory animals and appears to be confined to humans.

Lee (1874) in England first described a case of creeping disease characterised by an advancing linear skin lesion but was not able to find the cause. Crocker (1893) reported another case, again without describing the causative organism but expressed the belief that it was due to the migration of an insect larva within the skin, he suggested the name "larva migrans" for the disease.

Samson Himmelstjerna (1897) in Russia isolated a parasite from a case of creeping eruption that was identified as the larva of

Gastrophilus, the horse bot fly. A review of the evidence for creeping eruption being caused by insect larvae was included by

Austman (1926) along with a full description of a case from

Manitoba. 138

Tracks typical of those of creeping eruption were found by

Morishita & Faust (1925) to be caused by nematode larvae of the species Gnathostoma spinigerum. Kirby-Smith, Dove & White (1926) defined "creeping eruption" in a rather restrictive sense as the disease that they found in Florida and which they investigated, other- forms of the disease were considered to fall into a group of creeping diseases. This limited usage of the term has not been maintained (see review by Beaver, 1956a) and both creeping eruption and cutaneous larvae migrans have been used interchangeably for any of the linear lesions formed in the skin following parasite passage. However, since the importance of hookworms as causative organisms of the complaint has been recognised, it has generally been assumed that the disease covered by either term is due to nematode rather than arthropod, migration. Indeed, in too many cases it has been assumed to be due to A. braziliense for no other reason than that A. braziliense has been shown to be one of the causative organisms and that adults of the parasite have been found in the neighbourhood cats (e.g. Londero & Fischman, 1960).

White & Dove (1928) managed to isolate a larval nematode from skin biopsies from patients with creeping eruption attending a clinic in Florida, this was given the provisional name Agamotema- todum migrans until the adult worm could be identified. Attempts at inducing creeping eruption in volunteers with infective larvae 139

of Strongyloides ratti and Nippostrongylus muris from rats were unsuccessful. They were unable to demonstrate any evidence of penetration by Nippostrongylus although Lee (1972) has recently described ready entry of larvae into human skin. Both A. braziliense and A. caninum were found in cats and dogs in Florida and pure cultures were set up in previously clean cats and dogs. Infective larvae from both species were applied to the skin of volunteers and it was found that although the larvae of A. braziliense caused typical linear lesions those of A. caninum did not. Dove (1932) provided further observations on the etiology of the disease and conducted additional experiments that were interpreted as showing that typical lesions were produced only by A. braziliense and that

A. caninum and N. americanus produced only short non-linear lesions. It has been suggested (Beaver, 1956b) that the human source of A. braziliense used by Dove (1932) was in fact a mixed infection of A. caninum and N. americanus. This together with the specific distinction between A. braziliense and A. ceylanicum shown by Biocca (1951) has cast some doubt over the species used for the early experiments. Hitch & Iralu (1960) have considered that both A. braziliense and A. caninum are common causes of creeping eruption in the southern states of America. Haydon & Bearup

(1963) found larva migrans with A. braziliense but no intestinal worms whereas with A. ceylanicum the reverse was found i.e., no creeping eruption but development to adults in the intestine. 40

McCarthy (1933) confirmed A. braziliense as a causative organism of creeping eruption. It was stated that A. braziliense exsheath prior to penetration and microscopic evidence was presented of larvae migrating within the epidermis. The migration tunnels were confined to the epidermal layers being bounded by the stratum granulosum and compressed, pycnotic cells of the rete malpighii, entry into the dermis was not observed. From the pictures published it is difficult to tell if epidermal cellular destruction had occured. The width of the tunnel was quoted as being about twice that of the worm, this is consistant with the shrinkage found as a fixation artefact (Table V p. 77 In view of the consistency of the dermis reported herein it is difficult to see how a larva that possesses an enzyme that can cause digestion of epidermal cells can be confined to the epidermis, unless the cells along the dermo-epidermal junction possess special properties, for example are more tightly bound than the upper cells.

If this is so, then the greater thickness of the epidermis in human skin may present difficulties to penetrating larvae,

It may not be possible for sufficient purchase to be obtained from the stratum corneum to allow separation of the lower epidermal cells. Tamura (1921) and Kirby-Smith et al (1926) have described similar lesions in which migrating larvae were confined to the epidermis and did not penetrate through to the dermis. 141

Using large inocula, Hunter & Worth (1945) found that unbroken skin was readily penetrated by A. caninum and linear migrations were produced. The response differed markedly between the two patients studied and the violence of the reactions found was put down partly to micro-organisms having penetrated the cutaneous sites with the larvae. Stumberg (1932) suggested that the generalized leucocytic infiltration found in all cases of multiple skin invasion by A. caninum into dogs could have been caused by bacteria carried on the surface of the larvae. Gharib

(1955) demonstrated that bacteria can be transported into skin by Nippostrongylus larvae and nematodes have been implicated in bacterial disease transmission on a number of occasions (Beveridge,

1934; Taylor, 1935; Smirnov & Kamalov, 1951; Stefanski, 1959).

The extent of bacterial contamination in cases of larval migrans is not clear from the literature which largely deals with clinical cases in which primary symptoms may have been lost. Exsheathment immediately prior to penetration may greatly reduce the number of bacteria transported by the worm. Poinar & Doncaster (1965) have shown that the larval sheath of Tripius sciarae is attached to the insect cuticle prior to penetration and forms a seal after larval entry, an almost aseptic entry mechanism is thus achieved.

Preliminary experiments with apparently clean, A. tubaeforme 142

larvae removed, with a minimum of fluid, from the condensation fluid from faecal cultures, have shown that bacteria are readily transported across nutrient agar plates. It has been shown that ensheathed A. tubaeforme larvae can penetrate intact skin and it might be expected that bacterial contamination might be more common with this species. However, in several years working with A. tubaeforme in the laboratory during which at least one suspected case of human invasion has occurred, no example of larva migrans has been found.

A. caninum lesions are often marked by extending a short distance before more or less disappearing, only to reappear some time later (Hunter & Worth, 1945). This phenomenon has been explained by suggesting that the larvae penetrate temporarily into the deeper layers of the skin where, presumably, they avoid any host reaction, or at least it is no longer apparent from the surface. The sub-dermal fat layers have been shown to act as a

"trap" for A. tubaeforme larvae, a result confirming that of

Stumberg (1932) for A. caninum in dogs, and this may explain retention within the skin. Shelmire (1938) found that larval migration could be stimulated by holding the involved part near a fire or placing it in hot water. This type of treatment may be sufficient to increase larval activity to such an extent that 143

they can break out of the fat globules. An increase in surface temperature may attract larvae back towards the surface. Even if they are able to enter the epidermis through the dermo- epidermal junction it seems unlikely that they would be able to

pass back through the stratum corneum, the physical characteristics of which are very different on the inner surface. Thus, unless they enter circulatory vessels, larvae are trapped within the thickness of the skin by two more or less impenetrable barriers.

The length of time spent migrating in the skin may reflect the ease with which the larvae can enter the vessels, this in itself may depend on the immune state of the host.

The exact pathology of any particular case is dependant on a great many factors of which the species of worm involved, previous exposure to the parasite and individual response all play a part. The related skin reaction to foreign schistosome cercariae, known as cercarial dermatitis, has been shown to be a sensitization phenomenon (Olivier, 1949). Primary infection with

Trichobilharzia stagnicolae resulted in mild reactions which became more severe with subsequent exposure to the cercariae.

Stumberg (1932) was unable to show any increased cutaneous retention or inflammatory response to repeated skin penetration by

A. caninum in dog skin and found greater variation between dogs than between treatments. However, Maplestone (1933) found typical creeping eruption lesions after repeated exposure of 144

volunteers to A. duodenale and N. americanus larvae, although none were found following early infections. He considered that creeping eruption is in large part a manifestation of sensitization rather than due to a particular species of larva. Beaver (1945) also found lesions typical of creeping eruption following the third-and subsequent infection of his own arm with up to 200

N. americanus larvae. It is interesting to note that Maplestone

(1933) found that only 4 of 10 multiple exposures to A. duodenale resulted in larva migrans whereas all of 15 N. americanus did.

The differences found between penetration by A. tubaeforme and

N. americanus may go some way to explaining this result.. If larva migrans is due to a sensitization reaction by the host, it might be anticipated that a secreted protein e.g. a proteolytic penetration enzyme, as has been proposed for Necator and schistosomes, would cause a more intense and more rapidly manifest reaction than an entire worm that did not secrete such an enzyme.

Honeycutt et al (1965) reported similar lesions caused by

N. americanus, A. braziliense and A. caninum but a different form was found for S. stercoralis. White & Dove (1928) and Dove (1932) found that Strongyloides spp. could enter human skin but did not observe linear lesions although papular elevations were formed at the point of entry. Caplan (1949) showed a correlation between the incidence of linear urticarial weals, especially on the buttocks, 145

and intestinal strongyloidiasis in patients returning from the

Far East. Sandosham (1952) was not able to unequivocally demonstrate

that S. stercoralis caused larva migrans but, as he used only

three volunteers and himself considered that linear eruptions may

only occur after sensitization of the buttock region by repeated

exo-._auto=infection, this is perhaps not surprising. A strain

of S. stercoralis was isolated from a patient with creeping eruption

by Galliard & Chabaud (1952) and passaged successfully into dogs,

and Arthur & Shelley (1958) fully described a case of Strongyloides

creeping eruption. These latter authors considered the pathology

to be sufficiently distinctive to suggest that it be termed

"larva currens". A number of authors have remarked on the

difference between creeping eruption caused by hookworm larvae

and that caused by Strongyloides, the most consistant difference

being the rate of formation of the linear track. Rates of

1.27 - 7.6 cm/day (McCarthy, 1933) and 2.5 cm/day (Schacher &

Danaraj, 1957) have been reported for hookworm migration and

5 cm/h (Ftilleborn, 1926) and 5-10 cm/h (Caplan, 1949) for Strongyloides.

Both Lewert & Lee (1954, 1956) and the present investigations have indicated the presence of an azocoll positive secretion from

Strongyloides species but it is difficult to imagine that larvae cap move at this rate by digesting their way through the tissue.

A far more likely explanation is that Strongyloides migration is 11+6

through the dermis rather than the epidermis. The dermis has been shown to cause little hindrance to the passage of hookworm larvae, the hourly rate of migration calculated from the filmed sequences of A. tubaeforme is 7.9 cm/h, a result in the same order as for Simagloides. The difference in response to

A. caninum and Strongyloides when penetrating through the dermis may be due to the presence of a more or less continuously secreted protein from the latter species. The hookworm may be able to pass through the acellular dermal tissue undetected by the host immune reaction, and only cause a visible response when damaging the epidermal cells; whereas the continuous release of an antigenic secretion from Strongyloides larvae causes a tissue response even if cellular damage is not occuring. One of the standard cures for creeping eruption is to freeze the progressing end of the track with an ethylchloride spray. When this method was tried by Arthur & Shelley (1958) with a case of Strongyloides migration it was unsuccessful suggesting that the larvae were situated below the superficial epidermal layers. 14

REFERENCES

AL-HADITHI, I.A.W. (1972). Locomotory responses of larval hookworms (Ancylostoma tubaeforme). Ph.D. Thesis. University of London. ALLEN, J.F. (1888). Parasitic haematuria, or bloody urine. Practitioner 40, 310-320.

ARCHER, R.K. (1965). Haematological technique for use on animals. Oxford : Blackwell Scientific Publications. AUSTIN, F.G., STIREWALT, M.A. & DANZIGER, R.E. (1972). Schistosoma mansoni : stimulatory effect of rat skin lipid fractions on cercarial penetration behaviour. Experimental Parasitology 31, 217-224.

AUSTMAN, K.J. (1926). Creeping Eruption. Journal of the American Medical Association 87, 1196-1200. BARRETT, J. (1969a). The effect of ageing on the metabolism of infective larvae of Strongyloides ratti Sandground, 1925. Parasitology 59, 3-17. (1969b). The effect of temperature on the development and survival of the infective larvae of Strongyloides ratti Sandground, 1925. Parasitology 58, 641-651.

BEAVER, P.C. (1945). Immunity to Necator americanus infection. Journal of Parasitology 21, (Supplement), 18. (1956a). Larva Migrans. Experimental Parasitology 2, 587-621.

(1956b). The record of Ancylostoma braziliense as an intestinal parasite of man in North America. American Journal of Tropical Medicine and Hygiene 2, 737-738. 148

BENJAMINI, E. & FEINGOLD, B.F. (1970). Immunity to Arthropods. In Immunity to Parasitic animals Vol. 2 (ed. G.J. Jackson, R. Herman & I. Singer), pp. 1061-1134. New York : Meredith Corporation.

BETTLEY, F.R. & DONOGHUE, E. (1960). Effect of soap on the diffusion of water through isolated human epidermis. Nature, London 185, 17-20.

BEVERIDGE, W.I.B. (1934). Foot-rot in sheep. Skin penetration by Strongyloides larvae as a predisposing factor. Australian Veterinary Journal 10, 43-51. BILLINGHAM, R.E. & MEDAWAR, P.B. (1951). The viability of mammalian skin after freezing, thawing and freeze drying. In Freezing and Drying (ed. R.J.C. Harris), pp. 55-62. Symposium, Institute of Biology. BIOCCA, E. (1951). On Ancylostoma braziliense (de Faria, 1910) and its morphological differentiation from Ancylostoma ceylanicum (Looss, 1911). Journal of HelmintholoRY 25, 1-10.

(1954). Ridescrizione di Ancylostoma tubaeforme (Zeder, 1800) parasitta del gatto, considerato erroneamante sinonimo di Ancylostoma caninum (Ercolani, 1859), parasitta del cane. Rivista di Parassitologia 15, 267-278.

BIRD, A.F. & BIRD, J. (1969). Skeletal structures and integument of Acanthocephala and Nematoda. In Chemical Zoology, (ed. M. Florkin & B.T. Scheer), Vol. III pp. 253-288. New York : Academic Press.

BREATHNACH, A.S. (1971). An atlas of the ultrastructure of the human skin : development, differentiation and post- natal features. Edinburgh : Churchill. BRUCE, J.I., PEZZLO, F., McCARTY, J.E. & YAJIMA, Y. (1970). Migration of Schistosoma mansoni through mouse tissue. Ultrastructure of host tissue and integument of migrating larva following cercarial penetration. American Journal of Tropical Medicine and Hygiene 17, 959-981. 149

BURROWS, R.B. (1962). Comparative morphology of Ancylostoma tubaeforme (Zeder, 1800) and Ancylostoma caninum (Ercolani, 1859). Journal of Parasitology _715-718. .

CAPLAN, J.P. (1949). Creeping eruption and intestinal strongyloidiasis. British Medical Journal No. 4600. 396. CLARK, F.E. (1969). Ancylostoma caninum : Food reserves and changes in chemical composition with age in third stage larvae. Experimental Parasitology 24, 1-8.

CLEGG, J.A. (1969). Skin penetration by cercariae of the bird schistosome Austrobilharzia terrigalensis : the stimulatory effect of cholesterol. Parasitology 22, 973-989. COOPER, A.F. & VAN GUNDY, S.D. (1971). Senescence, quiescence and cryptobiosis. In Plant Parasitic Nematodes (ed. B.H. Zuckerman, W.F. Mai & R.A. Rohde) Vol. II pp. 297-318. London and New York : Academic Press. CORT, W.W. (1921). The development of the japanese blood- fluke Schistosoma japonicum Katsurada, in its final host. American Journal of Hygiene 1, 1-38.

(1925). Investigations on the control of hookworm disease. XXXIV. General summary of results. American Journal of Hygiene 5, 49-89. CROCKER, H.R. (1893). Larva migrans. Annales de dermatologie 2, 1184.

CROLL, N.A. (1972a). Energy utilization of infective Ancylostoma tubaeforme larvae. Parasitology 64, 355-368. (1972b). Feeding and lipid synthesis of Ancylostoma tubaeforme larvae. Parasitology 64, 369-378. (1972c). Behaviour of larval nematodes. In Behavioural Aspects of Parasite Transmission (ed. E.U. Canning & C.A. Wright) pp. 31-52. London : Academic Press. 150

CROLL, N.A. & AL-HADITHI, I.A.W. (1972). Sensory basis of activity in Ancylostoma tubaeforme infective larvae. Parasitol.oR,g 64, 279-291.

CROLL, N.A. & MATTHEWS, B.E. (1973). Activity, ageing and penetration of hookworm larvae. Parasitology (In press).

CROLL, N.A. & SMITH, J.M. (1972). Mechanism of thermopositive behaviour in larval helminths. Journal of Parasitology 58, 891-896. CURTIS, A.S.G. (1972). Adhesive interactions between organisms. In Functional Aspects of Parasite Surfaces (ed. A.E.R. Taylor & R. Muller) pp. 1-21. Oxford : Blackwell Scientific Publications. DOVE, W.E. (1932). Further studies on Ancylostoma braziliense and the etiology of creeping eruption. American Journal of Hygiene 15, 644-711. DUKE, B.O.L. (1971). The ecology of onchocerciasis in man and animals. In Ecology and physiology of Parasites (ed. A.M. Fallis) pp. 213-222. University of Toronto Press. ELLIOTT, A. (1954). Relationship of aging, food reserves and infectivity of larvae of Ascaridia galli. Experimental Parasitology 2, 307-320.

ERLANGER, M. & GERSHON, D. (1970). Studies on ageing in nematodes. II. Studies of the activities of several enzymes as a function of age. Experimental Gerontology 5, 13-19.

FUJINAMI, A. & NAKAMURA, H. (1909). (The route of infection and the development of the parasite of Katayama disease (schistosomiasis japonica) in the infected animal.) (In Japanese). Kyoto Igaku Zassi 6, No. 4. 151

FULLEBORN, F. (1926). Hautquaddeln und "Autoinfection" bei StrongyloidestrUgern. Archly fUr Schiffs- und Tropeahaal.m 30, 721-732.

GALLIARD, H. & CHABAUD, A. (1952). Anomalies, s'eteignant par passage chez le chien, d'une souche de Strongyloides stercoralis, isolee d'un cas d'urticaire migrant. Annales de Parasitologie 22, 597-599. GAZZINELLI, G. & PELLEGRINO, J. (1964). Elastolytic activity of Schistosoma iriansoni cercarial extract. Journal of Parasitology 50, 591-592.

GERSH, I. & CATCHPOLE, H.R. (1949). The organization of ground substance and basement membrane and its significance in tissue injury, disease and growth. American Journal of Anatom 85, 457-522.

GERSHON, H. & GERSHON, D. (1970). Detection of inactive enzyme molecules in ageing organisms. Nature London 227, 1214-1217.

GHARIB, H.M. (1955). Some observation3on the transmission of bacteria by. infective larvae of Nippostrongylus brasiliensis. Journal of Helminthology 29, 27-32.

GOODEY, T. (1922). Observations on the exsheathed larvae of some parasitic nematodes. Annals of Applied Biology 9, 33-48.

(1925). Observations on certain conditions requisite for skin penetration by the infective larvae of strongyloides and ankylostomes. Journal of Helminthology 2, 51-62.

GORDON, R.M. & GRIFFITHS, R.B. (1952). Observations on the means by which the cercariae of Schistosoma mansoni penetrate mammalian skin, together with an account of certain morphological changes observed in newly penetrated larvae. Annals of tropical medicine and Parasitology L+2, 227-243. 152

HALEY, A.J. & CLIFFORD, C.M. (1960). Age and infectivity of the filariform larvae of the rat nematode Nippostrongylus brasiliensis (Travassos, 1914). Journal of Parasitology 46, 579-582.

HAYDON, G.A.M. & BEARUP, A.J. (1963). Ancylostoma braziliense and Ancylostoma ceylanicum. Transactions of the Royal Society of Tropical Medicine and Hygiene.57, 76.

HEAD, K.W. (1970). Pathology of the skin. Veterinary Record 87, 460-471.

HERLICH, H. & RYAN, B.M. (1970). Effects of cold storage on survival and infectivity of third stage larvae of the stomach worm Trichostrongylus axei (Cobbold, 1879). Journal of Parasitology 56, 200-201.

HITCH, J.M. &IRALU, V. (1960). Studies relating to creeping eruption. Southern Medical Journal 53, 447-453. HONEYCUTT, W.M., DILLAHA, C.J., JANSEN, G.T. & MORGAN, ' P.N. (1965) • Creeping eruption in a non-endemic area : a report of 12 cases emphasizing occupational origin. Southern Medical Journal 58, 62-64.

HUNTER, W.M. & GREENWOOD, F.C. (1962). Preparation of 1311 growth hormone of high specific activity. Nature London 194, 495.

HUNTER, G.W. & WORTH, C.B. (1945). Variations in response to filariform larvae of Ancylostoma caninum in the skin of man. Journal of Parasitology 31, 3 6-372.

JARRETT, A. (1958). The structure of collagen and elastic tissue in unprocessed skin. British Journal of Dermatology 70, 343-347.

JARRETT, A. & MATTHEWS, B.E. (1973). The physical nature of dermal collagen. Rheumatology and Physical Medicine (In press). 153

JARRETT, A. & SPEARMAN, R.I.C. (1964). Histochernistt n - Psoriasis. London : English Universities Press.

KEILIN, D. & ROBINSON, V.C. (1933). On the morphology and life history of Aproctoneinoham Keilin, a nematode parasite in the larvae of Sciara puliala Winn. (Diptera, Nematocera). Parasitology 25, 285-295.

KINOTI, G.K. (1971). The attachment and penetration of the miracidium of Schistosoma. JotunLslEflpjltls12Ex1 45, 229-235.

KIRBY-SMITH, J.L., DOVE, W.E. & WHITE, G.F. (1926). Creeping eruption. AzsityesolDerT ISIThjEX-101-0,. 13, 137-173.

KOPPISCH, E. (1937). Studies on schistosomiasis mansoni in Puerto Rico. IV The pathological anatomy of experimental schistosomiasis mansoni in the rabbit and albino rat. Puerto Rico Journal of Public Health and tropical Medicine 13, 1-54.

KOSUGE, I. (1924). Histologische Untersuchungen Uber'das Eindringen von Strongyloides stercoralis in die Haut von Versuchstieren. Archiv fur Schiff s- and Tropen- hygiene 28, 15-20.

KUNTZ, R.E. (1953). Demonstration of the "spreading factor" in the cercariae of Schistosoma mansoni. Experimental Parasitology 2, 397-402.

LANE, C. (1933). The taxies of infective hookworm larvae. Annals of Tropical Medicine and Parasitology 27, 237-250.

LAPAGE, G. (1935). The second ecdysis of infective nematode larvae. Parasitology 27, 186-206.

LARRIVEE, D.H., BENJAMINI, E., FEINGOLD, B.F. & SCHIMIZU, M. (1964). Histologic studies of guinea pig skin: different stages of allergic reactivity to flea bites. Experimental Parasitology 15, 491-502. 154

LEE, D.L. (1972). Penetration of mammalian skin by the infective larvae of Nippostrongylus brasiliensis. Parasitology 65, 499-505.

LEE, R.J. (1874). Case of Creeping Eruption. Transactions of the Clinical Society of London 8, 44-45.

LEIPER, R.T. (1915). Reports on the results of the Bilharzia Mission. Journal of the Royal Army Medical Corps. 25, 1-55, 253-267. (1916) Ibid 27, 171-190

(1918) Ibid 30, 235-268. LEVINE, M.D., GARZOLI, R.E., KUNTZ, R.E. & KILLOUGH, J.H. (1948). On the demonstration of hyaluronidase in the cercariae of Schistosoma mansoni. Journal of Parasitology J.12, 158-161.

LEWERT, R.M., HOPKINS, D.R. & MANDLDWITZ, S. (1966). The role of calcium and magnesium ions in invasiveness of schistosome cercariae. American Journal of Tropical Medicine and Hygiene 15, 314-323.

LEWERT, R.M. & LEE, C.L. (1954). Studies on the passage of helminth larvae through host tissues. I. Histochemical studies on extracellular changes caused by penetrating larvae. II. Enzymatic activity of larvae in vitro and in vivo. Journal of Infectious Diseases 95, 13-51.

(1956). Quantitative studies of the collagenase- like enzymes of cercariae of Schistosoma mansoni and the larvae of Strongyloides ratti. Journal of Infectious Diseases 22, 1-14. LONDERO, A.T. & FISCHMAN, O. (1960). Dermatose serpinosa no interion do Rio Grande do Sul, Brazil. Revista del Instituto Medicine Tropical Sao Paulo 2, 230-234. 155

LOOSS, A. (1911). The anatomy and life history of Agchlostoma duodenale Dub. Pt. II. Cairo Records of the Egyptian School of Medicine 4, 167-607.

MANDLOWITZ, S., DUSANIC, D. & LEWERT, R.M. (1960). Peptidase and lipase activity of extracts of Schistosoma mansoni cercariae. Journal of Parasitology 46, 89-90.

MAPLESTONE, P.A. (1933). Creeping eruption produced by hookworm larvae. Indian Medical Gazette 68, 251-257. McCARTHY, L. (1933). Creeping eruption due to Ankylostoma braziliense. Dermatology and 27, 490-497. MILLEMAN,.R.E. & THONARD, J.C. (1959). Protease activity in schistosome cercariae. Experimental Parasitology. 8, 129-136.

MIYAGAWA, Y. & TAKEMOTO, S. (1921). The mode of infection of Schistosoma japonicum and the principal route of its journey from the skin to the portal vein in the host. Journal of Pathology and Bacteriology 24, 168-174.

MORISHITA, K. & FAUST, E.C. (1925). Two new cases of creeping disease (Gnathostomiasis) in China, with a note on the infection in reservoir hosts in the China area. Journal of Parasitology 11, 158-

NORRIS, D.E. (1971). The migratory behaviour of the infective- stage larvae of Ancylostoma braziliense and Ancylostoma tubaeforme in rodent paratenic hosts. Journal of Parasitology 2, 998-1009. OAKLEY, C.L., WARRACK, G.H. & VAN HEYNINGEN, W.E. (1946). The collagenase (K Toxin) of Cl. welchii type A. Journal of Pathology and Bacteriology 58, 229-253.

OLIVIER, L. (1949). Schistosome dermatitis, a sensitization phenomenon. American Journal of Hygiene 49, 290-302. 156

OLIVIER, L.J. (1966). Infectivity of Schistosoma mansoni cercariae. American Journal of tropical Medicine and Hygiene 15, 882-885. PAYNE, F.K. (1923). Investigations on the control of hookworm disease. XXXI. The relation of the physiological age of hookworm larvae to their ability to infect the human host. American Journal of Hygiene 3, 584-597. IOINAR, G.O. & DONCASTER, C.C. (1965). The penetration of Tripius sciarae (Bovien) (Sphaerulariidae : Aphelenchoidea) into its insect host, Bradysia paupera Tuom. (Myceto- philidae : Diptera) Nematologica 11, 73-78. ROGERS, W.P. (1939). The physiological ageing of ancylostome larvae. Journal of Helminthology 17, 195-202. (1940). The physiological ageing of the infective larvae of Haemonchus contortus. of 18, 183-192. ROGERS, W.P. & SOMMERVILLE, R.I. (1963). The infective stage of nematode parasites and its significance in parasitism. Advances in Parasitology 1, 109-177. ROSE, G.G. (1954). A separable and multipurpose tissue culture chamber. Texas Reports of Biology and Medicine 12, 1074-1083.

ROSE, J.H. (1963). Observations on the free-living stages of the stomach worm Haemonchus contortus. Parasitology 53, 469-481. SAMSON,HIMMELSTJERNA, C.V. (1897), Ein Hautmaulwarf. Archiv fUr Dermatologie and Syphilologie 41, 367-372. SANDOSHAM, A.A. (1952). An investigation into the association of creeping eruption with Strongyloides infection contracted in the Far East. Journal of Helminthol% 26, 1-24. 157

SCHACHER J.F. & DANARAJ, T.J. (1957). Creeping eruption, a non-patent zoonotic helminthiasis in Singapore. Proceedings of the Alumni Association, Malaya. 10, 141-146.

SHELMIRE, B. (1928). Experimental creeping eruption from a dog and cat hookworm (A. braziliense). Journal of the American Medical Association 91, 938-944.

SIMMONDS, P.A. (1958). Studies on the sheath of fourth stage larvae of the nematode parasite Nippostrongylus muris. Experimental Parasitology 7, 14-22.

SMIRNOV, G.G. & KAMALOV, N.G. (1950). The susceptibility of amphibians to percutaneous infection with ancylostome larvae. (In Russian). Dokladi Akademi Nauk SSR 72, 437-439.

(1951). Transmission of Bacillus anthracis by the larvae of Necator americanus. Dokladi Akademi Nauk SSR 759-760.

SMITH, M.A. (1972). A collagenase-like enzyme secreted by the eggs of Schistosoma mansoni and its inhibition by a factor in serum. Project report for the degree of Bachelor of Technology in Applied Biology, Brunel University.

STANDEN, O.D. (1953). The penetration of the cercariae of Schistosoma mansoni into the skin and lymphatics of the mouse. Transactions of the Royal Society of Tropical Medicine and Hygiene 47, 292-298.

STEFANSKI, W. (1959). The role of helminths in the transmission of bacteria and viruses. Proceedings of the XVth International Congress of Zoology, London, pp. 697-699.

STIREWALT, M.A. (1958). Relation of skin reaction to penetration and to the development of local resistance to entry by challenging cercariae of Schistosoma mansoni. Proceed- ings of the VIth International Congress on Tropical Medicine and Malaria vol. II, 67-76. 158

(1959). Chronological analysis, pattern and rate of migration of cercariae of Schistosoma mansoni in body, ear and tail skin of mice. Annals of tropical Medicine and Parasitology 53, 400-413. (1963). Chemical biology of secretions of larval helminths. Annals of the New York Academy of Sciences 113, 36-53.

(1966). Skin penetration mechanisms of helminths. In Biology of Parasites (ed. E.J.L. Soulsby) pp. 41-59. New York and London : Academic Press.

STIREWALT, M.A. & KRUIDENIhR, F.J. (1961). Activity of the acetabular secretory apparatus of cercariae of Schisto- soma mansoni under experimental conditions. Experi- mental Parasitology 11, 191-21,1*.

STIREWALT, M.A. & UY, A. (1969). Schistosoma mansoni : cercarial penetration and schistosomuli collection in an in. vitro system. Experimental Parasitology 26, 17-28. STIREWALT, M.A., MINNICK, D.R. & FREGEAU, W.A. (1966). Definition and collection in quantity of schistosomules of Schistosoma mansoni. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 322-360. STUMBERG, J.E. (1932). Cutaneous retention of infective larvae of the dog hookworm, Ancylostoma caninum, and the inflammatory reaction to skin penetration. American Journal of Hygiene 15, 188-205. SUMMERLIN, W.T., CHARLTON, M.E. & KARASEC, M.A. (1970). Transplantation of organ cultures of adult human skin. Journal of Investigative Dermatology ',2„ 310-316.

SZENT-GYORGYI, A. (1969). Molecules, electrons and biology. Transactions of the New York Academy of Sciences. Ser II 31, 334-340. 159

TALIAFERRO, W.H. & SARLES, M.P. (1939). The cellular reactions in the skin, lungs and intestine of normal and immune rats after infection with Nippostrongylus muris. Journal of Infectious Diseases 64, 157-192.

TAMURA, H. (1921). On creeping disease. British Journal of Dermatology and Syphilology 33, 81-102.

TAYLOR, E.L. (1935). Do nematodes assist bacterial invasion of the host by wounding the wall of the intestinal tract ? Parasitology 27, 145-151.

TROWFT,L, O.A. (1959). The culture of mature organs in a synthetic medium. Experimental Cell Research 16, 118-147.

UITTO, J., LINDY, S., TURTO, H. & DANIELSON, L. (1971). Biochemical characterization of pseudoxanthoma elasticum: collagen biosynthesis in the skin. Journal of Investigative Dermatology 57, 44-48.

VAN GUNDY, S.D., BIRD, A.F. & WALLACE, H.R. (1967). Aging and starvation in larvae of Meloidogyne javanica and Tylenchulus semipenetrans. Phytopathology 57, 559-571.

VETTER, J.C.M. (1970). Skin penetration by hookworm larvae. Second International Congress of Parasitology, Washington 1970. Journal of Parasitology 56 Sect II Pt. 2 479.

VETTER, J.C.M. & LEEGWATER, M. (1971). Skin penetration of hookworm larvae. 1er Multicolloque Europien de Parasitology, Rennes, 1 an 4 Sept 1971. 163-164.

VOGEL, H. (1932). Hauterscheinungen bei Schistosomiasis : Beabachtungen fiber Zerkarien. Dermatitis, Kutan- reaktionen and ein Vulva-Granulom. Archiv fair Schiffs- und Tropenhygien 36, 384-399. WAJDI, N. (1966). Penetration by the miracidia of S. mansoni into the snail host. Journal of Helminthology 40, 235-244. 160

WELCH, H.E. (1964). Mermithid parasites of blackflies. Bulletin of the World Health Organization 21, 857-863.

WHITE, G.F. & DOVE, W.E. (1928). The causation of creeping eruption. Journal of the American Medical Association 90, 1701-1704.

WILSON, P.A.G. (1965). Changes in lipid and nitrogen content of Ilippc ylusbr .i.tasi infective larvae aged at constant temnerature. Experimental Parasitolo, 16, 190-194.

WILSON, R.A., PULLIN, R. & DENISON, J. (1971). An investigation of the mechanism of infection by digenetic trematodes : the penetration of the miracidium of Fasciola hepatica in its snail host Lymnaea truncatula. Parasitology 491-5o6. Reprinted from TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 66. No. 1. pp. 14-15, 1972.

HELMINTHOLOGY AND HELMINTHIC DISEASES An in vitro system for investigating the skin-penetrating mechanisms of larval helminths B. E. MATTHEWS Zoology Department, Imperial College of Science and Technology A modification of the floating raft method (GooDEY, 1922) has been developed to study the dynamics of penetration by larval helminths. The skin is held under light tension between two perspex plates, apertures in these plates allow a larval suspension to be applied to the upper surface and a suitable medium to the lower. This medium is retained by cover slips providing a sealed chamber in which any larvae that completely penetrate the skin are trapped and may be recovered. Beneath this is a constant temperature water jacket, which maintains a temperature gradient similar to that across the skin of a living animal. By localizing the larvae on a small area of skin it is possible to cut serial sections through the whole tissue and thus investigate the site of invasion and the penetration route. Skin freshly excised from an animal and material kept under deep freeze conditions has been used and has given comparable results, and in vivo tests have confirmed that the results obtained in vitro are applicable. Ensheathed Ancylostoma tubaeforme larvae have been shown to penetrate both the epidermis and the dermis and sections show both ensheathed and exsheathed larvae within the dermis together with discarded sheaths. The use by the worm of a powerful collagenase to digest its way through the skin appears unlikely as the sheath itself is composed of a form of collagen and would probably be digested in the presence of such an enzyme. Up to 50% of larvae that completely penetrate the skin membrane still retain their sheaths. Scanning electron micrographs of the surface of the skin have shown ensheathed larvae within the stratum corneum and entering hair follicles. It would appear that penetration can occur at any site on the skin surface at which a suitable fissure presents itself and where the worm • is able to obtain sufficient purchase.

REFERENCE GOODEY, T. (1922). Proc. R. Soc. Med., 15, 19.

Locomotory responses of infective A. tubaeforme larvae I. A. W. AL-HADITH Zoology Department, Imperial College of Science and Technology The distribution of Ancylostoma tubaeforme infective larvae on 1% ion agar plates has been studied with respect to gravity. Using various angles to the horizontal, the extent of the response increased consistently from 15° to 90°. At 75° and 90° larvae first moved downwards, but had moved upwards within 2 hours. Only larvae actively moving from the inoculation point were counted. When tested at temperature from 15°C to 30°C, it was found that larvae moved upwards at all temperatures, the greatest response (91%) being at 25°C, decreasing to 78% at 30°C. When testing the effect of larval age on the response to gravity (ageing at 20°C ± 1), an increasing response was found for the first 9 days, which was followed by a gradual decline. Infective larvae responded to heat gradients as small as 0.5°C over 4 cm. although linear gradients were not maintained. By tracking larvae and plotting their position at 1 min. intervals it could be seen that the response was more likely to be a form of taxis than a kinesis, and that their rate of progression increased as they approached the heat source. 15 LABORATORY MEETING By varying the angle of the plates, and measuring the effects of opposing and reinforcing gravitational effects with a heat gradient and varying light intensities, a stimulus hierarchy was established. Using a gradient of about 2°C over 4 cm., it was found that heat was significantly more attractive than gravity. An intensity of tungsten light at 1750 c/ft2 intensity, incorporating heat filters, was also more attractive than gravity. Significant results were obtained to demonstrate a reversal of the phototaxis after as little as 3 hours storage at 37°C, which increased up to 24 hours.

Energy relations of larval hookworms, Ancylostoma tubaeforme N. A. CROLL Zoology Department, Imperial College of Science and Technology Pre-infective bacteriophagous larvae, grow and synthesize an energy reserve. Upon moulting to an infective stage, an energetically closed system is reached, larvae ceasing to feed and grow, utilizing energy only for survival and behavioural activity. By measuring a2b the volume( V) where V = — (a is greatest width, b is length) and density in Ficoll 1.7 gradients, mass changes were calculated. A newly hatched larva has a volume of 58,823 V.3 and a mass of 0.646 x 10-7 g., an infective larva is 523,441 t/3 of mass 5.110 x 10-7 g. Total increase from hatch to second moult is 4.546 x 10—' g., equivalent to about 500,000 bacteria, each weighing 10-12 g. Assuming 20% conversion of bacteria to nematode tissue, about 2,500,000 bacteria must be consumed in two days at 30°C, 80% of which enter the second stage larvae. Lipid is synthesized exponentially with growth, and is available for morphogenesis in periods of starvation. Unbound neutral lipid available to the larvae was stained in oil red 0, and the absorption measured on a scanning microdensitometer at 517 nm. Hookworm larvae have a low Reynold's number of about 10-2. Reynold's nL2 p i number — (n is frequency, L is larval diameter, p is density and (..t, is viscosity) This means the viscous stress in movement is over 100 times the inertial stress, and for, organisms dominated by viscous forces, energy consumption (pm) increases with the square of the velocity (v) pm = A v° (A is a constant depending on the form of movement). Energy utilization is dependent on the rate of larval movement. In 0.1 mg./ml. neostigmine bromide (inhibiting cholinesterase and possibly depolarising muscle) larvae are very active and use lipid rapidly. Lipid is used fastest in aerobic, isotonic, neutral pH at temperatures between 25°-30°C. When in sublethal extremes outside these 'optima' lipid is used at insignificant rates, the tolerance of 'stress' does not require measurable amounts of lipid reserve.

Printed by F. J. PARSONS LTD., London, Folkestone and Hastings.

Parasitology (1972), 65, 457-467 457 With, 1 plate and 4 text-figures Printed in Great Britain

Invasion of skin by larvae of the cat hookworm, Ancylostoma tubaeforme

BY B. E. MATTHEWS Department of Zoology and Applied Entomology, Imperial College, London University, London S.W. 7

(Received 24 February 1972)

The in vitro study of penetration of infective nematode larvae into skin was initiated by Goodey (1922) with the introduction of the `floating raft' technique. Subsequently Rogers (1939), using the `cat strain' of Ancylostoma caninum, and Barrett (1969), using Strongyloides rata, measured infectivity as the number of larvae that completely penetrated a stretched skin membrane. Stirewalt, Minnick & Fregeau (1966) used a modified Rose chamber (Rose, 1954) to investigate the change from cercaria to schistosomulum during the penetration of Schistosoma mansoni through dried skin. This was further developed by Stirewalt & 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 of Austrobilharzia terrigalensis cercariae showing that skin lipids and especially cholesterol stimulate penetration. In all 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 investigation was aimed at providing an in vitro method of studying the penetration of hookworm larvae through skin that would give qualitative information about the course, route and mechanism of penetration comparable to that obtained in vivo and to quantify larval infectivity. This paper describes the apparatus used, the initial invasion of the skin by Ancylostoma tubaeforme and entry into the epidermis.

MATERIALS AND METHODS 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 factors affecting penetration by A. caninum and Strongyloides spp. were established. Although the presence of larvae within the skin was demonstrated (Goodey, 1925) no attempt was made to follow the course of penetration or to quantify the results. The modified apparatus (Fig. 1) was designed to answer questions relating as much to the host skin as to the parasite. The effective area of skin exposed to the larvae was 3 mm in diameter permitting serial sectioning of the entire area and 458 B. E. MATTHEWS

Penetration cells

Skin

8 '0 CD 9 Collecting cells

000 Cover-glasses ,e Q 4110 411, Rubber gasket n

Water jacket

Rubber gasket

Base plate 8

Fig. 1. In vitro penetration apparatus. subsequent investigation of the course of penetration. The size and shape were such that three penetration sites were established on a single piece of skin 5 cm by 1 cm which allowed replication while reducing to a minimum the inherent variation in skin. The average surface temperature of the cat measured, using a thermistor probe on various parts of the body, is 32.5 °C; there is thus a temperature difference of 10-15 °C between laboratory temperatures and the skin surface. This difference was simulated in vitro by adjusting the temperature of the water passing through the water jacket so that the skin surface was 32 °C. The self-contained nature of the assembled apparatus meant that movement for observation under a microscope or inversion to study gravitational effects could be readily achieved. Skin invasion by hookworm larvae 459 The skin was held under slight tension by pins passing through it and the top plate, finally being retained by bolts clamping the top two plates. The collecting cells were filled with normal saline buffered with ra/15 phosphate buffer to pH 6.8, care being taken to ensure that all air was excluded when the cover-glasses were slid into place. The first gasket was then fitted and the whole apparatus assembled. Water at 37 °C was pumped through the water jacket for 30 min before each test to equilibrate the skin at 32 °C. The flow rate through each cell of the water jacket was balanced to reduce temperature differences between the skin in the separate penetration chambers. Preparation of membranes Both natural and artificial membranes were used. The skin was either fresh, deep frozen prior to use or dried, plucked and sanded using the method proposed by Stirewalt et al. (1966). Tanned gelatine membranes, prepared using the method of Clegg (1969), membranes consisting of Whatman filter paper and laminates of filter paper with tanned gelatine were used as artificial membranes, in an attempt to reduce the inherent variability of whole skin. The grade of filter paper used is important. Hardened papers, while having the advantage of greater wet strength, were too firm and only small numbers of larvae were able to penetrate. The qualitative grade Whatman no. 4 proved suitable. The quoted mean pore size of this paper is 3.4-5.0 ,am and the mean larval diameter 27.0 ± 1.3 ,am so that activity is required for penetration. Laminated membranes were prepared either by casting 10 % (w/v) sheet gelatine in distilled water in a 1 mm deep oblong mould 75 x 25 mm and applying no. 4 filter paper to the almost set top surface, or by placing filter paper in the mould and pouring gelatine over it. The gelatine was subsequently tanned using the method of Clegg (1969). Soluble skin products were removed from closely clipped skin using redistilled chloroform on cotton-wool pads handled in forceps (Clegg, 1969). Half the chloroform extract was applied to half the area of skin from which it had been prepared and the chloroform allowed to evaporate, providing a membrane with the lipid replaced. The chloroform/lipid extract was added to no. 4 filter paper by capillary action and the chloroform allowed to evaporate.

Procedure Larvae were cultured from infected cat faeces in Petri dishes at 30 °C (Croll, 1972a) and were used between 4 and 8 days of culturing following separation on a, Baermann. funnel. Larvae were counted into cells of microtitre agglutination plates and allowed to settle. The volume of water was then reduced to about 0.2 ml and the larvae transferred to the top surface of the membrane by micro-pipette and left for the duration of the experiment. At the end of the experiment those larvae that had not entered the membrane were removed, initially by pipette and subsequently by thorough washing into a watch-glass. The apparatus was then carefully dismantled, the saline in the collecting cells removed and the under surface of the membrane also washed thoroughly, the saline and washings being collected. For histological examination the exposed area of skin was removed, fixed in 70 cy, 460 B. E. MATTHEWS ethanol for at least 18 h, dehydrated, cleared in cedarwood oil and embedded in Paraplast (Shandon) prior to serial sectioning. 10 ,am sections were stained routinely with haematoxylin and eosin. For quantitative experiments it was essential to know not only how many larvae had completely penetrated the membrane, but also how many had entered it. Skin membranes were cut into 1-2 mm squares and digested in a 1.1 mixture of 4 % pepsin and 0.5 % 1101 at 25 °C until completely broken down. Larvae were not affected by this treatment and could be removed from the digest. Paper membranes were teased apart in water and larvae lodged among the fibres removed. Larvae from different cultures have measurable variation (Croll, 1972a). To minimize such differences, experiments were conducted in pairs using skin and filter paper membranes as mutual controls. A slightly modified technique investigated the effect of gravity on penetration. The larvae were applied in water to the cold top surface of membranes in two separate pieces of penetration apparatus and left for 10 min to settle. The volume of fluid was reduced from 3 mm to about 1 mm with a micro-pipette, taking care not to remove the larvae. Cover-slips with a small quantity of vaseline on the corners were applied to the top of the penetration chambers and one apparatus inverted. The water supply was then connected and the experiment started. This method prevented larvae in the inverted cells having to move through 3 mm of water before reaching the membrane surface. In vivo tests demonstrated the applicability of the in vitro results. A cat was anaesthetized using Nembutal and the abdominal hair removed using fine cutters on electric clippers and finally carefully trimmed with fine scissors. Strips of zinc oxide tape coated with vaseline on the non-adhesive surface and from which 6 mm diameter holes had been cut, were stuck across the clipped area and drops of water containing infective larvae placed in the circle. After infection the animal was killed and the centre of each exposed area marked with a fine pin. The skin was then removed and processed for histological examination. Skin exposed to larvae in vitro was dehydrated in graded ethanol, allowed to air dry, mounted on aluminium studs using Silver Dag and coated with gold/palladium for examination in the Cambridge Stereoscan Microscope.

RESULTS Preliminary experiments were designed to establish a method of assaying the penetration process. The structure and thickness of the skin varies widely from one part of an animal to another and between individuals. Infective larvae of A. tubaeforme were not able to enter or penetrate rat skin in vivo, freshly excised in vitro, or dried, plucked and sanded after the method of Stirewalt et al. (1966). Penetration into cat and rabbit skin occurred, but the greater dermal thickness and more complex arrangement of hair follicles in these animals made it im- practical to prepare dried membranes of sufficient constancy for quantitative use. A suitable artificial membrane with constant characteristics was thus desirable. Gelatine membranes tanned to withstand the temperature used in penetration ficantly different entering themembranein2h,54.8%with gravityandonly5.1%against not abletopenetratethegelatine. result (Table1). it. Bymodifyingthetechniquesothatbothcontroland testlarvaewereina1mm tanned gelatinedemonstratedthatthelarvaecouldenterfilterpaperbutwere tests werenotpenetratedbylarvaebutfilterpaperwas.Laminatedmembranes paper didnotsignificantly affectthepercentageoflarvaeenteringthesemem- percentage oflarvaeenteringfilterpaper(Table1). Thisreductionwasnotsigni- penetrants recoveredfrombothskinandpapermembranes. before reachingthemembranecausedatenfold drop inthenumberoflarvae ship betweenpercentagesenteringthevariousmembranes.Thereweredifferences orientated. Theadditionofchloroformsolublecatskin productsdidnotaffectthis using bothcatandrabbitskinno.4filterpaper,showsalinearrelation- branes (Fig.3). between batchesoflarvaeandthesedifferenceswerereflectedintheresults water layer,penetrationagainstgravityresultedin onlyaslightreductioninthe consisting ofno.4filterpaperfirstlyontopof,andsecondlyimpregnatedwith, Fig. 2givesregressionlinesfordataextractedfrompairsofexperiments Inverting theapparatussothatlarvaehadtomigrate through3mmofwater Removing lipidsfromcat skinandreplacingthemineithercatorfilter y = Fig. 2.Relationshipbetweenentryof or rabbit(•—•)skinandno.4filterpaper.Regressionlinesu=1.78x+6.36 0.90x+3.67 respectivelyareplottedonthedata.

% entering paper 100 20 40 60 80 (P > • o Skin invasionbyhookwormlarvae 0.05) fromthecontrolsituationwithapparatusnormally 20

% enteringskin 0 A. tubaeforme 60

larvae intocat(0-0) 80

100 461 462 B. E. MATTHEWS

Table 1. Mean percentage larvae entering filter paper membranes in 2 h with and against gravity Exp. 1, Exp. 2, Membrane mean ± s .E. mean + S.E. No. 4 filter paper 44.8 + 5.0 58.9 + 3.6 No. 4 filter paper (apparatus inverted) 38.8 ± 2.8 51.6 + 5.6 No. 4 filter paper + cat skin lipids 52.3 + 6.0 60.1 + 3.2 No. 4 filter paper + cat skin lipids 46.2 + 6.5 53.9 + 5.4 (apparatus inverted)

100 —

Cat skin No. 4 filter paper

Control Control 80 I 1

Lipid Cat skin ®extracted lipid added ane

br Lipid

em 60 replaced m ing

enter 40 % n Mea

Exp. 1 Exp. 2 Exp. 1 Exp. 2

Fig. 3. Effect of chloroform soluble cat skin lipids on entry of A. tubaeforme larvae into cat skin and no. 4 filter paper in vitro. Each column represents the mean of six determinations. The vertical lines indicate standard errors of the mean.

The invasion process Larvae have been observed during the process of invasion on many occasions and the following description has been compiled from these observations. When larval suspensions are placed on the skin surface the worms move rapidly and actively towards it, at least 90 % being found on or very close to the skin surface within 10 min The approach to the skin surface follows a very similar pattern for all membranes tested, regardless of whether penetration through the membrane was possible. The mechanical disturbance inherent in transfer by pipette, together with the temperature difference, stimulate larvae that had been inactive in the agglutination plate cavities. As the larvae are slightly more dense than water (Croll, 1972 b) gravity may play a passive role in the approach to the membrane surface. Table 1 and Fig. 3 illustrate that neither gravity nor the presence or absence of skin. lipids significantly affected the percentage entering. Initial contact with the skin is normally made with the worm roughly perpendicular to the surface. The larvae remain at a steep angle to the skin surface for as Skin invasion by hookworm larvae 463 100

L 60

at ° .'171 40

0 20

1 2 4 Time (h) Fig. 4. Effect of time on the percentage of larvae entering cat skin (O - - 0 ) and no. 4 filter paper (1111—®). Each point represents the mean of four determinations. Regression lines y = 0.158x + 0.892 and y = 0.28x + 10.49 respectively are plotted on the data. long as they remain active and as long as there is a sufficient depth of water. If the water level is allowed to fall below the length of the larvae (about 650 ,am) the angle decreases as the worms are pressed closer to the substratum. The time spent searching for an entry site varies greatly. Larvae have been found in the epidermis within 5 min (Pl. 1 B), but there is continuous invasion of the skin for at least 4 h if the surface is kept moist (Fig. 4). Disappearance of the surface film causes cessation of movement and thus penetration. The surface of the skin is not smooth, the outer layers of the stratum corneum are thrown into folds and rugosities and the surface is broken by the apertures of the hair follicles and by numerous microlesions and desquamations of the keratinized cells (Pl. 1A). These provide the entry sites for the larvae. Plate 1 shows a larva that has passed into the outermost layers of the stratum corneum and come back on to the surface. This site of entry is in an area of the skin well away from the bundles of hair follicles. Entry appears to be possible at any site on the skin surface at which a suitably sized fissure is located and although hair follicles are also used (Pl. 1D, larvae are not restricted to this route. Once an entry site has been found, the larvae orientate parallel to the skin surface (Pl. 1B, D, E) following the line of least resistance into the skin. On no occasion has evidence been found for any destruction of the keratinized layers by larval enzymic activity. Infective third-stage hookworm larvae are surrounded by the retained cuticle of the second-stage larvae after the second moult. This sheath is not necessarily lost before penetration and is clearly visible in both the scanning electron micrographs and light microscope sections. DISCUSSION The success of an in vitro penetration system that sets out to provide results comparable to the in vivo situation must rely on the fact that the skin used in vitro possesses characteristics that are not essentially different from living tissue. In 464 B. E. MATTHEWS this respect skin is a particularly useful tissue. In vitro culture of skin cells is readily achieved in suitable media (Trowell, 1959), full thickness explants remaining viable for up to 6 weeks (Summerlin, Charlton & Karasek, 1970) and [14C]proline is incorporated into biopsy material for a number of hours after excision (Uitto, Lindy, Turbo & Danielson, 1971). Deep freezing or freeze drying appear to have little effect on the viability of skin as shown by subsequent grafting experiments (Billingham & Medawar, 1951), and the competence of the epidermis, as shown by diffusion of water through it, is retained for up to 19 days (Bettley & Donoghue, 1960). The stratum corneum is composed of non-nucleated, flattened, hollow cells with the peripheral cytoplasm keratinized and these are even less liable to change than the living cells of the deeper skin tissues. Although quantitative work on penetration has been conducted in vitro, as this was more reproducible, control experiments have been done in vivo, and on each occasion the same picture of penetration has emerged. The migration of larvae through a filter was shown by Looss (1911) to occur when the temperature was sufficiently high to permit larval mobility. This ability to penetrate blotting paper has been explained in terms of positive thigmotaxy (Lane, 1933). He suggested that larvae attempt to retain their anterior 'faces ' in contact with a solid surface but on irregular surfaces such as paper asymmetrical movement causes them to slip from the fibres into spaces. The normal undulatory movement then carries the worm forward until the anterior is in contact with another fibre when the process is repeated. Penetration through the paper is made up of a series of such stages. This mechanism may also explain the initial entry of hookworm larvae into the stratum corneum, the fissures of which act in the same way as the spaces between paper fibres. The direct relationship between entry into filter paper and entry into rabbit and cat skin (Fig. 2) suggests that entry may depend more on the surface structure of the membrane than on its chemical composition. The different slopes for the two skin types probably reflect differences inherent in the physical characteristics of the outer layers of the two animals. According to Goodey (1925), Kosuge (1924), working with Strongyloides larvae, showed that in the case of thin-skinned animals the entry was by epidermal scales, whereas in thick-skinned animals it was via the hair follicles. Although the skin types used were not mentioned it seems probable that cat and rabbit skin would be classed as thick in this context. The use of the scanning electron microscope, coupled with light microscopy, has shown that penetration by A. tubaeforme may be by either route but the former is the more usual. The question of exsheathment by infective larvae prior to penetration of the skin received some attention from earlier workers (Looss, 1911; Goodey, 1922) and it seems to have been tacitly accepted that it was essential in all cases before penetration could commence. Looss (1911), in his classic work on the life-cycle of the hookworm, suggested that exsheathment occurred when friction between the substrate and the sheath was so great that the sheath could no longer move but the enclosed larva was able to force an exit through the anterior end. If allowed to dry on an agar plate under constant conditions larvae of A. tubaeforme exsheath Skin invasion by hookworm larvae 465 as the sheath becomes held by the drying surface (Croll, 1972c). The organized exsheathment shown by Lapage (1935) for trichostrongyle larvae does not appear to occur for hookworm larvae nor is any specific exsheathing stimulus required. That penetration of ensheathed larvae could occur was considered unlikely by Rogers & Sommerville (1963), due to thes tructure of the sheath, and they felt that the frictional properties of the host skin would be insufficient to allow mechanical exsheathment. They concluded that penetration probably involves secretion of proteases and hyaluronidases and that these may first help to break down the sheath. The sheath of A. tubaeforme is not necessarily lost on entry into the skin either in vivo or in vitro and may be discarded at any stage in the penetration process. There thus seems to be less case to postulate the use of enzymes. The sheath includes a type of collagen which is partly keratinized and is unique to the nematodes (Simmonds, 1958; Bird & Bird, 1969). It seems unlikely that larvae would be able to retain their sheaths during penetration if an enzyme sufficiently powerful to soften the keratinized layers of the stratum corneum were produced. The direct relationship between entry into skin and non-living filter paper membranes also suggests that a mechanical rather than an enzymic mechanism may be operative. There is still the possibility that some enzymic secretion may assist penetration, but I have found no evidence of it so far. A. tubaeforme larvae were unable to enter and penetrate tanned gelatine membranes even when provided with a filter paper membrane to supply friction and initiate penetration. Larval trematodes have attachment organs that may also assist in concentrating enzymic secretions, but no such attachment organ has been found in skin penetrating larval nematodes. Goodey (1925) reported finding large numbers of discarded sheaths of A. caninum on the skin surface after penetration; these have not been found for A. tubaeforme, perhaps because Goodey used young rat skin rather than the definitive host skin. Using A. tubaeforme it has not been possible to demonstrate entry into rat skin. There is a possibility that Goodey transferred sheaths from the cultures, the age of the larvae used was not given and exsheathment within old cultures is not unusual. The difference in. skin structure may also explain Goodey's (1925) inability to demonstrate penetration against gravity or penetration from deep water drops. Qualitatively entry of larvae into cat skin has been demonstrated against gravity from 3 mm deep water drops; from 1 mm drops entry into filter paper did not differ significantly with or against gravity. That the edge of the penetration chamber was not providing mechanical assistance was shown by the fact that larvae were found in sections taken from all parts of the exposed area. The importance of skin lipids in the penetration of larval trematodes has recently been demonstrated. Clegg (1969) has shown that cholesterol, the largest fraction in chicken skin lipids, can stimulate the penetration of Austrobilharzia terrigalensis through tanned gelatine membranes. Wilson et al. (1971) have demonstrated that miracidia of Fasciola hepatica are stimulated to increase their time of attachment by host snail mucus and fatty acids of chain length C7_9. Skin lipids do not seem to play a part in the penetration process of A. tubaeforme as removal 466 B. E. MATTHEWS and replacement of the chloroform soluble fraction had no effect on the percentage of larvae entering skin or filter paper. The lipids appear to play no part in attracting larvae to the skin, in stimulating penetration behaviour or assisting in the process of penetration. N. A. Croll & J. H. Smith (1972, personal communication) have demonstrated the very marked response of hookworm larvae to temperature changes and the evidence at present points to this factor being the most important in attracting larvae to the skin surface and stimulating penetration behaviour. Stirewalt (1971) has come to a similar conclusion with regard to schistosome cercariae, chemical factors coming into play only when the skin has been contacted. The temperature difference between the environment and the host skin results in an increase in activity at the skin surface which, if penetration is mechanical as is postulated, is the time that it is most needed. Location of a suitable site and penetration before the surface moisture dries is of the greatest significance and depends on the worms being fully active. SUMMARY A modification of the 'floating raft' method of in vitro penetration study is described. This allows replication of results and additional conveniences in opera- tion. Filter paper membranes have shown characteristics similar to those of skin and have been used for quantitative studies. A method of digesting penetrating larvae from skin has been used to reduce the variability inherent in whole skin. Neither chloroform soluble skin products nor gravity were found to have a significant effect on the number of larvae entering membranes. Exsheathment of larvae was not essential prior to penetration and no specific stimuli for exsheathment appear to be necessary. Scanning electron micrographs have shown that entry may be by either hair follicles or desquamations of the stratum corneum. No evidence of enzymic activity during the invasion process has been found so far and the results suggest that a mechanical rather than chemical system obtains. My thanks are due to Dr N. A. Croll for his encouragement and advice, and to Professor T. R. E. Southwood in whose department this work was carried out. I should also like to thank Miss J. Finery and the Botany Department at Imperial College for the use of the stereoscan microscope. The work was supported by the Medical Research Council of Great Britain.

REFERENCES BARRETT, J. (1969). The effect of ageing on the metabolism of infective larvae of Strongyloides ratti Sandground, 1925. Parasitology 59, 3-17. BETTLEY, F. R. & DONOGHUE, E. (1960). Effect of soap on the diffusion of water through isolated human epidermis. Nature, Lond. 185, 17-20. BILLINGRAM, R. E. & MEDAWAR, P. B. (1951). The viability of mammalian skin after freezing, thawing and freeze-drying. In Freezing and Drying (ed. R. J. C. Harris), pp. 55-62. Sym- posium, Institute of Biology. BIRD, A. F. & BIRD, J. (1969). Skeletal structures and integument of Acanthocephala and Nematoda. In Chemical Zoology (ed. M. Florkin and B. T. Scheer), vol. III, pp. 253-88. New York: Academic Press.

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B. E. MATTHEWS (Facing p. 467) Skin invasion by hookworm larvae 467

CLEGG, J. A. (1969). Skin penetration by cercariae of the bird schistosome Austrobilharzia terrigalensis: the stimulatory effect of cholesterol. Parasitology 59, 973-89. CROLL, N. A. (1972a). Energy utilization of infective Ancylostoma tubaeforme larvae. Parasito- logy 64, 355-68. CROLL, N. A. (1972b). Feeding and lipid synthesis of Ancylostoma tubaeforme preinfeetive larvae. Parasitology 64, 369-78. CROLL, N. A. (1972c). Behaviour of larval nematodes. Symposium on Behavioural Aspects of Parasite Transmission. Linnaean Society (in the Press). GOODEY, T. (1922). Observations on the ensheathed larvae of some parasitic nematodes. Annals of Applied Biology 9, 33-48. GOODEY, T. (1925). Observations on certain conditions requisite for skin penetration by the infective larvae of strongyloides and ankylostomes. Journal of Helminth,ology 3,51-62. KOSUGE, I. (1924). Histologische Untersuchungen fiber das Eindringen von Strongyloides stercoralis in die Haut von Versuchstieren. Arch. fur Schiffs- and Tropenhygiene Bd. 28, 15-20. LANE, C. (1933). The taxies of infective hookworm larvae. Annals of Tropical Medicine and Parasitology 2'7, 237-50. LAPAGE, G. (1935). The second ecdysis of infective nematode larvae. Parasitology 27, 186-206. Looss, A. (1911). The anatomy and life-history of Agchylostoma duodenale Dub. Pt. II. Cairo Records of the Egyptian Government School of Medicine. ROGERS, W. P. (1939). The physiological ageing of ancylostome larvae. Journal of Helmintho- logy 17, 195-202. ROGERS, W. P. & SOMMERVILLE, R. I. (1963). The infective stage of nematode parasites and its significance in parasitism. Advances in Parasitology 1, 109-77. ROSE, G. G. (1954). A separable and multipurpose tissue culture chamber. Texas Reports of Biology and Medicine 12, 1074-83. SIMMONDS, R. A. (1958). Studies on the sheath of fourth stage larvae of the nematode parasite Nippostrongylus muris. Experimental Parasitology 7, 14-22. STIREWALT, M. A. (1971). Penetration stimuli for schistosome cercariae. In Aspects of the Biology of Symbiosis (ed. T. C. Cheng), pp. 1-24. Baltimore: University Park Press. STIREWALT, M. A., MINNICK, D. R. & FREGRAU, W. A. (1966). Definition and collection in quantity of schistosomules of Schistosoma mansoni. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 352-60. STIREWALT, M. A. & Uv, A. (1969). Schistosoma mansoni: cercarial penetration and schistosomule collection in an in vitro system. Experimental Parasitology 26, 17-28. Summ-ERLIN, W. T., CDARLTON, M. E. & KARASEK, M. A. (1970). Transplantation of organ cultures of adult human skin. Journal of Investigative Dermatology 55, 310-16. TROWELL, 0. A. (1959). The culture of mature organs in a synthetic medium. Experimental Cell Research 16, 118-47. UITTO, J., LINDY, S., TORTO, H. & DANIELSON, L. (1971). Biochemical characterization of pseudoxanthoma elasticum: collagen biosynthesis in the skin. Journal of Investigative Dermatology 57, 44--48. WILSON, R. A., PumN, R. & DENISON, J. (1971). An investigation of the mechanism of infection by digenetic trematodes: the penetration of the miracidium of Fasciola hepatica into its snail host Lymnaea truncatula. Parasitology 63, 491-506.

EXPLANATION OF PLATE A. Scanning electron micrograph of the surface of cat skin showing the bases of a number of hairs and the micro-lesions of the surface of the stratum corneum. x 450. B. Transverse section of ensheathed A. tubaeforme larva within the stratum corneum of the cat. Haematoxylin and eosin. x 1000. C. Scanning electron micrograph of an ensheathed A. tubaeforme larva that has entered the stratum corneum at one of the surface lesions but failed to penetrate. x 200. D. Scanning electron micrograph of ensheathed larva entering hair follicle. x 850. E. Scanning electron micrograph of ensheathed larva laying parallel to the skin surface after passage beneath the outermost layers of the stratum corneum. x 1500.

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