ANTIL YMPHOCYTIC SERA AND FASCIOLA INFESTATION 335

GREENHILL, J. P. . . . . . . . 1965. Obstetrics, 13th ed., Philadelphia and

MASTERS, M., AND CLAYTON, S . G. . . 1940. J . Obstet. Gynaec. Br. Emp., 47,437. NOVAK, E. R., AND WOODRUFF, J. D. . 1967. Novak’s Gynecologic and obstetric path-

ology, 6th ed., Philadelphia and London, p. 503.

London, p. 816.

WISLOCKI, G. B., AND DEMPSEY, E. w. . 1946. Endocrinology, 38, 90.

EFFECT O F ANTILYMPHOCYTIC SERA O N THE HISTOPATHOLOGY OF FASCIOLA HEPATICA INFESTATION I N RABBITS

KEVIN DODD AND TRAOLACH ONUALLAIN Department of Veterinary Pathology, University College, Dublin

PLATES CXLII AND CXLIII

1 M M A T U R E Fasciola hepatica induce extensive cellular damage in the liver parenchyma before they migrate to bile ducts to complete their life cycle (Urquhart, Mulligan and Jennings 1954; Urquhart, 1956). An intense cellular response occurs in the vicinity of the fluke and in the haemorrhagic migration tract produced by it. This cellular response is con- sidered to be involved in the repair process, and perhaps to limit the progress of the fluke (Urquhart, 1954).

The experiments recorded here were designed to investigate the effect of inhibition of the normal cellular response on the reaction caused by flukes as they move through the liver substance. Anti-rabbit-lymphocyte serum, prepared either in sheep or horses, was used to inhibit the cellular response.

MATERIALS AND METHODS

Animals. Healthy rabbits weighing 3 4 Ib. (1.5-1.8 kg) were used. Antilymphocyte sera. Horse and sheep anti-rabbit-lymphocyte sera were prepared by

inoculating 1 ml of minced rabbit spleen substance, suspended in a minimal volume of sterile normal saline, subcutaneously into a horse and a sheep on five occasions at intervals of a week. One week after the last inoculation, the animals were bled, and the serum was separated and frozen at - 20°C until required.

Parasites. Fasciola hepatica metacercariae were produced and handled essentially as suggested by Hughes (1959). The table shows the manner in which four groups of two rabbits were arranged. The rabbits were given subcutaneous injections of 2 ml of either horse (group I) or sheep (group 11) antilymphocytic sera on days -3, -2, - 1, 0, +1 , +2, +3, +4, + 5 and +6 of the experiment. Group-I11 rabbits served as serum controls. On day 0 approximately 200 metacercariae were administered in saline by mouth to all the rabbits excepting those of group 111. Group IV, which had received no serum, served as a control on the viability of the metacercariae. This group was also used to study the normal response of the rabbit to the young fluke.

RESULTS The animals showed no ill effects after injection of the sera. On day 22 post-infection,

one of the rabbits of group I1 died. During the next 4-5 days the remaining rabbits from groups I and I1 died. Groups I11 and IV were clinically healthy. The remaining rabbits were killed on day 28.

Received 15 July 1969; accepted 10 Aug. 1969.

336 KEVIN DODD AND TRQOLACH 0 NUAXLASIN

Species of origin of serum

Horse

Gross lesions In groups I and 11, the rabbits died from an acute hepatitis. The abdominal cavity was

filled with blood-stained fluid. All the livers were grossly swollen, with subcapsular haernor- rhagic tracts visible in every lobe. Heavy deposits of fibrin formed a continuous network on the liver capsule.

No gross lesions were present in group 111. The livers of group-1V rabbits were slightly swollen, and a small number of haemorrhagic

tracts were visible in the central lobe.

Number of Number of rabbits rnetacercariae

2 200

Histopathology Groups I and 11. Extensive haemorrhagic tracts are present. Hepatic cellular debris is

present in the tract, mixed with red cells. There is a minimal cellular response in the area (fig. 1). There is scanty cellular infiltration either around the fluke or in the vicinity of the tract (fig. 2). There is no evidence of a repair process in older tracts.

Group 111 shows normal liver morphology.

TABLE

Design of the experiments

Group

I

111

IV

Sheep Horse

None 1 i 1 -

Group IV. Haemorrhagic tracts are present in the central lobe. These tracts contain large numbers of leucocytes and macrophages (fig. 3). Each tract is ringed by layers of leucocytes including a large number of eosinophils. A distinct layer of rnacrophages, eosinophils and lymphocytes rings the flukes (fig. 4). There is evidence of repair processes in the older tracts (fig. 5).

DISCUSSION It is interesting that the rabbits from groups I and I1 died as a result of acute hepatitis.

The histopathology indicates that only a minimal attempt was made either to contain the flukes or to repair the damage caused by their migration. Both sheep and horse sera were effective in suppressing the cellular response of the rabbit to the invading fluke. In contrast, group-IV rabbits remained healthy; they showed an intense cellular response both in the tracts and in the vicinity of the fluke. These findings suggest that the cellular response served to limit the damage done by the fluke and that any damage was promptly repaired.

SUMMARY Horse and sheep anti-rabbit-lymphocytic sera were effective in suppressing the normal

cellular response of the rabbit to infestation with Fasciola hepatica. The lesions indicate

DODD AND 0 NUALLAIN

ANTILYMPHOCYTIC SERA AND FASCIOLA INFESTATION

PLATE CXLII

FIG. 1 .-Group4 rabbit, given horse antilymphocytic serum. Liver. Extensive spreading tracts are present with minimal cellular infiltration in the area. Haernatoxylin and eosin (HE). X 400.

FIG. 2.-Group-II rabbit, given sheep antilymphocytic serum. Liver. No cellular response in the vicinity of the fluke. HE. x 400.

DODD AND 0 NUALLAIN

ANTILYMPHOCYTIC SERA AND FASCIOLA INFESTATION

PLATE CXLIII

FIG. 3. - Group-IV rabbit; no serum given. Liver. There is an intense cellu- lar response both in and immediately around the tract. HE. X100.

FIG. 4.-Group-IV rabbit. Liver. There is a distinct cellular layer immediately around the fluke. HE. x loo.

FIG. 5.-Group-IV rabbit; no serum given. Liver. An old tract containing fibroblasts and a mix- ture of macrophages and eosinophils. HE. x 100.

CHANGES IN GROUND-SUBSTANCE OF HEALING WOUNDS 337

that the normal cellular response is essential in limiting the damage caused by Fasciola hepatica as it migrates through the liver parenchyma.

We would like to thank Professor Mullaney for the provision of animal facilities during this experiment.

REFERENCES HUGHES, D. L. . . . . . . . . 1959. M.Sc. Thesis, Univ. London. URQUHART, G . M. . . . . . . . 1954. Expl Parasit., 3, 38.

9, . . . . . . . 1956. J . Path. Buct., 71, 301. URQUHART, G. M., MULLIGAN, W., AND 1954. J. Infect. Dis., 94, 126.

JENNINGS, F. W.

C H A N G E S IN T H E GROUND-SUBSTANCE OF HEALING W O U N D S

J. JACQUES AND H. C. S . CAMERON The Department of Pathology, The Queen’s University of Berfast

PLATES C x L l v AND CXLV

THE non-fibrillary component of the extracellular substance in wounds is a continuous matrix, which, though optically homogeneous, has a highly heterogeneous chemical nature. This ground-substance, called “ Grundsubstanz ” or fundamental substance by the early German histologists, is a complex mixture containing a high proportion of polysaccharides, which Spiro (1966) divides into two groups. The first group includes the mucopolysacchar- ides, characterised by the presence of uronic acid, sulphate esters, or both, in addition to hexosamine; they are usually highly acidic and may be associated with protein by ionic or covalent links. Hyaluronic acid and the chondroitin sulphates are the most important members of this group (Meyer, 1959). The other main group defined by Spiro contains the glycoproteins, which usually contain neutral sugars but no uronic acid or sulphate esters.

The differing chemical compositions of these groups leads to varying staining affinities, so that the highly acidic mucopolysaccharides react positively with the metachromatic stains, and with Hale’s colloidal iron and with alcian blue, but they are not stained by the periodic acid-Schiff (PAS) method. The glycoproteins, on the other hand, are PAS-positive, but do not react with the other stains (Spiro). Dunphy and Udupa (1955) used these methods to study changes in the extracellular space during wound healing, and more recently Viljanto (1964) and Bentley (1967) among others have demonstrated that many of the described histochemical changes correlate well with direct chemical analysis.

Curran, Clark and Love11 (1965) and Lannigan and Zaki (1968) have extended the colloidal iron method to electron microscopy, but as yet this approach does not appear to have been applied to skin wounds. This paper correlates light and electron histochemistry in a study of the extracellular changes during healing. These changes may well be significant in the development of wound tensile strength (Bryant and Weeks, 1967) and of contraction (Schilling, 1968).

MATERIALS AND METHODS

Experiment. The backs of 10 young adult rats of the Wistar strain were closely shaved and, with the animals under ether anaesthesia, 2x 1 cm rectangular, full-thickness skin wounds were made over each scapula. The animals, with the wounds undressed, were then housed in individual cages and fed on a standard diet of calf nuts and water ad libitum. Animals were killed, by anaesthetic overdosage, each day during the 1st wk and then on

Received 28 July 1969; accepted 13 Aug. 1969.