In vitro culture of Fasciola hepatica and the immunology associated with the metabolic products of the trematode

This work is presented in three Sections, concerned respectively with the in vitro maintenance of Fasciola hepatica, production and properties of the metabolic antigens of F. hepatica and immunisation of the mammalian host with these antigens.In Section One a number of studies were made on the in vitro maintenance of adult F. hepatica, with a view to establishing a routine method for maintaining large numbers of flukes together, to facilitate subsequent studies on the antigenic properties of their metabolic products. A detailed description is given of a continuousflow culture apparatus for the axenic in vitro maintenance of groups of adult flukes. The suitability of various media and culture conditions was assessed by measuring the rate of production of ammonia, an end-product of trematode protein metabolism.It was shown that when the medium consisted of a simple balanced salts solution (modified Earle's salts) flukes showed 100% survival over a five day culture period, but the rate of ammonia production declined rapidly during the initial three days. However, when the medium was supplemented with increasing amounts of serum, the rate of ammonia production was maintained at levels more closely approaching the initial level. Medium 199 (a multi-component tissue culture medium) was shown to be slightly more favourable than the simple balanced salts solution, but less so than those media which contained high proportions of serum. It was shown that inclusion of clotted blood in the medium had no effect on ammonia production, but an increased flow-rate of the culture medium was associated with the output of significantly greater amounts of ammonia, presumably due to the more rapid dispersal of toxic metabolic waste products.A series of experiments was undertaken to investigate the possibility of maintaining juvenile flukes in vitro, from the metacercarial stage. It was shown that the survival period could be increased from two days, in a balanced salts solution, to 11 days by the inclusion of more complex nutrients such as serum, blood or liver extract in the medium. However, little or no growth occurred under these conditions. Survival and growth were maximal when the young flukes were able to feed on monolayers of living cells, but even under the most favourable conditions growth was limited and compared very unfavourably with that expected in vivo.In Section Two the continuous-flow culture apparatus was modified to form a recirculating system, in which large groups of adult flukes could be maintained for reasonably prolonged periods. Following concentration, the medium in which flukes had thus been maintained showed strong antigenic properties against sera from fluke-infected animals. When flukes were maintained in Medium 199» it was shown , that they continued to produce antigenic metabolites throughout a nine day culture period, although the amounts produced decreased with time.Subsequent studies indicated that the concentrated culture medium contained a variety of antigenic components, including both heatstable and heat-labile substances, which reacted to differing degrees with serum from fluke-infected rabbits, sheep, rats and bovines. These reactions were investigated using double diffusion immunoprecipitation in agar and the Enzyme Linked Immunosorbent Assay (ELISA), a serological technique which had not previously been applied to fascioliasis. It was shown that metabolic antigen could be used to follow the serological response developing in experimentally infected animals, but there was no evidence to suggest that it had any particular advantage over more readily obtainable somatic fluke antigen. ELISA was found to be a useful technique, which in some cases enabled the serological response to be detected at an earlier stage than by immunodiffusion.In view of the apparently complex nature of the metabolic antigen, it was concluded that in its present form it was not likely to be suitable for application to the serodiagnosis of fascioliasis in the field. However, since it is probably less complex than somatic fluke antigen, it may provide a more suitable starting material from which to attempt to isolate purified and species-specific antigens for this purpose.In Section Three a series of experiments was undertaken to establish whether laboratory animals immunised with metabolic antigens derived from F, hepatica maintained in vitro would respond with the production of antibodies and, if so, whether these were associated with any degree of protection against a subsequent challenge infection.Rabbits immunised with metabolic antigen derived from adult flukes produced antibodies, which showed certain features in common with those present in the serum of rabbits carrying a patent fluke infection. There was some evidence that these antibodies had an effect on the course of the challenge infection, since the mean size of the flukes recovered from the immunised animals was significantly smaller than that of the flukes recovered from non-immunised control rabbits. However, there was no significant difference between the two groups of rabbits in terms of the numbers of flukes recovered. The host response to the challenge infection, as indicated by the eosinophilia and increase in plasma glutamate dehydrogenase, wan consistently less in the immunised rabbits, but there were marked within-group variations in these parameters and the differences between the two groups were not of statistical significance,A series of experiments was then carried out, in which rats, of a strain known to be capable of developing acquired resistance to F. hepatica. were immunised with metabolic antigen prepared in various ways from either adult or immature flukes. In all cases the immunised animals produced antibodies to the immunising antigen, which again showed some features in common with those present in the serum of infected animals. However, it was also apparent that there were significant differences between the antibody components of the two types of sera. In particular, it was evident that the immunised rats were not responding well to the non-protein component of the metabolic antigen, in contrast to the infected animals, which showed a strong reaction with this component of the antigen.There was no evidence that the antibodies resulting from "the immunisation of rats with metabolic antigen of F. hepatica had any effect on the course of a subsequent challenge infection. In no case was there any significant difference between the immunised, and control rats in terms of the numbers or sizes of flukes recovered from the challenge infection, or the host response to challenge, as indicated by the eosinophilia and plasma glutamate dehydrogenase assays.An attempt was made to verify the claim of Lang (1976) that mice could be successfully proteoted against F, hepatica by immunisation with the medium in which 16-day-old flukes had been incubated for 24- hours. The design of this experiment was similar, but not identical, to that of Lang, but the results were in marked contrast, there being no differences between the immunised and control mice in terms of the infection rate, mortality rate or numbers of flukes recovered from the challenge infection.It was therefore concluded that metabolic antigen derived from adult or immature F. hepatica maintained in vitro as described was incapable of stimulating resistance to a challenge infection, even in a strain of animals known to be capable of demonstrating acquired immunity to this parasite. This indicated that either the metabolic antigens obtained in vitro were significantly different from those produced in vivo, to the extent that they had lost the ability to stimulate protective immunity in the host animal, or that there is a need for some alternative or additional stimulus to the host before such immunity can be initiated