Fernandez, Jr., Tomas Jorge.

2018-05-22T12:38:39Z

2018-05-22T12:38:39Z

1984

The main aims of this study were to determine the times after infection or reinfection that immune serum from bullocks infected with Fasciola hepatica would transfer protection to rats and to de¬ vise means of sensitising bullocks by which the animals could be stimulated to produce a strongly protective immune serum.

The serological response by enzyme-linked immunosorbent assay (ELISA), using either a somatic or a metabolic antigen, the serum glutamic dehydrogenase (GD) activities, and the peripheral eosinophil counts were monitored from both bullocks and rats during the infections.

Firstly, two bullocks of about 18 months of age were given an initial infection of 1,000 metacercariae of F. hepatica and immune sera (IBS) collected at three-weekly intervals and passively trans¬ ferred to rats, to monitor their protective effect. Serum obtained during the prepatent period, 6-9 weeks after initial infection, gave partial protection. In an attempt to obtain a more strongly protect¬ ive serum that could transfer a more effective resistance, the bull¬ ocks were given two more infections (22 weeks apart) of 1,000 meta¬ cercariae to each animal on each occasion. However, the IBS from previously infected bullocks gave only relatively weak protection. Indeed IBS from one of the bullocks (9U)> collected after repeated infections was not protective at all.

From this study it is clear that juvenile flukes are immunogenic, giving rise to humoral protective agents, since IBS collected at weeks 6-9 after initial infection was protective. Conversely, it is also clear that repeated oral infection is not a suitable method for enhancing the humoral protective response, probably because the challenge flukes are soon killed by the cellular response in previously sensitised animals.

It was thought that age might influence the ability of immune serum from bullocks to transfer resistance. Accordingly three adult mature bullocks (b-S years old) were infected with 1,000 metacercariae of F. hepatica and 30 weeks later another 1,000 metacercariae were administered. However the immune serum collected at week 9 from one of the bullocks was not protective while that from the other two was rather weak. After secondary infection, the immune sera collected from 204 appeared to give some protection but not that from 203 or M199.

The opposite hypothesis was then examined, namely that older, immunologically mature animals rely more on a cellular response and consequently have a weaker humoral protective response. Accordingly four immature bullocks (6 months old) were infected with 1,000 meta¬ cercariae once, IBS collected six weeks later and again when the animals were killed nine weeks after infection. However these sera were not strongly protective to naive rats.

Following initial infection the animals tended to show a biphasic reaction in all the monitored parameters. However, because of great variations between animals, the pattern for some of the parameters was not always clear in individual bullocks.

The first ELISA peak occurred before patency and the second after patency. This phenomenon of a biphasic' serological response was ob¬ served by Gundlach (1971) in rabbits, using metabolic or somatic antigen in complement fixation tests. It is suggested that the first ELISA. peak coincides with liver migration and the active feeding by vi. the juvenile flukes on the parenchymal cells, while the second peak, which is usually lower than the first peak, coincides with an immune response to products released from flukes in the bile duct.

Following repeated infections, the ELISA values tended to be higher than those following primary infection. There was, therefore, no evidence of any direct relationship between the ELISA value and the protective effect of the sera. At the same time the serum GD activities and the peripheral eosinophil counts tended to be lower and faecal egg counts were very low after repeated infections, showing that the animals had resisted the challenge infections.

The GE and eosinophil levels rose to an initial peak at week with the maximum values being reached at weeks 12-16^ in bullocks. Again it is suggested that the first peak results from the direct effect of the parenchymal liver migration by the juvenile flukes, while the second peak probably results from the combined effect of both the fibrotic healing process in damaged liver and the presence of antigen-antibody complex in the liver.

It therefore appears that, although the bullocks previously sensitised by oral infection had acquired strong resistance against challenge infections, this was not related to the increased concen¬ trations of antibodies in their immune serum but was more likely to be a cellular effect. Thus it was thought that other means than simply challenging previously infected,animals would be necessary to produce more strongly protective antisera. Implantation of encapsulated flukes, which might be protected from the cellular protective mechanisms in the hosts and so be able to release the protective-inducing immunogen for a more extended period, was therefore adopted,

A preliminary study on the effect of implanting flukes in diffusion chambers on the immune response of animals was carried out in rats. The ELISA results suggested that there was an elevated ELISA response in the rats following implantation but this was short¬ lived. In the definitive experiment in bullocks, for logistic reasons, the serum was collected eight weeks after implantation, when a shorter period might have been optimal. Nevertheless, in the associated passive transfer study it was found that immune serum from the im¬ planted bullocks tended to give better protection, except in one animal, than that from orally challenged bullocks. However, these results cannot be considered conclusive because of the limited numbers of experimental animals used.

The relatively short elevation in the ELISA value after implantation was attributed to encapsulation of the diffusion chambers "containing the flukes by the host's cellular reaction against diffusing metabolic antigens.

http://hdl.handle.net/1842/30190

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 19

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Studies on the passive transfer of resistance to Fasciola hepatica

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