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
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
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