Following the general introduction, chapter one, the thesis is divided into four
chapters, covering literature review, materials and methods, results and discussion and
conclusion.
Fasciolosis is a liver fluke disease, caused by Fasciola hepatica in temperate
regions and high altitude areas of the tropics and subtropics and by Fasciola gigantica,
which is restricted to the tropics and subtropics. Liver flukes have a wide range of
definitive hosts, including man and in particular domestic ruminants, but the various
hosts are known to differ greatly in their resistance to infection with these parasites. For
example sheep are considered susceptible to challenge infection while cattle develop
resistance.
F. hepatica secretes the enzyme cathepsin-Ll protease (Fh-cathepsin) which has
a molecular weight (MWt) of 27 kDa. It is considered to have a functional role in
parasite evasion of the host immune response, through cleavage of host
immunoglobulin. The enzyme, glutathione s-transferase (GST) is of 27.8-29 kDa MWt,
is also secreted by F. hepatica (Fh-GST) and is thought to be involved in the
detoxification of exogenous (xenobiotic) and endogenous derived toxic compounds.
Both enzymes form part of the fluke excretory/secretory (E/S) products and are of
interest as they are considered as vaccine candidates against fasciolosis.
This study investigated the immunoglobulin isotype responses of sheep and
cattle, chronically infected with F. hepatica and F. gigantica, to defined the fluke
antigens (F. hepatica E/S products (Fh-E/S) or F. gigantica E/S products (Fg-E/S), Fhxii
cathepsin and Fh-GST). It was decided to study the immune response in chronically
infected animals since immunity is considered to play a potentially more important role
in chronic infection, than in acute infection, which is characterised by the death of the
animal through anaemia and blood loss caused by the migrating flukes. Serum and
faecal samples were collected weekly while the severity of the infections were defined
using clinical, parasitological, haematological, biochemical and pathological
parameters.
Serum and faecal antibody (total Ig, IgGb IgM, IgG2 and IgA) responses to 24-
48 hour Fh-E/S and Fg-E/S, adult Fh-cathepsin and adult Fh-GST were determined by
indirect Enzyme-linked Immunosorbent Assay (ELISA). The antigen recognition profile
of the Fasciola spp. infected sheep and cattle to Fh-E/S and Fg-E/S was examined by
sequential Western blotting.
The general clinical and pathological pattern, combined with the parasitological,
biochemical and haematological data confirmed that, in the main, the sheep and cattle
were suffering from chronic fasciolosis. Lesions were most severe in sheep culled 11 to
20 weeks post infection (wpi) and calves with single infection, but culled 12 wpi, and
those calves with challenge infection. Overall, the calves had very light infection and as
a result they displayed a less severe chronic fasciolosis than that observed in sheep.
A reduction in serum glucose levels was detected in F. hepatica and F. gigantica
infected sheep from 3 wpi, especially in severely infected sheep. In contrast, there was
an increase in serum P-hydroxybutyrate (P-HOB) levels in infected sheep, from about 6
to 15 wpi. And the extent of the rise in P-HOB levels was associated with the severity of
the infection There was no serum glucose or P-HOB changes observed in infected
calves. This was considered to be due to the very light fluke infection. Thus, it would
appear from the serum glucose and P-HOB levels, that fasciolosis leads to energy
deficiency (low glucose) and ketosis (increased b-HOB) especially noticeable in more
heavily infected animals.
There was an early (2-3 wpi) total Ig response to Fh-E/S and Fg-E/S, Fhcathepsin and Fh-GST in both F. hepatica infected sheep and cattle. Although there was
an early (2-3 wpi) total Ig response to Fh-E/S and Fg-E/S, and Fh-GST by F. gigantica
infected animals, there was a slight delay (7 wpi) noted in the response to Fh-cathepsin.
The pattern of the IgGj response of cattle and sheep to these defined fluke antigens was
similar to that of total Ig. In fact the serum isotype response was predominantly IgG].
The IgM response to Fh-E/S and Fg-E/S, Fh-cathepsin and Fh-GST was early in both
species. In cattle the IgG2 and IgA responses to Fh-E/S and Fg-E/S were late and more
pronounced (11 wpi and 19 wpi respectively) in contrast to sheep (2 wpi for both
isotypes). The serum IgG2 and IgA isotype responses to Fh-cathepsin and Fh-GST
followed the same pattern in cattle, however in sheep, responses to Fh-cathepsin was
much less marked and a response to Fh-GST was not detected. A rise in total Ig and
IgG, responses to Fh-E/S and Fg-E/S and Fh-cathepsin were detected following
challenge infection in calves, but there was no increase in the response to Fh-GST. The
dominance of the IgGj response in Fasciola spp. infected sheep and cattle suggests an
associated Th2 response in both species. The late IgG2 response in cattle may suggest
late Th, involvement in bovine cellular responses to Fh-E/S and Fg-E/S products.
The detection of serum antibody responses to Fh-cathepsin and Fh-GST in F.
gigantica infected sheep and cattle confirmed antigenic cross-reactivity. However this
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cross-reactivity may be only partial, as suggested by the late total Ig and IgG, response
to Fh-cathepsin by F. gigantica infected sheep and cattle. Comparison of the isotype
responses to Fh-E/S and Fg-E/S products, Fh-cathepsin and Fh-GST suggest that there
is very little difference between the response to Fh-E/S, Fg-E/S and Fh-cathepsin,
however, there was a relatively poor response to Fh-GST in both sheep and cattle.
Western blot analysis ofF. hepatica infected sheep serum identified antigens of
14 kDa from 2 wpi and 54, 79 and 134 kDa MW recognised later. In F. gigantica
infected sheep antigens of 14, 88 and 152 kDa were identified from 7-9 wpi. There was
a clear shift, at patency, in the antigen recognition pattern of cattle from higher (134
kDa for F. gigantica infection and 142 kDa for F. hepatica infection) to lower (60 kDa
for both parasite species) but there was no clear antigenic shift observed in sheep. None
of these antigenic molecules represented Fh-cathepsin (27 kDa MWt) or Fh-GST (27.9-
29 kDa MWt) used as antigens in the ELISA assay. The lower protein concentration of
the E/S products used in this assay is, one possible reason for failure to detect these
molecules.
There was no faecal antibody response detected in cattle to any of the three
defined antigens. This might have been due either to the light infections observed in
cattle or to the larger volume of faecal material produced by cattle (i.e. dilution). There
was an early (2 wpi) faecal total Ig response to Fh-E/S and Fg-E/S, Fh-cathepsin and
Fh-GST in F. hepatica and F. gigantica infected sheep. In fact there was no difference
in faecal antibody responses to the different antigens by either F. hepatica or F.
gigantica infected sheep. The isotype response was mainly IgA while a slight IgG2
response could be detected in F. hepatica infected sheep. The total Ig and IgA responses
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to all three defined antigens in F. hepatica sheep was biphasic, in F. gigantica infection
however the phases were less defined. The 1st phase (about 2-10 wpi) is considered a
response to juvenile flukes antigens after oral infection and the second peak (13-17 wpi)
is considered to be in response to antigens released by adult flukes in the bile duct.
This study indicates that in serum, IgG( isotype responses predominate and in
faeces IgA isotype responses to the three defined antigens predominate, suggesting
preferentially stimulation of the Th2 T-cell subset in sheep and cattle fasciolosis. The
late IgG2 response to F. hepatica and F. gigantica E/S and Fh-cathepsin in cattle may
indicate a delayed Th, stimulation.
In order to elucidate the different immune mechanisms operating in infected
sheep and cattle there is need for more work on the cellular responses to Fasciola spp.
as these may be related to resistance. There was antigenic shift in cattle at patency but
not in sheep. The 60 kDa molecule being recognised by cattle after the antigenic shift. It
is possible that this factor may be related to acquisition of resistance by cattle to
secondary Fasciola spp. infection
Finally the findings relating to the glucose and b- HOB levels in infected sheep
may prove helpful in experiments involved in the interaction between fasciolosis and
the nutritional levels of Fasciola spp infected animals, of particular importance in
young growing animals.
