| dc.description.abstract | Various Echinococcus granulosus antigens obtained
from hydatid cyst fluid have been applied by several
research workers in diagnosis of livestock and human
hydatidosis using a variety of serological tests.
These tests, however, have suffered from lack of
sensitivity, specificity or both. These drawbacks have
been blamed partly on the cross-reacting components of
the crude antigens used in these tests. There have
been several attempts to purify and partially
characterize ~ granulosus antigens from hydatid cyst
fluid, but as yet the results obtained with serological
tests have not been satisfactory.
This project was therefore initiated with the
objective of purifying and carrying out a partial
characterization of one of the main parasite antigens
of hydatid cyst fluid (HCF). Further, the role this
antigen may play in serodiagnosis of hydatid disease in
livestock and man was to be investigated.
Specific immunoadsorbents were used to remove host
components and one lL. ~anulosus antigen, "Antigen 346"
from cattle HCF. After these absorptions, an lL.
granulosus antigen, "Antigen 880", was isolated from
the absorbed HCF by use of 2M phosphotungstic acid and
2M magnesium chloride solutions.
Analysis (J J". t.ho i s o Ia t.e ci by
:immunodiffusion, crossed immunoelec~rophoresis and disc
polyacrylamide gels revealed one protein species. The
mo.l e cuLa r wej girt of tho an c i gen was shownt.o be 24(), 000
daltons after analysis in polyacrylamide gels under
reducing conditions. The iso-elec~ric point (pI) was
shown to be 4.2. Staining wi~h pro~ein and lipid
staius revealed that "An t.Lgen 880" was a 1 .ipopr-ot.ei.n.
Treatment of "Antigen 880" wi1:.htrypsin, pepsln,
and phospholipase C and subsequent analysis for
activity by immunodiffusion showed that antigenic
activity W[1.S unaffec~ed by trypsinization,
pupsini~ClL:i.on and du.Lipidization. Ihis suggested cha1:.
activit.y of "Antigen 88G" resided in both the protein
and lipid moieties. Treatment of "Antigen 880" with
iodoacetamide and dithiothreital lDTI) did not affect
antigen activity. This implied that disulphide bonds
(8-5 bonds) were not a prerequisite to the antigenic
integrity of "Antigen 880".
The possibility or obtaining "Antigen 880"
devoid of other E~ gr~n~lQ§g§ and host antigens by
heating concentrated HeF at various temperatures and
incubation at various pH values was investigated. It
was found af t.e r immunodiffusion and immunoelectrophoresis
that ..Ant.a ge n 880" was the only
reactive Hei<' antigen a f t.e r i.nc ub a t i.o n r o r 10 minutes at,
temperatures between 1050C and 121uC. Further analysis
of the supernate heated at L10uC was carried out in a
Sephadex-G200 column.
Sephadex-G200 column.
Two peaks were obtained with
Antigenic activity was obtained
in Peak I. Peak I was analysed by ion-exchange
chromatography in a DEAE-cellulose column. Step-bystep
elution of protein using various molariLies of
pho s ph at.e buffer, pH 8.5 was applied. The only peak
obtained was eluted with 0.2M phosphate huiier. This
peak had "Antigen 880" activity. Analysis oi this
protein by disc gel electrophoresis showed one proLein
species. The "Antigen 880" was shown to be stable at
pH range of 1-11.
"Antigen 880" was trea1:,ed with EDTA and
subsequently heated at 1100C and investigated for
antigenic activity by use of immunodiffusion. The
antigen r e t a i n e d activi t.y after 't.ra-t.eme n t, with EDTA and
he at i.n g at, 110uC for 10 minutes. It, was concluded that
"Antigen 880" was not a metallo-protein.
'I'e s t s to det eruu.ne the p rimar y structures of
"Antigen 880" were carried
dimension paper chromatography,
out. Using the doubleleucine,
phenylalanine,
tyrosine and alanine were identified as the amino acid
constituents of "Antigen 880". The Biotronik 2,000
automatic amino acid analyzer revealed the presence of
the following amino acids: valine (0.85 umol./ml),
leucine (0.22 umol./ml), iso-leucine (0.18 umol./ml),
tyrosine (0.04 umol./ml), histidine (0.02 umol./ml),
aspartic acid, threonine, proline, alanine, cystine,
methionine, phenylalanine and lysine. By use of gasliquid
chromatography C8:0, C10:0, C12:0, C14:0, C16:0
and C18 were identified as the fatty acids found in
"Antigen 880".
"Antigen 880" obtained after heating concentrated
ReF at 1100C for 10 minutes and centrifugation at
2,000 x g was used as the source of antigen for serodiagnosis
of hydatid disease in man and livestock. In
the indirect hemagglutination (IHA) test, when a titre
of 1:64 and above was considered positive, sera from
Turkana individuals with and without hydatid cyst-like
masses showed a sensitivity of 73.7% and a specificity
of 76.0%. A predictive value of 86.7% was obtained
with these sera. A sensitivity of ·85.0% was obtained
with sera from individuals with surgically confirmed
hydatidosis. No false positive reactions were reported
with sera from healthy individuals who came from areas
known to be free from hydatid disease. With regard to
enzyme-linked immunosorbent assay (ELISA), when a titre
of 1:100 and above was considered positive, sera from
Turkana individuals with and without hydatid cyst-like
masses showed a sensitivity of 90.6% and a specificity
of 88.0%. A predictive value of 94.1% was obtained
with these sera. A sensitivity of 85.0% was obtained
with sera from individuals with surgically confirmed
hydatid disease. No false positive reactions were
recorded with sera from healthy individuals who came
from areas known to be free from hydatid disease.
By use of the t-test based on McNemar's method for
correlated proportions, no difference was found in
sensitivity and specificity of the IHA test and ELISA
in diagnosis of human hydatidosis.
With regard to livestock sera, an IHA titre of
1:64 and above was considered positive, while an ELISA
titre of 1:50 and above was considered positive. The
cattle sera showed an IHA test sensitivity of 77.4%, a
specificity of 75.0% and a predictive value of 80.0%;
while the ELISA showed a sensitivity of 45.2%, a
specificity of 58.3% and a predictive value of 58.4%.
By use of the t-test, the IHA test was shown to be more
sensitive than the ELISA in diagnosis of cattle
hydatidosis, while the two tests showed no significant
difference in their specificities.
The sheep sera gave an IHA test sensitivity of
45.4%,a specificity of 92.6% and a predictive value of
71.4%. A sensitivity of 68.2%, a specificity of 53.7%
and a predictive value of 37.5% were recorded with the
ELISA method. The IHA test showed a higher specificity
than the ELISA while the two tests were equally
sensitive in diagnosis of sheep hydatidosis.
An IHA test sensitivity of 81.3%, a specificity of
68.3% and a predictive value of 40.0% were obtained
with goat sera. The ELISA showed a sensitivity of
87.5%, a specificity of 41.3% and a predictive value of
27.5%. The IHA test showed a higher specificity than
the ELISA while the two tests were equally sensitive in
diagnosis of hydatid disease in goats.
The IHA test was used in examination of livestock
sera from Turkana District with a view to investigate
the possible application of the test in the hydatid
control programme in the district by identification of
animals with infective hydatid cysts. This would
facilitate selective removal of the positive reactors.
A specificity of 82.6% was recorded with these sera.
Cross-reactions were observed ~ith sera from animals
with the following infections: Stilesia hepatica
(11.1%), Oesophagostomum species (13.6%), Haemonchus
species (18.7%) and cysts of Taenia hydatigena (19.2%).
An overall sensitivity of 51.4% was obtained. However,
100% of the sera from animals with fertile hydatid
cysts gave positive reactions, 50% of sera from animals
with sterile hydatid cysts showed positive reactions
while 51.8% of sera from animals with either tiny or
calcified cysts gave positive reactions.
It was concluded that the IHA test is more useful
than the ELISA in diagnosis of hydatid disease in
livestock and man. Further, the IHA test may be usefully
applied in hydatid disease control programmes,
since 100% of the sera from livestock with fertile
hydatid cysts gave positive reactions. In addition, it
was concluded that the "Antigen 880" was a pH-stable
and thermo-stable lipoprotein with a molecular weight
of 240,000 daltons and pI of 4.2. The antigenic
activity of "Antigen 880" resided in both the lipid and
protein moieties and S-S bonds were not a prerequisite
to antigenic activity of "Antigen 880". | en |
