Purification and partial characterization of an echinococcus granulosus antigen and its application in serodiagnosis of hydatidosis in •livestock and man.
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".
CitationNjeruh,F.M(1987). Purification and partial characterization of an echinococcus granulosus antigen and its application in serodiagnosis of hydatidosis in •livestock and man.
Department of Public Health, pharmacology and Toxicology, University of Nairobi
Echinococcus granulosus antigen