Research in Veterinary Science 1992, 53, 133-135
Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera D. LIU, University of Melbourne, Veterinary Clinical Centre, Princes Highway, Werribee, Victoria 3030, Australia, M. D. R I C K A R D , CSIRO Division of Animal Health, Private Bag Number 1, Parkville, Victoria 3052, Australia, M. W. LIGHTOWLERS, University of Melbourne, Veterinary Clinical Centre, Princes Highway, Werribee, Victoria 3030, Australia
Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by deetrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B. I M M U N O E L E C T R O P H O R E S I S has proved to be a useful technique for the qualitative study of taeniid antigens, and various taeniid antigens have been identified by their formation of distinctive electrophoretic bands with antisera. Chordi and Kagan (1965) showed that a total of 19 different antigenic components were identified in Echinococcus granulosus cyst fluid, of which 10 were attributed to the parasite. The observation of an immunoelectrophoretic arc 5 and later an antigen B (AgB) band formed between antibodies in sera of patients infected with E granulosus and hydatid cyst fluid antigens (Capron et al 1967, Oriol et al 1971) have made electrophoresis a valuable tool for the diagnosis of hydatid disease in man. Although arc 5 based human hydatid diagnosis has been compromised by the fact that sera from patients infected with E multilocularis, E vogeli and Taenia solium cysticercosis also contained antibodies to E granulosus antigen 5 (Ag5) (Varela-Diaz et al 1977b, 1978, Yarz~tbal et al 1977, Schantz et al 1980), the demonstration of arc 5 forming antibodies in human patient sera still provides clear evidence of taeniid parasite infection, most probably hydatidosis. On the other hand, attempts to use arc 5 for specific diagnosis of
Reprint requests: D. Liu, Department of Agriculture, Regional Veterinary Laboratory, Hamilton, Victoria 3300, Australia
hydatid infection in animals have often been hindered by the coexistence of other taeniid parasites. Sera of sheep infected with Thydatigena and Tovis were shown to form arc 5 with E granulosus cyst fluid, and Ag5 was detected in cyst fluids of Thydatigena and probably of T ovis (Varela-Diaz et al 1977a, Yong and Heath 1979). Further, the resemblance of antigens isolated from cyst fluids of T hydatigena and T multiceps to E granulosus Ag5 and AgB was also noted (Daoud and Herbert 1982). This report describes a comparison of cyst fluid antigens of E granulosus, T hydatigena and Tpisiformis using homologous and heterologous hyperimmune rabbit sera in electrophoresis with a view to elucidating further the antigenic relationships between E granulosus and T hydatigena and T
pisiformis. Cyst fluids of E granulosus (SHCF), T hydatigena (THCF) and T pisiformis (TPCF) were collected, centrifuged, filtered and dialysed as described (Liu et al 1992a). Methods for the preparation of hyperimmune rabbit and sheep sera to these antigens (ie, RxSHCF, RxTHCF, RxTPCF and SxSHCF) a r e reported elsewhere (Liu et al 1992b). Immunoelectrophoresis was based on the method of Varela-Diaz and Coltorti (1976) except that the slides were stained with Coomassie blue. SHCF, THCF and TPCF were used at 50 mg m1-1 in electrophoresis. At least five bands were detected in SHCF by rabbit anti-sHCF and four bands by sheep anti-SHCF sera, all with characteristic arc 5 and AgB bands (Fig l a). Rabbit antisera to THCF and TPCF formed five and two bands with SHCF, one of which seemed to be similar to hydatid arc 5 (Fig lb and c). Serum from a rabbit immunised with THCF revealed at least five bands with TI~CF, two of which appeared to share identities with hydatid arc 5 and AgB bands (Fig ld). Rabbit anti-SHCF detected five bands with THCFincluding arc 5 (Fig le), but it was not certain that the band located close to hydatid AgB was identical to AgB. Serum from a rabbit immunised with TPCF formed two bands with THCF, one of which had a pattern similar but not identical to hydatid AgB (Fig lf). TPCF 133
D. Liu, M. D. Rickard, M. W. Lightowlers
34
15
\B
b B
~5 ~B
B
f
f
©
......
.
~ ii¸ ~ : ~ : ,
....
IB
B FIG 1: Immunoelectrophoresis of cyst fluid antigens of E granulosus, T hydatigena and Tpisiformis using hyperimmune rabbit sera, Antigens in the middle wells: a to c, SHCF; d tO f, THCF; g to i, TPCF. Top troughs: a,e,h, rabbit antiserum to SHCF; b,d,i, rabbit antiserum to THCF; C, f, g, rabbit antiserum to TF'CF. Bottom troughs B: reference sheep anti-sHcF serum
Immunoelectrophoresis of taeniid cyst fluid antigens TABLE 1: Summary of immunoelectrophoretic bands formed between taeniid antigens and hyperimmune rabbit sera Antigen
RxSHCF
Serum RxTHCF RxTPCF
SxSHCF
SHCF THCF TPCF
5(5,B)* 5(5,B?) 2(B)
5(5,B?) 5(5,B) 1 (B)
4(5,B) 2(5,B?) 1 (B)
2(5,B?) 2(5?,B) 1 (B)
* 5 or B indicates the presence of hydatid arc 5 or AgB band. The ? indicates some uncertainty in assigning the band to arc 5 or AgB band
seemed to have a predominant antigen similar to hydatid AgB as shown by its reactions with hyperimmune rabbit sera to TPCF, SHCF and THCF (Fig lg to i). The immunoelectrophoretic bands formed by taeniid antigens with hyperimmune sera are summarised in Table i. Previous studies have indicated that T hydatigena cyst fluid contains antigens similar to E granulosus Ag5 and also possibly AgB (Varela-Diaz et a11977a, Daoud and Herbert 1982). The present report confirms the existence of hydatid Ag5 in T hydatigena cyst fluid. In addition, T hydatigena cyst fluid might also have an antigen close to hydatid AgB. However, the AgB band formed by RxSHCH and THCF, or by RxTHCF and SHCF does not have a perfect hydatid AgB band shape compared to the control, suggesting that the physico-chemical property or antigenicity of AgB in SliCE differs slightly from that in THCE. The demonstration of arc 5 forming antibodies in the serum of a rabbit immunised with TlaCV further supports the finding of arc 5 formed by sera of sheep infected with T hydatigena (Yong and Heath 1979). On the other hand, Tpisiformis cyst fluid might contain mainly an antigen similar to hydatid AgB as detected by rabbit antisera to TPCF, THCF and SliCE. Tpisiformis cyst fluid does not seem to have an antigen close to hydatid Ag5 even though rabbit antiserum to TPCF formed a band around the position of hydatid arc 5 with SliCE and THCE. Perhaps there is an antigenic component in T pisiformis cyst fluid, which by itself is not able to form an arc 5-1ike band with rabbit antisera to TPCF, THCF or SHCF. However, this antigen becomes more visible when injected into a rabbit by the reaction of rabbit anti-TPCF with SliCE or THCF.
Acknowledgements The financial support of the University of Melbourne Veterinary Research Fund and the National Health
135
and Medical Research Council of Australia is gratefully acknowledged.
References CAPRON, A., VERNES, A. & BIGUET, J. (1967) Le diagnostic immuno61ectrophor6tique de l'hydatidose. Le kyste hydatique du foie, Journ6e Lyonnaises d'Hydatidologie, Lyon, SIMEPeditions. pp27-40 CHORDI, A. & KAGAN, h G. (1965) Identification and characterization of antigenic components of sheep hydatid fluid by immunoelectrophoresis. Journal of Parasitology 51, 63-71 DAOUD, I. S. & HERBERT, I. V. (t982) Isolation of two lipoprotein antigens from the metacestodes of Taenia hydatigena (Pallas, 1766) and Taenia multiceps (Leske, 1780) and their evaluation in serodiagnosis. Veterinary Parasitology 11, 155-164 LIU, D., LIGHTOWLERS, M. W. & RICKARD, M. D. (1992a) Evaluation of a monoclonal antibody based competition ~LISAfor diagnosis of human hydatidosis. Parasitology 104, 357-361 LIU, D., LIGHTOWLERS, M. W. & RICKARD, M. D. (1992b) Examination of routine antibody response to secondary hydatidosis using ELISAand immunoelectrophoresis. Parasite Immunology (in press) ORIOL, R., WILLIAMS, J. F., PEREZ ESANDI, M. V. & ORIOL, C. (1971) Purification of lipoprotein antigens ofEchinococeus granulosus from sheep hydatid fluid. American Journal of Tropical Medicine and Hygiene 20, 569-574 SCHANTZ, P. M., SHANKS, D. & WILSON, M. (1980) Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. American Journal of Tropical Medicine and Hygiene 29, 609-612 VARELA-DIAZ, V. M. & COLTORTI, E. A. (1976) Immuno-diagnostic techniques for human hydatidosis, Monograph number 7. Pan American Zoonosis Center, Pan American Health Organisation, Buenos Aires VARELA-DIAZ, V. M., COLTORTI, E. A. & D'ALESSANDRO, A. (1978) Immunoelectrophoresis tests showing Echinococcus granulosus arc 5 in human cases of Echinococcus vogeli and cysticercosismultiple myeloma. American Journal of Tropical Medicine and Hygiene 27, 554-557 VARELA-DIAZ, V. M., COLTORTI, E. A., RICKARD, M. D. & TORRES, J. M. (1977a) Comparative antigenic characterization of Echinococcus granulosus and Taenia hydatigena cyst fluids by immunoelectrophoresis. Research in Veterinary Science 23, 213216 VARELA-DIAZ, V. M., ECKERT, J., RAUSCH, R. L., COLTORTI, E. A. & HESS, U. (1977b) Detection of the Echinocoecus granulosus diagnostic arc 5 in sera from patients with surgicallyconfirmed E multilocularis infection. Zeitschrift fiir Parasitenkunde 53, 183188 YARZABAL, L. A., BOUT, D. T., NAQUIRA, F. R. & CAPRON, A. R. (1977) Further observations on the specificity of antigen 5 of Echinococcus granulosus. Journal of Parasitology 63, 495-499 YONG, W. K. & HEATH, D. D. (1979) 'Arc 5' antibodies in sera of sheep infected with Echinococcus granulosus, Taenia hydatigena and Taenia ovis. Parasite Immunology 1, 27-38 Received November 25, 1991 Accepted February 21, 1992
Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera D. LIU, University of Melbourne, Veterinary Clinical Centre, Princes Highway, Werribee, Victoria 3030, Australia, M. D. R I C K A R D , CSIRO Division of Animal Health, Private Bag Number 1, Parkville, Victoria 3052, Australia, M. W. LIGHTOWLERS, University of Melbourne, Veterinary Clinical Centre, Princes Highway, Werribee, Victoria 3030, Australia
Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by deetrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B. I M M U N O E L E C T R O P H O R E S I S has proved to be a useful technique for the qualitative study of taeniid antigens, and various taeniid antigens have been identified by their formation of distinctive electrophoretic bands with antisera. Chordi and Kagan (1965) showed that a total of 19 different antigenic components were identified in Echinococcus granulosus cyst fluid, of which 10 were attributed to the parasite. The observation of an immunoelectrophoretic arc 5 and later an antigen B (AgB) band formed between antibodies in sera of patients infected with E granulosus and hydatid cyst fluid antigens (Capron et al 1967, Oriol et al 1971) have made electrophoresis a valuable tool for the diagnosis of hydatid disease in man. Although arc 5 based human hydatid diagnosis has been compromised by the fact that sera from patients infected with E multilocularis, E vogeli and Taenia solium cysticercosis also contained antibodies to E granulosus antigen 5 (Ag5) (Varela-Diaz et al 1977b, 1978, Yarz~tbal et al 1977, Schantz et al 1980), the demonstration of arc 5 forming antibodies in human patient sera still provides clear evidence of taeniid parasite infection, most probably hydatidosis. On the other hand, attempts to use arc 5 for specific diagnosis of
Reprint requests: D. Liu, Department of Agriculture, Regional Veterinary Laboratory, Hamilton, Victoria 3300, Australia
hydatid infection in animals have often been hindered by the coexistence of other taeniid parasites. Sera of sheep infected with Thydatigena and Tovis were shown to form arc 5 with E granulosus cyst fluid, and Ag5 was detected in cyst fluids of Thydatigena and probably of T ovis (Varela-Diaz et al 1977a, Yong and Heath 1979). Further, the resemblance of antigens isolated from cyst fluids of T hydatigena and T multiceps to E granulosus Ag5 and AgB was also noted (Daoud and Herbert 1982). This report describes a comparison of cyst fluid antigens of E granulosus, T hydatigena and Tpisiformis using homologous and heterologous hyperimmune rabbit sera in electrophoresis with a view to elucidating further the antigenic relationships between E granulosus and T hydatigena and T
pisiformis. Cyst fluids of E granulosus (SHCF), T hydatigena (THCF) and T pisiformis (TPCF) were collected, centrifuged, filtered and dialysed as described (Liu et al 1992a). Methods for the preparation of hyperimmune rabbit and sheep sera to these antigens (ie, RxSHCF, RxTHCF, RxTPCF and SxSHCF) a r e reported elsewhere (Liu et al 1992b). Immunoelectrophoresis was based on the method of Varela-Diaz and Coltorti (1976) except that the slides were stained with Coomassie blue. SHCF, THCF and TPCF were used at 50 mg m1-1 in electrophoresis. At least five bands were detected in SHCF by rabbit anti-sHCF and four bands by sheep anti-SHCF sera, all with characteristic arc 5 and AgB bands (Fig l a). Rabbit antisera to THCF and TPCF formed five and two bands with SHCF, one of which seemed to be similar to hydatid arc 5 (Fig lb and c). Serum from a rabbit immunised with THCF revealed at least five bands with TI~CF, two of which appeared to share identities with hydatid arc 5 and AgB bands (Fig ld). Rabbit anti-SHCF detected five bands with THCFincluding arc 5 (Fig le), but it was not certain that the band located close to hydatid AgB was identical to AgB. Serum from a rabbit immunised with TPCF formed two bands with THCF, one of which had a pattern similar but not identical to hydatid AgB (Fig lf). TPCF 133
D. Liu, M. D. Rickard, M. W. Lightowlers
34
15
\B
b B
~5 ~B
B
f
f
©
......
.
~ ii¸ ~ : ~ : ,
....
IB
B FIG 1: Immunoelectrophoresis of cyst fluid antigens of E granulosus, T hydatigena and Tpisiformis using hyperimmune rabbit sera, Antigens in the middle wells: a to c, SHCF; d tO f, THCF; g to i, TPCF. Top troughs: a,e,h, rabbit antiserum to SHCF; b,d,i, rabbit antiserum to THCF; C, f, g, rabbit antiserum to TF'CF. Bottom troughs B: reference sheep anti-sHcF serum
Immunoelectrophoresis of taeniid cyst fluid antigens TABLE 1: Summary of immunoelectrophoretic bands formed between taeniid antigens and hyperimmune rabbit sera Antigen
RxSHCF
Serum RxTHCF RxTPCF
SxSHCF
SHCF THCF TPCF
5(5,B)* 5(5,B?) 2(B)
5(5,B?) 5(5,B) 1 (B)
4(5,B) 2(5,B?) 1 (B)
2(5,B?) 2(5?,B) 1 (B)
* 5 or B indicates the presence of hydatid arc 5 or AgB band. The ? indicates some uncertainty in assigning the band to arc 5 or AgB band
seemed to have a predominant antigen similar to hydatid AgB as shown by its reactions with hyperimmune rabbit sera to TPCF, SHCF and THCF (Fig lg to i). The immunoelectrophoretic bands formed by taeniid antigens with hyperimmune sera are summarised in Table i. Previous studies have indicated that T hydatigena cyst fluid contains antigens similar to E granulosus Ag5 and also possibly AgB (Varela-Diaz et a11977a, Daoud and Herbert 1982). The present report confirms the existence of hydatid Ag5 in T hydatigena cyst fluid. In addition, T hydatigena cyst fluid might also have an antigen close to hydatid AgB. However, the AgB band formed by RxSHCH and THCF, or by RxTHCF and SHCF does not have a perfect hydatid AgB band shape compared to the control, suggesting that the physico-chemical property or antigenicity of AgB in SliCE differs slightly from that in THCE. The demonstration of arc 5 forming antibodies in the serum of a rabbit immunised with TlaCV further supports the finding of arc 5 formed by sera of sheep infected with T hydatigena (Yong and Heath 1979). On the other hand, Tpisiformis cyst fluid might contain mainly an antigen similar to hydatid AgB as detected by rabbit antisera to TPCF, THCF and SliCE. Tpisiformis cyst fluid does not seem to have an antigen close to hydatid Ag5 even though rabbit antiserum to TPCF formed a band around the position of hydatid arc 5 with SliCE and THCE. Perhaps there is an antigenic component in T pisiformis cyst fluid, which by itself is not able to form an arc 5-1ike band with rabbit antisera to TPCF, THCF or SHCF. However, this antigen becomes more visible when injected into a rabbit by the reaction of rabbit anti-TPCF with SliCE or THCF.
Acknowledgements The financial support of the University of Melbourne Veterinary Research Fund and the National Health
135
and Medical Research Council of Australia is gratefully acknowledged.
References CAPRON, A., VERNES, A. & BIGUET, J. (1967) Le diagnostic immuno61ectrophor6tique de l'hydatidose. Le kyste hydatique du foie, Journ6e Lyonnaises d'Hydatidologie, Lyon, SIMEPeditions. pp27-40 CHORDI, A. & KAGAN, h G. (1965) Identification and characterization of antigenic components of sheep hydatid fluid by immunoelectrophoresis. Journal of Parasitology 51, 63-71 DAOUD, I. S. & HERBERT, I. V. (t982) Isolation of two lipoprotein antigens from the metacestodes of Taenia hydatigena (Pallas, 1766) and Taenia multiceps (Leske, 1780) and their evaluation in serodiagnosis. Veterinary Parasitology 11, 155-164 LIU, D., LIGHTOWLERS, M. W. & RICKARD, M. D. (1992a) Evaluation of a monoclonal antibody based competition ~LISAfor diagnosis of human hydatidosis. Parasitology 104, 357-361 LIU, D., LIGHTOWLERS, M. W. & RICKARD, M. D. (1992b) Examination of routine antibody response to secondary hydatidosis using ELISAand immunoelectrophoresis. Parasite Immunology (in press) ORIOL, R., WILLIAMS, J. F., PEREZ ESANDI, M. V. & ORIOL, C. (1971) Purification of lipoprotein antigens ofEchinococeus granulosus from sheep hydatid fluid. American Journal of Tropical Medicine and Hygiene 20, 569-574 SCHANTZ, P. M., SHANKS, D. & WILSON, M. (1980) Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. American Journal of Tropical Medicine and Hygiene 29, 609-612 VARELA-DIAZ, V. M. & COLTORTI, E. A. (1976) Immuno-diagnostic techniques for human hydatidosis, Monograph number 7. Pan American Zoonosis Center, Pan American Health Organisation, Buenos Aires VARELA-DIAZ, V. M., COLTORTI, E. A. & D'ALESSANDRO, A. (1978) Immunoelectrophoresis tests showing Echinococcus granulosus arc 5 in human cases of Echinococcus vogeli and cysticercosismultiple myeloma. American Journal of Tropical Medicine and Hygiene 27, 554-557 VARELA-DIAZ, V. M., COLTORTI, E. A., RICKARD, M. D. & TORRES, J. M. (1977a) Comparative antigenic characterization of Echinococcus granulosus and Taenia hydatigena cyst fluids by immunoelectrophoresis. Research in Veterinary Science 23, 213216 VARELA-DIAZ, V. M., ECKERT, J., RAUSCH, R. L., COLTORTI, E. A. & HESS, U. (1977b) Detection of the Echinocoecus granulosus diagnostic arc 5 in sera from patients with surgicallyconfirmed E multilocularis infection. Zeitschrift fiir Parasitenkunde 53, 183188 YARZABAL, L. A., BOUT, D. T., NAQUIRA, F. R. & CAPRON, A. R. (1977) Further observations on the specificity of antigen 5 of Echinococcus granulosus. Journal of Parasitology 63, 495-499 YONG, W. K. & HEATH, D. D. (1979) 'Arc 5' antibodies in sera of sheep infected with Echinococcus granulosus, Taenia hydatigena and Taenia ovis. Parasite Immunology 1, 27-38 Received November 25, 1991 Accepted February 21, 1992
