Immunoblot evaluation of the species-specificity of Em18 and Em16 antigens for serodiagnosis of human alveolar echinococcosis

TRANSACTIONS OFTHE ROYAL SOCIETY OFTROPICAL MEDICINE AND HYGIENE

lmmunoblot evaluation of the species-specificity serodiagnosis of human alveolar echinococcosis Nirosbini UK

Nirmalan

and Philip

S. Craig

(1997) 91,4&l-486

of Em18 and Em16 antigens

for

Department of Biological Sciences, University of Salford, Salford, MS 4m

Abstract An immunoblot study to confirm the species-specificity of the diagnostic antigens Em18 and Em16 of Echinococcus multilocularisprotoscolex extract showed that both antigens cross-reacted with sera from cystic echinococcosis (CE) patients. The 18 kDa component was detectable by 75% of the sera from active alveolar echinococcosis (AE) patients, while only 31% detected Em16. Western blot analysis also showed that AE sera recognized a band in the 18 kDa region of E. granulosus protoscolex extract, which was different from the 16/l 7 kDa subunit of antigen B. The results suggested that Em1 8 antigen is present inE. granulosus as well as E. multilocularis, and that some CE patients may have serum antibody against this antigen. Keywords: echinococcosis, Echinococcwmultilocularis, antigens, Em1 8, Em1 6, serodiagnosis Introduction Alveolar echinococcosis (AE) is a rare but potentially fatal zoonotic infection caused by the taeniid cestode Echinococcus mtdtilocularis. It is restricted to the northern hemisphere with endemic areas in Alaska, Siberia, northern China and Japan (BAUSCH, 1986; CRAIG et al., 199 1, 1992). In humans, primary localization of the metacestode results in rapid hepatic destruction. Although it shows similarities with cystic echinococcosis (CE) caused by E. granulosus, AE runs a more malignant course and radical hepatic resection is still considered the frontline therapy, as opposed to chemotherapy as well as surgery for CE (WHO, 1996). Thus establishment of a definitive differential diagnosis is important, especially in endemic areas where both species are present (VUI-ITON, 1990; WEN et al., 1993). One of the more specific serological methods currently available to confirm the diagnosis of AE is the Em2 enzyme-linked immunosorbent assay (GOTTSTEIN et al., 1993). However, the Em2 antigen, not being a protein (DEPLAZES & GOT-TSTEIN, 1991), poses problems for large scale production for standardized seroepidemiology in hyperendemic areas (IT0 et al., 1993a). In an attempt to identify species-specific protein components of E. multilocularis by Western blotting, IT0 et al. (1993a) demonstrated 2 previously undescribed putatively specific antigenic components of protoscoleces of E. multiIocularis, designated Em18 and Em1 6, which were readily detectable with sera from active AE patients exclusively. The 18 kDa component was recognized by sera from all active AE patients in this Japanese study and the 16 kDa antigen was detected by patients with advanced lesions. However, a subsequent study suggested that Em16 was also recognized by CE patients WEN et al., 1995). casting doubt on the snecificity of Em 16 for differential serodiagnosis of AE: In this study we demonstrate, using a range of CE sera, that both Em1 8 and Em16 antigens are probably not species-specific. Materials and Methods Serum samples Human serum samples were obtained between 1991 and 1994. Eiahteen sera from oroven CE natients from Uruguay and”UK (countries not endemic-for AE), and 19 from proven AE patients t?om China, were used in the studv. Serum samples from healthv staff members of the University of Salford, UK, were-used as controls. Rabbit hyperimmune sera against E. multilocularis protoscolex extract (EmP) or E. granulosus extract (EgP) were raised as previously described (ALIAN et al., 1992). Briefly, approximately 1 mg of protein in 05 mL volume was mixed with an equal volume of complete Freund’s adjuvant and injected subcutaneously and intramuscularly to rabbits. Four subsequent booster injec-

tions were given with incomplete Freund’s adjuvant. Rabbits were bled when antibody titres exceeded 1: 10000; serum was separated and stored at -20°C. Echinococcus extracts A crude extract of E. multilocularis protoscoleces was prepared according to a previously described method (WEN & CRAIG, 1994). Briefly, 1-O mL of packed E. multilocularis protoscoleces (approximately lOOOOO), obtained from secondarilv infected cotton rats (Siamodon hispidis), were washed-in 0.15 M phosphate-buffered saline at DH 7.2 (PBS). frozen and thawed 3 times in a -70°C freezer, and resuspended in 4 mL of PBS with 0.1 mg/mL aprotinin (Sigma, St Louis, Missouri, USA). The protoscolex suspension was sonicated on ice for 15 set (10 um peak to peak) on and 5 set off for 15 cycles, and the sonicate tested microscopically to ensure that no intact protoscolex remained. The sonicate was left standing on ice for 2 h before being centrifuged at 1OOOOg for 30 min at 4°C. The supematant (EmP) was recovered and stored as 200 ~.ILaliquots at -70°C. One mL of packed E. granulosus protoscoleces were obtained from ovine hydatid cysts collected at an abattoir in Manchester (UK) and used to prepare a crude extract (EgP) according to the technique described above. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blottino SDS-PA&E was carried out aidescribed bv IAEMMLI (1970) in a 12% minigel system (SE-250; Mighty Small SvstemTM. Hoeffer. USA). The ootimum antieen conc&ration was 500 ug of EgP or* 400 l.tg of EmP per minigel (6 cm width). Electrophoresis was for 3 h at 60 V. Immunoblotting of separated polypeptides was performed according to the method described by WEN & CRAIG (1994). Nitrocellulose paper (NCP; Sartorius), transblotted with parasite protein after transference at 35 mA overnight, was blocked with PBS containing 0.3% Tween 2eTM-and 5% powdered milk, and incubac ed with human natient or rabbit antisera (1:200 dilution) in a multmack incubating chamber‘ (Biometra, Gottingen, Germany) for 2 h at room temperature. Alkaline phosphatase-conjugated anti-human or anti-rabbit immunoglobulin G (Sigma) was diluted 1: 1000 and used as the secondary antibody with 5-bromo-4-chloro3-indolyl phosphate/nitro blue tetrazolium (BCIP/ NBT) as substrate solution. Results Best results were obtained with 12% gels loaded with 400 ug of sonicated and centrifuged EmP. AE sera recognized 6 major peptides with molecular masses of 55, 44,35,31,21 and 18 kDa. The 16 kDa peptide was not detected at the EmP concentration described above and

SERODIAGNOSIS

OF ALVEOLAR

ECHINOCOCCOSIS

Fig. 1. Western blot analysis of Echinococcus multilocukzris protoscolex extract (EmP) probed with sera from patients with alveolar echinococcosis (AE). Lane A: pooled AE sera; lanes B-O: individual AE sera; lanes R and S: normal human serum controls. The numbers on the right indicate M, values derived from sodium dodecyl sulphate low molecular mass markers (BioRad), 144-97.4 kDa.

Fig. 3. Western blot analysis of Echinococcus muln’locularis protoscolex extract (EmP) probed with sera Tom patients with cystic echinococcosis (CE) . Lanes A and B: pooled AE sera; lanes C and D: rabbit anti-EmP sera; lane E: pooled CE sera; lanes F-W CE sera; lane X: normal human serum control; lane Y: normal rabbit serum control.The numbers on the right indicate M, values derived from MultimarkM (Novex) molecular mass markers, 4-250 kDa.

was comparable to that seen with AE sera. The lanes containing normal human control sera were clear except for a single band in the 55 kDa area. To confirm the specificity of Em18 and Em1 6, EmP was also blotted against 18 separate CE sera, pooled AE sera, hyperimmune rabbit anti-EmP antibody, and a pool of CE sera (Fig. 3). At least 7 of the CE sera tested showed prominent banding in the 18 kDa region, and 3 in the 16 kDa region. The normal serum control lanes were clear. Discussion Effective

differential

serodiagnosis

of AE and CE de-

pends on the identification of an easily detectable species-specific antigen. Our results suggested that both the Em18 and Emi antigens desc&ed by IT0 et al. C1993a.

Fig. 2. Western blot analysis of Echinococcus granulosus protoscolex extract (EgP) probed with sera from patients with alveolar echinococcosis (AE) Lanes A and B: normal human serum controls; lane C: pooled cystic echinococcosis (CE) sera; lane D: pooled CE serum samples which recognized Em1 8 antigen in EmP; lane E: pooled AE sera; lanes F-X: AE sera. The numbers on the right indicate Mr values derived as in Fig. 1.

required higher concentrations of freshly prepared antigens (800 @gel). Of 16 human AI! serum samples tested, 4 (25%; lanes F, I, J, N in Fig. 1) failed to recognize

either Em18 or Em16 and only 5 samples (31%; lanes C, E, G, L, P in Fig. 1) recognized Em16. When EgP was tested, CE sera recognized peptides at approximately 12, 17, 22, 24, 31, 44 and 57 kDa (Fig. 2). When EgP was blotted with a range of AE sera, prominent banding was visible in the 18 kDa regions in 12 of the 19 samples used, but the bands were diffuse,

merging into the 17 kDa regions. There was also faint banding in the 16 kDa region with 3 AE sera. However, lane C (Fig. 2) on the same blot probed with pooled CE sera showed banding in the 17 kDa region which was different to the diffuse 18 kDa band recognized by the AE sera. Further,

when

lane D was probed

with

a pool

of 2 CE sera which had previously shown cross reactivity with .Em 18 in EmP, the banding in the 18 kDa region

1993b)

were not svecific

for E. mdtiloculati

as

they appeared to cross-rea& with CE sera. Of 16 AE serum samples immunoblotted against the homologous EmP antipens. 12 (75%) contained antibodies which recognized the Em 1‘8 antigen, while only 5 (3 1%) recognized Em16. Thus the Em16 antigen was not easily detectable, requiring fresh parasite extract at twice the concentration needed for Em18 detection. The immunodiagnostic specificity of the sonicated and centrifuged EmP preparation was generally better than a whole, non-centrifuged protoscolex sonicate. Correlation between Em1 8/Eml6 recognition and the stage of the disease was not possible as all patients tested in the study were in an advanced active stage of the disease. The Western blot analysis of EgP antigen from E. arunuZosus usina 19 clinicallv defined AE serum samvles showed that a definite but &I&e band occurred in-the 18 kDa region in 7 samples. It is difficult to comment on whether this band was identical with the Em1 8 band or whether it was the 16/17 kDa subunit of antigen B, described from E. granulosus cyst fluid (LEGGATT et al., 1992). However, the same gel nrobed with nooled CE sera did show banding of 17-kDa which was iower than the band identified usina AE sera. Furthermore. when probed with a pool of CE sera which had previously shown cross-reactivity with Em18 in EmP, the banding was similar to that obtained with the AE sera suggesting that the peptide recognized by AE sera may in fact be the Em1 8 antigen,

rather

than-the

16/l 7 kDa subunit

of

antigen B. Interestingly, AE serum samples which showed prominent banding in the 18 kDa region with EgP antigen were the same samples which displayed strong Em 18 recognition when incubated with EmP an-

486

NIROSHINI

AND

PHILIP

S. CRAIG

& Sato, S. (1993a). Serodiagnosis of alveolar disease by Western blotting. Transactions of the Royal Society of Tropical Medicine and Hygiene, 87, 170-172. Ito, A., Wang, X. & Liu,Y. (1993b). Differential serodiagnosis of alveolar and cystic hydatid disease in the People’s Republic of China. American Journal of Tropical Medicine and Hygime, 9,208-213. Laemmli, U. K. (1970). Cleavage of structural proteins during the assemblv of the bacterioohaaeT4. Narure. 227,680-683. Leggatt, G. R.iYang,W. & M&&us, D. P. (1992). Serological evaluation of the 12kDa subunit of antigen B in Echinococcus granulosus cyst fluid by immunoblot analysis. Transactions of the Royal Socitq of Tropical Medicine and Hygiene, 86, 189-192. Rausch, R. L. (1986). Life cycle patterns and geographic distribution of Echinococcus species. In: The Biology of Echinococcus and Hydatzd Disease, Thompson, R. C. A. (editor). London: George Allen & Unwin, pp. 44-80. Vuitton, D. A. (1990). Alveolar echinococcosis of the liver: a parasitic disease in search of a treatment. Hepawlogy, 12, 617-618. Wen, H. & Craig, P. S. (1994). Immunoglobulin G subclass responses in human cystic and alveolar echinococcosis. American Journal of Tropical Medicine and Hygiene, 51,741-748. Wen, H., New, R. R. & Craig, P. S. (1993). Diagnosis and treatment of human hydatidosis. British Journal of Clinical Pharmacolog;y, 35,565-574. Wen, H., Craig, P. S., Ito, A., Vuitton, D. A., Bresson-Hadni, S., Allan, J. C., Rogan, M. T., Paollilo, E. & Shambesh, M. (1995). Immunoblot evaluation of IgG and IgG subclass antibody responses for immunodiagnosis of human alveolar echinococcosis. Annals of Tropical Medicine and Parasitology, 89,485-495. WHO [informal working group on echinococcosis] (1996). Guidelines for treatment of cystic and alveolar echinococcosis in humans. Bulletin of the IVbrld Health Organization, 74, 231-242.

tigen. When EmP antigen was probed with 18 CE serum samples, 5 CE sera recognized Em18 antigen, in contrast to the observations of IT0 et d. (1993b). The work of WEN et al. (1995) was also confirmed as serum samples from 3 CE patients showed cross-reactivity with the Em 16 antigen. In conclusion, although the results suggested that Em1 8 and Em16 are probably not species-specific, refined methods of assessment of specificity, such as the development of cloned Em 18 and Em 16, need to be developed to confirm these findings. Acknowledgements We are grateful for financial support from the European Community (STDf-project no. TS3.CT.940270) and to Professor Wen Hao, Professor D. Shi, Dr E. Paolillo and Dr W. Bailey for help in providing serum samples.

References Allan J. C., Craig, I? S., Noval, J. G., Mencos, F., Liu, D., Wang,Y., Wen, H., Zhou, P., Stringer, R., Rogan, M. T. & Zeyhle, E. (1992). Coproantigen detection for immunodiagnosis of echinococcosis in taeniasis in dogs and humans. Parasitology, 104,347-355. Craig, P. S., Deshan, L. & Zhaoxun, D. (1991). Hydatid disease in China. Parasitology Today, 7,46-50. Craig, P. S., Liu, D., MacPherson, C. N. L., Shi, D., Reynolds, D., Barnish, G., Gottstein, B. &Wang, Z. (1992). A large focus of alveolar echinococcosis in central China. Lancet, 340, 826-831. Deplaxes, P. & Gottstein, B. (1991). A monoclonal antibody against Echinococcus multi~oculari~ Em2 antigen. Parasitology, 103,41-49. Gottstein, B., Jaquier, P., Bresson-Hadni, S. & Eckert, J. (1993). Improved primary immunodiagnosis of alveolar echinococcosis in humans by enzyme-linked immunosorbent assay using the Em2pku antigen. Journal of Clinical Microbt&gy, 13,373-376. Ito, A., Nakao, K., Kutsumi, H., Lightowlers, M. W., Itoh, M.

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Received 11 December 1996; revised 24 January 1997; accepted for publication 28January 1997

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