hremotional Journalfor Parasiro/ogy Vol. 21, No. 3, pp. 315-319, 1991 Primed in Great Britain 0
002&75i9/9l $3.00 + 0.00 Pergamon Press plc 1991 Aurtrdim Socieryjor Porosimlogy
SPECIFIC ANTIGEN OF GNATHOSTOMA SPINIGERUM FOR IMMUNODIAGNOSIS OF HUMAN GNATHOSTOMIASIS PRAMUAN TAPCHAISRI,*~
CHAMNONG NOPPARATANA,~
WANPEN CHAICUMPA*
and PRASERT SETASUBAN~ *Department of Microbiology and Immunology and fDepartment of Helminthology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand (Received 27 August 1990; accepted 25 November 1990)
Abstract-TAKHAISRI P., NOPPARATANA C., CHAICUMPA W. and SETASUBAN P. 1991. Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis. International Journal for Parasitology 21: 3 15-3 19. Sera from four patients with parasitologically confirmed gnathostomiasis, 15
patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite’s antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis. INDEX KEY WORDS: Gnathostoma spinigerum; nematode; diagnostic antigen; immunodiagnosis; human gnathostomiasis.
INTRODUCTION
HUMANgnathostomiasis
caused by the round worm Gnathostoma spinigerum is prevalent in certain areas of Thailand and many Asian countries. The disease is characterized by intermittent cutaneous and subcutaneous migratory swellings and, in some cases, cerebral involvement resulting in eosinophilic meningitis. However, these clinical manifestations cannot be readily distinguished from a few other parasitic diseases, i.e. angiostrongyliasis, trichinosis, cutaneous larval migrans, etc. Currently, diagnosis of human gnathostomiasis is only presumptive unless the parasites can be recovered and identified. Attempts have been made for specific immunodiagnosis of the disease using the crude aqueous somatic extract of advanced third-stage larvae of G. spinigerum (L3) as antigen in an enzyme-linked immunosorbent assay (ELISA), but cross-reaction was found in sera from patients with other parasitic infections and also in sera from normal healthy individuals (Suntharasamai, Desakom, Migasena, Bunnag & Harinasuta, 1985; Dharmkrong-at, Migasena, Suntharasamai, Bunnag, Priwan & Sirisinha, 1986). Furthermore, the antigen responsible for the induction of the human immune response has not been
t To whom all correspondence should be addressed.
defined. Our recent study on antigenic analysis by SDS-PAGE and Western blot analysis using sera from parasitologically confirmed gnathostomiasis patients revealed that the antigen is highly complex, consisting of more than 20 antigenic bands, a few of which cross-reacted with sera from healthy uninfected individuals (Nopparatana, Tapchaisri, Setasuban, Chaicumpa & Dekumyoy, 1988). The purpose of the present study was to further characterize the specific L3 antigen which is useful for the immunodiagnosis of human gnathostomiasis by comparing the serum reactivities between patients with gnathostomiasis and other parasitic infections against the L3 somatic extract using the same techniques described. It was found that a specific antigen with a mol. wt of 24k reacted only with the gnathostomiasis sera but not those of other parasitic infections. MATERIALS AND METHODS Patient sera. Sera were collected from four male patients
with parasitologically confirmed gnathostomiasis (Nopparatana et al., 1988). Presumptive gnathostomiasis sera were obtained from 15 patients attending the Out-Patient Department of the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. These patients had an age range of between 16 and 37 years, with a mean of 25.9 years. Diagnosis of gnathostomiasis in these patients was based largely on their clinical symptoms of 315
316
P. TAPCHAISRI, C. NOPPARATANA, W. CHAICUMPAand P. SETASUBAN
intermittent cutaneous migratory swelling and previous history of consumption of uncooked flesh which could be a potential source of G. spinigerum infestation. Sera were also obtained from patients with other parasitic infections diagnosed by parasitological examination of blood, stool, and/or sputum and consisted of the following numbers of cases: parasitologically confirmed angiostrongyliasis (one with ocular angiostrongyliasis and one with eosinophilic meningitis); trichinosis (5); sparganosis (I); cysticercosis (1); paragonimiasis (13) schistosomiasis (2); opisthorchiasis (4); filariasis (1); taeniasis (6); strongyloidosis (4); trichuriasis (1); hook worm infection (3); ascariasis (1); amoebiasis (2); malaria (two Plasmodium falciparum and two P. vivax infections); and 14 mixed parasitic infections (two or more of the parasites found including Plasmodium, hook worms, Opisthorchis, Strongyloides, Trichuris, etc.). Control sera were obtained from a healthy adult Swede and 18 healthy adult Thais who had no history of intermittent cutaneous migratory swelling and were negative for any parasitic infection at the time of blood collection. G. spinigerum antigen. G. spinigerum advanced third-stage larvae (L3) were obtained from livers of infected freshwater eels purchased from a local market in Bangkok as previously described (Nopparatana et al., 1988). The worms were washed in 0.9% NaCl until free of blood and tissue debris, pooled, lyophilized and kept at -2o’C until use. A crude aqueous L3 extract was prepared by grinding the worms with alumina (Sigma Chemical Co., MO, U.S.A.) in distilled water containing 0.1 mr+phenylmethylsulphonylfluoride (Sigma), 0.1 mM-tosyl-amide-2-phenylethyl-chloromethyl ketone (Sigma) and 10 mM-ethylene diamine trichloroacetic acid at 4°C. After the alumina was removed by centrifugation at 250 x g for 5 min at 4°C the worm suspension was extracted twice with ether at 4°C. The worm extract was centrifuged at 10,000 x g for 30 min at 4°C and the supernatant removed. The sediment was resuspended in distilled water, sonicated at
4°C for 10 min and centrifuged as above. The supernatants were pooled, dialysed with distilled water and kept at - 20°C for further analysis. The protein content was determined by the method of Lowry, Rosenbrough, Farr & Randall (1951) using bovine serum albumin (BSA) as the standard. SDS-PAGE and Western blot analysis. The electrophoresis was performed in a vertical slab gel apparatus using the method of Laemmh & Favre (1973) with some modification. Briefly, a 4% acrylamide stacking gel and a 10% acrylamide separating gel were used. Samples containing approximately 30 pg of the L3 extract were boiled at IOO’C for 3 min in sodium dodecyl sulphate (SDS) and 2-mercaptoethanol before loading onto the gel. The separated protein bands were visualized by staining with Cooma&ie brilliant blue R (Sigma). The mol. wts of unknown proteins were estimated by comparing the relative migration ratios against those of standard proteins (Pharmacia Fine Chemicals, Uppsala, Sweden). For Western blot analysis the SDS-PAGE resolved components were electroblotted onto a 0.45 pm nitrocellulose membrane (Bio-Rad Laboratories, CA, U.S.A.) according to the method of Towbin, Staehelin & Gordon (1979). After blotting, the unreacted sites on the membrane were ‘blocked by soaking the strips in phosphate buffered saline. uH 7.4 (PBS) containina 0.5% gelatin. 3% BSA and 0.04% NaN, at 26’C for 2 h with gentie rocking. The strips were then washed twice (10 min each) with washing buffer (0.05% Tween 20 in PBS) before being reacted with the patient’s sera diluted 1:200 in PBS containing 0.2% gelatin, 0.2% BSA and 0.04% NaN, (PBS-BSA) at 26’C for 1 h with gentle rocking. The strips were washed four times (20 min each) in the washing buffer and incubated with ‘*‘I-labelled sheep anti-human immunoglobulins (Amersham International PTY, U.K.) in PBS-BSA (2-5 x 10’ c.p.m. ml-‘) at 26°C for 30 min. The strips were washed as above, dried and autoradiographed onto the Kodak X-Omat RP films (Eastman Kodak Co., NY, U.S.A.) with light intensifying screens at - 70” C for 2448 h.
AGCOEFGHIJKLMNOPQRS FIG. 1. Serum reactivities of patients with parasitologically proven (A-D) and presumptive (E-S) gnathostomiasis against L3 extract of G. spinigerum by SDS-PAGE and Western blot analysis. Numbers at left indicate mol. wt x IO-‘.
G. spinigerum
diagnostic antigen
RESULTS The serum reactivities as revealed by SDS-PAGE and Western blot analysis against the L3 antigen of four parasitologically proven gnathostomiasis patients are shown in Fig. 1. It was found that the L3 antigen is highly complex, consisting of more than 20 antigenic bands with mol. wts ranging from 150k to 13k. The predominant antigenic bands were 150, 135, 120, 94, 49, 43, 42, 38, 35, 34, 33, 32, 24, 22 and 21k. One prominent antigenic band of 24k was found to give consistent reaction with sera from all of the proven cases. When sera from 15 presumptive gnathostomiasis patients were tested for their reactivities against the antigen, it was found that these sera had their reactivities mainly against L3 antigenic components with mol. wts higher than 30k (Fig. 1). Only five cases (33%) gave positive reactions against the 24k antigen. The specificity of the L3 antigen was further defined by comparing the serum reactivities between the gnathostomiasis patients and patients with other parasitic infections. Data in Table 1 summarize the serum reactions against the major L3 antigenic components. It was found that all sera from patients with other parasitic infections reacted mainly with the L3 components with mol. wts higher than 30k and only one patient with paragonimiasis gave a weak reaction against the 24k antigen. Most of the healthy individuals had very low to moderate reactivities against the 38k antigen while some of them had, in addition, reactivities against the 49k and 43k antigens.
317
The representative patterns of the serum reactivities of patients with gnathostomiasis, patients with other parasitic infections and the normal controls are shown in Fig. 2. DISCUSSION The present study has demonstrated a specific 24k antigen of G. spinigerum which consistently reacted with sera from gnathostomiasis patients but not with those from patients with other parasitic infections. This study confirmed our previous report that the G. spinigerum L3 antigen was highly complex and consisted of various antigenic components as revealed by SDS-PAGE and Western blot analysis (Nopparatana et al., 1988). Although ‘251-labelled anti-human immunoglobulins were used to detect the serum reactivities in the present study, their patterns were similar to those of the previous study in which radioiodinated protein A was used. However, our previous study failed to clearly demonstrate the specific 24k antigen and this is explained by the fact that the antigen is relatively unstable upon storage before use in the electrophoresis sample buffer containing the detergent SDS and the reducing agent 2-mercaptoethanol. It was observed in our study that prolonged storage of the L3 antigen in the electrophoresis buffer at - 20°C resulted in a significant loss of the specific 24k band. We have found that this 24k antigen is most stable in aqueous solution and could be purified from the crude L3 extract by gel filtration,
TABLE I-SUMMARY OF SERUMREAC~~VITIES 0~ PATIENTSWITHGNATHOSTOMIA~IS, PATIENTSWITHOTHERPARASITICINFECTIONSAND HEALTHYCONTROLSAGAINSTL3 EXTRACTOF G. spinigerum ASREVEALED BYSDS-PAGE AND WESTERNBLOTANALYSIS Predominant
Sera (total number) Confirmed cases (4) Presumptive cases (15) Angiostrongyliasis Trichinosis (5) Sparganosis (1) Cysticercosis (1) Paragonimiasis Schistosomiasis Opisthorchiasis Filariasis (1)
(2)
(13) (2) (4)
Taeniasis (6) Trichuriasis (1) Strongyloidosis (4) Hook worm infection Ascariasis (1) Amoebiasis (2) Malaria (4) Mixed infections
(14)
Healthy
(19)
controls
194
94
49
43142
38
35
34133
32
24
3. 15
4 15
4 13
4 14
3 12
3 12
3 13
3 12
4 5
1 4 1 1
1 4 1 1
2 4 1 1
2 5 1 1
2 3 1 1
2 3 1 _
2 3 1
-
11
11 _ 4
10
12
9
9
8
4
4 1
4 -
4 _
4
6
5 1 3 2
6 _ 4 2 1 2
5 1 2 2 1 _
5
4 12
2 6
4
8
4
(3)
* Numbers of patients - No reaction.
6 1 4 3 1 2
4 3 1 2
2 11
3 IO
I
_ showing
mol. wt x IO-’
positive reaction.
1 _ 2 6 2
1
5
1 1 1
-
8 _ 4
-
_
1
_
_
4
_
_ 2 _
4 2 _ _
_ 2
_ _
2 6
3 5
_
_
3 10 1 1 1 _
1
_ 2 4
2212 1
_ _
9 1 3 _ 2 _ _ _ _ 2 3 _
318
P. TAPCHAISRI, C. NOPPARATANA,W. CHAICUMPAand P. SETASUBAN
ABCBEF6HlJKLIYIIIOPORSTU FIG. 2. Representative serum reactivities of patients with gnathostomiasis (A), angiostrongyliasis (B,C),trichinosis (D,E), sparganosis (F), cysticercosis (G), paragonimiasis (H,I), schistosomiasis (J), opisthorchiasis (K), filariasis (L), taeniasis (M), strongyloidosis (N), hook worm infection (0), ascariasis (p), amoebiasis (Q), mahria (R), mixed infections (ST) and healthy individual (U) against L3 extract of G. ~pi~iger~ by SDS-PAGE and Western blot analvsis. Numbers at left indicate mol. wt x 10--s.
ion-exchange chromatography and chromatofocusing (Nopparatana, C., Setasuban, P., Chaicumpa, W. & Tapchaisri, P., in preparation). It was observed in the present study that most sera from presumptive gnathostomiasis patients gave positive reactivities against various L3 antigenic components with mol. wts of 30k and above but only five out of 15 (33%) were positive for the specific 24k antigen. The most likely explanation for the low percentage of positivity in these patients was that their diagnosis was not correct and that the positive reactions against the other L3 components might result from previous infections with other parasites. Likewise, the presence of the serum antibody against the 24k antigen in one patient with paragonimiasis would have resulted from previous infection with G. s~iniger~~. The present study clearly demonstrated that extensive crossreactions of the antigen occurred and that the use of crude, undefined L3 extract was not suitable for the specific immunodiagnosis of human gnathostomiasis. Suntharasamai et al. (1985) found that 33% of patients with eosinophilic meningo-myeloencephalitis typically due to gnathostomiasis, 56% of patients with cutaneous migratory swellings (CMS) and 23% of patients with other parasitic infections were positive by ELISA using the crude L3 extract as the antigen. Further improvement of the technique using the similar antigen preparation by Dhamkrong-at et al. (1986) resulted in a sensitivity of 100% in CMS patients but cross-reaction occurred with sera from
some healthy blood donors and angiostrongyliasis patients. Furthermore, the specificity of the reaction in patients with other parasitic infections was not clearly demonstrated. It would be appropriate for an ELISA using the crude L3 antigen to be employed as a screening test for human gnathostomiasis and for Western biot analysis to be used for confi~ation. A similar system has worked well for the diagnosis of patients with acquired-immunodeiiciency syndrome (Sarngadharan, Popovic, Bruch, Schupbach & Gallo, 1984; Weiss, Goedert, Sarngadharan, Bodner, The AIDS Seroepidemiology Collaborative Working Group, Gallo & Blattner, 1985). However, more data will be needed regarding the sensitivity of the Western blot analysis in relation to various clinical manifestations of the gnathostomiasis patients. The demonstration of specific 24k antigen of G. spinigerum would lead to the development of a specific diagnosis of human gnathostomiasis using a simple immunological test, namely ELISA, provided that the specific component of the worms can be appropriately prepared, either through biochemical processes or recombinant DNA technology. Our current work involves fractionation of the worm extract by sephadex and ion-exchange chromatographies and chromatofocusing. It has been found that the purified fraction containing the specific 24k antigen could be obtained and the sensitivity and specificity of such an antigen in the serodiagnosis of human gnathostomiasis are being evaluated.
G. spinigerum diagnostic Acknowledgements--We would like to thank Dr Savanat Tharavanij and Dr Santasiri Sornmani for their encouragement and suggestions; and Dr Danai Bunnag for the sera from patients with presumptive gnathostomiasis and other parasitic infections. This work was supported in part by a grant from Mahidol University.
REFERENCES DHAMKRONG-AT A., MIGASENA S., SUNTHARASAMAI P., BUNNAGD., PRIWANR. & SIRISINHAS. 1986. Enzyme-linked immunosorbent assay for detection of antibody to Gnarhostoma antigen in patients with intermittent cutaneous migratory swelling. Journal of Clinical Microbiology 23: 847-85 1. LAEMMLIU. K. & FAVRE M. 1973. Maturation of the head of bacteriophage T4. DNA packaging events. Journal of Molecular Biology 80: 575-599. LOWRY0. H., ROSENBROUGHN. J., FARR A. L. & RANDALLR. J. 1951. Protein measurement by the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. NOPPARATANAC., TAPCHAISRIP., SETASULIAN P., CHAICUMPA
antigen
319
W. & DEKUMYOYP. 1988. Antibody responses in human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 19: 2 19-224. SARNGADHARANM. G., POPOVICM., BRUCH L., SCHUPBACHJ. & GALLO R. C. 1984. Antibodies reactive with human Tlymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224: 56508. SUNTHARASAMAI P., DESAKORNV., MIGASENAS., BUNNAGD. & HARINASUTAT. 1985. ELISA for immunodiagnosis of human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 16: 274-279. TOWBINH., STAEHELINT. &GORDON J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proceedings of the National Academy of Science of the United States of America 76: 4350-4354. WEISS S. H., GOEDER~J. J., SARNGADHARANM. G., BODNERA. J., THE AIDS SEROEPIDEMIOLOGY COLLABORATIVE WORKING GROUP, GALLO R. C. & BLATTNERW. A. 1985. Screening test for HTLV-III (AIDS agent) antibodies. Specificity, sensitivity and applications. Journal of the American Medical Association 253: 22 l-225.
002&75i9/9l $3.00 + 0.00 Pergamon Press plc 1991 Aurtrdim Socieryjor Porosimlogy
SPECIFIC ANTIGEN OF GNATHOSTOMA SPINIGERUM FOR IMMUNODIAGNOSIS OF HUMAN GNATHOSTOMIASIS PRAMUAN TAPCHAISRI,*~
CHAMNONG NOPPARATANA,~
WANPEN CHAICUMPA*
and PRASERT SETASUBAN~ *Department of Microbiology and Immunology and fDepartment of Helminthology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand (Received 27 August 1990; accepted 25 November 1990)
Abstract-TAKHAISRI P., NOPPARATANA C., CHAICUMPA W. and SETASUBAN P. 1991. Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis. International Journal for Parasitology 21: 3 15-3 19. Sera from four patients with parasitologically confirmed gnathostomiasis, 15
patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite’s antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis. INDEX KEY WORDS: Gnathostoma spinigerum; nematode; diagnostic antigen; immunodiagnosis; human gnathostomiasis.
INTRODUCTION
HUMANgnathostomiasis
caused by the round worm Gnathostoma spinigerum is prevalent in certain areas of Thailand and many Asian countries. The disease is characterized by intermittent cutaneous and subcutaneous migratory swellings and, in some cases, cerebral involvement resulting in eosinophilic meningitis. However, these clinical manifestations cannot be readily distinguished from a few other parasitic diseases, i.e. angiostrongyliasis, trichinosis, cutaneous larval migrans, etc. Currently, diagnosis of human gnathostomiasis is only presumptive unless the parasites can be recovered and identified. Attempts have been made for specific immunodiagnosis of the disease using the crude aqueous somatic extract of advanced third-stage larvae of G. spinigerum (L3) as antigen in an enzyme-linked immunosorbent assay (ELISA), but cross-reaction was found in sera from patients with other parasitic infections and also in sera from normal healthy individuals (Suntharasamai, Desakom, Migasena, Bunnag & Harinasuta, 1985; Dharmkrong-at, Migasena, Suntharasamai, Bunnag, Priwan & Sirisinha, 1986). Furthermore, the antigen responsible for the induction of the human immune response has not been
t To whom all correspondence should be addressed.
defined. Our recent study on antigenic analysis by SDS-PAGE and Western blot analysis using sera from parasitologically confirmed gnathostomiasis patients revealed that the antigen is highly complex, consisting of more than 20 antigenic bands, a few of which cross-reacted with sera from healthy uninfected individuals (Nopparatana, Tapchaisri, Setasuban, Chaicumpa & Dekumyoy, 1988). The purpose of the present study was to further characterize the specific L3 antigen which is useful for the immunodiagnosis of human gnathostomiasis by comparing the serum reactivities between patients with gnathostomiasis and other parasitic infections against the L3 somatic extract using the same techniques described. It was found that a specific antigen with a mol. wt of 24k reacted only with the gnathostomiasis sera but not those of other parasitic infections. MATERIALS AND METHODS Patient sera. Sera were collected from four male patients
with parasitologically confirmed gnathostomiasis (Nopparatana et al., 1988). Presumptive gnathostomiasis sera were obtained from 15 patients attending the Out-Patient Department of the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. These patients had an age range of between 16 and 37 years, with a mean of 25.9 years. Diagnosis of gnathostomiasis in these patients was based largely on their clinical symptoms of 315
316
P. TAPCHAISRI, C. NOPPARATANA, W. CHAICUMPAand P. SETASUBAN
intermittent cutaneous migratory swelling and previous history of consumption of uncooked flesh which could be a potential source of G. spinigerum infestation. Sera were also obtained from patients with other parasitic infections diagnosed by parasitological examination of blood, stool, and/or sputum and consisted of the following numbers of cases: parasitologically confirmed angiostrongyliasis (one with ocular angiostrongyliasis and one with eosinophilic meningitis); trichinosis (5); sparganosis (I); cysticercosis (1); paragonimiasis (13) schistosomiasis (2); opisthorchiasis (4); filariasis (1); taeniasis (6); strongyloidosis (4); trichuriasis (1); hook worm infection (3); ascariasis (1); amoebiasis (2); malaria (two Plasmodium falciparum and two P. vivax infections); and 14 mixed parasitic infections (two or more of the parasites found including Plasmodium, hook worms, Opisthorchis, Strongyloides, Trichuris, etc.). Control sera were obtained from a healthy adult Swede and 18 healthy adult Thais who had no history of intermittent cutaneous migratory swelling and were negative for any parasitic infection at the time of blood collection. G. spinigerum antigen. G. spinigerum advanced third-stage larvae (L3) were obtained from livers of infected freshwater eels purchased from a local market in Bangkok as previously described (Nopparatana et al., 1988). The worms were washed in 0.9% NaCl until free of blood and tissue debris, pooled, lyophilized and kept at -2o’C until use. A crude aqueous L3 extract was prepared by grinding the worms with alumina (Sigma Chemical Co., MO, U.S.A.) in distilled water containing 0.1 mr+phenylmethylsulphonylfluoride (Sigma), 0.1 mM-tosyl-amide-2-phenylethyl-chloromethyl ketone (Sigma) and 10 mM-ethylene diamine trichloroacetic acid at 4°C. After the alumina was removed by centrifugation at 250 x g for 5 min at 4°C the worm suspension was extracted twice with ether at 4°C. The worm extract was centrifuged at 10,000 x g for 30 min at 4°C and the supernatant removed. The sediment was resuspended in distilled water, sonicated at
4°C for 10 min and centrifuged as above. The supernatants were pooled, dialysed with distilled water and kept at - 20°C for further analysis. The protein content was determined by the method of Lowry, Rosenbrough, Farr & Randall (1951) using bovine serum albumin (BSA) as the standard. SDS-PAGE and Western blot analysis. The electrophoresis was performed in a vertical slab gel apparatus using the method of Laemmh & Favre (1973) with some modification. Briefly, a 4% acrylamide stacking gel and a 10% acrylamide separating gel were used. Samples containing approximately 30 pg of the L3 extract were boiled at IOO’C for 3 min in sodium dodecyl sulphate (SDS) and 2-mercaptoethanol before loading onto the gel. The separated protein bands were visualized by staining with Cooma&ie brilliant blue R (Sigma). The mol. wts of unknown proteins were estimated by comparing the relative migration ratios against those of standard proteins (Pharmacia Fine Chemicals, Uppsala, Sweden). For Western blot analysis the SDS-PAGE resolved components were electroblotted onto a 0.45 pm nitrocellulose membrane (Bio-Rad Laboratories, CA, U.S.A.) according to the method of Towbin, Staehelin & Gordon (1979). After blotting, the unreacted sites on the membrane were ‘blocked by soaking the strips in phosphate buffered saline. uH 7.4 (PBS) containina 0.5% gelatin. 3% BSA and 0.04% NaN, at 26’C for 2 h with gentie rocking. The strips were then washed twice (10 min each) with washing buffer (0.05% Tween 20 in PBS) before being reacted with the patient’s sera diluted 1:200 in PBS containing 0.2% gelatin, 0.2% BSA and 0.04% NaN, (PBS-BSA) at 26’C for 1 h with gentle rocking. The strips were washed four times (20 min each) in the washing buffer and incubated with ‘*‘I-labelled sheep anti-human immunoglobulins (Amersham International PTY, U.K.) in PBS-BSA (2-5 x 10’ c.p.m. ml-‘) at 26°C for 30 min. The strips were washed as above, dried and autoradiographed onto the Kodak X-Omat RP films (Eastman Kodak Co., NY, U.S.A.) with light intensifying screens at - 70” C for 2448 h.
AGCOEFGHIJKLMNOPQRS FIG. 1. Serum reactivities of patients with parasitologically proven (A-D) and presumptive (E-S) gnathostomiasis against L3 extract of G. spinigerum by SDS-PAGE and Western blot analysis. Numbers at left indicate mol. wt x IO-‘.
G. spinigerum
diagnostic antigen
RESULTS The serum reactivities as revealed by SDS-PAGE and Western blot analysis against the L3 antigen of four parasitologically proven gnathostomiasis patients are shown in Fig. 1. It was found that the L3 antigen is highly complex, consisting of more than 20 antigenic bands with mol. wts ranging from 150k to 13k. The predominant antigenic bands were 150, 135, 120, 94, 49, 43, 42, 38, 35, 34, 33, 32, 24, 22 and 21k. One prominent antigenic band of 24k was found to give consistent reaction with sera from all of the proven cases. When sera from 15 presumptive gnathostomiasis patients were tested for their reactivities against the antigen, it was found that these sera had their reactivities mainly against L3 antigenic components with mol. wts higher than 30k (Fig. 1). Only five cases (33%) gave positive reactions against the 24k antigen. The specificity of the L3 antigen was further defined by comparing the serum reactivities between the gnathostomiasis patients and patients with other parasitic infections. Data in Table 1 summarize the serum reactions against the major L3 antigenic components. It was found that all sera from patients with other parasitic infections reacted mainly with the L3 components with mol. wts higher than 30k and only one patient with paragonimiasis gave a weak reaction against the 24k antigen. Most of the healthy individuals had very low to moderate reactivities against the 38k antigen while some of them had, in addition, reactivities against the 49k and 43k antigens.
317
The representative patterns of the serum reactivities of patients with gnathostomiasis, patients with other parasitic infections and the normal controls are shown in Fig. 2. DISCUSSION The present study has demonstrated a specific 24k antigen of G. spinigerum which consistently reacted with sera from gnathostomiasis patients but not with those from patients with other parasitic infections. This study confirmed our previous report that the G. spinigerum L3 antigen was highly complex and consisted of various antigenic components as revealed by SDS-PAGE and Western blot analysis (Nopparatana et al., 1988). Although ‘251-labelled anti-human immunoglobulins were used to detect the serum reactivities in the present study, their patterns were similar to those of the previous study in which radioiodinated protein A was used. However, our previous study failed to clearly demonstrate the specific 24k antigen and this is explained by the fact that the antigen is relatively unstable upon storage before use in the electrophoresis sample buffer containing the detergent SDS and the reducing agent 2-mercaptoethanol. It was observed in our study that prolonged storage of the L3 antigen in the electrophoresis buffer at - 20°C resulted in a significant loss of the specific 24k band. We have found that this 24k antigen is most stable in aqueous solution and could be purified from the crude L3 extract by gel filtration,
TABLE I-SUMMARY OF SERUMREAC~~VITIES 0~ PATIENTSWITHGNATHOSTOMIA~IS, PATIENTSWITHOTHERPARASITICINFECTIONSAND HEALTHYCONTROLSAGAINSTL3 EXTRACTOF G. spinigerum ASREVEALED BYSDS-PAGE AND WESTERNBLOTANALYSIS Predominant
Sera (total number) Confirmed cases (4) Presumptive cases (15) Angiostrongyliasis Trichinosis (5) Sparganosis (1) Cysticercosis (1) Paragonimiasis Schistosomiasis Opisthorchiasis Filariasis (1)
(2)
(13) (2) (4)
Taeniasis (6) Trichuriasis (1) Strongyloidosis (4) Hook worm infection Ascariasis (1) Amoebiasis (2) Malaria (4) Mixed infections
(14)
Healthy
(19)
controls
194
94
49
43142
38
35
34133
32
24
3. 15
4 15
4 13
4 14
3 12
3 12
3 13
3 12
4 5
1 4 1 1
1 4 1 1
2 4 1 1
2 5 1 1
2 3 1 1
2 3 1 _
2 3 1
-
11
11 _ 4
10
12
9
9
8
4
4 1
4 -
4 _
4
6
5 1 3 2
6 _ 4 2 1 2
5 1 2 2 1 _
5
4 12
2 6
4
8
4
(3)
* Numbers of patients - No reaction.
6 1 4 3 1 2
4 3 1 2
2 11
3 IO
I
_ showing
mol. wt x IO-’
positive reaction.
1 _ 2 6 2
1
5
1 1 1
-
8 _ 4
-
_
1
_
_
4
_
_ 2 _
4 2 _ _
_ 2
_ _
2 6
3 5
_
_
3 10 1 1 1 _
1
_ 2 4
2212 1
_ _
9 1 3 _ 2 _ _ _ _ 2 3 _
318
P. TAPCHAISRI, C. NOPPARATANA,W. CHAICUMPAand P. SETASUBAN
ABCBEF6HlJKLIYIIIOPORSTU FIG. 2. Representative serum reactivities of patients with gnathostomiasis (A), angiostrongyliasis (B,C),trichinosis (D,E), sparganosis (F), cysticercosis (G), paragonimiasis (H,I), schistosomiasis (J), opisthorchiasis (K), filariasis (L), taeniasis (M), strongyloidosis (N), hook worm infection (0), ascariasis (p), amoebiasis (Q), mahria (R), mixed infections (ST) and healthy individual (U) against L3 extract of G. ~pi~iger~ by SDS-PAGE and Western blot analvsis. Numbers at left indicate mol. wt x 10--s.
ion-exchange chromatography and chromatofocusing (Nopparatana, C., Setasuban, P., Chaicumpa, W. & Tapchaisri, P., in preparation). It was observed in the present study that most sera from presumptive gnathostomiasis patients gave positive reactivities against various L3 antigenic components with mol. wts of 30k and above but only five out of 15 (33%) were positive for the specific 24k antigen. The most likely explanation for the low percentage of positivity in these patients was that their diagnosis was not correct and that the positive reactions against the other L3 components might result from previous infections with other parasites. Likewise, the presence of the serum antibody against the 24k antigen in one patient with paragonimiasis would have resulted from previous infection with G. s~iniger~~. The present study clearly demonstrated that extensive crossreactions of the antigen occurred and that the use of crude, undefined L3 extract was not suitable for the specific immunodiagnosis of human gnathostomiasis. Suntharasamai et al. (1985) found that 33% of patients with eosinophilic meningo-myeloencephalitis typically due to gnathostomiasis, 56% of patients with cutaneous migratory swellings (CMS) and 23% of patients with other parasitic infections were positive by ELISA using the crude L3 extract as the antigen. Further improvement of the technique using the similar antigen preparation by Dhamkrong-at et al. (1986) resulted in a sensitivity of 100% in CMS patients but cross-reaction occurred with sera from
some healthy blood donors and angiostrongyliasis patients. Furthermore, the specificity of the reaction in patients with other parasitic infections was not clearly demonstrated. It would be appropriate for an ELISA using the crude L3 antigen to be employed as a screening test for human gnathostomiasis and for Western biot analysis to be used for confi~ation. A similar system has worked well for the diagnosis of patients with acquired-immunodeiiciency syndrome (Sarngadharan, Popovic, Bruch, Schupbach & Gallo, 1984; Weiss, Goedert, Sarngadharan, Bodner, The AIDS Seroepidemiology Collaborative Working Group, Gallo & Blattner, 1985). However, more data will be needed regarding the sensitivity of the Western blot analysis in relation to various clinical manifestations of the gnathostomiasis patients. The demonstration of specific 24k antigen of G. spinigerum would lead to the development of a specific diagnosis of human gnathostomiasis using a simple immunological test, namely ELISA, provided that the specific component of the worms can be appropriately prepared, either through biochemical processes or recombinant DNA technology. Our current work involves fractionation of the worm extract by sephadex and ion-exchange chromatographies and chromatofocusing. It has been found that the purified fraction containing the specific 24k antigen could be obtained and the sensitivity and specificity of such an antigen in the serodiagnosis of human gnathostomiasis are being evaluated.
G. spinigerum diagnostic Acknowledgements--We would like to thank Dr Savanat Tharavanij and Dr Santasiri Sornmani for their encouragement and suggestions; and Dr Danai Bunnag for the sera from patients with presumptive gnathostomiasis and other parasitic infections. This work was supported in part by a grant from Mahidol University.
REFERENCES DHAMKRONG-AT A., MIGASENA S., SUNTHARASAMAI P., BUNNAGD., PRIWANR. & SIRISINHAS. 1986. Enzyme-linked immunosorbent assay for detection of antibody to Gnarhostoma antigen in patients with intermittent cutaneous migratory swelling. Journal of Clinical Microbiology 23: 847-85 1. LAEMMLIU. K. & FAVRE M. 1973. Maturation of the head of bacteriophage T4. DNA packaging events. Journal of Molecular Biology 80: 575-599. LOWRY0. H., ROSENBROUGHN. J., FARR A. L. & RANDALLR. J. 1951. Protein measurement by the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275. NOPPARATANAC., TAPCHAISRIP., SETASULIAN P., CHAICUMPA
antigen
319
W. & DEKUMYOYP. 1988. Antibody responses in human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 19: 2 19-224. SARNGADHARANM. G., POPOVICM., BRUCH L., SCHUPBACHJ. & GALLO R. C. 1984. Antibodies reactive with human Tlymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224: 56508. SUNTHARASAMAI P., DESAKORNV., MIGASENAS., BUNNAGD. & HARINASUTAT. 1985. ELISA for immunodiagnosis of human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 16: 274-279. TOWBINH., STAEHELINT. &GORDON J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proceedings of the National Academy of Science of the United States of America 76: 4350-4354. WEISS S. H., GOEDER~J. J., SARNGADHARANM. G., BODNERA. J., THE AIDS SEROEPIDEMIOLOGY COLLABORATIVE WORKING GROUP, GALLO R. C. & BLATTNERW. A. 1985. Screening test for HTLV-III (AIDS agent) antibodies. Specificity, sensitivity and applications. Journal of the American Medical Association 253: 22 l-225.
