Inkmalional JournalforParasitology Printed in Great Britain
Vol. 22, No. 8,pp. Il5lL1156,
1992 0
002&7519/92 S5.M) + 0.00 Pergaman Press Ltd 1992 Auswalian Sociery for Parasitology
TOWARDS A SUITABLE ANTIGEN FOR DIAGNOSIS GNATHOSTOMA SPINIGERUM INFECTION CHAMMONG
NOPPARATANA,*
WANPEN
SETASUBAN* *Department
and
OF
PRAMUAN TAPcHAIsRI,t PRASERT RumGKuN,4Porwt
CHAICUMPA,~$ YUWAPORN
of Helminthology and TDepartment of Microbiology Mahidol University, 420/6 Rajvithi Road, (Received 10 September
and Immunology, Faculty Bangkok 10400, Thailand
of Tropical
Medicine,
1991; accepted 20 June 1992)
Ah&act-NOPPARATANA C., CHAICUMPAW., TAPCHAISRIP., SETASUBANP. and RUANGKUNAPORNY. 1992. Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection. International Journal for Parasitology 22: 1151-l 156. Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva. INDEX KEY WORDS: Gnathostoma spinigerum; diagnostic antigen; morphology; gnathostomiasis; extraction solutions; sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); Western blot analysis; electron microscopy.
INTRODUCTION
DIAGNOSISof human gnathostomiasis is largely based on the patient’s history of residing in the endemic area, consuming raw or undercooked meat (which might be contaminated with Gnathostoma spinigerum infective stage larva) and clinical features. However, a few other parasitic infections give similar background information and clinical pictures that cannot be readily distinguished from gnathostomiasis. Definite diagnosis of the disease can be done when the worm is recovered from the patient but this is very rare. Several attempts have been made to diagnose the disease by immunological methods which detect antibodies. However, most investigators used crude antigens prepared from the infective larvae of the parasite which contained many antigens shared by other worms, thus the assays were also positive for other parasitic infections. It is only recently that specific antigen of G. spinigerum has been identified, purified
$ To whom all correspondence
should be addressed.
and used for diagnostic purposes. The test used a purified antigen that is a glycoprotein with a mol. wt of 24,000 (24 kDa), sensitive, apparently specific and has good predictive values (Tapchaisri, Nopparatana, Chaicumpa & Setasuban, 1991; Nopparatana, Setasuban, Chaicumpa & Tapchaisri, 1991). The specific antigen was extracted and purified from the advanced third-stage larvae of the parasite. This communication demonstrates the morphology of the infective larvae, organs where the diagnostic 24 kDa antigen is located and the conditions suitable for extracting the antigen from the worms. MATERIALS
AND METHODS
Infective third-stage larvae of G. spinigerum. The larvae were collected from two sources. The first source was from livers of naturally infected freshwater eels. These eels were purchased from local vendors in Bangkok. However, the eels were originally collected from various provinces of Thailand including Samut Prakan, Nakhon Sawan, Ayutthya (central Thailand), Ubon Ratchathani (northeastern Thailand) and Surat Thani (southern part of Thailand). The livers were sliced, pressed between two glass plates and examined for the
1151
1152
FIG. 1. Morphology
FIG. 2. Morphology
C. NOPPARATANA er al.
of L3E as revealed by light microscopy. a: Encysted larva; b: excysted larva; c: head-bulb oesophagus (E); d: lips (L), hooklets (0), muscular tube (MT) and glandular tube (GT).
of L3E as revealed by scanning electron microscopy. a: Whole body; b: head-bulb covered with spines (S); d: terminal end showing anus (A).
(H) and
and lips (L); c: body
Diagnostic
antigen
of G. s~~ig~r~
1153
67 67 -
30-
ABC
D
E
F
G
FIG. 3. SDS-PAGE and Coomassie brilliant blue staining patterns of crude L3E antigens extracted with water(B), Triton X100 (C) and sodium deoxycholate (D); A = molecular weight standards; E-G = reaction patterns of the three extracts with serum of a patient with gnathostomiasis as analysed by Western blotting, respectively. Numbers are mol. wt ( x lo-‘).
presence of the encysted larvae. The cysts were collected and dissected individually under a dissecting microscope. The living larvae were identified by light microscopy using the criteria described by Daengsvang (1980). The morphology was also confirmed by random selection of the larvae for scanning electron microscopy which was performed 10 times on different larvae. The second source of the advanced thirdstage larvae was from ex~~menta~ly infected mice. The mice were previously infected orally with the early third-stage larvae of G. spinigerum which developed in cyclops. The cyclops were infected experimentally with the first-stage larvae of the parasite obtained from eggs collected from faeces of cats bearing adult worms in stomach tumours. The cats were previously infected orally with advanced third-
stage larvae obtained from eels. The mice were sacrificed 30 days after the infection and all organs were examined for infective larvae. The larvae were collected in the same manner as from the livers of eels. The larvae from the eels (L3E) were washed with normal saline solution and distilled water, then lyophilized. The dried worms were ground in a paste of alumina (Sigma Chemical Company, U.S.A.) made with extraction solution (distilled water: 1% Triton X-100, or 1% sodium deoxycholate) containing phenyl-methyl-sulphonylfluoride (PMSF), tosyl-amide-2-phenylethyl-chloromethyl ketone (TPCK) (each 0.1 mM; Sigma Chemical Company, U.S.A.) and 10 mM- EDTA to minimize proteolysis. After grinding, a small volume of the extraction solution was added to the preparation and the alumina was removed by centri-
C. NOPPARATANA et al.
1154
(2) the oesophagus and intestines and (3) the remaining carcasses (body cuticles). The three parts were homogenized separately in distilled water containing the protease inhibitors. The fluid leaking out during dissection was also collected (body fluid). SDS-PAGE,
protein staining and Western blot analysis.
The techniques were performed as previously described (Nopparatana et al., 1991). Serum specimen. A serum sample of a patient from whom the G. spinigerum larva was recovered from skin of the abdomen was used for antigen analysis by SDS-PAGE and Western blot analysis.
-
67 67-
30
30 24a
24 20
A
B
C
D
FIG. 4. SDS-PAGE and Coomassie brilliant blue patterns of crude water extracts of L3E (A) and L3M (B); C and D are Western blot patterns of the serum of a patient with gnathostomiasis and the two extracts, respectively. Numbers are mol. wt (x lo-‘).
fugation at 75 x g at 26°C for 5 min. The preparation was subjected to centrifugation at 10,000 x g at 4’C for 30 min. The supernatant was collected. The sediment was resuspended in the extraction solution, sonicated at 20 kHz at 4°C for 10 min and centrifuged at 10,000 x gas above. All of the supernatants were pooled, dialysed against distilled water at 4”C, lyophilized and kept at - 2o’C for further analysis. The third-stage larvae collected from mice (L3M) were washed and extracted with distilled water. Protein contents of all preparations were determined by the method of Markwell, Hass, Bieber & Tolbert (1978). One hundred and fifty L3E were dissected under the dissecting microscope into three separate parts, namely (1) the head-bulbs and cephalic sacs,
RESULTS The infection rate of G. spinigerum infective stage larvae was found to be highest in eels obtained from the central areas of Thailand. Approximately 19% of the 2350 eels in these areas examined were infected. The infection rates in Ubon-Ratchathani Province (Northeast) and Surat Thani Province (South) were 12 and 8%, respectively. The L3E cysts collected from livers of naturally infected eels were approximately 1 mm in diameter with the coiled larva inside and surrounded by fibrous tissue (Fig. la). The released larva was about 0.4 x 0.7 mm in size (Fig. lb). The morphology of the larva could be divided into three parts (Fig. lc, d), namely lips, head-bulb and body with constriction between the head-bulb and the body (neck). The cervical sacs were composed of two pairs of muscular and glandular tubes located next to the oesophagus (Fig. Id). Lateral lips were equal in size. The hooklets on the head-bulb were well-developed and increased in numbers posteriorly from row 1 to row 4. The body was covered with transverse rows of single-pointed spines which gradually reduced in size and density towards the posterior end (Fig. 2). The posterior end was rounded with a terminal opening (anus). SDS-PAGE and Coomassie brilliant blue staining of the crude extracts of L3E (30 pg) are shown in Fig. 3, lanes B-D. Although the three extraction solutions, i.e. distilled water, Triton X-100 and sodium deoxycholate, resulted in extracts with similar protein compositions, the quantities of the proteins were different. Distilled water yielded the highest amount of the diagnostic 24 kDa (Nopparatana et al., 1991) antigen. The amount of the 24 kDa protein obtained from the Triton X-100 was reasonable while the sodium deoxycholate yielded the least quantity. Strongest reactivity between the patient’s antibody and the 24 kDa antigen was observed in Western blot analysis when the fractionated crude water extract was used in comparison to the reactions of the Triton X100 and sodium deoxycholate extracts (Fig. 3, lanes E-G).
Diagnostic antigen of G. spinigerum
1155
6767-
30 24
24 t
20 -
20 -
A
B
C
D
E
F
G
t-i
FIG. 5. SDS-PAGE and Coomassie brilliant bIue patterns of crude water extracts of head-bulbs and cephalic sacs (A), body cuticles(B), oesophagus and intestines(C) and body fluid (D); E, F, G and H are Western blot patterns of the four extracts, respectively with serum of a patient with gnathostomiasis. Numbers are mol. wt ( x 10e3). SDS-PAGE and Coomassie brilliant blue staining patterns of L3E and L3M are shown in Fig. 4. Extracts from the two separate sources yielded very similar patterns of proteins. However, careful examination of Fig. 4 shows a number of minor but distinct differences, e.g. eel bands of M, 17,000, 22,000 and 49,000 (lane A); mouse bands of 1%19,94 and 150 kDa (lane B). Nevertheless, both preparations contained relatively equal amounts of the 24 kDa component which is known to be a specific, diagnostic antigen of C. spinigerum (Nopparatana et al., 1991). The finding was confirmed by Western blot analysis (Fig. 4, lanes C and D).
The three homogenates prepared from different parts of the L3E, namely the head-bulb and cephalic sac homogenate, the oesophageal and intestinal homogenate and the body cuticle homogenate and the body fluid, at equal protein content, were subjected to SDS-PAGE and Coomassie brilliant blue staining. The head-bulb and the cephalic sac homogenate gave rise to protein patterns similar to those of the body cuticle homogenate, though with smaller quantities (Fig. 5, lanes A and B). These two homogenates contained negligible amounts, if any, of the diagnostic M, 24,000 antigen. The antigen appeared in the homogenate of the oesophagus and intestine and in the
1156
C.
NOPPARA~ANAet al.
body fluid (Fig. 5, lanes C and D). The finding was confirmed by Western blot reaction between the three homogenates and the body fluid and the serum of the patient with gnathostomiasis (Fig. 5, lanes E-H, respectively). DISCUSSION The antigens used in most studies on the immunology of gnathostomiasis have been prepared from the third-stage larvae of G. spinigerum. Two main sources were from naturally infected eels (L3E) and experimentally infected mice (L3M) (Dharmkrong-at, Migasena, Suntharasamai, Bunnag, Priwan & Sirisinha, 1986; Suntharasamai, Desakorn, Migasena, Bunnag & Harinasuta, 1985). In the present communication, the protein components of the crude extracts of L3E and L3M and their reactivities with serum antibodies from an infected human were compared. The two extracts contained similar as well as different components. However, both preparations contained an antigen of M, 24,000 which has been found to be a specific, diagnostic antigen for human gnathostomiasis (Nopparatana et al., 1991). Thus, the third-stage larvae of G. spinigerum obtained from either of these two sources seem equally suitable for preparing extracts containing this particular antigen. The surface and other somatic antigens of many nematodes have been successfully solubilized using water, normal saline solution or phosphate buffered saline. Non-ionic as well as anionic detergents such as Triton X-100 and sodium deoxycholate have also been used (Egwang, DuPont, Akue & Pinder, 1988; Nichol & Masterson, 1987). In this study, the efficacies of water, 1% Triton X-100 or sodium deoxycholate in solubilizing protein components from homogenized L3E were analysed under identical conditions on an equivalent protein weight basis. The water extract contained the highest amount of the 24 kDa antigen, while the sodium deoxycholate extract contained the lowest quantity. Thus water should be used for extracting this diagnostic antigen. The proteins in the extract of oesophagus and intestines of the larvae were more immunogenic in man than those of other parts of the worm (Fig. 5, lanes E-H). This was consistent with the previous observation of Morakote, Nateewatana, Maleewong
& Uthong (1989). A 24 kDa protein was also present in the oesophagus and intestine of the larvae. Acknowledgements-The
work was financially supported by grants from Mahidol University and the Faculty of Tropical Medicine, Bangkok, Thailand. The authors thank Professors Natth Bhamarapravati and Santasiri Sommani for their encouragement. REFERENCES DAENGSVANGS. 1980. A monograph on the genus Gnathostoma and gnathostomiasis in Thailand. SEAMIC Southeast Asian Medical Information Center, Tokyo, Japan. DHARMKRONG-AT A., MIGASENA S., SUNTHARASAMAIP., BUNNAGD., PRIWANR. & S~RHNHA S. 1986. Enzyme-linked immunosorbent assay for detection of antibody to Gnafhostoma antigen in patients with intermittent cutaneous migratory swelling. Journal of Clinical Microbiology 23: 847-851. EGWANG T. G., DUPONT S., AKUE J. P. & PINDERM. 1988. Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae. Molecular and Biochemical Parasitology 31: 251-261. MARKWELLM. A. K., HASSS. M., BIEBERL. L. & TOLBERT N. E. 1978. A modification of
protein determination
the Lowry procedure to simplify in membrane and lipid samples.
Analytical Biochemistry 87: 206210. MORAKOTEN., NA~EEWATANA N., MALEEWONG W. & UTHONG
T. 1989. Anatomical localization of Gnathosroma spinigerum larval antigens by an indirect fluorescent antibody test. Southeast Asian Journal of Tropical Medicine and Public Health 20: 291-295. NICHOL C. & MASTERSONW. J. 1987. Characterization of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance. Molecular and Biochemical Parasitology 25: 29- 38. NOPPARATANA C., SETASUBAN P., CHAICUMPA W. & TAPCHAISRI P. 1991. Purification of Gnathostoma spinigerum specific
antigen and immunodiagnosis of human gnathostomiasis. International Journalfor Parasitology 21: 677-687. SUN~HARASAMAI P., DESAKORNV., MIGASENAS., BUNNAGD. & HARINASUTAT. 1985. ELISA for immunodiagnosis
of human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 16: 274-279. TAPCHAISRIP., NOPPARATANAC., CHAICUMPAW. & SE~ASUBAN P. 1991. Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis. Iniernational Journal for Parasitology 21: 3 15-3 19.
Vol. 22, No. 8,pp. Il5lL1156,
1992 0
002&7519/92 S5.M) + 0.00 Pergaman Press Ltd 1992 Auswalian Sociery for Parasitology
TOWARDS A SUITABLE ANTIGEN FOR DIAGNOSIS GNATHOSTOMA SPINIGERUM INFECTION CHAMMONG
NOPPARATANA,*
WANPEN
SETASUBAN* *Department
and
OF
PRAMUAN TAPcHAIsRI,t PRASERT RumGKuN,4Porwt
CHAICUMPA,~$ YUWAPORN
of Helminthology and TDepartment of Microbiology Mahidol University, 420/6 Rajvithi Road, (Received 10 September
and Immunology, Faculty Bangkok 10400, Thailand
of Tropical
Medicine,
1991; accepted 20 June 1992)
Ah&act-NOPPARATANA C., CHAICUMPAW., TAPCHAISRIP., SETASUBANP. and RUANGKUNAPORNY. 1992. Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection. International Journal for Parasitology 22: 1151-l 156. Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva. INDEX KEY WORDS: Gnathostoma spinigerum; diagnostic antigen; morphology; gnathostomiasis; extraction solutions; sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); Western blot analysis; electron microscopy.
INTRODUCTION
DIAGNOSISof human gnathostomiasis is largely based on the patient’s history of residing in the endemic area, consuming raw or undercooked meat (which might be contaminated with Gnathostoma spinigerum infective stage larva) and clinical features. However, a few other parasitic infections give similar background information and clinical pictures that cannot be readily distinguished from gnathostomiasis. Definite diagnosis of the disease can be done when the worm is recovered from the patient but this is very rare. Several attempts have been made to diagnose the disease by immunological methods which detect antibodies. However, most investigators used crude antigens prepared from the infective larvae of the parasite which contained many antigens shared by other worms, thus the assays were also positive for other parasitic infections. It is only recently that specific antigen of G. spinigerum has been identified, purified
$ To whom all correspondence
should be addressed.
and used for diagnostic purposes. The test used a purified antigen that is a glycoprotein with a mol. wt of 24,000 (24 kDa), sensitive, apparently specific and has good predictive values (Tapchaisri, Nopparatana, Chaicumpa & Setasuban, 1991; Nopparatana, Setasuban, Chaicumpa & Tapchaisri, 1991). The specific antigen was extracted and purified from the advanced third-stage larvae of the parasite. This communication demonstrates the morphology of the infective larvae, organs where the diagnostic 24 kDa antigen is located and the conditions suitable for extracting the antigen from the worms. MATERIALS
AND METHODS
Infective third-stage larvae of G. spinigerum. The larvae were collected from two sources. The first source was from livers of naturally infected freshwater eels. These eels were purchased from local vendors in Bangkok. However, the eels were originally collected from various provinces of Thailand including Samut Prakan, Nakhon Sawan, Ayutthya (central Thailand), Ubon Ratchathani (northeastern Thailand) and Surat Thani (southern part of Thailand). The livers were sliced, pressed between two glass plates and examined for the
1151
1152
FIG. 1. Morphology
FIG. 2. Morphology
C. NOPPARATANA er al.
of L3E as revealed by light microscopy. a: Encysted larva; b: excysted larva; c: head-bulb oesophagus (E); d: lips (L), hooklets (0), muscular tube (MT) and glandular tube (GT).
of L3E as revealed by scanning electron microscopy. a: Whole body; b: head-bulb covered with spines (S); d: terminal end showing anus (A).
(H) and
and lips (L); c: body
Diagnostic
antigen
of G. s~~ig~r~
1153
67 67 -
30-
ABC
D
E
F
G
FIG. 3. SDS-PAGE and Coomassie brilliant blue staining patterns of crude L3E antigens extracted with water(B), Triton X100 (C) and sodium deoxycholate (D); A = molecular weight standards; E-G = reaction patterns of the three extracts with serum of a patient with gnathostomiasis as analysed by Western blotting, respectively. Numbers are mol. wt ( x lo-‘).
presence of the encysted larvae. The cysts were collected and dissected individually under a dissecting microscope. The living larvae were identified by light microscopy using the criteria described by Daengsvang (1980). The morphology was also confirmed by random selection of the larvae for scanning electron microscopy which was performed 10 times on different larvae. The second source of the advanced thirdstage larvae was from ex~~menta~ly infected mice. The mice were previously infected orally with the early third-stage larvae of G. spinigerum which developed in cyclops. The cyclops were infected experimentally with the first-stage larvae of the parasite obtained from eggs collected from faeces of cats bearing adult worms in stomach tumours. The cats were previously infected orally with advanced third-
stage larvae obtained from eels. The mice were sacrificed 30 days after the infection and all organs were examined for infective larvae. The larvae were collected in the same manner as from the livers of eels. The larvae from the eels (L3E) were washed with normal saline solution and distilled water, then lyophilized. The dried worms were ground in a paste of alumina (Sigma Chemical Company, U.S.A.) made with extraction solution (distilled water: 1% Triton X-100, or 1% sodium deoxycholate) containing phenyl-methyl-sulphonylfluoride (PMSF), tosyl-amide-2-phenylethyl-chloromethyl ketone (TPCK) (each 0.1 mM; Sigma Chemical Company, U.S.A.) and 10 mM- EDTA to minimize proteolysis. After grinding, a small volume of the extraction solution was added to the preparation and the alumina was removed by centri-
C. NOPPARATANA et al.
1154
(2) the oesophagus and intestines and (3) the remaining carcasses (body cuticles). The three parts were homogenized separately in distilled water containing the protease inhibitors. The fluid leaking out during dissection was also collected (body fluid). SDS-PAGE,
protein staining and Western blot analysis.
The techniques were performed as previously described (Nopparatana et al., 1991). Serum specimen. A serum sample of a patient from whom the G. spinigerum larva was recovered from skin of the abdomen was used for antigen analysis by SDS-PAGE and Western blot analysis.
-
67 67-
30
30 24a
24 20
A
B
C
D
FIG. 4. SDS-PAGE and Coomassie brilliant blue patterns of crude water extracts of L3E (A) and L3M (B); C and D are Western blot patterns of the serum of a patient with gnathostomiasis and the two extracts, respectively. Numbers are mol. wt (x lo-‘).
fugation at 75 x g at 26°C for 5 min. The preparation was subjected to centrifugation at 10,000 x g at 4’C for 30 min. The supernatant was collected. The sediment was resuspended in the extraction solution, sonicated at 20 kHz at 4°C for 10 min and centrifuged at 10,000 x gas above. All of the supernatants were pooled, dialysed against distilled water at 4”C, lyophilized and kept at - 2o’C for further analysis. The third-stage larvae collected from mice (L3M) were washed and extracted with distilled water. Protein contents of all preparations were determined by the method of Markwell, Hass, Bieber & Tolbert (1978). One hundred and fifty L3E were dissected under the dissecting microscope into three separate parts, namely (1) the head-bulbs and cephalic sacs,
RESULTS The infection rate of G. spinigerum infective stage larvae was found to be highest in eels obtained from the central areas of Thailand. Approximately 19% of the 2350 eels in these areas examined were infected. The infection rates in Ubon-Ratchathani Province (Northeast) and Surat Thani Province (South) were 12 and 8%, respectively. The L3E cysts collected from livers of naturally infected eels were approximately 1 mm in diameter with the coiled larva inside and surrounded by fibrous tissue (Fig. la). The released larva was about 0.4 x 0.7 mm in size (Fig. lb). The morphology of the larva could be divided into three parts (Fig. lc, d), namely lips, head-bulb and body with constriction between the head-bulb and the body (neck). The cervical sacs were composed of two pairs of muscular and glandular tubes located next to the oesophagus (Fig. Id). Lateral lips were equal in size. The hooklets on the head-bulb were well-developed and increased in numbers posteriorly from row 1 to row 4. The body was covered with transverse rows of single-pointed spines which gradually reduced in size and density towards the posterior end (Fig. 2). The posterior end was rounded with a terminal opening (anus). SDS-PAGE and Coomassie brilliant blue staining of the crude extracts of L3E (30 pg) are shown in Fig. 3, lanes B-D. Although the three extraction solutions, i.e. distilled water, Triton X-100 and sodium deoxycholate, resulted in extracts with similar protein compositions, the quantities of the proteins were different. Distilled water yielded the highest amount of the diagnostic 24 kDa (Nopparatana et al., 1991) antigen. The amount of the 24 kDa protein obtained from the Triton X-100 was reasonable while the sodium deoxycholate yielded the least quantity. Strongest reactivity between the patient’s antibody and the 24 kDa antigen was observed in Western blot analysis when the fractionated crude water extract was used in comparison to the reactions of the Triton X100 and sodium deoxycholate extracts (Fig. 3, lanes E-G).
Diagnostic antigen of G. spinigerum
1155
6767-
30 24
24 t
20 -
20 -
A
B
C
D
E
F
G
t-i
FIG. 5. SDS-PAGE and Coomassie brilliant bIue patterns of crude water extracts of head-bulbs and cephalic sacs (A), body cuticles(B), oesophagus and intestines(C) and body fluid (D); E, F, G and H are Western blot patterns of the four extracts, respectively with serum of a patient with gnathostomiasis. Numbers are mol. wt ( x 10e3). SDS-PAGE and Coomassie brilliant blue staining patterns of L3E and L3M are shown in Fig. 4. Extracts from the two separate sources yielded very similar patterns of proteins. However, careful examination of Fig. 4 shows a number of minor but distinct differences, e.g. eel bands of M, 17,000, 22,000 and 49,000 (lane A); mouse bands of 1%19,94 and 150 kDa (lane B). Nevertheless, both preparations contained relatively equal amounts of the 24 kDa component which is known to be a specific, diagnostic antigen of C. spinigerum (Nopparatana et al., 1991). The finding was confirmed by Western blot analysis (Fig. 4, lanes C and D).
The three homogenates prepared from different parts of the L3E, namely the head-bulb and cephalic sac homogenate, the oesophageal and intestinal homogenate and the body cuticle homogenate and the body fluid, at equal protein content, were subjected to SDS-PAGE and Coomassie brilliant blue staining. The head-bulb and the cephalic sac homogenate gave rise to protein patterns similar to those of the body cuticle homogenate, though with smaller quantities (Fig. 5, lanes A and B). These two homogenates contained negligible amounts, if any, of the diagnostic M, 24,000 antigen. The antigen appeared in the homogenate of the oesophagus and intestine and in the
1156
C.
NOPPARA~ANAet al.
body fluid (Fig. 5, lanes C and D). The finding was confirmed by Western blot reaction between the three homogenates and the body fluid and the serum of the patient with gnathostomiasis (Fig. 5, lanes E-H, respectively). DISCUSSION The antigens used in most studies on the immunology of gnathostomiasis have been prepared from the third-stage larvae of G. spinigerum. Two main sources were from naturally infected eels (L3E) and experimentally infected mice (L3M) (Dharmkrong-at, Migasena, Suntharasamai, Bunnag, Priwan & Sirisinha, 1986; Suntharasamai, Desakorn, Migasena, Bunnag & Harinasuta, 1985). In the present communication, the protein components of the crude extracts of L3E and L3M and their reactivities with serum antibodies from an infected human were compared. The two extracts contained similar as well as different components. However, both preparations contained an antigen of M, 24,000 which has been found to be a specific, diagnostic antigen for human gnathostomiasis (Nopparatana et al., 1991). Thus, the third-stage larvae of G. spinigerum obtained from either of these two sources seem equally suitable for preparing extracts containing this particular antigen. The surface and other somatic antigens of many nematodes have been successfully solubilized using water, normal saline solution or phosphate buffered saline. Non-ionic as well as anionic detergents such as Triton X-100 and sodium deoxycholate have also been used (Egwang, DuPont, Akue & Pinder, 1988; Nichol & Masterson, 1987). In this study, the efficacies of water, 1% Triton X-100 or sodium deoxycholate in solubilizing protein components from homogenized L3E were analysed under identical conditions on an equivalent protein weight basis. The water extract contained the highest amount of the 24 kDa antigen, while the sodium deoxycholate extract contained the lowest quantity. Thus water should be used for extracting this diagnostic antigen. The proteins in the extract of oesophagus and intestines of the larvae were more immunogenic in man than those of other parts of the worm (Fig. 5, lanes E-H). This was consistent with the previous observation of Morakote, Nateewatana, Maleewong
& Uthong (1989). A 24 kDa protein was also present in the oesophagus and intestine of the larvae. Acknowledgements-The
work was financially supported by grants from Mahidol University and the Faculty of Tropical Medicine, Bangkok, Thailand. The authors thank Professors Natth Bhamarapravati and Santasiri Sommani for their encouragement. REFERENCES DAENGSVANGS. 1980. A monograph on the genus Gnathostoma and gnathostomiasis in Thailand. SEAMIC Southeast Asian Medical Information Center, Tokyo, Japan. DHARMKRONG-AT A., MIGASENA S., SUNTHARASAMAIP., BUNNAGD., PRIWANR. & S~RHNHA S. 1986. Enzyme-linked immunosorbent assay for detection of antibody to Gnafhostoma antigen in patients with intermittent cutaneous migratory swelling. Journal of Clinical Microbiology 23: 847-851. EGWANG T. G., DUPONT S., AKUE J. P. & PINDERM. 1988. Biochemical and immunochemical characterization of surface and excretory-secretory antigens of Loa loa microfilariae. Molecular and Biochemical Parasitology 31: 251-261. MARKWELLM. A. K., HASSS. M., BIEBERL. L. & TOLBERT N. E. 1978. A modification of
protein determination
the Lowry procedure to simplify in membrane and lipid samples.
Analytical Biochemistry 87: 206210. MORAKOTEN., NA~EEWATANA N., MALEEWONG W. & UTHONG
T. 1989. Anatomical localization of Gnathosroma spinigerum larval antigens by an indirect fluorescent antibody test. Southeast Asian Journal of Tropical Medicine and Public Health 20: 291-295. NICHOL C. & MASTERSONW. J. 1987. Characterization of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance. Molecular and Biochemical Parasitology 25: 29- 38. NOPPARATANA C., SETASUBAN P., CHAICUMPA W. & TAPCHAISRI P. 1991. Purification of Gnathostoma spinigerum specific
antigen and immunodiagnosis of human gnathostomiasis. International Journalfor Parasitology 21: 677-687. SUN~HARASAMAI P., DESAKORNV., MIGASENAS., BUNNAGD. & HARINASUTAT. 1985. ELISA for immunodiagnosis
of human gnathostomiasis. Southeast Asian Journal of Tropical Medicine and Public Health 16: 274-279. TAPCHAISRIP., NOPPARATANAC., CHAICUMPAW. & SE~ASUBAN P. 1991. Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis. Iniernational Journal for Parasitology 21: 3 15-3 19.
