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Factor(s) in the venom of scorpions toxic to Schistosoma mansoni (intestinal belharzia) cercariae
roxkiorr, vol. Is . ~. 71 tau. O Perptoon Prey Ltd. 1980 . Printedin GreatBritain.
0041-0101/80/110I-0711 S02.ODi0
FACTORS) IN THE VENOM OF SCORPIONS TOXIC TO SCHISTOSOMA MANSONI (INTESTINAL BELHARZIA) CERCARIAE* M. F. EL-ASMAR,t N. SWELAM,t T. M. ABDEL AAL,$ I{H. GHONEIMt and S . S. HODHODt tDepartments of Biochemistry and $Parasitology, Faculty of Medicine, Ain Shams University, Cavo, Egypt (Acccpttd for publication 4 Merck 1980)
ZLOTKIN et al. (1972 a, b) identified substances in scorpion venom which are toxic to insects and crustaceans, and are different from the mammalian toxin . LAZAROVIA and ZLOTKIN (1979) reported the presence of toxins specifically active on arthropods and mammals from the venom of the scorpion Scorpio mourus . In this communication we demonstrate the presence of factors) which were toxic to Schistosoma mansoni cercariae (final free swimming larval stage of parasite). The Schistosoma mansoni cercariae used in this investigation were Egyptian in origin and have been maintained in our laboratory by serial passages through the snail Biompholaria alexandria and mouse (GHAREEB et al., 1975). Cercariae were collected just prior to their use from snails contained in dechlorinated tap water. Venom obtained from mature Leiurus quinquestriatus, Buthus minor, Buthus occitanus and Androctonus amoreuxi scorpions, by electrical stimulation of the telson as described by EL-ASMAR et al. (1972), was immediately freeze dried and kept in a dessicator over CaClp at room temperature . Chromatographic separation of Leiurus quinquestriatus venom was performed using Biorex 70, analytical grade cation exchanger (Biorad Laboratories, Richmond, California). The Biorex 70 was prepared for chromatography in a glass column 1 x 15 cm and elution of the venom (40 mg) was performed using linear gradient ammonium acetate buffer from 005 M, pH 5~8-0~5 M, pH 6~5 (250 ml of each at a flow rate of 35 ml/hr, 5 ml fraction). The active fraction was lyophilized, gel filtered on Sephadex G50column (1 x 54 cm), eluted with distilled water, (3~5 ml fractions), and lyophilized . Protein was determined in crude venoms and column eluates by absorbance measurement at 280 nm assuming that a 1 mg/ml solution of the venoms or their fractions has an average Al o = 1 ~0 when measured in a cell of 1 ~0 cm light path . For observation of cercaricidal and pericercarial envelope phenomena, slide preparations were made by mixing one drop of the tested venom solution or fractions, diluted to the proper concentration, with one drop of aged tap water containing approximately 20 cercariae, and a cover slip sealed in place (STIRWALT and EVANS, 1955). The preparations were kept at room temperature in a Petri dish with a moistened filter paper on the bottom . Examination of the microscope slides was performed using ordinary light after 24 hr . The 'Supported by grant from the National Academy of Science and Technology .
71?
Short ('ommunications
cercaricidal effect was demonstrated by the spastic behavior of the cercariae and the granular or globular precipitate surrounding the cercariae . The sequence of events in the formation of a pericercarial envelope about the larvae when observed under ordinary microscope was as follows; cercariae had orally secreted large amounts of sticky material, which included refractive granules of varying sizes. The interspace between the outer and inner limits of the cuticle seemed to thicken at the level of the widest body diameter, as though a fluid were collecting there or as if the cuticular material were swelling . The surface of the swollen area then appeared to become detached so that the cercariae were ensheathed in thin, tight, transparent envelopes which appeared to restrict the movement of the contained organism . The envelopes gradually hardened or 'set' and their elasticity was lost . The pericercarial envelope freed from the body began to loosen and balloon away until the cercaria wasencased in a wrinkled, loose-fitting structure similar in appearance to plastic cercarial molds. Crude venoms of Leiurus quinquestriates, Bathos minax, Buthus occitanes and Androctonus amoreuxi were active showing a positive pericercarial envelope test at a minimal concentration of 10 iu,g per slide. Partial purification of cercarial toxins was attempted by the elution of proteins of Leirus quinquestriatus venom from a Biorex 70 column as shown in Fg . 1 . Of the 12 fractions separated, fractions III, N and VI were active on Schistosoma mansoni cercariae. The minimal active concentrations were 8, 14 and 23 N"g per slide, respectively . Fraction VI, obtained from Biorex, when filtered on a Sephadex G 50 column gave a chromatographic pattern as shown in Fig. 2. Tubes collected and designated as subfraction A showed activity to cercariae at a minimal concentration of 9 Fcg per slide. The formation of a pericercarial envelope and secretion of sticky material as shown in Fig. 3 is considered as a simple test giving constant results indicating toxicity or an unfavourable environment for cercariae. 0~05M ammorim aoefate bufier pli 5~8-0~5M~~ pH 6~
0'9
0~7
w
I
0~5
0~5
0~4
0'4
0~3
03
02 0~1 0 0
0~2
rm
10
20
30
40
0' I 50
60
Tube Na (5 ml each)
70
BO
O 90
X 1S Cm) AND PlO. 1. CIßtOMATOGRAPHICPATTERN OFL. (j[ltnQYG4VlOrIL4 VENOM (40 mg) ON BIOREX iOCOLIJFIIY (1 ELIICED BY LINEARGRADIEN'rAMMONIUM ACETATE BUFI£R (0 " OS M,pH 5 " 8-0 " 5 M,pH 6 " 5) AT35 ml/hr .
Fraction 1 causes death to mouse whcn injected i .p . Fractions III, N and VI show cencaricidal activity .
Short Communications
F§G . 3 . CERCARIA SHOWING PERICERCARIAL ENVELOPE REACTION .
The arrow indicates pericercarial envelope .
713
Short ('ommunicati~ms
715
0~6
o~s o~a
.
~az _ oI 0
I 5
A
I 10
I 15
rti
~
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I 20
C
I 25
I 30
I 35
`
I 40
45
Tube Nn ( 3~5 ml each ) FIG. ?. GEL FILTRATION PATTERN OF LYOPHILIZED FRACnON VI (FIG. 1 . ) ON SEPHADEX G 5l1( I X 54 Cnl ) AND
ELl7IED WITH DISI7LLED WATER. Tubes 0-13 were pooled and designated as subfraction A which shows cercaricidal activity .
EL,-ASMAR et al. (1977) reported the presence of anticholinesterase activities in the venom of the scorpion Buthus minax while MANSOUR (1979) found acetylcholinesterase in schistosomes and other parasites . We tested for the presence of anticholinesterase activity in the active fractions we obtained from Leiurus quinquestriatus, but none was found . This eliminates the possibility that the pericercarial envelope response of cercariae is due to the presence of an anticholinesterase activity . No proteolytic activity, using casein as a substrate, was detected in the crude venom or the active fractions . Preliminary experiments indicated that the anticercarial compounds) are nondialysable as a dialysed crude venom showed the same activity on cercariae . Acid hydrolysis of the active compound (subfraction A), followed by paper chromatography, has given us a preliminary indication of the amino acid composition of the compound . The high frequency of schistosomal infection in many countries justifies a nontraditional approach for seeking potential antischistosomal compounds .
REFERENCES F1.-A .SMAR, M. F., IBRAHIM, S . A . and RABm, F. (1972) Fractionation of scorpion (L.tiurus quinquestriaAv H and S) venom . Tosicon 10, 73 . EUAshtwR, M . F., ISMAIL, M ., GHONEBri, KH . and Q4MAN, O . H . (19?7) Scorpion (Budrusminax L. Koch) venom fractions with anticholineaterase activity . Toxionn 1S, 63 . GHAREEB, A. M., EL-GUINDI, S ., SALEH, A., EL-SHERIF, M. alld EL-ASMAR, M . F, (19~I5) Endocrine changes in hepatosplenic schistosotniasis (I-LS .S). An experimental study . Ain Shmns Med. !. Z6, 81 . LwzAROVtw, P . and Zt.oltcnv, E. (1979) Toms specifically active to arthropods and mammals from the venom of scorpion Scorpiomm~rus pabrtaaar (soorpionidae) . Tasicon 17, suppl. 1, 98 . MANSOUR, T . E . (1979) Chemotherapy of paraaitic worms: new biochemical strategies . Scialce 205, 462. STIRWALT, M, A. and EVAI~, A. S . (1955) Serologic reactions in Sdtirtoaornu mma'soni infections. I . Cercaricidal, precipitation . agglutination and CHR phenomena . F.zpl Parasit . 4 , 123. ZLO'nCIN, E ., MntAntnA, F. and IJSSnûCY, S. (1972 a) Proteins toxic to mammah and insects in six scorpion venoma . Tazicon 10, 207. Zi.oTtcu~t, E., MIRANDA, F. and LtssrracY, S . (1972 b) A toxic factor to crustacean in the venom of the scorpion ~ mradahx Hector. Tosicon 1" , 211.
0041-0101/80/110I-0711 S02.ODi0
FACTORS) IN THE VENOM OF SCORPIONS TOXIC TO SCHISTOSOMA MANSONI (INTESTINAL BELHARZIA) CERCARIAE* M. F. EL-ASMAR,t N. SWELAM,t T. M. ABDEL AAL,$ I{H. GHONEIMt and S . S. HODHODt tDepartments of Biochemistry and $Parasitology, Faculty of Medicine, Ain Shams University, Cavo, Egypt (Acccpttd for publication 4 Merck 1980)
ZLOTKIN et al. (1972 a, b) identified substances in scorpion venom which are toxic to insects and crustaceans, and are different from the mammalian toxin . LAZAROVIA and ZLOTKIN (1979) reported the presence of toxins specifically active on arthropods and mammals from the venom of the scorpion Scorpio mourus . In this communication we demonstrate the presence of factors) which were toxic to Schistosoma mansoni cercariae (final free swimming larval stage of parasite). The Schistosoma mansoni cercariae used in this investigation were Egyptian in origin and have been maintained in our laboratory by serial passages through the snail Biompholaria alexandria and mouse (GHAREEB et al., 1975). Cercariae were collected just prior to their use from snails contained in dechlorinated tap water. Venom obtained from mature Leiurus quinquestriatus, Buthus minor, Buthus occitanus and Androctonus amoreuxi scorpions, by electrical stimulation of the telson as described by EL-ASMAR et al. (1972), was immediately freeze dried and kept in a dessicator over CaClp at room temperature . Chromatographic separation of Leiurus quinquestriatus venom was performed using Biorex 70, analytical grade cation exchanger (Biorad Laboratories, Richmond, California). The Biorex 70 was prepared for chromatography in a glass column 1 x 15 cm and elution of the venom (40 mg) was performed using linear gradient ammonium acetate buffer from 005 M, pH 5~8-0~5 M, pH 6~5 (250 ml of each at a flow rate of 35 ml/hr, 5 ml fraction). The active fraction was lyophilized, gel filtered on Sephadex G50column (1 x 54 cm), eluted with distilled water, (3~5 ml fractions), and lyophilized . Protein was determined in crude venoms and column eluates by absorbance measurement at 280 nm assuming that a 1 mg/ml solution of the venoms or their fractions has an average Al o = 1 ~0 when measured in a cell of 1 ~0 cm light path . For observation of cercaricidal and pericercarial envelope phenomena, slide preparations were made by mixing one drop of the tested venom solution or fractions, diluted to the proper concentration, with one drop of aged tap water containing approximately 20 cercariae, and a cover slip sealed in place (STIRWALT and EVANS, 1955). The preparations were kept at room temperature in a Petri dish with a moistened filter paper on the bottom . Examination of the microscope slides was performed using ordinary light after 24 hr . The 'Supported by grant from the National Academy of Science and Technology .
71?
Short ('ommunications
cercaricidal effect was demonstrated by the spastic behavior of the cercariae and the granular or globular precipitate surrounding the cercariae . The sequence of events in the formation of a pericercarial envelope about the larvae when observed under ordinary microscope was as follows; cercariae had orally secreted large amounts of sticky material, which included refractive granules of varying sizes. The interspace between the outer and inner limits of the cuticle seemed to thicken at the level of the widest body diameter, as though a fluid were collecting there or as if the cuticular material were swelling . The surface of the swollen area then appeared to become detached so that the cercariae were ensheathed in thin, tight, transparent envelopes which appeared to restrict the movement of the contained organism . The envelopes gradually hardened or 'set' and their elasticity was lost . The pericercarial envelope freed from the body began to loosen and balloon away until the cercaria wasencased in a wrinkled, loose-fitting structure similar in appearance to plastic cercarial molds. Crude venoms of Leiurus quinquestriates, Bathos minax, Buthus occitanes and Androctonus amoreuxi were active showing a positive pericercarial envelope test at a minimal concentration of 10 iu,g per slide. Partial purification of cercarial toxins was attempted by the elution of proteins of Leirus quinquestriatus venom from a Biorex 70 column as shown in Fg . 1 . Of the 12 fractions separated, fractions III, N and VI were active on Schistosoma mansoni cercariae. The minimal active concentrations were 8, 14 and 23 N"g per slide, respectively . Fraction VI, obtained from Biorex, when filtered on a Sephadex G 50 column gave a chromatographic pattern as shown in Fig. 2. Tubes collected and designated as subfraction A showed activity to cercariae at a minimal concentration of 9 Fcg per slide. The formation of a pericercarial envelope and secretion of sticky material as shown in Fig. 3 is considered as a simple test giving constant results indicating toxicity or an unfavourable environment for cercariae. 0~05M ammorim aoefate bufier pli 5~8-0~5M~~ pH 6~
0'9
0~7
w
I
0~5
0~5
0~4
0'4
0~3
03
02 0~1 0 0
0~2
rm
10
20
30
40
0' I 50
60
Tube Na (5 ml each)
70
BO
O 90
X 1S Cm) AND PlO. 1. CIßtOMATOGRAPHICPATTERN OFL. (j[ltnQYG4VlOrIL4 VENOM (40 mg) ON BIOREX iOCOLIJFIIY (1 ELIICED BY LINEARGRADIEN'rAMMONIUM ACETATE BUFI£R (0 " OS M,pH 5 " 8-0 " 5 M,pH 6 " 5) AT35 ml/hr .
Fraction 1 causes death to mouse whcn injected i .p . Fractions III, N and VI show cencaricidal activity .
Short Communications
F§G . 3 . CERCARIA SHOWING PERICERCARIAL ENVELOPE REACTION .
The arrow indicates pericercarial envelope .
713
Short ('ommunicati~ms
715
0~6
o~s o~a
.
~az _ oI 0
I 5
A
I 10
I 15
rti
~
.. .. .
I 20
C
I 25
I 30
I 35
`
I 40
45
Tube Nn ( 3~5 ml each ) FIG. ?. GEL FILTRATION PATTERN OF LYOPHILIZED FRACnON VI (FIG. 1 . ) ON SEPHADEX G 5l1( I X 54 Cnl ) AND
ELl7IED WITH DISI7LLED WATER. Tubes 0-13 were pooled and designated as subfraction A which shows cercaricidal activity .
EL,-ASMAR et al. (1977) reported the presence of anticholinesterase activities in the venom of the scorpion Buthus minax while MANSOUR (1979) found acetylcholinesterase in schistosomes and other parasites . We tested for the presence of anticholinesterase activity in the active fractions we obtained from Leiurus quinquestriatus, but none was found . This eliminates the possibility that the pericercarial envelope response of cercariae is due to the presence of an anticholinesterase activity . No proteolytic activity, using casein as a substrate, was detected in the crude venom or the active fractions . Preliminary experiments indicated that the anticercarial compounds) are nondialysable as a dialysed crude venom showed the same activity on cercariae . Acid hydrolysis of the active compound (subfraction A), followed by paper chromatography, has given us a preliminary indication of the amino acid composition of the compound . The high frequency of schistosomal infection in many countries justifies a nontraditional approach for seeking potential antischistosomal compounds .
REFERENCES F1.-A .SMAR, M. F., IBRAHIM, S . A . and RABm, F. (1972) Fractionation of scorpion (L.tiurus quinquestriaAv H and S) venom . Tosicon 10, 73 . EUAshtwR, M . F., ISMAIL, M ., GHONEBri, KH . and Q4MAN, O . H . (19?7) Scorpion (Budrusminax L. Koch) venom fractions with anticholineaterase activity . Toxionn 1S, 63 . GHAREEB, A. M., EL-GUINDI, S ., SALEH, A., EL-SHERIF, M. alld EL-ASMAR, M . F, (19~I5) Endocrine changes in hepatosplenic schistosotniasis (I-LS .S). An experimental study . Ain Shmns Med. !. Z6, 81 . LwzAROVtw, P . and Zt.oltcnv, E. (1979) Toms specifically active to arthropods and mammals from the venom of scorpion Scorpiomm~rus pabrtaaar (soorpionidae) . Tasicon 17, suppl. 1, 98 . MANSOUR, T . E . (1979) Chemotherapy of paraaitic worms: new biochemical strategies . Scialce 205, 462. STIRWALT, M, A. and EVAI~, A. S . (1955) Serologic reactions in Sdtirtoaornu mma'soni infections. I . Cercaricidal, precipitation . agglutination and CHR phenomena . F.zpl Parasit . 4 , 123. ZLO'nCIN, E ., MntAntnA, F. and IJSSnûCY, S. (1972 a) Proteins toxic to mammah and insects in six scorpion venoma . Tazicon 10, 207. Zi.oTtcu~t, E., MIRANDA, F. and LtssrracY, S . (1972 b) A toxic factor to crustacean in the venom of the scorpion ~ mradahx Hector. Tosicon 1" , 211.