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Effects of X-irradiation on the antigenic character of Agkistrodon piscivorus (cottonmouth moccasin) venom
Tosirnn, 1966, Vol. 3, PP. 30104. Pert
EFFECTS OF X-IRRADIATION ON THE ANTIGENIC CHARACTER OF AGKISTRODON PISCIVORUS (COTTONMOUTH MOCCASIN) VENOM HERSCHEL H . FLOWERS
U.S . Army Medical Research Laboratory, Fort Knox, Kentucky (Acceptedfirrpublicalian 19 November 1964)
Abstract--Cottonmouth moccasin venom with its necrotizing toxin inactivated 50 to 75 per cent by X-irradiation was studied to determine the alteration of its antigenic character. immune globulin of rabbits immunized with both normal and X-irradiated venom were compared. The globulin of rabbits immunizxd with X-irradiated venom showed little difference in its neutralizing titer from the globulin of rabbits immunized with normal venom. A group of animals immunized with normal venom suffered 20 per cent mortality, sloughing of tissue, and severe local reactions around the area of injection. The group of rabbits immunized with X-irradiated venom suffered no mortality or sloughing of tissue . Local reactions around the site of injection were less severe.
many serious problems encountered in individuals bitten by snakes of the family CrotaGdae, certainly one of the foremost is produced by the hemorrhagic and locally necrotizing properties of the venom. These properties are of such importance in the overall poisoning that correlations have been made between the degree of hemorrhagic activity and the lethal effect . Many investigators have searched for a method of treatment which will minimize the destruction of tissue that commonly accompanies the bite of crotalids. The problem, however, is not confined to the treatment of snakebite victims . It has also created diffi culties for the researcher who is endeavoring to produce 1t more effective antivenin[1] . It has been suggested that the local hemorrhagic and necrotizing effects exhibited following injection of crotalid venom are mainly due to the action of the enzymes[2]. SLOTTA[3] regards this damage as being attributable to the joint action of proteases and phosphatases . GOUC'HER and FLOWERS[4] have demonstrated that when these enzymes are destroyed or their action inhibited, lesions from intradermal injections of venom are greatly reduced . The effects of X-irradiation of cottonmouth moccasin venom with respect to the alterations of local effect on tissues, activity of certain enzyme systems, and lethality have been discussed in a previous publication[5] . X-irradiation was found to decrease local hemorr hage and necrosis 50 to 75 per cent. Lethality was reduced to where it necessitated five times the amount of X-irradiated venom to provide the mouse i .D ; . These toxic principles were effected with little alteration of the immunodiffusion and immunoelectrophoretic pattern of antigens . Material presented in this paper deals specifically with the quantitative alteration of important venom antigens. OF THE
301
IiERSCHEL
302
H.
FLOWERS
MATERIALS AND METHODS Venom was obtained from a colony of two hundred cottonmouth moccasins maintained at the U.S. Army Medical Research Laboratory . Immediately after extraction, venom was lyophilized and stored at 4°. A composite pool of lyophilized venom was then prepared from the quantity collected during a four-month period . Venom used in this study was taken from this composite pool. The X-ray machine employed was a North American Phillips 50 kV unit. No filter was used in order that maximum absorption of soft rays could be effected. The venom solution was exposed in 50 ml aliquots in a shallow glass dish. The depth of solution was maintained at 0'9 cm. A magnetic stirrer was employed to circulate the solution and provide even exposure . The exposure time was 120 sec. Ferrous sulfate dosimetry was used in determining the total absorbed dose. For the 120-sec exposure the total absorbed dose was calculated to be 18, 375 rad . :ï- 263 rad . [5J. Two groups of 10 New Zealand rabbits, weighing 3~ kg, were selected for the immunization procedure . X-irradiated venom was used in one group, non-irradiated venom in the other. A total of eight injections of venom, increasing in amount, were given each animal as suggested by RUSSELL.[6] (Table 1). The venom for injection was dissolved in normal TABLE 1 .
IMMUNIZATION SCHEDULE FOR PRODUCTION OF ANTISF7tUM IN RABHTTS
Vemom/kg (mg)
Day 0 4 8 12 16 21 28 35 45
0~2 0~3 0~6 1-0 2-0 3~0 4~0 4~5
Collect blood
saline and in a concentration of 10 mg per ml and added to an equal quantity of 4 per cent sodium alginate adjuvant.* The injection site was the subcutaneous region of the shoulder. Ten days following the final injection the animals were exsanguinated and the serum collected. The globulin fraction was separated by ammonium sulfate precipitation and then lyophilized . For testing purposes it was reconstituted in normal saline in a concentration of 100 mg per ml . The presence and titer ~f antibodies were determined by three methods : (1) Immunodiffusion (2) Neutralization of the local reactivity of venom (3) In vitro neutralization ofA. piscivorous venom by mouse LDS O . Immunodiffusion was accomplished by placing normal cottonmouth venom in a 10 mg per ml dilution in a well cut in petri dishes containing 15 ml of 1 per cent washed nutrient agar. The globulin from rabbits immunized with both the normal and X-irradiated venom * Algivant, Consolidated Laboratories, Chicago Heights, Illinois .
FIG. I . COMPARATIVE PRECIPITATION BANDS FORMED IN AGAR BY ANTLSERUM PRODUCED IN RABBITS INJECTED WITH X-IRRADIATED VENOM (XVG) AND BY UNTREATED VENOM (NVG) AGAINST UNTREATED VENOM (NV) .
Tox. f.p . 302
F1G . T. COMPARATIVE PRECIPITATION BANDS FORMED IN AGAR BY ANTISERUM PRODUCED IN RABBIT'S INJEC'T'ED WITH X-IRRADIATED VENOM (XVG) AND BY UNTREATED VENOM (NVG) AGAINST UNTREATED VENOM (NV).
FICi .3 . NEUTRALIZATION OF LOCAL REACTIVI'T'Y OF VENOM BY ANTISERUM PRODUCED BY INJECTIONS OF X-IRRADIATED (XVG) VENOM AND UNTREATED VENOM (NVG) AS COMPAREp WJTH A CONTROL OF UNTREATED VENOM (NV).
Effects of X-irradiation on the Mtigenic Character of A,gkis~raian pisci~»rus Venom
303
was deposited in wells around the venom. The concentration of globulin was 100 mg per ml. The petri dishes were then incubated for 72 hr at 22'. Zones of precipitation of diffused proteins were usually seen after 24 hr however, precipitation was not considered cc,mplete until after 72 hr. The results were then photographically recorded. Estimation of the neutralization of the local reactivity of the venom was accomplished by a test in the skin of rabbits[7J. Venom solutions of different concentration were added to a standard dilution of globulin and incubated at 37'' for 30 min ; 0'05 ml of each sample was then injected intradermally in rabbits. The rabbits were sacrificed in 24 hr, the skin removed, and the results recorded from observation of the undersurfacc . Neutralization of the mouse 'tu so was accomplished in the manner described by CitN(iIRtCH and HOFIENADEL[K]. The titer of the antibody from the rabbits immunized with X-irradiated venom was compared to that of the rabbits immunized with normal venom . R t :S U L .TS
fhe imntuuization procedure resulted in 20 per cent deaths in the group of rabbits immunized with normal venom . Sloughing and tissue reaction were common in this group. The group of rabbits immunized wish X-irradiated venom evidenced no deaths nor sloughing . Tissue reaction was greatly lessened, but did occur in all animals . Immunodiffusion indicated little or no visible difference in the precipitating antibodies in the globulin of the two groups of rabbits (Figs . I and 2). Precipitation bands were identical in clarity and joined smoothly at the corners. The globulin from the rabbits immunized with normal venom neutralized 20 mouse i .t~ ;,o., of cottonmouth moccasin venom . The globulin of rabbits immunized with Xirradiated venom neutralized 16 mouse t .t) ; o~, . Comparative neutralization of the Iexal toxicity of cottonmouth venom by intradermal titration in the skin of rabbits demonstrated little visible difference. Une hundred mg ~f the globulin from both groups of rabbits cf%ctively neutralized the local etTects ~f I mgc,f cottonmouth moccasin venom (Fig. 3). U[SCUSSIUN
The value of X-irradiation in the production of tuxoids has been demonstrated[9] . However, its use in the attenuation of certain activities of venoms has not been previously exploited . Since venom is a combination of complex substances, chiefly proteins, it would not be surprising if it were inactivated by very high doses of X-irradiation . Changes in its antigenic character would also be expected . Fortunately, X-irradiated venom, which has been reduced in toxicity by 50 to 75 per cent looses very little of its antigenic properties . ~fhe importance of such detoxification is demonstrated in the immunization of the animals . A group of rabbits immunized with normal venom suffered 20 per cent mortality as well :+s severe local reactions in sloughing . 'The group of animals immunized with X-irradiated venom suffered no mortality and nu tissue sloughs . Local reactions were lessened :+l,preciably . 'fhc antibcxly titer c(rmwmtratcd by thr animals immunized with X-irradiated ~en~,m was clTectively high . In comparison with t.irc titer uC globulin of rabbits immunized will+ normal venom there was little difference except in the neutralization of the mouse Lu~ o . In this, it demonstrated :+ tiler c,f Rtl per cent c,f that ~f the normal 4~enc,m immuni~xd
304
HEItSCHEL H. FLOWERS
rabbits. From preliminary experience with chemical agents it appears possible that X-irradiation plus chemical means of detoxification may remove much of the local toxic effects while retaining effective antigenicity .
[1]
REFERENCES
CRIIEY, B. R., Development of a multivalent antivenin for thefamily Crotalidae . Venorns, pp. (BUCKLEY, E. and Poeaes, N., Eds) A.A .A.S., Publication No . 44, Washington, D.C., 1956.
373-380
[2] . 7c, r ~ E. A. Enzymes of snake venoms and their biological significance, Adsances in Enrynrology (Noxn, F. F., Ed .) Interscience Publishers, New York, 8, 459, 1948. [3] Swrrn, C. H. Chemistry and biochemistry of snake venoms. Progress in the Chernistr.r of Organic Natural Prmlucts, 12, 406, 1955. [4] Goucf~e, C. R. and Fcowexs, H. H. The chemical modification of necrogenic and proteolytic activities of Crotalidae venom by EDTA to produce a venom toxoid . USAMRL Report No . 599, 1964 . [5] Hawses, H. H. The effects of X-irradiation on the biological activity of cottonmouth moccasin (Agkistrodon pisclvorous) venom. Toxicon, 1, 131, 1963 . [6] Ru CCF~i - F. E. Uso of Crotalus monovalent antivenin from rabbit serum. Grrr. Cher. Res. 3, 438, 1961 . [7] Korroo, H., Karma, S., hcszewn, H ., MURATA, R., and OHSAKA, A. Studies on the quantitative methods for determination of hemorrhagic activity of Habu snake venom. Jap. J. nred. Sci. Biol. 13, 43, 1960. [8] (itxcxtcx, W. C. and HO~rranE~, J. C. Standardization of polyvalent antivenin, pp . 381-385, Venoms, (Bucxr,~r, E. and PORGES, N., Eds.) A.A .A.S ., Publication No. 44, Washington, D.C ., 1956 . [9] taONCHARENKO, I. M. The use of ionizing radiations for the sterilization of tetanus toxoid-II . Zh. Mikrobiol. Épidemiol. Inrmunobiol. 32, 97, 1961 .
EFFECTS OF X-IRRADIATION ON THE ANTIGENIC CHARACTER OF AGKISTRODON PISCIVORUS (COTTONMOUTH MOCCASIN) VENOM HERSCHEL H . FLOWERS
U.S . Army Medical Research Laboratory, Fort Knox, Kentucky (Acceptedfirrpublicalian 19 November 1964)
Abstract--Cottonmouth moccasin venom with its necrotizing toxin inactivated 50 to 75 per cent by X-irradiation was studied to determine the alteration of its antigenic character. immune globulin of rabbits immunized with both normal and X-irradiated venom were compared. The globulin of rabbits immunizxd with X-irradiated venom showed little difference in its neutralizing titer from the globulin of rabbits immunized with normal venom. A group of animals immunized with normal venom suffered 20 per cent mortality, sloughing of tissue, and severe local reactions around the area of injection. The group of rabbits immunized with X-irradiated venom suffered no mortality or sloughing of tissue . Local reactions around the site of injection were less severe.
many serious problems encountered in individuals bitten by snakes of the family CrotaGdae, certainly one of the foremost is produced by the hemorrhagic and locally necrotizing properties of the venom. These properties are of such importance in the overall poisoning that correlations have been made between the degree of hemorrhagic activity and the lethal effect . Many investigators have searched for a method of treatment which will minimize the destruction of tissue that commonly accompanies the bite of crotalids. The problem, however, is not confined to the treatment of snakebite victims . It has also created diffi culties for the researcher who is endeavoring to produce 1t more effective antivenin[1] . It has been suggested that the local hemorrhagic and necrotizing effects exhibited following injection of crotalid venom are mainly due to the action of the enzymes[2]. SLOTTA[3] regards this damage as being attributable to the joint action of proteases and phosphatases . GOUC'HER and FLOWERS[4] have demonstrated that when these enzymes are destroyed or their action inhibited, lesions from intradermal injections of venom are greatly reduced . The effects of X-irradiation of cottonmouth moccasin venom with respect to the alterations of local effect on tissues, activity of certain enzyme systems, and lethality have been discussed in a previous publication[5] . X-irradiation was found to decrease local hemorr hage and necrosis 50 to 75 per cent. Lethality was reduced to where it necessitated five times the amount of X-irradiated venom to provide the mouse i .D ; . These toxic principles were effected with little alteration of the immunodiffusion and immunoelectrophoretic pattern of antigens . Material presented in this paper deals specifically with the quantitative alteration of important venom antigens. OF THE
301
IiERSCHEL
302
H.
FLOWERS
MATERIALS AND METHODS Venom was obtained from a colony of two hundred cottonmouth moccasins maintained at the U.S. Army Medical Research Laboratory . Immediately after extraction, venom was lyophilized and stored at 4°. A composite pool of lyophilized venom was then prepared from the quantity collected during a four-month period . Venom used in this study was taken from this composite pool. The X-ray machine employed was a North American Phillips 50 kV unit. No filter was used in order that maximum absorption of soft rays could be effected. The venom solution was exposed in 50 ml aliquots in a shallow glass dish. The depth of solution was maintained at 0'9 cm. A magnetic stirrer was employed to circulate the solution and provide even exposure . The exposure time was 120 sec. Ferrous sulfate dosimetry was used in determining the total absorbed dose. For the 120-sec exposure the total absorbed dose was calculated to be 18, 375 rad . :ï- 263 rad . [5J. Two groups of 10 New Zealand rabbits, weighing 3~ kg, were selected for the immunization procedure . X-irradiated venom was used in one group, non-irradiated venom in the other. A total of eight injections of venom, increasing in amount, were given each animal as suggested by RUSSELL.[6] (Table 1). The venom for injection was dissolved in normal TABLE 1 .
IMMUNIZATION SCHEDULE FOR PRODUCTION OF ANTISF7tUM IN RABHTTS
Vemom/kg (mg)
Day 0 4 8 12 16 21 28 35 45
0~2 0~3 0~6 1-0 2-0 3~0 4~0 4~5
Collect blood
saline and in a concentration of 10 mg per ml and added to an equal quantity of 4 per cent sodium alginate adjuvant.* The injection site was the subcutaneous region of the shoulder. Ten days following the final injection the animals were exsanguinated and the serum collected. The globulin fraction was separated by ammonium sulfate precipitation and then lyophilized . For testing purposes it was reconstituted in normal saline in a concentration of 100 mg per ml . The presence and titer ~f antibodies were determined by three methods : (1) Immunodiffusion (2) Neutralization of the local reactivity of venom (3) In vitro neutralization ofA. piscivorous venom by mouse LDS O . Immunodiffusion was accomplished by placing normal cottonmouth venom in a 10 mg per ml dilution in a well cut in petri dishes containing 15 ml of 1 per cent washed nutrient agar. The globulin from rabbits immunized with both the normal and X-irradiated venom * Algivant, Consolidated Laboratories, Chicago Heights, Illinois .
FIG. I . COMPARATIVE PRECIPITATION BANDS FORMED IN AGAR BY ANTLSERUM PRODUCED IN RABBITS INJECTED WITH X-IRRADIATED VENOM (XVG) AND BY UNTREATED VENOM (NVG) AGAINST UNTREATED VENOM (NV) .
Tox. f.p . 302
F1G . T. COMPARATIVE PRECIPITATION BANDS FORMED IN AGAR BY ANTISERUM PRODUCED IN RABBIT'S INJEC'T'ED WITH X-IRRADIATED VENOM (XVG) AND BY UNTREATED VENOM (NVG) AGAINST UNTREATED VENOM (NV).
FICi .3 . NEUTRALIZATION OF LOCAL REACTIVI'T'Y OF VENOM BY ANTISERUM PRODUCED BY INJECTIONS OF X-IRRADIATED (XVG) VENOM AND UNTREATED VENOM (NVG) AS COMPAREp WJTH A CONTROL OF UNTREATED VENOM (NV).
Effects of X-irradiation on the Mtigenic Character of A,gkis~raian pisci~»rus Venom
303
was deposited in wells around the venom. The concentration of globulin was 100 mg per ml. The petri dishes were then incubated for 72 hr at 22'. Zones of precipitation of diffused proteins were usually seen after 24 hr however, precipitation was not considered cc,mplete until after 72 hr. The results were then photographically recorded. Estimation of the neutralization of the local reactivity of the venom was accomplished by a test in the skin of rabbits[7J. Venom solutions of different concentration were added to a standard dilution of globulin and incubated at 37'' for 30 min ; 0'05 ml of each sample was then injected intradermally in rabbits. The rabbits were sacrificed in 24 hr, the skin removed, and the results recorded from observation of the undersurfacc . Neutralization of the mouse 'tu so was accomplished in the manner described by CitN(iIRtCH and HOFIENADEL[K]. The titer of the antibody from the rabbits immunized with X-irradiated venom was compared to that of the rabbits immunized with normal venom . R t :S U L .TS
fhe imntuuization procedure resulted in 20 per cent deaths in the group of rabbits immunized with normal venom . Sloughing and tissue reaction were common in this group. The group of rabbits immunized wish X-irradiated venom evidenced no deaths nor sloughing . Tissue reaction was greatly lessened, but did occur in all animals . Immunodiffusion indicated little or no visible difference in the precipitating antibodies in the globulin of the two groups of rabbits (Figs . I and 2). Precipitation bands were identical in clarity and joined smoothly at the corners. The globulin from the rabbits immunized with normal venom neutralized 20 mouse i .t~ ;,o., of cottonmouth moccasin venom . The globulin of rabbits immunized with Xirradiated venom neutralized 16 mouse t .t) ; o~, . Comparative neutralization of the Iexal toxicity of cottonmouth venom by intradermal titration in the skin of rabbits demonstrated little visible difference. Une hundred mg ~f the globulin from both groups of rabbits cf%ctively neutralized the local etTects ~f I mgc,f cottonmouth moccasin venom (Fig. 3). U[SCUSSIUN
The value of X-irradiation in the production of tuxoids has been demonstrated[9] . However, its use in the attenuation of certain activities of venoms has not been previously exploited . Since venom is a combination of complex substances, chiefly proteins, it would not be surprising if it were inactivated by very high doses of X-irradiation . Changes in its antigenic character would also be expected . Fortunately, X-irradiated venom, which has been reduced in toxicity by 50 to 75 per cent looses very little of its antigenic properties . ~fhe importance of such detoxification is demonstrated in the immunization of the animals . A group of rabbits immunized with normal venom suffered 20 per cent mortality as well :+s severe local reactions in sloughing . 'The group of animals immunized with X-irradiated venom suffered no mortality and nu tissue sloughs . Local reactions were lessened :+l,preciably . 'fhc antibcxly titer c(rmwmtratcd by thr animals immunized with X-irradiated ~en~,m was clTectively high . In comparison with t.irc titer uC globulin of rabbits immunized will+ normal venom there was little difference except in the neutralization of the mouse Lu~ o . In this, it demonstrated :+ tiler c,f Rtl per cent c,f that ~f the normal 4~enc,m immuni~xd
304
HEItSCHEL H. FLOWERS
rabbits. From preliminary experience with chemical agents it appears possible that X-irradiation plus chemical means of detoxification may remove much of the local toxic effects while retaining effective antigenicity .
[1]
REFERENCES
CRIIEY, B. R., Development of a multivalent antivenin for thefamily Crotalidae . Venorns, pp. (BUCKLEY, E. and Poeaes, N., Eds) A.A .A.S., Publication No . 44, Washington, D.C., 1956.
373-380
[2] . 7c, r ~ E. A. Enzymes of snake venoms and their biological significance, Adsances in Enrynrology (Noxn, F. F., Ed .) Interscience Publishers, New York, 8, 459, 1948. [3] Swrrn, C. H. Chemistry and biochemistry of snake venoms. Progress in the Chernistr.r of Organic Natural Prmlucts, 12, 406, 1955. [4] Goucf~e, C. R. and Fcowexs, H. H. The chemical modification of necrogenic and proteolytic activities of Crotalidae venom by EDTA to produce a venom toxoid . USAMRL Report No . 599, 1964 . [5] Hawses, H. H. The effects of X-irradiation on the biological activity of cottonmouth moccasin (Agkistrodon pisclvorous) venom. Toxicon, 1, 131, 1963 . [6] Ru CCF~i - F. E. Uso of Crotalus monovalent antivenin from rabbit serum. Grrr. Cher. Res. 3, 438, 1961 . [7] Korroo, H., Karma, S., hcszewn, H ., MURATA, R., and OHSAKA, A. Studies on the quantitative methods for determination of hemorrhagic activity of Habu snake venom. Jap. J. nred. Sci. Biol. 13, 43, 1960. [8] (itxcxtcx, W. C. and HO~rranE~, J. C. Standardization of polyvalent antivenin, pp . 381-385, Venoms, (Bucxr,~r, E. and PORGES, N., Eds.) A.A .A.S ., Publication No. 44, Washington, D.C ., 1956 . [9] taONCHARENKO, I. M. The use of ionizing radiations for the sterilization of tetanus toxoid-II . Zh. Mikrobiol. Épidemiol. Inrmunobiol. 32, 97, 1961 .