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In vivo ability of antimyotoxin a serum plus polyvalent (Crotalidae) antivenom to neutralize prairie rattlesnake (Crotalus viridis viridis) venom
7~ va . 24 . No . 2 . pp . 197-200,19@6 . Prlntea in 0~ ainlu.
0041-010118653 .W+ .00 ® 1986 Pe~ Pres Ltd.
SHORT COMMUNICATIONS IN VIVO ABILITY OF ANTIMYOTOXIN a SERUM PLUS POLYVALENT (CROTALIDAE) ANTIVENOM TO NEUTRALIZE PRAIRIE RATTLESNAKE (CROTALUS VIRIDIS VIRIDIS) VENOM V.
CHARLOTTE L. OwNBY,' TERRY R. COLBERO' and GEORGE ODELL2 Departments of 'Physiological Sciences and 'Biochemistry, Oklahoma State University, Stillwater, OK 74078, U .S .A . (Accepted tor publication 23 1u(y 1985)
C . L . Ownax, T . R. CoLmmG and O. V . OmLL . In vivo ability of antimyotoxin a serum plus polyvalent (Crotalidae) antivenom to neutralize prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon 24, 197 - 200, 1986.-A mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenom was injected i .v . in mice either 5 min before or 5 min, 30 min, 1 hr or 3 hr after i .m. injection of venom . Neutralization of the local myotoxicity of a sublethal dose (1 .5 Mg/g) of C. v . viridis venom occurred if the antisera were injected 5 min before or 5 or 30 min after venom, but not if injected 1 or 3 hr after the venom. Hemorrhage was neutralized when the mixture was injected either 5 min before or 5 min after injection of venom, but not when injected 30 min after injection of venom . Previous results showed that the mixture of antisera neutralized the same amount of venom (1 .5 yg/g) when mixed with the venom prior to injection. Thus it is not possible with these two antisera to neutralize myonecrosis if the time interval between injections is greater than 30 min .
consequence of rattlesnake venom poisoning in the United States is local tissue damage, such as hemorrhage and myonecrosis. Such local tissue damage may lead to loss of function or deformity of an extremity (CLEMENT and PIETRUSKO, 1979) . Although polyvalent (Crotalidae) antívenom has been effective in neutralizing the lethal activity of rattlesnake venoms, it seems to be less effective in neutralizing local tissue damage unless given immediately after venom (RUSSELL et al., 1973 ; OwNBY et al., 1983) . We have been investigating methods of improving antiserum treatment of local myonecrosis induced by Crotalus viridis viridis venom. Recently we demonstrated that a mixture of polyvalent (Crotalidae) antivenom and antiserum to myotoxin a from C. v. viridis venom was more effective in neutralizing the myotoxicity of C. v. viridis venom in mice than either antiserum alone (OwNBY et al., 1985) . The purpose of the present study was to determine the ability of the mixture of antisera to neutralize local myonecrosis and hemorrhage induced by C. v. viridis venom when antisera and venom were injected separately. Female white mice (CD-1, Charles River) weighing 25 - 28 g (myonecrosis) or 30 - 35 g (hemorrhage) were used in all experiments. Crude prairie rattlesnake (C. v. viridis) venom was purchased in lyophilized form from Miami Serpentarium Laboratories, Miami, Fl. THE MOST COMMON
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TIME [mini FIO .
1 . EFFECT OF A MUMIRE OF POLYVALENT (CROTALIDAE) ANIIVENOM AND ANnMYOTOXW a SERUM ON THE AMOUNT OF MYONECROBIS INDUCED BY CJnrahO Vb1d& V1rJ& VENOLI.
Time (min) indicates that antisem was injected before (-) or after (+) venom injection . Values are the mean t S.E . (n=4) . AS andsera; PSS 0.85% NaCl. 'PM03 compared to crude venom followed by Injection of physiologic saline (0.85Sí NaCI).
The same lot (CI13SZ) of venom was used for all experiments. Antimyotoxin serum was prepared in rabbits as previously described (OWNBY et al., 1979). For these experiments a pool of three different bleedings from one rabbit was used. Wyeth's lyophilized polyvalent (Crotalidae) antivenin (lot No. 17601, expiration date 21 June 1987, Wyeth Inc., Marietta, PA) produced in horses and subsequently called polyvalent antivenom was kept at 4°C and reconstituted to 10 ml with distilled water before use. Crude venom was dissolved in 0.85% NaCl to give a solution with a concentration of 0 .6 mg/ml . Upon injection of 0.05 ml/20 g body weight of mouse, this provided a dose of 1 .5 gg/g (one half an LD ). This dose was selected from previous in vitro neutralization experiments (OwNBY et al., 1985). The antiserum mixture, containing equal volumes of polyvalent antivenom and the pooled antimyotoxin a serum was injected i.v. Venom was always injected i.m. into the dorso-lateral aspect of the right thigh, as reported previously (OwNBY et al., 1976). In one experiment the antiserum mixture was injected 5 min prior to the injection of venom. In other experiments venom was injected first, followed by the injection of the antiserum mixture either 5 min, 30 min, 1 or 3 hr after venom. For each experiment three types of controls were used. As a control for the amount ofmyonecrosis or hemorrhage induced by venom alone, 0.85% N$CI was injected before or after venom, rather than the antiserum. mixture. Sodium chloride (0.85% i.m .) followed by antiserum mixture (Lv.) and 0.85% NACI (i.m.) followed by 0.85% NaCl (i.v.) served as controls for effects of j.v. injection of antiserum and tissue processing artifacts, respectively, and to obtain a control level of hemoglobin . Myonecrosis was measured by obtaining a myonecrosis index according to the method of OWNBY et al. (1983) . Hemorrhage after the i.m. injection was determined by the corrected mean hemoglobin method, as previously
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described (OwNBY et al., 1984b) . Since only one dose was used with only one treatment and a control, the Student's t-test was used to determine the significance of the difference between the two means. A significance level of PG0.05 was used. As can be seen from Fig. 1, there was a significant neturalization of myonecrosis when the antiserum was injected 5 min before venom or 5 or 30 min after venom injection. The neutralization was not significant when the time between venom injection and antisera injection was 1 or 3 hr. Figure 2 shows the results from the neutralization of hemorrhage experiments . There was significant neutralization when the antiserum mixture was injected either 5 min before or 5 min after venom injection, but not when injected 30 min after injection of venom. Our results demonstrate that the mixture of antisera can neutralize myonecrosis even when injected 30 min after venom injection. The amount of venom neutralized was twice that neutralized by antimyotoxin a serum alone in the same type of test (OviNBY et al., 1984a) . This indicates that there is some contribution by hemorrhaic toxins to the total myonecrosis of crude venom, since polyvalent antivenom can neutralize the hemorrhaggc activity of substantial (up to 24 FAg/g) amounts of crude venom (OWNBY et al., 1984b) . The results on the neutralization of hemorrhage also support this idea, since there was significant neutralization of hemorrhage when the antiserum mixture was injected 5 min after venom injection. Yet our results also point out that, at least with C. v. viridisvenom, components other than those that induce hemorrhage are responsible for a large part of the myonecrosis induced by the crude venom, i .e. myotoxin a. This is supported by the
z
á 0 W F V W V
TI IE I"dnl Fio. 2. Bnacr of mctvas oar PoLYVAt.HNr (CamAuam) Axnamom AND emuaxomxnr a smeum oar THE AMOUNT OF t03MOAAHACE INDUCED BY CnokAff virif VhIdis VENOM. Time (min) indicates that antisera was injected before (-) or after (+) venom injection . Values are the man t S.B. (n-4, unless otherwise indicated in parentheses). AS andsere ; PSS 0 .85% MCI . "PC0.05 compared to crude venom followed by injection of physiologic saline (0.85Vs NaCI).
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results of the neutralization of myonecrosis experiment in which neutralization was significant at 30 min after venom injection. However, the major problem of in vivo neutralization is still the lack of neutralization of hemorrhage or myonecrosis when the interval of time between venom and antiserum injection is greater than 30 min. So far there have been no reports of significant neutralization of local tissue damage induced by rattlesnake venom at longer time intervals. RUSSELL et al. (1973) reported prevention of local necrosis induced by C. v. helleri venom if antivenin (polyvalent Crotalidae) was injected (i .v.) simultaneously, some local necrosis if antivenin injection was delayed 10 min, and severe local damage if the delay was 20 or 30 min. One factor contributing to our inability to solve this problem is the rapidity of the hemorrhaggc action of these venoms . Both C. v. viridis and C. atroxvenoms induced local hemorrhage within 2-5 min after either i.m . or s.c. injection (unpublished results). Thus damage to blood vessel walls occurs long before antiserum can be injected. Some myotoxic components are extremely rapid in their action on muscle cells. For example, delta lesions and clumped myofibrils indicating necrotic muscle cells can be observed within 15 min after the i.m . injection of either C. v. viridis or C. atrox venom (Ownby, unpublished results). Acknowledgements-The authors thank VINITA PILL and TEtmA KENNmwER for typing the manuscript and MARY BooER for critically reading it . This work was supported by Public Health Service Grant 5 ROI AI16623-06 from the National Institute of Allergy and Infectious Diseases . C.L .O . is the recipient of a Research Career Development Award (KO4 AI00474) from this Institute. REFERENCES CLEMBrrr, J. F. and Piumuseo, R. O. (1979) Pit viper snakebite in the United States . J. Fancily Prwtice 6, 269. OwNBY, C. L., CAm aON, D. and Tu, A. T. (1976) Isolation of myotoxic component from rattlesnake venom: electron microscopic analysis of muscle damage. Am. J. Path. 85, 149. OwNBY, C. L., WooDs, W. M. and ODELL, 0. V. (1979) Antiserum to myotoxin from prairie rattlesnake (Crotales viridis virid1s) venom. Taxiton 17, 373. OWNBY, C. L., ODELL, O. V., WooDs, W. M. and CoL mRa, T. R. (1983) Ability of antiserum to myotoxin a from prairie rattlesnake (Crotales vk0s viridis) venom to neutralize local myotoodcrty and lethal effects of myotadn a and homologous crude venom. Toxicon 21, 35 . OwNHY, C. L., CoLBERo, T. R., CLAYPooL, P. L. and ODE T , O. V. (1984a) In vivo test of the ability of antiserum to myotoxin a from prairie rattlesnake (Crotahis vbidis vhidis) venom to neutralize local myonecrosis induced by myotoxin a and homologous crude venom. Toxicon 22, 99. OwNHY, C. L., Cot.smta, T. R. and ODELL, 0. V. (1984b) A new method for quantitating hemorrhage induced by rattlesnake venoms : ability of polyvalent antivenm to neutralize hemorrhagic activity. Taxkon 22, 227. Owxay, C. L., CoLSmta, T. R. and ODELL, 0. V. (1985) Ability of a mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenin to neutralize myonecrosis, hemorrhage and lethality induced by prairie rattlesnake (Crotales viridb viridls) venom. Toxiron 23, 317. RussELL, F. E., Rtmc, N. and Oowz uaz,'H. (1973) Effwdvenesi óf antivenin (Crotalidáe) polyvalent following injection of Crotahn venom. Tax*on 11, 461.
0041-010118653 .W+ .00 ® 1986 Pe~ Pres Ltd.
SHORT COMMUNICATIONS IN VIVO ABILITY OF ANTIMYOTOXIN a SERUM PLUS POLYVALENT (CROTALIDAE) ANTIVENOM TO NEUTRALIZE PRAIRIE RATTLESNAKE (CROTALUS VIRIDIS VIRIDIS) VENOM V.
CHARLOTTE L. OwNBY,' TERRY R. COLBERO' and GEORGE ODELL2 Departments of 'Physiological Sciences and 'Biochemistry, Oklahoma State University, Stillwater, OK 74078, U .S .A . (Accepted tor publication 23 1u(y 1985)
C . L . Ownax, T . R. CoLmmG and O. V . OmLL . In vivo ability of antimyotoxin a serum plus polyvalent (Crotalidae) antivenom to neutralize prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon 24, 197 - 200, 1986.-A mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenom was injected i .v . in mice either 5 min before or 5 min, 30 min, 1 hr or 3 hr after i .m. injection of venom . Neutralization of the local myotoxicity of a sublethal dose (1 .5 Mg/g) of C. v . viridis venom occurred if the antisera were injected 5 min before or 5 or 30 min after venom, but not if injected 1 or 3 hr after the venom. Hemorrhage was neutralized when the mixture was injected either 5 min before or 5 min after injection of venom, but not when injected 30 min after injection of venom . Previous results showed that the mixture of antisera neutralized the same amount of venom (1 .5 yg/g) when mixed with the venom prior to injection. Thus it is not possible with these two antisera to neutralize myonecrosis if the time interval between injections is greater than 30 min .
consequence of rattlesnake venom poisoning in the United States is local tissue damage, such as hemorrhage and myonecrosis. Such local tissue damage may lead to loss of function or deformity of an extremity (CLEMENT and PIETRUSKO, 1979) . Although polyvalent (Crotalidae) antívenom has been effective in neutralizing the lethal activity of rattlesnake venoms, it seems to be less effective in neutralizing local tissue damage unless given immediately after venom (RUSSELL et al., 1973 ; OwNBY et al., 1983) . We have been investigating methods of improving antiserum treatment of local myonecrosis induced by Crotalus viridis viridis venom. Recently we demonstrated that a mixture of polyvalent (Crotalidae) antivenom and antiserum to myotoxin a from C. v. viridis venom was more effective in neutralizing the myotoxicity of C. v. viridis venom in mice than either antiserum alone (OwNBY et al., 1985) . The purpose of the present study was to determine the ability of the mixture of antisera to neutralize local myonecrosis and hemorrhage induced by C. v. viridis venom when antisera and venom were injected separately. Female white mice (CD-1, Charles River) weighing 25 - 28 g (myonecrosis) or 30 - 35 g (hemorrhage) were used in all experiments. Crude prairie rattlesnake (C. v. viridis) venom was purchased in lyophilized form from Miami Serpentarium Laboratories, Miami, Fl. THE MOST COMMON
197
198
Short Communications
TIME [mini FIO .
1 . EFFECT OF A MUMIRE OF POLYVALENT (CROTALIDAE) ANIIVENOM AND ANnMYOTOXW a SERUM ON THE AMOUNT OF MYONECROBIS INDUCED BY CJnrahO Vb1d& V1rJ& VENOLI.
Time (min) indicates that antisem was injected before (-) or after (+) venom injection . Values are the mean t S.E . (n=4) . AS andsera; PSS 0.85% NaCl. 'PM03 compared to crude venom followed by Injection of physiologic saline (0.85Sí NaCI).
The same lot (CI13SZ) of venom was used for all experiments. Antimyotoxin serum was prepared in rabbits as previously described (OWNBY et al., 1979). For these experiments a pool of three different bleedings from one rabbit was used. Wyeth's lyophilized polyvalent (Crotalidae) antivenin (lot No. 17601, expiration date 21 June 1987, Wyeth Inc., Marietta, PA) produced in horses and subsequently called polyvalent antivenom was kept at 4°C and reconstituted to 10 ml with distilled water before use. Crude venom was dissolved in 0.85% NaCl to give a solution with a concentration of 0 .6 mg/ml . Upon injection of 0.05 ml/20 g body weight of mouse, this provided a dose of 1 .5 gg/g (one half an LD ). This dose was selected from previous in vitro neutralization experiments (OwNBY et al., 1985). The antiserum mixture, containing equal volumes of polyvalent antivenom and the pooled antimyotoxin a serum was injected i.v. Venom was always injected i.m. into the dorso-lateral aspect of the right thigh, as reported previously (OwNBY et al., 1976). In one experiment the antiserum mixture was injected 5 min prior to the injection of venom. In other experiments venom was injected first, followed by the injection of the antiserum mixture either 5 min, 30 min, 1 or 3 hr after venom. For each experiment three types of controls were used. As a control for the amount ofmyonecrosis or hemorrhage induced by venom alone, 0.85% N$CI was injected before or after venom, rather than the antiserum. mixture. Sodium chloride (0.85% i.m .) followed by antiserum mixture (Lv.) and 0.85% NACI (i.m.) followed by 0.85% NaCl (i.v.) served as controls for effects of j.v. injection of antiserum and tissue processing artifacts, respectively, and to obtain a control level of hemoglobin . Myonecrosis was measured by obtaining a myonecrosis index according to the method of OWNBY et al. (1983) . Hemorrhage after the i.m. injection was determined by the corrected mean hemoglobin method, as previously
Short Communications
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described (OwNBY et al., 1984b) . Since only one dose was used with only one treatment and a control, the Student's t-test was used to determine the significance of the difference between the two means. A significance level of PG0.05 was used. As can be seen from Fig. 1, there was a significant neturalization of myonecrosis when the antiserum was injected 5 min before venom or 5 or 30 min after venom injection. The neutralization was not significant when the time between venom injection and antisera injection was 1 or 3 hr. Figure 2 shows the results from the neutralization of hemorrhage experiments . There was significant neutralization when the antiserum mixture was injected either 5 min before or 5 min after venom injection, but not when injected 30 min after injection of venom. Our results demonstrate that the mixture of antisera can neutralize myonecrosis even when injected 30 min after venom injection. The amount of venom neutralized was twice that neutralized by antimyotoxin a serum alone in the same type of test (OviNBY et al., 1984a) . This indicates that there is some contribution by hemorrhaic toxins to the total myonecrosis of crude venom, since polyvalent antivenom can neutralize the hemorrhaggc activity of substantial (up to 24 FAg/g) amounts of crude venom (OWNBY et al., 1984b) . The results on the neutralization of hemorrhage also support this idea, since there was significant neutralization of hemorrhage when the antiserum mixture was injected 5 min after venom injection. Yet our results also point out that, at least with C. v. viridisvenom, components other than those that induce hemorrhage are responsible for a large part of the myonecrosis induced by the crude venom, i .e. myotoxin a. This is supported by the
z
á 0 W F V W V
TI IE I"dnl Fio. 2. Bnacr of mctvas oar PoLYVAt.HNr (CamAuam) Axnamom AND emuaxomxnr a smeum oar THE AMOUNT OF t03MOAAHACE INDUCED BY CnokAff virif VhIdis VENOM. Time (min) indicates that antisera was injected before (-) or after (+) venom injection . Values are the man t S.B. (n-4, unless otherwise indicated in parentheses). AS andsere ; PSS 0 .85% MCI . "PC0.05 compared to crude venom followed by injection of physiologic saline (0.85Vs NaCI).
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results of the neutralization of myonecrosis experiment in which neutralization was significant at 30 min after venom injection. However, the major problem of in vivo neutralization is still the lack of neutralization of hemorrhage or myonecrosis when the interval of time between venom and antiserum injection is greater than 30 min. So far there have been no reports of significant neutralization of local tissue damage induced by rattlesnake venom at longer time intervals. RUSSELL et al. (1973) reported prevention of local necrosis induced by C. v. helleri venom if antivenin (polyvalent Crotalidae) was injected (i .v.) simultaneously, some local necrosis if antivenin injection was delayed 10 min, and severe local damage if the delay was 20 or 30 min. One factor contributing to our inability to solve this problem is the rapidity of the hemorrhaggc action of these venoms . Both C. v. viridis and C. atroxvenoms induced local hemorrhage within 2-5 min after either i.m . or s.c. injection (unpublished results). Thus damage to blood vessel walls occurs long before antiserum can be injected. Some myotoxic components are extremely rapid in their action on muscle cells. For example, delta lesions and clumped myofibrils indicating necrotic muscle cells can be observed within 15 min after the i.m . injection of either C. v. viridis or C. atrox venom (Ownby, unpublished results). Acknowledgements-The authors thank VINITA PILL and TEtmA KENNmwER for typing the manuscript and MARY BooER for critically reading it . This work was supported by Public Health Service Grant 5 ROI AI16623-06 from the National Institute of Allergy and Infectious Diseases . C.L .O . is the recipient of a Research Career Development Award (KO4 AI00474) from this Institute. REFERENCES CLEMBrrr, J. F. and Piumuseo, R. O. (1979) Pit viper snakebite in the United States . J. Fancily Prwtice 6, 269. OwNBY, C. L., CAm aON, D. and Tu, A. T. (1976) Isolation of myotoxic component from rattlesnake venom: electron microscopic analysis of muscle damage. Am. J. Path. 85, 149. OwNBY, C. L., WooDs, W. M. and ODELL, 0. V. (1979) Antiserum to myotoxin from prairie rattlesnake (Crotales viridis virid1s) venom. Taxiton 17, 373. OWNBY, C. L., ODELL, O. V., WooDs, W. M. and CoL mRa, T. R. (1983) Ability of antiserum to myotoxin a from prairie rattlesnake (Crotales vk0s viridis) venom to neutralize local myotoodcrty and lethal effects of myotadn a and homologous crude venom. Toxicon 21, 35 . OwNHY, C. L., CoLBERo, T. R., CLAYPooL, P. L. and ODE T , O. V. (1984a) In vivo test of the ability of antiserum to myotoxin a from prairie rattlesnake (Crotahis vbidis vhidis) venom to neutralize local myonecrosis induced by myotoxin a and homologous crude venom. Toxicon 22, 99. OwNHY, C. L., Cot.smta, T. R. and ODELL, 0. V. (1984b) A new method for quantitating hemorrhage induced by rattlesnake venoms : ability of polyvalent antivenm to neutralize hemorrhagic activity. Taxkon 22, 227. Owxay, C. L., CoLSmta, T. R. and ODELL, 0. V. (1985) Ability of a mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenin to neutralize myonecrosis, hemorrhage and lethality induced by prairie rattlesnake (Crotales viridb viridls) venom. Toxiron 23, 317. RussELL, F. E., Rtmc, N. and Oowz uaz,'H. (1973) Effwdvenesi óf antivenin (Crotalidáe) polyvalent following injection of Crotahn venom. Tax*on 11, 461.