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Clinical use of antivenin prepared from goat serum
Toxtcoa, 1970, Vol. 8, pp. 63-65. Peraamon Prag Printed in Great 13riWn
CLINICAL USE OF ANTIVENIN PREPARED FROM GOAT SERUM FiNDLaY E. RUSSELL, W. Fxsn Tn~xMArr* and PAUL, E. MEADOWS Laboratory of Netuological Research, University of Southern California School of Medicine, Los Angeles County University of Southern California Medical Center, Los Angeles, California, U.S .A. (acceptedjorpublication 28 December 1969)
of choosing an adequate form of treatment following bites by venomous patients sensitive to horse serum and requiring antivenin is not uncommon (ALDEN, snakes in In experiences with 241 cases of snake venom poisoning, 41 patients were found 1969). our slightly positive (1 to have to 2 plus) to markedly positive (3 to 4 plus) skin or eye tests when horse serum. In such cases, other means of therapy are usually sought but, in exposed to general, these have been less satisfactory (RUSSELL, 1961 ; MINTON et al., 1967; RussELL, 1969a) . In 1961, a report was published on the use of an antivenin prepared in rabbits for administration to patients found to be sensitive to horse serum, and requiring antivenin (RUSSELL, 1961). Although good results were obtained in two patients with this antivenin, the need for larger amounts of sera than a colony of rabbits could provide, and the technical problems associated with maintaining large numbers of these animals, necessitated consideration of a larger source animal . It was decided to use goats, and subsequently the clinical use of an antivenin prepared from goat sera has been mentioned in several of our reports (RUSSELL, 1963, 1966,1967, 1969a-c; RUSSELL and LAURITZEN, 1966 ; Tn~RMAx and RU SSFiL; 1969). The present preliminary report deals with the preparation of this antivenin and its use in 9 patients at this Medical Center. Monovalent Crotalus Antivenin goatt was prepared by injecting goats with one ofseveral venoms : Crotalus viridis helleri, C. scutulatus scutulatus, C. atrox or C. r~ber rober. The snakes were milked in the laboratory and thevenom immediatelyquick frozen and lyophilized In several preparations the lethal fraction of the venom, separated on Sephadex columns, was added to the immunizing material (TIIMMERMAN and RUSSELL, 1969). The dried venom was dissolved in saline and was injected i.m . and/or s.c . with a vehicle that retarded absorption . The young goats were started on an initial dose of 0~ 1 mg per kg body weight and the amount increased at 1-week intervals over a 3-month period, or until the animal could withstand a 100 mg dose without deleterious systemic effects. Maintenance doses were then given at 3- to 4-week intervals. A small amount ofblood was taken from each goat at designated periods throughout the immunizing program to determine the level of antibody production . When protection against the lethal and cytolytic properties of the venom were TxE P1tosLEM
*Trainee in Toxinology University of Southern Californie, U.S.P .H .S . Training Grant GM 01700. tIdentification, Public Health Service, Dl3S-IND 408. 63
64
FINDLAY E. RUSSELL, W. FRED TTMMERMAN and PAUL E. MEADOWS
equal to or better than those of acommercially prepared standard, the goat was bled periodically and the sera prepared for processing. The various sera were processed in the following manner. One thousand ml of the crude serum was diluted with 2000 ml of sterile distilled water. Ammonium sulfate, 156 g, was added to each liter of diluted serum and the mixture allowed to stand overnight. The precipitate, containing residual fibrin and inert material, was collected by centrifugation or filtration and discarded. One hundred and forty-four g of ammonium sulfate per liter of diluted serum was then added, and the mixture allowed to stand overnight. The precipitate containing the active globulins was collected and the supernatant discarded. The precipitate was then dialyzed until the ammonium sulfate content was negligible. The dialyzed material wascollected and filteted. A sodium chloride solution of 0~85 per cent was then added and the antivenin concentrated, ifnecessary, by ultrafiltration. Preservatives were added, and the antivenin concentrate was sterile-filtered through ceramic candles or 025 ~. Millipore filters. The first patient given this antivenin was a 29-year-old male, who on 1 September 1963, had been bitten twice on the right thumb by a 1 m Crotalus viridis helleri and who on skin testing with horse serum was found to be 3-plus sensitive. The patient was then skin tested with goat serum and found not to be sensitive to this protein. He was given 300 mg of the species-specific goat antivenin i .v. in 5 per cent glucose over a 30-min period . Following administration of the antivenin, the tingling around the mouth and the metallic taste disappeared, the rate of increased swelling was markedly reduced and pain subsided . Figure 1 shows the patient's hand 24 hr following the bite . The second patient to receive the species-specific antivenin, (this was in 1963) has been described elsewhere (RUSSELL, 1969b) . Case number three was a 28-year-old male who was bitten by a 1~3 m Crotalus viridis oregam~s on the distal third of the right middle finger . The patient had a known sensitivity to horse serum. On two previous admissions he had been treated with a total of eight vials ofAntivenin (Crotalidae) Polyvalent, Wyeth. On the latter occasion he had a severe reaction to this antivenin, necessitating 4 day's hospitalization following discharge for the acute Crotalus envenomation . On the third admission for snake venom poisoning he was in shock on arrival. On the way to the Medical Center he had vomited and had complained of severe tingling about the mouth and at the finger tips. Swelling and edema had progressed over the dorsum of the hand, and there was left axillary lymphadenopathy. The patient vomited again while being skin tested for goat serum. Blood pressure at that time was 80/40. He received an i.v. dose of 400 mg of the species-specific C. viridis helleri antivenin over a 15min period, vasopressor agents, 300 ml of 5 per cent glucose in water and oxygen . His condition rapidly improved, although blood pressure remained low for 9 hr. Coagulation time was prolonged for 16 hr and platelets fell approximately 50 per cent during the first 10 hr after the bite . Again in this case, administration of the antivenin resulted in abeyance of the edema and its subsequent decrease over the next 24 hr, and cessation of the oral parasthesia. There were no complications and the patient was discharged 6 days after the injury. Figure 2 shows the injured finger 4 days after the bite. The small area of necrosis granulated in without defect . The fourth patient to receive the goat antivenin was a 34-year-old herpetologist who was bitten by a 1 ~5 m Crotalus rober rober on the dorsum of the left thumb (Fig. 3). He was seen within 20 min of the bite and complained of pain at the site of the bite, weakness and some nausea . Swelling was limited to the thumb and adjacent part of the hand . The patient
l, PATIENT BITTEN TWICE BY 1 m RATTLESNAKE Crotalus viridis %1CllCri oN RIGHT THUMB. Treated with monovalent species-specific antivenin prepared from goat serum. Photograph at 24 hr . FI(4.
FI(i . 2. PATIENT BITTEN Treated with monovalent C.
l'3 m CiOÍQ/US Vlr(!%(S OrCgRRdS ON RIGHT MIDDLE FINGER . vrridis he!leri antivenin prepared from goat serum. Photograph
BY
at 4 days .
FIG. 3. PATIENT BITTEN BY l'S m C. rllbCr rUi)Gr ON DORSUM OF LEFT THUMB. Treated with monovalent species-specific antivenin prepared from goat serum. Photograph at 48 hr.
Tox, f.p. 64
Clinical use of Antivenin Prepared from Goat Serum
65
had a 3-plus reaction to horse serum and was thus given 300mg ofthe goat antivenin. Following injection of the antivenin the edema slowly receded and there were no other significant findings or complaints . The patient was discharged 4 days after the bite . The fifth case (1964) has also been noted in the literature (Russsl.1.,1966). Ofthe remaining four cases, two received C. viridis hellerl antivenin, one received C. scutulatus scutulatus antivenin and one received C. atrox antivenin. In all of these cases the patients were found to be sensitive to horse serum but not to goat serum. The goat antivenin was given i.v. in each case. There were no deleterious side effects or delayed serum reactions. One patient complained of `gushing' and nausea during administration of the antivenin but this was thought to be associated with the rate of infusion . The present series ofcases indicates thatgoatsare good source animals for the preparation of species-specific antivenin. In one goat we attempted to produce a Crotales polyvalent antivenin but the titer of this produce was not sui%cient to warrant its clinical use. The series also indicates that there is a need for either species-specific or polyvalent antivenins prepared in animals other than horses . Although the number of persons now being exposed to horse serum, for reasons other than snake venom poisoning, is declining, so that at the present time we seldom find a child or adolescent who is sensitive to horse serum, the preparation of such a therapeutic agent seems indicated. We find a good number of herpetologists, snake handlers and zoo personnel who are sensitive to horse serum. Of our nine patients, five were in one way or another associated with the handling of snakes and were sent directly to us because of their known sensitivity to horse serum. Four of these persons had previously been exposed to antivenin. While the ideal Crotales antivenin would be that prepared from human serum, this has not yet been done, although there have been several popular reports stating that some individuals have been immunized, orhave become immune to this venom because ofrepeated bites by Crotales species. We have tested the sera of four of these individuals and have found no evidence of protection in their sera. An immunization course in humans for the production of a Crotales antivenin is fraught with many dangers, which, at the present, would, in our opinion, preclude our attempting such a program . Until such a time as a human serum becomes available, the preparation of a limited supply of antivenin from goat serum by those commercial agencies already producing antivenins from horse serum would not seem impractical or a financial hardship. Acknowledgements-The authors acknowledge the technical assistance of HexRY GONZALEZ . REFERENCES Ai.nErr, N. C. (1969) Treatment of rattlesnake bite. J. Am . med. dss. 207, 159. MtrrroN, S. A. Jtt., Dowi .nva,H. G. andRtrsset.t,, F. E. (1967) PoisonousSnakes ojthe World, p. 212. Washington, D.C . : U.S. Government Printing lice. RussEtL, F. E. (1961) Use of Crotalus monovalont antivenin from rabbit serum. Current tlrerap. Res. 3, 438. Rtrsset.t., F. E. (1963) Snake venom and antivenins . Med. News 1. Rtisseu,, F. E. (1966) Metronidawle in snake venom poisoning. Mems. Inst. Butantan Sirr{p. Internac.33, 845. Russets., F. E. (1967) Gel diffusion study on human sera following rattlesnake venom poisoning.Toxlcon S, 147. Russsts,, F. E. (1969x) Treatment of rattlesnake bite . J. Am. med. Ass. 207,159. Rt>sssu., F. E. (1%96) Clinical expects of snake venom poisoning in North America. Toxicon 7, 33. Rt ~a- F-r t - F. E. (1969c) Snakebite. In : Crrmru 77rerapy, p. 873, (Cotta, H. F., Ed.). Philadelphia : Saunders. Russst.t., F. E. and L.eutetTZtnv, L. (1966) Antivenins . ?bans. R. Soc. trop . Med. Hyg. 60, 797. ~, W. F. and Russtus., F. E. (1969) Comparative studies on the effectiveness of an antivenin and its fractions. Toxlrnn 6, 311 .
CLINICAL USE OF ANTIVENIN PREPARED FROM GOAT SERUM FiNDLaY E. RUSSELL, W. Fxsn Tn~xMArr* and PAUL, E. MEADOWS Laboratory of Netuological Research, University of Southern California School of Medicine, Los Angeles County University of Southern California Medical Center, Los Angeles, California, U.S .A. (acceptedjorpublication 28 December 1969)
of choosing an adequate form of treatment following bites by venomous patients sensitive to horse serum and requiring antivenin is not uncommon (ALDEN, snakes in In experiences with 241 cases of snake venom poisoning, 41 patients were found 1969). our slightly positive (1 to have to 2 plus) to markedly positive (3 to 4 plus) skin or eye tests when horse serum. In such cases, other means of therapy are usually sought but, in exposed to general, these have been less satisfactory (RUSSELL, 1961 ; MINTON et al., 1967; RussELL, 1969a) . In 1961, a report was published on the use of an antivenin prepared in rabbits for administration to patients found to be sensitive to horse serum, and requiring antivenin (RUSSELL, 1961). Although good results were obtained in two patients with this antivenin, the need for larger amounts of sera than a colony of rabbits could provide, and the technical problems associated with maintaining large numbers of these animals, necessitated consideration of a larger source animal . It was decided to use goats, and subsequently the clinical use of an antivenin prepared from goat sera has been mentioned in several of our reports (RUSSELL, 1963, 1966,1967, 1969a-c; RUSSELL and LAURITZEN, 1966 ; Tn~RMAx and RU SSFiL; 1969). The present preliminary report deals with the preparation of this antivenin and its use in 9 patients at this Medical Center. Monovalent Crotalus Antivenin goatt was prepared by injecting goats with one ofseveral venoms : Crotalus viridis helleri, C. scutulatus scutulatus, C. atrox or C. r~ber rober. The snakes were milked in the laboratory and thevenom immediatelyquick frozen and lyophilized In several preparations the lethal fraction of the venom, separated on Sephadex columns, was added to the immunizing material (TIIMMERMAN and RUSSELL, 1969). The dried venom was dissolved in saline and was injected i.m . and/or s.c . with a vehicle that retarded absorption . The young goats were started on an initial dose of 0~ 1 mg per kg body weight and the amount increased at 1-week intervals over a 3-month period, or until the animal could withstand a 100 mg dose without deleterious systemic effects. Maintenance doses were then given at 3- to 4-week intervals. A small amount ofblood was taken from each goat at designated periods throughout the immunizing program to determine the level of antibody production . When protection against the lethal and cytolytic properties of the venom were TxE P1tosLEM
*Trainee in Toxinology University of Southern Californie, U.S.P .H .S . Training Grant GM 01700. tIdentification, Public Health Service, Dl3S-IND 408. 63
64
FINDLAY E. RUSSELL, W. FRED TTMMERMAN and PAUL E. MEADOWS
equal to or better than those of acommercially prepared standard, the goat was bled periodically and the sera prepared for processing. The various sera were processed in the following manner. One thousand ml of the crude serum was diluted with 2000 ml of sterile distilled water. Ammonium sulfate, 156 g, was added to each liter of diluted serum and the mixture allowed to stand overnight. The precipitate, containing residual fibrin and inert material, was collected by centrifugation or filtration and discarded. One hundred and forty-four g of ammonium sulfate per liter of diluted serum was then added, and the mixture allowed to stand overnight. The precipitate containing the active globulins was collected and the supernatant discarded. The precipitate was then dialyzed until the ammonium sulfate content was negligible. The dialyzed material wascollected and filteted. A sodium chloride solution of 0~85 per cent was then added and the antivenin concentrated, ifnecessary, by ultrafiltration. Preservatives were added, and the antivenin concentrate was sterile-filtered through ceramic candles or 025 ~. Millipore filters. The first patient given this antivenin was a 29-year-old male, who on 1 September 1963, had been bitten twice on the right thumb by a 1 m Crotalus viridis helleri and who on skin testing with horse serum was found to be 3-plus sensitive. The patient was then skin tested with goat serum and found not to be sensitive to this protein. He was given 300 mg of the species-specific goat antivenin i .v. in 5 per cent glucose over a 30-min period . Following administration of the antivenin, the tingling around the mouth and the metallic taste disappeared, the rate of increased swelling was markedly reduced and pain subsided . Figure 1 shows the patient's hand 24 hr following the bite . The second patient to receive the species-specific antivenin, (this was in 1963) has been described elsewhere (RUSSELL, 1969b) . Case number three was a 28-year-old male who was bitten by a 1~3 m Crotalus viridis oregam~s on the distal third of the right middle finger . The patient had a known sensitivity to horse serum. On two previous admissions he had been treated with a total of eight vials ofAntivenin (Crotalidae) Polyvalent, Wyeth. On the latter occasion he had a severe reaction to this antivenin, necessitating 4 day's hospitalization following discharge for the acute Crotalus envenomation . On the third admission for snake venom poisoning he was in shock on arrival. On the way to the Medical Center he had vomited and had complained of severe tingling about the mouth and at the finger tips. Swelling and edema had progressed over the dorsum of the hand, and there was left axillary lymphadenopathy. The patient vomited again while being skin tested for goat serum. Blood pressure at that time was 80/40. He received an i.v. dose of 400 mg of the species-specific C. viridis helleri antivenin over a 15min period, vasopressor agents, 300 ml of 5 per cent glucose in water and oxygen . His condition rapidly improved, although blood pressure remained low for 9 hr. Coagulation time was prolonged for 16 hr and platelets fell approximately 50 per cent during the first 10 hr after the bite . Again in this case, administration of the antivenin resulted in abeyance of the edema and its subsequent decrease over the next 24 hr, and cessation of the oral parasthesia. There were no complications and the patient was discharged 6 days after the injury. Figure 2 shows the injured finger 4 days after the bite. The small area of necrosis granulated in without defect . The fourth patient to receive the goat antivenin was a 34-year-old herpetologist who was bitten by a 1 ~5 m Crotalus rober rober on the dorsum of the left thumb (Fig. 3). He was seen within 20 min of the bite and complained of pain at the site of the bite, weakness and some nausea . Swelling was limited to the thumb and adjacent part of the hand . The patient
l, PATIENT BITTEN TWICE BY 1 m RATTLESNAKE Crotalus viridis %1CllCri oN RIGHT THUMB. Treated with monovalent species-specific antivenin prepared from goat serum. Photograph at 24 hr . FI(4.
FI(i . 2. PATIENT BITTEN Treated with monovalent C.
l'3 m CiOÍQ/US Vlr(!%(S OrCgRRdS ON RIGHT MIDDLE FINGER . vrridis he!leri antivenin prepared from goat serum. Photograph
BY
at 4 days .
FIG. 3. PATIENT BITTEN BY l'S m C. rllbCr rUi)Gr ON DORSUM OF LEFT THUMB. Treated with monovalent species-specific antivenin prepared from goat serum. Photograph at 48 hr.
Tox, f.p. 64
Clinical use of Antivenin Prepared from Goat Serum
65
had a 3-plus reaction to horse serum and was thus given 300mg ofthe goat antivenin. Following injection of the antivenin the edema slowly receded and there were no other significant findings or complaints . The patient was discharged 4 days after the bite . The fifth case (1964) has also been noted in the literature (Russsl.1.,1966). Ofthe remaining four cases, two received C. viridis hellerl antivenin, one received C. scutulatus scutulatus antivenin and one received C. atrox antivenin. In all of these cases the patients were found to be sensitive to horse serum but not to goat serum. The goat antivenin was given i.v. in each case. There were no deleterious side effects or delayed serum reactions. One patient complained of `gushing' and nausea during administration of the antivenin but this was thought to be associated with the rate of infusion . The present series ofcases indicates thatgoatsare good source animals for the preparation of species-specific antivenin. In one goat we attempted to produce a Crotales polyvalent antivenin but the titer of this produce was not sui%cient to warrant its clinical use. The series also indicates that there is a need for either species-specific or polyvalent antivenins prepared in animals other than horses . Although the number of persons now being exposed to horse serum, for reasons other than snake venom poisoning, is declining, so that at the present time we seldom find a child or adolescent who is sensitive to horse serum, the preparation of such a therapeutic agent seems indicated. We find a good number of herpetologists, snake handlers and zoo personnel who are sensitive to horse serum. Of our nine patients, five were in one way or another associated with the handling of snakes and were sent directly to us because of their known sensitivity to horse serum. Four of these persons had previously been exposed to antivenin. While the ideal Crotales antivenin would be that prepared from human serum, this has not yet been done, although there have been several popular reports stating that some individuals have been immunized, orhave become immune to this venom because ofrepeated bites by Crotales species. We have tested the sera of four of these individuals and have found no evidence of protection in their sera. An immunization course in humans for the production of a Crotales antivenin is fraught with many dangers, which, at the present, would, in our opinion, preclude our attempting such a program . Until such a time as a human serum becomes available, the preparation of a limited supply of antivenin from goat serum by those commercial agencies already producing antivenins from horse serum would not seem impractical or a financial hardship. Acknowledgements-The authors acknowledge the technical assistance of HexRY GONZALEZ . REFERENCES Ai.nErr, N. C. (1969) Treatment of rattlesnake bite. J. Am . med. dss. 207, 159. MtrrroN, S. A. Jtt., Dowi .nva,H. G. andRtrsset.t,, F. E. (1967) PoisonousSnakes ojthe World, p. 212. Washington, D.C . : U.S. Government Printing lice. RussEtL, F. E. (1961) Use of Crotalus monovalont antivenin from rabbit serum. Current tlrerap. Res. 3, 438. Rtrsset.t., F. E. (1963) Snake venom and antivenins . Med. News 1. Rtisseu,, F. E. (1966) Metronidawle in snake venom poisoning. Mems. Inst. Butantan Sirr{p. Internac.33, 845. Russets., F. E. (1967) Gel diffusion study on human sera following rattlesnake venom poisoning.Toxlcon S, 147. Russsts,, F. E. (1969x) Treatment of rattlesnake bite . J. Am. med. Ass. 207,159. Rt>sssu., F. E. (1%96) Clinical expects of snake venom poisoning in North America. Toxicon 7, 33. Rt ~a- F-r t - F. E. (1969c) Snakebite. In : Crrmru 77rerapy, p. 873, (Cotta, H. F., Ed.). Philadelphia : Saunders. Russst.t., F. E. and L.eutetTZtnv, L. (1966) Antivenins . ?bans. R. Soc. trop . Med. Hyg. 60, 797. ~, W. F. and Russtus., F. E. (1969) Comparative studies on the effectiveness of an antivenin and its fractions. Toxlrnn 6, 311 .