Preparation of a potent anti-scorpion-venom-serum against the venom of red scorpion (Buthus tamalus).RC Kankonkar, DG Kulkurni, CB Hulikavi
Anti Scorpion-Venom-Serum Project, Haffkine Biopharmaceutical Corporation Limited, Mumbai.
A number of children and adults, especially pregnant women succumb to the sting by red Scorpion (Buthus tamalus) in Konkan region--particularly on the coastal line. No specific antiserum or any other antidote is available to treat a victim of scorpion bite and hence the need to prepare a potent antiserum. Red Scorpion (B. tamalus) venom is a mixture of a number of protein moieties and neurotoxins of low molecular weight. Therefore, the venom is poor in antigenic composition and it is difficult to get antibodies specific to neutralise lethal factor/factors. Using Bentonite as an adjuvant and extending the period of immunization a potent antiserum has been prepared capable of neutralising the lethal factor/factors. In vivo testing carried out in albino mice, guinea pigs, dogs and langurs confirms this finding and shows that the antiserum is quite effective in neutralising the scorpion venom to save the life of envenomated animals.
Keywords: Adult, Animal, Antivenins, therapeutic use,Arachnidism, therapy,Child, Dogs, Female, Guinea Pigs, Human, Immunization, Passive, methods,Mice, Myocarditis, prevention &control,Pregnancy, Pulmonary Edema, prevention &control,Scorpion Venoms, antagonists &inhibitors,toxicity,Scorpions, Support, Non-U.S. Gov′t,
Red scorpion (Mesobuthus tamulus concanesis, Pockock earlier called Buthus tumulus) and Black scorpion (Pelamneous gravimanus) are the two scorpion - species that are most commonly found in the State of Maharashtra. It is reported that a number of children below 12 years of age and adults, especially pregnant women succumb to the sting of red scorpion in the Konkan region - particularly on the coastal line. While the red scorpion proves highly fatal in this part of the country, the black scorpion hardly causes any death. As early as 1928 Prater the Curator of the Prince of Wales Museum, Bombay had reported that scorpion sting claimed several lives in the Central Provinces. Caius and Mhaskar observed that though a few children below 12 years of age died following sting by the red Scorpion, fatalities in human beings were very rare. They were of the firm opinion that scorpion sting could not be fatal to the man. However their opinion was invariably challenged. Basu in Calcutta, reported 5 deaths, all in children, out of 19 scorpion-sting cases, he studied. Santhanakrishnan and Raju studied 301 cases of scorpion stings in this country that included children admitted to paediatric institutions in Madras during 1971-72, out of which 7 children died. Bawaskar observed that every month about 11 to 12 cases of red scorpion sting occur in this country. In his other published work, he reported a high mortality due to refractory pulmonary oedema following scorpion sting.
Indian red scorpion stings produce acute myocarditis,, initial transient hypertension followed by hypertension and shock, disseminated intra-vascular coagulation (DIC), acute pancreatitis, increased osmotic fragility changes of RBC causing haemolysis,,, hyperglycemia,, and lipolysis resulting in increased free fatty acid levels,,. All these cardiovascular haemodynamics and haematological alterations associated with the various biochemical abnormalities could be due to an autonomic storm resulting in a massive release of catecholamines, counter-regulatory hormones (glucagon and cortisol) , angiotensin-II, thyroxine and tri-iodothyronine, and a reduced insulin secretion,,.
Serotherapy for scorpion envenoming syndrome is irrationally convicted without trial. Appropriate serotherapy with the appropriate antivenom doses is unsurpassed in reducing morbidity and morality caused by venomous scorpion stings,.
So far no specific anti-dote or antiserum is available to successfully combat the envenomation of Indian red scorpion sting. All this time, the treatment is non-specific and symptomatic with limited success. Hence the need to prepare a potent antiserum that can specifically neutralise the red scorpion venom and thereby save the life of envenomated patient is acutely felt.
Red scorpion venom was supplied by Haffkine Institute for Training, Research and Testing (HITRT), Mumbai, India. A large number of red scorpions were individually electrically stimulated using distal coil and ensuing venom was pooled and lyophilised. Red scorpion venom was also purchased from Irula Snake Catchers Industrial Co-operative Society Ltd. (ISCICS), Chennai, India. The venom supplied was dried under vacuum.
The animals used were albino mice (NIH strain, 18-20gms), guinea pigs (English short haired strains, 18-20gms) stray dogs (9-15kg) and langurs (5-6 kg).
Stray dogs and langurss were first dewormed and kept under observation in quarantine. Only healthy animals were taken for the study.
Bentonite, used in experiments, was of B.P. Evans Medical Ltd., Speke, Liverpool.
In case of albino mice, the certain lethal dose (CLD) value was found out by injecting 0.5 ml. of different dilutions of red scorpion venom solution by intravenous (iv) route and observing the mice for 24 hr. For each dilution three mice were used.
The CLD values of guinea pigs, dogs and langurs were found out by injecting different dilutions of red scorpion venom by subcutaneous (sc) route and observing the animals for 24 hours after the administration of injection. Three guinea pigs and three dogs were used for each venom dilution while only two langurs were used for each venom dilution.
For raising antibodies against the red scorpion venom ponies were used. They were administered the venom solution by intramuscular (im) route. The first dose was extremely low viz. 200 ?g. The doses were than gradually increased. The interval between the first two doses was of two weeks. The subsequent doses were administered at the interval for one week. Eighteen weeks after the commencement of immunization, 2% Bentonite was injected along with the venom as an adjuvant. Again the interval was increased to two weeks. When the dose increased to 50 mg. the interval was further increased to three weeks. During this period, the animals were bled randomly to check whether antibodies in detectable quantity appeared in the blood of the animals. For the purpose of immunization venom obtained from HITRT only was used.
Once the animals showed detectable antibodies in their blood, they were bled and plasma separated. It was then processed using ammonium sulphate precipitation method and treated with Pepsin (1:10,000) to get purified and refined antiserum.
The neutralising ability and efficacy of the antiserum was tested in vivo by challenge method in mice by iv route. While in other animals the venom was injected by sc route and the antiserum was administered by iv route at different intervals of time following the administration of the venom.
Further the CLD value of black scorpion was determined as described earlier and experiments were carried out in vivo by challenge method to see if it could neutralise the Black scorpion venom.
CLD value in mice using Indian red scorpion obtained from HITRT, Mumbai and ISCICS, Chennai is shown in [Table:1A], [Table:1B]. CLD value using black scorpion venom obtained from HITRT, Mumbai is shown in [Table:1C]. CLD value of guinea pigs, dogs and langurs using Indian red scorpion venom by S.C. route is shown in [Table - 2], [Table - 3], and [Table - 4].
The neutralizing ability and efficacy of the antiserum enhanced as doses of the venom injected in the ponies were increased. On giving sufficient boosters with adjuvant the titre of the anti-venom further increased.
[Table - 5] shows the neutralizing capacity of different antiserum lots tested by challenge method in mice.
Seras of all the above mentioned lots were pooled. The neutralising capacity of the pooled serum was 2400 ?g/ml, which was then adjusted to 1200 mg/ml by dilution. The protein content was found to be 14.6%. The pooled batch was then Seitz filtered and lyophilised.
The pooled batch was then used to study the efficacy of the antiserum by administering it to guinea pigs, stray dogs and langurs at different intervals of time.
[Table - 6] shows in vivo neutralization of venom in guinea pigs. Thus while the untreated guinea pigs (control) died within 70 to 80 minutes, the treated ones survived when 1.5 ml of antiserum was administered at the end of 60 minutes.
[Table - 7] shows in vivo neutralisation of venom in dogs. Thus while the untreated dog (control) died within 3 hours, the treated dogs survived when 10 ml of antiserum was administered at the end of 150 min.
[Table - 8] shows in vivo neutralisation of venom in langur. Thus the untreated langurs (control) died within 6 hours while the treated langurs survived when 20 ml of antiserum was administered at the end of 2 hours. Beyond the time-interval, the veins collapsed and it became very difficult to administer anti-scorpion-venom-serum by iv route. However, the same anti-scorpion-venom serum could not neutralise even one CLD value of the Black scorpion venom. The CLD value of venom of black scorpion in mice weighing between 18-20g. was 300 mg. by iv route. In other words, the antiserum was not para-specific and indicated the antigenic make up of red scorpion venom and Black scorpion venom are quite different.
It is a well-known fact that the scorpion venom is mixture of protein moieties and neurotoxins of low molecural weights and that its antigenicity is poor. Naturally different chemicals have been used to enhance the antigenicity of the scorpion venom. Mohammed used atropine and ergotoxin and later Mohammed et al used purified picrate venom obtained from dried telsons to prepare a potent antivenin against Egyptain scorpion venoms. At Haffkine, Kapadi et al attempted to prepare anti-scorpion-venom-serum using ground telson extract with Freundís incomplete adjuvant. The resulting antiserum, which contained antibodies against the scorpion venom (as revealed by antigen-antibody perception lines using the gel diffusion technique), it was found to be inefficacious when tested in vivo by challenge method. Demagalhaes claimed that the toxicity of the telson extract is less stable than that of pure venom. Therefore, it was decided to use the venom ďmilkedĒ by electric stimulation and use Bentonite instead of Freundís incomplete adjuvant.
The difference in the CLD value of the venom supplied by HITRT, Mumbai and that purchased from ISCICS, Madras as well as their neutrailisation by the antivenin indicates variation in toxicity because of geographical variation. Such variations have been reported by Balozet. He found that the venom of A australis, obtained from Box-Saada region had an LD 50 value while the venom of the same species from Chella - Laghout had a value of 67 ?g and that from Etoned showed value of 51.5 ?g. Such geographical variations in the Russellís viper (Vipera russelli) venom have been noted by Jayanthi and Veerabasappa Gowda.
The different CLD value or guinea pigs, dogs and langurs show variation in species susceptibility.
The efficacy study conducted at different intervals of time shows that the antivenin is quite potent and life saving.
Our own finding shows that 1 ml of reconstituted anti-scorpion-venom-serum neutralises 1.2 mg of red scorpion venom by iv route in in-vivo study of mice. Antiserum of the same batch was also tested independently by the Quality Control Department of Corporation as well as by HITRT, Mumbai. The report of the Quality Control Department showed that one ml of reconstituted antiserum neutralised 1.2. mg of the venom while HITRT, Mumbai report said that it neutralised 3.0 mg of the venom. The antiserum also passed the abnormal toxicity as well as acute intravenous toxicity test in mice. Only 10% mortality was observed with the maximum administrable dose of 50 ml/kg.
Bucheral emphatically states that all severe cases of scorpion poisoning, particularly in small children must be treated with anti-scorpion-venom serum. According to him the serum dose must be large enough to neutralise at least 2 mg dry scorpion venom. Whereas content of one vial prepared at HBPCL can neutralise more than 10 mg dry venom.
Scorpion antivenins are rather specific. That is the reason why the antivenin against red scorpion venom does not neutralise black scorpion venom. Raw of Instituto Butantan found that HBPCL scorpion antivenin did not neutralise Brazilian scorpion venoms and Instituo Butantanís scorpion antivenin did not neutralise the Indian red scorpion venom (Personal communication).
However, Balozet is skeptic about use of scorpion antivenin if not administered within 2-3 hr. after the sting because according to him, by that time the venom without serum would have already produced irreversible lesions.
According to Gureon and Ovsyshcher antivenin is of little value in the treatment. However, Freire Maia and Campos and Ismail, do not agree with his statement as they have demonstrated that intravenous injection of scorpion antivenin before scorpion toxin almost totally prevented cardio-vascular and respiratory effects induced by intravenous toxin in the rat. The present authors wish to work further in this field with the anti-scorpion-venom-serum that they have developed and study it thoroughly before entering into finer intricacies of serotherapy.
The authors are grateful to the Government of Maharashtra for providing funds to carry out the present work and to Shri S. V. Joshi and Shri A. Ramkrishan, former Managing Director of Haffkine Bio-Pharmaceutical Corporation Limited, Mumbai - 12, for their keen interest in the work and kind co-operation. They also wish to keep on record the co-operation extended to them by Dr. M V N Shirodkar, former Director, Haffkine Institute for Training Research and Testing, Mumbai-12 during the experimental work on langurs.
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7], [Table - 8], [Table - 9], [Table - 10]