Studies on the venom of South African scorpions (parabuthus, hadogenes, opisthophthalmus) and the preparation of a specific anti-scorpion serum

'L(RANSACTIoNS 01' 'THE RoYAL SocIKl'Y 01: 1'ROPIcAL i\~ll:Ul~I;vs AXI) HYGIENE. Vol. XXSIS. No. 5. Xpril, 1916.

S’l’UL)113S OK ‘1’1ILi \~ESOhI 01: SOU’l’Il AL~KICAN (PAHABWHUS, HAIMXENES, OPJS7’1~0PH[I%IAL~‘US) PREPARATIOX 01: ~4 SPECIFIC AKTI-SCORPIOn;

SCORPIONS AXI> ‘I’IIE SERUM.

BY E.

GRASSET,

A.

scHAAFsMA,

M.D., B.SC.

AND

‘l’hcsc investigations were initiated in 1940 in rcsponsc to a rcqucst from the Union Defence authorities for the preparation of an anti-scorpion skrum, on-ing to the presence of large numbers of scorpions in certain districts w-hew troops were encamped. This measure was of a precautionary nature as very few severe cases of scorpion sting had been reported in the Army. Although various species of scorpions are widely distributed throughout South Africa * We wish to acknowledge our thanks to the following persons for their valuable assistance in these investigations : Dr. I. H. POLE-EVANS (Irene), Mr. BLACKBEARD (Serowe), and the TRANSVAAL PONCOLA DEVELOPMENT COMPANY, for collecting thousands of scorpions ; Dr. R. F. LAWRENCE, Director of the Natal Museum, Pietermaritzburg, who classified the scorpions sent to him ; Dr. J. H. MASON, Research Veterinary Officer of the Serum Department; Mr. J. CHALMERS, Veterinary Officer of the Serum Dcpartmerit : Miss K. M. WOOLDRIDGE and Mr. F. A. BRANDT for their invaluable technical assistance : and finallv to our colleagues Dr. Il. A. LOUW (Brandvlei), Dr. Cr. HOLTZHALTSES (Pofaddder), Dr. A. ‘P. 13. H. I~OI)ENST.~R (Groblersdal), Dr. D. MALHERBE (Philipstown), Dr. B. BOVET (Johannesburg), Drs. J. v.4~ EDEN and J. ~'MALLEY IRWIN (Thabazimbj) for supplying us with clinical data on the effects of anti-scorpion serum.

and scorpion stings are not infrequent, especially among the Native l>ol)ulation, fatal cases are exceptional and are observed usually in children. No statistical data on this subject could be obtained either from the Union Public Health Department or the Director General of Medical Services in the Union. Furthermore, the almost complete lack of scientific data on the relative toxicity of the various South African scorpions, apart from a study on O~~isthophthalmus caper&s (EMDIN, 1933), made it necessary to undertake a study of the venom of the main representatives of South African scorpions. In CoJltrast, comprehensive studies have been made of the zoological taxonomy and systematics of South African scorpions. All our zoological references have been taken from HEWITT’S (1925) important monograph on South African scorpions and the more recent contribution of LAWRENCE (1942) which refers chiefly to Natal and Zululand scorpions. The investigations detailed in the present paper are limited to the following three genera : (1) Parahuthus. (2) Hadogenes. (3) O)~isthophtlzal~nus. ‘I’hese genera are the most common representatives of the South African scorpion fauna and constitute by far the vast majority of the specimens sent to this Institute by various collectors. A certain number of liroplectes (Ruthidae) and Cheloctonus and Opisthacanthus (Scorpionidae, sub-family : Ischnurinae) were received but in too small numbers to be of csperimental value. GEOGRAPIIICAL

DISTRIBUTION.

Puvabuthus. (Family, Buthidae).-This occurs throughout Africa and Arabia but is apparently absent from Natal and Pondoland. Our specimens were sent from the National Pongola Reserve, Northern Transvaal and Bechuanaland Protectorate. Most of the specimens were of the two following species : P. transvaalicus and P. trivadulattts. Average weight of adult specimen == 4.5 grammes, average length from anterior margin of carapace to tip of telson :- 7.2 cm. Parabuthus belongs to the same family as Buthzcs c/~rinquestriatus commonly found throughout the whole of Northern Africa and responsible for most of the severe stings reported in Egypt. (TODD, 1909.) These two genera present similar zoological characteristics. In contrast to the O~isthophthalmus and Hadogenes which are armed with powerful pincers (pedipalps) but have a slender tail and elongated telson, the Buthidae have thin pincers but possess a massive tail ending in a powerful chitinous telson. (See Photqqraph 1.) OpisthophthaZmus. (Family, Scorpionidac ; sub-family, Scorpioninae).-‘l’his occurs abundantly throughout South Africa, extending northwards to the Cunene river in the West and equatorial regions in the East. Most of our specimens came from the Bechuanaland Protectorate and two species were received, 0. zcahlbergi and 0. glabrifrons. ‘I’hc average weight of adult specimens received was 6.7 grammes, the average length 8.2 cm. Hadogenes. (Family, Scorpionidae ; sub-family, Ischnurinae).-This occurs in South Africa and Madagascar, North as far as the Feira district, Portuguese East Africa, West to Congo territory. It is, apparently, absent from the coastal strip from Capetown to Port Elizabeth. The maioritv of suecimens received belonged to the same suecies. Hadogexes troglodytes denta&, a;d were sent to us from the Eastern Transvaal. Ai,eragc weight of adult specimen .: 6 grammes, average length =:m 10.11 cm. (The maximum weight recorded was 18.45 grammes for a Hadogenes female, 15 cm. in length.) h’~RACTION l’he method of extracting large scale is based on mechanical

OF

VENOM.

scorpion venom usually crushing of dry telsons

adopted followed

by

various by aqueous

workers on maceration.

a

Opistlzophthnlirllls Averatre

~‘1101.0~:R.A1’H

1 .-Specimens

of South

African

Scorpions

dealt

wth

in this

lenrrth

paper.

8.2

cm.

PHOTOGRAPH

2.--T

4 hours after Opisthophthalmus (20 mg.

ypical lesion intracutaneous venom. above ; 4 mg.

in a guineq injection below).

S.-Lesion in a guineapig 24 hours intracutaneous injection of Hadogenes showing necrosis (20 mg. above; 4 mg. below).

PHOTOGRAPH

after venom,

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According to ‘I’onu (1909) telsons are dried in the sun and then placed in a desicWhen the venom was required the dry telsons were ground cator over calcium chloride. jn a Turkish coffee mill and added to 0.8 per cent. saline in the proportion of one sting to 1 ml. saline. Algiers, dried the cut telsons at 37” C., SERGENT (1938), at the Pasteur Institute, which he kept thereafter at room temperature in sealed tubes until they were required. ground up, macerated in physiological saline For experimental work the telsons were SHCLOV (1939),in Palestine ; MAHOMMED and extraction was completed with glass beads. in Brazil, used similar methods. (1942), in Egypt ; and MACALHAES (1989), C. PHIS~LIX and R/I. PI~ALIX (1923) obtained the venom by applying an electric While this method undoubtedly was an improved current to the telsons of the scorpions. SFRGEKT, who tried the Phisalix method on 397 technjque for individual specimens,’ Prionztrus uzcstrulis, states that the small yield of venom (an average of 1.3 mg.) so obtained A similar method was used does not advocate its use for large-scale collection of venom. by EMDIN (1933) in South -4frica on OpisthophthaZwms cup~~sis. Our method of extracting We endeal-oured to collect scorpion scorpion venom differed from the above techniques. \‘cnom on a large scale in as pure a form as possible. For Pavab~rhz~s possessing hard cheratinous telsons, the best method was to extract venom from the telson by aspiration with a fine capillary pipette, after the tip of the telsoll ‘J’hc thin layer of licluid venom collected on a xvatch glass was left at room had been cut. For Hadogelles and Opisthophtemperature exposed to air and dried Ivithin a few hours. thnlmus, the chitinous telsons of which arc easily collapsib!c, the method was to squeeze the telson glands between the arms of a pair of forceps and collect the liquid venom on a watch glass. After desiccation, the potency of the pooled venom was estimated in relaThe only reference tion to its dry weight in mjlligrammes, as in the case of snake venoms. \vc have found of a similar tcchniquc is that mentioned by KRAuSS (I
;Ifter

eatraction,

the

liquid

venom

had

the

appearance

of

;1 white,

milky

No appreciable difference was observed between the venoms MXS determined electrometrically of the three genera. ‘I’hc pI1 of the venoms with a Beckman pH meter ; the \-alues were : ~1-1 64 for Parabuthus venom, pH 5.5 for OpisthophthaIwu \xnom and pi-1 6.2 for Hadogenes venom. After

viscolls

fluid.

desiccation, to

the

of

fi11c

the watch

glass,

venom

from

took

the

which

it

Form could

of 1~

;L

white,

tletached

flaky with

product ;L neeclle

adhering in

the

form

scales.

of the ~cnom was dctcrmined by the difference betwecll the watch glass bcforc collection and after desiccation of the VCI~OIIL ‘I’hc \veight of the liquid venom immediately after collection was also taken, to record the percentage loss of moisture 011 drying. ‘l’he total number of’ scorpions used for each collection was noted ; dually 50 to 100 for each watch glass, which allowed for the determination of the aI-crage figure of dry venom per scorpion collected. 1. Parabuthzu--Venom was collcctcd from 338 specimens. ‘I’he total byeight of venom collected was 1~634% grammes. The average yield was 4-S mg. of dry venom per scorpion. Insufficient scorpions arrived at any one time to allow Lveight of liquid venom being taken with accuracy-. ‘l’he

\I-eight

3.

from

weight

of

the

OpisthophtlzalmzLs.~--For

5,985

specimens.

The

a

total

period

of

S+

years

Lveight of venom

venom

collected

was

collected

was 8.6225

grammes. The average amount of wet venom per scorpion was 6.5 mg. ; the average yield of dry venom was I.4 mp., giving a 78.5 per cent. loss of weight on drying. This figure compares closely with that given by EMDIN (1933) for the average dry weight of venom obtained per scorpion from 0. cajensis, i.e.. 1-3 mg. 3. Hadqelles.-During the same period venom was also collected from 9,603 specimens of this genus. The total weight of venom collected was 26.2791 grammes. The average amount of wet venom per scorpion was 12 mg. ; the average yield of dry venom was 2.7 mg., giving a 77.3 per cent. loss of weight on drying. No significant seasonal difference was detectable either in the appcarancc or the weight of the venoms collected. ‘I’he total number of scorpions used for all experiments was 16,946.

For experimental work, a given amount of saline was added to dry scorpion venom to obtain a solution’ containing 10 mg. per C.C. Solution was facilitated by mechanical stirring with a glass capillary pipette. The solution was left to stand to allow debris to settle. The supernatant venom solution was then used without filtration or centrifugation. In order to minimize possible individual differences, pooled venom samples have been used in each csperimcut. Experimental intoxication was studied on mice, guineapigs, rabbits and pigeons injected by various routes : intravenous, subcutaneous, intramuscular, Fresh venom solutions were used in all intracutaneous and intracerebral. All experiments, it being found that the venom was unstable in solution. experiments were completed on the day in which venom was put into solution. Parabuthus

VENOM.

intravenous I’arabuthus verlom. White mice (a:! to 22 gvammes).--‘l’he injection of 0.1 to 0.4 mg. of Panrabuthus venom (about l/40 to l/IO t&on) into white mice in a volume of 0.5 C.C. was followed immediately by acceleration of movement --hind of respiration, dyspnoea, staggering and into-ordination legs stretched in forced extension, with tail flexed over back ; in other C~SCS mice sat erect, gasped, jumped about uncontrollably, and fell paralysed. Death occurred after signs of asphyxiation from 30 seconds to 5 minutes after injection. In some cases death was delayed up to 30 minutes, after progressive paralysis and dyspnoea. Injection of sublethal doses resulted in the same type of symptoms, followed by gradual recovery. Subcutaneous injections of (le.5to 2.0 mg. of Pwabuthus venom arc necessary to produce death. In spite of the high dosage, onset of the symptoms is often delayed up to 30 minutes, death occurring in 2 to 3 hours after progressive dyspnoea and paralysis.

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Intracerebral injections were carried out with a venom concentration of 40 mg. venom per C.C. to minimize the volume of inoculum to 095 C.C. The Doses up to 0*0003 mg. venom injected varied from O*OOOl mg. to 0.2 mg. Injection of did not affect mice and produced only mild transitory symptoms. doses from 0.002 to 0.1 mg. were followed by immediate convulsions, trembling, paralysis with the hind legs stretched out. Death occurred from within a few seconds to 3 hours, according to dosage. Injection of O-1 mg. to 0.2 mg. resulted in almost immediate death. Guineapigs (X0 to 400 gmmmes).--Guineapigs were I’arabuthus venom. ‘I’he lethal dose of Parnbuthr~.s injected subcutaneously and intramuscularly. venom was 1 to 2 mg. Immediately on injection, the animals showed signs of acute discomfort and pain, by squeaking, biting the area of injection, coughing and All had a white exudate gasping for air. ‘l’here was distension of abdomen. around the eyes and symptoms of extreme salivation before death, which takes In some cases the heart continued beating for several place in 1 to :I hours. ‘l’herc \\-ere no haemorrhagic lesions. minutes after failure of respiration. Guineapigs were also injected intracutaneously to ascertain haemorrhagic action of venom. Thirty minutes after injection of 0.5 to 1.0 mg., erection of hairs is observed, followed by symptoms described above. At the site of the injection there were signs of vase-constriction with a 10 mm. area ol blanching. Death occurs in 1~: to 3 hours after progressive prostration and asphyxiation. KnhMs (2,000 g~rmznre,~).----~~rabbit injected intral’arabuth~ls 7~~tmrn. cutaneously in three places in the depilated skin of the abdomen with 3 mg., 2 mg. and 1 mg. of Parablithus venom gave similar symptoms. ‘l’he 4 mg. injection resulted in a very slight haemorrhagic infiltration of 20 mm. diameter, which enlarged to %O mm. after 5 hours. The 2 mg. injection gave a slight haemorrhagic infiltration, while the 1 mg. injection showed no visible altcration in the tissue. The rabbit developed paralysis in the hindquarters after 5 hours and succumbed in 6 hours with signs of progressive asphyxiation. ‘I’he intravenous injection of 5 mg. in a volume of 4 C.C. was followed by rapidly increasing signs of dyspnoea and gasping. ‘l-he rabbit lvas most sensitive to touch, cringing away. ‘l’his \vas followed by trembling and uncontrolled rolling on the floor. I>eath ensued in 5 to 10 minutes, with signs (Jf asphyxiation. At autopsy immediately after failure of respiration, the heart was found to be still beating. There were no apparent lesions of the internal organs nor haemorrhage of the muscles. Parabuthus 7~10112. Pigeons (400 to 450 ganzmes).-The intravenous injection of 0.8 to 1.5 mg. of Purabuthus \-enom in a volume of 2 C.C. resulted in the appearance of immediate symptoms of intoxication, the birds lying on their chests or side, with legs extended under the tail, gasping, sneezing, choking and with the head arched over the back. During the following minutes fhrs:c. ncllromotor symptoms wert- a,ygravated, respiration became ,gradually

more difficult and the bird died with signs of asphyxiation from 5 to 20 minutes after injection. The intravenous injection of sublethal doses, i.e., 0.5 to 0.7 mg. of venom, was followed by immediate paresis, inability to fly, and choking. After a few hours these symptoms gradually lessened and the bird recovered. The injection of doses up to 0.4 mg. did not result in apparent disorders. ‘rhe miaimum lethal doses of Parabuthus venom are given in Table 1. TABLE

Animnls. MiCe

Intrwenous Injection.

Intramusculnr Injection.

Subcutaneow Injection.

0.1-0.4 mg. -

0..5-24 mg. (l-2 hr.) I .o-2.0 mg. ( I-3 hr.)

5 mg. (8 min.) 0.8-1.5 mg. (5-20 min.)

15-20 mg. (5-10 hr.)

0.5-2.0 mg. (I -3 hr.) I dL24) mg. (q-3 hr.) -

(S-10

Guineapigs Rabbits Pigeons

I.

min.)

Intracerebml

Injection. 0~001-0~002mg. (.Y-I20 min.) -

From these experiments it is seen that the symptomology following injections of Purabuthus venom into animals, by various routes, is essentially the same. Parabuthus venom contains a neurotoxic substance which exerts a specific action on the neuromotor and respiratory centres. After a period of neuromuscular hyper-excitability, paralysis ensues ; death is due to asphyxiation by paralysis of the respiratory muscles. This action is very rapid when venom is introduced intracerebrally or intravenously-delayed and progressive where The toxic action of injections are made subcutaneously or intracutaneously. Parabuthus appears to be similar to that of Buthus occitanus and B. quinquestriatus (PHISALIX, 1922). Opisthophthalmus

VENOM.

intravenous injection of mice in Opisthophthalmus venom. M&.-The doses of 2 to 3 mg. (1 to 2 telsons) of Opisthophthalmus venom resulted in similar symptoms described above : increased rhythm of respiration, gasping soon followed by progressive paralysis and death in 1 to 20 minutes. Subcutaneous injections from 5 to 17 mg. were followed in 26 hours by oedematous swelling at the site of injection, which in the higher doses spread Death to the abdomen with evidence of pain, squeaking and irritability. ensued in 24 to 36 hours. Intracerebral injections of OMO2 mg. to 0~002 mg. gave no apparent

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symptoms. Injections of O-005 to 0.007 mg. resulted in immediate convulsions but did not cause death. With injections of 0.01 to 0.02 mg. the mice rolled from side to side, with convulsive movements, death following from 1 to 5 minutes after injection. Opisthophthalmus venom. Guineapigs.-Guineapigs injected subcutaneously with doses from 20 to 80 mg. of venom showed a haemorrhagic oedema at the site of injection, after 1 hour ; the intensity of the reaction was proportional to the dose, After 16 to 24 hours the subcutaneous tissue of the abdomen was distended by a gelatinous haemorrhagic fluid. This area, which may be as large as 40 mm. in diameter, became necrotic and sloughing occurred, followed later by the formation of a hard, deep scab with well delimitated edges. In no case was involvement of the peritoneal cavity observed. _Uotwithstanding the severity of the lesions the animals recovered. Guineapigs were injected intramuscularly in the internal portion of the thigh with doses of 20 to 80 mg. in a volume of 0.5 to 2 CC. Within 10 minutes after injection of 20 mg. there was a retraction of the leg injected, which was maintained in semi-flexion. During the following Z3Ominutes the limb became swollen, showing tense haemorrhagic infiltration extending to the subcutaneous tissue ~. . After I! to 3 hours, the whole leg wa s distended from the groin to the extremity with purple-\,iolet ecchymosis and complete loss of all active movement of-the limb. In spite of the set.crity of the local lesions, the animals remained in relatively good condition with no apparent dyspnoea or paraiysis of the limbs not injected. After 24 hours hacmorrhagic fluid oozed from several places in the distended limb. This \\-as soon followed by sloughing of the whole necrotic mass of muscles and skin, leaving the bone exposed, ‘l-he zrnimals eventually recovered after loss of the leg by spontaneous amputation. With the injection of higher doses of 40 to 80 mg., the haemorrhagic swelling involved the flank and abdomen with estensive oozing. After Joughing of the leg tissues, the limb remained hanging in a disarticulatcd position. ‘I’his was followed in 36 hours by spontaneous amputation accompanied by a very otfensive smell. The whole picture resembled experimental intoxication hy gas-gangrene toxins, i.e., I3. perfriqens and l3. histo+icm toxins. Death cnstlcd \\*ithin 36 to 48 hours, :jt autopsy the whole of the abdominal wall and adjacent thoracic portion were infiltrated with a haemorrhagic gelatinous oedema ; this was more intense in the groin and in the remaining necrotic upper part of the limb. ‘l’he peritoneal cavity was not involved. ‘l’here were no apparent lesions in the internal organs nor any generalized haemorrhagic syndrome. Intracutaneous injection in the guineapig resulted in a haemorrhagic patch of tegument appearing immediately at the site of injection, followed by a lesion differentiated into two zones-a whitish avascularized centre surrounded by a haemorrhagic ring (Photograph 2). Opisthophthalmzu venom clif%rs mainly from thxt of Hadoprrps it) its higher activity. With 2 loiv (jos(a

of 2 to 12 mg. of Opisthophthalmus venom the cutaneous lesion is about twice the diameter of the lesion formed when Hadogenes venom is employed. With a higher dose, the same results hold and, in addition, necrosis starts earlier than it does when Hadozenes venom of the same dosage is used. The lesion, however, remains localized. After a large scab has formed, healing ensues leaving a wide, thickened scar with permanent loss of hair. Opisthophthalmus venom. Rabbit.-Rabbits given an intravenous injection of 10 mg. of Opisthophthalmus venom showed signs of discomfort after 5 minutes, but no further signs were observed. Other rabbits when injected with 15 and 20 mg. showed signs of staggering after 1 minute. This was followed immediately after by uncontrolled jumping and rolling and the hindlegs were paralysed in forced extension. The animals died with signs of asphyxia in 2 minutes. Opisthophthalmus venom. Pigeons.-Pigeons were submitted to intramuscular injections of Opisthophthalmus venom in the pectoral muscle with doses from 10 to 50 mg. in a volume of 1 C.C. A dose of 10 mg. did not produce any apparent symptoms except local haemorrhage. ‘l’he injection of 20 to 50 mg. was followed 2 to 3 hours later by into-ordination of movement, staggering and inability to stand or fly. Birds lay on their chests with legs and claws in full extension under the tail, and wings flapping ineffectually. With higher doses such as 50 mg. death occurred in 3 hours ; occasionally delayed death was observed with the injection of 20 to 30 mg. On sectioning, apart from a haemorrhagic area in the pectoral muscles, no internal lesions were found. Pigeons injected subcutaneously with doses of 10 to 60 mg. in the pectoral region showed within 2 to 3 hours a haemorrhagic infiltration at the site of injection with some swelling. Although no actual paralysis was observed as in the case of intramuscular injection, the same inability to fly was shown and birds remained standing. Death was observed with the highest doses. Similar symptoms, using the venom of 0. capensis, have been described by EMDIN (1933). The minimum lethal doses of Opisthophthalmus venom are given in ‘I’able IT. Hadogenes

VENOM.

Hadogenes venom. Mice.-Mice injected intravenously with doses of O-2 to O-3 mg. (l/l0 telson) did not show apparent symptoms. With doses of 0.4 to 0*6 mg. (l/S to l/3 telson) dyspnoea was observed after a few minutes followed by signs of paralysis, with the hind-legs in forced extension. Death occurred within 1 to 15 minutes. At autopsy, no lesions were to be seen. ‘rhe clotting of the blood was normal. Mice injected subcutaneously with the surprisingly large lethal doses of 30 to 40 mg. showed, during the minutes following injection, an increased rhythm of respiration, accompanied by irritability, running about, erratic

jumping and biting of each other, with obvious severe pain at the site of injection. ‘I’his first period of 1 to 2 hours of hyper-excitability was followed by tremhling and progressive prostration, the animal lying on its abdomen or side with increasing dyspnoea. Death occurred 3 to 40 hours after injection. Lntracerebral injections of 04011 to 042 mg. of venom were necessary to produce signs of intoxication such as staggering, a hunching rip of the body and general paresis. After a period of 24 hours’ immobility, the animal gradually recovered. With doses of 0415 to 0.1 mg. the following symptoms appeared almost immediately : animal turned from side to side, or lay on its back with extremities stretched out and head in forced contraction forwards on the chest. It remained immobilized with occasional spells of into-ordinate movement. Death took place in from 5 minutes to 2 hours. Finally, with doses of 0.1 mg., cleath followed within 4 to S minutes. Hadogenes zmom. [email protected]‘l’hc subcutaneous injection of guineapigs with the still more surprising lethal doses of 100 to 1% mg., gave much the same picture as that seen in the subcutaneous injection of mice. Here, however, at the site of injection, a large saccular oedema containing about 10 C.C. of haemorrhagic fluid was formed. In a surviving pig this gradually dried and became covered with a hard necrosis. I)cath occurred in ahoirt 22 hours. ‘I’he intramuscular injection of SO mg. of venom into the leg of a guinenpig only caused swelling and redness, the pig remaining alive. Detailed experiments on the intracutaneous action of Hadogenes venom in guineapigs have been done with doses covering the very wide range of 2 to 20 mg. White, depilated pigs of 600 grammcs were injected with four decreasing doses in the flanks (a maximum of two on each side to avoid overlapping). ‘l’he injection of 2 mg. was sufficient to produce within the following minutes a haemorrhagic patch of 5 mm. which was still evident after 24 hours. Injections of 1 mg., H mg. and 12 mg. reslrlted in haemorrhagic reactions of 10 mm.,

406

SCORI’IOS

\-IiNOXI

15 mm. and 20 mm. respectively. On touching them, there were signs of pain. These haemorrhagic lesions do not disappear on prolonged pressure of the finger, proving the permanent nature of the lesion. They are followed by necrosis and scab formation, With doses of 16 to 20 mg. the haemorrhagic patch of 25 mm., which rapidly followed after the injection of the venom, soon differentiated into two zones-a whitish central avascularized zone of 20 mm. diameter, surrounded by an external, violet haemorrhagic ring of 2 to 3 mm. After 24 hours the central white area became necrotic and sloughed off in a mass leaving the normal subcutaneous tissue exposed. Regeneration took place from the periphery of the wound by granulation. (See Photograph 3.) During the whole evolution of the lesions the animals remained in good condition. The only general symptom observed soon after the injection, apart from obvious pain, was one of nervous excitability. Hadogenes venom. Rabbits.-The intravenous injection of 25 mg. of Hadogenes venom in 8 C.C. of saline into rabbits did not produce any apparent symptoms of intoxication. The injection of 50 mg. resulted in death within 15 minutes. There were no visible lesions at autopsy and the clotting of the blood was normal. A freshly depilated rabbit was injected intracutaneously with 4 mg., 2 mg. and 1 mg. At the site of the 4 mg. injection there appeared within the following minutes an area of marked congestion and hypervascularization of 40 mm. diameter, without haemorrhagic phenomena. After reaching its maximum during the following 30 minutes, it gradually faded away, and had practically disappeared in 5 hours. The 2 mg. and 1 mg. injections did not produce any appreciable skin reaction. were submitted to intravenous Hadogenes venom. Pigeons.-Pigeons injections of Hadogenes venom in doses of 2 mg. to 10 mg. in a volume of 2 C.C. of saline. The average M.L.D. was found to be 8 mg. With sublethal doses of 2 to 4 mg. the main symptom was the extension of the legs backwards immediately after the injection, accompanied by thk inability of the bird to fly. These early symptoms disappeared after a minute or two ; the bird was able to stand and recovered soon after. With lethal doses of 6 to 10 mg. the same signs appeared while the injection was still taking place. The birds remained lying on their chests or sides with shaking of the head which was sometimes flexed backwards, gasping, trembling of the whole body and violent into-ordinated movements of the wings and body. This first phase was followed by progressive prostration, death supervening in 2 to 10 minutes. At autopsy, there were no external haemorrhages, visible lesions of the organs, or any abnormality in blood-clotting. Pigeons were injected intramuscularly with 40 mg., 60 mg. and 80 mg. Only with the highest doses were symptoms noted. of venom, respectively. Here the leg injected was retracted and swollen and there was a serous exudate

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It was necessary to inject at the site of injection. All the birds survived. 125 mg. to cause death which then took place after 10 minutes. The minimum lethal doses of Hadogenes venom are given in Table III. ‘CABLE

III.

Subcutaneous Injection.

Intramusculnt Injection.

Intracerebrd Injection.

I”.;, mu. (Ill

‘TESTS

WITH

SHEEP

min.)

ERYTHROCYTES.

In a first experiment, decreasing amounts of venom, from 20 mg. to i/100 &g. per cc., in a volume of 1 C.C. were added to a 2 per cent. suspension of washed sheep erythrocytes. The final concentration of sheep red cells was thus 1 per cent. ‘I’he mixtures were placed ill the incubator at 37” C. for 1 hour after which they were read with the naked eye. For Pnrahuthzrs venom slight haemolysis (about 3 per cent.j was observed in the tube containing only the highest dose of venom. F’or Opisthophthalmus venom 5 to H per cent. haemolysis was observed-again only in the tube containing 20 mg. venom. No haemolysis was present in any mixture containing Hadogenes venom. In a second test with 0.1 per cent. concentration of sheep cells, 20 mg. of Purahuthus venom had no haemolytic effect, while the same amount of Opisthophthalnlzls venom caused complete (~’ 1 ; ) haemolysis ; the same large dose of Hadocqencs venom caused only a trace of haemolysis. COMPARATIVE

TESTS

‘WITH

SHEEP

AND

GI:INEAI’IG

liRYTHROCYTFP.

In the following comparative experiments with sheep and guineapig red cells, venom solutions over a range of 10 mg. to 1 mg. per C.C. were used. Readings u-ere taken after 1 hour incubation at Z3’7’C. Owing to the insufficient quantity of Purahuthus venom available, comparative tests with this venom could not be undertaken ; however, experiments performed with it demonstrated only a slight haemolytic action.

SCORPIOS

\‘I’ivO~?

IV.

TABLE Hadogenes

10’I II Sheep Cell Suspension. -/ + f

Amount

of Venom. mg. 20 mg. 10 mg. 1 mg. control 40

I I

venom.

o.““;, Sheep Cell Suspension.

1‘I,{, Guineapig Cell Suspension.

+ ++

+ ++

O.i?“,, Guineapig Cell Suspension. -____f +

f

0

++

++

0 0

0 1)

+++ 0

+++ 0

I TABLE

Amount of Venom. ______ 40 mg. 20 mg, 10 mg. 1 mg. control

1”x0 Sheep Cell ~ Suspension. __~--____++ ++ + ~ f 0 i I ,

V.

(,..JO’ / Sheep &ll SUSpenSiOll.

,--___

1

f f ++ + 0

i

, r,’ I).““(, Guinea& Cell Guineapi,q Cell Suspension. Suspension. -____ -_----+ ++ ++ + +++ + +a +++ ++ II 0

The experiment was repeated with guineapig red cells, using venom solutions in dilutions of 20 mg. to 041 mg. per C.C. to determine the end point of haemolytic action. Conclusions. From these experiments it appears that a haemolytic action is exerted by the three venoms under test only when used in relatively high concentrations. All conditions being equal, the tests show the greater sensitivity of guineapig erythrocytes to haemolysis, as compared with sheep red cells. It is also more marked on 0.2 per cent. cell suspensions than in the case of the 1 per cent. suspensions. These findings confirm those of EMDIN (1933) working on Opisthophthalmzts capensis, who also found that guineapig cells were the most sensitive. A similar haemolytic action has been observed by KUBOTA (1918) with the venom of the Manchurian scorpion Buthus martensis, and the venom of the Mexican (Jentrzrrirs e.xlicanda (1918a). In contrast, no haemolytic action co~dcl he

I..

(~I~\ssI~:‘I‘.

1.

\

.\hI,

.I,

\.

Ccl1

Suspension,

Guineapig Cell Suspension.

II)!1

111)1~~;so\

0.2”,,

1 ”, 0

Guinezpig

S(.II\.\I’SYI

I (’

Guineapi; Ccl1 Suspension.

O.?,,

Guineapig Cell Suspension.

++

+

++

+

++

++

+++

++

-I-++

++

+++

+++

+++

+++

i

-t++

++ + 0

+ f’ f 0

0

0

(I

0

0 0

0

0

0

0

0

0

0

0

observed by JkIOUSSAY (1919) (as quoted by AI. PIIISALIx, ncgati\:c reslllts have been scorlGon venom (apparently

0

on the venom of the African &thus yuinyuestviatus 1922) even after the addition of lecithin. Similar reported by ‘I’ODD (19309) \vith pooled Egyptian Uzrthus and P~iurz~uzu).

.1n c.\pcrilncnt was carried out to ascertain the effect of heat on a satnl)lc ITractions of a 10 mg. lw c.c. solution of the venom were heated in a water hath to (u) 70” C. for 30 minutes, (6) boiling iwint, which is 93” C. at 6,000 feet altitude, for 30 minutes, (c) boiling point for 90 minutes. 3licc u-u-c injected intra\,enorlsly with O-3 -kg. of each of these fractions. (‘l’his was the average h1.L.D. of this batch of venom, the mice dying I to -i minutes after injection.) IIeating the venom to 70” C. delayed the avcragc time of death to 8 minutes after injection ; heating the venom to boiling ljoint for 30 minutes delayed death for 10 minutes ; and heating the venom at boiling point for 90 minutes delayed the average time of death for 13 minutes after injection. Intracutaneous injections of 5 mg. of these venom fractions into guineapigs produced oedematous, hacmorrhagic local reactions of 20 mm. diameter, which rcachcd their maximum intensity in 38 hours. These later altered to dry necrotic scabs. ‘l’here was no difference in the intensity or in the final effect SC’CII ljet\\~cn the tInheated and heated fractions of the venom. of

\c‘110111.

A similar experiment was carried out on another sample of venom. Fractions of a 40 mg. per C.C. solution of the venom were heated to SO” C., 60” C. and 70” C. for 15 minutes each ; another fraction to SO” C. for 30 minutes. An unheated fraction was used as a control. Of each of these fractions, three doses, corresponding to 4 mg., 12 mg. and 20 mg. of venom, were injected intracutaneously into depilated guineapigs. The injection of 4 mg. of unheated venom was followed 4 hours later by a haemorrhagic patch 10 mm. in diameter. ‘The haemorrhagic areas resulting from the injection of 12 mg. and 20 mg. of venom, were respectively 30 mm. and 40 mm. in diameter. Although these injections were done on the same animal at a distance of 4 to 6 cm. apart, an extensive oedema enveloping the whole of the abdomen was caused. Injections of similar doses of the fractions of venom heated to 50” C., 60” C., 70” C. and SO” C. produced reactions of 10 mm., 20 mm., and 20 mm. diameter. Notwithstanding the comparatively smaller reactions, they were still definitely of a haemorrhagic nature. No appreciable difference in the intensity of the reactions could be observed in the four heated fractions. ‘Ihe oedema caused by the injections of the heated venoms was also comparable, though this was less than that caused by the unheated venom. These experiments tend to show the limited, destructive action of heat on the toxic and haemorrhagic principles of Opisthophthalmus venom. EMDIN (1933) found that the venom of 0. cape& loses its toxicity on prolonged heating to 100” C. but does not say to what extent this occurs. Hadogenes venom. Experiments to ascertain the effect of heat on Nadogenes venom were also carried out. Fractions of a 10 mg. per C.C. solution of venom were heated to (a) 70” C. for 30 minutes, (b) boiling point for 30 minutes, and (c) boiling point for 90 minutes. Amounts of 0.2 mg., 0.3 mg., 0-S mg. and 1 mg. were injected intravenously into mice. Similar amounts of unheated venom were ‘I’he results are set out in Table VII. injected into mice to act as controls. Guineapigs were injected intracutaneously with 5 mg. of each of the unheated and heated fractions of venom. An early oedematous reaction, followed by a haemorrhagic and necrotic lesion, was caused at the site of injection. ‘I’his was of an equal degree of intensity in all four pigs. Another sample of venom was dissolved in saline to obtain a 40 mg. per C.C. solution. In this case fractions of the solution were heated to 50” C., 70” C. and boiling point for 1 Three doses, corresponding to 4 mg., 12 mg. and 20 mg., hour respectively. Similar amounts of were injected intracutaneously into depilated guineapigs. unheated venom were used as controls. The results are set out in Table VIII. Forty-eight hours after the injection the haemorrhagic reactions described above were transformed in all cases into necrotic lesions. These all healed later by granulation from the sides. ‘The pigs were not affected. These

I,..

!;I1 \hhl,.

I’,

\.

kl.ll\

\I \,\I

\

\\I)

.I.

.\.

1101)(~\0~

111

Venom

______

for 30 min. . ..____ ~.-~_--~-.-I_--~__--_----.I~~_ Sul-vi\ctl Sun 1,etl Sur\-i\ cd Survived Survived IIietlfi min. IIiecl-I S min. SLirvixul Died--3 min. IIird2 min. Died-3 min. Died-I.5 min. DiedI min. DiedI min. Died--1 min. DieclS min.

7’.WLC

Venom --

-~-_-

fvr

30 min.

Suni\ied8 Died-13 Died2 IIied5 Died-.i I>ieclL;i

min. min. min. min. min. min.

for

!)tI min. __SurCvetl Survi\ ed i Survivrd ~ Diedti min. Died--S min. Died--1-j min. ~ Died-I min. LXed-- 3 min.

VIII.

Injected.

_-_____

experiments again show the limited principles of Haclogenes venom. PROTEOLYTIC

ACTION

action of heat on the toxic and haemorrhagic

OF SCORPION

VENOMS

ON GELATIN

Zk Z&O.

‘1’0 determine the proteolytic action of scorpion venom we used the same technique as in our studies on snake venoms (GRASSET and SCIIAAFSMA, 1940). ‘l’o a series of tubes containing O-3 C.C. of 20 per cent. gelatin in physiological

‘1I 2

SCOI1I’I(IS\‘IlAOhl

saline (kept in water-bath at 40” C.), decreasing amounts of a 40 mg. per C.C. venom solution were added, i.e., 30 mg. to 0.1 mg. The volume in all tubes was brought up to 1 C.C. by the addition of saline. ‘l’he final concentration of gelatin was, therefore, 6 per cent., which was found sufficient for the control tubes to solidify at 6” C. after 30 minutes. After addition of the venom solutions, the mixtures were kept at 37” C. for 5 hours and then placed in the refrigerator at 6” C. to allow the gelatin to solidify. Readings of the tubes were taken after 30 minutes and again after 12 hours. In all cases comprehensive pooled mixtures of venom were used. The results obtained are given in ‘I’able IX.

I

Amount of Venom. ----30 mp. 20 mg. IO mg. 6 m’g. 2 mg. I mg. 0.5 mg. 0.1 my. control

/

Pambuthus

---

Venonl.

T,iquefaction ++- + ~ I,iquefaction + + Liquefaction + ~ Softening 0 0 0 0 0

Of&hophthulmus

Venom. ~-~-------~ No licluefaction No softening I 0 0 0 0 0 0 I 0

Iludogews

I

.-

Venom.

-__ No liquefaction No softening II 0 0 0 0 0 0

I Repeated experiments carried out with other mixtures of venoms led to similar results. Thus only Pa~abuthus venom was found to exert a definite proteolytic action on gelatin, and that only when used in relatively high concentrations. ‘I’his is in contrast to the findings of HOUSSAY(1919), quoted by I’HISALIX (19221, who found that the glandular extracts of Buthus spp. have no enzymic action on gelatin, starch and casein. IMMUNOLOGICAL INVESTIGATIONS. NEUTRALIZATION OF SCORPIONVENOM I3Y POLYVALENT Naia jlava-Bitis arietans ANTIVENENE. With a view to ascertaining the possible neutralization of South African scorpion venom by snake antivenene, tests were carried out with the polyvalent antivenene prepared at this Institute. One C.C. of this concentrated antivencne neutralizes either 1 mg. of the venom of N. Java or 10 mg. of the venom of B. arietans. In a first experiment, 0.1 mg. (1 M.L.L).) 0.f a p oo 1e d mixture of Pavabuthus venom was mixed with 0.4 C.C. of antivenene. After 1 hour contact at 37” C.

E. GRASSET.

.A. SCH~AFSJIA

4ND

d. .\. HODGSON

413

white mice of 20 grammes were injected intravenously with the mixtures. Five out of the six mice injected died within 6 to 20 minutes. Of six control o f venom alone, four died within 5 mice injected with O-1 mg. (1 M.L.D.) . minutes. Experiments carried out with Opisthophthalmus and Hadogenes venom gave similar results. No protection thus appeared to be exerted by this polyvalent antivenene. Anticobra serum was also found to have no protective action against the venoms of Heterometrus mawus (NICOLE and CATOUILLARD, 1905), Buthus guinquestriatus or Tityus bahiensis (HOUSSAY, 1919). The same absence of group neutralization has been observed when using antilachesis and anticrotalic serum. Although clinical data of persons stung by South African scorpions and treated by injection of our polyvalent N. jlava-B. arietans antivenene tended to show that the pain symptoms were minimized, there were no active proofs of venom neutralization. In view of these facts it was decided to undertake the preparation of a specific anti-scorpion serum from the venom of the scorpion species available. PREPARATION

OF SPECIFIC

ANTI-SCORPION SERUM. in several countries where the incidence and gravity of scorpion stings warranted the preparation of a specific antidote. In Egypt, an anti-scorpion serum active against &thus quinquestriutzcs and Prionurus was prepared by TODD (1909) and recently perfected by MOHAMMED (1942). A similar type of therapeutic serum against the venom of B. quinquestriutus has also been manufactured by the Lister Institute (from dry telsons sent from Egypt to England), and also by SHULOV (1939) in Palestine. In Algiers, SERCENT (1936) prepared an anti-scorpion serum against the venom of Prionurus australis, the most toxic species in Northern Africa. This anti-serum In Brazil, is also active against the venoms of Prionurus lionvilleri and Buthus occitans. the preparation of a polyvalent anti-scorpion serum has been evolved by VITAL BRAZIL at the Butantan Institute and at the Ezequiel Dias Institute by MAGALHAES against species of South American scorpions (Tityus bahiensis and T. sewulatus).

Anti-scorpion sera have been prepared

DETOXICATION

OF SCORPION

VENOM.

Considering the satisfactory results obtained in the preparation of various snake antivenenes by the use of formolized venoms or anavenoms (GRASSET & ZOUTENDYK, 1932 and 33) attempts were made to apply similar methods to the preparation of anti-scorpion serum. As in the case of snake venoms, the scorpion venom concentration used for detoxication was 10 mg. per C.C. The yield of venom from 378 scorpions of the genus Opisthophthalmus, i.e., 673 mg., was dissolved in 67.3 C.C. of saline. After sedimentation of the organic debris, the solution was slightly centrifuged. The supernatant Auid had a white, turbid appearance and the pH, which was determined electrometrically, was 5.52. Determination of the toxicity of a sample of this venom solution showed a M.L.D. of 3 mg. by the intravenous route for mice. (Death in 5 to 20 minutes.)

414

SCORPION

VENOM

This 10 mg. per C.C. solution was split into two fractions-one had 0.5 per cent. and the other 0.8 per cent. of 40 per cent. form01 added. The t\vo fractions were incubated at 37” C. After a period of 15 days’ incubation the two formolized venoms were tested. As preliminary tests by means of intravenous injections given to mice tended to show that the residual form01 was lethal for these animals, the two products were dialized for 24 hours. The intravenous injection of 5 to S mg. of the dialized, detoxicated venoms? representing two to three lethal doses of the original venom, was not followed by anyapparent symptoms of toxicity. A similar method of detoxication was used for Hadogenes and Parabuthus venoms. The resulting detosicated antigens were used for the immunization of a horse. Immunization

of a horse with detoxicated and unaltered scorpion venom.

Horse 394 was submitted to the following series of subcutaneous injections of detoxicated scorpion venoms at 3- to S-day intervals. The first doses of 10 mg. and 20 mg. of formolized Opisthophthalmus venom were progressively increased to 100 mg. Then Hadogenes detoxicated venom was substituted in doses of 25 mg. to ZOO mg. The immunization was then continued with Parabuthus venom. At the end of 3 months the horse had received an amount of 2,210 mg. of detoxicated scorpion venom comprising respectively 1,235 mg. Hadogenes, 195 mg. Opisthophthalmus and 680 mg. Parabuthus venom. The immunization was completed with injections of unaltered Hadogenes venom up to a total of The total amount of scorpion venom 872 mg. spread over four injections. injected during the course of immunization from 8.3.41 to 29.5.41 was just under 3 grammes. No undue reactions were observed subsequent to the injection of detoxicated and unaltered venoms. Owing to the lack of venom, the immunization could not be continued. The horse was bled 9 days after the last injection. iVeutralization Considering

tests with immunized horse serum.

the fact that immunization was to the greater part carried out with the assays on the activity of serum dealt mainly with that venom. Mice In all these assays, scorpion venom and pigeons were used as experimental animals. solutions were used within the hours following preparation, as it has been our experience that, for Hudogenes venom, the toxicity of the original venom drops very quickly after dissolution. For each experiment the lethal dose by intravenous injection into pigeons weighing 350 grammes was determined before the neutralization tests were started. One cc. of serum from Horse 394 was then mixed with increasing multiples of 1 to 4 M.L.D. of Hudopenes venom for intravenous injection into the pigeon. Details of the results of the tests are given in the following table :One C.C. of anti-scorpion serum therefore protected against 24 mg. venom (three lethal doses for the pigeon). Similar neutralization tests with the immune serum from Horse 394 carried out with ten pooled samples of Hudogenes venom on pigeons, gave analogous results : 1 C.C. of immune serum was again found to be neutralized from 2 to A total of twelve pigeons were usuaily 3 M.L.D. of Hadogenes venom in the pigeon.

Hadogenes venom

LI~CY~ per test. As in the case of titration on prL>ceded by the determination of the M.L.D. iniection into white mice weighing 20 grammrs. Table XI.

Vol.

of

Xniount

Serum Horse 394.

Venom

in mg. used Test.

ir

pigeons, neutralization tests on mice were of Hadogenes venom b? subcutaneous Examples of such a titration are given in

Corresponding Number of Subcutaneous

__-

~__

Result?.

-I

1 C.C.

35

I

1 c.i. 1 C.C. Control

71, IO;i

.I

So symptoms, survived Death in PO hr.

3

Death

in

35

I 1

Death Death

in 7 hr. in 6 hr.

>>

f

?\I!CC. R1.L.D.

35

12 hr.

Repetition of this neutralization test against the same sample of venom and two other pooled samples of Hadogenes venom showed that in the case of subcutaneous mice titration, the neutralization power of the serum is found to be comparatively lower than in the case of the pigeon. It is limited to 1 M.L.D. although partial neutralization, reflected in a delay of 12 to 20 hours before death took place, was observed in the mixtures containing 2 and 3 M.L.D. as compared with the control injections of 1 M.L.D. of Hadogenes \.enom, when death occurred 6 to 7 hours later. No appreciable activity of the serum against the venoms of Parabuthus and Opisthophthalmus scorpions could be demonstrated, as was to be expected from the limited amount of these antigens used during horse immunization.

Intracutaneous

tests in the neutralization of scorpion venom by spec$ic antivenene, in the guineapig.

As shown in previous chapters, the intracutaneous injection of Opisthophthalmus Hadogenes venom in the guineapig resulted in the rapid formation of local haemorrhagic lesions. The following experiments were done with a view to ascertaining whether haemorrhagic action of these venoms would eventually be modified by anti-scorpion

and the

416 serum neutralization. Depilated guineapigs were injected intracutaneously respectively with :1. 8 mg. of Hadogenes venom in a volume of 0.3 cc. 2. 8 mg. of the same venom previously put in contact with 0.2 C.C. anti-scorpion serum for 1 hour ; this proportion was found, by subcutaneous tests in mice, to correspond approximately to the neutralization point. Whereas marked haemorrhagic reactions were observed at the sites of injection in the control animals, reactions of a different character were shown in the guineapigs when injected with the same dose of venom plus specific serum. For the dose of 8 mg. of venom alone, the haemorrhagic patch of 20 mm. diameter soon observed at the site of injection, was followed by the formation of a central livid, necrotic zone which became later a hard scab, with permanent loss of hair. In the case of the venom-anti-scorpion serum mixture, the local inflammatory reaction observed soon at the site of the injection did not devolve into a necrosis. It remained more superficial as evidenced by the persistent suppleness of the skin and the growth of depilated hairs after 4 days-a sign of the vitality of the deeper layers of the teguments. The same techniaue when followed in the neutralization of O~isthobhthalmus venom produced similar rest&s. The above differences in the reactions produced from the use of venom alone and the use of venom anti-scorpion serum mixtures, were also observed here. The results of these tests tend to show a definite, sub-total neutralization of the haemorrhagic principles of these two venoms by specific anti-scorpion serum.

PROTECTION

AFFORDED

BY HETEROLOGOUS

ANTI-SCORPION VENOMS.

SERA AGAINST

Hadogenes AND Parabuthus Lister Institute

anti-scorpion

serum (M.P.H.).

Two 1 C.C. ampoules of this concentrated serum (Batch No. 83 ; date of preparation, June, 1939 ; date of expiry, June, 1943) were obtained through the courtesy of Dr. N. KOVACS of the Serum Institute, Cairo. One C.C. was stated to be sufficient to neutralize one sting (species not specified but presumably Buthus) ; O-5 C.C. of this serum was mixed with 12 mg. of Hadogenes venom Soon after the injection, the pigeon (1.5 M.L.D. for pigeon, intravenously). showed severe symptoms of intoxication which lasted for several hours, but gradually recovered. 0.5 C.C. of the same serum was mixed respectively with 1 and 4 M.L.D. of Parabuthus venom for mice intravenously. The mouse injected with the mixture containing 1 M.L.D. survived, while the one injected with the 4 M.L.D. mixture died in 15 minutes. Although these tests could not be repeated, due to the limited amount of serum, they tend to show only a limited heterologous neutralization against the two venoms used in these tests. Institute Ezequiel Dias (Brazil)

anti-scorpion serum.

One C.C. of this serum (received March, 1941 ; date of expiry, 1945) is stated to neutralize l-5 lethal doses of Tityus scorpion venom for the mouse Neutralization tests on mice using 0.5 C.C. of this serum mixed (camondongo). with 0.2 mg. Parabuthus venom (1 M.L.D.) were followed by survival of the animals. Mixtures containing O-5 C.C. serum with 2 and 3 M.L.D. Parabuthus

E.

Cl&ASSET,

A.

SCHAAFSMA

AKD

,I.

417

.4. HODGSON

venom failed to protect the mice. Similar tests were done on pigeons injected intravenously. One c.c. of the serum mixed with 2 M.L.D. (16 mg.) caused It does not appear, therefore, that antithe death of these birds in 3 minutes. Tityus scorpion serum confers any appreciable protection against Parabuthus venom. ANTI-SCORPION

SERUM

IN THE TREATMENT

OF SCORPION

STING.

Scorpion stings in South Africa, although usually very painful and responsible in some cases for severe symptoms of intoxication, are seldom fatal even among children whose symptoms are more severe. The fact that no statistical data dealing with scorpion stings or fatalities have been made the subject of special records by the Union Public Health Department, nor by the military authorities, speaks for the relative benignity of scorpionism in this country, as compared with the high death-rate reported by TODD (1909) in Egypt. The mortality from scorpion stings in eight great Egyptian towns for the period 1901 to 1907, before the use of anti-scorpion serum, ranged annually from 0.036 (in Cairo) to 0.64 per 1,000 (in Assouan). This represents 1.6 per cent. of the total death-rate in the latter town. Death occurred almost entirely among children. In northern French African territories, the severity of scorpion stings inflicted by Prionurus australis appears, from data given by SERGENT (1940), to be equally grave. In the absence of any official data for South Africa, we have tried to obtain personal information from district surgeons in areas where scorpion stings are particularly frequent. From information kindly supplied by Dr. Lovw, Brandvlei district, Cape Province, we learnt that many cases had been severely ill in this region and some, mostly children, had died. The genus of scorpion found here is usually Parabuthus. The main symptom was an immediate and intense burning pain in the area of the sting. If the sting occurred on the leg, the pain sometimes extended up to the groin. In 1 or 2 days this was followed by stiffness of the lower limbs and joints and the patient found great difficulty in walking. In some cases, the stiffness was generalized and affected the entire body for a few days. These symptoms were accompanied, in some cases, by “ pins and needles ” in the hands and feet and a pricking sensation in the face. In other cases there was rapid and feeble heart action, while some patients complained of a severe headache and difficulty in talking. In adults this paresthesia and stiffness gradually passed off in the course of a few days. Usually no outward signs could be found, except some oedema at the site of the sting. Similar symptoms have been described in case records sent by district surgeons in other areas. The following case record supplied by Dr. HOLTZHAUSEN, district surgeon, Pofadder, Bechuanaland border of the Cape Province, gives an idea of the condition observed in some patients.

418

SCORPION

VENOM

Cases. A

Native mine worker of Caboop Mines was stung by a scorpion on both legs at night during sleep. The mine official was called only on the following afternoon. The patient’s condition resembled chronic rheumatism. His arms and legs were drawn up in flexion. His limbs were stiff and muscles hard. He could not talk. In desperation the mine official injected some snake antivenene. The man slowly recovered and 3 days after could walk slowly. The scorpion which stung the Native was sent to us by Dr. HOLTZHAUSEN for identification and proved to be a large Parabuthus granulatus. In children the symptoms are usually severe and may eventually result in death within a few hours as exemplified in the following case reported by DR. Louw : Boy of 7 years, Severe pain up the leg and in the was stung under the hollow of the left foot at 7 p.m. epigastrium resulted. Shortly afterwards he became powerless and later paralysed. He could not vomit even when emetics were given. He died at 1 a.m. the following night presumably from respiratory paralysis.

On the whole, these symptoms resemble those following stings by Buthus and Prionzms, as reported by TODD (1909), PHIS~LIX (1922) and SERGENT (1939) from North Africa. Critical conditions have been observed in some cases which were stung by Hadogenes as illustrated below in the case of a child treated with antiscorpion serum. Symptoms following stings by 0. capensis, referred to by EMDIN (1932) are usually not severe-local swelling, redness and pains from 1 to 3 days ; other symptoms are free perspiration, lassitude and sometimes loss of sleep. Fatalities are rare even in children. TREATMENT

OF SCORPION STING ANTI-SCORPION

CASES WITH SERUM.

SOUTH

AFRICAN

The anti-scorpion serum prepared, as described above, at this Institute was supplied to medical practitioners and district surgeons in the areas where Clinical records so far scorpions were reported as frequent or dangerous. received, on the effects of this serum, although they report favourably on the treatment, are still too limited to have any statistical significance. It is more difficult to interpret the influence of the serum on adult patients than on children, owing to the milder symptoms exhibited by adults. When administered soon after the sting, the serum is reported to minimize the pain considerably, and to reduce or inhibit the development of general symptoms. This is exemplified in the following case record supplied by Dr. HOLTZHAUSEN, Kakamas, C.P. :male, aged 40 years, was stung on the hand by a medium-sized : European The site of the sting was burning Patient’s condition was shocked. intensely. Ten minutes later, 10 C.C. of anti-scorpion serum were administered in the One hour later, only slight upper arm and the patient was kept well under observation. burning remained at the site of the sting and no other symptoms developed. Patient

Opisthophthalmus.

Records of cases occurring

in children

report that the serum had a definite

therapeutic nesburg :-

action.

The following

case was supplied

by Dr.

BOVET,

Johan-

European child, 12 years old, stung at 6.30 p.m. on 15th March, 1943, by a scorpion 2 inches long (apparently Opisthophthnlmus) on the lateral side of the right knee. The burning pain at the site of the puncture was accompanied by moderate oedema and redness. Next morning there was slight dizziness with local, burning pain on the lee, slight oedema, but no necrosis. The child went to school. At 9.30 a.m., during a lesson, During the following hour he went into the child fainted and was carried out of class. one faint after another, not totally unconscious, however, but could not articulate syllables although he tried to pronounce words. He complained of diffuse abdominal pain. Stiffness was noticed in the hands and fingers. The first clinical examination of the patient by Dr. ROVET at 11 a.m. found the patient in a collapsed condition but not completely unconscious, with dysarthria, no excess salivation, pulse normal at 96, heart and lungs normal, respiration regular. He continued to fall into one faint after another. Answers were given with difficulty and in monosyllables, his ideas were confused but his reflexes continued normal. At 1.35 p.m. intramuscular injection of 10 C.C. of anti-scorpion serum was given. Xt 3 p.m., i.e., 1: hours later, the child became more rational and was able to talk although his ideas were still confused. At 6 p.m. the patient seemed quite normal and awake, speaking without any difficulty. He had no recollection of being carried out The following day the child was normal and of school or of subsequent happenings. his recovery was uneventful. CONCLUSIONS

AND

SUMMARY.

The main toxic and biological characteristics of the venom of the most common genera of South African scorpions have been investigated ; i.e., Hadogenes, Opisthophthalmus and Parabuthus. The average yield of dry venom obtained per specimen was 0.0027 gramme from 9,603 specimens of Hadogenes, O*OO14 gramme from 5,985 specimens of Opisthophthalmus, and 0.0048 g ramme from 338 specimens of Pambuthus. Experimental intoxication with the venom of these three genera was studied on mice, guineapigs, pigeons and rabbits. The M.L.D. of the respective venoms for these animals have been determined by intravenous, intramuscular, subcutaneous, intradermal and intracerebral routes. The injection of Parabuthus venom by these various routes to the animals, resulted in neuromotor symptoms, dyspnoea and paralysis ending in asphyxiation. Similar neuromotor symptoms followed the intravenous injections of Hadogenes and Opisthophthalmus venom, but higher doses were necessary to kill the animals. The M.L.D. of Parabuthus venom by intravenous injection for the pigeon is 0% to l-5 mg. ; for mice 0.1 to 0.4 mg. ; and for the rabbit, 5 mg. The intravenous b1.L.D. of Hadogenes venom for the pigeon is 6 to 8 mg. ; for mice, O-2 to 0.6 mg. ; and for the rabbit, 50 mg. The intravenous R,I.L.D. of Opisthophthalnzusvellomfor the pigeon is 3 to 5 mg. ; for mice, 1 to 3 mg. ; and for the rabbit, 15 to 20 mg. The subcutaneous injection of the venoms required considerably higher doses (four to six times higher or more) to produce death. The animals all showed progressive paralysis and prostration ending in asphyxiation after several hours.

420

SCORPIOS

VENOM

The intramuscular injection of doses of Hadogenes or Opisthophthalmus venom resulted in intense haemorrhagic infiltration followed by sloughing of the necrotic muscular mass, and eventual spontaneous amputation of the leg which was injected. The intracutaneous injection of the venoms of Hadogenes and Opisthophthalmus into the guineapig in doses of 4 mg. was sufficient to produce rapid haemorrhagic reactions followed by local necrosis and the formation of hard scabs. Intracerebral injections of the venoms required doses 25 to 100 times lower than those injected intravenously, to kill mice. M.L.D. of Parabuthus for mice is 0.001 to 0402 mg. ; of Opisthophthalmus 0.01 mg. and of Hadogenes 0.05 mg. The facts observed point to two main toxic principles in these venoms :(a) Neurotoxin, chiefly developed in Parabuthus venom and the cause of the nervous and paralytic symptoms observed in small animals. It appears to be mainly responsible for the fatal cases of scorpion sting in man. (6) Haemorrhagin, responsible for the local haemorrhagic, necrotic lesions observed at the site of injection. This was confirmed histologically. If the relative total toxicity of the three venoms studied is expressed in terms of milligrammes necessary to produce death by intravenous injection in mice of 20 grammes, then Parabuthus venom is two times more active than Hadogenes venom and ten times more active than Opisthophthalmus venom. A haemolytic action was exerted in vitro by the three venoms in very high concentrations, in washed erythrocytes. This was more marked with Opisthophthalmus and Hadogenes venoms than with Parabuthus. Guineapig red cells appeared more sensitive than sheep cells to haemolysin. No coagulant action was observed with these three venoms on horse blood in vitro, but an anti-coagulant action was observed with high concentrations of Opisthophthalmus venom. Parabuthus venom was found to have a proteolytic action on gelatin in vitro, only when relatively high concentrations of venom were used. Heat (1 hour at 93” C.) exerted but a limited destructive action on the haemorrhagic principles of the venoms. No heterologous neutralization of these venoms was observed with polyvalent anti-Naia jlava-Bitis arietans serum. A limited heterologous neutralization was found to be exerted by the Egyptian (Buthus) and the South American (Tityus) anti-scorpion serums. Immunization of the horse with a view to preparing a specific anti-scorpion serum was started with formolized, detoxicated scorpion venoms, and continued with unaltered venoms. One C.C. of the serum so obtained neutralized in vitro 3 M.L.D. of Hadogenes venom intravenously injected into the pigeon. Preliminary results from the use of this anti-scorpion serum in the treatment of cases in South Africa, although of no statistical value, indicate that this specific serum has a definite therapeutic value.

E. GRASSET,

A.

SCHAAFSiW.~

AXD

.I. .A. HODGSOE

421

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W. African GRASSET, E. (Dispholidus GRASSET, E. venimeux C.R. Sot.

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