Protection against sea snake envenomation: comparative potency of four antivenenes

roxrca., t97s,

vo~. t4, pv. ~~-ass . ra~on r~ . r~oooa ~n c~eu a~to.

PROTECTION AGAINST SEA SNAKE ENVENOMATION COMPARATIVE POTENCY QF FOUR ANTIVENENES E. H. Bexr>at and Hr~~ A. GALL1l~II0 Commonwealth Savm Laboratories, Parkvilla, Victoria, 3052, Auat~alia (Acceptedfor pubikntton S February 197 E. H. iaNrrea and H . A. G~ .ucmo . Protection against sea snake enveaomation : comparative potency of four antiveneaea. Toxiaon 14, 347-355, 1976.~ubcutaneoua end i.v. in, o wero determined on all venoms used in the study. Statistical examination revealed that the lethality by the l.v, route was significantly greater (9S ~ confidence levee in only six of the eleven eea shako epodes. The neutralizing pot~cy of four antîvwenes, Tiger Snake Antiveoene (N. scutatus), monovalent Sea Snake Antivenene (E. achfstosa), oommacial Sea Shako Antivenene (N, scutatvs-E. achistoaa), and polyvalent Sea Snake Antivanene (L. hardwkkli, L. aen~ jasciata and E. cyareocürctua) was determined against all elev~ veaoms by as in vivo passive protection method. insults were oomparod with those obtained by rx vitro nwtralizatIon. In oniy 4 of tho 33 venonrantivenene combinations tested was there a statistically significant difference in potency determined by the two methods. Little difference in neutralization potency was seen why result of tests using the 2LDs o and Sz .ns . level of envenomation were statistically examined. Antivenene was more effective in protection whey given early after envenomation. Tiger Snake Antivenene proved to be the antivenene of choice for protection against sea snake envenomation. INTRODUCTION

Tt~ ~aroluTY of studies on cross-neutralizing potency of antivenenes against sea snake venoms, including one by ourselves (Tu and GANTHAVORN, 1969; Oxoxocz et al., 1972; Tu and $ALAFRANCA, 1974; BexT~ and GnLi tcfuo, 1974), have been made using in vitro combinations . C~xsst and Wiucx~r (1960) claimed that fre vitro combination is not necessarily a guide to the possible therapeutic value of the antiserum for treating a snake bite . It has always been commercial practice to assay antivenenes by in vitro combination methods. Earlier investigations in this laboratory using these methods indicated that antivenene capable of neutralizing the venom of Enhydrina schistosa, the most commonly occurring sea snake in Australian waters, could be readily produced using a mixture of Enhydrina schistosa venom and tiger snake (Notechis scutatus) venom as antigen. In vivo protection studies of the kind employed by MIIVTOx (1967) should be a better guide to possible therapeutic value of an antiserum than in vitro studies. The purpose of the present studies was to investigate whether in vivo protection against a range of sea snake venoms by Enhydrirta schistosa antivenene, Notechis scutatt~ts antivenene, and Enhydrina schistosa-Notechis scutatus antivenene paralleled the neutralizing values found for the monovalent antivenenes in the fn vitro studies previously reported (BexT~t and GaiLlcFno, 1974). It was of interest to study concurrently the relative protective value of a sea snake antivenene prepared using a mixture of the venoms of representatives of three genera of sea snakes (Oxoxoci et al., 1972). At the same time, in view of the rapidity of action of sea snake venoms, opportunity was taken to study the effects of increasing time interval between envenomation and antivenene administration, and the effect, if any, of level of envenomstion on protective value of the antivenenes. T~OXICON 1976 Yol. U

347

348

E. H. HAX'IBR aad H13ATHER A. CiALLICHIO

MATPRiAi Q AND METHODS Yenoma Venoma were made available to us by the Snake and Venom Research Institute, Panang Csenaral Hospital, Malaysia ; Burma Pharmaceutical Industries, Rangoon, Burma; Dr. Anthony Tu, Colorado, U.SA. ; Miss vat Lawsan of Cairns and Mr . Eric wornell of Ciosford. N. acutatrv antivenene was commercial Tiger SnakeAntivenene batch no. 028-1, E. sclFistoro-N.scutataa antivenene wanoommardatSeaSnake Antivenene batch no . 0241 and in aminority of ~batchno.026-1, and E. acJdatoaa antivenane was commercial Sea Snake Antiveaene batch no. 013-1, all prepared by the Commonwratth Serum Laboratories. The N. scutatrcr aativraene was derived from a pool of plasma obtained from horses which had not mrparienoed hyperimmuni7ation with any other venom or in some cases had been I>,ypaimmunized against other vanom(s) 2 yr previoua~y. The E. acJrfstowo-N. sc)ttatus antivenene was derived from plasma obtained frn4Yr horaea whkh had been i>,yperimmunized with a mixturo of N. aartatwr and E. scbittaia veaoms, and the E. :chlstosa aativenene was derived from plasma obtained from horeea which had not experlenoed hypaimmunfzation with any other venom. Aativenme units in all cease are deßned as follows: the amount of anttveneae narttalizing 10 i)g of the stated venom ea deta~mtned at the date of manufadrrro. No international antivenene standard preparations of these species earist. Venom toxicity of any apeclea and venom capacity to combine with the homologous antivmene may vary somewhat from batch to batch of venom, doepite the latoer being largepools. The Japanese Sea Snake Antivenene, produced from serum from a haaso which had ban )>Ayperimmunized with LaPenrLr l)mdwlaFfl, ZatiQOxrlo ~aaclata and Eydrophir cymacbechu vennms, wan kindly made availabb to ua by Dr. Takaahi Okonogl, of the ~b'o Ca~mpagy, Tokyo. TAaiB Venin

1 . I~so a~r v>nvoses sx s.c. At~ i.v. RoiTlBa Subwtaneoua X 50 (N9)

intraveaout X 50 lu9)

6 .SS (5 .50 - 7 .73)

3 .88 13 .41 - 1 .38)

6 .50 (5 .71 - 7 .35)

4 " 10 (3 .57 - 4 .71)

1 .77 (1 .30 - 3 .15)

1 .19 (1 .91 - i .70)

11 .41 (10 .13 - 13 .13)

6 .87 16 .18 - 7 .63)

NydROpkia etayana

6 .SS (5 .70 - 7 .53)

a .OS (S .l1 - 6 .BU

NydtoPki.a ~aJot

4 .84 (4 .01 - 5 .84)

3 .58 (3 .01 - 4 .11)

" HydROpkla alg+toc.tac .tua

8 .59 17 .73 - 9 .53I

6 .40 (5 .5T - 7 .34)

"Hydaopkia a Lc .Cet.CeottCa

4 .10 (3 .44 - 4 .84)

1 .71 (1 .38 - 3 .06)

13 . 53 (11 .13 - 14 .37)

7 . 57 (6 .18 - 9 .16)

e .11 (4 .74 - 9 .83)

7 .16 (6 .44 - 7 .95)

" A~LPya uan a taav .Ea

Eak dRLwa ac~ .(stoaa " HydtopkCa cyaaoc.twctw

" LapuiJ kaedwlekü Lat+.aauAa autbwc .iata N.(eROCepGatopkta y M~jt,(,a " Aoteckis aca.tatw

11 .00 (10 .44 - 13 .80) 6 .40 (5 .38 - 7 .10)

N" D " 1 .94 (1 .71 - 1 .19)

Att values arc expressed as Ng per mouse (22-25 g weight). N.D. ~ not determined . Limits calculated by the Spearman-ICarber method. 95 ~ confldenoe limits within 83-121 ~ of cxperimeatal valuus. 'Venoma showing vahres signißcantly different using the two injection routes. roxrcoN rsra vor 1~

349

Protection Against Sea Snake Bavmomation T~ 2. In vivo ~ to vitro rwrlr.~zru~rlor or vst~osa >n Vlv011

Dalta Notee "ia scataLa anti~aaaoa " aaotraliain" 30 v9 i"DO. 1R vWf

la vLüo""

A(ryauar 4ev.la

Y1.K ( " .1S - 17 . "1)

i .1" (4.43 - " .S])

Mttat4 abfea.ü

S.i7 13 .4] - " .7f)

5 .73 (4 .13 - 7 . " fl

Ea a

13 .0! l " .70 - li .i" 1

13 .Of ( " .70 - li .i ")

toaa

Daib E . aci4aloaa - N . aeatatw antlvaoawt naatraliaia" 10 4" ~aao,! ia v4ve

la vttao"

Onlta Eakydataa aeklatoaa " Dt!~aaaea4 naatrallainy 10 P9 ~or la vtvo 1.i3 (1.19 - 1 .17) 1 .13 (0, "] - L3!) 1 .74 (1 .SS - 3 .40)

O .fO - 1 .31)

3 .31 (1 .71 - ] .i " )

U .04 (! .]! - U .00) l.li (0 .74 - 1. "0)

0 .71 (0 .11 - 0 .41)

0 .13 (0 .1i - 0 .70)

l.u - 1 .f")

S .io (1 .77 - ] . "0)

:.lf (1.40 - ].]!)

o .s7 (0 .]" - 0 .77)

o .u (" .f] - 0 .71)

1] .7] U .60 - 1".401 1 .39tt (1 .47 - ] .54)

7 .17 (5 .1i - f .l01

13 . "3+t (9 .35 - 17 .74)

13 . " Stt (l .1S - 17 .74)

1 .14 1 .14 (0 .77 - 1 .i4) (0 .77 - l.ii) 0 .f7 " 0 .47 l " . "7 - 1 .3f) L0 .l4 - O .i4) "S 1. "4 1. (1 .]] - 3 .SS) (1.]] - 1 .SS)

" .01 (5 .77 - 11 .07) 17 .0f (10 .f4 - 3i .SO)

4 .17 (4 .44 - ".51)

Nyraarua atyaaa NyLtaykCa aafoa

7 .f7 (s .7! - 11 .57)

Ny/aa~kL a4YSLaa.taa

1.i1 <1 .17 - 3 .34)

7 . "] (S .i4 - 10 . "11 1 .1f (1 .47 - 7 .34)

Nylafrkta aLete.Ueftti.a

11 . "3 (l .1S - 17 .74)

11 . " 3 (9 .35 - 17 .74)

S

(1 .04 l~lsff) f U .7f ~ " S,7S)

(1 .041-4 1 .!!1

(1 .47 1 1~74)

(0 .1! ~~0~4i)

LatLeaa h aeallaae tata

" .47 (i .07 - ll .f ])

" .!3 (4 .f " - l .SS1

3 .43 (1 .74 - 3 .74)

11/,aaeaap4atep04 ";aa(!,(a

" .47 (i .07 - ll .é]) 3 . "0 (3 .03 - ] . "i)

"ataaLLa a" -r.r- .

0 .45 (0 .]3 - O .i3)

wy~.~

0 . "9 (0 .44 - 1 .13)

i .i7 <4 . "0 - f .11)

1 .11 (1.3] - 3 .f71 4 (0 . " 1 ] .o! (3 .17 - 4 .17) 4 (1 .07

Nylaa/kG efaao"~+s"

Sa vttto"

11 .4f (" .3S - 13 . "1)

13 . " ] p .14 - 17 .711 N .D. 0 .45 (0 .33 - 0 .411

3."0 (1 .03 - ] . " il 0 .45 (0 .13 - 0 . " ])

l .ittt (1 .17 - 1 .34)

".D. 0 .l7 (0 .70 - 1 .]4)

0 ." 0 (0 .S" - 1 .10) 0 .77 (0 .37 - O .S7)

O .f " (O .fi - 1 .47) 1 .77 (1 .15 - 1 .]!)

(O .1f~370 .]7) 1 .]0 h.ii - 7.1") ".D. 0.31 10 .]7 - 0 .73)

Tests conducted at S iDs " level. "Tiger Snake Antivenene Hatch 028-1, 380 units per ml . j~Sea Snake Antiv~ene Batch 024-1, 410 anti-N. acutatlrs units per ml, 28 anti-E. achirtara units per mL ttHatch026-1, 410 anti-N,acutatrrsunitsper ml, 30 anti-E,scholars unitsperml used forthesecombinations. ~ Snake Antivenene Batch 013-1, 27 .3 anti-E. scMstara units per ml. 9Figurea expressed in anti-N. scluatus units. "'Based on Hexane and Cï!ei ucmo (1974) for monovalent antivenenas. N.D. = not determined. Results eapreaaed as »SO calculated by the Spearman-Korber method. 95 ~ confid~ce limits within 53-183 ~ of exPalmental vahles. #Combinations showing values aigniflcantly d~erent using the two injxtion rouiea. In vivo neutrollmtfae White mice (Commonwealth Serum Laboratories white) each weighing 22-25 g were need for these e)lperimeata and physiological saline was used as diluent for veaoma and aativeaenes . Thes.c.andi.v. r u3 "'a of oath venom were deba~miaed by injecting into groups of 4 mice, 0.5 ml vohmxa of venom dilutions decreasing by 20~ (v/v) intervals. Deaths were recorded within the observation period of 48 hr ; IDSO'a were calculated by the Spearmaa-Korber method (Sr$e~urr, 1908 ; Kex»e, 1931) and 95 ~ confidence limits (Hteowtv, 1961) estimated. For the in vtvo neutralization, bent groups of mix were injected aubcutaneoualy with a constant amount of venom, and immediatelyy afterwards intravenously with varying emoemta of aativeaene in order to determine the neutralizing capadty of the antiveae~e. The method used was based on that of Mn~r1oN (1967) . N tlon teats were performed at two levels of venom, approximately 2 and S s.a IDS o (in the case of N. scutatus venom, 4 Ipso in the majority of experiments). Neutralizatia~n with the Japanese Sea Snake Antiveame was dcbaminedusingthe 2 LDs o venom level only.Groups of 4mixwens injected aubcutaneoua~y into the shoulder region with 0.05 ml quantities of venom solution and immediately afterwards (within 5-13 sec) injected intravwously with 0.2 ml quantities of serial doubling dihltio~ of the anttvenenea. The aativenene dilution neutralizing the test v~om done, whmtheanimalswere observed for48 hr, wasnocorded . The effect of increasing the time inteaval between envenomation and antivenene adminiatratlon was examined using N. scut4rtus, E, schirtara and N. nu;/or venoms andN. scdatus antiveaeae. aronps of 4 mice wore injected aubcutaneously at one of the two levels of venom (approx. 2 and 4 >:~s o), and at varying times . up to 2 hr later injected intravenously with serial doubling d>Yutionn of antiv 8D3 é s were cakvlated by the Spearman-Kartier method and 95 ~ oom8denoe limita estimated. 70XICON 1976 Yd. lI

a g. BAxT$R and I~AT~aR a aA1.r icHIo

350

In vitro mutroliwtion Tests wea~e performed by in vitro combination of antivenene d(7utions with the test venons, subsequently injecting mixtures ~ntaining approximate>,y flue lethal doses into mice as previously desc~bed (BextEa and ßuucmo,1974) . Calclrlatiml of venom >'eeutralked

Neuhaliud venom figutea weae derived, for both to vitro and in vivo determination by calculatIoa in the form : Venomneutralized by theantibody dose =venom teat dose minus 1 i nse . This is wnsideredto be the most realistic expression of neutralizing potency of an antivenane . A similar method of expressing this parameter was employed as long ago as 1933 by Moaonx (193:1). T~$ 3.

EFrBCr

ov

~zmx LsveL ox rxo~cnve roa~rcsr or N. scutartw sxnv~e SEBT OODWCT® A2

YYOII

91aDll OOBE APpsm. ~SO (p9)

71a1,~ Ii9R.

6D SG

VI~JIOII04B6

m30

(3171 SU laval) a

APOxO~ " (M9) ~SO

(SLD.~ Laval) ~

1 nl M<"t~ nancra]SSU"~ 1

5LD50 I89EL

u ML1"aYags na4trallua"

49

49

Alprsa .W 4a44

17

s.77

]o

a .n

3oe (777 - 175)

1]7 (739 - 15e)

Aataetla a2oiulC

1B

7.7e

10

19 .00

1517 (971 - 7751)

670 (179 - 10]9)

EaA~a,Laa acA4tela

1D

6.77

2S

7B .e7

109 (795 - Se4)

311 (776 - 1]])

Nrdxepi4 eruaelACtw

30

].]6

77

1] .11

7079 (1197 - 7e19)

1797 (1790 - 74731

NrltepALa tte~saa

70

3.36

SO

13 .11

1371 (1095 - 70991

177e (ee1 - 1e551

Y. rd1e~ACa

30

7.1i

15

17 .9e

N~~ pa~Laetw

70

l.fe

SO

i.77

NrfaoPAla

10

6.77

73

76 .e7

lap~~`~C

]1

l.fe

s0

8.77

7]ee (1705 - ]76e1

767e (lb7 - 3677)

Latleaada aul(uetata

li

S.sS

10

7e .e7

S30 (3B0 - 771)

151 (3]S - e77)

NteaoelphaLepAta ru..n7n

7f.D .

".D.

f0

1] .11

0.17

7S

O.B1

wLUa

YotaeAta aeniatua

17 .5

(170 5,1BOS)

()7f 1~9699)

(leSeSe13363)

flBe6 3/1 3737)

331

(711

161)

ti.O.

5519 (]756 - 9377I

(71]

396

1091

1357 (977 - Le73) e111 (sOSe - 11e11)

"units N, acutatus antlvenene (T'iger snake Antiv~e Batch 028-1, 380 units per ml). Results exprossed as im5B calculated by the Spearman-Barber method, 95 ~ confidence limits 59-169 ~ of experimental values. ~Reaulta çalculatod from BDso values above. Figures is parentheses are 95~ confidence limits (sea snakes 64-1 SS % N. acutatrcs 59-169 %~. N.D. ~ not determined. RESULTS

The results found in determining L.nsa by both s.c. and i.v. administration routes on all venoms used in this study are shown in Table 1 . Neutralizing potency of the three Australian antivenenes against a range of sea snake venoms investigated by in vivo protection tests and a comparison with previously obtained in vitro combination tests appear in Table 2. Effects ofenvenomation level on the protective potency of the three Australian antivenenes tested against a range of sea snake venoms are shown in Tables 3-5 . These tables also report the calculated in vivo neutralizing potency of the three antivenenes at the two levels . Table 6 shows the effect of varying time interval between envenomation and administration of antivenene. As could be anticipated, there is a relationship between delay and effectiveness of antivenene. TOXICON 1976 Vot. l1

Protection Against Sea Snake Bnvenomation TABLE 4. F.FFSCr OF

331

E. Jch~ftOJa-N . JCIltatuJ

HNVSNOiNA1ION IBV~" ON FROTSCrivB P018NCY OF

AN1iVSN~f¢

xaer eoeroucrm Ar vaw~

vnaa~ Doss

®s4

vamaK Doss

7ûy~ 1u91

( 1~s10 ) "

~0 Iu91

ALp,aaAaa LtavU Aaüa .Ua at0lea41

17

7rasD rsvet I la~ll 1 "

smsD uv~.

u Antltanw namu~3la~y 1 u aativavw~ n~uls~11au1 u0

uf

é .77

70

71 .f7

777 (710 - 1f0)

75f (i5f - If1)

l]fl b)f - 701f)

Sll 17if - 705)

iii (17f - 3f7)

i10 (151 - 7ri)

lffl (lifl - 77511 1f11 ülfi - iif5)

1f74 (11D1 - 7371) 1577 (107i - 1707)

370 (7f0 - 771j

" 70f (777 - 177)

1fq (175f - 7017) iss (la - 7fsJ iS55 (1f10 - 75if)

17f7 (1114 - i770)

101 (775 - Sf0)

IK (750 - f711

lé

7.f7

10

71 .f7

E~kriltwa aa tesa

10

11 .)0

75

7f .00

xfüePk4 apfwfaLsaLw

70

4 .00

75

1i .00

NpdAepil] etyw spQ."eP%.U ~ajae

30

7.7f

SO

31.70

30

7 .ff

15

7f.f7

NpdwPfJ+ .yaeetaatss

i0

i .lft

f0

f .50t

ffdaapf4 acuaua .ctu

u

f .wt

ls

a.f7f

lapu4 Aaad~LaYL.C L" s~"" -w" aut{ue .(a.4 r~ """ eep(u.fopkG fü~La ~ Notte44a aasdtfa

i1

l,if

{0

f.71

lf

7 .f9

10

if .f7

fLD.

" .D .

f0

S1.Ilt

17

0 .47

75

O .f1

t.D. SIf7 (77li - l77fI

alf (770 - Iul if7S (7041 - 7fIi1

14f1 (1051 - i0i01 f079 (Iff7 - 11i151

'Units Sea SnakeAntivonene (Batch 024-1, 410 anti-N. JcutatuJ units perml, 28 anti-E. schirtosaunits perml . tHatch 026-1, 410 aati-N.JcutatuJ unitsper ml, 30 anti-E. JchLstosa unitspa ml used for these combinations). Results expn~sed as aDSf calculated bY the Spearman-Karba mothod. 95 ~ confidence limits SS-183 ~ of experimental values. Results expressed in anti-N. JcutatuJ units. Results calculated from BDsf values above. Figures in parentheses are 93 ~ confidence limits (sea snakes 64-133 % N. acutatrts SS-183 %~. N.D. m notdetermiaed . The Japanese Sea Snake Antivenene neutralized all venoms tested . Results are shown in Tabla 7. DISCUSSION

For all venoms tested by the comparative routes, the 1,D6o values appeared lower for the i.v . route; however, statistical examination showed that in only six of the sea snake species was the i.Dbo by the i.v . route significantly lower than by the s .c . route (95 ~ confidence levee. It was of interest to note that the ratio of s.c . to i.v. lethal dose did not depart far from unity for the sea snakes, whereas the one land snake included (Tiger Snake, Notechis scutatus) had a ratio of approximately 3 times, comparable with the ratio of 7 times found by MORGAN (1956) for the Taipan (Oxyuranus scutellatus). another terrestrial snake. CABBY and WluaFrr (1960) claimed that, in neutralization of E. schlstosa venom by the homologous antiserum, in vitro neutralization was at least twice as effective as in vivo neutralization . With this venom antivenene combination, using larger numbers of animals and analysing the results statistically, we were unable to confirm this finding (Table 2) . In only four of the 33 venom-,antivenene combinations tested was there a significant difference between in vitro and in vivo combination. In three of the four, in vitro combination resulted in better neutralization than by the in vivo method . There are numerous combinations in this table where, on the basis of experimentally determined values there may appear to be a difference in results between the two methods, but when examined the differences are not T02TCON 1976 Yol. I~

332

E. H. BAXTER TASt$ S.

EFFECl' OF

and

HEATHER A. C3ALLICEIIO

BNVE1W01~lA7iON LEVEL ON F"ROTPCr1VE FOIENCY

O! E.

aCI1Lf10.fR AN'rIVENEtVB

!LS! f701m0(.Tlm AT ZIDSO L6yR.

VF]1011 VR1011 ÇO$ç

®SO

V771011

2~~ (v91

(27D 30 laval)

Appsos. S~SO (v91

OOSIL

~s9 (SID SO lral)

1 al AntLialroa aaotrallarai

S1oS0 12YB. 1 al Aotüaosa naotralluri H9

1.9

Alyyaaaaa 4avi1

1]

1.37

JO

].Of

105 (70 - 1f91

lif (130 - 339)

41ta0tia atetaali

1f

0.f0

10

1.0f

]tl (244 - 5911

277 (171 - 327)

Eah~d.Wa IahLatraa

10

0.97

23

].tf

707 (14f - 3001

137 I11] - 7171

NydxrphU eyaaee.inetua

30

0.40

75

1.97

7]t 1473 - 1144)

t!7 1f4f - L77t1

Nydaoplw atataaa

30

0.97

SO

7 .30

]79 (77] - 321I

Slf (331 - 7S])

NydLephll aa(uL

10

0.40

23

3 .]0

397 ß11 - 4041

3]! (lil - 7191

Nyd4rphia algaotiwatua

30

0.77

SO

l .tf

]31 1271 - 443)

397 (311 - 404)

NydtepkU aLL-letleo[Lu

10

1.97

25

1 .06

t] (60 - 1131

140 (107 - 304)~

Lapaala haadri. atli

31

0.39

f0

1.]7

!tf (f70 - 14401

93f (5!7 - 1433)

LatLtaada ItaL(4 " %""^

1f

3.77

40

7 .73

!4 l44 - 1371

117 (tl - LSf)

YLUOaaphaLrph4" BxaaLLG

l1 .0 .

D .O .

60

l.tf

N.D .

]7! (344 - Iftl

NOtechia atatatu

13

0.34

30

O.ft

f]7 (45f - t79)

947 (fOf - Slft)

Units Sea Snake Antiveaene (Batch 013-i, 27 .3 units per ml). iuaulta expressed as ~9o calculated by the Spearman-Karber method. 95 ~ oonfldenoe limita 64-135 ~ a~f cxperlmental values . ~Reaulta calculated from ED9o valses above. Figures in parentheses are 95~ oonSdenoe Wnita (all specks

64-155 %~. N.D. ~ not determined . TAa>~

6.

LdPW ..a

Tlaa of a~inlatratioa o! antiVwana altar aaraaoaatlm (aln)

OF

VARYBVß T'n~ IN1~tVAL HErVVBBN SNVENOMA170N AND ADII~rRA'IION

Nrt:eüA aeutatul ya0
0

0.42

NotacüA acutatul Vaoaa VaoO" doaa 7S y9 " Delta N. aeatatu antinnw aaatrallaioq 7S p9 " O.t4

Eahyd24a 4cüateas Vmo. Vaao" doaa 10 pq. Unita N. 4eatatW antlTaeasa aautral]alaq 10 p9" 0.73

2

f.73

S

4.73

7

scutatrcr

NydLOphG aa1~04 Vamo~ Weoa doaa 10 u9 " un1U N. Iea4Lu aatlVaaaaa aaatsalislnq 10 pg . ].99

f.73

10

f.77

13

0 .50

1.19

30

0.70

1.00

f0

l.ft

170

OF N.

"

"

Tiger Snake Antivenene Batch 028-1, 380 units per ml, was used in all studies Venom doses given aubcutaaeously, antiv~ doses given intravenously. 'Mice moribund or dead at time duo for antivenene administration.

statistically significant. We strongly urge that, in reports of this type of work, investigators treat data in this way, and avoid findings apparently inconsistent with those of other workers. 7bX1CON 1976 Yd. 11

Protection Against Sea Snake l nvenomation

353

TABLE %. IR YIIO NEITfRALiZA170N 9Y P4LYVALBNr 38A aNAKS AMIVENENE' V~iOl1 Ai.pyauxue fatvLa

1 al Sea 8naka nntivaneaa Nantralised 49

~SO

16 (71 - 67)

7

76B (190 - 377)

11

102 (77 - 141)

37

Hydxapki.a cyaaecia~c#w

570 (369 - 733)

IS

Hydxaph.i.a elegaiu

4S7 (307 - 660)

69

Hydxopl~La ~ajox

116 (101 - 706)

30

HydxopA.i.a a.ipxeciac.tw

457 (792 - 708)

S3

17 (30 - 59)

10

Aa,ttotia aloLüü fakydx.i.aa aek.iatoaa

Hydxopkia atx.LcticotUa Lapu.Ea Aaxdrrick .li.

1314 (933 - 1~53)

97

La.Ucauda aut{ue .(ata

s6 (10 - 79)

7

Neteak .U acu.ta .tua

56 (33 - 95)

!

Venom doses given aubcutaneously, antiveneae doses given intravenously. Teats conducted at the 2 iDse venom done level. Rewlta expraaed as ~, o calwlated by the 3pearman-K~rbe~ method. 95 ~ conûdence limits within 64 and 1SS ~ of experimental values for sea snake veaoma, and 59-169 ~ of experimental vahua for N. scrrtatus venom. 'L. hardwickit, 8 . cym)octnetrer and L. sem(fmdata venoma used u hyperfmmuni~ng antigens. No consistent level of envenomation has been adopted by investigators in assessing the protective value of snake antivenenes, and in the course of this present work, opportunity was taken to compare the effects of conducting the tests at the 2rnao and Si.nao levels . It was thought that the effects of such rapidly acting venoms (mice dosed at the higher level with sea-snake venoms show severe signs of envenomation within a few min of injection) might not bo proportionally reversed by antivenene when the higher dose was applied. As is evident in Tables 3-5, in only one combination (dstrotia stokesü venom with E. scMrtosa-N. scutatus antivenene) of the 33 tested in this way was a statistically significant difference seen. The result for the combination of this venom with N. scutatus antivenene is very close to significance, and perhaps adds to the uniqueness of this venom in this series. The occurrence of the significantly different result suggests that it would be advisable to conduct these tests at two envenomation levels, and again emphasizes that results should be statistically examined, as in six combinations outside the significant one, experimental results obtained at the two levels appear to differ by a factor of 1"5 to 2 until so analysed. As could perhaps be anticipated, antivenene is somewhat more effective, unit for unit, the earlier it is administered during the course of envenomation, and this could be argued as a case for giving antivenene without waiting for development of envenomation signs and symptoms before assessing the nced for antivenene . However, the difference is not so marked as to outweigh the responsibility of the clinician in deciding whether the foreign protein sensitivity risk is greater than the risk to life by the venom. Comparison of the Japanese antivenene (sea-snake venom derived) with the Australian 71~XICOIV 1976 YoL 14

E. H. BAXTER end HEATHER A. QALLICHIO

334

SLVx~ xxnvswaxa xrro Sax Sx~

Txsr~ 8. Couu~xturlva rorer)cr or Tm®t

sex sNxs~ va)vo~

~m xsvlxxcJZUVo

Xeutralisation pq Vanom aautralized by 1 al oi Ratiw 6ea Saaks Sea Baalca iiger Snaka Antivenene~ nntivenene 1lntivaaaaa Venus Bpeeiee TB S8 (T8) (EakgdR .i.na (POlyval.eat)+ ~$ J~$ dCkl .itOda)? (J88) (88) Ai.pydutiud latv .i.d

2.8

6.7

4.0

5.7

2.0

4 .0

2 .8

4 .0

4 .0

3 .4

2.0

4 .0

457

8.0

S.7

83

42

4.0

8.0

2368

986

1314

2.4

1.8

530

94

5.6

9.5

109

46

381

268

203

102

738

520

1521

379

452

584

393

>:46

2581

321

334

308

Aa .ttot+.a dtokedü

1517

Hydtiopkda cyaaottactud

2079

Hydaopk.i,a cltgaaa HgdROpki.a aLg+toc.Eactud

Eakgdetaa dch.tdioea

Hydxopki.a safoa

Nyda.opkC6 ataittitoLlLa Laptata kaldwEch,ii

La#i.tauda deeE4aacia .ta

409

56

Tests conducted at 2 IDSO venom dose level Columns 2, 3 and 4 figures extracted from Tables 3, 3 and 7. 'T'iger Snake Antivenene Batch 028-1, 380 anti-N. acutatur units per ml. tSea Snake Antivenene Hatch 013-1, 27 .3 anti-E. sclrtstosa units per ml. Sea Snake Antiven~o (Japanese) prepared using L. lwrdwickü, H. cyanocinctus and L. semifasciata venoms .

antivenenes, particularly those derived from terrestrial venom, is difl~cult. It can only be expressed as relative effectiveness against different species of venom of 1 ml of each of the materials (Table 8). This can be achieved by comparing the experimentally determined neutralization figures at the 2 Ln5 u venom dose level, and selecting as base the ratios for neutralization of the principal venom used as antigen in preparing the Japanese Sea Snake Antivenene, Lapemtr hardwickit. It is evident that the Australian terrestrial snake antivenene (Tiger Snake Antivenene) is comparatively more effective against all the remâining venoms tested than the polyvalent sea snake antivenene. A similar comparison reveals that the terrestrial snake antivenene is comparatively more effective against the majority of the venoms tested than the monovalent (E. schistosa) sea snake antivenene . These studies show the superiority of Tiger Snake Antivenene in protecting against sea snake envenomation, and that no advantage derives from a product resulting from hyperimmunization with a mixture of N. scutatus venom and E. schistosa venom. Therefore there would seem to be mounting evidence that there is no case for including venom of a sea snake species in antigen used for producing sea snake antivenenes. Immuncelectrophoresis of E. schistosa venom against each of the three Australian antivenenes reveals exactly the same precipitation pattern, and this seems to further indicate that the monovalent anti-terrestrial material should be adequate. .lck~eowleulgements-The authors are grateful to Drs. H. A. R.am, Ar)rsornr Tv, Burma Pharmaceutical Industries, Miss VAL Lxwsor) and MI. Enlc Wolelu~, for the supply of venoms, to Dr. Txxxs~ O>roxoal for the gift of the antiveneae, to Mr. W. Pbwosa for statistical advice and to Mr. K. Cixxvnn~ and Mrs. M. Dowrrav for technical assistance. R F.FFRF.1~jCE,S

Bexras, E. H. aad (3xlucmo, H. A. (1974) Cross-neutraliation by tiger snake (Noteclris scutatus) antivenene and ace snake (Enh~drfms schlstosa) antivenene against several sea snake venoms. Toxlcnn 12,-273. TOXICON 1976 Yo1. I~

Protection Against Sea Snake Envenomation

353

B. W. (1%1) Somo properties of the Spearman estimation in bioassay. Biometrika 48, 293. Cexssr, J. E. and Waxeex~r, A. 3. (1960) The toxicity and immunological properties of some eeasnake venons with particular reference to that of Fthydrüee schistose. Timrs. R. Soc. trop. Mad. Syg. 54, S0. Iüa~y Ci. (1931) Reitn3g zur kollektiven behandlung pharmakologisher reihenversuche. Archs exp. Path. Phar»mc. 162, 480. Mrrrc+ox, 3. A. (1967) Paraspecißc protection by elapid and sea snake antivenenea Toxicoet S, 47. Moaowx, F. d. (1938) The venom of Notechis sceuatres var eeiger (Reeveaby Island). Proc. Roy. Soc. Yicr . S0, (In, 394. Moao~uv, F. Ci. (1956) The Australian taipan Oxyeeranur acuteJlatussceetellatees (Peters). In : Vereoms, p. 359, (Buce~Br, E. E. and Poxae3s, N., Eds.) . Washington : American Association for the Advancement of Science. OYOreoor, T., Ii~z-roAr, Z., Ezavgo, A. S~w~r, Y. and Iüwnetv~, Y. (1972) Studies of immunity against tho venom of Lapemtr /eandwickil-with a special referonce to a pilot production of therapeutic antiveaene horse serum. The Snake 4, 84. SPBARMAN, C. (1908) The method of "right and wrong cases" ("constant stimuli's without Claus's formulae. Br. J. Psychol. 2, 227. Tv, A. T. and Cixxrsavoxx, S. (1969) Immunological properties and neutralization of sea-snake venons from southeast Asia. A»e. J. trop. Mad. Hyg.1 8 (1), 151 . T[r, A. T. and S~xca, E. S. (1974) Immunological properties and neutralization of sea snake venons (II). Am . J. trop. Med. Hyg. 23 (1), 135. BseowN,

TO.TICON 1976

Yoe. l~