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Tua~cnn
1966, Vol
3
pp
297 100
Per~anwn
Press Ltd
Pri nted
i n Gr cet Ar i t ai n
QUANTITATIVE PROTOZO NBIO-ASSAY
METHOD
FORDETERMNNG
VENOM
POTENCIES
BosDJ ol l xs ox, J oxxCTULLAR
and
H
L
STAxxx
Ar i zona
State
Uni versi t y,
Tempe
Ar i zona
Accept ed j or
pebl l cat l on
30J ul y 1965
Abstract A
si mpl e,
r a pi d
pr ot ozoan
assay
method requi r i ng
smal l
amounts of venom s
descri bed
L e t h a l i t i e s
of
Crot al es
atrox, Cr otal es
s c u t u l a t u s
Cent r ur of des
s c ul p t r v a t e s
Hadrurus a r l z o ne ns i s and Hel oder ma
suspect um
venom deter m ned by
t h i s
method
are
compared
w t h those obtai ned
i n
m t x
INTRODU TION
STATISTICALLY
V a l i d
t o x i c i t y
t e s t s
usi ng
mammal s
r e qu i r e r e l a t i v e l y l a r g e amount s of
venom
The
use of
smal l q u a n t i t i e s of venomo
det er m ne
pot ency
s
necessary i n t e s t i n g r a r e
venoms
and
venom
r a c t i o ns
PH LPOTT [1-4] u t i l i z e d
protozoa
i n measur i ng t he r e s i s t a n ce and
potency
of venoms
and
t o x i n s
and t h e i r
ant i bodi es
LACALLADE [ 5] measur ed
venom
potency
l
t erms of Par ameci um caudat unt Ehr enber g c y t o l y s i s I n c o n t r a s t MNTON
[ 6]
used
P c a u d a t t t t t l
s u r v i v al t i me s t o determ ne venomp ot e nc i e s : These s t u d i e s
wer e
sem - quant i t at i ve
Thep o s s i b i l i t y of
s i m l a r r esponses bet ween protozoa
and
met azoa t o ot her
t o xi c ol o gi c al
s t i mu l i such as car ci nogens
and
a n t i h i s t a m n es
has
a l s o
mot i vat ed
t he
use
of
protozoa
i n
scr eeni ng
t e s t s
measur i ng r e l a t i v e
potency,
determ ni ng
mode, and s i t e
of a ct i o n
and
i d e n t i f y i n g
s i de
r e a c t i o ns [ 7 9 ] I n t e r p r e t at i o n of
dat a
i n
t erms
of l i m t a t i o n s andval ues
of
t o x i c ol o gi c al pr otozool ogy
s
di scussed by HuTxER
[10]
W
r e p o r t
here
a r e l a t i v e l y
r api d pr ot ozoan assay
met hod desi gned
t o measur e l e t h a l i t y
L e t h a l i t i e s measur ed
by
t h i s assay met hod are compar ed
w t h
those
obt ai ned i n
m ce
M TER LS NMETHODS
Sampl es
f r om
l a r g e pool s of l y o p h i l i z e d
venom
f r om t he r a t t l es n ak es Cr o t a l u s atrox
Bai r d andG r ard, and Cr otal us s c ut ul a t u s
Kenni cot t , andf r o mt he
G l a
monst er
Hel oder ma
s u s p e c t t n n
Cope,
and
f r om the scorpi ons Hadr t r r us a r i z on ens i s Ew ng,
and
Cent r ur oi des
s c u l p t t t r a t u s
Ew ng,
were used
Sampl e wei ght
was
cal cul at ed by aver agi ng wei ght s f r o m
t h r e e
consecut i ve wei ghi ngs at 30- m n i n t e r v a l s usi ng a Met t l er
a na l y t i c al
bal ance The
venoms
wer e
r e c on s t i t u t e d
i n
s t e r i l e wheat bro th medi umor d i s t i l l e d wat er i mmedi at el y
pr ecedi ng
each
exper i ment
Al l
LD60 wer e c al c ul a t e d by t he Thompson
and
Wei l
t echni que The Par ameci um
n t u l t i m c r o t : ucl eat umPower s andM tchel l LDaO
at 6 hr
expr essed as
mg
per m of medi um
was
deter m ned by
i nocul at i ng
f o u r
par ameci a i n t o
each
dosage l e ve l
of venom
e co ns t i t ut e d
i n
s t e r i l e
wheat
bro th
The
mouse
LDo at 24 hr expr essed as mgof venom
e co ns t i t u t e d
Suppor t ed
byNat i onal I n s t i t u t e s
of Heal th Gr ant
NB 02330
297
G
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? ~l HO
t )
JOHNSON JOHNc t ULLARand
H
1 STAHNKE
i n de- i oni zed
d i s t i l l e d
wat er per
kg of
mouse,
was
determned
by
i nt raperi t oncal I P I
i nj ect i ons [11-13]
1 he
c l onal cul t ures [ 14J
of P
nur l r i nl i r r o r J UCl ec r l um wer e
mai ntai ned1
i n : mucl avcJ
wheat broth
medi um
i nocul at ed w t h Pse~udurr rurr cr s ueru ~nusa
p hi s b r o t } i consi sts of
h
wheat
grai ns
per 100
m of
synt heti c
spri ng wat er
I S]
Asepsi s
of
cul t ure
medi um
was
mai nt ai ned
up t o
t he
poi nt of
subcul t uri ng Al l
cul tur i ng
and
t e s t i n g of parameci a w~~s
conduct ed
a t
24- 25 Al l
gl assware was
c l eaned accord i ng
t o
procures out l i ned by
SoNN_twttN [ l 6 ] and aut ocl aved bef ore use
Pr i or
t o
each
exper i ment t he
P r u r r l t i -
n~ crorurcl eatum
wer e washed f our t i mes
i n
s t e r i l e wheat broth by cent ri f ugat i on at sl ow
speeds f or 6
mn After washi ng,
m of concent rated washedparamec i a was pi pet t ed
i nto : ~
Maxi mov
depressi on
s l i d e
Wt hi n
3 mn
these
par ameci a usual l y
aggregat ed
i n t o
a
r i ng
j ust
bel ow
t he
sur f ace
near t he
edge
of t he medi um Rapi d
transfer of 4 par ameci a from
t h i s aggregate t o u depressi on s l i d e
cont ai ni ng
0
~2 m
of
t e s t
medi um
w~ s done
w t h a
n t i c rol oop [ 1 7 j
Each
bi o-assay consi sted of
a
mni mum
of 4
dosage
l e v e l s
w t h 3
r e p l i -
cat i ons per l e ve l I nocul at i on of
each venom
dosage l e ve l was
compl et ed
before i nocul at i on
of
t he
next
hi gher
l e ve l
was
begun
Thus,
64
paramec i a
( 4
per
s l i d e )
were used
i n
each
bi o-
assay Two s e t s of cont rol s
wer e used
The l owest
dosage l e ve l i n whi chno
par ameci a
di ed
served as t he t e s t cont rol s
Paramec i a
i n venomdi l uent
s t e r i l e
wheat broth) served as
cont rol s of experi ment al condi t i ons
Resul t s wer e recorded af t er a 6 hr i ncubat i on
peri od
Four heal t hy
al bi no
mce
( 2 mal es
and
2
f emal es)
wei ghi ng
from
15- 23 g
wer e
used
per
dosage l e ve l
i n determni ng
t he t
. v
b The
mce
had
no
food
f or
24 hr ~t n d no wat er f or 2 hr
bef ore
wei ghi ng
After
wei ghi ng,
t he
mce wer e
i nj ect ed ( i P)
I nocul at i ons
wer e
made
fromvenomstock sol ut i ons of 5 mg per m Food and wat er were made avai l abl e 2 hr
a f t e r
i nj ec t i on
Observat i ons of mce a f t e r
i nj ect i on
wer e
f or
24 hr
These
exper i ment s
wer e
conduct ed
at
24- 2i ° and 30 per cent r e l a t i v e
humdi t y
RESULTS
Mce
responded t o
ol eos
venom
w t h rapi d i ur c e d respi rati on and hypoact i vi t y
Edema at t he
s i t e of
i nj ec t i on
was
observed
Autopsy showed t i s s u e dest ruct i on and
hemor r hagi ng
P
rr ur l t i rn i cranucl eat~urc responded
t o
t h i s venomw t h avoi di ng react i ons
f ol l owed by
c y t o l y s i s
and c e l l f r agmentat i on
I n both
par ameci a and mce, C scutul atus
venomcaused responses
si ml ar
t o
those
produced by C atro. ~
venom Less
venom
however ,
was
needed t o
produce
t he responses
M ce became hypoact i ve a f t e r
i n j e c t i o n
w t h H
srrspechrm
venom
aut opsy showed
venous di stensi on and hemor r hagi c l ungs
No
noti ceabl e par ameci a
responses wer e
observed except
w t h hi gh concent r at i ons of t h i s
venom
These concent r nt i ons of
venom
caused
hypoact i vi t y
;
sw mmng
gradual l y decreased
unt i l
death
C
scul pt rr ratus venomcaused convul si ons
and
copi ous sal i vat i on i n
mce
No
gross
t i s s u e dest ruct i on was
observed after
aut opsy
The
responses of P r r rul t i mc ronuc
l eatum
t o
t h i s venom
wer e
t he
f ormat i on
of l engt hy
prot opl asmc
ext rusi ons
and
an e r r a t i c swmmng
behavi or f ol l owed
by cessat i on of movement
Subsequent
mechani cal
sti mul at i on
i nduced
br i e f r esumpt i on
of swmmng
I n mce,
l l
ar i z onc r rs i s venomcaused
spasms,
i ncreased sal i vat i on, and
paral ysi s
of
l i mbs ;
aut opsy
showedhent orrhagi c l ungs
P
rncr l t i r ni c roni r c l eatur r t responded
w t h
avoi di ng
react i ons,
e r r a t i c
sw mmng behavi or, and f i n a l l y ,
f r agment at i on
I n Tabl e
l
venom
e t h a l i t i e s
f or
mce
are
compared
w t h
those obt ai ned by usi ng P
rnrr l t i mcrornecl eatr ur~
I n
mce, t he order of decr easi ngvenome t h a l i t i e s
i s
Csctt l pt t rratus,
C
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Quant i tat i ve Prot ozoan Bi o- assay
Methodf or Det enm ni ngVenomot enci es 299
s c ut ul a t u : suspectum a t r o x and a r i z o ne ns i s Wt hP mul t i m c r o nuc l e at ur n
t he
order of decreasi ng
l e t h a l i t i e s
i s
a r i z o ne ns i s
s c ut u l a t u s
a t r o x
s c ul p t u r a t u s
and
suspect um
Tnar a 1
Coedrnrurt va v s r ~ o e t
rsr rt wurn s
P m
r
. n
and I
ar e i nmgper m ; musecn and I ar e
i n
mgper kg
t
Parameci ummul t i mcr onucl eat urn
DS USSION
Due t o t he hygroscopi c
nat ure of
l y o p h i l i z e d
venoms t he wei ghi ngs
wer e
per f ormed
i n a
l ow
humdi t y chamber
t o
mni mze
wei ght f l uc t u at i ons
I n
both
mce
and parameci a Cs c ut u l a t u s
venom
wasmor e l e t ha l
t han
atrox
venom
Except f or t h i s
r e l a t i on s hi p no c o r r e l a t i o n amng a t r o x C s c ut u l a t u s suspectun
s c ul p t u r a t u s and
a r i z on ens i s
venome t h a l i t i e s
i n parameci a and
mce seems
evi dent
At
p r e s e n t
i t woul d be
pr esumpt uous
t o
concl ude why
t h e s e
condi t i ons
e x i s t
However
t he
s i mp l i c i t y
of
t echni ques
i n vo l v ed and t he hi gh consi st ency of r e s u l t s obt ai ned
duri ng
t he
p a s t
year w t h
t h i s
prot ozoan assay met hod seemt o i n d i c a t e i t s val ue i n f u t u r e s t udi e s
concerned
w t h
determ ni ng
modes of acti on
and
l o c a t i n g s i t e s of act i ons of
venoms
SUMM RY
1 e l a t i v e l y
rapi d
prot ozoan
assay
met hod
was
devel oped
and
used to q u a n t i t a t i v e l y
measur e
a t r o x
s c ut u l a t us a r i z o ne ns i r
s c r r l p t
: r r a t i t
s
and
s u s p e c t t t m venom
p ot e nc i e s
2 O
t he 5 venoms s t u di e d s c ul p t u r a t u s
venom
was
t he mos t
l e t h a l t o
mce
and
N
ar i z o ne ns i s
venom
was t he l eas t l et hal
3
I n
parameci a
a r i z o ne ns i s
venomwas
t he
mos t
l e t h a l
whi l e s c ul p t n r a t u s
venomwas onl y
moderat el y l e t h al
4
H
suspect um
venom
was r e l a t i v e l y nonl et hal t o P
mul t i m c r o nu c l e at u r n
~I ckrrow edgeneent- W
t hank N Ser
. saNO
f or hi s advi ce
Our
appreci at i on goes t o
M
T t r t t
A t t
HRJ OHNSONandGL Lrr t s f or t h ei r a s s i s t a n c e
REFEREN ES
I l l PampoTr E f f o c t
of
t oxi ns
andvenomoa prot ozoa
J
exp Zool
56 167 1930
[ 2 ]
P F I I L P 01 r Usa
of
prot ozoa i n measur i ng t he n e u t r a l i z i n g
val ue
of
cobra
ant i serum
Sci ence N
Y 74
157
1931
[ 3]
P t u t rorr
Rel at i vo
r e s i s t a nc e
of
fourteen
s p ec i e s of prot ozoa t o t ho act i on
of
Crotal es
a r r o r
and
cobra
venom Mart r e Bi o l Bul l Mar t he
Bl o l Lab
Woods Hol e 60 64 1931
Venom
T e s t
ani mal
r . n
95
Conf i dence
i n t e r v al
Route
of
i gj xt i on
Hadrurus
Pmt 030 0
25- 0
35 Medi um
a r l z on en si s
Mouse 168
0
99
0- 285
I P
Crotal us
P
. m 5
049- 0
55 Medi um
s c u t u l a t u s Mouse 1 8 1
2 25
I P
Crotal us
P
. m
0
67
062- 0
73 Medi um
atrox Mouse 6 54 67
I P
Cent rur ot des P. m 1 3 1 12- 1 5 Medi um
s c u l p t u r a t u s Mouse
1~5
1 1- 1 9 l
: P
Hel oderma P. m 24 7 20 8- 29 3 Medium
suspectum
Mouse
30 2
1- 4
2
I P
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300 HOB JOHNSON
JOHN
CTULLAR
and
H
L STAHNKE
[ 4] PHLPUTT
CH Nat ural and acqui r ed r e s i s t a nc e
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t o
t he
a c t i o n o f
anti geni c
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J
exp Zool
. 63, 553, 1932
[ 5]
L C I LL DE C
W
J r
The determ nati on
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t he pot ency
of
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venomn v i t r o
nr
J
Phys
103,
251, 1933
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MNTON S A Some
propert i es
of north amer i can
pi t
v i p er venom and
t h e i r corr el at i on
w t h
phyl ogeny
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E
BuctcLeY
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[ 8] WEST R A J r B RBER PW
KoLnR
J Rand
MURRELL
CB The agar l a ye r method f or
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[ 1 0 ] HUTNER S H Protozoa a s t o xi c ol o gi c al tool s
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Protozcw
11 , I , 1964
[ l l ]
THOMPSON
R
Use o f movi ng
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dose
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Rev
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[ 1 2 J
THOMPSON
W
R
and
WEI L C
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On
t he
const ructi on o f t a b l e s
f or movi ng average
i nterpol at i on
Bi onretr i cs, 8,
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[ 1 3 ] Wts
CS
Tabl es fo r
conveni ent
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e f f e c t i v e
dose
w Or
ED
T
and
i n s t r u c t i o n
i n
t h e i r
use
Bi onretr i cs, 8,
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l a WCHTERMAN
R
TheBi ol ogy
oj Parm rec i unr
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1953, p 104
I S ]
K RBY
H
J l ~a t er i a l s
andAf elhorl s
i n t hr
Strrdy
ojProtozoa
Berkel ey
Uni versi ty
of
C a l i f o r n i a ,
p
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[ 1 6 J
SONNEaORN T
M
Methods
i n
gener ; t l bi ol ogy
and
g en et i c s of Par urr recl wr r
a r r r e l i a
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[ 1 7 ] TULL R
J C JOHNSON
B
andST HNKe HL Rapi d
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