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THE REMOVAL OF BOWUNUS TOXiN
FROM WATER

Them for {€19 Qwaas of in. 8.
MéCHIGAN S?A?§ COLLEGE
Aifsad M. Wafibrsfik
E9733

This is to certify that the

thesis entitled

THE REMOVAL OF BUIULINIB TOXIN FROM WATER

presented by

Alfred M1118 Wall'bank

has been accepted towards fulfillment
of the requirements for

M degree in W8?

9 ' _ l. .
K1431 %u\\.- '\ 1 M» \L\,' y \_1

Ma or professor

Date July 93 1953

0-169

 

 

THE REE ‘AL OF EOTULINUS TOXIN FROM U‘TER

By

Alfred M. Wellbank

Submitted to the School of Graduate Studies of Hichigen
State College of Agriculture and Applied Science
in partial fulfillment of the requirenents
for tne degree of

MASTER OF SCIEKOE

Department of Eacteriology and Public Health

Year 1953

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Acknowledgehent

The author wishes to eXpress sincere appreciation to
Dr. W. L. Hallmann for his interest and guidance in these

studies.

Appreciation is also extended to Dr. J. P. Newsan and

Irving L. Dahljelm.

To Richard Hetherington of the Rhom and Hass Company
for the strong base anion exchange resin XE-QB, and
T. L. Gresham of the B. F. Goodrich Company for Beta-
PrOpiolactone the author is indebted for generous samples

to carry out the following exgeriments.

I. PURPOSE

During the past war, crystalline botulinus toxin,
type A was develOped as a bacterial warfare weapon.
Statements have been made that the use of botulinus
toxin as a weapon could be as effective as an Atom
Bomb as far as destruction of humans is concerned.

Vany methods of dissemination are possible sucn
as dusting over heavily pOpulated areas and con-
taminating water reservoirs and water distribution
systens.

Inasmuch as there is little data available on the
destruction of botulinus toxin in water an eXploratory
'study of chemical and physical methods of destruction

ap;eared advisable (1). If a chemical method of des-
truction could be evolved tiat would fit into a water
treatment process, preventive measures would be

possible in case of enemy attach.

II.

REVIEN OF TTE LITLRATJFE

The crystallization and isolation of type A toxin
was accomplished simultaneously and indegendently by
two different groups of workers. Lamanna et. al. (2,3)
reported crystallir e toxin with an L350 of 239.9 x 106

per milligram of nitrogen. Toxin prepare by Abrams

C);

et. al. (4) contained 220 x 105 n.L.D. per milligram of
nitrogen. Eoth toxins, prepared by different methods,
met the usual criteria of protein purity.

Botulinus toxin is 15,003 times as active on a weight
basis as the most toxic drug known, aconitin, and a mole-
cule of toxin is 200 million tiies as tcxic as a molecule
of the dru3 (5). The K.L. D. for a 20 gram mouse is
3 x 10'll grams of crystalline toxin, and if man is as

susceptible on a body wei;ht basis s the mouse, 0.25

91

micr03rams of the pure toxin would kill a 70 kilogram
man (6). Finally, to ex;ress its activity in terms re-
cently used by an official of an international health or-
ganization, seven ounces cistribu ted prOperly WCLlld kill
the entire pOpulation of the world (5).

he toxin mole mile has a molecular wei ht of 900,000.
This brin3s up the question: how does this toxin injure
the tissues of hi3her er Lima.ls? Chemical analysis has failed
to answer this question. The toxin is made up of proteins

composed of the szme amino aciis found in the normal tissue

proteins of the host itself. In the case of type A bot-
ulinus toxin, a complete amino acid analysis has revealed
no unusual chemical groupings that mi3ht provide a clue
as to why it is toxic.

(i

The calculated elementary iormula of tue toxin is:
C40.298 H62.679 N10,472 012,534 P15—17 S123'

Its amino acid composition is represented by the ex-
pression:

Glycine166, Alanine394, Valine406, Leucine708’ Iso-
leucineggo, Prolineeoj’ Phenylalanine64, Cystine SH20'
(Cystine 5-)40, Methionine64’ TryptOphaneag’ Arginine239,
Histidine6o, Lysinep77’ Asparagin61370' Glucine953’
Serine374’ Threonines42’ Tyrosine672 (7).

Comparison of the effects of'botulinus toxin and
curare (8) has appeared in the literature for many years
but Guyton and VacDonald (6) have recently presented work
which indicates teat its action is different from that of
curare. Acetylcholine and nicotine injected intra-arteri-
ally still caused contraction of the muscle after botulism
poisoning. With curare poisonin3 such is not the case.
This indicates a fundamental difference between curare
and cotulinus toxin. Eridence is presented which indicates
that the principal action of botulinus toxin is probably
at the myoneural Junction, thou3h possibly in the tirminal

nerve fibrils.

About the treatment of botulism poisoning Guyton
and MacDonald (6) said,

"Treatment of botulinus poisoning consists

of massive doses of antitoxin, the use of
artificial respiration and in cases of severe
poisoning, the administration of vasoconstric-
tor drugs. The fact that poisoning lasts for
many months makes the results of such treat-
’ment discouraging. The use of artificial
respiration for several months or longer is
not practical, and if a patient is poisoned
sufficiently to require vasoconstrictor drugs
he will probably die anyway. The only real
salvation seems to be the early use of anti-
toxin in doses greater than 100,000 units of
multivalent serum. Though antitoxin has been
shown to be of value for guinea pigs as long
as two days after poisoning, it is still true
that its effect decreases eXponentially with
time. One must remember that once toxin has
reached the nerve ending and produced its
damage this action is irreversible for many
months."

Lamanna (9) observed that type A botulinus toxin will
cause hemagglutination of a red cell suspension. After
this work was published many believed that this method of
determining toxin activity would replace the eXperimental
animal. Since then Lamanna (10) has found a lack of iden-
tity between hemagglutination activity and toxicity of the
toxin.

There have been previous reports in the literature
of attempts to inactivate tie toxin. Abrams (4) found
that at room temperature the toxin was most stable between
pH 1.0 and 6.0 with maximum stability between pH 4.0 - 5.0,
while above pH 7.0 the toxin was rapidly destroyed. He

found also that a temperature of 60 C. at pH 5.0 was

'sufficient to destroy its activity. Their method was
most peculiar in that they treated the toxin wits form-
alin for one hour at room temperature and then refrig-
erated it for 18 hours before testin3. Samples treated
with hydrochloric acid were kept at room temperature for
one hour and a half and tien refrigerated for 18 hours.
This may have been done because they were testing
hemagglutinating activity of the toxin as well as toxicity.
Jude et. al. (ll) reported on their efforts to inactivate
the type D botulinus toxin. Type D toxin was sensitive to
potassium permanganate, to an organic form of iodine, and
a quaternary ammonium derivative.

Bellinger et. al. (12) reported that after standing
for 12 hours at 18 C. neither patulin, strepomycin,
penicillin, hydroquinone, benzalacetonethiosemicarbazone,
patulin thiosemicarbazone, 4-formy1antipurine thiosemi-
carbazone, antihestimic drugs nor vitamin D2 had'any
effect on the botulinus toxin. The oxidizing agents
potassium permanganate and a solution of elementary bro-
mine in distilled water neutralized this toxin while
quinone, quinhydrone, and hydrOgen peroxide as well as
aldehyde compounds failed to do so. 0f the numerous dyes
tested, only crystal violet detoxified botulinus toxin

after one hours combination.

A recent communication by Littauer (13) indicates that
five percent c0pper sulfate and silver ions were used
with no adverse effects on the type A toxin. Potassium
iodomercurate in concentrations of one and two-tenths

percent caused inactivation.

III.

MATERIALS AND METHODS

 

A.

Medium and Organism

The work by Lewis and Hill (14) has shown that
clarified corn steep liquor (two-tenths to four-tenths
percent total solids), two percent powdered skim
milk or one-half percent casein (technical grade),
two-tenths to six-tenths percent commercial grade
glucose (cerilose) at a pH of 6.8 to 7.6 gives a high
yield of toxin. This medium is inoculated with two
percent of an actively growing culture of the "Hall
strain" of Clostridium botulinus, type A, and in-

cubated at 35 C. for 48 to 72 hours.

EXperimental Animals

The mouse was selected because of its sensitivity
to the toxin. The mice used were from 16 to 24 grams
for preliminary work and 18 to 22 grams for final de-

terminations.

Dosage
Five-tenths of a ml. inoculated intraperiton-
eally was used because it is believed that this is

the most sensitive method for determining toxicity.

D.

Dilution of Toxin for Injection

A buffer made up of two-tenths percent gelatin
phosphate solution adjusted to pH 6.5 was used in
the early eXperiments. In the later experiments all
dilutions were made with sterile distilled water be-
cause under the conditions of these experiments no

appreciable loss in toxin titer was observed.

Toxicity

The toxicity was determined on a LD50 basis
rather than the M.L.D. because it is statistically
more valid (3). Eight animals were used at a given

point but for preliminary work three animals were

used. The animals were checked daily for five days.

Time of Contact of Agent with Toxin

All inactivating agents except the methylene
blue chloride, ultra-violet light, and ion-excnange
resins were allowed to remain in contact with the
botulinus toxin for 30 minutes at room temperature
(22 - 25 0.). Samples that were treated with chlorine
were kept at room temperature for 30 minutes with
enough chlorine to give a solution that had a no
chlorine demand, then they were treated with the
experimental dosage and allowed to stand for 30

minutes.

In all eXperiments except with the use of active
carbon and ion-exchange resins the chemicals were
added to 10 ml. of toxin-water solution.

To 100 ml. of toxin-water solution active car-
bon (activated Charcoal) was added to give the desired
concentrations. The solution was shaken in a dilution
blank 25 times, then poured into centrifuge bottles
and centrifuged at 100 x G for 30 minutes.

Kethylene-blue chloride photodynanic effect was
determined by eXposing the toxin-dye solution in a
Bioassay Petri dish to a 200 watt electric light
bulb at a distance of 15 centimeters for 15 minutes.

Tee ultraviolet light (General Electric Germi-
cidal Lamp - 15 watts) was placed two centimeters
away from the toxin in a Eioassay Petri dish and
was exyosed for periods of one and ten minutes.

The column technique was used for most of the
experiments with ion-exchange resins. Thirty grams
of resin was placed in tme column and then regenerated.
The resin was tsen wasged well with water to get rid
of the excess regenerant. The toxin-water solution
was then poured through the column very slowly.

In the batch technique 10 grams of resin was re-
generated, then washed well with water, and all of the

excess water poured off. The toxin-water solution

was added to the resin in a dilution blank and hen
shaken 25 times. The supernatant was withdrawn for

testing after 15 minutes.

Controls
All chemical compounds were tested intraperitone-
ally the saie as he toxin. Controls were allowed

to stand for 30 minutes and the hiyhest concentration

used in tie exneriment was used as a control.

Chlorine Determinations

Concentrations of the chlorine solutions were de-
termined by the amperonetric method of titration (15).
Calorine demand of tne toxin was found by the Ortho-

Tolidine-Arsenite Test (16).

 

hcccal

Chlorine

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Effect of Chemicals on 500 Mouse LDSQ'S

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The firs t cherica l givei cor siferation was chlorine oe-
cause it is being used in this country for treating m'1_y water
supplies. Tables I, I, and III show that it would take 200
ppm to inactivate the toxin. this would be too much chlorine

to use in water treatient.

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beca se it has bee h used in some countries for water treat-
ment. Tables I, II, and III show that 10; Lim had sone

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200 ppm did not inactivate botulinus toxin.

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ihe results in Iables I, II, and III reveal th:

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- 15 -

Table VI
Effect of 400 ppm of Beta-PrOpiolactone on 10,000 Mouse LLBO'S

Dilution
of Sample 10"1 10‘2 10—3 10-4
Deaths 3/3 3/3 3/3 3/3

Beta-PrOpiolactone was of interest because it has been
shown to be effective against bacteria and viruses in plasma
and blood (17). It is an acylating agent possessing a low

degree of toxicity (13). However, it had no activity agairst

the toxin at the concentration used in these experinents.

Photodynumic Effect of hethylene glue Chloride on 10,000
va '5
D

 

So
hetnylene :lue Chloride 1:2,000
Dilution 1 _q j
of Sample 10‘ 10 L 10-3 10“+
Deaths 3/3 3/3 3/3 0/3

Hethylene Blue Chloride l:lO0,000

Dilution

of sample 10-1 10-2 10-3 10-4
Deaths 3/} 3/3 3/3 3/3

Kethylene blue chloride was shown to be capable of in-
activating tetanus toxin by an oxidation phenomena (19). It
showed a slight effect against botulinus toxin at a concen-

tration of 1:2,000 of the dye.

- l7 -

 

Effect of Active Carbon (Aqua Luchar A) on 5,012 Xouse LDJO's

./

70 ppm of Active Carbon

Dilution
of Sample 100 io‘1 10'2 10'3
Deaths 3/3 3/3 3/3 3/3

140 ppm of Active Carbon

Dilution
of Sample 100 10-1 10-2 10-3
Deaths 3/3 3/3 3/3 3/3

Under the experimental conditions active carbon did

not remove any of the botulinus toxin from water.

The use of active carbon was of particular interest be-
cause it is used in some water treatment plants to remove

particulate matter and excess chlorine.

-18-

Table IX
Effect of Ultraviolet Light on 1,000 Mouse LD50'S
One Minute Exposure at a Distance of Two Centimeters

Dilution
of Sample 100 lO 1 10-2 10"3

Deaths 3/3 3/3 3/3 3/3

10 Kinutes Exposure at a Distance of Two Centimeters

Dilution
of Sample 100 10‘1 10"2 10‘3
Deaths 3/3 3/3 3/3 3/3

Ultraviolet light was used because it was hought it

might denature the protein but it had no apparent effect.

_ 19 -

Table X
Effect of Passing 100 milliliters of 1,000 House LD5O's of

Toxin Through 30 grams of Strong Base Exchange Resin-
Hydroxy Form (Amberlite XE-98) in a Column.

Dilution

Of Sample 100 10-1 10-2 10-3 10-4
Deaths 0/3 0/3 0/3 0/3 0/3
Table XI

Effect of Passing 100 milliliters of 10,000 House LD5O’s of
Toxin through 30 grams of Strong Base Exchange Resin-Hydroxy
Form (Amberlite XE-98) in a Column.

Dilution
of Sample 100 10‘1 10‘“ 10‘3 10"4

Deaths 0/3 0/3 0/3 0/3 0/3

An anion exchange resin was reported by LoGrippo (20)
to absorb the Lansing strain of the Poliomyelitis virus

and Theiler virus (T0).

- 20 -

Final Results

Table XII

Effect of Passing 100 milliliters of 10,000 Mouse LD‘O's of
Toxin Through 30 grams of Strong Base Exchange Resin-fiydroxy
Form (Amberlite XE-98) in a Column.

Dilution
of Sample 100 10‘1 10'2
Deaths 0/8 0/8 0/8

Table XIII

Effect of Using the Batch Method of Ion Exchange on 100 milli-
liters of 10,000 Mouse LD 's with 10 grams of Strong Base
Anion Exchange Resin-Hydr xy Form (Amberlite XE-QB).

Dilution
of Sample 100 10"1 10'2
Deaths 0/8 1/8* 0/8

* Not a typical botulism death

A strong base anion excnanger used in the hydroxy form
proved to be successful in removing the toxin from water.
Since strong base anion exchange resins are used in water
conditioning for the removal of silica from water fed to high-
pressure boilers (21) this should prove to be a practical

method for removing type A botulinus toxin from a water supply.

V.

v V“- A. «.9
Sf —

l.

. _, I
. -A-A‘L

s in an

FJ

A study has been made of a number of cremica
attempt to inactivate botulinus toxin type A. lone of
the chemicals used were effective in low enough con-
centrations to be of practical value for use in treat-
ing a water supply because of toxicity. Chlorine was
of prihary interest because it is used by gany water

treatment plants.

Evidence is given which indicates that a strong base
anion exchange resin removes type A botulinus toxin from

water.

Further research should be done to determine if ion
exchange resins could be used to concentrate the toxin
and purify it. T43 concentration of the toxin would
be helpful in detection in case of attach during war-

time.

Rosebury, Theodor and Rabat, Elvin.A.
Bacterial Warfare
Journal of Immunology 56; 7-96, 1947

Lamanna, Carl; McElroy, Olive E.; a Eklund, Henniig W.

The Purification and Crystallization of Clostridium
botulinum Type A Toxin

Science 105; 613-614, 1946

amanna, Carl; Eklund, Kenning U.; XcElroy, Olive 2.
Botulinum Toxin (Type A); Including a Study of Shaking with

Chloroform as a Step in the Isolation Procedure
Journal Bacteriology 52; 1-13, 1946

Abrams, Adolph; Kegeles, Gerson; & Hottle, George A.

The Purification of Toxin from Clostridiun botulinum
Type A

Journal of BiolOgical Chehistry 164; 63-79, 1946

T1

va Heyningen, W. E.

Bacterial Toxins

Blackwell Scientific Publications, Oxford, England
133 pp. 1950

‘fr‘

Guyton, Arthur C. and IacDonald, marshall A.
Physiology of Botulinus Toxin

Archives of Feurolcgy and Psychiatry 57; 378-592, 1 47

KO

Buehler, Henry J.; Schantz, E. J.; & Lamanna, Carl

The Elemental and Amino Acid Comrosition of Crystalline
Clostridium botulinum Tyne A Toxin

Journal of 3i010gica1 Cnenistry 169; 295-302, 1947

8.

10.

ll.

12.

13.

- 23 -

Edmunds, Ch.rles J. and Lon , :3rr'

Contribution to tfld Pat4010”ic Physio 0gv 0P sotulism

Journal of tAe An-ric:* ' " ‘s 03
- ..1 ,- .

Lamanna, Carl
Hemagglutination by Lotulinal Ionin

Proce e0 .i.1: 01' the Society 0; Bz-Lgeri...ent:.l Biology an;
7 —v':' .\},r_')
Ledicine 69; 532-330, 1940

Lamamia, Sarl and Lowentix‘l, Josegh .c.
The Lalo: of Identity Bet..e31- hemagvlutior and tie 101.111
of Type A Eotulinal 01.1n1sm

Journal of Bacteriology 61; 751-752, 1951

Jude, A.; Gi arf, P. and Ctrrot, I.

Destructive Action In vitro of Certain Chehical Agents
on Botulinus an; Tetanus To: 1

s1t~s Ren;is de la Societe de

:13
142; 318-319, 1949

ellinger, 3.; Xoernlein, I. and a he, A.

as erhslten des Toxins von Clo ridiu:. bot linum und
Sal: onella enteritidis geg enuoer chemotherapeutica,

Antibiotica und Earbstoffen

Zentralblatt fur Bakteriolo ie Parasite1.liunde,

Intentionsir :heiten und Iyg ine, I Orig

153; 450-445, 1951

[‘1 U

Litta zer, Uriel
Ooservations on *he Tyge A Toxin of 310: ridium ootulinum
Tature 107; 994, 1951

Lewis, K. H. and Hill, 3. V.
Practical Iedia and Control easures for Producing Highly
Toxic Cultures of Clostridium botulinun, Type A

1\

Journal of Bacteriology 53; 215-220, 1947

15.

l7.

19.

20.

21.

- 24 -

Griffen, A. E.

The Breai {point Process

Technical Publication E0. 215, 25 pp
"allace a Tiernan Co., Inc.

.Stan dard Iethods for the Examination of Hater and Sewage
Ameiic n Public Health Association, Yew York

2C6 pp: 9ti edition, 1946

Hartman, F. W. ; Piepes, S. L.; 0nd .allba n:, A. I.
Virucidal and OBac tericidal Pro1erties of B- -Pr0piolactone
Federationr9100eedings 10; 553, 195

Kelly, A. 3., and Hartman, E. H.
Beta-PrOpiolactone; Its Toxicity, Degradation Products aid
Comparison with litro Etrusteid

O¢Cfl
Federation Proceedin 10; 561, 1951

Lippert, Karl n.
he Photody namic Effect of lethyle ne Blue on Tetanus Toxin
Journal of Immunology 28; 195-205, 1955

LoGrippo, Gerald A., and Berger, Bernard

Use of Ion Exchange Resins in Partial Purification and
Concentration of Poliomyelitis Virus

Journal of Laboratory and Clinical 1 Iedicine

39; 970-973, 1952

Ton Eyeiang e

The fiohm and Hess Cthany

The Resinous Products Division
23 pp. 1950

 

 

    
 
 
  
   
 
  
    

   
    

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