COMBINED PREVENTIVE PREPARATIONS AGAINST WOUND INFECTIONS.

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• Document Page Count: 229
• Document Creation Date: December 27, 2016
• Document Release Date: February 24, 2014
• Sequence Number: 1
• Publication Date: February 8, 1956
• Content Type: REPORT

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INTELLIGENCE TRANSLATION
Wound infection prevention
Pols(Title page)g ACADEMY OF THE MEDICAL SOINYCES0U0S0S.R0G-
c..INSTITUTE OF EPIDEMIOLOGY AD MICROBIOLOGY in the name of the respected
STAT
academician NO3?0GAIILLEIO.
Combined preventive preparations against wound infections
* * * * fit * * * *W * * * * * *
-
(Materials according to the exchangs of experience of the scienttfic.industrial
works of the N?F.GAMALEI Institute for r.pl?erlicology and Microbiology? of the Acad..
emy of the Medical Sciences of the U05080R )
FIRST ISSUE,
MOSKVA?19590
p?28 THE EDITORIAL BOARDS
Pc 3
Director of the institute 0
O ? 0 Prof S011,-; MUROMTSEV
Deputy director for science 0, 0 Prof? V0V0ANAN0IN
Deputy director
AWA
the science of production? 0 . Oandpmedoecio Z0M0VOLKOVA
Active member of the 11S?S0RoMed,Sciences Academy 0 0 Prof0G?V0VIGODCHIKOV
G0V0VYGODCRIKOV1, :1-4,n6WILKOVA? S,AZZELEVINSRLYA.
(110F0Gamaleirnstitute (JA -4'Fiptilemiology and Microbiology. U?S R.
0S.0 Academy of the
.
Medical Sciences?. Directors Prof0S0N0Muromtsev)
COABINED PREPARATIONS FOR Thin ACTIVE PREV7.7TION OF WOUND INFACTIONS (p03.18)
= = CO CO CC
0 0 0 CO OW CP CP CO CM
Numerous data in the literature of recent times on the liathogenesis and im.
munity in case of wound infections prove the fact that the most effective and pro
soectiva direction in the combat of these infections is the solution of the prob.
lems of active immunization, If we reckon that in the pathogenesis of tetanus? of
gas gangrene and of the staphylococcic wound infection the leading factor is the
poisoning by the corresponding toxins? this determines of course not only the path=
way of immunization and the immunogenic factor but also the end result of immuni.
tatiou.. the creation of a permanent and stable immunity.
The gas gangrene itself is a quickly spreading necrosis of the muscles,and it
is accomanied by considerable edema which gave reason for OKLI to call tis process
an edematous myonecrosis0 AIKET and DIBL(1956) think that the basic lesion is loro.
yoked by the exotoxin of the 01?rerfringens0.. the myonecrosis is provoked by the
action of the alpht.to71n(lecithinase). The myonecrosisodue to the direct action
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Wound infection prevention
PAGE NUMBER
2
upon the muscular fiber by the exotoxin of the Oloperfringens, but not to the action
?
Of the-collagenase upon the reticular structure and upon the fibrous tissue of the
STAT
endomysium, The disarenearance of the endomysial collagen connective tissue is the
result of the action of hyaluronidase, The reticulin is stable. In the injured mue.,
cles, the nutrition of the tissue with blood is partly or completely impaired. The
anast=otic blood surly is insufficient to prevent a necrosis of the muscles which
develops in the anoxic parts. The amoint of haemoglobin at the sites of the blood
extravasation is quickly diminished, the aerobic oxidation stops rand the oxidation.
.reduction potential is lowered, and the pH of the muscles will drop, Favorable con,,
ditions arise for the growth of the anaerobic bacteria*
The gravity of the evolution of the process is complicated by the following
changes in the white blood countt. the polymorphonuclear leukocytes (p04) pPrish
in the anoxic tissues. The serum therapy and the medicamentous theraPy is of small
effectiveness under such conditions, since, in consequence of the anoxia, of the
edema and of the thrombosis of the vessels, the introduced specific antibodies and
medicinal substances are unable to penetrate in sufAcient amount to the site of
the lesion and they cannot stop the development of the pathological process. Under
such conditions, the active immunization may show itself as the most effective and
most prospective means,
In this way, for the prevention of wound infections(at least,for the most im=
portant ones among them),the most prospective method seems to us the active immun.
isation with anatoxins for the ultimate end to produce Chiefly an antetoxic,but
also an antibacterial immunity, As it is well known, the employment of the tetanus
anatoxin for active immunization meant a new stage in the combat against tetanue,
Numerous works of domestic and foreign investigators have shown the feasibility of
a reinforced active immanization,against tetanus by means of the tetanus anatoxin,
and its advantages above the serum prophylaxis( G.RAMON et al,g E017GLOTOVit &
0.Y.A.OSTROVSKLYA; A0V.PONOMAREV et al.g P.F.ZDRODOVSKII; B.V.VOSKRESVISFII &
NIKOLAEVA,I,I0ROGOZI% D,WATITONA0and others), The effectivenePs of active im7nun1.
zation with the
tetanus anatoxin was corroborated during the Second World Warg.
among 100,000 wounded persons who had received full courses of vaccinations only a
few cases of sickness were observed due to tetanus.
In the pathogenesis of gas gangrene,, caused by Clostridium perfringens, the
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, INTELLIGENCE TRANSLATION .
Wound infection prevention
??^????1101NOMICIIIIIIIM1101.04....
I PAGE NutABER
3
basic importance is attached to the toxin of this microbe? HowevPro as it had been
establishpd by.a number of investigators(S?NoMUROMTSEVAKLI), the generalized de..
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velopment of the infection with serious universal poisoning is accomanied by a
vehement proliferation of the microbPs at the site of their intrusion and by their
quick penetration into the bloodoand later.. almost into all the organs and tissues
of the organism? In connection with thiso the question arises about the significance
of the antimicrobic UNIM factor in the mechanism of immunity against the Clostri.
dium perfringenso and about the appropriateness of including microbic components in
an antigen predestined for active immunisation against gas gangrene caused by the
perfringeite bacIllus
The litPrature is not very large on this question WEINBERG and other eo in a
series of investigations( 2927. 1929) had obtained the reinforcement of anti,infeccp
tious substances of the antipprfringens serums after the inclusion of microbic bodie
of the Type A Clostridium perfringens in the antigen meant for immunization
ar results were obtained by LAKHIERI(1938) and VINOENT(1939)0
The cited works do not determineohowevero the role of the antibacterial factor
in the mechanism of the passive antiperfringens immunity (1)05) since the different
effectiveness of the obtained sera could be conditioned not only by the presence
or the absence of the antibacterial factor? but also by their different contents
in antitoxino which has been also later convincingly shown by 0,I0LETKOVICH(1945)
LEVICOVICE proved that the effectiveness of the antiperfringens sera which were pre.,?
pared by means of introducing various antigens(of microbic bodiesf, of toxin and
anatoxin) had depeneed only upon the level of the obtained antitoxic titre.
G,BMGODCHIKOV0Z014,VOLICOVA08,.A0ZELEVINSRAYA and I,LLARINA set themselves the
task to study the possibility of increasing the immunogenic properties of the ?er.
fringens anatoxins by mans of including in them different protein fractions of the
microbic cell of Clostridium perfringens and to show the role of the antibacterial
factor in the active immunity against gas gangrene which was provoked by the Tyros A
01.perfringenso As antigens for the immuniration of the animalsothey uaed concen.
trated purified sorbed perfringens anatoxinz different protein fractions of the
-microbic bodies of the Type A Cloperfringens; a mixture of anatoxin with microbic
fractions which they prepared by the methods elaborated by NeV?KHOLC1rEV(1952) for
the obtaining of the protein fractions of staphylococci.
if
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Wound infection ,revention
PAGE NUMESTAT
4
For getting the first protein extracted dry microbic bodies
three times bi means of cold distilled water. They cooled the obtained extract at
4000and subjected it to precipitation with a 20% solution of trichloracetic acid
at pH 3,7 e, 4?0. They collected the obtained precipitate of protein by means of
centrifugation in a little volume of water(distilled) at PH 7000 and then subject.
ed it to lyophc exeiecation(first fraction)* This -procedure is similar to the
precipitation of the toxin from the bouillon filtrates of the cultures of staphylo.
cocci?
For getting the second fraction, they poured a weak solution of alkali( 0.05c
normal solution of NaOH) ever the microbic mass which remained after the extraction
of the first fraction? at the rate of 50.100 ml per 1 gram of dry mass.
They precipitated the fxtract with acetic acid at pH 400 4,Z,The outfallen
precipitate vas collected by centrifugation0dissolved in a 0.0&N solution of a0H,
and subjected to a second precipitation at pH 400 Qg. 4,2. Again, the obtained nre.
cipitate was separated by mans of centrifugation; a euspension was made of it in
a small volume of distilled water at pH 700,and it was exsiccated by the lyonhilic
method(second fraction).By the extraction method, this fraction hathers the part of
the microbic nucleoproteids,
For getting the third fraction to the centrifugate which was left over after
the removal of the second fraction, 20% trichlora'acetic acid was added at pH 100
. 009.0 and with the same the rest (p.6) of the-protein contained in the centrifug.
ate was precipitated The obtained precipitate was collected by centrifugation, tlt
was dissolved in a small volume of distilled water, and exsincated by the lyophil.
ic method(third fraction), The method of isolation of this fraction is similar to
the obtaining of the specific protein of staphylococci( VEHVEI)b
In all the prepared protein fractions the physicochemical properties were
studiedome the toxicity was determined on white mice by means of intravenous injec.
tionpand on rabbits by means of intracutaneous injection.
All prepared microbic fractions and concentrated sorbed anatoTins were utilized
for the immunization of animals. Fortyfive rabbits were arranged for the immuniza=.
tion experiment, The immunization was done twice(two.shot) at an interval of twenty
days,
Fifteen days aftPr the second injectionothe content G-14 aniatoxin was deter.
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Wound infection prevention 5
mined in the blood of all animals. Five months after the immunization? a siseekre
revaccination tollowed with the came doses of antigens which had been introduced
at the first injection. Fifteen days after the revaccinationothe antitoxin content
of the blood of the revaceinated animals was determixtedpand the strength of immun=
ity was tested by a direct experiment? by means of infecting a spore culture of the
perfringens bacillus. The infection was done intramuscularly into the rear paw,with
the spores of B.nerfringens, activated with 0.1 ml of a 50% solution of Ca0120
The conducted studies permitted to draw the following conclusions
1) The Immunization of the rabbits with the concentrated sorbed perfringene
anatoxin causes in the organism of the animals the formation of a considerable
amount of antitoxin which fully protects them from experimentil gas gangrene caused
by the perfringens bacillus The addition of various microbic fractions to the ana
toxin provokes the formation of bacterial antibodiespbut it does not help the in..
crease of the antitoxin production.
2). The immunieation of the rabbits with anatoxic protein fractions of the
microbic cells of the perfringens bacillus will provoke in the immunired animals
the formation of agglutinin-,..-precipitins0 of complement.binding antibodies and,
evidently? it imparts a slightly marked anti-soinfectional immunity to lait separate
animals,
3)0 The basic prot6ctive factor in the immunity against gas gangrene due
to the perfringens bacillus appears to be the antitoxin. The antibacterial
which are detectable to a slight degree are just playing a secondary role.
factors
Safi.
cient content in antitoxin in case of the abrence of IN antibacterial components
(1e7) will fully protect the animals from experimental gas gangrene? even under
the harsh conditions of the experiment( infection with eporal culture, activated by
O1 ml of a 50% solution of calcium chloride).
The firet investigations about the active imeunization of people have been
carried out in 1915 by WEINBERG who had prepared an antiperfringens vaccine, and
had used thee vaccine for the treatment of wounds ia ease of phlegmone(cellulitis)
of an indolent cousec. In
40,1,41,
subsequent years, witusEm and his coworkers had
used? in ad.dition to the vaccine? anacultures and anatoxins for immunization. The
results which the WEINBERG School achieved had been just slightly satiefactory.
WEINBEBG thought that the immunization against gas gangrene is ' a. d fficult task",
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Wound infection prevention 6
pAGE N u LS nasa
In further works of ZELEVINSKATA(1935). YENFOLuD and TOLKHORST(1937)? PLUM
(1939)0 .KOLME1(1942)? STEWART(1942)? ROBERTSON and IMPI(1949), DOUDI(1942)o MAW.
INSKAYA0VOLKOVA & KONSTANTINOVA(1944)0 ZELEVINSKLYA & EBERT(1944)0 MILAN0L0GAN &
TAITL(1944), LOGAN & TAITL(1945)0 KOMOVA(1946), ALTHEYERMLBERTSON0TAITL & LOGAN
(1947)0 BERNREMR(1947)? WATERS AND MOLON(1949)0 PLETNEVA(1952)0TOVTUMOVICH(1953.
1954)0 and of other authors,it was proved thatoin the majority of cases? the im.
muni ration with native (g natural) anatoxins gave unsatisfactory results in relation
to the immunity against B.perfringensotoo. ZELEVINSKAYA0 VOLKOVA and BULATOVL es.
tablished that the purification and concentration of the perfringens anatoxins with
the aid of-ammontum sulfate or with the salt of heavy metal(Cd) permits the ob=
taming of considerably stronger antigens than with tie native anatoxins, The pos. /
sibility has been also found that immunity can be experimentally produced against
tetanus and gas gangrene due to the perfringens bacillus and to the edematlens ba,
cillus by the use of a composite preparation for the immunization.
As it can be seen from the available literature? the slight efficiency of re=
search in relation to the specific prevention from gas gangrene is connected with
the difficulties of getting preventive preparations( anatoxins) which would have
sufficient antigenicity and immunogenicity,? However? ADAMS(1947)0 TAITL & LOGAN
(1947)0 thanks to the bivalent immunization and the revaccination of volunteers with
perfringens plus edematiens anatoxinso have detected perfringens antitoxin In
the strength of from 0.05 to 2 antitoxic units, and edematiens antitoxin it the
strength of from 0.1 to 1 antitoxic units(1947)0 in the blood of the inoculated
persons., BEREHEIVIER(1947)? as a result of a combined imeunization with the anatox.
ins of perfringens and edematiens and septicus0 after the revaccinations had ob.
tallied the titres of perfringens antitoxin from 0.1 to 2u0 A,11(antitosic units),
of the edematiens antitoxin from 002 to 200 AeX0, and of the antitoxin of Vibrio
septicus from 0.1 to 100 AoTJa in the blood of the inoculated individuals(o08) Om
the other hand? according to the data of ADAMS(1947)0 TAITL & LOW(1948), and
BERNHEIMMR(1947)0 the use of precipitated and concentrated anatoxins allowed to get
positive results under the condition of a compulsory performance of revaccination
in a period of 6 to 12 months after the immunization. This has been also pointed
out by MAUS() who thought that a stimulating dose is required some time after
the initial injection for a high level of immunity.
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twatarmerspamegim4marammaNaw.uoniorwompeolowie.s..,...cornetrom;rwaxemaimpwrin
Wound infection prevention I PAGE NUNI 7BESTAT
INTELLIGENCE TRANSLATION
The modern status of lenowledge about the antigens cnd the. broad development Of
immunochemicale research makes it possible to considerably strengthen the range of
action of the preventive preparations by employing purified and concentrated preper
arations simultaneously against several infectioni, This circumstance has a pro.
found importance for the active immanizationft against wound infections?
What kind of coneiitions are then required and which are satisfactory for the
development of this really new stage in the field of practical immunology?
The production of immunogenic complex preparations for active immunization
against wound infections is tied up with 1) the investigation and study of new
nutrient media which are best for the formation of toxins, and the production of
the toxins; with 2) a study of the process of detoxication of the toxins which are
obtained on the above indicated media, for the purpose of getting anatoxins; with
3) the elaboration of the methods of purification and concentration of the toxins
and anatoxine whichoin the presence of a Small amount of the preparation, will per.
mit to reach the maximum immunological effect as a result of the active immuniza=
tion with 4) the study of the process of sorption of the anatoxins for the purpose
of strengthening their antigenic and immunogenic properties.
Considerable interest is concentrated on the results of the elaboration of a
new method for getting anaerobic toxins by means of the breeding of a culture of
anaerobe germs in cellophane bags; the method had ben undertaken by S,A,ZELEITINS,
KATA, E,A,GIL2GUT, N0S,XASHIYTETVA, I,B"LANOVALE4V,VIAS0VA and GFRMCKINA?
It was shown that at the cultivation of toxigenic strains of anaerobes, the
culture in the cellophane bag may get "dialyzed" toxins, considerably exceeding
in strength the toxins which can be obtained in case of the direct seeding of clan
tares into the nutrient mPdivan The tetanus toxins which are prepared by this meth.
od were ten to hundred times stronner, the edematiens toxin 10 to 40 times stronger,
and the septic vibrio toxin 5 to 10 times strongerethe perfringesn toxins 2 to
4 times stronger than the toxins made by the usual methods? It has been established
that the maximum.peak of the toxin formation in the cellophane bag comes later than
In the case of a cultivation of the strains (p09) on ordinary nutrient median. for
the tetanus and the botulinas toxin of Type B..on the 9th day; for the edematines..
on the 6th day; for the perfringentan on the 5th day0 In the process of incubation
a lysis of the mocrobic bodies takes place,At the end ofthe incubation, their al.
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1.401111031.1111?????????.???????
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Wound infection prevelition
.A24.1x:41:1114coure, Aown*,;.,.,,?;434.
MININIONormaimpommINIMMONIAMMOMMI -Ang~Atow.W.00-4101MMIUMMLWOOPOOM
most comPlete disintegration has been observed.
At staitthg by MORCZOVes method. flagella(cilia) are easily deseeTted in the
Clostridium botulinum and the Clostridium edematiens. Among the lysed forms punc.
tuate ciliated forms have been observed. It has been proved that the "dialyzed" tox.
ins, at the addition of 0.3 Q. 0.4% formalin0 will change into enatoxins in a short.
er period of time than the ordinary toxins. The anatoxins obtained from the "dia.
lyzed" toxins possess higher antigenic and immunogenic reoperties. Their immunolog.
ical effectiveness is several times higher than of tt ordinary anatoxins. A. seri.
ons shortcoming of the elaborated method has been the difficulty of creating the
conditions which are reouired for getting "dialyzed" toxins in large volume for the
purpose of wholesale production.
The native preparations.even those of considerable antigenic power.which are
prepared on meat media of hardly definable and of changing chemical composition0
are sufficiently effective in themselves( for instance. the native tetanus anatoxin)
Yet.they do not satisfy the basic requirements that can be claimed for the compon.
ents of the complex preparations.which reeuirements consists above all in high pu.
? rity and high concentration so that in a small volume the highest possible amount
? of fullevalue antigens should be included.
In connection with the study of the indicated questious, the collective team
of the Department of Wound Infections of the N,F0GAMALEI Institute of Voidemiology
and Microbiology of the Medical Science Academy of the U.Sb.S.R.0 with the jointly
combined work of the Biochemical Department(Chiefe. V4A01314/1GOVESTICHTITSKII) and the
Department for the Preparation of Culture Media(Chiefg. I.V.VINOGBADOVA) have sub.
jected to elaboration and have studied the immreiring properties of the combined
preparations for active immunizatioa against tetanus0 gas gangrene and stanhyl000c.
dc infection.
We are now proceeding to the descelption of the investigations the purpose of
which has been the comearative evaluation of the nutrient media used for the prod=
action of the toxins and anatoxins.
The organization of the scientific works in this direction has been entirely
necessary since the ordinarily uzed culture media composed of expenetve meat prod.
ucts0 in addition to this factor of cost0 are very inconvenient when it is the
Iquestion
to purify the produced. toxins and anatoxins from the ballast substances. ,
Moreover the meat nutrient media are vertheterogenous as regards their
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Wound infection prevention
and, owing to their complexity, they can be hardly standardized.
(p.10) ----For the production of the tetanus toxins, KASPITTSMVA, GIL0GTIT and HUM.
STAT
NOVA used the media of RAMON and GIEZMAN for the production of the perfringens
toxins, ZELEVIITSWk0VOLKOVA and LARPIA used a medium of the tryptic digestion of
.410.11.0?4??
meat and the meat.fungal medium of ZELEVINSMA and VINOGRADOVA in which the amount
of meat was considerably decrease and a fungal protease has been used as a fermene4
for the obtention of the edematiens toxins, VIAerVA used a medium of the mentic Oi.
gestion of meat,
By using for the production of toxins such media which had a caseine hydrolys.
ate for their basic substa- :s the following reeulte have been obtained:
When the toxicity of tt 211trates of the tetanus bacillus cultures which can
be prnduced on Ramonvs and GI .etangs media are compared with the toxicity produced
at seeding the very same strain(KOLLE No.8) on bouillon of a caseine hy:Irolysatea
then one may become convinced of the advantage f the latter medium.
The average titre of the produced toxins reached 3,000,000 in one ml of the
filtrate of the culture a exceeding six times the titres of the toxins which can be
produced on Ramongs medium and three times the titrPs of the toxins produced on
Gluzman s medium. The maximum formation of the toxin on the meatless mrdia is af.
VW11.- U=if
days of incubation
at ree?
4a amma,Atai-
0 43. 0 0 4411.?
tha,n
%lir% aft Iva 4
te.54,1u tair-Ot.
media on
which the peak of toxin production had freouently occurred already aftPr 3 to 5
1
days of cultivetione It is possible that this can be explained with a retardaVon
1
1
of the commencement of the prowth(in comparison with the cultures on the meat media)
For the prodnAion of the perfringene toxins we delayed with two variants of
the meatless medle. 1) fish.casein hydrolysatea and 2) caseine hydrolysate.
The titres of the toxins obtained on these mediaain case of seeding upon them
the strains BR6Ka were 2.5 times higher than those obtained on POUP9s media, and
lo5 times higher than on the meat.funga, media. With the cultivation on caseine
hydrolysatesa the growth of the culture is extremely intensive, and it is accompan.
led by an abundant fernation of gas.
The same regularities wrre noticed at the pro&iction of the edematiens toxins.
On the caseine mediapin case of the seeding of the No.794 Straina the toxins were
c+
izALA
times stronger than those toxins obtained on a medium of the peptic digest of
meata and three times stronger than on the fish hydrolysa e. Edematiens toxins of
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Wound infection prevention
weeXana000000,41?011slitir.....
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high power were also produced on media which did not contain corn extract, However,
in suc17a case.; the multiplication of the culture developed very slowly; sometimes,
STAT
visible growth was noticed 2.3 days after the seeding only, At the addition of corn
extract to the medium, the intensity of the growth was not less than on the meat
media.
(p.11) The neutralization of the toxin.stimulators of gas gangrene and of teta.
nus produced on different nutrient media occurs quicker in case of the cultivation
on caseine media. The neutralization of tetanus toxins produced on meatless media
occurs in a considerably shorter periled of time. For their full neutralization, with
the addition of 0.40 forma4n0 a 15.day? stay in the thermostat
at 370 is suffici.
ent instead of the 21.25 days required for the neutralization of toxin obtained on
meat media.
The perfringens toxins,as well as the tetanus toxin, when prepared on meatless
media, are rather quickly Changed into anatoxins, particularly when media of the
fungal protease ferment are used for their preparation. While for the full neutra,
lization of the toxins which are obtained on pancreatic meat media and on meatless
media,after the aedition of formalin v it is necessary that they should stay in the
thermostate at 37()0 for a period of 21 days, then for the neutralization of the
toxins which are produced on eeeeineepfungal mPdia nine days are sufficient,
The quick change into anaoeins is of considerable advantafe since under these
conditions,of course, the produee anatoxin is less liable to denaturation.
The harmlessness of the anaeuleins as tested on guinea pigs,and thPreafter
the
anatoxins were subjected to purifIcetion and to eoncentration. The purification was
carried out according to the motho eorked out by V.A.BLAGOVESHNE"SKII and by his
coworkers, namely by the precipitation with HC1 at the isoelectric point,after a
preliminary salting out with a 20=254 solution of common salt. The furthPr purifica.
tion of the concentrated tetanus anatoxins was achieved by adsorption to aluminium
hydroxideowith the subseqe:ent elution of the anatoxins. The ap7lication of this
method allowed the production of highly purified tetanus anatoxins containing
20,000 . 50,000 binding units(ES) per 1 mg of protein nitrogen. Later on, it has
been proved that the method of precipitation with aceteac In the cold,with strict
control of the pH and of the ionic potency, a highly purified perfringens,edemati.
ens and tetanus anatoxin was successfully produced, completely pigmentless and of
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a high level of binding units(BS) per 1 mg of protein nitrogen.
The anttgenic properties of the native and of the concentrated anatoxins were
STAT
determined in experiments in vivo on mice for the tetanus and for the edematiens
toxins andoin a reaction with the emulsion of erg yolkOin exriments in vitro
for the perfringens anatoxins.
The anatoxins received from toxins which were produced on meat media contained
considerably smal!er amounts of binding units(ES) than the anatoxins received from
toxins produced on meatless media. 4s an average, the tetanus native anatoxins which
were prepared (p.12) on meat median contained 33.50 binding units the native ana?
toxins on meatless medis.. 100.700 binding unites the purified concentrated tetanus
anatoxins.. 2000 . 5000 binding units. The edematiens anatoxins which were produced
on meat media contained 4 binding units, on meatless media.. 15 . 30 binding units,
the concentrated ones.. 400 . 800 binding units. The perfringens anatoxins on meat
media contained 2 binding units 0 on meatlees media.. 2 4 binding unit s0 the con.
centrattd ones.. 60 . 100 binding units.
The sterile anntoxinsncontaining 608O binding units of perfringens0 400 . 800
binding units of edematiens0 2000 . 5000 binding units of tetanus were sorbed to
aluminium hydroxide.
From the purified concentrated and a1uminium.hyroxide.sorbed anatoxins 0the
triAnatoxin was prepared by mane of their combination at definite ratios so that
one ml should contain 25 binding unitz of perfringenso 4 binding units of edema.
tiens0 200 binding units of tetanus The A1203 content of the ready preparation was
equal to 5 mg %.
As a preservative0 0.25 phenol or merthio1ate(1g100000) has been added to the
trianatoxin. The trianatoxin 0after being poured into vials? was checked for ster.
ility and for harmlessness by mr=ans of injecting it into two guinea pige(each
receiving 5 ml subcutaneously).
The immunizing properties of the trianatoxini were investigated on a great
number of laboratory animals(small animals). guinea pigs ,mice and rabbits.
The guinea pigsaboth those who were immunized with a single shot of 3 Ea tri.
anatoxin and a double shot(two shots) each time with 1 ml of trianatoxin0proved to
be resistant to 1 and 2 M.L.D.(8minimum lethal dose; in Russian sDlm)of perfringcans
toxin. Five of the control pigs which receibed 1 M014Do of toxin died in 24.48
hour The guinea pieLyhich were twice inoculated with 1 ml of trianalulAst
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INTEWGENCE TRANSLATION
Wound infection prevention
11140.1.1raWANIMI
interval of 20 days and0 30 days after the second injection were intramuscularly
infected with -1.2 and 5 M4L.D. of the Boperfringens culture in a 50% solution of
STAT
0ael20 proved to be completely protected from 1 and 2 M.L.D. of the culture; from
15 pigs which were infected with 5 M,10.D. of the culture ten remained alive, five
died in 48.72 hours under the symileroms of gas ganciene; five control pigs which
were infected with 1 M.L.D. of the culture died in 24. 48 hours.
All guinea nig; which were immunized either with a single shot of 3 ml of
trianatoxin or with two shots of 1 mI trianatoxin provPd to be resistant to So and
100 M.L.D. of the edematiens toxin.
The guinea pigs which were immunized with 1 ml of the trianatoxin with two
successive shots proved to be resistant to 1 and 5 M.L.IL of the B.edematiens cul.
tare, The control pies died in 24.48 hours*
With the research of immunity at the inoculation of the tetanus toxin, the
guinea pigs which were imeunized with a single 3 ml injection (p013) of trianatoxin
or with two shots of 1 ml of trianatoxin proved to be resistant to the inoculation
of 500 to 100000 MeL0D of the tetanus toxin.
In this mannera the immunization of the guinea pigs with trianatoxin Droved
to be effective in regard to all thrPe of its components.
The immunization of white mice with trianatoxin which was sorbed to aluminium
hydrozide show-d its hi* immunokenic properties. The white mice which were inocmla.
ted with a single shot of the sorbed trianatoxin in the amount of 0.5 ml and were
exemieed 30 days
later for their resistance to the perfringens toxin(intravenously)0
to the edematiens toxin(intrammscularly) and to the tetanus toxin(subeutaneously)
were resistant at the rate of the fo1lowing8 .to 10 M.L.D. of perfringens toxin,
to 1 M.L.D of odematiens toxin, and to 10 M.L.D. of tetanus toxin.
The white mice which were inoculated with two shots of trianatoxin at en inter.
vl of Pfl 411" IN'41-"" 4" ""A"t'"^""' -A""ftt. 4.'4640-74. being
e.,eee.1 e". .,.deeeeeme., wacA.41 4?,r-AoiNJ. O5 alp proved to be
resistant to the intravenous introduction of 5 . 10 . 20 . 50 M.L.D. of the Derfr1n.
gene toxin. All mice which received 5 10= 20 M.L.D. remained healthy. Out of
5 mice which received 50 M.L.D., 2 died and 7 remained healthvg all the 5 mice which
were given 100 M,11.D. died. Positive results were also reached in resDect to the
creation of an immunity against an infection with cultures of the correstonding
agents. The white mice which were given two shots of 0.5 ml of sorbed trianatoxin
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Wound infection prevention
at ain interval of 20 days,one month after the
sistant to an-infection with 1 a 5 MolaD0 of
M?110D0 of the Boedematiens culture? and to 5
bacillus,
For the determination of the optimum dosages of the trianatoxin and of the most
advantageous intervals between the injections whiCh will contribute to the peak
accumulation of the antitoxins in the blood of the inoculated animals ? a few groups
of rabbits were ales immunized.
The results of the observation about the Avskamies of the antitoxin formation
in the rebbits,which were immunized with two shots of trianatoxin and then revacci=
nated, have proved that, twenty days after the injection of 1 ml or 0?5 ml of tri.
anatoxin, formation of antitoxin takes place against all the three components of
the trianatoxins Five days after the first injection ? the antitoxins cannot be de.
tected even in a minimum amount? Fifteen days after the second injection, the aver.
age titre of the perfringens antitoxin in the blsod serum of the rabbits is consid.
1100.11MMINMA~01.1.0.0.4.10.*411.1000.4r....0.4r,
I PAGE PLUMBER
13
last injections were found to be re.
the 1Lperfringens culture, to 1 . 5
STAT
M?L,D? of the culture of the tetanus
erably raised,and 4. to 2 1.
even reaches 25 AaiL(antitoxit
ita Rassiana AB)in one ml
the titre of the tetanus antitoxin was equal to 4 antitoxic units? and the titre
of the edematiens antitoxin reached 8 AU,
Fortyfive days after the second injections, a reduction of the antitoxin level
was noticed against perfringens and against edematiens(p,14) and a slightly smaller
drop in the titre of the tetanus antitoxin.
The obtained data indicate that the second injection at the initial immanisa.
tion has a great importance in regard to the increase of the antitoxin level in
the blood of the Inoculated animals, It could be also successfully established that
1) the reduction of the dose of the antigen to its half also decreased the titre
of the antitoxins in the blood of the inoculated animals, especially against per.,
fringPns as the weakest cemponfant which enters into the composition of t1Le triana.
toxin 2) a more favorable interval between the injections seemed to be the intera
val. of 20 days,in contrast to the five.day interval?
The revascination of the rabbits which have been immunised with trianatoxin
was done six months after the initial immunisation, All together 50 rabbits were
sublected to revannination Before the injection blood was taken from all rabbits,
and in a mixture of the sera the titres of the antitoxins were determinee before
Ilsgasgrasossissattans_a_.
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Wound infection prevention
1 PAGE NUI4131.1TAT-
14
The average titre of the zingens antitoxin eoualled 0.15 A.U. in 1 ml, the
ant-
titre of tho..demAtIone fir//, .a was..0.75 A*U? that of the antitetenne statie^ele
Was 0.15 A,U. in one ml, starting from the data in the literature which show that
the revaccination..wk,en can be done with non.sorbed antigens.. is accompanied by
a higher formation of antitoxin, for the revaecination of this series the rabbits
were separated into two portions. One group was inoculated with one ml of non.
eitorbed trianatoxin, and the other group.. with one ml of sorbed anatoxin. After
the revaccination, blood was taken from the rabbits at the 3rd07th and 15th days
And 102 and A months after the revaccination*
The conclusion can be made that,three days after the revaccination with non.
.esorbed trianatoxin, the antitoxin level in the blood of the revaccinated rabbits
was almost twice as high as in the rabbits inoculated with the sorbed antigen; 7 to
15 days after the vaccination, the antitoxin level was at its peak, and it remained
??
at an identical level in both groups; after a month, a reduction folowed in both
groups. After two months, in the rabbits revaccinated with non.sorbed antigen, the
perfringens antitoxin titre was twise as low as in those rabbits which were revae...
cinated with sorbed antigen; after 6 months, the titres of thti perfringens, edema.
tiens and tetanus antitoxins were twice as low In the animals revaccinated with
sorbed antigen as in those animals revaccinated with non.sorbed antigen. The re.
salts of the study of the immunizing properties of the trianatoxin in the animals
allowed usoand also other investigators, to study the reactogenicity and the anti.
genic properties of this preparation on volunteer persons. The initial immunization
was done with two shots of trianatoxinyeach of 1 m1 amount, and containing 25 bind.
Ad
ing units of perfringenso 40 binding units of edematieus and 200 bi ing units of
tetanus .
(p015) The results of vaccination and revaccinatien of the human volunteers with
the purified sorbed trianatoxin proved that the reactt;genicity of this prepare
I s but plight; serious local and general reactions were absent; moderate arW. 7.ht
reactions did not exceed those which have been noticed in persons inocula . with
other bacteriological preparations. The immunization of trianatoxin is accompanied
by the formation of the corresponding antitoxins in the blood of the inoculated in.
dividualsoThe perfringens antitoxin is detected in 92/4 of the inoculated persons
15 days after the inoculations,with amounts from 0.05 to 1 A.U.; the eematiens
ACM 1.7()Lii4
8 FEB. fief
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1:41,M160?111010.11O111?~41?1110011.17111
INTELLIGENCE TRANSLATION
Wound infection prevention
OOMMWWW.WMVWOWIQWghe...,
I PAGE NUMBIsTAT
15
antitoxin( from 0005 to 1 A01;0) in 78% of persons; the tetanus antitoxin(from 0025
to 1 AX0 or. in 100 of the inoculated individuals.
Sixty days after the inoculation,at a titre of from 0005 to 1,0 AX?the per.
fringens antitoxin has been detected in 78% of the inoculated personsg the edematia
ens antitoxin( from 0,05 to 1 A0U0) in 98%3 and the tetanus antitoxin(from 0.5 to
.1 A011.) in 98%0 (sic),
After the revaccinationowhich is done 6 months later with I ml of trianatoxin,
the perfringensoedematiens and tetanus antitoxins were found in all investigated
sera the perfringens antitoxin from 0.1 to 3 A0U, in 1 ml; the ode matiens,from
0.5 to 10 Ajo in 1 ml; and the tetanus antitoxinofrom 0.5 to 20 AVM. in 1 ml,
In this wayoit was 'Droved 8e; the revaccination which was done after 6 months
has considerably increased the titre of the antitoxins0It can be assumed that the
antitoxin content in 1 ml of the blood serum is sufficient in the majority of cases
to protect the individual from infection with tetanus or with gas gangrene,
As a result of the researches of ZELEVIITSKAYA,VOLKOVA0GIIOGUT,LARPA,VIASOVA,
,KASHINTSEVA and BLAGOVESHOTTENSYII0 from the purified concentrated anateetins a the
perfringens,edematiens and tetanus toxins, preparations of triartatOxin sorbed to
aluminium hydroxide have been made which possess high antigenic and immunogenic
properties
The facts which were established as the result of the mentioned Investigations
about the absence of an immunological competition between the components that enter
into the composition of the trianatoxino and the good immunological effectivenees
of the combinPd preparations and their transmissibility in the imMunization experim.
eat: permitted that we, after using and perfecting the elaborated methods, suge.est
a preparation for the active immunisation against tetanus and against the agents
of gas gangrene(B.perfringens? edematiensjibrio septicus)a.this is the tetranatoxin
(tetraaanatoxin). The dynamics of the antitoxin formation were studied in rabbits
immunized and revaccinated with sorbed tetra.anatoxin.
(P016) Rabbits were immunized with one shot of 1 ml of tetra.anatoxin and with
two shots of the same amount given at an interval of 20 days.This amount of 1 ml
contained 25 bimiing units of perfringens anatoxino and 15 binding units of Vibrio
septicus anatoxino 40 binding unite of edematieue anatoxin and 200 binrling unite of
tetanus anatoxin? itevaccinatiOn was made after 6 months.
L.
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Twenty day ,fter the first injection, the perfringens antitoxin reached a
titre of 0.25-A.U.0 the septicus antitoxin reached 0.5 A.U.D the tetanus antitoxin
STAT
itre reached 200 A0.0 the fedimatiens antitoxin titre reached 300 A.U. On the
15th day after the second injection0 the individual titres of the antioxins rose
very similarly to that which we had already observed in the examination of the dy.
namic function of antitoxin formation after the immunization with the trianatoxin,
At the 45th day? the titre of all antitoxins was reducedo and it continued to drop
until the time of revaccination which was done 6 months later. Between the 3rd and
.the 7th days after the revaccination? a sharp increase was discovered in the titre
of alL antitoxins; the smallest elevation of the titre was observed in the Bosenti.
cus amtitoxin the titre of the perfringens antitoxin increased considerably, and
the titres of the edematiens and tetanus antitoxins increased very sharply, It shoul
be also underlined that the revaccination showed an identical influence upon the
elevation of the antitoxic titres(edematiens0tetanus and perfringens) in both grouts
of the rabbits whether immunized with one shot or with two shots.
We have investigated the immunogenicity of the tetraanatoxin on guinea pigs
and on white aice,, We immunized 63 guinea pigs with a single shot of 3 ml of tetra.
anatoxinpand we examined them as to the strength of immunity 30 days after the im.
munization0 by mans of toxins and cultures of the nerfringenspedematiens 'septic
vibrio and tetanus bacilli.
It was proved that all pigs inoculated in the exporiment tolerated 1 MOLDr.
f the perfringens toxin Pigs receiving 5 Mels.D, of the perfringens toxin died
as well as the controls; guinea pigs proved to be resistant to 50 M.L.D, of the
edsmatiens toxin and to 1 M.L.D. of the Vibrio septicus toxin,
Guinea pigs examined in regard 21 to resistance to the tetanus toxin tolera=
ted from 500 to 1000000 M.L.D. of the toxin, with the death of the controls at
1 14?1J.D. of the toxin.
Forty guinea pigs imnunized with the tetraanatoxin were grouped in 4 groups,
and examined 30 days after the immunisation concerning their resistance to 1 ).111.1).
of the corresponding cultures. All pigs remained alive? while all the controls died,
After the immunization of white mice with a dose of 0,,5 ml of tetraanatoxin
which dose contained 12.5 binding units of perfringens anatoxin0 20 binding units
of edematiens anatoxin, /,5 bidning units of Vibrio septicus anatoxin0 and 100 bind.
tug units of tetanus anatoxita....ta...talair...;.mai.c..Uance.ta.-...
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I INTELLIGENCE TRANSLATION
1. gnfect
AMN1111101?01111?1111.11.1144101011110111PON
M.0011.00?111FINMI
various amounts of minimum lethal doses of the mynropriate toxins. The mice Showed
resistirine to-5 M.L.D.(p017) of the perfringens toxin, to 2 M.L.D&stilso-the edema.
tiens toxin, to 2 M.L.D. of the vibrio septicus toxin, and to 50 M.L.D. of the te.
tanus toxin. At the examination of white mice which were immunized with two shots
(at 22.4ay interval) 01t was found that the mice were resistant to 20 M.L.D. of the
oerfringens toxin, to 10 MoL.D. of the edematiens toxin, to 10 14,1J.D., of the Vib.
rio
the
the
septicu toxin, and to 100 LL.D. if the tetanus toxin.
At the investigation of the white mice immunized under the same conditions,
following was discovered in regard to their resistance to from 1 to 5 M,L,D? of
corresponding cultures, After a single shot immunization, the mice showed resis.
tance to 5 M,L.D. of the perfringens culture() to 1 M?L,D, of the edematiens culture,
to 1 M.L.D, of the sopticus culture, and to 1 M.L.D.
Ir 1
the culture of the tetanus
bacillus. After two.shot imtunizationspat 20 days 2 interval, the mice were resistant
to 50 M?L.D? of all the above mentioned cultures? The control mice died,from 1 M
InD? of the culture.
In this manner, the immunization of the guinea pigs and of the white mice with
the tetra.anatoxin has created in them an immunity of sufficient power both against
the corresponding toxin and against the corresp3nding culture.
In a study of the preparation on volunteers, the transmissibility (tolerability
of the tetra.anatoxin provPd to be the same as that of the trianatoxin. The immun01.,,
ogical researches of the blood sera of the vaccinated persons showed that the dyna.
mica of the antitoxin formation were the same as at the immunization with the tri.
anatoxin in respect to the components of perfringensveeematiens and tetanus anntox.
ins. The level of the titres of the antitoxin of the Vibrio septicus was the same
as that of the perfringens antitoxin?
It must be said that we were unable to obserTe any immnologioal competition
between the antigens under these conditions?
The results obtained with the tetraanatoxin gave us a possibility to study also
another combined preparation...= the purified sorbed pentakmanatoxin in the compoai.
tion of which we find the anatoxins of perfringens, edematiens, tetanus( in the
same dosage at for the trianatoxin)as well as Types A and B of the botulinus anatox.
lux?
were
For the study of the dynamics of the Antitoxin formation, the nenta.anatoxins
introduced subcutaneously in two Shots at 20 days between the injections.
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I PAGE NURSER
18
?
1?1111110.
As a result of this research it has been established that the dynamism of
antitoxin formation in regard to the components perfringens,edematiens and tetanus
STAT
was the same as we have earlier observed at the immunization with the tlece and
tetra.atatoxins; however, in regard to the botulinus components the titres of the
antitoxins were small, which was evidently in direct connection with the insuffi,
cient dosage
of the botulinus antigens included by us in the composition of the
penta.anatoxina
(p28) For the elucidation of the question whether here we were dealing with an
immunological competition between antigens or our dosage of the botulinus nomnonent
was insufficient, we arranged an experiment under other conditions when, with the
maintenance of the earlier dosage of the perfringensvedematiens and tetanus anatoxp
Ins as components of the pentaeanatoxin? the amounts of the botulinus A and B ana=
toxins were raised up to 200 binding units for an injection.
The following results were obtained, Twenty days after the first injection of
the pentaaaanatoxinothe blood serum of the rabbits contained 3 AJF0 of the antitox,
anti
ins of tetanus and edematiens as an average; the titre of the perfringens/Opoxin
reached Ca25 Aat],,, the same were also the titres of tbe botulinus A and B anti=
toxins. Fifteen days after the second injection, the titres of the nntitneins of
tetanus and edematiens were up to 10 AJ10; the titre of he botulinus B antitoxin
reached 5 AcI%, the titre of the botulinus A reached 4 AAr,,, and the titre of the
nerfringens was 1 AOIL In this way, it could be successfully shown that, with suffi
cient dosage(of the antigen) the introduction of the botulinus A and B anitoxins
into the composition of the pentaaanatoxin assures a considerable increase in the
titres of the botulinus antitoxins and it does net opnress the formation of the
ether antitoxins called for by the other components of the penta.anatoxinO
The obtained data permitted for us to develop the researches for the study of
the immunogenicity ef the tril and pesta.anatoxins in combined preparation ? with
the polyantigen of the Gamalei Institute for Epidemiology and Microbiology which
contains antigens of the microbes of the group of intestinal infections?
caacrusims8
1) The utilization of less complex(non.meat) nutrient media permits to obtain
toxins of sufficient power which are capable to be quickly neutralized and to
change into anatoxins.
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wound inxection prevention
MI
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2) The workrd out methods of purification and concentration of the anatoxins
permitted the-nroduction or preparations of high degrPe of purity ?which contain
STAT
sufficient immunogenicity in a small amount*
3) The separate components in the combined preventive preparations(with the
dosages suggested by us) do not show the phenomenon of immunological competition
in the compositions of the tri;=D tetra,- and pentamanatoxins
? 4) The WW2= farther trends of the investigations are:
a) study of other polyvalent preparations for active immun17ation;
b) reinforcement of the separate components ill the combined preparations
against wound infections(Lperfringens and Bosmpticus).
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V.11.00?11111mr.
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vonlinmr.~0/rOIMMPPimilbr:AP.M100,mbrobs11.
I PAGE NUMBER
20
? INTELILI' GENCk: TRANSLATION
(p.19)
ZMoVOLKOVA, -A,P6GINDIN & E.V.VLASOVA.
STAT
(Department of Wound Infection; Chief! GVVy htkovanA Pat oor
Laboratory.Chiefs A0PaGIFDIN; N.F.GW!ALEI I7STITUTE of Epidemiology and Microbiology
Academy of Medical Sciences? U,S.SO10)
CELLULAR REACTIOF AND REPRODUCTION OF RIBONUCLEIC ACID IN THE LYMPHATIC
* * tie * aft * * * * * * * * * 4 111' 13' *
NODES IN CASE OF IMMUNIZATION WITH COMBIrED PERMEATIONS AGAINST INFECTION (p.194,..28)
* * * * * * 41 att * * * 4 * * * 44 * * * * * 3i1 0
f? 4^%
%1,03.vj The study of the reaction of the regional lymph nodes to the subcutaneoua
injection of the new combined preventive preparations against wound infection, the
reaction to the trianatoxin and to the penta.anatoxin which were worked out in the
N,F0GAMALEI Institute, is of interest from both a practical and a theoretical paint
of view.
The illumination of this question has an important meaning for the judgment
en the reactogenicity of the preparation and for the study of the processes which
occur in the lymphatic nodules at the time of iamunogenesis.
By the investigations of VYGoDCHIKOV,VOLKOVA,ZELEVrFSKAYA,KASPINTSEVAJLASOCA
and GIL0GUT(1957) the high immanogenic.propertiee of tha trianatoxin and of the
tetraanatoxin have been proved, and their practical lack of reactogenicity was
shown in experiments on animals and at imiranization of vo1unter The obtained
rPsults allowed the use of the trianatoxin for the immunization of people with 'nos,
itive findings(PONOMAREV0BRYZGALOVA01956; KONDEAMV0.956),
From the large number of published works,it is known that the formation of the
antibodies(antitoxins) happens basically in the lymphatic aode s which are regional
4m urle alIe Of
injection of the antirens?
and in the spleen,? MCMASTER and HUD,
ACK(1925)0and thereafter EHRION and HARRIS(1942) have already established that the
injection of an antigen will provoke the
lymph nodes
Later on, many investigators corroborated these findings by utilizing various
methods;their confirmations were direct or indirect(cf0 the review articles 'OESY.
TER01955 PA1NES01957; UCHITEL0?1957; EHEICH,01955 and others). In recently published
formation of agglutinins in the regional
works(GINDIN and FORSETER01958; (17020)
STENDER0STRAUCH & WINTER01958) new data were
brought forward about the formation of 'agglutinins in the lymph nodes.
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Wound infection prevention
trodowinsommomiliwompara.
A work which is devoted to the study of the reaction in the regional lymph
nodes after the introeuction of nolyanatoxins against wound infectious does not ex.
STAT
1st in the literature. The present investieation is devoted to this problem,
Materials_and_methods..
For the immunisation of the animals two combined prfTarations against wound
infection have been used:. the trianatoxin.composed of the anatoxins of tetanus,
nerfringens and edematienso and the tetra.anatoxin which is composed of the trianac
toxin end the anatoxins of botalinus Type A and Tyne B, In one ml of trianatexin
there are 25 binding units of perfrtneens anatoxin, 40 binding units of edematiens
anatoxin, and 100 binding units of tetanus anatoxinaOne ml of penta.enatoxin con.
tains 25 binding units of perfringenen, 40 binding units(B.C.) of edematienso 100
2.t% of tetanus,snd 100 Botio
of botulinus A and B anatoxins.
The immunization was carried out subcutaneously into the right lateral our_
face of the trunk of the rabbits.
In the experiment of immunization? 42 rabbits were used.each weighing about
2.5 to 3 Kg. The animas were divided into six Proupso The rabbits of Group I ob.
tamed, a single subcutaneous shot of 1 ml of nentaanatoxin.The. rabbits of Group II
received correspondingly a single injection of 1 ml of trianatoxin.The rabbits of
Group III served as controlspane they received a subcutaneous shot of 1 ml of stan,
dard aluminium hydroxide, For the purpose of studying the nature of the reaction to
a second immunizationwhichoas well known gives reinforcement to the formation
of tha antitoxin..,the remaining three groups received correspondingly the same
pre7.larations but in two ihots.keeping a one.month interval between the injections,.
On the 2nd,254 5th and 10th dayeand after 3 and 4 weeksa a rabbit of each
group was killed by means of air embolism? and after 5 weeks two rabbits were kill-
ed in eaah group.
The lymph nodes regional to the site of the injection( the right axillary nodes
and the right inguinal nodes) and the homolateral controls to these nodes( left
axillary and left inguinal nodes) were extracted ad fixed in alcehol,or in a mix.
ture of alcohol/formalinoand they were treated with the usual histological methods,
andjor the detection of ribonucleic acid(RNA) they were treated according to the
method of BRASCRE,
Owa investigations.
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-T.R.ANSIAT'iON PAGE NtIAT3ER4-""--1-4.7
Weland infection prevention 22
2wn investieatione.
TW5 days After the single shot of the immunizing preparations, a distinctly
STAT
manifest reactive process is observable in the regionalalymph nodes .,1 The lymphatic
(13021) glands are enlarged? slightly coligested and edematous. During the following
three days0these processes continued in develonmentga the lymph nodes became en.
larged still more, and their medullary substance became swollen. At the tenth day,
this process has become so strong that the size of the lymph nodes was almost
larger than the control nodera, At this time,the hyperemia was reduced.
4,4....440516M
During the three following weeks,the dimensions of the regional lymph nodes
gradually diminished? but they did not return to their normal condition, not even
35 days after the injection( period of the observation time).
FIG.1g INITH NODE4 Small lumps of ribmacleic acid( on the photograph in black)
in the cytoplasm and in the nucleoli of the reticalum cells(indicated with arrows),
Methyl greenapyronin staining.
In the control animals which were even aluminium hydroxide only the (4evelopa
ment of the mentioned processes wae.delayed,and it did not reach such an intensity
as in the animals subject to experimentation.
By the histological and histochemical examinations, it was discovered that
already two days after the immunieing injection, parallel with the hyperemia and
the dilatation of the sinusesD a marked proliferation of the reticulum cells ac
curred in the parenchymal cordspand they were transformed into large(coaree) little
differentiated cell with large nuclei? with _r chromatin, and with a contracted
thin (narrow) belt ef the cytoplasm., saturated with ribonucleic acid, and with muca
leoli rich in ribonucleic acid.
(p22) These cells were similar to the hemocyteblasts or plasmoblasts. The re,
active centers of the secondary nodules Were still Small at this time, and their
lymphoblasts and reticulum cells were rich in ribonucleic acid.
During the following three dayspall these processes became strengthenedg. in
the parenchymal cords the number of.hemocytoblasts and of nlasmoblasts increased,
and the ripening of many of them-into lymphoblastic and plasma cells was noticedu
At this time( on the 5th day after the immunization) the size of the preexisting
follicles sharply increasedeand a large :number. ofanew felliculi apneared. In them,
Intensive proliferation of the lymphobleets 44a of ;4.-!-X'4,tictilum cells occurred,
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Wound infection prevention
I PAGE NUMBER
23
which is accompanied by,or which conditions, a heavy reproduction of ribonucleic
acid in their cytoplasms and nuclei. The number of the small lymphocytes becomes
STAT
less on this account, and the folliculi are transformed into very large reactive
centers.
On the 10th day after the immanization, further enrichment of the parenchymal
cords occurred, with lymphoblasts, plasma celle,and small lymphocytes rich in riboa
aucleic acid? The reactive centers remained strongly enlarged as before()
FIG.2t LYMPH NOM plasmoblasts with eytoplasm and nucleoli rich in ribonuc.
lei e acid(indicated by the doubleacontour arrows), and plasma cells(indicated by
arrows).The ribonucleic acid is intensively black on the photograph, Staining with
methyl.greenapyronin.
CV GED f=, CC!
(m.23) Between the 10th and the 24th days after the immunization, the prolifera.
tient of the lymphoblasts and plasmoblasts in the parenchymal cords was less marked?
In these cords, the number of Plasma cells has somewhat increased, and the number
of small lymphocytes has sharply increased, with a narrow thin belt of the eyto.
plasm, containing more or less ribanucleic acid. At the end of the observational
period(after 35 days), reduction of the size of the reactive centers occurred, but
their cells remained rich in ribonucleic acid as before. The entire lymph
mained hyperplastic chiefly due te the small lymphocytes with their cytoplasm rich
in ribonucleic acid, and due to the reactive centers.
FIG.38010 legend).
IV% cs as,
In the control rabbits whieh received eely aluminium hydroxide the above
Aee,
scribed changes were at first manifested very little, and they became more noticea
.4:1= 10th day of immunisation?, but even at that time they did
brie,Li
able ou "- A?t- and
not reach such a size as they had reached in the rabbits which were given immuniz=
ing preparations, on the 2nd and the 5th day after the injection.
The second immunization(revaccination) which was performed one month after the
first has again provoked an intensive reproduction of the ribonucleic acid, a aro.
(p.24)liferation of the lymphoblasts and of the plasmoblests in the at this time
still hyperplastic tissue of the lymph node. Therefore, on the second day after the
re.immunization?the lymph node in its size was roughly the same as it hAe been t/t
the 5th day and later after the initial immunization. One the 10th to POth day
eat
ash
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1 PAGE NUMBER
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INTELLIGENCE TRANSLATION.
Woupd infection prevention
The second inlection of the aluminium hyiroxide, as the first one also, proe
yoked a.by.far_weaker process in the lymph nodes *However, this process war also
STAT
ManifPeted in the reproduction of the ribonucleic acid(in a small number of cells),
in the formation of a small number of plasmobla te -and of plasma cells, and in a
Blight increase in the size of only a few separate reactive centers.
From the described observationsvit is evident that in the regional lymph nodes
a single.shot immunisation with the pentap.anatoxin or the trianatoxin has provoked
a proliferative process which was accompanied(or which conditioned) by au intensive
synthesis of the ribonucleic acid in the cytoplasm and in the nucleoli of the lymph.
eid and of the reticulum cells, and by its further maturation to lymphocytes and to
plasma cells
The second immunizing shot after 30 days has preveked a similar process which
however diffuse over a large area since at the time of the second immunizatien
the lymph aode itself was still considerably enlaie7ed and its cells,including eve
the small lymphocytes, were enriched with ribonucleic acid.
The obtained data permit to rate the queetien2. 14110LJJaLILIIAMAilaag-tig
the indicated proceeses in the gtAtgis of immunity?
In recent times it has been established that the synthesis of the protein sub.
stances which are required for the [-rowth and for the division of the cells as well
as the secretion of the protein gubstances by the cells are achieved with the par-
ticipation of ribonucleic acid. For the synthesis of the proteins a preliminary
multiplication of the ribonucleic acid is required( BEL0ZERSKII01948 BRATTET,1942,
1950,1955; l? A rrr1T? .0 CY nIt-r 1 OA1 0
Wiak;04-4;,LIQ?dup.i.wz.a.v
T-MTINFInN & PAVLOVA01S49t BRACRET019420195001955; ROSYIN
1951; MAKA,R0V01956,and others)0
From the data quotPd in this work it is evident that the introduntion of the
polyanatoxins has produced an intensive reproduction of the ribonucleic acid in
the cells of the lymph nodes? EHRIOH,DRABKIN and FORMAN(1949) as well as HARRIS
and HARRIS(1949)0 by using biochemical and histochemical methods, have established
that, after the immunisation with different antigens, the amount of the ribonucleic
acid will increase in the regional lymph nodes.
Our researdhes have indicated that the immunisation with peutaanatoxin and
with trianatoxinD side by side with the reproduction of ribonucleic acid? also
Provoked a hynerplastic processowith the new formation of lymphoblasts and of plas.
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PAGE NUM3ER
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I INTELLIGENCE TRANSLATION
Wound infection prevention
moblasts, with their subsequent maturation into lymphocytes and plasma cells.
(p025).-. The electron microscopic researches(BRIMSTBINER & PARTSCH01955), the re..
STAT
searches with the phase...contrast microscope(MOMILIW01951)0 and the researches with
fluorescent antibodies(COONS,LEDUO & KONNOLY?1957) indicated that the plasma cells,
particularly their young forms(aHRAWS01948) are producing the antibodies? The come
parison of these data with the results of our investigations permits the conclusion
that the ribonucleic acid which was newly formed after the immunization took part
in at least two processesee in the hyperplastic process(multiplication of the eel..
lular elements of the lymph nodes) and in the synthesis of the antitoxins. GINDIN
and FORSHT741958) drew a similar conclusion on the basis of their study of another
modeles- the formation of agglutinins in mice by the soluble antigens derived from
cultures of the Breslau bacillus
The titration of the blood serum of the immunized rabbits showed thatin 2=5
days after a single.shot immunization? antitoxins could not be detected in the lee.
ripheral blood; the histochemical research has also showed that by this time the
synthesis of ribonucleic acid was already going on at an energetic manner,, XI other
researches, with another avetieen(diphtheria anatoxin) and on other objects of ex.
perienntatinu(om
horses),it was shown that, after the second immunization(revaccinae
tion), the process of intensive synthesis of the ribonucleic acid could be seen
not only in the lymphoid organs but also in the lymphocytes of the peripheral blood
to such a degree as the antitoxin titre was being increased in the blood(GINDIN &
OGIENK0,1959),
In the present investigation, it was established that the antitoxins apneared
roughly on the tenth day is titrable amounts in the blood; that they continued to
increasepand they reached their peak on the 24th to 30th deys?
Five days after the second immunization(revaccination) the titre of the anti.
toxins has grown several times (see Tables 1 and 2)0
The comparison of the morphological and histochemical findings with the immun.
()logical data Droves that OA the Oay of the second immunizatio* the regional nodes
were still hynerplastie and rich in cells which were saturated with ribonucleic
acide The second immunizing shot leads to the synthesis of ribonucleic acid at a
larger scale, to the reinforcement of the hyperplastic process and, evilently as
a seouela to this, to a more intensified formation of the antitoxins? It can be
II
a,
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1.000eM16504004~0....0000604,
4 INTELLIGENCE TRANSLATION
TIGT;i511557*-""*-1
........
Wound infection ',prevention 26
surmisrd that this also includes the cause of the increase of immunogenesis after
the second shot of immunisation?
STAT
The introduction of pure aluminiUM hydroxide into the control animals provoked
a weak proliferative process and a weak multip ication of ribonucleic acid, It is
possible that these processes were conditioned by the absorption
4'
%I .11
the products of
cellular disintegration and of the products(p026) (contoon p027 of original),
TABLE/
ACCUMULATION OF A?TTITOXIIN IN THE BLOOD OF RABBITS AFTER IMMUNIZATION WITH PEYTA.
g.? 4.0/0 ?Wmo
Day of
examination perfringens edematiens tetanus botulinus A botulinus B
ANATOMi
Titres of antitoxin in A0U.
avielstemociummaimrte
A.?Ingle.shot immunization
2 41 (VI 0.05 41e00001 4:0.1 001 1.0 < 2.0 1.0
30 >0.140.25 Y2 6
35 0?1
10
21
RC, .:432 Ca6 esn
2
1.0
B. Two. oho t immunization
>0.25 2 1,0 4:2 )? 10ioo < 2 )1OO0l1 Aalja
The complex preparation composed of native anatoxins, by, its Immanogenicity,
has yielded to the combined Preparation into which the purified sorbed anatoxine
were mixed, nevertheless the accumulation of the antitomin in the animals was sati
factory, None the lessowe do not consider RI It adequate that the combined native
preparations can be produced in view of the possible sensitization of the organ.
isms with their employment.
I 1
angsimmanoloeical effectiveness is the APDT which includes the purified aluminium
hydroxide adsorbed anatoxins0 which is completely in accordance with the findi
available in the literature on this subject.
a
The quoted exaeriments showed, that the best antigen which has the greatest
I
tione?as to the pertussis components is carried out (p.88) by
The determination of the immunological propertees of
11011.1V
combined preparae
the method of XEN.
DRIWU.S.A.) by means of a single.shot intraabdominal immunization of mice with
subsequent intracerebral infection(5tock of Kenerick:Nca,16323).
The pertuesie vaccine was checked before its addition, and mixture with the
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Wound infection prevention
.leamoisserroamasiona
011111WWWWWWWWWWWWWWWW100.000111W.CD.1400011,
PAGE NUMBER
77
01,11080001,1010,000001000,0800/1010,0100001100,0?000,10(S400001111110100000
complex preparatons SP well as the immunogenity of the combined vaccine* PDT and
APDT has-been studied among whose
was also present?
(P088)
composing parts this series of pesiATeste vg,ccine
'
TABLE 4"
I tn,11114I ZATION OF GUINEA. PIGS. WITH:PDT AND VDT
PREPKRATION CONCENTRATION
OF ANTIGEN IN
aan ML
PDT
z
APDT
d e f g
READIling a...amount of inoculated vacoineumlofor the checking
of the diphtheria componeilt; bo..amaant of inoculated vac.,
cine0ml0for the checking of the ,tetanus component; c000num.
ber of guinea pig; (10.0AA; 00,0140L.D.(minimum lethal doe;
f000tL:rvived guinea pigs 8 0,000dead guinea pigs
P suspension
40 bill0
D anat060
T anat0200 BU
P 40 bill?
D 60 10'.
T 200 BX.
005 17 0?03 0500 4 2
005
.0095 T500 5
Simultaneou4ly
T500 5 3
0500
? 19 0025D500 4
D2000 1
0.5 19 1,05 T500 4
T500 2
Simultaneously
5
T500
0500
To tan,us
Purif.sorbed
anatoxin
100 B,U0
2
10
2 1000
Purified 100 Boil
1
10
0.1 100
Diphtheria
Parited sorbed
10
0.25 500
anatoxin
30 180f.
.01,5
Purified
10
0.015 30
30 L.fo
cA)003
ate 911 gi =
= - WI CD CO oiti WO CIS 010 CD W W CO 010
2
E. 2
3
The experience showed that the immunogenicity of the pertassis component in
the PDT and the APDT has been the same as in the initial series of the nertussio
vaccine,
On the:, other bandu it Should be remarked that at the present time there
Is no reliable method
J.VA
the determination of the Immunogenic properties of the
pertaseis vaccine nd only by the findings of the observation can authentic data
be obtained on the effectiveness of the pertussis antigen?
In the preliminary exneriments we wished to find whether an inhibition of
I
shown in Table 4(above)0
combined prenarationie0Tor this purpose an experiment was arranged which is
ns4:30 ki
?
any of the components would :exist which components were aded to the samples of
our
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101mill'infection prevention
11110,1111401111?1111111111417
We immunized the animals with the compex vaccine preparation made from puri.
fled anatoxinsowith the addition of the pertussie vaccine to it(PD1ATT as well as
S T
immunization was made on guinea pigs with a combined vaccine composed of the purif.
ted sorbed anatoxinsowith the addition of the formalinized pertassis microbes to
it(APDT),
As controls we have used guinea pigs which had been separately immunized with
the purified and with the purified sorbed teteelle anatoxino and we also used anim.
ale which were given injections of both the native and the purified sorbed diphc
theria anatoxin.
The conducted experiments did not rermit to notice a marked comretition of
the anatoxins which were put into the combined prevaration.
This problem? in regard to the PDT and the A:ODT was left open by us since it
requires serious elaboration which could not be done during the preliminery ex.
nerimentse It should be remarked that the competition will be necessarily visible
and noticeable if the'quantitative ratios of the antigens will not matCh. To cor.
rectly balance the antigens in the combined samples Is not an easy matter; how.
ever we endeavour to do this in our further work.
CONCLUSTONS
1) the rertussieediphtheria.tetanus vaccine(PDT) can be produced from native
and purified anatoxinsowith the additon of the pertussis vaccine, The adsorbed
(p090)pertussis.diphtheria.tetanus vaccine(APDT) Is produced from purified sorbed
anatoxins with the subsequent tr'elition to it of the pertussis vaccine.
2) The harmleesness of the PDT and of the APDToin regard to the pertussis
componento is checked on white mice in compliance with the Instruction for the
testing of the harmlessness of the vertussis veccine. The harmlessness of the PDT
and of the APDToin regard to the tetanus and the diphtheria components? is checked
on guinea pigs by means of inoculation of 3 mi, of the vaccine under the skIn
04`
ee
the flank( in two times eArth with 1.5 ml)o
3)0In comparison with the PDT? the APDT possesses a great immunogenic effec=
tiveness in regard to the tetanus and the diphtheria components?
4) The immunogenicity of the pertursis component in the PDT and in the APDT
has been the same which is also in the initial series of the vertu sis vaccine.
LITERATURE.
(over)
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itipivond infection prevention
PAGE NUMBEA
79
1
LI TBRATURE 8
PAUL A, J0Pediatr,0 19470 vo1.318
STAT
j0Exp0Pathe 0 19530 348 203{,
c,lrjESSELVICK Be Acta pediatrop 19540 43a 1p...21; 22.260
10 ,)20 ). 40
ANATOXIN 2O 000'4o000
2000 c4000
251
45m1
20041 The surrounding medium
germineoed in one day.
251
25 ml
20m1
2.5 1
100 ml
150 ml The surround ng medium
germinated Um two days?
5 >400 C1000 2.5 1
10 4>40 00044 60 000 100 ml
10 >I0 000 4,20 000 150 ml The surrounding medium
germinated in 3 days.
10 )40 000 4,60 000 50 ml
4 ). woo 10 000 A20 000 150 ml
7 >10.000 (20 000 120 ml
m
4
r )20 000 4J4-0 "In
200 mi
c25 q a a a a a a a a
TABLE 2.
d*RAROTEBISTIOS OF THE MDMATIENS DIALYZED TOXINS.AND AUTOXINS
No.og Number
series f bags
qz
Strenght of
in the bags
= =
188 10 qY 20000 440000
193 16 40000 A60000
194 8 w000 cet*.40opo
195 12 )4poolo 460o00
toxin Strength of toxin after Amount % of No.of
Dliniml addition of physiol0 of form.
solution after washing toxin alin
ef bags ? Dim/mi In 1
a a 0
IT.= a
az 0 a 0
C ?????? o ??? r ????
> 6000 datlOciOD 3.8 0.4 20
(Extent of harmlessness for all 43h)
>40000 X60000 1.2 0.4 80
1.75 0.4
2.0 0,4
)5000 20000 44040000
cez ize co mr.
O 010 a 0 Ile a
40
These data-also showeil.that the dialyzed toxins are considerably superior
in their quality to the ordinary ones and the anatoxins which are made of them
contained 2 to 8 times larger numl4y14 combining(binding) units. In the three sec
ries of eiAlyzed Anatoxins And in one
eries of ordinary_ anatoxin
AM FORM
8 FEB. 56
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the contents of
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4INTELLA4RICE TRANSLATION
Wound infection prevention
PAGE NUVISrat
120
the total of the proteino and of the amino nitrogen has been determined*(FOOTNOTEz
ThPse determinations were done by G.F.SREMANOVA0 collaborator at the Biochemical
STAT
Department o for which we thank her very much). The results are given in Table 3.
TABLE 3.
CONTENT OF2MTALAAIIINO4VID PROTEIN NITROON IN THE REGULAR. AND THE DIALYZED
ANATOXINS 07 THE EDEMATI7N8.
No.of Number Type of anatoxin Total Protein Amiao Number Number of
series of Blj. N N N of BU/mg of
PER ml0 mg mg mg BU/ng protein N
of total
CAA
183 10 Regular on fish 3.78 0.50 2,308 2,6 20
hydrolyzate
188 20 Dt.alyzate 2.02 0.27 1.85 10 74
193 80 Dialyzate 2.86 0.77 1.72 28 104
194
195 40 Dialyzate 1.51 0.30 1.72 26 133
From these data it follows that,with the evaluation in units of extivity,
the dialyzed anatoxins have contained 4=6 times leemballast proteins thim the reg.
ular anatoxins.
The immunogenic properties of the three series of dialyzed anatoxins incon
parison with the first series of ordinary anatoxin r were determined in exnerime,ts
on guinea pigs. The animals(12 guindea pigs in each group) were immunized with onl
shot of one ml of anatoxin precipitated by 0.51, potassium alum. Moreover, 20 guinev,
pigs( 5 in each group) were immunized with one ml of native anatorin0 both dialyzo.
and ordinary.
The anatoxin content was determined 10.20,and 30 days after the immunization.
The results of the experiments are given in the illustration.
The exneriments proved that the immunizing effect of the native dialyzed ana.
toxin is smaller than that of the ordinary one notwithstanding the large content
of antigenic units within it. The immunogenic properties of the precipitated dia.
lyzed anatoxins proved to be somewhat better than those of the ordinary ones, More.
overOthese differences(text continues later...)...
(p.13?) FIG.1;(Full.page figure) =spasm OF THE ACCUMULATION OF THE ArTITOXIN
in Amines pigs immunized. with 41alyzed and with ordinary anatoxins of the edematiens
(o*Pr)
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. . . .
...19.W.Id...,=1=2.4.121,2,72.11Man,
bacillus?
PAGE NUMBER
121
galIMIRMAI.M.M.warlowsemomignorm
ORDINATA; ABSCISSA& daYel n DI4GEAMg inoculated 1.0 aaatOxin without
STAT
alum f4 aeries 183 10 BXd(ordinarY); - series 194 plus 1950 40 B.U.
(p0134) FIG 2; (full,.page illustration) COIrARISON OF THE INITUNOLOGICAL EFFECT*
IVENESS of the different concentrations of aluminium hydroxide.
ORDITTATAg ABSCISSAg daysw. On DIAGRAM8 inoculated 10o anatoxin, plus
0055; alum,m series 1820,010 B lj.(ordinary); Series 188,20
Bet
CoMo
Series 1930.,80Bat% ;
Cr2.02) ISO
CID CD
Series 194 and 195,? .40 UT,
(text continues
_.00).0. are not very greatoSuch results0 evidently, are explained so
that A enngliaerrtblo
part of the activity was left in the supernatant fluid,and only
an insignificant part was precipitated by the alums.
The produced dialyzed anatoxins were used by 00A,K0MK0VA for the immunization
of horses. Three horses have been given dialyzed anatoxin precipitnted with 0054
of alums, during 2.3 cycles.
The advantage of these anatoxins0when compared with the ordinary anatoxins,
could not be seen.
-
CONCLUSIONS*
1) At the cultivation of glostridium edemntiens cultures in cellophane bags
which are immersed in nutrient media, o toxins were obtained which were 20 to 50
times superior to those of the cultivation with the ordinary method and which con.
tamed 10 000 to 100 000 Dlm/ml,
2) HowevPr, notwithstanding the great power? the dialyzed toxins are trans.
formed into anatoxins with the addition of O44 of formalin,and with keeping them
in the thrrmostat at 3700 for the same length of time as the ordinary toxins(48 h,).
3) The dialysed anatoxins contained UX46 times less ballast proteinpand 4.10
times/ less total nitrogen per unit of effectiveness of the preparttion.
4) In the work, data were introduced about the study of the antigenic and im
* ? r ?
munogenic properties of the dialyzed anatoxins.
1
Q.. 1
LITERATURE,
ZELEVINSKAYA S.A.,ATIMOVA0V.V.,GIL0GUT E 4.0 VLASOVA E.Vo01cASHINTSEVA LS)&?
.4iA
BULANOVA I.V? & BALAYAN,L.B. New method of production of the toxins of anaerobic
P*- " 1: ?
- ?
.;-?
and aerobic bacteria. Sborn.Seientific bases for the Productioa
ACS 1 FORM
8 FEB. 56 -
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tiel-17157-'"-1
ilf.-"171c91G9JA prevention
5era019550p0100.1150
011J8ONIK Ts0B0Comparative study of edematiens toxins and anatoxins in eel.
STAT
lophane bags and under ordinary conditions. Doctor Dissert00
M = C
4ri.J.J0
= VLASOVA BoV00 VINOGR&DOVA0I0N0 &PnTRNIC00 V0A0 Production of the edematiens
anatoxin on a nutrient medium made from the hydrolyzate of fish.bone meal its
antigenic and inmunogenic properties(Manuscript)019530
. FUSON A, & STrRFM tto Production of potent botulinum toxin and formol tow.
oid0 Nature0 19460 1588 2980
? WENTZEL Lor4.0 & STFRNE 140 A simple double.surface dialyzing membrane0Science9
19490 1108 2590
Col IC:.-CM.7g==/A,
4A.,17
method for large scale production of high.titre botulinum for=
mol.toxoid types 0 Doctor Dies JoImmunol
gp =OE W & KALA N Do A simple method
mo J0Immunol0? 19530 708 1-5,,
* *
ACM FORM
8 FEB. 56
O1
00 19500 658 1755.1830
for obtaining highly potent tetanus tox.
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PAGE NUER
1 --FiFTELLIGENCE IVAN$LAVQN
Waund inrection prevention
1
(p0140gBlank)
123
(p0141)-
V0A,,BLAGOVFSHCHETTSICII0 A0P.ICUZ0MINA.
(Department of BiochemistryoN.F.Gamalei Tnetttnte for Epidemiology and Microbi
logy, Academy of Nedical sciences ? U.S.S.RcoeChiefa Vok.Blagoveihchenskii),
OONCI-NTRATION AND PURIFIOATION OF THE GANGRENE ArATOXIN OF THT; CLOSTRIDIUM
0EnE1ATIENS(p.141 . 148)
STAT
As it is known, the native gangrene anatoxins have small effectiveness; in
the Cl.oedematiensolt makes 5 =5O/ Bounits0 Moreover? they contain a large amount
of ballast protein pigment substances? and other remnants of the nutrient media.
These prnperties of the native anatoxins hinder their utilization for the pure
pose of immunization against infection? and they force us to submit them to puri.
fication and concentration.
The methods of purification and concentration of the Clooedematiens anatoxins
are not elucidated in the literature? although generally there are descriptions of
the diverse methods applied for the production of purified toYins and anatoxins of
the pathogenic microbes of the anaerobe group g," the precipitation with alvohol at
the isoelectric po1nt(IPGAIT)0 the method of adsorPtion and elution(VAN HIPITMEv)o 1
the purification of the anatoxin with the; use of the fractionation of proteins by
salting oat with ammonium salfate(NH4!? SO3 (VOROB0EV0VAN HErnivGrn)0 the purifica.
tion with methanol and ethanol(PILLEIR) and others..
For the purification of the 010oectemat1ens anatoxin we accepted the method of
precipitation with 1.normal hyd.rochloric acid in the presence of table salt.
The material of investigption was the native anatoxin of Cl.oedematiensocul.
tivated on casein hydrolyzate with corn extract All twenty series of the exnerim.
ental material were different from each other in regard to effectiveness and amoun
of the protein nitrnpen. For this reamno the work over each series had started
with the determination of the isoelectric point of the anatoxin() the pH value of
which varied from 303 to 4,9 for the different series.
AiNte.s. th detrmtnatton plj LIA,
of the lectrie
salt was put into the
anatoxin at the rate of 20% by the solution of which I.normal hydrochloric acid
outTy-Too- :
was flooded..at the rate of the test.tube analysis apnroximately from 60 to 80 ml
of acid_ Rex.2.2m_14:S.mx..thLzzx.y4Ljin&t22dz)_,_..........,
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1 PAGE NUlkISER
7INTETaraliCTiRANSIATION
Wound infection prevention
(p.142) .
No, NoF.of 10INITIAL ANATOXIN
P/P series a b c d
HEAMNGSg
T I1V (No heeding of table)
/ 2.00VCENTRATED kNATCg787
f /abcd
124
la...activity0B.U.per 1 ml; 00.00totel nitrogen in n%; lc..,protein N,
nisp,%; ld...chargeo B,U, perl mg of protein 21; le...concentrate by to.
tal nitrogen; 11.0.activit7D'B.U. in 1 ml.
2a.00total N in me+) 2b...protein N in me; 2o0.0dhargeo 1.).V. per lmg
of protein N; 2&6 per cent of purification by protein 2e.0.percent
of yield of antigen.
1 4 5 265.1 37.8 13.0 25 100 113.1 84.8 117.0 88.8 80.0
9 25 274.1 11.2 223.0 30 600 175.0 11100 541.0 66.6 80.0
3 11 25 30606 12.6 198.0 30 600 161.0 126.0 477.7 9607 77.7
4 12 25 345.8 40.6 '61.3 30 600 294.0 12608 473.2 89.5 80.0
5 41 10 267,4 18.2 55.0 21 200 120,1 66.4 701.0 82.6 95.0
6 201 10 278.6 19.6 51.0 25 200 118.7 97.6 204.0 80.0 80.0
7 Mixture
of 208n
210*214 30 315.0 1504 129.8 28 800 128.8 91.0 879.0 78.8 93.6
212 50 347.2 32.2 155.0 30 800 90.4 67.0 1212.0 94.5 5303
214 50 383.6 18.2 274.0 30 900 203.0 107.8 841.0 82.2 60.0
0.1 tl .WO MO oft .11. OM =0 PO 1712.
Then the mixture was 4 for the night at plus 40 to plus 6?00 and on the next
day it was centrifuged or senarated depending upon tie amouilt of the
mftte-041,1 tt
be treated. The separated sediment was dissolved in buffer phosphate of pH 7.40or
in physiological solution with alkalization by Ea0H(caustic sodium) up to s pH 6.8.
...700,The solvent was taken in an ambunt equal to 1/30 part of the native anatoxin
which was taken for concentration.
The degree of purification of the anptoxin was Checked as to its content in
total and protein nitrogen by means of / Kjeldahlis micromethod? at which the con
tent of the protein nitrogen was determined according to the difference between
the total nitrogen and the residual nitrogen.
The comparative results of the Analyses of the native 01.oedematiens anatoxin
and of the from it obtained first concentrate are given in Table 1.
From the Table It
can be seen thatoAlready at the first concentration of the
anatoxinoits considerable purification has been also taken p1ace8. in ten of the
twenty experimental series? the content in protein nitrogen did not exceed 100
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after the concentration;. the content inr?tein nitrogen has increased 2 to 10
10L.O0O210
I PAGE NUMER
125
times at the concentration ,of the anatoxin at A rate of 30 times of its original
t":17"17 ?. STAT
volume.
:17 '
As a result of the anatoxin conpentratienpas the Table indicates? the activity
e_7-7?4 ? 1?1P
.44
has also been considerably strengthened expressed in units of bindings for ins.
tance0 in the experiments Nos.105,607,e the activity in one ml of the native anatox=
ins was 5010,10030 B.U.0 and in the concentrates, correspondingly, 100,200022M 200,
and 800 B.U.0 i.e., the output of antigen was 80 to 9p% in thPse series as to B.U.
The concentrated anatoxino after the first purification, had preserved it
L
activity completely for 6-5.8 months when it was kept in the refrigerator. The con.
?
centrates which were obtained due to the first precipitation of the anatoxin with
the 1.normal hydrochloric acid werestrongly contaminated in the majority of
cases, and their further purification was necPssary.
The repeated purification of the anatoxin was carried out in different ways.
1.0VERPRECIPITATI01! WITH.1,..1".0111vIAL HYDROCHLORIC ACID.
r ' ? ?
The second and the Ithird repeated precipitations of the concentrates were
done with a minimum amount of hydrochloric acid at the rate of 1.3 ml per 100 ml
of the concentrateowith addition of table salt up to 510% Na014, each time the
_
precipitate wai dissolved in physiological solutlonowithout Change of the concen.
tratinnr,
Thekresults of the purification of the anatoxin with triple overprecipitation
by means of the 1.normal hydrochloric acid are given in Table 2.
TAIILE. 2.
No. Number of series ' Agtivi*i; -Protein N
Beil. in 1 me;
ml
Pip
o o o
Concentrate of ana=
toxin of mixture of
ser. 20802100214
before purific.
After 2.precipit. 800
After 3.precipit. 1,100
mo *W. OM 1011, WO
800 saeo
2. Anatox.concentrate
ser02140beforepinf900 ,
After 20PrettP1C; 900
After 30precipit0 000
3. Conc.No0212 anatox,,
before pu.rif,
After 20preciPit.
after 30precipi,
800
- 800
?
81,2
3505
?
,107-0-0
? 9104'.
? 6.110-iP
r
6609
64005,
40025 -
CO 40 eil ons
(No specific heading)
Charge in % of purification
Bett. per 1 I.conc. Native
mg-of prot. of
nitrogen anatoxin
879
1212
lg81
anatoxin
C:Z1
1:3?
62 0
0.
92.8
-
(VD
4107
88.4
95.9
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Wound infection prevention
? ????!.....? ????? ?
..esswerissmaura.wisarrowswammiews.
I PAGE NUMBER
126
(P0144 cont.) 2,PURIFICAT/Oi 07 THE CONCENTRATES OF ANATOXIN WITH N/10
BUSTER ACETATE.
STAT
After the second precipitation of the concentrate with 1.normal hydrochloric
acid9 the precipitate is washed with buffer acetate at pH 4,0. In the case? if
the first concentrate was strongly pigmented ? the precipitate is washed with buf.
fer acetate even after the third repetition of precipitation. This wrisotpitate is
dissolved in physiological solutionDwithout a change of the concentration?
UndPr such conditioned, the anatoxin of Cloedematiens is not dissolved in the
buffer acetate?
The results of the work are given In Table 30
30 PURIFICATIO7 WITH ALCOWL IN THE COLD.
After the second repetition of precipitation with 1IN RO10 the precipitate is
twice wahsed with buffer acetate0and dissolved in the ivaitic.1 amount of the physi.
?logical solutoon0 and thereafter the concentrate was precipitated with an equal
amount of alcohol at pH 595D and at a temperature of cp5? to .7000 and with a rate
of ionic strength (both the alcohol and the concentrate of the anatoxin was cooled
down in advance to SE =500). After an hours settling in a cold room? the precip.
itate is centrifugedo and dissolved in the initial amount of the physiological
Oolution,
(see Tablas A????-?? A.. . ti
? n other e4Am)
__, A
49 PURIFICATION WITH AMORE.
1
The cooled concentrate of anatoxin is poured out into centrigugal tubule sn coot
ed off in advance? and then? with a constant stirring with the aid of a glass rod,
the acetone is poured into it which was cooled off to .10Q Co in half of the amount
of the anatoxi40 In avoidance of the denaturation of the protein? this operation
was carried out at the rate of ionic strength from 0,07 to 0020 with a pH of 5,7 .
GR 6.30 and at a temperature of .50C to .7000 At this temperatureothe mixture was
left for 30 minutes? after which the tubes were centrifuged with their content for
the length of 10=20 minutes. The precipitation was dissolved in physiological sol.
ution without a change of the concentrat4on0
Jer
The results of the puraIleav1Rn eye gummarized in Tbi-
-
a 4.17
4.1* teem
further -mites)
5. PWIFICATIONOW*MMUM HTDROXITM,
1.
The concentrate of the anatOxin is acidified with an m/60 mono .substituted
potassium phosphate of pH 5959 and then one half amount of an Al(OH)3 suspension
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(from 145) p.145) TABLEL.3,
a b" e. f g QTAT
Headings a000NulWr-P/P; b000Number of series; inclu8 1.1.0.
concentrate of anataxin in i mixture of aeries 208?210D2140before
purification 20,0-3M41ATI1:41fter thefl second prectpitationowashed
with buffergIX tA:ooconcintrate of anatoxin of series 214.before
purification 2.2.00.m4pitate after the second precipitation, wash=
ed with buffer 301o''.'.-eoitCentrate of anatoxin in the mixture of aerie
183 and 184nbefore PuTAficationg 3.2.0.precinitate0after second and
third precipitation wished With buffer./ c...activityg B.U. in 1 ml;
do.oprotein Noin meke.p.charge0B.U. per 1 mg of protein Nt
PERUNT OF PURIFICLTIOtoin relation to the first concentrate, g. oo
sameoin relation to the native anatoxino
a b
1 1.1
1 1.2
2 2.1
2,2
3 3.1
3 3.2
= = = OW Om Oa OD we
-
y60 We 021
gCla ... . 00, WI, We Ow CW
d e f g
800 91.0 879
700 46.2 1515 AgoA 895
? 906 107.8 841
900 ?42 1340 31.2 86.3
400?152,, 6 263
G;Z)
SOO 33.6 892 77,9 938
TAKX4cx
(NOT Eg Headings codpeof.g the same as for Table 3)
1 Anatoxin concentrate of tA100
series 150before purif. a a 0 0 0 0 a ...
FIRST8 Ammonium sulfate
SECONDg
55.6 99.05
?
209 23?4
217 39,1*
0.0000 0 0 0 0 0 0
504 892
554.3
536,0
80.6
84.0
0, a 4,i14
404 515 ?57?7
15.72
11.45
8001
7.13
4=
C:=P
98092 203 253 33,8 2007
........... a .
FIRST Trichloroacetic acid
SECOND Acetone 209 3307 99.81 11EM 1680
i= 0 a 0 0 0 0 a ......
168.9
a
4.47
* ThPse figures^are lowered on account of the loss of antigen
at the steriliiing filtration?
a = a a a a me
CU M. a 0 a a a a a a CO
(text from p.15$ cont0)?00tieni anatoxin the! most accessib1e! and the most effective
proved to be the method of tireci-ortating the antigen with hydrochloric acid and
with the mixture of phosphates. It gave a possibility to produce preparations cona
taining- from 564 -to 2472 330,U0 Iieic---tiiie'..mg-Tot:.tOtalitiammx nitrogen,. .and from 1604
to. 4890 LU, per :one mg of prOtein-iiitibe
The eotparative ? itiCOitty of A1(OE)3 a
na.' of Al phog,
T71P-7.1A.
phite was carrird out. with the edenatiens anatoxin of Series No.40purified with
CS3 FORNi
817E13.-513:---
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136
hydrochloric acid and with the mirtire of the phosrhatesoand containing 475 B.U.
in one-ml. The A1(0103 suspension was prepared in the Department of Biochemistry,
STAT
according to the modified prescription(formula) of WILLSTETTER0 the aluminum phos4;
!Oate suspension was prepared by us indepeneently According to the method of
HOLT(1950).
To the separate portions of the antigenoequal volumes of the sorbents were
added? containing different amounts of Al(01)3(from 1 to 10.5 mg) and of aluminum
phosphate(from 2 to 13.6 mg) per one ml. In this way o in the samples of the alum=
inum hydroxideowe produced 218 R.U.
4vt
Aniftelb
mlo elvsg.4
oft 57.
41170.1.00
1 n R
.DDOc&4Aown
mg of the sorbentoand in the samples of aluminum phosphate.. also 238 B.U.of an.
tigen-oand 1,203050 and 6.8 mg of the sorbent per one ml, The adsorption prepara.
tion with the aluminum hydroxide was made at pH 607o with the aluminum phosphate
at pH 6,1. After 18 hour:to in the supernatant fluidothe amount of the non.sorbed
anatoxin was determinedo
It was detected that for a complete sorption %/a. -' the antigPno 3 mg of A1(014)3
and 608 mg of aluminum phosphate was necessary. Oonseouently0 the aluminum hyeroxi
de proved to be a more active sorbent than the phosphateowhich is in agreement wit
the data of a number of authors? data which were obtained in experiments with
other anatoxins( HALORM & JA B501955 PETROSTAN and caworkerso 1956; BERGOLo.
TSEVA01957).
For the complete charateristice 4f the different methods of purification of
the edematiens anatoxin it was necessary to conduct a comparative study of the
immunogenicity of the preparations purified and concentrated by the various me=
thods.Ne conducted this work with the antigen of Series No.2170purified by three
methods ,cs 1) by precipitation with hydrochloric acid and phosphateso 2) by -orect.
pitation with metaphosphoric acid and with(p0154) phosphates; 3) double out.saltin
with ammonium sulfate. AftPr the purificationoall preparations were completely
sorbed on Al(OR)3,0 with a concentration of the latter of 5 mg in one ml.
Rabbits of six groupsoweighing each 2.0 . 203 Kg(four rabbits in each group)
were immunized with a single-Shot under the skin of their flankowith two doses
.of each of three preparations. . 10 and 75 B.U.c. introduced in the same amount
(one ml)pand with a conctant content of 5 mg of A1(OR)3 in one *10 After two
monthsoall rabbits were revaccinated with the same doses of the corresponding
,ftwow..a./Wamlisamor
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I PAGE NUMBER
237
?
1 ttrrELISPICE TRANSIATION
Vound'infebtion prevention
preparations? In the animalsofor the,determination of the content of antitoxin in
their-blood? blood was taken on the 15th,25th and 60th day after thes.4pmunisati0n,
and on the 5th015th and 118t4 day after the revaccination0Each serum was titratPd
separately, The obtained data were submitted to a statistical treatment which con.
misted in the fo1lowing3. during .the entire DPriod of investigationofor each group
of rabbits0the geometric average of the edematiens antitoxin titres and its log.
arithm,with the standard deviationowas calculated?
For the elucidation of the authenticity(significance) of the difference be.
tween the antitoxin levels in the different groups, a comparison was made of the
s ?
logarithms of the geimetrical averages of the titresowith the aid of thP t.test
with a level of significance of 00050
In the course of the experimentoit came out that not a single one of the EIT.
amined preparations had a true superiority above the other in regard to immunoFe.
nicity,In the group of rabbits immunized with 10 B,DF?, the average(geometric) of
the titres after the immunization had reached a maximum of 3,42 . 5,12 AO? af.
ter revaccination.. 18054 . 49,33 AO1T0; in the groups
oculated with 75 B0U0corresponding1rothe values were
of animals which were in.
6?90 15030 ILL, and
43,09 . 84,26 AU 0 Four months after the revaccination, all rabbits were complete.
ly resistant to the intramuscular inoculation of 100 005,11, of a 2day culture of
Clooedematiens efore the inovalation? the antttoxin level in their blood varied
from 1,25 to 1700 AdDr?
In this way, the method of purification of the edematieus toxoidowith thv? aid
of hydrochloric acid and of phosnhateso having proved itself the simplest and the
most effective in regard to its chemical indices, hatf allowed the production of
a preparation which is not less immunogenic than the Preparations purified by
other and more complicated methods?
The following sedtion of our work was devoted to the elucidation of how the
amount of Al(oH)3 ingluences the iMmAnogenicity of the oedematiens anatoxin If
the sorbent is in surplus in oormarisqn with the, needs for a full sorption,This
question gained special interest in connection eith the researches of FISL'R and
t31(l953)which revealed that th 14111mWrItqgics1 effectiveness of the sorbed ana 1
toxins),aPart frot the_ comPlertene.r.s of the sorption of the antigens is also deter.?
1
mined by the otrength:of its _compation with the adsorbent() and the latter,
as
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PAGE NUMBER
138
INI'ammiarr476104 E TRANSLATION
Wound infection prevention
(p0155)
FIG.(fall.page) COMP&R1SON OP THE IMMUNOLOGIOAL EFFECTIVIWTSS OF
THE DITFERTINT COTTOSINTTRATIONS Or 41mna1M HYDROXIDE.
OP.DP111.Tits Titres in A.U. and their loperithms;
ABSCISSA.: Days( 0 to 129)
On DIAGRItivis Revaccination(arrow)0
STAT
p1.56)0it has been indicated by DEEMBL(1949) and HOLT( 1950)0 depends upon the
content of the depot substance in the preparation.
The rabbits of three groups0wighing each 2.0 to 2.3 Kg(three rabits in each
group) were submitted to a single.shot immunization under the skin of the flank
with preparations purified with the Series No.4 sorbed edematiens anatoxin, con=
taming one and the same dose of antigen(25 n.U)0but different amounts of A1(OH)3
. .0.560500 and 10096 mgoin one ml of the preparation. The degree of sorption
of the antigens in all cases has been complete(full)0 The revaccination of the
animals was done two months later The content of edematiens antitoxin was exam=
ined in the blood of each rabbit dynamically. The obtained data were submitted to
the usual statistical treatment(see above). Its results are given in the Pigure.
After the first immanization0the preparrAions which contained 5.0 and 10,96
mg of Al(OH) revealed definite immunological superiority over the antigen with
0,56 mg of aluminum hydroxide. Between the first two preparations, no notice We
differences could be found in the immunogenic capecity. After revaccination, the
effectiveness of all three antigens was roughly identical. In this manner, the
results of the first immunization of the rabbits indicated that the increase In th
amount of A1(OR)3 in the preparation over the amount whiCh was required for the
full sorption of the antigen(frem 0.56 to 5.0 mg) has increased the immunogenicIty
of the purified sorbed edematiens anatoxin taken in the dose of 25 B.U. Farther
increase in the concentration of the aluminum hydroxide(up.to 10.96 mg) proved
to be ineffective.
In Connection with thie?we suggest that,at the Choice of the dose of the ad.
sorbent for the purified sorbed anatoxins0 one should not stop at that minimum
concentration of the adsorbent which gives a full sorption of the antigen, It is
necessary to bring to light the optimum dose of the adsorbent which assures the
easiest bondage of the latter(adsorbent) with the antigens andocousequentlyo also
7-
-n
the highest immunogenicity of the preparationo not raisingohoweverp thegeby the
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IONNOWROMPIMM111001...
"threshold of reactogenicity?
I PAGE NUMBER
139
C(iliCLITSTolto
STAT '
1) among the methods which we have 'examined for the purification of the ede=
matiens toxoid the simplest and the most effective proved to be the method of pre=
ctpitating the antigen at the firit stage of purification with a linormal hydro.
chloric acids with the addition of 20% ItaC1 to it, and at the second stage with
a mixture of a 305 M solution of KW04 and Y HP0
2) At the comparism of the study of the sorption activity of Al(OH)3 and
of aluminum phosphate,introduced into the experiments for the purification of
the edematiens toxoid it wag reveled that the aluminum hydroxide is a more
active sorbent than the phosphate*
(1:0157) 3) The idematiens anatoxin which was purified with the aid of hydroch10.
ric acid and of phosphates, in regard to its immunogenic power,was not inferior
to the preparations which were purified by other, more complicated methods?
4) The increase in the concentration of the A1(OH)3 above the amount which
is needed for be fall sorption of theXii antigens has caused,within known def1.
nit e limits, an increase in the imtuOgenicity of the purifiedssorbed edematiens
anatoxino
LITERATURE0
*wBERGOWTVArk Loko Trudy KharkovollotoIcVoSo Mechnikov Inst1tute)019570
242 193.2010
BLAGOVESPOMNSKII V,A00 BOJANOVA I0V., ZAKHAIOVA No 00 ISPOLATOVSKAYA 1. 3V
KUVMIFA A0P00 MAIOROVA. IoP00 MAt4ALEVS11YA Lon. Theses Dokl InterinstoScient,
Conference on Probloof AnaerobesoMoskva, 1956, p036.370
- VTGODOITIKOV G0V00ZELEVIrSKAYA SoA00VOLKOVA Z M00 KAVINTSEVA :NoSeJLASOVA
PAIT? & GIL0GUT BoA. G10uor0I0V0S0 Materials for exchange of exper019570 1/19530
85.1050
* .PETROSYAN BoAo,ZELIKOVA LEO?& IPTEKA.REV1L FoL-(Moskva NIIVS Mechnikov)
Theses of the ScientoWorks done in 19550Moskva019560p0250
- CHERIKAS GoPo J,Microb epidemoimmun0, 19580 73 60.65,
AMOUREUX Go& YEN Fo Ann Inst Pasteur, 1950079?M:9069 po913.9140
DEHMEL H,Zschro Immun00 194,9D-10,68 No 2 13,174.1840
- EISLER L4 EIBLL:110 ZsOhr0,7 JA0.9p? 1E3, No020 po12qp133,
?
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(Literature cont.)
21240
PAGE NIXIBER
140
HALONEN Po& JONES Lo Anne medoerp o& biolo firma 0
19550 WTHoolm,20 p,5.110
HOLT LoBo Development in diphtheria prophylaxis. London, 1960,
-.BAUM Mo0TUWIN AA NICOL Lo Corend0Acad0sc00 Parc o 19530 2368 21: 2122.
44 WENN A00RMINVELD EoH00 PILLET Jo& BATNAUD M, Ann0inetPasteurc,0 1954,
voloIrs No020 D0185.1930
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WWIRMIPPerillIkerfallgritriOrf,:ti
iNTELLIGENCE TRANSLATION
Wound infection prevention
p0158)(Blank)
(p0159)
EeV.VLASOVA, 0.4..KOMYOVA0 S.Y.SOKOLOV0 M0Kh0VOLESNIKOVA
(Department of Wound Infection? CrIettamalei Institute for Microbiology and Npide.
miology? Academy of Medical Sciences0U0S.S.R)(Chiefs G.V.Vygodchikov)
ACTIVE IMMUNIZATION OF THE Erlurio. PRODUCTORS OF THE ANTITETANUS SERUM WITH
I PAGE NUMBER
141
.1101.MONIMY4~011M~~4..01?1
mompomommomore ? ?
TAT
EDVATIENS ANATOXIN(D.159 . D.166)
Among the equine productors of therapeutic sera0sometimes cases of gas gangrene
are observed? Thus0ZANNOLLI and CATIWO(1925) describe a few cases of gas gangrene
in equine productors of various sera, In the majority of the cases0 these authors
have distinguished Clohistolyticum as well as other anaerobes(O10septicum0C10per.
fringens, Clpoedematiens). These anaerobes were sometimes isolated in association
with each other as well as with other aerobes not identified by the authors0 knaeroc
bie microbes were also isolated from abscesses at the site of the inoculation of an
antigen. In a number of cases? upon the inoculation of 01.histolyticumea specific
serum was used which showed a therapeutic effectiveness.
In the native literature0we were unable to detect data about the gas gangrene
in equine Droductors of therapeutic sera. One of the
^1.141101nirso
swab, :AA,.
ef the present work
(0.K.KOMKOVA) had observed eases of gas gangrene in equine productors of theraDeut.
sera.At the bacteriological investigation of the materialoin a number of cases,
a culture of the Clooedematiens was isolated; in some cases the specific serum show.
ed therapeutic action.
From 1951 to 19550 among the equine productors of the antitetenus serum two
cases of gas gangrene were observed? In both instances0 a highly toxigenic culture
of the Cl.oedematiens was isolated. One of the isolated strains(79) is used in our
Institute and in a number of other institutes as the production strain for the mak.
ing of edematiens anatoxin.
The above indicate cases prempted us to immunise the livestock of the equine
producers of the tem antitetams serum=(p0160) with edematiens anatoxin -In this man.
ner0 at our disposalp a considerable livestock of horses was found on which we
were able to investigate the imminogenie proDerties of the native and of the eon.
centrated,edematiens anato*ins prodUcediTin.the Gamalei?Institute for -Epidemiology
and Microbiology,
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Wound infection prevention
PAGE NUMBER
142
...41,11MON/NOPIN/MO.OfOtingarIllOagalia.a.
In the years 1954.19550 in the G?et Institute of Epidemiology and Microbiol.
ogyofaraulaa of nutritive media were worked out whose Dasis was the acid hydrolys.
STAT
ate of aaseinCVTNO(RAirVA,011LASOIPeoPALXINA 1956). On these nutrient mediapedematiens
toxins were obtained of the power of 2000 . 10 060 Dlmo and anatoxins in which 10.60
B.Ii&?s were contained(VLASOVADVINoGRAnOVA"LWINA01957). The native anatoxins were
purified and concentrated in the Department of Biochemistry of the rramalei Institute
of Epidemiology and Microbiology( BLAGOVESHOHMSXII and others 1956). The produced
preparations contained 600 . 1500 B U. Per mlo depending upon the quality of the
initial preparation and upon the degree of concentration.
The antigenic and immunoFenic properties of the native and of the concentrated
preparations were examined on small laboratory animals( VLASOVAMILEVIIMVIA0VOL.
KOVA.1957). The conducted experiments showed the good immunogenic properties of the
produced preparations. All the above discussed data permitted us to take the indica.
ted preparations for active immunization of horses against gas gangrene provoked
by the Cl.oedematiens. All together two experiments were set mo(one in 19550 and
the other in 1957)0and 226 horses were immunized.
EXPERIMENT Noolg
* * * * * * * * In the first experiment0154 horses were immunized. The im.
munizatioa was carrind out with native edematiens anatoxin of Qarime N00203 contain.
ing 40 B.U. per m10 The immunization was done with 5 ml of anatoxin which was pre.
cipitated with 0.51 of potassium alumoor sorbed on 54 WOW30In this manner, for
the immuniaationo 300 B.U. of anatoxin were taken? or 0.4 . 0.5 B.U. per Kg of
body weight. A, portion of the horses was immunized with a single ihoto a portion
was immunized with two shots. The samples for the determination of the titre of
antibodies were taken at different times...from 15 days to 3 months after the immun.
ization. The results of the exneriments are given in Table 1 and 2.
As it can be seen from Table 10 at the taking of the samples 15.30 days after
A.S?
tiLIC LiumunizauLtmo J.0 VL
majority W
Irless...iA2 eignA RCN 41ftm 4.4.11-4Am
await:1%70%-m., =aim& Low' wic VA VAC: was "al Ac.U.0 and
in 8 of them it was7110 A.U00 and only in 4 horses was the titre 0 001 A.U.
In the groups in which the samples were taken at a more advanced period of
time? the number of horses with low titres was increased; thus? at taking the sam=
pies after 2.3 monthsoin half of the horses(in 8 out of 15) the titre was lower
than 0.1 haU.
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1
1 The fact calls attention that at the immunization with the same antipen but
1 ?
/ 11
sorbed on 51 Al(OH) the refractory group was missing. These data were confirmed
1 3'
1
1 in the subsequent exprriment(See Table 4 Group 1).Therefore0 we think them possible. 1
i
1 notwithstanding the small number of this group compared with the previous one, atten-
ition should be paid to these results.
II i
I presented.
In Table 20the results of 1?10 two.shot ImMunization at different intervals are
?-- TKIXt.1-
3
I
(p.161)
I ME NUMBER
143
SINGLE IMMUNIZATION 07 HORSES WITH EDPIATIEffS TOXOID
NAME OF ANTIGEN
& DOSAGE
'S TAT
TIMX OF Number OF THEM.o.DHAVING A TITRE0A,M0
TAKING plaPLE, of
tiolises 1 A,U, has ac=
cumulated?Only individual horses(4 out of 56) did not have 001 A.U,
2) Unon a single shot immunization with the native and concentrated edAmati.
ens anatoxin, sorbed on Al(01030identical results were obtained0but somewhat in.
ferior to those received upon the immunization with native alum.precipitated ans,
toxin,
3) After a two.shot immunization with the same antigen(twice0 each 200 LU.),
In the blood of the majority of horses(53 out of 56) In A0U0 was found; in half of
them 0?, )10 A0U and only in 3 horses
000 `). 001 10 A0110 has accumulated; in th lood
of half of them?? A,U,
n S,
(Literature0over)
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..110.11.11MOINIMINI?01111
PAGE NUMBER
148
LITERATURE*
VINOGRADOVA IN., VLASOVA 1101,00 PALKIWA N0A08 Casein medium fr-A7 the produc.
tion of edematiens anatoxino.Materials by exchange of experience 19560 To2/52ono
61.67,
/......./Production of edematiens anatoxin on nutrient medium from caseine
hydroly7ate0(Manuscript)19570
BLLGOVESHOREYSKII0V,A, Concentration and purification of anatoxins of the
agents of anaerobic infection? prenared on casein
(n_166) mediaoTheses Dokloof
the InterInstit0Scient0Conf0on Problems of the Anaerobes019560036.370
VLASIVA. B0V00 ZELVIINSKAYA SoA00 VOLKOVA ZoM? Study of the antigenic and
immunogenic properties of the edematiens anatoxin prepared on casein media o Mater,
for Xxchoof ExperoGlouproI0V0S0 19570 (63) po75.840
VYGODCHI7OV GoVo and others, Antigenic and immunogenic properties of Othe
trianatoxinso prepared on meatloss media? Ibidop P085.105.
PLETENEVA Iola? To the active immunization against gas gangrene? DoctoDisso
Moskva019500
..,TOVTINOVICR LG o Comnarative study of the immulogenic pronerties of the depot
and of the rattye anatoxinso of the perfringens and edematiens in experimento Docto
Dissert0019530
= SRMRSON Aolfo Perfringens anatoxin and edematiens anatoxin as antigens for
the combined immunization and revaccination in eroerimento DoctoDissoo 19540
KHOWROVA ZoN,, Study of a few conditions of producing edematiens toxin and
of immunizing effect of the edematiene anatoxin o DoctoDissoo 19540
ZIMINA 0010 On the production of toxin of the Cloedematiens of high titre?
Trudy KhargkovoInstoVaccines & Sera(Mechnikov Ineto) 1957, 24; po 203.209,,
BORISONIK Te130 Comparative eti.Ay
of edematiens toxins and anatoxin r in cel.
lophane bags and under ordinary conditions? Doct0Dissoo 19550
* ZARNOLLI Co & CATIVO No Anaerobic flora of the horses(French)000rend0Socbiol0
19250 928 p0817.8190
ROWERTSON & KEPPIE0 Gas gangrene active immunisation by means of concentr&.
ted toxoids o Lancet? 19430 2458 p0311.314,
. AMMER W0A0 WITH Roo SHERMAN Roo 1/000 M.A.0 TYTELL La Toxoid immuniza.
tion of experimental gas gangrene? AMA Archo Surgoo 19520 658 633-6400
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(Literature cont.)
1 PAGE NUMBER
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- ALTMEIER W.A., LURSTL W.M. OULBERTSnNoW.R00 WADSWORTH 0.7ff.TYTELL A., LO.
Ann0Surg0
GAR M.A.08 Toxoid immunization in experimental gas gangrene./INIMIX 1947, 1262 509.
522.
TUIPTICLIFF E0A,0 MARSH H.A.g Amul precipitated toxoid an immunizing agent
against infectious necrotic hepatitis in sheep. J0AmoVet.M.Ass.. 19390 943 98.110.
- TUNNIOLIFF0 B.A. Bleck disease immunization?, Ibid, 1940, 968 105.106.
- /=/Persistance of immunity against infectious necrotic henatitis
sheen vaccinated with alum.precipitated toxoid0 'bid? 1943) 103s 368.370,
GUILLEUMIEL & KIMIGER A,? & DEVISn M. Immunization with edemptiens aria.
toxin(French) Ann.Inst.Pasteur 1936 (sic; 1956)091g 459.571.
* * *
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130167
A?K1LICATOV0
(Department of Wound Infections
?,,**.???; ,
?:
PAGE NUMBER
150
v,
f
tr,Ajil le/
STAT
N PAAMAplI Institute, for Epidemiology and Microb.
? rp
tOlogyo hcademy of Medical Sci ce ?proper; (Ai ?,91,4ket gr,,491,01710Vygod chi kov)
INDIVIDUAL PECULIARITIES OF ANTITOXIN PliODUZIION.AT THE ONE.SHOT IMMIPTIZAT1014
rf rie - 4".6 "*"' aqa?
-'
WITH PURIFIED SORBED ANAT0XINS(p.167.17.4)
'A'477%
The wide perwoectives of the practi(41 use of purif,ied sorbed anatoxins are
?
creating the necessity for the study fit the immunological laws which amply to the
vaccination with these preparations The investigatIons of this ?type of study are
always timelyosince up to now the basic idea! pPotyt
the laws of antitoxic immunity
had been chiefly founded upon data ?Obtained :in eXneripents,with native anatoxinso
A big step forward in the matter of, the studyof the immukological peculiar1.
ties of the sorbed preparationiis thiO4drk6itifi A
llatedin!4ur Departmett(1958) in
r s s,
which, together with other problemso-the*enOtal laws -of the dynamism of antitoxic
-
he dynamism of the individual Im=
immunity were investigated in detail, HoWeve.ro
munologi cal reactions upon vaccination with
studied for the present6
thesdrbed
antigen is still very little
The study of the dynamism of the acciumulation, of the antitoxin in the blood of
the animals is conveniently conducted by means of the constructemn(construction)
of graphic curves of the relationship of "TIMPlatFFECTII0 ? in which on the abscissal
axis the time is mut down that has past from themoment of immunization and on
the ordinatal axis,. the height of
the antito?ictreso? The juxtaposition of the
"
individual graphs of antitoxin mpbehrimniation and the attempt at grouping them accord.
ing to separate typesp starting from the immunological similarity or differenceo
may represent certain theoretical
4 ? .
At the use of the purified serbeciant0X*ng the peculiarities of the immun.
?logical reaction of the organism are discerned in the best way after a single...shot
, .
initial immanizstiono The second inoculation of ,the antigenocarried out under con.
ditions of raised immune reactivity w111(p.108) level out to a considerable degreo
'TT ?Qr.AF
the individual differences of antitoxic respons00
We have studied the dynamiSm of antitoxin formation at single shot immuniza.
'T4 P'
tion of rabbits with purified sorbed antitoxins7plf the oeeeMatienso and the tetanus
-
, .
? ,, r .
bacilli 81 rabbits
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PAGE NUMBER
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Pill..100?1400.1???
.1(
26 were inoculated with, varioui,.. 9S
? Ar.,
.ens
-aatoxin( from 5 to 160 B,U4);
t'tfit:,?;;
27 ,were in.,opulated. with 4ifferej?t,,slosex*f.,.the,.... tetanus. anAtpxin.(from ?() to 200 LU
STAT
28 were submi.tted,.. to a apaibined nriltia0,24%tti,011 liftth thpsme arkti,gens(yith doses of 25
ixr;./fozzi.7 c,01,4,1zx
and 160 B,tic, of the ed.ematiens anatoxin_. and 40A 200 ILL, of the tetanus a.nrktox.
.
ino taken in different combinations),.? A,11 ditties 01' the_antjgens vore,k_ introduced into
ATITto-TE131. Tla-fo?,!- ?? ? -
rabbits at, a constant concentration. pftbe 4102_11,03D 1..9 5 mg in one The content
? ..,4:0% Y.7 ?
of the blood in antitoxin was examined. in Or Animals 5015?25,45 and 80 days after
,t
the immunization,For each rabbi_toa grath of the,apcumulation of the antitoxin
.eur, orr ? '4
was drawn(for the rabbits which were wublected to the combined immunizationD two
motrOpt*CSIT
graphs were marlif.)n?n th- basis of the averaste(arithrleAtislo. of the titrespa common
-
curve was also plotted of the accumulation of both antitoxins.. individually for
vr'ia .? ?% ?
the
rabbits which eere Inoculated with one antigen and for the animals which were
vtirrTv)r-,-- ??
immunised with the sorbed prey)aration,A separate _Rlottinr.twas made? Therebyoit was
u- 7 - " ? ? '
discovered that the fortnis ,of: the ,(7,-"Cbmnicii; lb_ t,4, t,o7a1nis comole tiny
44
identi cal at the -stroarate. and at the 'co-fob-tiled s-immunteatiOni.--- Con se.Ouently , the t3
-L:71 -
second antigen Into the organism did. not influence thp
,21e, r ?
tome ou rie
v) Viet / + 4
%co hf .1' AL WA. a
dynamism of the devplopment of the, tromunr.)1ogi.oalresetio7i, in respect to the other
of the .anti0;ens.
/ There is ireason to suppose. . that-the- amOttnt of -- the_ produceable antitoxin after
cr-t .
S
a single.shot 1"1""4 'Patton with sorbed- anatoxin at each given Moment de-oend.e
? r f T -14- - - -
to the utmost degree upon three factiorstit= the' levels of the immune reactivity
eto.tizt
of the organisms 2) the -amount of 'the antigen *itch approaches the cells that prod.
(A, tvk-r
u.ce the antitoxins and. 3) the concentration of the antibodies in the tissues and
fluids of the organismocapable to some degree tp-n-e'Ut'rali.se the action of the arsti,_
? _ T.; ;;;.,, ?
gent, By the rectorocal interaction,. of -these if!!c:tormlLthe shape of, the curve of the
- *
?? tt t- 7'? ?
antitoxin '66ritient th6 hi -h 1414. bse'j4iltisib a4termined,
r , ?- :=
On VI 1 ' the nrirtirrihiot carvefifr the A'cctt1on fvf'
'the..tatiLimri.....tiens antitoxin
cur r:14-47c7.7 :715 ? ? ?
presented() We see that the antitoxin agoears in the blood of the rabbits 5 days af.
1
ter (the immunization) pond it has alire Y reached its maximum content on the 15th
-
day,,Thereafterpits level rems.in! ?:7dioxfiv.....a01frftsfyo7 .t!ie 15th to the 45th)0
which is probably conditioned by.* ptiripod 01, i1Zibrium between the acting factors.
After 45 daysoa gradual reduction -in :the antitoxins level is observed,. It is prpb.
- IvoSi
able that it is provokezd by the decrease in the ASOlallt of the antigen which goes
to the cellgutiall_PLIS1110.1ttJUALtIELDA.AMLAILAWL-tMMULAJULexameAuLmay.ta?........
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,poreviasipassoawr,30114,10,410S,.
PAGE NUMBER
152
INTELLIGENCE' TRANSLATION
Wound infection revent!on
partly explained by the drop in the concentration of the antibodies in the tissues
(p0170) and fluids of the organismgWe cannot,say whether at this tiaeAT the immune re.
ST
activity in regard to the edematiens anatoxin is lowered or notasince there are no
data in the literature about this problem,In the experiment of KHAINAPIWL and co.
corkers(195401958) with the diphtheria and the tetanus anatoxins,such a reduction
'?T"
was not observed?
FIGl(full page illustration on p169)t DYNAMISM of the accumulation of the
antitoxin
edematiens antitoxin (4 graphsamarked A0B.VoGO(Ift low left; Gs low right),
1= ccp i=o at, ......
In this manner, the common carve of the accUmttlation of the edematirns anti.
toxin is characterized by the following phasee:. quick risea prolonaed
zationa and gradual reduction,
At the study of the individual antitoxinic response we have found thatain 36
rabbits out of 54 inoculated with edematiens'anatoxina the curve of the accumula.
tion
resembled the common curve to a larper or smaller degree, We called this the
Type I Graph; it is presented in Fig010B0 with all its variants, For variant a,
subsequent decrease at the 45th dayawithoat an expressed period of stabilization; 1 1
characteristic is the slight rise of the antitoxic level at the 25th dayoand its
for variant b, characteristic is the reaching of the teak on the 25th day and a somel II
what shortened stabilisation in connection with this phase; for variant vacharaca
Is the beginning of the decrease of the level with the 25th dayoand also
4 a.44
a shortened phase of stabilization; for variant so characteristic is the maintena
ance of the maximum level of the antitoxin up to the 80th daya 1.e.0 a prolonaation
of the phase of stabilisation, In our (minion? all these variants are not indepen.
dent types of the curve, but only different shape's of one type characteristic for
animals with a stable form of Amtuhological reaction? at,eadily;paintaining, for a
shorter or longer period of timea the tivel of iiti4ne-activity which they reached
The grouping of the rabbits according to:the variants was the following:
Tye I?.09 rabbits; variant aa?,?; viiriantli., 6g vpriant v?0.9; variant gm
In XXI 8 rabbits a graph was found whia' wasshitiWly distinct from the granh of
Type Iowith all its variants, We calfieit grataII-curve(See Fig010V)0For this
carveacharacteristic is the quick risi'of.Aheiiittoxin level at the 15th dayawhich
is followed by a shprp drop of the lei rlif theirnth, day and then a very slight de.
III"
ACaSi FORMcrse at the 45th and 80th days,Stith: a- 4iiirie must be characteristic accord-
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pahtettarrimmigumvirlmmttawrv,rtt
Wound infection revention.
? $4.....110110.11.01.1
MINOIMIIMPIMMOMOW101.8?11...ir."......MPIMM411..W ?
I PAGE NUMBER
1=a
.16.5)0
ing to our opinion 0for animals with: e.icomo weak and unsteady character
of the immunological reaction() antmili 1n dW tetatus of increosed immune re.
STAT
activity is ou.ictrly changed into a isioldiveita 1i?f 'relv.tive immunological inertia
Finally() in 10 rabbits we discoverieTis flV Of the cu.rve(See Fig01) for whic
the characte,ristic features were two iiiiiieor'the'- antitoxin levelCto
on the 15th and the 45th days(on a vartlicintWie'':ekrOia? "peaks was noted in a later
period of time.. on the 80th day). The? secOnUrise iot the graph can be explained
wo,5,7
the presence of a second "stimulush4GLINNY4& BAR101931) which ampears upon immun.
izations with the depot preparations. tWirldarneei and the vigor of this phenomen.
on must d epend to a large degree upon rn 'off 'the immune reactivity of the
animals. In connection with this%) the turve Of Tetie .111s '11 -r-cteristic,, accord.
lag to our opinion, for animals in which'i-brief iteriod of stabilita.tion or of some
reduction of the level of immune reactitiity is replaced by its sharp riseowhich
will also give the 'nios'sibility ,of a distinct Inanitettation, of a "second stimulus",
These animals are characterized by the 'highest activity of the immunological reac.
tion. Their distribution according to the'- Variantii was the following
TYPE I110008 rabbits; variant a
In this mannerothe study of the individual graphs of the accumulation of the
edematiens anntoxin permitted to .eparate -thEf inOtulated animals into three groupeo
according to the three types of graphs'. first(laTabbits),,with high intensity of
the immunological reactions second(36 -reibbits)0.'a*ith mod.erate intensity And rrskekr.
ed stability of this reaction; third(8 rabbits)seni;with not su.fficient intensity and
with an unstrble character of the imanidotogioal rels:Ction.
The general common curve of the accumulation of the tetanus anti toxin is 're.
eented onFig020A. It shows that the antitOxini in the blood, of the rabbits
aP!....;eatIS
5 days after uthe: oh) and t ontentT on the 45th day,
at which the tempo of growing of its level is visibly lessened after the 26th clay.
At the BOth dayosome reduction of the antitoxin level is noted:, Although herr. also
a clear phase of the stabilization of the titres is not seen nevertheless the dif.
ference between the amount of antitoxin an the 25th.45th and 80th days is so small
_7 .- -
that this period slay be considered the phase or the relative stabilization of the
nn.- n in%in
level of the tetanus antitoxin.
t 14- 1:7 7 a
:
The study of 55 individual graphs allows to collect them into three basic types
_ rnTinninnf: nt-1,
TYPE 1(seP Fig029B) with its *variants to a lAttutz.=5raller dere
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Wound infection prevention
PAGE NUMBER
154
the common carve of acsnmulation of lotiOiatts antitoxin? For variant aocharacter.
$...una Ve...,,-.44MM46 Waal M s.? ?
istic is the stabilization of the tf*ii4ilirliftei7A8adymo but in sinclig,,T eases there
may be also their slight increase 'fo183itVolltaiiszlicteristic is the reaching of
the maximum pPak level already on thei.'25thrWayo its stabilization before the 45th
day O and a gradual lowering at the BOW diOt vo characteristic is a
maximum -peak level at the 25th dayoand 10Wilds;Untehande until the 80th day. The
tribution
of animals according-to theaii ttevethe following:. Type 100.24 F&b.
bite g variant ao0.4 rabbits; variant b'eabbit'=; 6,riant vo
0 0
8 rabbits? On the
wholeothe curve of Type I was observed?Id(45 rE:.bwits? We assume thatohere as well
as at the immunization with the edematiens anatoYino the Type I Curve defines (ifl.
cludes) animals of a stable character Ortheir immunological reaction.
For the Type 11 Curve(See Fig02 V),Icharacteristic isoafter the rise of the
antitoxin level at the 25th days its iharp drop at'the 45th day and/6.
FIG02(Full.page illustration on pag 172)0ITNAMISM OF TH1,1, ATIUMULA...
TION of the tetanus antitoxin0(4 graphsomarked A0B0V0G.?(V8 left low; G8 right low)
Ordinatas carry the hatIo.s; abscissas000days after the immunization)
(p0173)
sali am am am =
pthe f"rthe,,- gdu1 reduction -rat the 80th day(for variant ao the curve
of the drop,of titres is somewhat leveled out)00n the wholeo the Type IIGraph waz
seen in 7 rabbits; from themo variant la-was present in 20 Such a curve may be char.,
acteristicoin our opiniono for animal:1-o -n unstable character of the immunolog.
ical reaction
The Type III Graph(See Figo2o0) with t;tio *peak " of the antitoxin accumula.
tion..on the 25th day
and
10 eft
the 80th day(for variant ao on the 15th and 45th days)
..is characteristic for animals having a, hieh immunological reactivity? The carve
of this type was present in R rnhhitn(One-Of them showed variant
In this manner? the rabbits immaniseiewfth titanus anatoxino according to the
character of the immunological reaction; ibUld be eellarated into the same three
groups as the animals which were immunited'with the edemntiens anatoxin. It is true
that here the quantitative correlationiqa 'tire iiPUps 'were somewhat different(3045,
rabbitsorepectively).
41m Qcgc-uu4,,,,Tg.i.1;
We were unsuccessful in showing 44601 iitiiVeinressed relationship between
the values of the immunizing doses1;Fifti iiitikiefittliiiid the types of the graphs of
the accumulation of the antitoxins0
? ACSI FORM
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1 TABLE
antitoxin had much guleker reached the.igiver4416iflexiI4 it.
WWI( SO reduced on the 80th day
considerably more strongly than the Vititius antitoxin(See Figt,101k,
and 20/00The same is shown by the juitititioftion Of the times of maximum accumla.
tion of the antitoxins in the different 'ratitifts-Ftlits?is given in the table.
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FaTialigErfiasinsota
..:Kaumi.kaaption revention
edesIMINIMION.YOW.????????????1100.0.01.? s./..111.0.1.0001.00?110.1.? ? ?
PAGE NUMBER
155
It must be said that in the rabbit1,4aLch;:we're`:-*thjected. to the combined imma.
nisation the coincidence of the graph types as observed of the twosv-rtito.xins in
17 out of 28 cases Eleven raboits r? tr?t ways to the 'simultaneous
inoculation of the two antigens. Thai Citkirsbl Wititniiirby the differences ii the
antigenic and immun.ogenic properties of the edematiens and tetanus anatoxins. In
regard to the edematiens anp.twrinD therfaianoabgical processes differed by a greater
level
lability than in respect t
,
the tetaritis'aiatotilatii' The. ow of the edematiens
:
TIMES OF PEAK ACCUMUL&TION OF THE'AnITOICIN'tf-APTER A. 0112.7..SHOT IMUNIZA,TION
rms.,
WITH PURIFIED SORBED:LICATOXINS.
TIKE IN DAYS
.M.Prit3ER OF' RA.BNITg' WVITIPPAIC TITRES OF ANATOXIN
of edernattisnif
OM .110 MS Ma
f tetanus
,
15 27 0
,
25 14
DA
ices
45 11 " 27
80 2 J:,---,--,y- - ..- 4
- ,._.., - -4,-
. . . . . . ma. 1' _,, ,; :,.- .1 .,....1?
I( P p 174 ) It is possible that, during AhemeXaniinfi&pi=!riod? the immune -A reactivity
I
1 of the animals was not kert on a constant Iiirler- ili-4'eti szl
trd to the eematiens ar.
atoxin? while this was the case in resTieTctqlre thtfttanus anatoxin but? a cer,-
t,
1
1 1
I i
1 of edematiens and tetanus? it was founct'ItartiiillAcndividtial curves of the acct=
1
ii lation of anatoxins could be batiicalli talifeffethree types.3 'The type of the
1
0
graph is determined by the strength AKE r-Vat4iil itlibilYVY: of the immunological rec-_,
tam n tendency to drop was detected ? = 1114
"
1) At the Sinale.shot immunisati6i,icor eitbtli bf with Purified
sorbed ana to3:in
actions character1stic for the givenTimifialliel-' 5? 3
2) There is a reason to stilitiose,Alsifarstit iritlgOriartti'Ihe edetratiens
anatorin.,_
the iumuno logi cal processes are diatiL ftr1abi1ity than in regar6.
AC55 FORM
8 FEEL 56
111
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urr:7
Wound infection prevention
to the tetanus anatoxin.
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trOILLIIGENCE TRANSLATION
,
LI,TERATUBE.
PAGE NUMBER
156
,
STAT
ACS! FORM
8 FM_ 56
KRAINAPINA
GLENFY koT
droTo VoproInfectopat.A; imnunoo 19540 No02. p0723,
Rt, BERM M,,J" J PathBactpb- 1%71 :34; Noolo 118.1190
-43 t'1,! riv
Rsbq
:10
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--r-
VETELLIGENCE TRANSLATOON
Wound infection prevention
pe175
No SXASHINTSEVAOI,VIBULANOVAo
??????01111MIMII ?110.00.0.110?611111.11116.1.1.1011,04... 111. 650101.0?1409/4?01
IPAGE NUMBER
157
..() Lt.;
?STAT
(N.F.GAMALEI Institute for Epidemiology and Microbiology. Academy of ledical Sci.
ences, U0S.S.R; Dir.; Prof0S0N0MURO4TSTO
PRODUCTION oF TF.TANUS TOXIYS WITP THE tra OF CELLOPHANE BAGS FOR THE GROWTH
OF THE CULTURE AND STUDY OF TH3 PROPERTIES OF THE OBTAInD ANTIGE1TS(p0175.181)
The obtainin,;- of powerful toxins has a great practical Importance,
u:ne
of a few authors showed thatowith the use of cellophane bags for the growth of the
culture, toxins of good quality can be produced.
Vaftwo
XWA
the purpose of producing powerful toxins of the tetanus bacillus, we have
also employed the cellonhane bags.
At the growing of a culture in cellophane bags we used various utensils which
we modified into corresponding suitable Shapes. Into a 5.1iter flAstkowith 3,5 ii.,
tere of nutrient medium in it, we let down a doubly everted bag one end of which
we tied tightly and fastened to the neck of the flask0 and the other end we put on
the neck of the flask, Then, we poured physiological solution into the cellophane
bagoup to the level of the nutrient mediump and we inserted a siphon into it0 with
a cotton plu, For taking a sample from the nutrient medium, we also inserted a tu.
bule into the flask; the tubule was placed between the cellophane bag and the neck
of the flask,
We have arranged the fourth (flask) slightly differently0 Into the fourth
we poured two liters of nutrient medium0 and we inserted a siphon Into it to which
we tightly fastened a doubly everted cellophane bag, and we filled it with phyaol
ogical solution, We sterilized the thus adjusted vessels together with the medium
,
at 110oCo after which we cnrried out the seeding in the cellophane bags, For this
IDUrnmseo
100 ml of a liquid culture of the tetanus bacillus was diluted in 3 liters
of physiological solution; with the aid of the siphon.we poured it into the bago
to the level of the nutrient medium0 and we ELNEME114 incubated it for 9 days at
rt
35?C.
In
growing
14AN 0and
PICS1 FORM
8 FEB. 56
this way, nine bags were seed ed Each ba g was controlled with a culture
T
by the ordinary method0 Por Sp01?6ye used the media of RAMON,GLUZ.
To c,
a medium prepared on hydrolyAate of fish meal, The determination of the
g;T: Jkfti4n.1:' ?
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rat
1
1
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Wound infection Prevention
0-Fr9afr....4 7
I PAGE NUMBER
158
strength of the toxin as well as of the antitoxin combining capacity of the anato.
xins was done by means of titration on white mice. The results of the experiments
STAT
are given in Table 1,
TABLE 1
PRODUCTION OF DIALYZED TFTANUS TOXIN ON DIFFERENT NUTRIENT MEDIA
MEDIUM TOXIN NUMBER OP Dlm in RATE BY WITICH DIALYZED TOXIN
. 11
1 ml of toxin' is stronger than the normal
1000000000 200
II
500 000
1000000000 :iir. , 10
1?0000000 .
Same 500000 000 100
500 000
FISH HYDRO. Dialyzed 10.000.000 10
11 . .
lyzate fishy 100000000 -
a DialyEed 50?000?000 100
regular 500'0000 - .
Same 50600 000'
N 100
500 000
,
4 II Same
Same 5
1?000?000
0.0000000
50
GTITMANus 10?0000000 10
1,0000000
5000000000 50
.
11
Samm
From the conducted experiments it is evident that the strength of the dia.
.
m
10000,000
lysed toxin is 10 to 200 times higher than the a renr.th of the toxin pre-rmred in
i 11
the ordinary way.
ed in cellophane bags? was slowed down lifi comparison with the growth of the culture
in the flasks. Therefore? experimints were arranged for the study of the dynamism
of the toxin production under thPse circubstinci-s For this purpose? we seeded 4 1
Daring the experiment.4 was noted that the growth of the tetanus bacillus.see
I2. I.
Ii
RAMON 9 9
Dialyzed
regular
Some
the cultureof the tetarals
?
U....41.41111ma.
titv ""^phane bags immersed in Ramon's
medium. As control? a culture was used' diatriated
m-th-A
=
by 41ftw,. An.ftwAS.km
WL1.44.1SabA4
The tetanus cultureppoured out traiiCtfielagt,and the flasks? was checked for
3.66.9 days for growth. In the toxinAii;deti4iined (p.177) the amount of the lethal
Tr'7c7f7,Pc,
?
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14.444110SlerettP*R4V-41.1fral"Attt79.'
.0.....1111.0.?????????????????????????????????????????????.4 n I
I PAGE NUMBER
159
doses and the units of combining(BoIT )1), _1ti4i) kt the same times we measured
the pH of the toxinswe examined the glucose consumption and under STAT microscnne
we observed the tetnnus bacilli for attiiittbilii"
Table 2 indicates howoaccordine toilieCiadei:;of the growth of culture on the
Ramon medium in the cellophane beigso the reastneth of the toxin and its combining
capacity had gradually increased, reaching t eak'between the 6th and the 9th day.
In the control flasks, the optimum toxin f6rtatifilit was observed on the SX 3rd(third)
day of growth of culture. The pH of the dialy*eo^ltin and of the ordinary one in
creased from pH 7.2 to pH 7.5, The glucoseaaAed'io the nutrient medium in the
amount of 0.754(750 mr) ?was determiniia' bytheriiethcid of BERTWD. The most energ.
etic exioenditure of glucose was noted in the culture seeCed with the ordinary
thod in which it was consumed by the 6th 'day 'of the growth of the culture. In the
medium, too, surrounding the cellophane bags? traces of glucose could be noted at
the 9th day of the cultivation of the Culture,
mn text cont.) It should be remarked that on the first day of the growth of
the culturesin the smears under the microscopeslong threads of the tetanus ba_cile
lus were seen. It is evident that the
-Ilture had gwown so violently that it could 1
not succeed in dividing, forming threads which were well stained with methylene.
blue, On the 6th day the culture had the form of bacilli with a small number of
spores of the tetanus microbes? weakly eUsceptible to .staining. On the 9th day a
large portion of the microorganisms hist Undergone lysiso
With cultivation of the tetanus bacilli by an ordinary method, the anDearance
of the lysiz will come Later.
The nutrient "medium surrounding the-bag has not contained any amount of te-_,
tanus toxin. The inoculation of 1 ml of the nutrient medium under the skin of ';112
paw of guinea pigs did not cause the stofts of tetanus.
The dialyzed toxin sin comparisonith the ordinary ones is rather puiekly
transformed into toxoid. For its complete neutraliration it ie enough to let it
n
sbay in the thermostat at 3703Ors = for days and to add to the toxin 34 of for.
malin,
11"
?14
..
Raving obtained anatoxins from the dialyred toxin ? we decided to verify their
effectiveness on animals.
It has-been noted that our further investigationse both with the ordinar-
ftur
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INTELVIGENCE TRANSIATiON
Wound infection preventtbon
-
xins and with the dialyzed toxinsoweredpne
CIA-RDP81-01043R004200160001-3
MMIMMOMMORMmemMft?mae....0 a 2 mdlowes.yreM7
I PAGE NUMBER
160
140ipMenimayromMimgelIMMOMIMMAOMMtle
ter)..their filtration through a Zeiss
fi1ter0Thereforepin view of the accumulation of a large amount of m4lrobic bodies
STAT
uNG
in the cellophane bags,at the preparation of dialyzed toxin we dissolved it in
physiological solution.
CHANGE OF THE PROPERTIFS
1.Y
TABLE 2
CM:MoMmmenOMMIMMIMM,
'
OF TETANUS BACILLI IN WE PROCESS OF GROWING IN cmul.
PHANE BAGS.JVID_AT
DAYS OF INCU.
Bag 1o01 BATION
BagoN001 3
Bag No02
fe.msw*Amilmal
WWIJOJA.W.A.
Bag No01 6
Bag No,2
Control
CUT-TIVATION BY THE ORDINARY MTHOD
TYP1 OF aff. Dim 1fl 3,. BoU.in one
TURE ml ml
Thread form
Same
Spores &
many lyzed
bacilli
Bacilli &
spores
Same
Spores &
many lyzed
bacilli
Bag Noul 9 Spores &
lyzed tetan,
bacilli
Bag N002
Control
Same
Spores &
lyzed tetan,
culture
s7,7 mm
10000 000
15 I
>5000000
aooD000
4.1 ......
.1 .
274'?.:. :,..,..;??,t, (.7.7 ci Sir '!.,. 1'2 ... :.r. - 3100.000 .
22 10.000
21' ',:'(-.413 crl
21 5.000
20 i- :. .7fGarit7',.: UC.1 "'7.171; 'Cir 7000 ' .-
20 2O00
:)
:19 - -::' - O ., uw....- -, f' -i-... -';'7.-i'''zi :10..' 000;
19 100000
'16. :. . . - 1 . i W rillIM; 1. .4r . 112T: 13?0 0 00 ' Y
16
16 50000
15 50000
15-?':.1 :.' .e.T.41.3' A.si.i'..' u':.'150'0.
15 5.050
- 15 :.:: -.!,:lj i:1 r'14-13-.' ?'":' '''.fai, OW..
15 10 .000
13 -- i--3*.Porr.1:' ;..476 P:.1. '4:1'12.0000
- CZ2 p 4 4 4 4 4 4 4 4 ................. 4
cpv r : .
TABLE 30
smooth
Is
V.
ft
01
to
moo .7rna
?:-.Q WM p
Smooth
RESISTANCE OF GUINEA PInS IM9UNIZED WITH PURIFIM NON.SORBED TETANUS TOXOID
IV 4::4.) 0 .J E
PY SI STA:rCE TO TnkhrtiS TrIXIN)
y$6.14 Arfp vis-,1%poxr
Day on which Dl. of
' tO3C1ik mai givictt
Number of of Anatoxin.
pigs per ml
11
10 ,000
^ 4 wisp ego 4 41.? CIO 4
Qi1 C
30
11..^444
warW.A.A.44.
? l 000
Result
10 healthy
1 died
10 - 5.7 ? 10: D 0 00 ? Wrir 1*. 41Y? Xi ? 1.1 00 0 ? bit
4 tetanuJ of
.4A4. gp . sozspbq c.rnyrrf:. ";-:
-IX.anei III
degree,
nsi FORM
8 FEEI. 56
? 7
,
puoi4Attvg.Tolvc
4 m o 4 ?
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[.....
I NTEIILIGENCE TRANSLAT PAGEE N UMBER
ION ,
Wound? infection prevention I
172
(p.191) In the following experimentowe have observed for eight months the dy.
namism of the accumulation of the antitoxin in the guinea pigs whieh were immun.
STAT
ized with two shots of the native and of the sorbed tetanus toxeids(Table 4).
TABLE 4.
TWO.
SHOT IMMU"IZATION OF THE GUINEA PIGS WITH NATIVE AND WITH SnRBED TETOUS
ANATOXIN
ANTIGEN MONTHS AFTYR 1st. AFTER THE SEG OND
Injection/A.U.
Native,
limo.after 5 months Aftsbr e months after
75 B,U, 8/
Sorbed , 15/
3 B.U0
;O l13
14/5.3
030 00 CI21
13/15.
30
14/14.4
13/11.2
REVACCINATION
r400
11/2.8 I.
9/1.4 5/0.5
8/4.5
8/52. 6/4
(see below)
44
(from above
RFAVAICIN.
M fl
.00V0
3 B.U.
3 B.U.
3 BdUo
AMOUNT OF ANTITOXIN AFTER REVAICIN.
10 days 5 months 13 months
A/15.5 5/13.1 2/20
2/45 1/10 1/15
4/27.5 1/20
5/56 1/30
ANNOTA,TIONs Numerator,
i=Za
0 number of guinea pigs;
denominAtor0 0
0 amount of antitoxin(in originalgslanatoxin")
........
CO CD
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440001111001000.0000000.
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INTELLIGENCE TRANSLATION
Wound infection revent ion
i
I I
.1
1
I 11
I I
1.00?10?01.1.1111.
PAGE.. NUMBER
17'5
In case of further investigations of the titres after 6 and 9 months, in
the animals of this group the amount of antitoxin was maintained mighly at the
same level.Thus, 6 months after the first injection, in 8 guinea pigs of Groun III
it
MIK equalled 4.5 A.U., and in 8 pigs of Group IV,..it was 5.2 A.U. After in
monthsoin seven guinea pigs of Group III the titre of the serum dronned to 3,5
A.U., and in the pigs of Group IV it
In the animals of Group I which
30 B.U.
drooped to 4.0 A.U. per one ml of serum,
had been immunized with a sinkle shot of
of sorbed anatoxin, 2.5 months after the first injection,
pigs the amount of antitoxin was eoual to 5.3
tbso antitoxin level droned down to 2.8 A.U.; after 9 months?
reached 2,5 A.U.
In the pigu of Group II which received a single shot of 3 B.U. of
in 14 guinea
after 6 months? in two plgq
in seven pigs0it
genvat the e7amination it armeared
105 A,U. in 13 animals; after
9 months it was 0.5 A.U. in 5
after 2.5 months the antitoxin
falai
6 months? it was 14 Acti in 9
animals.)
the anti.
level was
animals? and after
(p.194) By comparing the obtained results tt can be shown that, in ease of a
single shot immunization with sorbed anatoxin the lsrge amounts of antigen im-
part a higher immunity to the animals than the one we have noticed in thl? preced=
lug works.
As it can be seen, the amount of antitoxin in the two following grotrps as
almost identical.
It is evident that in case of a two.shot injection it is not necessary to
strive for large doses of antigen. It is likely that there esists some kind cf
limit in this respect; it can be also thought that the amount of antitoxin dues
not accumulate in proportion with the Increase in the antigen? which is almady
nointrd out by TOPLEY0WILSON(1936) and EDSALL(1953)0
The revaccinf)tion of the an mals
f the fourth group 9 months after the L'"ir tl
s
injectionomade with 3 11J). of the sorbed antigeno_has given the best result: in
the animals which were immuni?ea wIth ngle ,shots of small doses of the anigen,
Thus.10 days after inoculatigns_Of 3 Bab_ of the sorbed anAtoxin?, 6 guiea
pigs of Group I had an average of 15.5 IL pigs of Groum 11.45 A011. 4
pigs of Group III...27.4 A.IL0 andrkPigs Pi 016.09.1) IV.00.56 A0U.
Later on,we had a possibility to track down the reffults of the revaccirtion
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IMTEWGENCE TRANSLATION
Wound infection prevention
on a very small number of animals from 13 months?
Man.C.1...11.1011111~1.0,14.?-?s atoev..~.MMI4ONM."
PAGE NUMBER
176
Five months after revaccination,, 5 pigs of Group I had 1301 AXerv and after
STAT
13 monthsvin 2 pigs of this group the antitoxin level was an average of 20 &X, In
one pig of Group II at the same timesvit was 10 AX, and 15 &X, In each of the
Groups III and IV ? only one pig remained alive? and 5 months after the revaeelne.
tion the pigs of ()roux) III had 20 &X" and those of Group IV had 30 fh0U.
The completed work shows the high immunizing activity of the purified sorbed
tetanus anatoxin v its a0vantages in compnrison with the native preparation,
CONCLUSIONS?
1) The findings obtained with the mass preparation of the native and of the
purifird sorbed tetanus anatoxins attest the great effectiveness of the latter,
2) In case of the two.shot immunization of the guinea pigs with native(75 BX,
and purified aorbed(3 110U0) tetanus anatoxinsv in a long period of tiTe(8 months)
the animals retain higher immunity in ease of their immunization with the purified
sorbed rreparation( by 3 to 45 times higher).
I3) The potential rower of small doses of the sorbed tetanus anatoxin is high.
er than the potential power of large doses of the native antigen,
fi 4) In ease of a sinele.shot immunizelion with sorbed anatoxinv larger amounts
of the antigen impart higher immunity to the animals,
5) In case of the twoeshot immunization with 30.and 3 BX0 of the sorbed ana.
toxin? the pigs respond with an almost equal immunizatory stimulation, The increase
In the antigen up to 30 BX. does
2'. Aft
JimViO1
yield a larger accumulation of antitoxin,
6) After revaccination? thr amount of antitoxin is maintained
high level for 13 months?
at a sufficiently
LIMATURE8
VOROBDEV 410&u(Doctor dissertation)Leningrad019510 Byull0eken0biolomed00 19520
98 0043.47; also J?),LE.I? 1954v 6s 660
- /...../ & GONCHARelV BOLO & LUTWANSKII 1,?13,
med00 1957v 28 63.660
& MARKOVIOB A0V0 ByullOekepobiol,
- GOLINWICH E0M0 AcBjcp 19380493 (firkhobiolonauk?)
. =SALL G, Sympos,N,York Acad.M001953069.890
? PRIWY. H, Zbl0Bact00 Abt.Or1g0.1949015S: 0/5 . 171.
.1,
. TULEY & wILSON,Principles of BactA DMA.? 19360
ACSI FORM
8 FEB. 56
40.11?411111?10!????.,
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*!","...e..4.1."?"..r..60814571"0.1""".."
INTELLIGENCE TRANSLATION
Wound infection prevention
1
i
r
q
;
4 i III
i
...16 pigs each of which was inoculated with 7 ml of purified non.sorbed te ,laus
4
3
N anatoxin; into the third group... 31 pigs each of which received 5 ml of Durifit,
t
;
1 1
sorbed anatoxin, !al together 60 seri os of tetanus anatoxin were checked.
Twentytwo days after the inoculation of the antigen0in.50 Of the animals which
were under the experiment of testing the harmlessnesso we determined the AnniimalAtion,
of the antitoxin. I I
1
II
42cC1/100 AO., in 4 lolgc 1/100 A0U,S0 and in 2 pigs > 1/1001/l001/100
4DL/10
3 1/10
5 3= 5 24
(1000)
=0 C=1 .22, 00 02. 00 CO 00 02 sis ............
was
SPORES OF
1.
Amount Ronnic
2 5=5 death'
on
40.8
day
205.5 deathi
day
125 no
in
25 of
tetan,
1 died in
2.3 hours
(p0199) But in 16 guinea pips which were under the experiment of examining the
harmlessness of the sorbed anatokin and each of which had received 5 ml of the brep.
aration (1000 B.U.)o at the determination of the antitoxinoit hapnened to be)30-Z5
AO.0 in on of the blood serum.
Twentyfaur days after the inoculation of the antigenospores of the tetanum bac.
Illus were intramuscularly injected into all animalsoThe spores were
tion of calcium chloride,
in tilb rytrhLrr
The preliminary titration of the spores on guinea pigs showed the.t the determin=
seeding
ation of 1 M,L0D, of the spores is very difficult. At the sowing of the snores aDon
an agar column the following results were obtaineds
M0L0D0 contained 3.5 snores;
5 M0140D0
25 M0L0D0
.0.1.0.000.10001. MinAmmolowl10000,01?0
ACSI FORM 13A DISSEMINATION
8 FEB. 56
5.15 spores;
25.75 spores,
FORM FOR INTELLIGENCE TRANSLATION
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?
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INTELLIGENCE TRANSLATION
Wound infection prevention
aassersismosammeratorrawr*Smiatisistfhw
PAGE NUMBER
179
ata..xt wonon. rte:ro?
In view of the fact that the guinea Digs are very sensitive to the microbe of
tetanus, it is likely that one spore..inoculated with an apnropriate_irritant in the
STAT
form of calcium chloride.= proves to be auf,icient to provoke disease. But together
k.
with this tube to be sure thatoin ease of its intrnAlictinn 'Intel the animal? onlY
one spore will get in, does not eem to be possible. Hence, it is necessary to tre.
pane such a suspension that 1 M.11.1), vOuld Contain not less than 5 spores(at the
seeding of a medium) which also will assure a 1001; mortality of the control anfmals.
" A. ,???>6 ifth
14
Lite EllinP'".
c w lotrn
de/04
c4.1.1ru-,aa.
yyraa Noyes VA far
and purified tetanus anatoxin,
at their intramup.cular injection with 2.5 . 5 M.L.D. of the spores have died of te..?
tanus
Oa Ira
%dr
.VinAlm A 0
Wa0.7 "1.7.,5=C1.
-
zen-i7 ??????-???
St.niiUW/Lar
survivi--eli
sa r ur artimnia
But the guinea pigs which completed the experiment of testing the harmlessnoss
of the purified sorbed tetanus
cular injection of the tetanue snores in individual dozes from 12.5 to 25 MaL.DL,
all remained alive for 14 days without the clinical symptoms of tetnnua
The intramuscular introduction of the snores in a 101 solution of calcium chlor.
ide allowed us in all oases to noticeaafter 2.3 daysa painful infiltration which
caused contractures (of the muscles) in the animals.
In such oases when the guinea pigs did not die the infiltrated tissue becp:.me
necrotic on the 10, . 140day.
As it can be seen from the exPer1ment0 the inoculation of a large amount of
concentrated and purified tetanus anatoxin had not protect the animals from 25=
5 MJ4.1), of the tetanus !mores. This evidently is explained so that the purified
anatoxin Ana had received subsequently an intermus..
preparation is quicIdy eliminated from the
organism and thata for the time
of
In the organism(p.200) of the animal? the immunizatory ap-aratus does not elaborate
wafficient amount of antibodies,which would be Powerful to cope with the developing
infection Apparently.the single.shot inoculation %J.,41. c^n^d%ntrntea And nuri'Ipe,
anatoxin in large amounts is not enough. Since the antitetanus serum does not
sess either
a ba^t.4.4.4.4
,
Go*
hoe.tabri^4140 effect(DANTOFJEODENS)0thereforz)
in case of infectionoin the organism of the animal the phagocytes are
protective role? by phagocy0ing and lysing the snores. In eases of
of the chaef
deficiency in
the ant.toxin, however, in ease of a heavy trauma,with large necrosis of the times
and with the presence of blood clots which facilitate the reproduction of the xac.
teria and hinder the phagocytosi.s the_increased develspment of the microbes and the
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Wound infection nrevention
mloomim0WW06,444ofitillgr!IA-ttp..4,4.0.7;Drz.t.,
I PAGE N6MBER
180'
the excretion of a large amount of toxin by the bacteria it possible, and the antt.
toxin may not show a satisfactory effect?
STAT
In our exaeriments with the concentrated and purified anatoxino as it has been
already indicatedoin the exnerimental animals we' had a small amount of antitoxin
which finally0 in such an acute experiment and in the presence of such a massive dose
of the infection could not protect them from tetanus. Thereforeoin the present case,
it is impossible to talk of a change in the structure of the anatoxin in consecuence
to its purification. At the same timeo the 7arifie1 adsorbed anatoxin has imparted
a high immunity to the animalsowhich immunity nermitted to protect them from /205 -
25 M.L.D. of the tetanus snore.
Since the effectiveness of the native anatoxino for the preparation of 41nh
Men
the Stock Kolle No.8of ea. 4-_,Inotincirvi
FJ.11 40 0 %Zs V WavaiWit
leas
-hear_
usedo was epidemiologically apnroved,
the following experiment was staged for the purpose of a comparative study of the
Immunising activity and effectiveness of the native sorbed and of the purified or
bed tetanus anatoxins in regard to the tetanus spores?
The native sorbed tetanus anatoxin was used for the reason because the effect.
iveness of the non.eorbed anatoxins is considerably lower than that of the sorbed
native antigenso which was found by us in the preceding work.
For the comparative study of the antigens an experiment of single.shot and two.
.shot imilunizationsowith Wentical immunising doseso was arrangedowith eubseeuent
infection of the animals with the snores.
In one ml o the solaced tetanus anatoxin of Series No.30 contained 200 BJJ" com.
pletely sorbed on 2 mg of A1207. The supernatant fluid had less than I Both in one mle
The native tetanus anatoxin of Series Mo.709 had 150 B0U, in one mlo and after
sorntions on 2 mg of A1203 the sunernatant fluid contained 5 B,U, of the antigenn
In the exneriment of the single.shot imeunivationoeach of eIx guinea pigs re.
ceived purified sorbed anatoxin in the amount of 20 BM. One month after(n201)the
injectionoin one ml of the blood serum of the animalso three pigs had4;P0,5 A.U.
and three had 0.1 AuU,
Two months after the inoculation in five Digs the amount of anatoxin in^rebagleil
to 11-4::3 AM.(TABLE 2).
1115YINDI Six pigs received the same amount of sorbed tetanus native anatoxin
(20 B.U.).0ne month after the injection in one of the pigs the titre was4esp.01 in
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Wound infection prevention
I PAGE NUMBER
181
one ml of the serum? in another guinea pig it was 00014;0.1 A.U. and in another.n.
4> 0 140 5 A.Udg another contained exactly 1 AU., and one 13 g yielded)14C3 U0
STAT
(11104.12Ti% MN
NAAJJaaiii Mio,
Later on? two months after the introduction of the antigen? the amount of anti.
toxin increasedn..in three guinea pigs it was; 0.11:1 A.T.J.0 in one pig it wae
)1147.3 ?and in another pig it was 2 A.U.
Two month after the injection each animal was intramascularly &noculated with
12.5 to 25 MtL.D. of the spores of the tetanus bacillus/
Out of five guinea pigs immunised with purified sorbed tetanus anatoxin) ne
1
died from tetanus(on the 4th day)g the rest remained healthy for 14 days(see TOL-4:2)
Out of 5 pigs which were given sorbed native tetanus anatoxin0 one pig aleo
died of tetanus. The remainder had no symptoms of this infection up to the 14th day
(T.A.BLIi 3)0
In this wayD the experiment with the single.shot immunisation of the pigs with
the purified sorbed and with the native sorbed tetanus anatoxin dtbd not show any ad.
vantage of any of the antigens.
Thenowe immunized five guinea pigs with two shots of the purified sorbed an
toxin, and seven pigs with the native sorbed anatoxin ? by giving 5 13,uu of antigen
at each injectionowith an interval of 30 daysobetween the injections(TABLT 2 and 3), 1
One months after the injection the animals each of which received 5 B.U, ef the
1
purified sorbed tetanus anatoxinJn one ml of the serum0had a slight accumulation of 1
the antitoxinp4;? in four pigs it was 5 0.:11cX A.T.Te0 4 pigs had>rc3 AetTc;
Then-again each of the animals was given 5 B,U, of antigen more. Thirty days
one pig had
"31c5 A.U.(TABLE 2 and 3) and one had 5 AeU,Two months after the first injection and
ACS FORM
rcri ce
1-- r..i"
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LNTELLIG`plar. TRANSIATION
Wound infection prevention
M116.1.4?11014100
(P.202)
I PAGE NUMBER
eik.-1
ADZ
TABLE
SINGLE.SHOT. AND TWO.SHOT_-W
IWIZA.TION OF GUINEA. PIGS WITH rURIFIE".0_searrlED TETANUS
ANTI 'roily ova TH.THEII4_50 sE.21..TENT
Serial B.U.OF
No0 first
inject,
C=.
3.
20
2
20
3
20
4
20
5
20
20
CA
10
5
11
5
12
5
13
5
INFECTION WITH SPORES OP TETANUS BACILLI
TWO MOFTHS AFTER
first injection
ONE ZAONTHS AFTIM
first injection
ou
>0.541
>00544.
lb005t4C.J.