does gaertner »endotoxin« administered parenterally to white rats and mice engender immunity to a...
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DOES GAERTNER )) ENDOTOXIN ADMINI- STERED PARENTERALLY TO WHITE RATS
SEQUENT PERORAL INFECTION WITH LIVING GAERTNER BACILLI?
AND MICE ENGENDER IMMUNITY TO A SUB-
By L. Bahr, V. M. D.
In (9-14 days) old Gaertner broth cultures that have been sterilized by means of heating there will - as is known - be formed substances that have a poisonous effect on ex- perimental animals (mice, rats, guinea pigs, rabbits etc.) when the route of administration is parenteral. These sub- stances are often termed >endotoxins<< as they are found in the bacteria themselves.
When a sufficiently large dose of these Dendotoxinsc is injected subcut. or intraperitoneally into the experimental animal, the latter will generally die in the course of about 24 hours (or less) from septicemia, without it being possible to demonstrate living bacteria in the organs. In a few exceptional cases ordinary intestinal bacteria (e. g., B. coli) can, however, be demonstrated (in spleen and liver); but they have then invaded these organs during the stage of agony. If smaller doses than the lethal ones are injected into the experimental animals, only comparatively few of them die and as a rule after 2-3 days, or they are taken ill for some days and are in good health afterwards. Still smaller doses do not seem to affect the animals.
The Bendotoxinsc are found in the greatest amount in the
bacilli themselves, for which reason boiled broth culture con- taining the killed bacteria is of the greatest toxic effect. In the experiments referred to below I have, therefore, constant- ly used broth cultures, that were not filtered and which had been sterilized by heating in steam at looo C. 20-30 minutes.
I t was a common belief formerly that these >endotoxins<, which are termostabile, were of an analogous poisonous ef- fect also when administered per 0s ; but more recent experi- ments performed by L. Bahr,l) L. Bahr & Aage Dyssegaard2, 3 ,
and a series of subsequent investigations by other au tho r~4 ,~ ) have proved this view to be incorrect. The >>endotoxin<< must be given by parenteral route in order to engender the toxic effect; if ingested per 0s - even in very large doses - it does not affect the animal.
By means of experiments SavageB) has recently tried to prove that any of the salmonella bacteria, especially the >>ge- nuine meat poisoning bacteria<, besides the said >endotoxins<< produce another poisonous product that also was effective when ingested per 0s . I shall return to these examinations in a future publication; I only want to draw the attention to them here.
The question to which it in my opinion was interesting to get a reply was: Is a non-lethal dose of Gaertner (BRatin<)- endotoxin administered by means of parenteral injection to suitable experimental animals (white mice and white rats) able to protect the animals against living virulent ratin cul- ture given per 0s at a proper length of time after the endo- toxin injection?
I chose ratin bacillus culture for these experiments, be- cause when taken together with the feed (absorbed in wheat bread) it is able to engender an unquestionable food infection in rats and mice, from which infection all mice and a suffi- ciently large percentage of rats die when the requisite dose is administered.
For the purpose of producing the ,enddtoxins<< the follow- ing procedure was employed: Inoculation from the newly iso- lated (and virulent) ratin agar culture was made in sterile
Liebig's-extract peptone broth (with 2 O/oo glucose), pH: 8,O. The broth culture was then allowed to stand in the incuba- tor (at 30-37O C.) for 9-14 days when it was examined for the purpose of ascertaining whether it was a pure culture. If so, it was sterilized (for 20-30 minutes) in steam (sterilizer). Then 1 cc of the killed culture was inoculated (by )means of sterile pipette) into 100 cc of glucose peptone broth, which was kept for 3 days in the incubator at 37O C. If the broth remained sterile, the toxicity of the dead culture was deter- mined by means of subcut. injection into white mice and rats. The paracolon bacillus >>endotoxin<< referred to in one of the experiments was obtained and tested in the same manner.
The cultures and their endotoxins with which I 'have worked in these experiments were, besides a paracolon culture isolated from a calf, ,Ratin 241<< and >>Ratin 425q both of them newly isolated from 2 rats that died of ratin infection after peroral infection with ratin cultures.
Before use the tested >>endotoxins<< and the cultures were kept continuously in refrigerator at + 3O C.
Toxicity test on white mice with the ratin endotoxins (subcut. inject.). A dose corresponding to 0,5 cc. >>endotoxin<< per 15 gm. mouse killed the mice within 24 hours, 0,25 cc. per 15 gm. mouse killed only about 25 per cent. of the mice; about 75 per cent. of the mice were frequently taken ill for a couple of days but then recovered. Doses corresponding to 0,l cc. and 0,05 cc. per 15 gm. mouse were apparently without any effect.
Toxicity test on white rats with the ratin endotoxins (sub- cut. inject.) showed that 0,5-1,O cc. >>endotoxin<< per 100 gm. rat did not affect the animals, at any rate not as a rule; larger doses, 2-2,5 per 100 gm. rat, sometimes killed the animals (in the course of 1-3 days) ; still larger doses, 3-4 cc., as a rule killed the rats, most frequently within 24 hours.
The paracolon culture had been isolated by Mejlbo, V. S. (The State VeteriAary Serum Laboratory, Copenhagen), who had isolated it from a calf that died of paracolon infection. The
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paracolon >>endotoxin<< prepared fro,m this culture was some- what weaker than the ratin endotoxin. 0,5 cc. paracoli Ben- dotoxincc per 15 gim. mouse (subcut. inject.), however, as a rule killed white mice after 24 h-3 days. 0,25 cc. per 15 gm. mouse did not affect the mice to any great extent; 0,l cc. per 15 gm. mouse was of no effect whatever.
As regards the virulence of the said two ratin cultures with respect to white mice and white rats by peroral infec- tion with glucose broth culture, 24h old and at 37O C., (ad- ministered absorbed in wheaten bread) this will appear from the below experiments with %Ratin 2 4 1 ~ (>>Ratin 425<< was of approximately the same degree of virulence) : -
I. 4 white mice (weight 15-19 gm.) got each 2 cc. ratin culture dilution 1 : I00 per 15 gm. mouse. All 4 mice died of ratin infection in the course of 7-11 days.
11. 4 white mice (weight 15-19 gm.) got each 2 cc. ratin culture dilution I : 200 per 15 gm. mouse. All 4 mice died of ratin infection in the course of 7-9 days.
111. 4 white mice (weight 15-20 gm.) got 2 cc. ratin culture dilution I : 500 per 15 gm. mouse each. All 4 mice died of ratin infection in the course of 6-11 days.
IV. 4 white mice (weight 20-22 gm.) got each 2 cc. ratin culture dilution I : I000 per 15 gm. mouse. 2 mice died of ratin infection in the course of 9-11 days. 2 mice survived the infection (observation: 30 days).
V. 4 white mice (weight 14-21 gm.) got each X cc. ratin culture dilution I : 2000 per 15 gm. mouse. 2 mice died of ratin infection in the course of 8 days; the remaining 2 mice survived the infection (observation: 1 month).
The minimal lethal dose was thus 2 cc. ratiir culture dilu- tion 1 : 500 per 15 gm. mouse.
Infection experiments with rats.
1. 11 white rats (weight 87-156 gm.) got each 5 cc. undiluted broth culture per 100 #n. rat. 6 rats died of the ratin infection in the course of 4-10 days, whilst 5 rats survived the infection (observation : 1 month).
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11. 10 white rats (weight 90-110 gm.) got each 10 cc. un- diluted broth culture per 100 gm. rat. 9 rats died of ratin infection in the course of 7-10 days; one rat survived the infection (observation : 1 month).
111. 10 white rats (weight 80-120 gm.) got'each 15 cc. un- diluted broth culture per 100 gm. rat. All the rats died of ratin infection in the course of 7-17 days.
5 cc. ratin bouillon culture thus showed a questionable, 10-15 gm. an unquestionable effect in the case of rats.
After these preliminary tests, the main experiments could be carried out as plamed.
I. Experiments with rats: The reords of the three series of experiments was presented in Table Nr. 1: I (A, B), I1 (A, B, C) and I11 (A, B, C) will give sufficient information as to how the experiments were performed and about the results arrived at. In experiment I A each of the 16 rats got a subcut. injection of a uery large dose of ratin >>endotoxin<<, so large that 5 of the rats died from the toxic effect. The was made so large because the object of the experiment among other things was to examine whether one verg large dose of ,endo- toxin< - so large that the danger of death owing to poisoning was not completely excluded - would be able to immunize the surviving rats against a comparatively slight infection per 0s with living ratin bacilli ( 5 cc. broth culture per 100 gm. rat). Simultaneous with the remaining 11 experi- mental rats 11 controls were also infected per 0s with the same dose of the broth culture (per 100 gm. rat) . The infection of the experimental rats took place 13-18 days after the ,endotoxin<< injection. The result was that about 18 per cent. of the experimental rats died of the ratin infection and about 55 per cent. of the controls died.
As, however, so large a dose of endotoxin involved the danger of death owing to poisoning, experiment I1 A was per formed in such a manner that the 15 experimental rats once got a subcut. injection of 0,5 cc. of the same ratin ,endotoxin< per 100 gm. rat; in I1 B the 15 experimental rats used for this purpose got a subcut. injection of 0,5 cc.
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of paracolon >>endotoxinu per 100 gm. rat. As moreover a dose of 5 cc. of ratin broth culture per 100 gm. rat according to the above experiments only killed about 50 per cent. of the infected rats, which must be considered a rather too low mortality, all the 30 experimental rats (I1 A B) and the 14 controls*) infected simultaneously per 0s (I1 C) were fed with a dose of ratin culture each, correspon- ding to 10 cc. per 100 gm. rat. The results of these three experiments (I1 A, B, C) were that 28 of the 30 experi- mental rats died of the ratin infection (in the course of 4-17 days) = about 93 per cent.; whilst only 8 of the 14 controls died of the infection = about 57 per cent. Thus we cannot in this case speak of any immunity whatever in the rats treated with endotoxin, it is rather the reverse. In the last series of experiments, 111, 10 white rats (111 A ) got subcut. injections twice (at intervals of 8 days) with >>Ratin 425(( - >>endotoxin((, the first time 1,0 cc. and the second time 3,O ccm. per 100 gm. rat, and 5 white rats (111 B) got subcut. injections of the same >>endotoxinu 3 times at intervals of 8-9 days (the first time 1,0 cc., the second time 3,O cc. and the third time 4,O cc. per 100 gm. rat) . The infection per 0s of the experimental rat (I11 A ) and the controls (I11 C) took place on Oct. 3rd, 1933, and in the case of the experimental rats (111 B) on Oct. 12th, 1933. Each rat got 10 cc. ratin broth culture (425) per 100 gm. rat.
The result was that 7 of the 10 experimental rats that had been treated twice in advance with >>endotoxin(( died of ratin infection = about 70 per cent. - 3 of the 5 experimental rats that had been treated 3 times in advance died of ratin in- fection = about 60 per cent., and 12 of the 14 controls (that had not been treated with >endotoxinu) died of ratin infection = about 86 per cent. - These mortality rates are not so far from one another as to allow us to speak of a distinct turning of the scale.
*) Originally 15 controls were also chosen for this experiment but one rat died before the infection.
3
Experi- menls
Case re- cord No.
I A. No. 10 and No. 11
I R. No. 14 control
I1 A. No. 41
I1 B. No. 42.
11 c. No. 69 control
111 A. No.52(1)
I11 B. No. EX2 (11)
I11 c. No. 53 control
-- Weight
of the rats
100- 120 gm
105- 145 gm
80- 152 gm
90- 120 gm
90- 120 gm
Date of subcut. inject.
Endotoxin
6 white rats 9/6/33 ; lowhite rats 14/6/33
1/8/33
2/8/33
1) 1219 1933
2) 2019 1933
I) 1219 1933
2) 2019 1933
3) 2919 1933
34
uEndo- toxincc
Ratin 241 1 Inj.
N o inj.
Ratin 241 1 Inj.
Para- colion 1 Inj.
N o inj.
Ratin 425
Ratin 425
N o inj .
Dose per 100 gm
rat
2 cc.
0
0,5 cc.
0,5 cc.
0
1 Inj. l,o cc. 2 Inj. 3,O cc.
1 Inj. l,o cc. 2 Inj. 3,O cc. 3 Inj. 4 cc.
0
Table Experiments with rats
Result
5 t of E.T. 11 survi- ved the in- jection
All 15 rats survived the injec- tion
All 15 rats survived the injec- tion
All 10 rats survived the injec- tion
All 5 rats survived the injec- tion
Number of white rats
infected per 0s with G . B. C.
11 white rats (rest No. 10 and 1l)ratin 241
11 white rats ratin 241
15 white rats ratin 241
15 white rats ratin 24 1
14 white rats ratin 241
10 white rats ratin 241
5 white rats ratin 241
14 white rats*) ratin 241
C . B. C. = glucose broth culture 24 h 37' C . E. T. = -f from Ratin ))Endotoxin((.
35
No. 1. treated with ))endotoxin((.
Dose per 100 gm
rat
5 ccm
5 ccrn
10 ccm
10 ccm
10 ccm
10 ccm
10 ccm
10 ccm
Date of Infektion
2716 1933
2716 1933
1618 1933
17/8 1933
1818 1933
311 0 1933
12/10 1933
3/10 1933
Case record
NO.
No. 14
No. 15
No. 67
No. 68
No. 69
No. 52 (1)
No. 52 (11)
No. 53
Weight of
the rats
11 0-1 7a gm
87-156 gn'
120-150 gm
90-160 gn'
70-140 gm
100-140 gm
100-170 gm
90-150 gm
Number of rats t
of the infection
2 (7-14d) 9 R. +-
6 (4-10d) 5 R. f
14 (6-17d) 1 R. +-
14 (4-1 4d) 1 R. +-
8 (6-13d) 6 R. +- 7 (8-13d) 3 R. f
3 (5-1 Id) 2 R. f
12 (6- 1 Id) 2 R. f
Mortality Per
cent.
about 18
about 55
about 93
about 93
about 57
about 70
about 60
about 86
Surviving rats
observed
1 montt
do.
do.
do.
do.
do.
do.
do.
Comments
' ) + - l R t i / l O 1933 not of ratin)
Experi- ment No.
IV a No. 6
IV b No. 7
IV c No. 17
VI a No. 57 (1)
VI b No. 57 (11)
VI c No. 56
Experi- ment No. 22
Weight of
the mice
16-19 gm
17-19 gm
20-25 gm
17--20 gm
12-17 g 111
10-14 gm
13-14 gm
15-17 gm
20-24 gm
20-25 gm
Date of in- ject. ))Endo-
toxinu
I. P. In'. 816 1934
Subcut. inj 8/6 1933
No. inj. (Control)
1st subcut inj. 12/9 1933 2nd 20/9 1933 1st subcut inj. l2/9 33 2nd 2019 33 3rd 291933
No inj. (Control) 10/7 1933
do.
do.
do.
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*Endo- toxinu
Ratin 241
do.
Ratin 425
do.
Ratin 241
do.
do.
do.
Table Experiments with mice
Dose per 15 gm mouse
a 0,5 cc. b 0,25 D
d 0,lO * j
a 0,5 cc. b 1.0 n \ c 0,25 B j d 0,lO 3
c 0,lO \
1. Inj. 0,2 cc. 2. Inj. 0,4 cc.
1. Inj. 0,2 cc. 2. Inj. 0,4 cc. 3. Inj. 0,6 cc.
0,5 cc.
0,25 cc.
0,l cc.
0,05 cc.
Result
t 24 h. RE t 3 d . R E in good healt all the lime t 13 d. RE ill:
t 12 d. RE 1-3 d
Obs. up to 3/10 1933 all micc in good health Ohs. up to 12/10 1933 all mice in good health
2 mice (ab) t
24 h RE 2 mice (cd) f
3 mice f 1 mouse t 1117 RE 4 mice +
4 mice f
~
Number of white mice inf.
per 0s with GBC
By experiment VI abc living broth culture Ratin 425 was used; by >> IV abc and No. 22 living broth culture Ratin 241.
37
,
No. 2. treated with endotoxin.
(
ca. 100
~~
Dose pr. 16 gm mouse
Dilution 1:750 2 cc. each
do.
do.
Dilution 1:500 Each mouse 2 cc.
do.
do.
Undilu- ted cul- ture l cc. per mouse
do.
do.
do.
Date of infektion
3016 33
3016 33
3016 33
3/10 33
12/10 33
12/10 33
2617 33
2617 33
2617 33
2617 33
- __
Case recorc
No.
- 16
16
17
57 (1)
57 (11)
56
23
23
23
23
-
Weight of the mice
c 19 gm d 20 gm
b 23 gm c 19 gm
20-25 gm
19-22 gm
14-20 gm
10-14 gm
15-17 gm
18-20 gm
22-26 gm
23-26 gm
Number of mice
t of the
infektion
2 c -f 8 days d t l 0
2 b t 9 days c t 1 6
3 (8-9 days)
5 (5-7 days)
5 (3-8 days)
5 (5-7 days)
2 (9 days)
3 (7-9 days)
4 (8-11 days)
4 (7-9 days)
__-
Mortalit) SurvivinE
mice observed
b. sur- vived obs. 26 days
__- ~
Comments
Control for IV a, b
Control for VI a, b
G. B. C. = 24 h 37O C. glucose broth culture. Ratin 241. R. E. = .i. from Ratin-))Endotoxin((.
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If all the rat experiments (Table No. 1) are viewed as a whole 40 out of the 56 rats - treated with ,endotoxin< - died of ratin infection ( = about 71,4 per cent.), whilst 16 survived the infection. 26 out of the 39 controls died of ratin infection (= about 66,7 per cent.), whilst 13 rats survived the infection.
These figures are perhaps the correct mortality figures to be used in arriving at an opinion of the matter, but the slight difference that is present is a strong case against the supposi- tion that an immunization of real importance has taken place after injection of rather large doses of ratin >>endotoxin<< performed once or several times.
The same applies to a still greater extent to the experi- ments with mice (Table No. 2 ) , as all the mice treated with >>endotoxinu ( 1-3 times) on succeeding peroral infection with ratin broth culture (even in small amounts, see Table Nr. 2, IV a b and VI a b) succulmbed to the ratin infection and approximately in the course of the same time as the controls.
The principal result of the experiments performed must, therefore, be said to be that mice did not become immunized by means of subcut. or i P . injection (1-3 times) of ratin->>en- dotoxin(< in such a manner that they could resist a subsequent peroral infection with living ratin bacteria; and I am of opi- nion that the same conclusion must be drawn from the ana- logous experiments with rats, when all the experiments (in Table I) are viewed as a whole in order to exclude accidental circumstances, which doubtless must be said to be the proper thing to do.
A s far as mice are concerned this result will not be sur- prising; for the experiments hitherto made (including my own experiments) with living ratin culture on white mice seem to show that mice are not immunized. If mice for in- stance survive a too slight peroral ratin infection, it may well happen that a subsequent ingestion per 0s of a somewhat larger amount of ratin bacilli give them a lethal infection.
In the case of the rats the result was more surprising, as
only a small percentage of rats that have survived peroral infection (a t any rate up to 6 months afterwards) succumb to a subsequenf infection of the same nature. The offspring of such rats has, however, in my experiments and in experi- ments performed some years ago by H. Raebiger (Halle a. d. Saale) proved to be susceptible to the ratin infection.
Maybe then that the conditions of immunity are not the same when the rat ingests living ratin bacilli per 0s as when a dead culture is injected into the animal. The above examinations might be indicative hereof. On subcut. injection of increasing doses of ratin ))endotoxin(( at intervals of 8 days mice as well as rats were able to tolerate constantly increasing doses (Table No. 1 : TI1 A-B and Table No. 2: VI a-b) without any inconvenience - doses that would have killed the experimental animals in the course of about 24 hours, i f used for the first injection.
In this connection I have made a few orientating examina- tions of the agglutinin contents in the blood of such mice and rats (see Table No. 3).
Thus it was only possible to demonstrate ( a rather great) agglutinin production in the blood serum of one rat; aggluti- nin, or only traces hereof, could not be demonstrated in the blood of the other mice and rats. On subsequent infection with living ratin bacilli all these mice and rats died of the infection except one, viz. the rat whose blood serum agglu- tinated the ratin culture to a great extent.
Thus a certain immunization takes place in rats after 3 injections (subcut.) of ratin >)endotoxinu; but is chiefly an immunization against the >endotoxins<< only and as it is not so strong as to be able to protect the animal against a sub- sequent peroral infection with living bacteria - except in rare cases - it is in my opinion only of slight importance.
Instinctively one comes to think of the subcut. protective inoculation of typhoid >)endotoxinu, even if the experiments with ratin >endotoxinu on rats and mice reported here of course do not justify a comparison. Lunge and Kauffmann,7) too, have recently touched on the question: protective inocula-
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Table No. 3.
--I++
~ -~
Experi- mental animal
0 0
0 0
0 0
0 0
0 0
0 0
++++
0 0
Subcut. inj. R-uEndotoxin(<.
Number of times at inter- vals of 8 days
Rlood sample; number of days
after last inject
12 days
12 m
12 D
15 w
13
12
13 n
15 D
Agglutination experiments with ratin culture
Serum dilution
1 40 ~
- 0
0
0
0
0
0
-++-!
0
-
1 80 ~
- 0
0
0
0
0
0
I+++
0
-
- 1 i40 -
- 0
0
0
0
0
0
--t
0
- tion against typhoid fever on another basis, viz. when refer- ing to the importance of the 0-agglutinins in their experi- ments with mouse typhoid immunization.
Summary. A number of experiments have been performed on white
rats and mice with ratin- and - in a single experiment - with paracolon Bendotoxina with a view to examining whether one or more Bendotoxincc injections protected the experimental animals against a subsequent peroral infection with living ratin culture.
- 1 280 -
- 0
0
0
0
0
0
+
0
-
41
As far as the mice are concerned the result was absolutely negative and in the case of the rats it was negative or doubtful,
A certain immunity against the endotoxins was observed, as both mice and rats after repeated subcut. injections of >endotoxins<< were able to tolerate constantly increasing doses. This immunity was, however, not so great as to be able to protect the animals against a subsequent peroral infection with living culture.
LIST OF LITERATURE.
1. Bahr, L.: Ober das EndotoBin der Ratinbazillus und einiger Glirtnerstlimme (Zeitschr. f. Fleisch- und Milchhygiene Jg. 36, 1926).
2. Bahr, L. und Aa. Dyssegaard: Die Endotosine der Paratyphus- Enteritis-Bakterien (Centralbl. fur Bakter. etc. I Abt. Orig. Bd. 102, 1927).
3. Bahr, L.: Kedforgiftning eller Kedinfektion? (Beretning fra 3. nor- diske Veterinaerm~te, Oslo, 9.-11. Juli 1928. Oslo 1928).
4. Duck, G. M., Jordan E. O., Wood, W. L.: #Food poisoningcc pro- duced in monkeys by f eed ing living Salmonella Cultures (The Journal of preventive medicine, Vol. 3, No. 2, March 1929).
5. Duck, Gail, Y., Hamzan, Paul H. and Jarra, Irene E.: Negative results obtained on introducing heatkilled cultures of Sal- monella Enteritidis into the intestinal tract of monkeys and other animals (The Journal of preventive medicine, Vol. I1 No. 6, November 1928).
6. Savage, Will iam G.: Esperimental evidence of a heat-resistant gastro-intestinal irritant produced by bacilli of the Salmo- nella-Group (The Journal of Hygiene Vol. XXXIII No. 2, 1933).
7. Lunge, B. und Kauffmann, F.: Experimentelle Untersuchungen iiber die Immunitiit beim Mausetyphus (1-11: Zeitschr. fur Hy- giene Bd. 114, 215, 1933).