NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
10
of rabies virus which we have found under certain definite conditions to be
capable of producing distinct paresis in a rabbit within six or seven days after sub-
dural inoculation. It will be readily understood that very exact numerical results
are almost impossible of attainment, where we are dealing—as in the case of rabies
—with an entirely unknown virus. The actual amount of the organism present
is only determinable by indirect means. We have, however, been able to obtain
certain limits of infectivity of rabies virus, variously treated, which to us appear
to be very much more definite than any which have hitherto been published.
Within certain limits of unavoidable error, which for practical purposes may
be neglected, we hold that we have been able to demonstrate a method by which
any system of treatment may be approximately estimated in standard units and
so become capable of direct comparison with any other system. We proceed
to describe our method and the results obtained in detail.
Technique. —The amount of rabies emulsion used for subdural inoculation has
been invariably 0.2 cc. of a definite dilution. The virus was fixed virus. The
animals used were all rabbits ; the inoculation made subdurally. The preparation
of the emulsion in the case of fresh rabies material needs scarcely any descrip-
tion. A definite quantity of the fresh rabies material is ground up as finely as
possible in a mortar ; to it is added a sufficiency of physiological salt solution
to make a 1 per cent. emulsion ; this is strained through wire gauze of 0.3 mm.
mesh. From the emulsion so made others are obtained by simple dilution.
Where the emulsions were of greater strength than 1 in 100 the same procedure
was followed.
In the case of dried rabies material the making of an emulsion of definite
strength is not so simple a matter. Suppose we were to take the spinal cord of
a rabies rabbit and divide it up into small lengths of equal weight. One length
would suffice for the preparation of an emulsion - of fresh virus. The other lengths
each after a definite period of drying would be ground up to make their re-
spective emulsions. The quantity of fluid added to each of these latter would be
exactly the same as in the case of the fresh virus, irrespective of the fact that
drying had diminished their weight. All these emulsions would then be exactly
comparable. But as the spinal cords used in our experiments are dried in entire
length and not in these small pieces of originally equal weight, we must make a
calculation to determine what the weight of any given dried portion would have
been in the fresh condition. Then we make up our emulsions as if the portion
possessed this latter weight ; otherwise our results would not be comparable. An
example will make this clear. The weight of a fresh spinal cord when removed
was 4.0282 grm. A portion weighing 0.153 grm. was removed for the test of
the infectivity of fresh rabies virus. The remainder was then 3.8752 grm. This
remainder weighed only 1.9986 grm. after one day's drying, and a portion
weighing 0.1079 grm. was then removed From it for the test ' of infectivity of
one-day dried cord. The weight, therefore, of the portion taken would, in its.
fresh state, have been
3' 8752/1.9986x0 . 1 0 7 9
= 0 : 2 0 9 2 g r m .
and it is on the basis of this latter weight, and not of 0.1079 grm., that the
necessary dilutions are made. This calculation is essential to any method of
estimation in which more or less dried material is to be compared with fresh
moist material.
Results .
—
Altogether 128 rabbits were used in' the test. Of these a very
large number were expended in trials with fresh rabies material, as it was some
considerable time before a definite decision upon limits could be reached. The
numbers were
—
F r e s h m a t e r i a l . . . . . . .
9 1
One-day dried cord. . . . . . .
4
Two-day dried cord.
.
. . .
7
Three-day dried cord.
.
.
.
.
13
Five-day dried cord
.
.
1 0
Nine-day dried cord.
. .
3
TOTAL
.
128
(1) Fresh material. —It was exceedingly difficult to get any definite limits
in this case. The M. I. D. ranged from certainty at 1/8,000 dilution up to tolerable
certainty through successively greater dilutions to 1/40,000 The possibilities of
experimental error here were very great, especially in the high dilutions. A
1/20,000 dilution contains only .00001 grm. of rabies material per 0.2 cc. of emul
-
sion used, and therefore a still less quantity of the pure rabies organism. It is
not easy, then, to differentiate between such small amounts as, say, 0.2 cc. of
1/20,000 and 0.2 cc. of 1/16,000 dilution of rabies material containing respectively
grm. and .0000125 grm. of solid matter in suspension. Some of our later trials
were more definite than the earlier, and we found, e.g., that whereas 0.2 cc. of a
1/8,000 dilution produced, upon subdural inoculation, rabies in the fixed time of 6 or-
7 days, the same amount of 1/10,000 dilution and 1/15,000 dilution gave rabies with an
incubation period of some 9 days or longer or did not give rabies at all. We
decided therefore to fix our unit for fresh rabies material at 0.2 cc. of
/8.0 dilution with this qualification that dilutions very much higher would in many
instances afford examples of the M. I. D. in quantities of 0.2 cc.
(2) One-day cord—
Number of experiments 4.
Dose 0.2 cc.
i. Dilutions of 1/4,000 and 1/1,000. In all, four tests made : paresis was
distinct by the 6th or 7th day.
C 2
12
Limit reached—nil. M. I. D. not greater than 0 . 2 cc. of a 4000-fold
dilution.
(3) Two-day cord—
Number of experiments 7.
Dose 0.2 cc.
i. Dilutions 1/1,000 ; 1/500 ; 1/250 ; 6 experiments. In all cases
paresis by the 6th or 7th day.
ii. Dilution 1/2,000 ; 1 experiment. Paresis after 34 days.
Limit.—0.2 cc. of a 1,000-fold dilution.
(4) Three-day cord—
Number of experiments 12.
Dose 0.2 cc.
i. Dilutions 1/200 ; 1/100 ; 1/50 ; 8 experiments. In all cases
paresis by the 6th or 7th day.
ii. Dilutions 1/500 ; 1/1,000 ; 4 experiments. Paresis in 6 to 13 days.
Limit—0.2 cc. of a 200-fold dilution.
(5) Five-day cord—
Number of experiments 10.
Dose 0. 2c.c.
i. Dilutions 1/50 ; 1/100 ; 8 experiments.
In all cases paresis
by the 6th or 7th day.
ii. Dilution 1/200 ; 2 experiments. Paresis doubtful ; certainly
well over the limit of 6 or 7 days.
Deaths after 23 and 33 days.
Limit—0.2 cc. of a 100-fold dilution.
(6) Nine-day cord—
Number of experiments 3.
Dose 0.2 cc.
Dilutions 1/10 and 1/5
Result—no rabies ; progressive emaciation ; death.
Limit (if any)—some concentration greater than is represented by
0.2 cc. of a 5-fold dilution.
The M. I. D 's obtained with fresh, three-day, five-day, and nine-day
dried material give us four well separated points on a curve convex
to axes representing respectively the degree of dilution of the
material used and the time of desiccation undergone. This number
of points, well interspaced as they are, is sufficient to enable the
curve to be plotted with fair accuracy. We have, however, as
described in the text, also carried out a certain number of experi-
13
ments with one-day and two-day cords, which, although limits
have not been reached in both cases, still go to confirm the results
obtained with those other experiments in which limits were
obtained. We think certain conclusions may be drawn from these
results,
These are—
(1) That emulsion of nine-day cord is little if at all infective in a dose of
0. 2 cc. of a 1 i n 5 emul si on.
(2)That emulsion of five-day cord is infective in minimal time in a dose of
0.2 cc. of 1 in 100 emulsion, but becomes less so or not at all in a dose of 0.2 cc.
o f 1 i n 2 0 0 e m u l s i o n .
(3)That in the same way the M. I. D. for an emulsion of three-day cord is
0.2 cc. of a 1 in 200 emulsion.
(4)That the M. I. D. of two-day cord is not less than 0'2 cc. of a 1 in
1,000 emulsion and probably not so great as 0.2 cc. of a 1 in 2,000 emulsion.
(5)That the M. I. D. of one-day cord is not less than 0.2 cc. of 1 in 4,000
emulsion and almost certainly not greater than 0.2 cc. of 1 in 8,000 emulsion
(the lower accepted limit of fresh material).
(6)That fresh material is infective (M. I. D.) in a dose of 0.2 cc. of a 1 in
8,000 dilution and may be so in considerably higher dilutions even up to 1 in
40,000, but that with such high dilutions the experimental errors become so great
as to preclude any more exact fixation of the M. I. D.
Although these observations may be wanting in mathematical exactness,
still we are enabled to deduce from them certain conclusions regarding the
infectivity of rabies material according as it has undergone desiccation to a
greater or less degree. It is evident that by the third day of drying the rabies
cord has lost very much of its original infectivity. Thus, let us take as
our M. I. D. of fresh material 0.2 cc. of 1 in 8,000 dilution. The fall in
infectivity is a rapid one. If 0.2 cc. of 1 in 8,000 fresh material be the
M. I. D., then the M. I. D. of three-day cord is at least 40 times greater,
whilst that of five-day cord is about 80 times greater. By the 9th day the cord
has lost all or nearly all infectivity. The infectivity between the 5th and 9th
days of drying diminishes very slowly and gradually. Our results may now be
plotted for the sake of clearness in the shape of a curve (fig. I). The numbers
along the vertical axis represent therelative degrees of dilution which the particular
rabies material corresponding to a given amount of desiccation can undergo
and still remain fully infective. Thus, for example, fresh undried material may
be diluted eight times more than two-day dried material and will then be infective
to an equal extent.
The curve (fig. I) shows the very sharp drop in infectivity which takes place
in the first three days and the very gradual diminution thereafter. The inferences
which it would seem justifiable to make from a particular study of this curve
are
—
(1) Sharp differences in infectivity between fresh, one, two, and three-day
cords.
(2) A difference of infectivity amounting to about one half between three-
day and five-day cords.
(3) Exceedingly gradual diminution in infectivity from five-day cord down
to little or none in the nine-day cord.
Are we to conclude from the non-infectivity of nine-day cord that it is of no
service in immunisation ? The answer to this question depends on the nature
of the material which is left after the death of the living virus and the usefulness
of this non-living substance. We take up this subject in full later (vide existence
of rabies toxin, p. 27).
II.—The rate of desiccation of rabies spinal cord by the ordinary
procedure and its bearing upon the use of the cord for
immunising purposes.
The material used for desiccation was caustic potash in sticks. The rabies
cords were dried in the usual way over caustic potash, in air, in the dark, and at
a temperature of 22°— 24ºC. The following is a typical instance
—
one of six
observations—of the results obtained :—
TABLE IV.
Day of drying.
Weight.
Temperature.
o.
• . .
4.0282 grm.
. . .
•
I.
•
•
2.0775 ,,
22 º
—
2 4°C .
2. .
.
1 . 6 2 3 , ,
, ,
3. •
.
1 . 4 4 0 1 , ,
, ,
4.
•
•
•
.
1 . 3 8 1 4 , ,
, ,
5. .
1.3540 ,,
, ,
6. .
.
•
1 . 3 3 8 9 , ,
, ,
15
TABLE IV.— Continued.
Day of drying.
Weight.
Temperature.
7 . . . . . .
1.3297 grm.
22
º—24ºC.
8. . . . . . .
1. 3193 , ,
,,
9 . . . . . .
1.3127 ,,
,,
10..
.
.
1.3103 ,,
, ,
11. . . . .
1.3057 „
, ,
12..
.
. .
1.3018 ,,
, ,
13. . . .
.
.
1.3002 „
, ,
The loss of weight continues milligramme by milligramme for about 60 days.
It is of course a loss of weight of water, for the solid basis material does not lose
weight. We may plot the figures given above in the form of a graph (fig. II).
We now proceed to compare the curve of drying with the curve of infectiv-
ity (fig. III). But before doing so we may observe that in instituting this com-
parison it is the loss of weight of water only in desiccation which is taken into
account ; that is to say, that the solid unchanging basis material is excluded from
the comparison. Moreover, infectivity with the doses which we used disappeared
by the 9th day, but the loss of weight of water was not actually complete until
about the 60th day. We may conclude from this, that the small amount of water
remaining in the cord from the 9th day onward is insufficient to keep the virus
infective. We may state this perhaps more correctly as follows :—The quantity
of living virus which may possibly be present in. the cord by virtue of the small
amount of water remaining at the 9th day, is an insufficient amount in the doses
used to produce infection. This statement amounts to this, that the very small
quantity of water remaining in the rabies cord after the 9th day may be neglected
so far as it affects the question of infectivity in our comparison. This quantity re-
presents, therefore, so much dead weight and may be added to the dead weight of
basis nerve-material already alluded to. We may show this in fig.II by moving
the abscissa upwards so as to cut the curve of desiccation where it meets the
ordinate corresponding to the 9th day of drying. This is equivalent to saying that
the practical zero from our point of view is reached by the drying curve on the 9th
day. We utilise this fact in making the calculation of the actual loss of weight
of water which is to be the subject of comparison with loss of infectivity. Thus
in fig. II the area below the line A B C represents the dead weight of solid basis
nerve-materal, which does not undergo any change in weight, plus the small
16
amount of non-effective water which is present after the 9th day of desiccation.
We now proceed to plot the two curves on the same chart, reducing the figures in
each case so as to make the graph start from unity. In this way we obtain
fig. III. Our figures for the drying curve are averages obtained from four
different sets of observations. The two sets of figures to be plotted are here given.
It is to be noted that the weight of water remaining in the cord after the
9th day together with that of the solid basis material of the cord has been duly
deducted from the actual weights obtained, and the result is shown in column 3
as ' l os s of wat er . '
TABLE V.
(1)
(2)
(3)
Desiccation.
Infectivity.
Loss of water.
Fresh
.
.
1.2
1 . 2
1st day
.
. . .
0.392
2nd day ...
0.125
0.115
3rd day .
. . . .
0.025
0.049
5th day .
.
0.0125
0.013
9 t h d a y .
0 . 0
0.0
These curves (fig. III), we think, show a very marked correspondence,
experimental error being allowed for. Both curves show a marked and rapid fall
to the third day and both show extremely gradual subsidence after the fifth day
of drying. We believe, cæteris paribus, the two curves to be identical and indeed
that they represent two parallel effects of the same causal action.
If we take this case as proven we may state this proposition:—
The rate of loss of infectivity of rabies cord undergoing slow desiccation is
directly proportional to the rate of loss of water contained in that cord.
In other words, as slow desiccation means death of the rabies virus, and as
we consider dead rabies virus to be comparatively speaking valueless for immunis
- a t i o n , w e m a y s t a t e h e r e p r o v i s i o n a l l y t h i s c o r o l l a r y t o t h e a b o v e p r o p o s i t i o n
The immunising power of a given portion of rabies cord is a function of the
unkilled remnant of rabies virus which is contained in that cord.
17
If this be so, it follows that a cord which has been drying for nine days or
longer has little or no value for immunisation. With regard to less dried cords
a greater or smaller quantity will be required to produce a sufficient immunity to
rabies infection according to the greater or less degree of desiccation. The ex-
periment which we give in detail below gives, by means of a particular instance,
support to this view and, by extension, to the view that immunisation against
rabies might conveniently be carried out with a cord which had dried for a fixed
time only, instead of with a graded series of dried cords. In our experiment we
show that whereas nine-day cord has probably little immunising power, five-day
cord has considerable power and might probably be used by itself 1 as the immunis-
ing substance from commencement to termination of a course of treatment,
It may be as well to say here that, even if such a method as this were elevated
into the position of a system of anti-rabic immunisation, we should still regard it
only as a temporary compromise between the use of dried material and of
fresh rabies virus. The method suggested by Nitsch amongst recent authors
we regard likewise as simply a step towards the more general use of fresh
material.
Experiment.—Two dogs were inoculated in the abdominal region, the one
with nine-day cord alone, the other with five-day cord alone. After prolonged
treatment with this material and after allowing a definite interval for the full
development of immunity, these two dogs along with a control dog were proved
by subdural inoculation. The following table gives all details together with the
results obtained:
—
TABLE VI.
Dog.
Weight.
Cord
used for
immunis-
ing.
Total
amount
used.
Number of
days occupied
in the
immunising
process.
Subdural
inoculation
after an
interval of
Amount of
dose inoculated
subdurally.
Result.
REMARKS.
1.
20 lbs. . 5-day
. 3.4045 grm.* 20 days
27 days
0.3 cc. of a 5
per cent.
emulsion.
Remained
well.
Immune.
II.
18 lbs. . 9 day
3.3809 grm.*
Do.
Do.
Do.
Developed
rabies 9 days
after subdu-
ral inocu-
lation.
Not immune.
III. C o n t r o l
21.5 lbs. .
..
...
...
Inoculated
subdurally at
same time as
I and II.
Do.
Developed
rabies 8 days
after subdu-
ral inocu-
lation.
Not immune.
* This is actual weight of cord used in its dry state.
1
Since this was written we find that Nitsch has recommended a method of treatment in which a com
m-mencement is made from five-day cord. Extract Bull de l'Inst. Past., 1906, p. 1,057.
D .
18
The five-day inoculated dog showed itself entirely immune to the subdural
infection. The nine-day dog and the control dog both succumbed to rabies,
the former showing paresis nine days after subdural inoculation and the latter
eight days after. The nine-day cord inoculations therefore produced no effective
immunity, and it would seem that the incubation period of the disease had not
been to any degree extended.
We have spoken above of "slow desiccation" (p. 16) ; this is merely in a
comparative sense to distinguish it from the rapid desiccation of rabies material
in vacuo and over sulphuric acid 1 b y m e a n s o f w h i c h , w i t h i n2
4
h o u r s , a p o w d e r
may be obtained which, although dry, still retains its infectivity. This pheno-
menon is not unknown in the case of other organisms, e.g., vaccine virus, Sta-
phylococcus pyogenes aureus, B. diphtheriæ. The periods to which such
organisms will survive drying vary greatly. We have not made very many
experiments to compare the infectivity of rapidly desiccated rabies virus with
fresh material, but such as we have made would lead us to the opinion that,
although the potency of the former may not be denied, it is yet very greatly less
than that of the latter.
I I I . — A n a t t e m p t a t r e d u c t i o n o f d i f f e r e n t s y s t e m s o f t r e a t m e n t
to a uniform standard by means of a unit of comparison
obtained by experiment.
We have to some extent worked out the correspondence between the dried
cord system and the fresh rabies material system. We have seen that 0.2 cc.
of /8,000 dilution is by our method the M. I. D. for fresh rabies cord material, while
0.2 cc. of 1/200 dilution is somewhere near the M. I. D. for three-day cord and
0.2 cc. of 1/100 dilution for five-day cord. We have also found that 0.2 cc. of
1/5 dilution of nine-day cord had no infectivity. A point which has to be taken
into account in estimating these values is the varying virulence of different
regions of the central nervous system. Thus it is said 2 that cortical material
is 10 times more virulent than spinal cord. Except in some of our earlier experi-
ments with fresh material we have used spinal cord material throughout. We may
now take the values which we have found as actual and endeavour to interpolate,
by means of curve No. I (fig. I), other values for those days of desiccation for
which no observations at all are available. Thus roughly for the 4th day we take
the mean value of the 3rd and 5th days : so also for the 7th day the mean value
between the 5th and 9th days : for the 6th and 8th days the mean values between
the 5th and 7th days and the 7th and 9th days, respectively.
1 V a n s t e e n b e r g h e
C o m p t . R e n d . S o c . d e B i o l .t
. L V . 1 9 0 3 , p p . 1 6 4 6 - 4 7 .
2 Frosch quoting Nitsch Handbuch f. Path. Mikr. Supplement., 2nd part, Art. Lyssa, p. 646.
19
Let then the M. I. D. by this calculation be for
(1) Fresh rabies material
. . .
0.2 cc. of 1/8,000 dilution.
(2)
3rd-day desiccated material
. . .
,, 1/200 ,,
(3) 4th-day
,, ,,
. . .
, , 1 / 1 5 0 , ,
(4) 5th-day
,, ,,
. . .
, , 1 / 1 0 0 , ,
(5) 6th-day
,, ,,
. . .
, , 1 / 7 5 , ,
(6) 7th-day
,, ,,
. . .
, , 1 / 5 0 , ,
(7) 8th-day
,, ,,
. . .
, , 1 / 2 5 , ,
(8) 9th-day ,, ,,
...
,, 0 ,,
We do not proceed further than the 9th day because we regard the immun-
ising value of material which has been drying for nine days or more may be neglect-
ed in practice. Let now 0.2 cc. of 1/100 dilution of fresh rabies material be our
arbitrary standard for the comparisons of infectivity, and equal to 1,000 units ;
then 0.2 cc. 1/8,000 dilution is obviously equal to 12.5 units. So with the other.
M. I. D's, which are of course all equal to one another, e.g., 0.2 cc. of 1/200 dilu-
tion three-day material = 12.5 units and so also 0.2 cc. 1/100 dilution five-day material
=12.5 units. The estimation of Höyges' system of immunisation is easy upon the
basis of our arbitrary standard or indeed any arbitrary standard. With the
Pasteurian methods we must know what the strengths of the emulsions used are.
In the Kasauli Pasteur Institute 1 cm. in length of dried cord constitutes a dose
for an adult person. This is emulsified in 3 cc. of physiological salt solution and
the resulting emulsion is one of, on an average, 5 per cent.* Under this system
the dose for an adult is 3 cc. of a 5 per cent. emulsion throughout the whole
course. Calculated on this basis the following two systems (light schemata)
work cut in units to
–
25,028 units
. Kasauli.
25,287
„.
. Höyges.
The respective schemata are here given.
* 015 grm. in 3 cc.
D 2
20
TABLE VII.
III. HÖYGES ' METHOD LIGHT SCHEME.1
Dilution.
Dose in cc.
Units.
1 m.1/10,000+1/8,000
3 – 3
.
.
887.5
3 – 3
. .
e . 1 / 6 , 0 0 0 + 1 / 5 , 0 0 0
2 m. 1/5,000 . . . .
3
. . . .
800
2
.
.
.
e. 1/2,000 . . . .
3
m.1/2,000 . . . .
2
. . .
1,250
1.5 .
. . .
e. 1/1,000. . . . .
4
m. 1/1,000 . . . .
1.5 . . .
1,750
1
.
.
.
.
e. 1/500 . . .
5
m. 1/200 . . . .
1
.
. . .
2,500
6 m. 1/6,000+1/5,000 . . . . .
3–3 . . . .
1,050
2
.
.
.
.
e. 1/1,000 . . . .
7 m. 1/2,000 . . . . .
2
. . . .
1,250
1.5 .
..
.
.
e. 1/000 . . . . .
8 m. 1/1,000 . . . . .
1.5 .
.
.
.
1,750
1
. . . .
e. 1/500 . . . . .
9
m. 1/200 . . . . . .
1
.
.
.
2,500
1 0 m. 1/6,000+1/5,000 . . . .
3 – 3
. . .
1,050
2
.
.
.
e . 1 / 2 , 0 0 0 . . . . . .
1 1
m . 1 / 2 , 0 0 0 . . . . .
2
.
. . •
1,250
1.5 . . . .
e . 1 / 1 , 1 0 0 0 . . . .
12 m. 1/1,000 . . . . .
1.5
.
.
.
1,750
1 . . . . . .
e . 1 / 5 0 0 . . . . .
13
1 / 2 0 0 . . . .
1.
.
.
.
2,500
14
1 / 1 0 0 . . . . .
1.
. . .
5,000
TOTAL
.
25,287.5
1 Deutsch & Feistmantel Impfstoffe and Sera, p. 154.