NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
DIAGNOSIS OF JOHNE'S DISEASE
217
prepare for examination smears from fragments taken from two or three
different parts of the rectum, on account of the unusual distribution of
the bacilli. At least one hundred fields of the microscope should be
examined in each smear, after staining in the usual way for acid fast
bacilli. The bacilli when they are found are usually rare and occur
as short, delicate, scattered single rods. Mistakes frequently occur
in the diagnosis of smears containing rare bacilli. Observers are apt to
confuse other acid-fast bacilli such as normally found in fæces (the
so-called "manure bacillus") and even bacterial spores with the true
Johne's bacillus. This error can usually be avoided if the decolourising
agent employed in the Ziehl Neelsen method consists of an alcoholic
acid solution (such as three per cent. hydrochloric acid in methylated
spirit) applied for a long time, about 5 minutes or longer, instead of
the usual watery solution of acid, for the other organisms are usually
not " alcohol fast " although they are acid-fast ".
This method is the best one available when the lesions are in the lower parts
of the bowel. Very often, however, as already mentioned, the lesions exist higher
up the intestine and diagnosis cannot then be established by this method. When
this method fails, the bowel-washing method described below should be carried out.
The bowel-washing method employed by Mr. V. Krishnamurti is described in
his article entitled " A note on the treatment of Johne's disease by intravenous
injections of formalin " (Proceedings of the Indian Science Congress, Madras, 1929).
The method employed is described by him as follows:—
" After lubricating the forearm with soap and water, the hand is intro-
duced into the rectum and the rectal contents emptied. A long
rubber tube is then introduced per rectum as far anterior as possible
and by an enema attached to its other end, the bowels are irrigated
until water commences to escape from the rectum. While this is
being done, such portions of the mucous membrane of the rectum as
can be reached by the hand are also washed by the palm of the hand.
The fluid that escapes as a result of the irrigation is collected into a
basin and allowed to sediment for a few hours. The supernatant fluid
is then drained off and a few c.c. of the fluid with the sediment are
then centrifuged. Smears are then made from the topmost layers of
the deposit and examined for the presence of acid-fast bacilli. If
Johne's bacilli are present they are found either in clumps or in stray
rods and can be detected very easily."
Our experience of this method has shown that, provided the preliminary
emptying and washing of the bowel has been effectively carried out, a slightly
218 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, III
blood-tinged fluid containing a few mucus shreds will be obtained, and after
centrifuging the topmost layer of the deposit will often consist only of small
particles of mucous membrane. Since the organisms are contained in the mucous
membrane the material thus obtained is ideally suited for the purpose of diagnosis.
It has also been found that a long period of centrifugation is necessary as in some
cases examination after 5 to 10 minutes yielded negative results, while after further
centrifugation organisms were detected in the same samples. Centrifuging must,
therefore, be continued for at least 15 to 20 minutes where an ordinary hand
centrifuge is employed.
The two methods of diagnosis described above have recently been employed
in a number of animals at the Muktesar Institute and the results obtained are
recorded below.
A batch of nine cows and two bullocks which had all given definitely positive
reactions to avian tuberculin was received at Muktesar from a dairy farm in July
1930. In order to confirm the existence of the disease in these animals by
demonstrating acid-fast bacilli of Johne's disease, rectal smears were taken and
examined, but in every case with negative results. Smears were taken again the
next day and examined in every case with the same result. The bowel washing
method was then tried and all of them eventually revealed acid-fast organisms,
although in two or three cases the examination had to be repeated more than once
before they were found. The details of these examinations are given in the
appended Table.
After a period of nearly two months from the first examination, six of the
animals began to reveal the bacilli also by the rectal smear method. This fact
indicates that the disease was in its early stage with lesions only high up in the
intestine at the time of the first examination.
It is of interest to note that when housed under good conditions at Muktesar
and with a liberal diet the majority of these animals began to improve greatly in
condition and diarrhœa stopped in all cases within a period of 2 months, and it
was then very difficult to realize that they were cattle infected with Johne's
disease.
An opportunity was afforded to confirm the diagnosis of Johne's disease in
this batch of animals by post-mortem examination when cow 199 died, though not
of Johne's disease, on 6th November 1930. The lesions of the disease were not
very extensive, but there was appreciable thickening and corrugation at the ileo-
cæcal valve, cæcum, the last portion of the ileum and the beginning of the large
intestine. Smears from these areas as well as from some of the mesenteric glands
revealed numbers of Johne's disease bacilli. The value of the bowel-washing
method is amply demonstrated from the results recorded, but it will not be used
COLON BACTERIA IN THE MILK SUPPLY AT PUSA
223
A positive reaction for B. coli was given by 1 c. c. milk on only four out of
twelve tests in October 1930 and three out of fifteen in November 1930, and was
absent in all the tests in December 1930 and January 1931.
We see from the table that in the months November to February members
of the VP+MR—group were found much less frequently than those of the VP—
MR+group ; in the next three months, March to May, this disparity grew less,
and from June to September the number of VP+forms was found to equal or
surpass that of the VP—forms.
Further examinations of the VP+forms enabled two-thirds of them to be
identified as Aerobacter œrogenes (Kruse) Beijerinck, and the remainder as Aerobacter
cloacœ, (Jordan) Bergey et al.
Identification of the VP—forms was more difficult. Using the classification
of Bergey's Determinative Bacteriology (3rd Ed.), the three species Escherischia
formica, vesiculiformis, and acidi lactici made up 30 per cent., E. anindolica 7 per
cent., E. enterica 1.3 per cent. and E. communior 0.75 per cent. of the cultures
isolated. The other sixty per cent. were not identified ; thirty per cent. had no
action on salicin, but produced gas from sucrose, sixteen per cent. had no action
on sucrose, but produced gas from salicin, ten per cent. produced gas from both
sucrose and salicin, three per cent. produced acid and gas from sucrose and gas
from salicin, and the remaining forms produced acid from sucrose and had no action
on salicin.
It was noticed that Escherischia coli (Escherisch) Castellani and Chalmers, the
type species of the genus, was never found.
220 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, III
TABLE I—contd.
Showing the comparative results of the rectal smear and the bowel-washing methods for
the demonstration of acid-fast bacilli in the early stages of Johne's disease—contd.
Animal
Cow 195 . {
Cow 196 . {
Cow 197
.{
Cow 198 .{
Cow 199 . {
Bullock 524 . {
Bullock 523 . {
Rectal smear examina-
tion
Date
9-7-30
10-7-30
31-8-30
9-7-30
10-7-30
30-8-30
9-7-30
10-7-30
30-8-30
4-9-30
9-7-30
10-7-30
29 8-30
4-9-30
9-7-30
10-7-30
25-8-30
9-7-30
10-7-30
..
..
28-8-30
9-7-30
10-7-30
27-8-30
4-9-30
Result
Negative .
Negative .
+ V. rare .
Negative .
Negative .
Negative .
Negative .
Negative .
Negative .
+ V. rare .
Negative .
Negative .
Negative .
+ Few
Negative .
Negative .
Negative .
Negative .
Negative .
..
..
+ Few
Negative .
Negative .
Negative .
+ Few .
Bowel-washing
examination
Date
..
12-7-30
31-8-30
. .
12-7-30
30-8-30
..
12-7-30
30-7-30
4-9-30
..
12-7-30
29-8-30
4-9-30
..
11-7-30
28-8-30
...
11-7-30
16-7-30
22-7-30
18-8-30
. . .
11-7-30
27-8-30
4-9-30
Result
..
+ Rare .
+ Rare .
. .
+ V. rare .
+ V. rare .
..
+ V. rare .
Negative .
+ V. rare .
..
+ V. rare .
Negative .
+ Rare .
..
+ V. rare .
+ V. rare .
..
Negative .
Negative .
+ Rare .
+ Rare
. .
+ V. rare .
Negative .
+ Rare
Clinical condition
Poor ; no diarrhœa.
„ „ „
Improved in condition.
Poor ; with offensive diarrhœa.
„ „ „ „
Condition much improved.
Poor.
„
„
Fairly good.
Poor ; diarrhœa.
„ „
„ „
Fairly good.
Poor.
„
Died 6-11-30. Confirmed Johne's
disease, post mortem.
Poor ; diarrhœa.
„
„
Fair.
. . . .
Poor ; diarrhœa.
„ „
„ „
Fairly good.
THE COLON BACTERIA OCCURRING IN THE MILK SUPPLY
AT PUSA.
BY
J. H. WALTON, M.A., M.Sc.,
Imperial Agricultural Bacteriologist, Pusa.
(Received for publication on 14th April 1931.)
In the course of investigations for the purpose of raising the standard of
cleanliness of the Pusa milk supply, it was found that both the plate and the coli
counts of bacteria per c.c. milk rose with the onset of the rainy season. At other
times of the year the plate counts could, by proper management in the dairy, be
kept down to very low figures, but the coli count was much more difficult to
control, and though this might rise with the plate count, yet anomalous instances
were frequent. In one sample giving a plate count of 6,000 bacteria per c.c., colon
bacteria were found in 0.1 c.c. and two days later, in milk giving a plate count
of 29,000, the colon bacteria were absent in 0.1 c.c. On other occasions colon
bacteria have been found in 0.01 c.c. milk with a total plate count of under 10,000
colonies per c.c.
After some preliminary experiments in December 1928 and January 1929, the
samples of milk were examined regularly in order to form an opinion regarding the
types of colon bacteria that might be found, and also to find out whether the
prevailing types varied with the season of the year.
The tests for the presence of coliform bacteria were made by inoculating 1 c.c.,
01 c.c. and 0.01 c.c. milk into tubes of a medium composed of 20 grms. peptone,
5 grms. lactose, 5 grms. sodium taurocholate, 1 litre water, and coloured blue with
litmus. The tubes contained a Durham fermentation tube. The presence of
coliform bacteria (Coli +) was indicated by the presence of gas in the fermentation
tube and reddening of the litmus.
Plates were made on Endo Agar from a tube of the highest dilution giving
a Coli + reaction. A sample of the red (i.e., Coli) colonies, generally nine, was
taken from a portion of the plate, subcultured on ordinary agar, and the agar
cultures examined for motility and inoculated into tubes of a glucose broth, consist-
ing of 5 grms. each glucose, peptone, and dipotassium phosphate per litre water.
The broth tubes contained Durham fermentation tubes.
( 221 )
222 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, III
After incubation for 48 hours at 37°C., the reaction of the broth medium to
methyl red was observed, and the Voges Proskauer test applied. The latter test
was carried out by pouring about 2 c.c. of the medium into a watch glass and
adding an equal volume of 20 per cent. caustic potash. A pink colour, generally
developing in 20 minutes to half an hour, indicated a positive Voges Proskauer
reaction (VP+). The positive Voges Proskauer reaction is associated with an
alkaline reaction towards methyl red (MR—), a negative Voges Proskauer reaction
(VP—) with acidity to methyl red (MR+).
Very rarely, cultures gave both VP+, MR+, but these cultures were impure
and could be separated into VP+ MR— and VP—MR+ forms. The table gives
the number of days in each month on which the VP+ MR— and VP— MR+
forms were found, and also the number of days for each form on which its cultures
outnumbered those of the other.
Month
1929.
February.
March .
April .
May .
June .
July .
August
September
October
November
December
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1930.
January
.
.
.
.
February
March
April .
May .
June .
July .
August
September
October
November
.
.
.
.
.
.
.
.
. .
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Number
of
samples in
month
1
17
9
13
14
12
12
13
13
5
12
10
13
8
13
13
15
13
13
11
12
3
4
Number
of
samples in
which VP—
MR+type
occurs
2
16
8
11
11
8
3
2
8
4
12
8
11
8
12
13
13
7
8
5
6
2
3
Number
of
samples in
which VP—
MR+type
predomi-
nates
3
16
6
11
11
6
3
0
7
4
12
8
10
8
11
12
12
5
5
5
6
2
2
Number
of
samples in
which VP+
MR—type
occurs
4
2
4
7
4
9
11
13
6
2
3
2
3
0
4
6
8
9
11
9
7
1
2
Number
of
samples in
which VP+
MR—type
predomi-
nates
5
1
3
2
3
6
9
13
5
1
0
2
3
0
2
1
3
7
8
6
6
1
1
COLON BACTERIA IN THE MILK SUPPLY AT PUSA
223
A positive reaction for B. coli was given by 1 c. c. milk on only four out of
twelve tests in October 1930 and three out of fifteen in November 1930, and was
absent in all the tests in December 1930 and January 1931.
We see from the table that in the months November to February members
of the VP+MR—group were found much less frequently than those of the VP—
MR+group ; in the next three months, March to May, this disparity grew less,
and from June to September the number of VP+forms was found to equal or
surpass that of the VP—forms.
Further examinations of the VP+forms enabled two-thirds of them to be
identified as Aerobacter œrogenes (Kruse) Beijerinck, and the remainder as Aerobacler
cloacœ, (Jordan) Bergey et al.
Identification of the VP—forms was more difficult. Using the classification
of Bergey's Determinative Bacteriology (3rd Ed.), the three species Escherischia
formica, vesiculiformis, and acidi lactici made up 30 per cent., E. anindolica 7 per
cent., E. enterica 1.3 per cent. and E. communior 0.75 per cent. of the cultures
isolated. The other sixty per cent. were not identified ; thirty per cent. had no
action on salicin, but produced gas from sucrose, sixteen per cent. had no action
on sucrose, but produced gas from salicin, ten per cent. produced gas from both
sucrose and salicin, three per cent. produced acid and gas from sucrose and gas
from salicin, and the remaining forms produced acid from sucrose and had no action
on salicin.
It was noticed that Escherischia coli (Escherisch) Castellani and Chalmers, the
type species of the genus, was never found.
SELECTED ARTICLES
ON TREATMENT OF STERILITY IN COWS WITH WHEAT
GERM OIL (VITAMIN E).
BY
P. VOGT-MOLLER, M.D., AND F. BAY, D.V.M.
Denmark.
(Reprinted from the Veterinary Journal (London), Vol. 87, No. 4, April 1931.)
The existence of a fat soluble anti-sterility vitamin—vitamin E—must now be
considered established. The literature concerning this vitamin up to the year 1927
is to be found in Evans and Burr's (1927, 1) monograph; we would, however,
mention here that the first communication about such a vitamin is due to Evans
and Bishop (1922).
Vitamin E is found in certain vegetable oils, in largest quantities in wheat
germ oil, but, as mentioned, also in many other oils of vegetable origin, e.g., in
crude maize oil (Sure, 1926), but not in the commercial product of maize oil; it is
also to be found in plant leaves, e.g., in lettuce leaves. Little or no vitamin E
is found in animal substance : it is, for instance, not found in cod-liver oil.
As yet the importance of vitamin E to the organism has only been elucidated
as far as rats are concerned, the above works only comprising experiments with
these animals ; Beard (1926) has, however, proved that mice, too, need vitamin E.
As far as we know the literature does not comprise any works on the need
for vitamin E of other animals than those mentioned, nor do we know of any
communications concerning the therapeutic usefulness of vitamin E preparations
in domestic animals.
The sterility occurring when this vitamin is lacking in the food is of a different
pathogenesis in the two sexes: in the male animal the vitamin seems to play a
part in the spermatogenesis, histologic changes of the testis being found in
avitaminosis E, especially a more or less marked degeneration of the spermatogonia
or even complete disappearance of the latter. In the female animal, on food
without vitamin E, ovulation and implantation take place in the normal manner,
the reproductive system being unimpaired and the sterile condition being due to
the fact that the implanted ova became resorbed at an early stage of the pregnancy.
In male as well as in female animals vitamin E may be stored to a certain extent.
( 224 )
TREATMENT OF STERILITY IN COWS
225
In 1927, Evans and Burr (1927 (2) (3)) showed that various fats, e.g., lard,
when mixed with vitamin E (wheat germ or wheat germ oil) were capable of
depriving the latter of its value as a vitamin; this vitamin destroying property
of the fat increased with the growing rancidity of the latter, and Evans and Burr
(1928) drew the attention to "the possibility of destroying vitamin E by the
development of rancid substance in wheat germ oil itself " ; it is then of importance
to use fresh preparations. We shall not go into further details, but as far as
researches of recent years are concerned, we refer to the monographs of McCollum
and Simmonds (1929), and Jackson (1929).
The preparation used as vitamin E source in the investigations referred to
below was wheat germ oil; by courtesy of Miss M. H. Roscoe (Lister Institute,
London), the wheat germs used, a very pure preparation, were delivered by Messrs.
I. and H. Robinson, Ltd., Deptford Bridge Mills, London, and the wheat germ oil
was made from the said wheat germs by the following simple procedure :—500 gm.
of wheat germ, heated beforehand to 100° C. for two hours, were extracted in
percolator for forty-eight hours with two litres of peroxide ; free ether, the ethereal,
yellowish extract, was mixed with the fluid squeezed out from the moist wheat
germs, the whole of the extract was filtered, and then evaporated in a vacuum
distillation apparatus to 10 per cent. of the weight of the wheat germs used, and
the clear yellowish oil that was employed was then obtained. For the reasons men-
tioned above, a frequent renewal of the preparation must be considered necessary.
According to Evans (1928-29) wheat germ oil does only contain minimal amounts
of vitamin A.
The dose of wheat germ oil necessary for rats is only a few drops daily, and
according to Evans and Burr (1927(1)) vitamin E (wheat germ oil) is efficacious
both on enteral and parenteral application. That wheat should contain toxic
elements extractive in ether, as was maintained by Hart, Miller and McCollum
(1916) and by Hart, McCollum, Steenbock and Humphrey (1917), has, as is known,
been thoroughly refuted by Osborne and Mandel (1919), and by all researchers of
recent years.
As we had worked for years with the question of vitamin E and reproduction
in small experimental animals (rats and mice) without, however, reaping essentially
new experience, but still with the result that Evans and Burr's investigations could
be verified in every respect, it was natural to try whether the experience gained
could be used in practice, the more so as we had thought for some length of time
that according to all probability the cause of sterility in cows should as a rule not
be sought in pathological changes of the ovaries or the uterus as such, but had a
complicated causative relation possibly associated with the manner in which cows
D
226 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, III
are kept and fed in this country. For through generations, these animals have
gradually been selected almost exclusively according to the demand—more milk
and fatter milk; and at the same time it has been an ever increasing demand that
these animals should consume and assimilate sundry, otherwise worthless, waste
products from various industrial undertakings (coco, soya, cotton, rape, sugar, etc.).
In order to render it still more difficult for the cows to perform this task, to turn
the above products into fat milk, they are fettered with a chain round the neck, and
have to remain at the same spot upto nine months of the year, being prevented
from getting exercise and sufficient access to the direct influence of sun, light, and
fresh air. No wonder then that under these conditions, so abnormal to the primary
nature of the cow, a loss of equilibrium or complete absence of certain substances
necessary to impregnation and propagation may occur, but it is difficult to
state which substances are lacking ; to the general practical observer, however, it
seems as if there is a reason to direct the attention, especially to the absence of
three substances : viz., protein, minerals, and/or vitamins, and of the latter one
may perhaps be justified in thinking especially of vitamin E.
In the experiments referred to below we administered, in the rather rough
dosage stated, the wheat germ oil intramuscularly to cows that had often been in
heat, but could not become pregnant despite repeated leaps. Before each treatment
we explored per rectum, and we did only inject the preparation in cases where the
anatomical conditions were apparently normal, i.e., where there were no palpable
alterations of the uterus, the ovaries, and the oviducts, and, of course, where there
were no marked pathological changes in the animal in general. The experimental
animals, all of the red Danish milk race, were selected gradually, as in practice we
found subjects that were considered well suited for the experiments, and were in
the custody of owners or holders willing to allow the experiment, and to undertake
not to dispose of the animals until we had had an opportunity to investigate into
the result of the treatment, i.e., two to three months after the leap.
The cows have been selected irrespective of age, some were treated the day on
which they were in heat, others outside the period of heat, as far as possible the
former were covered on the same day, whilst the latter were covered the first time
they were in heat after the treatment. The animals are mentioned below in
chronological order with information about dates of calving, and leaps, and about
the dose of wheat germ oil employed; we remark, however, that in case nothing
else is stated, the delivery was normal, and the cow then became normally clean.
EXPERIMENTAL ANIMALS.
(1) Owner, R. R., farmer of S. Five-year-old cow. Calved 1-4-30. In heat,
and covered 10-5, then quiet for two months, again in heat, and covered, and then