NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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APPENDIX TO REPORT OF THE LEPROSY COMMISSION:
All these growths in bouillon, gelatine and agar were pure and
undoubtedly identical, and the bacilli stained excellently, and resisted
decolorisation with 10 per cent., 15 per cent. and 20 per cent. nitric
acid. This resistance varied at different periods of growth, being
greatest in the early stages. Subsequent generations, though retain-
ing the shape of the original, did not resist the action of the acid so
well, but still stained to some extent. The individual bacilli were
shorter and slightly thicker than those obtained in alcohol hardened
ieprous tubercles. They also had a distinct tendency to aggregate into
clusters and lines.
This series of experiments was repeated and the details were as
follows :—
2. Expressed tubercle juice of Bishan Singh, and fluid from blister over sound
skin of the same leper, was collected in a sealed capillary tube on April 14th ;
examined on May 6th and 7th, stained bacilli were found in strings and in groups.
No admixture of other bacilli found. A bouillon tube was inoculated and a growth
commenced. This culture however fouled a week later.
3, Expressed tubercle juice of Bishan Singh, and fluid from a blister over
the sound skin of the back of the same leper, was taken on April 14th ; examined on
May 8th, numbers of stained bacilli, which had the appearance of a culture, were
found. No other bacteria present.
The tube was resealed and again examined
on May 14th; no bacilli were found. A bouillon tube, inoculated on May 14th,
was the commencement of a series similar t o Series I.
Cultivation experiments with Excised Tubercle in Bouillon.
Rahim Khan, a Muhammadan, formerly resident in Thibet, has
very marked tubercular leprosy. The disease commenced two and
a half years ago, and the patient is at present in the full height
of the disease ; the lobes of both ears are enormously pendulous and
thickened, The whole aspect of the man indicates that he is ex-
tensively diseased. The uvula is destroyed and there is extensive
ulceration of the fauces and the raucous voice indicative of laryngeal
trouble. His saliva and sputum abound in nests of Leprosy Bacilli.
It was determined in this case (and the results of the treatment
were a great improvement in the health of the patient) to excise the
tuberculated masses in the ears. Accordingly, on May 22nd. 1891,
the whole lobe of the left ear was excised with all antiseptic pre-
caution. The mass was placed in a sterile plate, and with sterilised
knives, one cut being made with only one knife, pieces from the
centre were placed in bouillon, gelatine and on glycerine-agar.
Nine tubes were prepared in this way, seven being bouillon. The
pieces of tissue abounded in bacilli. From none of these experi-
ments were Leprosy Bacilli obtained. The growth on agar about a
Appendix I.
4.29
week later showed streptococcus chains, and the gelatine presented
the same growth ; the other tubes, with one exception, became
turbid and foul, but in this isolated instance a bacillus shown in
Fig. 7, Plate III, was obtained, and successive pure cultivations
raised upon gelatine agar and bouillon. The bacillus, which was
certainly obtained from the tissues, and was never seen in numerous
accidental foulings, we believe is probably the third variety figured
by Neisser. It stains exactly like the bacillus of the tissues with
Ziehl's solution, and resists powerful decolorisation with acids.
Gram's method makes its structure very evident, and it stains also
by the method employed by Giacomi for the Syphilis Bacillus (?)
of Lustgarten. Aqueous methyl-blue and most of the common ani-
line dyes stain this vacuolated bacillus perfectly.
This vacuolated bacillus is absolutely immotile even in the early
stage. At this period a rod-shaped organism, in length and breadth
exceeding the typical Bacillus Lepræ, but smaller than Bacillus
Subtilis, is seen aggregated into clusters and now shows no trace of
vacuolation. This subsequently occurs when the culture is about
one week old, and one to four clear unstained spaces are to be
seen in the rod, which is slightly increased in length. Bacilli,
shorter than Bacillus Lepræ, with only one ovoid space, are also
to be seen, and occasionally a club-shaped form with a terminal
space. At all periods of growth this bacillus stains admirably by
the methods employed as diagnostic of the Bacillus Lepræ of the
tissues. We do not believe these clear spaces are spores. In no
instance were isolated spores seen. The study of the bacillus in
hollow ground glass slides did not throw any light on this question.
The growth of this vacuolated bacillus in bouillon is very like
that formerly described for the bacillus obtained by the blister
method. In gelatine the appearance is, however, so characteristic
that it was easily possible to at once detect the culture. A speck
growing to a white disc appears after twenty-four hours, the needle
track at the same time commencing to form a stocking-like lique-
faction in the gelatine. At the end of sixty hours a tenacious pellicle
forms on the surface of the gelatine, and a flocculent deposit settles
down to the lower part of the liquefied stocking-like tract. Sub-
sequently the pellicle becomes still denser, and by the end of ten
days the growth has almost ceased, all the gelatine is fluid, and a
flocculent loose deposit occurs at the bottom of the tube. The growth
on glycerine-agar is much slower, and there is nothing very typical
about this.
On June 20th the lobe of the right car of the same leper was
430
APPENDIX TO REPORT OF THE LEPROSY COMMISSION:
excised, and with all precautions, pieces of tuberculated tissue were
placed in six tubes of bouillon and two of gelatine. The cultures
were kept at the room temperature, which averaged 25°C. On the
eleventh day in two of the bouillon tubes distinct turbidity was
noticed, and both tubes showed bacilli closely resembling the Leprosy
Bacilli of the tissues in shape and staining reactions. No other
bacilli were present. The vacuolated form was not met with. A
pellicle formed on the surface three days later and the culture
was still found to be pure. Generations from these original growths
were carried on in bouillon and gelatine and agar, and these re-
mained quite pure for some weeks, when great fouling occurred in
many tubes.
This bacillus, as far as we could judge, exactly resembled the
one obtained in our earliest experiments. It stained excellently with
Ziehl's solution and resisted decolorisation with 26 per cent. nitric
acid. As is figured in Fig. 2, Plate III, a hull becomes very apparent
around the bacilli, and this is capable of being well shown with
watery methyl-violet and to a less extent by the method employed
for showing the capsule of Friedländer's Pneumococcus. 50 The
aggregation of these bacilli into groups is most peculiar, and the
appearance figured in Plate III, Fig. 4, recalls very much the arrange-
ment within the tissues, masses of bacilli being collected together
into clusters, due possibly to the nature of the hull of the bacillus.
Cultivation Experiments with Juice from Leprous Tubercle.
On July 8th an unbroken tubercle on the elbow of the same leper
was scraped with a knife, washed with carbolic soap and perchloride
solution, and after puncture with every precaution, leper juice was
collected in a sterile capillary tube and dropped into glycerine-
bouillon. Six tubes were prepared in this way, of which four rapidly
became fouled with growth of Bacillus Subtilis and staphylococci ;
the other two remained pure and were kept in the incubator at 37°C.
In both these tubes free bacilli, undistinguishable from Leprosy Bacilli,
were found about the tenth day. We did not have the opportunity
of continuing this culture on agar.
As will be seen from the next section, all attempts to establish
the pathogenic character of the three bacilli described above were
without success, and consequently it is impossible to affirm with
certainty that the cultivation of the Bacillus Lepræ has been accom-
plished by us. We do, however, claim to have obtained the same
( 50 ) Friedländer, op. cit.
Appendix I.
431
bacillus from the tissues of lepers by three different methods.
Allowing that the form of this cultivated bacillus is slightly broader
than that met with in the tissues, this alone would not constitute
a serious distinction, for, as is well known, bacilli, which are specific
pathogenic parasites, vary greatly in size, according to the nature of
the medium 51 on which they grow, and further the same specific
bacillus varies in size according to the animal in which it occurs. 62
Lastly, the aspect of the cultivated Bacillus Lepræ is unknown to us.
Also, as far as staining reaction and measurements can serve as
criteria, the bacillus we have figured does correspond with that
of the tissues. In the course of numerous foulings of cultures neither
this form nor that of the vacuolated bacillus were ever found as a
contamination, and we are convinced that both these forms were
obtained from tubercular masses. No skin was adherent to the
pieces used as a source of culture. It is on the staining properties
that we rely, and we believe that we have cultivated two bacilli of
different morphological form which respond to the staining methods
used as diagnostic of the Bacillus Lepræ. However, the rapidity of
the growth of the subcultures greatly exceeds that of the original.
This has surprised us, for we should have expected that the rate of
multiplication of the specific bacillus of a disease such as leprosy,
which is so essentially chronic, would have been slower.
Quite recently Campana 53 found in cultivations of tissue from
tuberculous leprosy a bacillus which morphologically entirely resem-
bled Bacillus Lepras. Treated by Ehrlich's method this did not take
on a double staining. In two cases this bacillus grew in agar-ao-ar
and in meat peptone to which 3 per cent. of grape sugar was added.
The bacillus did not grow in fluid media. Inoculation experiments
on rats were negative. Campana does not infer that this bacillus is
identical with Bacillus Lepræ, since he only found a resemblance
between the two. It is perhaps possible, though we do not incline
to this view, that the bacillus described by Campana may resemble
that of our cultures.
We refrain from definitely asserting that our culture is the Lep-
rosy Bacillus, since it apparently possesses no pathogenic power.
Possibly leprosy, like syphilis and cancer, 64 is an essentially human
( 51 ) See Buchner's figures of Bac. Anthracis in Zopf's Die Spaltpilze .
( 52 ) K. Huber, Deutsche Med. Wochen. 1881, No. 8. Bac. Anthracis is largest in mice,
smallest in the cow, and of a mean between these in rabbits and guinea-pig's.
( 53 ) Campana (Reforma Med. No. 14, 1891), quoted from British Medical Journal of June
20th, 1891.
( 54 ) Hanau's rats became affected with cancer, not in consequence of the introduction of
cancer from the human subject, but of a cancerous growth taken from another rat.
432
APPENDIX TO REPORT OF THE LEPROSY COMMISSION:
disease. We propose to continue these cultivation experiments as
opportunity offers,
VI.—TRANSMISSIBILITY OF THE DISEASE BY EXPERIMENT.
Keanu, a Hawaiian man in Oahu Gaol, in consideration of the
commutation of his sentence of capital punishment, voluntarily sub-
mitted to inoculation of a leprous tubercle under the skin of the left
fore-arm. This was performed by Dr. Arning on September 30th,
1884, 55 and at this time no symptom of leprosy was present, and no
family history of the disease could be obtained. In four weeks' time
the patient suffered from rheumatic pains in the left shoulder, and
subsequently also in the elbow and wrist joints
Accompanying
these symptoms there was a painful swelling of the ulnar and median
nerves ; fever was absent. By the end of six months the neuritis had
gradually decreased, and a small leprous tubercle had formed on the
keloid growth at the site of inoculation. Leprosy Bacilli could be
detected in the tissues of this scar sixteen months after inoculation.
Definite symptoms of leprosy existed in September 1887, and in an-
other year the patient was at the full height of the disease.
In estimating the value of this experiment it must be remembered
that Keanu's son, nephew, and maternal first cousin were lepers
either before or after the experiment. 50 But apart from this, which
we are not inclined to use as a strong argument, Arning's experi-
ment, in order to be scientifically free from objection, needs to be
performed with reference to the question of race and habitat. Haw-
aiians become lepers with great frequency, and the disease may be
regarded as endemic at the present time in the Sandwich Islands.
The possession of a criterion of the living Bacillus Lepræ is also of
the first importance in experiments of this nature.
Attempts to transmit the disease to man were made more than
thirty years ago by a Norwegian physician, who endeavoured to in-
oculate himself and other individuals with leprosy by the introduction
under the skin, of leprous tubercles, blood, and pus from lepers.
These efforts to transmit the disease uniformly failed.
Profeta 57 has similarly failed to inoculate himself or others with
leprosy, and Cagnina 58 met with the same result. Numberless phy-
( 55 ) Journal of the Leprosy Investigation Committee, No. 2, 1891, page 132, where
Arning's paper in Archiv. für Dermat. und Syph. January 1891, is translated.
( 56 ) Report on Leprosy in Molakai, by Dr. Swift, who attended Keanu's relations.
( 57 ) Profeta " Sur l'Elephantiasis des Grecs "—Giornale intern. dell. sc. med., 18S4. quoted
by Leloir.
( 58 ) Quoted by Leloir, p. 238,
Appendix I.
433
sicians and surgeons during the performance of autopsies, or opera-
tions on lepers, have accidentally wounded themselves, and there is
no case recorded of the subsequent appearance of leprosy.
Experimental inoculations upon animals have never succeeded in
establishing leprosy as it is clinically seen in man. . A visceral lep-
rosy, if such a term may be employed, has however been obtained by
some experimenters, though a far greater number of observers have
failed.
In Neisser's observations, 59 a freshly extirpated leprous tubercle
was introduced into the peritoneal cavity, subcutaneous tissue, and
anterior chamber of the eye of rabbits and dogs. The value of these
experiments, in some of which there was perhaps a local lepra, as
well as some subsequent negative observations in which "cultures"
and leprous tubercles were used, is thus summarized after a criticism
of the work of others in this direction. " Leprosy in animals has not
yet been established by certain convincing experiments." 60
Köbner 61 and Hansen 62 (both of whom inoculated monkeys)
obtained negative results. Damsch, 03 after implantation of leprous
tubercle in the anterior chamber of the eye of rabbits, described an
infiltration of the iris, ciliary body, and Descemet's membrane, with
cells containing Leprosy Bacilli. In consequence of the enormous
number of bacilli, he believed that a definite multiplication of these
had occurred within the body. But it is the case that pieces of tuber-
cle left in ordinary water for some weeks similarly shows the water to
be full of Leprosy Bacilli, which no doubt are washed out of the mass,
and it is quite conceivable that the results obtained by Damsch may
be a simple mechanical process, especially since a distribution of the
bacillus within the body did not occur. The implantation experiments
beneath the skin and into the peritoneal cavity of cats do not appear
to have led to more than the results obtained by Neisser, that is, a
possible local lepra. Campana's 64 experiments consisted in numerous
inoculations of leprous tubercle on the vascular comb and beard of
fowls. A local tumour frequently formed at the seat of inoculation,
and large cells produced in the inflammatory process took up the
Leprosy Bacilli in the same manner as inorganic particulate foreign
bodies would be ingested by white blood corpuscles. Campana
( 59 ) Virch. Archiv. Vol. 4, 1881, p. 534.
( 60 ) Virch. Archiv. 1886, Vol. 103, p. 384.
( 61 ) Virch. Archiv. 1882, Vol. 88, p. 282.
( 62 ) Congress de Copenhague, 1884, quoted from Leloir.
( 63 ) Virch. Archiv. 1883, Vol.90, p. 20.
( 64 ) Campana, Archiv, suela. Scienza Med. 1883, and Clinica Dermatopatica, 1883, quoted
from Neisser.
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APPENDIX TO REPORT OF THE LEPROSY COMMISSION :
believes that the results obtained by Damsch aire capable of [the same
interpretation. Vossius, 65 by inoculation in the anterior chamber of
the eyes of rabbits, found an extensive accumulation of Leprosy
Bacilli in the cornea, iris and ciliary body, but a distribution of these
bacilli throughout the body, and the establishment of visceral or
cutaneous lesions, is necessary before these can be considered other
than implantation experiments,
Hillairet and Gaucher 66 inoculated a pig with leprous tubercle ; E.
Vidal repeated' the experiment, and the implanted mass a year later
showed numbers of Leprosy Bacilli, though none were to be found in
the surrounding tissue ; these observations may therefore be regarded
as negative. Thin, Kaurin, 67 Arning, Rake, 68 Bordoni-Uffreduzzi (who
failed to inoculate with his cultivated Bacillus Lepræ) have hitherto
met with failure to establish leprosy in animals.
Hitherto the transmissibility of the disease to animals has suc-
ceeded only in the experiments of Melchor and Ortmann. 69 In rabbits
they describe an intestinal and a lymphatic gland lepra as developed
four months after the introduction of freshly extirpated leprous tuber-
cle into the anterior chamber of the rabbit's eye. After inoculation
a subacute general infective disease is established, and the autopsy
showed almost all the viscera (caecum, lymphatic glands, spleen,
lungs) to be infiltrated with nodules varying in size from a pin's head
to a pea. In these nodules were quantities of typical Leprosy Bacilli.
Numbers of observers, 70 who have seen the specimens obtained from
these disseminated nodules, are satisfied that a genuine visceral
leprosy was established by the experiments of these observers, and
the conclusion that under certain circumstances leprosy is an infective
disease transmitted by the agency of the Bacillus Lepræ, may fairly
be made from these experiments. At the same time it must be
stated that, although a few animals became acutely infected, a far
greater number escaped infection.
With the exception of the last-mentioned experiments, it may be
considered that the proof of the inoculability of leprosy has not yet
( 65 ) Bericht der XVI Vers. der Opthal. Gesell. Heidelberg, 1884, quoted from Neisser.
( 66 ) Société de Biologie, 1881, quoted from Les Bacteries, p. 760, by Cornil and Babés.
( 67 ) " In spite of several experiments I have not been able to prove Dr. Ortmann's inocula-
tion of the Bacillus Lepræ on rabbits." Journal of the Leprosy Investigation Committee, No 2
1891, p. 6S.
( 68 ) Reports of Trinidad Leper Asylum, 1884, 1885, 1886, 1887, 1888, 1889.
( 69 ) Berl. Klin. Wochen. 1885, No. 13, and Ibidem, 1886, No. 9.
( 70 ) Among others Arning (p. 117, Journal of the Leprosy Investigation Committee No. 2),
who writes " there is no doubt to my mind that Dr. Ortmann of Königsberg has succeeded
in inoculating animals with leprosy. I have seen his specimens and am fully convinced of the
accuracy of his results."
Campana, Wesener (Munch. Med. Wochen. 1887, No. 18) and others have adversely
criticised the above experiments.
Appendix I.
435
been given. Had Melchor and Ortmann worked with a pure culture
of Bacillus Lepræ and again recultivated the bacillus found in the
organs, the proof would have been complete, and Koch's postulates
for the specific pathogenic nature of a bacillus would have been satis-
fied. As mentioned above, Bordoni-Uffreduzzi failed to inoculate
with his cultivated Bacillus Lepræ.
Our experiments were commenced at Almora on April 3rd, 1891.
Pieces of freshly extirpated tubercle from the wrist were removed
with all antiseptic precautions, and a piece of the jejunum of a dog
about 4 inches in length, was resected, washed out with sterilized
warm water, then closed at one end, the piece of tubercle introduced,
and the other end closed. The mesentery was left attached. The
gut was now joined by double rows of Lembert's suture and the
abdomen closed. Two dogs were operated on in this way ; the first
died on the sixth day. Considerable peritonitis was found. The
sutures were in place, and there was no obstruction of the bowel at
this point. The resected loop contained a yellowish faecal liquid. In
this liquid the piece of tubercle lay unchanged. All the viscera ap-
peared healthy. The fluid in the resected bowel and the mucous
membrane were examined microscopically. Plenty of Leprosy Bacilli
were found in half the specimens of the former; the latter were nega-
tive. The second dog died on the sixth day. Resected ends of the
bowel had almost separated. In the resected loop, which was gan-
grenous, the piece of tubercle lay unchanged. Cover-glass specimens
of the tubercle, the contents of the loop, and the submucous layer of
the bowel wall were taken. In the first, were quantities of Bacillus
Lepræ, in the second, three specimens contained Bacillus Lepræ, and
one showed none, in the third, no bacilli were found. These two
experiments were performed chiefly with a view to estimate the pos-
sibility of success in the operation. The piece of tubercle would have
remained in the loop, and had distribution of the bacilli occurred
these would in all probability have been found either in the lymphatic
glands or in the fluid of the thoracic duct. The admixture with
digested food and mixed intestinal secretions, such as the bile, which
might affect the activity of Bacillus Lepræ, would also have been
obviated. The bacilli found in the contents of the resected loop were
probably only mechanically washed out of the introduced tubercle.
Further experiments were carried on in the laboratory built by the
Indian Government for the Commission at Simla. Cultures obtained
from blister fluid and leprous tubercle were mixed with about twenty
times their volume of .65 per cent. saline solution, and the mixture so
obtained was used for all the inoculations at Simla.
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