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of several communications by him in 1874 and later.
Further researches, first by Neisser in 1879, and afterwards
by observers in various parts of the world, agreed in their
main results, and confirmed the accuracy of Hansen's
observations. The bacilli as seen in scrapings of ulcerated
leprous nodules, or in sections, have the following characters.
They are thin rods of practically the same size as tubercle
bacilli, which they also resemble both in appearance and
in staining reaction. They are straight or slightly curved,
and usually occur singly, or two may be attached end to
end ; but they do not form chains. When stained they
may have a uniform appearance, or the protoplasm may be
fragmented, so that they appear like short rows of cocci.
They often appear tapered at one or both extremities ;
occasionally there is slight club-like swelling. Degenerated
and partially broken down forms are also seen. They take



262 LEPROSY.

up the basic aniline stains rather more readily than tubercle
bacilli, but in order to stain them deeply a powerful stain,
such as carbol-fuchsin, is necessary. When stained, they
strongly resist decolorising, though they are more easily
decolorised than tubercle bacilli. The best method is to
stain with carbol-fuchsin as for tubercle bacilli, but to use a




FIG. 70. Superficial part of leprous skin; the cells of the granulation
tissue appear as dark patches, owing to the deeply-stained bacilli in
their interior. In the upper part a process of epithelium is seen.

Paraffin section ; stained with carbol-fuchsin and Bismarck-brown,
x 500.

weaker solution of sulphuric acid, say 5 per cent, in de-
colorising ; in the case of films and thin sections, decolor-
ising with such a solution for fifteen seconds is usually
sufficient. Thereafter the tissues are coloured by a contrast
stain, such as a watery solution of methylene-blue (vide
p. 113). The bacilli are also readily stained by Gram's



POSITION OF THE BACILLI. 263

method. Regarding the presence of spores practically
nothing is known, though some of the unstained or stained
points may be of this nature. We have, however, no
means of testing their powers of resistance. Leprosy bacilli
are non-motile.

Position of the Bacilli. They occur in enormous num-




FIG. 71. High-power view of portion of leprous nodule showing the
arrangement of the bacilli within the cells of the granulation tissue.

Paraffin section ; stained with carbol-fuchsin and methylene-blue.
x 1 100.

bers in the leprous lesions, especially in the tubercular
form. In fact, so numerous are they that the granulation
tissue in sections, properly stained as above, presents quite
a red colour under a low power of the microscope. The
bacilli occur for the most part within the protoplasm of the
round cells of the granulation tissue, and are often so



264 LEPROSY.

numerous that the structure of the cells is quite obscured
(Fig. 70). They are often arranged in bundles which con-
tain several bacilli lying parallel to one another, though the
bundles lie in various directions (Fig. 71). The appearance
thus presented by the cells filled with bacilli is very character-
istic. Bacilli are also found free in the lymphatic spaces,
but the greater number are undoubtedly contained within
the cells. They are also found in spindle-shaped con-
nective tissue cells, in endothelial cells, and in the walls of
blood vessels. They are for the most part confined to the
connective tissue, but a few may be seen in the hair follicles
and glands of the skin. Occasionally one or two may be
found in the surface epithelium, where they probably
have been carried by leucocytes, but this position is, on
the whole, exceptional. They also occur in large numbers
in the lymphatic glands associated with the affected parts.
In the internal organs liver, spleen, etc., when leprous
lesions are present, the bacilli are also found though in
relatively fewer numbers. In the nerves in the anaesthetic
form they are comparatively few, and in the sclerosed
parts it may be impossible to find any. There are few
also in the skin patches referred to above as occurring in
this form of the disease.

Their spread is chiefly by the lymphatics, though distri-
bution by the blood stream also occurs. They have been
said to be found in the blood during the presence of fever
and the eruption of fresh nodules, and they have also been
observed in the blood vessels post mortem, being chiefly
contained within leucocytes. Recent observations (e.g.,
those of Doutrelepont and Wolters) show that the bacilli
may be more widely spread throughout the body than was
formerly supposed. A few may be detected in some cases
in various organs which show no structural change, especially
in their capillaries. The brain and spinal cord are practically
exempt.

Relations to the Disease. Attempts to cultivate the
leprosy bacilli outside the body have so far been unsuccess-
ful. From time to time announcements of successful culti-



RELATIONS OF BACILLI TO THE DISEASE. 265

vations have been made, but one after another has proved
to be erroneous. A similar statement may be made with
regard to experiments on animals. If a piece of leprous
tissue be introduced subcutaneously in an animal, such as
the rabbit, a certain amount of induration may take place
around it, and the bacilli may be found unchanged in appear-
ance weeks or even months afterwards, but no multiplica-
tion of the organisms occurs. The only exception to this
statement is afforded by the experiments of Melcher and
Orthmann, who inoculated the anterior chamber of the eye
of rabbits with leprous material, the inoculation being
followed by an extensive growth of nodules in the lungs
and internal organs, which they affirmed contained leprosy
bacilli. It has been questioned, however, by several
authorities whether the organisms in the nodules were
really leprosy bacilli, and up to the present we cannot say
that there is any satisfactory proof that the disease can be
transmitted to any of the lower animals.

It would also appear that the disease is not readily
inoculable in the human subject. In a well-known case
described by Arning, a criminal in the Sandwich Islands
was inoculated in several parts of the body with leprosy
tissue. Two or three years later, well-marked tubercular
leprosy appeared and led to a fatal result. This experiment,
however, is open to objections, as the individual before
inoculation had been exposed to infection in a natural way,
having been frequently in contact with lepers. In other
cases inoculation experiments on healthy subjects, and
inoculations in other parts of leprous individuals have given
negative results. It has been supposed by some that the
failure to obtain cultures and to reproduce the disease
experimentally may be partly due to the bacilli in the
tissues being dead. That many of the leprous bacilli are
in a dead condition is quite possible, in view of the long
period during which dead tubercle bacilli introduced into
the tissues of animals retain their form and staining reaction.
There is also the fact that from time to time in leprous
subjects there occur attacks of a certain amount of fever,



266 LEPROSY.

which are followed by a fresh outbreak of nodules, and it
would appear that especially at these times multiplication of
the bacilli takes place more actively.

The facts stated with regard to cultivation and inocu-
lation experiments go to distinguish the leprosy bacillus all
the more strongly from other organisms. Some have
supposed that leprosy is a form of tubercle, or tubercle
modified in some way, but for this there appears to us
to be no evidence. Both from the pathological and
from the bacteriological point of view the diseases are
distinct. It should also be mentioned that tubercle is a
not uncommon complication in leprous subjects, in which
case it presents the ordinary characters.

The mode by which leprosy is transmitted has been the
subject of great controversy, and is one on which authorities
still hold opposite opinions. Some consider that it is a
hereditary disease, or at least that it is transmitted from a
parent to the offspring ; others again that it is transmitted by
direct contact. There appears to be no doubt, however,
that on the one hand leprous subjects may bear children
free from leprosy, and that on the other hand, healthy
individuals entering a leprous district may contract the
disease, though this rarely occurs. Of the latter occurrence
there is the well-known instance of Father Damien, who
contracted leprosy after going to the Sandwich Islands. In
view of the fact that we must regard the bacillus as the
cause of the disease, it is highly probable that in certain
conditions it may be transmitted by direct contact, though
its contagiousness is not of a high order.

In leprosy, therefore, there is an organism which is
invariably present in the disease, and has a special relation
to the changes in the tissues. This organism can be
distinguished from all other known organisms, and is found
in no other condition. Further, all the tissue changes in
leprosy can be readily explained by the presence of a low
form of irritation, such as is afforded by this organism.
The evidence stated must be accepted as to its being the
cause of the disease, though absolute proof is still wanting



METHODS OF DIAGNOSIS. 267

owing to failure to cultivate the organism outside the
body.

Methods of Diagnosis. Film preparations should be
made with the discharge from any ulcerated nodule which
may be present, or from the scraping of a portion of excised
tissue, and should be stained as above described. The
presence of large numbers of bacilli situated within the
cells and giving the staining reaction of leprosy bacilli,
is conclusive. It is more satisfactory, however, to make
microscopic sections through a portion of the excised tissue,
when the structure of the nodule and the arrangement of
the bacilli can be readily studied. The points of difference
between leprosy and tubercle have already been stated,
and in most cases there is really no difficulty in distinguish-
ing the two conditions.



CHAPTER XL

GLANDERS AND RHINOSCLEROMA.
GLANDERS.

THE bacillus of glanders (bacillus mallei ; Fr., barille de la
morve ; Ger., Rotzbacillus) was discovered by Loffler and
Schutz, the announcement of this discovery being made
towards the end of 1882. They not only obtained pure
cultures of this organism from the tissues in the disease,
but by experiments on horses and other animals conclu-
sively established its causal relationship. These have been
fully confirmed. The same organism has also been culti-
vated from the disease in the human subject, first by
Weichselbaum in 1885, who obtained it from the pustules
in a case of acute glanders in a woman, and by inoculation
of animals obtained results similar to those of Loftier and
Schutz.

Within recent years a substance, mallein, has been
obtained from the cultures of the glanders bacillus by a
method similar to that by which tuberculin was prepared,
and has been found to produce corresponding effects in
animals suffering from glanders to those produced by tuber-
culin in tuberculous animals.

The Natural Disease. Glanders chiefly affects the
equine species horses, mules, and asses. Horned cattle,
on the other hand, are quite immune, whilst goats and sheep
occupy an intermediate position, the former being rather



NATURAL OCCURRENCE OF GLANDERS. 269

more susceptible and occasionally suffering from the
natural disease. It also occurs in some of the carnivora
cats, lions, and tigers in menageries, which animals are
infected from the carcases of animals affected with the
disease. Many of the small rodents are highly susceptible
to inoculation (vide infra).

Glanders is also found in man as the result of direct
inoculation on some wound of the skin or other part by
means of the discharges or diseased tissues of an animal
affected, and hence is commonest amongst grooms and
others whose work brings them into contact with
horses.

In horses the lesions are of two types, to which the
names "glanders" proper and "farcy" have been given,
though both may exist together. In glanders proper the
septum nasi and adjacent parts are chiefly affected, there
occurring in the mucous membrane nodules at first firm and
of somewhat translucent grey appearance. The growth of
these is attended usually by inflammatory swelling and pro-
fuse catarrhal discharge. Afterwards the nodules soften in
the centre, break down, and give rise to irregular ulcera-
tions. Similar lesions, though in less degree, may be
found in the respiratory passages. Associated with these
lesions there is usually implication of the lymphatic glands
in the neck, mediastinum, etc. ; and there may be in the
lungs, spleen, liver, etc., nodules of the size of a pea or
larger, of greyish or yellow tint, firm or somewhat softened
in the centre, and often surrounded with a congested zone.
The term " farcy " is applied to the affection of the super-
ficial lymphatic vessels and glands, which is specially seen
where infection takes place through an abrasion of the
skin, such as is often produced by the rubbing of
the harness. The lymphatic vessels become irregularly
thickened, so as to appear like knotted cords, and the
lymphatic glands associated become enlarged and firm,
though suppurative softening usually follows, and there
may be ulceration. These thickenings are often spoken of
as "farcy buds" and "farcy pipes." In farcy also, secondary



270 GLANDERS.

nodules may occur in internal organs and the nasal mucous
membrane. In the ass the disease runs a more acute
course than in the horse.

In man the disease is met with in two forms, an acute
and a chronic ; though intermediate forms also occur, and
chronic cases may take on the characters of the acute
disease. The site of inoculation is usually on the hand or
arm, by means of some scratch or abrasion, or possibly
along a hair follicle, sometimes on the face, and occasionally
on the mucous membrane of the mouth, nose, or eye. In
the acute form there appears at the site of inoculation
inflammatory swelling, attended often with spreading
redness, and the lymphatics in relation to the part also
become inflamed, the appearances being those of a
" poisoned wound." These local changes are soon followed
by marked constitutional disturbance, and by an eruption
on the surface of the body, at first papular and afterwards
pustular, and later there may form in the subcutaneous tissue
and muscles larger masses which soften and suppurate, the
pus being often mixed with blood ; suppuration may occur
also in the joints. .In some cases the nasal mucous
membrane may be secondarily infected, and thence inflam-
matory swelling may spread to the tissues of the face ; in
others it remains free. The patient usually dies in two or
three weeks, sometimes sooner, with the symptoms of rapid
pyaemia. In addition to the lesions mentioned there may
be foci, usually suppurative, in the lungs (attended often
with pneumonic consolidation), in the spleen, liver, bone-
marrow, salivary glands, etc. In the chronic form the local
lesion results in the formation of an irregular ulcer with
thickened margins and sanious, often foul, discharge. The
ulceration spreads deeply as well as superficially, and the
thickened lymphatics have a great tendency to ulcerate
also, though the lymphatic system is not so prominently
affected as in the horse. Deposits form also in the sub-
cutaneous tissue and muscles, and the mucous membrane
may become affected. The disease may run a very chronic
course, lasting for months, and recovery may occur, though,



THE GLANDERS BACILLUS.



271




on the other hand, the disease may take on a more acute
character and rapidly become fatal.

The Glanders Bacillus Microscopical Characters. The
glanders bacilli are minute rods, straight or slightly curved,
with rounded ends,
and about the same
length as tubercle
bacilli, but distinctly
thicker (Fig. 72).
They show, however,
considerable varia-
tions in size and in
appearance, and their
protoplasm is often
broken up into a
number of deeply-
stained portions with
unstained intervals
between. These
characters are seen
both in the tissues
and in cultures, but,
as in the case of many organisms, irregularities in form
and size are more pronounced in cultures (Fig. 73), short
filamentous forms 8 to 12 /A in length being sometimes
met with, but these are on the whole rare. The organism
is non-motile.

In the tissues they usually occur irregularly scattered
amongst the cellular elements ; a few may be contained
within leucocytes and connective tissue corpuscles, but the
position of most is extracellular. They are most abundant
in the acute lesions, in which they may be found in con-
siderable numbers ; but in the chronic nodules, especially
when softening has taken place, they are few in number,
and it may be impossible to find any in sections. They
have less powers of persistence, and disappear in the tissues
much more quickly than tubercle bacilli.

There has been dispute as to whether or not they



FlG. 72. Glanders bacilli amongst broken-
down cells. Film preparation from a glanders
nodule in a guinea-pig.

Stained with weak carbol-fuchsin. x 1000.



272



GLANDERS.




contain spores. Some consider certain of the unstained
portions to be of that nature, and it has been claimed that

these can be stained
by the method for
staining spores
(Rosenthal). But it
is very doubtful that
such is the case ; the
appearances cor-
respond rather with
mere breaks in the
protoplasm, such as
are met with in many
other bacilli which
do not contain spores,
and the compara-
tively low powers of
resistance of glanders
containing
these so-called spores
is strongly against their being of that nature. The powers
of resistance is after all the important practical point.

Staining. The glanders bacillus differs widely from the
tubercle bacillus in its staining reactions. It stains with
simple watery solutions of the basic stains, but somewhat
faintly (better when an alkali or a mordant, such as carbolic
acid, is added), and even when deeply stained it readily
loses the colour when a decolorising agent such as alcohol
is applied. Loffler and Schutz recommended staining of
sections in an alkaline solution of methylene-blue for five
minutes and then decolorising for a few seconds in water,
10 c.c., to which were added ten drops of a concentrated
solution of sulphurous acid and one drop of a 5 per cent
solution of oxalic acid. We have, however, obtained the
best results by carbol-thionin-blue (p. 109), and we prefer to
dehydrate by the aniline-oil method. In film preparations
of fresh glanders nodules the bacilli can be ready found by
staining with any of the ordinary combinations, e.g., carbol-



FIG. 73. Glanders bacilli, from a pure
culture on glycerine agar. Stained with
carbol - fuchsin and partially decolorised to bacilli
show segmentation of protoplasm. x 1000.



CULTIVATION OF GLANDERS BACILLUS. 273

thionin-blue or weak carbol-fuchsin. By using a stain of
suitable strength no decolorising agent is necessary, the film
being simply washed in water, dried, and mounted. Gram's
method is quite inapplicable, the glanders bacilli rapidly
losing the stain in the process.

Cultivation. (For the methods of separation vide infra.)
The glanders bacillus grows readily on most of the ordinary
media, but a somewhat high temperature is necessary,
growth taking place most rapidly at 35 to 37 C. Though
a certain amount of growth occurs down to 21 C., a
temperature above 25 C. is always desirable.

On agar and glycerine agar in stroke cultures growth
appears along the line as a uniform streak of greyish-white
colour and somewhat transparent appearance, with moist-
looking surface, and when touched with a needle is found
to be of rather slimy consistence. Later it spreads laterally
for some distance, and the layer becomes of slightly brownish
tint. On serum the growth is somewhat similar but more
transparent, the separate colonies being in the form of
round and almost clear drops. In subcultures on these
media at the body temperature growth is visible within
twenty-four hours, but when fresh cultures are made from
the tissues it is not visible till the second day. Serum,
however, is much more suitable for cultivating from the
tissues than the agar media ; on the latter it is sometimes
difficult to obtain growth.

In broth, growth forms at first a uniform turbidity, but
soon settles to the bottom, and after a few days forms a
pretty thick flocculent deposit of slimy and somewhat
tenacious consistence.

On potato the glanders bacillus flourishes well and
produces a characteristic appearance, incubation at a high
temperature, however, being necessary. If inoculation is
made to potato from another medium, growth proceeds
rapidly, and on the third day has usually formed a trans-
parent layer of slightly yellowish tint, like clear honey in
appearance. On subsequent days, the growth still extends
and becomes darker in colour and more opaque, till about
18



274 GLANDERS.

the eighth day it has a reddish-brown or chocolate colour,
while the potato at the margin of the growth often shows
a greenish-yellow staining. The characters of the growth on
potato along with the microscopical appearances are quite
sufficient to distinguish the glanders bacillus from every
other known organism (sometimes the cholera organism
and the B. pyocyaneus produce a somewhat similar
appearance, but they can be readily distinguished by their
other characters). The potato is also a suitable medium
for starting cultures from the tissues ; in this case minute
transparent colonies become visible on the third day and
afterwards present the appearances just described.

Powers of Resistance. The glanders bacillus is not
killed at once by drying, but usually loses its vitality after
fourteen days in the dry condition, though sometimes it
lives longer. It is not quickly destroyed by putrefaction,
having been found to be still active after remaining two or
three weeks in putrefying fluids. In cultures the bacilli
retain their vitality for three or four months, if, after growth
has taken place, they be kept at the temperature of the
room ; on the other hand, they are often found to be dead
at the end of a month when kept constantly at the body
temperature. They have comparatively feeble resistance to
heat and antiseptics. Loffler found that they were killed
in ten minutes in a fluid kept at 55 C, and in two to
three minutes by a 5 per cent solution of carbolic acid.
Boiling water and the ordinarily used antiseptics are very
rapid and efficient disinfectants.

We may summarise the characters of the glanders
bacillus by saying that in its morphological characters it
resembles somewhat the tubercle bacillus, but is thicker,
and differs widely from it in its staining reactions. For its
cultivation the higher temperatures are necessary, and the
growth on potato presents most characteristic features.

Experimental Inoculation. In horses subcutaneous
injection of the glanders bacillus in pure culture reproduces
all the important features of the disease. This fact was
established at a comparatively early date by Loffler and



EXPERIMENTAL INOCULATION. 275

Schutz, who, after one doubtful experiment, successfully
inoculated two horses in this way, the cultures used having
been grown for several generations outside the body. In
a few days swellings formed at the sites of inoculation,
and later broke down into unhealthy -looking ulcers.
There was the usual involvement of the lymphatic vessels
and glands, and symptoms due to affection of the nasal
mucous membrane also appeared after some time, there
being the characteristic discharge. One of the animals
died; after a few weeks the other, showing symptoms of
cachexia, was killed. In both animals, in addition to
ulcerations on the surface with involvement of the
lymphatics, there were found post mortem, nodules in the
lungs, softened deposits in the muscles, and also affection
of the nasal mucous membrane, nodules, and irregular
ulcerations. The ass is even more susceptible than the
horse, the disease in the former running a more rapid
course, but with similar lesions. The ass can be readily
infected by simple scarification and inoculation with glanders
secretion, etc. (Nocard).

Of small animals, field-mice and guinea-pigs are the
most susceptible. Strangely enough, house-mice and white



Online LibraryRobert MuirManual of bacteriology → online text (page 23 of 47)