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1904. — No. 15.

DEPARTMENT OF THE INTERIOR.

BUREAU OF GOVERNMENT LABORATORIES.

BIOLOGICAL AND SERUM LABORATORIES.

3 1/2*. //J

REPORT ON BACILLUS VIOLACEOUS MANM:
A

By Paul G. Woolley, M. D.

MANILA:

BUREAU OF PUBLIC PRINTING.
1904.

18108

LETTERS OF TRANSMITTAL.

Department of the Interior,
Bureau of Government Laboratories,
Office of the Superintendent of Laboratories,

Manila, P. I., May IS, 190Jf.
Sir : I have the honor to transmit herewith a "Report on Bacillus
Violaceus Manilas, a Pathogenic Microorganism," by Dr. Paul G.
Woolley, Assistant Director of the Serum Laboratory.
I am, very respectfully,

Paul C. Freer,
Superintendent Government Laboratories.
Hon. Dean C. Worcester,

Secretary of the Interior, Manila, P. I.

Department of the Interior,
Bureau of Government Laboratories,
Biological Laboratory,
Manila, P. L, May 12, 190k-
Sir: I have the honor to submit herewith and recommend for
publication a "Eeport on Bacillus Violaceus Manilas, a Pathogenic
Microorganism," by Dr. Paul G. Woolley, at present Assistant
Director of the Serum Laboratory.
Very respectfully,

Richard P. Strong,
Director Biological Laboratory.
Dr. Paul C. Freer,

Superintendent Bureau of Government Laboratories,

Manila, P. I.
3

REPORT ON BACILLUS VIOLACEUS MANILA: A PATHOGENIC
MICROORGANISM.

By Paul G. Wooixey, M. D..
Assistant Director Serum Laboratory.

I can find no account in literature of a pathogenic organism
similar to the one of which a description follows, but there are
one or two bacilli which resemble it so closely in morphologic and
cultural characteristics that it seems wisest to consider the present
germ as simply a variety of these well known but at least usually
nonpathogenic forms.

The group to which reference is made is the one, the individual
members of which are discussed collectively by Migula (System der
Bakterien, 1900, II, 939) under the title of Pseudomonas violacea,
and which are separately described by other authors as B. violaceus,
B. violaceus berolinensis, B. violaceus lutetiensisyB. violaceus lau-
rentius, etc.

The organisms are described by these authors as bacilli, which
are short or of medium length, sometimes somewhat bent, and with
rounded ends. The measurements are about 1 by 0. 65 /*, although
some individuals may attain a length of 3 m. In cultures on agar
and potato, formations much resembling spores, but which are like
these reproductive bodies in appearance only, are seen within the
individual rods. These pseudospores are considered to be either
plasma globules or vacuoles, and true spores have not been demon-
strated. The rods are motile — sometimes, as in cultures kept at
body temperature, quite actively so. They possess a single polar
flagellum. Deep colonies in gelatin have an irregular, rounded
appearance, with projecting filaments. On the surface the colonies
form small depressions, in which blue-gray masses of bacilli are
seen. The color gradually becomes more intense, and the strag-
gling filaments grow out into the surrounding medium. On agar
the growth is blackish violet. Blood serum is liquefied — pepton-
ized. On potato the growth spreads over the surface of the

5

medium and forms a rich pigment, which does not stain the whole
fragment of the vegetable but only that portion immediately beneath
the growth. Broth is clouded, and a dirty white sediment falls to
the bottom of the tube and takes on a pale violet color. No pellicle
is formed on the surface. The organisms grow fast at room tem-
perature, but more slowly than most water bacteria, and they do
not grow beneath the isinglass. They are most readily stained
with hot carbol-fuchsin, and are decolorized by Grain's method.

B. violaceus manilce corresponds with the above description in
some details, but in some it does not. The organism is a short
rod, measuring approximately 0.5 by 1 to 1.5 m. Occasional rods
are even longer. It stains with the usual aniline dyes, best per-
haps with carbol-thionin, or with carbol-fuchsin or gentian-violet
diluted five times with water. It is not stained by Grain's method,
and is not "acid fast." When well stained by any of the above-
mentioned solutions, it appears as a short, thin bacillus, very fre-
quently slightly bent, and with rounded ends. It does not, as a
rule, stain uniformly, and may show one or more clear sjDaces
which are not tinted and which appear remarkably like spores.
These clear spaces can not be stained by the usual methods used in
coloring spores. The organisms are generally single, sometimes
in pairs, occasionally arranged in short chains of three or four
individuals. They are motile, usually sluggishly turning and
twisting, but frequently single rods may be seen to cross the field
of the microscope with a more rapid, wriggling motion. Each
organism possesses one polar flagellum. In rare instances two
flagella may be distinguished springing from the same pole of the
bacillus.

On agar plates, within twenty-four hours at 37° C, colonies
appear as small, round, violet-gray spots. These slowly enlarge
and become deeper in color. The maximum depth of color is
attained in from forty-eight to seventy-two hours. As the colonies
grow, their margins become slightly irregular and with a rather
indefinite concentric arrangement of layers, which are somewhat
thicker toward their peripheries, with the result that the centers
of the colonies have a less intense blue color than the edges. These
masses of organisms are slighty tenacious, but after removal from
the medium are readily dissociated in water. The growth on agar
slants is similar to that on plates, extension being slow but rather
even, resulting in a very slight crenation. The water of conden-

sation is clouded and bluish, with a blue sediment. In stab cul-
tures in agar the growth is scant, and no pigment is formed below
the surface.

Gelatin is liquefied slowly at the temperature of the ice box, the
growth forming a funnel-shaped area with a cup-shaped upper por-
tion. The sediment is bluish.

Bouillon is diffusely clouded. A delicate pellicle is formed,
which, upon 1 per cent alkaline (to phcnolphthaline) material, is
but slightly pigmented, but which is better developed and bluer
on a 1 per cent acid liquid. The sediment in 1 per cent acid bouillon
is pale violet blue and rather viscid.

Dunham's peptone solution is also clouded, and a thin, pigmented
pellicle is produced. A sediment is deposited in this medium which
resembles that in broth but in which more pigment is present. The
whole medium becomes diffusely bluish within forty-eight hours.

On potato, the growth extends over the whole surface of the
medium and to the water of condensation. The pigment produc-
tion is luxuriant. The superficial layers of all the solid media
are stained by the soluble blue pigment. In sugar containing
media — glucose and lactose — no gas is formed.

The reaction of milk, after seven days, is slightly acid, but there
is no coagulation. However, the casein is peptonized, and the
upper third of the tube becomes almost clear and faintly violet.

The organism thrives better and produces pigment somewhat
more freely at 37° C, than at lower temperatures. It does not
grow at all well and does not produce any pigment below the surface
of a solid medium. It is almost an obligatory aerob. It is killed
by boiling for one minute, by a temperature of 67° C. after an
exposure of five minutes, and by a temperature of 57° C. after one
hour. It does not form spores.

The pigment is soluble in alcohol, giving a deep, rich, violet
solution. It is slightly soluble in water, barely .dissolved by ether,
and insoluble in chloroform.

The organism described above has been isolated from three cara-
baos, which died suddenly with such noticeable absence of clinical
symtoms that acute hemorrhagic septicaemia was suspected. In
two of these cases Dr. Jobling obtained cultures of this organism
from the lymph glands and lungs, and in the third case the writer
found it predominating in cultures from the same organs. Later
on, in the press of work, the cultures from the first cases were lost,

/

and only those from the third were used in the following investi-
gation. However, Dr. Jobling had injected his organism into
guinea pigs, producing the same kind of lesions which I shall
describe. The details of the first autopsies are meager. Little can
be said, except that the prescapular glands resembled those to be
described in connection with the third case.

Like the first ones, the third animal died suddenly. It had had
no rise in temperature, as far as the records of the corral show, and
symptoms of rinderpest were absent. On removing the hide none
of the gross lesions of hemorrhagic septicaemia or surra, were
encountered, neither hemorrhages nor cedemas. The prescapular
glands were enlarged and intensely injected, but neither showed
true hemorrhages nor necrotic areas. There were a few small
peteeehise under the visceral pericardium and under the endocar-
dium of the left ventricle. The lungs were not collapsed, but
were for the most part crepitant. The surfaces of these organs
were dotted with small, pale areas varying in size from that of
a pinhead to a small hazelnut. These areas were firm and
projected slightly above the surface of the pleura. They were
not round, but irregularly stellate, and each was surrounded by
an appreciable zone of congestion. On section they appeared
granular and gray, and with no indication of caseation or suppura-
tion. Other noncrepitant areas which occurred chiefly along the
anterior margins of the lungs had much the appearance of red
infarcts. They were dark red and raised above the general surface
of the organ in which they occurred. On section they were dark
and moist, simulating the stage of red hepatization in pneumonia.
The lungs on section were generally pale and cedematous, and in
their substance contained large numbers of the small miliary
nodules similar to those seen upon their surface. The spleen
appeared to be normal. The liver showed no macroscopic changes.
The gall bladder was somewhat larger than normal, but the stomach
shoAved no pathologic lesions visible to the naked eye, nor were any
found in the trachea, pharynx, or tonsils. The kidneys were per-
haps a trifle pale. The intestinal tract was normal.

Smears from the prescapular glands showed a few very small
organisms, which appeared as diplococci or polar-stained bacilli,
and a large number of somewhat larger rods ; some of these irregu-
larly stained, and others curved. Cover-glass preparations made

from the nodules of the lungs showed almost nothing but small
bacilli, which stained unevenly. Smears from the heart showed
no organisms.

Cultures from the blood gave no growth after some days, and
those from the lung lesions produced a few colonies of a large thick
bacillus, resembling B. subtilis, and other colonies in very much
larger numbers (in one plate, these latter only), which developed
a blue color after twenty-four hours at 37° C. and which were com-
posed of the organisms which have been described. In cultures
from the prescapular glands two organisms were demonstrated.
One was identical with the chromogenic one isolated from the
lungs ; the other, a small coccus, arranged mostly in clumps, and
which, after several days, produced a very faint yellow color. • Both
of these organisms — the coccus and the chromogenic bacillus — -were
studied carefully. One was in all probability a modified Staphylo-
coccus aureus, which produced a minimum of pigment, coagulated
milk very slowly with coincident reduction of the litmus, and which
did not kill guinea pigs in reasonable doses, either when injected
into the peritoneal cavity, or under the skin. The other has been
described in its cultural and morphological character, and corre-
sponds quite closely with Bacillus violaceus (Schroter), or Pseudo-
monas violacea (Migula). In none of the books to which we have
access have I been able to find any reference to the pathogenicity of
Bacillus violaceus. This makes the following experimental study
of some interest:

One cubic centimeter of a forty-eight-hour-old culture from the
lung of the carabao (No. 431) was injected under the skin of a
guinea pig (No. 329). The animal became quite ill within the
next twenty-four hours and a large semifluctuating mass ajipeared
at the site of the inoculation. Coincidently the temperature, which
previously had been running between 36.5° and 38.4° C, rose to
39.8° C. and then dropped gradually until the animal died. Death
occurred on the fifth day after infection.

At autopsy a large area of necrosis was found under the skin
about the point of inoculation. This was surrounded, by tissue
in a state of coagulation necrosis, in which were occasional lacuna?
filled with a dark, gelatinous material. There was no true pus
and no hemorrhages appeared, although the adjacent tissues showed
extensive congestion. The peritoneal cavity contained a small
amount of a clear fluid. The lungs were the seat of large and small

10

hemorrhagic infarcts, the lower lobes of- both sides being almost
completely infarcted. Tbe spleen was markedly enlarged, was very
dark, soft, and mottled with miliary gray spots, resembling focal
necroses. The liver was also thickly studded with similar areas.
There were no macroscopic lesions in the heart or kidneys, though
the latter were pale. The adrenals were markedly enlarged, their
medulla? congested, and with small areas of necrosis in the cor-
tices. The lymph glands of the axilla? and groins were enlarged
and injected. Nothing abnormal Avas remarked in the intestines,
stomach, or bladder.

Smears were made from the subcutaneous tissues at the site of
the primary lesions from the lungs, spleen, liver, lymph glands,
and heart. All save those from the heart showed numbers of rods
morphologically identical with those which had been injected. The
organism was recovered in pure cultures from the liver, peritoneal
cavity, lungs, the primary lesion, and the heart.

A second guinea }3ig (No. 458) was inoculated intraperitoneally
with one-half of a cubic centimeter of a bouillon culture obtained
from the carabao. It survived the operation.

From the cultures obtained from the first guinea pig a second
(No. 327) was inoculated with one-half cubic centimeter of an
emulsion made with three loopfuls of a twenty-four-hour-old agar
culture in 5 cubic centimeters of a normal salt solution. The ani-
mal died on the third day after inoculation. The anatomical pic-
ture was the same as that in the first guinea pig, except that in the
lungs small miliary necroses were present in place of the infarcts
noticed before. Pure cultures of the experimental organisms were
obtained from the heart, liver, and the subcutaneous lesions.

With 1 cubic centimeter of an emulsion made with three loopfuls
of the organisms obtained from this last animal and 5 cubic centi-
meters of salt solution, a rabbit (No. 474) was inoculated subder-
mally. It died within thirty-six hours. Autopsy showed a bloody
fluid in the peritoneal cavity, a widespread hemorrhagic lesion at
the site of inoculation with no suppuration, but with necroses in
the liver. The organisms were recovered in pure culture from the
peritoneal cavity, liver, and heart. At the same time a guinea pig,
inoculated with an equal amount of the same material, died within
twenty hours. The only macroscopic lesions were miliary abscesses
of the liver. Cultures were obtained from the heart, liver, and
peritoneal cavity.

11

A cat (No. 366) was inoculated under the skin of the belly with
1 cubic centimeter of an agar suspension of a culture obtained from
guinea pig No. 327. The succeeding day a large semi fluctuant
mass surrounded the point of inoculation. The second day after
infection this abscess was discharging externally by a sinus, the
edges of which were ragged and about which the skin was semi-
necrotic. Eventually all the skin about this first sinus sloughed,
leaving an ulcer measuring 5 by 5 centimeters, whose base was on
the subjacent muscles and whose edges were regular, indurated, and
undermined. This ulcer gradually healed, and the animal showed
a complete skin on the thirtieth day after inoculation. AYhen re-
covery had been established, a. second dose of a suspension of the
organisms from Xo. 47-1 was introduced hypodermically. No
lesion was produced. The serum from this animal taken at this
time agglutinated the specific organisms in a dilution of 1 to 60
after about forty minutes, in dilution of 1 to 200 after about one
hour and fifteen minutes.

Five rabbits were inoculated with different amounts of the organ-
isms to show, if possible, what variations occurred in the lesions.
The first (No. 454) received one-half loopful; the second (No.
455), one; the third (No. 436), two; the fourth (No. 435), three,
and the fifth (No. 433), four, of a culture obtained from rabbit
No. 474. All of these animals died — Nos. 454, 455, and 436 in
three days, No. 435 in four days, and No. 433 in five days after
inoculation. From all, the organism was recovered from the tissues
and heart's blood, except in two cases (No. 435 and 436), in which
the cultures from the heart were negative.

The progressive changes in the organs of these animals were
noticeable and interesting. In animal No. 454 the subcutaneous
jelly-like oedema was present; there were miliary abscesses in the
liver, none in the spleen, none in the lungs, which were simply in-
jected. In animal No. 455 there were a few nodules in the lungs
and in the liver; the other organs appeared like those in animal
No. 454. In animal No. 436 there were large abscesses in the
lungs, and but a few small ones in the liver. Animal No. 435
showed similar lesions. In No. 433 there were a few very small
abscesses in the liver and spleen, while the lungs were generally
consolidated, showing comparatively large areas composed of collec-
tions of miliary abscesses. In other words, the lesions varied as
the dose of the organisms, and while with small doses the liver was

12

more prone to be affected, with large ones the lungs were more
prominently diseased. This may or may not be true for other
animal species. At any rate, the principal feature of the patho-
genic action of the bacillus is its necrotizing power.

A dog (No. 515) was inoculated subcutaneously with 1 cubic
centimeter of an agar suspension of a culture from No. 474. Sev-
eral days later a local lesion appeared, which resembled that pro-
duced in the cat, but which was less extensive and healed more
rapidly. About three weeks afterwards this dog received 1 cubic
centimeter of similar material in the femoral vein. Following this
injection the animal was irritable, eating but little for a few days.
On the third day, and continuing for four days, tremors were
noticed in the head and limbs, the animal lying quietly without
seeming to suffer. He subsequently became entirely well.

In the case of another dog (No. 516), in which the organisms
were introduced into the trachea, no illness followed.

A calf was inoculated in the dewlap with 2 cubic centimeters of
the same material which was used with the clogs. The day follow-
ing inoculation there was a large, painful, cedematous mass visible
in the region of infection. This enlarged until it reached the
size of a large fist, and then gradually disappeared.

Inoculation into the peritoneal cavity of 1 cubic centimeter of a
forty-eight-hour-olcl agar culture suspended in salt solution caused
the death of a rabbit (No. 492) within twelve hours. At autopsy
there was nothing to be seen except evidence of an acute peritonitis.
Cultures from heart and peritoneal cavit}'' were positive and pure.

Intravenous inoculation of one-third of a cubic centimeter of a
forty-eight-hour-old agar suspension killed a rabbit (No. 490)
within ten hours. No lesions were visible to the naked eye.
Cultures made from the heart, peritoneal cavity, and pleura showed
the organisms in uncontaminated growths.

Feeding experiments were negative. Large quantities of viru-
lent agar cultures were fed to two monkeys (Nos. 488 and 489)
with no ill effects.

Experiments upon serum reactions were begun with a monkey
(No. 468), which had received a dose of 1 cubic centimeter of an
emulsion made from a twenty-four-hour-old agar culture of the
organisms isolated from the carabao. The monkey did not suc-
cumb to this first subcutaneous injection, and four days later a
second one was made with !-£■ cubic centimeters of an emulsion

13

made with a twenty-four hour culture of the organisms isolated
from the second guinea pig (No. 327). The day following this
second injection a small quantity of blood was withdrawn and its
agglutinative powers tested. In dilutions of 1 to 20 a complete
agglutination was present at the end of fifteen minutes. In 1 to
40 a complete reaction was given in half an hour. In a dilution
of 1 to 200 the result was positive and complete in one hour.

Later this animal was inoculated subcutaneously with 1^ cubic
centimeters of an agar suspension made with a culture of the
organisms recovered from animal No. 474. Two days afterwards
a large slough appeared on the abdomen about the point of inocula-
tion, and the animal was very ill. It was killed and the blood
drained off into a sterile tube. The serum obtained from this
blood was tested for its agglutinative and bactericidal powers. In
a dilution of 1 to 200 agglutination was complete in twenty min-
utes, 1 to 400 in thirty minutes, 1 to 600 in forty-five minutes, 1 to
1,000 in fifty minutes, 1 to 2,000 incomplete after one hour, 1 to
4,000 was negative.

This experiment was repeated in small tubes, so that the reaction
could be watched with the naked eyes. The tubes were allowed to
stand overnight, The next day all the organisms were contained
in a precipitate, except in the control tube in which the liquid was
still cloudy. Cultures made from all these tubes gave luxuriant
growths. No appreciable bactericidal action was present.

To determine whether or not a soluble toxin was produced, a
four-clay-old culture in peptone was filtered through a Pasteur-
Chamberland bougie F. The filtrate was kept at 37° C. overnight
and no growth occurred. The next day 5 cubic centimeters of the
material was injected under the skin of a monkey (No. 509). Fol-
lowing this there was no sign of toxamea, no rise of temperature,
nor any sign of altered health in the animal.

After it had been proved that even very small amounts of the
living organisms would cause death of small animals and at the
same time produce the specific lesions without the appearance of
any appreciable immunity, cultures were heated to 57° C. for one
hour, and these dead cultures, suspended in normal salt solution,
were used for injection.

This material was used subcutaneously in a guinea pig (No. 486)
and a monkey (No. 411). In the case of the monkey one cubic
centimeter of an agar suspension was used for the first injection.

14

There was no appearance of a reaction until the fifth day, when the
temperature rose one degree above the normal one for the animal.
The day following this reaction a second injection of the same
amount of the same material was given. On the fourth day follow-
ing, the blood was tested for its agglutinating powers. Complete
agglutination was accomplished in dilutions of 1 to 400 in fifteen
minutes. A third inoculation was then made with 0.75 cubic centi-
meter (intraperitoneal) and 0.75 cubic centimeter (subcutaneous)
of the same material which had been used for the other inoculations,
and four days later a fourth inoculation was made with 2-| cubic
centimeters subcutaneously. Two days after the last inoculation
the monkey was found dead. At autopsy there was a small pocket
of a bluish material at the site of the last inoculation. Cultures
from this lesion, from the heart, liver, and spleen were entirely
negative, and the media was perfectly sterile after five days. The
serum taken from the heart at autopsy gave a perfect agglutination
in a dilution of 1 to 1,000 in forty minutes. The guinea pig suf-
fered no harm from the inoculations.

Later, using cultures of the organisms from animal No. 474, and
which had been killed by heating to 70° C. for forty-six minutes,
a dog was inoculated with a series of injections. He remained well
throughout the time. However, after the third inoculation his
blood agglutinated in a dilution of 1 to 20 only, after one hour.

The essential lesions produced in experimental animals are found
at the site of inoculation, in the lungs, liver, lymph glands, and
spleen.

At the site of inoculation, when infection has been caused sub-
cutaneously, there is a wide area of necrosis with local and circum-
ambient oedema resembling the lesions produced by the diphtheria
bacillus. The whole area may undergo necrosis, become gangren-
ous, and slough away.

In the rabbits the oedema has been more marked than the ne-
crosis. In the cat the necrosis occurred with but little oedema.
The monkeys showed oedema as well as necrotic and gangrenous
processes, as did the guinea pigs.

The lesions in the parenchymatous organs are miliary abscesses,
which may show a suppurative stage. In the lungs and liver the
surface of these abscesses may be covered with a fibrinous exudate.
The bacilli are found in all the lesions.

The losses in stock due to infection with B. violaceus manilce are

15

not extensive, and there seems to be little clanger of an epidemic.
In all our work we have seen but these three cases. Each was in
a different herd, and these herds were widely separated from each
other.

So far as treatment is concerned there is little to be said. All
the animals have died so suddenly and unexpectedly that there
was no time either for experiment or speculation on this subject.

BOSTON PUBLIC LIBRARY

AJ>999 06724 919 1

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