Part 55

Thigh striped Wallaby (_Macropus thetidis_). Streptothricosis of soft tissues of jaw. Early bronchopneumonia. Acute fermentative gastritis. Acute general infiltrative enteritis. Cloudy swelling of myocardium. The general condition of coat and of nutrition is good. The jaws are wide and the maxillocervical region full, both due to an indurative inflammation of the gums, tongue, floor of mouth and upper cervical tissues. At either side of the tongue and running around body of maxilla both sides, the inflammatory tissue becomes softer and there is an area about one inch long where it is soft, gray and contains yellow gray bodies in a grumous matrix. The teeth seem sound as do the external buccal tissues. The nasopharynx is free from induration. The bone on the left side shows a periosteitis with involvement of the superficial layers of bone, while on the right side the periosteum is swollen and opaque but the bone is free. The thyroid is imbedded in the edematous infiltration of the lower cervical tissues. The pleuræ are free of fluid and adhesions. Lungs are collapsed, uniformly pink somewhat emphysematous at places but give the impression of being lumpy. On palpation numerous nodular areas are detected. These prove to be peribronchial areas of gray-red solidity which swell out on section. The bronchus contains a gray and bloody thick mucoid matter. There is distention of the mesenteric vessels especially near the enteric insertion. The liver surface is smooth, edges very sharp, consistency firm, tough, resilient, color deep red, the section surface is glistening, moist, opaque, architecture probably normal. The gall-bladder is distended with viscid green bile; the common duct is patulous. The spleen has a rough, thin capsule, consistency tough and resilient, the section surface is mottled red with purple points; on section two small, pale objects seem to be squeezed out. The kidney capsule is smooth, strips easily leaving a smooth, deep red surface, the consistency is soft, the cortex is deep red, then a purple line between it and the red medulla, striæ invisible. The stomach contains frothy grayish mush. The mucosa is finely mammillated, deep pink until the last third when it becomes deep red, deeply injected and somewhat thickened. The pylorus is closed. Externally the gut is congested, in places translucent but for most part seems thickened by reddish swelling of both external layers and mucosa. The mucosa is granular or pebbly with here and there a small bloody suffusion. The histological section of lung shows alveoli open, septa relatively thin but somewhat congested, bronchi mostly open and connective tissue not increased. Some few bronchi, especially the larger, show a slight catarrhal bronchitis but mostly an infiltrative peribronchitis. The nearby veins and arteries show the most striking change, there being in nearly all of them a distinct thrombosis without circumferential pneumonia. In one place a distinct peribronchial pneumonitis was found. The kidney shows very marked congestion of all parts, causing compression, cloudiness and granularity of the epithelium. Glomeruli and connective tissue about normal. The intestinal serosa is negative save for congestion. Submucosa is densely infiltrated with mononuclears, some in definite groups. Section does not show areas mentioned in notes but these could be accumulations of cells with congestion. No streptothrix forms. Section from the infectious focus of face consists of active granulation tissue, densely injected and filled with mononuclears of two types, one the lymphoid cell, the other of the young connective tissue type. Areas of grouping like abscesses are seen and some necroses. Streptothrix in small numbers in the cellular collections.

Nail tailed Wallaby (_Macropus unguifer_). Kangaroo disease of nasal region. Necrotizing bronchopneumonia (Aspergillus fumigatus and Micrococcus albus). Acute diffuse splenitis. Congestion of liver and kidney. The general condition of coat and nutrition is good. The face is wide just below the eyes. About the “hare lip” and the nose the soft tissues are soft, gray, necrotic. All the internal nasal tissues seem swollen, gray-red. There is subcutaneous edema, bloody in places, around the right face, eye and jugular angle. Tissues of nasopharynx swollen, deeply injected and covered by a thick mucus. Pharyngeal and buccal cavities negative. Tonsillar areas pink and flat. Larynx and trachea slightly swollen but pale on mucosa. Salivary glands and cervical glands normal in size and pale pink. Pleuræ pale and empty. Lungs swollen out uniformly, quite cottony except at lower right base where there is a nodule about 3 × 5 cm. firm and doughy. On section it is found to be a peribronchial consolidation of pale reddish gray color and indefinite outline. The bronchus itself is deeply congested and contains a grumous mass. The peribronchial lymph nodes are small, soft, pink, homogeneous. The heart is negative. The liver is large, surface smooth, edges sharp, color deep purple, consistency soft. Section surface is glistening, smooth, moist, very dark purple with obscure markings. The gall-bladder contains fluid brown bile; common duct is patulous. The spleen is soft, tough, capsule pebbly, section surface is mottled, light and deep pink, follicles and trabeculæ not distinguished. The kidney capsule is smooth, strips easily leaving a smooth purple surface, section surface is glistening, deeply congested, striæ obscure but seem normal, glomeruli not visible, organ is soft. The gums and teeth are not involved in the mycosis. The stomach contains mushy digesting food. The mucosa is mottled pink, soft, digesting, at lower half submucosa is deep pink, a few small ecchymoses. From pylorus to ileum, serosa is deeply injected, edematous, mucosa swollen and edematous, deep pink, loosened in places, but translucent. Below this the mucous membrane becomes smooth, flat, pink-yellow. Lower ileum and colon contain rather firm fecal balls. Follicles nowhere prominent. The pancreas is small, soft, yellow pink. The follicles of the mesentery are small, pink gray and homogeneous. Smears from the bronchopneumonia show a threadlike Gram- positive form and a few Gram-negative rods. Cultures from lung show _Aspergillus fumigatus_ and _Micrococcus albus_. Nose too foul for culture. Histological section of lung shows the alveoli mostly open but the septa widened by congestion. Blood vessels are open and contain recent clots; one vessel near lesion below is thrombotic. The two large bronchi in section show catarrhal bronchitis and infiltrative peribronchitis of which the latter is more severe and advanced. Beside the larger is a necrotizing pneumonitis from which nearly all the architecture has disappeared. The exudate is chiefly mononuclear around the edges; centre no cellular identity. Another mononuclear process not connected with bronchus in section is found with an early necrosis. Streptothrix strands may be found in the bronchial exudate and near the margin of the necrotic patch. They do not grow in colonies however. The spleen shows general congestion without pigmentation. Follicles large, solidly lymphoid. Connective tissue about normal. The kidneys show marked congestion everywhere. Capsule and intrarenal fibrous tissues about normal. Very severe congestion which seems to have caused compression and granularity of the epithelium.

The last case, judging by stained smears, is one of pure nasosinusitis from streptococci and streptothrices. Cultures were not tried because of the enormous bacterial flora.

Robust Kangaroo (_Macropus robustus_). Acute purulent ethmoiditis. General acute purulent anterior cranial sinusitis. Acute necrotizing glossitis and pharyngitis. Cloudy swelling of kidney. The face seems a little full and the subcutaneous tissues slightly edematous. The nasopharynx contains a thick tenacious mucopus. Ethmoid and frontal sinuses and turbinate spaces contain a thick purulent matter, the mucosa being densely injected, swollen and velvety. Pharyngeal wall and right half of posterior half of tongue are involved in a dull brown and necrotizing process, quite sharply outlined by zone of congestion. This process is comparable to the necrotizing gingivitis seen in front of jaw in kangaroos. Larynx, trachea and lungs seem uninvolved save for slight generalized congestion. Cervical lymph nodes especially those about the larynx are definitely enlarged, soft, moist and brown. Mediastinal nodes slightly enlarged, soft and pink. The heart is negative. Liver normal. Spleen is soft, homogeneous dull red. The capsule of the kidney is smooth, strips easily leaving a purple surface. The glistening section surface swells slightly, vasa recta are congested, striæ wide and pale, glomeruli not visible; consistency is resilient. The mouth and teeth are not involved in the process mentioned above. There is a small quantity of properly digesting food in the stomach. Stomach and intestines negative. Brain not involved. No extension from anterior cranial sinusitis. Smears from the mucopus confirm the gross appearance and contain short chains of streptococci and large diplococci. Smear from cut surface of tongue shows innumerable small bacilli and diplococci but especially mycelia with rather heavy clubbed ends but without true branching. One group was found arranged like ray fungus. It is noteworthy that there is no aspiration pneumonia and very slight evidences of septicemia.

BIOLOGY OF N. MACROPODIDARUM.

The original discovery of the streptothrical forms was made in stained smears from necrotizing lesions. They were considered as secondary invaders until repeated observations of a similar character aroused the suspicion that they stood in some important relationship to the lesion. Early attempts at their cultivation were made under anaerobic precautions, a method now known to be almost certainly doomed to failure because a strain long under cultivation requires two to three weeks to make an appreciable growth in the absence of air. Finally in 1911 a successful cultivation occurred by searing the surface of an unopened mass in a freshly dead animal and planting bits of the interior upon aerobic blood serum plates. Colonies grew after three or four days and from them the first strain was started. It grew for several generations, long enough for the preparation of a vaccine, which will be described later, when by mischance it was lost. In 1920 another successful cultivation occurred, this time by incising a mass in the soft sublingual tissue and plating in the same manner; upon this culture the biology is described. Smear preparations offer no more than has already been mentioned.

Colonies develop upon blood serum plates as opaque, pale yellow, circular, discrete masses with a slightly depressed uneven centre, but without umbilication. They remain smooth and slightly glistening for several days, then become slightly wrinkled and twisted with a more definitely raised edge and a tendency to an uneven sinking in of the centre. Transfers to agar slants show wrinkled continuous opaque, dull yellow, sharply outlined growths which soon wrinkle, fold, and twist like certain tubercle bacillus cultures. Spreading occurs, but is slow after forty-eight hours. As medium becomes drier it is possible to see a thin, colorless, wrinkled film stretching out from the main growth. If the medium be dry or old or if only a small portion of seed material be used and this scattered over the surface of the slant, discrete colonies arise. These are circular, seldom exceeding 3 mm., dirty yellow-white, distinctly umbilicated and without clear film of spreading around them.

In nearly all quite old cultures, a white chalky efflorescence appears over the surface.

The morphology of the young agar culture is chiefly mycelial or filamentous, whereas from a culture on dried media and those showing efflorescence, the organisms are short, heavy, deeply granular and of the mycobacterial type.

Glycerine agar.—Corresponds to agar.

Blood agar.—Similar to agar but much less luxuriant.

Blood serum.—Limited dirty yellow, raised, dull, wrinkled and granular, tightly adherent to the medium.

Potato.—Spreading, dirty yellow, much wrinkled, friable, tightly adherent.

Gelatine.—Limited growth as a wrinkled, tough scum only on surface.

Litmus milk.—No change for six days, then beginning slight alkalinity which increases very little, shows digestion of the caseinogen, slight, thin filmy growth on surface.

On media such as litmus lactose agar and old Endo it grows slowly on surface and assumes the color of the medium.

Broth.—Only surface growth appearing during early generations, after 3–6 days as a wrinkled, pale yellow scum very much like the tubercle bacillus growth; later generations grow perceptibly in one to three days. Medium perfectly clear. If a large mass be seeded into neutral broth there is a perceptible increase in the growth after ten days. The medium thereafter tends to a faint turbidity. Titration of broth growth after twelve days shows alkalinity requiring 0.3 cc. decinormal acid, while the control tube incubated same length of time showed an acidity requiring 0.57 cc. of decinormal NaOH.

On the following sugars there is a slight surface growth without change in the color, Andrade indicator—dextrose, lactose, saccharose, maltose, mannite, dextrin, galactose, salicin.

Cultures observed on two per cent. neutral agar.

A.—Stained by Loeffler’s stain.

Twenty-four hours.—Shows threads growing out from a central amorphous mass, but the whole does not retain the regularity or parallelism of actinomyces. Threads are poorly stained and rather disconnected but not jointed. Small number of metachromatic bodies apparently in older individuals, certainly in the better formed ones. No intercalary spores, unless the metachromatic bodies be so considered. Individual threads measure from one-third to one micron in width. Metachromatic bodies measure on the average one micron.

The threads in the forty-eight hour preparation seem distinctly wider, up to one micron and possibly become heavier toward the end, but do not have a distinct bulbous extremity.

In three days the threads are much longer, show distinct branching and a tendency to transverse segmentation. More than one metachromatic body may be present in one segment.

Four days.—Still coarser, short segments have appeared separately. Metachromatic body is coarser and blacker; some of the masses have gone to pieces and show only a diffusely staining smudge of metachromatic bodies. The short segments show a tendency to grow out into threads.

Fifth day.—Condition is much the same plus many young, delicate, poorly staining threads.

Sixth day.—The same but all seem to be somewhat wider and diffusely staining.

Seventh day.—More diffuse staining and decidedly fewer metachromatic bodies.

A.—Stained by Gram’s stain.

Twenty-four hours.—All forms are light purplish. The threads stain much more clearly than by Loeffler’s and show distinct transverse segmentation of rather uniformly long bacilliform shape. Metachromatic bodies not so distinct but seem larger where found. Coarser threads have swellings in some of the areas which are not segmented and this type seems to have more branching and metachromatic bodies; in other words it would seem that this is a form that reproduces by budding or intercalary spore formation.

Forty-eight hours.—Much the same, more long threads with transverse division, somewhat more delicate, generally fewer coarse threads with swellings and spores. Still pale purple and not distinctly Gram- positive.

Three days.—Condition much the same.

Four days.—Two forms present—definitely Gram-negative delicate slender threads, nearly Gram-positive, and heavier, curved and twisted long bacillary forms, some streptococcoid threads and a few bulbous short threads. Very few metachromatic bodies.

Five days.—Condition much the same except that the delicate threads are inconspicuous and the darker purple bacilli have increased. Metachromatic bodies increased as have swellings in coarser threads.

Six days.—Much the same but for the appearance of young, delicate definitely Gram-negative threads. There are fewer metachromatic bodies and internal spores.

Seven days.—The same.

B.—Grown on Loeffler’s blood serum.—Loeffler’s stain.

Twenty-four hours.—Delicate, poorly stained short threads, few tiny metachromatic bodies.

Two days.—Not well stained, relatively short threads show numerous metachromatic bodies varying from exceedingly tiny dots to coarse granules wider than the thread. These may be numerous in the same segment and form a row from six to ten. Many short bacillary forms.

Three days.—Poorly stained, metachromatic bodies apparently more numerous but much smaller.

Four days.—Almost entirely short, heavy bacillary forms, some of which are very like diphtheria bacillus in the irregularity of width; many metachromatic bodies, distinct branching, some of the small heavy ones have fusiform swellings; practically no long, heavy threads.

Five days.—Essentially the same, individual elements slightly larger, fewer but coarser metachromatic bodies, more numerous round forms suggesting large pale cocci.

Six days.—Much the same but elements shorter, smaller and some more segmented.

Seven days.—More long forms of uniform staining but still a majority of coccoid or short bacillary forms with irregular staining and metachromatic bodies; no long threads.

B.—Gram’s stain.

Twenty-four hours.—Pale purple, almost Gram-negative, long, slender but well outlined threads, a few coccoid forms, practically no granules.

Two days.—Very pale, almost Gram-negative threads, very many coccoid forms and short rods, considerable segmentation of the longer threads.

Three days.—Increase in short, heavy bacillary forms with bulbous ends, deeply stained ones and the granules being lightly Gram- positive; long, slender threads are disappearing.

Four days.—Almost exclusively short, heavy forms with bulbous ends with coccoid forms, heavier forms almost definitely Gram-positive, granules Gram-positive.

Five days.—Much the same but more segmentation in the bacillary forms, coccoid forms become more numerous.

Six days.—Individuals are somewhat longer but there are many rods with fusiform swellings containing granules; coccoid forms present in chains sometimes.

Seven days.—More long rods or short threads, pure coccoid and bacillary forms.

The morphology upon bouillon depends somewhat on age and upon the location. Upon the surface the long branching mycelial type appears early and persists until the whole surface is covered whereupon the segments divide into coccoid elements with metachromatic bodies. If heaping-up develop the coarse grains on the mass consist of granular or coccoid rods. When growing in the depth the coccoid form is the predominant one, only a few delicate mycelia, usually Gram-negative, being found.

The Gram character of the organism should be emphasized. The young, delicate mycelia are negative or take a very feeble blue stain. The heavy bacillary forms are Gram-positive. Like the ray fungus the heavy ends are sharply Gram-positive, but unlike it, there has never been seen a Gram-negative bulbous capsule around this end.

The determination of this organism was undertaken from the classifications of Petruschky (Kolle-Wassermann), of Castellani in Castellani and Chalmers’ _Tropical Medicine_, and of the Society of American Bacteriologists. In the first classification it corresponds in some ways with _Streptothrix hominis_, and in some ways with _Streptothrix capræ_. As for the second authority it falls into the Nocardiaceæ, section parasitica, subsection I, in that a distinct earthy odor is absent and that there is no liquefaction of coagulated protein. It resembles several of the species given in this subsection, but does not correspond exactly with any of them. Consultation of the classification of the American Bacteriologists would place it among Mycobacteriaceæ. The facts that it is strongly aerobic, produces whitish efflorescence which may possibly be aerial hyphæ and breaks up into short segments, place it in the genus Nocardia. It seems, however, to belong to a division of Nocardia which is close to the Mycobacterium since the short elements are swollen, cuneate and usually heavy, which is unusual in the more typical Nocardia. It is not, however, acid fast and therefore cannot be classified among the Mycobacteria. This culture seems to be a variety not heretofore described, and since its association with the disease is so definite, whether or not it be the cause, the name NOCARDIA MACROPODIDARUM is proposed, because the kangaroos belong to the order Marsupialia, family Macropodidæ.

The discovery of these organisms within tissues is by no means easy even though the larger colonies may be located by staining. If Loeffler’s method be used the central mass stains quite diffusely and the spreading mycelia around the edge stain faintly. For study purposes this stain is preferable to Gram-Weigert, since despite the positivity of the cultures, the blue dye can be removed very easily from sections and only with great care will enough remain to permit tracing of the separate threads; with Gram stain no detail can be made out in the centre of the colony, it being a diffuse blue. Careful search near the edge of these necroses will usually succeed in the discovery of a few mycelia stretching in between the mono- and polynuclears of the low grade inflammation. This is best seen in the margin of gastric ulcers, but may also be found in the cervical masses. When searching in the pulmonary tissues the organisms are to be found in the bronchial exudate or at the edge of pneumonias. In one nasal mucosa the mycelia were dispersed, not growing in colonies as in localized inflammations.

EXPERIMENTS AT THE REPRODUCTION OF THE DISEASE.