ON THE PATHOLOGY AND PATHOGENESIS OF SPIROCHBTOSIS ICTEltOHBMOItRHAGICA.

By CARLO BASILE, D.T.M. (London). Libero Docente-University of Rome.

From the Bacteriological Department of the Lister Institute, Lo.ndo?z.

INTRODUCTION.

THE study of the clinical symptoms of spirochaetosis icterohzmorrhagica shows that this disease presents a different symptomatology in different (3888s. The impossibility of carrying out anatomo-pathological re- searches in all cases of human patients has led me to believe that the pathogenesis of the symptoms and the alterations in the tissues caused by this spirochste may be rendered intelligible by a study of the disease experimentally produced in the guinea-pig, which is a susceptible animal.

The term Xpiroch&osis icteroh~morrhagica (Weil’s disease) is based not only on historical grounds but also on the main clinical symptoms (jaundice and hamorrhage) caused by this spirochaete when it manifests its full pathogenic action in a susceptible organism.

Material and Methods. The virus of which I have made use in these researches was kindly placed at

my disposal by Dr Ledingham, to whom I am extremely grateful. This virus was exceedingly virulent, guinea-pigs inoculated intraperitoneally dying after five or six days, though a few of them lived as long as twenty days after inoculation. The guinea-pigs were dissected immediately after death. Some were killed with chloroform, in order that the changes induced by the spirochste might be followed approximately day by day. The organs were fixed in 10 per cent. formaldehyde, in alcohol, in Schaudinn’s fluid, and in Zenker. A portion of each was treated by the original Levaditi method, or by the Volpino modification, or again by the Volpino-Bertarelli method.

Course of the Disease in Guinea-pigs. Healthy guinea-pigs were inoculated in traperitoneally with the

blood from the heart of infected guinea-pigs ; the blood, as soon as it was drawn, was diluted in 5 c.om. of a 2 per cent. citrate of soda solution previously sterilised ; in some of the animal5 the inoculation was effected with a liver or lung emulsion.

The clinical side of spirochztosis ic terohaemorrhagica in guinea-pigs 5 2 JOUHN. O F PATH.-VOL. XXIV.

278 CARL0 BASZLE

has been fully dealt with by previous writers, the course of the fever, the changes in the blood picture, the hmnorrhages, the alburninuria, and the jaundice being the principal symptoms. As previous authors have observed, the clinical picture is not always complete; jaundice may be absent in experimentally infected guinea-pigs. I n my own work, which has been chiefly directed to the study of the pathology, I have almost constantly observed both in the serious and in the slight cases of experimental spirochztosis, the presence of hceernorrhages in the internal organs. In all niy investigations this lias been the most constant characteristic of the disease.

NAKED-EYE PATHOLOGY. In acute cases the naked-eye pathology is almost always the same. Jaundice shows itself in the skin of the regions without hair, and in

its internal surface. The lymphatic glands of the groin and the axilla are hypertrophied. The hsmorrhages are the leading characteristic in all cases, acute or slight; those in the skin, the muscles, and the internal tissues may be very diffused; when localised they niay be small, almost petechial, or they may be considerably larger. I have paid particular attention to the hzemorrhagic spots in the Icngs, which are characteristic; Inacla (1917 7 and Kaneko and Oknda (1911 lo)

have already described these lungs as having the appearance of the wings of a butterfly of the genus Vanessa. The hEmorrhage may attack the whole of a lobe of the lung. Sections show the hamorrhagic spots to be situated round the bronchi and in the vicinity of the pleura. Hmnorrhagic spots are also seen in the serous niembranes, in the stomach, in the intestine, in the region of the kidneys, and in the epiclidyinis and vas deferens of the male guinea-pig. These hrcmorrhagic spots seen with the naked eye show well-defined limits.

The liver to the naked eye showed nothing characteristic, and in the majority of cases its cut section was normal; in some guinea-pigs, however, I found hmiorrhagic spots.

The spleen in a few cases showed hypertrophy, but the cut section was of normal consistency.

The kidneys were in various stages of congestion, both of the cortex and the medulla ; the suprarenal capsules were congested.

HISTOLOGY. Lung.-Microscopically as well as to the naked eye the most constant and

most characteristic lesions found in the guinea-pig were in the lungs. The cutaneous and subcutaneous haemorrhages, the hariorrhages in the serous membrane of the peritoneum, hemorrhages in the stomach and intestines may be absent or unimportant, the changes in the liver and spleen may be slight if not negligible, but a constant feature was the hemorrhages of the lungs and pleura.

I think that the hamorrhagic state of the lungs, which may be very extensive,

SPZRO CHAZTOSIS ICTE ROHAZMOR RHA GZ CA 279

is the most frequent cause of death of the infected guinea-pigs. The hsemorrhages in the lungs may occupy all the pulmonary alveoli ; the smallest foci are formed of two or three pulmonary alveoli ; the most serious hemorrhagic foci are seen round the bronchi and may involve a large part of the pulmonary surface. All the blood-vessels are congested and full of red corpuscles.

The anatomical structure of the lung where there are no hzemorrhagic foci is usually well preserved. I n preparations of the tissues fixed in formalin or sublimate, and stained with hemotoxylin-eosin, or by the methods of Van Gieson, Hansen, and Twort, it is possible to recognise that some of the alveoli are distended and enlarged, and in these the alveolar wall is thin and may contain red corpuscles.

In the hemorrhagic foci the quantity of blood is so great that it is not possible to distinguish the alveolar structure. I n the foci, however, in which the process of reabsorption of the blood is going on, the structure is intact. In a guinea-pig which died seventeen days after inoculation 1 was able to distinguish the earlier hemorrhagic foci from those which were formed subsequently. The first were found round the large bronchi and the blood was there in process of reabsorption, so that the soundness of the pulmonary tissue was readily distinguished. The more recent hzemorrhagic foci were in the neighbourhood of the pleura. Alveoli, even those a t a distance from the hemorrhagic foci may contain red corpuscles, and may also show internally a granular deposit suggesting cedema.

The cells of the walls of the alveoli shorn a well-stained nucleus, and no change is appnren t in their protoplasm. Mononuclear cells containing phagocyted red corpuscles and polymorphonuclear leucocytes are occasionally seen.

In the epithelium of the blood capillaries mitotic figures inay be detected. The epithelium of the bronchi and of the bronchioles shows proliferation. The bronchioles may also contain blood.

In some guinea-pigs I have also noted well-developed lymphatic nodules and lymphoid infiltrates mixed with red corpuscles.

These phenomena of lymphocytic reaction were more obvious round the bronchi and bronchioles and the pleura.

I n all my preparations I have noted no conspicuous reaction of the connective tissue, which always appeared to me to be normal. The quantity of hzemosiderin waa in relation to the state of degeneration of the red corpuscles. I have not observed fatty degeneration.

Spirochsetes were very rare and there was no relation at all between the seventy of the lesions and the number of spirochetes observed in lung sections.

Liver.-The presence of jaundice in spirochzetosis icterohsmorrhagica has led students of this subject to turn their attention to the liver.

The observations hitherto published all agree in recognising that the changes in this organ are very varied. They differ in various patients, and are not in relation to the gravity of the jaundice. Garnier and Reilly (1917, 1918,27) have noted that serious jaundice may run its course without the discovery of important lesions of the hepatic cells, while in slight jaundice serious changes in these cells may be discovered.

My own observations in regard to the changes in the liver confirm thosc made by others. The liver is not an organ which invariably presents histological lesions in experimental spirochsetosis icterohaemorrhagica. I group my observa- tions in three series.

(a) In guinea-pigs which died from an acute form of the disease after five or six days, and in which the brain, lungs, and kidneys were highly congested, the liver appeared normal to the naked eye. Microscopically the congestion

280 CARL0 BASZLE

was not remarkable. The chief finding was a lymphoid reaction around the blood-vessels. The hepatic cells showed minute vacuoles, and there were many cells with two nuclei; the necrotic foci were minute and surrounded by lymphoid cells. Fatty degeneration was not evident, and the connective tissue was normal.

( 6 ) In other guinea-pigs which also died after five or six days from an acute experimental form of the disease, it was not possible to distinguish the lobules clearly. The cells partly retained their arrangement in columns. They were dissociated and were not swollen ; the cellular plasma showed minute vacuoles. The nuclei of the liver cells were generally well preserved ; some cells, however, showed two or three nuclei and other signs of degeneration with scattered granules of chromatin. The biliary capillaries were dilated, indicating bile stasis. The blood capillaries were full of red corpuscles and some poly- nuclear cells. Phagocytosis of red cells and polymorphonuclear corpuscles by macrophages was very prominent; these were rounded and might be of large size ; they may show a degenerating nucleus with scattered granules of chromatin.

Infiltration of lymphoid cells is also observable round the portal and arterial vessels but never in the central endolobular vessels. Again I have not noted fatty degeneration. Haemosiderin was present in proportion to the quantity of red corpuscles and showed itself massed in small groups.

The endolobular connective tissue, in preparations well stained by Hansen’s method, showed itself in the form of extremely fine fibres.

(c) I n guinea-pigs which died sixteen days after inoculation, the liver, to the naked eye, was normal. Microscopically the arrangement in columns of the hepatic cells was not invariable; the hepatic cells were swollen, and their protoplasm showed vacuoles ; the nuclei might be deeply stained or also swollen and showing signs of karyolysis. The necrotic foci were extensive and consisted of a residuum of cytoplasm in which there appeared remains of nuclei. The bile capillaries were dilated. The endolobular and perilobular connective tissue was very evident in the necrotic foci.

By the nitrate of silver method I was able to find in the liver spirochates usually isolated and extracellular. Very rarely they were in cells which were in process of degeneration.

KicZnqs.-The most interesting microscopic changes were observed in the convoluted tubules, the ascending loop of Henle, and in the glomeruli. These changes might indicate congestion or also necrosis. The cells which clothed the tubules were for the most part degenerated ; in severe cases they were completely necrotic, and the tubules were delimited by the connective tissue which appeared to be sound, and which enabled the renal structure to be recognised. In some of the tubules the degeneration was so great that the lumen of the tubules was full of residua of nuclei, of red globules, and of leucocytes ; these results which I have noted explain the presence of the characteristic cylinders which are found on examination of the urine in spirochstosis icterohsmorrhagica. I have observed also tubules containing hEmoglobin and red corpuscles. The blood vessels and the intertubular capillaries were congested. I have also found macrophages with included red corpuscles and leucocytes.

I often found them also in small groups.

Mitosis in the uriniferous tubules did not appear to me to be very marked. The changes in the glomeruli might be equally serious; the glomerular

capillaries were much congested, the cells showed degeneration, their cytoplasm was vacpolar or granular, and their nuclear chromatin was reduced to granules. I n the interior of the glomeruli also there were macrophages with red corpuscles and leucocytes. The endocapsular space showed itself in some glomeruli full of red corpuscles and leucocytes. The cells of the flattened Bowman’s capsule were unchanged, although red corpuscles might be found in their midst.

SPZROCflA?TOSZS ZCTEROHLEMORRHAG~CA 281

In some kidneys I also noticed perivascular infiltration. I observed also hsmosiderin not only in the tubules but also in the glomeruli.

The interstitial connective tissue, stained by Hansen’s method, was very plainly seen round the tubules and glomeruli.

In the different guinea-pigs which I examined I very rarely found spirochaetes in the kidneys ; when present they appeared to me to be chiefly localised in the lumina of the tubules.

Spleen.-Garnier and Reilly (1917-18 2-7, and Martin and Pettit (1919 11), have already called attention to the function of the spleen in cases of icterohaemor- rhagia ; the spleen is the principal seat of haematophagic reaction.

Monti (191613) was the first to recognise, on microscopic examination, that the lymphatic nodules of the spleen were frequently enlarged.

I have been able clearly to distinguish the enlargement of the lymphatic nodules. In the microscopic examination of the spleen I observed congestion, phagocytosis of red cells, and necrosis. When the nodules were surrounded by congested blood-vessels the phenomena of haxnatophagia were predominant ; each cell might contain twenty or more phagocyted red globules in various stages of degeneration. Around the lymphatic nodules were also to be found foci of necrosis formed of a granular substance containing red corpuscles and cellular residua.

The examination of preparations treated by the nitrate of silver method showed that the spirochztes are very rarely found in the spleen.

Suprarenal Capsules.--In my observations of these organs I have noted not only diffuse congestion but also a marked lymphocytic reaction.

Brain.-With the naked eye I have in all cases noted meningeal congestion. In fact a t the microscopical examination the blood-vessels in the meninges as in the brain substance properly so called were deeply congested. The lymphocytic infiltration did not appear to me to be remarkable. Spirochaetes were frequently to be found especially in the meninges.

Testicle and Epididymis. -The epididymis is the seat of a characteristic hsemorrhage; seen by the naked eye it is strongly haemorrhagic, and, as has been described by Martin and Pettit (191g11), the testicle would appear to possess a‘red cap.

On microscopical examination the tubular structure was found to be unaltered ; the intertubular blood capillaries were deeply congested ; in the intertubular spaces I have also noted a veritable collection of red corpuscles. The tubular epithelium was not much changed, and I have not observed any reaction in the connective tissue.

PATHOGENESIS OF TEE SYMPTOMS. The most important symptoms in man and in guinea-pigs are

fever, hamorrhage, and jaundice. The hsematological data are not without interest, and, in fact, ansemia may be noted clinically in the second period.

The fever corresponds to the cycle of the spirochste, or, more precisely, to the invasion of the organism.

Jaundice is not aIways present. Martin and Pettit classify the spirochaetoses in this respect as icteric and non-icteric. Between these two forms there is a whole series of steps.

The hsemorrhages are less variable; sometimes there is simple congestion of the vessels, and this was found in all the guinea-pigs

282 CARLO B A S ~ L E

experimentally infected, even in those which showed slight clinical symptoms. It is very probable they always occur also in man but that they are not always visible.

The jaundice and the hsmorrhages are the pathological data upon which is based all the symptomatology that is to be met with in spirochaetosis icterohsmorrhagica. The haernorrhages, which are at times exceptionally serious, do not appear to be determined by the direct action of the parasite ; there is no intimate connection, in fact, between the violence of the h%morrhage and the presence of spirochetes in the tissues.

In the lungs especially I have rarely been able to observe in the large hzmorrhagic foci well-preserved spirochaetes, which are also very rarely present in the spleen and liver even when these organs are profoundly hemorrhagic.

To explain this haemorrhage, therefore, we must imagine some influence of a toxic nature on the endothelial cells of the blood capillaries. From the investigations carried out up to the present it does not appear that the spirochstes produce an exotoxin. Martin and Pettit believe that the hemorrhages may be produced by an endotoxin contained in the body of the spiroch&e, which is liberated by the destruction of the spirochaete following phagocytosis by the cells of the organism; in fact it is not infrequently found that these phagocyted organisms break down and appear as granules enclosed in the cells. This phagocytosis would explain the disappearance of the spirochetes from the circulating blood and subsequently from the organs.

The degenerat,ion and death of the spirochete cause the liberation of toxic substances in the body of the cell, and owing to their action the cell is not long in showing phenomena of degeneration and necrosis. This condition which is repeated in the various organs leads us to think that it may be repeated also in the endothelial cells of the blood capillaries thus causing the hamorrhagic state.

The phagocytic reaction, upon which I particularly insist, helps not a little to explain the jaundice.

Indeed the jaundice shows itself in the second stage of the disease; in experimental spirochaetosis it is observed a few days before the animal dies. When the jaundice has appeared it is dificult to find spirochetes in the circulating blood-evidently they have been phago- cyted. Inada (191V9) has demonstrated that the infectivity of the blood in spirochaetosis decreases gradually until it finally disappears

* I n 1916, during my military service, I was able to follow the case of a soldier in hospital for jaundice. He had bloody sputum, very like the sputum in tuberculosis, suggesting a tuberculous infection in a jaundiced patient. For several successive days I made careful search for the tubercle hacillus, but without success; but later I found in preparations of the same sputum spirochetes morphologically identical with the spirocheta icterohsmorrhagica. This observation was described in a report on spirochatal jaundice presented to the Health Department of the Third Army Corps of the Italian Army in the field.

Haemoptysis is, in fact, well known.*

SPIR 0 CHAZ T O 5’2s IC T E R O H X M O R R H A G I C A 283

with the appearance of the jaundice. Garnier and Reilly (19172) proved that three days after the appearance of the jaundice not only inoculations with peripheral blood but also inocuiatioiis with fluid from the spinal cord of spirochztosis patients gave negative results in guinea-pigs.

In the majority of cases the jaundice follows the hzmorrhage; the phagocytic reaction destroys the spirochaetes whose poisons cause necrosis of the phagocytes, and hence also of the endothelial cells, thus causing congestion and hzmorrhage.

The red corpuscles are disintegrated and destroyed by phagocytes and by the action of the toxic substance set free by spirochaetes.

The greater part of the haemoglobin is transformed into bili- rubin, and I think that this transformation can take place not only in the liver but even in the blood-vessels themselves and in other organs. I do not know how otherwise to explain why in severe cases of jaundice the bile capillaries of the liver are not dilated and no other serious lesion is found in this organ, while in slight cases of the disease we have serious histopathological manifestations in the liver. That the hzmoglobin can be transformed into bile pigment not only in the liver but also in the peripheral circulation has been demonstrated by the experiments of Hooper and Whipple (191614), who have proved that hzmoglobin can be rapidly changed into bile pigment in the local circulation of the head, of the neck, of the thorax, and of the pleural and peritoneal cavities without the participation of the liver. These writers also consider it very probable that the endotheliuni and also the mesothelium (serous cavities) may be able to transform haemoglobin into bile pigment ; they also advance the hypothesis that this property may also exist in cellular protoplasm in general.

We may therefore hold the opinion that in spirochaetosis ictero- haemorrhagica the haemoglobin, produced by the marked phagocytic action and by the action of the toxic substance set free by spirochstes above described, may be converted into bile pigment without the assistance of the liver.

Garnier and Reilly (19175) (who were the first to observe the contrast between severe jaundice with the liver histologically almost normal, and slight jaundice with serious histopathologic manifestations in the liver, being supported by the fact recognised by themselves that bile in vitro has a lytic action on the spirochztes) have consideyed that the bile stasis, by injuring the vitality of the spirochztes, hinders their pathogenic action on the hepatic cells, and therefore that the slight change or the absence of change in the hepatic cells in severe cases of jaundice is in relation to the bile stasis, while in those cases in which the bile stasia is absent, the spirochztes can develop all their pathogenic action on the hepatic cells. But if this fact explains the action of the bile on the spirochstes i t does not explain the cause of the jaundice. The jaundice in spiroehaetosis icteroh8morrhagica niay

284 CARL0 BASILE

even have a mechanical cause, namely, inflammation and swelling of the duodenum and the papilla of Vater.

Beside the haematophagic reaction we also find the lymphocytic reaction. This occurs not only in the spleen and in the principal lymphatic glands but also in all the lymphatic nodules of the organs, as in the lungs, in the liver, in the kidneys, etc. The lymphoid reaction joined with the phagocytic reaction contributes to the destruction of the spirochetes and to the defence of the organism. These two reactions together with the relative absence of nucleated red globules in the spleen and in the peripheral blood explain to us the anaemia which so frequently presents itself in the secondary period of spiro- chetosis icterohaemorrhagica, and it also explains the leucocytosis which is so constantly revealed a t the haematological examination.

Addendum. Since this paper was completed I have seen the recent communica-

tion by M‘Nee (1920l2) on the morbid anatomy and mechanism of production of the icterus in spirochaetal jaundice in. man. I appreciate his conclusions ao far as the human infection is concerned, but believe that the hemorrhages which in infected guinea-pigs are far more abundant and striking than in man, play a very important part in the pathogenesis of the icterus.

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