THE FUNDAMENTAL PATHOLOGY OFINFECTIOUS MYXOMATOSIS

FRED W. STEWART, M.D.

Pathological Laboratory, Memorial Hospital, New York

Infectious myxomatosis of rabbits was first described bySanarelli in 1898 (1). Despite the peculiar features of the diseaseand the interest which it merits, studies on infectious myxomatosishave resulted in but thirteen papers and in several discordantstatements as to its fundamental pathologic manifestations. Theterm" myxomatosis" by common usage implies that the processis neoplastic. Yet Aragao (2) states that the myxomatous tumorsare not truly neoplastic, but merely remarkable collections ofedematous fluid due to the presence of an infectious agent. Rivers(3) believes that in certain respects the infectious myxomatosisof rabbits resembles the Rous sarcoma of fowls, whereas in othersit is quite different. It may serve to bridge the gap betweenvirus tumor and vesicular, destructive virus diseases.

It occurred to the writer that the early stages of the diseasestill merited purely morphologic investigation. A number ofrabbits were inoculated, intradermally at first and by scratchinoculation from rabbit to rabbit later on. To secure as manyinvolved lymph nodes as possible, the infection was usuallyintroduced at more than one point, i.e. the thoracic skin near theaxilla, near the groin, and the ear, to make certain of spread tothe preauricular and cervical nodes. The animals were usuallykilled by air embolus at stages between the development of orbitaland conjunctival lesions and the intense dyspnea preceding deathfrom the disease itself. All organs, the subcutaneous tissues andskin, and as many lymph nodes as possible, were preserved forstudy. Tissues were fixed in absolute alcohol for mucin, in 10per cent formalin, and in Zenker's fluid. The stains employedwere mucicarmine, hematoxylin and eosin, eosin-methylene blue,Wright's blood stain which was found to give excellent results onformalin-fixed tissues, Giemsa stain, and Mallory's phosphotungsticacid hematoxylin.

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Observations on the character and distribution of the grosslesions add nothing to those of prior investigators and do notmerit further space. Although the writer has been particularly

FIG. 1. INFECTIOUS MYXOMATOSIS: SUBCUTANEOUS LESION

Much of the mucinous-looking material is fibrinous. Phosphotungstic acid staining.

concerned with the lymph nodes, a brief description of the cutane­ous lesions seems advisable.

First of all, it may be stated that the inclusion bodies describedby Rivers (4, 5) have been found in all animals. This is contraryto the findings of Findlay (6). The virus employed was obtainedfrom Rivers' laboratory. Moreover, Findlay states that" the nod­ular lesions are thus due, not to an active proliferation of the tissueelements, but simply to the myxomatous changes in the tissues."This is contrary to the report of Rivers and to the writer's ownobservations.

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Aside from the inclusions and the epidermal vesiculation, theearly changes in the skin and subcutaneous tissues consist incapillary engorgement, hemorrhage, and severe edema. The fluid

Fro. 2. INFECTIOUS MYXOMATOSIS: SUBCUTANEOUS LESION, SHOWING NECROSIS,

COLLAGEN SWELLING, AND MUCINOUS EXUDATE

expressible from the cut surface of the lesion is decidedly mucinous,but no more so than the exudates of certain inflammatory processes,as, for example, certain pneumonias. Microscopically the exudatestains a faint bluish color, as does mucin. Phosphotungstic acidstains show, however, that most of the mucinous material can beresolved into denser or finer fibrinous meshwork (Fig. 1). This

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fibrin is interspersed between swollen collagen fibers (Fig. 2).An acute inflammatory cellular exudate is most abundant in andjust beneath the epidermis. It consists mainly in polynuclearleukocytes in various stages of fragmentation and necrosis. Theedema and fibrin deposit extend throughout the skin and sub­cutaneous tissue down through the superficial muscle and intothe adjacent deeper fascial layer.

The proliferative changes occur most markedly about hairfollicles, cutaneous glands, and in the loose areolar tissue, butparticularly about the smaller blood vessels. Endoneurium andmuscle sheath cells appear to escape. The proliferating cells arespindle or polyhedral, with branching processes. Fibroglia fibrilsare absent. The nuclei are large, the chromatin is coarselyclumped, often in pseudobacillary formation, and the cytoplasmis pale and frequently vacuolated. Mitotic figures are common,and there can be no question but that an active proliferation isoccurring. Certain of the cells exhibit a tendency to round offor to assume an ameboid form. The evidence convinces us thatwe are dealing with an active proliferation of the clasmatocytesof the areolar tissues and the perivascular sheaths. The fixedendothelium does not appear to contribute "myxoma" cells.Some of the spindle cells are invaded by leukocytes. The actualnecrosis appears confined to the epidermal structures. Subcu­taneous fat, muscle, and nerve elements escape.

In describing the early stages of lymph node involvement itmay be practical to consider the changes in the cortical lymphoidfollicles and in the medullary cavernous areas and pulp cordsseparately. The early lesions of the lymphoid follicles consistfirst in a moderate hyperplasia. Whether or not there is anactual increase in the numbers of small lymphocytes is difficult toascertain. There is evidence that at least part of the increase insize of the cortical lymph nodules is the result of the appearanceof plasma cells to a varying extent within the follicle. Theprincipal cause for the increased size of the lymphoid noduleseems to be the very marked stimulation of cells of the folliclereticulum. It is not unusual to find from 20 to 30 large reticulumcells per oil immersion field. Numerous mitotic figures are present.These reticulum cells are four or fives times the size of the smalllymphocyte, and are round or ovoid in shape. Their nuclei arelarge and clear. The cytoplasm is bluish or faintly violet, foamy,vacuolated, and contains scanty granules. These cells are scat-

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tered diffusely throughout the follicles. All evidence indicatesthat they originate locally within the follicles. They do not arisefrom any special germinal centers, for the latter show no constanthypertrophy. Occasional polynuclear leukocytes and eosinophilesinvade the follicles.

FIG. 3. INFECTIOUS MYXOMATOSIS: EARLY FIBRINOPURULENT EXUDATE IN THE LYMPH

NODE MEDULLA

In the cavernous medullary area the larger vessels are unalteredexcept for occasional clusters of polynuclear leukocytes in thelumina. The tissues making up the adventitial sheaths, however,are markedly edematous, and contain loose fibrin strands (Fig. 3),between which are interspersed numerous polynuclears, eosino­philes, and large mononuclear leukocytes. There is evidence ofmarked stimulation of the clasmatocytes of the loose adventitia.These show a tendency to rounding off and are often indistin­guishable from the hypertrophied reticulum cells of the follicles.

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Fibroglia fibrils cannot be demonstrated with phosphotungsticacid stains. Mitotic figures are very numerous. Certain of theinvading polynuclears appear deficient in granules; their nucleiare small, very irregular and hyperchromatic, and there is adecided tendency toward karyorrhexis. The fixed endotheliumof the larger vessels is unaltered.

This marked proliferative activity in the adventitial sheathsof the larger vessels is carried into the lymph sinuses surroundingthe smaller capillaries of the pulp, where the whole process becomesintensified. Mitoses are more abundant, polynuclears and largemononuclears and plasma cells are numerous. The fibrin networkbecomes thicker, and eventually many of the lymph sinusesbecome plugged with dense aggregates of fibrin (Fig. 4). Thecollection of polynuclear leukocytes, some of which undergokaryorrhexis and necrosis, in the thrombosed sinuses, convertssuch areas into tiny miliary abscesses.

The disease so far as the nodes are concerned seems anatomi­cally practically confined to the node proper, although there maybe moderate stimulation of the clasmatocytes of the surroundingloose areolar tissue and scanty polynuclear and plasma-cellinfiltration. For some unexplained reason the infectious processin the nodes may show wide variations in distribution. Portionsof the node may entirely escape, i.e. a definite, advanced myxo­matous process may involve one part of a node, whereas theremainder may show little or no change from the normal. Inother instances a single node may exhibit all stages of the disease,ranging from the early inflammatory manifestations to the ad­vanced myxomatous changes. This feature is peculiar, butlocalized node manifestations are no more obscure, so far asinterpretation is concerned, than are localized skin lesions. Thewriter has observed nodes where the definitive myxomatousprocess was largely confined to the peripheral sinus, producingthe appearance of a myxomatous sheath around the node. Evi­dence indicates that the early acute inflammatory lesions tend toinvolve the peripheral sinus last. This much may be said, namely,that there is nothing whatever in the early node lesions to justifythe use of the term "myxoma" or to suggest that the process hasany neoplastic characters. Necrosis, inflammation, and reactionwould appear to explain the histology fully.

Following these earlier disease manifestations, the lymphocytesare rapidly lost from the nodes or from portions thereof. The

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mechanism of this disappearance is not entirely clear. They mayvanish partially as a result of simple pressure atrophy, since thetime of the disappearance of the lymphocytes coincides with themetamorphosis of the follicles and pulp cords into masses ofproliferating reticulo-endothelial cells, some rounded and phago­cytic, others preserving a branched reticular structure producing

FIG. 4. INFECTIOUB MYXOMATOBIB: FIBRIN THROMBI IN THE MEDULLARY LYMPH

SINUBEB

an appearance suggestive of embryonal mesenchyme. Yet simpleatrophy cannot explain the thoroughness and the acuteness of thedisappearance of the lymphocytes, and the virus must be assumedto possess specific lymphocytotoxic properties. The lymphocytesshow evidence of pyknosis and fragmentation, and the reticulum ofthe lymphoid follicles, as well as the sinuses, becomes infiltrated withvery numerous polynuclear leukocytes and eosinophiles (Fig. 5).

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At this stage there is an intense phagocytosis of lymphocytes andpolynuclears by the reticulo-endothelial cells of the node. Plasmacells may be fairly abundant both in the follicles and pulp cords.

FIG. 5. RETICULUM-CELL HYPERPLASIA, DISAPPEARANCE OF LYMPHOCYTES, AND

POLYNUCLEAR INVASION OF A CORTICAL LYMPH NODULE

The latter may actually be replaced by small sheets of plasmacells of typical morphology. One derives. the impression thatthese plasma cells are developed from the residual lymphocytesof the node. Many of the follicles, now essentially devoid oflympl).ocytes, become centers of leukocytic attack, and the frag-

FIG. 6. THE DEFINITIVE "MYXOMATOUS" NODE: HYPERPLASTIC RESIDUAL RETICULUM

INTERSPERSED WITH MULTIPLE ABSCESSES

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mentation of the leukocytes and lymphocytes converts the areasinto small abscesses. Other abscesses' are formed in the pulpand about the larger vessels. The larger vessels are still clearlyrecognizable. Their adventitia, although involved early, showsless intensification of the sheath cell proliferation than do thereticulo-endothelial constituents of the pulp and follicles. Themore advanced proliferative and necrosing processes in the nodeproper are not accompanied by comparable increases of activityin the surrounding areolar tissues. Adjacent organs-as, forexample, the salivary glands-are normal, when a very activeprocess is present in the attached or actually imbedded lymphnode. In other words, the disease lacks the aggressive featuresof a true neoplasm.

Still later stages are characterized by the subsidence of theexudative, necrosing, lymph node lesions. The proliferated,reparative tissue becomes the principal feature. Rare, scatteredfocal abscesses may still be present, but the bulk of the node nowconsists of a massive reticulo-endothelial overgrowth, whichtransforms the node into a spongy reticulum (Fig. 6). Thesereticulum cells are partially fixed, and partially mobilized aswandering phagocytes, rather large and atypical, usually withsingle, pale nuclei and often filled with fibrin and leukocyticdebris. The appearance of the tissue bears certain resemblancesto washed splenic pulp. The fibrin strands described as pluggingthe sinuses now show evidences of liquefaction and are gatheredtogether in coarse, granular clumps throughout the tissue spacesof the node. The disappearance of most of the exudated cellsand of the normally present lymphocytic constituents of the nodemay lead to the appearance of small cysts, lined by reticulum andfilled with coarse fibrin granules. Some of the nodes now consistof a spongy framework of larger and smaller blood vessels sur­rounded by cuffs of more or less fragmented leukocytes. Through­out the intervening spaces are many large phagocytic cells whichevidence indicates are derived from the reticulum (Figs. 7 and 8).The lymph nodes of infectious myxomatosis should prove anexcellent material for the study of the pluripotentialities of thenodal reticulo-endothelial apparatus.

At or before the transformation of the node into a semi-cysticreticulum the animal succumbs to the disease, and there is nomethod at present available for determining what would havehappened, had the animal survived. In later stages certain

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reticulum cells surely assume a morphology suggestive of fibro­blasts, leading one to believe that, did time permit, the eventualresult might be sclerosis of the node. At the time the animal diesthere is no evidence of collagen deposition nor do fibroglia fibrilsstamp the reticulum cells as fibroblastic. One might summarize

FIG. 7. PROLIFERATIVE REACTION IN THE LYMPH NODE, SHOWING THE TRANSITION

BETWEEN THE PROLIFERATION OF RETICULUM CELLS AND PERIVASCULAR CLASMATO­

CYTES, AND LOOSENING UP OF TISSUE WITH TENDENCIES TOWARD CYST FORMATION

the sequence of events iIi the node in the following order: (1)acute exudative inflammation, (2) fibrin thrombosis of the lymphsinuses, (3) necrosis of lymphocytes, (4) abscess formation incortical lymph nodules, pulp cords, and sinuses, (5) extraordinaryproliferation of the reticulo-endothelial apparatus, (6) cysticdegeneration in the resulting syncytial reticulum.

In the writer's series of animals the changes in the spleen

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have been much less extensive than in the nodes. This has beentrue even after intravenous inoculation. Yet the type of lesionis quite similar. The splenic lymph nodules often contain numbersof plasma cells and show evidence of moderate reticulum cellhyperplasia, not nearly so extensive as in the nodes draining aprimary skin nodule. The bulk of the splenic lesions occupies

FIG. 8. A LATE STAGE OF INFECTIOUS MYXOMATOSIS: NETWORK OF BLOOD VESSELS

BETWEEN WHICH LIE PHAGOCYTIC CELLS ATTACKING THE FIBRIN RESIDUE

the tissues surrounding the larger blood vessels. The vesselsthemselves are not thrombosed, but the surrounding tissues reveala marked proliferation of spindle-shaped perivascular and re­ticulum cells (Fig. 9). Their nuclei are large, clear, and swollen,and contain coarse chromatin. These cells are interspersed withlymphocytes, many plasma cells, and eosinophiles. Clusters ofpolynuclear leukocytes occupy many of the pulp vessels, but

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thrombosis has not been observed. Other similar clusters arefound about the pulp sinuses. Much phagocytosis of leukocytesis in progress. The proliferat.ive reaction about the larger vesselsis continued into the pulp sinuses. The spleen is enlarged andhyperemic. The capsule is normal. The actual myxomatous stagehas not been encountered to any extent in the spleen.

FIG. 9. PERIVASCULAR PROLIFERATIVE PROCESS IN THE SPLEEN

Rivers has noted the finding of myxomatous nodules in thelungs. In our own series the lung changes have not progressedto this point. No intratracheal insuffiations have been done.It is usual to find the trachea and bronchi injected, although nothemorrhagic. The epithelium is unaltered. The subepithelialconnective tissue of the bronchi may be edematous and containrecent fibrin deposits recalling the early changes in the subcu­taneous tissues. There may be slight leukocytic invasion. Thebronchial lymphoid nodules and the small lymphoid nodules ofthe lung may show hyperplasia and slight reticulum cell prolifer­ation, but the lesions at death are early and limited.

Kidneys, pancreas, liver, and ovaries have shown no lesions.It is interesting to note that no evidence has been seen of the

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participation of the thymic lymphocytes in the necrosing process,although the number of examinations of the thymus has been toosmall to permit of definite conclusions. Sections from the stomachand intestines have shown no disease, but the terminal -analregion may be involved by direct extension upward from the in­fected perineal tissues.

In all of the animals the testicular tunicae are involved. Theprocess resembles that in the cutaneous nodules and does notmerit separate description. There is an interstitial epididymitisand often an acute interstitial orchitis. In the testis itself theproliferative aspects of the lesion are not notable. A fibrindeposit and a polynuclear infiltration may constitute the onlyevidence of infection. When the skin about the genitalia isinvolved, the edematous inflammatory lesion may extend to theperiurethral tissues and the bladder neck. The penile tissue maybe included.

The bone marrow has been studied in all animals. Marrowhas been secured from all of the long bones. The degree ofmarrow involvement varies greatly. Early stages show clustersof polynuclear infiltration in small foci throughout the marrow.More advanced stages reveal diffuse hemorrhage in various regionsand the appearance of areas of bone marrow necrosis. Not all thebone marrow of the long bones is simultaneously involved and theareas in the marrow of an individual bone may be quite discrete.Many of the marrow lymphocytes show the morphology of plasmacells. Following cutaneous infection, up to the lethal terminationof the disease, the writer has seen no actual "myxomatous"proliferation in the marrow. The changes at death are still inthe acute, necrosing stage. Nevertheless, there is slight butdefinite evidence of stimulation of long spindle-shaped cellsgathered in a narrow zone along certain of the marrow capillaries.Thus minute spindle-cell foci are formed. The early picturesuggests that, were the inoculation more direct or did the animallive long enough, extensive marrow degeneration followed bymyxomatous replacement might result.

DISCUSSION

Infectious myxomatosis is a disease difficult to classify withprecision, but in that respect it does not stand alone. In humanpathology the border zone between chronic productive inflam­matory hyperplasias and neoplastic processes, as for example in

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chronic productive mastitis, or in the field of lymph node tumors,is very narrow. The ultimate fate of the myxomatous tissue ofinfectious myxomatosis, were the disease in some manner prolongedinto a chronic productive phase, might entirely alter our interpre­tation. Nevertheless, there are certain reasons why one musthesitate to regard these tissue changes as neoplastic.

First of all, we have the early phase, which has been shown tobe characterized by a very acute necrosing inflammatory process.Here the mucinous exudate recalls that of certain acute infectiousdiseases. The concomitant or ensuing proliferative change is anacute process, occurring in the reticulum cells and in the peri­vascular clasmatocytes. Where the proliferative reaction involvesthe node reticulum, the resultant picture is not that of the re­ticulum-cell sarcomas of lymph nodes. Rather, the node istransformed into a loose cytoreticulum of separate or anastomosingspindle and polyhedral elements. It suggests that the picture is aresult of the rapid disappearance from the nodes or portions of thenodes of the lymphocytes, leaving only the blood vessels and thestimulated reticulum. This myxomatous reticulum does not showindependent proliferative, invasive properties. The outlines ofthe cortical follicles are preserved and limited by the peripheralsinus. The cell arrangement within the node is not entirelydisorderly, but shows distinctly a relation to node structures.When the proliferative process extends to perinodal tissues thecell arrangement is not that of a diffuse, invasive permeation ofthe surrounding tissue, but of a loose, mucinous, node capsule.The appearance is that of a hyperplastic reticulum in a nodewashed free from lymphocytes.

The cell activity as judged by numbers of mitoses is of con­siderable interest. Mitoses are very numerous during the earlieracute manifestations of the disease, but with the disappearanceof the lymphocytes and the assumption of the myxomatousappearance, there is a marked slowing down of the process andevidence of mitotic activity is practically absent. In other wordsthere is an acute proliferative wave of reticulum cell activityfollowed by a more or less indolent stage after the myxomatousreplacement of the lymphoid structures has occurred. With theestablishment of this phase the aggressive features are wanting.

It is of interest to note that at times, for example in the spleen,the early change is more of the proliferative type than the acutenecrosing form. The property of the virus to excite proliferation

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in the absence of marked necrm'lis suggests that, were its actionin some manner attenuated, the proliferative phase of the diseasemight overbalance the necrosing, and the resemblance to theneoplastic assume a greater prominence. The similarity to theRous sarcoma might then be more apparent.

REFERENC};S

1. SANARELLI, G.: CentralbI. f. BakterioI. 23: 865, 1898.2. ARAGAO, H. B.: Mem. do Inst. Oswaldo Cruz 20: 225, 1927.3. RIVERS, T. M.: J. Exper. Med. 51: 965, 1930.4. RIVERS, T. M.: Proc. Soc. Exper. BioI. & Med. 24: 435, 1926-27.5. RIVERS, T. M.: Am. J. Path. 4: 91, 1928.6. FINDLAY, G. M.: Brit. J. Exper. Path. 10: 214, 1929.