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STUDIES OF COCCIDIOIDES IliivITIS RIXFORD ET GILCHRIST

by

P. Negroni, D. Vivoli, and H. Bonfiglioli

From Revista del Instituto Bacteriol6gico (Journalof the Bacteriological Institute), Vol XIV, No 1,Buenos Aires, 1949, pages 273-286.

VII. Immuno-Allergic Reactions in ExperimentalInfection of the Guinea Pig

Continuing our investigations of Coccidioides inmitis(1,2,3,4,5,6,7,8), we present on this occasion a study of mani-festations of allergy and immunity in experimental infectionof the guinea pig. We chose that animal as being sensitive toinoculation with the fungus in question and easy to handle andto obtain.

This work was planned and carried out by Drs. Negroniand Bonfiglioli, with Dr. Vivoli in charge of the histopatho-logical study.

Iork plan. We proposed to investigate the followingma~ters: l) at what moment of the infection cutaneous sensi-t vization to the coccidioidin appears; 2) what is the reac--Cion of infected and allergic guinea pigs (sensitive to cocci-dioidin ), when exposed to a second inoculation with a) materialfrom a live culture and b) the same material after its vitalityhas been destroyed by heating; and 3) the appearance and courseof circulating antibodies.

Methods and techniques. As shown in th. .ccompanyingtables, we used 5 groups of white guinea pigs jaing 350-400grams, which we designated with the first 5 capi .l letters ofthe alphabet.

Groups A. B, and C, consisting of 15 guinea s each,were inoculated via the routes indicated with an infecting0--

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Vt

dose of 10 mg (moist weight) of the product obtained by pro-longed trituration in a sterile mortar of cultures of C. inm-itis,Argentine strain N2 695, after incubation for 20 days at280 C in agar broth.

Five guinea pigs in each group were subjected to oub-cutaneous vaccination with a suspension of spores, in progres-sively increasing concentration, in a physiological solutionwith 0.3% carbolic acid added, heated in a water bath 30 minutesat 650 C. Sterility controls made it possible for us to verifythe fact that this temperature destroys the vitality of the"entospores" of the cultures. Four series of this vaccine were

' prepared, of which the most concentrated corresponded in

opacity to tube N2 3 of McFarland's scale and each of the otherswas prepared by dilution at 1/10. Each guinea pig was given 3weekly injections of 0.5ml during one month beginning nine daysafter the infecting inoculation.

The guinea pigs of group D were subjected to vaccinationwithout previous infecting inoculation.

The study of cutaneous allergy was made by intradermicinjection of polyvalent coccidioidin prepared from Argentineand US strains of C. immitis according to the technique recom-mended by C.B. Smith and "standardized" with the coccidioidintype (batch 29-31) kindly supplied by him.

It was decided to use the testicle in evaluating thereactions to a second inoculation because that organ, as hasbeen demonstrated by E.R. Long (9), reacts intensely to tuber-cular infection, to which coccidioidomicotic infection hasmuch similarity, and also with the object of getting uniformityin the literature. The material used in reinoculations con-sisted in a suspension of "entospores" in physiological solu-tion, the opacity of which corresponded to that of N2 3 ofMcFarland's scale. This suspension was divided into two frac-tions: a) heated in a water bath at 650 C for 30 minutes todestroy its vitality, and b) unheated. The guinea pigs wereinoculated with 0.2 ml and were killed after 18-20 hours andafter 5 days, with the object of observing tissue reactionsthat might afford us an index of allergy and immunity.

Finally, group E received only inoculation via thetesticle with live and dead material. Groups D and B, then,were taken as controls, the former for the variant introducedinto our experiments and the latter to appraise the reactionto testicular inoculation with "entospores" in " organismpreviously uninfected.

For the reactions of fixation of the complement we usedtwo distinct antigens. The first consisted in an emulsion ofthe lipides extracted from the mycelium and the second in coc-cidioidn, both conveniently diluted. In view of the negative

2

results obtained with the first, we continued our studies usingcoccidioidin exclusively for these reactions. This product wasalso used in the precipitin reactions, and for agglutinationreactions we used a suspension of "entospores" whose vitalityhad been destroyed by heating.

Results. The intradermal reaction with pure coccidioid-in gave positive results ++ at the end of one week of infectionand +0+ with coccidioidin diluted to 1/10. These results wereaccentuated in intensity in tests made a week later.

Serological tests made on four guinea pigs that wereinfected and four others that were infected and vaccinated, allof group A, at the end of a month gave negative results. Thesame results were obtained with two guinea pigs of group Bat the end of 20 days after infection and two of group C after17 days.

Serological reactions repeated at the end of threemonths after infection with three guinea pigs from group A andone from group B showed intensely positive fixation of the com-plement, but negative precipitin and agglutination reactions.

In guinea pigs inoculated with culture material in otherexperiments nine months to a year before and still surviving,we got intensely positive results in the complement fixation

O reaction from three of ten animals. The agglutination and pre-cipitin reactions were always negative.

The results of the tissue reactions are assembled in theaccompanying tables, which also contain notes of the modifica-tions that the parasite undergoes in microscopic appearance,number, and size.

Oranslator's note: The tables in the source cover atwo-page spread and cannot be readily reproduced here. Forconvenience, all the material contained in the tables is givenat this point in paragraph form.

Group A. Guinea Pies Infected Subcutaneouslyand Reinoculated Via the Testicle

Guinea pig A765. Reinoculated with dead material.Killed after 18-20 hours. Not vaccinated; reinoculated after23 days. Macroscopic lesions: testicle: slight congestion.Histological lesions: congestion and monocytic reaction.

A795. Reinoculated with dead material. Killed after 5days. Not vaccinated; reinoculated after 23 days. Macroscopiclesions: testicle: slight hemorrhage; granulations in thelung, spleen; hypertrophy of ganglion. Histological lesions:testicle without lesions. Pseudotuberculous granuloma withparasites in the spleen.

A789. Reinoculated with live material. Zilled after

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18-20 hours. Not vaccinated; reinoculated after 23 days.Macroscopic lesions: testicle: slight congestion. Histologic-al lesions: hemorrhagic focus and nodular monocytic reactionwithout parasites.

A7". Reinoculatzd with live material. itlled after 5days. Not vaccinated; reinoculated after 23 days. Macroscopiclesions: testicle of larger size, ganglion hypertrophied,granulations in the lungs. Histological lesions: testicle:nodular productive lesions and productive-exudative lesions.Tuberculoid granuloma with epithelial cells. A few parasites,without endospores and with radiate formations.

F28. Reinoculated with dead material. Killed after 18-20 hours. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle of larger size and congested.Histological lesions: Interstitial, discrete, non-speccificorchiepididymitis.

N8. Reinoculated with dead material. Killed after 5days. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle of larger size and congested;granulations in the spleen. Histological lesions: nodularproductive lesions and exudative lesions. Dropsical degenera-tion of the cells of the covering of the seminiferous tubules.

F26. Reinoculated with live material. Killed after 18-20 hours. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: orchiepididymitis with non-specificinterstitial orchitis without parasites.

M7. Reinoculated with live material. Killed after 5days. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: nodular lesion with productive predomin-ance. Very rare parasites, small, without endospores and withradiate formations.

F27. Reinoculated with dead material. Killed after 18-20 hours. Vaccinated and reinoculated at the end of 43 days.Macroscopic lesions: Testicle enlarged in size. Histologicallesions: exudative lesions. In the spleen, exudative product-ive lesions with a few parasites (cystic forms).

M6. Reinoculated with dead material. Killed after 5days. Vaccinated and reinoculated at the end of 43 days.I acroscopic lesions: testicle unchanged. Histological lesions:exudative lesions with abundant eosinophiles; dropsical de-ener-ation of cells of the seminiferous tubules. Discrete intersti-tial infiltration.

F25. Reinoculated with live material. Killed after 18-20 hours. Vaccinated and reinoculated at the end of 45 days.Macroscopic lesions: testicle enlarged in size and congested;

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nodular lesions in liver and spleen. Histological lesions:interstitial non-specific orchiepididymitis without parasites.

1M5. Reinoculated with live material. Killed after 5days. Vaccinated and reinoculated at the end of 43 days.icroscopic lesions: testicle enlarged in si7e and congested;hypertrophy of ganglion. Histological lesions: lesions withproductive predominance. Rare parasites, of medium size and

with endospores.

Group- B. Guinea Pigs Infected Intramuscularlyand Reinoculated Via the Testicle

A728. Reinoculated with dead material. Kialed after18-20 hours. Not vaccinated; reinoculated after 23 days., acroscopic lesions: testicle congested. Histological lesions:vasodilatation, monocytic inflitration.

A712. Reinoculated with dead material. Killed after 5days. Not vaccinated; reinoculated after 23 days. Nacroscopiclesions: testicle congested, ganglion hypertrophied. Histolo-gical lesions: vasodilatation, monocytic infiltration, anddiffuse productive lesions. Ganglion: small parasites withoutendospores.

A718. Reinoculated with live material. Killed afterO 18-20 hours. Lot vaccinated; reinoculated after 23 days.

Macroscopic lesions: testicle congested. Histological lesions:discrete monocellular infiltration. No parasites observed.t A736. Reinoculated with live material. Killed after 5

days. Not vaccinated; reinoculated after 23 days. Nacroscopiclesions: testicle hemorrhagic. Caseosis at the point of theinoculation. Nodule in lung. Histological lesions: necroticand productive granuloma of the testicle, with very rare para-sites of medium size with endospores.

P3O. Reinoculated with dead material. Killed after 18-20 hours. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: discrete orchitis.

1.2. Reinoculated with dead material. Killed after 5days. Not vaccinated; reinoculated at the end of 43 days.Ncroscopic lesions: testicle slightly hypertrophied. Histo-logical lesions: exudative nodular lesions.

P24. Reinoculated with live material. Killed after 18-20 hours. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: exudative orchiepididymitis with abund-ant eosinophiles. No parasites observed.

Mll. Reinoculated with live material. Killed after 5o days. Not vaccinated; reinoculated at the end of 43 days.

I

Macroscopic lesions: testicle enlarged in size and congested;granulations in lungs. Histological lesions: productiveorchitis and exudative and nodular interstitial epididymitis.Very rare parasites, small and with radiate formations.

F29. Reinoculated with dead material. Killed after 18-20 hours. Vaccinated and reinoculated at the end of 43 days.Xacrosccpic lesions: testicle enlarged in size and congested.Histological lesions: orchitis and non-specific interstitialepididymitis. Spleen: productive and exudative nodularlesions with a few parasites.

MlO. Reinoculated with dead material. Killed after 5days. Vaccinated and reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: none.

F23. Reinoculated with live material. Killed after 18-20 hours. Vaccinated and reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested;ganglion hypertrophied. Histological lesions: testicle:interstitial exudative and productive lesions without parasites.Ganglion: follicular productive lesions, parasites with cysticendospores.

M9. Reinoculated with live material. Killed after 5days. Vaccinated and reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: nodular exudative and productive epi-didymitis without parasites.

Group C. Guinea Pigs Infected Intraperitoneallyand Reinoculated in the Testicle

A737. Reinoculated with dead material. Killed after 18-20 hours. Not vaccinated; reinoculated at the end of 20 days.Macroscopic lesions: testicle congested. Histological lesions:hemorrhagic and nodular focus with monocytic reaction, withoutparasites.

A773. Reinoculated with dead material. Killed after 5days. Not vaccinated; reinoculated at the end of 20 days.Macrosoopic lesions: testicle atrophied, casoosis at the pointof the infecting injection, ganglion hypertrophied. Histologic-al lesions: testicle: exudative-productive lesions witheosinophiles; parasites rare and small. Ganglion: granulomawith sosinophiles and abundant parasites.

A723. Reinoculated with live material. Zilled after18-20 hours. Not vaccinated; reinoculated at the end of 20 days.Macroscopic lesions: testicle congested. Histolo,ical lesions:edema and hemorrhage with exudative necrotic lesions withoutparasites.

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A786. Reinoculated with live material. Killed after 5days. Not vaccinatcd; reinoculated at the end of 20 days.Macroscopic lesions: testicle hemorrhagic, caseosis at thepoint of the infection; ganglien hypertrophied; nodules inlungs. Histological lesions: testicle: encysted granulomawith productive and exudative reaction, the former predominat-ing. No parasites observed.

PQ6. Reinoculated with dead material. Killed after 18-20 hours. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle atrophied and contains pus.Ganglion hypertrophied and caseated. Granulations in theperitoneum and viscera. Histological lesions: testicle:exudative-productive granuloma with parasites in various evo-lutive states. Liver: granuloma without parasites.

F97. Reinoculated with dead material. Killed after 5days. Not vaccinated; reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size, with adhe-sions and caseum; abscesses in liver, spleen, and peritoneum.Histological lesions: specific interstitial orchitis with pre-dominance of fibrous reaction. Epididymus: productive andexudative lesions containing parasites of medium size withradiate formations and others with polyhedral endospores.

P94. Reinoculated with live material. Killed after 18-Q 20 hours. Not vaccinated; reinoculated at the end of 43 days.MIacroscopic lesions: testicle atrophied, with adhesions andcaseum; granulations in peritoneum and viscera. Histologicallesions: interstitial orchitis, exudative lesions in the epi-didymus with few parasites.

P95. Reinoculated with live material. Killed after 5days. Not vaccinated; reinoculated at the end of 43 days.acroscopic lesions: testicle atrophied and caseated. Histo-

logical lesions: specific interstitial orchitis with predomin-ance of fibrous reaction. Bpididymus: productive and exuda-tive lesions containing parasites of medium size with radiateformations and others with polyhedral endospoi.s.

P92. Reinoculated with dead material. Killed after 18-20 hours. Vaccinated and reinoculated at the end of 43 days.Macroscopic lesions: testicle enlarged in size and congested.Histological lesions: discrete orchiepididymitis.

P93. Reinoculated with dead material. 2-_i!!ed after 5days. Vaccinated and reinoculated at the o.:d of 43 days..oroscopio lesions: testicle con.ested. EUs~olo Ical lesions:exudative lesions. Spleen: granuloma without p;raaies.

P90. Reinoculated with live material. Kll ed after 18-20 hours. Vaccinated and reinoculated at the end of 43 days.Maoroscopic lesions: testicle unchanged. Histological lesions:

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necrotic exudative lesions and interstitial orchitis withoutparasites. Ganglion: few parasites, of medium size and with-out endospores.

F91. Reinoculated with live material. Killed after 5days. Vaccinated and reinoculated at the end of 43 days.Macroscopic lesions: testicle atrophied and caskated. Histo-logical lesions: exudative lesion with few parasites, one withpolyhedral endosporos.

Group D. UninfectedGuinea Pips, Vaccinatedin Various. Ways. andInocula.ted in the Testicle

R22. Inoculated with dead material. Killed after 18-20hours. Vaccinated subcutaneously. Xacroscopic lesions: tes-ticle congested. Histological lesions: con~estion, edema, anddiscrete monocytic infiltration.

R28. Inoculated with dead material. Killed after 5 days.Vaccinated subcutaneously. Macroscopic lesions: none. Histo-logical lesions: none.

R21. Inoculated with live material. Killed after 18-20hours. Vaccinated subcutaneously. Macroscopic lesions: tes-ticle enlarged in size and congested. Histological lesions:edema with monocytic filtration without parasites.

Q R27. Inoculated with live material. Killed after 5 days.Vaccinated subcutaneously. Macroscopic lesions: testicle en-larged in 3ize and congested. Histological lesions: nodularinterstitial infiltration of monocytes and a great number ofpolynuclears; hemorrhagic foci with a great number of parasitesin the infecting phase.

R24. Inoculated with dead material. Killed after 18-20hours. Vaccinated intramuscularly. Macroscopic lesions- tes-ticle congested. Histological lesions: congestion, edema, anddiscrete monocytic infiltration.

R30. Inoculated with dead material. Killed after 5 days.Vaccinated intramuscularly. Macroscopic lesions: none. His-tological lesions: none.

R23. Inoculated with live material. Killed after 18-20hours. Vaccinated intramuscularly. Macroscopic lesions: tes-ticle enlarged in sise and congested. Histological lesions:edema with monocytic infiltration without parasites.

R29. Inoculated with live material. Killed after 5 days.Vaccinated intramuscularly. Macroscopic lesion3: tezticlo en-larged in volume and congested. Histological lesions: nodularinterstitial infiltration of monocytes and a graat n.-ber ofpolynuclears; hemorrhagic foci with a great number o: parasitesin the infecting stage. Young and adult parasites almost all

OD infectious sporangia, only one vith cystic spores.

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II __ II II] I I I I I I I I I I L

R26. InocuJlat~d with dcad matcziZAI. KRillXd _,ftcr l8-

20 hours. Vaccinated intraperitoneally. M~acroscopic lozions:testicle coni~ested. HIistological lesions: Zonal infilt:atioaof polynuclears.

R25. Inoculated with live material. Killed aftczr 18-20 hours. Vaccinated intrap~ritoncally. IXacroscopic lesiona:testicle unc'han~ed. Histological 1esio.a: testicle unc2.anjedad without parasites.

R31. Inoculated with live caterlal. Killed after 5days. Vaccinated intraperitoncally. !-"acroscopic les.Lons:tezticle enlarged in voluace and conr-sted. Histologica. le-sions: nodular Interstitial infiltra.tion of raonocytes and agreat nu-ber of polynuclear's; hcrmorrhal-ic foci with a greatnumber parasites in the infectin,- plhse.

Grou, "S. Uninfectod Gn'nea Pi7qz InculaTted Via AVhe T'esticle

L4,2. Inoculated w' 'th dead -material. Killed after 18-20 hours. M1acroscopic lesions: discrete con,-estion. Uisto-logical lesions: slight interstitial nodular reaction withoutparasites.

L43. Inoculated %4:ith dead material. Killed after 5days. ANacroscopic lesions: none. Histological lesions: none.

0 140. Inoculated v~ith live =aterial. Killed after 18-20 hours. 11-acroscopic lesioas: slight congestion. Histolog-ical lesions: congestion and discrete monocytic infiltration.One parasite of 10 microns.

141. Inoculated with live material. Zilled after 5days. Macroscopic lesions: testicle enlaraed in size and con-gested. Histological lesions: nodular productive lesionsalternating with exudative ones; a great number of eosinophileaand of parasites in the infecting phase.

Discussi a. The experimzental study ue have junt de-scribed enables u~s to confirm the facts scientifically discov-ered by Posad as in 1892 and interpreted in the liCght of our

lesont isole of teraulomaou dy. Immuniyb the Italndylclischols Cvialeraog (1 ad to1F Realli (12),ao .nd en a

p Cnmeo. Frn ctdb Cavallero) r14)i i td ero ueda aned factsnityeinta yo e ad o tentied t to he rfancu teati Gchrit'

Vpareater tne seh dy tahe rinoculete;raesuK -9-

a cellular reaction which passes through the followin twophases: 1) a reactive phase of granulocytic character and2) a resolving phase of granwalomatous aspect with giant cells.

C. Cavallero (1943) encounters identity of coccidioido-mycosis with tubercular infection, as had already been noted byPosadas and later in the US by C.E. Smith, Jacobson, Chipma.n,and Templeton. In coccidioidomycosis, besides -he chronic forzin which a granulomatous reaction similar to tuberculosis isobsexred, there exist the acute forms discovered by Gifford

and Dickson (1936-1937) in which exudative-vascular phenomenapredominate.

Redaelli and Ciferri (1942) described the reaction ofthe host to the material inoculated in experimental coccidioido-mycosis in the following terms: a reaction of non-specificcharacter is first produced, which tends to eliminate theforeign matter by digestion, and in which polynuclears, mono-cytic elements, and non-specific histocytic cells appear.There are elements of the gera that resist, "anamalize," mul-tiply, and diffuse the infection. Then there begins a reactionof the conjunctive vascular tissue and particularly of thelocal reticuloendothelial system, which is characteristic ofmycotic granuloma, the specificity of which is shown solely bythe existence of a particular parasite, in this case C. immitis.

Hyperreceptivity and allergy put a particular stamp onthe histogenic reactions of the animal infected. In hyperre-ceptive individuals, in a state of negative allergy, an exuda-tive vascular reaction is observed followed by a series ofregressive processes finally becoming necrotic and hemorrhagic,while in hyperresistant, hyperergic animals we observe theprevalence of hyperplastic reactive activity with the formationof granulomata with epithelial and giant cells and with a greatquantity of plasmacellular and lymphoid elements arranged inthe periphery, accompanied by a succession, varying in extent,of the fibroblastic series.

According to our studies intratesticular inoculation ofmaterial from cultures of C. immitis into a guinea pig notpreviously infected with coccidioidomycosis brings on in 18-20hours a granulocytic and monocytic non-specific reaction whichtends to eliminate the inoculated material as a foreign body;this iz comzpletely accomplished by the end of five days whenmaterial hilled by heat has been inoculated. When on the otherhn living material has been inoculated, the spores of thegerm that have resisted this first non-specific defensive reac-tion evolve into the infectious phase, forming sporangia witha very fine peridial membrane which attain a diameter up to83.5 )a and contain numerous very small polyhedral endospores(Posadas's granulose forms). The histogenic reaction isexudative-productive with abundant eosinophiles, as observed in

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spontaneous infection in man (Wickoff and Usighi, cited byC.E. Smith, 1943).

Intratesticular reinoculation performed 26 to 43 hoursafter the first infecting inoculation gives rise after 18-20hours to a reaction characterized by congestive-hemorrhagicphenomena and in some cases a necrotic exudative-productivereaction, which disappears by the end of five days " deadmaterial has been inoculated but leads to a predominance of theproductive reaction with formation of a granuloma when livematerial has been injected. This histogenic reaction, indicat-ing a certain degree of immunity, is manifested by an almostcomplete eisappearance of the parasites, and when these persistthey appear in modified morphological and biological form. Infact, the parasites are medium to small, some 30 U in diameter,without endospores and with the thick membrane frequently sur-rounded by an acidophilous areole or by radiate or claviformformations of the same staining properties.

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Summary. Inoculation of material from cultures of Coc-

cidodes immitis in the guinea pig in various ways leads to astate of hypersensitivity and partial immunity. The cutaneoushypersensitivity is quite evident on the seventh day and is ac-centuated on subsequent days.

Reinoculation with culture material killed by heat Dro-yokes at 18 to 20 hours an intense congestive-hemorrhagic reac-tion and histologically discernible exudative-necrotic phenomenawhich disappear toward the fifth day. Reinoculation with livingmaterial, on the other hand, induces productive phenomena whichlead to the total or partial disappearance of the material in-jected, depending on the degree of immunity existing. Thisreaction, similar to Koch's phenomenon, also follows the biolo-gical law established by Levandowsky and Jadassohn in the caseof leprosy.

In a guinea pig not previously infected, 18-20 hoursafter inoculation we observe a non-specific granulocytic andmonocytic reaction which leads to total elimination of deadmaterial by the fifth day. After inoculation with live material,on the other hand, the endospores that have resisted this firsttissue reaction evolve toward the formation of primary infectionsporangia, which are characterized by their enormous size (up to83.5 in diameter), their fine membrane, and their content ofSQ granulose endospores without any apparent membrana of their own.

Vaccination with material killed by heat does not createcutaneous or tissual hypersensitivity, nor does it seem to modi-fy the course and the reactivity of the organism in the face ofa new inoculation with either living or dead material. Neitherhave we observed any variations due to the manner of the infect-ing inoculation L.e. subcutaneous, intramuscular, intraperi-tonealj.

The parasite which develops within an organism in astate of allergy and partial immunity exhibits the followingcharacteristics: its volume is generally less, the productionof endospores is notably restricted, and when such productiondoes take place it leads to the production of cystic endospores(Posadas's vegetative or cystic phase). The peridial membraneis always thick and frequently surrounded with an acidophilousareole or with radiate or claviform formations of the samestaining properties.

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I __ ___ ___ ____ ___ ___ ___ ____ ___ ___ ____ ___ ___ ___ ____ ___ ___ ___

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Figure 1.. Sporangia with 'grarniok.e endosporez.

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Figure 4. Empty vade by yopo4es.

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Fi.gure 3. 8~poranpan wit cicedospleuores.

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10 F±gure 5.Cystic parasite with radiate formations.

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Bibliograohy

1. Negroni, P. and Radice, J.C.: Rev. Are. Dermatosif.(Argentine Journal of Dermatology and Syphilology),Vol 30, 1946, page 219.

2. Negroni, P. and Radice, J.C.: Rev. Arg. Dermatosif., Vol31, 1947, page 573.

3. Negroni, P.: Rev. Arg, Dorratosif., Vol 32, 1948, page 50.

4. Negroni, P.: Rev. Arg. Dormatosif., Vol 32, 1948, page 58.

5. Negroni, P.: Report to the Argentine Association of Derma-tology and Syphilology, 13 May 1948.

6. Negroni, P. and VIvoli, D.: Report to the Arg. Assoc. ofDermatology and Syphilology, 13 May 1948.

7. Negroni, P., Daglio, C.A.N., and Briz de Negroni, C.: Re-port to the Arg. Assoc. of Dermat. and Syph., 7 July1948.

8. Negroni, P. and Briz de Negroni, C.: Report to the Arg.Assoc. of Dermatology aud Syphilolog, 3 September 1948.

9. Long, E.R.: American Review of Tuberculosis, Vol 9, 1924,page 215.

O 10. Cavallero, C.: Mycopathologia, Vol 3, 1941, page 1.

11. Cavallero, C.: Xyvcopathologla, Vol 3, 1943, page 310.

12. Redaelli, P.: XvooDathologia, Vol 3, 1943, page 280.

13. Redaelli, P. and Ciferri, R.: Le Granulom'atosi FunRine.etc.(The Granulomatous Fungi, etc.), S.E.8., Florence, 1942.

14. Smith, C..: Med. Clin., North Amer., Vol 27, 1943,790.

15. Posada, A.: Obras Oompletas de (Complete Works of),Buenos Aires, Imprenta de la Universidad (UniversityPres), 1928.

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