Revista da Sociedade Brasileira de Medicina Tropical 18(2): 101-108, Abr-Jun, 1985 EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN THE MOUSE. III. HISTOPATHOLOGICAL AND IMMUNOLOGICAL FINDINGS AFTER INTRAVENOUS INFECTION IN THE PRESENCE OR ABSENCE OF PREVIOUS IMMUNIZATION Maura Moscardi B acch i1 and Marcello Franco1 Fifty male white Swiss mice aged 4 weeks were inoculated with 5 x 10$ viable yeast form s o/Paracoccidioides brasiliensis (strain 18). Ten o f these anim ais had been previously im m unized withparticulate P. brasiliensis antigenfor 4 weeks by intradermal injection. The cõntrols consisted o f 10 anim ais that were only im m unized and 10 anim ais subm itted to no treatment. The anim ais were sacrificed 2, 4, 7 ,11 and 16 weeks later. We studied: 1) the anti-P. brasiliensis delayed hypersensitivity response measured by the foo tpad test 24 hours prior to sacrifice; 2) the specific antibody production measured by double immunodiffusion in agar gel; 3) the histopathology oflungs, liver, spleen, adrenals and kidneys. We observed that: a) the im m unized animais developed more intense cell-immune responses than the infected ones; b) infection reduced the cell- im m une response o f the im m unized animais; c) intravenous infection o f mice with P. brasiliensis was characterized by a systemic and progressive granulomatous in- flam m ation. The anim ais infected after previous im m unization showed less extensive lung inflammation, with sm allergranulom as andfew erfungi. The results indicate that the present murine model mimics some findings o f the human subacute form o f paracoccidioidomycosis (systemic disease with depressed cellular im m unity) and that the extrapulmonary im m unization scheme was able to induce a certain degree o f protection o f the lung from infection with P. brasiliensis Key words: Murine paracoccidioidomycosis. Immune response. Immunization. In previous papers2021, we studied the histo­ pathology and the humoral and cellular immune response of mice experimentally infected by intrape- ritoneal inoculation of yeast forms of Paracoccidioides brasiliensis (P. brasiliensis). The peritonitis induced was characterized by a non-specific, localized and self- healing inflammation accompanied by moderate antibody production and persistent leveis of cell- immune response. The course, intensity and nature of the peritonitis were similar in infected animais in the presence or absence of previous specific immuniza­ tion21. Some patients with paracoccidioidomycosis (Pbmycosis) show a localized and benign disease, with adequate immunological response and evolution to ‘ cure ’ after treatment. This clinicai and immunological presentation of the human disease is comparable to 1 Departamento de Patologia, Faculdade de Medicina de Botucatu da U N E S P , 18600 Botucatu, São Paulo, Brâzil. Research supported by F IN E P (grantB /76/80/231/00/00). Recebido para publicação em 18/7/84. that observed in the mouse-intraperitoneal inoculation experimental model2^ 21. In contrast, other patients present a progressive and systemic disease with depressed cell-immune response, which may be fatal even when treated. In order to standardize an experimental model of Pbmycosis with a parallel course to that observed in those patients, we have studied the evolving histopathology and immune response of intravenous infection in mice. W ith the aim of looking at protective mechanisms against the disease, the infection was further studied in animais that had been previously immunized with P. brasiliensis. M A TER IA L A N D M ETH OD S Experim ental groups 1. Infection: Thirty white Swiss male mice aged four weeks were used. Groups of 6 animais were sacrificed 2, 4, 7, 11 and 16 weeks after infection. 2 .Infection after immunization: Forty mice similar to those described above were distributed into 4 101 Bacchi M M , Franco M . Experim ental paracoccidioidomycosis in the mouse. III . Histopathological and immunological find ings after intravenous infection in thepresence orabsence ofprevious immunization. Revista da Sociedade Brasileira de M edicina Tropical 18:101-108, Abr-Jun, 1985 experimental groups: a) 10 infected animais; b) 10 immunized animais; c) 10 animais infected after immunization; d) 10 Controls that were neither im­ munized nor infected, injected only with sterile saline. Subsequently, 5 mice from each group were sacrificed at week 2 and 7. Imm unization Immunization was carried out by the protocol of Rifkind et al21 24. Briefly, each mouse received a 0.025 ml intradermal weekly injection of a P. brasi- liensis particulate antigen for 4 weeks. The animais infected after immunization were inoculated one week after the last immunizing dose. Inoculum Strain 18 of P. brasiliensis was cultured in F ava N etto’s medium7, at 37°C for 10 days. Each animal was infected through the caudal vein with 0.25 ml of fungai suspension in sterile saline containing 5x105 colony-forming units25. The Controls were inoculated with the same volume of sterile saline. Anti-P. brasiliensis im m une response Cellular immunity: it was evaluated in vivo 24 hours prior to sacrifice by the footpad test, as previously described20 21. Humoral immunity. it was quantitated at sacrifice, by double immunodiffusion in agar gel, as previously standardized20 21. Histopathology Ali animais were dissected and the gross findings recorded. Fragments of lung, liver, spleen, adrenals and kidneys were fixed in 10% formalin, embedded in paraffin, cut into 4 uim sections and stained with hematoxylin-eosin (H E) and Gomori-Grocott, by routine procedures. The intensity of the granulomatous reaction (number and size o f granulomas) and number of fungi in lesions were semiquantitated on a scale to 0 to 4 + in mice with or without previous specific immunization. RESU LTS Cellular im m unity Table 1 shows the indices of the footpad test in infected, immunized, immunized and infected, and control animais. Table 1 - Result o fthe foo tpad test expressed as the difference in volume between the control and the testfootpad, in infected, immunized, im m unized and infected, and control animais. Statistical differences between means are expressed by dijferent letters. Sacrifice Footpad test indices N.° o f M ean ± Statistical analysis (W eek) Groups Anim ais s.d. Infection Infection vs. Im m unization 2 Infection 0.4 Infection 0.4 2 Immunization 1.5 Immun. + Infection -0.4 Control -0.1 4 Infection 0.6 7 Infection 0.5 Infection 0.9 Immunization 1.4 7 Immun. + Infection 0.2 Control 0.1 11 Infection 0.4 16 Infection 0.5 AI = accident during inoculation 102 0.5 0.7 0.7 0.9 1.1 6 0.5 0.6 0.8 0.8 5 1.7 1.8 2.0 2.2 5 0.2 0.2 0.4 0.6 5 0.0 0.1 0.1 0.2 5 0.8 0.8 1.0 1.3 1.6 6 0.8 0.8 1.0 1.0 1.0 6 1.0 1.6 1.8 2.2 5 1.6 1.8 2.7 AI* 5 0.9 1.3 1.5 AI 5 0.2 0.3 0.4 0.4 5 0.5 0.7 0.7 1.0 1.2 6 0.6 0.7 1.0 1.2 1.6 6 0.7 ± 0 .2 a 0.6 ± 0.2 a 1.8 ± 0 .2 b 0.2 ± 0.3 a 0.1 ± 0 .1 b 1,0 ± 0.3 a 0.8 ± 0.2 a 1.5 ± 0 .5 a 1.8 ± 0 .5 a 0.9 ± 0.5 a 0.3 ± 0 .1 b 0.7 ± 0.3 a 0.9 ± 0.4 a Bacchi M M , Franco M. Experim ental paracoccidioidomycosis in the mouse. III . H istopathological and immunological findings after intravenous infection in the presence or absence o f previous immunization. Revista da Sociedade Brasileira de Medicina Tropical 18: 101-108, Abr-Jun, 1985 Statistical analysis of the results was performed on the basis of three different approaches: 1) infection throughout the experimental period; 2) infection ví. immunization (2nd week); and 3) infection vs. im­ munization (7th week). The Kruskall-Wallis non- parametric testfor independent variables2 was applied in each case, considering K = 6 groups in approach 1, and K = 3 groups in approaches 2 and 3. The H statistical parameter was calculated with distribu- tion, with K = 1 degree of freedom, under Ho, where Ho: there is no effect of treatment. The tests were performed considering a = 0.05; in case of rejection of Ho, contrast between pairs of means was performed. The control mice showed footpad test indices between - 0.1 and 0.4, with mean values of 0.2 ± 0.1. The infected animais showed positive cell-immune response from the 2nd to the 16th week of infection, with indices ranging from 0.6 to 1.5, with mean values of 0.8 ± 0.3. In general, the control groups indices were statistically different from those of the infected groups, which however did not differ amongst themselves. Immunized animais, both at weeks 2 and 7, developed a more intense cell-immune response (mean — 1.8 ± 0.4) than infected animais (mean = 1.0 ± 0.3) with a significant difference at week 2. The cell-immune response in mice infected after previous immunization (mean = 0.5 ± 0.4), was depressed compared to that of the immunized group (mean = 1.8 ± 0.3), both at week 2 ' and 7. A significant difference was observed at week 2. H um oral Im m unity Table 2 shows the serum titers of anti-i’. brasiliensis antibodies detected in ali mice. The infected animais showed low specific antibody produc­ tion starting at the 7th week and persisting up to the 16th week. Animais infected after immunization developed a humoral anti-P. brasiliensis response at higher frequency and with higher titers than only immunized mice. Histopathology 1. Infection: Since the 2nd week, the animais showed a diffuse and progressive involvement of the lungs characterized by a loose ill-defined granulomatous inflammation, made up by macrophages, epithelioid cells and neutrophils, around great number of fungi Table 2 - Results o f the immunodiffusion test, expressed as the reciprocai o f the m axim um serum dilution showing a precipitation band, in infected, immunized, im m unized and infected and control animais. Sacrifice (W eek) Groups Titers N p o f anim ais M edian 2 Infection 0 0 0 0 0 0 6 1 Infection 0 0 0 0 P l 5 0 Immunization 0 0 0 0 P 5 0 2 Immun. + Infection 0 0 p P 8 5 P Control 0 0 0 0 0 5 0 4 Infection 0 0 0 0 0 0 6 0 7 Infection 0 p p 2 4 12 6 p Infection 2 8 8 16 16 5 8 Immunization 0 0 0 0 2 5 0 7 Immun. + Infection P P 8 8 16 5 8 Control 0 0 0 0 0 5 0 11 Infection 0 0 P P 2 2 6 P 16 Infection 0 p 2 2 2 8 6 2 1 = Undiluted serum 2 = Insufficient serum volume 103 Bacchi MM, Franco M. Experim ental paracoccidioidomycosis in the mouse. III . H istopathological and immunological findings after intravenous infection in thepresence or absence o f previous immunization. Revista da Sociedade Brasileira de M edicina Tropical 18: 101-108, Abr-Jun, 1985 (Figure IA). The inflammation affected both the alveolar septa and lumina. From week 7 onwards, the epithelioid granulomas became more compact, well- defined and confluent, with Langhans giant cells, surrounded by lymphocytes and plasmacells (Figure 2A). Fig. 1 - Histopathology of the lung at week 2. A - Infectíbn: loose ill-defined granulomatous inflammation with neutrophils and moderate number of P. brasiliensis. (H E 400X). B - Infection in the presence of previous immunization: inflammation with similar pattem , showing smaller granulomas and diminished number o f fungi. (H E 400X ). Throughout the infection, extrapulmonary dis- semination occured to the liver, spleen, kidney and adrenal, with histological pattern similar to that in lungs. The dissemination lesions were Progressive, reaching great proportion at week 16 and showing numerous viable and actively multiplying fungi. 2. Infection after previous immunization'. Dur- ing the 2nd week, the histological pattem of the inflammation was similar in infected animais in the presence or absence of previous immunization. However, in previously immunized mice, the lung granulomas showed a tendency to be smaller and to present a significantly reduced number of fungi (Figure 1B). A t week 7, infected animais showed lung gran­ ulomas which were more compact, extensive and with a greater number of fungi when compared to those of previously immunized mice (Figure 2B). J+Z'' B Fig. 2 - Histopathology of the lung at week 7. A - Infection: well-defined, compact and confluent granulomata with numerous fungi and halo o f lymphocytes and plasma cells. (H E 200X). B - Infection in the presence o f previous immunization: small ill-defined loose granuloma with reduced number of fungi. (H E 250 X). 104 Bacchi M M , Franco M . E xperim ental paracoccidioidomycosis in the mouse. III. Histopathological and immunological findings after intravenous infection in the presence or absence o fprevious immunization. Revista da Sociedade Brasileira de Medicina Tropical 18: 101-108, Abr-Jun, 1985 The extent, pattem and number of fungi of the dissemination lesions were similar in both groups. D ISCU SSIO N Intravenous infection of mice with P. brasiliensis as standardized in the present study is a chronic, progressive and systemic granulomatous disease that predominantly involves the lungs and the reticuloen- dothelial system. U p to the 16th week of infection, the mice showed neither clinicai signs of the disease nor mortality. However, in view of the evolving character of the lesions and the extent of lung in- volvement, we believe that the infection would have eventually determined the animais’ death. W hen we compare the present results with those obtained by other investigators who reproduced the P. brasiliensis intravenous infection in mice (Table 3), we note an agreement as regards the systemic, evolving, granulomatous and eventually fatal nature of this Pbmycosis experimental model. As reported for m ice13 17 ancj other animais^, the intensity and nature of the P. brasiliensis experi­ mental infection vary according to the fungai strain Table 3 - Paracoccidioides brasiliensis intravenous infection in mice. Author (year) N.° o f mice Strain treatment Inoculum P. brasiliensis strain Characteristics o f the infection Organs involved Survival Lacaz et al1^ (1949) 9 7 to 12-day culture at 37°C Systemic Granulomatous Lungs Liver Spleen Lymph nodes 6 to 45 days Mackinnonló (1959) 18 Swiss white Strain IHM/1438 Strain IH M /1448 8 to 14-day culture Systemic Slowly evolving Progressive Lungs Liver Adrenal Skeletal muscle Tongue Central nervous system ■ 18 days to 5 months Del Negro & Britcr (1963) 15 2 non-specified strain Systemic Adrenal N o mortality Sacrifice between 25 and 145 days Conti-Diaz & Furcolow* 30 Swiss white Strain IHM/1438 50-day mycelial culture 190 and 950 viable fungi/animal Absence of lesions Absence of lesions N o mortality Sacrifice between 30 and 40 days 10 Swiss white Strain IHM/1438 4-month culture 7650 viable fungi/animal Absence of lesions Absence of lesions N o mortality Sacrifice between 30 and 35 days Linares & F riedm an^ (1972) 110 Swiss white Strain CDI Corticoid treatment Strain 3 (New Orleans) Strain 17 (Caracas) Strain 1, 7, 9, 10, 11, 12, 19 (Medellin) 5-day culture at 37°C, 1x10^ and 5x10^ viable colonies/animal Systemic Slowly evolving Liver Spleen Skeletal muscle Heart Kidneys Skin Pancreas Intestine 10% mortality without corticoid treatment and 27% with corticoid Linares A F riedm an^ (1972) 10 Swiss white Strain Alvarez (Medellin) Culture at 37°C, 2x10* viable colonies/animal Systemic Granulomatous Lungs Liver Spleen Lymph nodes Intestine No mortality Sacrifice between 50 and. 60 days H ay & ChandlerlO (1978) 80 Swiss white Strain 2460 (Atlanta) Culture at 37°C, 1x10^ and 1x10^ viable forms/animal Systemic Granulomatous Early hematogenic dissemination Emphasis on head and neck lesions N o mortality Sacrifice between 1 and 16 weeks Present work 70 Swiss white Strain 18 (Sflo Paulo) 10-day culture at 37°C, 5x10^ viable colonies/animal Systemic Granulomatous Slowly evolving Lungs Liver Spleen Adrenal Kidney No mortality Sacrifice between 2 and 16 weeks 105 Bacchi M M , Franco M . E xperim ental paracoccidioidomycosis in the mouse. III . H istopathological and immunological find ings after intravenous infection in the presence or absence ofprevious immunization. Revista da Sociedade Brasileira de M edicina Tropical 18:101-108, Abr-Jun, 1985 utilized, the inoculum and the infection route. Our present and previous data2 0 2 !, taken as a whole, demonstrate the importance of the inoculation route. The systemic, progressive, granulomatous charac- teristics of the intravenous infection contrast with the localized, benign, non-granulomatous and non-progres- sive nature of the intraperitoneal infection20 21. j n both, early specific cell-immune response occurred and was maintained throughout the infection. The humoral immune response was delayed and less intense in the intravenous infection. Few studies have been carried out on the immune response of mice experimentally infected with P. brasiliensis. As observed in the present investigation and in previous studies® 19 20 21, cell-immune response is detectable 2 to 8 weeks after infection, persisting or decreasing according to the inoculation route. Leveis of circulating specific antibodies tend to be detected later®. The immune behavior of fruit-eating bats (Artibeus lituratus) in response to intraperitoneal and intranasal infection with P. brasiliensis9 parallels that of mice. In the present study, intravenous infection caused a depressed cell-immune response in previously im­ munized mice (Table 1), contrasting with the intra­ peritoneal model, where infection acted as abooster21. Thus the intravenous model mimics the subacute diffuse juvenile form of human Pbmycosis^ which hallmarks are diffuse, evolving infection with depressed immune response. The presence of large number of actively multiplying P. brasiliensis in lesions of both the present experimental model and the human subacute diffuse Pbmycosis represent intense antigen overload, which is probably one of the mechanisms leading to immunologic depression of the host22. The most interesting histopathological finding was the decrease in the number of fungi and intensity of pulmonary granulomas in mice infected after im­ munization when compared to animais only infected. These data indicate that active immunization with intradermal injection of P. brasiliensis antigen was able to induce a certain levei of protection in animais subsequently infected by the intravenous route (infec- tious “challenge” ). In these mice, fungi were destroyed more promptly and effectively and as a consequence the pulmonary lesions were less extensive. Similar to this approach, several studies have demonstrated that immunization schemes using extrapulmonary routes induce humoral and cell-immune response in the respiratory tract1®. Furthermore, the immunization may be protective, as observed in guinea pigs challenged, by the aerogenous route, with Pseudomonas after specific intramuscular immunization23. In the present study, in contrast to what was observed in lungs, the extrapulmonary lesions of mice in the presence or absence of previous immunization were of similar intensity. This appears to indicate a greater ability of the lung to deal with the infection, suggesting a distinct compartimentalized pulmonary response to infectious ‘ challenge ’ after previous immunization. Reports on active immunization as a form of protection against P. brasiliensis are scarce and date back many years1. This is a m atter o f great medicai importance that is only now beginning to be further explored. R E SU M O Cinqüenta camundongos suíços, brancos, com quatro semanas de idade, foram inoculados com 5 x l0 5 form as leveduriformes, viáveis de Paracocci- diodes brasiliensis (cepa 18). D ez destes anim ais tinham sido previamente im unizados com antígeno particulado de P. brasiliensis, durante quatro sema­ nas, por injeção intradérmica. Os controles consisti­ ram de 10 anim ais que fo ram somente im unizados e 10 inoculados com solução salina estéril. Os animais fo ram sacrificados após 2, 4, 7, 11 e 16 sema­ nas. Estudamos: 1) resposta de hipersensibili- dade retardada medida pelo teste do coxim p lan ­ tar, 24 horas antes do sacrifício; 2) anticorpo- gênese específica avaliada pelo teste de imunodifusão dupla em gel de ágar; 3) histopatologia dos pulmões, fígado, baço, supra-renal e rins. Observamos: 1) os anim ais im unizados desenvolveram resposta imu- necelular mais intensa que os infectados; 2) a infec­ ção deprimiu a resposta imunecelular dos anim ais imunizados; 3) a histopatologia da infecção endo- venosa revelou inflamação granulomatosa sistêmica e progressiva. Os anim ais infectados após im uniza­ ção prévia apresentaram inflamação pu lm onar me­ nos extensa, com granulom as menores e com redu­ zido número de fungos. O presente modelo murino de paracoccidioidomicose m im etiza alguns achados da form a hum ana subaguda da micose (doença sistêmi­ ca com depressão da im unidade celular). O esquema de im unização extrapulmonar utilizado fo i capaz de induzir certo grau de proteção do pulm ão contra um desafio infeccioso pelo P. brasiliensis. Palavras chaves: Paracoccidioidomicose mu- rina. Resposta imune. Imunização. 106 Bacchi M M , Franco M . E xperim ental paracoccidioidomycosis in the mouse. III . Histopathological and immunological find ings after intravenous infection in the presence or absence ofprevious immunization. Revista da Sociedade Brasileira de M edicina Tropical 18:101-108, Abr-Jun, 1985 A CK N O W L E D G E M E N T S The authors are grateful to Mr. L u is Gastão Chamma andM rs. Sonia Orsifor technical assistance and Dra. Sheila Z. Pinho fo r the statistical analysis. R E FE R E N C E S 1. Almeida FP. Vacina contra o granuloma paracoccidiói- dico. Folia Clinica et Biológica 10: 195-197, 1938. 2. Campos H. Estatística experimental não paramétrica. Piracicaba, Departamento de M atem ática e Estatística. E SA L Q -U SP, 3? ed„ p. 343, 1979. 3. Coelho KIR, Defaveri J , Iwasso M TR, Peraçoli MT, M ota N G S. Paracoccidioidomicose experimental. In: Paracoccidioidomicose. São Paulo. D el Negro G , Lacaz LC, Fiorillo A M (Eds.) Editora Sarvier-EDUSP, p. 69-84, 1982. 4. Conti-Diaz IA, Furcolow ML. Susceptibility of hamsters and mice to Paracoccidioides brasiliensis using different routes of inoculation. Mycopathologia et Mycologia Applicata 47: 73-79, 1972. 5. Del Negro G , Brito T. Adrenal lesions in mice due to experimental infection by Paracoccidioides (Blastomyces) brasiliensis. In: Annals of VII Congresso Internacional de Medicina Tropical, Rio de Janeiro, p. 118, 1963. 6. Defaveri J , Rezkallah-Iwasso M T, Franco M F. Experi­ mental pulmonary paracoccidioidomycosis in mice: morphology and correlation o f lesions with humoral and cellular immune response. Mycopathologia 77: 3-11, 1982. 7. Fava Netto C. Estudos quantitativos sobre a fixação do complemento na blastomicose sul-americana com antí- geno polissacarídico. Arquivos de Cirurgia Clínica e Experimental 18: 197-254, 1955. 8. Franco M F, Montenegro M RG. Anatomia Patológica. In: Paracoccidioidomicose. São Paulo. Del Negro G, Lucaz LC, Fiorillo AM (Eds.). Editora Sarvier-EDUSP, p. 97-117, 1982. 9. G reer D L, M cM urray D N . Pathogenesis and immune response to Paracoccidioides brasiliensis in the fruc- tivorous bat, Artibeus lituratus. Sabouraudia 19: 165- 178, 1981. 10. H ay RJ, Chandler Jr. FW . Experimental paracoccidioi­ domycosis: cranial and nasal localization in mice. British Journal o f Experim ental Pathology 59: 339-344, 1978. 11. Kaltreider HB. Expression of immune mechanisms in the lung. American Review of Respiratory Disease 113: 347-379, 1976. 12. Lacaz CS, Iaria ST, Ferreira M, Martins A A , Vega VS. Blastomicose experimental. O Hospital 36: 341-349, 1949. 13. Lacaz CS. Micologia Médica. 6? ed. São Paulo, Editora Sarvier, 1977. 14. Linares LI, Friedman L. Experimental paracoccidioi­ domycosis in mice. Infection and Immunity 5: 681-687, 1972a. 15. Linares LI, Friedman L. Pathogenesis of paracoccidioi­ domycosis in experimental animais. Scientific Publica- tion Pan American Health Organization 254: 287-292, 1972b. 16. Mackinnon JE . 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Journal of Clinicai Investiga- tion 68: 1140-1148, 1981. 24. Rifkind D, Frey JA , Peterson EA, Dinowitz M. Delayed hypersensitivity to fungai antigens in mice. I. Use of the intradermal skin and footpad swelling tests as assays of active and passive sensitization. The Journal of Infec- tious Diseases 133: 50-56, 1976. 25. San-Blás F , Cova LJ. Growth curves of the yeast-like form of Paracoccidioides brasiliensis. Sabouraudia 13: 22-29, 1975. 108