CHLAMYDOSPORE FORMATIO N B Y Paracoccidioide s brasiliensis MYCELIAL FOR M

Marcello FRANCO(l) , Ayako SANO (2), Keiji KERA (2), Kazuk o NISHIMURA(2), Kanj i TAKEO(2) & Makot o MIYAJK2 )

SUMMARY

To investigat e th e rol e o f som e advers e environmenta l condition s i n chlamy - dospore formatio n b y th e mycelia l for m o f P. brasiliensis, we culture d fou r P . brasi- liensis isolate s (18 , Bt4 , 1183 , Pb9 ) a t 25° C withi n soli d aga r mediu m eithe r ric h or poo r i n nutrients . Isolate s 1 8 an d 118 3 wer e als o culture d unde r anaerobiosi s in a nitrogen atmosphere . Isolat e 1 8 produced grea t numbe r o f terminal and interc a lary chlamydospor e afte r 7-1 0 day s o f cultur e i n a mediu m poo r i n nutrient s (2 % agar with 0.1 % dextros e an d polypepton) . The thre e othe r isolate s als o produce d chlamydospore s unde r th e sam e condi - tions, bu t i n lowe r numbers . Chlamydospore production b y isolat e 1 8 was abolishe d when th e fungus was cultured i n two aga r media rich in nutrients (brain heart infusio n and potat o dextros e agar) . Anaerobi c incubatio n o f isolat e 1 8 under a n atmospher e of N 2 showe d smal l mycelia l outgrowt h wit h numerou s chlamydospores . A t th e electron microscopica l level , the chlamydospore s showe d on e o r variou s nucle i an d numerous mitochondria , indicatin g grea t potentia l fo r furthe r development . Accor - dingly, chlamydospore s produce d multipl e buddin g afte r onl y 2 4 h incubatio n a t 35°C. The results demonstrate tha t under advers e environmental condition s P . brasi- liensis mycelia l for m produce s chlamydospore s within a short perio d o f time .

KEY WORDS : Chlamydospore ; Paracoccidioides brasiliensis.

INTRODUCTION

Paracoccidioides brasiliensis, the causativ e habitat o f th e fungus . Most author s believ e tha t agent o f paracoccidioidomycosis, is a thermall y infection i s acquired b y inhalation , an d tha t c o dimorphic fungu s producin g a mycelial for m a t nidia ma y b e th e infectiou s propagules 8 20 . Re- room temperatur e an d yeast form a t 37°C . In th e cently th e substrat e an d cultura l requirement s mycelial phase , i t ma y giv e ris e t o eithe r fe w were me t t o th e productio n o f variou s type s o f nonspecific sporulatin g structure s o r charact e P. brasiliensis conidia. Using culture medi a poo r ristic spores 19 20 . in nutrient s an d incubatio n tim e rangin g fro m 3 t o 6 weeks, RESTREPO an d co-workers descr i The exac t mechanis m o f infectio n i s no t bed th e developmen t o f arthroconidia , singl e known, because of uncertainty abou t th e natura l celled conidia and aleuroconidia 3 2 2 23 . The sam e

(1) Departmen t o f Pathology, Botucat u Medica l School , Sã o Paulo Stat e University , Botucatu, Sã o Paulo, Brazil . (2) Researc h Cente r o f Pathogenic Fung i & Microbia l Toxicoses, Chiba University , 1-8-1 , Inohana, Chib a 280 , Japan. Address fo r correspondence: Marcello Franco , M.D. , Departamento d e Patologia, Faculdad e d e Medicina UNESP . CEP 1861 0 Botucatu, São Paulo, SP, Brazil. authors wer e abl e t o separat e an d quantitat e te. The suspension was further homogenize d b y the arthroconidi a an d successfull y infect mic e b y vortexing an d the n filtere d throug h cotto n gau - intranasal instillation , producin g activ e diseas e ze. The inoculu m wa s adjuste d t o a concentra - in health y animals 13. Despite th e fac t tha t chla - tion o f 10 5 t o 10 6 funga l cells/ml . Soli d culture s mydospore formatio n ha s lon g bee n describe d in 9 0 x 2 0 mm petr i dishe s wer e the n se t u p i n at early stage s i n th e mycelia l growth o f P. brasi- duplicates: on e m l o f th e yeas t cel l suspensio n liensis4 5 7 1 7 18 ' 1 9 24 , an d tha t chlamydospore s was carefull y mixe d wit h 2 5 m l o f 2 % aga r (im - seem t o b e th e transitiona l for m i n th e mycelia l munodiffusion aga r fo r Immunoelectrophoresis , to yeas t conversion 4 16 , little attentio n has bee n Oxoid, England), with 0. 1 c /< polypepton (Wak o P u given t o it s optima l cultura l condition s an d po - re Chemicals Ind., Osaka, Japan) an d 0.1 % anh y tential infectivity . CARBONEL L & RODRI - drous dextros e (Wako) (0.1 % IDAgar), a t 40-43°C . GUEZ4 gav e indication tha t chlamydospore for - The culture s wer e prepare d i n duplicate fo r eac h mation i n P. brasiliensis mycelial form occurre d strain. After solidificatio n o f th e agar , the plate s under advers e condition, namel y poo r oxygena - were incubate d a t 25° C fo r 5 , 7 an d 1 0 days . A t tion; usin g slan t cultures , th e author s observe d day 10 , th e plate s wer e incubate d a t 35° C fo r 5 an outermost layer , outside th e cultur e medium , days. consisting of slender hyphae without spores, and a middl e layer , possibl y unde r lo w oxyge n a t For direc t examinatio n o f th e cultures , a mosphere, with abundant intercalar y an d termi - small sample of the medium wa s cut with a razo r nal chlamydospores . blade an d place d o n a microslide togethe r wit h a dro p o f lacto-pheno l cotto n blue ; a cove r sli p While attemptin g t o grow P. brasiliensis my - was the n lai d ove r it , carefull y pressed , and th e celia insid e th e aga r o f a cultur e mediu m poo r specimen wa s observe d directl y unde r a ligh t

16 in nutrients , w e observe d tha t withi n a shor t microscope. For each dish, 2 slides were prepared . period o f time , th e mycelia l growt h produce d The number s o f chlamydospore s presen t i n 1 0 great numbe r o f intercalar y an d termina l chla - to 1 5 high powe r field s (HPF; 400X) o f each slid e mydospores. We hypothesized tha t th e resistan t selected a t random wer e counte d i n a blind way . cells wer e bein g forme d subsequen t t o starv a For histologica l examination , simila r sample s tion and low oxige n content. Th e present investi - from eac h dish were formali n fixed , paraffin em - gation wa s aime d a t studyin g chlamydospor e bedded, cu t i n 1 5 /Lt m sections an d staine d wit h formation amon g differen t isolate s o f P . brasi - PAS, accordin g t o routin e procedures . liensis i n medi a wit h differen t nutritiona l con - tent an d differen t level s o f 0 2 atmosphere . 2. Chlamydospor e formation i n poo r an d ric h culture medi a — Isolat e 1 8 an d thre e differen t MATERIAL AN D METHOD S agar medi a wer e used , namel y 0.1 % IDAgar , brain hear t infusio n an d potat o dextros e aga r 1. Chlamydospor e formation b y P. brasiliensis (Difco). In al l media, th e aga r concentration wa s isolates — Fou r isolate s wer e examined . The y 2%. On e m l o f yeas t cel l suspensio n was mixe d were selecte d a t rando m fro m a collectio n o f with 2 5 ml o f each melted mediu m (appro x 40°C ) stock culture s obtaine d worldwide , namel y and culture d i n duplicate s a t 25° C fo r 1 0 day s strain 1 8 (Department o f Microbiology, Sã o Pau as described above. For the microscopical obser - lo Stat e Schoo l o f Medicine , Brazil) , strain Bt 4 vation, lactopheno l cotto n blu e preparation s (Department o f Pathology , Botucat u Medica l were made with a loopful of each plate; th e num - School, Sã o Paulo, Brazil), strain Pb 9 (Institut o bers o f chlamydospores wer e counte d a s descri - Venezolano de Investjgaciones Cientificas, Cara bed above . cas, Venezuela ) and strai n 118 3 (Center s fo r D i sease Control , Atlant a Georgia , USA). Fres h 3. Chlamydospor e formatio n i n anaerobi c transfers wer e don e t o slant s o f brain hear t infu - condition — Th e techniqu e employe d fo r cultu - sion agar (Difco, Detroit, Michigan), supplemen - ring isolate s 1 8 an d 118 3 o f P . brasiliensis wa s ted wit h 1 % dextrose , an d incubate d a t 35° C fo r the sam e describe d fo r th e previou s experi - 5 t o 7 days, th e growt h wa s harveste d i n steril e ments. Th e cultur e mediu m use d wa s 0.1 % phosphate buffere d salin e (PBS; p H 7.4 ; 1/1 5 M ) IDAgar. Cultures were kept a t 25° C for 7 days unde r by carefull y scrapin g the yeas t mat wit h a pipet - anaerobic condition s i n a nitroge n atmospher e produced a s follows. A BBLGa s Pa k plasti c ja r The number s o f chlamydospore s produce d (Becton, Dickinson Co., Cockeysville, USA) was by th e fou r P . brasiliensi s isolates , expresse d placed inside a vinyl-isolated chambe r fo r hazar - by th e media n o f spore s presen t i n 6 0 HPF (1 5 dous experiments , containin g a sealed entranc e HPF i n eac h cotto n blu e preparation : 2 micro - and tw o vinyl-isolate d window s fo r handling . A slides pe r plate , 2 plate s pe r isolate ) a t da y 10 , continuous flow o f pur e nitroge n (Niho n Sans o are show n i n Fig . 1 . Isolate 1 8 produced a grea t Co. Ltd, Tokyo, Japan) was then produced insid e number o f bot h intercalar y (Fig . 2A, B, C ) an d the chambe r an d th e plasti c jar . Afterward s th e terminal (Fig . 2A , B , D , E , F) chlamydospore s petri dishe s containin g th e soli d culture s wer e after 1 0 day s o f cultur e a t 25°C . A s th e spore s rapidly introduce d insid e th e ja r togethe r wit h were counte d a t randoml y selecte d microscopi - steel woo l coate d wit h coppe r fo r eliminatio n cal fields , th e numbe r o f spore s per HPF show n of th e residua l oxygen . The jar wa s the n sealed . by isolat e 1 8 varie d fro m 9 t o 49 . However, th e process o f sporulation wa s homogeneous through - For coatin g th e stee l wool , approximatel y out th e funga l colonie s insid e th e soli d cultur e 50 g stee l woo l wer e dippe d twic e int o 30 0 m l medium (Fig . 3a) . The chlamydospore s range d of a 1 % cupri c sulfat e solutio n unti l decoloura - in diamete r fro m 5 t o 2 0 /m i an d presente d a tion o f th e solution . Th e copper-coate d stee l thick cel l wal l (Fig . 2). In th e cytoplasm , ther e wool was squeezed to remove th e exces s o f wate r were intensel y blue-staine d area s (Fig. 2). At da y and place d int o th e plasti c jar . Th e coppe r i n 10, when th e plate s containin g numerou s spore s the surfac e o f th e woo l react s wit h th e residua l oxigen wit h th e subsequen t productio n o f cop - per oxide . T o ensur e th e presenc e o f a atmos - phere o f N 2 insid e th e jar , a balloo n fille d wit h the ga s was connecte d t o th e ja r throug h th e lid an d clampe d whe n n o furthe r shrinkag e o f 50- the balloo n wa s observed .

For th e microscopic examination, th e proce - c o dures wer e th e sam e a s used in th e previou s ex - g c periments. 2 4 0 o(/) Q 4. Electro n microscopi c morpholog y o f chla - > • mydospores — Isolat e 1 8 was cultured a s descri- < bed abov e i n 0.1 % IDAga r a t 25° C fo r 1 0 days . i 30 - Afterwards, sample s from th e soli d cultur e me - ^ dium wer e fixe d i n 2.5 % glutaraldehyd e i n O Hank's solutio n (p H 7. 2 - 7.4 ) a t 4° C fo r 4 days , £ j post-fixed i n 1 % osmiu m tetroxid e i n Hank's so- ® 20 - lution fo r 2 h , dehydrate d i n a grade d serie s o f | > ethanol an d embedde d i n Epon-81 2 resin .

Ultrathin section s were doubl y staine d wit h 10- 2% urany l acetat e in 60% ethanol, and lead citra - te. They wer e examine d wit h a Hitachi H-700 H electron microscop e a t 10 0 kV .

RESULTS 18 118 3 Bt 4 Pb 9 ISOLATE Fig. 1 — Chlamydospore formation by four P. brasiliensis isol a We define d chlamydospores , as proposed b y tes culture d i n 2% agar wit h 0.1 % polypepto n an d dextros e AINSWORTH10, a s thick-walle d nondeciduou s for 1 0 days a t 25°C Column s ar e the medians o f the numbers of spore s i n 6 0 high powe r field s counte d i n 4 microslides ( 2 intercalary an d termina l asexual spores forme d slides per plate; 2 plates per isolate). Bars represen t the range by th e roundin g u p o f preexisting cells . between the highest an d lowest numbers . were incubated a t 35°C , chlamydospores alread y The histologica l examinatio n o f th e soli d showed multipl e buddin g afte r 2 4 hour s (Fig . 2 culture mediu m o f bot h matur e an d immatur e G. H , I) . Chlamydospore formatio n b y isolate s chlamydospores showe d simila r finding s t o 1183, Bt 4 an d Pb9 was significantly les s frequen t those describe d abov e (Fig . 3). than b y isolat e 1 8 (Student's t test ; p < 0.001) .

Sporulation b y isolat e 18 showed a positiv e correlation wit h th e poo r nutritiona l conten t o f the cultur e medium . Th e mediu m poo r i n nu - trients (0.1 % IDAgar) yielde d a greate r numbe r of spores (median o f spores per HPF = 22 ; lowes t Sporulation b y isolat e 1 8 was already obser - count = 14 ; highes t coun t = 35) . The tw o ric h ved a t da y 7 , althoug h i t wa s significantl y les s media (BH I an d PDA ) di d no t induc e chlamy - frequent tha n a t day 10 , as demonstrated b y th e dospore formation . median count s o f 4 an d 2 5 spore s i n 6 0 HPF, respectively (Student' s t test ; p < 0.001) . No chla - mydospore formatio n wa s detecte d a t da y 5 . Anaerobic incubatio n o f isolat e 1 8 and 118 3 under a n atmospher e o f N2 resulte d i n a limite d In addition t o typica l chlamydospores, ther e outgrowth a s observed grossl y by th e smal l siz e were numerou s segmentar y swelling s wit h dia - of th e colonie s i n th e soli d medium. Cotto n blu e meter les s tha n 5 fim an d thi n cel l wal l (Fig . preparations o f th e culture s o f bot h isolate s re - 2 A , B, C) ; these structure s wer e interprete d a s vealed hypha l growt h wit h chlamydospor e for - immature chlamydospores . mation (Fig . 4). reticulum, vesicle s an d mitochondria . Th e cel l wall wa s characteristicall y thic k ( = 26 0 nm) an d consisted o f a n electron-translucen t inne r laye r and a n electron-dense granula r oute r layer cont i nuous wit h th e oute r laye r o f th e suspenso r cel l wall.

The transformatio n proces s from hyphae t o terminal chlamydospore s o f isolat e 1 8 culture d in 0.1 % IDAgar fo r 1 0 days was studie d b y ele c DISCUSSION tron microscopy . A t th e earl y stag e intercalar y and termina l cellula r component s o f th e hypha l It i s well known tha t severa l fungi unde r a d form bega n t o roun d u p (Fig . 5). These irregula r verse conditions , suc h a s starvation , exposur e swellings o r immatur e chlamydospore s contai - to membrane-alterin g o r enzyme-activatin g ned on e o r more nuclei , and showe d al l th e orga - agents, develo p unit s o f hypha l differentiatio n nelles presen t i n th e hypha e counterpart ; th e produced b y isotropi c growth 9 25 . Thes e resi s cell wall had als o the same morphological appea - tant cell s ar e calle d chlamydospore s an d the y rance an d thickness . The fully develope d spore s need dissolution o f the supporting hypha e befor e were round structure s wit h one o r several nuclei, dispersal i s possible 6 9 . Th e releas e ca n b e a c numerous vacuoles , lipid droplets , endoplasmi c complished b y mechanica l fractur e o f a non-dif - ferentiated cel l wall o r by natural breaking down " under extremel y advers e conditions t o th e fun - of th e supportin g hypha e throug h bacteria l ac - gus9. I t i s worth pointin g ou t tha t th e biologica l tion, funga l lysis and/o r weathering 9 25 . behaviour o f th e chlamydospore s seem s t o b e close t o that o f yeast cells in latency 21, since the y Isolate 1 8 o f P. brasiliensis, when grow n a t started t o sho w multipl e buddin g afte r 1 da y 25°C insid e a soli d aga r mediu m wit h lo w nutri - of incubation a t 35°C . tional conten t an d poo r availabilit y o f oxygen , produced afte r on e wee k incubatio n a grea t The fin e structur e o f P. brasiliensis chlamy- number o f terminal an d intercalar y chlamydo s dospores showed findings similar t o ungermina -

24 pores, as well as irregular hyphal swellings, inter- ted spore s o f thi s species an d o f othe r patho -

11 1 5 26 preted a s immature chlamydospores . The phe - genic fungi . The presenc e o f on e o r mor e nomenon was not restricte d t o on e isolate, sinc e nuclei an d numerou s mitochondri a ar e indica - three othe r P . brasiliensis isolates were als o abl e tors o f potentia l fo r promp t furthe r develop - to sporulat e under th e same cultural conditions , ment. although with significantly less frequency. As al l isolates teste d wer e simila r a s tha t the y wer e Further experiment s t o produc e P . brasi - originally obtaine d fro m clinica l source s an d liensis chlamydospore s unde r cultur e condi -

12 have bee n kep t i n vitr o fo r lon g perio d o f time , tions tha t woul d allo w thei r isolation an d us e the presen t result may indicat e that , i n additio n to anima l infection ar e now unde r investigation . to environment , chlamydospor e formatio n i s probably unde r geneti c control . RESUMO

The positiv e correlatio n betwee n poo r nutri - Formação d e clamidósporo s pela fas e micelia l tional condition s an d chlamydospore formatio n do Paracoccidioide s brasiliensis by isolat e 1 8 was furthe r checke d in a n experi - ment wherei n th e isolat e tha t produce d abun - O pape l d o conteúd o nutritiv o d o mei o d e dant chlamydospor e wa s culture d i n tw o ric h cultura e de oxigêni o na produção d e clamidós - media an d i n a poo r medium . Sporulatio n wa s poros pel a fas e micelia l d o Paracoccidioides restricted t o th e cultur e mediu m wit h greate r brasiliensis fo i investigado . Quatr o cepa s d o restriction o f nutrients . Thi s findin g reinforce d fungo (18 , Bt4, 1183 , Pb9) fora m cultivadas , a the suppositio n tha t whe n th e mycelia l for m o f 25°C, em mei o sólid o ric o e pobre em nutrientes . P. brasiliensis meets advers e nutritional condi - As cepa s 1 8 e 118 3 foram també m cultivada s e m tion, th e fungu s defend s itsel f b y th e formatio n anerobiose e m atmosfer a d e nitrogênio . A cep a of spores with thic k wall s which wil l eventuall y 18 produzi u grand e númer o d e clamidósporo s stay i n a dorman t stat e unti l improvemen t o f terminais e intercalares após 7-1 0 dias de cultur a the environmenta l conditions . em mei o sólid o pobr e e m nutriente s (aga r 2% , com dextrose epolipeptona 0,1%) . As outras trê s cepas produzira m númer o significativament e Based o n our pilot experiments, we hypothe - menor d e esporos. A cep a 1 8 não produziu clami - size tha t chlamydospor e formatio n i n P . brasi - dósporos quand o cultivad a em doi s meio s rico s liensis might b e multifactorial dependin g on nu - trition an d o n th e oxyge n atmosphere . Accor - em nutriente s (infusã o d e cérebr o e coração , e dingly, w e carrie d ou t a furthe r experimen t t o agar dextros e d e batata) . A incubaçã o anaeró - analyze th e phenomeno n unde r anaerobi c con - bica da cepa 1 8 em atmosfera de nitrogênio apre - dition. sentou pequeno crescimento micelial com a pre - sença d e numeroso s clamidósporos . À nive l ul -

Like othe r dimorphi c pathogeni c fungi 1 14 , traestrutural, o s clamidósporo s apresentara m P. brasiliensis showed growth unde r anaerobio - um o u mai s núcleos e numerosas mitocôndrias , sis althoug h i n a smal l scale . Th e growin g hy - indicativos de potencial para posterior desenvol- phae o f bot h isolate s teste d rapidl y presente d vimento. Assim , o s esporo s produzira m gemu - rounding u p o f som e cells , wit h th e formatio n lação múltipl a 1 dia apó s incubaçã o a 35°C . O s of chlamydospores. This findin g represent s fur - resultados demonstrara m que , so b condiçõe s ther evidenc e for th e rol e o f thes e structure s a s ambientais adversas , a fase micelial d o P . brasi - asexual spore s primarily involve d i n perenatio n liensis produ z clamidósporo s em curt o períod o de tempo. É possível que o fungo encontr e condi - 12. LIU , P. & NEWTON , A . — Rapi d chlamydospor e form a tion b y Candid a albicans i n a buffered alkalin e medium . ções semelhante s n o solo , produzind o o s espo - Amer. J . clin . Path., 25: 9 3 97 , 195 5 ros, que poderia m desempenha r pape l na propa - gação d a paracoccidioidomicose . 13. McEWEN , J. G.; BEDOYA, V. ; PATINO , M . M. ; SAL A ZAR, M . E . & RESTREPO , A . — Experimenta l murin e paracoccidioidomycosis induce d b y th e inhalation of con i ACKNOWLEDGEMENT dia. J . med . vet . Mycol. , 25 : 16 5 175 , 1987 .

14. McVEIGH , I . & HOUSTON , W . E . — Factor s affectin g Dr. Marcell o Franc o wa s a Guest-Professo r mycelial t o yeast phase conversion and growth of the yeas t at th e Researc h Center o f Pathogenic Fungi an d phase o f Histoplasma capsulatum . Mycopathologi a (De n Microbial Toxicoses , Chiba University , unde r Haag), 47: 13 5 151 , 1972 . the auspice s o f Educatio n Ministr y o f Japan . 15. MILLER , S . E.; SPURLOCK, B . O . & MICHAELS , G . E. — Electro n microscop y o f youn g Candid a albican s chl a mydospores. J . Bact. , 119 : 992-999 , 1974 .

REFERENCES 16. MIYAJI , M . & NISHIMURA , K. — Experimenta l funga l infections. In : MIYAJI, M, ed . — Animals model s i n medi - 1. BARTNICK I GARCIA , S . & NICKERSON , W . J. — Thi a cal mycology. Boca Raton, Florida, USA, CRC Press , 1987 . mine an d nicotini c acid : anaerobic growt h factor s fo r Mu - p.1-52. cor rouxii . J . Bact. , 82: 14 2 148 , 1961 . 17. NEVES , J . A . & BOGLIOLO, L . — Researches on th e etio - 2. BARTNICK I GARCIA , S. & NICKERSON, W . J. — Nutr i logical agent s o f th e America n blastomycosis . I . Morph o tion, growth, and morphogenesis o f Mucor rouxii. J. Bact. , logy an d systemati c o f th e Lutz' s disease agent. Mycopa - 84: 84 1 858 , 1962 . thologia (De n Haag) , 5 : 13 3 142 , 1951 .

3. BUSTAMANTE , B. ; McEWEN, J. G.; TABARES, A . M. ; 18. POLLAK , L . — Aleuriospore s o f Paracoccidioide s brasi - ARANGO, M . & RESTREPO , A . — Characteristics o f th e liensis. Mycopathologia (De n Haag), 45 : 21 7 219 , 197 1 conidia produce d b y th e mycelia l for m o f Paracoccidioi - des brasiliensis . J. med . vet . Mycol. , 23 : 40 7 414 , 1985 . 19. RESTREPO , A . — A reappraisa l o f th e microscopica l a p pearance o f the mycelia l phas e of Paracoccidioides brasi - 4. CARBONELL , L . M . & RODRIGUEZ , J. — Transform a liensis. Sabouraudia, 8 : 14 1 144 , 1970 . tion of mycelial and yeast forms o f Paracoccidioides brasi - liensis i n culture s an d i n experimenta l inoculations . J. 20. RESTREPO , A . — The ecology o f Paracoccidioides brasi - Bact., 90 : 50 4 510 , 1965 . liensis: a puzzl e stil l unsolved . J. med. vet . Mycol. , 23 : 323 334 , 1985 . 5 CARBONELL , L . M. & RODRIGUEZ. J . — Mycelial phas e of Paracoccidioide s brasiliensi s an d Blastomyce s der - 21. RESTREPO , A. ; BEDOUT, C ; CANO , L . E. ; ARANGO , matitidis: a n electro n microscop e study . J. Bact., 96 : M. D . & BEDOYA , V . — Recover y o f Paracoccidioide s 533 54 3 1968 . brasiliensis fro m a partiall y calcifie d lymp h nod e lesio n by mycroaerophili c incubatio n o f liquid medium . Sabou - 6. CARMICHAEL , J . W . — Blastospores, aleuriospores, chl a raudia, 19 : 29 5 300 , 1981 . mydospores. In: KENDRICK, B. , ed. — Taxonomy o f fun - gi imperfecta Toronto , Universit y o f Toronto Press , 1971 . 22. RESTREPO , A. ; SALAZAR, M . E. ; CANO, L . E. & PAT I p. 5 0 70 . NO, M . M . — A techniqu e t o collec t an d dislodg e conidi a produced b y Paracoccidioides brasiliensis mycelial form . 7. CONANT , N . F . & HOWELL , A . — The similarit y o f th e J. med . vet . Mycol. , 24 : 24 7 250 , 1986 . fungi causin g South America n blastomycosi s (paracocc i dioidal granuloma ) an d Nort h America n blastomycosi s 23. RESTREPO , B . I. ; McEWEN , J. G.; SALAZAR, M . E . & (Gilchrist's disease) . J. invest. Derm. , 5 : 353-370 , 1942 . RESTREPO, A . — Morphologica l developmen t o f th e c o nidia produce d b y Paracoccidioides brasiliensis mycelia l 8. FRANCO , M. ; MONTENEGRO , M . R. ; MENDES, R . P. ; form. J. med . vet . Mycol. , 24 : 33 7 339 , 1986 . MARQUES, S . A.; DILLON, N . L . & MOTA , N . G . S . — Paracoccidioidomycosis: a recentl y propose d classific a 24. SA N BLAS, F . — Ultrastructure o f spor e formatio n i n Pa- tion o f it s clinica l forms . Rev . Soc. bras. Med . trop. , 20 : racoccidioides brasiliensis . J. med. vet . Mycol. , 24 : 129 132 , 1987 . 203 210 , 1986 .

9. GRIFFITHS , D . A. — The origin , structur e an d functio n 25. SZANISZLO , P . J.; JACOBS , C . W. & GEIS , P . A. — Di - of chlamydospor e i n fungi.Nov a Hedwigia , 25 : 50 3 546 , morphism: morphologica l an d biochemica l aspects . In : 1974. HOWARD, D . H . & HOWARD , L . F. , ed. — Fungi patho - genic fo r human s an d animals . Part . A : Biology . Ne w 10. HAWKSWORTH , D . L., SUTTON, B . C. & AINSWORTH , York, Marce l Dekker , 1983 . p. 32 3 436 . B. C. — Ainsworth & Bisby ' dictionary o f th e fungi . Hu d dersfield, England , H. Charlesworth, 1983 . 26. TAKEO , K . — Ultrastructur e o f polymorphi c Muco r a s observed by means o f freeze-etching. III . Dormant spora n 11. JANSONS , V . K. & NICKERSON, W . J. — Induction, mo r giospore. Arch. Microbiol., 99 : 9 9 107 , 1974 . phogenesis, an d germinatio n o f th e chlamydospore s o f Candida albicans . J. Bact. , 104 : 91 0 921 , 197 0 Recebido par a publicação e m 2/11/1988 .