University of Nigeria Research Publications

OKAFOR, Josephine Ifeyinwa A

utho

r

PG/Ph.D/79/521

Title

Some Aspects of the Ecology, Taxonomy and

Physiology of Basidiobolus Species and Conidiobolus Coronatus

Facu

lty

Biological Sciences

Dep

artm

ent

Microbiology

Dat

e

September, 1984

Sign

atur

e

SOME A S P E C T S O F T H E ECOLOGY, TAXONOMY AND PHYSIOLOGY

O F E A S I D I O B O L U S S P E C I E S AND C O N I D I O B O L U S CORONATUS

J O S E P H I N E I F E Y I N W A OKAFOR (MRS.)

T H E S I S S U B M I T T E D T O ?HE DEPARTMENT O F MICROBIOLOGY,

U N I V E R S I T Y O F N I G E R I A , NSUKKA, I N P A R T I A L F U L F I L M E N T

O F T H E HEQUIHEMCNTS FOR THX AWARD O F T H 6 DEGRXB O F

DOCTOR O F P H I L O S O P H Y I N H I C R O @ I O ~ ~ .

, .

S U P E R V I S O R :

P R O F E S S O R H e C. GUGNANI

DWARTMENT O F MICROBIOLOGY

U N I V E R S I T Y O F N f G E F t I k

NSUKKA.

CONTENTS

LIST OF FIGUHZS * I . LIST OF TCLHLXS --. . . . . . . . -. INTRODUCTION + w

LITEItATURE REVIEW . 0 0. I 0. I.? Discove ry of B a s i d i o b o l u s and Con id iobo lus

and t h e i r taxonomy -. . . . . . . . . 1.2 Epidemiology of human i n f e c t i o n s due t o

B a s i d i o b o l u s h a p t o s p o r u s - . . . . . . . 1.3 Epidemiology o f i n f e c t i o n s due t o

Con id iobo lus c o r o n a t u s . . . . . . . . 1.4 Therapy o f i n f e c t i o n s due t o - B. h a p t o s p o r u s

and 2. c o r o n a t u s . . .... . . . . . . 1.5 Pa tho logy o f phycomycosis due t o g .

h a p t o s p o r u s and 2. coroncltus . . . . . . 1.6 B iochemica l s t u d i e s on B n s i d i o b o l u s and

Con id iobo lus s p e c i e s .. . . ... . . . .

E c o l o g i c a l and Taxonomic s t u d i e s . . . . "' ,",.*f. 0 . .> * . I . ? ' .

Trapp ing o f a n i m a l s .. . . . . . . . . I s o l a t i o n of UasidioboLus from a n i m a l s . . I s o l a t i o n o f Uas id iobo l u s and Con id iobo lus - from s o i l . , s n u f f and d ropp ings -- of .+ b a t s . . I d e n f i c a t i o n o f i s o l n t c s . . . . . . Thermotolerance of i3as id iobolus i s o l a t e s - P h y s i o l o g i c a l t e s t s f a r c h a r n c t e r i z a t i o n o f D ~ s i d i o b o l u s i s o l a t e s .. . . . . 8

PAGE

i

iii

v i

I

5

Haernolysin p r o d u c t i o n

G e l a t i n l i q u e f a c t i o n

D i g e s t i o n of c a s e i n

Urca h y d r o l y s i s . . L e c i t h i n n s e p r o d u c t i o n

Pigment p r o d u c t i o n by B a s i d i o b o l u a i s o l a t e s on t y r o s i n e a g a r medium .. . . .. . Xxper imentn l i n f e c t i o n c f l i z a r d s i n c a p t i v i t y

PHY SIOLOGICr,L STUlJIZS 0. 0.

D e t e c t i o n o f e x t r a c e l l u l a r enzyme p r o d u c t i o n on s o l i d media . . . . . . . . . - .

Amylase p r o d u c t i o n .. . . d . . P r o t e a s e p r o d u c t i o n . . . . . . 9

LipaSu p r o d u c t i o n . . . . . . .. .. Deoxyr ibonuc lense p r o d u c t i o n . . .. .. Hibonuc lease p r o d u c t i o n . . . . .. . P e r i o d i c measurements o f l i p a s e a c t i v i t y o f B a s i d i o b o l u s and Con id iobo lus s p e c i e s

D a i l y maasurements o f t h e p r o t e a s c a c t i v i t y o f i 3as id iobo lus ,~q ,11~~ . C&i.@iobolus s p e c i e s

Chromatographic p u r i f i c a t i o n and some p r o p e r t i e s of t h e m o t e a s e s from U a s i d i o b o l u s and Con id iobo lus s p e c i e s .. - p s s a y o f p u r i f i e d p r o t e n s e .. . . . .

I' .. .L

E f f e c t o f pH and t e m p e r a t u r e on t h e a c t i v i t y of t h e p u r i f i e d p r o t e a s e . . . . . . N o l e c u l a r we igh t d e t e r m i n a t i o n by g e l f i l t r a t i o n . . . . . . . . . . . .

VITHO SENSITIVITY ANTIFUNGAL DRUGS

I V . 1 Fungal i s o l a t e s t e s t e d .. . . . . . . IV.2 Media .. . . . . . . . . . . . I V . 3 A n t i f u n g a l d r u g s t e s t e d and p r e p a r a t i o n of

drug s o l u t i o n s . . . . . . . . . . I . . I Hmphotericin B . . . . . . . . . . I V . 3 . 2 Miconnzole .. . . . . . . . . . . I V . 3 . 3 Ketoconazole .. . . . . . . . . . . IV.3.4 5 -F luorocy tos ine . . . . . . . . . . I V . 3 . 5 Potass ium i o d i d e . . . . . . . . . .

1 V . 4 P r e p a r a t i o n o f i n o c u l a . . . . . . I V . 5 Broth d i l u t i o n method .. . . . . .

i < c 0 1 0 g i c d and Taxonomic s t u d i e s .. . . I s o l a t i o n of B a s i d i ~ b o . 1 ~ . 5 2nd Conidiobolus .... from n a t u r a l s o u r c e s .. . . . . I d e n t i f i c a t i o n of t h e Uas id iobo lus i s o l a t e s

I d e n t i f i c a t i o n of -- Conidiobolus c o r o n a t u s .. T h e r ~ n o t o l e r a n c e of ~ a s i d i o B o ~ u s i s o l a t e s .. C h a r a c t e r i z a t i o n o f t3as id iobolus i s o l a t e s .. Pigment p r o d u c t i o n on t y r o s i n e medium . .

V.8 Expcr i rncnta l i n f e c t i o n o f l i z a r d s i n c a p t i v i t y wi th B a s i d i o b o l u s . . . . . . . .

VI.1 E x t r n c c l l u l a r enzyme p r o d u c t i o n of C a s i d i o b o l u s and Con id iobo lus i s o l a t e s L.-.*. - ... o n s o l i d media . . . . . . . . . .

VI. 2 P e r i o d i c measurements o f l i p a s e a c t i v i t y of . . . . U:.sicliobolus and Con id iobo lus s p e c i e s _*.. -..-----

VI.2.1 Gffcc-I; of pH and t e m p e r a t u r e on l i p a s e x t i v i t y .. . . . . . . . . . . ....

I D a i l y mensurements o f p r o t e n s e a c t i v i t y .... VI. 4 Enzyr.1~ ; > ~ ~ r i f i c a t i o n . . . . . . . . . . VI.4.1 a f f e c t of t e m p e r a t u r e on a c t i v i t y o f p u r i f i e d

p r o t e a s e .. . . . . . . . . . . .... VI.4.2 E f f c c t o f pH on a c t i v i t y o f p u r i f i e d p r o t e a s e

VI. 4.3 i4o lccular weight d e t e r m i n a t i o n . . . . . . Ib i VITRO SENSIBIPITY TO ANTIFUNGAL DRUGS .. Thc e f f e c t o f age o f f u n g a l c u l t u r e s on t h e i r s u s c c i ~ t i b i l i t y t o d rugs . . . . . . . . E f f e c t of t h e medium on t h e a c t i v i t y o f t h e clrue .. . . . . . . . . . . . . . . S e n s i t i v i t y t o d r u g s .. . . . . a * . .

,, ,, .> . * T . ,t' .> * . ? a

The c f f c c t o f d r u g on t h e u l t r a s t r u c t u r e o f a d n s i d i o b o l u s i s o l a t e . .

. I -..-_*__1 .. . . . . . .

DISCUSSION .

REVERENCES . . .

S i x hundred and s e v e n t y s e v e n a n i m a l s be long ing t o I 3 s p e c i e s

of r e p t i l e s and amphibians namely, Agama agama ( A f r i c a n l i z a r d ) ,

Ano l i s c a r o l i n e n s i s ( g r e e n l i z a r d ) , An, a g r e i (cuban l i z a r d ) ,

r i c r i s ~ r y l l u s ( c r i c k e t f r o g ) , I-=? c i n e r e a ( g r e e n t r e e f r o g ) , - Rana u t r i c u l a r i a ( t r u e f r o g ) , Hemidacty lus b r o o k e i (>va l l gecko) , - Bufo bufo ( ~ f r i c a n t o a d ) , 2. marinus ( g i a l t t t o a d ) , 2. ~iefi..a,nuu -- ( t r u e t o a d ) , - 3. t e r r e s t r i s ( c o n t i n e n t a l t o a d ) , Gopherus ~0,1yphemus

( l a n d t u r t l e ) and --- Che lon ia w d a s - ( g r e e n t u r t l e ) t r a p p e d from

Y i g e r i a and F l o r i d a , USA were i n v e s t i g a t e d f o r i s o l a t i o n o f

B a s i d i o b o l u s s p e c i e s . The fungus w a s r e c o v e r e d from t h e i n t e s t i n a l

c o n t e n t s of n i n e o f t h e s e an ima l s p e c i e s . Some of t h e s p e c i e s

v i z , n n o l i s c a r o l i n e n s i s , &. s a g r e i , A c r i s p y l l u s , I h f o ~ x ~ i n u s ,

B. nmericanus and g, t e r r e s t r i s have n o t been known b e f o r e t o habour - t h e fungus i n t h e i r i n t e s t i n a l c o n t e n t s . The i s o l a t e s o f -Bmidio-

bolus t h u s o b t a i n e d were i d e n t i f i e d on t h e b a s i s o f d e t a i l e d - morpho log ica l s t u d i e s under l i g h t microscope as w e l l as s c a n n i n g

e l e c t r o n microscope. Out o f t h e 378 B a s i d i o b o l u s i s o l a t c s o b t a i n e d

from t h e s e a n i m a l s , 358 (9576) wcre - I3. h a p t o s p o r u s and o n l y 12 (3%) ., ,, . ..f. ,I. .> .i* .

were g. ranarurn wh i l e t h e r e r ~ a i n i n g 2% were g, magnus and R. ... h c t e r o s p o r u s .

One hundred and twent: s o i l s amples , 80 samples oi' s n u f f and 1' - ..

50 of b a t d ropp ings were a l s 3 , s t u d i c d f o r p o s s i b l o iso1:;t ion o f

B a s i d i o b o l u s and & r n i d i o b o l ~ z s p e c i e s . A h i g h percentage of

o c c u r r e n c e of Con id iobo lus c o r o n a t u s i n t h e s o i l samples examined, i

probab ly accoun t s f o r many c a s e s o f rhinoentornophthoromycosis among

f a rmkrs and o t h e r ou tdoor

Biochemica l t e s t s on

h y d r o l y s i s , p r o d u c t i o n of

& s i d i o b o l u s i s o l a t e s which i n c l u d e d case in

u r e n s e and l e c i t h i n a s e , g e l a t i n

l i q u e f a c t i o n and pigment p r o d u c t i o n i n t y r o s i n e medium d i d n o t

q u a l i f y a s d i a g n o s t i c c r i t e r i a f o r s p e c i e s i d e n t i f i c a t i o n . The

haemoly t i c a c t i v i t y o f t h e s e i s o l a t e s on e r y t h r o c y t e s oT an ima l s

p a r t i c u l 3 r l y t h o s e of chicken, r e q u i r e s f u r t h e r i n v e s t i g a t i o n f o r

i t s p o s s i b l e u s e a s a c r i t e r i o n i n s p e c i a t i o n .

X x t r a c e l l u l n r enzyme p r o d u c t i o n s t u d i e s r e v e a l c d t h a t

i s o l a t e s o f - B. h a p t o s p o r u s , 2. ranarum and 2. c o r o n a t u s r e l e a s e

l i p a s e and p r o t e a s e i n s i g a i f i c a n t q u a n t i t i e s . Chromntographic

p u r i f i c a t i o n o f t h e p r o t e a s e o f t h e s e t h r e e s p e c i e s showed t h a t

B. h a p t s o p a r u s and 2. ranarum s e c r e t e o n l y one t y p e of a c t i v e p r o t - - ease i n t o the medium w h i l e - C. c o r o n a t u s r e l e a s e s two types . The

p o s s i b l e r o l e o f l i p a s e and p r o t e a s e i n t h e p n t h o g c n i c i t y o f 2,

h a p t o s p o r u s and - C. c o r o n a t u s i s d i s c u s s e d and t h e p r o b a b i l i t y of

E. ranarum b e i n g a l s o a pa thogen of man i n i n d i c a t e d o - C

F i n a l l y , i n v i t r o s e n s i t i v i t y of r e p r e s e n t a t i v c s p c c i c s of

B a s i d i o b o l u s and Con id iobo lus i n c l u d i n g ,lTCC sfrc-?rcS. t o some , , r r . , r t . . , . . i * ' .

a n t i f u n g a l d r u g s namely, po ta s s ium i o d i d e , a m p h o t e r i c i n B,

miconazole , k e t o c o n a z o l e , 5 - f l u o r o c y t o s i n e and t o a n a n t i b a c t e r i a l

d rug , s e p t r i n , showed t h a t miconazole and p a r k i c u l a r l y k c t o c o n a z o l e

a r e most e f f e c t i v e . ~ l t r a s t r u c t u r a l ' * e f f e c t of ke toconnzo le on a n

i s o l a t e o f 2. h a p t o s p o r u s conf i rmed d e t e r i o r a t i o n o f s n b c c l l u l a r ,

o r g a n e l l e s which l e d t o c e l l n e c r o s i s . ~ m p h o t e r i c i n B and s e p t r i n

were a l s o found t o have s i g n i f i c a n t i n v i t r o a n t i f u n g a l a c t i v i t y . -- The s i g n i f i c a n c e o f t h e s e f i n d i n g s ;s d i s c u s s e d .

LIST OF F I G U H U S

FIGURE

3 a s i d i o b o l u s co lony i s o l a t e d by i n v e r s i o n o f a Petri d i s h of SDA o v e r f a e c e s of a l i z a r d on t h e c o v e r of t h e P e t r i d i s h .

A l i z a r d i n c a p t i v i t y f o r s t u d y i n g $he l i f e c y c l e o f 3 a s i d i o b o l u s .

Con tamina t ion o f meal worrils w i t h

S t a n c l ~ r t l c u r v e f o r p r o t e i n e s t i m a t i o n .

Smooth-walled zygos , lo res o f Yas id ioboLus h a p t o s p o r u s i s o l a t e d from a N i g e r i a n , l i z a r d -

Smoothrwalled zygosl lores o f Has ic t ioh? lus h n g t o s p o r u s i s o l u t e l l f rom a n American l i z a r d .

Rough-wnlled z y g o s p r e s o f R a s i d i o b o l u s ranarum.

Scann ing e l e c t r o n micrograph o f B a s i d i o b o l u s h e t e r c 3 s p o r u s zy[<ospores .

La rge g l o b o s e c o n i d i a r3f 3 a s i d i o b o l u s mngnus . Scannint{ e l e c t r o n mic r )graph o f a smooth- w a l l e d zygospore o f Y,. h n p t o s p o r u s i s o l a t e d from Agama agama 0

Scann ing e l e c t r o n micrograph o f a smooth- w a l l e d z y p ) s p o r c ! ~ ~ ' n P + g . h a p t o s p o r u s i s o l r ~ t e d from rrno:iis s a p e i .

Ycnnnin); e l e c t r o n microcraph o f a rough- u a l l e d zy/;ospore ) f :. ranarum.

I P r imary con i r i i a of , A n s i d i ~ b o l u s h a p t o s p o r u s o b t a i n e d i n lnrgc. numbers by f o r c e d d i s c h a r g e from n young c u l t u r e . 6 2

iv.

PAGE FIGURE

13be Subglobose c o n i d i n a t t a c h e d t o s u b c o n i d i a l v e s i c l e .

Scann ing e l e c t r o n micrograph o f a d e v e l o p i n g r e p l i c a t i v e c o n i d i l ~ m o f B h a p t osporus . - E l o n g a t e passively de tached a d h e s i v e conidiutn r ~ f 2. hap tosporus .

Scann ing e l e c t r o n micrograph o f a n a d h e s i v e conidium o f - d. hap tosporus .

M e r i s t o s p o r e s from a young c u l t u r e o f l3. hnp tosporus . - Discha rged pr imary c o n i d i a o f Con id iobo lus c o r o n n t u s

R e p l i c a t i v e c o n i d i a of 2. c o r o n a t u s i s o l a t e d from s o i l .

V i l l o s e conidium from a n o l d c u l t u r e o f C. co ronn tus . - Blood a g a r p l a t e showing h a e m o l y s i s o f humnn e r y t h r o c y t e s by B a s i d i o b o l u s s p e c i e s a f t e r 24 h r s . o f i n c u b a t i o n .

Pigment p r o d u c t i o n by B a s i d i o b o l u s s p e c i e s on t y r o s i n e ngar medium.

A p l a t e o f g e l a t i n medium showing c l e ~ z r zones a round t h e a g a r d i s c s c o n t a i n i n g hyphae of z... -boapbr'us.

C o l o n i e s of B a s i d i o b o l u s s p e c i e s on -. S i e r r a medium s u r r o u n d e d by v i s i b l e p r e c i p i t a t e due t o t h e f o r m a t i o n o f c r y s t a l s o f t h e ca l c ium s a l t o f l a u r i c a c i d . , I- .+

P e r i o d i c measurements o f l i p a s e a c t i v i t y o f t h e c u l t u r a l f i l t r a t e of B a s i d i o b o l u s h a p t o s p o r u s , i. rnnarum and ~ c n i d i o b o l u s c o r o ~ a t u s , -- --

FIGURE

26. l i f f e c t o f pH o n l i p c ~ s e a c t i v i t y o f c u l t u r e f i l t e r a t e o f ! 3 a s i d i o b o l u s and C o n i d i o b ~ > l u ~ i s o l n t e s * G G

l i f f e c t o f t e m p e r a t u r e o n l i p a s c a c t i v i t y o f c u l t u r e f i l t e r a t e of l l q s i d i o b o l u s a n d C o n i d i o b o l u s i s o l a t e s ,

D a i l y m e a s u r e m e n t s o f i ~ r o t e a s e a c t i v i t y o f c u l t u r e P i l t c r a t e of 21 h a p t > ~ s n o r u s , 2. r n n a r u m a n d g. c o r + ) n a t u q , 88

Yephadex G-10.0 column chromatography of p r o t e a s e o f 2. h a y t o s p o r u s (A'I'cI; 3 6 6 0 0 ) ~ 9 1

S e p h n d e x G-100 column c h F o r n a t o p a p h y o f p r o t e a s e o f E. ranarurn (MHI3 ).

1 92

S e p h a d e x (3-100 column ~ h r o r n n t o ~ p - a p h y o f p r o t e a s e o f 2. c o r o n a t u s , 33

X f f e c t o f t e m p e r a t u r e o n n c t i v i t y o f p u r i f i e d p r o t e a s e n f l 3 a s i d i o b o l u s a n d C o n i r i i o b o l u s is ~ l n t e s , 9 5

X f f e c t o f pH o n a c t i v i t y o f p u r i f i e d p r o t e a s e o f B a s i d i o b o l u z a n d C o n i d i o b o l u s i s o l a t e s . 96

E s t i m a t i o n o f m o l e c u l a r w e i g h t s o f p r o t e a s e o f 2. h a p t ' x . p o r u s , g. rannrum rund g. ~ O r O n a t u S by t h e m e t h ( ~ i o f Andrews ( 1 9 6 4 ) . 97

E l e c t r o n microgr,a& , ,af a c r o s s s e c t i o n of a h y p h a l c e l l o f 13. h a p t [ > s p o r u s n o t e x p o s e d t o a n t i f u & n l d rug .

The L i f e c y c l c :>f l 3 a s i d i o b o l u s a s was p L ) s t u l a t c d by L e v i s o h n ( 1 9 2 7 ) - 111

LIST OF TABLZS

10 D i s t r i b u t i o n of H a s i d i o h o l u s s p e c i e s among r e p t i l e s and amphib ians i n N i g e r i a , 48

2. D i s t r i b u t i o n of 3 a s i d i o b o l u s s p e c i e s among r e p t i l e s and amphib ians i n F l o r i d a . U. S. A. 49

3. D i s t r i b u t i o n of 13as id iobo lus h a p t o s p o r u s and C o n i d i o b o l u s c o r o n a t u s i n s o i l s amples from Nige r i a . 5 1

4. The f r equency of o c c u r r e n c e of U a s i d i o b o l u s and Con id iobo lus i n a n i m a l s , s o i l , s n u f f and b a t d ro2p ings . 5 2

50 The e f f e c t o f t e m p e r a t u r e on c d o n y s i z e ( a v e r a g e d i a m e t e r i n mm) o f U a s i d i o b o l u s i s o l a t e s a f t e r 48 h o u r s o f i n c u b a t i o n . 71

6 . Pigment p r o d u c t i o n o f d i f f e r e n t s p e c i e s o f B a s i d i o b o l u s on t y r o s i n e a g a r medium. 74

7. Xxper imen ta l i n f e c t i o n o f caged l i z a r d s w i t h s p o r e s of 13asidiobolus. 77

8. L x t r a c e l l u l a r enzymes of U a s i d i o b o l u s and C o n i d i o b o l u s i s o l a t e s on s o l i d media ( p ~ 6 ) at 23%. 80

3. P u r i f i c a t i o n o f d a s i ! i obo lus h a p t o s p o r u s , E. ranarum and Coni d i o b o l u s g p g ~ n a t u s p r o t e a s e s . ----I!

10, Comparat ive s u s c e p t i b i l i t y of hyphae/ zygospores t o a m p h o t e r i c i n D.

1 7 * The e f f e c t o f mcdium on t h e a c t i v i t y of . ket . , connzolc on B s s i d i o b o l u s i s o l a t e s .

IT .- .+ 101

12- The e f f e c t o f v 3 r i s ) u s a n t i f u n g a l nirugs on i s o l a t e s o f B a s i d i o b o l u s and Con id iobo lus . 103

730 I n v i t r o s u s c e l ~ t i b i l i t y t o s e p t r i n ( T r G o p - i m . ~ n d su lpharne thoxazole , 1 : 5). 104

14, -- I n v i t r o s u s c e p t i b i l i t y t o h i g h c o n c e n t r a - t i o n s o f po ta s s ium i o d i d e . 1 05

INTRODUCTION

B a s i d i o b o l u s and Con id iobo lus a r e f u n g i which be long t o

t h e c l a s s zygomyce tc?~ , o r d e r Entomophthorn les , f a m i l y Antorno-

p h t h o r a c e a e ( f t l exopo lus , 1962; Waterhouse, 1973). T h e i r

v e g e t a t i v e growth i s c h a r a c t e r i z e d by t h e p r o d u c t i o n of b r o a d

hyphae and s i m p l e c o n i d i a which i n b o t h g e n e r a :Ire f o r c i b l y

d i s c h a r g e d ( I n g o l d , 1971) . The s i g n i f i c a n t d i s t i n g u i s h i n g

ch:lractr:rs of t h e genus B a s i d i o b o l u s a r e t h e developrncni; 01

zygospor e s w i t h c h a r a c t e r i s t i c "beaks" and t h e mode o f l i b c r a t i o n

o f t h e f o r c i b l y d i s c h a r g e d c o n i d i a ( I n g o l d 1934). A 1 1 spcc i e s of

B n s i d i o b o l u s p roduce r e p l i c ~ i t i v e ( s e c o n d w y ) c o n i d i a , and dl w i t h

t h e e x c e p t i o n o f 2. microsporus produce p a s s i v e l y d e t a c h e d

e l o n g a t e a d h e s i v e c o n i d i a ( D r e c h s l e r , 1947). The genus LC_oCiobolus

r a r e l y p roduces zygospores b u t forms c h a r a c t e r i s t i c replicative

c o n i d i a iMacleod, I9&). The un ique v i l l o s c s p o r e d i s t i r l ~ u i s h . e s

C. c o r o n n t u s from o t h e r s p e c i e s of Con id iobo lus ( ~ i n g , 1374). ..- l j a s i d i o b o l u s , s i n c e i t s f i r s t i s o l a t i o n by Bidan i n 1886

, L ,, .,. "!. ,t. " > '

i s now known t o be a s a p r o p h y t e o f t h e decay ing v e g e t a t i o n

( D r c c h s l e r , 1947) as w e l l :is n commensal o r g m i s m o f t h o

i n t e s t i n a l t r a c t s of r e p t i l e s , amphib ians and f i s h e s ( I Iu t ch i son

and h i c k e r s o n , 1970; Coreman$ - - - P c l s e n e e r , 1973; T i l l s , 1377;

P o r t o and Milonez , 1979 ; Gugnani and Okaf o r , I 980) . ~~o~~<d~-~o>-,

f i r s t i s o l a t e d by C o n s t a n t i n i n 1897 i s a common s o i l s a p r o p h y t e

found i n l e a f d e t r i t u s ( U r e c h s l e r , 1952). It i s a l s o a !!ell known

p3thogen of s e v e r a l a r t h r o p o d s i n c l u d i n g s p i d e r s xlcl i n s e c t s

(Brobyin and U i l d i n g , 1977; H e m u d i e r e e t a l , 1976)

B a s i d i o b o l u s hnp tosporus and Conidiobolus c _ o r c l i ~ ~ ~ ~ a r e e t i o l o g i c

s g e n t s of subcutanuous phycomycosis and rhinoentomophthoromycosis

r e s p e c t i v e l ~ , two impor tan t human mycotic d i se : l se s cndei:lic i n

t r o p i c a l Africa. and Asia ( J o e - e t -$ a 1 1956; C la rk , 1968; Froment in

and H a v i s s e , 1977; Uksfor and Gugnani, 1983). Uas id iobo lus -Y-----

a t t a c k s most ly c h i l d r e n under 1 5 y e a r s of age, wh i l e ~ o n i d i o b o l u s - - i n f e c t s a lmos t e x c l u s i v e l y a d u l t s 20 t o 60 y e a r s o l d (13ippon,

1982)- I n f e c t i o n s of t h e two f u n g i have a l s o been r c p o r t e d i n

h o r s c s ( Overesm, 1925; Emmons and B r i d g e s , 196-1 ; Hutch ins

and Johnsons , 1972; Cannole, 1973; M i l l e r and P o t t , 1980;

M i l l e r , 1983).

Subcutaneous phycomycosis and r h i n o e n t o m o p h t h o r o i ~ ~ y c o s i s a r e

r e l a t i v e l y benign i n f e c t i o n s and t h e forrner somet incs c l e a r s

spon taneous ly ( J o e - e t -9 a 1 1956)- Most p a t i e n t s , however, r e q u i r e

t r e a t m e n t wi th a n t i f u n g a l d rugs l i k e potass ium i o d i d e and

anphot e r i tin B b e f o r e r ecovery i s ach ieved (Onuigbo and Gugnnni , ,4 ,?.. ..T. ,B. ., % , I # .' .

1976; Karnalarn, and Tharnbinh, 1979; Froment in sncl Xav i s se , 1977;

B i t t e n c o u r t e t a 1 1979, 1382; K e l l y and G i l l , 1980). Some - -' p a t i e n t s were r e p o r t e d t o show no recovery a f t e r pro longed t r e a t -

I

ment wi th t h e s e d rugs ( ~ e s t r e p b -- e"t ‘b1, 1967, I<amal;un and Thambiah,

1978; Nathan e t n l , 1982)- k few d e a t h s a t t r i b u t e d d i r e c t l y t o

t h e s e d i s e a s e s have a l s o been r e p o r t e d ( d d i n ~ t o n , 196Lt; Cameron, et & -

1973; Froment in and Hnvisse , 1977; Greenham, 1379; Aguinr 1 1 9 8 0 ) -

3 There h a s been a l o t o f c o n t r o v e r s y on t h e taxonomy o f

13csidiobolus, The c r i t e r i a u sed f o r s p e c i e s d i f f c r e n t i z t i o n - i n c l u d e , zygospore w a l l u n d u l a t i o n (Drechs l t t r , 1952) ; o p t i ~ l u m

t e m p e r a t u r e f o r g r m t h ( ~ r e e r and Fr iedman, 1 9 6 6 4 , p r o d u c t i c n o f

n c r i s t o s p o r e s ( ~ r e c h s l e r , 1955~1) ; mic rospores fo r inn t ion

(Benjamin, 1962) and S t r e p t o m y c e s - l i k e odour p r o d u c t i o n

( D r e c h s l e r , 1953). S r i n i v a s a n and Thi rumnlachnr (1967) d ismissed

t h c v a l i d i t y of optimum t e m p e r a t u r e f o r growth and n e r i s t o s p o r c

f o r m a t i o n a s d i f f e r e n t i a t i n g c r i - t e r i a s i n c e many o f t h e human and

s a p r o p h y t i c i s k ? l n t e s grew e q u a l l y w e l l a t 2 5 " ~ and 3 7 ' ~ and a l s o

produced m e r i s t o s p o r e s i n K n c i b o r s k i ' s medium. Hu tch i son -a c t - cl

(1972) i n a comprehens ive s t u d y , obse rved much v a r i a b i l i t y among

s p e c i e s o f B a s i d i o b o l u s w i t h r e g a r d s t a t h e s e c h a r a c t e r s . Thus

t h e o n l y s p e c i e s u n e q u i v o c a l l y r e c o g n i z e d a t p r e s e n t as n s c p w n t e

s p e c i e s i s B. microsporus by v i r t u e of i t s p r o d u c t i o n o f u n i q u e

mic rospores ,

The problem o f taxonomy o f B: i s id iobolus s p e c i e s remains

unsolved , Also i n f o r m a t i o n on t h e phys io logy of s p e c i e s o f .<,,.... q. , t . " , s . l * ' .

B a s i d i o b o l u s and Conidiqbo. lus w i t h p a r t i c u l a r r e f e r e n c e t o t h e i r

e x t r a c e l l u l a r f>nzymes i s s c a n t y , The p r e s e n t s t u d i e s werc

t h e r e f o r e u n d e r t a k e n to I - . 3 ' - -L

( i ) c h a r a c t e r i z e i s o l a t e s o f B a s i d i o b o l u s employing

S O R P P ~ ~ ~ e l e c t r o n microscopic a s w e l l as c e r t a i n

b i o c h e m i c a l etu,dies.

4.

( i i ) i n v e s t i g a t e some a s p e c t s o f t h e ecology and phys io logy

of U a s i d i o b o l u s and Con id iobo lus w i t h s p e c i a l rcf wence

t o t h e i r e x t r n c e l l u l a r enzyme p roduc t ion .

( i i i ) de t e rmine i n v i t r o s e n s i t i v i t y of t h e s e f u n g i t o some -- newly developed zint imycotics s o as t o recornmerid b e t t e r

and more e f f e c t i v e d rugs f o r t h e t r e a t m e n t of i n I o c t i o n s

caused by them.

5.

LITXRHTURE RZVIEW

1.1 Discovery o f B a s i d i o b o l u s and C o n i d i o b o l u s and t l ~ e i . T a x o n o m ~

Eidam (1886) f i r s t e s t a b l i s h e d B n s i d i o b o l u s as a new genus o f -1-

s a p r o p h y t i c dn tomophthoraceae and t h i s he c a l l e d D. ranarum. He "". - i s o l a t e d t h i s fungus from t h e i n t e s t i n a l c o n t e n t s o f two s p e c i e s

o f f r o g s , Hana e s c u l e n t a and 2. oxyrh ina . He a l s o i s o l a t e d

a n o t h e r s p e c i e s which h e c a l l e d U. l a c e r t a e f rom t h e dung o f a - l i z a r d , L a c e r t a a g i l i s .

I n 1899, F r i e s d e s c r i b e d a n o t h e r s p e c i e s , -g. jI?y_ophilus

from z o o g l o e a a s s o c i a t e d w i t h a r o t t i n g c o n i f e r o u s stump.

D r e c h s l e r (1952 , 1953) d e s c r i b e d two s p e c i e s o f E a s i d i o b o l u s

which h e i s o l a t e d from l e a f c u l t u r e s from desiduous woods o f

v a r i o u s a r e a s i n t h e S t a t e o f Maryland, UoS,A. Bc o b s e r v e d t h a t

t h e two deve loped numerous zygospores o f a b o u t 30 i n d i a m e t e r and r '

each w i t h p a i r e d "beak- l ike" p r o t u b e r a n c e s ( r e m n a n t s o f t h e

c o p u l a t i o n tubes) . . He a l s o n o t e d t h a t one o.T t h e s p e c i e s p o s s e s s e d

on ly smooth-walled zygospores and t h i s h e c a l l c d 2. h s p t o s p o r u s .

The o t h e r s p e c i e s , h e named 2. ranarum b e c a u s e it had rough-wal led

z y g o s p o r e s and a l s o p o s s e s s s e b . s & r o n ~ ."earthy1' o r "st;reptomyces-

l i k e 1 ' odour s i m i l a r t o that of a n i n s e c t i c i d e benzene h e x a c h l o r i d e . I n

1955 h e d e s c r i b e d a n o t h e r s p e c i e s , H. ~ i e r i s t o s ~ r u ~ -+ which

r e s e m b l e s - B. kinptosporus i n a l l a e s p & t s e x c e p t t h a t , i n a d d i t i o n ,

i t p rodncee numerous m e r i s t o s p o r e s by c l e 2 v a g e o f c o n i d i n l contents.

Benjamin (1962) i s o l a t e d from dungs o f r o d e n t s , . a new s p e c i e s ,

B. m i c r o s p o r u s which h a s rough-wal lcd zygosporec and un ique - microspo re s .

k new s p e c i e s , 2. magnus was i s o l a t e d and d e s c r i b e d by

U r e c h s l e r ( 1964) . It r e s e m b l e s 2. rnnarum i n odour p r o d u c t i o n

and possession of rough-wal led zygospores b u t d i f f e r s from i t

by t h e f a c t t h a t i t p roduces l a r g e r c o n i d i a and z y e o s p o r c s

(60p i n d i a m e t e r ) .

S r i n i v n s a n and Th i ruma lacha r (1967) d c s c r i b c d rrnothey s p e c i e s ,

13. h e t e r o s p o r u s which p roduces b o t h smooth and rough- - w a l l e d zygospores .

J o s u e :md Quimio ( 1976) d e s c r i b c d i l s p e c i e s , A. ~ ~ h > J , i j ~ i n e i l s i ~ ,

which l i k e - :3. h a p t o s p o r u s p o s s e s s e s srnooth-walled zygo,sporcs.

S e v e r a l o t h e r s t u d i us h:ive shown t h a t 13 ,mid iobo lus i s wor ld - -* . wide i n d i s t r i b u t i o n . Apart from i t s f r e q u e n t i s o 1 n t i o ; l f rom

dccny ing v e g e t a t i o n i t is a l s o r e a d i l y i s o l a t e d from t h e

i n t e s t i n t r l c o n t e n t s o f r e p t i l e s , a tnphibians and f i s h i n c c v e r a l

c c u n t r i o s ( H u t c h i s o n and Nicke r son , 1970; Corernms-Pelocnccr ,

1980; I r r lw id thsy~ - e t -9 a 1 1982) . I t h a s r t - c c n t l y 5ecn isol,..l;ed

from t h e i n t e s t i n a l c o n t e n t s o f an i n s e c t i v o r o u s b a t (Clic.,l;urvedi

c t a1 1984) . - -1 . l . '

A f t e r t h e d i s c o v e r y o f i 3 a s i d i o b o l u s l t h e r e w:xs z l o t o f

c o n t r o v e r s y w i t h r e g a r d t o i t s taxonomy. The c r i t c r i a Por i t s

d i f f e r c n t i n t i o n i n t o s p e c i e s i n c l u d e zygospore w a l l ( s m o o t h v e r s u s , I .. .L

r o u g h ) ; optimum t e m p e r a t u r e f o r g rowth; p r o d u c t i o n 01 inc.1-inti:spor :s ,

exogenous rn i c rospo rc s and s t r e p t o m y c e s - l i k e :>dour. j 3 ~ ~ & i i ~ J - o l u s

was i n i t i n l l y d i v i d e d i n t o t h r e e fl;roups b a s e d on t h c zy;;os],ors.

wn l l . The f i r s t g roup p roduces rough-wal led zygs j spo rc s ? s w e l l

.is :; t r cp tomy c, .s- l ike odour ; ~ n d i t i n c l u d e s ;.rsnnrum_ (3-elm 1886) ,

2. p ? . ( U r e c h s l c r i 1947) and 2. micronporus (Bcnjamin 1962).

The zecond group which produces on ly smooth wa l l ed zygosporcs

i r~c luc les 2. haptt-,sporus ( b r e c h s l e r , 1947) , go l ne r i s tosporus

( D ~ c c h s l e r , 1955a) and - 3. p h i l i p i n e n s i s ( J o s u e and d u i n i o , 1976).

Tile t h i r d group which c o n t a i n s on ly 2. h e t c r o s p o r u s ( S r i n i v a s a n

aid Thirumalachar , 1967) produces both rough- and smooth-wallcd

Locwcnthal (1903) , Levisohn (1927) ; C i f e r r i , 1958 and Benjan:iu

(19G2) i n t h e i r s e p a r a t e s t u d i e s c o n Bas id iobo lus i s v l n t c s

ccilcluded t h a t - B. l a c e r t r . ynd B. myxophilus (Xidam 1886)a re

:synonymous wi th 2. rnnnrum, King (1979) a l s o found no

d i i f e r c n c e s between - 2. p h i l i p i n e n s i s and 2. haptosporus .

J o e Gt (1956) d e s c r i b e d a human pa thogen ic Bas id iobo lus

which they i s o l a t e d from subcutanevus granuloma of t h e s k i n as

l3. rnnx-urn, Morphologica l o b s e r v a t i o n of many o t h e r human . - -.-,-

i s o l a t e s showed t h a t they a l l produced smooth - wal led zygosporcs.

Greer and Priedman ( I 966n) c a r r i e d ou t a therrnotolernnt s tudy on

n nul,lbcr o f i s o l a t e s of Bas id iobo lus and observed t h a t i s o l a t e s

., ,, . . * a . \t. > ' . 0 0 wi th smooth-walled zygospores grkw both a t 25 C and 37 C whi le

(3 t h o s c wi th rough-wallad zygospores grew on ly a t 25 C. They

f i n a l l y concluded t h a t t h e pa thogenic s p e c i e s shou ld be s e p a r a t e d

from t h e s a p r o p h y t i c specicsl'act!dik.ding t o t h e i r a b i l i t y t t ) grow

0 2.t 37 C.

S r i n i ~ ~ ~ s a n and Thirumalachar (1967, 1968) c a r r i e d o u t a

coliiparative s tudy on l a r g e number :)f i s : , l a t o s o f Bns id iobo lus

Proin p l a n t d e t r i t u s and i n t e s t i n a l a ~ n t e n t s of f r o g s and l i z a r d s

8.

i n o r d e r t o de t e rmine whe the r , t h e taxonomic s t a t u s g i v c n t o 2.

~ ~ c r i s t o s p ~ r u s was v a l i d . They obse rved t h a t - 13. ---A meri - tor . )olaus __.-- c l o s e l y resembled - 2. hnp tosporus excep t f o r t h e s l i g h t difference

i n s i z e of t h e pr imary c o n i d i a and a l s o f o r t h e f a c t t h a t t h e

former p roduces rne r i s tospores , However, t hey f ( ~ u n d t h n t

rne r i s tospore p r o d u c t i o n c o u l d be induced i n any o f t h e two s p e c i e s

by i n c q r p o r a t i n g a n ammonium s a l t ( e g g ammonium c h l o r i d e ) i n t o

t h e growth medium' ( S r i n i v a s a n 2nd 9'hirumnlachar,

1965). They] t h e r e f o r e included t h n t B. e e r i s t , s ?o rus i s I -

synonymous wi th - B. h a p t o s p o r u s and t h a t t h e fo rmnt i9n o f

m o r i s t ~ s p o r e s was nr:t o f any tnxunomic s i g n i f i c a n c e . In 90 ~f er

s/-lrdy they a l s o compared some p ~ t h o z e n i c i s o l a t e s o f ---).I-P- Basid. iobolus

from I n d i a and Uganda w i t h s a p r o p h y t i c ones from p l a n t d c t r i t u s

and found t h a t t h e y a l l p o s s e s s z d smooth-walled zygosporcs ?.nd

a l s o grew e q u a l l y w e l l a t b o t h 28"~ and 3 7 ' ~ w h i l e i s o l e t c s from

0 h i l l y a r e a s ( w i t h t e m p e r a t u r e a t 5- 20 C f ,>r most o f t h e y e s )

f a i l e d t o grow a t 37'~. They t h e r e f o r e concluded t h a t t c n p c r c t u r c

of b e s t growth s h o u l d n o t be used i n s e y a r n t i n g t h e p a t h o g e n i c

.,,, .," .. .%J , ;C. .>;'..,M .'. . s p e c i e s from t h e sap roph 'y t l c ones. But b ~ c ( ; t , ~ c fAe p a t h o g e n i c

s p e c i e s ! p o s s e s s e d smooth-walled zy g a s p o r c s , O r e - /O I&>- - r e c o g n i z e d as B. h a p t o s p o r u s and n o t 3. runarum a s ~ g c s e a r l i e r - - -.I- i d e n t i f i e d by J o e e t a1 (1956) .-- -bugnuni and Oknfor (1980) found -- t h a t a l l t h e i r i s o l a t e s of b a s i d i q b o l ~ s w i t h smooth-walled

zygosporcs (g. haptr ,s!)orus) from r e p t i l e s and amphibians , krcw

w c l l a t 3 7 O ~ ( a l t h o u g h growth a t 25% w a s g e n e r a l l y b c t t c r ) and

some o f them formed norietospores. They, t h e r e f o r e concluded

0 0 t h n t a b i l i t y t o grow b e t t z r a t 37 C t h a n a t 25 C m d f o r m a t i o n

of rne r i s tospores canno t be c o n s i d e r e d v a l i d taxonomic c r i t e r i a

f o r s e p a r a t i o n o f t h e spr:cies of Uns id iobo lus .

C u t l e r and Swatek (1969) i n a a t t e m p t t o f i n d a s o l u t i o n

t o t h e taxonomy of B a s i d i o b o l u s s t u d i e d 29 i s L > l a t e s u h i c h i n c l u d e d

9 human and 20 s e p r o p h y t i c s t r a i n s . They obse rved t h a t a l l human

i s o l a t e s produced a b l a c k pigment i n t y r o s i n e aga r mecliun w i t h i n

48 h o u r s a t 3 0 ' ~ whi l e a l l t h e 20 s a p r o p h y t i c s t r a i n s pror!uced

e i t h e r p i n k pigment o r none. h r e g u l a r s u b c u l t u r i n g o f t h e s e

i s c l a t e s o v e r a p e r i o d of one y e a r as w e l l as passage t l i rough t h e

g u t s of t o a d s and f r o g s d i d n o t r e s u l t i n any change i n pigment

p r o d u c t i o n of each i s o l a t e . They, t h u s , concluded t h n t p i e r e n t

fo rmnt ion is 21 r e l i a b l e c r i t e r i o n f o r s e p a r a t i n g pat1iol;cnic s p e c i e s

from t h e s a p r o p h y t i c o n e s , and t h a t a d e f ~ . n i t e c n r r e i n t i o n e x i s t s

between b l a c k pigment p r o d u c t i o n and p a t h o g e n i c i t y . Thcy , howcver , n ~ t e d t h n t pigment p r o d u c t i o n had no c o r r e l a t i o n wi th t h e

zygospor e w a l l .

Hu tch i son c t a1 (1972) s . tudiqd , 169 i s o l a t e s from ior ic r v c r t e - -- .. ,,+. '1. ,t .,.,I2

b r n t e s and 8 ATCC i s o l a t e s , u s i n g s e v e r a l c h a r a c t e r s ;? rcvious ly

employed i n t h e taxonomy o f Bas id iobo lus . They found among t h e s e

s p e c i e s much v a r i a b i l i t y and l a c k of c o r r e l a t i a n i n %hc t:ixonornic I' - .L

c h a r a c t e r s such as .~bse r i ca of odour 2nd l o s s of a b i l i t y t o produce

zygospores i n some s t r a i n s a f t e r some p e r i o d s of s u b c u l t u r e . They

concluded t h a t t h a o n l y s p e c i e s t h a t can be u n e q u i v o c a l l y identified

i s C. ~m,i,crosporus by v i r t u e o f t h e p r e s e n c e o f microspores .

They, howcver, s t r e s s e d t h a t u n t i l f u r t h e r s t u d i e s were under t aken

t o c l u c i c l i t e more dependable c r i t e r i a , t h e c h a r a c t e r of t h e

zygospore w a l l appeared t o be t h e most r e l i a b l e c r i t e r i o n t o be

employed i n t h e taxonomy of t h e fungus.

Thc Genus Con id iobo lus is r c p r e s e n t e d by twenty seven s p e c i e s

(King, 1377) i n c l u d i n g n w e l l r e c o g n i z e d human pathogen, - C ,

coronntus. It was f i r s t i s o l a t e d from p l a n t d e t r i t u s by -- - -a -- C o n s t a a t i n ( 1897) who d e s c r i b e d i t as B o u d i e r e l l a c o r o n a t a , The

n a t u r a l h a b i t a t i s t h e s o i l and decaying v e g e t a t i o n ( D r e c h s l e r ,

1952, 1960). L ike 13ns id iobo lus , i t i s a l s o world wide i n

d i c t r i b u t i o n ( G u s t a f f s s o n , 1965; Hutchison, 1963; I" l i l le r , 1983).

Ho i~cvcr , i t h a s s o f a r not been ism3lnted from r e p t i l e s and

a m p h i b i r ~ l s 2nd i ts n s s o c i n t i o n wi th an ima l s is p u r e l y p a r a s i t i c ,

u s u a l l y c m s i n g disease. I t h a s a l s o been i s o l a t e d from a

chimnpnzce (Roy and Cameron, 1972); from h o r s e s (Emmons and

U r i d g c ~ , 7961 ; M i l l e r , 1983) and from n mule (Carva lho , 1976).

Apart from C. c o r o n a t u s , a n o t h e r s p e c i e s o f Con id iobo lus , v i e , - . , , . . . , I '

C , i nconvruus h a s been r e c e n t l y r e c o g n i z e d as a human pathogen - ..--k---

(King aild Jong, 1976). Yhih s p e c i e s was f i r s t i s o l a t e d from p lan ( ;

d e t r i t u s ( D r e c h s l e r , 1967) id l a t e r from man ( G i l b e r t e t a1 - - 9

1970; i k k c r t e t a 1 1972). !a .- .& - - 9

I d e n t i f i c a t i o n o f C. c o r s m t u s h a s n o t been a r e a l problem; - -- howavcr, t h c r e h a s been much c o n f u s i o n wi th r e g a r d t o i t s

c o r r e c t name and tnxnncir~iic p o s i t i o n . A f t e r i t s f i r s t ic'.c.ntificn-

t i o n by C o n s t a n t i n :IS I- loydierelJa c o r o n a t n , l(evor1rinn (1337)

renamed i t khtorno;~hthorn c o r o n n t a and t h i s name wz:;. r c t a i n ~ d

u n t i l 1964 when i t Was f i n a l l y c a l l e d Conidiob > l u s c ~ l r o n ? ~ t u s by -- - Q C ..--a

S r i n i v n s a n and Thi rumalschar . The main problem i n Je k c rhl ining

t h e c o r r e c t name of t h i s s p s c i e s h a s r e s u l t e d f rom the l a r g e r

problem of s e p a r a t i n e t h e gene r? lhtornophthora an(? c r J ~ ; $ , i ~ ~ i c ~ b o l ~ s .

For many y e a r s t h e c r i t e r i o n used t o s e p a r a t e t h e s e c c n c r n was

p a r a s i t i s m on i n s e c t s by &nt,-imophthora and t h e s a p r c $ t i c h a b i t

o f Con id iobo lus (McLeod, 1963; Waterhouse, 1973). T h i s

d i s t i n c t i o n i s now n o t t e n a b l e f w many Conidiqbo1,u. :,;~ocit.s

a r e b e i n g found i n a s s o c i a t i o n w i t h i n s e c t s ( i iemaudicrc g,

1976). S r i n i v a s n n -- e t a 1 (1964) proposed t h o s e p n r z t i o n of

t h e s e g e n e r a by t h e ch r i r ac t e r of t h e pr imary conicl iophorcs:

macr oner~lous i n L'ntornophthora 2nd rnicrnnemous i n Coniclic;bolus. --

T h i s c r i t e r i o n w a s l a t e r found n o t v a l i d s i n c e MacLuod and

Muller -Kc~gler ( 1973) cha rnc t e r i z e d a t l e a s t s even E n t ~ r ~ ~ o l ~ h t h o r a -.----

s p e c i e s :is hav ing ~ n ~ i a i u , p k ) r e s t h a t fire n o t d i s t i n c t l y s w o l l e n

w h i l e some s p e c i e s of Con id iobo lus (2. a d i a o r e t u s ~ r c c h s l e r )

have l a r g e pr imary coni d iophores . King ( 1974, 1376) c:cvelopecl

rnethods.anJ a key f o r i l ie l1 l ; t i f i ta t ion - . o f t h e s p e c i e s 0;' t h i s gcnus

Con id iobo lus and twenty seven s p e c i e s a r e p r e s e n t l y : e c o p i z e d

King (19763) nbservecl t h a t s p e c i e s o f ConidiL3Lo&s, do n o t

t end t o l o s e t h e i r a b i l i t y t o s p o r u l a t e as r a p i d l y as .those o f

B a s i d i o b o l u s . HI-. s t a t e d t h a t m u l t i p l e c o n i d i a l for!iw.tidn is

n o t nf g r e a t impor t ance i n i d e n t i f y i n g 2. coronatu2- b u t s h o u l d

b e obse rved b e f o r e an i s o l a t e is i d e n t i f i e d ss 2. i . i i ~ c i - i ~ u g

w h i l e v i l l o s e s p o r e s must be obse rved t o i d e n t i f y a n i s o l a t e

a s go coronn tus .

1.2 Spiderniology of Human I n f e c t i , ~ n s Due t o 3 a s i d i o b o l ~ ~ ~ ~ l i > t _ o s l ~ o r u s 3

I n f e c t i c n due t n - B. h n l ~ t o s p n r u s i s termed s u b c u t ~ . n e o u s

phycomy c o s i s (entomophthoromycosis bas id iobo1:~e) . Thc f i r s t

r e 2 o r t o f t h e d i s e a s e supported by myco log ica l conf i r i i l a t ion cane

from I n d o n e s i a i n 1956 by ~ o e e t n l . Previously, the

:!isease was r e p o r t e d i n a h o r s e (Van Overeern, 1925) :.ud p o s s i b l y

i n a man ( C a s s g r a n d i , 1931) b u t no c u l t u r a l s t u d i e s were made.

The e t i o l o g i c a g e n t was i d e n t i f i e d as - B. ranarum but s i n c e t h e n

i t h a s been r e i d e n t i f i e d a s 2. hap tosy~orus . .,,,... I . . 1 . . > . . , * .

A f t e r t h i s f i r s t r e p o r t by J n e , s e v e r a l more c a s e s were

1962)- I n Sntyland, two c n s e s were obse rved (S~rnmers , ,1960, 1964) - .+.

~ n d one n f t h e p a t i e n t s was fount1 t o have l i v c d i n TnJoncs in ,

The m a j o r i t y o f r e p o r t e d cases came from ~ f r i c a i n c l u d i n g U ~ a n d a

13

1976) and Ni l ;e r ia ( L l e b u t e ant! (jkubade j o , 1962; ~arrnan.52 2,

1964; Eclingtun, 1964; Mar t inson and C l a r k , 1967; O n u i ~ b o and

Gugnaui, 1976). O t h e r s a r e fror?, Cameroon ( d l a c h e c-i; n l 1961; -*1 .-*I-

Garnet nn~! Hrc>ttcs , 1963) , Sudan (Lynch and ;lusbaiid, 19621,

Kenya (Comcron e t a 1 1973; Greenham, 19791, Scnepl asset - -1

e t a1 1963; S n r r a t and Irnbert , 1973)$ I v o r y C O L ? S ~ ( f a g a t e e t a l , - -9 -- 1963; Loub ie rc -- e t n l , 1976) , Ghana ( B u r k i t t -. e t .-- nl 1964.1, Upper

Voltn (Lasch & al, 1965) anti S i e r r a Leone ( K e l l y 2nd G i l l ,

1980). Many c a s e s were a l s o found i n o t h e r t r o p i c a l c o u n t r i e s

l i k e I n d i a ( ~ u k e r j i -- e t a l , 1962; Basse t etg, 1963; i toshi et 2,

c o u n t r i e s l i k e Burma ( S t o c k d a l e , 1964; Grcer and Fr iedimn, 1966)

and I r a q ( R i d l e y and h i s e , 1965). A few c a s e s wcrc uncountered

i n s,)me s u b t r o p i c a l c o u n t r i e s i n c l u d i n g Columbia nnd 12rozi l

B i t t e n c r ~ u r t , 1979, 1980, 1982). Hecent ly olio c m c w a s r e p o r t e d

i n t h e Uni t ed S t a t e s o f America (Uworznck -- c t a l , 1378). The

d i s c n s e :i?pears t o occur predominant ly i n t h e t r o p i c s and. . , , , . . . w s, ,*. ' .

o c c a s i o n a l l y i n t h e s u b t r o p i c s c iespi te t h e spori~2,i.c world-wide

d i s t r i b u t i o n of t h e fungus. M a j c r i t y o f t h e cases u c r e r e p o r t e d

i n c h i l d r e n under 15 y e a r s of age and e s p e c i a l l y i n ride c h i l d r e n , ,t - -L

The d i s e a s e o c c u r s p r i m a r i l y i n t h e subcutnncouc t i s s u e

i n v o l v i n g u s u a l l y t h e t r u n k , b u t t o c k s , t h i g h ( J o c . c t - -. n l , 1960;

Lynch and husband, 1962; IIxrnnn - c t - v a 1 1964; C l a r k , 1968; Ke l ly

: m c l G i l l , 1980) and o c c n s i o n d l y t h e s c a l p ( ~ a ~ i h a k r i s h n a m u r t h y , 0 I n a few o t h e r c a s e s , involvement of under ly ing o rgans

l a hcen r e p o r t e d . I n Nit;erin, Xdington (1964) r e p o r t e d n

cnsc which invo lved t h e l a r ~ e i n t e s t i n e whi le Onuigbo and

Cugnani (1976) observecl a n involvement <)f the stomach wi th

c i i l ~ r ~ e m e n t of many c ~ n t i g u o u a lymph nodes. Lymph node invnsiolz

11ns a l s o been - r e p o r t e d i n o t h e r p a r t s of t h e

world (Vanbreuseghem, 1970; Kamnlam and Thambinh, 1975). il c a s e

of ~ a s t r o - i n t e s t i n a l i n v a s i o n was r e p o r t e d i n B r a z i l by q u i n r

(1960) and pulmonary i n v a s i c ~ n wi th v i s c e r a l involvement was

I - c p s r t c d by R i t t e n c o u r t (1980). I n U.S., Dworzack -- e t a1 (19783

r e p o r t e d :i c a s e i n v o l v i n g t h e m a x i l l a r y s i n u s and p a l a t e i n

a r~licldle aged man.

N a t u r a l i n f e c t i o n s a l s o occur i n h o r s e s (Cannole, 1973;

Pi-illcr and P o t t , 1980; M i l l e r , 1'382, 1983) and i n f r e s h water

f i s h (Cyprinus c a r p r i o ) (Yank:, 1962; Chien, 1981).

Attempts a t e x p ~ r i m e n t a l i n l e c t i o n of s e v e r a l l a b o r a t o r y

nilimals v i a in t r a t l e rmal l .4wtmVenous, i n t r a p e r i t o n e a l o r

:;dbcutaneous r o u t e s f z i l e d t o i n i t i a t e p r o g r e s s i v e d i s e a s e

(Clrcer and Priedman, 1966; Coremans-Pclseneer , 1974 ; Okofor

1977; Tio and V r i e s , 1977, a i t t e n c b u r t Gal, 1982). However,

X a h s and Corbel (1979) ]~ro luced prc;t:rossive d i s e a s e when conir'tin

o f .- E n s i d i c b ~ l u s were i n j e c t e d i n t r a c e r e b r a l l y i n t o mice.

The p o r t a l o f e n t r y o f t h i s organism i n t o human t i s s u c i s

no t w e l l untierstoorl, Some workers b e l i e v e t h a t i t p i n o ~xcccss

t o t h e t i s s u e by d i r e c t i n o c u l a t i o n from t h e ve, :etat ion o r

p o s s i b l y th rough i n s e c t and m i t e b i t e s (Baker e t al, 1962).

They a rgued t h a t t h e h i g h f requency o f l e s i o n s arnund t h e

b u t t o c k s and t h i g h s i s due t o b i t e s by i n f e c t e d m i t e s when t h e

i n d i v i r l u a l l i e s down t o s l e e p . C l a r k (1968) sug, . ;ested t h a t Lhc

o r ~ c m i s m g a i n s a c c e s s i n t o t h e body throuil;h t h e a c t o f defaecntion.

She a rgued t h a t most of t h e a f f e c t e d c h i l d r e n were unlil .cly t c

u s e napk ins o r sl,f t t i s s u e s f o r c l e a n i n g up a f t e r d e f a e c a t i o n ~

I n s t e a d t h e y most o f t e n u s e h a r d l e a v e s i n t h e v e g e t a t i o n which

o f t e n c a r r i e d organisms, S i n c e s k i n t rauma is f r e q u e n t dur ing

c l e a n i n g up wi th t h e s e h a r d l e a v e s , organism on them g e t ,lnntecl

i n t c t h e t i s s u e , Female c h i l d r e n , however, t e n d t o bc more

m e t i c u l o u s i n t h e i r h a b i t s and t h i s , a c c o r d i n g t o h e r , might

cx1,lain t h e s e x d i f f e r e n c e i n t h e d i s t r i b u t i o n of t h e d i s e a s e .

Accordink; t o Harman (1364) h p o s s i b l e method of i n f e c t i o n ic

N i g e r i a n c h i l d r e n he encoun te red was by d i r e c t i n o c u l a t i o n o f ,,,,... k q . , t . , ~ , ~ a a .

s p o r e s i n t o t h e s k i n from i n f e c t e d f a e c e s of a n i m a l s s c a t t e r e d i n

t h e environment . Most houses e s p e c i a l l y i n t h e t r o p i c s , havc

l o t s o f l i z a r d s anti g e c k l ~ s millint:; around them and dropping t h e i r I t "- --

f a e c e s a l l ove r t h e p l a c e s . S i n c e most c h i l d r e n , p a r t i c u l ? c l g

i n t h e r u r a l a r e a s go about nsked most of t h e t i m e , t h e y of.Lcn

p;et i n f e c t e d e a s i l y wh.ile playin:; i n a r e a s h e x v i l y contruilinated

w i t h s p o r e s of t h e fungus. A r a r e p o r t a l o f e n t r y f o l l o w i q

i n t r a n ~ u s c u l n r i n j e c t i o n was r e c e n t l y r e p o r t e d by Karnzlam and

Thambiah (1982).

1.3 Epidemiology of I n f e c t i o n dug t~ C c n i d i o b o l u s co r . , n a t z

I n f e c t i o n caused bg -. C . c w o n a t u s i n man i s c a l l e d

rhinoento~llophthoro~~~o~~s(entomo~~hthornm~ c i , s i s c .>nicl iobolne) .

T h i s was f i r s t r e p o r t e d i n h o r s e s by Xrnrnons and B r i d g e s (1961 1.

The f i r s t human c a s e w i t h s u b s t a n t i a t e d mycolofi icnl evidence was

r e p o r t o ( : by B r a s & 2 ( 1965). L i k e s u b c u t a n e o u s ;3hycomgcosis,

i n t r o i ~ i c ~ a l and s u b t r o p i c a l c o u n t r i e s of t h e worl:l. I n A f r i c a

many c a s e s were from N i ~ e r i t i ( M a r t i n s o n and C l a r k , 1967;

Mar t in son , 1971; Harrnan, -- e t a 1 1964; Cockshot ets, 196%;

0nuik;bc) and Gugnnni , 1976 ; S u s c e l n n and Mukher j e e , 1973; Oliafor

c t a1 19831, Cameroon (Ulache e t a1 1961 ; I i a v i s s e kt p ~ , 1976; - -9 - -9

Floch - e t -9 a1 ( 1 9 7 9 ) , S e n e g a l ( ~ a s s e t e t a l , 1963) , and Conco

K i n s h a s a ( H e n o i r t e e t a l , 1965). Ihny c a s e s were a l s o r c p o r t c d ,,,,..%,m. \?. > , la

from ~ n d i a ( ~ i a r n a l a m and Thambiah, I 9 7 9 ) , T h a i l a n d (F romen t in .e& &,

1981 ) , Cos ta H i c a (Segurn 2 &, 1 9 8 1 ) , Colurnbia ( ~ e s t r c p o .eJ al,

1967; Hedoya, 1981) and U r n z i l (i+ntlrade e t a l , 1967). 14 - ...

The d i s e a s e i n v o l v e s t h c n a s a l mucosn and is c h a r : ~ c t c r i c ~ d

face i n c l u d i n i ~ t h e ; ~ a r n n ; ~ s a l s i n u s e s , pharynx m d t h e su5cut nncous

17.

i n f e c t i o n s i n v o l v i n g t h e pulmonary ~ ) r g a n s 2nd lymph nodcs hnvc

been d e s c r i b e d (Kzrnalam and 'Thambinh, 1979). Very r c c c n t l y , a

c a s e of 1nrynl;enl involvement was r e p o r t , < d i n N i g e r i a S y O h f o r

e t a 1 (1983). A new s p e c i e s o f C n n i d i o b o l u s , 2. inco; i&l~yl . was -- found t o ccluse dncp s e a t e d i n f e c t i o n i n man ( E c k e r t & :;I-, ,1972;

King and J a n g , 1976).

N a t u r a l i n f e c t i o n s have a l s o been obse rved i n o t h e r anin?.]-s

i n c l u d i n g h o r s e s ( H u t c h i n s and Johnson , lV72) , n d o l p h i n

(Sveency and Mi[;aki , 19761, a chimpanzee (lioy nnc: Cameron, '1972),

i n s e c t s (Urobyin and 'Yi ld ing , 1977; Hemaudiere e t a l , 1976) ,

ant1 a mule (Ca rva lho 1976).

k i t t emp t s a t ex;)er i rnental i n f e c t i o n o f : ;cveral l a b o r a t o r y

a n i m a l s h;?ve s o f a r been u n s u c c e s s f u l (F r?men t in , 1976; Lo;?ez-

Mar t ine e t a l , 1978). -- Over 80% o f t h e c a s e s o f rhin<>c:ntomophthoromycosis r e p o r t e d

have been i n males and c o n t r a r y t o s u b c u t a n e o u s phycanycos i c ,

most of t h e c a s e s were i n a d u l t s between t h e :qes of 20 id 6 0

y e a r s ( C l a r k , 1968). M a j o r i t y ~f t h e c a s e s occu r amon3 ,, ,, -. 4 "{. \?. " >

a g r i c u l t u r a l and outd{;;or workers . C l a r k ( 1976) f dune! 2. ~cr.ronntus t c b e common i n decay ing v e s s t a t i o n , p a r t i c u l a r l y d u r i n ~ t h e

r a i n y seasop . I n f e c t i o n p robab ly o c c u r s by i m p l a n t a t i o n o f t h e , t "- .L

i n h a l e d s p o r e s of t h e fungus i n nasal mucosn o r t h r o u z h minor

t rauma ( i n s e c t b i t e s ) .

I .4 Thexapy of I%f-n&.. due , t o -Basi&iobo&~@ h a p t o s p ~ r u s ant Conidiobo Lus-c,oronat u,6

The two mycoses a r e r e l a t i v e l y benign i n f e c t i o n s and

subcutaneous phycomycosis sometimes c l e a r s spontaneously ( Joe

e t a1 1956; Ernmons $t al, 1957). However, most cases r e q u i r e - -9

treatment with an t i funga l drug before complete cure is achieved.

Therapeutic t r i z l s with n y s t a t i n and g r i s e o f u l v i n have been

unsuccessful and t h e organisms a r e also i n s e n s i t i v e t o t hese

drugs 2 Gr? (Tio 2 al, 1966, 1977). Potassium iodide therapy

(30 mg per kg body weight, given i n iocrsmentr t o a t t a i n 2 t o 3 g

per day f o r a month o r more) has been remarkably succes s fu l i n

majority of t h e ccascs (Mukerji, 1962; Harman e t all 1964;

Fromentin and Ravisse, 1977; Kamalam and Thambiah, 1979, 1982.

Vismer - e t -* a1 9 1980; E i t t encour t e t all 1982). ~imphoter ic in U has

been adminstercd to a number of p a t i e n t s who had taken iod ide

f o r up t o nine months with l i t t l e o r no e f f e c t (Burk i t t et al,

1:)63; 1964; Emnons and Bridges, 1361). Dramatic success i n

t reatment o f t h e two mycoses with s e p t r i n , an a n t i b a c t e r i a l drug

has been r e p o r t e d by many workers i n . MSgeria (Marttnrron, < , I . . . t \ t . . > % . , , .

1971, 1972; Pomufod and Antia , 1971; Aowernimo, 1974; Ladapo, 1982;

Okafor and Gugnani 1983).

A few deaths have been d i r e c t l y a t t r i b u t e d t o subcutaneous I ' - .*.

phycomycosis (&lington, 1964; Garnet and B r o t t e s , 1963; Greenham,

1979; Aguiar, 1980) and rhinoentomophthorornycosis (Fromentin and

Havisse, 1977). I n most o f t h e chronic and f a t a l cases, t h e

i n f e c t i o n s were found t o be r e s i s t a n t t o a l l a v a i l a b l e

t he rapeu t i c drugs i nc lud ing potassium iod ide , amphotericin B and

s e p t r i n (Kamnlm and Thambiah, 1978; Hers tof f 3 2, 1978; Nathan

1.5 ,P&\-oLogy of Yhycomycosis Due t o Basidiobolus haptosporus and

Conidiobolus coronatus -1- LO--,+

The h i s t o l o g i c a l f e a t u r e s of subcutaneous phycomycosis and

rhinoentomophthoromycosis are t h e same. At t he onse t of each of

t h c two d i s e a s e s t h e r e i s migra t ionof bo'irt's eos inoph i l s , plasma

c c l l s and lymphocytes t o t he i n f e c t e d s i t e ( ~ l i l l i a m s , 1969). L C L ~ C ~

t h c r c i s d i s i n t e g r a t i o n of t h e hos t c e l l s immediately surrounding

t h c fungi i nc lud ing t h e i r cytoplasmic con ten t s t o form a precipita: : .

This r e a c t i o n which r e s u l t s i n t h e formation of a p r e c i p i t a t e is

ref crred t o as "Splendore-Hoeppli" phenomenon (Hoeppli, 1932; I i e ~ ; r o . i

and Prndo, 1951; Splendore, 1963). . The p r e c i p i t a t e was found t o bc

composed of cytoplaswic and n u c l ~ a r deb r i s der ived from tho

cos inophi la , plasma c e l l s , macrcphages and lymphocytes. It s t a i n s

i n t e n s e pink (eosin~g~~$~~c.)..,ySJi.tb hematozylin and eos in and it was;

suggested t h a t t h e presence of t h e p r e c i p i t a t e i s due t o an

antipen.-antibody r e a c t i o n (Ksppisch, 1941). Histochemical t e s t s /

i on it demonstrated t h e preFencevof phosphol ipids , n e u t r a l l i p i d s ,

I - ... b

PAS-posi t i v e ma te r i a l s and g lobu l in s ( Liilliarns e t a1 1969). The - -' p r e c i p i t a t e has a yellow o i~ to f luo re scence which is u s e f u l f o r

observing the organisms wl.en they a r e s p a r s e i n t i s sue .

1.6 Biochemica l S t u d i e s o n B a s i d i o b o l u s and Con id iobo lus s z a

L i t t l e i n f o r m a t i o n w i t h r e g a r d t o t h e b i o c h e m i s t r y of

Baf i id iobolus and Con id iobo lus s p e c i e s is a t p r e s e n t n v a i l a b l e .

T y r e l l (1967) s t u d i e d t h e l i p i d c o n t s n t o f s e v e r a l spec i e s

o f B a s i d i o b o l u s and found them t o be t h e same, Thc chcmica l

a n a l y s i s o f t h e p o l y s a c c h a r i d a s o f d i f f e r c n t s p e c i e s o f

B a s i d i o b o l u s by G r e e r and Bnrbosn (1969) d i d n o t r e v e a l any - d i g n i f i c a n t d i f f e r e n c e s among them.

B i e v r e (1974) s t u d i e d t h e l i p i d s o f - B, r n e r i s t o s . o r u s - -.- and

C, c o r o n n t u s and c h a r a c t e r i z e d n i n e i m p o r t a n t p h o s p h o l i p i d s - u s i n g t h i n l a y e r chromatography, He found t h a t (3-6 , C ~ O ~ , O > I , ~ ~ , U .

one s t r ~ i n s c o n t a i n one s p h i n g o l i p i d ancj/phosphatidyl N-N-

d i rne th~r l e thano lamine which were a b s e n t i n 2. m e r i s t o s p o q u ~ ~

s t r a i n s . d s o t h e p h o s p h a t i d y l c h o l i n e c o n t e n t was much

h.tykrr i n - C. c o r o n a t u s t h a n i n g. meris toeponus . . ins lya i s

o f t h e CGII wall c o m p o s i t i o n of C. c o r o n a t u s showed t h n t i ts -,

s u p e r f i c i a l l a y e r was composed c f a c i d i c p o l y ~ a c c h a r i d c s ~ .

glucomannan and g l u c a n and t h e n a i n l a y e r c o n t a i n e d g l u c a n , & ,l..i.l. d. .> . . I . ) .

and mannan whi l e i n n e r l a y e r c o n t a i n e d g l u c nn and c h i t i n

( ~ i e v r e ct &, 1978). Anotrler s t u d y of t h e compos i t ion o f t h e

c e l l w a l l s u g a r s of 13 s t r a i n s o f g. c o r o n a t u s and one of 2.

i ncongruus by B i e v r e e t a1 ('1978j r e v e a l e d t h a t g l u c o s e and -- -- -- mannose were t h e main s u g r r s . ~ a n n o s o / g l u c o s e c o n t e n t s f o r

C, c o r o n a t u s from man and chimpanzee were c h a r a c t e r i s c d by -

:! r a t i o l y i n g between 0.92 and I .3; s t r a i n s from h o r s e s and

~ o i l h n d r a t i o between 2.7 and 4.1 w h i l e f o r 5. i ncongruus ,

i t w a s 0.44.

, i t t empt s t o e s t a b l i s h e x t r a c e l l u l a r enzyme p r o d u c t i o n

i n .-a C. .- c o r o n a t u s showed tha t t h e human s t r a i n s produced more

c l a o - t a s e , e s t c r a s e and c o l l a g e n a s e t h a n t h e s a p r o p h y t i c

ones ( z r o m e n t i n et; d., 19781.

ECOLOGICAL ANP T.AYONOM~,C,. $TUG JLS 11.1 ,*.-

Trn&ng . o f Animals - +.-

One hundred and f i f t y s a m a agama ( ~ f r i c a n l i z a r d ) and

e i g h t y Uufo bu fo (Common t o a d ) were t r a p p e d from t h e v i c i n i t y -- of r e s i d e n t i a l houses and s c h o o l s i n Nsukka town of Anambra

S t : ~ t e , N i g e r i a , Twenty Hemidacty lus b r o o k i i ( w a l l ~ e c k o ) wcro

cau,yi~t i n s i d e t h e houses i n t h e same a r e a , E i g h t Che lon ia &

(?;reen t u r t l e ) were Sought from a v i l l a g e mcirket i n t h e s u r b u r b

of Nsukka, One hundred and f i f t y s e v e n A n o l i s s a g r e i (Cuban

l i z a r d ) , 93 &. c a r o l i n e n s i s ( g r e e n l i z a r d ) , 10 Gopherus

23,1&:111!mu7 ( l a n d t u r t l e ) , 3 Bufo mar inus ( g i a n t taar i ) , 78 30 - t c r r e s t r i s ( c o n t i n e n t a l t o a d ) , 75 13. amer icanus , ( t r u e t o a d ) , .a- - I ---- A c r i s ~ r y l l u s ( c r i c k e t f r o g ) , 'I Yyln c i n e r e a ( c r c e n t r e e f r o g )

and ? Rana u t r i c u l a r i n ( t r u e f r o g ) were collected from t h e

s u r r o u n d i n s s of r e s i d e n t i a l houses and a t t h e beaches i n Tampa

and Liiami towns of F l g r i d a , USA.

11.3 I s o l a t i o n o f H a s i d i o b o l u s from Animals

?he f a e c a l d r o p p i n t l ; ~ of some r ) f t h e a n i m a l s were c o l l e c t e d

2nd cul%uret i . The r e s t of t h e Bni-rna-ls were s a c r i f i c e d and t h e i r

i n t e s t i n a l c o n t e n t s a e a p t i c i l l y removed anii c u l t u r e d f o r

i s ~ l l n t i o n of H a s i d i o b o l u s u s i n s a m o d i f i c a t i o n nf t h e method o f

G r c e r and Friedmsn (1966). The i n t a s t i n a l c o n t e n t s of each of

t h c an ima l s o r t h e f n c c a l 1Iroj2~;)ings mois tened w i t h a few d r c y s

of s t e r i l e d i s t i l l e d wa te r was p l n c e d o n a P e t r i - d i s h l i d .

The bottom o f t h e l i d which c l>ntn ined Snbouraud d e x t r o s e n g ~ v

(SUA) supplemented w i t h ch ln rnmphen icc~ l W i l S i n v e r t e.1 o v e r t h e

l i d and i n c u b a t e d a t 30'~. With in 48 t o 72 hours c o l o n i c s o f

E n s i d i o b o l u s ( F i g , 1 ) which developed from f o r c i b l y d ischarscc l - .- c o n i d i a were s u b c u l t u r e d on SDA s l a n t s t o a v o i d contaminat ion .

Thcy wcre s tore(-1 a t room t e m p e r a t u r e (25 - 3 0 " ~ ) and s u h c u l t u r c d

One hundred and twenty s o i l s amples which i n c l u d e d r i v e r

sand, f o o t p a t h s o i l and garden s r . i l were c o l l c c t e i : f r o n

v a r i o u s parts of Idsukka. F i f t y samples o f f a e c a l ciroppings of

b a t s were o b t a i n e d from t h e f l o o r o f trees a t Enugu which are

i n h a b i t e d by hundreds o f b a t s . Human i n f e c t i o n due t o

Cun id iobo lus c o r o n a t u s m a n i f e s t s 2s n a s a l [;ranulomatous l e s i o n ,

S i n c e s n u f f t a k i n g i s c,o,lg~,q. gwng .Ni l :e r inns , s n u f f samples

wcre t h u s i n c l u d e d i n t h e s t u d y t o a s c e r t a i n p o s s i b l e p r c s c n c e

of t h i s fungus i n t h e marketed s t u f f . EighOy s n u f f sanlples

b o u l ~ h t i n s m a l l . q u n n t i t i e s (10gm) f r o n v a r i o u s l o c a l markets I' - .L

i n Nsukka, Gnugu, Owerri and iLwlra were i n v e s t i i ~ a t e d . A l l of

the above samples were c u l t u r e d s e p a r a t e l y usin[; t h e u s u n l

t echn ique , f o r t h e i s o l a t i o n r ~ f !3nsidiol,olus ,ind C o n i d i o b o l s .

11.5 I d e n t i f i c a t i o n o f 1 s : ) l d t e s

A 1 1 t h e i s . 1 1 3 t c s of 13nsidinbnlus and C<7nidiob&u- o b t a i n e d

ware examined m i c r o s c ~ ~ p i c n l l y w i t h 1 i l ; h t r n i c r o s c o i ) ~ (Olymyus)

and i d e n t i f i e d on t h e b s s i s o f t h e c h a r a c t e r i s t i c c o n i d i n ,

zygospores and v i l l o s e s p o r e s . For d e t a i l e d m o r ~ d i o l o ~ i c a l

exr-imination, some representative isc>liltt.s nf Bctsj .cliobo~us were

p u t up on s l i d c c u l t u r e and L-iter examined unLler scnnnin1y

e l e c t r o n microscope ( ~ 1 2 ~ ) . The f u n g a l m a t e r i a l was p r e p a r e d

f o r t h e SXM i n t h e fo l lowink ; manner. A f t e r 1 0 days df

i n c u b a t i o n on a g a r b l o c k , t h e c o v e r s l i p contain in^ g o w t h of

a t e s t i s o l a t e was g e n t l y removed w i t h a p a i r o f c l c n n f o r c e ~ s ,

and t h e n p l a c e d f a c e up i n s i d e a P e t r i p l a t e c o n t a i a i n g a

f i x i n g m a t e r i a l , 3% parnforrnci ldehyde - g l u t a r n l d e h y d e f i x a t i v e

( E l e c t r o n l11icrosc,)py S c i e n c e s ) f o r a t l e a s t 2 hours. T h i s was

f o l l o w e d by a 176 usmiurn ; m s t - f i x and subsequen t c lchyckat ion

i n d i f f e r e n t g r a d e d c o n c e n t r a t i : > n s ( 10-100%) oP a l c o h o l .

Fo l lowing t h i s , t h e cove r s l i p w a s t r a n s f e r r e d t o ncc tone /

e t h a n o l ( 1 : 1 ) and f inq l ; ly , , .$~ , , 100% ace tone . The d e h y J r n t i o n

was done q u i c k l y t o p r e v e n t d r y i n g i n a i r . The c o v e r s l i p was

t h e n q u i c k l y t r a n s f e r r e d i n t o a c r i t i c a l p o i n t clrjrer ( S a n d r i

PVT-3) a n d f i n a l l y c u a t e d w i t h g t ~ l d f o r 3 rninutcs at 1 O m i ~ i n I ' . .L

a m i n i - c o s t e r (Cornrnon-wealth S c i e n t i f i c Comp.) U l t i m a t e l y a l l

t h e p r e p a r e d specimens were s t o r e d i n x desiccator tuic! l a t e r

exalninetl w i t h a scctnninil: e l e c t r o n microsco~oe (NOVLLSCG~ 3 0 ) and

p h o t o p a p h e d w i t h T r i X Or tho 4 x 5 f i lm .

11.6 Therrnoto le rance of B a s i d i o b o l u s Isolates

R e p r e s e n t a t i v e i s o l a t e s of U a s i d i o b n l u s spec ies were first

grown on SlIi.1 p l a t e s f o r 5 days , Us ing s t e r i l e c o r k h o r t r (brim

diar f le te r ) , a n inoculurn w a s removed from each t e s t c u l t u r e

t h c n ' l > l a c e c i a t t h e c e n t r e of a p l a t e o f SDA. T r i p l i c a t e

plates o f each i s o l a t e were t h e n i n c u b a t e d a t 20, 30, 37 and

45 '~ . The d i a m e t e r t,f c o l o n i a l g rowth was t a k e n f o r each

i s r l n t c a t 24 h o u r s i n t e r v a l s f ~ : ~ r 7 days ,

11.7 B f oohsnrical T e s t s f l w C h a r a c t e r i z a t i o n o f ;insidi-)bo_lu?

I s o l a t e s

f i r hnemolys ln p roc iuc t ion , ( : . e la t in liquefaction, c a s e i n li : e s t i n n ,

u r e a h y J r o l y s i s and l e c i t h i n a s e 1 , roduc t ion u s i n g t h e t c c h i l i q u e ~

d e s c r i b e d h9 Cowan ant1 Y t c ~ l (1955). The i ~ l o c u l n c o n s i s t c d of

ai;m d i s c s c o n t a i n i n g myce l in of t h e t e s t ordanisrn 311d

0 i n c u b a t i o n was at 30 C.

.< ,, . . .I, .=' ! a .>*

11.7. 1 Haemolys in P r o d u c t i o n

Haemolys i s was d e t e r m i n e d on b l o o d afar b a s e (0xoicl)

containing 5% a s e p t i c a l l y c ' ) l l e c t e d f r e s h b l o o d from h u r ~ ~ a n , ( I - -.

g o a t , s h e e p o r c h i c k e n , C l e a r i n g a round t h e co1 , )n i c s a f t e r

24 - 48 h o u r s o f i n c u b a t i o n i n d i c a t e d hacrnolysis .

11.7.2 ,(..*eeL,?tin L i q u e f a c t i o n

G e l a t i n q;ar medium was d i s p e n s e d i n 10ml volumes i n t o 1nr i ; o

t e s t t u b e s (18mm x 175mm). A f t e r i n c ~ c u l a t i o n wi th t h e t e s t

0 or&:misms, t h e t u b e s were i n c u b a t e d a t 3 0 C f o r a. maximum p e r i o d

o f .two weeks. Xvery 2 - 3 days , t h e i n o c u l a t e d t u b e s were coolcc!

i n n refr ic; i . rat l ' ) r f o r 2 h(.lurs.and examined f o r l i q u e Faction. A

c o n t r o l t u b e o f unins-lculnted medium was s e t up and i n c u b a t e d nlonii;

u i t h t h e t e s t s .

11.7.3 Di&?-stion of C a s e i n

P l a t e s of c a s e i n af::ar ( D i f c o ) were i n o c u l a t e d wi th t h e t e s t

o r @ m i s i n s and examined d a i l y f o r up t o 7 days, A c l e n r i n z nrourrc! !

t h o colony i n d i c a t e d c a s e i n d i g e s t i o n .

11.7.4 !?-%a lIyctrc,lysis

The t e s t i s o l a t e s were i n o c u l a t e d o n t o s l o p e s o f C h r i s t c i l s c n 1 c

urea meciiurn (Oxoid) and i n c u b s t c d f o r a coup le of days- A chance

t o ; ) ink :)r r e d c ' d o u r indiccr ted u r e a h y d r o l y s i s . ,',,.. . , T . , t , . , * . ? * ' .

11 .7.5 .LA- L e c i t h i n a s e -- P r o d u c t i o n /

S t e r i l e n u t r i e n t aj:-ir, pH &, i n t o which was added 1% f r e s h l y \

m c ! a ~ e p t i c s l l y p r e p n r c d etS,;''yollic (as s u b s t r a t e ) wns t h e nieiliu~i~

usci! i n d c t c c t i n t ; l e c i t h i n a s e . A c l e a r i n g around t h e colony

i n c l i c a t c ~ l e c i t h i n a s e p roduc t ion .

11.8 Pilzfient P r o d u c t i o n by E c s i d i o b o l u s I s o l a t e s on T y r ( ~ s i . n . i . y _

Medium

R e p r e s e n t a t i v e i s c i l a t e s of Uns id iobo lus s p e c i e s werc

i n o c u l a t e d o n t o 0.5% L - t y r o s i n c a:;ar medium (0.5% L-tyrosrinc + 0

2% ~ m r i f i e d al:nr - Difco) nnd i n c u b a t e d a t 30 C f d r 5-10 J.?as.

The c o l o u r o f t h e p i p e n t prc3duced i n t h e q;ar medium w a s noted.

11.9 Lxper imen ta l I n f e c t i o n :)f L i z a r d s i n C a p t i v i t ~

Twelve f r e s h l y caught l i z a r d s ( iknol i s SJJ) were i n c l i v i c l u d l y

housed i n cai:es (Fi!: 2 ) which were p r e v i o u s l y swabted w i t h 70$

e t h a n o l and t h e i r f a e c e s c u l t u r e d ff.)r 3 a s i d i o b o l u s . D u r i u ~ this

p e r i o d , t h c a n i m a l s were s t a r v e d u n t i l t hey s t o p p e d defaecating.

;,bout a week l a t e r , t hey werc f e d wi th men1 worms, '&uubrio

m o l i t o r , rind t h e i r f a e c e s a g a i n c u l t u r e d . When t h e y stopped

defaecating f o r t h e second t ime , t hey were t h e n f e d wi th 111cnlworms

which had been r o l l e d around i n pu re c u l t u r e s o f !3asidiA3,oJ,u>

h n p t o s p o r u s (Fi t ; 3) anti t h r $ i r f a e c e s were z!;:lin culturecl. Thc

f i r s t f e e d i n g w i t h rneal.yu$sa\,.w,~ done t o r u l e o u t n a t u r z l p r c s c n c c

o f E n s i d i o b o l u s on t h e meal worms.

111.1 Screening f o r I . ;x t race l lu lar Znzyrne P r o d u c t i o n on S o l i d Ncdin

- - .. AX? t h e i s ~ l n t e s o f Eas i t l i obo lus a n d - Con id iobo lus o1,tninecl

from c l i f f e r e n t s o u r c e s d u r i n g t h c p r e s e n t i n v e s t i g a t i o n

i n c l u d i n g t h o s e from ATGC were t e s t e d on s o l i d n c d i n f o r t h e

e x t r a c e l l u l a r p r o d u c t i o n of amylase , p r o t e a s e , l i ; )nse ,

de : ,xyr ib( ,nuclease ( D l ~ ~ a s e ) and r i b o n u c l e a s e (81\:iia~o) u s i n g t h e

methods d e s c r i b e d by Hankin and Ana[;nos t s k i s ( 1975). 'J1ri1!li c a t e

p l a t e s were i n o c u l a t e d f [ ~ r n p a r t i c u l a r t e s t f o r i s o l a t e .

The inoculum f o r each p l a t e comprised a n agar d i s c (6mm i n

d i a m e t e r ) u b t n i n e d wi th a c o r k b o r e r from a 5 .lag-old c u l t u r e o f

t h e t e s t i s , l a t e on SUB p l n t e .

111-1. 1 Amylase P r o d u c t i o n

The c r i t e r i o n used t o ( le te rmine t h e a b i l i t y o f n fungus t o

pruduce amylase enzyme was based on i t s * a b i l i t y t o d c p a d e s t a r c h .

The medium was p r e p a r e d by i n c o r p r a t i n g 0.2% soluble s t a r c h i n t o

n u t r i e n t a(;ar ( D i f c o ) and t h e pH a d j u s t e d t o 6. A f t e r 3-5 days

o f i n c u b a t i o n , t h e i n o c u l a t e d p l a t e s were flooc!ecl with an iur i ine

s o l u t i o n , c l e a r zone around t h e funr:nl ,;rotrth which was

d i s t i n c t from t h e o t h e r b l u e a r e a , in(:icatecl a l . iy lc~ly t ic a c t i v i t y -

. . , , . . < L I I . + " , . $ * ' .

111.1-2 F r o t e a s e P r o d u c t i o n

To d e c t e c t y r o d a c t i o n o f p r o t e o l y t i c enzymes, J i f c o n u t r i e n t

qya r medium ( p ~ 6 ) c o n t a i n i n g 0 ~ 4 % . t i e l a t i n as t h e a i jurce of p r o t e i n , I - .i

was used. The p r e s e n c e o f p r o t c a s e w a s s e e n a s n c l c a r i n g i n t h e

somewhat opaque ar;ar nrountl t h c c o l o n i e s . $i:d confirmation was

done by f 1 o o : l i n ~ t h e plate with a n aqueous s a t u r a t e d s o l u t i o n

of ammonium s u l p h n t e which would enhance any c l c n r zone around

t h e c o l o n i e s o

111.1.3 L i p n s e P r o d u c t i o n

To d e c t e c t t h e p r o d u c t i o n o f l i p o l y t i c enzymes, t h e medium

c lescr ibed by S i e r r a (1957) was used. The medium contninecl ;>cr

l i t r e : 106 B i f c o pe l ) t one , 51; N a C 1 , O.lg C a C l 2 H 0 , 2 0 ~ ngnr , 2' 2

pH 6. 1% p o l y o x y e t h y l e n e s o r b i t a n mono lau rn t e ween 80) o r

l ~ ~ l y o x y c t h y l e n e s : l r l - ) i t an monoolea te (Tween 20) from F i s h e r

S c i e n t i f i c Comp. N. J. was u sed as t h e l i p i d s u b s t r a t e . T h i s

was s t e r i l i z e d s e p a r a t e l y anil mixed w i t h t h e s t e r i l e and cooled

b a s a l medium. The f o r m a t i o n of l i p o l y t i c enzymes w a s d a t c r n i n e d

by t h e v i s i b l e p r e c i p i t a t e nrountl e ach co lony due t o t h e

f o r m a t i o n of c r y s t a l s o f t h e c a l c i u m sa l t of t h e l a u r i c ( o l c i c j

a c i d r e l e a s e d by t h e enzyme. A c l e a r i n g of such p r e c i p i t a t e duo

t o comple t e d e g r a d a t i o n o f t h e s a l t o f t h e f a t t y a c i d c o u l d 21130

h e u s e d as a p o s i t i v e i n d i c a t i o n o f t h e p r e s e n c e o f l i p s o .

r+

I . 4 Deoxyrit~c-~nucl~aese~-V~~~duOtion

The d e p : t d a t i o n o f t l eoxyr ibonuc le i c a c i d by t h e ~ r o c ! u c t i o n

o f DNhase was t e s t e d by [p-owing t h e i s o l a t e o n D N ~ a s e ascv

( D i f c o ) a d j u s t c d t o pH 6. ,,,rfte,t. 3-5 days o f i n c u b a t i o n , t h o

i n o c u l a t e d l j l a t e s were f l o o d e d w i t h I N HCL and examined f o r

c l e a r zones a round t h e c o l o n i e s which would i n d i c a t e d e , g a ~ l a t i u n

of t h e d e o x y r i b ~ ) n u c l c i c acicl i i l t h e t e s t rnc-d' lum.

111.1.5 R i l -~onuc lease P r o d u c t i o n

X medium f o r m u l a t e d hy J e f f r i e s e t a1 (1957) w z s u s c d t o -- t l e t c c t t h e a b i l i t y of t h e i s o l a t e s t o llegradt2 r i b o n u c l c i c a c i d .

T h i s medium c o n t a i n e d l ) e r Utre : 5@ g l u c o s e , 513 jl if co v i t a m i n -

w a s u s e d as t h e t z s t s u b s t r a t e , 2g of t o r u l a y e a s t was

d i s s o l v e d c a r e f u l l y i n I N NaOH and added t o t h e b a s a l imdium

p r i o r t o au toc lav inr ; . A f t e r i n c u b n t i n ~ f o r 5-7 clays, t h c

p l a t e s were f l o o d c d w i t h I N HCL 3nd r e a d as i n DNAasc t z s t .

111.2 P e r i o d i c Measurements o f L i p c s e ~ c t i v m o f Uasitli-d~~Q~- -

a n ~ l ConidioboAue- species

S i e r r a medium ( b i e r r a 1957) supplementeJ . w i t h 1% (wt /vo l )

polyoxyethylene s o r h i t a n monc~ lau rn t e (Tween 8 0 ) b u t which c!i:! n o t

c i r n t a i n aL;ar was u s e d a s t h e b r o t h medium f o r m c n s u r i n ~ l i p w e

A ~ t i ~ 3 , t y . T h i s medium was c l i s ~ i e n s e d i n 50ml volumes i n t o

p u r e c u l t u r e s

and - C. c o r o n a t u s (C1) i n S2,i p l a t e s were u s e d f o r i n o c u l a t i n g

t h e broth .medium. Two a g a r d i s c p (6mm i n t l i amc te r ) b o a r i n g I 4 - _C

mycelium o f t h e t e s t i s o l a t e were i n o c u l a t e d i n t o each o f t h e

f l a s k s and t r i 2 l i c a t e f l a s k s were p u t up f o r each t e s t . The

0 i n o c u l a t e d f l a s k s were f i n a l l y i n c u b a t e d a t 30 C. 24 l~uu:

i n t e r v z i l s f o r t h e f i r s t f o u r days and s u b s e q u e n t l y z t 48 1 2 0 ~ 1 .

i n t e r v a l s ( u i ) t o 1 2 d a y s ) t h r e e r e p l i c a t e c u l t u r e f lasks o f e a c h

i s o l a t e were t a k e n o u t o f t h e i n c u b a t o r ant1 t h e rngcel-iuiil ill

e ach f l a s k removed by T i l t . r a t i n n . The growth o f e a c h i s o l a t c ,

expressed a s a v e r a g e Cry wei[;ht o f t h e mycelium i n t h c

t r i p l i c a t e c u l t u r e f l a s k s was r e c o r d e d a t each i n t e r v a l a f t e r

washin[; t h e mycelium w i t h t l i s t l l l e d w a t e r and t h e n 1J.rying it

0 f o r 24 h o u r s a t 80 C b e f o r e weigh ing . The v a l u e s o b t a i n e d were

r e c o r d e d as mg p e r 50 r n l o f c u l t u r e f i l t r a t e . The f i l t r a t c

f rom e a c h f l a s k w a s t h c n u s e d f o r t h e l i p a s e a s say .

The q u a n t i t y c f l i p a s e i n t h e c u l t u r e f i l t . r ~ ~ t e o f e a c h

f l a s k w a s a s s a y e d t i t r i m e t r i c n l l y by employ ing a medication

o f t h e t e c h n i q u e s d e s c r i b e d by 1)cploey ( I* . ) )$ E&p~fea

a n d V i n c e n t ( 1 9 8 1 ) , U: i s and l i a n e r j e e (1982) . Thc r e a c t i o n

m i x t u r e c o n t a i n o d 5ml r>f t h e c x t r n c e l l u l n r f i l t r a t e nnJ 5 m l

o f 576 Tween 80 i n 0.1M sodium i ~ c e t a t e b u f f e r a d j u s t e d t o a pH

. , , . . * l . .I. . , . I + ' . o f 5.5. A c o n t r o l mixture was a l s o p r e p a r e d which cant:-.ined 5al

o f t h e c u l t u r e f i l t - r a t e ; ) l a c e d i n a b o i l i n g w a t e r b z t b f o r 10lliin-

u t e s t : ) d e n a t u r e t h e enzyme b e f o r e 5ml o f Tween 80 w a s a d d d t o

i t . The ween 80 s e r v e d as" t h e ' k u b s t r a t e f o r t h e l i p n s o

a c t i v i t y . ' i l l t h e t e s t m i x t u r e s were i n c u b a t e d i n n w a t c r

b a t h o f 3 5 " ~ f o r 3 h r s . , i f t e r t h i s , 20ml o f a c e t o n e : c t h a n a l

m i x t u r e (1 : 1 ) was i-tddeti t o e a c h t e s t m i x t u r e inclurXn[; t h c

c o n t r o l m i x t u r e 5 and t h e l i b e r a t p c t f a t t y a c i i l s t i t r n t c d w i t h

O,Of;M aqueous NaOH. The a c t u a l amount o f NaOH usecl i n t i t r a t i n g

t h e l i h c r n t c d f a t t y a c i d s was o b t a i n e d by s u b t r a c t i n : ; t h o t i h a t i o n

v a l u e o b t a i n e d from t h e c o n t r o l m i x t u r e o f each t e s t %om t h e

v a l u e f o r t h e r e a c t i o n m i x t u r e , The amount o f l i p a s e produced

was t h e n e x p r e s s e d a s u n i t s o f cnzyne a c t i v i t y . One cnzync u n i t

i s t n e amount o f enzyme n e c e s s a r y t o l i b e r a t e f a t t y o .c ids

e q u i v a l e n t t:, 0.01ml ~ j f 0.05M IdaOH from t r i g l y c e r i c l e ~ (i'wcen ,801

i n 1 h r (Uas and f i n n e r j e e , ~ 9 8 2 ) .

I n t h e same a a n n c r , t h e t e m p e r a t u r e and pH o p t i m z f o r t h e

l i p a s e a c t i v i t y o f each i s : ) l n t e were d e t e r m i n e d by ml ju s - tnen t o f

t h e a s s a y m i x t u r e t o t i i f f e r e n t pH v a l u e s (4-8) and by i n c u b a t i c g

a t d i f f e r e n t t e m p e r a t u r e s ( 3 0 - 6 0 ' ~ ) .

111.3 D a i l y Measurements of t h e P r o t e s s e ( ~ a s e i n ~ l ~ t i c ) A - ~ t s t .

o f i 3ns id iobo lus and C o n i d i o b n l u s speqies

The p r v t e a s e a c t i v i t y of 2. ha ; , t d spo rus (.rTCC 3 6 6 0 0 ) ~ - 3.

rancmum ( 1 l H 3 ~ ) and - C. c r ~ r o n a t u s (C ) were de t e rmined urii?.g t h e I

methods d e s c r i b e d by F romen t in (1376) and Gimpkin - e t ,- , ~ 1 (19801, .',,.,. C , . ,' .>'. '* I .

The inocula were p r e p a r e d a s i n l i ~ ~ a s e d e t e r m i n a t i o n t:iicl t h e b r o t h

medium used was Sabournud l i q u i d ~nediurn, which w a s d i s j2enscd i n

501x1 q u a n t i t i e s i n volurnetrie f l a s k s . The i n o c u l a t e d f l a s k s

, - ... G were t h e n i n c u b a t e d i n a s t a t i o n a r y i n c u b a t o r a t 3 0 C. A ' L ~ 24 hou:

i n t e r v a l s up t o 7 d a y s , t h r e e r e p l i a a t e c u l t u r e f l a s k s of each

i s o l n t e were t a k e n o u t o f t h e i n c u b a t q r an? a s s a y e d f o r p ro tec i se

a c t i v i t y ,

The mycelium i n each f l a s k was removed by f i1ter ; : l ; ion and

t h e f i l t e r a t e was assayed . The a s s a y m i x t u r e consiskccl of 1 m l

o f f i l t e r a t e and 4ml o f 1% c a s e i n i u s e d as s u b s t r a t e ) i l l 0.1M

sodium a c e t a t e b u f f e r , pH 5.5. A c o n t r o l m i x t u r e war : a l s o

p r e p a r e d which c o n t a i n e d 1 m l of c u l t u r e f i l t z r a t e p l ~ c c d i n a

b o i l i n s w a t e r b a t h f o r 1 0 min t o l e n n t u r e t h e enzyme l ~ c f o r c 4

m l o f c a s e i n i n b u f f e r was added t o i t . dl t h e t e s t m i x t u r e s

were i n c u b a t e d i n a w a t e r 113th a t ~ O * C f o r 1 hr . The r c n c t i o n

was s t o ~ p e d by addin[; 5 m l o f 5% t r i c h l o r a c e t i c acic! and t h e n

k e p t f o r 3 0 min. Fo l lowing t h i s , t h e p r e c i p i t a t e d ;3ro tc in

was removed by f i l t t ' r s t i o n usint.; 2 l a y e r s of Yhatrnan f i l t e r

p a p e r and t h e f i l t e r a t e ( d i c e s t e d p r o t e i n ) was a s sayc~ l . f o r

s o l u b l i z e d m a t e r i a l usin;; F o l i n - C i o c a l t e a u r e x ; c n t (Lowry

e t a 1 1951). The difference between t h e abs l~ r ' uancc of cncli -- sample mcl t h e con t r c , l was u s e d f o r c a l c u l a t i n g t h e enzyme

a c t i v i t y . The enzyme a c t i v i t y was e x p r e s s e d a s p o f s o l u l ~ l i z e d

p r o t e i n p e r hour and this.wrfs~ ~ d c u l n t e d from t h e stnizclnrd c u r v e

f a r p r o t e i n e s t i m a t i o n (Fi[:. 4) .

111.4 Chromntoi;rafihic P u r i f i c a t i o n and"shme P r o p e r t i e s of t h z

P r o t e a s e from f i a s i d i q l m l u s and C o n i d i o b o l u s species

The e x t r n c e l l u l c * r p r i s t ea scs from 3 , h n p t v s p o r u s 9.. r:Lnarum - ant1 C, c r r o n n t u s were p u r i f i e d by chromxtol?raphy u s i n g t h e - methods d e s c r i b e d by Somkuti and 3 a b e l (1968) ; Uiarnoiid and

S t a n d x d cu rve f o r p r o t e i n e s t i m a t i o n

(Lowry & &, 1()5?)

38b

ijenman ( 1966 1. Sephatlex G-100 powder (Pharmacia P i n e Chcillicnls)

was a l lowed t o s w e l l i n e x c e s s s o l v e n t (0.1M sL~tf ium a c e t c i t e

b u f f e r pH 5.5) i n a d a r k cuphoard f o r 3 dsys. ,L chr?mnto::raphic

column (60cm lon[< x 2.5cm i n t e r n a l t l i amute r j , was mounted

v e r t i c a l l y i n a r e t o r t s t m d . A s m a l l p l u g of !;lass wool ims

p o s i t i o n e d nC t h e bot tom o f t h e column us in^ a l ~ n , : g l a s s rod.

The s w o l l e n sephndex !:el w a s poured [gent ly i n t o t h e c o l u n n

u n t i l a bed hei!:ht o f 50 cm was o b t a i n e d ,

The rnadium f o r t h e p r o t e n s e p r o d u c t i o n was Sabourr~.ud l i q u i d

medium dis1)cnsed i n 250rfl1 volumes i n t o 500ml brlenmeyer f l a s k s .

Each f l a s k was i n o c u l a t e t i w i t h 10 q ; a r d i s c s c !~n ta in i i i~ ; mycelium

o f t h e t e s t i s o l a t e . The i n o c u l a t e d f l a s k s were t h e n incub:lted

f o r 8 days i n a n nr ln i t - r l i n c u b a t o r (Gallenkamp) opc ra t inp , a t 200

o c y c l e s p e r minute a t 35 C. ,,t t h e cnd of i n c u b a t i o n t h e mycc l i a

i r ? e ach f l a s k were removed by f i l t e r a t i o n . To l 5 O m l of each

c u l t u r e f i l t e r a t e , j O O m l o f c h i l l e d a c e t c n e were added c:ently

t o p r e c i p i t a t e t h e yjrotease protluced. The erzyme p r e c i p i t a - t e

" $ , , ... " ! * , ? . . , , . , a ' . was t h e n c o l l e c t e d hy c e n t r i f u g a t i o n a t 25,000 x g f o r 20 nin.

i n a neckman MoJel 5-27 r e f r i ! ; c r a t e d c e n t r i f u g e . T h e p r c c i p i t n t e

bras t h e n d i s s o l v e d i n 10ml of 0.1M sodium a c e t a t e bu f fo r .

Be fo re c a r r y i n g ~ u t t h c ch~nrn5t36[ ;~aphic purif ication of each

enzyme, t h e ::cl column wns washed p r o p e r l y wi th t h e c l ~ i t i o i i

b u f f e r (0.1iq sociiurn a c e t a t e ) by opening t h e o u t l e t and d l c i i i n @

t h z s f - l v e n t t o r u n thrc.ugh f i r abou t 2 h o u r s , t o c q u i 1 i b r : y . t ~ t h e

c(-,lurnn, J u s t b e f o r e s e p a r a t i o n , t h e s o l v e n t was a l lowed t o r u n

a f f u n t i l t h e l e v e l c n i n c i d e d wi th t h a t of t h c upper s u r f a c e of

t h e sephntlex [:el. About 10ml of t h e enzyme c ~ f t h e t e s t i s o 1 a . L ~

Chromnto,;raphic sep:-mation was t h e n c a r r i e d o u t usin]: t h c

~ c e t n t e b u f f e r f o r e l u t i o n , a t a f low r a t e o f 0.251n1 p e r nliiiutc.

F i f t y (5ml each) f r a c t i o n s were c ~ l l e c t e t l mannually wi th c l e n n

tubes. The ] , r o t e i n c o n t e n t o f each f r a c t i o n w a s es t im'a ted by

de te rmin ing t h e o y ~ t i c a l d e n s i t y (OD) a t 280nm u s i n g Unicam Sp

800 UV s l ! ec t ro~>ho tomete r . A ~ r a p h of CL) a t 280nm a,.;ainst t h o

f r a c t i o n number f o r each i s o l a t e was t h e n p l o t t c d .

111.5 -- Assay of t h e P u r i f i e d P r o t e a s e

The p r o t e o l y t i c a c t i v i t y of ench f r a c t i o n was measured

u s i n g n modifi~acl method of McI)onalli and Chen (1965). Thc

r e a c t i o n mix tu re which c o n t a i n e d Iml of each f r a c t i o n 2nd 4nl

o f 1% c n s c i n s u b s t r a t e i n 0.7M a c e t a t e b u f f e r was t h e n incubn tcd

f o r 60 mins. a t 3 0 ' ~ . The r e a c t i c n w i i s stol3ped by qddinc 5 m l o f

,, ,,*."!. ,* ., * ,2

5% t r i c h ~ o r n n c e t i c a c i d t o t h e n i x t u r e , and i t was a l lowe6 t o

s t a n d f o r 30 min. The mix tu re w a s t h e n f i l t e r e d t h r m g h two

l a y e r s of Iihattnnn f i l t e r paper No. 42. The amount of t r i c1 i l . 0~0-

acc t i c a c i d - s o l u b l e mat t c r '( t y " ~ ~ i s i n e ) was measured a t 700nm

u s i n g t h e ne thod of Lowry (1951). A p a p h of p o t e a s e a c t i v i t y

( exl i r txsed a s Oil a t 700nm/hr) was p l o t t e d a g a i n s t t h e f r a c t i o n

numbcr f o r each isr-,)lat e .

111.6 Z f f e c t c f pH and t e m p e r a t u r e on i i c t i v i t y o f P u r i f i e d is- -..-iWIU.4 P r o t e a s e

A l l t h e f r n c t i o n s c o n t a i n i n i g a c t i v e p r o t e i n were pooled c a d

c o n c e n t r a t e d by f r e e z e - dry ing . The concentra.%c f rom each

i s o l a t e was t h e n used t o t ie tcrmine t h e e f f e c t o f hydrogen i o n

0 c o n c e n t r a t i o n (pH range,3-8) a n ~ 1 t e m p e r a t u r e (20 - 60 C ) on t h e

p r o t e a s e a c t i v i t y ( e x p r e s s e d a s pt; o f s o l u b l i z e d p r o t e i n p e r h o u r ) .

The molccu ln r we igh t s c,f t h e p r o t e a s e o f -- A. hz;,>t o , ~ p r u s , 3.

rai~arurn and C. co ronn tus were t le termined from t i1c . i~ d u t i o n - p r n f i l e on t h e sephatlcx G - 100 cclurnn accorc!irL< t o t h e methods

G r d d e s c r i b e d by Androws (1964) Mantle (1978). Cytochroac C (MW

(Nw 24,000) were used as prL.,tein rnorkers. The e l u t i o n volume

of each marker was c a l c u l a t e d and t h e p l o t of t h c i r e l u t i o n

volumes a g a i n s t t h e 1ot;nrithm of t h e i r mo lecu la r i ~ c i ~ h t s was used

t o e s t i m a t e t h e molecu la r w e i z h t s n f t h e aat ive protease of kho ,',,.. " f . \ t ' " > * . > & .

t e s t organisms.

I v I N V I T R O SLNSITIVITY TO ANTIFUNGAL DRUG8 I t - .-

IV. 1 Fungal I s o l a t e s T e s t e d :

The f o u r t e e n i s o l a t e s of Unsir i iobolus t e s t e d i n c l u d e d s e v e n

i s o l a t e s o b t a i n e d durin;; t h e ; )resr?nt s t u d y , The l n t t c r ccmpr ised

s i x i s o l a t e s o f 2. f o u r

L L ) ; rjne each from R n o l i s s n g r e i (GA5) and !3ufo bu fo ( T ? ) 25' 27 - .---.

and one i s o l s t e of 2. rnnw-um (MHD1) f r m A n o l i s c a r o l i n c , n ; ~ i ~ .

The r ema in ing s e v e n H a s i d i n b o l u s i s ( ; l n t e s were o b t a i n e d from

,.merican Type C u l t u r e Ct . r l lec t ion an3 they compr ised 2. T~>~~UL.

(L,TCC 32277) , - 3. rnq;nus (ATCC 15379) bo th from p l a n t c l c t r i t u s ;

13. micr(jsyorus(ATC0 14708) from r f ~ d e n t dun^ and f o u r human - i s o l a t e s o f 2. halLtpsp!~ru,s (ATCC 36600, 14052, 24670, 3k122).

The f o u r Con id iobo lus i s o l a t e s u s e d i n t h e t e s t i n c l u d e d t h r e e

i s o l a t e s o f 2. c o r o n a t u s compr is ing one sc~il i s o l a t e ( c? ) nil< two

human i s o l a t e s (ATCC 42063, 32867) and one human i s o l x t e ol 2.

1 2 Media

I n i t i a l l y f o u r media, v i z u n b u f f e r e d y e a s t n i t r o g e n base

( D i f c o ) , a n t i b i q t i c medium Wo. 3 ( U S . Food a n d Drug A d m i n i s t r a t i o n ) ,

Sabouraud l i q u i d medium ( b i f c o ) and E a p l e s minimum e s s c n t i c l medium <. ( , I f I

. I , I" ."(. .I' . , i* ' ( ~ 1 3 ~ 0 Labora t o r i c s ) which was su$plernent ed w i t h 10% f ctal serum 7 were t r i e d f o r inves t i ( :a t in i ; t h e nnt i funl ;a l a c t i v i t y o f r e i ~ r e s e n t n -

t i v e drugs . T h i s s t u d y demonstrated t h a t a n t i b i o t i c mec!iuril >io. 3

was b e s t f < ) r ~ i e t c r m i n i n g M I C " (in-inirnal i n h i b i t o r y c o n c e n t r a t i o n )

t h i s medium ( r e f e r r e d t r ) a s b r o t h ) was employed f o r t h e .i> ,~,i't;,r~

drut: s e n s i t i v i t y s t u d i e s .

I !p t i funga l Urut;s T e s t e d and P r e p a r a t i o n o f I)rui: S o l u t i o n s

1 ~ ~ 3 . 1 & p h a t e r i c i n 3: The sample used f ~r t h e s t u d y was a n i n j e c t i o n

v i a l o f a m p h o t e r i c i n n (Funl;izone) manufac tured by Squibb X L ~

Sons , P r i n c e t o n , New J e s s e y . Each v i a l c o n t a i n e d a s t e r i l e

lypllc,lyzer! cake provic t ing 50mg a m p h o t e r i c i n 2 and 4mg s ~ ~ d i u r f l

c lcsoxychnlate b u f f e r e d w i t h 20.2mg sodium phosphate . I t W c l s

r c c 3 n s t i t u t e d by add ing a e e p t i c a l l y 10ml s t e r i l e d i s t i l l e d

w a t e r and s h a k i n g v i c o r o u s l y u n t i l t h e s o l u t i o n was c l e a r .

T h i s cave a 50X c , n c a n t r a t i o n . To o b t a i n IX, O . l m l o f 50X tins

mixed wi th 4,gml o f b r o t h medium ( a n t i b i o t i c medium IJo. 3 ) and

s o cnch m l o f t h i s hrc.th f i n a l l y c o n t a i n e d 100 g of t h e r l r u ~ . r t l f t c r r e c ~ ~ n s t i t u t i o n , t h e drug was s t o r e d i n t h e d z r k i n a

o ? c f r i c , e r a t o r a t 4 C f o r a mnxinum p c r i o d of 7 days.

IV.3.2 I l i conazo le : The form used was a w h i t e powder from J a n s e e n - -- P h a r m a c e u t i c a l s , Hew l3runswick, N . J . S i n c e t h e powder i s

i n s o l u b l e i n w a t e r , i t was r e c o n s t i t u t e d u s i n g d i m e t h y l s u l ~ h o x i d e

(DflS0). The po tency o$ , , , th i s . , b u g i s 98%. Hence t o o b t a i n 1000

1 0.01022 w a s weighed nu t w i t h a M ? t t l e r h a l a n c e (H 51 &dl)

and d i s s o l v e d i n 10ml 9MSO. T h i s was t h e n c o v e r e d w i t h

aluminium f o i l and l e f t t o s t a n d f o r 30 minu te s t o p e r m i t 41 -- .L

a u t o s t e r i l i z a t i o n . The f i n d c o n c e n t r a t i o n of 200 p d m l used i n

t h c t e s t was o b t a i n e d by addint ; 8 m 1 o f s t e r i l e b r o t h mecliuil! t o

2nd of t h e c!ru,; i n '3MSO. The r e c o n s t i t u t e d d r u g can keep i n a

0 deep f r e e z e r ( -30 C ) f o r a lon,: p e r i o d o f t ime.

1v.3.3 Kctoconzzo1.e: The form used was a l s o a w h i t e powdcr f r L ) n t h e

Jnnseen P h a r m n c c u t i c a l s and i t h a s t h e same p o t e w y 2s :G.conazole.

The 13rncedure f o r r e c o n s t i t u t i o n was similar t o t h a t of miconazole.

IV-3.4 5 -F luorocy tos ine : T h i s was p rocured as a w h i t e pow:cr form

Hoffmann-La Hochc Inc. Nu t l ey , N. J. S i n c e t h e c l r q i s s o l u b l e

i n wnter and h a s 100% po tency , 0.01,-~ qf i t was ~ S i s s o l v c J i n lorn1

d i s t i l l e d w a t e r t(1 o b t a i n n c ~ n c c n k r a t i o n of 1000 p / n L T h i s

was t h e n f i l t e r - s t e r i l i z e d u s i n g Gelman A C H O D I S C disposable

f i l t e r assembly. A f i n a l c o n c e n t r a t i o n o f 200 g/1nl w n s 0 3 t n i n c d P by addint; 8 m l of s t e r i l e b r o t h t o 2rnl o f t h e r e c o n s t i t u t e d drug.

0 It a l s o keeps f o r a lon;; p e r i o d a t -30 C.

IV.3.5 Potass ium I o d i d e : C r y s t a l s o f i ~ o t a s s i u m io t l i de (DDII) were weighed

o u t and d i s s o l v e d i n c i i s t i l l e i wa te r a c c o r d i n g t o t h c r e q u i r e d

c o n c e n t r a t i o n (5000 t o 30 ,000 g jml) . I t was f i l t c r - s t e r i l i z e d P b e f o r e use.

,' ,, .~ .!.a. ,,' > $ 3 ,

IV.go6 S e p t r i n : T h i s drug marketer1 hy h'ellcome Labso Z n ~ l n n h comprises

t r i m e t h o j ~ r i r n and su lphzmethoxazole and were used i n n l : T r a t i o ,

The two t i r u p were r cce ive 'h l i l - p u r e f o r n s i n separn'cd p a c k c t s

from t h e manufac tu re r s , Tr imethoyr im, 0.001g and 0 , 0 0 5 ~ o f

su lphnmothoxazole were mixed i n a c l e a n b o t t l e :lnd t h c n d i s s o l v e d

i n IOml of c l i s t i l l e d water t o a t t a i n a f i n d c , > n c e c ~ t r n t i o n 6000

p / m l . T h i s was f i l t e r - s t e r i l i z e d and used in t h c tilst.

IV.4 P Y e p n ~ n t i o n o f I n o c u l a

~ 1 1 t h e t e s t i s m l a t o s wort? grown on SDA p l n t c s a t ~ O O C f o r 48

t o 72 h o u r s t o ; ~ b t a i n young a c t i v e l y ,;rowing c u l t u r c s c o n s i s t i n g

of only rnycel ia and c o n i d i a b u t no zyi~;osj2ores. The fun:;al p c w t h

of ench i s o l a t e was a o > y t i c n l l y sc rapped o f f from t h e pla.te a11d

pu t i n t o a c o r k sc rew ( u n i v e r s a l ) b o t t l e c o n t a i n i n s IOml of

s t e r i l e s a l i n e and i;lass heads . The fun:;.al rilnss was t h e n broken U;J

i n t o small co lony forming u n i t s (CFU) u s i n g v o l t c x mixer (58220

by t h e . m e r i c a n S c i e n t i f i c l J r o d u c t s , McGraw P a r k , ~ l l i n o i s ) .

I1aer11ocytnrnctor was used t o s t anc la rd ize ench inoculum s o nr t o

6 o b t a i n 10 CFU p e r m l . The s t a n ~ l a r d i z e d inoculum was cross-

checked usin(; pour l ? l a t s countink; method. The p u r i t y 02 each

inoculum f o r b a c t c r i n l c o n t a m i n a t i o n was t e s t e d by i n o c u l a t i n z

Oe05ml of each on n u t r i e n t a g a r and i n c u b x t i n g f o r 24 h o u r s i?t

37'~. A 48 hour b r o t h c u l t u r e of Czndida a1 l ) i cans (ATCC 2.8516)

, f o r which t h e e f f e c t of c l i f f t - ren t d rugswas a l r e a d y known, w a s used

as a. c o n t r o l .

IV.5 i3roth D i l u t i o n Mcthod

The macro-broth d i l u t i o n metl?,\) i: of Shadomy an.1 X s i ~ i n c l -

Iny;roff (1980) w a s u sed i n t h i s ~+stutly. For t e s t i n g each j . so l z t c ,

n t o t a l of 1 4 s t c r i l c t u b a s were p l a c e d i n a t e s t - t u b e rzck. One

mil l i l i t re of t h e h i g h e s t c , n c e n t r a t i o n o f each drui; i n t h c b r o t h

medium w a s as tic tic ally 1~lz.ced i n t o t h e f i r s t tube . The d rug- f r ee

b r o t h medium w a s t h e n ~ o u ~ e d i n t o t u b e s 2 - 14 s o t h ~ t c:xb t u b e

c o n t a i n e d 0.5ml o f b r o t h , From t h e f i r s t t u b e , 0.5ml w?.s

t r a n s f e r r e d t o t u b e 2 usin[: s t e r i l e p i p e k t e attached t o p ipe t te -a id

(UrummrJd S c i e n t i f i c Comp.). The c o n t e n t s of t u b e 2 vas mixed

tho rough ly and 0.5ml from i t was t r a n s f e r r e d t o t u b e 3. The

p r o c e s s was c o n t i n u e d u n t i l t h e 1 2 t h t u b e a f t e r which 0.5111

from i t was d i s c a r d e d . T h i s gave a s e r i a l dilution rniiging i n

c o n c e n t r a t i o n from 100 pg/ml t o 0.05 @ m l f o r n n p l i o t o r i c i n B;

200 t o 0.098 p / m l f o r m i c m a z o l e , k e t o c o n s z o l e and 5-f luorocy-

t o s i n e ; 6000 t o 2.94 G / r n l f o r s e p t r i n . Tuhes 1 3 nnd 14 c n n t a i n e : i r no drui; ant1 si) server? a s c o n t r o l s . ; i l l t h e 14 t u b e s wcro f i n a l l y

i n o c u l a t e d w i t h 0. l m l o f t h e f r e s h l y p r e p a r e d f u n g x l suspension

6 ( c o n t a i n i n g I 0 Cm/ml) and i n c u b a t e d f o r 48 h o u r s at 3 0 ' ~ .

A f t e r ' t h i s , when v i s i b l e ~ r o w t h ( s e e n as clumi? o f mycelium)

o c c u r r e d i n t h e two c o n t r o l t u b e s , t h e minimal i n h i b i t o r g

c o n c e n t r a t i o n (MIC) fdr each druk; on t h e t e s t i s i ; l n t c 112s

r eco rded . PIIC i s t h e lowes t cc incen t r a t ion of n C r u ~ v h i c h

i n h i b i t s v i s i b l e growth o f t h e , t e s t organism. Thc m i n i m 1 ., ,,-.. "!. +' , ,< ,a

funl ; ic i (la1 c o n c e n t r a t i o n (MFc) w a s a l s o de te rmined f o r each drug

by s u b c u l t u r i n g 0.05~11 from each ne , ;a t ive t u b e and from t h e

p o s i t i v e o o n t r o l t u S e s on to SDA p l a t e s w i t h s u b s e q u c c t i ncubcz t ion I) "- .-

a t ~ O O C f o r 48 h r s o r u n t i l );rowth o f t h e subcu l tu rc?s form t h e

c o n t r o l t u b e s were a p p a r e n t . The lowes t c o n c e n t r a t i o n o f d rug

from which subcultures were neL;ative o r which y i e l d c d Scwcr t h a n

t h r e e c o l o n i e s was ref;ardt:d a s t h e MFC o f a drug.

1v.6 U l t r a s t r u c t u r a . 1 Sf f e c t of Kotoconazolc on I l a s i d i o h o l ~ . ~ ~

A q e t s i l c d e f f e c t of one o f t h e : in t i fun , ;a l d r u d s

( k e t o c o n a z o l e ) on t h e u l t r a s t r u c t u r e of a h n s i d i o b o l u ~ i s o l a t e

(2. h a p t o s ; ~ o r u s , G A 5 ) was s t u J i e d un( le r t r a n s m i s s i o n c l c c t r o n

k e t o c o n a z o l e and a n o t h e r j - j repara t ion of t h e i s o l a t e in a drug-

f r e e b r o t h medium, which s e r v e d a s c o n t r o l , were i n c u b z t c d c.t

3 0 ' ~ f o r 48 h r s and t h e n f i x e d i n 3% g l u t a r a l t l e h y d c i n O e l U

caco t ly l a t e b u f f e r , pH 7.0. They were stort>c-i a t 4 ' ~ f o r 48 h r s

a f t e r which t h e y were r i n s e d i n t h e same b u f f e r and p o s t - f i x e d

i n O s O 4 (Osmium t a t r o x i d e ) f o r 1 h r . S u b s e q u e n t l y t h e f i xed I

spec imens were r i n s e d i n 1 ;u f f e r a n d d e h y d r n t c l i n a ;rndcd

e t h a n o l c o n c e n t r a t i o n s e r i e s , f o l l o w e d by 100% a c e t o n o ( ~ n w e s I

1979). A l o w - v i s c o s i t y embedfling medium, forrnulntc?. 1~;. S p u r r

(1969) was used , w i t h hyphze f l a t l y emhedde(1. ~ireriz' f o r

s e c t i o n i n g were c u t o u t and mounted on micrntome s t u b s , xnd t h i n -

s e c t i o n e d w i t h a s o r v a l MT-2 u l t r a m i c r o t o m e Pant 2s , & , , . .a "*. \?. " >

Nernaurs 2nd Coo Inc . ) . The s e c t i o n s were s t a i n e d f o r 15 mine i n

0.5% aqueous u r a n y l a c e t a t e and i n l e a d c i t r n t e f o r 5 minutes

(Xeynold, 1 9 6 3 ) , and t h e n exnmined wi th a JLOL 100-C G I - e c t r o n i i .L

microscope ( J a p a n X l e c t r o n o p t i c s ) .

RXSULTS

'Je 1 ECOLOGIC{iL AND TAXONOMIC STUDILS

V-2 I s o l a t i o n of H a s i d i o b o l u s and C:)nitiiobolus from N a t u r & l ~ o u r c c s

Uas id iobo lus w a s r e c o v e r e d from t h r e e gu t of f o u r s lpecies of

an ima l s examined from Nsukka a r e a i n Ni[:eria (Tab le 1). The

h i g h e s t f requency of i s o l a t i o n (48%) was o b t a i n e d f rcln t h e

A f r i c a n l i z a r d (4gam.a agama) fo l lowed by t h a t from t h e common

t o a d (3ufo bufo) (40%)- Only 1@b of t h e w a l l geclros ( ~ c m i d a c t y l u s -7

b r o o k i i ) y i e l d e d p o s i t i v e c u l t u r e s o f t h e fun,:us whi1.c no

i s o l a t e s were o b t a i n d from t h e e i g h t p e e n t u r t l e (Cj i

mydas) examined,

T h i s fun,:us w a s a l s o i s o l a t e d from 6 o u t o f 9 s p e c i e s o f

an ima l s from Tampa and M i a m i towns o f F l o r i d a (Tab le 2) . A l l t h e

t h r e e g i a n t t o a d s (23ufo mar inus) and one c r i c k e t f r o g

f i r y l l u s ) examined, s a v e p o s i t i v e c u l t u r e s of I3nsiclioboLus~ -_--a A

very h igh p e r c e n t a g e of i s o l a t i o n was a l s o o b t a i n e d from t h e

c o n t i n e n t a l t o a d , ( ~ u f o t e r r e s t r i s ) (78%) ; Cubnn l i z n r c l ( A n o l i s * & ,, ., . d. >r .? '14,

s a g r e i ) (7.6%) and t r u e t o a d ( ~ u f o amer icanus) (61%). However, - f a e c e s of t h e 10 l a n d t u r t l e (Gopherus polyphemus) ; one creel1

t r e e f r o g (Kyla c i n e r e a ) a n d nne t r u e f r o g (Hnna utric$-mia) ,, .- ...

f a i l e d t o ~ { i v e p o s i t i v e c u l t u r e s . It is noteworthy thst

13asidiobolus w a s i s o l a t e d from an ima l s c o l l e c t e d a t t h e bcaches

i n Tampa and F l o r i d a ,

z c a G- (D 9

E ' D 0 I-'.'.

2 0. -

Y 'z: 0 0 '-5 9

W h;: P 0 0) 0) P. I-'. r i d d- r . l-'. 0 < U' (D 3 I-' c 0) - ? 2 0) '-5 P' 0 rt- (C r . ;i d c+

Distr ibut5.cn o f , - . s i d i o b o l u s S p e c i e s anong f i b i ~ t i l e s and ~gphibians i n .--.--. ------- I Percentage of I s o l a t i o n i n D i f f e r e n t I Izb i - -.* *

T o t a l No. Zxarnined ---

R e s i d e n t i a l R e s i d e n t i a l i

- -. u a m i Beach Tot

( - Areas i n Tzmpa Areas in Niani

Bufo marinus_ - Bufo arnericanus - V L L - d

Bufo t e r r e s t r i s

Of t h e 120 s o i l samples c c l l e c t c d from t h r e e d i f f e r e a t s i t e s ,

namely, f o o t p a t h , g2 rdcn and r i v e r , Con id iobo lus occurrecl i n

t h c t h r e e s i t e s w h i l e 3 a s i d i o b o l u s w a s i s c ~ l a t e d from o n l y the

(;cl.rden s o i l ( T a b l e 3 ) . Conid iobo lus was n o t i s o l a t e d from nny

cf' t h e a n i m a l s i n v e s t i y a t e r l . i iga in none of t h e samples o f

s n u f f and b a t d r o p p i n g s examined, ,:ave i m s i t i v e c u l t u r e s of

c i t h e r U a s i d i c ~ b o l u s o r Conid iobolus .

V - 3 ? d e n t i f i c a t i o n c f t h e . h ~ i d i ( ~ b o l ~ ~ I s o l a t e s

Fo l lowing d e t a i l e d m o r p h o l ~ ~ g i c a l o b s e r v a t i o n s under t h e

l i c h t microscope and v e r i f i c a t i o n wi th scannin l ; e l e c t r o n

mic roscope , a l l t h e i s o l a t e s of U a s i d i o b o l u s from t h e v a r i o u s

s p e c i e s o f a n i m a l s i n v e s t i g a t e d a s w e l l as i s o l a t e s from :;oil

were i d e n t i f i e d t o s p e c i e s l e v e l ( ~ a i ~ l e 4) . A l l t h e 108

i s o l a t e s from Ay;ama n,:arnn, 3 u f o k u f o , I iemidac ty lus broolc i i - =

and s o i l from N i g e r i a , had smooth-walled z y i p s p o r e s and s o were

i d e n t i f i e d a s - B, h a p t , ~ s y ~ r ? u s , , ( ~ i , ; . 5 ) . Out o f t h e 274 i s o l a t e s

f r orn A n o l i s c a r o l i n e n s i s , A. Buf o - 3. t e r r e s t r i s and A c r i s p y l l u s cau,;ht from F l o r i d a , 250 (91%) - wore n c c o ~ d i n g l y i d e n t i f i e d , a s H6 h a p t o s p o r u s ( ~ i g 6). R n 1 0 i l ~

I .-c.h

t h e r e m a i n i n g 24 i s o l a t e s , 12 (4$5) c;f t h e n which had r o u ~ h -

w a l l e d zyLTospores (Fig- 7) were i d e n t i f i e d as ~. rnnarum ant! - 2 (Oe7%) w i t h b o t h r,>u.l;h-walled and smooth-walled zy,;os;mrcs

( F I s . 8) a s 2, heterosporv .2 , Ten o t h e r s (4%) w i t h i n f k t e d

b.J I 0 0 rt

'a T J cb v m 0 v- I-' A

ul 0 V

- 0 P Y a 8 E; Pa P

A

V! 0 V

Y - z P . 4

8 t; D 3 a A

IU 0 w

-

ff and 32t Droppings he Frequency of --. -A - s i d i o h o l u s a 1

e o f L

Speci e s

Bas id iobo lus hap tosporus

B, h e t e r o s p o r u s - Conidiobolus c o r o n a t u s

= None = No of samples exanined = The numerals i n each column i n 4 i c a t e p e r c e n t c g e o f ~ s o i ~ t i o n . = ~igarna agama = x n o l i s c a r o l i n e n s i s = m o l i s s a g r e i = Bufo bufo -- = Bufo marinus - = Bufo arnericanus - = Bufo t e r r e s t r i s - - - , ~ c r i s g r y l l u s - = Hemidactylus br-ookii = Bat droppings = Snuff = Soi l .

Ti[;. 5: Smooth-walleJ zyigospores o f Wasid iobolus

h a p t J s y o r u s i s o l a t e l l from a 1 J i ~ ; e r i a n l i z a r d

(h!>;arna a~ama) . Note t h c "beak- l ike" ,* ,, .. . , , 3 +' ., ' . I #

p r o t u b e r a n c e s which were o b s e r v e d on mast of

t h e z y s o s p r e s . X 300.

(Fig.= 9 ) were i d e n t i f i e d as 2. m a p u s .

scanning e l e c t r o n riiicroscopy (SGM) conf i rmed t h e rough-

wa l l ed and smooth-walled c h a r a c t e r i s t i c o f t h e zy[;os&oyes

( F ~ K . 1 0 , 1 1 , 1 2 ) t h a t were obse rved under t h e l i q h t

rnicrc>scope. The re were no o t h e r a p p r e c i a b l e morpholo,.;icnl

d i f f e r e n c e s among t h e hyphae and c o n i d i a of t h e different

s p e c i e s under SGM. Most o f t h e i s o l a t e s o f 2, h a p t o s ~ u l ~ had

t h e d i s t i n t p i s h i n g ch :wncters of t h e genus. ' i he se inclucl.c t h e

f o r c i b l y t l ischnrr;ed g l i ~ b o s e o r sub i ; l o l~ose c o n i d i a ( F i g 1 3 ) ,

r e p l i c n t i v e c o n i d i a ( F i g 1 4 ) and p a s s i v e l y d i s c h a r g e d c l o n ~ n t e

a d h e s i v e c o n i d i a ( F i g 1 5 & 16) . A few i s o l a t e s devc lopcd

m e r i s t o s p o r e s (Fit: 1 7 ) on SDx medium.

Llas id iobolus h a p t o s i ~ o r u s was t h e s p e c i e s most f r e y u c n t l y

i s o l a t e d . Out o f t h e 378 i s o l a t e s o b t a i n e d from t h e a n i m a l s

i n v e s t i g a t e d , 358 (95%) were 2, h x p t o s p o r u s and o n l y 1 2 (3%)

were 2. ransrum w h i l e th.e*f.~'eneiriing 2% were 2. rna[r;nus

h e t e r o s l w r u s . T h i s fun:;us was alsr-, found t o be w e l l ~1.i.s i ; r i l ~ t e c !

ammi; t h e an ima l s p e c i e s , It was i s , ~ l a t e d from 9 o u t of 13 i 'I

(69%) s p e c i e s o f a n i m a l s f r o q Ni&ria and F l o r i d a (':'nl)lc Lt)a

V-4 I d e n t i f i c a t i o n o f Con id iobo lus c o r o n a t u s

I s o l a t e s o f C. c o r u n a t u s wsre i d e n t i f i e d by comp~ . r ing t h e i r - rnorpholo({ical c h a r a c t e r s w i t h t h e s t a n d a r d d e s c r i p t i o n s ::-ivcu by

c o i ) u l . : t i ; i n tub(- .?) , !'r. ,) ;><r.~t i o n is f r o m . , . I .

. .. . . ..

i 0 1 u j l t ( I ; : I : x 'I500 -A

i . ? S c a n n i n g e l e c t r o n rnic:ograph o f :I d e v e l o p i n g

r e p l i c n t i v e c.)riic?i.urn o f Ui.i,.;itfiobolus h n p t o a p o r ~ ~ s

s h o w i n g :

- P r i m a r y , ~ , J o b o s c : , c o n j t l i i l l n , , , I 4 . .

. <

3 - slender / :oniciiophore a r i s i n g f rom ri

C - X l o n g a t e ; ,lhc!siva con id ium.

Effect o f Tem2era ture on Colony_ 48 house of Iacubation

S p e c i e s ( R e f e r e n c e No,)

B. h a p t o s p o r u s * l (ATCC 14052) - B. ranarum - (ATCC 32277)

3. h a p t o s ~ o r u s * 2 (ATCC 35600) - B. magnus - ( a r c c i j 3 7 9 ) 5. microspcmus (ATGG 14708) - ,

3. h a p t o s y o r u s * l (ATCC 24670) -

) o l u s I s o l a t e s r i f t e r

v. 6 C h a r a c t e r i z a t i o n of i3as id iobolus I s o l a t e s . ". Thu r e s u l t ,,f some s t a n d a r d b iochemica l t e s t s con2uctccl

showecl t h a t a l l t h e t h i r t e e n i s \ ; l a t e s n f B : ~ s i d i o b c ~ l u s s ; ~ e c i c c

s t u d i e d had s i l n i l m b iochemica l p r o p e r t i e s . They a l l protluccd

u r c a s e on u r e a qgxr axcep t t h a t w h i l e most Gave p o s i t i v e r e s u l t s

w i t h i n ei:;ht h o u r s o f i n c u b a t i o n , o t h e r s t ~ o k t h r e e days (:o

so. A ~ : t i n a l l o f t h e n produced l e c i t h i n a s e , hydrn lysed c c s e i n

anti l i q u e f i e d [ ; e l a t i n .

They a l s ~ hnemolysed human, sheep and , p a t e r y t h r o c y t e s

complc tc ly w i t h i n 24 - 72 h o u r s of i n c u b a t i o n (E ' i tS 21). It is

notewcr thy t o mcnt ion t h a t w h i l e a n i s o l a t e of 2. rannruc: ( l i ~ ~ ~ ) ,

hnemnlysed t h e e r y t h r o c y t e s c f c h i c k e n c ~ m p l e t e l y w i t h i n 24

h o u r s , t h e r e n 3 i n i n g 12 i s o l a t e s corn'prising 11 2. h n , , ! t o s ; ~ r u s

?.nd one 3. ranarum (ATCC 32277) on ly p a r t i a l l y hacmolyced chiclccn - erythrocytes a f t e r '7 ~ l i ~ y s o f i n c u b a t i o n .

v.7 . P i i y e n t P r o d u c t i o n on Tyrooine i,,;ar Medium

2 n s i d i o b o l u s t e s t e d f e l l i n t o two mnjnr : ;roups, F i f t y s i x o f . -- than ( a l l - N, hap tosporus )which i n c l u d e d 2 human, 53 from &gyL

ngnmn, one , f rorn A n o l i s c a r o l i n e n s i s , and one i s o l a t e o f 2 , -- - I ) * ...

rnnmum (ATCC 32277) produced b l a c k l i i p e n t (Tab le 6 ) . The = ---

rc!aainintl; i a n l * i t e s c o ~ ~ i p r i s i n ~ 2 - 3. h a p t r ~ s p o r u s from human, 10

I;. ranarum, 5 2. mixenus, 95 D. haptos ; ,orus and one i s o l n t c o f .. . -

T a b l e 6

P i g r n e ~ t P r o d u c t i o n by D i f f e r e n t S p e c i e s o f - B a s i d i o b o l u s on T y r o s i n e f t p r Medium

Human I

IS I

Hodent d u n g 1

%ant d e t r i t m

'1-t d e t r i t u

n e n s i s I &, c a r o l i n e n s i s -----7'

.Ljwfa-, t l e r r e s t r i s 5 - U. amer icanus 12 - f t n o l i s sn!;rei 3

smooth

smoo t h

smooth

smooth

rough

rough

rough

m o o t h

smooth

smooth

rough

smooth

r out.;h

smooth

smooth

b l a c k

p ink

,>xcept ion of o n e , p roduced p i n k pi<:rnent. A,;ain t h e r e was 110

c o r r e l a t i o n be tween zyi:os;mre w a l l u n d u l a t i o n o r s p e c i e s O r

n u r c e of an i s ) l a t e and ;:i,;ment p r o d u c t i o n . The p i n k ant!

' ) l a c k pigrnvnt yrociuc t i o n by t h e i s f l l n t u s of 2. hr i j~ tos ;~c? ru -~

i s i l l u s t r a t u d i n F i , ; u r e 22.

V.8 *, kxpe r i rnen tn l - I n f e c t i o n o f L i z a r d s i n Ca j : t i v i t y w i t h t l n s i : ! i i l l J~~ l~u~

Seven o u t o f 12 ca/:;cd l i z a r d s u s e d i n t h e e x j ~ e r i r n c i ~ t !icre

; e r i o i l o f 2 - 1 8 days ( T a ? ~ l e 7). lvhen t h e s e s t n r v c d nniinnls ric;.c ,.,,..a k t . 7 , . .,*,,.? . .

l n t c r fccl w i t h u n t r e a t e d mealworms , t h e y s t a r t e d defaecmting again

: ) ~ x t none o f them y i e l d e d p o s i t i v e c u l t u r e s from t h e i r faeces.

5 'h i s obse rva t i : ; n i n r i i c a t e d t!lnt t h e mcalw\)rms d i d n o t nzturz!.l.; 11 . ..

il:?rbour t h e funl;us. However, when t h e 1 2 l i z a r d s were l-.i;cr Led

:!il;h menlworrns r o l l e d i n c u l t u r e s c o n t a i n i n i : youn:: s p o r e s o f

, ) r ,~duce t l nec;:itive c u l t u r e s ) y i e l d e d I3as i t l iobolus from t h c i r Lr-.eccs.

c 8 A g. 22: Pigment p r o d u c t i o n by i3;;sidiobolus i s o l a t e s on

t y r o s i n e a g a r medium.

A , r e p r e s e n t c u l t u r e s o f l d igc r l an i s o l 2 t e s n n ~ !

t h e y all produced b l a c k pigment.

R , r e p r o s e n t . ,cu&-ta~w nf F l o r i d a i s c j l a t e s m t !

t h e y a l l ~ ) r o d u c e tl ; , ink pil;rnent.

C , represent c u l t u r e s o f F l o r i d a i s o l a t e s t h a t

f a i l e d t o produce pigment even a f t e r 10 r!:tys

i n c u b a t i o n . I { 1 .*

0, I n o c u l n t e d t u b a s l ~ r i o r t o pigment p r o d u c t i o n

(Top row).

UJ

1 p : Y CD CD

P I-'. P.

F P ( D a m P P. P.

Ti le d u r a t i o n uf t h e p a - l o d o f r e i n f e c t i o n was 15 - 16 " q c ,

:~rob:~I) ly influenced by t h e q u a n t i t y of t h e fun@ S i ! O r G S

i n g e s t e d by each a n i m a l -

V i PfIY S IbLOGICclL STUDIES

Vx. 1 ? :x t r ace l lu l a r Enzyme P r o d u c t i o n of 11asidiobolu~-~~11,i~

Con id iobo lus I s o l a t e s on S o l i d l ledia

A l l t h e 383 i s o l a t e s of j j a s i ~ i o b o l u s s p p ho rn r e p t i l e s

and anph i l j i ans from I i i g e r i a and F l o r i d a and t h e o t l l c r 7 from

t h e Arnorican Type C u l t u r e C o l l e c t i o n (ATCC) proil.uccd on s o l i d

ai:ar media, e x t r a c e l l u l a r p r o t e a s e and l i y a s e b u t f a i l e d t o

produce amylase , r i b o n u c l e a s e ancl deoxyr ibonuc lensc ( l i 'n l~le 8).

P r o t e n s e and 1ip:ise o n l y were a l s o produced by the 36 i s o l a t e s

of Con id iobo lus c o r o n a t u s o b t a i n e d durint ; t h e p r c s e n t s tudy .

P r o t e a s e p r o d u c t i o n by a l l t h e i s o l a t e s of &l~;f~oj>-o,I .~~ and

Con id iobo lus t e s t e d , was ral : id and p o s i t i v a r c , ~ u l t s I l x e s e e n

w i t h i n 24 h o u r s of i n c u b a t i o n (Fig . 2 3 ) . iIowevcr, l i ~ j n s e

p r o d u c t i o n was slow and t h e v i s i l j l e p r s c i : ~ i i ; a t e (F ' i g 24) o f

t h e ca lc ium s a l t s o f l a u r i c acic, ( f rom Tween 80) o r o l c i c

a c i d (from Tween 20) was obsz rved a f t e r 5 days of i:ici.tbntion

f o r most i s o l a t e s but,,.,&tg~~,,,lO :lays f o r t h e rezit.

A s e m i q u a n t i t a t i v e a n a l y s i s of t h e l i p ~ s e , c c t i v i t y of

U a s i d i o b o l u s i s o l a t e s on s o l i d ap;nr medium was o h t a i u e d by

measuring t h e d i a m e t e r o f t h e zone o f p r e c i p i t n t e c-ild I ' I ..

r e l a t i n [ : t h i s t o t h e colon;{ s i z e . The ratio:; s o o b t a i n c d

r anged from 2 - 5.7 ( T a b l e 8). There was, however, no

c o r r e l a t i o n between t h e s e r a t i o s and t h e s ; )ec ies of i s o l a t e s ,

3. rnn$?rum and Conidiobolus coronn tus , (8xprcssccl -

Fig. 25 o f Culture ( Days )

X f f e c t o f t e m p e r a t u r e a n l i p n s e a c t i v i t y

o f c u l t u r e f i l t r a t e o f 13nnid iobolus and

Conid iobo lus i s o l a t e s .

Fig. 27

D a i l y measurements of p r o t e n s e a c t i v i t y o f c u l t u r e

f i l t e r a t e o f 3 a s i d i o b o l u s h a p t o s p o r u s , 2. ranarum,

~ n d C o n i d i o b o l u s c c r o n a t u s ,

Fig. 2 8

t h e h i g h e s t enzyme a c t i v i t y (66 pg o f s o l u b l i z e d pco te in /ml /h r )

was r e c o r d e d on t h e t h i r d day and i t remained s o uj? t o t h e

s e v e n t h day. 3. ranarum had a s t e a d y i n c r e a s e o f enzyme - a c t i v i t y u p t o t h e s i x t h day when a peak o f a c t i v i t y (114 v g o f

s o l u b l i z e d pro te in / rn l /hr ) was reco rded . 2. _corqnatus c o n t i n u e d

t o show a s t e a d y i n c r e a s e i n enzyme a c t i v i t y up'co t h e s e v e n t h

day when t h e peak of a c t i v i t y (124 pg o f s o l u b l i z e f i p:.otein/ml/

h r ) w a s r eco rded .

V I . 4 Snzyrne P u r i f i c a t i o n

The s t e p s i n v o l v e d i n t h e p u r i f i c a t i o n of t h e p o t e a s e of go

h a p t o s p o r u s (ATCC 366001, - Be rannrwn ( I ' I I I D ~ 1 and . C. , :>zonatus

(GI) a r e shown on T a b l e 9. Cn sephadex G-100, t k c !)rotease of

B. h a p t o s p o r u s was p u r i f i e d 50 - fo ld wi th 35% r e c o v c r y w h i l e t h a t - o f - B. ranarum was p u r i f i e d 38-f01(i w i th 20% recover ; and 43- fo ld

, w i t h 3374 recove ry f o r - C. c o r o n a t ~ p r o t e a s e .

The e l u t i o n p a t t e r n s of t h e p r o t e a s e s o f ~ ~ i ~ ~ t ~ o ~ s ~ p o r u s , g . ranarurn and 5. c o r o n a t ~ ~ " ~ ' r ~ " r & s ~ e ' c t i v e l ~ shown on f i [ ;u re s 29,

3 0 and 31. Two p r o t e i n peaks were obse rved f o r c ~ c h 01 t h e t h r e e

S ~ z c I e-s, a small peak ( A ) w i t h i n f r a c t i o n s 10-12 :u14 a h i g h peak

(B) w i t h i n ' f r a c t i o n s 22-32. ~ r a c t h o n s w i t h i n ped: il had no

p r o t e a s e a c t i v i t y , The re was o n l y one a c t i v i t y p:>d: r e c o r d e d

i n each c a s e f o r go h a p t o s p o r u s and go ranarut! bu t Tor .a- C .

c o r o n a t u s , two a c t i v i t y peaks were observed ,

3. ranarum - (MliB1)

C. c o r o n a t u s - (c ,

3u r i . _ " i ca t i c jn o f B s s i d i o b o l u s h?;,tos;-,orus, U. r a w m and Conidiol;olfL3 c o r o n a t u s P r o t e a s e s =- - *.* z A-

T o t a l A c t i v i t y T o t a l . P r o t e i n T o t a l Sl1ecL5.c . - c t i v i t y Y i e l d P P u r i f i c a t i o s Volume (mg o f A c t i v i t y Content P r o t e i n (i,~~/a;: of ? r o t e i n ) ( $ 6 )

S t el2 (d) s o l u b l i z e d (mg) (me/ml) (mt;) p r o t e i n /

L- ml -

C u l t u r e Filtrate ( Crud e 3ndxr-~e-I, 100 0.63 63 ; 6-12 612 --- S. --- 103 100

Acetone 1

P r e c i i !~t>-c*i~-

G - 100

C u l t u r e Filtrate

Sephadex G-1 .. -A % *

Cu.l tur e Filtrate \ C r u J e Enzyme) - - --.-A- --- - Acetone P r i c i ; > i t ~ t i o n

, ~ c p t l i n d c x G-100 column chromatography o f

Kl-uant : - 0.05M sodium : :cutate h u f f e r (pH 5.5).

F low rate: - 0.25rnl/rninute a t 3 0 " ~ .

Snphndex G-100 cdlumn chromatography of

R n s i d i o b o l u s

L l u a n t : - 0.051Jl s l ~ d i u m a c e t a t e b u f f e r (pH 5.5)

Sample l o a d : - 1 Om1

Gluant : - 0.05PI sodium a c e t a t e b u f f e r (pH 5.5)

Sample load : - 10ml

Flow r a t e : - 0.25rnl/minute a t 3 0 ' ~ .

V1.4.1 ;<f*II^~C~,t,t,tof Temperature on Yro tease Ac t iv i ty_

2'h.e optimum t e m p e r a t u r e f o r p r o t e a s e a c t i v i t y o f t h e

i ,ur if j .ed enzymes of - B. h n p t o s p o r u s , H. ranarum and 2. 0

c o r o n a t u s was 30 C (F ig . 32 ) . However, ove r 50% of t h e .. -.A-

L i ~ t i v i t y was r e c o r d e d a t 20'~: and 4 0 - 6 0 ° ~ ,

VI.4.2 C f i c c t of pH on P r o t e a s e A c t i v i t y . ,* a *. + . --=

The optimum pH f o r p r o t e a s e a c t i v i t y o f enzymes o f t h e

-Ill~-i?e s p e c i e s was 5.5 (P ig . 33 ) . Over 80% of t h e a c t i v i t y

:r;lS skil l r e c o r d e d a t pH 5 and 6.

E l e molecu la r w e i g h t s of t h e a c t i v e p r o t e a s e of Be - ll,! 7 t o s ; ~ o r u s (ATCC 36600) and U. ranarum (I4HB ) a r e t h e --**-A- - 1

ccmc and a r e approx ima te ly 23,000. go c o r o n a t u s (c,)

protlaccd two a c t i v e proteaess a n t t h e i r mo lecu la r w e i g h t s .,,,- . .bt. , t- . ,c. la '

x e a,?proximately 23,000 and 32 ,000 r e s p e c t i v e l y (Fig. 34).

X f f e c t cjf t e m p e r a t u r e on a c t i v i t y o f p u r i f i e d

p r o t e a s e of l i a s i d i o b o l u s and Conicl inbL, lus is',l:it es.

1 1 1 I I * 20 ?bO 40 $0 60

Te m p eroture ( O C ) Fig. 3 2

FIGUHX 34

S s t i m n t i o n ~f m o l e c u l a r w e i g h t s o f p r o t e a s e

oS G a s i d i o b o l u s hn;)t o sp , , r u s , 2. r ana rum and . -- C o n i d i o b o l u s c r ~ r o n a t u s by t h e method o f d m irews - - -... --.

( 1964)

fi = Cytnchrome C , i.113 1 2 , 5 0 0

1: = I* lyo@lnbul in , M!J 16,000

C = T r y p s i n , MW 24 ,000

D = Uovine se rum a l b u m i n , XW 6 5 , 0 0 0

E = , J d o l n s e , MJ 158 ,000

,. '*...b! 3,- , .>a ' Y = P r o t e n s e o f 4, h n 2 t c ~ s y , ~ , r u s ,

i;. rnnnrum and C. c o r o n a t u s - - Z = P r o t e n s e o f 2. c o r o n a t u s

. q.2 4.4 4.6 4.8 5.0 5.2 5.6 Lag 6f M o l e c ~ l a r Weights

Fig. 3b

VI I I N VITRO SENSITIVITY TO ANTITFUM~UL DRUG?

V I I - 1 . The .- E f f e c t of &ge of Fungal C u l t u r e s on t h e i r s u s c e ~ t i b i l i t ~

t o t h e Drugs -9

I n o r d e r t o determine t h e p roper age of an in~culun $0 be

used f o r t h e i n v i t r o s e n s i t i v i t y t e s t , a p r e l i m i n a r y s tudy

was conducted u s i n g some r e p r e s e n t a t i v e isolates (L 21' L24*

GA ) a t d i f f e r e n t a g e s of growth. For t h e t e s t , only one drug, 5

alaphoter ic in I3 was employed, Ten day-old c u l t u r e s of t h e s e

th ree i s o l a t e s of 2, hap tosporus compr2sing zygospores and t h e i r

48 hour-old c u l t u r e s which con ta ined only hyphae and c o n i d i a

were s u b j e c t e d t o v a r i o u s c o n c e n t r a t i o n s o f arnphotericin B

(100 - 0.05pg/ml) f o r 48 hours. The hyphae of t h e s e i s o l a t e s

m r e observed t o be more s u s c e p t i b l e t o t h e drugs t h a n t h e i r

zygospores (Tab le 10). For L and L t h e MIC and range 21 24

rma 0.39 - 0+?8~g/rnl f o r their hyphae and 0.78 - 100pg/rnl f o r

t h e i r zygospores, While f o r GA , MIC and MFC range for its 5

byphae was 12.5 - 25pg/rnl and f o r i t s zygospores , 12.5 - 1 0 ~ g / m l .

S i n c e Bas id iobo lus hap tosporus and Conidiobolus corona tus occur .' ,, . Z . " !. ,b. .? < t, * .

as hypae i n t h e t i s s u e o f t h e i r h o s t s , i n o c u l a f o r t h e

s c n s i t i v i t y s t u d i e s were o b t a i n e d from 48 hour-old c u l t u r e .

Table 10

Susceptibility of hyphae and zygospores of Basidio&l>.s4

haptosporus to amphotericin B

Isolate

. .

%.%.-- -"

MFC

j ; f f e c t o f Medium on t h e A c t i v i t y o f t h e Drug VII.2 . ,,..,.-,-,

R p r e l i m i n a r y s t u d y c a r r i e d o u t t o d e t e r m i n e t h e b e s t

nc;.cl.iurn t o be u s e d f o r t h e s e n s i t i v i t y t e s t s showed t h a t t h e

l o w e s t MIC and MFC v a l u e s were o b t a i n e d i n t h e a n t i b i o t i c

r~c.cliu~n-3 ( T a b l e 11 ) . Seven o u t o f n i n e i s o l a t e s t e s t e d w i t h

t h i s n ~ d i u m g a v e MIC and NFC r a n g e o f 0.098 - 0. 195pg/ml

f o r k e t o c o n a z o l e . I n Sabouraud l i q u i t i medium t h e r a n e e was

0 . 0 ~ 3 - 0 . 7 8 p ~ / m l ; 0.098 - 1.56pc/rnl i n Yabournud rnedium

r-;u:)i)lcrncnted w i t h 10% c a l f se rum; 0.39 - 1.56pg/ml i n YIVl3;

.:>.nd 0.098 - 1.56 g/ml i n X a g l o ' s min imal e s s e n t i a l medium. r Al though t h e v a l u e s o b t a i n e d i n Sahou raud rnedium a n d Xagle ' s

iwdium were a l m o s t as low a s v a l u e s o b t a i n e d i n a n t i b i o t i c

i~cclium-3, t h e u s e o f L:1gl.eV s medium w a s , however ,

d i s c o u r a g e d b e c a u s e t h e f u n g a l g rowth i n t h e c o n t r o l t u b e s verc

, cfiin:)arotively poor . Snhournucl l i q u i d medium was 4/50 not

u-se-~i- b e c a u s e o f p r e v i o u s r e p o r t s o f i t s , ~ n t a ~ o : ~ i & l on some

.. ..,,., .,'. cl.inugs (Ho e p r i ch and fIu3't rbn, Tni?', ' I 976) .

VII.:3 ; i , : ~ ~ ~ ~ ~ i v i t y t o Drugs

A 1 1 t h e f o u r t e e n i s o l a t e s o f : 13as id iobo lu s and 3 o f . .. ..-. ..

Go:-iic.licbolus showed re r t i a rkab le s e i i s i t i v i t y t o nmph .o t e r i c i n 3 , .-- ------ :-.:,.(.! t h e i rn i i l azo le d e r i v a t i v e s , m iconazo l e and k e t a c a n a z o l e .

?or' - the U a s i d i o b o l u s i s a l n t c s , t h e M I C :and IWC r a n g e f o r

i o t r c i n 1: was 0.098 .- 25pg/ml; and f o r rn ic (~nazo l i . , i t w a s

W W * 0 0 \ D \ D u \ D \ D

m m M

0.098 - 1 2 . 5 ~ ~ / 1 n l w h i l e f o r k e t o c o n n a o l e i t was 0.096 - d1.55pg/ml

( T a b l e 1 2 ) . E'or t h e ( ; on id iobo lu s i s o l n t e s , t h e MIC nntl EI7C r a n g e

f o r n rnphote r in 13 was 0.05 - 0.78pg/ml, and f o r rricom::oli. i t was

0.098 - 1 2 . 5 p ~ / m l w h i l e f o r k e t o c o n a z u l r i t w a s 0.195 - -1.56pg/rnl

( T a b l e 1 2 ) . 5 - F l u o r o c y t o s i n e d i ( i n o t i n h i b i t t h e r<,:or:tb o f any

s u c c e s s f u l l y i n t h e t r e a t m e n t o f i n f e c t i o n s by C o n i i l i o l ~ o l u s L L - _ L I L .

c o r o n a t u s ( L n t t a ~ m , 1982 , Oks fo r 2nd cfugnani 1983 ) . I%:; An v i t r o

e f f e c t o f t h i s d r u g on 5 i s o l n t c s o f 3. hnp to s :mrus nLlc? one o f - C. c o r o n a t u s showed t h a t t h e d r u g h a s s i g n i f i c a n t i n h i l ~ i t o r y - and f u n g i c i d a l e f f e c t s o n t h e s e f u n g i , t hough a t nuch h i@cr

d e r i v a t i v e s ( T a b l e 1 3 ) ~

P o t a s s i u m i o d i d e ( K ) hnd no e f f e c t o n t h e g rowth v f i : l~ : j t 1

o f t h e s e i s o l a t e s even a t c o n c c 1 , t r a t l o n s as h i g h as 90,00Op;/ml

m o l e c u l a r i o d i n e (1000pe/rnl) , R1 at much l o w e r concenk r : ; t i on

(15i00yg/ml) h a d b o t h i n h i b i t , ; ~ r y anti f u n g i c i d a l e f f e c k s on a l l

t h e t h r e e i s o l a t e s o f i l a s i c ?~~d l~a . - fuk t e s t e d . --

V11*4 The Z f f e c t o f Drug o n t h e U l t r a s t r u c t u r e o f o Bns id ioboLus --a* --.A

I s o l a t e

The t e s t i s o l a t e , 3,. h a p t o s p o r u s ( G A ) d i d n o t sll.o:! any 5

r no ryho log i ca l c h a n g e s untl(,r t h e l i g h t m ic ro scope a f t e r c::;>osure

Ta?) le I 3

I n V i t r o S u s c e p t i b i l i t y t o S e p t r i n (Trimethoprim and

Sulphame t h o x a z o l e , 1 : 5 )

I s o l a t e (Heference No. )

h a p t o sporus ( ATCC

B. hap tosporus (ATCC 14052 - -

C. c o r o n a t u s (C1) -

M I C ( p d m l )

375

3 75

375

750

750

7.50

111 V i t r o S u s c e p t i b i l i t y t o High C o n c e n t r a t i o r . of ., -.--

i5,;:toslmrus ( ATCC 36600) . , *

2, l i , ~ ~ t o s ~ ) o r u s ( ATCC 14052) r . -

2. h n p t o s p o r u s (T1) " A - +

C. c o r o n a t u s (ATCC 42093) .-* .I---

S o u r c e

Human

Rodent dung

P l a n t d e t r i t u s

I ' : ....... i.

Human

10, OOC

pshl

+ = growth

- = no growth

I OG.

t o kctoconazc,le f i i r 48 h o u r s , though t h e drowth w : ~ i i l h i l j i t e d .

However, u l t r a s t r u c t u r a l s t u d i e s ( w i t h t h e t r a n s m i s s i o n c l c c t r o n

mic roscope ) o f a mount from t i l e c o n t r o l t u b e , SIIOWCL' z ! l?yi r~~I .

hyphn l c e l l w i t h i n t a c t n u c l e u s ( F i g 35 ) . T3ut t i le nouoi: f rom

t h e t u b e c o n t a i n i n g 0-75 PE o f ketoconazole p e r ml revealed a c e l l

i n a n e c r o t i c s t a t e w i t h t h e o r g a n e l l e s i n c l u d i n g t h e ! ~ n c l e u s

comple t e ly d i so r&:mized ( F i g 3 6 ) .

li~. 35: E l e c t r o n micrograph o f a c r o s s - s e c t i o n o f a

h y p h a l c e l l o f i 3 a s l ~ i i o h o l u s hnp tosporus ( i ; f I 5 ) -- no t cx?osed t o a n A p t i f u n g a l drug. ( C o n t r o l ) .

11 -- .i x I I500

i ; . 6 : X l e c t r o n rnicrogrnph o f a c r o s s - s e c t i o n o f a . ' , , - . v $ . ,b. > . I > '

hypha l cell o f ~ a s i t l i o l ~ o l u s haytos : ,orus

(GA5) exposed t o 0.78 g of kc toconnzo le p e r P m l f o r 48 hours . X 1 1 5 0 0

D I S C U S S I O N

The p r e s e n t s t u d y c o n t r i b u t e s v a l u a b l e i n f o r m a t i o n on t h e

11 llurc?S o c c u r r e n c e o f U a s i d i o b o l u s and Con id iobo lus . B a s i d i o b o l w .---- a

I!_;; r e c o v e r e d from a v a r i e t y of s p e c i e s of r e p t i l e s and arnphibi.?nc;.

Some of t h e s e a n i m a l s , namely Ano l i s c a r o l i n e n s i s , 4. s a g r e i , ?_-S,t!

*:!;?rinus B. t e r r e s t r i s , g. arnericanus and ,'i:ris g r y l l u s w e r e . - - *-* - -9 ,

r c c o r d c d f o r t h e f i r s t t i m e t o h a r b o u r l 3as id iobo lus i n t h e i r

i j l t c s t i n a l c o n t e n t s . A h i g h r a t e of r e c o v e r y o f l 3 a s i d i o b o l u ~

froi,? a n i m a l s i n Anambra A t a t e i n N i g e r i a (41%) and a l s o from

S l o r i d a S t a t e i n U,:.;. (65%) is a c l e a r ev idence of i ts wide

~ ~ ~ c o ~ r a l d i l c a l d i s t r i b u t i o n . The f a c t t h a t t h e an ima l s c o l l e c t e d 0

.froin ~ : o s i d e n t i a l p l a c e s a s w e l l as t h o s e from Tampa and M i a m i

i ~ o ~ : . c h e s i n F l o r i d a were found t o f r e q u e n t l y ha rbour t h e fungus

i n d i c a t e s i t s a d a p t a t i o n t o a wide r a n g e of e c o l o g i c a l c o n d i t i o n s .

3onc c o n c l u s i o n s c a n b e drawn from t h e s tudy on t h e experimen.l;r:.i

i a S e c t i o n o f caged a n i m a l s , F i r s t o f a l l t h e f a c t t h a t p r e v i o u s l : ~

i n i e c k e d a n i m a l s e v e n t u a l l y became devo id of U a s i d i o b o l u s i n lcz:; .,,, . . . b 4 . . t . " > + 4 * ' .

t lmn 3 weeks shows t h a t t h i s fungus is a c t u a l l y a t r n n c i t m t orga:zi::::

o:~: t h c i n t e s t i n a l t r a c t s of r e p t i l e s and amphibians. T h i s i s

c c n t r a r y t o t h e s p e c u l a t i o n t h a t i t is a commensal organism o f t l i c 11 .- .i

i n k c s t i n e o f t h e s e a n i m a l s (Coremans-Pelsneer , 1973). Ncver thc lcc ;

tL; i g o s s i b l e q e r m ; r l ~ / i o h o f t h e c o n i d i a o f t h e fungus i n tllo

i n to; ; t ine of t h e a n i m a l s (Levisohn. 1927) canno t be doubted,

.k!lereby e x t e n d i n g i t s p e r i o d o f i n f e c t i v i t y and hence i t s c o n t i n u o c ~ ;

s a r v i v n l i n any envi ronment . Aecondly t h e f i n d i n g t h a t r e i n f e c t i o c

v ~ i t h H a s i d i o b o l u s c o u l d be a c h i e v e d a f t e r f e e d i n g t h e l i ~ i l r d s

wi th mealworms con tamina ted w i t h t h e fungus , a l t e r s i n a i~ i ino r

tray t h e p r e s e n t l y p o s t u l a t e d l i f e c y c l e of U a s i d i o b o l u s

(F ig . 3 7 ) . I t i s e v i d e n t from t h e p r e s e n t work t h a t s p o r c s o f

C n s i d i o b o l u s do not n e c e s s a r i l y have t o p a s s th rough t h e ,- --

i n t e s t i n e of i n s e c t s ( e . g . b e e t l e s ) i n o r d e r t o i n f e c t t h e @%her

an ima l s , b u t c o u l d be t a k e n up d i r e c t l y by r e p t i l e s and

amphibians th rough f e e d i n g on con tamina ted m a t e r i a l s i n c l u d i n g

mcalwarrns. H c p t i l u s and amphibians f e e d mainly on i n s e c t s m d

s i n c e i n s e c t s a r e o f t e n a s s o c i a t e d wi th v e g k t a t i o n p a r t i c u l a r l y

d u r i n g i n s e c t p o l l i n a t i o n , t h e s p o r e s o f t h e fungus ( i o e

p a s s i v e l y d i s c h a r g e d a d h e s i v e c o n i d i a ) may g e t a t t a c h d d t o Lhc

c u t i c l e o f t h e s e i n s e c t s . Consequt:ntly a n i m a l s can be i n f e c t c d

:lifter f e e d i n g on c o n t a v i n a t e d i n s e c t s .

Out o f t h e 378 3 a s i d i o b o l u s i s o l a t e s from t h e aiiim?.ls

investigated, 358 (9556) were i d e n t i f i e d a s 2, haptospor5s- v h i l c

o n l y 12 (3%) were 2, ranarum. T h i s i n d i c a t e s t h a t 13, w ~ o : ; p r u s - . A* * - -7 -

,, ,t- . .<. 9,. < < , a * . i a far more preva)ent t h a n - b,, ranarum i n t h e i r n a t u r a l !i?hii;nt.

A 1 1 t h e i s o l a t e s o f B a s i d i o b o l u s from human c a s e s of i n f e c t i o n f

have been - B. haptooporus . TLe a l a r m i n g number o f l i z a r d s 01:tside

r e s i d e n t i a l houscs a s w e l l ss" t he+h igh i n c i d e n c e o f 3 , ha~t._o:~~~;f~~, - - ahong l i z a r d s demands t h a t we ( p a r t i c u l n r l y i n ~ i g c r i a ) siiould.

recognize t h e p o t e n t i a l h e a l t h h a z a r d t h a t e x i s t s i n our

immediate environment . T h i s t h e r ~ . f o r e c a l l s f o r c l e a n l i n c a s of

ou r s u r r o u n d i n g s and p rope r c l o t h i n g f o r ou r c h i l d r e n who 01teil

p l a y o u t s i d e t h e homes.

It is riot s u r p r i s i n g t o f i n d a h i g h i n c i d e n c e of - Coilir!iobolus - . - . . -. - .*.-- c o r o n n t u s i n N i g e r i a n s o i l s i n c e t h e organism i s kno;rn t o occur

f r e q u e n t l y o n v e g e t a t i o n ( D r e c h s l e r , 1957; C l a r k , 1 3 7 6 ; ! d l l e r ,

1980). Tht? o c c u r r e n c e o f t h i s fungus i n s o i l o f \r:-.rj-ing 11z.bitat

e x p l a i n s why i n f e c t i o n s a r e more i n a d u l t s p a r t i c u l a r l y i? - . l cs who

a r e engaged i n f a rming ( C l a r k , 1976; Okafor and Gugiinlli, 1383) .

I n most d e v e l o p i n g c o u n t r i e s , ~ g r i c u l t u r e i s n o t moch:.:iizi:d

t h s f a r m w o r k e r s , mos t ly a d u l t males a r e t h e r e f o r e ex:~o::cd .to

s p o r e s o f - C. c o r o n a t u s d u r i n g farm work. The most p r o b a b l e method

of p r e v e n t i n g i n f e c t i o n by t h e fungus is by p r a c t i s i n g good

p e r s o n a l hyg iene . I t i:j a l s o n e c e s s a r y t h a t n a s a l c i l l ; o ~ . ~ ~ p h t h o r o -

mycos is due t o - C. c o r o n a t u s s h o u l d b e d e t e c t e d e a r l y irlough by t h e

m e d i c a l p e r s o n n e l s o a s t o p r e v e n t s e r i o u s and c h r o n i c i x ~ c c t i o n s .

Absence o f - C. c o r o n a t u s i n s amples o f s n u f f ex:tmiiu:d doon n o t

r u l e o u t t h e p o s s i b i l i t y o f i t s p r e s e n c e i n t h e 1ocnl i .y ..tL:.rkc.tcd I

ones . Tobacco l e a v e s , i f n o t p ro :oer ly p r o c e s s e d , 1;iny skill

c o n t a i n s p o r e s o f t h e fungstd'mfd 'tW pones p o t e n t i d cla:igcr

t o consumers. Again t h , ? r e i s n bad h a b i t o f unsc rupu louc

t r a d e r s a d u l t e r a t i n g s n u f f by a d d i n g o t h e r d r i e d c,.nd p i 8 0 b ~ b l y

c o n t a m i n a t e d l e a v e s t o i n c r e a s e thair p r o f i t .

' d i t h r e g a r d t o t h e tqxonomy o f B a s i d i o b o l u s , pignieni; ] x o d u c t i o n

i n t y r o s i n e medium d o e s n o t ,seem t o p r o v i d e any a i d i n i t s

identification. I n n ~ r e e m e ! ~ t w i t h t h e observation^: of C u t l e r

cnd Swatek ( 1 9 6 9 ) , t h e r e wr:s no c o r r e l a t i o n b e t w ~ e n pi;_;l:?en.i;

p r o d u c t i o n and mygospore w a l l u n d u l a t i o n . An i n t e r c s t i n ~ ;

o b s e r v a t i o n i n t h e s t u d y was t h a t a l l i s o l a t e s from ?!i!;.orin

p roduced b l a c k pigment w h i l e a l l t h e ones from Florid::, : i i t h t h e

e x c e p t i o n o f o n e , p roduced p i n k pigment . I t i s no tewor thy t h a t

one o f t h e - human i s o l a t e s (ATCC 36600) which produced b l a c k

pigment was a c t u a l l y i s o l a t e d from n p a t i e n t i n 1Jiger i .x ~. i lc t (4tg)

d o p o s i t e d a t t h e American t y p e C u l t u r e ~ o l l e c t i o n by ~ l - r r l d It

is n o t c e r t a i n i f s t r a i n s o f U a s i d i o h o l u s w i t h b l n c k ;.?i[gacnt

have more p a t h o g e n i c i t y p o t e n t i a l t h a n t h o s e w i t h p i n k p i p l e a t

as was p o s t u l : ~ t e d by C u t l e r and Swatek (1969) . Blsclc ; ~ i ~ i . ~ c n t

p r o d u c t i o n is p r o b a b l y r e l a t e d t o t h e e c o l o g i c n l habi-i;r.t o f all

iso1: i te . Hence, t h o s e from a r e a s w i t h h i g h temper ,x ture u o c t o f

t h e y e a r ( e , g . N i g e r i a ) d e v e l o p s p e c i a l p r o t e c t i v e p i p l e n t which

i s b l a c k i n t y r o s i n e medium.B. h a p t o s p o r u s was found t o l )c . - f r e q u e n t l y i s o l a t e d from a n i m a l s i n F l o r i d a as i n i \J igcr ia , Eut

human i n f e c t i o n s a r e r a r e l y r e p o r t e d i n F l o r i d a d e s p i t e ~ u c h

b c t t e r d i n g n o s t i c f a c i l i t i e s w h i l e i n N i g e r i a i t i s n f r c q u c n t l y

'.N .it.'. ? W i

known d i s e a s e . I t m:if 'bc h p i ; s s i d l ~ t h a t s t r a i n s o f 3. :l;!~~!pg~~orus

from N i g e r i a have g r e a t e r p : ~ t h o g e n i c p o t e n t i 3 l . It w i l l h e

i n t e r e s t i n g t o t e s t t h i s p o s s i b l e r e l a t i o n s h i p by devc1o:~ing

a n e x p e r i m e n t a l model o f 13~s: i 'd : iobt~lus i n f e c t i o n i n a l a b o r a t o r y

an ima l .

The s t a n d a r d b i o c h e m i c a l t e s t s s u c h a s u r e a s e and IL~ci:i;ii.ineco

p r o d u c t i o n , h y d r o l y s i s o f c a s e i n and l i q u e f a c t i o n o f ,.;.cl::tiin d i d

n o t o f f e r any d i a g n o s t i c v a l u e a s t h e y were p o s i t i v c f o r dl t h e

s p e c i e s of l 3as id iobo lus t e s t e d . Likewise t h e t e s t s f o r cuzyi~ie

p r o f i l e s , v i z amylase , D ikasc , HNAase, p r o t e a s e arid lip::no d i d n o t

h e l p i n s p e c i e s d i f f e r e n t i a t i o n . The d i f f e r e n t Basidi.b-~>~~~.,~,

s p e c i e s comple te ly haemolyzed ma~nrnalian e r y t l i r o c y t e s wI~i!-c complete

haemolys i s o f c h i c k e n e r j t h r o c y t e s was o b t a i n e d on ly w i t h ?*,

ranarum. The mechanism of haemolys i s o f c?ry throcytcs h:m been

l i n k e d t o p h o s p h o l i p a s e a c t i v i t y ( B r e t s c h e r , 1972; Taguchi and

Ikezawa, 1976). Thc i n a b i l i t y o f zi hhnptosporus t o conlp1:~"icly

hnemolysc c h i c k e n e r y t h r o c ~ t z s may be due p o s s i b l y t o lac!^: o f

s p e c i f i c phospho l ipase , Thus rnore work on haemolys i s o f ,?ilinal

e r y t h r o c y t e s may r e s u l t i n f i n d i n g a new c r i t e r i o n f o r si?cci:i.tion

o f B a s i d i o b o l u s . I n view o f t h e present s t u d y and t h e nvt \ i ln?>le

i n f o r m a t i o n , t h e p r e s e n t l y a c c e p t a b l e c r i t e r i a f o r s p c c i c s

s e p a r a t i o n i n B a a i d i o b o l u s . i n c l u d e zygospore w a l l undu:lntj.or, and

p r o d u c t i o n of mic rospores ,

With r e g a r d t o t h e enzyme s t u d i e s , 2. h a p t o s p o r u s , E,, ;r;r2.narum

2nd - C. c o r o n n t u s produced v x t r n c e l ~ u l a r 1- ipase and p r o t 2 ~ 1 . s ~ i n . ' , " . . . . # . , t . . > < d a ' .

i h e i r growth media, : I ; x t r a c e l l u l n r p r o t e a s e p r o d u c t i o n L<!s been

r e p o r t e d e a r l i e r i n - C, c o r o n a t u s (Sornkuti and 8nrhc1 , ,1968~ ,-

Froment in , 1975; S r i n i v a s a n ct &, 1983) b u t n o t i n -- ~~ .n . i . c l iobo lus . -- -.- -- L i p a s e p r o d u c t i o n h a s been r e p o r t ~ b i n c e r t a i n zygomycctco like

Mucor p u s i l l u s and 3. meihei (Somkuti and B a b e l , 1968; O:;~:ndcro - 9

1980; Ueploey, 1981) b u t no t i n Con id iobo lus and B ~ ~ i d i o ~ ~ , o > , u ~ c j ~

s p e c i e s .

i i p a s e p r o d u c t i o n by 2. h n p t o s p o r u s , 2. ranarum and 2.

~ o r o w ~ t u s was o b t a i n e d i n s i g n i f i c a n t q u a n t i t i e s a s from t h e . - *.- .- -- -,*.-

f o u r t h day o f growth and i n c r e a s e d g r a d u a l l y beyond t h e t w e l v e t h

A . .~ ., ,. <,,.,, . i jeploey i 1981 ) working wi th Mucor rneihei dernonstrntcd l i p m e

.~ :~ roduc t ion 24 h r s . ~ f t e r i n c u b a t i o n w i t h a peak of a c t i v i t y on t h o

3 r d d a y , wh i l e f o r 1.lucor p u s i l l u s t h e peak of l i p a s e a c t i v i t y v2.s

2 c ~ ~ r ~ l e d on t h e 6 t h day of growth (Sornkuti and U a r b e l , 19661,

'11 ~ ~ l i s lshoi~s t h a t H a s i d i o b o l u s s p e c i e s and C , co ronn tus produce - c x i r n c e l l u l a r l i p a s e o v e r a l o n g e r p c r i o d i n a growth medium th:n

lAucor .* - . s p e c i e s and t h e r e is 3 p o s s i b i l i t y t h a t they a l s o p o s s e s s

i!r!:~~brnne-bound l i p n s e which may be l i b e r a t e d a t dea th . F u r t h e r

on p o s s i b l e p r e s e n c e o f nernbrane-bound l i p a s e s i n t h e s e

Zimgi i s t h e r e f o r e d e s i r a b l e .

P r o t e a s e p r o d u c t i o n was much f r ~ s t e r t h a n l i p n s e p r o d u c t i o n

-:IJO~I;; t h e H a s i d i o b o l u s and Con id iobo lus .kocr/c;es t e s t e d , ,

md wyj.s obse rved w i t h i n 24 h r s , of i n c u b a t i o n . A s i m i l a r

o '~~ . ; c rvn t ion w i t h r e g a r d s t o Nucor p u s i l l u s was r e p o r t e d e ~ r l i e r

. , . , , ' ( S o i k u t i and B a r b e l , 1967). "hey a l l r e a c h e d t h e i r pcnks of

2 . c t i ~ ~ i t y w i t h i n s i x days of i r c u b a t i o n .

Chromatographic p u r i f i c ~ 5 i o n o f t h e p r o t e a n c of B. hn-ptos);o~;~cj, - C. x n a r u m and C. c o r o n a t u s ' h ~ s not y e t bean r e p o r t e d , The two s.. - -- C:~.nidiobolus s p e c i e s under t F e c o n d i t i o n s o f c u l t i v a t i o n used ..a - .-.-- , z ec rc t ed o n l y one t y p e of p r o t e n s e i n t o t h e medium w h i l e - C. .-.. c o r o n r t t l ~ - .. .... . 7 .

3i.cr..eted two t y p e s , Only one t y p e of a c t i v e p r o t e n s e w m a l s o

produces the two a c t i v e eneymee, protease and l fpase i n

s i g n i f i c a n t quanti t ied. Apparent l ack of human in fec t ion due t o

t h i s fungus m a y be explained by the f a c t t h a t i ts frequer)#p i n

ecological h a b i t a t ie not s ign i f i can t .

Basidiobolus and Conidiobolue species t e s t e d were found t o be

more suscept ib le t o the imidazole de r iva t ives , mioonaeole and

ketoconazole than t o amphotericin B. The MIC and MFC range of

micanazoh was 0.098-1 2.5 pg/ml while f o r ketoconazole i t was

0.098-1.56 p g / m l . Over 62% and 68% of t h e isolate . were inh ib i t ed

by miconazole and ketoconazole respect ive ly a t a concentrat ion of

0.78 pg/rnl. The MIC and WC of these two drugs a r e within the

t o l e r a b l e therapeut ic serum l e v e l s which are 7-8 p / m l a f t e r

600~1000rng of intravenous dose of niconaeole (Stevens et nl,

1976) and 2-4 p / m l a f t e r a 200-400 m g / d y of o r a l dose of

ketoconazole (Bras et al, 1982).

Amphoterin B inh ib i t ed the growth of approximately 81%

of the t ee tcd isolate at a concentration of 0.78 @ml . This

concentrat ion is within the to le rab le therapeut ic serum l e v e l s . , , r - . . w 6 ~ . , . * n ~ .

which is 0.5-1.5 pg/ml a f t e r 3g/day of intravenous doae ( ~ i g p o n ,

19821, Three i s o l a t e s of - B. haptosporua were,however, r e s i s t a n t

t o t h i s drug. This might exy la in . ce r t a in r e p o r t s of c l i n i c a l I t - .-

f a i l u r e s of amphote?rLcr~ B i r l t h e treatment of dubcutaneous

phycoaycosis (Kamalam and Thambiah, 1978) and a l s o rhinoentomo-

phthorornycosis (Nathan e t al, 1982). - . -

'

M ~ u i m u m the rapeu t i c concentrat ion of 5-fluorocytosine d id

n o t h::vz . i d - - i b i t o r y e f f e c t a g a i n s t any o f t h e t e s t e d i s o l a t e s .

I n ~ ~ f T , c L l v e n c s s o f t h i s d r u g i n t h e t r e n t i g e n t o f rhino-entomo-

~ ~ h t l ~ o ~ o i s ~ y ~ o ~ i s h a s b e e n r ? c o r d e d ( H e r s t o f f et &, 1978) . The d rug

h n s , h o ~ : c v c r , been u sed s u c c e s s f u l l y i n t h e t r e a t m e n t o f i n f e c t i o n s

by Cnj~did,l, n l b i c n n s (Warmer e t 81, 1970, Utz a n d Shadomy, 1977; - W _.- - * "-

K o l d i n and i i cdo f f , 1983) .

Tile j~ y 1 t r o s u s c e p t i b i l i t y o f i s o l a t e s o f 2. h a p t o s p o r u s and

C. -.-.. coron,. : tus ----.. -.. t o s e p t r i n c o n f i r m s i t s s u c c e s s f u l u s e i n t h e

t r e a t m e n t of i n f e c t i o n s due t o 2, h a p t o s p o r u s and 2, c o r o n a t u s

(Fomufod ?l.n.d. r%nt ia, 1971 ; Fln r t i n son , 1971 ; 1972; Sowomimo, 1974;

ItI-xinun t h e r a p e u t i c c o n c e n t r 3 t i o n o f p o t a s s i u m i o d i d e (KI)

d i d n o t L u . ~ c any e f f e c t on t h e i s o l a t e s o f U n s i d i o b o l u s and

Con id iobo lus . The d i s p a r i t y be tween t h e i n v i t r o o b s e r v 2 t i o n s %and I- -_* _L S .-.I

t h e ~~II.o~.I;L .-_. ii.1 vivo , _ a c t i v i t y o f K I on s u b c u t a n e o u s phycomycosis

(I:ain::lafi~ ,-.;zd Thnmbiah, 1379; 1982 ; B i t t e n c o u r t -- e t a l , l 9 8 2 ) and

rhinoini ;oisopi-~t l iorornycosis ( F r o m e n t l n and R n v i s s e , 1977) i s o f

in tc rc : ;%. The r c s u l t s o f t h e i n v i t r o s t u d i e s w i t h K I a re q u i t e -- compzrnble t o t h o s e o b t a i n e d ii e n r l i c r s t u d i e s by T i o and V r i e s

(1977) who en$gee-tedthat K I a c t i v i t y is n o t due t o a d i r e c t e f f e c t

on t h c flin;;~l.s b u t r - i t he r t o a A n - s ~ c c i f i c a c t i o n . A s imi la r

phenomenon h ~ s b e e n o b s e r v e d w i t h S p o r o t h r i x ~ c h c n c l c i i i n which

t h e . i.11 -.. ..+ v i t r o - -..-.A r ; t u d i c s f a i l e d t o demons t ra . t e any d i r e c t a c t i v i t y

of K C on t h e fungus ( D a v i s , I g l ? ) d e s p i t e i t s h i g h c l i n i c a l

efficacy i n tl?c t r e a t m e n t o f s p o r o t r i c h o s i s ( ~ h i b u y a , (1959) .

T.7 ax1.y i n v e s t i g a t o r s s u g g e s t e d t h a t KI s t i m u l a t e s t h e h e a l i n g

pyoccss o f s n o r o t r i c h o s i s (Uavis , 1919). O t h e r s showed t h a t

in p r e s e n c e o f rnoleculnr i o d i n e , K I h;is a f u n g i c i d a l e f f e c t

(Sl i i l )uyn, 1959). I t was a l s o s u g g e s t e d t h a t m o l s c u l a r i o d i n e

Li:-idr enhance phngocy t i c a c t l v i t y , Subsequent s t u d i e s speculc . ted

ih?C f r e e o r molecular i o d i n e formed from t h e body may be

; ~ ~ . n ~ ; i c i d a l whereas 1{I may b e f u n g i s t a t i c (Urabe and Ngashim.,

5969; Sada , 1968). However t h e c o n c e n t r a t i o n o f K I t h a t was

conhincd w i t h molecu la r i o d i n e t o k i l l t h e t e s t e d i s o l a t e s of

3 x i d i o b o l u s and Con id iobo lus w a s 30 t i m e s t h e t o l e r a b l e serum .. - - - -- l cvcL (which i s 50 p g / m l a f t e r 2-3g/ml o f intr:+vanous doses) .

r-r I n u s , t h e e x a c t mechanism of t h e r a p e u t i c a c t i o n of K I i n

S ~ O ~ O tri c h o s i s , subcu taneous phy c6m:rcosis and rhino entomoph-

E l e c t r o n mic roscop ic ev idence of t h e morpho log ica l

o b l i t e r a t i o n and n e c r o s i s o f t h c c e l l u l a r s t r u c t u r e s o f 2.

; i L ~ p l ; o s ~ r ~ s i s o l a t e (GAS) exposed. t o 0.78 g of k e t o c o n n z o l e per ml ...4.--- - P i s 11.1 agreement wi th &mLY'n~'"s t'udy on Cocc id iodes i m m i t is

( ~ o r g e r s , 1981). T h i s conf i rms t h e mechanism of a c t i o n o f t h e

i i ; i idnzole d e r i v a t i v e s which were s a i d t o cause d e t e r i o r a t i o n 02

s u h c c l l u l a r o r g a n e l l e s r e s u ~ t i n g . : i n c e l l necros is . ( H o l l i n am!

Dorger s , 1976; f iorgers , 1980; i t ippon, 1982).

The r e s u l t s o f t h e i n v i t r o s c n s i t i v i t y s t u d y s u g g e s t t h ~ t -- the n i c o n a z o l e and o r a l l y a d m i n i s t e r e d k c t o c o n a z o l e cou ld bc

c o n s i d e r e d p o t e n t i a l t h e r a p e u t i c d rugs f o r t r e a t i n g s u b c u t n n e o ~ ~ s

i )?~ycotnycosis and rhino-cntomopkthorornycosis e s p e c i a l l y f o r paCIc-.i-~i;::

who f a i l t o r e spond t o K I and a m p h o t c r i c i n B. M i c o n ~ z o l c h a s

becn found t o i n h i b i t t h e growth of a lmost 211 patllof:enj.c :Cungi

(Van Custem and T h e i p o n t , 1972) and h a s been used as t : l c -npeu t i c

drug a g a i n s t s y s t e m i c f u n g a l i n f e c t i o n s ( S t e v e n s , 1977). J2i v i t r o

s e n s i t i v i t y s t u d i e s have shown t h a t Ketoconnzolc i s n l s o e f f e c t i v e

a g a i n s t s e v e r a l p a t h o g e n i c f u n g i . The r e s u l t s of c l - i i ~ i c d

e x p e r i e n c e w i t h Ketoconazole on 1 ,104 p a t i e n t s i n d u ~ o p o and

America proved t h a t t h e d rug i s h i g h l y e f f e c t i v e i n t h c t ~ c a t r n e n t

of bo th superficial and deep mycoses (Syrnoens 2 A;, 13301.

There i s no c l i n i c a l r e p o r t on t h e u s e of any of tnesc tluo d rugs

i n t h e t r e a t m e n t o f subcu taneous phycomycosis o r rhin0-cilt01~10ph-

thoromycos is . Thus i n view o f t h e p r e s e n t i n v i t r o ~ti:dj.?,.c i t i s

wor thwhi le t o i n v e s t i g a t e t h e e f f i c a c y of t h e s e d rugs in t l ~ c

t h e r a p y o f t h e s e two i m p o r t a n t d igenses .

APPENDIX

+*

1. DIFCO Sabourautl l l e x t r o s e i i r o t h ( l l e h y ~ i r n t e d ) I n g r e d i e n c e P e r L i t r e

Neopeptone - 1 O[;

To r e h y d r a t e , d i s s o l v e 3 0 ~ o f above powdered m i x t u r e i n 1000ml o f t i i s t i l l e1 . l water .

2. ?>PC0 , & n t i b i o t i c I&dium IJo. 3 ( i k h y d r a t u r ? ) I n p e d i e n c e P e r L i t r e

Ccef S x t r n c t - 1.5e

Y e a s t ldxtr:lct - 1 .St.

I-cptone - 5r~

D e x t r o s e - 1~

Sodium C h l o r i d e - 3 . 5 ~

Lipo tns s ium Phospha te - 3 . 6 8 ~

honcpotass ium .k:hnspb,a&e . - 1 3 2 ~

To r e h y d r a t e , d i s s o l v e 17.5g i n 1000ml of

d i s t i l l e d water .

I t "- .C

!Jo r c h y d r a t e c l i ssn lve 56 o f d e x t r o s e and 6 . 7 ~ o f

i'acto-Y e a s t l \ l i tro,;en j jsse i n 100ml o f d i s t i l l e d

~ : , l t c r t o o b t a i n 1 0 x s t r e n g t h .

Prof. H. C. Gugnani f o r h i s r e l e n t l e a s effoar.ts dm srscow~age

me t o reach t h e climax of my academic pursu i t . I thank

him f o r t h e d i l i g e n t supe rv i s ion and c r i t i c a l review of t h o

manuscript i n s p i t e of h i s b igh t academic commitments. I

wish a l s o t o thank Drs. U. T. Merner and 3. G. Yangco, both

of t h e Univers i ty of South F l o r i d a , Tampa, U. S., who

generously provided the f a c i l i t i e s used f o r a p a r t of thia

work and a l s o introduced me t o some new research techniques.

I a m equal ly g r a t e f u l t o t h e Univers i ty of Niger ia , Nsukka

f o r sponsoring m e on one yea r etudy leave t o t h e Univers i ty of

South Flor ida, I would a l s o l i k e t o express my g r a t i t u d e t o

Bet ty Loraam, Henry Mushinsky, Karen Rel i sh and o the r s , also

of U, S. F, as wel l a s t o D r . S. K. C. Obi, my present Haad

of Department, Dr. A. Anoaike of Biochemistry Department and of

s e v e r a l o t h e r s i n t h e D e p a r t m c n t ~ M i c r ~ b i o l o g y ~ Biochemistry

and Chemistry f o r t h c i r ass i s tance . I a m h igh ly indebted t o .* , " ; *WC, 9- ,,P u+ -

my husband, Prof. C. 0, Okafor and my c h i l d r e n and d e o t o

my f a t h e r Mr. J. 0. Igboko f o r t h e i r encouragement* Finally

I wish t o thank Mrs. R . ,inyogu f o r typ ing t h e manuecript. I t .- .L

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