Sabouraudia(1978), 16, 63-68

STUDY OF THE R O L E OF P I G E O N S IN T H E D I S S E M I N A T I O N OF
CR Y P T O C O C C U S N E O F O R M A N S IN N A T U R E

M. ABOU-GABAL AND *M. ATIA

Department of Microbiology and Preventive Medicine, College of Veterinary Medicine,

Ames, Iowa 50011, U.S.A.

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Cryptococcusneoformanswas recovered from droppings collected within the first 24 h from
pigeons experimentally fed with a dose of 5 x 106 cells. The fungus proved to multiply well
though differently in the sterilized pigeon and chicken excreta seeded with the organism. In
both unsterile types of droppings no viable cells of C. neoformanswere detected after 4 weeks
incubation. Isolated bacterial flora from the intestinal contents of apparently healthy pigeons
showed a complete inhibitory effect on the growth ofC. neoformsin vitro. It has been concluded
that pigeons do not favor multiplication of the fungus in their gut and consequently they do
not seem to play an active biological role in dissemination of C. neoformansin nature.

Cryptococcosis of h u m a n s a n d different a n i m a l s is a disease of w o r l d - w i d e dis-

tribution caused b y the yeast, Cryptococcus neoformans. M a n y studies have shown t h a t
excreta of pigeons and, less f r e q u e n t l y , t h a t of o t h e r birds serve as a n i m p o r t a n t
natural reservoir of C. neoformans infections. T h e recovery of C. neoformans from p i g e o n
excreta has been r e p o r t e d from m a n y countries, e.g., U . S . A . (12, 19, 28, 20, 28),
Australia (13), E n g l a n d (32, 38), J a p a n (43), S w e d e n (6), W e s t G e r m a n y (7),
Czechoslovakia (17, 21), P o l a n d (23), T h a i l a n d (40, 5), I n d i a (35, 15), Tunis (25),
and Nigeria (25, 14).
Apart from pigeons, C. neoformans was isolated from e x c r e t a of t h e J a p a n e s e
skylark (22), c a n a r y (36), chicken (41), s p a r r o w (31), p h e a s a n t (16) a n d swift (Apus
~#) (15).
Though pigeon excreta apparently play an important role in the dissemination
of the organism in nature, the role of the pigeon itself in that respect is not yet fully
understood. The intention of our investigation was to determine whether C.
neoformans can m u l t i p l y in the g u t a n d the possibility of pigeons p l a y i n g a n active
biological role in disseminating the fungus in n a t u r e .

]V[ATERIALS AND METHODS

Cultures tested
Four recently isolated pathogenic strains OfC. neoformans from cats (IV]V[ 21 ? and
IVM 320) and dogs (IVM 450 and IV]V[ 482) were used. The strains were first sub-
cultured on brain-heart infusion, Sabouraud glucose agar and incubated at 37°C
for 10 d. A suspension in sterile saline was made from each strain and the number of
cells per millilitre was determined using the slide hemocytometer (Bright-Line).
Two suspensions containing 5 x 106 and 6 × 104 eells/rnl were prepared from each
strain for the in vivo and in vitro investigations, respectively. A preliminary viable
count on the prepared suspensions was conducted directly before the experiments.
In vivo Studies
To investigate the v i a b i l i t y of C. neoformans in the g a s t r o i n t e s t i n a l t r a c t of pigeons,
20 birds (Columba livia) were used. A g r o u p of 4 pigeons was i n c l u d e d as a control.
*Postdoctoral scholarship, Department of Microbiology, College of Medicine, Asslut University,
Eygpt.
63
64 ABOU-GABAL AND ATIA

Excreta and feed of pigeons were checked to determine if they were free of C.
neoformans by culturing and mouse inoculation procedures. Pigeons were divided
into groups of 4 and each was inoculated with the same strain. One ml suspension of
C. neoformans (4 x 106 cells/ml) was then inoculated orally in each of the pigeons
tested. Oral inoculation was achieved by delivering the suspension directly into the
esophagus with the aid of a sterile syringe and then holding the beaks of the pigeons
closed to ensure swallowing. The inoculated groups were kept separately and their
excreta collected together in sterile cellophane bags every 24 h for a period of 4 weeks.
Droppings from each group were homogenized thoroughly in 500 ml sterile saline
directly after collection. Six serial ten-fold dilutions in sterile saline were prepared

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from the homogenized suspension. Triplicate plates were made from each suspension
using thistle extract, creatinine, Littman oxgall and brain-heart infusion agar.
Chloramphenicol was added in 0"05 mg/ml amounts to all media used. T h e average
n u m b e r of C. neoformans colonies on the 3 media was counted and the number of
viable cells in the homogenized excreta originated from each group of 4 pigeons was
calculated (10). In addition, 0'5 ml of the dropping suspension was inoculated
intraperitoneally into white Swiss mice to ensure reisolation Of C. neoformans. Also,
droppings frcm the control pigeons were checked for the occurrence of the fungus
by culturing and mouse inoculation technique. After 4 weeks, the pigeons were
killed and cultures, as well as mouse inoculations, were m a d e from the intestinal
contents and different internal organs.

I n vitro studies

Growth of C. neorfomans in pigeon and chicken excreta

A group of 5 pigeons and 5 chickens were kept in separate cages and fed on
Wayne pullet starter over a period of 4 weeks. Excreta were collected from each
group and distributed in 25 gm amounts in glass Petri dishes. A group of plates was
autoclaved at 121°C for 30 min and their sterility checked. T h e other plates were
left unsterile and were proved free ofC. neoformans. Five ml of the prepared suspension
(6 x 104) were seeded onto the droppings in each plate. Plates containing non-seeded
sterile and unsterile droppings were included as a control. The plates containing
sterile excreta and half of the unsterile ones were kept moist by adding sterile saline
from time to time wher~ it seemed necessary. T h e plates were incubated at 25°C for
4 weeks. The content of each plate was then homogenized in 500 mI sterile saline
and the n u m b e r of viable cells ofC. neoformans was calculated.

Determination of pH
The p H of pigeon and chicken excreta was measured before seeding with the
organism. Approximately 5 gm of the droppings were suspended in 20 ml distilled
water and the p H was determined using a Beckman p H meter.

Eiffect of the intestinal bacterialflora of pigeons on the growth of C. neoformans

For isolation of bacteria occurring normally in the gastrointestinal tract, samples
of the intestinal contents were obtained aseptically from apparently healthy pigeons.
Cultures were made on nutrient agar, blood agar, and MacConkey's agar medium
and incubated for 24-48 h at 37°C. T h e isolates were identified according to their
characteristics, microscopical appearance and biochemical activities (10). Two
 STUDY OF C. NEOFORMANS IN NATURE 65

colonies from each isolate were suspended together in 10 ml sterile saline. One ml of
the prepared C. neoformans suspension (6 x 104) and 0"1 ml suspension of the isolated
intestinal bacteria were mixed and transferred directly into tubes, each containing
10 ml brain-heart infusion broth media. Each strain was tested separately. The same
amount of C. neoformans suspension was inoculated into B.H.I. broth as a control.
Another control tube of B.H.I. broth inoculated with 0'1 ml of the bacterial sus-
pension was also included. Before culturing, the p H of tested and control tubes was
adjusted to that of the normal pigeon excreta. The inoculated media were incubated
at 41°C for 7 d. The number of viable cells of C. neoformans in the bacteria-fungus
mixture tubes was calculated and compared with both of the control bacteria free

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and the original inoculated yeast suspension. The viability of the bacterial mixture
in both test and control tubes was checked after the incubation period.

RESULTS
Survival of C. neoformans in the gastrointestinal tract
C. neoformans was recovered from the droppings collected within the first 24 h from
all groups of experimentally fed pigeons. The average number of recovered viable
cells was 16 x 10 s in each group of 4 pigeons. After this period no viable ceils of
C. neoformans were detected in any of the pigeon droppings throughout the period of
the experiment. The pigeons did not show any symptons of infection and cultures
as well as mouse inoculations from the intestinal contents and internal organs did
not reveal the organism.

Growth of C. neoformans in pigeon and chicken excreta

The fungus proved to grow well though differently in both sterile pigeon and
chicken droppings. A marked variation among the strains was not noticeable. The
average rate of multiplication of C. neoformans in the excreta is shown in Table 1.
In both unsterile moist and normal pigeon and chicken excreta viable cells of the
organism were not detected after the incubation period.

E6ect of pigeon intestinal bacterial flora on C. neoformans

Seven different species of bacteria were recovered from the intestinal contents of
pigeons. They were: Staphylococcus albus, Streptococcus faecalis, Bacillus subtilis,
Escherichia coli, Pseudonomas aeruginosa, Proteus mirabilis, and Klebsiella aerogenes. The
suspension comprising these isolates exhibited a complete inhibitory effect on the

TABLE 1 . - - I n vitro MULTIPLICATION OF C. neoformansIN STERILE

EXCRETA

Average number of viable cells

Originally inoculated
Type of dropping onto eachplate After 4 weeks

Pigeon 3 x 105 15 x 10s

Chicken 3 x 105 3 x 10s
66 ABOU-GABAL AND ATIA

growth of C. neoformans and no viable cells of any of the tested strains were detected
after one week incubation period. In the control bacteria-free tubes, the average
numbers of viable C. neoformans cells counted reached 96 x 104.

DISCUSSION
A correlation between the prevalence of C. neoformans in nature and bird excreta,
particularly of pigeons, is well established. However, the role of the pigeon itself
in disseminating the fungus is still obscure. Spontaneous cryptococcus in pigeons
or other birds is, so far, not known. O n the other hand, the susceptibility of pigeons

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to experimental infection with the organism has been proven (34, 19, 8). In our
study, recovery of C. neoformans from the droppings of pigeons within the first 24 h
agree with the findings of Littman & Borok (28). They reported that the fungus
survived passage through the gut of the pigeon and appeared in fresh droppings
within one hour and was still present for at least 24 h. Also, Swinne-Desgain (39)
claimed the appearance of C. neoformans within 24 h in the droppings of 4 out of 10
pigeons orally inoculated with the fungus. However, she mentioned the recovery of
the organism on the 22nd d in the droppings of one pigeon. A non-significant
variation in the number ofC. neoformans cells recovered was noticed among the tested
groups of pigeons inoculated with different strains. Such a difference was also
observed by Swinne-Desgain (39).
Our results showed that steIilized pigeon droppings support the growth of C.
neoformans. The fungus multiplied well when seeded in chicken excreta under the
same conditions. However, the rate of multiplication of the organism was 5 times
higher in pigeon than in chicken droppings. Walter & Yee (42) reported that
chicken droppings inhibited growth and this may explain the failure in attempts to
isolate C. neoformans from chicken excreta. On the cther hand, Tsubura (4), Mcnga
et al. (30), and Hergeg et aI., (19) reported the recovery ofC. neoformans from samples
of chicken excreta. Walter & Yee (42) attributed the growth inhibitory effect of
chicken droppings on C. neoformans to the presence of a high molecular growth
inhibitory substance and the high alkalinity of the droppings. In our study, a sig-
nificant variation in the p H of pigeon and chicken droppings was not observed (6"3, and
6-5 respectively). We assume that the difference in the multiplication rate of the fungus
in both excreta may be due to variation in their chemical composition, especially
in respect to creatinine.
In unsterile droppings, either of pigeon or chicken, the fungus did not survive
after the experimentation period. Failure of survival of C. neoformans in unsterile
droppings seems to be due to the effect of their bacterial flora. T h a t the intestinal
bacterial flora of pigeons may influence growth of C. neoformans was shown by the
in vitro studies. In this respect, the fungus failed to survive when it was cultured with
the intestinal bacterial flora over a period of one week. Whether this inability to
survive can be attributed to competition for space and/or nutrition, or is due to the
elaboration of toxic metabolites by one or more of the intestinal bacteria, still needs
further elucidation. Klitte & Gale (27) reported that Ps. aeruginosa inhibited the
in vitro growth of C. neoformans. In our study, Ps. aeruginosa constituted one of the
isolated intestinal bacteria from the pigeons which inhibited the fungus. Under
natural conditions, the rapid drying of pigeon droppings suppresses multiplication
of the bacteria (42) and this may explain the recovery of the fungus from old but not
fresh excreta (33, 14, 24, 37, 15).
In view of these observations, it may be concluded that the multiplication of
C. neoformans is not supported in the gut of pigeons. Recovery of 8"30/o of the orally
 STUDY OF C. NEOFORMANS IN N A T U R E 67

inoculated cells w i t h i n t h e first d a y a n d f a i l u r e to r e i s o l a t e t h e f u n g u s d u r i n g t h e

rest of the e x p e r i m e n t a l p e r i o d , seems to r u l e o u t a n a c t i v e b i o l o g i c a l r o l e of p i g e o n s ,
in the dissemination o f t h e f u n g u s in n a t u r e . H o w e v e r , t h e possibility t h a t p i g e o n s
may act as a m e c h a n i c a l c a r r i e r o f t h e f u n g u s o n t h e i r beaks a n d legs (28) a n d in
their crops (39) s h o u l d n o t b e r u l e d out. I t seems a g r e a t e r l i k e l i h o o d t h a t i n i t i a l
inoculum for c o l o n i z a t i o n o f p i g e o n d r o p p i n g s b y C. neoformans m a y o r i g i n a t e f r o m
other sources, m o s t p r o b a b l y t h e soil. I s o l a t i o n o f t h e f u n g u s f r o m soil, free o f p i g e o n
excreta, is well d o c u m e n t e d (11, 3, 18, 26, 9, 4, 1, 2). T h e c o i n c i d e n c e o f a c c u m u l a t e d
pigeon d r o p p i n g s in n a t u r e besides o t h e r f a v o r a b l e e n v i r o n m e n t a l c o n d i t i o n s m a y
support the i n c r e a s e d p r e v a l e n c e o f C. neoformans in s u c h locations.

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ZUSAMMENFASSUNG
C. neoformans wurde innerhalb den ersten 24 Stunden aus Tauben Kot nach oral Inokulation mit
lebenden Zellen Wiederisoliert. Die intestinal Bakterien-flora yon Tauben zeigten sich total in-vitro
Hemmwirkung fiber das Wachstum des C. neoformans. Der Pilz vermehrte sich wohl obgleich
unterschiedlich in beider Tauben und H~inchen Kot. In beider unsteril Kot typen waren C.
neoformans Zellen nach 4 Wochen Bebrfitung nicht entdeckbar. Es wird geschlossen dass Tauben in
ihren Darm die Vermehrung von C. neoformans nicht begfinstigen und infolgedessen keine aktive
biologische Rolls bei der Verbre~tung des Pilz in der Natur spielen.

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