Aim of Study

This study was aimed to obtain an overview of the presence

of C. neoformans in pigeon droppings in Tripoli urban area
as well as to identify the molecular type and the mating type
of the collected isolates

Back Ground
Cryptococcus neoformans is the principal etiological agent
of cryptococcosis that has a worldwide distribution. The
pathogen occurs as a saprobe in a wide variety of natural
substrates including avian excreta, soil and wood.
The yeast Cryptococcus neoformans has currently been
classified according to its perfect state, Filobasidiella.
Single species has been described, composed by two
varieties and five serotypes: Filobasidiella neoformans
variety neoformans (serotypes A, D, and A/D) and F.
neoformans variety bacillispora (serotypes B and C). The
varieties neoformans (serotypes A, D, A/D) and gattii
(serotypes B and C), which belong to teleomorph F.
neoformans,
have
been
correlated
(Cryptococcus
neoformans). More recently, the variety grubii (serotype A)
and the new specie, C. bacillispora (var gattii), have been
proposed. The yeast has been frequently recovered from
pigeon droppings in many countries of the world but not
Libya. Therefore, the present study was conducted on the
environmental distribution, variety and mating types of C.
neoformans in Libya.

Molecular typing of environmental Cryptococcus neoformans isolated

from pigeon droppings in Tripoli, Libya
1
Mohamed S Ellabib, 1Mohamed A Aboshkiwa, 2Roberta
DAmicis and Massimo Cogliati
1
Department of Medical Microbiology and Immunology Faculty
of Medicine, Tripoli University, Libya
2
Lab. MicologiaMedica, Dipartimento di ScienzeBiomediche per
la Salute, UniversitdegliStudi di Milano
Results
Cryptococcus neoformans was isolated from pigeon dropping samples collected from
two out of four sites selected. Out of 100 samples collected, 32 (32%) samples were
positive for Cryptococcus neoformans. The samples from site four were showed the
highest rate of contamination 24 out of 50 (48%), while site one 8 sample were positive
out of 35 (22.9%). Only 10 and 5 samples were obtained from 2 and 3 sites respectively.
These two sites did not show any contamination to Cryptococcus neoformans as shown
in Table 1 and Figure 1.

Site Number

NP

Positive %

35

23

10

50

24

48

Table.1 Distribution of C. neoformans isolated from pigeon droppings in Tripoli

region
N=Number of Sample, NP= Positive sample

Materials and Methods

Sampling Collection and Processing:
One hundred samples of dried and old pigeon droppings
were collected during year 2013 with wooden spatulas and
placed in clean polythene bags from four different locations
in Tripoli, the capital of Libya. They were processed in the
laboratory of Medical College. One gram of specimen from
each sample was suspended in sterilized glass bottle
containing 9 ml of sterile physiological saline (0.85% NaCl)
supplemented with chloramphenicol (10 mg/ml). The mixture
was left at room temperature for about 10-15 min, then
shaken manually for 4-5 min and later incubated at 37C for
one hour. Ten fold serial dilutions were made and 0.1 of each
dilution was plated onto duplicate plates of sunflower seed
agar supplemented with antibiotics and biphenyl. The plates
were incubated at 30C for one week.

Growth of Cryptococcus neoformans on

Sunflower Seed Agar shown brown
color

positive sample %

60%

Species Identification:
Identification of C. neoformans was performed on the
basis of melanin pigment production on sunflower seed
agar, presence of a capsule observed in India ink
preparation, urease production on urea agar medium, and
ability to grow at 37 C. Vitek2 compact system was used
to confirm C. neoformans identification.

The 32 strains molecular type and mating type were carried

out at Lab. Medical Mycology, Department of Biomedical
Sciences for Health, University of Milan by multiplex PCR
as described elsewhere (Cogliati et al., Med. Mycol. 2000;
38: 97-103, Esposto et al., Clin. Microbiol. Infect. 2004;
10:1089-1104).

Discussion:
This study provides for the first
time information concerning the
ecology and genotypes of C.
neoformans in the Tripoli region
of Libya. In this study, 32 isolates
of Cryptococcus neoformans
were obtained from samples of
pigeon feces of Tripoli, in which
all of them reveal that they were,
belong to molecular type VNI (C.
neoformans var. grubii) and
mating type alpha A.
Conclusion
This study provides for the first
time information concerning the
ecology and genotypes of C.
neoformans in the Tripoli region
of Libya

50%

40%

30%

% of positivity

Recommendation:

20%

10%

0%
0.5

Molecular Typing and Mating Type Allelic Pattern:

Urease test for Cryptococcus

neoformans (Positive) and Candida
albicans (Negative).

brown colonies of Cryptococcus

neoformans after incubation for
48 h at 30C from pigeon
dropping

1.5

2.5

3.5

4.5

Site number

Figure 1: Percentage of Cryptococcus neoformans in pigeon

droppings.

Molecular and Mating Type

Multiplex PCR amplifications reveal that all isolates
belonged to molecular type VNI (C. neoformans var.
grubii) and mating type alpha A, as shown in Figure 2.

Figure Legend
Determination of mating type and molecular type by
multiplex PCR.The pictures show the results obtained for
nine representative isolates from pigeon droppings. H99
strain was included as reference strain VNI aA.

Figure 2. PCR amplifications of isolate strains

Epidemiology surveying study on large

scale is needed for confirming
presence or absence of other
Cryptococcus neoformans varieties
and their genotypes
Acknowledgment:
I would like to express my very great
appreciation and thanks to Dr
Massimo
Cogliati
and
Roberta
DAmicis for their valuable and
constructive molecular analysis work. I
would also like to thank the staff of all
places for enabling me to collect the
sample from their sites.