Introduction
The Republic of Djibouti is a small country located in Eastern Africa on the Gulf of Aden and the Red Sea bordering Ethiopia, Eritrea, and Somalia. The country has an area of 21,783 square kilometer. The population of Djibouti is around 500,000 and they are largely of Somali, Afar, and Arab descendents. Two thirds of the country’s population lives in the costal capital, Djibouti city. The climate of country is hot and arid, and along the coast, it can get very humid. Different types of animals such as mammals, avians, and reptiles are found in Djibouti. Several food borne, water borne, and vector borne diseases are present. In 2003, there were 9100 cases and 690 deaths due to HIV/AIDS. There was no information about fungal infections in humans or animals (Mathew, 2009).

Cryptococcus neoformans, the principal cause of cryptococcosis, is an important basidiomycetic fungus, which produces life-threatening disease in humans as well as in a wide variety of animals (Pal, 2007). Globally, one million cases of cryptococcal meningitis occur every year in HIV/AIDS patients resulting in nearly 625,000 deaths (Pal, 2014). The yeast occurs as a saprobe in a wide variety of natural substrates including pigeon droppings, parrot excreta, poultry droppings, other avian faeces (budgerigar, lorikeet, munia birds), bat guano, soil, fruits, vegetables, and wood (Ajello, 1958; Pal and Mehrotra,1984: Pal, 1989; Pal et al., 1990; Ruiz et al., 1989; Pal et al, 1990; Swinne et al., 1991; Pal, 1995; Pal, 2005; Pal, 2007; Dave and Pal, 2015). However, pigeon excreta serves as the chief saprobic reservoir of C. neoformans, at it has been frequently recovered from pigeon droppings in many countries of the world (Pal, 1986; Ruiz et al., 1989; Pal, 1997; Cermeno et al., 2006; Pal et al., 2014; Dave and Pal, 2015). In this context, Staib (1962) demonstrated that pigeon manure, which contains uric acid, creatinine and other chemicals, provide an enrichment for the growth of C. neoformans. Moreover, it has been mentioned that avian excreta collected from open sites exposed to ultra violet rays of sun failed to yield the yield any isolation of C. neoformans, indicating that direct sun light is detrimental to the survival of this yeast in avian excreta (Pal, 2007). The internet search failed to reveal any information on the prevalence of C. neoformans in natural substrates in Djibouti. Therefore, the present study was undertaken to investigate the natural occurrence of C. neoformans in the environment of Djibouti by employing Pal’s sunflower seed agar, as a selective medium.

1 Materials and Methods
Sixteen samples of old and dried pigeon droppings were collected with the help of wooden spatulas and placed in clean polythene bags from different locations in Djibouti, the capital of Djibouti. About 5 gram of dry and old avian droppings were collected from different locations. In order to avoid the environmental exposure of C. neoformans, a facemask was used while collecting the samples of pigeon excreta. These were processed in the Laboratory of Microbiology, Debre Zeit, Ethiopia. One gram of specimen from each site 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 15-20 min, then shaken manually for 4-5 min, and later incubated at 37℃ for one hour. An aliquot of 0.1 ml of supernatant from the suspension was streaked onto duplicate plates of Sabouraud dextrose agar with chloramphenicol (0.05 mg/ml) and Pal's sunflower seed medium (Pal, 2007). It contained pulverized sunflower seed 4.5 g, agar 2.0 g, chloramphenicol 50 mg, distilled water 100 ml. The former medium was incubated at 37℃, while the later was kept at 25℃. Each inoculated plates were examined daily for yeast growth and the number of colonies showing brown colour were counted, and also sub-cultured on APRM medium for further confirmation (Dave and Pal, 2015). The new medium contained 4.0 g of marigold dried flower, 2.0 g agar, 50 mg chloramphenicol and 100 ml distilled water. The strains growing at 37℃ with capsule, urease positive, and negative for potassium nitrate and lactose can be identified as C. neoformans. The detailed morphology of the isolates were studied in Narayan stain, which contained 4 ml of glycerin, 0.5 ml of 3% aqueous solution of methylene blue and 6 ml of dimethyl sulfoxide (Pal, 2004).

2 Results
C. neoformans could be isolated from two of the 16 samples of giving a prevalence of 12.5%. The positive samples of pigeon droppings were dry, and old, and originated from two of the five sites, which were not exposed to direct sunlight. Both the isolations were made only on Pal's sunflower seed medium at 25℃ by observing few light brown to dark brown coloured colonies of C. neoformans. In contrast, the yeast could not be recovered from any of the pigeon droppings on Sabouraud medium, as all of the inoculated Petri dishes were badly contaminated with fast growing moulds that masked the growth of C. neoformans. The number of colonies, which grew from bird droppings on Pal’s sunflower seed medium varied from two to five. Both the isolates of C.neoformas grew well on Sabouraud medium at 37℃, hydrolyzed urea, but failed to utilize lactose and potassium nitrate. The sub-cultures of C. neoformans grown on APRM medium, when examined in Narayan stain under light microscopy showed many spherical to few oval thinly encapsulated yeast cells, with and without budding. Unfortunately, the laboratory did not has facility to determine the variety and mating types of C. neoformans.

3 Discussion
The natural habitat of C. neoformans was not known until 1955, when  Emmons from USA first isolated this fungus from the droppings of the pigeon (Columbia livia), and thus established the ecological relationship of this zoopathogenic basidiomycetous yeast. Later, this observation was substantiated by many investigators from different regions of the world (Pal, 1978; Pal and Baxter, 1985; Pal et al., 1990; Pal, 1997; Sasaki et al.,1999; Cermeno et al., 2006; Pal et al., 2014; Dave and Pal, 2015). The recovery of C. neoformans from the pigeon excreta for the first time in Djibouti established that avian habitats serve as an important saprobic reservoir for this opportunistic pathogen. However, further studies on the occurrence of C. neoformans in a wide variety of environmental materials should be undertaken to reveal the ecological niches of this medically important basidiomycetous yeast in Djibouti. The findings of this investigation indicated that the isolation of C. neoformans from environmental sources is extremely difficult on conventional media like Sabouraud agar. Therefore, a differential medium should be employed for investigating the prevalence of C. neoformans in saprobic environment. The success in recovering C. neoformans  from the samples of pigeon droppings was because of the use of Pal's  sunflower seed medium. The development of brown pigmented colonies on Pal's medium within 3-4 days at 25℃ helped in the rapid isolation and quick presumptive identification of the yeast from pigeon droppings. The efficacy of Pal’s sunflower seed agar as an excellent medium for rapid isolation and identification of this zoopathogenic yeast from clinical as well as environmental substrates has been reported by several other workers (Pal and Baxter, 1985; Baro et al., 1998; Sasaki et al., 1999; Cermeno et al., 2006). Very recently, Katiyar and co-workers (2011) studied the comparison of five media such as Henna agar, Mustard seed agar, Niger seed agar, Sunflower seed agar, and Tobacco agar for pigment production of C. neoformans. These investigators conclusively proved that sunflower seed medium was the best medium with the mean day of growth and pigment production being 1.25 and 2.8, respectively. As Pal's sunflower seed medium is less expensive, easily available, highly specific, and very sensitive, its routine application in microbiology and public health laboratories as the most valuable tool for the selective isolation and presumptive identification will help the scientists in ecological and epidemiological studies of C. neoformans that has emerged as an important opportunistic pathogen producing life threatening disease in immunocompetent and immunocompromised hosts particularly, HIV/AIDS patients  throughout the world. It is deduced from this investigation that immunocompromised persons should use face when dealing with avian excreta, as respiratory tract is the principal mode of entry of C. neoformans.

Acknowledgements
The author is highly grateful to his students who collected and brought the samples of pigeon droppings from Djibouti for this study. The technical help of the staff of Microbiology Laboratory is also thankfully acknowledged.

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