Journal of Veterinary Science & Medical Diagnosis ISSN: 2325-9590

All submissions of the EM system will be redirected to Online Manuscript Submission System. Authors are requested to submit articles directly to Online Manuscript Submission System of respective journal.

Research Article, J Vet Sci Med Diagn Vol: 4 Issue: 4

Lovebirds and Cockatiels Risk Reservoir of Cryptococcus neoformans, a Potential Hazard to Human Health

Elhariri M 1*, Hamza D2, Elhelw R 1 and Refai M 1
1Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Egypt
2Department of Zoonoses, Faculty of Veterinary Medicine, Cairo University, Egypt
Corresponding author : Mahmoud Elhariri
Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, PO Box 12211, Giza, Egypt
Tel: +201008854559; Fax: +20235725240
E-mail: [email protected]
Received: June 08, 2015 Accepted: July 23, 2015 Published: July 28, 2015
Citation: Elhariri M, Hamza D, Elhelw R, Refai M (2015) Lovebirds and Cockatiels Risk Reservoir of Cryptococcus neoformans, a Potential Hazard to Human Health. J Vet Sci Med Diagn 4:4. doi:10.4172/2325-9590.1000168

Abstract

 Lovebirds and Cockatiels Risk Reservoir of Cryptococcus neoformans, a Potential Hazard to Human Health

Lovebirds and cockatiels are potential carriers and/or transmitters of zoonotic diseases. Some of them could have an important impact for human health, In Egypt, the role of these birds in dispersing C. neoformans is not well documented, which evoked a high need to investigate the environmental ecology of this fungus in order to establish surveillance programs and applying the preventive measures for this pathogen infection. The C. neoformans prevalence and role of pet birds in spreading this fungal pathogen in Egypt was illustrated in this study. Two hundred Cockatiels and lovebirds excreta were collected from captive birds. The recovered isolates of C. neoformans species were identified by molecular identification using capsular gene specific primer CAP64. The subtyping of isolates was performed by multiplex PCR using CNa-70S/A -CNa-49S/A. Four isolates (3 and 1) from lovebirds & Cockatiels respectively were subjected to sequence analysis of Internal Transcribed Spacer (ITS) regions. Alternatively the fungal isolates were analyzed by PCR fingerprinting with uniplex PCR amplification using an oligonucleotide (GTC)5. From this study it was concluded that, the excreta of these birds can play a role as a risk reservoir of C. neoformans in domestic and public environments and enhance their zoonotic importance to human.

Keywords: C. neoformans; Egypt; Cockatiels; Lovebirds

Keywords

C. neoformans; Egypt; Cockatiels; Lovebirds

Introduction

The basidiomycetes yeasts of genus Cryptococcus include C. neoformans/C. gattii species complex, which is composed of two separate species C. neoformans and C. deneoformans, and five species within C. gattii. The most important pathogenic species C. neoformans and C. gattii show different geographical distributions. C. neoformans is globally distributed and has been recovered from various natural sources and with high incidence particularly in a wide variety of bird droppings, trees and soils [1].
In Africa, the isolation of 19,753 C. neoformans and C. gattii strains were reported from 25 of the 58 African countries and mainly in South Africa (79%). Environmental surveys, carried out in eight African countries (Tunisia, Egypt, Nigeria, Democratic Republic of Congo, Burundi, Zimbabwe, Botswana, and South Africa) revealed that these pathogens represented 1% of the total reported isolates in environment [2].
The most common isolate responsible for Cryptococcal infection is C. neoformans [3,4]. It causes mainly opportunistic infections in immunocompromised patients with underlying conditions, such as HIV, leukemia, and other cancers, or in those taking corticosteroid medications [5].
C. neoformans is primarily associated with nests and soils containing avian droppings, especially those of pigeons [1,6]. Allover, the bird guano may represent the main ecological niche for C. neoformans [7]. Many reports illustrated the role of the captive birds in promoting and disseminating the contamination of surrounding and public areas by Cryptococcus species [8,9]. It is suggested that the hosts acquired Cryptococcal infection via inhalation of contaminated air or excreta to the lungs with the potential to disseminate to the central nervous system and/or to other distant tissues [10].
Pet birds are bought individually or in couples, as families often do, which is a profitable business for pet shops or local breeders for their very high genetic or exotic value, these birds, commonly cockatiels and lovebirds, which are regularly sold at high prices. These birds are potential reservoir of fungal zoonotic diseases mainly Cryptococcal infection.
C. neoformans has an autochthonous environmental niche, is likely to be the source for outbreaks among various animals and humans. The worldwide increasing number of published studies for the ecology and health significance of C. neoformans isolates infection in human and animals. On the other hand, in Egypt there is scare information about the health significance of this fungal pathogen.
Therefore, this study was undertaken to investigate, the existence of C. neoformans in cockatiels and lovebirds in Egypt, depending on the CAP64 gene molecular detection and biotyping by CNa-70S/A - CNa-49S/A. In addition, we applied the sequence analysis of the ITS2 region of selected isolates and fingerprinting using (GTC)5 to determine the presence of C. neoformans from these types of bird excreta and provide insight into the environmental epidemiological life cycle of C. neoformans in Egypt.

Materials and Methods

Sample collection
A total 200 samples of cockatiels and lovebirds excreta was collected in clean paper envelopes from different pet bird houses and transferred directly to the laboratory. One gram from each sample was suspended in a sterilized glass bottle containing 99.0 ml of sterile physiological saline (0.85% NaCl) supplemented with chloramphenicol (10.0 mg/ml). The mixture was left at room temperature for about 10-15 min to complete dissolving of dropping, and then shaken vigorously for 4-5 min. The bottle was left for complete precipitation [11].
Isolation and phenotypic identifications
From the supernatant fluid of each prepared sample, a loopful was streaked onto plates of Sabouraud dextrose agar with chloramphenicol and incubated at 30°C for 48 hours. C. neoformans species were phenotypically identified by their color and microscopic morphology on Sabouraud dextrose agar (Oxoid CM41) and Eucalyptus leaves agar media [12]. The isolates were identified by classical mycological procedures of C. neoformans [6].
DNA isolation and molecular identification by capsular gene
The chromosomal DNA was isolated from C. neoformans isolates according to [13]. The molecular confirmatory identification of C. neoformans was done by using specific capsular gene primers CAP64. The primers for CAP64 were designed on the basis of DNA sequences according to [14].
Molecular subtyping of Cryptococcus neoformans isolates
All identified isolates by CAP64 gene were subtyped by multiplex PCR primer pairs as recommended by [15]. The two PCR primer pairs, which are specific for C. neoformans serotype A were used to amplify the 695-bp fragment CNa- 70S (5’-ATTGCGTCCACCAAGGAGCTC-3’) and CNa-70A (5’-ATTGCGTCCATGTTACG TGGC-3’). The other set of primer for serotype B is CNb-49S (5’-ATTGCGTCCAAGGTGTTGTTG-3’) and CNb-49A (5’ ATTGCGTCCATCCA ACCGTTATC-3’), which produce amplicon of 448-bp.
Sequencing of the ribosomal internal transcribed spacer regions
ITS1 and ITS2 regions DNA were amplified with 900nM primer ITS1 (5`-TCCGTAGGTGAACCTGCG-3`), 300nM primer ITS4 (5`-TCCTCCGCTTATTGATATGC-3`) [16]. The PCR amplification reaction was performed with a DreamTaq PCR Master Mix (2X) (Thermo, Fermentas), according to company instructions. The amplification parameters: were 95°C for 6 min, followed by 30 cycles at 95°C for 30s, 55°C for 30s, and 72°C for 30s, followed by one final extension at 72°C for 10 min. A MiniPro PCR thermal cycler (SWIFT, ESCO) was used. Negative control reactions without any template DNA were carried out simultaneously. Gel electrophoresis with 1.5% agarose gels was conducted with 1xTBE buffer (0.1 M Tris, 0.09 M boric acid, 1 mM EDTA) at 4.8 V/cm for 2 h. A 100-bp DNA ladder (JENA BioScience, Germany) was run concurrently with amplicons for sizing of the bands. Gels were stained with ethidium bromide- TBE solution for 20 min and the obtained bands were visualized using UV-trans-illuminator and photographed by a digital camera (Cleaver).
Internal Transcribed Spacer (ITS) sequence analysis
Bio edit software was used to obtain consensus sequences from aligned forward and reverse sequence reads. The obtained ITS sequences were analyzed for homology between the nucleotide sequences of the detected C. neoformans strains and other strains published on Gen Bank, which was done using BLAST 2.0 search programs (National Center for Biotechnology Information website. Species and genus identification were presumed for fungal isolates with scores designated in the BLAST search and The Barcode of Life Database (BOLD) [17,18].
Molecular genotyping using micro-satellite (GTG)5 specific primers
PCR fingerprinting using the microsatellite (GTG)5 was done by a modification of the method described [19]. PCR was performed with volumes of 50 μl containing 10 to 25ng of genomic DNA, 20 to 30ng of primer, Under the recommended buffer conditions, the PCR was performed as follows: 40 cycles of denaturation at 93°C for 20s, annealing at 50°C for 30 s, extension 72°C for 20 s and final extension 72°C for 6 min. Amplification products were analyzed by electrophoresis in 2.5 % agarose gels run in 1x TBE buffer and detected by staining with ethidium bromide under UV-transilluminator and photographed by a gel documentation (Cleaver microDoc). Electrophoretic bands were sized and compared with a scanner and gel image analysis software.

Results

A total of 15 (7.5%) isolates of C. neoformans were obtained from the 200 examined samples. Among these, 12 isolates were recovered from 143 lovebirds (8.3%) and 3 from 57 cockatiels (5.2%) (Tables 1 and 2).
Table 1: Collected samples isolation sources from lovebirds and cookatiel in Egypt.
Table 2: Number of C. neoformans isolates and their recovery rates from lovebird & cockatiel excreta.
As shown in Table 3, all the recovered Cryptococcus isolates were identified as C. neoformans strains based on all conventional and physiological characters of C. neoformans. Moreover, all tested isolates were positive by PCR for CAP64 the specific capsular gene. All the 15 positive isolates were produced a 695-bp amplicon with CNa-70S/A primer pair, while no products were produced with CNa- 49S/A (Figure 1).
Figure 1: Agarose gel electropherosis of CNa70A/S and CNa49A/S specific PCR of all the examined C. neoformans isolates with production of amplicons of 695 bp for CNa70A/S, Marker 100 bp DNA ladder plus (JENA BIOSCIENCE).
Table 3: Molecular typing of Cryptococcus neoformans isolates from lovebirds & cockatiel excreta..
However, the molecular subtyping of C. neoformans isolates was done by the sequencing of ITS region of four selected isolates representing the four major bird species in this study (Table 4). The results were compared with known sequences using the BLAST program and The Barcode of Life Database (BOLD). Three isolates of The sequenced C. neoformans strains were identified as C. neoformans (Serotype A) from Melopisittacus andulatus, Agapornis roseicollis and Psittacula krameri.
All isolates recovered from bird droppings belonged to serotype A. The comparison of ITS for the selected four strains (3 from lovebirds and 1 from cockatiel) revealed that 3 of the selected isolates were C. neoformans, except one isolates from Zebra Finch, whose serotype could not be identified (EGYST- KJ767782) (Table 3).
Regarding, the prevalence of C. neoformans in different birds species, 10 of captive birds belonging to 8 species differed in the incidence rate of C. neoformans isolation according to bird species, where it was recovered at the rate of 22.2 % from Psittacula krameri, 20% from Agapornis roseicollis, 16.6% from Agapornis pullarius, 9% from Agapornis fischeri and 8.3% from Taeniopygia guttata. Whereas, the lower rates were recovered from Nymphicus hollandicus (5.6%) and Melopisittacus andulatus (4.6%) (Table 4).
Table 4: Distribution & recovery rate of Cryptococcus neoformans according bird’s species
On the other hand, the oligonucleotide primer (GTG)5 was used to amplify the variable DNA fragments yielded from all the tested strains of C. neoformans. The fingerprint patterns by (GTG)5 revealed a two different banding profile in between different strains and even this variation detected belonged to the same serotype A. (GTG)5 produced amplicons with variable size ranged from 580 to 1131 bp for banding profile (I) ( 2). In case of banding profile (II) the PCR product varied between 587 to 1904 bp as shown in Table 5. Generally, all tested strains in the banding profile (I), were produced four bands and banding profile (II) were yielded five bands (Figure 2).
Table 5: The sizes of diagnostic bands for the two major banding profiles produced by (GTG)5.
Figure 2: Micro-satellite PCR banding fingerprint using the single primer (GTG)5 KJ767782 & KJ767783 represent the banding profile (I) . KJ767784 & KJ767785 are representing the profile (II), Marker Solis BioDyne 100-3000 bp.

Discussion

The yeast of C. neoformans is a highly potential basidiomycete fungal pathogen for humans and animals health. The major route of acquisition of this fungus is via inhalation of infective basidiospores. Environment plays a major role in dispersing the infection with C. neoformans in human and animal surroundings. The global isolates which are responsible for Cryptococcus infection are identifies as C. neoformans that is commonly recovered from pigeon droppings, soil and decaying wood in hollow trees [1,3,4,20].
Pigeons are known to be reservoirs of pathogenic yeasts, like C. neoformans, which is described to cause opportunistic infections in humans [21]. In Egypt, only one study focused on the role of pigeons, canaries and parrots in spreading this fungal pathogen in the environment via bird droppings and it recorded the prevalence of C. neoformans in bird droppings about 2.5% [22].
Role of captive and free-ranging birds as carriers and spreaders of potential pathogens for human beings is very critical, which contributes to environmental contamination and possibly to human and animal infection [23-25].
Unfortunately, in Egypt most studies on Cryptococcal infection did not correlate between environmental and clinical cases isolates. The first trial to isolate C. neoformans from the avian was done in Lower Egypt; C. neoformans was recovered by 15% of samples examined [26]. In other study, four cases of Cryptococcal meningitis in Egyptian patients were clinically diagnosed without any information about the subtype and/or variety of causative Cryptococcal agent [27].
On the other hand, with the beginning of the last century, the studies on fungal meningitis, received more attention and at 2003, a great improvement in the epidemiological data about Cryptococcus infection in Egypt, and laboratory diagnosis of Cryptococcal meningitis illustrating the role of C. neoformans in such infection [28-30].
In this study, 12 C. neoformans isolates were recovered from 143 (8.3%) Lovebirds samples and 3 isolates out of 57 (5.2%), the total of pet bird droppings yielded 15 C. neoformans isolates with a prevalence of 7.5% (Table 1 & 2). Also, the percentage of caged bird excreta samples which contained C. neoformans was comparatively lower than those detected in other study on birds with recent environmental sources associated [31,32]. Whereas our finding were in higher rate of incidence in comparison to previous work at the same area [22].
Currently, it was found that the number of C. neoformans positive samples were significantly recovered at higher rate from lovebirds than from cockatiel birds (Table 1). Australian or English budgerier (Melopisittacus andulatus) excreta had a large number of positive samples but not at significantly higher percent of recovery (4.6%) (Table 4). It is suggested that the uneven number of samples collected from lovebirds and cockatiels birds may explain the differences in number of positive samples from each order. The number of samples collected from each species was influenced by the number and types of bird’s availability in pet shops.
All the recovered Cryptococcus isolates were identified as C. neoformans strains based on all conventional and physiological characters of C. neoformans. Moreover, all tested isolates were positive by PCR for CAP64 the specific capsular gene. Molecular subtyping of C. neoformans isolates was done by two methods the CNa70A/S and CNa49A/S primer pair in multiplex PCR to determine the C. neoformans types (Figure 1). ITS region sequencing of selected four strains and comparing the results with known sequences using the BLAST program and the Barcode of Life Database (BOLD). ITS regions are widely used in taxonomy and molecular phylogeny because they are highly conserved region, what bring out the possibility of detecting and identifying fungi [16,33].
All examined C. neoformans strains were identified as C. neoformans serotype A according to CNa70A/S primer as shown in Table 3. These findings are similar to most of previous studies on molecular typing on this pathogen. C. neoformans the most dominant and prevalent worldwide [3,4,19,20] .
In Egypt, most of studies at beginning of 2003 investigated the major subtypes of C. neoformans either from environmental (Plant or Bird origin) or a clinical sample (Veterinary or human) is C. neoformans (Serotype A). [28,30,34,35]
It seems that the main reason for the high prevalence of C. neoformans in Egypt is attributed to its thermo-tolerance ability, the weather in Egypt is considered as temperate to hot climate, which may be reflected on isolation rate and recovery of other serovars of C. neoformans.
PCR fingerprinting by the microsatellite-specific sequences (GTG)5 was used globally as single primer in the PCR for its discriminatory index to differentiate among unrelated isolates [19,36]. Despite the high discriminatory power, which associated with (GTG)5 primer, the analysis of the PCR patterns grouped all C. neoformans into two major molecular types only. It is common the (GTG)5 oligoprimer is characterized by production of few bands (6 to 17) and to achieve reproducible fixed results with this oligoprimer, each sample was repeated triplicate with standardised conditioning in reagents and thermal cycling conditions [19].
It appears from Table 5 that all the examined C. neoformans produced a conserved banding in the 3rd and 4th bands, which give a characterized profile at range 890 to 1131bp. These data confirmed that, the major C. neoformans genotype in Egypt form different bird species is completely related even, is related to genotype (I) or (II).
Most of captive pet birds in Egypt are imported from different regions all over the world either from Australia, Africa and Asia (Table 2). This fact reflects on the potential risk of international trade of such birds species without international regulatory measures. Boseret and coworker mentioned on this point, exotic birds like greater psittaci forms (parrots, e.g. ara or cockatoo), legally or illegally traded from for example Asia or South America, remain high in the ranking of popular pets and are also profusely represented in zoos and parks [37].
Therefore , the increasing popularity of lovebirds & cockatiels as a pet bird, and the close relationship between human beings and their pets, which may help in exposure of people to potential pathogens that may belong to the normal microbiota of these birds [38]. The greater risk of this disease was detected in children, elderly people and immuno compromised individuals after exposure to pathogenic yeasts [24].
In conclusion, this study gave a highlight and awareness about the role of pet birds as risk reservoirs for disseminating the potentially pathogenic C. neoformans in the environment. Moreover, the pet birds may cause a hazard to human health, particularly to immune compromised patients, children and the elderly. Therefore, further studies are urgently needed to clarify the diagnosis of such infection and its recent treatments. Current periodical examination of environmental factors related to birds as water, excreta and air must be advised to devoid the zoonosis hazard infection to human health.

References

  1. Hagen F, Khayhan K, Theelen B, Kolecka A, Polacheck I, et al. (2015) Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol 78: 16-48.

  2. Cogliati M (2013) Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An Atlas of the Molecular Types. Scientifica (Cairo) 2013: 675213.

  3. Casali AK, Goulart L, Rosa e Silva LK, Ribeiro AM, Amaral AA, et al. (2003) Molecular typing of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul. FEMS Yeast Res 3: 405-415.

  4. Viviani MA, Cogliati M, Esposto MC, Lemmer K, Tintelnot K, et al. (2006) the European Confederation of Medical Mycology (ECMM) Cryptococcosis Working Group. Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECM survey of Cryptococcosis in Europe. FEMS Yeast Res. 6: 614-619.

  5. Mitchell TG, Perfect JR (1995) Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 8: 515-548.

  6. Granados DP, Castañeda E (2005) Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogotá, Colombia, and study of ecological conditions in the area. Microb Ecol 49: 282-290.

  7. Nielsen K, De Obaldia AL, Heitman J (2007) Cryptococcus neoformans mates on pigeon guano: implications for the realized ecological niche and globalization. Eukaryot Cell 6: 949-959.

  8. Filiu´WFO, Wanke B, Aguena SM, Vilela VO, MacedoRCL, et al. (2002) Avian habitats as sources of Cryptococcus neoformans in the city of Campo Grande, MatoGrossodeSul, Brazil. Rev Soc Bras Med Trop. 35:591-596.

  9. Abegg MA, Cella FL, Faganello J, Valente P, Schrank A, et al. (2006) Cryptococcus neoformans and Cryptococcus gattii isolated from the excreta of psittaciformes in a southern Brazilian zoological garden. Mycopathologia 161: 83-91.

  10. Lin X, Heitman J (2006) The biology of the Cryptococcus neoformans species complex. Annu Rev Microbiol 60: 69-105.

  11. Pal M, Baxter M (1985) Isolation of Cryptococcus neoformansusing a simplified sunflower seed medium. Microbiol Soc 29: 155-158.

  12. Refai M, Kotb M, Abo-Elyazeed H, Tawakkol W, Al–Arosi R, et al. (2005) Development of brown colonies and capsule of Cryptococcus neoformans on plant extract agar and media containing oils. Mycology forum Deutschsprachige Mycologische Gesellschaft 3: 25-27.

  13. Möller EM, Bahnweg G, Sandermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 20: 6115-6116.

  14. Chang YC, Penoyer LA, Kwon-Chung KJ (1996) The second capsule gene of cryptococcus neoformans, CAP64, is essential for virulence. Infect Immun 64: 1977-1983.

  15. Aoki FH, Imai T, Tanaka R, Mikami Y, Taguchi H, et al. (1999) New PCR primer pairs specific for Cryptococcus neoformans serotype A or B prepared on the basis of random amplified polymorphic DNA fingerprint pattern analyses. J Clin Microbiol 37: 315-320.

  16. Fujita SI, Senda Y, Nakaguchi S, Hashimoto T (2001) Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains. J Clin Microbiol 39: 3617-3622.

  17. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402.

  18. Kurtzman CP, Robnett CJ (1997) Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol.- 35: 1216-1223.

  19. Meyer W, Marszewska K, Amirmostofian M, Igreja RP, Hardtke C, et al. (1999) Molecular typing of global isolates of Cryptococcus neoformansvar. neoformansby polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA- a pilot study to standardize techniques on which to base a detailed epidemiological survey. Electrophoresis 20: 1790-1799.

  20. Meyer W, Castañeda A, Jackson S, Huynh M, Castañeda E; IberoAmerican Cryptococcal Study Group (2003) Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis 9: 189-195.

  21. Wu Y, Du PC, Li WG, Lu JX (2012) Identification and Molecular Analysis of Pathogenic Yeasts in Droppings of Domestic Pigeons in Beijing, China. Mycopathologia174: 203-214.

  22. Abo El-Yazeed H, Ezz-Eldin N, Tawakkol W, El-Hariri M, Kotb M, et al. (2006) Isolation and identification of Cryptococcus neoformans from bird droppings, with special reference to newly formulated differential media based on development of brown colonies. J Egy Vet Med Assoc 66:165-179.

  23. Mancianti F, Nardoni S, Ceccherelli R (2002) Occurrence of yeasts in psittacines droppings from captive birds in Italy. Mycopathologia 153: 121-124.

  24. Cafarchia C, Camarda A, Romito D, Campolo M, Quaglia NC, et al. (2006) Occurrence of yeasts in cloacae of migratory birds. Mycopathologia 161: 229-234.

  25. Cafarchia C, Romito D, Iatta R, Camarda A, Montagna MT, et al. (2006) Role of birds of prey as carriers and spreaders of Cryptococcus neoformans and other zoonotic yeasts. Med Mycol 44: 485-492.

  26. Refai M, Taha M, Selim SA, Elshabourii F, Yousseff HH (1983) Isolation of Cryptococcus neoformans, Candida albicans and other yeasts from pigeon droppings in Egypt. Sabouraudia 21: 163-165.

  27. Girgis NI, Farid Z, Youssef HH, Hafez A, Hassan MN, et al. (1985) Fatal Cryptococcal meningitis in four Egyptian patients. Ain Shams Med J 36: 93-99

  28. Abdel-Salam HA (2003) Characterization of Cryptococcus neoformans var. neoformans serotype A and A/D in samples from Egypt. Folia Microbiol (Praha) 48: 261-268.

  29. Mansour A, Sultan Y, Nakhla I, Morsy M, Frenck RW Jr (2003) Fluconazole for the treatment of Cryptococcus neoformans meningitis in an immunocompetent host. Neurosciences (Riyadh) 8: 126-128.

  30. Elias ML, Soliman AK, Mahoney FJ, Karam El-Din AZ, El-Kebbi RA, et al. (2009) Isolation of cryptococcus, Candida, Aspergillus, Rhodotorula and nocardia from meningitis patients in egypt. J Egypt Public Health Assoc 84: 169-181.

  31. Lugarini C, Goebel CS, Condas LA, Muro MD, de Farias MR, et al. (2008) Cryptococcus neoformans Isolated from Passerine and Psittacine bird excreta in the state of Paraná, Brazil. Mycopathologia 166: 61-69.

  32. Ferreira-Paim K, Andrade-Silva L, Mora DJ, Pedrosa AL, Rodrigues V, et al. (2011) Genotyping of Cryptococcus neoformans isolated from captive birds in Uberaba, Minas Gerais, Brazil. Mycoses 54: e294-300.

  33. Katsu M, Kidd S, Ando A, Moretti-Branchini ML, Mikami Y, et al. (2004) The internal transcribed spacers and 5.8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans species complex. FEMS Yeast Res 4: 377-388.

  34. El-harir M, Abo El-Yazeed H, Ezz-Eldin N, Tawakkol W, Kotb M, et al. (2007) Typing of Cryptococcus neoformansisolates recovered from droppings ofpigeons, parrots and canaries. Vet Med J Giza 55: 411-423.

  35. Saleh H, Moawad AA, El-Hariri M, Refai MK (2011) Prevalence of Yeasts in Human, Animals and soil sample in El-Fayoum Governorate in Egypt. Int J Microbiol. 2: 233-239.

  36. Escandón P, Sánchez A, Martínez M, Meyer W, Castañeda E (2006) Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in Colombia. FEMS Yeast Res 6: 625-635.

  37. Boseret G, Losson B, Mainil JG, Thiry E, Saegerman C (2013) Zoonoses in pet birds: review and perspectives. Vet Res 44: 36.

  38. Brilhante RS, Castelo-Branco DS, Soares GD, Astete-Medrano DJ, Monteiro AJ, et al. (2010) Characterization of the gastrointestinal yeast microbiota of cockatiels (Nymphicus hollandicus): a potential hazard to human health. J Med Microbiol 59: 718-723.

Track Your Manuscript