Classification of Cryptococcus neoformans and yeast-like fungus isolates from pigeon droppings by colony phenotyping and ITS genotyping and their seasonal variations in Korea

Cryptococcus neoformans (C neoformans) is a frequent cause of invasive fungal disease in immunocompromised human hosts. Ninety-eight samples of pigeon droppings were collected from the pigeon shelters in Seoul, and cultured on birdseed agar (BSA) and Sabouraud dextrose agar (SDA). One hundred yeast-like colonies were selected and identified via phenotype characteristics, such as colony morphology and biochemical characteristics. This was then followed with genotyping via sequencing of the internal transcribed spacer (ITS) region. The colonies were classified into four kinds of colony color types: brown type (BrT), beige type (BeT), pink type (PT), and white type (WT). Numbers of isolated BrT, BeT, PT, and WT colonies were 22 (22%), 30 (30%), 19 (19%), and 39 (39%), respectively. All BrT colonies were identified as C neoformans. BeT were identified as 19 isolates of Cryptococcus laurentii, 10 isolates of Malassezia furfur, and 1 isolate of Cryptococcus uniguttulatus. PT was divided into two colony color types: light-PT (l-PT) and deep-PT (d-PT). Eighteen of l-PT and one of d-PT were identified as Rhodotorula glutinis and Rhodotorula mucilaginosa, respectively. WT were identified as 34 isolates of Cryptococcus guilliermondii, 3 isolates of Cryptococcus zeylanoides, 1 isolate of Cryptococcus sake, and 1 isolate of Stephanoascus ciferrii. Most strains were classified identically with the use of either phenotype or genotyping techniques, but C uniguttulatus and C sake classified by phenotyping were Pseudozyma aphidis and Cryptococcus famata by genotyping. This rapid screening technique of pathogenic yeast-like fungi by only colony characteristics is also expected to be very useful for primary yeast screening. Additionally, we investigated the seasonal variations of C neoformans and other yeast-like fungi from 379 pigeon-dropping samples that were collected from February 2011 to March 2011. We isolated 685 yeast-like fungi from the samples. Almost all C neoformans and yeast-like fungi were isolated in the fall (298 strains, 43.5%) and spring (244 strains, 35.6%). A few yeast-like fungi were isolated in winter (98 strains, 14.3%) and summer (45 strains, 6%). These results would be used as an important indicator related to epidemiology and prevention of pathogenic yeast-like fungi infections transmitted through pigeon droppings.     

Isolation of saprophytic Cryptococcus neoformans

Isolation of Cryptococcus neoformans was carried out on sunflower seed agar medium (SFA) and Sabouraud dextrose agar (SDA). Out of 346 environmental substrates (133 fruits, 107 avian extreta, 91 vegetables and 15 wooden scrapings) tested, 3 specimens were positive for C. neoformans. The positive isolations came from the fruits of 2 banana (Musa sapientum) and a potato tuber (Solnum tuberosum). The pathogen could not be demonstrated in 107 samples of avian droppings and 15 of wooden materials. All the 3 isolates of the yeast were obtained on SFA, while they were not cultured on the plates of SDA with chloramphenicol which were badly contaminated with rapidly growing molds, yeasts and bacteria. To the present author's knowledge, this appears to be the first reports of the isolation of this pathogenic basidiomycetous yeast from contaminated fruits of banana. We suggest more comprehensive ecological surveys to search for environmental niche of C. neoformans var. neoformans and C. neoformans var. gattii as the latter variety is also implicated in the etiology of cryptococcosis.     

Isolation,molecular identification,and phylogenetic evaluation of Cryptococcus neoformans isolated from pigeon lofts,Psittaciformes, and Passeriformes in Ahvaz,Iran

Cryptococcus neoformans, the main pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from avian excreta, soils, and plant material. This study was carried out to determine the infection rate of pigeon lofts, Passeriformes, and Psittaciformes in Ahvaz, the capital of Khuzestan province in Iran and to determine varieties of Cryptococcus neoformans (C. neoformans). The 80 samples were collected from pigeon lofts. Also, 163 feces of captive birds (Passeriformes and Psittaciformes) which kept in Ahvaz pet shops, and the 70 cloacal swabs of pet birds (Passeriformes and psittaciformes) referring to the department of avian medicine (the faculty of veterinary medicine of Shahid Chamran University of Ahvaz) were analyzed. The samples were directly inoculated on niger seed agar (NSA) and also enriched in brain heart infusion broth and then inoculated on NSA. Dark brown colonies suspected to C. neoformans subcultured on saborouds dextrose agar and pure cultures subjected to molecular (polymerase chain reaction (PCR)) diagnosis. For detection of C. neoformans, primer sets that targeting the CNLAC1 gene were selected and nested PCR was conducted. For identification of C. neoformans varieties, a primer set targeting the STR1 gene was selected. For more accurate confirmation, the purified PCR products of some isolates were also sequenced, and based on the gene sequences, all of the isolates belonged to C. neoformans variety grubii (var. grubii)(serotype A). Totally 16 out of 80 pigeon samples (20%) were contaminated with C. neoformans. The results in pigeons disclosed a 98.64% identity when compared with other strains of C. neoformans (CN1525, T4, and T1) which were previously deposited in GenBank from Italy and Thailand. Also, 21 out of 233 samples from Psittaciformes (9.01%) were contaminated with C. neoformans. The results in Psittaciformes disclosed a 99.7% identity when compared with other strains of C. neoformans (TIMM1313, IFM5882, CN1525, etc.) which were previously deposited in GenBank from Japan and Italy, etc. In the present study, the samples belonging to the passerine order were free of C. neoformans infection. According to the results, C. neoformans is prevalent in pigeon flocks and pet birds including Psittaciformes in the Ahvaz area, and should be considered by pigeon and captive bird breeders, veterinarians, and public health organizations in Ahvaz. The cryptococcus species isolated from captive birds and pigeons could be potential pathogens in humans.     

Cryptococcus neoformans and Mycobacterium bovis causing granulomatous pneumonia in a goat.

An adult Blanca-Celtibérica doe originating from a goat herd with a high prevalence of tuberculosis was presented with respiratory signs. At necropsy, this goat had a diffuse and severe mycotic pneumonia associated with the presence of Cryptococcus neoformans concomitant with pulmonary focal caseous nodules from which Mycobacterium bovis was isolated. Microscopically, the mycotic lesion was a granulomatous pneumonia with many large foamy macrophages containing intracellular yeast bodies. The extensive mycotic changes, their granulomatous nature, and the lack of positive response to different immunologic tests for mycobacterial infection suggested an impaired immune status in this animal.     

Diagnosis and successful treatment of Cryptococcus neoformans variety grubii in a domestic ferret

A domestic ferret was presented for episodic regurgitation. Cytologic examination and culture of an enlarged submandibular lymph node revealed Cryptococcus neoformans variety grubii (serotype A). The ferret was successfully treated with itraconazole. This is the first documented case of Cryptococcus neoformans variety grubii in a ferret in the United States.     

Use of terbinafine in the treatment protocol of intestinal Cryptococcus neoformans in a dog

A 2.5 yr old sexually intact male vizsla was admitted to the Iowa State University Veterinary Teaching Hospital for persistent diarrhea, weight loss, and panhypoproteinemia. Examination revealed an emaciated condition and melena. Two masses were palpated in the cranial abdomen. Hematology and serum biochemistry exhibited a regenerative anemia and confirmed the presence of panhypoproteinemia, suggestive of a protein-losing eneteropathy. Distinct areas of thickened intestinal wall and enlarged mesenteric lymph nodes were found on abdominal ultrasound. Cytology from those nodes showed the presence of suspected Cryptococcus spp., and infection was confirmed utilizing a cryptococcal antigen titer. Medical therapy with lipid-complexed amphotericin B and fluconazole was unsuccessful. Two surgical procedures were performed to remove the affected areas of intestine and lymph nodes, but the disease persisted as evidenced by a persistently elevated cryptococcal antigen titer. Terbinafine was prescribed, which resulted in complete resolution of clinical signs and a steadily decreasing cryptococcal antigen titer. Very few cases of intestinal cryptococcosis have been reported. In this case, infection resulted in a protein-losing enteropathy. In addition, this article describes the use of terbinafine in the treatment of intestinal cryptococcal infection in the dog, which has not been previously reported.     

The excretion of Cryptococcus neoformans (Sanfelice) VUILLEMIN 1894 by pet birds

A survey on taxonomy, diagnosis, distribution and pathogenicity of Cryptococcus neoformans is given. The investigation presented here concerns 1046 fecal samples of avian species, which can be kept in the living area of humans. All together only 15 strains have been isolated, 9 of which originated from the same breeder.     

The occurrence of Cryptococcus neoformans in fecal samples from birds kept in human living areas]

With help of Guizotia creatinine agar (syn. bird seed agar) Cryptococcus (Cr.) neoformans var. neoformans was isolated from 71 (7.7%) of 925 investigated droppings of birds kept within human living area. Cr. neoformans was detected in droppings of 1.2% from 164 psittacines, in droppings of 1.7% from 118 budgerigars and in droppings of 18.4% from (carrier) pigeons. This yeast was not isolated from droppings of 13 small parrots, 10 eclectus parrots, 13 rosellas, 21 cockatiels, 6 Polytelidus sp., 32 Cacatua sp., 13 Carduelis sp., 5 waxbills, 5 starlings, 120 chicken, 9 turkeys, 2 geese, 2 Turdus sp., 1 duck and 1 gull.     

In vitro responses of cheetah mononuclear cells to feline herpesvirus-1 and Cryptococcus neoformans.

In vitro T cell function by domestic cats and cheetahs to two common pathogens, feline herpesvirus-1 (FHV-1) and Cryptococcus neoformans, was assessed. Peripheral blood mononuclear cells (PBM) were stimulated with two strains of UV-inactivated FHV-1, whole heat-killed organisms or capsular antigen of Cryptococcus neoformans, and proliferative responses measured. As a group, cheetah PBM responded significantly poorer than domestic cat PBM when cultured with FHV-1. However, individual cheetah responses varied widely. Supplementation of cultures with exogenous interleukin 2 (IL-2) significantly increased the level of response of individual cheetahs to both strains of FHV-1. Cheetah sera contained slightly higher neutralizing antibody titers to FHV-1 than did domestic cat sera, suggesting that B cells function adequately in cheetahs. When stimulated with Cryptococcus neoformans, both species had similar incidences of positive proliferative responses. These data demonstrate that cheetahs exhibit heterogeneous responses to specific antigens, similar to domestic cats. However, a lower group response to FHV-1 in cheetahs suggests species differences occur. In addition, level of variability in major histocompatibility complex (MHC) class I-like genes, as determined by Southern blot hybridization, does not appear to correlate with a uniform response in in vitro functional assays. Therefore, additional mechanisms influence the final outcome of the immune response.     

Farnesol inhibits in vitro growth of the Cryptococcus neoformans species complex with no significant changes in virulence-related exoenzymes

Farnesol is a sesquiterpene alcohol that modulates cell-to-cell communication in Candida albicans. In recent years, several studies have shown that this molecule presents inhibitory effects against non-albicans Candida species, Paracoccidioides brasiliensis and bacteria. The present study aimed at determining the effect of farnesol on the growth of strains of the Cryptococcus neoformans species complex, through microdilution assays. In addition, the effect of farnesol on the synthesis of phospholipase and protease - important virulence-associated enzymes - by C. neoformans and Cryptococcus gattii was also investigated. A total of 36 strains were studied, out of which 20 were from veterinary sources, 8 were from human cases and 8 were from a reference collection. The minimum inhibitory concentrations (MICs) were determined in accordance with the M27-A3 protocol as described by the CLSI and farnesol was tested at a concentration range of 0.29-150μM. Phospholipase and protease activities were evaluated through growth on egg yolk agar and spectrophotometry, respectively, after pre-incubating the strains at different farnesol concentrations (MIC/4, MIC/2 and MIC). It was observed that farnesol presents an inhibitory activity against C. neoformans and C. gattii (MIC range: 0.29-75.0μM). Although farnesol did not significantly alter phospholipase activity, a tendency to decrease this activity was observed. Concerning protease, no statistically significant differences were observed when comparing the production before and after pre-incubation at different farnesol concentrations. Based on these findings, it can be concluded that farnesol has in vitro inhibitory activity against C. neoformans and C. gattii, but has little impact on the production of the analyzed virulence factors.