Occurrence and Distribution of Cryptococcus species in Environmental Sources from Lower Assam Belt of india

© The Author(s) 2020. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Islam et al. | J Pure Appl Microbiol | 14(4):2781-2800 | December 2020 Article 6651 | https://doi.org/10.22207/JPAM.14.4.54

Cryptococcus species are commonly found in dropping of pigeons that apparently harbors the organism in normal commensal form 6 . In addition, the digestive tracts of parrots and canaries also harbour the fungus 7 . Pigeon droppings, are available in most unlikely places such as roofs and ventilations of abandoned buildings, cornices, leaves and branches of trees that serve as ecological niche for adaptation, dispersion of Cryptococcus replication and transmission. Other environmental sources such as fruits and vegetables retain the fungi as saprophyte that may cause infection in man and animals either by inhalation of spores of the organism or through subcutaneous inoculation 3,[8][9][10] .
Excreta of pigeon as saprobic reservoir of Cryptococcus species have been frequently recovered from various countries of the world 3,11,12 . Isolation of Cryptococcus from many tree species in America 13 Brazil 14 , India 15 , Iran 16 have been reported.
Among the systemic and opportunistic mycoses, cryptococcosis continues to exacerbate severe health risks, especially in high risk groups and immuno-compromised patients. It has been estimated that the global prevalence of cryptococcosis among AIDS patients stands at 2.33% and 6.8% both worldwide and within India respectively 3 . However, incidence of cryptococcosis in recent years is alarmingly increasing at a global scale amounting to one million infections and approximately 6,25,000 deaths annually, rendering the disease to be considered as an most important fungal diseases 17 .
Cryptococcosis is a highly infectious and enigmatic mycotic disease that affect a variety of animals too 18,19 . The disease occurs in acute, sub-acute or in chronic forms and have global significance 20 . The fungi causes respiratory as well as neurological problems and is often sporadic in nature 19,21,22 . Incidentally, the exact epidemiological data on the incidence and prevalence of the disease is not readily available as the disease is not a notifiable one although several reports of considerable morbidity and mortality in human as well as in animals are well documented 21 .
The first report of Cryptococcosis in India was documented in 1952 and subsequently many other investigators reported its occurrence from time to time in various parts of the country 23 . Although numerous studies on the zoonotic importance and epidemiology of Cryptococcus neoformans have been reported, yet there is lack of proper information as literatures are scanty on the non-neoformans species like Cryptococcus albidus, Cryptococcus laurentii or Cryptococcus uniguttulatus 7 . In recent years, a burst of increase in opportunistic infection by these non-neoformans pathogenic yeasts have been observed 24 . Environmental sources including canopy leftovers from some trees are regarded as the main sources of Cryptococcus albidus, Cryptococcus laurentii, and Cryptococcus uniguttulatus that lead to infection in human and animals 16,25,26 .
Epidemiology of non-neoformans Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus uniguttulatus, Cryptococcus luteolus and some other species of the genus are highly relevant as these species often turn out to be pathogenic and thereby increases the risk of infection. A case of encephalitis in an HIV patient due to Cryptococcus albidus was reported in China 27 while fungal keratitis due to the same species was reported by Huang et.al, (2015) 28 . Earlier, pulmonary Cryptococcosis due to Cryptococcus laurentii in diabetis and patients suffering from ganglio-neuroblastoma were reported by Averbuch et.al, (2002) 29 and Shankar et.al, (2006) 30 respectively. Reports are also available on Cryptococcal meningitis, Cryptococcal myelitis and multiple skin lesions in HIV patients infected with Cryptococcus neoformans var. grubii from Assam having a history of occupational exposure to pigeons and chicken 31 .
Such findings are of greater significance to places like Assam where domestication of pigeons, fowls and various birds of the finch family are common among the inhabitants at a large scale both as household pets and for meat. A proper surveillance of the sites of occurrence of the fungi as well as characterization of its diverse forms for creating a database is a primary need since the practice of rearing pigeons is non-scientific, traditional and sporadic reports of pulmonary diseases (including cryptococcal meningitis) have been recorded from time to time amongst the common mass. There is a remarkable gap of information on this aspect in the entire northeastern part of India, including Assam. This is a preliminary attempt for identifying the hot spots of Cyptococcus occurrence in environmental samples representing the lower Brahmaputra valley of Assam and characterizing the isolates for their proper identification to generate a baseline data. It is envisaged that the study will generate information for future planned research activities on the opportunistic systemic mycotic disease causing fungi.

MAteRiAls AND MethOD
In the present investigation, a total of 435 samples of pigeon droppings and droppings of other avian species, barks and leaf litter of natural stands of eucalyptus trees as well as environmental soil from different sources from five different distinct regions of the lower Brahmaputra Valley of Assam were collected. These included the districts of Bongaigaon, Dhubri -(both north bank extending up to West Bengal border and south bank extending upto the border of the state of Meghalaya along the river Brahmaputra), the district of Kokrajhar BTAD region, the district of Goalpara extending up to the border with Meghalaya, the district of greater Kamrup (including both urban and rural extensions along the north bank of the river Brahmaputra) and finally the district of Nalbari of western lower Assam. Samples collected were transported to the laboratory in ice cooled boxes packed in polyethylene bags. However, cloacal samples of pigeon were collected using sterile cotton swab in a transport media (Eurotubo r , Rubi Barcelona, Spain). After collection, all samples were either processed on same day or stored at 4 0 C until being processed for isolating Cryptococcus species. In the meantime, basic environmental characteristics of the collection areas were noted for understanding the ecological and epidemiological relevance of the organism collected from different sources (Table.1, Fig.1, Fig.2 and Fig.3). The marked samples were processed as per standard methods mentioned elsewhere with slight modifications 14,32 .

Isolation and Identification
Processing of samples was done in the Department of Biotechnology, Gauhati University. Two grams (2g) of the samples was diluted in 20 ml of sterile physiological saline and the mixture was stirred for five minutes in a vortex apparatus and allowed to stand for 15-20 minutes for Fig. 1. Representation of sample collection areas developed from GPS coordinates and using GIS software prepared by Aaranyak, Guwahati, Assam decantation. After this, 1 ml of the supernatant was inoculated onto 9 ml of sterile physiological saline supplemented with chloramphenicol (0.05mg/ml) and then incubated at 37 0 C for an hour. An aliquot of 0.1ml of each supernatant of the processed samples were aspirated and streaked onto duplicate plates of Sabouraud Dextrose Agar (SDA) and a Bird Seed Agar (BSA) supplemented with chloramphenicol (0.05mg/ml).
Cultures with SDA media were incubated at 37 0 C while that of Bird Seed Agar (BSA) were incubated at 25 0 C for 15 days and monitored daily from 2 nd day onward for observing growth and presence of colony and to evaluate colony morphology. Brown pigmented colonies were identified as Cryptococcus neoformans while colonies showing cream colouration with smooth and mucoid appearance were identified as non-neoformans species coglomerate. Results evaluated on the basis of morphological data were expressed as the average nos. of viable yeast cells per gram of sample (CFU/g). For comparisons, Cryptococcus laurentii MTCC 2898 (procured from CSIR Institute of Microbial technology, Chandigarh -1600361, India) was used as control. Identified isolates were sub cultured, purified and further subjected to an array of morphological, cultural and bio-chemical tests such as carbohydrates, nitrite reduction, urease production and phenol oxidation 11,15,16,20,24 .

Capsule Identification
For capsule identification of the probable Cryptococccus spp., a drop of inoculum from newly inoculated isolates grown on SDA was added onto India ink stain on a sterile glass slide with a cover slip and observed under a bright  field microscope (Olympus CX33) at 100 and 400 times magnification (Kwon-Chung and Bennett, 1992) and also phase contrast microscopy (Leica DM750) under 1000 fold magnification. Presence of distinct wide round to oval gelatinous capsule, with or without hyphae was considered as positive observation [33][34][35][36] .

Molecular Characterization DNA extraction
For DNA extraction, isolates from pure cultures were inoculated onto 1.5ml Eppendorf tubes containing 0.5 ml of Sabouraud Dextrose broth supplemented with chloramphenicol and incubated overnight in an orbital shaker at 150 rpm and 30°C. After 24 hours, the fungal suspensions with predetermined concentrations were centrifuged at 5,000 rpm for 10 minutes, and the pellet was frozen at -20°C for 1 hour with further incubation at 65°C for 1 h in 0.5 ml of extraction buffer containing 50 mM Tris-HCl, 50 mM EDTA, 3% sodium dodecyl sulfate and 1% 2-mercaptoethanol (Ferrer et al., 2001). The lysate was finally extracted with phenolchloroform-isoamyl alcohol (v/v) in the ratio 25:24:1. To this, 65μl of 3M sodium acetate and 75μl of 1M NaCl were added and the resulting volume was incubated at 4°C for 30 minutes. DNA was recovered by isopropanol precipitation and washed with 70% (v/v) ethanol. Concentration of DNA was measured at 260 nm in a UV-VIS Spectrophotometer (Shimadzu) and stored at -20°C until further use [37][38][39] .

PCR amplification
For characterization of Cryptococcus species, isolated DNA was amplified in a gradient PCR (Eppendorf Nexus Gradient). The primers    used for amplification included the D1/D2 regions targeting ITS1 and ITS4 with expected fragment length of 600 bp. Details of the primers are given () in Table 2. The program for amplification was set for 1 cycle of initial denaturation for 30 minutes at 94 0 C followed by 35 cycles of denaturation for 1 minute at 94 0 C, primer annealing for 1 minute at 55 0 C, chain extension for 1 minute at 72 0 C and a final extension for 7 minutes at 72 0 C respectively. The PCR products after amplification were resolved in a 1.5 % agarose gel subjected to electrophoresis and was visualized under UV gel documentation system (UVitec Cambridge, Genei). The amplicons were later stored at -20 0 C for further analysis [40][41][42][43] .

Sequencing
Sequencing of PCR products were done at Xcelris Labs Limited, India through outsourcing. Chromatogram files obtained were analyzed for nucleotide-BLAST on NCBI portal for identification of the species. Phylogram and dendrogram was prepared using PhyML (Phylogenetic Maximum Likelihood) (http://www.atgc-montpellier.fr/ phyml/).

Statistical analysis
A very brief descriptive analysis was carried out to interpret the data as they were mostly qualitative attributes. However, positive results obtained from different sources were compared through k-proportion test through Monte Carlo / Marascuilo methods to assess their homogeneity across sources with the help of XLSTAT software [44][45][46] .
During the investigation of 57 (22.61%) Cryptococcus species isolated from pigeon droppings from sites A & B, 34 (13.49%) were identified to be Cryptococcus albidus while 23 (9.13%) represented Cryptococcus laurentii. The highest recovery percentage of Cryptococcus (57) was observed in pigeon droppings and the least (2) was from avian species other than pigeon. In terms of relative isolation of positive cultures of Cryptococcus, 14 (15.73%) were from attics / ventilations / old and abandoned buildings (Site A), 43 (26.38%) were from houses of pigeon fanciers / dovecots (Site B), 3(4.84%) were from contaminated environment / soil (Site C), 5 (9.80%) were from the debris / barks / leaves of eucalyptus trees (Site D) while 2 (2.86%) were from other avian sources like chicken, duck, parrot etc (Site E). The results are presented in Table 3.
Relative and absolute percentage of isolates of Cryptococcus (Naganishia albida and Papiliotrema laurentii) from each collection site was also analyzed. Prevalence rate of isolates from Site A (3.22%), Site B (9.89%), Site C (0.69%), Site D (1.15%) and Site E (0.46%) was recorded (Table  4). Results interpret that there exist a significant difference at 0.05% among the variants of different proportions obtained from various samples during the study. Values having the superscript across different rows do not differ significantly.
The overall prevalence of Naganishia albida, across all dry and most environmental samples was 9.43% (41 of 435). However, the prevalence of the species was 35 (11.07%) in dried specimens in comparison to 6 (5.04%) from moist specimens. Details of prevalence of Naganishia Table 3.  albida is presented in Table 5 Table. 6, Fig. 6(A) and Fig. 9 alongwith the comparable datasheet of reference strain (Cryptococcus laurentii MTCC 2898, Fig. 5).
Result correlate a significant difference at 0.05 % level among the representative proportions obtained from different sources during the study. Details of genetic identity of the isolated strains of Naganishia albida and Papiliotrema laurentii including sequenced data of the rDNA ITS region and their accession numbers obtained from NCBI after submission of the sequences are presented in Table. 7 and Fig. 10 respectively. Comparisons pertaining to the prevalence of Naganishia albida and Papiliotrema laurentii across the studied geographical locations depict an overall 11 (18.64%), 16 (17.58%), 9 (16.98%), 16 (15.38%). 10 (13.33%) and 5 (9.43%) numbers of positive isolates representing the districts of Bongaigaon, Dhubri, Kokrajhar, Goalpara, Kamrup and Nalbari (Table. 8; Fig. 11). We could not establish any significant difference in the results observed across different districts. Moreover, the prevalence of pathogenic Cryptococcus neoformans could not be observed from any of the tested samples indicating absence of the species in the specimens collected which may be due to unfavorable habitat niche for the species along the lower Brahmaputra valley of Assam resulting in complete absence of the most feared representative form of the family of Cryptococcus.

DisCUssiON
Cryptococcosis is a major public health concern in India. Epidemiology and pathogenicity of this enigmatic myco-zoonosis in man and animals have been well studied [18][19][20][21] . Isolation and identification of Cryptococcus from the environment demonstrates the importance of biodiversity and environmental niche of the pathogenic     of different biovars of this pathogen, includes both pathogenicity and antifungal resistance which have tremendous implications. Ecological relationship of Cryptococcus was not known until Emmons (1955) 10 found the fungus in droppings of pigeon and soil colonized by the genera. Later, his observation was substantiated to be true by many researchers 2,3,17,20,24 . Although many studies have been conducted on the epidemiology and pathogenicity of Cryptococcus neoformans, yet there is scarcity of current information on the prevalence, epidemiology and pathogenicity of the nonneoforman species. The present study defines the incidence of non-neoforman species of the genus Cryptococcus inherent in pigeon droppings, ecological niche of eucalyptus trees and in other microfloral sources of the environment at a specific geographical region along the lower Brahmaputra valley of Assam.
Our observation confirmed the presence of two non-neoforman species of the genus Cryptococcus, viz., Naganishia albida and Papiliotrema laurentii. To our knowledge, this is the first report on the isolation of these two species from pigeon droppings and other environmental sources of the northeastern region of India and this may be considered as an established fact that the environmental sources of the studied areas serve as ideal saprobic reservoirs of these two opportunistic pathogens. Considering the importance of the species at the backdrop  In the current study, 13.49% (34) and 9.13% (23) of Naganishia albida and Papiliotrema laurentii could be recovered from pigeon droppings from attic ventilations of old buildings, dovecotes houses of pigeon fanciers and from cloacal swabs. Earlier, Rosario et. al. (2009) 24 reported a lower isolation rate of 23 (6.9%) Cryptococcus albidus and 2 (0.6%) Cryptococcus laurentii from droppings of pigeons in Spain which may be due to differences in environmental and climatic conditions as mentioned above. Studies have also been reported from Nigeria whereby Pal (2015) 47 , had isolated 16 representatives of Cryptococcus neoformans from environmental samples of pigeon droppings with a prevalence of 12.5%. In the present study, the prevalence of Naganishia albida (13.49%) and Papiliotrema laurentii (9.13%) in pigeon droppings and in eucalyptus tree specimens were comparable to previous reported findings, despite the absence of Cryptococcus neoformans.
Although Cryptococcus neoformans is regarded as the most commonly occuring species of the Cryptococcus family, its absence in probable specimens collected from this part of the country is quite intriguing and it reflects the status of overall persistence of the species under humid conditions exposed to abrupt change in temperatures, a feature commonly observed in the studied locations. . The effect of seasonal variations on the persistence of non-neoforman Cryptococcus isolates cannot be nullified and it suggest the need of proper screening through a robust molecular approach concomitant to the behavior and food sources of the birds questioned, not to mention the presumable role of temperature and other edaphic factors that needs further appreciation.
Isolation of several species and varieties of the genus Cryptococcus from different sources, soils, atmospheric airs, dust and even from other avian species i.e chicken, ducks, parrots and munia had been previously reported by many authors. In a study conducted in Brazil, Leite et. al, (2012) 48 found the occurrence of 18 (21.4%) Cryptococcus species from 84 specimens of dust collected from public libraries. The isolation rate of Cryptococcus albidus 2 (4.6%) in their study was found to match our findings from dry soil 2 (4.88%). Furthermore, the percentage of isolation of Cryptococcus species in this study was 18.33% (58) as compared to 7.56% (9) from a total of 435 collected specimens in dry and moist conditions. The observations were in congruence with the number of isolates from pigeon droppings whereby 25.12% (51) and 12.24% (6) Cryptococcus isolates could be recovered from dry and moist specimens.
While studying the frequency of isolation in context of regional characteristics, no specific trend in terms of frequency was observed as the number of isolates varied from 9.43% (in Nalbari region) to 18.64% (Bongaigaon region), despite close proximity of the study areas that share the same environmental conditions. It may be recalled that factors like humidity, rainfall, temperature and pH of a definite geographical location do affect population parameters of domesticated birds like pigeons which need further evaluation. Ecological relevance of the non-neoformans species of Cryptococcus (4.84% in soil, 9.80% in eucalyptus tree samples) was the most important findings of this study with profound influence on replication and transmission of the pathogenic species from the ideal sources to the susceptible population at risk. Such situation is very imminent and highly dangerous for crowded and highly populated areas like Nalbari and Kamrup as the route of entry of such microorganisms is either through inhalation or direct skin contact.
From this investigation, it may be concluded that Naganishia albida and Papiliotrema laurentii are the most prevalent Cryptococcus species in the lower Assam belt compared to the more pathogenic Cryptococcus neoformans indicating the need of extensive epidemiological studies for establishing proper identify of the different strains that perpetuate, thrive and adapt to changing environmental conditions which are bound to challenge public health and pose as potential hazards in the near future. It may be stated that to ensure safety of the people, mass awareness programmes on the prevalence of the fungus need to be arranged whilst proper prevention, control and management of such disease causing pathogens be prioritized to prevent future outbreaks so as to minimize the risk of transmission of opportunistic Cryptococcus infections.

ACKNOwleDgMeNts
The authors duly acknowledge the support and contribution provided by Department Fig. 11. District wise distribution of positive isolates of Cryptococcus species of Biotechnology, Gauhati University, Guwahati, Assam to carry out the research activity. The authors also duly acknowledge the support and cooperation received from state department of Animal husbandry and Veterinary, Govt of Assam, Xcelris Labs Limited and other personnel involved during the data collection / processing to carry out task effectively.