Abstract

The aim of this study is to report the regional distribution of Cryptococcus. gattii and Cryptococcus. neoformans in decayed wood inside trunk hollows of Syzygium cumini trees (Java plum, Indian black berry) investigated in Amritsar (Panjab), Meerut Cantt. and Bulandshahr (Uttar Pradesh) and Delhi, in north-western India. Two hundred and seventeen wood samples collected from 74 S. cumini trees were investigated. This includes 7 known positive S. cumini trees in Delhi subjected to a mycological surveillance for perennial colonization by C. gattii and C. neoformans. Cryptococcus gattii showed the highest prevalence (89%) in S. cumini trees in Delhi, followed by 27%, 12.5% and 9% prevalence in Bulandshahr, Amritsar City and Meerut Cantt., respectively. In contrast, C. neoformans had the highest prevalence (54%) in Amritsar, followed by 44% in Delhi, 9% in Bulandshahr and 0% in Meerut Cantt. Furthermore, 44% of the S. cumini trees in Delhi, 9% in Bulandshahr and 8% in Amritsar were concomitantly colonized by both C. gattii and C. neoformans. A mycological surveillance over 4.8–5.2 years of 7 selected S. cumini trees in Delhi revealed perennial colonization by both the Cryptococcus species. In addition, air samples taken close to the decayed trunk hollows of 4 of the perennially colonized S. cumini trees contained strains of the C. neoformans species complex. Of a random sample of 48 isolates serotyped, 26 (54%) were C. neoformans, serotype A, and 22 (46%) C. gattii, serotype B. Determination of mating type alleles was done in 44 of the isolates, comprising 31 of C. neoformans, serotype A and 13 of C.gattii, serotype B. All of them proved to be mating type α (MATα). The data on high prevalence, fungal population density, perennial colonization and aerial isolations indicate that decayed wood in trunk hollows of S. cumini trees is to-date the main well documented primary environmental niche of C. gattii and C. neoformans in north-western India. Attention is drawn to the likely health hazard posed by the environmental reservoirs of C. gattii and C. neoformans occurring in tree trunk hollows in proximity to human and animal habitations.

Introduction

Cryptococcosis is a major systemic mycosis of worldwide distribution with predilection for the central nervous system. It is potentially fatal unless diagnosed and treated at an early stage with appropriate antifungal therapy 6–13. The natural habitat of C. gattii was unknown until Ellis and Pfeiffer from Australia in the early 1990s reported its association with debris of Eucalyptus trees 16–18. Our laboratory reported recently that the most important host tree for C. gattii in the Delhi/New Delhi area was Syzygium cumini (Indian black berry, Java plum, black plum), harbouring the fungus in decayed wood in its trunk hollows 19–21 and not Eucalyptus camaldulensis or Eucalyptus tereticornis as in Australia. In this communication, we report the results of a wider environmental survey of C. gattii and C. neoformans in S. cumini trees extending to Amritsar City (Panjab), Meerut Cantt. and Bulandshahr (Uttar Pradesh), situated about 450 km in north-west, 70 km and 60 km east of Delhi, respectively. The study also includes the results of attempted aerial isolations of the two pathogens from inside trunk hollows of several trees of S. cumini in Delhi known to harbour both or either of the species and demonstrates their perennial colonization in this ecologic niche during the 4.8 to 5.2 years of sampling.

Material and Methods

Collection sites and wood sampling

One hundred and eighty-five decayed wood samples collected from trunk hollows in 74 Syzygium cumini living trees were investigated. The distribution of investigated trees was as follows: 24 in Amritsar City, Panjab, 23 in Meerut Cantt., 11 in Bulandshahr, Uttar Pradesh, and 16 in Delhi. The sampling included reinvestigation of 7 S. cumini trees from Delhi, designated as TT-28, TT-41, TT-44, TT-47, TT-110, TT-115 and TT-151 that had been shown to harbour C. neoformans or C. gattii in trunk hollows during our previous study in 2000–2002 20. These trees have been under surveillance for perennial colonization by C. neoformans and/or C. gattii. The S. cumini trees selected for sampling had generally large canopies and appeared to be very old as apparent from the large girth of their extensively decayed trunks.

Sampling of decayed wood was done with an in-house swabbing technique using simplified niger seed agar (NSA) as earlier described by Randhawa et al.21. The plates of NSA inoculated with the swabs were incubated at 28°C and observed up to 7 days for isolation of C. gattii and C. neoformans. To determine a rough estimate of viable population density of the C. neoformans species complex in a given test sample, one swab from each trunk hollow was immersed in 10 ml of sterile physiological saline and vortexed for 2 min on a cyclomixer (Remi, Mumbai, India). The swab was taken out and discarded. The sample suspension was allowed to sediment for 30 min, followed by inoculation of duplicate plates of NSA with 100µl aliquots of the supernatant. After 3–4 days of incubation at 28° C, the number of chocolate brown yeast-like colonies compatible with the C. neoformans species complex appearing on NSA plates was counted visually. The number computed was multiplied by 102 in order to determine the colony count per swab.

Aerial sampling

For aerial isolations of the two Cryptococcus spp. from trunk hollows, 4 S. cumini trees (TT-28, TT-110, TT-115 and TT-248) were selected. These trees had been shown to be repeatedly positive for C. gattii/ C. neoformans in their decayed wood. Ten petri plates containing NSA were exposed one by one for 30 seconds in each tree trunk hollow. After removing its lid, each medium plate was held facing the decayed trunk while aerosols were created by repeated manual pressing of an egg-shaped rubber bulb, measuring 90 mm long and mid-point circumference of 150 mm, that was attached to a 160 mm long rubber tubing (inside diameter 5 mm) ending in a plastic nozzle having an opening with 2 mm diameter. At its proximal end, the rubber bulb was fitted with a metallic valve that controlled one-way flow of air through the nozzle when it was pressed. The exposed media plates were incubated and observed as in the isolation of fungi by swabbing from decayed wood.

Identification and serotyping

Initial screening of C. gattii and C. neoformans on the inoculated plates was done by microscopic examination of variably brown yeast-like colonies developing on NSA. The suspected colonies were purified by dilution plating and identified by their morphological study and verification of salient physiological characteristics, employing the VITEK 2 System (bio-Mérieux, Marcy-l'Etoile, France) 19, 20, 22. Serotyping of the isolates was done by the slide agglutination test based on monoclonal antibodies specific for the variable capsular polysaccharide, employing the commercially available kit, Crypto-Check (Iatron Laboratories, Tokyo, Japan). The number of isolates serotyped was 48.

Mating type determination

The mating types of strains were determined as described previously (Yan et al.) 23. Briefly, genomic DNA was isolated from each strain by the method described by Xu et al. 24. A total of five pairs of primers were used in PCR reactions to screen the mating types. One was the MATα specific STE12α primer pair. PCR using this primer pair was found to be able to amplify a product from all MATα strains, but none from MATa strains, regardless of their serotype (A, B, C, D, or AD) (Yan et al.) 23. The other four pairs of primers targeted the STE20 gene and they were serotype (A, D) and mating type (a and α) specific. While these four primers have been shown to be effective for identifying the mating types of serotypes A and D strains, they were found to be less effective in serotypes B and C strains. Details of PCR reaction, gel electrophoresis and scoring followed those of Yan et al. 23. The combined results from these PCR reactions were used to interpret the mating type of individual strains. The number of isolates investigated for mating type was 44; 26 of these originated from S. cumini and 18 from Ficus religiosa decayed wood in Delhi. Thirteen of the S. cumini isolates were C. gattii, serotype B whereas all of the remaining 31 were C. neoformans, serotype A.

Results

Strain isolation

The trunk hollows of S. cumini trees that harboured C. gattii and C. neoformans in their decayed wood varied widely in their dimensions depending upon the extent of decay. Some of the trees were extensively damaged with their hollows vertically extending several meters up their trunks. In primary cultures of wood samples on NSA, smooth and variably mucoid, yeast-like colonies of C. neoformans species complex were distinctly seen after 2–4 days of incubation, exhibiting variable shades of a chocolate brown pigment that darkened with age (Fig. 1). At this stage, we occasionally observed chocolate brown, glistening colonies of black yeast associated with decayed wood that resembled the macroscopic features of C. gattii / C. neoformans (Fig. 2) with which it could be confused but it was readily differentiated by its microscopic morphology.

Fig. 1

Innumerable tiny, chocolate brown, yeast-like colonies of C. gattii on a simplified Staib's niger seed agar (NSA) plate, isolated from decayed wood inside trunk hollow of a S. cumini tree in Amritsar, seen after 6 days of incubation at 28° C. Interspersed among them are also many cottony white, fluffy mold colonies.

Fig. 1

Innumerable tiny, chocolate brown, yeast-like colonies of C. gattii on a simplified Staib's niger seed agar (NSA) plate, isolated from decayed wood inside trunk hollow of a S. cumini tree in Amritsar, seen after 6 days of incubation at 28° C. Interspersed among them are also many cottony white, fluffy mold colonies.

Fig. 2

Brown to dark brown, glistening colonies of a black yeast isolated from decayed wood of a pine tree, simulating those of the C. neoformans species complex in macroscopic morphology on a NSA plate after 6 days of incubation at 28°C.

Fig. 2

Brown to dark brown, glistening colonies of a black yeast isolated from decayed wood of a pine tree, simulating those of the C. neoformans species complex in macroscopic morphology on a NSA plate after 6 days of incubation at 28°C.

Serotyping and mating type determination

Of the 48 isolates of the C. neoformans species complex randomly serotyped, 26 (54%) were found to be C. neoformans serotype A, and 22 (46%) C. gattii serotype B. PCR using the mating type α-specific STE12α primers amplified an expected fragment for all of the 44 strains investigated for their mating type. For serotype A strains, PCR using the STE20Aα primers produced expected products. None of the other three STE20 primer pairs produced any PCR product for any of the strains. In addition, negative controls of mating type strains in our collection produced no amplification product (Data not shown). Therefore, our results indicate that all analyzed strains were mating type α (MATα).

Regional distribution

The data on prevalence of C. neoformans and C. gattii in decayed wood inside trunk hollows of S. cumini trees in Amritsar, Meerut Cantt., Bulandshahr and Delhi are presented in Table 1 and depicted in Fig. 3. The highest prevalence of C. gattii, serotype B (89%), was found in Delhi where 8 out of the 9 S. cumini trees investigated were positive. Concomitantly, 44% of these trees yielded C. neoformans, serotype A. In strong contrast, C. gattii had a prevalence of only 12.5% in Amritsar, 9% in Meerut Cantt. and 27% in Bulandshahr. The predominant species found in S. cumini trees in Amritsar was C. neoformans, serotype A, showing a prevalence of 54% as against 44% in Delhi where it occurred concomitantly with C. gattii, serotype B. Samples from Meerut Cantt. did not yield any isolation of C. neoformans. Overall, concomitant occurrence of C. gattii and C. neoformans in the same trunk hollows was seen in 7 of the 67 (10%) S. cumini trees investigated.

Fig. 3

Comparison of percent prevalence of Cryptococcus gattii and Cryptococcus neoformans in decayed wood inside trunk hollows of Syzygium cumini in Amritsar, Delhi, Meerut Cantt. And Bulandshahr, 2004–2006.

Fig. 3

Comparison of percent prevalence of Cryptococcus gattii and Cryptococcus neoformans in decayed wood inside trunk hollows of Syzygium cumini in Amritsar, Delhi, Meerut Cantt. And Bulandshahr, 2004–2006.

Table 1

Prevalence of Cryptococcus gattii and Cryptococcus neoformans in decayed trunk wood of Syzgium. cumini trees in north- western India, 2004-2006.

Place / locality Collection period No. trees positive for C.gattii alone / No. examined No. trees positive for C.neoformans alone / No. examined No. trees positive for both species No. wood samples positive / No. examined* Highest Colony count / swab 
Amritsar, Panjab.       
Duni Chand Road and Ram Bagh Nov., 2004 to Jan., 2005 1/24 (4.1%) 11/24 (45.8%) 2/24 (8.3%) 21/30 (70%) 3.2×104 
Meerut Cantt, U.P. May, 2005 2/23 (9%) 0/23 0/23 2/74 (3%) 
Bulandshahr, U.P. Mar., 2006 2/11 (18%) 0/11 1/11 (9%) 5/22 (23%) 2.1×105 
Delhi       
Roshanara & Gulabi Bagh Gardens May-July, 2004 4/9 (44.4%) 0/9 4/9 (44.4%) 13/16 (81.2%) 3.5×103 
       
Total:  9/67 (13%) 11/67 (16%) 7/67 (10%) 41/142 (29%)  
Place / locality Collection period No. trees positive for C.gattii alone / No. examined No. trees positive for C.neoformans alone / No. examined No. trees positive for both species No. wood samples positive / No. examined* Highest Colony count / swab 
Amritsar, Panjab.       
Duni Chand Road and Ram Bagh Nov., 2004 to Jan., 2005 1/24 (4.1%) 11/24 (45.8%) 2/24 (8.3%) 21/30 (70%) 3.2×104 
Meerut Cantt, U.P. May, 2005 2/23 (9%) 0/23 0/23 2/74 (3%) 
Bulandshahr, U.P. Mar., 2006 2/11 (18%) 0/11 1/11 (9%) 5/22 (23%) 2.1×105 
Delhi       
Roshanara & Gulabi Bagh Gardens May-July, 2004 4/9 (44.4%) 0/9 4/9 (44.4%) 13/16 (81.2%) 3.5×103 
       
Total:  9/67 (13%) 11/67 (16%) 7/67 (10%) 41/142 (29%)  

*Data inclusive of both C. gattii and C. neoformans

The viable population density of the C. neoformans species complex varied widely in decayed wood of S. cumini trees not only from place to place or tree to tree in the same locality but also from site to site in the same trunk hollow. For example, the count was found to be as low as 1 CFU/swab inoculated with material from trunk hollows in S. cumini trees investigated from Meerut Cantt. whereas it ranged from 3.25×103 CFU /swab in Delhi to 2.1×105 CFU/swab in Bulandshahr. In a long-term surveillance study on some selected S. cumini trees from Delhi (Table 2), decayed wood was found to be colonized by both C. gattii and C.neoformans during our 4.8–5.2 years of investigation.

Table 2

Surveillance of selected Syzygium cumini trees in Delhi for perennial colonization by Cryptococcus gattii and Cryptococcus neoformans, 2000–2005.

Locality Tree No. No. wood samples positive / No. examined (%) Collection dates of positive wood samples Observation span (yrs.) Species isolated 
New Police Lines, Kingsway Camp TT-28 8/13 (61.5) Nov. 28, 2000, May 3, 2003, Sept.13, Nov. 28, 2005,Feb. 21, 2006 5.2 C. gattii and C.neoformans 
 TT-41 7/10(70.0) Dec.7,2000, May 13 and August 5, 2003, Nov. 28, 2005, March 11, 2006 5.2 C. gattii 
 TT-44 6/7(86.0) Dec. 7, 2000, Oct. 18, 2002, March 13, 2006 5.2 C. gattii and C. neoformans 
 TT-47 8/9 (88.8) Dec. 7, 2000, May 19, 2003, Sept.13, Dec. 7, 2005,Feb. 21, 2006 5.2 C. neoformans 
 TT-151 10/11 (90.9) Dec.21, 2000, May 19, 2003, Sept. 13, 2005, Feb. 21, 2006 5.1 C. neoformans and C. gattii 
Delhi University, North Campus TT-110 10/12 (83.3) May 6, 2001, Oct. 6, 2003, Nov. 6, 2004, July 2, Sept.13 and Oct. 22, 2005, Feb. 21, 2006 4.8 C. gattii 
 TT-115 12/13 (92.3) March 6, 2001, April 26, 2003, April 24 and Nov. 6, 2004, July 2 and Sept.13, 2005, Feb. 21, March 7, 2006 5.0 C. gattii 
Locality Tree No. No. wood samples positive / No. examined (%) Collection dates of positive wood samples Observation span (yrs.) Species isolated 
New Police Lines, Kingsway Camp TT-28 8/13 (61.5) Nov. 28, 2000, May 3, 2003, Sept.13, Nov. 28, 2005,Feb. 21, 2006 5.2 C. gattii and C.neoformans 
 TT-41 7/10(70.0) Dec.7,2000, May 13 and August 5, 2003, Nov. 28, 2005, March 11, 2006 5.2 C. gattii 
 TT-44 6/7(86.0) Dec. 7, 2000, Oct. 18, 2002, March 13, 2006 5.2 C. gattii and C. neoformans 
 TT-47 8/9 (88.8) Dec. 7, 2000, May 19, 2003, Sept.13, Dec. 7, 2005,Feb. 21, 2006 5.2 C. neoformans 
 TT-151 10/11 (90.9) Dec.21, 2000, May 19, 2003, Sept. 13, 2005, Feb. 21, 2006 5.1 C. neoformans and C. gattii 
Delhi University, North Campus TT-110 10/12 (83.3) May 6, 2001, Oct. 6, 2003, Nov. 6, 2004, July 2, Sept.13 and Oct. 22, 2005, Feb. 21, 2006 4.8 C. gattii 
 TT-115 12/13 (92.3) March 6, 2001, April 26, 2003, April 24 and Nov. 6, 2004, July 2 and Sept.13, 2005, Feb. 21, March 7, 2006 5.0 C. gattii 

Aerial isolations

Air-sampling of trunk hollows of S. cumini trees, TT-28, TT-110, TT-115 and TT-248 investigated from Delhi yielded isolates of the C. neoformans species complex on both of the dates when tested, i.e. 31 October and 11 November, 2005. Of the 40 NSA plates exposed altogether, 21 (52.5%) were found to contain isolates of the C. neoformans species complex. The frequency of aerial isolation and the number of colonies recovered per plate varied widely from tree to tree. Thus, TT-28 yielded a 90% isolation frequency, followed by 70% in TT–110, 30% in TT–248 and 20% in TT-115. Likewise, the highest mean colony count ranged from 1.5–4.2 in TT–28, followed by 1.2–3.1 in TT–110 and 0.1–0.5 in TT –115 and TT–248. It appeared from these results that the isolation frequency of C. neoformans species complex and the number of colonies recovered per plate was higher from trees with deep-seated hollows than from trees with open-faced decay of their trunk. The highest colony count recorded was 10 on a NSA plate exposed to the trunk hollow of S. cumini tree, TT-110.

Discussion

The results presented above reinforce and extend our earlier observations 20 that decayed wood in trunk hollows of S. cumini living trees is an important environmental niche of C. gattii, serotype B, and C. neoformans, serotype A, in Delhi and possibly elsewhere in India wherever this tree species may be distributed. It is apparent from the data that the association of C. gattii and C. neoformans with S. cumini as a host tree is not restricted to Delhi but has a wider distribution in northwestern India. It is interesting to note that the prevalence of C. gattii vis-à-vis that of C. neoformans in tree trunk hollows of S. cumini varies widely from place to place. The divergent prevalence of C. gattii or C. neoformans in various places as noted above underscores the complexity of a number of abiotic and biotic factors governing the distribution of the two species in a given environmental niche. Outside of India, C. neoformans had been previously reported from rotten wood and other plant debris inside the tree trunk hollow of a S. cumini (S. jambolana) tree in Rio de Janeiro, Brazil 25. However, it was interpreted as a transient contamination because the investigators failed to repeat the observation with wood material from the same tree or other S. cumini trees in the area.

It seems pertinent to point out that contrary to the literature reports from Australia 14, 15, C. gattii has been only sporadically reported from plant debris of Eucalyptus species in India. The first report was by Chakrabarti et al 26 who reported C. gattii, serotype B, from flowers of 3 Eucalyptus camaldulensis trees in Ferozpur area, Panjab, whereas the results were negative with 623 additional samples of plant debris of E. camaldulensis, E. tereticornis and E. citriodora. In a subsequent study covering 390 bark samples collected in north-western India from miscellaneous Eucalyptus species, including E. camaldulensis and E. tereticornis, Randhawa et al.19 reported a solitary isolation of C. neoformans from an unidentified Eucalyptus tree in Amritsar. As no information was provided in either of the afore-mentioned studies on the number of C. gattii or C. neoformans colonies isolated from the positive samples nor was any attempt made to repeat the isolations from positive sites, the epidemiologic significance of these observations remained unclear. More recently, Gugnani et al.27 have reported two isolates each of C. gattii, serotype B and C. neoformans var. grubii from flowers and bark collected under canopies of E. tereticornis and E. camaldulensis trees in Delhi and Chandigarh, respectively. The significance of Eucalyptus trees in the epidemiology of cryptococcosis in India will, however, continue to be elusive until data of a long-term surveillance study of the positive trees including information on population density of the two fungi in the test samples is available. Going by our data on the high prevalence, population density, aerial isolation and perennial colonization (already demonstrated for over 5 years) by C. gattii and C.neoformans in decayed wood of tree trunk hollows of S. cumini, we believe that this tree species is to date the main primary ecological niche for C. gattii and C.neoformans in north-western India. Further investigations are under way to probe the possible role of a number of other tree species as additional hosts to C. gattii and C. neoformans.

It is notable that isolates of the C. neoformans species complex were found in air samples taken close to the decayed trunks of all of the four perennially colonized S. cumini trees investigated. This observation indicated that dispersal of C. gattii and C. neoformans from one colonized tree could possibly occur to other trees by their aerosols which may be generated artificially or by natural disturbances of decayed wood surfaces due to a variety of agents such as wind, insects, birds, etc. Previously, C. gattii had been demonstrated by air sampling conducted under the canopy of Eucalyptus camaldulensis host trees in Australia, and in air samples collected near the hollows of two pink shower (Cassia grandis) trees in Brazil 13, 14. On the other hand, attempts at aerial isolation of the fungus from the vicinity of host trees were unsuccessful in two instances, namely, one from India and one from Australia 26, 28. It is generally believed that cryptococcosis in humans and animals is acquired by inhalation of such aerosols from the environment. It is pertinent to mention in this context that a large scale outbreak of cryptococcosis due to C. gattii, serotype B, involving at least 59 laboratory-confirmed cases in humans and 45 in terrestrial or marine mammals occurred during 2000–2003 on southern Vancouver Island, Canada. The outbreak was related to colonization on Vancouver Island by C. gattii of a large number of local trees such as Douglas fir (the majority), cedar, arbutus, alder, maple, spruce, Garry oak, etc, thus highlighting the risk of cryptococcosis developing in humans and animals following their exposure to such environmental reservoirs of the pathogen 29, 30.

Until recently, information was lacking on the etiologic role of C. gattii in cryptococcosis in India. Attention to the occurrence of C. gattii in this country was first drawn in 1993 by Padhye et al. 32–35. Keeping in view our observations on the frequent and widespread association of C. gattii with decayed wood of S. cumini trees that are cultivated throughout India, it seems enigmatic that C. gattii has been rarely reported so far in the etiology of cryptococcosis in this country. This may be largely attributable to inadequate awareness in general about the importance of accurate speciation of the isolates of Cryptococcus encountered in diagnostic microbiology laboratories.

It appears that MATα allele is highly prevalent in the environmental populations of C. neoformans, serotype A and C. gattii, serotype B, in north-western India. This is in conformity with previous studies of Yan et al. 23 with the isolates from the USA and Kidd et al. 36 on the isolates from British Colombia. While highly biased sex ratios in the C. neoformans species complex might suggest that clonal reproduction could be the main mode of reproduction in natural populations of these two species in India, for two reasons our current data cannot preclude the possibility that sexual reproduction may also occur. First, the analyzed sample is relatively small. A larger sample might identify MATa strains from those locations in India. Second, a recent study identified that same-sex mating can occur in C. neoformans (Lin et al.) 37. Interestingly, there was ample evidence in the present study for co-existence of both serotypes A and B strains in the same tree hollow. This observation indicates that these two serotypes may frequently co-exist on the same substrate in nature. Further analysis of a large sample using established molecular markers such as polymorphic gene sequences (Xu et al. 23; Kidd et al. 36; Xu et al.38) should shed light on these and other important issues.

Acknowledgements

We thank the Indian National Science Academy, New Delhi, for the award of an Honorary Senior Scientist position to H.S.R., and the Department of Science and Technology, Govt. of India, for a Young Scientist research project to A.C. We greatly appreciate the help from Profs Sukhbir Singh and Iqbal Singh Randhawa, Khalsa College Amritsar, Col. A. S. Bali and the Commanding Officer, Meerut Cantt., for their invaluable cooperation in the collection of S. cumini wood samples.

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