Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 47 Cryptococcus gattii Survey in an Israeli Zoo
Environmental Survey for Cryptococcus gattii in an Israeli Zoo
Populated with Animals Originating from Australia
Gilad, A.,
1#
Bakal-Weiss., M.,
2
Blum S.E.,
1
Polacheck, I.
3
and Elad, D.
1
*
1
Dept. of Clinical Bacteriology and Mycology, Te Kimron Veterinary Institute, Bet Dagan, Israel.
2
Te Israeli Veterinary Field Services, Ha’Amakim District Veterinary Ofce, Israel.
3
Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
#
In partial fulfllment of the requirements for the degree of Doctor in Veterinary Medicine at the Koret School of Veterinary
Medicine, Te Hebrew University in Jerusalem.
*
Corresponding Author: Prof. Daniel Elad, Dept. of Clinical Bacteriology and Mycolog, Te Kimron Veterinary Institute, P. O. Box 12 Bet Dagan, 50250 Israel.
Phone +972-(0)3-9681688, Fax: +972-(0)3-9601578. Email: daniel.elad@gmail.com
ABSTRACT
Cryptococcus gattii is an emerging human and animal fungal pathogen. Te frst isolation of C. gattii from
an environmental source was from organic material adjacent to eucalyptus trees (Eucalyptus camaldulensis)
in Australia but subsequently the yeast was isolated from decaying wood of other tree species. It has been
suggested that a unique infectious cycle, between koalas (Phascolarctos cinereus) and the eucalyptus trees
they live and feed on, facilitates the persistence of the fungus in the environment. Since C. gattii has not
been reported in Israel, a zoo populated with animals originating from Australia, including koalas fed with
eucalyptus leaves, was deemed a suitable place to conduct the frst environmental survey for the presence of this
microorganism in this country. Te survey was conducted in two seasons (winter and summer). Environmental
samples were collected from diferent sites inside and around the koala’s cage. Fur and nail samples and
nasal swabs of the koalas were cultured on dopamine agar and sera were tested for the presence of specifc
cryptococcal antigen. All environmental samples and samples taken from the koalas were negative, with no
evidence of Cryptococcus spp. colonies. Serum samples were negative for cryptococcal antigen. Additional
environmental surveys in Israel, focusing on diferent ecological niches and especially on a larger variety of
tree species are suggested.
Keywords: Cryptococcus gattii; Koala (Phascolarctos cinereus); Eucalyptus; Israel; Zoo
INTRODUCTION
Te genus Cryptococcus comprises 37 basidiomycetous yeast
species, among which Cryptococcus neoformans and Cryptococcus
gattii, are the main human and animal pathogens (1). While
C. neoformans usually requires a status of immunosuppression
to infect a patient, C. gattii does meet this requirement (2). C.
gattii is an emerging pathogen, frst reported in 1970 in the
Congo (3) as C. neoformans var. gattii. Subsequently it was
reclassifed as a distinct species as C. gattii (4).
While originally limited to tropical and subtropical envi-
ronments (5, 6), C. gattii has been involved since 1999 in an
ongoing outbreak that started in Vancouver island in Canada
and then spreading through the Pacifc Northwest of the
United States, albeit with some variability of the genotype
involved (6). An interesting hypothesis as to the source of
these highly pathogenic strains from the Amazon basin has
been proposed (7).
Both pathogenic C. neoformans and C. gattii are character-
ized by being surrounded by a capsule (8). In vitro they pro-
duce of melanin on media containing diphenol compounds
such as 3,4-dihydroxyphenylalanine (L-DOPA), resulting in
dark tan colonies, which is considered as an enhancing factor
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Gilad, A. 48
of the fungus’ ability to survive in the environment (9) and
as a virulence factor (10, 11). Two teleomorphs Filobasidiella
neoformans and Filobasidiella gattii have been described for
C. neoformans and C. gattii, respectively. Canavanine-glycine
bromthymol blue (CGB) agar is successfully used to difer-
entiate between the two species (12).
Te source of infection with C. gattii is mostly envi-
ronmental. Te importance of environmental “hot spots”
was demonstrated frst in Australia by Ellis and Pfeifer
(13), showing the fungus to be associated with organic
material around eucalyptus (Eucalyptus camaldulensis) trees.
Subsequently such associations were shown in other niches
as well (14, 15, 16, 17, 18). Moreover, during the blooming
period of the eucalyptus trees, the environmental dissemina-
tion of C. gattii has been found to be increased (13).
Direct transmission between humans or animals is rare
(19, 20). Clinically, infection may vary from asymptomatic
carrier states to disseminated, lethal ones (1, 21).
C. gattii was has been found in a variety of other animals,
wild and domestic (22, 23), showing disparate clinical signs
(6). Being endemic in Australia, the interaction between C.
gattii, koalas (Phascolarctos cinereus) and the eucalyptus trees
they live and feed on has generated special interest. Due to
their habitat and feeding pattern, these animals are at high
risk of contracting C. gattii, resulting in clinical infections
or carrier states where the yeast may colonize the upper
respiratory tract and skin (24). Te prevalence of carrier
animals was found to be directly proportional to the yeast’s
presence in the environment, possibly indicating a unique
infectious cycle that facilitates the persistence of the fungus
in the environment. Antigenemia, sometimes transitory, was
found in carrier animals (25).
A survey was undertaken to study the possible presence of
the zoonotic agent C. gatti in Israel. For this purpose, a zoo
populated with animals originating from Australia, including
koalas fed with eucalyptus leaves was surveyed.
MATERIALS AND METHODS
Environment and animals
Te survey was conducted over the years 2010-2011 in a zoo
(“Gan-garoo”), with an area of about 1.2 hectares, located
in the north of Israel. It was populated by various animals
originating from Australia, some of them, notably diferent
species of kangaroos, roaming freely. Te zoo’s fora’s com-
position has been devised so as to mimic the one the animals
were used to in their original environment and was based
mostly on eucalyptus trees. At the time of the survey there
were 3 koalas in the zoo. A male and a female kept in one
cage and an additional male, separately, in a second cage. Te
koalas had no direct contact with the visitors. Te details of
the koalas are presented in Table 1.
Table 1: Details of the koalas present at the zoo at the time of
the survey.
Name Sex Subspecies Origin Birth At zoo since
Mindy Female Victor Kyabram 1998 2002
Didge Male Cinereus Sydney 2002 2002
Milo Male Victor Ballarat 2006 2006
In the koalas’ cages, dry eucalyptus branches, serving
the animals’ activities were replaced once every few months,
Fresh eucalyptus leaves of various species (E. camaldulensis, E.
viminalis, E. robusta, E.maculata) were grown at a designated
orchard and were kept refrigerated after harvesting for no
more than 2 days before feeding them to the koalas. Te
cages’ foor was mostly covered by concrete and washed daily.
Sampling
To assess an impact of the weather on the presence of C.
gattii, the cages’ environment was sampled twice, once in
January 2010 and then in September 2011. In the frst sam-
pling cycle, 37 sterile swabs were inserted into nooks of the
dry eucalyptus branches and recesses on the cages’ foor. In
the event that resampling would be necessary, the sites were
marked (Figure 1). In addition eucalyptus leave samples from
the animals’ feed were taken. In the second sampling cycle
(September 2011), 25 environmental swabs were taken as
previously described. In addition, fresh eucalyptus leaves from
the trees adjacent to the cages but not in direct contact with
the koalas, were sampled, about 30 on each occasion.
In April 2010, swabs from the coat, nails and nares and
blood samples for cryptococcal antigenemia testing of two
koalas (Mindy and Didge) were taken by the zoo personnel
(Figure 2). Sampling of the third koala, Milo, was not un-
dertaken as he was too aggressive to be manipulated without
anesthesia.
Laboratory examinations
Environmental swabs from the frst cycle were inoculated
onto dopamine agar (26, 27) in situ after being transported
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 49 Cryptococcus gattii Survey in an Israeli Zoo
under refrigeration to the laboratory. Swabs from the second
cycle were inoculated in the laboratory, onto Niger Seed
agar (28). Swabs from the koalas were inoculated in situ di-
rectly after suspension in 5 ml sterile saline onto dopamine
agar. Leaves were transported to the laboratory, where the
leaves were immerged in 400 ml sterile saline and shaken
continuously, at 100 rpm, at room temperature, for 7 days.
Subsequently the saline was distributed to 8x50 ml tubes
and centrifuged at 500 x g for 15 minutes. Te supernatant
was inoculated onto dopamine agar (the frst cycle) or Niger
Seed agar (the second cycle). Te pellet was inoculated onto
the same media undiluted and suspended in 25 ml or 45 ml
saline. All media were incubated at 30°C for 14 days and
examined daily.
Te koalas’ sera were examined for Cryptococcal anti-
gen using a commercial kit (Cryptococcal Antigen Latex
Agglutination System (CALAS®), Meridian Bioscience,
USA).
RESULTS
All samples (environmental and those from the koalas)
were negative for Cryptococcus spp. in general and C. gattii
in particular.
DISCUSSION
Tis unique survey, aimed at assessing the presence of C.
gattii in an Israeli zoo populated with animals originating
from Australia, is the frst one of its kind performed in Israel.
To the best of our knowledge C. gattii has not been isolated
in this country, neither from humans nor from animals or the
environment, and thus the likelihood of fnding it in the zoo
was low. However, being a sanctuary of animals originating
from Australia where the fungus is endemic, the surveyed zoo
was especially suitable to perform this frst survey. Moreover,
considering the zoonotic potential of C. gattii and the large
number of visitors at the zoo, especially children, it was im-
portant to exclude its presence, at least at levels detectable by
the methods employed.
Te fact that we did not fnd C. gattii in our survey may
be the result of its absence in the zoo or due to lack of a
more extensive sampling. Te number of animals present
at the time of the survey was very low and all koalas were
thoroughly examined before being exported from Australia.
Even in the case of a carrier animal being introduced, the
likelihood of the fungus completing its life cycle is low due
to the fact that there is no contact between the koalas and
live eucalyptus trees.
Te isolation of C. gattii from the environment is dif-
Figure 1: Sampling spots on dry eucalyptus branches.
Figure 2: Sampling the koalas.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 Gilad, A. 50
fcult and the rate of positive samples is relatively low: in the
Australian Northern Territories, for instance, it took 2000
samples to isolate the fungus (13). Similarly, in India only
3 samples out of 696 (0.4%) (28) were positive, whereas in
Brazil the corresponding numbers were 1 out of 260 (0.38%)
(29). In the Middle East and Mediterranean Basin several
attempts have been made to isolate C. gattii from the envi-
ronment in Turkey, Jordan, Egypt, Italy and Spain (18, 31,
32, 33). It was isolated in Egypt from 3 samples out of 245
(1.2%), in Italy from 6 out of 255 samples (2.35%) and in
Spain from 14 samples out of 479 tested (2.9%). Interestingly,
in Spain C. gattii was found in association with carob trees
(Ceratonia siliqua) (18, 34), thus indicating that future en-
vironmental surveys in Israel should include additional tree
species as well.
Te above-mentioned reports showed that C. gattii may
be present in our region and that the number of samples
we examined provided a fair chance of detecting the yeast,
were it present. Moreover, the presence of C. gattii in the
environment is usually linked to human and/or animal cases
of infection (18, 29, 32, 33,35 ). Te fact that the fungus was
thus far not isolated in Israel further substantiates the likeli-
hood that its has a very low prevalence or is absent in Israel.
Nevertheless, its presence in the Middle East and
Mediterranean Basin indicates that this environment is
suitable as a niche and consequently it may be introduced
in the future, for example by marine mammals, as has been
previously reported (36). Such animals that are stranded
on Israeli beaches are routinely inspected for infectious
organims, including fungi (37). Moreover, human and vet-
erinary mycological laboratories must be alert to identify C.
gattii should it emerge in Israel.
DECLARATION OF
CONFLICTING INTERESTS
Te authors declare no potential conficts of interest with
respect to the research, authorship, and/or publication of this
article.
REFERENCES
1. Ma, H. and May, R.C.: Virulence in Cryptococcus species. Adv.
Appl. Microbiol. 67: 131-190, 2010.
2. Mitchell, D.H., Sorrell, T.C., Allworth, A.M., Heath, C.H.,
Mcgregor, A.R., Papanaoum, K., Richards, M.J. and Gottlieb, T.:
Cryptococcal disease of the CNS in immunocompetent hosts:
inf uence of cryptococcal variety on clinical manifestations and
outcome. Clin. Infect. Dis. 20: 611-616, 1995.
3. Gattii, F. and Eeckles, R.: An atypical strain of Cryptococcus neo-
formans (San Felice) Vuillemin 1894. I. Description of the disease
and of the strain. Ann. Soc. Belges Med. Trop. Parasitol. Mycol..
50: 689-693, 1970.
4. Kwon-Chung, K.J., Boekhout, T., Fell, J.W., Diaz, M.: Proposal to
conserve the name Cryptococcus gattii against C. hondurianus and
C. bacillisporus (Basidiomycota, Hymenomycetes, Tremellomycetidae).
Taxon. 51: 804-806, 2002.
5. Kwon-Chung, K.J. and Bennett, J.E.: High prevalence of Cryp-
tococcus neoformans var. gattii in tropical and subtropical regions.
Zentralbl. Bakteriol. Mikrobiol. Hyg. A. 257: 213-218, 1984.
6. Harris, J., Lockhart, S. and Chiller, T.: Cryptococcus gattii: where do
we go from here? Med. Mycol. 50: 113-129, 2012.
7. Hagen, F., Ceresini, P.C., Polacheck, I., Ma, H., van Nieuwer-
burgh, F., Gabaldón, T., Kagan, S., Pursall, E.R., Hoogveld, H.L.,
van Iersel, L.J., Klau, G.W., Kelk, S.M., Stougie, L., Bartlett, K.H.,
Voelz, K., Pryszcz, L.P., Castañeda, E., Lazera, M., Meyer, W.,
Deforce, D., Meis, J.F, May, R.C., Klaassen, C.H. and Boekhout,
T.: Ancient dispersal of the human fungal pathogen Cryptococcus
gattii from the Amazon rainforest. PLoS One. 8: e71148, 2013.
8. Mitchell, T.G. and Perfect, J.R.: Cryptococcosis in the era of
AIDS-100 years after the discovery of Cryptococcus neoformans.
Clin. Microbiol. Rev 8: 515-548, 1995.
9. Rosas, A.L. and Casadevall, A.: Melanization afects susceptibility
of Cryptococcus neoformans to heat and cold. FEMS microbiol. Lett.
153: 265-272, 1997.
10. Casadevall, A., Rosas, A.L. and Nosanchuk, J.D.: Melanin and
virulence in Cryptococcus neoformans. Curr. Opin. Microbiol 3:
354-358, 2000.
11. Kwon-Chung, K.J. and Rhodes, J.C.: Encapsulation and melanin
formation as indicators of virulence in Cryptococcus neoformans.
Infect. Immun. 51: 218-223, 1986.
12. Kwon-Chung, K.J., Polacheck, I. and Bennett J.E.: Improved
diagnostic medium for separation of Cryptococcus neoformans var.
neoformans (Serotypes A and D) and Cryptococcus neoformans var.
gattii (Serotypes B and C). J. Clin. Microbiol. 15: 535-537, 1982.
13. Ellis, D.H. and Pfeifer, T.J.: Natural Habitat of Cryptococcus neo-
formans var. gattii. J. Clin. Microbiol. 28: 1642-1644, 1990.
14. Pfeifer, T.J. and Ellis, D.H.; Environmental isolation of Cryptococ-
cus neoformans var. gattii from Eucalyptus tereticornis. J. Me. Vet.
Mycol. 30: 407-408, 1992.
15. Kidd, S.E., Chow, Y., Mak, S., Bach, P.J., Chen, H.M., Hingston,
A.O., Kronstad, J.W. and Bartlett, K.H.: Characterization of envi-
ronmental sources of the human and animal pathogen Cryptococcus
gattii in British Columbia, Canada, and the Pacifc Northwest of
the United States. Appl. Environ. Microbiol. 73: 1433-1443, 2007.
16. Romeo, O., Scordino, F. and Criseo G.: Environmental isolation
of Cryptococcus gattii serotype B, VGI/MATα strains in southern
Italy. Mycopathologia. 171: 423-430, 2011.
17. Mseddi, F., Sellami, A., Jarboui, M.A., Sellami, H., Makni, F. and
Ayadi, A.: First environmental isolations of Cryptococcus neoformans
and Cryptococcus gattii in Tunisia and review of published stud-
ies on environmental isolations in Africa. Mycopathologia. 171:
355-360, 2011.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (3)  September 2015 51 Cryptococcus gattii Survey in an Israeli Zoo
18. Colom, M.F., Hagen, F., Gonzalez, A., Mellado, A., Morera, N.,
Linares, C., García, D.F., Peñataro, J.S., Boekhout, T. and Sánchez,
M.: Ceratonia siliqua (carob) trees as natural habitat and source of
infection by Cryptococcus gattii in the Mediterranean environment.
Med. Mycol. 50: 67-73, 2012.
19. Sorrell, T.C. 2001. Cryptococcus neoformans variety gattii. Medical
Mycology 39: 155-168.
20. Duncan, C., Stephen, C., Lester, S. and Bartlett, K., H.: Follow-
up study of dogs and cats with asymptomatic Cryptococcus gattii
infection or nasal colonization. Med. Mycol. 43: 663-666, 2005.
21. Galanis, E. and Macdougall, L.: Epidemiology of Cryptococcus
gattii , British Columbia, Canada, 1999-2007. Emerg. Infect. Dis.
16: 251-257, 2010.
22. McGill, S., Malik, R., Saul, N., Beetson, S., Secombe, C., Rob-
ertson, I. and Irwin, P.: Cryptococcosis in domestic animals in
Western Australia: a retrospective study from 1995-2006. Med.
Mycol. 47: 625-639, 2009.
23. Centers for Disease Control and Prevention (CDC).: Emergence
of Cryptococcus gattii – Pacifc Northwest, 2004-2010. MMWR
Morb. Mortal. Wkly. Rep. 59: 865-868, 2010.
24. Krockenberger, M.B., Canfeld, P.J. and Malik, R.: Cryptococcus
neoformans in the koala (Phascolarctos cinereus): colonization by
C. n. var. gattii and investigation of environmental sources. Med.
Mycol. 40: 236-272, 2002.
25. Krockenberger, M.B., Canfeld, P.J., Barnes, J., Vogelnest, L., Con-
nolly, J., Ley, C. and Malik, R.: Cryptococcus neoformans var. gattii in
the koala (Phascolarctos cinereus): serological evidence for subclinical
cryptococcosis. Med. Mycol 40: 273-282, 2002.
26. Polacheck, I., Hearing, V.J., and Kwon-Chung, K.J.: Biochemi-
cal studies of phenoloxidase and utilization of catecholamines in
Cryptococcus neoformans. J. Bacteriol. 150 :1212-1220, 1982.
27. Staib, F.: Cryptococcus neoformans and Guizotia abyssinica (syn. G.
oleifera D.C.) (Farbreaktion für C. neoformans). Z. Hyg. Infektion-
skr. 148: 466-475, 1962.
28. Chakrabarti, A., Jatana, M., Kumar, P., Chatha, L., Kaushal, A.
and Padhye A.A.: Isolation of Cryptococcus neoformans var. gat-
tii from Eucalyptus camaldulensis in India. J. Clin. Microbiol. 35:
3340-3342. 1997.
29. Fortes, S.T., Lazera, M.S., Nishikawa, M.M., Macedo, R.C.L. and
Wanke, B.: First isolation of Cryptococcus neoformans var. gattii from
a native jungle tree in the Brazilian Amazon rainforest. Mycoses.
44: 137-140, 2001.
30. Ergin, C., Ilkit, M. and Kaftanog Lu, O.: Detection of Cryptococ-
cus neoformans var grubii in honeybee (Apis mellifera) colonies.
Mycoses. 47: 431-434, 2004.
31. Mohammad, A., Hamasha, S., Yildiran, S.T., Gonlum, A., Saracli,
M.A. and Doganci, L.: Cryptococcus neoformans Varieties from ma-
terial under the canopies of eucalyptus trees and pigeon dropping
samples from four major cities in Jordan. Mycopathologia 158:
195-199, 2004.
32. Romeo, O., Scordino, F. and Criseo, G.: Environmental isolation of
Cryptococcus gattii serotype B, VGI/MAT alpha strains in southern
Italy. Mycopathologia. 171: 423-430, 2011.
33. Mahmoud, Y.A.G.: First Environmental Isolation of Cryptococ-
cus neoformans var. neoformans and var. gattii from the Gharbia
Governorate, Egypt. Mycopathologia. 148: 83-86, 1999.
34. Lazera, M.S., Cavalcanti, M.A.S., Trilles, L., Nishikawa, M.M.;
Wanke, B. 1998. Cryptococcus neoformans var. gattii: evidence for a
natural habitat related to decaying wood in a pottery tree hollow.
Medical Mycology. 36: 119-122.
35. Bartlett, K.H., Kidd, S.E. and Kronstad, J.W.: Te Emergence of
Cryptococcus gattii in British Columbia and the Pacifc Northwest.
Curr. Infect. Dis. Rep. 10: 58-65, 2008.
36. Rotstein, D.S., West, K., Levine, G., Lockhart, S.R., Raverty, S.,
Morshed, M.G. and Rowles, T.:Cryptococcus gattii vgi in a spinner
dolphin (Stenella longirostris) from Hawaii. J. Zoo Wildl. Med.
41: 181-183, 2010.
37. Elad, D., Morick, D., David, D., Scheinin, A., Yamin, G., Blum,
S. and Gofman, O.: Pulmonary fungal infection caused by Ne-
oscytalidium dimidiatum in a Risso’s dolphin (Grampus griseus).
Med. Mycol. 49: 424-426, 2011.
Research Articles