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Prevalence of Chytrid Fungus (Batrachochytrium
dendrobatidis) in Wild Amphibians at Nam Nao
National Park, Thailand
Nuntita Ruksachat1, Kanit Chukanhom1*, Fanan Suksawat1, Michael G. Levy2
1
Faculty of Veterinary Medicine, KhonKaen University, Muang, KhonKaen. Thailand, 40002
2
College of Veterinary Medicine, North Carolina State University, USA
*
Corresponding author Email: [email protected]
Abstract
Objective To study the prevalence of Batrachochytriumdendrobatidisin wild amphibians at Nam Nao
National park, Thailand
Materials and Methods Theprevalenct study of chytrid fungus (Batrachochytrimdendrobatidis) in
wild amphibians was employed in 2013. The sampling site was in the buffer zone used by humans,
domestic animals and wildlife at Nam Nao National Park, Thailand. One hundred and sixty-one wild
amphibians of 3 families; Megophryidae, Microhylidae and Ranidae were collected by swabbing from
amphibian skin. Real time Taqman PCR was used to detect Chytrid fungal DNA.
Results The real time Taqman PCR results of one hundred sixty-one samples were all negative for
chytrid fungus.
Conclusion The study is useful and necessary for surveillance of chytrid fungus, the emerging
infectious disease in wild amphibians.
Keywords: Chytrid fungus, Batrachochytrimdendrobatidis, Amphibian, Nam Nao National Park
Introduction
The globalization has affected on the biological of amphibians; for example, biological condition,
metabolism, fertilization, habitat, and food sources. In addition, the climate changes are likely to affect the
transmission of the emerging infectious diseases causing the amphibian population declines for all regions
of the world [1]. Chytridiomycosis is the important infectious disease in amphibian caused by Chytrid
fungus (Batrachochytriumdendrobatidis) [2]. The fungus originated in Africa and then found to be causing
problems in Australia, North America, South America, Europe and Asia. The African clawed frogs (Xenopuslaevis) have been exported from Africa since 1930, which could be the beginning and spreading of the
chytrid fungi to the other regions of the world [3]. The first detection of Chytrid fungi in Asia was from the
native and imported amphibians in Japan, 2006[4]. In Southeast Asia [5] reported the chytrid fungal
detection from native amphibians in the Peninsula Malaysia including 6 families and 47 species, the results
showed 7.87% (10/127) of native amphibians were infected. In China, [6] detected chytrid fungus from
wild and market amphibian in 10 provinces of China, the prevalence of fungal detection were 13.5%
(89/659) in wild amphibians and 7.6% (157/2,075) in market amphibians. In Thailand, there was
preliminary study on Chytrid fungus infection in Thailand in 60 years kept specimens from amphibians
kept in museums. Histopathologic diagnosis was employed for 123 samples from 23 species of
amphibians. However, the result was all negative [7]. During July 2009 to August 2010 [8] had done the
molecular studyforchytrid fungus in quarantine amphibians and in wild amphibian at KhaoKheow Open
Zoo, the resulting prevalence were 11.9% (20/168) in quarantined amphibians, but 492 samples of wild
amphibians were negative.
Chytrid fungal infection can be diagnosed by histopathological examination from the carcasses of
amphibians but not from the living amphibians. Therefore, it is not applicable for surveillance and
monitoring. In this study, real-Time Taqman PCR method was used since it can detect the Chytrid fungal
DNA at the first phase of infection in the amphibians infected with only one zoospore [9]. In Thailand,
many amphibianspecies have been increasingly imported as food and also raised as exotic pets. This may
lead to the dissemination of chytrid fungal infection in native species either raised in farms or in wild
amphibians. Suitable climate and efficient factors in national parks in Thailand may facilitate the chytrid
fungal growth. Therefore, the survey of chytrid fungal prevalence of in natural areas is very important for
disease surveillance in wild amphibians.
Materials and Methods
Sampling site: It was the Nam Nao National Park in Phetchabun Province located in the lower Northern
part of Thailand in contact the Northeastern part. The Northern and Northeastern parts of Thailand in this
area are connected by the route number 12. Before sampling, 8 areas of Nam Nao National Park had been
observed including Haew-Sai Waterfalls, HuaiSanamsai Point, Sammakhao Unit, Wildlife Observation
Tower, Phrom Song Unit, PhromLaeng Unit, Fai Nao Unit and Nam Nao National Park Office. Nam Nao
National Park Office area was chosen as the sampling site. This area has been exploited by humans and
wildlife. There are the natural trail, the stream flowing through and the campsite for tourists. In addition,
the height of this area is of 845 meters above sea level and the transportation is convenient that it is only
500 meters away from the route number 12. So it is suitable for the sample collection.
The sample collection: One hundred and sixty-one wild amphibians from 4 families including
Megophryidae, Microhylidae, RanidaeandRhacophoridae were collected at Nam Noa National Park
(Table1). The sampling was variable, not limited by age, gender, or weight. Hand nets were used to
capture the amphibians in the sampling site with gentle catching, and non-powder gloves were used and
had been changed every time before the next capturing begun. In addition, the swab test was done Hyatt
methods as previously described [10]. According to Hyatt methods, cotton swabs were applied to 7 areas
of amphibian skin including stomach, abdomen, both sides of hind legs on and toes by smearing for 5
times on each area. The first point was two sides of stomach followed by abdomen, the inside area of the
hind legs and toes, respectively. Each amphibian was applied with a cotton swab soaked in the tube which
contained absolute ethyl alcohol, and the tube was closed firmly for sending to laboratory, to be detected
by real time Taqman. Subsequently, the examination and the data were recorded in the amphibian
sampling forms. The photos were also taken, and the identification of amphibian species was done by
following the manual of amphibians in Thailand [11].
Real time Taqman PCR
DNA extraction: DNA was extracted from swabs by PrepMan Ultra. Fifty μl of PrepMan Ultra was added
to each sample along with 30-40 mg of Ziriconium/silica beads 0.5 mm diameter. The sample was
homogenized for 45 sec in a bead-beater. After brief centrifugation (30 sec at 13000×g in microfuge) to
settle all materials to the bottom of the tube, homogenization and centrifugation was repeated. The
homogenized sample was immersed in a boiling water bath for 10 min, cooled for 2 min, then centrifuged
at 13000×g for 3 min in a microfuge tube; 20 μl of supernatant was recovered and stored -80 ◦C.
TaqMan assay: The mater mix is prepared with primer 1 (Forward primer), primer 2 (Reverse primer) and
the probe. Add 5 μl DNA at 1/10 dilution was included in the master mix of 1 well of the 3 triplicates.
Detection system was set as the following programme: 50 ◦C for 2 minutes 1 cycle; 95◦C for 10 minutes
1cycle; 95 ◦C for 15 seconds, 60 ◦C for 1 minute; 45 cycles.
Interpretation of data: Positive results are the presence of specific amplicons, indicated by a
characteristic amplification curve similar to the positive control with a CT value less than or equal to 39 (in
all three wells). Negative results are defined by the absence of specific amplicons, indicated by no
characteristic amplification curve and having a CT value greater than 41(in all three wells). Inconclusive
result was the presence of specific amplification curves similar to the positive control but with a CT value
between 39 and 41 or a low number of zoospore equivalents in only one or two wells. Such results
necessitate a repeat of the assay.
Results
All samples were negative for chytrid fungus by real time Taqman PCR (Table 1 and Table 2).
Discussion
Chytridiomycosis has affected the amphibian population declines around the world due to the difficulty to
determine and to cure. The diagnosis can be done mostly in late-stages of the diseases. The molecular
biology method (real time Taqman PCR method) has been developed to diagnose the beginning stage of
infection by only one zoospore that the examination could be done effectively and expeditiously. For this
reason, the disease treatment, prevention and surveillance in amphibians were implemented effectively
[12]. The diagnostic method which suggested by OIE applied in this research study was real-time Taqman
PCR method in order to diagnose chytrid fungal infection, effectively. The growth of Chytrid fungus is
well developed in low temperature about 17-25 degree Celsius and at pH 6-7 [12] which is closely to the
amphibian study in the natural areas of Nam Nao National Park, Petchabun Province. The height above sea
level of the National Park office area is 845 meters, and the average annual temperature is 25 degree
Celsius. According to all negative results from 161 samples by real time TaqmanPCR,this is correlated
with [7] studied 123 samples of amphibians which have been kept in museums in Thailand for more than
60 years. There was no chytrid fungal infection in all the samples either. [8] was no chytrid fungal
infection in wild amphibians. The result also suggested that there was no chytrid fungal infection in natural
areas. In addition, from the result in Chiang Dao wildlife reservation area, it was also found that there was
no chytrid fungus infection in 661 samples of wild amphibians. Furthermore, the Department of national
parks, wildlife and plant conservation also survey chytrids in PhuLuang wildlife reservation area and the
area of Halabala wildlife reservation area. From the results, chythid fungus in wild amphibians was not
found. Moreover, according to PimchanokSuwanthada et al. (the data has not been published yet) detected
chytrid fungus in captive frog which raised in farms in KhonKaen province by real time Taqman PCR, and
the result also showed that there was no chytrid fungal infection. According to the information discussed
above, it can be concluded that there is no incidence of chytrid fungus infection in both wild amphibians
and captive amphibians. Thus, it could be assumed that climate change is not the factor affecting the
incidence of the disease in amphibians. However, [8] found 11.9% (20/ 168) of imported living
amphibians kept in the quarantine area were positive by PCR method, and 2.0% (6/ 50) of dead
amphibians were positive to chytrid fungal infection by histopathological examination. According to [12],
it was discovered that the chytrid fungus could be examined in some amphibian species which lived in
forests without clinical signs or death. However, it cannot be obviously explained how they become
disease carrier in long period of time. This was correlated with [5] found chytrid fungus in native
amphibians without clinical sign. It could be suggested that chytrid fungus was endemic pathogen in this
region. The surveillance of chytrid fungus in wild amphibians should be implemented especially during
breeding season which chytrid fungus infection causing the highest death rate of crawfish frog were found.
However, the death rate was gradually decline in summer [14] .
Acknowledgements
We would like to thank Department of national parks, wildlife and plant conservation, all staffs of Nam
Nao National Park for help us provide facilitation, assist and sampling collection of wild amphibians in
this research.
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