Batrachochytrium salamandrivorans is the predominant chytrid fungus in Vietnamese salamanders.

The amphibian chytrid fungi, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), pose a major threat to amphibian biodiversity. Recent evidence suggests Southeast Asia as a potential cradle for both fungi, which likely resulted in widespread host-pathogen co-existence. We sampled 583 salamanders from 8 species across Vietnam in 55 locations for Bsal and Bd, determined scaled mass index as a proxy for fitness and collected environmental data. Bsal was found within 14 of the 55 habitats (2 of which it was detected in 2013), in 5 salamandrid species, with a prevalence of 2.92%. The globalized pandemic lineage of Bd was found within one pond on one species with a prevalence of 0.69%. Combined with a complete lack of correlation between infection and individual body condition and absence of indication of associated disease, this suggests low level pathogen endemism and Bsal and Bd co-existence with Vietnamese salamandrid populations. Bsal was more widespread than Bd, and occurs at temperatures higher than tolerated by the type strain, suggesting a wider thermal niche than currently known. Therefore, this study provides support for the hypothesis that these chytrid fungi may be endemic to Asia and that species within this region may act as a disease reservoir.

Globalization has led to the emergence of infectious diseases that threaten biodiversity and contribute to the current 6th mass extinction, a recent example being amphibian chytrid fungi: Batrachochytrium dendrobatidis (Bd), which has caused declines and extinctions within Anura, Urodela and Gymnophiona throughout many parts of the world1234567 and B. salamandrivorans (Bsal), which is causing massive declines in salamander populations in Europe89. Bsal appears to be restricted to urodelan hosts, causing erosive skin lesions with subsequent death in diseased individuals, and the type strain has lower thermal growth tolerance limits than its sister species Bd8. A recent screening for Bsal revealed the presence of the fungus during disease outbreaks in wild salamander populations within the Netherlands, Germany and Belgium9, and in captive animals from the UK and Germany1011. The presence of Bsal in several urodelan species in Thailand, Vietnam and Japan, coupled with clinical infection trials indicating three Asian salamander species were capable of persisting with the infection or clearing the infection completely, gave rise to the hypothesis that Bsal is endemic to eastern Asia and the salamander species inhabiting this area may act as a reservoir for the fungus12. This hypothesis has been proposed for Bd within Asia as well, due to the fact that a high diversity of lineages is found in eastern Asia1314 and, throughout this region Bd has a low prevalence with no signs of disease outbreak in many amphibian species151617181920. A reservoir, in relation to disease, has been defined as “a passive host or carrier that harbors pathogenic organisms without injury to itself and serves as a source from which other individuals can be infected”21. In regards to Bsal and Bd, we thus predict suitable reservoirs to sustain the fungus in the absence of mortality and disease events that negatively affect the host populations. Here we conducted the first large scale screening for Bsal, along with Bd, in Southeast Asia to test this hypothesis. In order to do this, we sampled 583 salamanders belonging to 8 urodelan species of the salamandrid genera Paramesotriton and Tylototriton across 55 localities (ponds or streams) in Vietnam to test for the presence of Bsal and Bd. We then looked into associations of pathogen prevalence and load with potentially influential environmental factors and the presence of associated disease.

Results

Across 11 provinces located in northern Vietnam, 44 ponds and 11 streams were sampled and 583 individuals from 8 species, belonging to the genera Paramesotriton and Tylototriton, were sampled for the presence of Bsal and Bd and their body condition determined (Table 1). A total of 17 individuals belonging to 5 species (T. vietnamensis, T. ziegleri, T. asperrimus, P. deloustali and P. sp.) tested positive for B. salamandrivorans resulting in an overall prevalence of 2.92% (95% CI: 1.80%, 4.65%). Two of the three species which did not contain positive individuals had small sample sizes (T. anguliceps (8), T. sp. (17) and P. guanxiensis (54)) and as the prevalence is low within this area more samples need to be gathered to definitively determine if these populations contain infected individuals. The average prevalence of the 14 positive sites was 17.56% (SD ± 25.27, 95% CI: 3.06%, 32.24%). Four individuals of T. ziegleri, in one pond, tested positive for Bd resulting in an overall prevalence of 0.69% (95% CI: 0.20%, 1.83%) and a prevalence of 30.77% (95% CI: 12.35%, 57.96%) within the positive pond. The Bd samples amplified with the global pandemic lineage (BdGPL) probes indicating a BdGPL-like lineage.

Table 1

Bd and Bsal prevalence per province/district, indicating the number of ponds or streams sampled, the species found, the total number of animals sampled, the number of Bd and Bsal positive individuals and the corresponding prevalences.

Province, District	Sampling locations	Species	Water temperature range (°C)	Animals swabbed	Bsal positive	Bd positive	Bsal prevalence	Bd prevalence
Bac Giang	11 Ponds	T. vietnamensis	22.7–24.8	184	3	0	1.6%	0.0%
Qiang Ninh	2 Ponds	T. vietnamensis	23.8–24.9	11	0	0	0.0%	0.0%
Lang Son	1 Pond	T. vietnamensis	31.9	12	0	0	0.0%	0.0%
Cao Bang, Bao Lac	4 Ponds	T. ziegleri	16.07–22.53	30	2	4	6.7%	13.3%
Cao Bang,	4 Streams	P. guanxiensis	20.6–22.97	54	0	0	0.0%	0.0%
Nguyen Binh	1 Pond	T. ziegleri	19.57	8	0	0	0.0%	0.0%
Ha Giang, Bac Me	2 Ponds	T. ziegleri	18.7–19.7	13	0	0	0.0%	0.0%
Ha Giang, Quan Ba	9 Ponds	T. ziegleri	16.37–20.73	33	2	0	6.1%	0.0%
Ha Giang, Bac Quang	2 Ponds	T. ziegleri	21.6–23.37	26	0	0	0.0%	0.0%
Lai Chau	4 Ponds	T. sp.	18.53–23.9	17	0	0	0.0%	0.0%
Son la	1	T. anguliceps	20.6	8	0	0	0.0%	0.0%
Hoa Binh	7 Ponds	T. asperrimus	20.07–26.43	46	3	0	6.5%	0.0%
Bac Kan	2 Streams	P. deloustali	21.2–22.47	62	1	0	1.6%	0.0%
Lao Cai	4 Streams	P. sp.	20.5–22.07	61	4	0	6.6%	0.0%
Tam Dao	1 Stream	P. deloustali	18.8	18	2	0	11.1%	0.0%
Total	55	8 species	16.37–31.9	583	17	4	2.9%	0.7%

Bsal infection prevalence did not differ between provinces or species (Fisher’s exact tests, P = 0.34 and P = 0.25, respectively). There were no correlations between infection intensity of Bsal or Bd and scaled mass index (SMI) for any of the eight species (Spearman’s correlations < ±0.18). Elevation, province, species, scaled mass index, water temperature and ambient temperature were not significant predictors of infection intensity for Bsal (Generalized linear model, P > 0.05).

Discussion

Our study provides the first large scale screening for Batrachochytrium salamandrivorans (Bsal) within Southeast Asia, specifically Vietnam. We found that Bsal was much more widespread throughout salamander populations in Vietnam than its sister species B. dendrobatidis (Bd), occurring in 14 of the 55 locations compared to the one pond in which Bd was detected (Fig. 1), corroborating the predilection of Bsal for urodelan hosts12 and providing further evidence for a Bd prevalence that is low throughout Asia151617181922. We did not find any signs of Bsal associated disease such as decreased body condition, lesions, or death in the infected populations. The overall low prevalence for both Bsal (2.92%) and Bd (0.69%), despite presence of Bsal in Vietnamese salamander populations at least since 201312, combined with the absence of disease signs indicates endemism of both species of chytrid fungi within Vietnamese salamander populations, adds support to the hypothesis that South East Asian salamanders constitute a Bsal reservoir. Similarly low prevalence but widespread occurrence has been demonstrated for Bd in regions where Bd is considered endemic in the absence of epidemic disease132324. If compensated, co-existence does not exclude an actual cost of Bsal on the Vietnamese salamander populations, as has been demonstrated for Bd2325, especially given the potential of both pathogens to cause juvenile mortality under experimental conditions1222.

Figure 1

Map of sampling locations within Northern Vietnam.

The size of the pie charts corresponds to the sample size ranging from 1 to 44 individuals. The pie chart indicates the prevalence of Bsal (Red) or Bd (Blue) within that population. This map was generated in ArcMap 10.1 (http://www.esri.com/software/arcgis/arcgis-for-desktop).

Through the use of lineage specific qPCR, we found that the Bd we detected amplified as global pandemic lineage (BdGPL) indicating it could be the true BdGPL, endemic or non-endemic BdGPL-like, or a hybrid of an endemic and BdGPL cross26. BdGPL is associated with all of the known epizootic events and has experimentally been shown to be more virulent than other lineages27. As Bd seems to occur in an endemic state within Asia and our study site of Vietnam, it is likely an endemic BdGPL-like lineage, further testing should be done to determine the strain as well as the virulence of the Bd within Vietnam.

The thermal optimum for the type strain of Bsal is 15–20 °C28, whereas the majority of infected individuals we found in Vietnam were in ponds or streams with water temperatures between 20–25 °C, even reaching 26.43 °C in one positive location (Fig. 2). This might have serious implications as differential thermal preferences of strains could significantly expand the invasive niche if the fungus is vectored into a naive region (Yap et al.29).

Figure 2

Prevalence of Bd and Bsal positive salamanders at each positive location (pond or stream), and its corresponding water temperature.

*Only one individual was sampled at this location.

In conclusion, due to the low prevalence of Bsal and BdGPL and lack of correlation between any of the environmental or physical parameters, we provide evidence supporting the hypothesis that these chytrid fungi may be endemic to Asia and that species within this region may act as a disease reservoir for naive regions. We found that Bsal is much more widespread within northern Vietnam, occurring at temperatures higher than those tolerated by the fungus’ type strain, which suggests a wider thermal niche than currently known. Further screening should be conducted to determine the range of Bsal and which species carry the fungus. These results reinforce the notion that there should be strict biosecurity measures in place, in order to prevent the further spread of Bsal and avoid a global impact similar to that of Bd.

Methods

Study Species

Eight salamander species were sampled for the presence of B. salamandrivorans: Tylototriton vietnamensis (207 individuals), T. ziegleri (110 indv.), T. sp. (17 indv.), T. anguliceps (8 indv.), T. asperrimus (46 indv.), Paramesotriton deloustali (80 indv.), P. guangxiensis (54 indv.) and P. sp. (61 indv.). Nets were utilized to capture salamanders within the stream or pond. Each salamander was weighed, using a digital scale (MS-Series,G&G GmbH), and measured for snout to vent and tail length, using a 0–150 mm digital caliper (Fixpoint). A scaled mass index was calculated following the method developed by Peig and Green30.

Data collection

Since the Bsal type strain has a low tolerance for high environmental temperatures, these were predicted to drive Bsal infection. Therefore, at each pond or stream water temperature and ambient temperature were measured using an HHC201 thermocouple thermometer, with a hermetically sealed thermocouple (OMEGA®). Prevalence and infection loads of Bsal and Bd in the captured amphibians was determined by collecting non-invasive skin swabs29. All methods were carried out in accordance with Vietnamese guidelines and regulations. Experimental procedures were approved and research was carried out under permits obtained for each province of Vietnam through the corresponding provincial government and the Ministry of Agriculture and Rural Development (MARD) (Lang son: 224/SNgV-LS, Bac Giang: 866/UBND-XD, Quang Ninh: 1781/UBND-MT, Cao Bang: 998/UBND-NC, Lai Chau: 663/UBND-NC, Hoa Binh: 1444/VPUBND-TH, Ha Giang: 107/SNgV-NVLS, Bac Kan: 1293/UBND-NV, Vinh Phuc: 104/BTTNVN, Lao Cai: 1887/UBND-NC and Son La: 304/BTTNVN).

Sample processing

DNA was extracted from the swabs using 100 μl of Prepman Ultra DNA extraction buffer. Extracted DNA samples were diluted 1/10 with HPLC water to reduce PCR inhibition31 and then stored at −20 °C until processing. Samples were processed using the B. salamandrivorans and B. dendrobatidis specific duplex real-time PCR procedures described by Blooi et al.32 on CFX96 real-time system (Bio-Rad Laboratories, Hercules, CA). Real-time PCR results (genomic equivalents (GE) of B. salamandrivorans and B. dendrobatidis zoospores) were corrected for the applied dilution factor. Lineage specific qPCR was conducted utilizing Taqman MGB probes that are able to discriminate single nucleotide polymorphisms in the Bd mitochondrial genome which are diagnostic for the globalized pandemic lineage (BdGPL), following the protocol of Bletz et al.26.

Data analysis

The modified Wald method used to calculate the 95% CI for proportion of infected individuals. Fisher exact tests were used to compare prevalence between species and provinces. We examined how GE load was affected by species, SMI, SVL, water temperature and elevation using generalized linear models (Gaussian functions) and spearman correlations in R (version 3.2.3), due to the non-normal distribution of the data.

Additional Information

How to cite this article: Laking, A. et al. Batrachochytrium salamandrivorans is the predominant chytrid fungus in Vietnamese salamanders. Sci. Rep. 7, 44443; doi: 10.1038/srep44443 (2017).

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.