A historical overview of Batrachochytrium dendrobatidis infection from specimens at the National Zoological Collection Suriname

The amphibian skin disease chytridiomycosis, caused by the pathogenetic fungus Batrachochytrium dendrobatidis (Bd) has become one of the major contributors to global amphibian population declines and extinctions. This fungus has spread globally and has caused mortalities in nearly every continent. In South America, Suriname, Guyana and Paraguay are among the remaining three countries where Bd has not been detected to date. To complete the assessment of the possible presence of Bd in Suriname, 205 specimens from the Zoological Collection of Suriname, compromising 6 frog families and 15 genera were sampled for chytrid fungus. No specimens were found to be infected by this fungus and as such the outcome strengthens the previous result of field sampling that there is no support that Bd has spread to Suriname.


Introduction
Amphibians are generally regarded as good indicators for habitat or ecosystem health due to their dependency on both terrestrial and aquatic habitats for their life history [1]. With 40% of global amphibian species currently at risk of declines or extinctions [https://www.iucnamphibians.org/; accessed 8 June 2020], concerns for amphibian threats are increasing, with human activities at the forefront of risks [2]. The decline in amphibian populations is not something occurring locally but was demonstrated by [3,4] to be on a more global scale than once thought. In addition to rapid habitat change from anthropogenic factors, a top contributor to amphibian losses includes the human-mediated introduction of invasive alien species throughout the world including emerging infectious diseases such as chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) [5]. Originating in South Korea [6] this disease-causing skin fungal pathogen has taken proportions of a global epidemic with disease affecting species on all continents with amphibians. In South America, Bd has been detected in amphibian communities within all the countries apart from Suriname, Guyana and Paraguay (Bd-maps. net, accessed December 2019). In Suriname, tests for Bd presence were initially conducted by sampling Atelopus hoogmoedi at the Brownsberg Nature Park [7]. In a second study, Pipa pipa frogs collected from Suriname and deposited in museums abroad were also been tested for Bd

PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0239220 October 2, 2020 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 by Soto-Azat et al. [8]; this research was done to verify the possibility of African Xenopus spp. as a source of Bd, and also tested other frog species in the family Pipidae from Guyana, Venezuela, French Guiana, and Africa [8]. No Pipidae specimens of these South American countries collected between 1844 and 1994 tested positive for Bd [8]. Although Bd has not been detected in Suriname to date, it has been detected in many places in French Guiana [9] with one location being approximately 7 km away from the border of Suriname where frog infection rates ranged between 1-5% [9]. Though Bd has just recently gathered a lot of attention by the international community, the existence of this fungus can be found in museum specimens dating back as far as the 1970's [10]. Knowledge of verified gaps in Bd occurrence is of paramount importance, as enhanced biosecurity may forestall inadvertent human-mediated transmission, aiding amphibian conservation. Although initial investigations of selected taxa including one field study [7] and one museum study [8] have not supported Bd presence in Suriname, additional sampling is warranted for broader spatial and taxonomic coverage. Herein, we report findings of a broader geographic and taxonomic study in Suriname, conducted by checking museum specimens housed at the National Zoological Collection Suriname (NZCS) for the presence of Bd. The advantages of testing museum specimens for the presence of Bd includes a provision of an historical overview of the time that the fungus is present in a particular species and in a particular location. Despite the fact that most museum specimens are formalin fixed, successful studies have still been able to check for the presence of Bd [11][12][13][14]. The few museum specimens from Suriname tested by Soto-Azat et al. [8] in a single family support the efficacy to test more species using this approach. Additionally, it allowed us to examine the Bd presence across widespread locations that could be logistically constrained today due to budgetary and time limitations for sampling species in the wild. The research presented herein gives an overview of specimens tested for Bd stored at the NZCS in 6 families and 15 genera compromising 205 specimens.

Materials and methods
The NZCS houses approximately 2500 specimens of several amphibian species and is the only zoological institute in Suriname equipped to store specimens and disseminate knowledge on the diverse amphibian taxa present in Suriname. Species that were selected from the database for Bd testing were in the following families; Bufonidae, Dendrobatidae, Aromobatidae, Leptodactylidae, Ranidae and Pipidae. These families were selected due to past studies showing their members can be vulnerable to Bd infection (e.g., Bufonidae [15,16] Dendrobatidae [17,18] and Aromobatidae [9]) or have associations with water resources for parts of their life cycle (Leptodactylidae [19,20] Ranidae [21] and Pipidae [8]), hence could encounter flagellated zoospores of the aquatic fungal pathogen Bd if it were present [22]. Temporal and spatial considerations also affected selection for sampling (Fig 1 and Table 1). Of the species that were selected in the families mentioned above some of the oldest specimens were collected in 1905 and consisted of 2 Leptodactylus specimens. Some specimens were chosen from just a few years back (2014 to 2016) targeting the location where collected. Waypoints on the map were selected based on their singularity of depicting a new geographic area. Although it might not be evident on the map for some areas, a selection for depicted waypoints was done using a minimum distance of 3 km from the nearest waypoint. The main purpose for this was to present all the different geographic areas sampled instead of cluttering the map with waypoints of every specimen. All species mentioned in the table are sorted starting with the oldest specimens collected on top.

Specimen sampling
Amphibian specimens used for this study were selected by querying the NZCS database.
To swab for Bd we followed the same procedure as Rodriguez et al. [23] where each individual frog was first rinsed with clean uncontaminated 70% ethanol and allowed to dry on a clean sheet of tissue paper. We then swabbed each specimen using Medical Wire swabs as per the standard swabbing methods described by in Hyatt et al. [24] and Cheng et al. [13]. We swabbed and pooled up to eight specimens to minimize the cost of lab analyses keeping swabs of the same species in one vial as much as possible. The vial number on each vial was linked to the NZCS no of the specimens that were swabbed with that swab. Pooling swabs or swabbing multiple specimens has become an accepted method of verifying Bd in presence absence studies [24,25]. After swabbing the specimens, swabs were cut to desired length and placed in sterile 1.5ml screw top centrifuge tubes with O rings. Swabs were allowed to dry completely before the tubes were closed and stored in a refrigerator with a temperature of 4˚C until shipped to be processed.

Sample preparation
All swabs were processed using the following procedure. One ml of 70% ethanol diluted to 70% final concentration with deionized H 2 O) was added in the lab to each sample tube. After vigorous mixing the liquid to dislodge any zoospores/skin tissue from the swabs, the entire volume from each sample was transferred in two different microfuge tubes. The tubes were spun in a microcentrifuge at~16,000 x G for 3 minutes. Next, the supernatant was drawn off and discarded since B.d zoospores are negatively buoyant in 70% ethanol and therefore will pellet upon centrifugation. Lysis buffer (180 uL of Qiagen ATL buffer + 20 uL Qiagen Proteinase K) was added to the tubes and any pellet present was resuspended by vortexing. Ten μg of carrier DNA was added to the lysis buffer. Total DNA was extracted from all samples using a silica membrane spin-column DNA purification procedure (Qiagene DNeasy, blood and tissue kit). qPCR assay. The sample DNAs were assayed for the presence of the Batrachochytrium dendrobatidis ribosomal RNA Intervening Transcribed Spacer (ITS1) region by 45 cycle PCR amplification using a qPCR assay developed at Pisces and a Stratagene MX4000 real-time PCR instrument. Primer and probe base sequence are as follow: Primer Forward: 5' TGGATGGGAGTTTTATTGATGTGTA Reverse: 5' TCGTGACATATGGCACACTGTATT and the probe 5'-FAM -TGG AAT GAC CCA TTG TT-BHQ1 plus. The reaction master mix contains ROX as passive reference dye for normalizing variations in individual reaction total volumes, and a VIC-labeled internal positive control (IPC) (Life Technologies TaqMan Positive Control, catalog #4308323) to detect PCR inhibition. The detection sensitivity of this assay is three target sequence molecules (approximately 0.02 zoospore equivalents). Each PCR run included the following controls: Positive DNA: DNA prepared from a plasmid constructed at Pisces containing the B. dendrobatidis ribosomal RNA Intervening Transcribed Spacer (ITS1) region. Serial ten-fold dilutions of this plasmid DNA from 2.9 x 10 6 to 2.9 x 10 0 molecules per reaction were used to generate the standard curve. Serial dilutions controls were done since these spanned the Ct values observed for all positive samples observed while using this assay.
Control. Water in place of template DNA. This reaction remains uncapped during addition of sample DNA to the test reactions, and serves as a control to detect contaminating DNA in the PCR reagents or carryover of positive DNA during reaction set-up.

Results
Using the 0.02 threshold for any sample that gave clearly observable, exponential (log-linear) fluorescent signal increase we found no museum specimens to be positive for the chytrid fungus. However, any weak or questionably positive samples were retested in a second independent qPCR run. Only samples which retested positive in this second run were scored as positive. In the study herein all the samples were tested negative. The specimens used for swabbing also showed no aberrant skin condition to indicate a possible chytrid infection.

Discussion
The negative results for the museum specimens tested in this study supports that Bd has not infected any frogs in Suriname. Many South American countries have had specimens tested positive for this fungus including countries bordering Suriname. The data presented herein and the specimens sampled in the field by the author gives a first country wide overview of this chytrid fungus sampling and as such serves as a baseline study for future Bd presence/absence research. The inclusion of museum specimens in this chytrid sampling overview for Suriname, apart from the historical point of view enabled us also to sample specimens that were not sampled in the field survey due to a lack of funds to be able to visit those places. The research presented herein of the 15 different species sampled added to the number of species sampled during the field survey gives us a total of 52 frogs species sampled for Suriname and a total of 555 specimens. One particular reason for the Bd free status of Suriname could probably be due to the relatively high mean temperature of Suriname which varies between 26.2˚C for the coldest month to 28.2˚C for the warmest time period [26] thus making conditions not favorable for Bd to get established [27,28]. However, the inhibition by temperature should not be ruled out completely as lowland sites have also been reservoirs of species carrying the pathogen [29,30] so chances are that in due time and with few existing precautions chytridiomycosis caused by Bd might eventually set foot in Suriname. The wide eastern and western river border of Suriname could be yet another reason that this chytrid fungus has not yet reached here. Evidence that Bd infection spreading into different countries seems to be mostly coupled with human activities [31] and that possibly infected animals or substrate with the zoospores could make it into Suriname. Although this fungus has spread due to the pet trade in many countries [32] it is highly unlikely that the amphibian pet trade in Suriname will develop to such an extent that frogs will be imported from other countries to keep as pets. In the same vein it is not envisaged that frogs will be imported from other countries to be consumed or bred here to serve as part of the Surinamese diet. Ornamental fish however are imported in Suriname and have shown to be carriers of this chytrid fungus [33].
Regular surveys sampling for Bd in Suriname should be a mandatory activity to constantly monitor the status of this pathogen.