BATRACHOCHYTRIUM DENDROBATIDIS OCCURRENCE IN DEAD AMPHIBIANS OF CENTRAL MEXICO: A REPORT OF AMBYSTOMA ALTAMIRANI AND LITHOBATES MONTEZUMAE PRESENCIA DE BATRACHOCHYTRIUM DENDROBATIDIS EN ANFIBIOS MUERTOS DEL CENTRO DE MÉXICO: UN INFORME DE AMBYSTOMA ALTAMIRANI Y LITHOBATES MONTEZUMAE

— Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), is an infectious disease of amphibians linked to mass amphibian die-offs worldwide. In this study, we sampled four dead and dying individuals of Ambystoma altamirani and Lithobates montezumae to detect the presence of Bd and Bsal. By real-time PCR (qPCR) and histopathology methods, we found the presence of Bd and the absence of Bsal in all individuals sampled. Our study indicates that chytridiomycosis may act as a threat for these species and highlight that future surveys are urgently needed to evaluate the Bd infection on populations of A. altamirani and L. montezumae.

Chytridiomycosis is cataloged as the worst infectious disease in vertebrates due to the great extent of affected species and the mass amphibian die-offs caused worldwide over the last century (Gascon et al., 2007). The disease is caused by two fungal pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Chytridiomycosis causes hyperkeratosis and hyperplasia, which can cause death and even catastrophic declines in susceptible species' populations (Voyles et al., 2009;Martel et al., 2013). In Mexico, Bd has been found in 83 amphibian species (Basanta et al.,  Between January-July 2019 (winter and summer seasons) we found two dead and one dying individuals of Ambystoma altamirani, and one dead individual of Lithobates montezumae in the municipalities of Villa del Carbón, and Jilotzingo (at San Miguel Tecpan locality), Estado de México, all located in the northern part of Sierra de las Cruces in Central Mexico (Fig. 1). All specimens were found without obvious external causes of death or damage (e.g., predation or injury), and the dying individual showed chytridiomycosis signs such as lack of reflexes, stiffness, and extreme skin shedding (Fig. 2). The specimens of A. altamirani (N = 3) and L. montezumae (N = 1) were swabbed with a synthetic cotton swab following the protocol by Hyatt et al. (2007). All the individuals were fixed and stored in neutral 10% formalin. In the laboratory, DNA extraction from swab samples was performed using Prepman or Qiagen Blood and Tissue Kit DNA extraction (Table 1). Then, samples were assayed using realtime TaqMan PCR assays according to Boyle et al. (2004) and Martel et al. (2013) to detect Bd and Bsal presence, respectively. Each sample was run in duplicate with a negative control (5 uL sterile water) and four standards of DNA Gblocks (1, 100, 1000, and 10000 genome equivalents, GE) for separate assays of Bd and Bsal.
Multiple skin samples from fixed individuals were obtained for histological examination according to Berger et al. (1999). Briefly, skin samples were dehydrated in ethanol of increasing gradation, from 40% to 100%, clearing with xylene, and embedded in paraffin using a Tissue Embedding Center-Tissue-Tek®. Microtomy with disposable blades was carried out in a microtome Leica RM2125RT to obtain 4-6 µm thick sections which were stained with hematoxylin and eosin (Berger et al., 1999) or Schiff periodic acid histochemistry (PAS) and analyzed with an Olympus BX50 microscope equipped with a Lumenera digital camera and Infinity Analyze 6.3.0 software. The search  for Bd zoosporangia was carried out at a total magnification of 400X. All specimens were deposited in the Colección Nacional de Anfibios y Reptiles, Instituto de Biología, UNAM (IBH).
The analyses of qPCR showed Bd presence and Bsal absence in all swab samples of A. altamirani and L. montezumae (Table 1). Dead individuals showed lower Bd infection loads than the dying individual ( Table 1). Histopathology of skin showed evidence of fungal infection in all A. altamirani individuals. Spherical and ovoid zoosporangia, empty or containing zoospores, were identified in the superficial and partially detached keratinized cell layers of the epidermis (Fig. 2) with irregular thickening of the epidermis due to hyperplasia. The infected areas included zoosporangia ranging from 5 µm to 10 µm in diameter, mild to moderate hyperkeratosis, and areas of focal erosion adjacent to the infection. These observations agree with Bd infection as described by Berger et al. (1999). Skin histopathology of L. montezumae skin showed diffuse epidermal detachment related to postmortem changes, so the search for fungal infection was not feasible.
Our finding constitutes the first record of Bd in dead or dying amphibians of Central Mexico. The presence of dead and moribund specimens on different occasions and seasons of the year of A. altamirani suggests that these species could be susceptible to Bd infection. The low Bd infection load found in dead individuals may have been due to DNA degradation as a cause of the deteriorating state of the specimen, while the high infection load found in the two dying individual suggests that Bd may be one of its causes of death. Ambystoma altamirani and L. montezumae are threatened and endemic species of Mexico. The axolotl A. altamirani has a restricted distribution in Central Mexico, considered "Endangered" by the IUCN (IUCN, 2020a) and identified as "Threatened" by the Mexican law (NOM-059;SEMARNAT 2015). Meanwhile, the frog L. montezumae has a wide distribution in Central Mexico and it is considered a species of "Least Concern" by the IUCN (IUCN, 2020b) and subject to  "Special Protection" by Mexican law (NOM-059;SEMARNAT, 2015). The main threats to both species are habitat loss, pollution of the streams where these species are distributed, and the presence of invasive fish species (Lemos-Espinal et al., 1999). Our study indicates that chytridiomycosis is an additional threat for these native species from Central Mexico. Previous studies of Bd detection on wild Ambystoma and Lithobates species in Mexico have found medium to high Bd prevalence, but without any dead individuals or those with signs of the disease chytridiomycosis (Frías-Alvarez et al., 2008;García-Feria et al., 2017;Peralta-García et al., 2018;Basanta et al., 2019).
Based on our results, future surveys are urgently needed to evaluate the prevalence and infection intensity in populations of A. altamirani and L. montezumae across their respective distributions, so that proper conservation strategies can be implemented for these species.

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