Introduction

In recent years, the threat to conservation posed by emergent wildlife diseases has become increasingly apparent. Factors include anthropogenic-driven environmental destruction, climate change, and emerging infectious diseases (EIDs) (Fisher et al., 2012; Allender et al., 2016b; Franklinos et al., 2017; Chandler et al., 2019; Long et al., 2019; Walker et al., 2019). Fungal EIDs have been widely recognized as one of the causes of population declines and, in some cases, species extinction. Mycotic diseases affect multiple taxa including plants, mammals, fish, corals, and amphibians, among others (Sutherland et al., 2014). For instance, Pseudogymnoascus destructans causes white-nose syndrome (WNS) in bats (Blehert et al., 2009; Gargas et al., 2009), Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) cause chytridiomycosis in frogs, salamanders, newts, and caecilians (Allender et al., 2018; Chandler et al., 2019; Lastra Gonzalez et al., 2019), Aspergillus sydowii has been associated with disease in soft corals (Fisher et al., 2012), and Nosema spp. affect bees (Fisher et al., 2012). While these pathogens can cause clinical disease, subclinical cases may also threaten fitness, reproduction, and ultimately species survival (Lind et al., 2019a; Lind et al., 2019b).

Ophidiomyces ophidiicola (Oo), the pathogen causing ophidiomycosis (also referred to as snake fungal disease, SFD), is a keratinophilic fungus that can affect wild and captive snakes (Franklinos et al., 2017; Ohkura et al., 2017; Long et al., 2019; Paré, 2019). Molecular studies suggest that the fungus has been circulating since 1985, when it was classified as part of the Chrysosporium anamorph of Nannizziopsis vriesii complex (CANV) and later assigned to the genus Ophidiomyces (Lorch et al., 2016; Ohkura et al., 2016; Paré and Sigler, 2016). The geographical distribution of Oo has been reported to be broader in captive snakes than in wild ones (Lorch et al., 2016), and it includes the countries and host species listed in Table 1.

Table 1. Known host range of Ophidiomyces ophidiicola by continent.

View Fullscreen

Table 1. Continued.

View Fullscreen

Ophidiomyces ophidiicola has been detected in different Serpentes families. Within the genus Python, cases have been reported in ball pythons (Python regius) (Lorch et al., 2016; Franklinos et al., 2017; Picquet et al., 2018) and African rock pythons (Python sebae) (Lorch et al., 2016; Ohkura et al., 2016; Paré and Sigler, 2016).

Some recent studies have focused on the role of individual and environmental factors regarding survival and spread of the fungus (McCoy et al., 2017; Lind et al., 2019b; Long et al., 2019). Walker et al. (2019) found that snake skin bacterial taxa not only differ from the environmental bacterial assemblage but also vary throughout wider geographical spaces and seasons. They concluded that Oo infection is predictive of the bacterial taxa on the snake skin that associate with Oo.Allender et al. (2018) evaluated the snake–host microbiota relationship in eastern massasauga rattlesnakes (Sistrurus catenatus) as a predictor of the emergence of pathology, variability in snake health, therapeutic intervention, and reduction of disease impact. Although Oo infection was associated with shifts in microbiome composition, the authors did not find a quantitative correlation between the two; this contrasts with Bd in frogs where a quantitative correlation exists. This difference was attributed to difficulties in measuring Oo abundance and to the fact that Oo spreads deep to the skin–microbiome interface.

Skin microbiota is not only altered by the presence of Oo because some bacteria may also act as probiotics against Oo.Hill et al. (2018) hypothesized that skin microbiota might manifest differently in the presence of Oo and that some bacteria may produce an anti-fungal effect. The authors identified 16 different bacterial strains that demonstrated antifungal effects. Among them, Morganella morganii, a snake skin commensal, elicited a potent anti-Oo effect.

Clinical signs of ophidiomycosis have been linked to circulating corticosteroid concentrations, which can vary depending on body condition, environmental temperature, and metabolic functions (e.g., pregnancy and vitellogenesis). Poor body condition, due to starvation and other diseases, as well as low environmental temperatures, negatively affect circulating stress hormone concentrations. It also appears that pregnancy not only influences the ability to feed but could also lead to clinically significant disease and eventually death (Lind et al., 2018b; Licitra et al., 2019). Moreover, concerning the pregnancy status, Lind et al. (2018a) suggested that the postgravid energetic state predisposes a snake to the infection.

Apart from an individual's predisposition to the disease, environmental components may also influence the pathogenicity of this fungus. Knowing the ecology of Oo in relation to the environment and time (season) is essential when setting mitigation and conservation actions against this EID. All published studies support the theory that environmental temperature and humidity play significant roles in the expression of this disease. According to Long et al. (2019), Oo exhibits optimal growth at temperatures around 25°C (77°F), which corresponds to spring temperatures in the eastern United States. Temperature-driven pathogenicity has also been observed in other fungal diseases such as Bd in amphibians (Latney and Klaphake, 2013). Identifying the effects of temperature on pathogenicity would help in narrowing the surveillance period for this EID. Snakes are believed to be predisposed to the development of ophidiomycosis during brumation (Steeil et al., 2018). The most severe pathological changes could coincide with the winter months when the physiological capability of an individual to fight a disease is low. When environmental conditions are not suitable, ectotherms may have to tradeoff an effective metabolic rate to down-regulation of immune functions to promote a positive energy balance (McCoy et al., 2017; Lind et al., 2019b). Lind et al. (2019b) suggest that seasonal tradeoffs between host defense, reproduction, and behavior may affect population-level responses to disease.

In addition to temperature and humidity, other specific environmental conditions may also affect the persistence or transmission of the fungus. Some snake species share environmental niches with other species of reptiles (e.g., gopher tortoises [Gopherus polyphemus]) (Chandler et al., 2019). Gopher tortoise burrows present a stable temperature and high humidity, which are favorable conditions for fungal growth; however, the relationship between this mycosis and other species requires further study. It is, however, fair to hypothesize that the concentration of the pathogen, either due to multiple snakes sharing the same environmental niche or because of the use of another species' shelter, may serve in maintaining and propagating the disease (Steeil et al., 2018; Long et al., 2019).

Kadoorie Farm and Botanic Garden (KFBG), in collaboration with the Agriculture, Fisheries and Conservation Department (AFCD) of the Government of the Hong Kong Special Administrative Region, initiated the Wild Snake Rescue Project in 1999 to mitigate human–snake conflict by providing a resource for snakes captured following police call outs in Hong Kong. Snakes received by the KFBG generally strayed into human habitation and were subsequently captured by local snake catchers under the authority of the police. Once at KFBG, snakes are identified to the species level and subject to a full physical examination; native species are then released back to the wild in suitable habitats. To date, over 14,000 snakes have been received via the project, with the majority (88%) being released. As part of this project, KFBG and AFCD started an Oo surveillance study in 2018. About 170 snakes have been sampled since the study was initiated. Clinically healthy snakes and snakes that present with any skin alterations are included in the survey, which uses single swabs rubbed along the body length or in the oral cavity for screening. In snakes with skin lesions, the lesions are swabbed. This brief communication represents the first report of Oo in a Burmese python (Python bivittatus).

Case Report

A 1.7 kg, 1.75-m female wild caught Burmese python was admitted to the KFBG Wild Animal Rescue Center in November 2019. The snake appeared dull, but was alert and responsive when stimulated. Her body condition score was rated as “moderately underconditioned” and a physical examination revealed areas of necrosis on the left side of the head and mid-body (Figs. 1–3). The head lesions were compatible with focal ulcerative dermatitis around the left eye and upper lip, and facial disfiguration was apparent. There was necrotic oral mucosa lateral to the upper row of teeth. Some scales of the lower lip appeared to have become thickened and yellow-brown, but no ulcers were visible. The left eye had a diffuse opacity, possibly a sign of ophthalmitis, and the globe rim displayed an irregular shape. The body lesions, with an irregular pattern and color, extended about 20 cm in length and were around the whole girth of the snake. Some areas had prominent caseous material and necrosis. A sterile cotton-tipped applicator (Medical Wire & Equipment, Corsham, Wilt-shire, United Kingdom) was used to collect DNA from the affected areas on the head and body. Due to the severity of the lesions, the snake was not deemed suitable for release and was subsequently euthanized. A postmortem was not performed.

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

Nucleic Acid Extraction: A bacterial genomic DNA extraction kit (TianLong, Xian, China) was used to extract DNA from the swab using a modified protocol. The swab was immersed in 250 µl of bacterial digestion buffer (TianLong) and pretreated with 250 U lyticase (Sigma-Aldrich, St. Louis, MO, USA) at 37°C (98.6°F) for 30–60 min with occasional vortexing. After a brief centrifugation at 18,800 g, 200 µl of supernatant was transferred to the sample well for extraction. Automatic nucleic acid extraction was performed using a TianLong nucleic acid extractor (TianLong). Nucleic acids were eluted in elution buffer (TianLong) at the end of the extraction.

Real-time Polymerase Chain Reaction (PCR): TaqMan™ real-time PCR was used to detect Oo from the swab. Three sets of primer pairs and probes were used to target the internal transcribed spacers (ITS) 1 and 2 and intergenic spacer (IGS) loci of Oo (i.e., OphioITS-F, -R, -P; Oo-rt-ITS-F, -R, -P; and Oo-rt-IGS-F, -R, -P, respectively) (Table 2). For the OphioITS PCR, a hot-start Taq polymerase and probe with conjugated minor groove binder (MGB) was used to enhance the specificity of the PCR. For the Oo-rt-ITS and Oo-rt-IGS PCRs, hot-start Taq polymerase and probes with conjugated FAM/BHQ1 were used. In addition, a snake endogenous gene (the β-actin gene) real-time PCR was performed to ensure the presence of enough genetic material and the absence of PCR inhibition. Real-time PCR was performed using the Bio-Rad CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA), and data were analyzed using Bio-Rad CFX Maestro™ Software. The OphioITS, Oo-rt-ITS, and Oort-IGS PCRs were performed in a total volume of 20 µl, which was comprised of 5 µl DNA template, 10 µl Bio-Rad SSo Advanced Universal Probe Supermix, and a final concentration of 400 nM for primers and 200 nM for probes. The thermal profile consisted of 3 min at 95°C (203°F), followed by 45 cycles of denaturation at 95°C (203°F) for 10 sec, and annealing/elongation at 60°C (140°F) for 30 sec. The real-time PCR for the detection of the β-actin gene was also performed in a total volume of 20 µl (2 µl DNA template, 10 µl Bio-Rad SSo Advanced Universal Probe Supermix, and a final concentration of 300 nM for primers and 100 nM for the probe). The thermal profile consisted of 2 min at 95°C (203°F), followed by 50 cycles of denaturation at 95°C (203°F) for 10 sec, and annealing/elongation at 60°C (140°F) for 60 sec.

Table 2. Information on sets of primers and probes used for the detection of Ophidiomyces ophidiicola and endogenous β-actin genes.^a

View Fullscreen

Synthetic Positive Control: A synthetic DNA template of the target loci was used for the generation of standard curves to evaluate the detection limit and amplification efficiency of the real-time PCR assays. Two plasmids containing the IGS and ITS sequences were used for the generation of standard curves (GenScript, Piscataway, NJ, USA). The ITS and IGS fragments were selected with reference to accession numbers KF225599 and KP691510.1 in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Both IGS and ITS plasmids carried vectors 2,752 base pairs (bp) and inserts 290 bp long. Therefore, 1 fg of plasmid DNA yielded 305 copies of Oo IGS/ITS (i.e., 3.27 pg plasmid DNA = 1.0 × 10⁶copies of Oo IGS/ITS). The plasmid DNA (both ITS and IGS) at a concentration of 1.0 × 10⁶copies/µl was prepared using Tris-EDTA (TE) buffer (Thermo Fisher Scientific Waltham, MA, USA). Concentrations at ten-fold dilutions ranging from 1.0 × 10⁶copies/µl to 1.0 × 10¹copies/µl were used for the generation of the standard curve.

Sequencing: The Oo-rt-IGS PCR was performed on the positive sample using the Oo-rt-IGS primer set and PCR conditions listed above. The amplified PCR product was sequenced using the Oo-rt-IGS forward and reverse primers. Sequencing data was compared to sequences deposited in GenBank using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi). The swab from the Burmese python was positive in the OphioITS, Oo-rt-ITS, and Oo-rt-IGS PCRs, with Ct values of 26.24, 26.83, and 29.22, respectively. Oo-rt-IGS (133 bp) was sequenced and the result was 100% identical to the corresponding O. ophidiicola-IGS sequence (GenBank accession no. KP691514.1). The sequence has been submitted to GenBank (accession no. MT459829).

Discussion

This article describes a case of Oo in a Burmese python. Since the Wild Snake Rescue Project commenced in 1999, very few snakes have shown clinical signs of sickness, and sampling for Oo only started in 2018. The results of this case suggest more testing is needed to measure the prevalence of Oo in Hong Kong.

Seasonality has been implicated as a contributing factor in ophidiomycosis outbreaks, and low environmental temperatures may negatively influence the immune system of the infected snakes (Lind et al., 2019b; Long et al., 2019; Walker et al., 2019). Hong Kong is located closer to the equator than other countries in which Oo has been previously detected (e.g., United States, Europe, Australia, and Asia). The climate of Hong Kong is considered subtropical, and with climate change the average temperatures have been increasing in recent years. Winters have become milder and the number of very cold days has decreased in the past decades; 2019 was found to be the warmest year in Hong Kong since recording began in 1884, with 11/12 months warmer than average and characterized by an annual mean temperature of 24.5°C (76.1°F), which is 1.2°C (2.2°F) above the average. In the same period of time, the average rainfall and number of tropical cyclones were also found to increase (Hong Kong Observatory, 2020). These changes in climate could influence the epidemiology of this disease in Hong Kong and warrant further study.

The PCR tests used in this study targeted the multicopy ITS or IGS of Oo and are extremely sensitive and highly specific. These assays can be used to confirm a diagnosis of ophidiomycosis in both wild and captive snakes, in addition to providing an important research tool for better understanding the biology of the fungus and ecology of this disease (Bohuski et al., 2015).

The case described represents the first Oo-positive in a group of sampled snakes. Only one snake was found to be Oo-positive out of 170 (0.5%) snakes sampled, suggesting a low prevalence of disease. Asymptomatic and clinically sick snakes were sampled in random numbers, and a single swab was collected for every snake. Recent literature reports that the presence of lesions is the best predictor of Oo infection, and failure in sampling infected tissues in clinically healthy snakes is possible because of the nature of the disease affecting the deeper layers of the skin (Chandler et al., 2019; McKenzie et al., 2019). Unfortunately, no necropsy or histopathology was performed for this case; thus, a direct link could not be proven between the skin lesions and the pathogen, and other possible causes of the lesions could not be ruled out.

Future sampling efforts should aim to 1) increase the sensitivity of the screening protocol, and 2) evaluate the connection between clinical signs and pathogen presence. Sensitivity would likely be increased by modifying the swabbing technique to include larger body areas and by performing PCR on skin biopsies as well as swabs. Skin biopsies can, via histopathology, also be used to confirm lesion etiology (Allender et al., 2016a; Hileman et al., 2018; Chandler et al., 2019; Long et al., 2019). The KFBG's Wild Animal Rescue Center receives many Burmese python hatchlings in September and October and, in view of the possibility of vertical transmission (Stengle et al., 2019), samples could also be taken from hatchlings. Including this age range would contribute to the expansion of the sample size and potentially provide data on whether hatchlings may be more predisposed to infection due to their immature immune system.

The recent appearance of Oo infection in snakes in various countries suggests that this mycosis is becoming a global conservation concern (Lorch et al., 2016; Franklinos et al., 2017; Takami et al., 2021). Its emergence could be caused by individual and/or environmental variables that could be linked (Franklinos et al., 2017; Steeil et al., 2018). Climate change (Paré et al., 2019), other pathogens (Allender et al., 2018), and population dynamics (Chandler et al., 2019) may all contribute to various degrees in propagating the disease. Detection techniques are also becoming more precise and sophisticated, which may lead to earlier detection. Snakes are regarded as important models for evolutionary and ecological investigations; they are indicators of ecosystem health and may yield potential benefit to humans as agents of pest control (Lind et al., 2005; Walker et al., 2019).

Conclusions

The findings reported here highlight the significant role wildlife rescue centers serve for monitoring wildlife diseases and ecosystem health. Considerable information can be gained by analyzing the data collected from rescue center admissions and collaborating with different stakeholders regarding disease surveillance (Trocini et al., 2008). It is important that veterinarians, conservationist biologists, and governments jointly participate in the collection and analysis of data and develop conservation strategies and mitigation actions that are aimed at minimizing disease in populations.