Leveraging preserved specimens of Nerodia to infer the spatiotemporal dynamics of Ophidiomyces ophidiicola via quantitative polymerase chain reaction

Abstract Ophidiomyces ophidiicola (Oo) is a fungal pathogen and the causative agent of ophidiomycosis that has affected multiple snake taxa across the United States, Europe, and Asia. Ophidiomycosis has often been referred to as an emerging infectious disease (EID); however, its status as an EID has recently come under debate. Oo infections have been confirmed in wild snake populations in Texas; however, it is unknown if the pathogen is novel (i.e., invasive) or endemic to the state. To address this knowledge gap, we conducted surveys for Oo among preserved Nerodia deposited at three university museums in Texas. First, we visually assessed snakes for signs of infection (SOI), and if SOI were present, we sampled the affected area. We then used quantitative polymerase chain reaction to diagnose the presence of Oo DNA on areas with SOI and used these data to evaluate spatiotemporal patterns of Oo prevalence. We also tested for significant spatial clusters of Oo infenction using a Bernoulli probability model as implemented in the program SatScan. We found that the proportion of snakes exhibiting SOI was constant over time while the prevalence of Oo DNA among those SOI increased across space and time. Within these data, we detected an incidence pattern consistent with an introduction and then spread. We detected six spatial clusters of Oo infection, although only one was significant. Our results support the hypothesis that Oo is an emerging, novel pathogen to Texas snakes. These data narrow the knowledge gap regarding the history of Oo infections in Texas and establish a historical record of confirmed Oo detections in several counties across the state. Thus, our results will guide future research to those areas with evidence of past Oo infections but lacking confirmation in contemporary hosts.


| INTRODUC TI ON
Emerging infectious diseases (EIDs) are defined as diseases that are novel to science, novel to a population, or are known to be naturally occurring yet suddenly increase in prevalence or range owing to some factor or set of factors (Daszak et al., 2000;Morse, 1995). EIDs caused by fungal pathogens have emerged as threats to plants and animals across the globe (Fisher et al., 2016;Gurr et al., 2011) and are a concern for wildlife because of their potential to trigger the collapse of afflicted populations (Berger et al., 1998;Frick et al., 2010;Lips et al., 2003). Notably, Batrachochytrium dendrobatidis (Lips et al., 2006;Vredenburg et al., 2010) and Batrachochytrium salamandrivorans Spitzen-van der Sluijs et al., 2013) were linked to amphibian population declines worldwide, while Pseudogymnoascus destructans has been linked to the collapse of some North American bat populations (Blehert et al., 2009;Thogmartin et al., 2012).

In snakes, Ophidiomyces ophidiicola [formerly ophiodiicola] (Oo)-
the causative fungal pathogen of ophidiomycosis-was first described in 2009 (Rajeev et al., 2009) but may have contributed to the decline of viper populations in New Hampshire in 2006 (Clark et al., 2011) and Illinois in 2008 (Allender et al., 2011). Since then, the pathogen has been detected in wild snakes throughout the Midwest and eastern U.S. (Chandler et al., 2019;Glorioso et al., 2016;Guthrie et al., 2016;Last et al., 2016;. Recently, Oo was detected in Idaho (Allender et al., 2020) and California (Haynes et al., 2021). Thus, it now seems to be distributed across the contiguous U.S. In light of these observations, Oo has been referred to as an emerging fungal pathogen of snakes (Allender, Raudabaugh, et al., 2015;Franklinos et al., 2017;Grioni et al., 2021;Guthrie et al., 2016;McKenzie et al., 2019;Ohkura et al., 2017). However, a recent study proposed that Oo should be viewed as naturally occurring rather than novel, and an unrecognized yet common fungal pathogen of snakes as opposed to a newly emergent pathogen (Davy et al., 2021).
Historically, there are reports of "hibernation blisters" or "hibernation sores" on snakes that emerge from brumation (Clark et al., 2011;. Other infections may have been responsible for these observations; however, these reports provide anecdotal evidence for the possibility that Oo may be naturally occurring or at least has maintained a historical presence in some snake populations. Recently published retrospective surveys for Oo in preserved snake specimens corroborated these possibilities by showing evidence that Oo infected wild snakes as early as 1945 in the eastern U.S. (Lorch et al., 2021) and 1959 in Europe (Origgi et al., 2022).
Using population genetics, Ladner et al. (2022) proposed Oo has been introduced to North America multiple times between 1731 and 2012, which could support established historical presence in some populations and potentially, pathogen novelty in others.
In Texas, there is a paucity of data for Oo. Presently, six con-  , and another estimated 15% prevalence among terrestrial and aquatic snakes via SYBR Green qPCR in northeast Texas (Lizarraga et al., 2023). Thus, there is a knowledge gap regarding Oo infection dynamics in this region, and it is unknown if Oo is naturally occurring and previously unrecognized, naturally occurring but emerging, or has recently spread into Texas (i.e., a novel pathogen).
Evidence supporting these hypotheses could be evaluated by testing for two expected epidemiological patterns. If a pathogen has been introduced and spread, the spatial distribution pattern will be such that there are relatively few cases isolated to only a few areas, then followed by increases in prevalence and distribution across space and time (Cheng et al., 2011;Childs et al., 2000;Guerra et al., 2003;Lips et al., 2008). If a pathogen is naturally occurring, or from a disease ecology perspective-endemic, then evidence of dynamic equilibrium measured as nonchanging pathogen prevalence across space and time would be expected (Becker et al., 2016;Rodriguez et al., 2014).
However, a challenge to addressing historical pathogen dynamics (i.e., support for natural occurrence, endemicity, or novelty) is the availability of samples that retrospectively span several decades. For this purpose, museum collections are advantageous because they provide access to preserved specimens collected at different points in space and time. In the B. dendrobatidis system, museum surveys of preserved amphibians have been used to show pathogen emergence concomitant with host population declines in Central America (Cheng et al., 2011); and pathogen endemicity in South America (Becker et al., 2016, Rodriguez et al., 2014. These retrospective studies were useful because they elucidated the contrasting spatiotemporal dynamics of B. dendrobatidis infections in Central and South America over several decades and may help explain contemporary patterns in these regions.
Similarly, the goal of our study was to derive support for whether Oo is a previously unrecognized, naturally occurring pathogen to Texas snakes, or if it is a recent invader (i.e., novel). To achieve this goal, we inspected preserved snakes for potential SOI and used molecular analyses to determine the presence of Oo within this subset.
Using these data, we investigated the spatiotemporal dynamics of Taxonomically, we aggregated snakes by species; except N. harteri, which was identified to the subspecies level (N. h. harteri and N. h. paucimaculata). We focused primarily on Nerodia spp. for three reasons: (1) the first confirmed report of Oo infection in Texas was from N. h. harteri, (2) the state contains several widespread species, and (3) to gain historical insight for Oo infection associations observed in contemporary Nerodia populations by Harding et al. (2022).
Before surveying, the snakes were removed from their jar and placed onto a dissecting tray disinfected with 95% EtOH and wiped clean with fresh paper towels. Then, we visually inspected each snake for potential signs of Oo infection (SOI) and assigned a negative (0) if no signs were present or positive (1) if SOI were identified.
We defined SOI as the presence of scale abnormalities consistent with signs of ophidiomycosis (e.g., signs of inflammation, dermatitis, gross lesions, crust, or nodules) (Allender, Baker, et al., 2015;Lorch et al., 2015). If a snake showed SOI, it was completely rinsed with fresh 50% EtOH to remove debris. We then sampled affected areas using a single sterile cotton-tipped swab (Medical Wire, MW113); afterward, we immediately placed the swab into a labeled, sterile screw-cap tube with an O-ring. We photographed the dorsum, venter, and lesions for all snakes exhibiting SOI.
To control for false positives, we randomly swabbed one snake with no SOI (i.e., asymptomatic negative controls) for approximately every 10 snakes showing SOI. To assess cross-contamination between snakes in jars, we selected negative control snakes from jars that also contained snakes showing SOI. Thus, swabs from asymptomatic snakes were collected during the same session(s) and inbetween swabs taken from snakes with SOI. To maximize the area sampled for asymptomatic snakes, we swabbed the entire body starting at the dorsal, anterior end of the snake and then moved towards the posterior using a back-and-forth motion. We then repeated this method for the ventral surface. We used a chi-square test of independence to evaluate the relationship between the presence/absence of SOI and the detection of Oo.

| DNA extraction, qPCR, and molecular analysis
To extract DNA from the swabs, we used the PrepMan Ultra Sample Preparation Reagent (Applied Biosystems) protocol followed by Harding et al. (2022). Specifically, prior to DNA extraction, the screw-cap tubes were opened and swabs were allowed to dry for 1 h to ensure that any residual ethanol evaporated. After which, we added 50 μL PrepMan Ultra reagent to each sample, vortexed the tubes for 30 s, and then briefly centrifuged each tube. Then the tubes were boiled at 96-100°C for 10 min and cooled for 2 min. We then centrifuged the samples for 1 min at ≥12,000 g. We aseptically inverted the swabs with flame sterilized tweezers in the tubes and centrifuged for another minute at the same velocity to pull the extract out of the swab. The swabs were then aseptically removed and the tubes were centrifuged for 10 min (≥12,000 rcf) to pellet precipitates that might inhibit PCRs. Then, we carefully transferred the supernatant to a new sterile tube leaving behind any precipitates and stored the aliquot at −20°C until processing.
We extracted swabs taken from negative control snakes in the same session as those taken from snakes with SOI. In general, we treated all negative control DNA extracts as experimental samples and included them in the same reaction plate(s) as the DNA extracts from swabs of snakes with SOI. Before conducting qPCR reactions, each sample was diluted 1/10 with nuclease-free water to reduce the concentration of potential PCR inhibitors. We conducted our reactions utilizing the primers and probe designed by Allender, Bunick, et al. (2015). We used the standards, thermal cycling profile, and reaction protocol described by Harding et al. (2022). We ran all unknown sample reactions in triplicate. We considered a sample positive for Oo DNA if the calculated quantity was ≥10 fg (see Harding et al., 2022). Samples that showed no amplification, had calculated quantities <10 fg, or showed aberrant amplification curves were considered negative. We used the Thermo Fisher Connect Cloud Dashboard Software (Thermo Fisher Scientific) for qPCR data processing and analysis.
To control for false negatives caused by low DNA quality owing to the preservation method or other unknown factors, we also con- Reactions were processed similarly to microbiome PCRs in that they were made in a UV and 10% bleach sterilized biosafety cabinet using dedicated pipettes and barrier tips. All plastic consumables were also UV sterilized prior to use and only unopened reagents were used to minimize the potential for environmental contamination. To test for successful amplification, we electrophoresed the PCR reactions on a 2% agarose gel made with 1X TAE (w/v) in 0.25X TAE buffer at 200 V for 15 min. Reactions were scored based on the presence or absence of the expected band size.

| Spatiotemporal analysis
Among the total snakes surveyed, we reported the number that exhibited SOI. For each county and species, we reported the number of Oo detections among those with SOI (Table A1 in Appendix 1; Tables 1 and 2). We calculated the proportion of Oo detections (i.e., Oo prevalence) among all snakes with SOI and for each species surveyed with SOI-and, when applicable, subspecies. We evaluated the strength of detection by estimating the probability of a falsenegative using the equation (1 -P) S (Cheng et al., 2011), where P was an assumed true Oo prevalence value of 5%, 10%, and 20% for a time interval during which Oo was not detected and S represents the number of qPCR samples (i.e., the number of snakes swabbed).
To visualize temporal patterns of SOI and Oo prevalence and spatiotemporal patterns of Oo detections, we aggregated snakes by the year collected in the following manner: 1905-1954, 1955-1959, and then at 10-year intervals afterward. We tested for significant Oo infection clusters across space using a Bernoulli probability model implemented in SaTScan v9.6 (Kulldorff & Nagarwalla, 1995). Because several snakes (N = 99) did not have a date of collection associated with their catalog number and the sampling distribution was not even across time, we did not conduct a temporal cluster analysis.
We defined the maximum spatial cluster size as 50% of the population with a maximum radius of 50 km. To maximize the sample size for each county, we aggregated data points into time intervals of 10 years.

The proportion of Oo detections among preserved Texas Nerodia
with SOI and the spatial distribution of those detections increased TA B L E 1 Texas county of record for preserved Nerodia that tested positive for Ophidiomyces ophidiicola infection via qPCR, the sample size for each county (N), the number of snakes that showed signs of infection (SOI), the number that tested positive of O. ophidiicola DNA (Oo +), and the earliest year an Oo + snake was captured.

County
N SOI Oo +   1905-1979, Oo prevalence increased between 1980-1989, 1990-1999-2009, and 2010-2019 ( Figure 2b). Our estimated probability of a false-negative (i.e., failure to detect Oo when it is present) for samples collected between 1905 and 1954 with an assumed true prevalence of 5%, 10%, and 20% was <0.001% for all three assumptions. Spatial analysis of Oo prevalence resulted in six clusters; however, only one was significant (Figure 3).

| DISCUSS ION
By surveying preserved Nerodia spp. from three museum collections, we have shown that Oo has been widespread and infecting snakes in Texas since at least 1955-approximately 53 years before first being detected in wild snake populations in the eastern U.S. (Allender et al., 2011;Rajeev et al., 2009). In general, Oo prevalence among our samples increased (Figures 1 and 2b) over time, while the prevalence of SOI was consistent (~11%) (Figure 2a). The spatiotemporal patterns for Oo prevalence in our study are consistent with a pattern of introduction-or multiple introductions-and spread rather than a pattern indicative of long-term presence (i.e., endemism or natural occurrence). We also detected evidence of past outbreaks within Nerodia harteri, a Texas endemic species of conservation concern.

| Historical pathogen dynamics
We might expect that SOI would also increase temporally if Oo was an invasive pathogen; however, we observed a stable prevalence of SOI over space and time. This pattern could have been due to TA B L E 2 Species summary of the number of preserved Nerodia (N) surveyed for signs of infection (SOI), the prevalence of SOI (SOI/N), 95% binomial confidence intervals (SOI CI), the number of Oo + snakes, Oo prevalence among those with SOI (Oo +/SOI), and 95% binomial confidence intervals (Oo + CI).  1905 -1954 1955 -1959 1960 -1969 1970 -1979 1980 -1989 1990 -1999 2000 -2009 2010 -2019 sampling bias when these snakes were initially collected, or it could have also resulted from our inclusive approach to surveying for the presence of Oo. During the early stages, Oo infection is sometimes indicated by mild dermatitis or a subtle crust. Therefore, we did not constrain our methods to target snakes with specific lesion types or the severity or number of lesions. Consequently, it is possible that we identified and sampled snakes with skin wounds or lesions not infected with or caused by Oo. If Oo invaded this region, prior disruption of the dermal layer via naturally occurring injuries may have served as opportunistic pathways for infection given that experimental infections are sometimes initiated via dermal abrasion (Lorch et al., 2015). In this case, SOI, as we broadly defined it, would remain relatively constant, yet prevalence of Oo would increase.
Nonetheless, our observations are consistent with other retrospective studies that have shown Oo infections have been present in wild snakes in the eastern U.S. since 1945 (Lorch et al., 2021) and in Europe since 1959 (Origgi et al., 2022). Thus, ophidiomycosis has indeed gone unrecognized or overlooked in Texas and other regions for decades. However, our results showed Oo prevalence among our samples increased significantly starting in 1980 (Figure 2), and snakes across more counties were infected when compared to the period from 1905 through 1979 (Figure 1). This spatiotemporal pattern of increasing prevalence is consistent with epizootic outbreaks shown in studies of B. dendrobatidis (Brem & Lips, 2008;Cheng et al., 2011;Lips et al., 2006)   this study support the conclusions of Ladner et al. (2022). Another study comparing the genetic similarity between Oo isolates collected in Texas, the eastern U.S., and Europe showed evidence of shared genotypes between Texas, Massachusetts, Maryland, and New York (Harding, 2022). Collectively, these results are consistent with the hypothesis that Oo spread across North America, possibly via human-mediated transport, and that Oo is a recently introduced pathogen in Texas.
Regarding false negatives owing to a failed extraction of Oo DNA, when we assumed a true prevalence (20%) comparable to our observed estimated prevalence (~22%), our estimated probability of a false negative for our sample size was low (<0.001%).
Comparatively, our methods reported here were based on our Oo sampling methods for live snakes where our estimated false negative rate was ~15% . Explicitly, we surveyed 599 snakes collected in Texas from 1905 to 1954, of which 52 showed SOI. Considering the probability of false negatives in these results and our aforementioned false-negative rate, we estimated up to 8 samples from this period are true positives but tested negative.
Thus, we assume that SOI observed on snakes collected before 1955 were caused by something other than Oo, and our failure to detect Oo DNA is more likely owing to the absence of Oo infections rather than false negatives.

| Conservation implications
Subspecies of N. harteri are taxa of conservation concern characterized by restricted ranges, low abundance, and low genetic diversity (Janecka et al., 2021;Rodriguez et al., 2012). Our retro- and should be considered in future management decisions for this species .
Our Oo prevalence estimates for Nerodia likely do not reflect the true prevalence of Oo infections in Texas snakes across either space or time. Indeed, wide confidence intervals reflect the uncertainty in some of our estimates (Figure 2b;

ACK N OWLED G M ENTS
We would like to thank Toby Hibbitts, Travis LaDuc, and Greg and Jared Oakes for their assistance with data collection. We thank Utpal Smart for computational assistance, manuscript review, and figure design.

FU N D I N G I N FO R M ATI O N
This project was funded by startup funds to D.R. from Texas State University.

CO N FLI C T O F I NTER E S T S TATEM ENT
All authors have no competing interests to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Supporting data and additional photographs documenting signs of infection on specimens are available at our Zenodo repository: 10.5281/zenodo.7775088.

TA B L E A 1 (Continued)
A PPE N D I X 1