OPHIDIOMYCES DETECTION IN THE EASTERN MASSASAUGA IN MICHIGAN
Report prepared by:
1 Wildlife Epidemiology Lab, Department of Comparative Biosciences, College of Veterinary Medicine, University
of Illinois Urbana-Champaign, Urbana, IL
2 Illinois Natural History Survey, Prairie Research Institute, University of Illinois Urbana-Champaign, 1816 South
Oak Street, Champaign, IL
3 Department of Biological Sciences, Northern Illinois University, Dekalb, IL
4 Biology Department, Grand Valley State University, Allendale, MI
5Indiana-Purdue University Fort Wayne, Fort Wayne, IN
Fish and Wildlife Service, East Lansing Field Office
The combined effects of habitat fragmentation, infectious diseases, and toxicological exposure
on wildlife populations are of increasing concern, especially for endangered species already
persisting at small population sizes (Bender et al., 1999; Daszak et al., 2000; Golden and Rattner
2003; Grillitsch and Schiesari 2010). Assessing the overall wellness of wild populations will aid
in forming conservation goals and developing recovery strategies to minimize population level
disease threats and enhance individual health (Allender et al., 2006; Sleeman, 2013). Monitoring
pathogen prevalence is becoming increasingly important as the number of published case studies
for disease outbreaks that cause population declines or extirpations is rapidly growing (Thorne
and Williams, 1988; Cunningham and Daszak, 1998; Woodroffe, 1999; Schoegel et al., 2006).
However, intervention and management strategies to mitigate the effects of disease outbreaks are
hampered after a pathogen becomes established in the environment. Consequently, continuous
health monitoring and disease investigations are needed to provide valuable insight into the
overall ecological health of natural systems (Brand, 2013).
Disease outbreaks are especially concerning for endangered species already contending with
small populations. In the eastern US, the Eastern Massasauga (Sistrurus catenatus), a federal
candidate species for endangered listing, is specifically vulnerable to disease due to
Ophidiomyces fungal infections. Ophidiomyces infections have been identified as the primary
cause of mortality in infected individuals (Allender et al., 2011; Allender et al., 2013). This is
unusual for a fungal pathogen, as they are typically more opportunistic, infecting already
compromised individuals. The emergence of Ophidiomyces fungi (Snake Fungal Disease; SFD)
recently documented from the skin, muscle, and bone of Timber Rattlesnakes (Crotalus
horridus, Clark et al., 2011) and Eastern Massasaugas (Allender et al., 2011) and suspected in
Black Ratsnakes (Pantherophis obsoletus, Rajeev et al., 2009), Pygmy Rattlesnakes (Sistrurus
miliarius, Cheatwood et al., 2003), and other reptiles (Pare et al., 2003; Sigler et al., 2013;
Sleeman, 2013) is alarming because of its broad geographic and taxonomic distributions.
Extinction events due to disease, while rare in wildlife, have been confirmed in a species of land
snail due to a parasite infestation and the sharp-snouted day frog (Taudactylus acutirostris)
(Schloegel et al., 2006) due to chytridiomycosis (Cunninghman and Daszak, 1998). In both
cases, disease outbreaks led to rapid catastrophic declines such that populations could not
recover. Neither study described the health of individuals in the population prior to the outbreak,
which may have allowed a more concerted effort to mitigate disease impact.
Populations of massasaugas in Michigan, the only state that the species is not listed as threatened
or endangered, have been well-studied, but the presence of this pathogen has never been
investigated. Thus, our objectives for this study were to: 1) determine the prevalence of
Ophidiomyces at three sites in Michigan and 2) characterize differences in sex, age class, and
location between positive and negative snakes.
MATERIALS AND METHODS
GENERAL METHODS.– During 2014, from spring egress through fall ingress, visual
encounter surveys were conducted of three wild Eastern Massasaugas populations: 1) Edward
Lowe Foundation (ELF) in Cass County, MI), 2) Pierce Cedar Creek Institute (PCCI) near
Hastings (Barry County, MI), and, 3) Camp Grayling (CG) near Grayling (Crawford County,
MI). All individuals were marked via PIT-tagging. We recorded sex, sex and age class, snout-
vent length (SVL, cm), tail length (cm), mass (g), and behavior for all individuals. After
processing all snakes were returned to their initial points of capture. Sterile handling and
equipment protocols were used at all times.
FIELD HEALTH ASSESSMENT.– All animals were assessed for clinical signs consistent with
SFD, i.e. generalized dermatitis, such skin lesions, facial swelling, or discharge. Presence of any
of these signs was assigned as present/absent. Using sterile cotton-tipped or nylon-flocked
applicators, facial sites on each animal were swabbed. Samples were stored in 2 ml eppendorf
tubes, frozen at -20C, and batch sent to the Wildlife Epidemiology Laboratory at the University
of Illinois. DNA extraction and quantitative PCR amplification (qPCR) were performed as
previously reported (Allender et al., 2015). Briefly, qPCR was performed in triplicate on an ABI
7500 real time thermacycler. Samples were considered positive if all three replicates had a lower
cycle threshold (Ct)value than the lowest detected standard dilution.
STATISTICAL ANALYSIS.–Prevalence of O. ophiodiicola was estimated in Eastern
Massasaugas by calculating the 95% binomial confidence interval (Wilson, 1927). We calculated
the mean, median, standard deviation and range for each morphological parameter measured
above. To assess the normality of data we used a Shapiro-Wilk test. For normally distributed
data, we used ANOVA to test if there was a difference between group (by sex, age class,
location, and disease status) then used a Tukey’s test to determine within group differences. For
non-normally distributed data, we used a Kruskall-Wallis test for between group differences, and
a Mann-Whitney U for within group differences. We used Fisher’s exact test to determine if sex,
age class, or location was significantly associated with disease status. We assessed statistical
significance at α=0.05 and conducted all statistical analyses using IBM Statistics 22.0 (SPSS
Inc., Chicago, IL).!
GENERAL SURVEY RESULTS.– A total of 112 swabs were collected from 100 Eastern
Massasaugas in 2014 from three sites in Michigan. There were 25 individuals sampled at the
ELF, 34 at CG, and 41 at PCCI. In adults, SVL (mean: 53.2 cm; SD: 5.3) was not significantly
different between sites (p=0.449). Similarly, there was no difference in adult mass (mean:
214.5g; SD: 76.5; p=0.07) between sites. Each site sampled the ratios of males and females
(p=0.140) and adults and juveniles (p=0.601) equally, despite a trend of more females at ELF
OPHIDIOMYCES DETECTION.– Skin lesions were observed at each site, with an overall
prevalence of 13.3%, but the number of individuals with lesions was not significantly different
between sites (p=0.274; Table 1). Ophidiomyces DNA was detected in samples from snakes at
each of the three sites (Table 1), but with no difference in prevalence between sites (p=0.578).
Presence of dermatitis (Figures 1-4) was significantly associated with presence of Ophidiomyces
detection (p=0.014). A total of five positive samples were detected, with two each at ELF and
CG and one at PCCI. Confidence intervals for prevalence were broad at all three sites (Table 1).
Fungal copy numbers in the five positive snakes ranged from 11 – 308 copies/qPCR reaction,
with no significant difference between sites (p=0.121).
There was no significant difference in Ophidiomyces prevalence between males (n=3; 6%) and
females (n=2; 4.7%; p=0.572) or between age classes (p=0.573), despite all positive snakes being
adults. Similarly, snakes that tested positive or negative for SFD were not significantly different
in SVL (p=0.326) or mass (p=0.843).
The emergence of SFD mortalities has potentially serious consequences for the viability of the
Eastern Massasauga in Michigan. In 2013, two cases of SFD were confirmed at the Camp
Grayling site through diagnostic testing of clinically ill snakes and resulted in death for both
individuals. In 2014, prospective sampling revealed that SFD was present at two additional
Michigan sites and persisted at Camp Grayling . The overall prevalence of the three sites was
5.0%, but the true prevalence lies anywhere between 2 and 11%, which is consistent with
previous reports in this species using qPCR(Allender et al., 2011; Allender et al., 2015). There
was no significant difference in prevalence between sites, but this may be a related to lack of
statistical power, subtle differences in swabbing techniques between sites, or differences in
detection rates among sites between infected and uninfected snakes, rather than a biological
SFD lesions in Eastern Massasaugas are associated exclusively, to date, with lesions of the skin.
Skin lesions were not uncommon in this study (13.3% of all sampled snakes), and three of the
five positive snakes had associated clinical signs. Twenty-three percent of snakes with clinical
signs had detectable Ophidiomyces, indicating snakes that are demonstrating clinical signs have a
greater chance of having the causative agent of SFD. However, the lack of clinical signs does not
preclude infection and surveillance studies should continue to survey all snakes in a population.
The two snakes with no clinical signs in this study also had the lowest fungal copy number. It is
possible that these were early infections that had not yet elicited clinical signs. Following these
individuals over time may reveal the development of clinical SFD symptoms. Additionally, 10
individuals had dermatitis, but tested negative. This may demonstrate that swabs and qPCR may
underestimate the true prevalence. This has been proposed previously, as this fungus is a tissue
fungus and aggressive swabs or biopsies may be needed to confirm diagnosis. However, a recent
study in experimentally challenged animals demonstrated that swabs were able to detect
Ophidiomyces DNA in all snakes that had visible fungi on histopathology (unpub data). The
higher prevalence of dermatitis than Ophidiomyces may represent other processes, injuries, or
pathogens are playing a role in these lesions. Further emphasizing the need to test and confirm
the causative agent. Continued work is needed to determine the pathogenesis in order to
determine how fungal numbers influence clinical signs and progression of disease.
In addition to direct mortality from infection, O. ophiodiicola may also indirectly cause
morbidity through behavioral changes, decreases in overall health, or reduced reproductive
effort. In reptiles, metabolic rate is directly tied to body temperature (Kleiber, 1971).
Immunosuppressed individuals may spend extended periods basking to control pathogens by
inducing a fever response (Vaughn, 1963). Prolonged periods spent basking result in higher
metabolic rate and faster consumption of resources. More time spent basking also reduces the
amount of time the individual can spend on other activities such as foraging or mate searching
(Hertz et al., 1988). Increased metabolic rate combined with less time spent foraging can lead to
severe declines in body condition or death if body condition is already poor. These indirect
mortalities are difficult to quantify, as we are unlikely to recover the bodies for disease
screening. Thus, prevalence estimates will likely be underestimated unless this undetected
proportion of the population is accounted for.
SFD epidemiologic investigations have required a collaborative effort between biologists,
veterinarians, and land managers; however, it is not the only threat, and may not even be the only
disease facing the Eastern Massasauga. Wildlife diseases have been increasingly more important
for wildlife populations and public health. The need to detect early, or, ideally, to prevent the
next disease event, has never been more important, as 60% of emerging diseases are zoonotic.
Wildlife are commonly associated as reservoirs for these diseases, with recent examples of Nipah
virus, avian influenza, and West Nile virus (Randall et al., 2012; Daszak et al., 2013; Olson et
al., 2013). Future health assessments, pathogen detection, and assessment of contaminant
exposure in these Eastern Massasaugas populations may allow us to identify trends and new
CONCLUSIONS AND RECOMMENDATIONS.– Eastern Massasauga populations in Michigan
are under several potential threats including habitat loss, road mortality, and disease. In general,
as habitat loss occurs, populations are forced into smaller areas, which increase potential disease
transmission and environmental persistence. Future efforts to curb a disease epidemic may
require aggressive intervention and therapy, and efforts are underway to identify therapeutic
options. The approaches to wildlife diseases have historically been to describe outbreaks rather
than to manage or prevent them. However, management and prevention is the only way to
proactively address these emerging issues. To mitigate the mortalities caused by ongoing
infections, an understanding of the natural disease ecology of this fungus is needed. This should
be multifaceted, but can be initiated with environmental sampling (through eDNA), radio-
telemetry or capture-recapture studies of known infected populations, habitat quality and
composition assessments that exist in known Ophidiomyces areas, and following appropriate
biosecurity protocols. The detection of this pathogen in Michigan indicates that field protocols
and procedures should be required for each site. While this surveillance did not address the
disinfection, continued efforts and dialogue are needed at each site to develop appropriate,
practical, and effective plans that minimize disease transmission.
It is imperative for land managers to document the extent of pathogens across the landscape so
that future efforts may focus on environmental control. Furthermore, sick or dead animals with
associated clinical signs should be evaluated and tested for this pathogen. Developing a proactive
approach to dealing with this disease will require an innovative and multi-disciplinary team to
develop management plans to maintain the health of the population, mitigate current disease
threats, and prevent the next major disease threat.
We stress the importance of continuing annual monitoring programs to document both
population size and disease prevalence. Future work with these data will include looking for
temporal trends in health parameters, linking health data to body condition indices for individual
snakes, and conducting a “hotspot” analysis to examine health on a landscape scale. The
presence of abnormalities in a single year gives a “snapshot” indication of individual health at a
single point in time; However, monitoring populations through time may allow for the early
detection of deteriorating population health and identification of possible mechanisms for the
emergence of SFD.
We thank all the staff of the Edward Lowe Foundation, Pierce Cedar Creek Institute, and Camp
Grayling as well as the many students and volunteers that assisted in snake capture, sample
collection, and processing.
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Table 1: Descriptive statistics for Ophidiomyces surveillance in eastern from Michigan in 2014
2.2 - 25.0%
1.6 - 19.1%
0.4 - 12.6%
! ! ! ! !
Figure 1. Eastern massasauga with a skin lesion identified as positive for Ophidiomyces by
Photo credits to Jennifer Moore
Figure 2. Eastern massasauga with a skin lesion identified as positive for Ophidiomyces by
Photo credits to Jennifer Moore
Figure 3. Eastern massasauga with a skin lesion identified as positive for Ophidiomyces by
Photo credits to Sasha Tetzlaff
Figure 4. Eastern massasauga with a skin lesion identified as positive for Ophidiomyces by
Photo credits to Sasha Tetzlaff