The diamondback terrapin (Malaclemys terrapin) is a coastal turtle with a range from Massachusetts to Texas and is the only exclusively brackish water turtle in North America. Two populations of wild terrapins from Maryland (n=55) and Georgia (n=7) were examined and tested for potential reptile pathogens. Whole blood and a mucosal (combined oropharyngeal and cloacal) swab from each animal were evaluated by quantitative PCR for 15 potential pathogens including frog virus 3, box turtle Mycoplasmopsis, Mycoplasma agassizii, Mycoplasma testudineum, Salmonella Enteritidis, Salmonella Typhimurium, Borrelia burgdorferi, Anaplasma phagocytophilum, tortoise intranuclear coccidia, testudinid alphaherpesvirus 2, terrapene herpesvirus 1, and terrapene adenovirus. Swabs were positive for a DNA segment 100% homologous to M. testudineum in both populations, with Maryland animals 87% (48 of 55) positive and Georgia animals 86% (6 of 7) positive. Although Mycoplasmopsis spp. are important respiratory pathogens for members of the order Testudines, none of the animals in the study showed any sign of upper respiratory disease. Our data suggest that M. testudineum may survive in non-Testudinidae turtles without causing clinical sigs of disease and suggesting appropriate precautions should be taken in facilities that house multiple species of turtles simultaneously.

The diamondback terrapin (Malaclemys terrapin) lives within the brackish-water marsh environments along the eastern coast of the US, plus there is a small population in Bermuda (Roosenburg and Kennedy 2018). Terrapins are listed as lower risk and near threatened on the International Union for Conservation of Nature red list of threatened species (Roosenburg et al. 2019). Major historical and current sources of mortality in diamondback terrapins are anthropogenic (Roosenburg 1991). In the 19th and early 20th centuries, the largest cause of mortality was commercial harvest (Kennedy 2018). Currently, the most significant threats to the terrapin are habitat destruction, drowning in crab pots (Roosenburg et al. 1997), and motorized vehicle strikes from boats for all terrapins (Roosenburg 1991) and from cars for nesting females (Wood and Herlands 1997; Butler and Roosenburg 2018). Infectious diseases are not known as a risk for the species, and previous studies of terrapins in the wild and under human care have found few bacterial pathogens (Werner et al. 2002). However, infectious diseases are increasingly becoming a concern for chelonian populations throughout the world, with multiple documented outbreaks in both wild populations and those under human care (McLaughlin et al. 2000; Brown et al. 2004; Feldman et al. 2006; Marschang 2011; Allender et al. 2013; Sim et al. 2016).

A population of diamondback terrapins around Poplar Island (PI) in Sherwood, Maryland, has been intensively studied, with thousands of animals individually identified and monitored since 2002. This population has high nesting success and recruitment (Roosenburg et al. 2014), and the juveniles in the island's protected marshes have high survival rates (W.M.R. pers. comm.), indicating that this population is growing and robust (Converse 2016). The health of this population, combined with its long-term monitoring, suggest it is an appropriate wild reference population. The second study group consisted of animals being rehabilitated following automobile trauma, at the Georgia Sea Turtle Center (GSTC) on Jekyll Island, Georgia. Our goals were to identify pathogens present in the terrapins, and to compare prevalence between wild populations from different geographic locations.

Animals

A total of 55 terrapins (39 females, seven males, and 10 juveniles, with one animal initially classified as a juvenile before later being reclassified as female) from PI were collected from near-shore brackish waters around the island as part of an ongoing population study. Animals were collected and sampled for 2 d in 2015 and 1 d in 2016. In 2015, 14 terrapins were captured, and in 2016, 43 were captured, with two animals captured in both years. Nets and traps were set and checked daily to remove and sample terrapins. Animals were taken to a field laboratory on PI, where they were identified, measured, and received examinations prior to sample collection. All animals were processed, sampled, and returned to the area where they were captured within 8 h.

Sex of terrapins was determined using secondary sexual characteristics, primarily the size of the tail and the position of the cloaca relative to the posterior edge of the carapace. Any individual over 750 g was considered female, because of the sexual dimorphism in size of terrapins. Smaller, younger (<3–4 yr) individuals that were not sufficiently sexually dimorphic were considered juveniles. Age was determined by counting growth annuli (keratinized growth rings) from the plastral scutes either at the time of capture or extracted from the database for older, no longer ageable, recaptures. Age was not determined for individuals initially captured when they no longer had annuli.

Seven female terrapins hit by automobiles on Jekyll Island, Georgia, were brought to the GSTC for treatment of traumatic injuries. At the time of sampling in 2015, all animals had completed rehabilitation and were considered healthy and ready for release.

All work on PI was approved by the Institutional Animal Care and Use Committee through protocol 13-L-023 under W.M.R. at Ohio University prior to the initiation of sampling. Scientific collecting permit 53958 issued by the Maryland Department of Natural Resources covered terrapin captures at PI. Work with the Jekyll Island animals was performed under the Georgia Department of Natural Resources scientific collecting permit 29-WJH-15-161 and wildlife rehabilitation permit S4-WJH-15-88 under customer number 19717.

Sample collection

Blood from the study animals was collected to examine clinicopathologic parameters; we took the opportunity in this study to additionally evaluate blood for the presence of pathogens. Blood was collected under manual restraint from the cranial subcarapacial sinus using 22-ga, 2.5-cm (1-in) needles and 3-mL syringes (Covidien Monoject, Medtronic, Minneapolis, Minnesota, USA) and stored in lithium heparin anticoagulant vials (Sarstedt Ag and Co., Nümbrecht, Germany). Sterile swab samples were taken from each animal, collecting a single pooled oropharynx and cloacal sample. All samples were stored on cold packs for a maximum of 8 h until transfer to a –80 C freezer at GSTC or the National Aquarium in Baltimore, Maryland, for <48 h and shipped to the University of Illinois, College of Veterinary Medicine, in Urbana, Illinois.

Sample processing

We extracted DNA independently for whole blood and the oropharyngeal-cloacal swab samples with a DNeasy Blood & Tissue Kit (Qiagen Inc., Redwood City, California, USA), following the manufacturer's protocol with the following exception: 50 µL of whole blood was used instead of the recommended 15-µL volume for samples with nucleated red blood cells. The larger volume was used to increase sensitivity. Quantity (nanogram per microliter) and quality (A260:A280 ratio) of DNA was evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, Massachussetts, USA). Quantitative PCR (qPCR) was performed in a multiplex format to evaluate 15 pathogens (frog virus 3, box turtle Mycoplasmopsis, Mycoplasma agassizii, Mycoplasma testudineum, Salmonella Enteritidis, Salmonella Typhimurium, Borrelia burgdorferi, Anaplasma phagocytophilum, tortoise intranuclear coccidia, testudinid alphaherpesvirus 2, terrapene herpesvirus 1, and terrapene adenovirus) simultaneously, using published or in-house primer-probe assays (Table 1). Specific target amplification was performed on each sample with pooled pathogen TaqMan assays and PreAmp Master Mix (Thermo Fisher Scientific). Each reaction was performed under the following cycling program on the GeneAmp PCR System 2400 (PerkinElmer, Foster City, California, USA): 95 C (10 min), 14 cycles of 95 C (15 s), and 60 C (4 min). The qPCR assay was then performed in triplicate using 2.25 µL of amplified DNA from the first reaction on a Fluidigm 96.96 Gene Expression Integrated Fluid Circuits and amplified on the Fluidigm Biomark HD Real-Time PCR thermocycler (both Fluidigm, San Francisco, California, USA) using the following cycling protocol: 70 C (30 min), 25 C (10 min), 95 C (1 min), followed by 35 cycles at 96 C (5 s), and 60 C (20 s). Serial dilutions of positive controls for FV3-like ranavirus, terrapene herpesvirus 1, box turtle Mycoplasmopsis, terrapene adenovirus 1, M. agassizii, and M. testudineum were prepared from 107 to 101 copies per reaction. A non-template control was included on each plate. All reactions were then analyzed using Fluidigm Real-Time PCR analysis software to determine the number of copies of pathogen DNA recovered from each sample.

Table 1

Quantitative PCR primers and probes (TaqMan, Thermo Fisher Scientific, Waltham, Massachussetts, USA) used to determine the prevalence of pathogens and copathogens in diamondback Terrapins (Malaclemys terrapin) from Maryland and Georgia, USA, in 2015 and 2016.

Quantitative PCR primers and probes (TaqMan, Thermo Fisher Scientific, Waltham, Massachussetts, USA) used to determine the prevalence of pathogens and copathogens in diamondback Terrapins (Malaclemys terrapin) from Maryland and Georgia, USA, in 2015 and 2016.
Quantitative PCR primers and probes (TaqMan, Thermo Fisher Scientific, Waltham, Massachussetts, USA) used to determine the prevalence of pathogens and copathogens in diamondback Terrapins (Malaclemys terrapin) from Maryland and Georgia, USA, in 2015 and 2016.

Statistical analysis

We compared number of individuals infected versus uninfected between the two populations using a Fisher exact test. For the PI population only, we used logistic regression to evaluate the effect of sex and year on M. testudineum infection status (positive or negative) of individuals. We tested qPCR reaction counts for normality of with the Shapiro-Wilk test. To compare M. testudineum counts among sex and year, we used a Scheirer-Ray-Hare nonparametric two-way analysis of variance (ANOVA), followed by the Mann-Whitney U-test to compare between individual groups. All statistical analysis was performed using R software (R Core Team 2021). The Scheirer-Ray-Hare test was run using the package rcompanion version 2.4.13 (Mangiafico 2022).

Using qPCR, we detected only M. testudineum in 56 of 64 of the pooled oropharyngeal-cloacal swabs (Table 2). We did not detect any DNA of the other pathogens for which we had primers. No DNA from any pathogen was observed in the whole blood samples from any animal. The two terrapins from PI caught both in 2015 and 2016 tested positive both years. One was initially identified as a juvenile in 2015 but later classified as female in 2016 when it was old enough to determine sex. The second animal was identified as female in both years.

Table 2

Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamond-backed terrapins (Malaclemys terrapin) sampled on Poplar Island (PI), Maryland, USA, and at Georgia Sea Turtle Center (GSTC), Jekyll Island, Georgia, USA, in 2015 and 2016.

Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamond-backed terrapins (Malaclemys terrapin) sampled on Poplar Island (PI), Maryland, USA, and at Georgia Sea Turtle Center (GSTC), Jekyll Island, Georgia, USA, in 2015 and 2016.
Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamond-backed terrapins (Malaclemys terrapin) sampled on Poplar Island (PI), Maryland, USA, and at Georgia Sea Turtle Center (GSTC), Jekyll Island, Georgia, USA, in 2015 and 2016.

We did not detect a difference in the proportion of individuals infected between PI and the GSTC (Fisher exact test; P>0.05; Table 2), suggesting that infections by M. testidineum remain high across a substantial portion of the terrapin's range. We did not notice any adverse effects associated with the high prevalence of M. testidineum, suggesting that the bacteria may naturally survive in terrapins without causing clinical signs of disease. The logistic regression model with the best fit (Akaike information criterion [AIC] 25.202) was a sex-only model, but the sex effect was not significant (Z=0.007; P>0.05). The next best model included sex and year (AIC, 27.106), but again the factors in the model, year (Z=0.0; P>0.5), and sex (Z=0.0; P>0.5) were not significant, suggesting that neither model accounted for significant variation in the data. We pooled our adults to investigate potential differences in the number of individuals infected by age (adults versus juveniles), and discovered that juveniles had lower infection rates than adults (Tables 3 and 4). Logistic regression with the best model fit included only age (AIC, 27.139) and age was significant in this model (Z=–3.53; P<0.01). The second-best model included year and age (AIC, 29.066), but only the age term was significant (Z=–3.22; P>0.01), indicating that both models were similar in only detecting an age effect and that there was no year effect. Summary statistics for proportion of M. testudineum animals based on sex, age, and year are listed in Tables 3 and 4.

Table 3

Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamondback terrapins (Malaclemys terrapin) by sex, sampled on Poplar Island, Maryland, USA, in 2015 and 2016. One animal was sampled as a juvenile in 2015 and adult female in 2016, resulting in combined juvenile and adult counts adding to 56 despite only 55 total animals being sampled.

Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamondback terrapins (Malaclemys terrapin) by sex, sampled on Poplar Island, Maryland, USA, in 2015 and 2016. One animal was sampled as a juvenile in 2015 and adult female in 2016, resulting in combined juvenile and adult counts adding to 56 despite only 55 total animals being sampled.
Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamondback terrapins (Malaclemys terrapin) by sex, sampled on Poplar Island, Maryland, USA, in 2015 and 2016. One animal was sampled as a juvenile in 2015 and adult female in 2016, resulting in combined juvenile and adult counts adding to 56 despite only 55 total animals being sampled.
Table 4

Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamondback terrapins (Malaclemys terrapin) sampled on Poplar Island, Maryland, USA, comparing 2015 and 2016. Two animals were sampled in both 2015 and 2016, with both animals being positive for M. testudineum in both years.

Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamondback terrapins (Malaclemys terrapin) sampled on Poplar Island, Maryland, USA, comparing 2015 and 2016. Two animals were sampled in both 2015 and 2016, with both animals being positive for M. testudineum in both years.
Prevalence of Mycoplasmopsis testudineum determined by quantitative PCR in diamondback terrapins (Malaclemys terrapin) sampled on Poplar Island, Maryland, USA, comparing 2015 and 2016. Two animals were sampled in both 2015 and 2016, with both animals being positive for M. testudineum in both years.

We did detect effects of year and sex on the number of copies of M. testudineum DNA (Scheirer-Ray-Hare two-way nonparametric ANOVA: sex, H=11.00, P<0.01; year H=14.16, P<0.01) but failed to detect an interaction effect (year × sex, H=0.42, P>0.05). Copies of M. testudineum for PI animals were higher in 2016 (median 201,105 per reaction) than 2015 (median 2,890 per reaction; P<0.01). Further analysis using the Mann-Whitney U-test revealed that copies of M. testudineum were significantly lower in juveniles (median 0 per reaction) than adult females (median 172,767 per reaction; P<0.01) and adult males (median 156,410 per reaction; P<0.05) for PI animals. No statistical difference was observed in copies of M. testudinatum between female and male animals (P>0.05). Summary statistics for qPCR-measured viral loads for each group are listed in Table 5.

Table 5

Viral loads (mean, median, and standard error of number of DNA copies) of Mycoplasmopsis testudineum found by quantitative PCR in diamondback terrapins (Malaclemys terrapin) from Poplar Island (PI), Maryland, USA, and the Georgia Sea Turtle Center (GSTC), Jekyll Island, Georgia, USA, sampled in 2015 and 2016. Animals are grouped based on location, sex, and year of sampling. Only data from PI animals are displayed in the sex and year sections. Sample numbers for sex and year add to 57 because two animals were sampled in both 2015 and 2016, with one animal characterized as juvenile in 2015 and female in 2016, and the second animal female in both years.

Viral loads (mean, median, and standard error of number of DNA copies) of Mycoplasmopsis testudineum found by quantitative PCR in diamondback terrapins (Malaclemys terrapin) from Poplar Island (PI), Maryland, USA, and the Georgia Sea Turtle Center (GSTC), Jekyll Island, Georgia, USA, sampled in 2015 and 2016. Animals are grouped based on location, sex, and year of sampling. Only data from PI animals are displayed in the sex and year sections. Sample numbers for sex and year add to 57 because two animals were sampled in both 2015 and 2016, with one animal characterized as juvenile in 2015 and female in 2016, and the second animal female in both years.
Viral loads (mean, median, and standard error of number of DNA copies) of Mycoplasmopsis testudineum found by quantitative PCR in diamondback terrapins (Malaclemys terrapin) from Poplar Island (PI), Maryland, USA, and the Georgia Sea Turtle Center (GSTC), Jekyll Island, Georgia, USA, sampled in 2015 and 2016. Animals are grouped based on location, sex, and year of sampling. Only data from PI animals are displayed in the sex and year sections. Sample numbers for sex and year add to 57 because two animals were sampled in both 2015 and 2016, with one animal characterized as juvenile in 2015 and female in 2016, and the second animal female in both years.

Of the pathogens for which we tested, only Salmonella spp. have previously been reported in the diamondback terrapin (Otis and Behler 1973; Harwood et al. 1998; Werner et al. 2002). The diamondback terrapins in this study showed a high prevalence of M. testudineum–like bacteria, and affected animals had a high number of quantitative DNA copies of these bacteria. Interestingly, none of the other potential pathogens tested for were observed in any animal in the study, suggesting that terrapins, in general, have a low pathogen microbiome. We did not observe any clinical signs or adverse effects associated with M. testudineum, suggesting that the bacterium may survive in some species of aquatic turtles without causing harm, or that M. testudineum has coevolved in terrapins and is a symbiont. Furthermore, our data suggest that this Mycoplasmopsis sp. is horizontally transmitted, because younger individuals, juveniles, had both lower prevalence and lower qPCR counts. It is most likely that the bacterium observed was either M. testudineum or a closely related Mycoplasmopsis sp., given the lack of cross reaction with M. agassizii or box turtle Mycoplasmopsis sp. Although our methods could not differentiate between pathogen presence in the oropharyngeal versus the cloacal mucosa, given the use of a single swab for both, it is most likely that M. testudineum was present in the oropharyngeal space, because this is the normal environment for this genus of bacteria (McLaughlin et al. 2000; Brown et al. 2004; Feldman et al. 2006; Lecis et al. 2011; Braun et al. 2014).

Previous studies have found Salmonella spp. in the gastrointestinal flora of both free-living diamondback terrapins and those under human care (Otis and Behler 1973; Harwood et al. 1998), but none were identified in this study. Prevalence of Salmonella spp. in free-living chelonians varies greatly between both species and locations (Readel et al. 2010), with previous studies in terrapins generally showing a low prevalence in feces (Harwood et al. 1998). Sampling method can influence the detection of Salmonella spp. in reptiles, with cloacal swabs potentially underestimating the true prevalence of fecal Salmonella spp. (Harwood et al. 1998). These factors may account for the lack of observed shedding of Salmonella spp. from animals in this study, but is unlikely, given the relatively large total number of animals sampled.

In susceptible chelonian species, Mycoplasmopsis spp. are often associated with upper respiratory disease. Clinical signs generally include oculonasal discharge and conjunctival swelling, although asymptomatic Mycoplasmopsis spp. infections are possible (McLaughlin et al. 2000; Feldman et al. 2006). The PI terrapin population is intensely monitored, and recapture rates exceed 50% annually (W.M.R. unpubl. data), but no clinical signs of upper respiratory disease have been observed in this population. The GSTC animals also received general veterinary care as part of rehabilitation from traumatic injury (T.M.N. pers. comm.). None of the animals sampled showed any signs of upper respiratory disease. For this reason, although specific screening for upper respiratory infections was not performed on any animal, substantial morbidity or mortality as a result of M. testudineum in these diamondback terrapin populations is unlikely. Mycoplasmopsis spp. have been reported to show similar patterns of high prevalence with low to no evidence of pathogenicity in other turtle species in the eastern US, leading to speculation that some of these Mycopolasmopsis spp. may be part of the normal commensal oral flora of some emydid turtle species (Ossiboff et al. 2015). However, histologic study was outside the scope of this study; thus, subtle lesions associated with M. testudineum could easily have been missed.

Given the small sample sizes of some of the individual populations in this study, the observed differences in M. testudineum prevalence and individual animal bacterial load between the two study populations may have been due to random chance or variability in sampling between years. However, some differences could be explained by the expected behavior of M. testudineum. Horizontal transmission would be expected for M. testudineum (McLaughlin et al. 2000; Brown et al. 2004) and would explain the lower qPCR count median M. testudineum copies observed in juveniles than counts in adult females or adult males, due to less time to become infected.

Interestingly, although no difference in the proportion of animals infected were observed between PI and GSTC animals, differences in median copies of M. testudineum per animal were observed between the different locations. Environmental factors may play a role in this difference, with PI and GSTC animals differing in local origin climate and relative anthropomorphic environmental contamination. Another important difference is that GSTC animals received veterinary care prior to sampling, with all animals having received systemic antibiotic therapy during rehabilitation. This might have artificially lowered the bacterial load for M. testudineum in the animals sampled. However, it is also possible that the difference in DNA copies may be attributable to variations in sample collection. Significant further study is needed to determine if the differences in DNA copies of M. testudineum between these populations were due to differences in the sampling procedure or represent true differences in individual bacterial load.

Despite these complications, note that the overall prevalence of M. testudineum was statistically indistinguishable in both populations regardless of the large geographic distance between them. Further study is required before concluding that M. testudineum is truly benign in the diamondback terrapin. Nonetheless, the high prevalence of M. testudineum in free-living terrapins suggests that quarantine of such animals is appropriate and that care be taken to not comingle, in captive settings, such as zoos, aquaria, or other facilities housing a diversity of turtle species, species that may not naturally occur together.

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