# Veterinary medicine in reintroduction and reinforcement of the European pond turtle Emys orbicularis: a review

Carolin Eichert, Kathrin Theissinger, Benoît Quintard, Georges Jean-Yves

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Carolin Eichert, Kathrin Theissinger, Benoît Quintard, Georges Jean-Yves. Veterinary medicine in reintroduction and reinforcement of the European pond turtle Emys orbicularis: a review. BMC Veterinary Research, 2025, 21 (1), pp.668. 10.1186/s12917-025-05097-w. http://hal-05381121

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1 Veterinary medicine in reintroduction and reinforcement of the European pond

2 **turtle *Emys orbicularis*: a review**

3

4 Eichert Carolin₁@, Theissinger Kathrin₂, Quintard Benoît₃*, Georges Jean-Yves₄*

5

6 ₁ Department of Biogeography, University of Trier, 54296 Trier, Germany

7 ₂ Department of Ecology, faculty of Biology, Rheinland-Pfälzische Technische Universität

8 Kaiserslautern-Landau, Germany

9 ₃ Parc Zoologique et Botanique de Mulhouse, 68100 Mulhouse, France

10 ₄ University of Strasbourg, CNRS, IPHC UMR7178, F-67000 Strasbourg, France

11

12 @ corresponding author: caroeichert@t-online.de

13

14 * shared last authors

15

16
17 Abstract

18 The European pond turtle *Emys orbicularis* is among the most endangered reptile species in
19 Europe, facing severe population decline due to habitat loss and environmental degradation.

20 Conservation efforts increasingly rely on translocation-based strategies, including species
21 reintroduction and reinforcement. Veterinary medicine plays a crucial role in ensuring the
22 health and survival of captive-bred and translocated individuals, yet standardized health
23 assessment protocols remain scarce. This review based on 418 publications synthesizes
24 current veterinary practices related to freshwater turtle conservation, focusing on health
25 monitoring before, during, and after translocation. We evaluate key medical concerns across
26 different life stages, including prevention of zootechnical issues, pathogen screening,
27 microbiome health, and adaptation challenges in captive and wild populations. Additionally,
28 we propose standardized health assessment guidelines to improve diagnostic accuracy and
29 long-term monitoring, and provide a global overview of the common health issues as well as
30 normal blood reference values. By fostering transdisciplinary collaboration between
31 veterinarians, researchers and conservationists, this study aims to refine best practices and
32 ensure sustainable, evidence-based conservation efforts for *E. orbicularis* and other
33 freshwater turtle species.

34

35 **Keywords:** Conservation management process, species translocation, contagious disease
36 control, freshwater turtles, health monitoring, captive breeding, quarantine regime

37
## 1. Background

In the context of the ongoing sixth mass extinction crisis, population reinforcements and species reintroductions are increasingly recognized as effective nature-based solutions to mitigate biodiversity loss [1–3]. Reinforcement involves the translocation of individuals to bolster existing local populations [4], whereas reintroduction aims to restore a species to its former natural habitat from which it has disappeared [5]. Both approaches are ex situ conservation strategies whose success depends on the suitability of the release habitat and the ability of translocated individuals to survive and reproduce in the wild after being, in most cases, raised in captivity under artificial conditions [6].

Zoos and breeding facilities, both institutional and private, play a crucial role in such conservation efforts. The European Association of Zoos and Aquaria (EAZA) reported 240 reintroduction or translocation projects involving 156 species across Europe, with zoological parks actively participating [7]. Moreover, zoos provide critical expertise in veterinary science and medicine to ensure optimal health conditions for individuals before and after translocation [8]. Health assessments serve as essential tools integrating veterinary medicine and wildlife ecology to enhance conservation planning and species protection [9]. These assessments provide key indicators at individual, population, and ecosystem levels, aiding in the refinement of management strategies [9–11]. Veterinary monitoring is essential to establishing healthy, self-sustaining populations in both captivity and the wild, while minimizing the risk of infectious disease transmission to newly established and existing populations [12–15]. Additionally, private breeders have developed specialized and effective practices; however, these are often restricted to specific species or not aware of certain health issues concerning the species and are rarely disseminated beyond individual projects or citizen science initiatives [16–19]. As a result, numerous veterinary and husbandry practices exist worldwide, yet their limited dissemination makes it challenging for new projects to identify standardized and appropriate protocols.
A key issue in veterinary medicine is the complex definition of health, especially in turtles and the need of compliance of translocated and recipient populations' health states, making a on-site situation-adjusted approach inevitable in chelonian species [20,21]. At a first stage it is critical to determine a turtles' individual health, which has to be evaluated in the context of external and internal influencing factors like reproductive state, the concomitance of co-infections and cumulation of environmental stressors [22]. Besides, with chelonians as patient with little signs of externally detectable disease often advanced techniques of investigation appear inevitable, which in turn means more intense financial and temporal effort [21]. Even if those additional tests have been made, the results have to be interpreted with caution, as blood parameter shifts can have various physiological reasons or be absent, antibody titers can increase with delay and many latent infectious agents exists [23]. Added to this, for several infectious agents, which continuously emerge newly, molecular diagnostic techniques still have to be adapted and improved for different species [24]. In a next step, the health screening has to be widened to an appropriate number of individuals in a population to define the communities' health state [25]. Finally, the potentially detected health impairments of either the translocated or the receiving group of animals should not harm each others health in terms of facultatively pathogenic infectious agents or non-infectious health issues that become clinically relevant in case of co-morbidities or other health deteriorations that are not clinically aberrant or relevant in captivity only and in the wild respectively (e.g., [26]).

Notably, for chelonian species the health and wellbeing of individuals involved in reintroduction initiatives, needs to be acknowledged to a greater extent than in other species due to their high life expectancy, slow pace of life and reproduction cycle and low effective population sizes inherent to endangered species [27].

This review synthesizes key health concerns and veterinary practices related to translocation-based conservation initiatives for the European pond turtle (*Emys orbicularis*, hereafter referred as *Emys*). Freshwater turtles face significant threats globally, primarily due to habitat loss and degradation of wetlands, their primary habitat [28,29]. *Emys* (Box 1) has
91 experienced one of the most severe declines among European reptiles, reaching extinction

92 or near-extinction in several regions [30,31]. In response, numerous *ex situ* conservation

93 initiatives, including both reinforcements and reintroductions, have been established

94 throughout Europe [32–34]. Both headstarting, where turtle eggs are collected in the wild, so

95 contamination of the captive facilities by theses eggs, as well as contamination of eggs

96 during collection in the wild, but also contamination of captive facilities by those eggs

97 collected in the wild has to be prevented, and captive breeding involving breeder animals

98 held under controlled conditions in captivity and artificial egg incubation are approved

99 procedures (e.g., [35]). Few more steps in preparing and accomplishing headstarting

100 initiatives differ from reintroduction via captive breeding, like the genetic considerations in

101 selection of breeder animals in the wild, the impact of climate conditions on wild populations

102 or urban-wildlife interactions [36].

103 While many of these initiatives include health checks, detailed descriptions of these protocols

104 are often lacking, with most studies limited to biometric assessments [37–39].

105 Based on an extensive assessment of 418 scientific articles published until April 2025, this

106 review aims to: 1) summarize the current literature on health assessments in translocation-

107 based ex situ conservation initiatives focusing on *Emys*, 2) examine the methodologies and

108 specificities of health checks at different life stages of captive-bred freshwater turtles at large

109 and throughout the translocation process, and 3) provide recommendations for future

110 research needed to standardize health assessments before and after translocations, with a

111 specific focus on *Emys*. By compiling and evaluating best practices, this review seeks to offer

112 critical insights for present and future freshwater turtle *ex situ* conservation initiatives.

113

## 2. Freshwater turtle veterinary issues by age class

115 Freshwater turtle reintroductions rely either on headstarting protocols where eggs are

116 collected from the wild for being hatched in captivity for limiting natural mortality, or on
117 captive breeding populations that usually consist in a relatively small pool of breeders. Since
118 headstarting protocols and benefits have been extensively described, especially for the
119 Blanding's turtle ([19,35,40]), this review focuses on protocols based on captive breeding.
120 The key concerns associated with multigenerational captivity include: i) reduced
121 immunogenetic diversity due to potential inbreeding, ii) genetic and behavioural adaptations
122 to captive environments, and iii) the absence of exposure to native or exotic parasites due to
123 the controlled and isolated nature of captive habitats [41]. To address the long-term genetic
124 and behavioural consequences of multigenerational captivity, several management strategies
125 have been proposed. These include conducting comprehensive assessments of genetic
diversity within the captive population to guide the selection of maximally diverse breeding
126 groups [42], as well as promoting the exchange of genetically compatible individuals among
127 institutions to reduce inbreeding and maintain adaptive potential [43]. Here, it is mandatory
128 along the IUCN (International Union for Conservation of Nature) guidelines for species
129 reintroductions [3] to guarantee breeding animals are of the same subspecies (for Emys
130 there are currently 14 subspecies described in Europe) [32], as animals from the receiving
131 population [44,45]. They thus should be pure-bred and not genetically admixed. This allows
132 best adaptability to local environmental conditions and consequently higher chances of
133 persistence of the population in the wild based on historically adapted phenotypic
134 characteristics [44], i.e., overall increased conservation value of the translocation [46].
135 Currently, genetic assignment of subspecies is determined using a mitochondrial sequence
136 marker (cytochrome B) and, because mtDNA is only inherited from females, also using
137 microsatellites which permits to exclude hybrids between subspecies or genetic lineages
138 [45]. Within such a pure genetic lineage, preferably high levels of genetic diversity are
139 desired, holding the opportunity for further adaptation to novel external conditions under
140 global climate change (e.g., [47]). In addition, removing reproductively dominant males –
141 whose disproportionate mating success can skew genetic contributions – has been
142 suggested as a mean to equalize reproductive output. However, this latter approach requires
143 detailed knowledge of individual reproductive success, which can be obtained through either
144
145 genetic parentage analysis or systematic observations of mating behaviour [48–50]. In the
146 following, we present the varying health issues of captive-bred *Emys* per age class.

147

148 2.1. Egg collection and incubation

149 Optimizing conditions for egg collection, incubation, and subsequent hatchling management
150 is critical for ensuring the health and survival of individuals intended for reintroduction. While
151 zootechnical aspects have been addressed in previous studies [51–54], this review focuses
152 on key health-related considerations. First, when collecting eggs from nests, it is essential to
153 preserve the maternal mucus coating on the eggshell, as it plays a vital role in supporting the
154 external microbiota that develops during incubation. This has been demonstrated in
155 freshwater turtles, sea turtles, and lizards [55–58]. Mechanical or chemical cleaning of eggs
156 should therefore be strictly avoided. Second, artificial incubation requires precise control of
157 temperature and humidity to prevent developmental abnormalities. Excessively high
158 temperatures can accelerate embryonic development, leading to morphological defects (e.g.,
159 scutellation abnormalities, missing digits, vertebral deformities, anophthalmia), neurological
160 impairments, and even sterility [59]. Elevated humidity levels can promote mold, fungal, and
161 bacterial infections, which may result in embryonic mortality, immunodeficiencies, and
162 congenital disorders [59–64]. Conversely, insufficient humidity can lead to limb deformities,
163 missing toes, and reduced body size [59]. The effects of fluctuating incubation temperatures
164 remain debated. Some studies suggested benefits for immune function in snapping turtles
165 *Chelydra serpentina* [65], while others reported reduced hatching success in western saw-
166 shelled turtles *Myuchelys bellii* [66]. Notably, freshwater turtles incubated near their optimal
167 temperature range exhibit higher post-release survival rates compared to those incubated at
168 intermediate temperatures [67]. Based on this, we recommend incubating *Emys* eggs in
169 moistened vermiculite at 28–30°C with a nocturnal temperature drop of 4–6°C and
170 maintaining 80–90% humidity [68,69]. Finally, eggs may harbour pathogens (e.g., viruses,
171 bacteria, and parasites such as astigmatic mites [Acaridae]) that remain undetected during
incubation. Pathogens can infiltrate eggshells (e.g., *Salmonella* sp. [70]) or be maternally transmitted (*Testudinid herpesvirus 3*, [71]). For several other bacterial and viral pathogens this route of vertical transmission has also been proposed, however it is not yet proven (e.g., *Nidovirus*, *Adenovirus*, *Turtle Fraser Virus 1* [TFV1], *Bohle iridovirus*, and blood parasites like *Haemogregarina stepanowi* [72–78], *Mycoplasmosis* sp. [73,79]). For the latter pathogen a vertical route of transmission has been described for mammals and birds, so that a potential analogy in reptiles and freshwater turtles becomes more likely [80–82]. To mitigate these risks, we recommend: i) daily inspections during incubation to promptly remove infertile, dead, or decaying eggs; ii) immediate removal of hatchlings; iii) systematic sterilization (e.g., autoclaving) or replacement of vermiculite between incubation cycles (see also [54]).

182

2.2. Health aspects of hatchlings, neonates, and juveniles

Hatching is a critical phase for newborn turtles, presenting challenges in both immunity and nutrition. Immunologically, hatchlings primarily benefit from maternal antibodies provided through the yolk sac [83,84] and from the maternal microbiota deposited on the eggshell during oviposition, which plays a crucial role in immune defence [56,85,86]. Additionally, the incubation substrate influences immune development. Since the turtle immune system is largely adaptive and takes time to fully develop after exposure to specific pathogens [23], young freshwater turtles are particularly vulnerable to infectious diseases (e.g., Stromsland and Zimmerman, 2017 [87], for sea turtles see: Flint et al., 2010 [88]). Rana-, Adeno- and Herpesviruses, secondary bacterial septicaemia (*Aeromonas* spp. and *Pseudomonas* spp.), Mycoplasmosis, and opportunistic fungal (*Saprolegnia* spp., *Aspergillus* spp.), as well as protozoal (*Hexamita* spp., *Eimeria* spp.) and parasitological (Spirurida, Trematoda) infections are considered particularly dangerous for young freshwater turtles [89–93].

196

2.2.1. *Hatching and early developmental issues*
One common issue in neonate freshwater turtles is yolk sac retention. If not fully absorbed, it can lead to infections by opportunistic bacteria (e.g., Aeromonas sp., Citrobacter sp.) or fungi, potentially resulting in septicaemia and systemic infection [59,94]. To prevent infections, hatchlings should be removed from the incubation substrate immediately after complete plastron closure. If the retained yolk appears abnormal in texture or colour, indicating infection, surgical intervention is recommended [59]. A rarer but problematic condition is the persistence of the chorioallantoic membrane (CAM), often associated with the absence of an egg tooth, which can delay hatching significantly [95]. The occurrence of persistent CAM in multiple eggs within a clutch may indicate maternal health problems or genetic factors, potentially influenced by environmental pollutants or hereditary defects [96-98].

### 2.2.2. Growth and metabolic disorders

Continuous growth during the first year of life is considered to be an indicator of good health and should be ensured. Growth is primarily influenced by temperature (through ectothermy), natural light (for vitamin D synthesis and bone mineralization), and diet [86,99-101]. A common health issue in young freshwater turtles is nutritional secondary hyperparathyroidism, a metabolic bone disease (MBD) caused by calcium deficiency or inadequate UV-B exposure [102,103]. Symptoms include paralysis, muscle tremors, shell deformations, and pathological fractures [104]. Hypocalcaemia can also lead to prolapses (e.g., cloacal or urogenital prolapse), while in adult females, it may result in egg-laying issues such as follicular stasis or dystocia [105]. Preventive measures include providing a near-natural diet with adequate calcium supplementation (e.g., cuttlebone) while maintaining an optimal calcium-to-phosphorus ratio [106]. Daily exposure to appropriate UV-B light (290-320 nm wavelength) is strongly advised [107]. UV-A light (320-400 nm wavelength) is also required for freshwater turtles kept indoor, as it contributes to the Previtamin D3 synthesis, regulates circadian rhythm and therefore reproduction along with positive effects on
225 phenotype and behaviour [108–110]. Another potential cause of MBD is impaired calcium
226 absorption due to intestinal disorders caused by high endoparasite loads, which increase
227 calcium consumption [111]. Other dietary inhibitors of calcium absorption include phytates,
228 oxalates, high-fat diets, and high dietary acid contents [112]. Diagnosis is based on
229 anamnesis, clinical examination, and blood calcium levels (<2.4 mg/dL or <0.6 mmol/L;
230 reference range: 1.3–2 mmol/L) [113,114]. In addition to infusion therapy (hereafter fluids),
231 vitamin, and mineral supplementation, husbandry conditions should be corrected, and
232 parasitic infections treated accordingly.

233 In detail, the species-specific environmental temperature should be appropriately adjusted
234 along seasonal and daily variations (generally preferred optimal temperature zone between
235 24°C and 28°C [115]; details for *Emys* temperature recommendations in [116]), as it
236 influences (calcium) metabolism via vitamin D synthesis [117]. Basking in UV-B light (see
237 also [69]) should be enabled for the conversion of pre-vitamin D3. UV-B lightning and
238 sufficient humidity should be maintained to promote calcification during neonate development
239 [117].

240

241 **2.2.3. Parasitic load and hygiene**

242 Young freshwater turtles are more susceptible to high endoparasite loads compared to
243 adults, similar to other testudines [118–120]. Clinical signs of endoparasitic infections include
244 inappetence, regurgitation, diarrhea, intussusception, weight loss, and cloacal prolapse [59].
245 Diagnosis should be confirmed via microscopic faecal analysis [121,122] to ensure
246 appropriate treatment (see Supplementary Table 1). To reduce parasitic risks, good hygiene
247 and the avoidance of overcrowding are recommended [123,124]. Implementing these
248 measures helps optimize the health of hatchlings and juveniles, ensuring better survival rates
249 and preparing them for eventual reintroduction into the wild.

250
251 2.3. Freshwater veterinary issues in all age classes

252 2.3.1. *Endoparasites*

253 A comprehensive overview of parasitic infections in European freshwater turtles is provided
254 in Supplementary Table 1. A recent global review on infectious agents in freshwater turtles
255 identified Platyhelminthes (n = 719), Trematoda (n = 440), Monogenea (n = 272), and
256 Cestoda (n = 5) as the most common endoparasites [125]. Studies on free-living

257 Mediterranean pond turtles *Mauremys leprosa* have documented helminths (Oxyuroidea and
258 Ascarididae) and protozoa (Entamoebidae) [122], while Trematoda, such as Spirorchiidae
259 (blood flukes, which can cause peracute death) and Polystomoidea (which attach to mucosal
260 surfaces in various body cavities) have been recorded in *Emys* [126,127]. Additionally,
261 species like *Trachemys venusta venusta* in Mexico have been found to host Ascarididae,
262 Ancylostomatidae, Physalopteridae, and Trichuridae [128], with potential spillover and host-
263 switching events occurring in translocated individuals of native European species [129].

264 Haemoparasitic infections, particularly *Haemogregarina stepanowi*, are commonly reported
265 in *Emys* but are often subclinical [130,131]. In contrast, intranuclear coccidiosis in tortoises
266 (TINC) remains rare and has not yet been documented in wild European turtle populations
267 [132,133].

268 Parasites can cause localized or systemic infections, affecting the digestive tract, lungs,
269 heart, liver, bile ducts, gallbladder, connective tissue, muscles, kidneys, urinary bladder, or
270 blood. Host susceptibility depends on several factors, including immune status, co-infections,
271 and environmental stressors such as inadequate husbandry or overcrowding. For example,
272 *Spirorchis* sp. infections may remain asymptomatic but can also be fatal [126]. Similarly,
273 *Myxidium* sp. infections can lead to severe necrotizing nephritis, while nematodes such as
274 *Serpinema rispinosus* and *Siroxys contorta* can cause granulomas in the small intestine
275 [123,134–139].

276
277 **2.3.2. Ectoparasites**

278 Ectoparasitic infestations in freshwater turtles are relatively rare, with leeches of the genus

279 Placobdella being the most frequently observed, also in *Mauremys leprosa* [140]. High

280 infestation levels, such as those caused by *Placobdella costata*, can lead to lethargy, circular

281 skin lesions, anaemia, restlessness, and restricted limb and tail mobility in *Emys* [141]. Ticks,

282 such as those of the genus Amblyomma, have been recorded in Colombian freshwater

283 turtles of the genus Rhinoclemmys [142], though reports of pathological effects are scarce.

284 However, these ectoparasites may serve as vectors for haemoparasites [143].

285 To effectively manage parasitic infections, proper species identification and post-treatment

286 monitoring are essential. Diagnostic methods typically include light microscopy of fresh

287 faecal samples or cloacal flushes using direct smear, flotation, and sedimentation

288 techniques, both before and after treatment. All infected individuals within the same

289 enclosure or water circulation system should be treated simultaneously, and husbandry

290 conditions must be optimized to prevent reinfection. This includes thorough cleaning of

291 enclosures and equipment, water and substrate replacement, and increased water changes

292 following treatment to eliminate medication residues and parasitic debris [144]. Infected

293 individuals should be quarantined and managed according to established protocols (see

294 Section 6.1).

295

296 **2.3.3. Viruses**

297 Herpesvirus infections have been documented in various freshwater turtle species, including

298 *Chelonid Herpesvirus 2*, 3, and 4, as well as *Glyptemys herpesvirus 1* and 2 [145,146], and

299 *Emydoidea herpesvirus 1* in *Emydoidea Blandingii* [147]. *Emydid herpesvirus 1* is particularly

300 relevant for *Emys* [148], with additional reports of uncharacterized herpesvirus strains

301 (*Emydid herpesvirus 2*) in Pacific pond turtles *Actinemys marmorata*, painted turtles

302 *Chrysemys picta*, and map turtles *Graptemys pseudogeographica* [138,146,149–152]. In
303 many cases, chronic herpesvirus infections remain asymptomatic or present with stomatitis,
304 rhinitis, blepharedema, subcutaneous oedema, lethargy, and anorexia. However, severe
305 cases can result in lethal lesions in the liver, kidneys, and spleen, as seen in *Graptemys* sp.
306 [78].

307 Similar to fibropapillomatosis in marine turtles, which is caused by Herpesviruses,
308 Siadenovirus has been implicated in lymphoid leukaemia in Sulawesi turtles *Indotestudo
309 forsteni* [153]. Additionally, a novel herpesvirus strain (*Emydoidea herpesvirus 2*) was
310 detected in a wild Blanding's turtle *Emydoidea blandingii* with concurrent squamous cell
311 carcinoma [154] (see also Section 3.4 Neoplasia). Moreover, as Herpesviruses tend to cause
312 more severe clinical diseases in aberrant hosts [155] an infection with *Trachemys*
313 herpesvirus 1 in case of shared enclosures or water pipelines, as well as contact with
314 infected invasive Trachemys turtles in wild environments represent potential sources of
315 infection and clinical disease [156].

316 Adenovirus infections have been associated with cachexia, anorexia, lethargy, ulcerative
317 stomatitis, nasal and ocular discharge, and diarrhea, with survival rates influenced by
318 environmental temperature [157,158] and have also recently been investigated in *Emys*
319 (Marschang et al., 2025 published [159]). A novel Adenovirus strain, related to *Sulawesi*
320 tortoise adenovirus 1, was identified in ornate box turtles *Terrapene ornata ornata* from
321 Hungary [160]. *Sulawesi tortoise adenovirus 1* infections have recently been reported in
322 multiple turtle species (Blandings' turtle, painted turtle, red-eared slider (*Trachemys scripta*
323 elegans) [161].

324 Indeed, different Adenoviruses like Box turtle Adenovirus 1 have next to ornate box turtles
325 (*Glyptemys muhlenbergii*), [162] been detected in Blanding's turtles and red-eared-slider, so
326 it is likely not a host-adapted adenovirus [158], which might point to the general potential of
327 host-jump.
328 Ranaviruses (family Iridoviridae) generally induce severe systemic effects, including
329 cutaneous and mucosal ulceration, skin abscesses, stomatitis, pharyngitis, glossitis,
330 tracheitis, pneumonia, hepatitis, splenomegaly, conjunctivitis, esophagitis, enteritis,
331 pancreatitis, vasculitis, and cystitis [132,163–165]. *Ranavirus rana* 1, a Ranavirus
332 subspecies, has been linked to cutaneous abscesses, oral ulceration, respiratory disorders,
333 anorexia, and fatalities in captive Eastern box turtles *Terrapene carolina carolina* [166].
334 Furthermore, co-pathogens, such as *Terrapene Herpesvirus 1*, Adenovirus, *Mycoplasmopsis*
335 sp., and polymicrobial necrotizing bacterial infections, were identified as contributors to
336 morbidity and mortality events in wild *T. c. carolina* populations in Illinois [167]. For viral
337 detection usually PCR from oral-cloacal swabs and or blood samples are favoured [168].
338 *Turtle fraservirus 1 (Tosoviridae)* has been detected in Florida softshell turtles (*Apalone*
339 ferox), but also Florida red-bellied cooter (*Pseudemys nelsoni*) and Peninsular cooter
340 (*Pseudemys peninsularis*) causing oral and or cloacal plaques, respiratory and neurological
341 disorders, ocular and skin defects and anorexia with a low rate of mortality [76,169].
342 Since specific antiviral treatments are unavailable or need further practical testing (Lysine
343 and Lactoferrin [170]), management strategies focus on addressing co-infections,
344 implementing strict quarantine protocols, and providing supportive care. Fluid therapy (like
345 lactated Ringer's solution with 5% dextrose, for details see e.g., Gibbons, 2009 [171]),
346 assisted feeding (see [172] for feeding tube placement in turtles), prophylactic broad
347 spectrum antibacterial treatment like Enrofloxacin 10 mg/kg sc./im./po. q48; [173]) to prevent
348 secondary septicaemia and antiviral agents such as Valacyclovir (40 mg/kg every 24 hours
349 [174]), Famciclovir (e.g., 20 mg/kg po. q24h [173]), or Acyclovir (≥80 mg/kg q48h [175]) may
350 offer some therapeutic benefit.
351 Many viruses are known to be stable for a long time period, even under extreme climate
352 conditions like freezing and /or are supposed to be shed intermittently (e.g., Ready, 2025
353 [158]), Ranavirus, for example, is suspected to be spread by several pathways including
354 insect vectors and is known to stay in the environment for weeks to months [176,177]. Strict
and appropriate disinfection regime of field items as well as enclosures and equipment used in a captive setting is required (2% chlorhexidine gluconate, soak items overnight in 5% sodium hypochlorite and UV light; [151]). Adenovirus disinfection regime includes the use of formaldehyde, chlorine, bleach, and quaternary ammonium compounds as well as intense UV light [178], Herpesvirus disinfection includes high heat (56°C, 5 - 10 min, pH < 5), or sodium hypochlorite (3%) [151].

361

### 2.3.4. Bacteria

*Mycoplasmosis* sp. is a well-documented pathogen responsible for Mycoplasmosis, which is often triggered by environmental stressors such as cold temperatures, aging, or immunosuppression, particularly in combination with co-pathogens [179]. Initial symptoms include conjunctivitis and rhinitis, which can progress to nasal obstruction, erosion, and depigmentation of the nares, leading to upper respiratory tract disease (URTD [180]; Three-toed box turtle *Terrapene carolina triunguis*). In the USA an *Emydid mycoplasmosis* sp. has been found in wild bog, spotted, and eastern box turtles, representing another subspecies of *Mycoplasmosis*, which was considered as a commensal bacterium in the given setting [181]. Mycoplasmosis infections are typically chronic, with mortality resulting from secondary infections and multimorbidity rather than the pathogen itself [167,182].

Mycobacterial infections, most commonly caused by *Mycobacterium marinum* and *M. chelonae*, lead to chronic granulomatous disease affecting the lungs, coelomic organs, skin, bones, and joints [183]. Acute manifestations include haemorrhagic limb and pulmonary lesions, coelomic effusion, disseminated intravascular coagulation, and nasal bleeding, with lethargy as a common symptom ([184] for Eastern spiny softshell turtle *Apalone spinifera* spinifera; [185] for Eastern box turtle).

*Salmonella* sp., often considered an opportunistic pathogen, has been widely detected in both wild and captive aquatic turtles worldwide and is of significant zoonotic concern due to
381 turtle-associated human salmonellosis (TAHS [186,187]). With up to 85% of chelonians
382 harbouring multiple serotypes [188], turtles can intermittently shed Salmonella without clinical
383 symptoms. However, in some cases, infections result in shell and skin lesions, septicaemia,
384 anorexia, pneumonia, and enteritis [183].

385 *Dermatophilus chelonae* is a common bacterial pathogen affecting turtle skin, leading to
386 cutaneous lesions and abscesses. In severe cases, the infection can spread systemically,
387 causing granulomas in the liver and coelomic cavity as well as septic arthritis [74,183].

388 Macroscopically, lesions present as raised, yellow, hyperkeratotic nodules on the head and
389 neck [189].

390 Although not yet confirmed causing clinical disease neither in wild nor captive freshwater
391 turtles, reports of *Leptospira* sp. infestations from several freshwater turtle species including
392 *Emys* in Europe indicate turtles as potential vectors for the zoonotic infectious agent, thus
393 further investigation regarding the clinical relevance for freshwater turtles becomes apparent
394 [190–193].

395 Routine testing for *Mycoplasmosis* sp. is recommended during pre- and post-release
396 health screenings to prevent disease transmission. For Eastern box turtles in captivity Daleo
397 et al., 2025 [194] recently provided guidelines for optimal pathogen detection via oral-cloacal
398 swabs either monthly in case of shorter stays or in a three month rhythm respectively. This
399 can be assumed for a recommended course of action for other freshwater turtles as well.

400 Mycobacterial and *Dermatophilus* sp. infections should be investigated when skin lesions, or
401 ocular and or nasal discharge, as well as systemic infections are present. Salmonella
402 screening should be prioritized in systemic infections and zoonotic outbreaks but should not
403 replace comprehensive bacteriological and fungal diagnostics in infected wounds or organ
404 systems. As always, antibiotic treatment should be guided by resistance testing, with dosing
405 and duration determined based on the latest literature (e.g., [175]).

406
407 2.3.5. Fungi

408 Mycoses can be classified into superficial dermatomycoses (SD) and visceral or systemic
409 mycoses (SM) (like Phaeohyphomycosis [79,183,195,196]). SCUD-like conditions are
410 referred to as necrotizing scute disease (NSD [197]). Secondary fungal infections, often in
411 conjunction with bacterial infections, may develop in areas where scutes fail to dry
412 completely or have been compromised due to previous trauma [198,199]. In the wild,
413 septicaemic cutaneous ulcerative disease (SCUD) has been associated with environmental
414 pollution or parasites as contributing factors [200–202], whereas in captivity stressors that
415 weaken the local and systemic immune system and therefore promote disease progression
416 are predominantly bad water quality, nutritional imbalance, superficial traumata and
417 overcrowding (e.g., [203]). Fungi such as *Mucor* sp., *Paecilomyces* sp., *Fusarium* sp.,
418 *Ochroconis* sp., and *Exophiala* sp. have been associated with the disease complex
419 [183,204]. Also, bacterial pathogens, like *Citrobacter freundii*, *Pseudomonas* sp. (e.g.

420 *Pseudomonas aeruginosa*) and *Aeromonas* sp. (e.g., *Aeromonas hydrophila*) (e.g.,
421 [203,205]) are commonly involved in the disease complex. Besides, next to algae
422 (*Chamaesiphon* sp., *Phormidium* sp., *Oscillatoria* sp.), ectoparasites such as leeches and
423 even Herpesvirus associated dermatosis had been isolated [201]. In the United States of
424 America, invasive lesions in freshwater turtles have been documented due to
425 Nannizziopsiaceae (*Emydomyces testavorans*), resulting in coalescing areas of scute
426 ulceration, keratin discoloration, deformation, and necrotizing osteomyelitis [206–208].

427 Common symptoms include irregular scute ecdysis, carapace and plastron pitting, and in
428 severe cases, signs of septicaemia such as erythema, anorexia, lethargy, limb paralysis, and
429 cutaneous abscesses [183,209]. Additionally, systemic fungal infections caused by
430 *Nannizziopsis* sp. have been reported in African side-neck turtles *Pelomedusa subrufa* in
431 Illinois [210]. Systemic dissemination by fungi such as Nannizziopsiaceae with synergistic
432 bacteria (e.g., *Citrobacter freundii*, *Serratia* sp., *Beneckea* sp.) can lead to necrosis in the
433 liver, spleen, kidneys, coelomic cavity, bones, and heart [183].
434 Diagnostic testing involves physical examination, PCR or fungal culture from swabs from skin
435 lesions, histopathological investigations from biopsies, as well as radiographs or even CT, as
436 especially *E. testavorans* is known to cause deep defects and coelomic inclusion cysts
437 [206,208,211].

438 In captivity, fungal overgrowth can be prevented by providing sufficient dry retreat areas.
439 Additional preventive measures include maintaining high water quality and avoiding
440 overcrowding and overfeeding [203,212]. If fungal infections occur, husbandry conditions
441 should be optimized, and vitamin A supplementation should be considered [175]. Local
442 treatment options include antiseptic solutions such as ethacridine lactate (Rivanol 0.1%),
443 propolis, medical honey, povidone-iodine (Braunol® 7.5%), or black tea baths [213]. Besides,
444 infected wounds should be subjected to topical treatment with clotrimazole or silver
445 sulfasalazine based ointments along resistance testing [214,215]. More severe lesions may
446 necessitate systemic antibiotics and antifungal agents [205,216,217]. In critical cases,
447 surgical debridement, or the application of extracellular matrix powder for wound healing may
448 be beneficial [218–220]. Against *E. testavorans* effective disinfection regime involves bleach
449 3% or 10%, Betadine, undiluted Lysol All-Purpose Cleaner, F10 SC, F10 SCXD, Roccal-D
450 Plus, Rescue, chlorhexidine, as well as heating to 60°C [221].

451

452 2.3.6. Algae

453 Algae of the families Methylomonaceae, Saprospiraceae and Nostocaceae are commonly
454 found as external epibionts on the carapace and, to a lesser extent, on the skin of freshwater
455 turtles [222]. Nevertheless, algal infestations can also promote shell disease [223,224] or in
456 the case of algal blooms induce severe internistic disorders [225]. Macroscopic skin lesions,
457 including degenerative, inflammatory, or neoplastic diseases, have been observed in
458 approximately 50% of individuals in a wild *Emys* population in Serbia [226]. Degenerative
459 and inflammatory shell injuries were the most frequently detected, while the skeletal system
460 was predominantly affected by mechanical injuries and osteopathies of various origins. About 461 two-thirds of examined turtles exhibited abnormalities of the skeletal system and shell. In 462 France, a study on diatoms found on the carapace of *Emys* suggested a neutral relationship, 463 viewing algae as commensals and, to some extent, mutualistic organisms contributing to 464 turtle camouflage [227]. Consequently, the application of a broad spectrum anti-parasitic 465 should be carefully considered, as disrupting the co-evolutionary adaptation and resistance 466 to parasites provided by algae may lead to long-term issues, potentially leaving future 467 generations vulnerable to natural parasites in the wild.

468 In captivity, preventing overcrowding and overfeeding, maintaining regular water changes or 469 water flow, and providing access to direct (or artificial) sunlight with sufficient drying time for 470 the shell should help prevent excessive algal growth on external body surfaces.

471

472 **3. Health impacts of non-infectious and environmental factors in captive-bred turtles**

473 Wild turtles and captive-bred individuals released into natural habitats are subjected to 474 markedly different non-infectious and environmental health challenges compared to those in 475 controlled captive conditions. These include factors such as fluctuating environmental 476 parameters, causing for example severe drought, variable food availability, and exposure to 477 novel abiotic stressors like environmental toxic load, all of which can significantly influence 478 physiological health and survival (e.g., [228–230]).

479

480 **3.1. Kidney disorders**

481 The most common non-infectious health impairments in both captive and wild freshwater 482 turtles include nutritional and chronic kidney diseases, such as chronic kidney disease- 483 metabolic bone disorder (CKD-MBD). Renal disorders, including reduced renal perfusion, 484 dehydration, and excessively protein-rich diets, may lead to gout in freshwater turtles [231].
485 Other predisposing factors of the disease that occurs in a visceral or articular form are
486 vitamin A deficiency, nephrotoxic substances (antibiotics, heavy metals), nephritis for
487 example due to a hexamitiasis or too low rearing temperatures [232,233]. While more
488 frequently observed in tortoises, gout has also been reported in cold-stunned wild sea turtles
489 [234]. Unlike the precipitation of urate salts in tissues, pseudogout is characterized by the
490 deposition of other crystal types, such as calcium hydroxyapatite, typically within joints [235].
491 Underlying causes of nephrocalcinosis again comprise traumata, hypercalcemia due to
492 hypervitaminosis D, primary or secondary hyperparathyroidism and osteolysis [232].

493 To mitigate these risks, breeding and husbandry practices should aim to replicate the natural
494 and diverse diets of the species in accordance with current literature (e.g., [236]; Meyer et
495 al., submitted article). Dietary supplementation with vitamins and minerals [237,238] is
496 recommended to prevent nutritional imbalances. Toxic aetiologies, such as medication
497 overdose, misuse, or chemical contamination of water sources, should be avoided in
498 captivity. While congenital or age-related degenerative processes may require symptomatic
499 management, infectious causes of renal impairment may be treatable with pathogen-directed
500 therapy, depending on the extent of tissue damage.

501

502 3.2. Metabolic disorders

503 In captivity, overfeeding, imbalanced diet (low fiber, high protein content), or reduced activity
504 levels can contribute to obesity and hepatic lipidosis, particularly during reproductive periods
505 or hibernation [204]. However, fatty liver syndrome particularly can also be the consequence
506 of non nutrition-related predispositions like a follicular statis or dystocia, chronic infections,
507 wrong hibernation temperature, dark rearing conditions, low essential amino acid
508 supplementation, iron-rich diets, intoxications, or thyroid dysfunctions [239-243]. Conversely,
509 anorexia can lead to fat mobilization, resulting in hepatic lipidosis. Chronic conditions such as
510 hepatic inflammation, heart failure, biliary obstruction, or hepatic lipidosis may lead to hepatic
511 fibrosis and hepatocellular nodular hyperplasia (cirrhosis). Protein malnutrition, hepatic
512 trauma, and systemic disorders have been reported in captive populations of *Emys* [244].

513 Fish-eating freshwater turtles are susceptible to thiamine (vitamin B1) deficiency, which can
514 result in neurological disorders such as torticollis, opisthotonus, muscle tremors,
515 incoordination, blindness, and jaw gaping, potentially with fatal outcomes if fed frozen fish
516 containing high levels of thiaminase [111,245]. To prevent thiamine depletion, proper fish
517 storage and rapid thawing methods are crucial. Fast thawing in 80°C water for five minutes
effectively denatures thiaminase, while thawing in a refrigerator prevents its activation.
518

519 Supplementing thawed fish with 20 mg of thiamine per kilogram or small amounts of brewer's
520 yeast is recommended. Additionally, fish species with high thiaminase content, such as
521 goldfish *Carassius auratus* and fathead minnow *Pimephales promelas*, should be avoided in
522 turtle diets.

523 As previously mentioned, MBD is a common disorder in captive freshwater turtles, affecting
524 both juveniles and adults [246]. In older turtles, renal secondary hyperparathyroidism
525 represents an additional aetiology, wherein kidney dysfunction disrupts calcium-phosphorus
526 homeostasis, leading to severe neurological symptoms such as seizures and collapse
527 [103,247]. Nutritional management for MBD includes a dietary calcium content of 2–3
528 mg/kcal, with approximately 1.4% calcium and 0.7% phosphorus on a dry matter basis. If
529 exposure to unfiltered sunlight for 12–14 hours per day is not feasible, full-spectrum lighting
530 including UV-B radiation (285–320 nm) should be installed, with appropriate placement
531 according to voltage output [116]. Initial treatment in severe cases involves fluid
532 supplementation (Jarchow's modification: two parts 2.5% dextrose in 0.45% saline and one
533 part lactated Ringer's solution, 278 mOsm/L, administered at 10–30 mL/kg q24h [248].

534 Supportive care includes temperature regulation, seizure control, and stabilization of bone
535 fractures. Calcium gluconate (100 mg/kg) can be administered intravenously or
536 subcutaneously every six hours until clinical improvement, followed by oral calcium gluconate
537 (23 mg/kg q12h). Vitamin D3 supplementation (100 IU/kg, administered intramuscularly or
538 subcutaneously once weekly for two weeks) may be required. If normo-calcaemia is not achieved after 3-7 days of oral calcium therapy, synthetic calcitonin (50 IU, administered intramuscularly twice weekly) can be introduced, alongside assisted feeding if necessary.
539
540
541 Long-term management requires individual monitoring of blood calcium and vitamin D3 levels.
542

543 Aural abscesses are relatively common in freshwater turtles, often resulting from inadequate husbandry and poor sanitary conditions, particularly vitamin A deficiency. Secondary colonization by gram-negative aerobic bacteria occurs via the Eustachian tube [249]. Vitamin A deficiency leads to squamous metaplasia of the lacrimal and Harderian glands, cyst formation in the third eyelid, and persistent eyelid closure [235]. However, periocular swelling may also result from systemic disorders involving the liver, kidneys, or heart [250,251].
544
545
546
547
548
549 Therefore, biochemical blood analyses are recommended for individuals presenting with generalized weakness. Therapeutically surgical removal of the abscess mass and capsula as well as applying of tests of antimicrobial resistance for further associated treatment are indicated. Thyroid disorders and goiter, though rare, have been reported in response to dietary iodine deficiencies, excessive iodine intake, or consumption of goitrogenic plants such as kale, broccoli, and cabbage [252].
550
551
552
553
554

555 Prolapse of urogenital organ structures, the rectum, or cloacal mucosa can occur due to various underlying causes, including calcium deficiency, imbalanced nutrition with low crude fibre content, parasitic overloads leading to tenesmus, dystocia, or the presence of foreign bodies [232]. It should be considered a differential diagnosis to cloacal neoplasia, which is relatively rare. Additional contributing factors include debilitation, neurological dysfunction, excessive libido, trauma, constipation, or cystic calculi [253]. Therapeutic management is contingent upon the specific diagnosis and underlying aetiology. In addition to initial stabilization of vital functions through fluid therapy, pain management, and wound care, treatment focuses on addressing the primary cause. This is followed by either repositioning the prolapsed structures or, if necessary and unavoidable, their surgical amputation [253].
556
557
558
559
560
561
562
563
564
Intoxications are observed from time to time in captive freshwater turtles, although such incidents occur less frequently in well-managed husbandry settings. Several factors contribute to the risk of intoxication. First, exposure to hazardous chemicals, including chlorinated water, cleaning agents, pesticides, insecticides, coumarin, metaldehyde, and nicotine, can induce severe clinical symptoms. Second, an inappropriate diet, particularly the ingestion of toxic plants such as avocado and rhubarb, as well as spoiled food, poses a significant risk. Third, heavy metal toxicity from lead, copper, or cadmium, along with medication overdoses (especially involving potentially nephrotoxic antibiotics such as gentamicin, amikacin, or metronidazole) can result in severe intoxication. Additionally, the administration of inappropriate medications, such as levamisole in tropical terrapins, may have detrimental effects. Lastly, environmental toxins, including ammonia or nitrate accumulation due to poor tank hygiene, represent a substantial health hazard. Common clinical signs of intoxication include lethargy, anorexia, impaired swimming ability, swelling of the extremities or eyes, skin lesions, seizures, and other neurological disorders [204,233,254,255]. General treatment regimes include the removal of the turtle from water and cleaning of body surfaces with water. Possibly anaesthesia can be necessary if a gastric lavage or endoscopic removal of toxic material should be performed. Supportive care including fluid therapy, assisted feeding and oxygen therapy can help recovery as well as in more specialized cases forced diuretic medication, activated charcoal, or specific antidotes [204,233,254,255].

### 3.3. Traumatic disorders

Various aetiologies can lead to traumatic injuries in captive freshwater turtles, including bite wounds from conspecifics (especially in cases of overcrowding), shell abrasions due to rough surfaces on basking spots, or sharp edges on hiding structures in the tank [256]. In the wild, bite wounds from predators, human interactions (collisions with boats, fishing gear, garden tools, unattended dogs), or rodent bites during hibernation are common causes of trauma
592 and even mass mortality events (e.g., in *Graptemys pseudogeographica* and *Sternotherus odoratus* [257–259]). In critically ill patients, stabilization of blood circulation and pain
593 management should be prioritized. In later stages, microbial pathogen identification may be
594 performed, followed by wound debridement and closure, including standard wound
595 management practices, administration of anti-inflammatory and analgesic medications, and
596 anti-infective therapy based on resistance testing [260,261]. In severe cases of shell necrosis
597 with wide or deep defects of the carapace of plastron, tamponades soaked in iodine or
598 ethacridine solution, as well as gelatine sponges coated with silver ions can be placed into
599 the wound cavity and changed at the beginning of treatment daily to less frequent exchanges
600 while more granulation tissue is building up. The use of negative pressure to enhance
601 granulation via vacuum sponges can be installed, however technical requirements can
602 exceed their use [248,262,263]. Additionally, defects can be treated with therapeutic laser
603 light [264], small defects can temporarily be closed with coral glue or intra oral bandages
604 from human medicine (ora-aid®). Proteolytic enzymes containing ointments as well as
605 preparations including insulin [265] may support continuous wound care and debridement.

607 Promising treatment methods for *Trachemys scripta elegans* with deeper shell defects and
detachment of scute segments besides include the use of two-component epoxy glue [260],
608 photopolymerizable nano-hybrid dental composite [218], and advanced techniques such as
609 photobiomodulation via low-level laser therapy [266].
610

611

612 3.4. Neoplasia

613 In addition to previously reported fibropapillomatosis (see Section 3.2. Viruses), other
614 neoplastic disorders described in freshwater turtles include dermal fibroma, cutaneous mast
615 cell tumour, squamous cell carcinoma, thyroid adenoma and carcinoma, parathyroid
616 adenoma, pulmonary fibroma and fibroadenoma, gastric carcinoma, intestinal
617 lymphosarcoma, gallbladder papilloma, testicular interstitial cell tumour, and renal
618 adenocarcinoma. These conditions have been documented in species such as the Chinese
619 box turtle *Cuora flavomarginata* [267], the common snapping turtle *Chelydra serpentina*
620 [268], and the Matamata turtle *Chelus fimbriatus* [269].

621 In wild populations, incidences of neoplasia remain sporadic and are more frequently
622 observed in older captive breeding animals. External abnormalities, such as mass lesions or
623 appendages, should be surgically removed and subjected to histopathological examination.
624 However in a case of substantial suspicion, animals can undergo diagnostic imaging like
625 sonography or x-rays in the process of diagnosis confirmation. Captive animals diagnosed
626 with neoplastic conditions should be isolated, especially in case of Herpesvirus-associated
627 neoplastic lesions, like detected in a wild Blanding's turtle [154], or in case of intense
628 individual care needed, and/or provided with supportive therapy, if necessary (see for
629 example [270]).

630

631 3.5. Cold stunning

632 Cold stunning may occur in turtles overwintering in outdoor enclosures from which they
633 cannot escape to warmer areas [241]. It leads to deviations in blood cell counts and
634 composition, potentially causing irreversible damage to the neurological and respiratory
635 systems (e.g., negative buoyancy, ataxia [271]).

636 Treatment approaches are adapted from sea turtle medicine, although they often yield limited
637 success. Affected individuals should be gradually warmed and subsequently provided with
638 prophylactic antibiotic therapy, fluid therapy, and nutritional support. This includes correcting
639 hyperkalaemia via calcium and sodium bicarbonate administration, ideally monitored through
640 blood gas and electrolyte evaluations [272]. Mechanical ventilation may be required to
641 compensate for respiratory acidosis, while metabolic acidosis can be managed with small
642 doses of sodium bicarbonate, with necessary carbon dioxide monitoring [273].

643
644 **4. Health checks in freshwater turtles**

645 Health assessment methods should always strive to be as accurate and effective as possible
646 while minimizing invasiveness and handling time for the animals [274,275]. General
647 information on methods and protocols for reptile examination and diagnostic testing can be
648 found in current literature [196,235,276–278]. The following section summarizes the main
649 visual, biochemical, haematological, and microbiological assessments in freshwater turtles.

650

651 **4.1. Non-invasive visual examinations**

652 Pre- and post-release health assessments of freshwater turtles regularly include a physical
653 examination, preceded by an evaluation of available background information (e.g., individual
654 history, former captive conditions, previous tests [279]). Visual examinations are prioritized
655 non-invasive methods for regularly documenting the general health status (body mass or
656 condition, physical abnormalities), posture, and behaviour [279–281].

657 As a first step the investigation consist in observing the individual animal behaviour [282]. For
658 instance, normal behaviour comprises hiding/predator avoidance, exploratory behaviour,
659 feeding and basking, yet with potential variations related to individual personality, which in
660 turn can have consequences for wild animals and/or on survival after translocation [283–
661 285]. Abnormal behaviours have been reported in captivity, including stereotype behaviours
662 e.g., repeated swimming along tank boundaries [286,287]. Due to the cryptic behaviour of
663 freshwater turtles, recent technologies such as animal-borne behaviour data-recorders can
664 be used in the wild [288]. Especially in the captive environment stereotype behaviours as
665 repeated swimming along tank boundaries are documented in freshwater turtles as abnormal
666 behaviour [282,286]. In a second step, in a physical examination normal locomotion
667 (including buoyancy changes), other body functions (breathing changes, any abnormal
668 sounds, defecation), normal evasive behaviour including vision and hearing, and absence of
669 neurological disorders (like tremor and opisthotonos) can be documented. However, as all
670 other reptiles, freshwater turtles do not follow rules of routine neurological check ups like
671 performed in mammal patients (e.g., Xiao et al., 2025 [289]), so interpretation of absence of
672 certain reflexes is not necessarily pathologic [271]. All these changes can indicate husbandry
673 and nutritional problems as well as clinical diseases, indicating the need for more detailed
674 investigations, convoking for more detailed investigations, but should also be considered in
675 the context of seasonal and reproductive stage to avoid misinterpretation of generally
676 abnormal behaviour as loss of appetite or courtship behaviour [290,291].

677 External examinations should focus on the carapace and plastron, as well as the skin
678 (condition and integrity), the musculoskeletal system (trauma, signs of infection such as
679 parasites, ulcers/plaques, haemorrhaging, or swelling), and the limbs and tail. Additional
680 assessments should include an inspection of the oral cavity (mucosal coloration, plaques,
681 foreign bodies, haemorrhaging), the eyes (discharge, hydration/nutritional condition,
682 parasites, trauma), respiratory and cardiovascular parameters (nasal discharge, signs of
683 unusual respiratory effort), and the genitourinary system.

684 Furthermore, palpation of the coelomic cavity through the hind limb openings can be
685 performed to estimate overall fat reserves as an indicator of body condition and, in females,
686 to check for eggs as a sign of active reproduction [292]. Pignatelli et al., 2023 [293]
687 emphasized the importance of documenting carapace damage in freshwater turtle population
688 monitoring, as it provides vital information regarding conservation concerns such as
689 predation pressure, population viability trends, intraspecific interactions, stress caused by
690 traffic and pollution, and pathogen exposure. This documentation can also help identify
691 individual- or population-specific issues.

692

693 4.2. Faecal examination

694 Sample-based methods are often more precise but may also be invasive. Faecal analyses
695 are a well-established, low-invasive approach for diagnosing endoparasites [148,294] and for
696 conducting microbial screenings (e.g., pathogen detection or microbiome composition analysis; [222,295,296]). However, especially for examinations of individual animal, the turtle has to be removed from water and placed into a lattice box, which constitutes a considerable amount of stress.

700

701 4.3. Tissue-based analyses

702 Samples obtained from the respiratory, intestinal, or reproductive tracts, as well as from the skin, enable microbiological testing for infectious agents or assessment of bacterial and fungal flora, including antibiograms for resistance profiling. This is particularly crucial in cases of infected wounds and serves as a diagnostic tool for guiding medical treatment of diseased individuals [297]. Explicit sampling of either the intestinal or reproductive tract via the cloaca is only feasible ante mortem through endoscopic techniques; thus, swab samples typically contain microbial communities from both body cavities.

709 Tracheal and/or pulmonary lavage in turtles follows a protocol detailed in [175]. Under anaesthesia, the animal is intubated, and a radiopaque catheter is inserted into the affected lung lobe. The lung is then infused with sterile saline solution at a dose of 5–10 ml/kg. The aspirated fluid is subsequently analysed for the presence of infectious agents.

713 When testing for infectious diseases, it is important to consider that disease susceptibility is influenced by host-related factors such as immunocompetence, pathogen-related virulence factors, and environmental factors including habitat connectivity and population structure, which may affect pathogen transmission dynamics [298].

717

718 4.3.1. Blood analyses

719 Blood samples provide valuable insights into general health status by enabling assessments of blood cell counts (e.g., via blood smear analysis [299]), genetic health impairment [300],
721 biochemical and nutrient/vitamin levels, endocrine parameters, toxic or pollutant loads, and
722 infection status [167,204]). Samplings comprise blood smears for leucograms and detection
723 of blood parasites, inclusion bodies or blood morphological abnormalities, haematology,
724 blood biochemistry and blood cultures of bacterial pathogens in septicaemic patients (e.g.,
725 [22,301]). However, blood biochemical and haematological parameters exhibit substantial
726 variation depending on population, sex, age, reproductive state, stress, food availability,
727 venipuncture site, season, and environmental factors [20,302,303]. Therefore, reference
728 values should be derived from individuals of the same population and sampled at the same
time of year. For *Emys*, reference biochemical and haematological values for both captive
729 and wild individuals are available in [148,300,304] (compare also Supplementary Table 2).

730 Generally, the collection site of choice is the dorsal coccygeal vein, but in smaller individuals
731 or those with an inaccessible tail the subcarapacial venous sinus can be selected, however
732 the risk of uncontrolled bleeding or damage of neck and cervical vertebrae have to be
733 considered (e.g., [305]). Additionally, the jugular vein puncture represents a well accepted
734 blood collection site, without lymphatic and/or cerebrospinal fluid dilution [306]. A safe blood
735 collection and regular parameters is possible in animals weighting over 80g [241] and serial
736 sampling of one population however, should follow an uniform setting with allowance of the
737 same blood collection site, equipment and consideration of intrinsic and extrinsic factors, as
738 mentioned above [306].

740 Although not yet widely established for all freshwater turtle species, studies on acute-phase
741 proteins, such as erythrocyte sedimentation rate (ESR) and haemoglobin-binding protein
742 (HBP), in Box turtles *Terrapene* sp. have shown promising results as indicators of non-
743 specific illness and inflammation in wild populations [307]. In Blanding's turtles inflammation
744 has been assessed via ESR using both microhematocrit tubes and commercial kits [308].
745 While the two methods yielded different reference intervals, they exhibited similar response
746 patterns to ecological impacts. Additionally, specific blood parameters—lactate, packed cell
747 volume (PCV), total solids, glucose, in conjunction with vital parameters such as temperature
and heart rate—has been identified as reliable prognostic indicators of mortality following traumatic injuries in five species of North American freshwater turtles [309]. The study found significant correlations between hyperlactatemia, elevated lactate levels over 24 hours (initial concentration > 5 mM/L), anaemia, and hypoglycaemia in males with increased mortality risk. Regarding blood lactate concentration, a point-of-care-testing has been recommended [310] to avoid inaccurate measurements. Furthermore, stress assessments in freshwater turtles can be conducted by measuring eosinophil concentrations, heterophil-to-lymphocyte (H:L) ratios, and plasma glucose levels [311–313].

## 4.4. Medical imaging

Medical imaging represents an advanced set of diagnostic techniques for the targeted investigation of specific health issues in freshwater turtles, including radiography, ultrasonography, endoscopy and computed tomography [148,271,272,314]. Radiographic imaging is commonly employed to assess conditions affecting the digestive, respiratory, and reproductive tracts, such as the presence of foreign bodies or constipation, as well as to evaluate musculoskeletal integrity, particularly in cases of fractures [315,316]. For the assessment of fractures or other traumatic injuries, craniodorsal-caudoventral oblique and caudodorsal-cranioventral oblique radiographic views taken at a 45° angle to the frontal plane can reveal fractures in the limbs that may otherwise be obscured by the shell [317,318]. Ultrasonography is particularly valuable for diagnosing preovulatory egg binding as well as congenital and acquired heart diseases [319]. Additionally, it is useful for planning targeted biopsy sampling [320]. Computed tomography has become an essential diagnostic tool in modern turtle medicine, allowing for the measurement of internal body fat as an index of individual condition [321] and for evaluating the severity and extent of traumatic injuries. It also aids in pre-surgical planning, such as in cases of shell fractures in *Trachemys scripta elegans* [322]. Additionally, coelioscopy has been highlighted as a valuable diagnostic tool for assessing internal organs for biopsy sampling in freshwater turtles [320]. Cystoscopy,
involving endoscopic examination via the urinary bladder, serves as a minimally invasive method for gender identification including assessment of reproductive state, visceral organ evaluation, and parasitic infestation assessment in turtles, including smaller post-hatchlings (e.g., 7-month-old Chinese three-banded box turtle *Cuora trifasciata*) [323–326]. Furthermore, endoscope-assisted minimal invasive surgery can be applied to neuter selected animals for example in captive breeding programs [327]. Lastly, digital image processing can support (regular) health monitoring of wild and captive specimen allowing for documentation of individual clinical history of shell lesions and growth performance in less time and therefore stress of handling for the animal than conventional records [328].

### 4.5. Necropsy

Post-mortem examinations serve as a critical tool for determining unknown causes of death, preventing the spread of infectious diseases, and identifying potential errors in captive management practices [329,330]. Technical guidelines and protocols for conducting necropsies in freshwater turtles can be found in [331].

### 4.6. Advanced health surveys

#### 4.6.1. Hormone-associated diagnostics

Serum androstenedione levels can serve as an indicator of reproductive fitness in male turtles, providing insights into both the quality of captive care and the individual's overall health status [332]. Similarly, it has been demonstrated, that correlations between threshold concentrations of oestradiol, testosterone, and progesterone, along with various blood chemistry parameters, in female *Chelonia mydas*, enable a predictive monitoring to enhance captive breeding success [333]. A non-lethal method for assessing hatchling fitness through yolk sample analysis has been developed [334], particularly for measuring steroid hormone
concentrations in *Trachemys scripta* sp. While approximately 70% of hatchlings survived this procedure, the range of parameters that can be analysed remains limited, suggesting that this method may be most applicable for scientific research rather than routine health assessments. In addition to non-invasive hormone assays for fitness evaluations, stress assessment could offer a valuable approach for post-release health monitoring. However, the application of chronic stress evaluation via non-invasive corticosterone sampling in freshwater turtles remains insufficiently explored. Other authors [335,336] investigated corticosterone accumulation in turtle claws, providing potential insights into habitat suitability, the presence of persistent stressors, and long-term reintroduction success. Studies on corticosterone metabolite analysis in faecal samples of lizards (*Smaug giganteus* [337]) and sea turtles (*Chelonia mydas* [338]) have shown promising results, though further research is needed to refine interpretations in ecological contexts. Conducting a non-invasive corticosterone assay using claw tips, researchers assessed chronic stress levels in midland painted turtles *Chrysemys picta marginata* living in proximity to roads [339]. The potential significance of the glucocorticoid-to-DHEA(S) (dehydroepiandrosterone and its sulfate ester) ratio has been emphasized as a more comprehensive measure of animal welfare and stress [340]. However, to date, standardized protocols for applying this approach to freshwater turtles remain unavailable.

818

### 4.6.2. Advanced infectious agent diagnostics

Whole genome sequencing (WGS) of infectious pathogens, particularly viruses, presents significant potential for elucidating epidemiological characteristics in pathogen screening, including emerging infectious diseases (EIDs) in animals [341]. Combining long-read sequencing technologies with target enrichment is recommended for optimal pathogen characterization. Sample types include swabs, faecal samples [342], and organ tissues [343] on which environmental DNA (eDNA) based methods can be applied [344,345]. Next-generation sequencing (NGS) is already applied in amphibian pathogen screening, such as
827 in monitoring the chytrid fungi *Batrachochytrium dendrobatidis* (Bd) and *Batrachochytrium salamandrivorans* (Bsal) [346]. Similarly, metagenomic or metabarcoding approaches could
828 be employed for pathogen detection in bulk samples of released turtles or through scanning
829 eDNA for potential pathogen presence or population monitoring [347–351].
830

831 The spectrum of screened pathogens can be expanded to include vector-borne diseases
832 detected in blood samples [352]. Studies have identified *Rickettsia* and *Ehrlichia* sp. in ticks
833 from wild invasive Yellow-margined box turtles in Okinawa, though the clinical consequences
834 remain unclear [353]. Other reports describe *Rickettsia* sp. in *Amblyomma dissimile* ticks
835 from Colombia [354], *Rickettsia helvetica* in *Amblyomma sabanerae* from freshwater turtles
836 in Mexico [355], and the transmission of *Eastern equine encephalitis virus* (EEEV) by
837 *Culiseta melanura* mosquitoes in the USA [356].

838 Deep sequencing of infectious agents' subspecies may be warranted when disease signs do
839 not align with known pathogens, allowing for the identification of novel or emerging
840 contagious diseases, such as new *Herpesvirus* and *Adenovirus* strains [145]. Similarly,
841 virulence assessments of bacterial strains, such as *Chryseobacterium indologenes* [357],
842 provide valuable epidemiological insights. A recently identified potential EID, systemic
843 Caryospora-like coccidiosis, was found post-mortem in *Trachemys scripta elegans*, with
844 related genotypes detected in sea turtles; further research is required to determine its
845 pathogenicity [358]. Strain-level investigations have also been conducted in farmed turtles
846 has been examined host-pathogen interactions and gut microbiome shifts in *Pelodiscus
847 sinensis* infected with *Morganella morganii* in aquaculture [359].

848

849 **4.6.3. Advanced diagnostic testing**

850 Advanced diagnostic methods infrequently used in wildlife species include immune gene and
851 biomarker analysis (e.g., bactericidal capacity and natural antibody agglutination) [299], and
852 pathohistological examinations for emerging diseases, unclear causes of death, and
853 pathological lesions from biopsy samples [329]. An emerging diagnostic approach, though
854 currently experimental in wild fish species, involves the development of an environmental
855 microbiota health index (microHI) based on external (water) and internal (gut) microbial
856 communities. This index has been proposed to identify key bacterial taxa indicative of
857 microbiome health disruptions in fish [360]. Lastly and possibly less relevant for wild living
858 individuals, except for monitoring vital parameters during general anaesthesia the technical
859 performance of electrocardiogram has been described [361].

860

861 **5. Euthanasia of freshwater turtles**

862 Following a medical examination, certain freshwater turtles may be deemed unsuitable for
863 release due to severe injuries or infections that are untreatable and not yet established in
864 wild populations. Immediate euthanasia is justified in cases where animals are near death,
865 beyond medical help, and suffering [362]. Additionally, severe injuries such as open fractures
866 or extensive wounds involving multiple body parts, particularly those affecting sensory
867 organs, warrant critical evaluation. Prolapsed or ruptured tissues exposing deeper structures,
868 especially chronic cases, should also be assessed carefully.

869 Some systemic infectious diseases, such as severe *Mycobacteria* infections, pose a
870 zoonotic risk and are often untreatable; in such cases, euthanasia is advisable [235]. Severe
871 myiasis with deep tissue invasion is frequently untreatable due to the rapid spread of toxins
872 and tissue damage [363]. Similarly, severely injured or moribund hatchlings, as well as
873 individuals with severe congenital deformities, require assessment to determine whether
874 treatment and recovery are feasible. Permanent disabilities that impair movement,
875 reproduction, or the ability to evade predators may also justify euthanasia if alternative long-
876 term care solutions, such as adoption by rescue centres, zoos, or responsible private
877 caretakers, are unavailable. However, if euthanasia is considered solely due to the inability to
878 return the turtle to the wild, alternative options such as captivity for breeding or educational
879 purposes should also be explored.

880 For severely injured or suffering turtles identified during routine monitoring or medical
881 interventions, recommended euthanasia protocols include deep anaesthesia—such as an
882 intramuscular injection of 120 mg/kg ketamine hydrochloride and 2 mg/kg diazepam [364]—
883 followed by an intravenous or intracoelomial injection of pentobarbital [365]. Alfaxalone (20
884 mg/kg intramuscular) is another option for general anaesthesia, though its analgesic efficacy
885 in major surgical interventions remains debated [366].

886 Euthanasia should always be performed by a veterinarian when possible and, at a minimum,
887 by trained personnel in accordance with the *Association Guidelines for the Euthanasia of
Animals* [367] or relevant national regulations [368]. Additionally, post-mortem examinations
888 by specialized veterinarians can provide valuable insights into disease monitoring, benefiting
889 the conservation of remaining wild populations [329,369].

891

892 **6. Health issues in the context of translocations**

893 6.1. Pre-translocation quarantine

894 Health assessment is crucial for optimizing captive conditions and ensuring successful
895 translocation and reintroduction into the wild [370,371]. Quarantine is essential for thorough
896 health evaluation and for preventing the introduction of pathogens at the release site,
897 protecting both resident and translocated populations. However, translocation-induced stress
898 can trigger clinical manifestation of subclinical diseases [372,373], such as infections with
899 Adeno- and Herpesviruses or Mycoplasmosis [374–376]. It may also facilitate co-infections,
900 leading to disease emergence in previously exposed but asymptomatic individuals [377,378].

901 Pre-release health checks have been emphasized as a fundamental component of
902 rehabilitation and rewilding, particularly since turtles frequently carry subclinical infections
903 that may threaten naïve wild populations [379].
904 Quarantine primarily serves to assess the health status of translocated individuals. The
905 duration should exceed the incubation period of monitored diseases and depend on the time
906 required for diagnostic test results [74]. For reptiles, quarantine recommendations vary from
907 90 days [13] to 9–12 months [380], allowing sufficient time for repeated pathogen testing.
908 Other authors suggests a minimum one-year quarantine period for turtles, including two
909 Herpesvirus antibody tests at a minimum six-week interval [74,232]. However, guidelines for
910 many pathogens remain incomplete, underscoring the need for expanded disease screening
911 in wild populations.

912 Reptiles rely significantly on innate immune responses, mediated by complement proteins
913 and antimicrobial peptides [83]. Their adaptive immune system exhibits a prolonged
914 response compared to mammals, with peak antibody production occurring 6–8 weeks post-
915 antigen exposure and persisting for up to 34 weeks [381]. Given that secondary antigen
916 exposure does not always enhance antibody titers or affinity, repeated testing is necessary
917 [381].

918 Preventing pathogen transmission from translocated individuals to resident populations is the
919 highest priority during reintroduction [382,383]. For *Emys*, a recommended protocol includes
920 faecal sample analysis on three consecutive days at both the beginning and end of
921 quarantine to detect bacterial, and parasitic pathogens as well as oral-cloacal swabs for viral
922 and bacterial pathogen screening such as *Herpesvirus and Ranavirus*, and *Mycoplasmosis
923 sp.* [69,384]. Haemoparasites can be identified via blood smears [54,372]. Others
924 recommend screening for protozoans and helminths, either through collective faecal samples
925 for larger groups or individual samples for smaller groups [232]. Specific parasite families of
926 interest include *Oxyuridae* [385], *Ascarididae* [386], *Camallanidae* [387,388],
927 *Gnathostomatidae* [389], and *Kathlaniidae* [390]. Additionally, trematodes have been reported
928 in *Mauremys leprosa* [126,391]. Although cestodes and pentastomids are rarely found in
929 freshwater turtles [128,392], their prevalence may increase with the introduction of exotic
species [127,294,388]. Necropsy of any individual that dies during quarantine is crucial for determining the cause of death and preventing the spread of subclinical infections [74].

Overcrowding also represents an issue, next to elevated stress levels leading to immune deficiency, also facilitating disease transmission due to increased contact rates (e.g., Rangel-Mendoza et al., 2014 [200]). Hence, recommended minimum tank sizes for adult single animals according to the Federal Ministry of Agriculture, Food and Regional Identity in Germany (BMLEH) should be at least five times the carapace length in length and 2.5 times in width for up to two turtles, with an additional 10% surface area for the 3rd-4th animal and 20% from the 5th onward, and a water depth of at least twice the carapace width (https://www.bmleh.de/SharedDocs/Downloads/DE/_Tiere/Tierschutz/Gutachten-Leitlinien/HaltungReptilien.pdf?__blob=publicationFile&v=2). In their first year, animals can be housed in 50L tanks filled with 5cm water at 25°C, with plants and cork for hiding and basking [54]. One adult animal can be kept at 300-400L water, with plus 150-200L per additional animal, whereas juveniles could be kept at ten animals per square meter water surface if filtering, water quality, hiding and basking space and enrichment is available to an appropriate extent [255,393].

Minimal handling of quarantined animals is recommended to reduce stress, disease transmission risks and encourage natural behaviours before reintroduction [74]. Strict hygiene protocols must be followed at all handling stages, including the use of disposable gloves, surface disinfection (e.g., l-propanol, ethanol, glyoxal), and heat treatment of equipment at 200°C for 15 minutes [394]. Enclosures should be minimally furnished to facilitate observation and capture when necessary [372]. Equipment should be designated for specific groups and disinfected after each use [74]. Additionally, turtles should be housed separately during hibernation and for 4-6 weeks post-hibernation to mitigate infection risk, as immune suppression at lower temperatures increases vulnerability to outbreaks, particularly Herpesvirus [74]. However, brumation is not recommended for previously sick or injured individuals, those that underwent chemical treatment during the last six weeks and animals
involved in reintroduction programs, where turtles should be released in early ages, so that a
continuous growth over winter is ensured [54,235] or an optimal body condition index during
hibernation of *Emys* see Carina et al., 2021 [280].

960

6.1.2, Veterinary management in soft versus hard release scenarios

Soft release consists in placing captive individuals in a temporary, natural-like place on the
translocation site before they are actually released, or let dispersing by themselves, into the
wild. In contrast, hard release consists in translocating captive animals directly into the wild.
For soft-release, extended holding periods facilitate repeated captures, health examinations
(followed by targeted treatments if needed), and implementation of preventive measures, as
animals remain readily available for clinical assessment [395]. This approach also allows for
gradual adaptation to environmental conditions, potentially improving growth rates,
behavioural acclimatization, and predator avoidance skills before complete release [396].

Furthermore, by retaining individuals near the intended release site, soft release can
increase the likelihood of settlement within suitable habitat containing adequate resources,
reducing the risk of immediate and/or widespread dispersal. In addition, the nesting or
breeding area can be maintained under closer observation, enabling more effective long-term
health and population monitoring. However, prolonged captivity may carry disadvantages,
such as increased risk of pathogen transmission in the case of overcrowding or high animal
exchange, unbalanced nutrition, stress-related behavioural disorders and modifications that
could impair skills survival in the wild [397].

978

6.2. Post-translocation monitoring

A key challenge in translocation efforts is ensuring adequate post-release monitoring while
minimizing disturbance to the individuals. In some cases, a deliberate decision to limit direct
interaction with the translocated population is made to reduce human impact and the risk of
983 pathogen introduction [398]. However, limited resources may also constrain the implementation of comprehensive long-term monitoring
984 (https://cinea.ec.europa.eu/programmes/life/nature-and-biodiversity_en). The IUCN recommends continuous health and mortality monitoring in reintroductions and conservation
985 translocations to detect potentially high disease prevalence or welfare impairments that could jeopardize reintroduction success or negatively affect neighbouring populations [3,287].
986
987
988
989 Population monitoring typically relies on standard capture-mark-recapture protocols [399]. Implanting PIT tags (Passive Integrated Transponder) subcutaneously between foreleg and
990
991 neck or subcutaneously into the hind limb for a permanent marking and cataloguing of individuals in conservation programs is mostly favoured, while shell notches are more
992
993 invasive and can become unreadable after severe shell necrosis or traumatic injuries [400].
994 Handling of translocated individuals for identification and biometric assessments provides an
995 opportunity for health evaluation. To minimize stress and handling time, such procedures should be conducted by experienced personnel following established veterinary guidelines
996
997 [401]. Fieldworkers are encouraged to refer to resources such as the World Organisation for Animal Health (WOAH) Training Manual on Wildlife Diseases and Surveillance, the European
998 Wildlife Disease Association (EWDA) Diagnosis Cards, and research projects like WildTech
999 (http://www.wildtechproject.com) and APHAEA (Harmonized Approaches in monitoring
1000 wildlife Population Health, And Ecology and Abundance) – a project to monitor species
1001
1002 abundance and pathogen distribution within a European network (http://www.aphaea.eu).
1003 The importance of educating future scientists in wildlife health monitoring has also been
1004 emphasized [401].
1005 For freshwater turtle reintroductions, health monitoring recommendations have been
1006 developed [204,402]. However, gaps remain in best practices, particularly concerning (i) standardized quarantine protocols for European turtle species, (ii) diagnostic procedures and
1007
1008 methodologies, and (iii) frequency and timing of pathogen testing. Limited research exists on
1009 the prevalence of relevant pathogens throughout different life stages [87,403]. Additionally,
1010 financial constraints have restricted regular screenings for less common, but potentially
1011 emerging infectious agents [404].

1012 Invasive turtle species pose significant threats to translocated populations through direct
1013 biotic interactions, such as predation and competition for resources, as well as indirect
1014 ecological impacts and disease transmission [294,405]. Invasive species often exhibit higher
1015 reproductive efficiency and aggressive behaviour, disrupting food webs and predator-prey
dynamics [406,407]. Hatchlings and adult turtles are particularly vulnerable to predation by
1016 invasive fish [408] and mammals, including raccoons (*Procyon lotor*) and raccoon dogs
1017 (*Nyctereutes procyonoides*) which pose increasing challenges for reintroduction programs
1018 [409]. Veterinarians play a crucial role in advising on eradication management while ensuring
1019 ethical and welfare considerations are met [410].

1021 Most importantly, the protocols and field methods as well as practical procedures before
1022 release have to be transferred, made coherent and applicable to all stakeholders involved in
1023 the knowledge transfer of the reintroduction/translocation effort, to ensure comparability and
1024 continuity of successful reintroduction management methods [3,411]. The synergistic roles of
1025 wildlife veterinarians lies in coordinating and educating the collaborative work of all
1026 veterinarians, scientists, field workers, zoo keepers, or managers involved in the
1027 reintroduction process, so that on the long run protocols are executed correctly along animal
1028 welfare claims, but also adjusted along new scientific findings (e.g., Sutton 2010 [412]).

1029

1030 **7. Proposed guidelines for pre- and post-translocation health surveys of Europe-native
freshwater turtles *Emys orbicularis* and *Mauremys leprosa***

1031

1032 Based on our review, a standardized pre-release health assessment—independent of
1033 specific diseases or site-related factors—should include a comprehensive physical
1034 examination, biometric measurements, and molecular diagnostics. At a minimum, PCR-
based testing should target Ranavirus, Herpesvirus, Adenovirus, *Mycoplasmosis* sp., and
1035
1036 *Spirorchis* sp. using combined oral and cloacal swabs, performed twice: once at the start of
1037 quarantine and again at least six weeks later, shortly before release. The suggested time
1038 period can be applied for animals originating from institution with high hygienic quarantine
1039 protocols, otherwise its duration has to be extended respectively.

1040 Additionally, bacteriological and microscopic faecal analyses are recommended to identify
1041 potential bacterial pathogens and parasitic infections. In cases of skin or shell ulcerations,
1042 swab or tissue samples should be submitted for microbiological examination, primarily for
1043 bacterial and fungal pathogens. Blood sampling should be considered in response to
1044 unexplained morbidity or mortality events, including toxicological, biochemical, and
1045 hematological analyses. Post-mortem examinations should be conducted in cases of
1046 unexpected deaths to determine underlying causes and potential epidemiological
1047 implications.

1048 Alongside clinical symptomatology, it is crucial to assess the significance and incidence of
1049 co-infections that may exacerbate disease progression. Ideally, long-term epidemiological
1050 monitoring should include representative population screenings over several decades (e.g.,
1051 [224,413,414]). Unlike emergency response protocols in human medicine [415], universal
1052 guidelines for systematic EID surveillance in turtles—such as standardized screening
1053 methods or optimal testing frequencies—are currently lacking. While specific
1054 recommendations remain unavailable, passive surveillance via molecular screening (e.g.,
1055 PCR-based assays) could facilitate the detection of novel strains or subspecies of known
1056 pathogens. At a minimum, such screening should be implemented in cases of cumulative
1057 mortality (e.g., [416,417]).

1058

1059 **8. Conclusion**

1060 In this review, we exhaustively synthesized scientific articles and proposed guidelines for pre-
1061 and post-release health assessments of *Emys orbicularis* as part of translocation-based
1062 conservation initiatives. Wildlife veterinarians are catalysts in the early phase of
1063 reintroduction or reinforcement, aiming at creating self-sustaining systems that run
1064 independently from any controls or interventions after translocation. Such conservation
1065 actions are implemented in a wide diversity of ecological, sociological and economical
1066 contexts, involving actors from private individuals to institutions, that disseminate their
1067 protocols and outcomes in their distinctive ways. This highlights the urge for better,
1068 systematic dissemination of conservation initiatives, and a strengthened bridge between
1069 herpetology and herpetoculture for ensuring best practices in future initiatives [418].
1070 Reintroductions would specifically benefit from operational transdisciplinary collaborations
1071 between veterinarians and involved stakeholders, including local populations, field scientists
1072 and land managers for broader engagement for nature conservation, best integrated
1073 monitoring in the wild and appropriate operational decision making [54]. Such goal can be
1074 achieved through participatory action-oriented research projects (e.g.,[34]), that consolidates
1075 existing network of stakeholders for best sharing of knowledge and expertise on wildlife
1076 monitoring and management and for long term sustainable, socially supported, effective
1077 conservation initiatives.

1078

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2310

2311 **List of abbreviations**

2312 EAZA: European Association of Zoos and Aquaria

2313 *Emys: Emys orbicularis*

2314 mtDNA: mitochondrial DNA
2315 MBD: metabolic bone disease

2316 UV-B: ultraviolet B

2317 CAM: chorioallantoic membrane

2318 TINC: intranuclear coccidiosis in tortoises

2319 URTD: upper respiratory tract disease

2320 SD: superficial dermatomycoses

2321 SM: visceral or systemic mycoses

2322 SCUD: septicemic cutaneous ulcerative disease

2323 NSD: necrotizing scute disease

2324 CKD-MBD: chronic kidney disease-metabolic bone disorder

2325 ESR: erythrocyte sedimentation rate

2326 HBP: hemoglobin-binding protein

2327 PCV: packed cell volume

2328 H:L: heterophil-to-lymphocyte ratio

2329 WGS: whole genome sequencing

2330 EID: emerging infectious disease

2331 eDNA: environmental DNA

2332 NGS: next-generation sequencing

2333 Bd: *Batrachochytrium dendrobatidis*

2334 Bsal: *Batrachochytrium salamandrivorans*

2335 EEEV: *Eastern equine encephalitis virus*
2336 microHI: microbiota health index

2337 PIT: Passive Integrated Transponder

2338 WOAH: World Organisation for Animal Health

2339 EWDA: European Wildlife Disease Association

2340 APHAEA: Harmonized Approaches in monitoring wildlife Population Health, And Ecology and

2341 Abundance

2342

2343 Supplementary Table 1:

2344 phys. exam.: physical examination

2345 BW: blood work

2346 expl. surg.: explorative surgery

2347 eutha: euthanasia

2348 husb.corr.: husbandry correction

2349 histo: histology

2350 LM: light microscopy

2351 sono: sonography

2352 bact: bacteriological

2353 supp. care: supportive care

2354 TEM: transmission electron microscopy

2355 poss.: possibly

2356 granulomat. inflam.: granulomatous inflammation
2357 sol.: solution

2358 spec.: specific

2359 suppl.: supplementation

2360 vit.: vitamin

2361 CT: computed tomography

2362 X-ray: radiography

2363 LC/MS technique: liquid chromatography (LC) tandem mass spectrometry (MS)

2364 GC/MS: Gas Chromatography/Mass Spectrometry

2365 PP2A: Protein Phosphatase 2A

2366 pot.: potentially

2367

2368 **<u>Supplementary Table 2:</u>**

2369 SD: Standard deviation

2370 RBC: red blood cell

2371 WBC: white blood cell

2372 MCV: mean corpuscular volume

2373 MCH: mean corpuscular haemoglobin

2374 MCHC: Mean corpuscular hemoglobin concentration

2375 AST: aspartate aminotransferase

2376 ALT: Alanine transaminase

2377 GGT: γ-glutamyltransferase

2378 LDH: Lactate dehydrogenase

2379 CK: creatine kinase

2380 UA: uric acid
2381 Glu: glucose

2382 tCa: total calcium

2383 Phos: phosphate

2384 TP: total protein

2385 Alb: albumin

2386 Glob: globulin

2387 Fe: iron

2388 Cl: chloride

2389 K: potassium

2390 Na: sodium

2391

2392 **Declarations**

2393 • <u>Ethics approval and consent to participate</u>

2394 Not applicable.

2395 • <u>Consent for publication</u>

2396 Not applicable. This manuscript does not contain any individual person's data in any form

2397 (including individual details, images, or videos) that would require consent for publication.

2398 • <u>Availability of data and materials</u>

2399 The supplementary material of the current manuscript are available in the figshare

2400 repository, 10.6084/m9 figshare.29095058

2401 10.6084/m9. figshare.29095070

2402 10.6084/m9. figshare.29095082

2403 10.6084/m9. figshare.29095103

2404 10.6084/m9. figshare.30126292

2405 • <u>Competing interests</u>
2406 The authors declare that they have no financial or non-financial competing interests
2407 that could have appeared to influence the work reported in this paper.

2408 • **Funding**

2409 Not applicable.

2410 • **Authors' contributions**

2411 KT, JYG, BQ and CE conceived and designed the study. CE performed literature
2412 research. CE wrote the manuscript, and all authors read, edited and approved the
2413 final manuscript.

2414 • **Acknowledgements**

2415 We acknowledge the Emys-R project (https://emysr.cnrs.fr) funded through the 2020–
2416 2021 Biodiversa+ and Water JPI joint call for research projects, under the
2417 BiodivRestore ERA-NET Cofund (GA N°101003777), with the EU and the funding
2418 organizations Agence Nationale de la Recherche (ANR, France, grant ANR-21-BIRE-
2419 0005), Bundesministerium für Bildung und Forschung (BMBF, Germany, grant
2420 16LW015), the State Education Development Agency (VIAA, Latvia, grant ES
2421 RTD/2022/2), and the National Science Center (NSC, Poland, grant
2422 2021/03/Y/NZ8/00101). KT is funded by the DFG Heisenberg programme (TH
2423 1807/10-1).

2424

2425 **Supplementary information:**

2426 **Supplementary Table 1:** Infectious and non-infectious health issues of *Emys orbicularis*

2427 - The table contains infectious and non-infectious health issues of *Emys orbicularis*
2428 including clinical findings, diagnostics and treatment according to the current
2429 literature.

2430 **Supplementary Table 2:** Biochemical and haematological blood values of *Emys orbicularis*
2431 - The table contains biochemical and haematological blood values of *Emys orbicularis*

2432 complied from three sources of the current literature.

2433 **Map 1:**

2434 - The map shows the countries of the present distribution of *Emys orbicularis* (mid

2435 grey) and sites of reintroduction projects along Fritz and Chiari, 2013 (light grey) in

2436 Europe.

2437 **Infobox 1:**

2438 - The box contains information on biology of life cycle, reproduction and lifestyle of

2439 *Emys orbicularis*.

2440 **Figure 1, Flow chart:**

2441 The flow chart shows the process of suggested health screening steps during the

2442 reintroduction process of *Emys orbicularis*.

2443