25 Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Accepted: 27.05.2021 | Available on line: 16.11.2022 1Department of Veterinary Medicine, University of Perugia, Perugia, Italy. 2Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ‘Togo Rosati’, Perugia, Italy. 3Institute of Veterinary Bacteriology, Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, Bern, Switzerland. 4Museo di Storia Naturale dell’Università degli Studi di Firenze, Museo ‘La Specola’, University of Florence, Florence, Italy. *Corresponding author at: Department of Veterinary Medicine, University of Perugia, Perugia, Italy. E‑mail: marialuisa.marenzoni@unipg.it. Maria Luisa Marenzoni1*, Valentina Stefanetti1, Emilia Del Rossi1, Alessia Zicavo2, Stefania Scuota2, Francesco Carlo Origgi3, Gianluca Deli1, Claudia Corti4, Massimo Trabalza Marinucci1 and Oliviero Olivieri1 Keywords Mycoplasma agassizii, Reintroduction, Salmonella, Testudinid alphaherpesvirus, Testudo hermanni, Free-ranging. Summary Testudo hermanni is included as near-threatened in the Red List of the International Union for Conservation of Nature, while T. hermanni hermanni is considered endangered in the Italian Red List. Appropriate management of smuggled or seized wild individuals is recommended before their reintroduction into the wild. Accordingly, a health monitoring study was carried out. During 2014-2016, 133 oral swabs and 121 cloacal swabs were collected from a total of approximately 180 free-ranging and rescued T. hermanni hermanni from eight different Italian regions to investigate the presence of DNA of Testudinid alphaherpesvirus (TeAHV), Chlamydia spp. and Mycoplasma spp. in the oral cavity, and Salmonella spp. isolates in the cloaca. Mycoplasma spp. was detected in 52  out of 87 (59.77%) of rescued and in 1 out of 46 free-ranging (2.17%) individuals; 33 out of 53 (62.26%) Mycoplasma  spp. positive samples were typed as M. agassizii by PCR. Salmonella spp. was isolated from 45 out of 121 (37.19%) cloacal swabs, typed into 14 serovars, and characterized for complete antimicrobial susceptibility. A significantly different distribution of Salmonella spp. isolates was found in 2016 in comparison with 2014 and 2015, without any difference between free-ranging and rescued tortoises. All the tested tortoises were negative for TeAHV and Chlamydia spp. These results are considered a baseline information critical to monitor the dynamics of these microorganisms in free-ranging and rescued populations of T. h. hermanni, and to correctly approach the management of rescued animals and possible relocation programs. Detection of Testudinid alphaherpesvirus, Chlamydia spp., Mycoplasma spp., and Salmonella spp. in free‑ranging and rescued Italian Testudo hermanni hermanni of Nature (IUCN), while the Italian Red List reports T. h. hermanni as endangered (Rondinini et al. 2013). The Balkan subspecies has been widely traded and many tortoises have returned to the wild, either by escape or by release from captivity, sometimes even hybridizing with autochthonous individuals. Despite the Italian distribution of T.  h.  hermanni is relatively well known, some autochthonous wild population may include captive individuals of the Balkan form (Corti et  al. 2013, Di Tizio et  al. 2016). The Western subspecies (T.  h.  hermanni) occurs irregularly in the Introduction The Hermann’s tortoise (Testudo hermanni), represented by two subspecies, is distributed in the northern Mediterranean area. The nominal form (T. hermanni hermanni) is found in the western Mediterranean, from Catalonia to Italy, while the eastern subspecies, Testudo hermanni boettgeri, inhabits the Balkans (Mazzotti 2016). The Hermann’s tortoise is considered as near-threatened in the Red List of the International Union for Conservation 26 Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Microbiological investigation in Italian Testudo hermanni hermanni Marenzoni et al. et  al. 2005, Kabeya et  al. 2015, Taylor-Brown et  al. 2015, Laroucau et  al. 2020). The infection has been reported both in clinically healthy and ill captive tortoises in Italy (Di Ianni et  al. 2015). However, no conclusive characterization of Chlamydiae responsible for reptile infections has been described, although C. pneumoniae and C. psittaci have been more frequently reported (Kabeya et  al. 2015). Recently, new species and strains of Chlamydia spp. have been recognized in tortoises, suggesting also a risk of interspecies and zoonotic transmission, although their pathogenic role remains unclear (Mitura et al. 2017, Laroucau et al. 2020). Mycoplasmas have been frequently detected in chelonians. Some species are considered commensal bacteria of the host (Farkas and Gal 2009, Di Ianni et al. 2015), whereas others can cause severe diseases of the upper respiratory tract, with chronic evolution and mortality. Mycoplasma  testudinis has been reported as a non-pathogenic agent of the excretory tract of T. graeca, whereas Mycoplasma agassizii and Mycoplasma  testudineum have been demonstrated to cause disease (upper respiratory tract disease) in both free-ranging and captive tortoises in the USA and Europe (Brown et  al. 1995, Brown et  al. 1999, Feldman et  al. 2006, Lecis et  al. 2011), and they are considered a threat for the management of wild tortoise populations (Jacobson et  al. 2014). M.  agassizzi has been reported in Italy in rescued tortoises, but its potential role as etiologic agent of disease has not been clarified in the infected tortoises (Lecis et al. 2011). Moreover, the isolation of specific species of Mycoplasma  has been reported for only a few species of chelonians in captivity conditions, which makes it very difficult to understand whether the virulence of the microorganisms and the severity of the infection are linked to the microbial species or to the host (Kolesnik et al. 2017). Therefore, their pathological role still requires clarification. Salmonella spp. are ubiquitous enteric Gram-negative bacteria, able to infect a wide range of host species, including mammals, reptiles, birds and insects. Reptiles are generally considered carriers and intermittent shedders of Salmonella  spp.; some serovars are more frequently isolated in reptiles and are therefore called reptile-associated Salmonella, RAS (Bertrand et  al. 2008, Harris et  al. 2010, Pees et  al. 2013, Bosch et  al. 2016). Some of these have been implicated in a number of outbreaks involving humans, and especially young children (Bertrand et  al. 2008, Pees et  al. 2013), so that a European Directive defines infection by RAS as an emerging zoonosis (European Commission 2003, Pees et  al. 2013). Although rarely, Salmonella  spp. can also responsible for disease in reptiles, working as opportunistic pathogens generally associated with predisposing factors such as inappropriate environmental temperature (Pasmans et  al. 2002, Western Mediterranean area, in particular along the coasts of Spain and France. Even if it is protected by national and international laws, human activities in particular those which may cause habitat loss or illegal collection of wild animals, haevily contribute to the decline of this species. Rigorous monitoring, including close surveillance of infectious diseases, and appropriate management of smuggled or confiscated individuals are supported by the Italian Ministry of the Environment (Ministero dell’Ambiente e Tutela del Territorio e del Mare, MATTM), as part of the efforts needed to preserve natural population. However, specific information to plan possible relocation programs, without impacting biodiversity and respecting the ecosystem, are still limited. A multidisciplinary project on free-ranging and captive Italian Testudo spp., with special attention to T.  h.  hermanni, has been carried out to characterize the genetic background of the tortoises candidate to be relocated, their eco-ethology, morphology, and health conditions (MATTM 2019). More specifically, selected relevant infectious agents of tortoises were considered critical for the health assessment of tortoises part of the recollocation program. Within this scenario, surveillance of infectious diseases becomes very important when performing relocation programs (Hartley and Lysons 2001). Specific selection criteria for the choice of the microorganisms to detect were: 1) their ability to cause disease threatening individuals or populations either in captivity or in the wild, 2) the presence of asymptomatic carriers in their transmission cycle, and 3) the zoonotic risk for humans who handle the animals. Accordingly, Testudinid alphaherpesviruses (TeAHVs), Chlamydia  spp., Mycoplasma  spp., and Salmonella spp. were investigated. Four genotypes of TeAHVs are known (TeAHV-1, TeAHV-2, TeAHV-3, TeAHV-4). The genotype TeAHV-3 is considered one of the most relevant pathogens of chelonians, especially in T.  hermanni, because of its potential epizootic spread and mortality rates (Origgi 2012, Marenzoni et  al. 2018). TeAHV-3 is rarely associated with fatalities in T.  graeca or T. marginata, whereas it is often lethal in T. hermanni. Additionally, surviving infected animals, although apparently clinically healthy, can harbor the virus and shed it following reactivation, from its dormant condition (latency), typical of herpesviruses. Post-hibernation seems to be the period of major risk for reactivation (Origgi et  al. 2015, Marenzoni et  al. 2018). The consequences of the infection on the biodiversity could be considerable (Marschang et al. 2009, Martel et al. 2009). The phylum Chlamydiae consists of intracellular bacteria, able to infect a wide range of animals and humans. The infection has been described both in free-ranging (Mitura et al. 2017) and captive reptilian hosts, including tortoises (Soldati et al. 2004, Hotzel 27Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Marenzoni et al. Microbiological investigation in Italian Testudo hermanni hermanni sampling) was applied and tortoises were sampled when found in the wild. Rescued T.  hermanni included smuggled or confiscated animals housed in 10 different recovery centers, located in the eight different regions of Italy, selected at national level because considered representative of other centers present in the country. The average number of animals housed in each center was approximately n  =  10, with two centers hosting much larger groups (>  100 animals); the management and the species of the hosted tortoises other than T. hermanni varied from center to center. All the animals were seized or confiscated in Italy (however, based on the genetic characterization, some animals were later found to be non-Italian, data not shown). Information concerning for how long the tortoises had been kept in the centers before testing was not available. The OS and CS samples were collected by rotating sterile polyester swabs, dipped in 500 μL of phosphate buffered saline (PBS, pH 7.2), against the mucosa of the mouth or the cloaca. All the swabs were stored at 4 °C in PBS until analysis, which was performed within 24-48 hrs. No repeated sampling was performed. Biomolecular investigation Total DNA was extracted from 200 μL of each OS using a commercial kit (QIAamp DNA Mini kit, Qiagen, Italy), according to the manufacturer’s instructions. A consensus nested PCR protocol was performed to amplify a 225 bp-long highly conserved region of the DNA polymerase gene across the family Herpesviridae to identify any possible herpesviruses present in the investigated tortoises (VanDevanter et  al. 1996). A nested PCR was used to identify TeAHV-3 in case of positivity (Marenzoni et al. 2018). A PCR protocol targeting the 16S rRNA gene, discriminating between C. pneumoniae from C.  psittaci, was used to detect Chlamydia  spp. (Messmer et al. 1997). A two-step protocol based on conventional PCRs targeting a fragment of 273 bp and 1,013 bp of the Gonzalez-Candela et  al. 2005, Sting et  al. 2013). Isolates of Salmonella can be used also as indicators of antibiotic-resistance because of its role and the capacity to respond to antibiotic treatments (European Food Safety Authority and European Centre for Disease Prevention and Control 2018). The aim of the present study was to investigate the presence of TeAHVs, in particular TeAHV-3, Chlamydia  spp., Mycoplasma  spp., and Salmonella  spp. in both free-ranging and rescued Italian T.  h.  hermanni, monitored during a period of 3 years, to determine the infectious status of free-ranging tortoises and to elaborate appropriate health management guidelines for the rescued individuals to be relocated. Materials and methods Sample collection From April to October of the years 2014, 2015, and 2016, oral swabs (OS) and cloacal swabs (CS) were individually collected from free-ranging and rescued T.  h.  hermanni. Collection of OS and CS was carried out only in individuals which could be sampled only with minimal restraint in compliance with animal welfare guidelines. Accordingly, not all the tortoises (approximately 180 individuals) were systematically sampled for both samples. Overall, 133 OS (from 46 wild and 87 rescued tortoises) and 121 CS (from 84 wild and 37 rescued tortoises) were sampled. Sampling type and year are described in Table I. A complete clinical examination was performed for each animal at the time of sampling, including a general health assessment, inspection of visible mucous membranes, and recording of visible gross lesions. Free-ranging tortoises were captured in eight different regions of insular and peninsular Italy (Emilia Romagna, Abruzzi, Campania, Molise, Apulia, Calabria, Sardinia, Sicily) across their distribution range. Areas of sampling were identified during previous surveys carried out in the field, which are described in detail in MATTM (MATTM 2019). A non-probability sampling method (convenience Table I. Description of the type and year of sampling of free-ranging and rescued Testudo hermanni hermanni. Year Oral swabs Cloacal swabs Free-ranging tortoises Rescued tortoises Total Free-ranging tortoises Rescued tortoises Total 2014 22 57 79 31 27 58 2015 18 30 48 22 10 32 2016 6 - 6 31 - 31 Total 46 87 133 84 37 121 28 Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Microbiological investigation in Italian Testudo hermanni hermanni Marenzoni et al. Statistical analysis The apparent prevalence of each microorganism detected in infected tortoises was defined as the proportion of positive samples (based on the applied test) out of the total number of samples analyzed. Postulating the hypothetical target population as infinite, the number of animals sampled was used to estimate: 1) the maximum expected prevalence of infection with all the samples testing negative; and 2) the expected prevalence, with the corresponding 95% confidence interval (CI), if positive results were present. Specific tables for sample size and calculations were used (Cannon et  al. 1982, Thrusfield 2005). The chi-squared test, with Yates continuity correction, and Fisher’s exact test were applied as appropriate to compare the proportions of positive samples based on wild or rescued origin and year of sampling for Mycoplasma  spp. and Salmonella  spp. A P value <  0.05 was considered statistically significant. Odds ratios (ORs) were calculated as a crude measure of the association between single risk factors and outcomes (isolation of Salmonella  spp./ PCR positivity for Mycoplasma spp.). OpenEpi (Open Source Epidemiologic Statistics for Public Health, Version 3.01., www.OpenEpi.com) was used for the analysis. Results None of the animal investigated showed detectable clinical signs. All OS were negative for TeAHV and Chlamydia spp. DNA (0/133, 95% CI: 0.07-3.52%), consistently with an estimated prevalence lower than 2.23% for a hypothetical infinite population. Mycoplasma spp. DNA was detected with an estimated prevalence of 39.85% (53/133, 95% CI: 31.57-48.72%). Results were reported in Table  II. Prevalence was statistically different between rescued (52/87 OS, 59.77%, 95% CI: 48.69-69.98%) and free-ranging tortoises (1/46 OS, 2.17%, 95% CI: 0.11-12.97%, P < 0.0001), with rescued tortoises being 66.86 times (95% CI: 8.80-507.76) more likely to be positive. No differences in positivity were observed over years. Test positivity was not associated with detectable clinical signs. Thirty-three out of 53 (62.26%) Mycoplasma spp. positive samples were typed as M. agassizii by the specific PCR-based enzymatic restriction. The only positivity to Mycoplasma  spp. detected in a free-ranging tortoise was determined not to be M. agassizii. The sequences of the PCR products of both 273 bp and 1,013 bp, obtained from three randomly selected tortoises, shared 98-99% identity with the homologous 16S rRNA gene sequences of M. agassizii (GenBank accession nos. KY212528-KY212536, 16S rRNA gene was used to detect Mycoplasma spp. (Vojdani and Franco 1999, Lierz et  al. 2007). In the case of positive samples, a PCR-based enzymatic restriction was used to confirm the presence of M. agassizii (Brown et  al. 1995, Brown et  al. 1999). Moreover, PCR products of the expected size were purified using a commercial kit (Qiaquick, Qiagen, Italy) and directly sequenced on both strands with the same primers as previously described (Biofab srl, Italy). The sequences were assembled and aligned using BioEdit (BioEdit 2009). The sequence similarity was checked against sequences available from GenBank using the BLAST software (https://blast. ncbi.nlm.nih.gov/Blast.cgi) to confirm the species specificity of the PCR. Screening of Salmonella serovars and antimicrobial susceptibility The CS were processed according to the procedures indicated by the World Organization for Animal Health (OIE) guidelines to isolate and identify Salmonella  spp. and its serovars (OIE 2015). Briefly, each CS was used to inoculate the enrichment medium, Rappaport-Vassiliadis broth (Oxoid, Italy), and incubated at 41.5 °C for 24-48 hrs. The samples were then subcultured at 37 °C on xylose lysine desoxycholate agar (XLD, Oxoid, Italy) and brilliant green agar (Oxoid, Italy) solid selective media. The results were read after 24 and 48 hrs of incubation. Identification of suspect colonies was performed using composite biochemical media and the commercial biochemical test API RAPID 20E (bioMérieux, Italy). Salmonella  spp. isolates were serotyped by direct slide agglutination using specific antisera (Statens Serum Institut, Copenhagen, Denmark), according to the Kaufmann-White-Le Minor scheme. Salmonella  spp. isolates were tested for antibiotic sensitivity using the standard disk diffusion method of Kirby-Bauer on Mueller Hinton Agar (Oxoid, Italy). The following antimicrobial molecules (Oxoid, Italy) were tested: amoxicillin-clavulanic acid (30 μg), ampicillin (10 μg), nalidixic acid (30  μg), cephalothin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), enrofloxacin (5 μg), gentamicin (10 μg), kanamycin (30 μg), streptomycin (10  μg), sulfamethoxazole-trimethoprim (25 μg), sulfonamides (300 μg), and tetracycline (30 μg). The zone of inhibition around each disk was measured after 24 hrs incubation at 37 °C for each isolate. Results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI 2016) and the isolates were classified either as susceptible, intermediate, or resistant. 29Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Marenzoni et al. Microbiological investigation in Italian Testudo hermanni hermanni community for further studies at the Regional Reference Center for Pathogenic Enterobacteria, Istituto Zooprofilattico dell’Umbria e delle Marche ‘Togo Rosati’. The distribution of Salmonella  spp. isolates was significantly different according to the years of sampling, especially in  2016 (OR for 2016 vs. 2014 = 2.75, 95% CI: 1.06-7.11, P = 0.03, and OR for 2016 vs. 2015 = 3.12, 95% CI: 1.02-9.51, P = 0.03), whereas no statistical difference was found between free-ranging and rescued tortoises. Discussion Our results confirmed the presence of Mycoplasma  spp. and Salmonella  spp. in rescued NR025954, AF060821). Additionally, homologies were observed also with M. agassizii sequences detected previously in T. marginata, T. graeca, and T. hermanni in Italy, although with limited coverage (different amplified intervals) (HQ326165-HQ326177, Lecis et  al. 2011). The three sequences were submitted to GenBank (accession numbers MF185252, MF185253, MF185254). Salmonella spp. were isolated from 45 out of 121 CS (37.19%, 95% CI: 28.72-46.49%). The results of the isolation and typing of Salmonella spp. based on year and group of tortoises are reported in Table III. The geographical origin of the isolates is reported in Table IV. The isolates were collected in an official repository and have been made available to the scientific Table II. Results of the PCR for Mycoplasma spp. and Mycoplasma agassizii in free-ranging and rescued Testudo hermanni hermanni. Year of sampling Number of Mycoplasma spp. positive Total (%) Number of Mycoplasma agassizii* positive Total (%)Free-ranging tortoises (%) Rescued tortoises (%) Free-ranging tortoises (%) Rescued tortoises (%) 2014 1/22 (4.54) 31/57 (54,38) 32/79 (40.5) 0/1 (0) 20/31 (64.5) 20/32 (62.5) 2015 0/18 (0) 21/30 (70) 21/48 (43.75) 0/0 (0) 13/21 (61.9) 13/21 (61.9) 2016 0/0 (0) ne 0/6 (0) 0/0 (0) ne 0/0 (0) Total (%) 1/40 (2.5) 52/87(59.77) 53/133(39.84) 0/1 (0) 33/52 (63.46) 33/53 (62.26) ne = not executed; *PCR performed only on samples already positive for Mycoplasma spp. Table III. Year of sampling, number of tortoises positive for Salmonella enterica subsp., apparent prevalence (Pr), and list of the corresponding serovars of Salmonella enterica isolated from free-ranging and rescued Testudo hermanni hermanni. Year of sampling Number of positive free-ranging tortoises (Pr) and isolated serovars Number of positive rescued tortoises (Pr) and isolated serovars Total (Pr) 2014 8 (8/31, 25.8%) 7 (7/27, 25.9%) 15 (15/58, 25.9%) Abony (n = 1) Abony (n = 5) Hermannswerder (n = 1) Miami (n = 1) Langford (n = 5) Newport (n = 1) Wedding (n = 1) 2015 5 (5/22, 22.7%) 3 (3/10, 30%) 8 (8/32, 25%) Abony (n = 1) Hermannswerder (n = 2) Hermannswerder (n = 1) Lindern (n = 1) Langford (n = 1) Richmond (n = 1) Salamae 17:b :enz15 (n = 1) 2016 22 (22/31, 71%) 22 (22/31, 71%) Abony (n = 1) Ferruch (n = 2) Halle (n = 4), Hermannswerder (n = 5) Kottbus (n = 3) Langford (n = 4) Mikawasima (n = 1), Newport (n = 1) Zadar(n = 1) Total 35 (35/84, 41.7%) 10 (10/37, 27%) 45 (45/121, 37.2%) 30 Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Microbiological investigation in Italian Testudo hermanni hermanni Marenzoni et al. get infected with lethal outcome, often secondary to the introduction of clinically healthy carriers, including T. marginata or T. graeca (Marschang et al. 1997, Origgi and Rigoni 2003, Martel et  al. 2009, Origgi 2012, Marenzoni et  al. 2018). Accordingly, avoiding the mixing of animals of different species and origins, as it can happen in a rescue center, is of critical importance; personnel of the rescue centers need to be educated in this direction. Repeated tests and frequent health checks should be performed, in particular on rescued tortoises, in order to increase the probability of detecting the virus, which can reactivate in unpredictable manner (Marenzoni et al. 2018). Investigations for Chlamydia  spp. suggest that Chlamydiae are not a common pathogen in Italian T.  hermanni. However, considering that novel Chlamydia  strains have been detected (Mitura et  al. 2017, Laroucau et  al. 2020), the protocol used in the current study, which was designed to detect in particular C. pneumonia and C. psittaci, might not be broad enough to identify these new microorganisms. Moreover, a limit of the study was that the investigations for Chlamydia  spp. were restricted to oral swabs to detect respiratory disease, whereas these recent studies used successfully cloacal swabs, that maybe represent a better sample to improve the detection of Chlamydia  spp. (Mitura et  al. 2017, Laroucau et al. 2020). Detection of M. agassizii DNA in rescued tortoises only, raises concerns for relocation programs. Mycoplasmoses, especially those caused by M. agassizii and M.  testudineum, are considered to have contributed to the decline of some wild populations of tortoises in the USA (Jacobson et  al. 2014). In this perspective, the clear difference found in the prevalence of Mycoplasma spp. and M. agassizii among free-ranging and rescued animals suggests that M. agassizii is not a common component of the tortoise microbiome in the wild and its spread among free-ranging animals might have unpredictable consequences. Accordingly, realease of positive animals in the wild is not recommended. A previous study reported the presence of novel mycoplasmas similar to the pathogenic M. agassizii in a group of T. graeca, T. marginata, and T.  hermanni, but it was limited to an Italian recovery center; the infection was weakly associated with respiratory signs (six animals with respiratory signs, of which three were Mycoplasma  PCR positive vs three Mycoplasma  PCR negative) (Lecis et al. 2011). Further investigations are needed to determine the actual pathogenic potential of M. agassizii in T.  hermanni and the significance of this agent in T. hermanni disease ecology. The estimated prevalence of Salmonella  spp. in tortoises of the present study was 37.19% (95% CI: 28.72-46.49%). However, it may have been underestimated because of the intermittent shedding of Salmonella  spp., considering that Italian T.  hermanni, as previously observed (Lecis et al. 2011, Pasmans et al. 2000, Corrente et al. 2004, Di Ianni et  al. 2015, Bertelloni et  al. 2016), and no molecular evidence of TeAHVs and Chlamydia  spp. DNA. On the contrary, it is the first description of Mycoplasma spp. in free-ranging Italian T. hermanni, although with a very low prevalence. As reported previously (Lecis et  al. 2011), M. agassizii was detected only in rescued Italian T. hermanni, but this is the first report documenting the lack of detection of M. agassizii in the free-ranging tortoises, raising biosecurity questions concerning the management of captive animals. The lack of detection of TeAHV DNA by PCR confirmed that the tested free-ranging and rescued T.  hermanni were not shedders of TeAHVs. Further serological investigations, not performed in this study, could be useful to complete the monitoring of the exposure to TeAHVs and eventually to exclude their circulation in Italian free-ranging T.  h.  hermanni. In the context of a possibly naïve population of free-ranging Italian tortoises, strategies to avoid virus circulation in captivity as well as the introduction of the virus into the wild must be implemented. T.  hermanni is notoriously poorly resistant to herpesvirus and highly prone to Table IV. List of the serovars of Salmonella enterica isolated in the present study, reporting the number of strains isolated and their geographical origin (Region). Serovar (n) Geographical origin and number of isolates in free- ranging tortoises Geographical origin and number of isolates in rescued tortoises Langford (10) Apulia (5) - Calabria (2) Calabria (1) Calabria (1) Molise (1) Hermannswerder (9) Apulia (5) Emilia Romagna (2) Abruzzo (1), Sicily (1) Abony (8) Apulia (2) Sardinia (4) Calabria (1) Sicily (1) Halle (4) Campania (4) - Kottbus (3) Campania (2) - Molise (1) Ferruch (2) Apulia (2) - Newport (2) Apulia (1) Sardinia (1) Lindern (1) - Emilia Romagna (1) Miami (1) - Basilicata (1) Mikawasima (1) Molise (1) - Richmond (1) Calabria (1) - Salamae, 17 :b :enz15 (1) Calabria (1) - Wedding (1) Apulia (1) - Zadar (1) Calabria (1) - 31Veterinaria Italiana 2022, 58 (1), 25-34. doi: 10.12834/VetIt.1915.13833.1 Marenzoni et al. Microbiological investigation in Italian Testudo hermanni hermanni All the Salmonella  spp. isolated were completely susceptible to the antimicrobials tested, as commonly observed for Salmonella  spp. isolated from wild animals (Corrente et  al. 2004, Percipalle et  al. 2011, Rubini et  al. 2016). This supports that tortoises can be relocate into the wild with a very limited risk of introducing antimicrobial resistance into the environment through this route. The characterization of the antimicrobial susceptibility of the isolates could be a useful tool to monitor antibiotic-resistance in these animals and their environment. The results obtained with this study, that analyzed and compared the different prevalences in free-ranging and captive tortoises, contribute to formulate and update guidelines for proper sanitary management and relocation of rescued tortoises, placing particular emphasis of the risk of introduction of new microorganisms into the wild (MATTM 2019). The different prevalence of relevant microorganisms (Mycoplasma  spp. and M. agassizii) found between free-ranging and rescued animals and the absence of others (Chlamydia  spp. and TeAHVs) suggest that monitoring of these infections is critical to prevent the spread of microorganisms in recovery centers and to preserve wild populations. In the future, the list of microorganisms to monitor should be extended to ranavirus, picornavirus, and ferlavirus, other known chelonian pathogens. The data regularly collected from this monitoring could be used to understand the relevance, the pathogenic potential, the host susceptibility to these infections, and draw a baseline to evaluate their possible evolution over time in these populations. only a single sampling was performed on each tortoise for practical reasons. A wide range of Salmonella prevalence has been previously reported in chelonians, varying from 0%-100%, probably depending on several factors including host species, feeding habits, environment, management, location and the Salmonella  serovars involved (Pasmans et al. 2000, Hidalgo-Vila et al. 2007, Lecis et al. 2011, Percipalle et  al. 2011, Pees et  al. 2013, Marenzoni et al. 2015). The majority of the 14 serovars identified in the present study have been previously reported in Italian Testudo spp. (Pasmans et al. 2000, Corrente et  al. 2004, Ebani et  al. 2005, Percipalle et  al. 2011, Dipineto et al. 2012, Bertelloni et al. 2016, Marenzoni et  al. 2015), and the presence of the same serovars has been confirmed in the 1970s (Corsalini 1975) and in later studies (Pasmans et  al. 2000, Corrente et  al. 2004, Ebani et al. 2005, Percipalle et al. 2011, Dipineto et al. 2012, Bertelloni et al. 2016). Statistical analysis showed that no difference exists in the prevalence of Salmonella  spp. infection between free-ranging and rescued tortoises, whereas differences are observed across time, with 2016 being associated with a prevalence of 71%. Many factors, that can stimulate differently a multiplication of bacteria, may have led to these results, such as temperature or environmental factors, or animal density, but they have not been considered herein as they were not relevant to the aim of the present work. Other studies reported differences in detecting also further microorganisms in chelonians over different years (Kabeya et  al. 2015, Archer et  al. 2017), but the factors influencing these trends are generally difficult to identify. 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