Skip to main content

Health assessment of wild speckled dwarf tortoises, CHERSOBIUS SIGNATUS

Abstract

Background

In free-ranging reptile populations, bacterial, fungal, viral and parasitic pathogens may affect hosts through impairment in movements, thermoregulation, reproduction, survival, and population dynamics. The speckled dwarf tortoise (Chersobius [Homopus] signatus) is a threatened species that is mostly restricted to the Succulent Karoo biome in South Africa, and little information on pathogens of this species is available yet. We derived baseline parameters for five males and five females that were captured to genetically enhance a conservation breeding program in Europe. Upon collection of the tortoises, ticks were removed and identified. Immediately upon arrival in Europe, ocular, nasal, oral and cloacal swabs were taken for viral, bacteriological and mycological examinations. Fecal samples were collected before and 1 month after fenbendazole treatment, and analyzed for parasites. A panel of PCR, aiming to detect herpesviruses, adenoviruses and iridoviruses, was carried out.

Results

Samples were negative for viruses, while bacteriological examination yielded detectable growth in 82.5% of the swabs with a mean load of 16 × 107 ± 61 × 108 colony forming units (CFU) per swab, representing 34 bacterial species. Cloacal and oral swabs yielded higher detectable growth loads than nasal and ocular swabs, but no differences between sexes were observed. Fungi and yeasts (mean load 5 × 103 ± 13 × 103 CFU/swab) were detected in 25% of the swabs. All pre-treatment fecal samples were positive for oxyurid eggs, ranging from 200 to 2400 eggs per gram of feces, whereas after the treatment a significantly reduced egg count (90–100% reduction) was found in seven out of 10 individuals. One remaining individual showed 29% reduction, and two others had increased egg counts. In five tortoises, Nycthocterus spp. and coccidian oocysts were also identified. Soft ticks were identified as Ornithodoros savignyi.

Conclusions

Our baseline data from clinically healthy individuals will help future studies to interpret prevalences of microorganisms in speckled dwarf tortoise populations. The study population did not appear immediately threatened by current parasite presence.

Background

Free-ranging reptiles may be infected by bacteria, fungi, viruses and parasites, which can affect hosts through impairment of movements, thermoregulation, reproduction, survival, and population dynamics [1,2,3]. Accordingly, pathogens can represent challenges for wildlife conservation, particularly when acting in conjunction with anthropogenic stressors [4, 5]. Assessing the composition and epidemiology of pathogen communities in wild host populations is, therefore, essential for the development of host conservation management.

Tortoise populations are declining world-wide, leading the IUCN [6] to categorize 65% of all extant species [7] threatened with extinction (i.e., categories Vulnerable and [Critically] Endangered). Primary causes for decline are habitat destruction, harvest for food, use of body parts for traditional medicine and pet trade, but some tortoise populations are also under threat of infections by some virulent pathogens [2]. Relatively few wild populations have been rigorously assessed yet.

The speckled dwarf tortoise (Chersobius [Homopus] signatus) is mostly restricted to a small range in the Succulent Karoo biome in South Africa, where its numbers are declining [8, 9]. The decline is primarily caused by habitat destruction and degradation, climate change and increased predation [9], but other factors may exacerbate their effects. About speckled dwarf tortoises, little information is available on infestations by mutual and commensal organisms. One study suggested that ticks might switch from mammalian hosts to speckled dwarf tortoises during drought [10], potentially increasing the impact of drought on the tortoises. Given the lack of knowledge concerning if and how pathogens may contribute to the decline of speckled dwarf tortoises, the aims of this study were: 1) to derive baseline data on mutual, commensal and pathogenic organisms found in C. signatus; 2) to investigate their distribution among sexes and anatomical district; 3) to assess if their presence may pose a threat to the sampled C. signatus population.

Results

Bacteriological and mycological examinations

All tortoises harbored bacterial species. Most of the collected swabs (82.5%, n = 40) yielded bacterial colonies with a significant difference observed among anatomical districts (χ2 = 11.60, P = 0.011, d.f. = 3). The anatomical districts most colonized were the cloacal (100%, n = 10) and the oral cavities (100%, n = 10), with less frequent colonization of the nasal cavity (50%, n = 10) and conjunctival sacs (80%, n = 10). The mean of total bacterial loads was 16 × 107 ± 61 × 108 CFU/swab. Cloacal (244 × 107 ± 794 × 107 CFU/swab), and oral cavities (158 × 105 ± 452 × 105 CFU/swab) yielded higher counts than conjunctival sacs (13 × 103 ± 25 × 103 CFU/swab) and nasal cavity (33 × 104 ± 51 × 104 CFU/swab), but differences among anatomical districts were not significant (F = 1.76, P = 0.173. d.f. = 36).

Overall, thirty-four different bacterial species were isolated (Table 1). Bacillus cereus (13 isolates), Mycoplasma spp. (9 isolates), Salmonella enterica subsp. salamae serovar 6,7:a:z42 (13 isolates) were commonly detected, especially in the cloacal and oral swabs. Aeromonas spp., Enterobacter sakazakii, Yersinia spp., Vibrio spp., Campylobacter spp., Helicobacter spp. lacked in all isolates. Gram positive represented the most frequent bacteria (49.6% of all positive isolates), followed by Gram negative bacteria (38.7%), Mycoplasma spp. (7.6%), and Ureaplasma spp. (4.2%) (χ2 = 72.70, P < 0.001, d.f. = 3).

Table 1 Frequencies of bacterial species and isolations from different anatomical districts in five male and five female speckled dwarf tortoises (Chersobius [Homopus] signatus). Absence of a frequency value indicates that the bacterial species was not isolated. Two ocular and five nasal swabs tested negatively

When Mycoplasma spp. and Ureaplasma spp. were combined, the frequencies of Gram positive, Gram negative, and Mycoplasma-Ureaplasma spp. isolates differed significantly among anatomical districts (χ2 = 35.032, P < 0.001, d.f. = 6; Fig. 1). More specifically, a difference between Gram positive and negative was observed in ocular swabs, (78.6% vs. 21.4%), where Mycoplasma spp. and Ureaplasma spp. were absent (Fig. 1). Mycoplasma spp. and Ureaplasma spp. accounted for a mean load of 504 × 103 ± 515 × 103 CFU/swab. They were cultured in nasal (28.6%, 1 × 106 ± 0 CFU/swab), cloacal (13.8%, 561 × 103 ± 520 × 103 CFU/swab) and oral swabs (9.1%, 1 × 103 ± 0 CFU/swab). No significant differences were recorded among anatomical districts, neither for prevalence (χ2 = 3.11, P = 0.795, d.f. = 3) nor for the average loads recorded (F = 3.25, P = 0.078, d.f. = 36).

Fig. 1
figure1

Bacterial frequencies (%) observed in five male and five female speckled dwarf tortoises (Chersobius [Homopus] signatus) in relation to anatomical district and Gram affinity. [*: χ2 = 8.74, P = 0.017]

The frequencies of Gram positive and Gram negative bacterial isolates in males and females were similar (χ2 = 0.64, P = 0.42, d.f. = 1). However, analysis performed within each gender sub-group showed different frequencies of isolates among anatomical districts (χ2 = 68.31, P < 0.001. d.f. = 3; Fig. 2), with the largest frequencies in cloacal and oral swabs. Both sexes had a similar mean bacterial load (150 × 107 ± 697 × 107 and 487 × 106 ± 217 × 107 CFU/swab for males and females, respectively, t = − 0.663, P = 0.511, d.f. = 38), with large individual variation within males.

Fig. 2
figure2

Bacterial frequencies (%) observed in five male and five female speckled dwarf tortoises (Chersobius [Homopus] signatus) in relation to sex and anatomical district. [*1: χ2 = 4.37, P = 0.034; *2: χ2 = 8.60 P = 0.003; *3: χ2 = 9.92, P = 0.002; *4: χ2 = 5.53, P = 0.019; *5: χ2 = 19.89, P < 104; *6: χ2 = 10.29, P = 0.001]

Yeasts and fungi were observed in 25% of swabs with a mean fungal load of 3.5 × 103 ± 13 × 103 CFU/swab. Yeasts represented 78.6% of positive swabs (mean load 7 × 103 ± 14 × 103 CFU/swab), and fungi 21.4% (20 ± 10 CFU/swab; t = 0.77, P = 0.456). Rhodotorula rubra, R. mucilaginosa, Candida spp. and Alternaria spp. were over-represented in ocular and oral swabs (Table 2). In ocular swabs, yeasts resulted 13 × 103 ± 20 × 103 CFU/swab (n = 5), in cloaca 2 × 103 ± 1 × 103 CFU/swab (n = 3), and in mouth 20 ± 17 CFU/swab (n = 3). Penicillium spp. was identified only in nasal swabs.

Table 2 Frequency/swab of fungal species isolated from different anatomical districts in five male and five female speckled dwarf tortoises (Chersobius [Homopus] signatus)

Virological examination

None of the samples revealed the presence of detectable amounts of herpesvirus, iridovirus or adenovirus DNA.

Parasitological examination

All tortoises were infested by morphologically similar ticks, although at different stages of development. Based on morphology, number of limbs and absence of the typical shield of the hard ticks, they were identified as larvae and nymphs of soft ticks. Light brown nymphal stages (Fig. 3a) had an oval body, with a wider posterior gently rounded to form a sub-triangular shape and four-paired legs of moderate length and slender. They measured about 0.988–2.457 mm in length and 0.936–2.340 mm in width. Larvae have a semicircular and dark brown body and three pairs of legs (Fig. 3b). They are 1.10 mm in length and 1.0 mm in width. Morphological and metric characteristics of collected ticks permitted their identification as Ornithodoros savigny.

Fig. 3
figure3

Parasites identified in 10 speckled dwarf tortoises (Chersobius [Homopus] signatus). a Ornithodoros savignyi nymph (scale bar 0.45 mm); b) Ornithodoros savignyi larvae (scale bar 0.4 mm); c) Oxyurid egg (scale bar 35 μm)

All fecal samples collected before the treatment were positive for oxyurid eggs (Fig. 3c) with a FEC ranging from 200 to 2400 EPG. Egg dimensions ranged from 83 to 125 μm in length and 52–70 μm in width. A FECR higher than 90% was observed in 7 out of 10 treated animals (Table 3). Moreover, few Nycthocterus spp. ovoid cysts of about 50 μm in length and trophozoites, about 30–40 μm in length, and subspherical coccidian oocysts of about 20–23.4 × 17–23.4 μm were identified in five tortoises at flotation test, before and after the fenbendazole treatment.

Table 3 Fecal egg count reduction (FECR) of pinworm eggs observed in 10 wild speckled dwarf tortoise (Chersobius [Homopus] signatus) fecal samples prior and one month after deworming with Fenbendazole (75 mg/kg, repeated with 50 mg/kg after 10 days)

Discussion

This study provides the first comprehensive analysis of microorganisms and parasites in free-ranging speckled dwarf tortoises, and expands previous recordings of nematodes [11] and ticks [10] in the same species.

All sampled individuals appeared clinically healthy, suggesting that encountered microorganisms might be commensal and accordingly, not posing an immediate threat to the population in absence of specific stressors. “Commensalism” is defined as a state of infection that results in either no damage or clinically inapparent damage to the host, though it can elicit an immune response [12]. Evolutionary co-existence has equipped both the micro-organisms and the immune system of the host with similar mechanisms of defense and selection [13], however the interaction with microbes can be harmful for the host and opportunistic infections can occur [14]. These definitions and these scenarios, are becoming more complex at the light of the role of the environment as a critical third player, when it comes to host-pathogen interaction in free ranging individuals. It is becoming more and more evident that the delicate balance and equilibrium existing between host and the components of its microbiological community may be challenged and subverted by environmental factors, which may impact the host immune response in many ways, from the availability of critical resources as food to, in case of poikilotherms, as tortoises are, anything that could influence the environmental temperature and regularity of seasonality [15]. Accordingly, the significance of the identified organisms, within the disease ecology of C. signatus, needs to be evaluated also at the light of the possible role that emerging environmental stressors might have and on the information that will be collected in follow up studies.

The ongoing “-omic” revolution, provides now very powerful investigation tools, which allow a very complete and thorough characterization of very complex microbiological communities present in specific hosts. Our investigation was based on conventional culture-based, approach, which although might appear reductive at the light of the currently available technology, was considered ideal for an initial screening of the C. signatus microbiological environment and functional to the isolation of viable organisms, which might be critical for future in vitro and in vivo, and follow up studies, which we plan to enrich as well with a Next Generation Sequencing (NGS) approach.

Along with the considerations made above, even when considered part of the natural microbiota of the tortoises, some bacterial species may have clinical significance, mostly as opportunistic pathogens, both for animals and humans. Mycoplasma are frequently found in chelonians, where are often cause of severe disease [16, 17], but not are systematically associated with obvious pathology [18]. We found the presence of Mycoplasma spp. in four out of 10 examined animals. Our results showed a prevalence higher than that observed in a previous research [19], that reported a prevalence of 15.8% for Mycoplasma agassizii in captive tortoises (Testudo spp.) in the United Kingdom. In Italy, in particular on the Sardinia island, a similar prevalence of Mycoplasma spp. (37%) was found in tortoises belonging to the genus Testudo [20].

A clear predominance of Gram-positive species can be noticed, in accordance with data reported for Geoffroy’s side-necked turtle (Phrynops geoffranus) [21], in particular in the conjunctival flora. Only one species of S. auricularis was recorded in this anatomical district and it could be considered part of saprophyte microflora, although in few studies this species has been considered responsible of ocular lesions in some reptiles [22, 23]. To the best of our knowledge, this study is the first report of Kytococcus areolatus and Kytococcus schroeteri in chelonians. K. aerolatus was first identified from an indoor air sample and has never been reported to cause human infections [24], whereas K. schroeteri was identified in bacteraemia in a human patient [25]. A strain of Listeria spp. was also detected, but it was not identified as L. monocytogenes. However, this bacterial genus was reported as nonpathogenic in European pond turtles (Emys orbicularis) [26, 27].

Several studies analyzed microorganisms in free ranging wild reptiles and investigated different anatomical sites. In Brazil, the Pantanal alligator (Caiman crocodilus yacare) and the “jacarétinga” (Caiman crocodilus crocodilus) carried Aeromonas spp., Acinetobacter spp., Citrobacter freundii, Escherichia coli and Pseudomonas spp. [28], while Salmonella spp. was found in 39.1% of pet reptiles, including chelonians [29]. In the same country, Escherichia coli, Klebsiella pneumoniae, Enterobacter agglomerans, Citrobacter freundii and Bacillus spp. were identified in oral samples of Geoffroy’s side-necked turtles [21].

Although disease associated with Gram-positive bacteria is sporadic, Gram-negative bacteria are commonly reported as pathogens of reptiles [30, 31]. Different Gram-negative bacteria were identified in this study. Acinetobacter calcoaceticus, detected in oral and conjunctival swabs for C. signatus, has previously been reported in the oral cavity of Caretta caretta in poor general conditions [32], and in nasal swabs from Testudo graeca showing rhinitis [33]. Citrobacter spp. has been identified as the main microorganism associated with septicemic cutaneous ulcerative disease (SCUD) or other necrotic or ulcerative changes [34]. Nevertheless, the three C. signatus found positive for Citrobacter freundii in this study were asymptomatic. Stenotrophomonas maltophilia may cause several opportunistic infections as sepsis, pneumonia, urinary tract infection, meningitis, endocarditis, septic arthritis, and peritonitis in humans [35]. This bacterium was isolated from Red-footed tortoise (Geochelone carbonaria) with a respiratory infection [36] and in captive snakes with oral bacterial infection [37], but in this study it was isolated from oral and cloacal cavities of two healthy tortoises and not associated to detectable lesions.

E. coli and P. aeruginosa, reported as the prevalent bacterial species in several previous studies [27, 38,39,40], were not detected in this study.

It is well known that chelonians can carry bacteria with zoonotic potential [29, 41, 42], mainly Salmonella spp., that is prevalent in high rates in both captive and free-ranging turtles [43]. In our study, the prevalence of Salmonella spp. was relatively low (10.9%). This result is similar to data reported in exotic and native turtles in Spain [44], and in Polish European pond turtle (Emys orbicularis) [27]. In contrast, 100% incidence of Salmonella was recorded in free-ranging Spur-thighed tortoises (Testudo graeca) living in a National Park in southwestern Spain [45]. The ingestion of feces or contaminated water is considered a probable way of transmission [46].

In previous studies, several bacteria including Burkholderia cepacia were found associated with exudative bronchopneumonia and/or granulomatous pneumonia and other bronchopneumonic lesions of marine turtles [32, 47, 48]. In our investigation Burkholderia cepacia complex (Bcc) was identified in two tortoises, in one female from conjunctival, nasal and cloacal swabs, and in one male only from cloacal cavity. Bcc is a group of Gram negative opportunistic pathogens that can be found in soil and water [49, 50], in healthy fishes [51], but it has also been detected in humans with necrotizing pneumonia [52].

Usually, reptiles colonized with yeasts do not show any clinical sign, as in our case. Rhodotorula spp. are considered emerging opportunistic pathogens in humans [53]. R. mucilaginosa is a species frequently found in plastrons and, together with Cryptococcus laurentii, in oral cavities of two chelonian species in Brazil [54]. Most of the soil yeasts and fungi, belonging to Hyalohyphomycetes (e.g. Fusarium) and Zygomycetes (e.g. Alternaria), are implicated in infections in human patients, ranging from colonization and localized infections in immunocompetent individuals to fungemia and disseminated diseases in immunocompromised patients [55].

Oxyurids (pinworms) are generally the most frequent internal nematodes identified in tortoises. These nematodes, localized in the large intestine, are generally considered to be almost commensal [56]. The low pathogenicity [57] and the monoxenous life cycle [58] of these parasites are considered the main reasons for their large distribution among tortoises. However, in heavily infested tortoises, pinworms may be a potential cause of intestinal obstruction, anorexia and death after hibernation [56]. Oxyurid infection showed a prevalence of 100% in the tortoises examined here. Thirty days after the treatment with fenbendazole, 100% efficacy was observed in seven out of ten treated animals. However, four tortoises remained positive and in two animals the number of eggs was higher than it was prior the treatment. This result could depend on incomplete efficacy of the therapeutic protocol used, but it cannot be completely excluded that this result may also depend on a new infection. In any case, although in some previous studies the therapeutic protocol used has been proven to be effective for the treatment of pinworms of other turtle species [59] and despite fenbendazole is commonly used in veterinary medicine [60], in Hermann’s tortoises (Testudo hermanni) it can cause hematological and biochemical changes that may indicate transient immune suppression [61]. As our results suggest that the protocol used for the treatment of oxyurids was not totally effective and the clinicopathological changes reported as possible side effects [61] were not examined in this study, further investigations are needed to establish the risk/benefit ratio in oxyurid treatment in speckled dwarf tortoises.

Nycthocterus spp. are ciliated protozoans of the chelonian digestive-tract flora that may play an important role in the digestion of cellulose. Therefore, in most cases, these protozoan cause little or no damage to reptiles [62]. However, in heavy infections or in stress conditions Nycthocterus infections may cause severe irritation of the intestinal wall and colitis, diarrhoea, dehydration, weight loss and passage of undigested food in the feces have been recently reported in infected captive turtles [63].

Eimeriid coccidia commonly infect turtles and might contribute to morbidity and mortality under captive conditions. These protozoan parasites typically show tissue specificity, usually being limited to the epithelium of the gut. However, disseminated infections have been reported in vertebrates (i.e. birds and mammals) as in turtles [64, 65]. Eimeria spp. are the most common coccidia species in chelonians. However, the morphological identification of coccidian oocysts found in positive C. signatus was not possible because this would require live parasites.

This study is the first report of protozoan infections, i.e. Nycthocterus spp. and coccidia, in the speckled dwarf tortoise. The tick species that we identified, O. savigny, is distributed throughout Africa and has already been reported in C. signatus, mainly on the hind limbs, forelimbs and neck of infested tortoises [10].

Conclusions

Speckled dwarf tortoise populations are undergoing a dramatic decline due to habitat destruction, climate change and other threats [9]. The deteriorating conservation status of the taxon requires a pro-active approach to assess and understand emerging threats before they exacerbate threats to populations. We showed that speckled dwarf tortoise populations may host a wide range of microorganisms and metazoan species. Although sampled individuals appeared clinically healthy despite their bacterial and fungal loads, other stressors such as drought [10, 66] may induce shifts in parasites that could exacerbate negative effects in speckled dwarf tortoise populations [67].

This study identified no immediate conservation threats associated with microorganism infestations in examined speckled dwarf tortoises. Because changes in parasite populations, through stressors enacting on parasites or tortoises, might induce new threats, we recommend that the prevalence of actual and potential pathogenic species hosted by speckled dwarf tortoises should be considered when developing conservation measures for the taxon. Despite the small sample size is a limitation of this study, this health assessment provided very initial preliminary baseline data that we consider critical for future similar studies in this and other tortoise species. Further studies will be necessary to collect samples from C. signatus individuals in other locations and/or at different times of year, contributing to provide a more complete health assessment of the C. signatus free-ranging tortoise population and further understand the disease ecology of this species.

Methods

Animals

Five adult male and five adult female speckled dwarf tortoises (Chersobius [Homopus] signatus) were included in the study. The tortoises were collected between 11 and 19 September 2015 near Springbok, South Africa (permits FAUNA 053/2015, CITES 148487 and 15NL226435/11), to genetically enhance a European conservation breeding program. Ticks were immediately removed from each specimen at collection, and preserved in alcohol for further identification. Fenbendazole (Panacur, MSD Animal Health S.r.l., USA) was administered preventively 75 mg/kg, repeated with 50 mg/kg after 10 days, because of the large number of nematodes that had previously been observed in wild C. signatus [11]. Before and 1 month after the treatment [59], individual fecal samples (n = 10) were collected and preserved in 70% alcohol to be later examined for parasites. Immediately after capture, tortoises were transported in individual and isolated containers in order to prevent cross-contamination of pathogens among individuals. Samples for bacteriological, mycological and virological examinations were collected after the arrival in Europe to minimize the stress related to handling in the field conditions. Tortoises were held for a maximum of 10 days in the dark, at room temperature to reduce metabolic activity and stress related to handling and transport. After the study, the tortoises are maintained in captivity in Europe, for a conservation breeding program managed by the Dwarf Tortoise Conservation.

Bacteriological and mycological examinations

Upon arrival in Europe, swabs from conjunctival sacs (n = 10), nasal districts (n = 10), oral cavities (n = 10) and cloacas (n = 10) were taken for bacteriological and mycological examinations. Swabs were immediately shipped to the laboratory on dry ice, and cultural examinations started as soon as the samples arrived at destination. Standard diagnostic protocols [68] and selective culture media were used for the detection of bacteria, including Salmonella spp., Enterobacter sakazakii, Aeromonas spp., Listeria spp., Mycoplasma spp., Ureaplasma spp., Burkholderia cepacia complex, Yersinia spp., Vibrio spp., Campylobacter spp., Helicobacter spp., Bacillus cereus, and Clostridium spp.

To evaluate the total fungal and bacterial loads, each swab was filled with 1 ml of sterile saline solution (ThermoFisher Oxoid, Italy) for 30 m at room temperature and then vortexed for 30 s. Aliquots of 100 μl were spread onto Sabouraud Dextrose Agar with chloramphenicol (Liofilchem®, Italy) and Mycosel Agar (Liofilchem®, Italy), for 7 d incubation at 30 °C, and on Plate Count Agar (Liofilchem®, Italy) for bacterial count after incubation at 37 °C for 24–72 h.

For qualitative bacteriological investigations, the extracted swabs and the suspensions were enriched in Tryptic Soy broth (ThermoFisher Oxoid, Italy) for 6 h at 37 °C and then cultured onto Columbia Blood Agar, Columbia CNAM Agar, Mannitol Salt Agar, MacConkey Agar, Hektoen Enteric Agar (Liofilchem®, Italy), and Yersinia CIN Agar (ThermoFisher Oxoid, Italy). Selective and chromatic media (Liofilchem®, Italy) were used to detect Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, Bacillus cereus, Salmonella spp., after pre-enrichment in Rappaport Vassiliadis broth, Burkholderia cepacia complex, Listeria spp., after pre-enrichment in Half-Frase broth and Frase broth, Vibrio spp., Enterobacter sakazakii, Helicobacter pylori, Campylobacter spp., Clostridium spp., following standard protocols [68]. Plates were incubated for 24 h at 37 °C aerobically, anaerobically or in microaerophilic atmosphere (CampyGen Oxoid, Italy). In all cases in which bacteria growth was not observed, plates were incubated for a further 24 h before being classified as negative or, in the case of subsequent growth, underwent bacterial identification. Moreover, for each anatomical district, the qualitative and quantitative Mycoplasma spp. and Ureaplasma spp. detections were carried out using Mycoplasma System Vet (Liofilchem®, Italy). For the detection of yeasts and fungi, Sabouraud Dextrose Agar with chloramphenicol (Liofilchem®, Italy) and Mycosel Agar (Liofilchem®, Italy) plates were incubated at 30 °C aerobically, and examined daily over a 14 d period before being classified as negative.

Bacterial isolates were identified using standard microbiological procedures, as growth and colonial characteristics, Gram staining, cellular morphology, catalase and oxidase reactions, coagulase test (Coagulase Test, Liofilchem®, Italy), and hemolysin production. Species identification was carried out using the biochemical gallery systems (Remel Oxoid, Italy; API bioMérieux, France; Liofilchem®, Italy). Serological identification of Salmonella spp. was performed by a slide agglutination method following the Kauffmann-White-Le Minor scheme [69].

Identification of filamentous fungi was achieved at the genus or species level. Yeast colonies were identified by macro- and micro-morphologic characteristics and based on morphological and biochemical characteristics, such as the presence of capsule by India Ink testing, urease production at 25 °C, and the germ tube test. Biochemical identification was performed by Integral System Yeast (Liofilchem®, Italy).

For each plate, the number of colony forming units (CFU), was converted into number of bacteria or fungi or yeast per ml of saline solution, equal to the number of microflora per swab, following standard methods [70].

Virological examination

Swabs from oral cavity (n = 10) and cloaca (n = 10) were collected for virological examination. DNA was extracted using the DNeasy kit (Qiagen, Hombrechtikon, Switzerland) following the manufacturer’s instructions. The obtained DNA (assessed for purity and quantified with a spectrophotometer (Nanodrop, Thermofischer, Reinach, Switzerland) was used as a template (100 ng total, per sample) in distinct PCR reaction mix that was prepared and tested to detect the presence of herpesvirus, adenovirus and iridovirus DNA according to established protocols (panherpesvirus, [71]; panadenovirus, [72]; iridovirus, [73]) together with the proper reaction control samples.

Parasitological examination

Ticks collected from each tortoise were mounted in Hoyer medium and microscopically examined for their identification at species level, by using the taxonomic keys [74,75,76]. Fecal samples were analyzed macroscopically for helminths (e.g., adult nematodes, proglottids of cestodes, and worm fragments), and then microscopically for helminths and protozoa. For microscopical analysis, we used a flotation test with a low-density solution (saturated NaCl solution, specific gravity 1.2).

In order to evaluate the efficacy of fenbendazole treatment against oxyurids, a fecal egg count reduction (FECR) test was performed on samples prior and about 1 month after deworming with Fenbendazole (75 mg/kg, repeated with 50 mg/kg after 10 days) by using a low density solution (specific gravity 1.2) McMaster technique with a sensitivity of 50 eggs per gram (EPG) [62]. Pre- and post-treatment fecal egg counts (FEC) were used to calculate the reduction of fecal egg counts (FECR) according to the formula FECR = 100 * (1 - FEC post-treatment/FEC pre-treatment); FECR ≥90% indicated the efficacy of the treatment [59].

Statistical analysis

Frequencies of isolated microorganisms were compared among anatomical districts, sexes and, in the case of bacteria, Gram affinity, using Chi-square (χ2) and Fisher’s exact tests. Continuous variables are reported as means ± standard deviations and were analyzed using Student’s t-tests or ANOVA followed by Holm-Sidak post-hoc test when indicated. Statistical tests were performed using software STATA version 13 (STATA Corporation, College Station, Texas, US). P-values less than 0.05 were considered significant.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ANOVA:

Analysis of variance

Bcc:

Burkholderia cepacia complex

C:

Degrees celsius

CFU:

Colony-forming unit

CIN:

Cefsulodin-irgasan-novobiocin

CITES:

Convention on international trade of endangered species

CNAM:

Colistin and nalidixic acid modified

d:

Day

d.f.:

Degrees of freedom

DNA:

Deoxyribo nucleic acid

EPG:

Eggs per gram

e.g.:

For example (lat: Exempli gratia)

F:

Result of ANOVA test

FEC:

Fecal egg count

FECR:

Fecal egg count reduction

h:

Hour

IUCN:

International union for conservation of nature

kg:

Kilogram

m:

Minute

mg:

Milligram

ml:

Milliliter

mm:

Millimeter

μl:

Microliter

μm:

Micrometer

n:

Number

P :

P-value

PCR:

Polymerase chain reaction

s:

Second

SCUD:

Septicemic cutaneous ulcerative disease

STATA:

Software for statistics and data science

t:

Result of student’s t-test

χ2 :

Chi-square

References

  1. 1.

    Jacobson ER. Infectious diseases and pathology of reptiles. Boca Raton, Florida, USA: CRC Press; 2007.

    Google Scholar 

  2. 2.

    Jacobson ER, Brown MB, Wendland LD, Brown DR, Klein PA, Christopher MM, Berry KH. Mycoplasmosis and upper respiratory tract disease of tortoises: a review and update. Vet J. 2014;201(3):257–64.

    PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    McGuire JL, Smith LL, Guyer C, Yabsley MJ. Effects of mycoplasmal upper-respiratory-tract disease on movement and thermoregulatory behavior of gopher tortoises (Gopherus polyphemus) in Georgia. Usa J Wild Dis. 2014;50:745–56.

    Article  Google Scholar 

  4. 4.

    Fournié G, Goodman SJ, Cruz M, Cede V, Vélez A, Pati L, Millins C, Gibbons LM, Fox MT, Cunning AA. Biogeography of parasitic nematode communities in the Galápagos Giant tortoise: implications for conservation management. PLoS One. 2015;10(9):e0135684.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  5. 5.

    Thompson RCA, Lymbery AJ, Smith A. Parasites, emerging diseases and wildlife conservation. Int J Parasitol. 2010;40:1163–70.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    International Union for Conservation of Nature [IUCN]. 2019. The IUCN Red List of Threatened Species. Version 2019–1. <http://www.iucnredlist.org>. Accessed 22 Apr 2019.

  7. 7.

    Turtle Taxonomy Working Group, Rhodin AGJ, Iverson JB, Bour R, Fritz U, Georges A, Shaffer HB, van Dijk PP. Turtles of the world: annotated checklist and atlas of taxonomy, synonymy, distribution, and conservation status (8th Ed.). In: Rhodin AGJ, Iverson JB, van Dijk PP, Saumure RA, Buhlmann KA, Pritchard PCH, Mittermeier RA, editors. Conservation Biology of Freshwater Turtles and Tortoises: A Compilation Project of the IUCN/SSC Tortoise and Freshwater Turtle Specialist Group. Chelonian Res Monogr. 7:1–292. Arlington; 2017. https://doi.org/10.3854/crm.7.checklist.atlas.v8.2017.

  8. 8.

    Loehr VJT. Unexpected decline in a population of speckled tortoises. J Wildlife Manage. 2017;81:470–6.

    Article  Google Scholar 

  9. 9.

    Hofmeyr MD, Loehr VJT, Baard EHW. Chersobius signatus. The IUCN Red List of Threatened Species 2018;e.T10241A115650943. doi:https://doi.org/10.2305/IUCN.UK.2018-2.RLTS.T10241A115650943.en. Downloaded on 09 May 2020.

  10. 10.

    Loehr VJT, Henen BT, Hofmeyr MD. Tick infestations in the Namaqualand speckled padloper, Homopus signatus signatus (Gmelin, 1789). Afr Zool. 2006;41:170–7.

    Article  Google Scholar 

  11. 11.

    Loehr VJT. Diet of the Namaqualand speckled padloper, Homopus signatus signatus, in early spring. Afr J Herpetol. 2002;51:47–55.

    Article  Google Scholar 

  12. 12.

    Casadevall A, Pirofski LA. Host-pathogen interactions: basic concepts of microbial commensalism, colonization, infection, and disease. Infect Immun. 2000;68(12):6511–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Boma H. Innate immunity and the normal microflora. Immunol Rev. 2000;173:5–16.

    Article  Google Scholar 

  14. 14.

    Tlaskalová-Hogenová H, Štěpánková R, Hudcovic T, Tučková L, Cukrowska B, Lodinová-Žádnıková R, et al. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett. 2004;93(2–3):97–108.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  15. 15.

    Marenzoni ML, Santoni L, Felici A, Maresca C, Stefanetti V, Sforna M, et al. Clinical, virological and epidemiological characterization of an outbreak of Testudinid Herpesvirus 3 in a chelonian captive breeding facility: lessons learned and first evidence of TeHV3 vertical transmission. PLoS One. 2018;13(5):e0197169.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. 16.

    Brown MB, Schumacher IM, Klein PA, Harris K, Correll T, Jacobson ER. Mycoplasma agassizii causes upper respiratory tract disease in the desert tortoise. Infect Immun. 1994;62(10):4580–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Brown DR, Merritt JL, Jacobson ER, Klein PA, Tully JG, Brown MB. Mycoplasma testudineum sp. nov., from a desert tortoise (Gopherus agassizii) with upper respiratory tract disease. Int J Syst Evol Micr. 2004;54(5):1527–9.

    CAS  Article  Google Scholar 

  18. 18.

    Ossiboff RJ, Raphael BL, Ammazzalorso AD, Seimon TA, Niederriter H, Zarate B, Newton AL, McAloose D. A mycoplasma species of Emydidae turtles in the northeastern USA. J Wildl Dis. 2015;51:466–70.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Soares JF, Chalker VJ, Erles K, Holtby S, Waters M, McArthur S. Prevalence of Mycoplasma agassizii and Chelonian herpesvirus in captive tortoises (Testudo sp.) in the United Kingdom. J Zoo Wildl Med. 2004;35:25–33.

  20. 20.

    Lecis R, Paglietti B, Rubino S, Are BM, Muzzeddu M, Berlinguer F, Chessa B, Pittau M, Alberti A. 2011. Detection and characterization of mycoplasma spp. and Salmonella spp. in free-living European tortoises (Testudo hermanni, Testudo graeca, and Testudo marginata). J Wildl Dis. 2011;47(3):717–24.

    PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Ferronato BO, Marques TS, Souza FL, Verdade LM, Matushima ER. Oral bacterial microbiota and traumatic injuries of freeranging Phrynops geoffranus (Testudines, Chelidae) in southeastern Brazil. Phyllomedusa. 2009;8(11):19–25.

    Article  Google Scholar 

  22. 22.

    Reavill D, Schmidt RE. Pathology of the reptile eye and ocular adnexa. In: Proc Annu Conf Assoc Rept Amph Vet 2012;87–97.

  23. 23.

    Di Ianni F, Dodi PL, Cabassi CS, Pelizzone I, Sala A, Cavirani S, Parmigiani E, Quintavalla F, Taddei S. Conjunctival flora of clinically normal and diseased turtles and tortoises. BMC Vet Res. 2015;11:91.

    PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Kampfer P, Martin K, Schafer J, Schumann P. Kytococcus aerolatus sp. nov. isolated from indoor air in a room colonized with moulds. Syst Appl Microbiol. 2009;32:301–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Jihye H, Keon-Han K, Jung-Ok K, Jun-Sung H, Seok HJ, Kyungwon L. Bacteraemia caused by Kytococcus schroeteri in a pneumonia patient. J Med Microb Diagn. 2015;4:4.

    Google Scholar 

  26. 26.

    Ebani V, Fratini F. Bacterial zoonozes among domestic reptiles. Ann Fac Med Vet. 2005;58:85–91.

    Google Scholar 

  27. 27.

    Nowakiewicz A, Ziołkowska G, Zieba P, Majer Dziedzic B, Gnat S, Wójcik M, Dziedzic R, Kostruba A. Aerobic bacterial microbiota isolated from the cloaca of the European pond turtle (Emys orbicularis) in Poland. J Wildl Dis. 2015;51(1):255–9.

    PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Serafini PP, Guarrido MM, Biesdorf MS, Vianna VO, Klostermann DZ, Oczkovski FI, Machado D. Microbiota oral e cloacal de jacaré- do-Pantanal (Caiman crocodilus yacare) e jacaré-tingá (C. c. crocodilus). In: Proc of XXVI Congresso Brasileiro de Zoologia (Brazilian Conference of Zoology). Londrina: PR; 2006.

    Google Scholar 

  29. 29.

    Abalem de Sá IV, Solari C. Salmonella in Brazilian and imported pet reptiles. Braz J Microbiol. 2006;32(4):293–7.

    Google Scholar 

  30. 30.

    Rosenthal KL, Mader DR. Microbiology. In: Reptile medicine and surgery, ed. Mader DR. 1996;117–25.

  31. 31.

    Joyner PH, Brown JD, Holladay S, Sleeman JM. Characterization of the bacterial microflora of the tympanic cavity of eastern box turtles with and without aural abscesses. J Wildl Dis. 2006;42(4):859–64.

    PubMed  Article  Google Scholar 

  32. 32.

    Glazebrook JS, Campebell RSF. A survey of the diseases of marine turtles in northern Australia. II. Ocenarium-reared and wild turtles. Dis Aquat Org. 1990;9:97–104.

    Article  Google Scholar 

  33. 33.

    Martinez-Silvestre A, Mateu-de-Antonio EM. Bacteriological features of rhinitis in captive Greek tortoises, Testudo graeca. Bull Assoc Reptilian Amphybian Veterinarians. 1997;7(2):12–5.

    Article  Google Scholar 

  34. 34.

    Chuen-Im T, Areekijseree M, Chongthammakun S, Graham SV. Aerobic bacterial infections in captive juvenile green turtle (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata) from Thailand. Chelonian Conserv Biol. 2010;9:135–42.

    Article  Google Scholar 

  35. 35.

    Chang YT, Lin CY, Chen YH, Hsueh PR. Update on infections caused by Stenotrophomonas maltophilia with particular attention to resistance mechanisms and therapeutic options. Front Microbiol. 2015;6:893.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Yang PY, Lee AH, Chi CH. Case report: the first report of respiratory infection by Stenotrophomonas malthophilia in a red-footed tortoise (Geochelone carbonaria). Taiwan Vet J. 2010;36(2):177–81.

    Google Scholar 

  37. 37.

    Draper CS, Walker RD, Lawler HE. Patterns of oral bacterial infection in captive snakes. J Am Vet Med A. 1981;179:1223–6.

    CAS  Google Scholar 

  38. 38.

    Oettle EE, Steytler YGM, Williams MC. High mortality in a tortoise colony. S Afr J Wildl Res. 1990;20(1):21–5.

    Google Scholar 

  39. 39.

    Dickinson VM, Duck T, Schwalbe CR, Jarchow JL, Trueblood MH. Nasal and cloacal bacteria in free-ranging desert tortoises from the western United States. J Wildl Dis. 2001;37:252–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Santoro M, Hernández G, Caballero M, García F. Aerobic bacterial flora of nesting green turtles (Chelonia mydas) from Tortuguero National Park, Costa Rica. J Zoo Wildl Med. 2006;37(4):549–52.

    PubMed  Article  Google Scholar 

  41. 41.

    Percipalle M, Giardina G, Lipari L, Piraino C, Macrì D, Ferrantelli V. Salmonella infection in illegally imported spur-thighed tortoises (Testudo graeca). Zoonoses Public Hlth. 2011;58(4):262–9.

    CAS  Article  Google Scholar 

  42. 42.

    Bruce HL, Barrow PA, Rycroft AN. Zoonotic potential of Salmonella enterica carried by pet tortoises. Vet Rec. 2018;182(5):141.

    PubMed  Article  Google Scholar 

  43. 43.

    Gaertner JP, Hahn D, Jackson J, Forstner MRJ, Rose FL. Detection of salmonellae in captive and free-ranging turtles using enrichment culture and polymerase chain reaction. J Herpetol. 2008;42:223–31.

    Article  Google Scholar 

  44. 44.

    Hidalgo-Vila J, Díaz-Paniagua C, Pérez-Santigosa N, de Frutos-Escobar C, Herrero-Herrero A. Salmonella in free-living exotic and native turtles and in pet exotic turtles from SW Spain. Res Vet Sci. 2008;85(3):449–52.

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Hidalgo-Vila J, Diaz-Paniagua C, de Frutos-Escobar C, Jimenez-Martinez C, Perez-Santigosa N. Salmonella in free living terrestrial and aquatic turtles. Vet Microbiol. 2007;31:119–23.

    Google Scholar 

  46. 46.

    Mermin J, Hutwagner L, Vugia D, Shallow S, Daily P, Bender J, Koehler J, Marcus R, Angulo FJ. Reptiles, amphibians, and human Salmonella infection: a population-based case-control study. Clin Infect Dis. 2004;38:S253–61.

    PubMed  Article  Google Scholar 

  47. 47.

    Glazebrook JS, Campbell RSF. A survey of the diseases of marine turtles in northern Australia I. Farmed turtles. Dis Aquat Org. 1990;9:83–95.

    Article  Google Scholar 

  48. 48.

    Orós J, Torrent A, Calabuig P, Déniz S. Diseases and causes of mortality among sea turtles stranded in the Canary Islands, Spain (1998–2001). Dis Aquat Org. 2005;63:13–24.

    Article  Google Scholar 

  49. 49.

    Rhodes KA, Schweizer HP. Antibiotic resistance in Burkholderia species. Drug Resist Updat. 2016;28:82–90.

    PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Coenye T, Vandamme P, Govan JRW, Lipuma JJ. Taxonomy and identification of the Burkholderia cepacia complex. J Clin Microbiol. 2001;39:3427–36.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Lemaitre C, Orange N, Saglio P, Saint N, Gagnon J, Molle G. Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpio). Eur J Biochem. 1996;240:143–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Saini LS, Galsworthy SB, John MA, Valvano MA. Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation. Microbiology. 1999;145:3465–75.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Tuon FF, Costa SF. Rhodotorula infection. A systematic review of 128 cases from literature. Rev Iberoam Micol. 2008;25(3):135–40.

    PubMed  Article  PubMed Central  Google Scholar 

  54. 54.

    PBD M, Pimenta RS, Tavares IB, de Garcia V, Rosa CA. Yeasts occurring in surface and mouth cavity of two chelonian species, Podocnemis expansa Schweigger and P. unifilis troschel (Reptilia: Chelonia: Pelomedusidae), in the Javaés river border of Araguaia national park in Brazil. Int J Microbiol. 2010. https://doi.org/10.1155/2010/504524.

  55. 55.

    Meletiadis J, Roilides E. Rare invasive fungal infections: epidemiology, diagnosis and management. Curr Fungal Infect Rep. 2013;7(4):351–60.

    Article  Google Scholar 

  56. 56.

    Dipineto L, Capasso M, Maurelli MP, Russo TP, Pepe P, Capone G, Fioretti A, Cringoli G, Rinaldi L. Survey of co-infection by Salmonella and oxyurids in tortoises. BMC Vet Res. 2012;8:69.

    PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Capelli G, Borsato E, Stancampiano L, Bozzolan G, Pietrobelli M. Epidemiology of gastrointestinal parasites of tortoises (Testudo hermanni boettgeri) in captivity. Parassitologia. 1998;40:29.

    Google Scholar 

  58. 58.

    Traversa D, Capelli G, Iorio R, Bouamer S, Cameli A, Giangaspero A. Epidemiology and biology of nematodofauna affecting Testudo hermanni, Testudo graeca and Testudo marginata in Italy. Parasitol Res. 2005;98(1):14.

    PubMed  Article  PubMed Central  Google Scholar 

  59. 59.

    Giannetto S, Brianti E, Poglayen G, Sorgi C, Capelli G, Pennisi MG, Coci G. Efficacy of oxfendazole and fenbendazole against tortoise (Testudo hermanni) oxyurids. Parasitol Res. 2007;100(5):1069–73.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  60. 60.

    Carpenter JW. Exotic animal formulary. 4th ed. St. Louis: Elsevier; 2013.

  61. 61.

    Neiffer DL, Lydick D, Burks K, Doherty D. Hematologic and plasma biochemical changes associated with fenbendazole administration in Hermann's tortoises (Testudo hermanni). J Zoo Wildl Med. 2005;36(4):661–72.

    PubMed  Article  PubMed Central  Google Scholar 

  62. 62.

    Taylor MA, Coop RL, Wall RL. Malattie parassitarie degli animali domestici (Parasitic diseases of domestic animals). First italian ed. from Garippa G, Manfredi MT, Otranto D. EMSI, Rome, Italy, 2010;665–672.

  63. 63.

    Satbige AS, Kasaralikar VR, Halmandge SC, Rajendran C. Nyctotherus sp. infection in pet turtle: a case report. J Parasit Dis. 2017;41:590–2.

    PubMed  Article  PubMed Central  Google Scholar 

  64. 64.

    Helke KL, Cooper TK, Mankowski JL, Poynton SL. 2006. Disseminated visceral coccidiosis in indo-gangetic flap-shelled turtles, Lissemys punctata andersonii. J Wildl Dis. 2006;42(4):788–96.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  65. 65.

    Schmidt V, Dyachenko V, Aupperle H, Pees M, Krautwald-Junghanns ME, Daugschies A. Case report of systemic coccidiosis in a radiated tortoise (Geochelone radiata). Parasitol Res. 2008;102(3):431–6.

    PubMed  Article  PubMed Central  Google Scholar 

  66. 66.

    Stringer AP, Linklater W. Everything in moderation: principles of parasite control for wildlife conservation. Bioscience. 2014;64:932–7.

    Article  Google Scholar 

  67. 67.

    Hing S, Narayan EJ, Thompson RCA, Godfrey SS. The relationship between physiological stress and wildlife disease: consequences for health and conservation. Wildlife Res. 2016;43:51–60.

    Article  Google Scholar 

  68. 68.

    NHS - United Kingdom Standard Units, Microbiology Services, 2014. Public health, England.

  69. 69.

    Grimont PA, Weill FX. Antigenic formulae of the Salmonella serovars. WHO Collaborating Centre Reference Res Salmonella. 2007;9:1–166.

    Google Scholar 

  70. 70.

    Ferguson AW, Scott JA, McGavigan J, Elton RA, McLean J, Schmidt U, Kelkar R, Dhillon B. Comparison of 5% povidone-iodine solution against 1% povidone-iodine solution in preoperative cataract surgery antisepsis: a prospective randomized double blind study. Br J Ophthalmol. 2003;87:163–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. 71.

    Vandevanter DR, Warrener P, Bennett L, Schultz ER, Coulter S, Garber RL, Rose TM. Detection and analysis of diverse Herpesviral species by consensus primer PCR. J Clin Microbiol. 1996;34(7):1666–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  72. 72.

    Wellehan JFX, Johnson AJ, Harrach B, Benkö M, Pessier AP, Johnson CM, Garner MM, Childress A, Jacobson ER. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the Atadenoviruses. J Virol. 2004;78(23):13366–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

    Marschang RE, Becher P, Posthaus H, Wild P, Thiel HJ, Müller-Doblies U, Kaleta EF, Bacciarini LN. Isolation and characterization of an Iridovirus from Hermann's tortoises (Testudo hermanni). Arch Virol. 1999;144(10):1909–22.

    CAS  PubMed  Article  Google Scholar 

  74. 74.

    Neveu-Lemaire M. Traitè d’entomologie Medicale et Veterinaire. Paris: Vigot Freres; 1983. p. 431–2.

    Google Scholar 

  75. 75.

    Abdel-Shafy S, Gabr HS, Abdullah HH, Mahmoud MS. Morphological and molecular description of immature stages of Ornithodoros savignyi (Acari: Argasidae). Parasitol Res. 2016;115(8):3033–40.

    PubMed  Article  PubMed Central  Google Scholar 

  76. 76.

    Bakkes DK, De Klerk D, Latif AA, Mans BJ. Integrative taxonomy of Afrotropical Ornithodoros (Ornithodoros) (Acari: Ixodida: Argasidae). Ticks Tick Borne Dis. 2018;9(4):1006–37.

    PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgments

Northern Cape Department of Enviromment and Nature Conservation for issuing permits to collect and export C. signatus. Volunteers Olda Mudra, Sérgio Silva and Sam Beales for their help to locate and capture C. signatus in the wild. Martijn Kooijman for his help collecting swabs.

Funding

This work has not been founded.

Author information

Affiliations

Authors

Contributions

VL and LG were responsible for the conception of the study. ARA, SP, FCO, AMT, LG, VL performed data analysis and wrote the manuscript. VC, MN, NAM, GP performed microbiological analysis. GR reviewed the manuscript and provided critical suggestions and comments. All authors discussed the results and approved the final manuscript.

Corresponding author

Correspondence to Livio Galosi.

Ethics declarations

Ethics approval and consent to participate

The animals included in this study were collected in South Africa respecting local rules (permit FAUNA 053/2015) and imported in Europe (export permit CITES 148487; import permit 15NL226435/11). According to the European Directive 2010/63/UE and the Italian Legislative Decree 26/2014, no ethical approval is needed for the examinations performed in this study as they were a good veterinary survey, without cause animal suffering. Written consent to participate was given by the owner of the animals, the Dwarf Tortoise Conservation, Netherlands.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Galosi, L., Attili, A.R., Perrucci, S. et al. Health assessment of wild speckled dwarf tortoises, CHERSOBIUS SIGNATUS. BMC Vet Res 17, 102 (2021). https://doi.org/10.1186/s12917-021-02800-5

Download citation

Keywords

  • Health assessment
  • Chersobius [Homopus] signatus
  • Reptile
  • Tortoise
  • Wildlife
\