Nutrition and husbandry conditions of Palearctic tortoises (Testudo spp.) in captivity

Mediterranean and Russian tortoises (Testudo spp.) are popular companion animals (pets), despite ongoing controversy concerning privately keeping reptiles. The arguments used during these controversial discussions have often been based on outdated facts. Therefore, a survey was developed to evaluate the current population structure, husbandry conditions, diet regime, and health status of Testudo species in captivity. More than 75% of the 1075 respondents housed their tortoises in an outdoor enclosure containing a greenhouse or cold frame, which is considered the most species-appropriate way of husbandry. Of the respondents, 67.7% fed their tortoises with the optimum diet of more than 80% grasses and weeds during the summer vegetation period. Only 8.2% of respondents owned a tortoise with a diagnosed disease. According to the results, the likelihood of tortoises developing pyramidal growth syndrome, which can be used as an indicator of the quality of tortoise husbandry, was high in tortoises kept in a terrarium and/or fed a diet of less than 80% grasses and weeds in summer. This likelihood varied among species, with a higher incidence in Hermann’s tortoises (Testudo hermanni).     

Pathology of upper respiratory tract disease of gopher tortoises in Florida

Between August 1993 and September 1995, 24 gopher tortoises (Gopherus polyphemus) were received for pathological evaluations from various locations in Florida (USA). All tortoises were examined for clinical signs of upper respiratory tract disease (URTD) including nasal and ocular discharge, palpebral edema, and conjunctivitis. Of the 24 tortoises, 10 had current or previously observed clinical signs of URTD and 14 did not. A blood sample was drawn for detection of anti-mycoplasma antibodies by ELISA, and nasal lavage samples were collected for culture and detection of Mycoplasma agassizii gene sequences by polymerase chain reaction (PCR). Of the 14 clinically healthy tortoises, eight were sero-, culture- and PCR-negative, and six were seropositive for antibodies against M. agassizii. Of those six, five were culture- and/or PCR-positive for M. agassizii, and one was culture- and PCR-negative. Of the 10 ill tortoises, nine were seropositive by the ELISA and one was in the suspect range. Nine of the ill tortoises, including the suspect tortoise, were culture- and/or PCR-positive for M. agassizii, and one was culture- and PCR-negative. For histologic evaluation and discussion, the eight sero-, culture-, and PCR-negative tortoises were designated URTD-negative, and the other 16 were classified as URTD-positive. Histologic evaluation of the upper respiratory tract (URT) indicated the presence of mild to severe inflammatory, hyperplastic, or dysplastic changes in 14 URTD-positive tortoises. Seven of eight URTD-negative tortoises had normal appearing nasal cavities; one had mild inflammatory changes. Transmission electron microscopy revealed an organism consistent with Mycoplasma spp. on the nasal mucosal surface of tortoises with clinical signs and lesions of URTD. Additionally, gram-negative bacteria were isolated more frequently from the nasal cavities of URTD-positive tortoises than URTD-negative tortoises. Because clinical signs of URTD were never observed in six of the URTD-positive tortoises, we also conclude that subclinical URTD can occur in gopher tortoises.     

Effect of hypophysectomy on plasma electrolytes and adrenal mineralocorticoid secretion in the terrestrial chelonian Testudo hermanni

Na, K, ACTH, corticosterone and aldosterone plasma levels were studied in Testudo hermanni after hypophysectomy and dexamethasone administration. Hypophysectomy as well as dexamethasone treatment caused a deep decrease in the plasma levels of electrolytes, corticosterone and aldosterone when compared to sham-operated or normal controls. ACTH plasma level was markedly lower in hypophysectomized or dexamethasone-treated tortoises when compared to the controls but never reached undetectable levels. Administration of ACTH significantly increased plasma electrolytes and hormone levels in hypophysectomized and dexamethasone-treated tortoises. The present data seem to indicate that the pituitary plays a significant role on electrolyte homeostasis and on the regulation of plasma mineralocorticoids level in terrestrial chelonians.     

Mycoplasmosis in free-ranging desert tortoises in Utah and Arizona

Upper respiratory tract disease (URTD) has been associated with major losses of free-ranging desert tortoises (Gopherus agassizii) in the southwestern United States. This prompted a clinical examination of 63 free-ranging desert tortoises for signs of URTD and sampling for Mycoplasma agassizii, the causative agent of URTD. Tortoises were sampled from three sites in the eastern Mojave Desert (1992-93), and from three sites in the Sonoran Desert (1992-94). Plasma samples were tested for antibodies to M. agassizii using an enzyme-linked immunosorbent assay (ELISA). Nasal aspirate samples from 12 Sonoran tortoises were tested using polymerase chain reaction (PCR) test directed at the 16S rRNA gene of M. agassizii. Nasal aspirate samples from all tortoises were cultured for M. agassizii. In the Mojave Desert, nine tortoises had clinical signs of URTD and eight were seropositive for M. agassizii. In the Sonoran Desert, there were no clinical signs of URTD, but two tortoises were seropositive, and two tortoises had positive PCR results.     

Mycoplasma and herpesvirus PCR detection in tortoises with rhinitis-stomatitis complex in Spain

Chelonid herpesvirus (ChHV) and mycoplasmal infections cause similar clinical signs in terrestrial tortoises and may be the most important causative agents of rhinitis-stomatitis complex, a common disease in captive tortoises worldwide. Currently, diagnosis of ChHV and Mycoplasma spp. infections is most often based on serologic testing. However, serologic results only detect past exposure, and the specificity of these tests can be reduced due to antigenic cross-reactions with other pathogens. Molecular-based techniques could help to define the causative agent and to better manage infected tortoises. Using polymerase chain reaction, we analyzed 63 tortoises (59 spur-thighed tortoise, Testudo graeca; three Greek tortoise, Testudo ibera; and one Russian tortoise, Agryonemys horsfieldii) with clinical signs of rhinitis-stomatitis complex to identify the causative agent. Molecular evidence of ChHV type I (24%), type II (3%), and Mycoplasma agassizii (6%) infections, as well as coinfection of Mycoplasma-ChHV and both types of ChHV, were detected. Both ChHV and M. agassizii are considered pathogenic in captive tortoises and both are a threat to wild populations. However, neither agent was detected from most of the symptomatic tortoises we evaluated, indicating that other agents could be involved in the rhinitis-stomatitis complex.     

Foraging behaviour and diet of an ectothermic herbivore: Testudo horsfieldi

Herbivorous vertebrates of arid regions are frequently faced with inadequate food quality, quantity or both. The time and energy devoted to foraging is vital to balancing their energy budgets. For desert ectotherms, a low metabolism should be advantageous, reducing their total energy requirement, but extreme ambient temperatures can strongly constrain these animals' activity periods. We provide the first data on the activity budgets, foraging behaviour and diet of a highly abundant, desert-dwelling, herbivorous ectotherm, the steppe tortoise Testudo horsfieldi . Extreme climatic conditions of Central Asia limit steppe tortoise's activity to only three months per year. They remain inactive most of their "active season" (90%), and spend very little time foraging (<15 min per day). This suggests that steppe tortoises can satisfy their energy requirements with modest feeding efforts. Interestingly, steppe tortoises avoid feeding on grass species and feed mostly on plant species that are usually highly toxic to mammals. This result suggests that steppe tortoises and ungulates do not compete for food.     

Seroepidemiology of upper respiratory tract disease in the desert tortoise in the western Mojave Desert of California

Several factors have combined with an upper respiratory tract disease (URTD) to produce declines on some population numbers of desert tortoises (Gopherus agassizii) in the western USA. This study was designed to determine the seroepidemiology of URTD in a population of wild adult tortoises at the Desert Tortoise Research Natural Area (DTNA) study site in Kern County (California, USA). Prior to initiation of the study, there was a dramatic decline in the number of individuals in this population. At each individual time point, samples were obtained from 12 to 20 tortoises with radiotransmitters during winter, spring, summer, and fall from 1992 through 1995. During the course of the study, 35 animals were sampled at one or more times. Only 10 animals were available for consistent monitoring throughout the 4 yr period. Specific antibody (Ab) levels to Mycoplasma agassizii were determined for individual tortoises by an enzyme-linked immunosorbent assay (ELISA) test. Specific Ab levels were not influenced by the gender of the tortoise. Levels of Ab and distribution of ELISA+, ELISA- and suspect animals were not consistently affected by season within a single year or for a season among the study years. Significantly more tortoises presented with clinical signs in 1992 and 1995. The profile of ELISA+ animals with clinical signs shifted from 5% (1992) to 42% (1995). In 1992, 52% of tortoises lacked clinical signs and were ELISA-. In 1995, this category accounted for only 19% of tortoises. Based on the results of this study, we conclude that URTD was present in this population as evidenced by the presence of ELISA+ individual animals, and that the infectious agent is still present as evidenced by seroconversion of previously ELISA- animals during the course of the study. There is evidence to suggest that animals may remain ELISA+ without showing overt disease, a clinical pattern consistent with the chronic nature of most mycoplasmal infections. Further, there are trends suggesting that the clinical expression of disease may be cyclical. Continued monitoring of this population could provide valuable information concerning the spread of URTD in wild tortoise populations.     

Phylogenetic relationships among the species of the genus Testudo (Testudines: Testudinidae) inferred from mitochondrial 12S rRNA gene sequences

To test phylogenetic relationships within the genus Testudo (Testudines: Testudinidae), we have sequenced a fragment of the mitochondrial (mt) 12S rRNA gene of 98 tortoise specimens belonging to the genera Testudo, Indotestudo, and Geochelone. Maximum likelihood and neighbor-joining methods identify two main clades of Mediterranean tortoises, one composed of the species Testudo graeca, Testudo marginata, and Testudo kleinmanni and a second of Testudo hermanni, Testudo horsfieldii, and Indotestudo elongata. The first clade, but not the second, was also supported by maximum parsimony analysis. Together with the genus Geochelone, a star-like radiation of these clades was suggested, as a sister-group relationship between the two Testudo clades could not be confirmed. The intraspecies genetic variation was examined by sequencing the mt 12S rRNA fragment from 28 specimens of T. graeca and 49 specimens of T. hermanni from various geographic locations. Haplotype diversity was found to be significantly larger in T. graeca compared with T. hermanni, suggestive of reduced genetic diversity in the latter species, perhaps due to Pleistocene glaciations affecting northern and middle Europe or other sources of lineage reduction. No ancient mt 12S rRNA gene haplotypes were identified in T. graeca and/or T. hermanni originating from islands in the Mediterranean Sea, suggesting that these islands harbor tortoise populations introduced from the European and African mainland.     

Renin-angiotensin system and osmoregulation in the terrestrial chelonian Testudo hermanni Gmelin

1. Renal renin activity, Na and K plasma urinary levels were studied in Testudo hermanni after salted diet, saline solution administration and furosemide injection. 2. Salt loading depressed renal renin activity whereas injection of the diuretic resulted in sodium loss and enhanced renal renin activity. 3. Results lead one to conclude the presence of a renin-angiotensin system with a direct action upon osmoregulation in these terrestrial chelonians.     

Variation in Growth and Potentially Associated Health Status in Hermann's and Spur‐Thighed Tortoise (Testudo hermanni and Testudo graeca)

Captive reptiles often show higher growth rates than in the wild, possibly due to higher feeding intensity. Although health problems are usually linked to inappropriate diets, fast growth itself, such as triggered by appropriate diets fed in high amounts, has traditionally also been considered unfavorable for tortoises. We document growth rates (based on age and mass) from private Testudo hermanni and T. graeca breeders, which are generally higher than those reported for free‐ranging specimens, but show enormous variation. Tortoise patients presented to an exotics clinic also covered the whole growth rate spectrum. To test whether fast growth was associated with diseases, the age–body mass relationship of these patients was tested, in a retrospective evaluation, for additional influence factors, such as dietary history and occurrence of certain diet and growth‐related diseases. No indication was found that animals particularly heavy for their age were more prone to diet/growth‐related disorders. In general, tortoises fed diets with meat/grain were heavier for their age than tortoises fed more appropriate diets; dietary history was not related to a particular disease. The results suggest the age–body mass relationship may not be suitable for testing effects of fast growth; an age–body length relationship would be more appropriate. Animals presented for a diet/growth‐related disorder were younger than animals presented for other reasons; there was a significant negative correlation between the severity of pyramiding and age, suggesting that growth‐related disorders may well limit the life expectancy of tortoises. Controlled clinical studies are required to fully test this hypothesis. Zoo Biol. 31:705‐717, 2012. © 2012 Wiley Periodicals, Inc.     

The evolution of island gigantism and body size variation in tortoises and turtles

Extant chelonians (turtles and tortoises) span almost four orders of magnitude of body size, including the startling examples of gigantism seen in the tortoises of the Galapagos and Seychelles islands. However, the evolutionary determinants of size diversity in chelonians are poorly understood. We present a comparative analysis of body size evolution in turtles and tortoises within a phylogenetic framework. Our results reveal a pronounced relationship between habitat and optimal body size in chelonians. We found strong evidence for separate, larger optimal body sizes for sea turtles and island tortoises, the latter showing support for the rule of island gigantism in non-mammalian amniotes. Optimal sizes for freshwater and mainland terrestrial turtles are similar and smaller, although the range of body size variation in these forms is qualitatively greater. The greater number of potential niches in freshwater and terrestrial environments may mean that body size relationships are more complicated in these habitats.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

Long-term occurrence of Hemolivia cf. mauritanica (Apicomplexa: Adeleina: Haemogregarinidae) in captive Testudo marginata (Reptilia: Testudinidae): evidence for cyclic merogony?

Blood smears from wild-caught, long-term captive tortoises, Testudo marginata, revealed the presence of gametocytes of a Hemolivia mauritanica-like hemogregarine in the erythrocytes of 72% tortoises examined. Significant parasitemia was also found in animals living several years in captivity. Experimentally infected tortoises showed no evidence of a decrease in parasitemia level more than 15 mo after infection. Morphologically, stages found in tortoises' erythrocytes were indistinguishable from those referred to by previous workers as H. mauritanica from Testudo graeca. Moreover, successful experimental transmission to Hyalomma aegyptium confirms the conspecificity with H. mauritanica. The occurrence of H. mauritanica gametocytes in tortoise living up to 8 yr in captivity is suggested to result from continuous, long-lasting cyclic merogony in tortoises' parenchymatous organs, which is an unknown phenomenon in the life cycle of Hemolivia spp.     

Female preference for fast-rate, high-pitched calls in Hermann's tortoises Testudo hermanni

The courtship and mounting behavior of promiscuous tortoisesis based on a multiple signaling system that includes visual,olfactory, tactile, and acoustic signals. Vocalizations relatedto mounting seem to be particularly intriguing because tortoisesvocalize mainly at this time. Vocalizations and courtship behaviormight be costly for males, and if these costs increase differentiallyfor different males, then the potential exists for vocalizationsand displays to reveal male individual quality on which femalescould base their mate choice. In the present study, by usingindividuals breeding in seminatural enclosures, we experimentallyinvestigated whether the mounting call of male Hermann's tortoises(Testudo hermanni) represents a relevant stimulus to both malesand females. We also looked for potential preference towarddifferent acoustic stimuli in order to determine which callfeatures, if any, played a role in attracting potential partnersor deterring rivals. Females were sensitive to call playback,whereas males apparently paid little attention to playback.In addition, females showed a significant preference towardhigh-pitched calls emitted at high rate. Also, females exhibiteda strong tendency toward short-duration calls. Because callrate is related to hematocrit in Hermann's tortoise males, byresponding to fast-rate calls females are also choosing high-qualitypartners as sires of their offspring. This is the first studyin which an attractive function of tortoise vocalizations hasbeen documented.     

Intranuclear coccidiosis in tortoises — discovery of its causative agent and transmission

Intranuclear coccidiosis of testudines (known as TINC) is an emerging disease in chelonians. Although endogenous stages were repeatedly detected in various tissues, attempts to find the oocysts in faeces failed, leaving the question of the transmission and classification of the causative agent of TINC unresolved. We recorded small spherical oocysts (∼6–7 μm in diameter) of an eimeriid coccidium in faeces of a leopard tortoise (Stigmochelys pardalis). Sporulated oocysts were used for the experimental oral inoculation of juvenile coccidia-free tortoises representing 5 species (S. pardalis, Testudo graeca, T. hermanni, T. horsfieldii, and Geochelone sulcata). The oocysts’ association with TINC was confirmed based on clinical signs, histopathological findings of intranuclear endogenous stages of the coccidium in many organs (including intestine), and by the partial 18S rDNA sequence analysis of the DNA isolated from organs of the experimentally infected animals and from a single naturally infected as well as from all experimentally infected tortoises. Breeding colonies of chelonians should be screened for this pathogen in order to prevent its further spread and unwanted introduction into endangered free-ranging chelonian populations.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway.

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.     

Hemolivia mauritanica (Haemogregarinidae: Apicomplexa) infection in the tortoise Testudo graeca in the Near East with data on sporogonous development in the tick vector Hyalomna aegyptium

Testudo groeca tortoises were collected in the northern and southern Golan Heights (Israeli occupied territory of south Syria), and various locations in Israel and Palestine. Hyoalomma oegyptium ticks were found only on Golan Height tortoises, and only the tortoises and ticks from the northern Golan Heights were infected with Hemolivia mouritanica. Tortoises became infected after ingesting infected ticks. Male ticks carrying sporocysts, which remain attached to tortoises for extended durations, apparently served as the source for dissemination of new infections among tortoises. Sporogenesis followed the pattern observed in the two other known species of Hemolivia, though there was some evident variation in fine-structura detail. The sutural slit detected in the H. mouritanica mature sporocyst wall was reminiscent of the suture characteristic of Coccidia of heterothermic vertebrate hosts; it could be a common ancestral character for both hemogregarines     

Effects of landscape features and demographic history on the genetic structure of Testudo marginata populations in the southern Peloponnese and Sardinia

Testudo marginata, the largest European land tortoise, is suffering habitat degradation and destruction. Some populations, in markedly degraded habitats, are characterized by divergent morphotypes. However, the evolutionary significance of these morphotypes is of debate. Using 11 polymorphic microsatellites, we studied: (1) marginated tortoises from Sardinia that display a divergent morphotype – this population was potentially introduced from Greece; and (2) an area in the southern Peloponnese that includes a small and degraded zone in which marginated tortoises are dwarf. Genetic analyses run without any a priori assignment clearly acknowledge the specimens sampled in the territory of the dwarf form as a single group whilst Sardinian specimens are clustered with other specimens from the northern part of the area sampled in Greece. Demographic analyses suggest that Sardinian tortoises originated recently from some of the populations sampled in the northern part of the area sampled in Greece. Over locations sampled in Greece, a landscape‐genetic analysis allowed us to detect potential landscape features that may reduce gene flow between the dwarf form territory and surrounding areas. Our results suggest that the territory of the dwarf form is particularly propitious for marginated tortoises and that conservation regulations in Greece should be reinforced to protect this area from increasing impact of human activities changing from traditional agriculture to mechanization and extensive use of chemicals. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 591–606.