HEALTH MONITORING: REPTILES

CROCODILES

Lovely C. J., Leslie A. J. (2008): Normal intestinal flora of wild Nile crocodiles (Crocodylus niloticus) in the Okavango Delta, Botswana. Journal of the South African Veterinary Association 79: 67-70.
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Bacterial and fungal cultures were performed from cloacal swabs collected from 29 wild Nile crocodiles, captured in the Okavango Delta, Botswana. Sixteen species of bacteria and 6 fungal species were cultured. Individual crocodiles yielded bacterial species, and 0-2 fungal species. The most commonly isolated bacteria were Microbacterium, Enterococcus faecalis, Aeromonas hydrophila, and Escherichia coli. No salmonellae were cultured. The most commonly occurring fungus was Cladosporium. Several of the bacterial and fungal species isolated have been implicated in cases of septicaemia in crocodilians. Knowledge of the normal intestinal flora will contribute towards the development of a crocodile-specific probiotic for use in farmed crocodiles.

Johnston M. A., Porter D. E., Scott G. I., Rhodes W. E., Webster L. F. (2010): Isolation of faecal coliform bacteria from the American alligator (Alligator mississippiensis). Journal of Applied Microbiology 108: 965-973.
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To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina’s coastal waters. Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most-probable-number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8·0×109 g−1 (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.

Charruau P., Pérez-Flores J., Pérez-Juárez J. G., Cedeño-Vázquez J. R., Rosas-Carmona R. (2012): Oral and cloacal microflora of wild crocodiles Crocodylus acutus and C. moreletii in the Mexican Caribbean. Diseases of Aquatic Organisms 98: 27-39.
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Bacterial cultures and chemical analyses were performed from cloacal and oral swabs taken from 43 American crocodiles Crocodylus acutus and 28 Morelet’s crocodiles C. moreletii captured in Quintana Roo State, Mexico. We recovered 47 bacterial species (28 genera and 14 families) from all samples with 51.1% of these belonging to the family Enterobacteriaceae. Fourteen species (29.8%) were detected in both crocodile species and 18 (38.3%) and 15 (31.9%) species were only detected in American and Morelet’s crocodiles, respectively. We recovered 35 bacterial species from all oral samples, of which 9 (25.8%) were detected in both crocodile species. From all cloacal samples, we recovered 21 bacterial species, of which 8 (38.1%) were detected in both crocodile species. The most commonly isolated bacteria in cloacal samples were Aeromonas hydrophila and Escherichia coli, whereas in oral samples the most common bacteria were A. hydrophila and Arcanobacterium pyogenes. The bacteria isolated represent a potential threat to crocodile health during conditions of stress and a threat to human health through crocodile bites, crocodile meat consumption or carrying out activities in crocodile habitat. We especially warn about the presence of Salmonella arizonae and S. typhi, which cause enteritis and septicemia in crocodiles and salmonellosis and typhoid fever in humans. The risk of bacterial contamination from crocodiles to humans could increase in the future because of the accelerated destruction of crocodile habitat, which could lead to an augmentation of human−crocodile interactions. Information on bacterial diversity reported here could help in the choice of antibacterial products in case of infections that are of crocodile origin.

Rudolf C. A., Jaimes A., Espinosa-Blanco A. S., Contreras M., García-Amado M. A. (2018): Oral and cloacal Helicobacter detection in wild and captive Orinoco crocodiles (Crocodylus intermedius) in Venezuela. Vector-Borne and Zoonotic Diseases 18: 567-569.
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Helicobacter species can colonize digestive tract of animals and humans and have been associated with gastrointestinal diseases; however, this genus has not been studied in crocodiles. Our objective was to detect by PCR Helicobacter genus and Helicobacter pylori in oral and cloacal swabs from Orinoco crocodiles of two wild (Cojedes River System and Capanaparo River) and two captive breeding centers (CBCs; Masaguaral Ranch and UNELLEZ) populations. Bacterial DNA was found in 100% of oral samples (10 wild and 10 captives), and in the 95% of cloacal samples (10 wild and 9 captives). In wild populations, Helicobacter spp. was not detected, whereas in CBCs, Helicobacter was detected in 10% of the oral samples, and 66.7% of cloacal samples. H. pylori was detected in two Orinoco crocodiles. Two cloacal non-pylori Helicobacter amplicons were sequenced, showing low similarity (≤97%) to Helicobacter sequences reported. This is the first report of Helicobacter species, including H. pylori in Crocodylus intermedius from CBCs.

Gholamhosseini A., Sharifiyazdi H., Rakhshaninejad M., Soltanian S., Salighehzadeh R., Kordestani H. (2021): A study on the oral and cloacal bacterial flora of Mugger crocodiles (Crocodylus palustris) in the Negour protected area, Iran. Veterinary Research Forum 12: 361-367.
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Mugger crocodile is the only crocodile existing in Iran. The present study was aimed to investigate the bacterial flora in oral and cloacal cavities of wild Mugger crocodiles in Negour protected area, Iran. The isolation and molecular characterization of oral and cloacal bacterial flora were performed in 22 Mugger crocodiles captured in Negour protected area, Iran. Ten bacterial species from all oral samples and six bacterial species from all cloacal samples were recovered. The most commonly isolated bacteria in oral samples were Burkholderia contaminans and Lactococcus garvieae, respectively; whereas, in cloacal samples, it was Lactococcus lactis. It is likely that the isolated bacteria would pose a threat to both crocodiles and humans health. It can threaten crocodiles during stressful conditions; while, humans would be susceptible if they are bitten by crocodiles, consume their meat or spend time near their natural environment. This study provides useful information about bacterial diversity which could help to select the most appropriate anti-bacterial when dealing with infections caused by crocodiles.

Rodrigo M. P., Javier M., Santiago M. (2016): Structural-and carotenoid-based throat colour patches in males of Lacerta schreiberi reflect different parasitic diseases. Behavioral Ecology and Sociobiology 70: 2017-2025.
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Host species in populations under strong pressures from parasitic diseases may evolve ornaments to signal individual host quality to conspecifics. Colour ornaments in lizards result from the interaction of different layers in the skin. When inner layers of melanin and well-arranged iridophores are combined, UV-blue structural colouration results. On the other hand, when layers of erythrophores are densely loaded with carotenoids, a UV-yellow colouration is seen. The expression of carotenoid-based traits has been frequently studied in relation to parasite infections. However, few studies have explored the relationship between parasitic diseases and structural colouration. In this study, we investigated the expression of UV-blue and UV-yellow throat colour patches in males of Lacerta schreiberi in relation to infection by haemoparasites, ixodid ticks and intestinal nematodes. The brightness of the UV-yellow throat patch (a carotenoid-based ornament) was positively correlated with body condition and negatively correlated with the number of attached ticks, supporting Hamilton and Zuk’s hypothesis. Additionally, individuals that passed nematode eggs in the faeces had UV-yellow throat patches with higher hue values (more greenish colouration). Strikingly, the individuals infected by haemoparasites of the genus Schellackia showed UV-blue throat patches (a melanin-based ornament) with higher values of both UV-blue chroma and hue (i.e., UV-biased throats) than did uninfected individuals, suggesting a key role for melanin in the nuptial colouration of this lizard species. Thus, the combined information from both UV-blue and UV-yellow throat patches may convey integrative information about individual quality in this lacertid species.

Jiang H. Y., Ma J. E., Li J., Zhang X. J., Li L. M., He N., Liu H. Y., Luo S. Y., Wu Z. J., Han R. C., Chen J. P. (2017): Diets alter the gut microbiome of crocodile lizards. Frontiers in Microbiology 8: 2073.
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The crocodile lizard is a critically endangered reptile, and serious diseases have been found in this species in recent years, especially in captive lizards. Whether these diseases are caused by changes in the gut microbiota and the effect of captivity on disease remains to be determined. Here, we examined the relationship between the gut microbiota and diet and disease by comparing the fecal microbiota of wild lizards with those of sick and healthy lizards in captivity. The gut microbiota in wild crocodile lizards was consistently dominated by Proteobacteria (∼56.4%) and Bacteroidetes (∼19.1%). However, the abundance of Firmicutes (∼2.6%) in the intestine of the wild crocodile lizards was distinctly lower than that in other vertebrates. In addition, the wild samples from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve also had a high abundance of Deinococcus–Thermus while the wild samples from Guangxi Daguishan Crocodile Lizard National Nature Reserve had a high abundance of Tenericutes. The gut microbial community in loach-fed crocodile lizards was significantly different from the gut microbial community in the earthworm-fed and wild lizards. In addition, significant differences in specific bacteria were detected among groups. Notably, in the gut microbiota, the captive lizards fed earthworms resulted in enrichment of Fusobacterium, and the captive lizards fed loaches had higher abundances of Elizabethkingia, Halomonas, Morganella, and Salmonella, all of which are pathogens or opportunistic pathogens in human or other animals. However, there is no sufficient evidence that the gut microbiota contributes to either disease A or disease B. These results provide a reference for the conservation of endangered crocodile lizards and the first insight into the relationship between disease and the gut microbiota in lizards.

Mockett S., Bell T., Poulin R., Jorge F. (2017): The diversity and evolution of nematodes (Pharyngodonidae) infecting New Zealand lizards. Parasitology 144: 680-691.
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Host–parasite co-evolutionary studies can shed light on diversity and the processes that shape it. Molecular methods have proven to be an indispensable tool in this task, often uncovering unseen diversity. This study used two nuclear markers (18S rRNA and 28S rRNA) and one mitochondrial (cytochrome oxidase subunit I) marker to investigate the diversity of nematodes of the family Pharyngodonidae parasitizing New Zealand (NZ) lizards (lygosomine skinks and diplodactylid geckos) and to explore their co-evolutionary history. A Bayesian approach was used to infer phylogenetic relationships of the parasitic nematodes. Analyses revealed that nematodes parasitizing skinks, currently classified as Skrjabinodon, are more closely related to Spauligodon than to Skrjabinodon infecting NZ geckos. Genetic analyses also uncovered previously undetected diversity within NZ gecko nematodes and provided evidence for several provisionally cryptic species. We also examined the level of host–parasite phylogenetic congruence using a global-fit approach. Significant congruence was detected between gecko-Skrjabinodon phylogenies, but our results indicated that strict co-speciation is not the main co-evolutionary process shaping the associations between NZ skinks and geckos and their parasitic nematodes. However, further sampling is required to fully resolve co-phylogenetic patterns of diversification in this host–parasite system.

Lewbart G. A., Cohen E. B., Hirschfeld M., Muñoz-Pérez J. P., García J., Fu A., Chen E. P., Lohmann K. J. (2018): Field-based radiographic imaging of marine megafauna: marine iguanas (Amblyrhynchus cristatus) as a case study. Frontiers in Marine Science 5: 40.
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Effective conservation of marine megafauna requires a thorough understanding of the ecology, physiology, population dynamics, and health of vulnerable species. Assessing the health of large, mobile marine animals poses particular challenges, in part because the subjects are difficult to capture and restrain, and in part because standard laboratory and diagnostic tools are difficult to apply in a field setting. Radiography is a critically important diagnostic tool used routinely by veterinarians, but it has seldom been possible to image live marine vertebrates in the field. As a first step toward assessing the feasibility of incorporating radiography into studies of vulnerable species in remote locations, we used portable radiographic equipment to acquire the first digital internal images of living marine iguanas, Amblyrhynchus cristatus, an iconic lizard endemic only to the Galápagos Islands of Ecuador. The radiographic machinery was powered by batteries and performed well on a rocky beach environment of an uninhabited island, despite high heat and humidity. The accuracy of radiographic measurements was validated by computing a snout-vent length (SVL) using bone dimensions and comparing this to standard measurements of SVL made externally with a tape measure. These results demonstrate the feasibility of using radiography to study animals in remote sites, a technique that may prove useful for a variety of physiological, ecological, and biomechanical studies in which reliable measurements of skeletal and soft-tissue dimensions must be acquired under challenging field conditions. Refinements are discussed that will help the technology reach its full potential in field studies.

Charruau P., Pérez-Juárez J. G., Medina M., de la Cruz F. R. M., Pérez-Flores J. (2020): Bacterial flora of wild black (Ctenosaura similis Gray, 1831) and green (Iguana iguana Linnaeus, 1758) iguanas from a Mexican Caribbean atoll. Herpetology Notes 13: 369-376. 
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Black (Ctenosaura similis) and green iguanas (Iguana iguana) occur in a great variety of habitats and could be a source of zoonotic diseases. Herein, we characterized the bacterial flora in oral and cloacal cavities of these species from Banco Chinchorro atoll, Mexico, where they are in close contact with humans. We obtained samples from 19 green and 10 black iguanas. We performed bacterial cultures on different growth media and identified bacteria by gross examination and biochemical tests. We isolated 25 bacteria species (15 genera from 6 families) from all samples. Twelve bacteria (48%) were common to both iguana species and 12 (48%) and 1 (4%) were only detected in green and black iguanas, respectively. Iguana iguana shows a greater richness of bacteria in cloaca (n = 15 vs n = 7) and oral cavity (n = 16 vs n = 11) than C. similis, likely due to different feeding habits. 72% of the detected bacteria have been associated with infections in humans. This information on oral and cloacal bacteria of wild green and black iguanas could help to provide adequate clinical assistance to sick animals and ideal antimicrobial therapy in cases of bites or infections.

Tang G. S., Liang X. X., Yang M. Y., Wang T. T., Chen J. P., Du W. G., Li H., Sun B. J. (2020): Captivity influences gut microbiota in crocodile lizards (Shinisaurus crocodilurus). Frontiers in Microbiology 11: 550.
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Captivity is an important measure for conservation of an endangered species, and it is becoming a hot topic in conservation biology, which integrates gut microbiota and endangered species management in captivity. As an ancient reptile, the crocodile lizard (Shinisaurus crocodilurus) is facing extreme danger of extinction, resulting in great significance to species conservation in the reserve. Thus, it is critical to understand the differences in gut microbiota composition between captive and wild populations, as it could provide fundamental information for conservative management of crocodile lizards. Here, fecal samples of crocodile lizards were collected from two wild and one captive populations with different ages (i.e., juveniles and adults) and were analyzed for microbiota composition by 16S ribosomal RNA (rRNA) gene amplicon sequencing. This study showed that the lizard gut microbiota was mainly composed of Firmicutes and Proteobacteria. The gut microbiota composition of crocodile lizard did not differ between juveniles and adults, as well as between two wild populations. Interestingly, captivity increased community richness and influenced community structures of gut microbiota in crocodile lizards, compared with wild congeners. This was indicated by higher abundances of the genera Epulopiscium and Glutamicibacter. These increases might be induced by complex integration of simple food resources or human contact in captivity. The gut microbiota functions of crocodile lizards are primarily enriched in metabolism, environmental information processing, genetic information processing, and cellular processes based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This study provides fundamental information about the gut microbiota of crocodile lizards in wild and captive populations. In the future, exploring the relationship among diet, gut microbiota, and host health is necessary for providing animal conservation strategies.

Du Y., Chen J. Q., Liu Q., Fu J. C., Lin C. X., Lin L. H., Li H., Qu Y. F., Ji X. (2022): Dietary correlates of oral and gut microbiota in the water monitor lizard, Varanus salvator. Frontiers in Microbiology: 3985.
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Numerous studies have demonstrated that food shapes the structure and composition of the host’s oral and gut microbiota. The disorder of oral and gut microbiota may trigger various host diseases. Here, we collected oral and gut samples from wild water monitor lizards (Varanus salvator) and their captive conspecifics fed with bullfrogs, eggs, and depilated chicken, aiming to examine dietary correlates of oral and gut microbiota. We used the 16S rRNA gene sequencing technology to analyze the composition of the microbiota. Proteobacteria and Bacteroidota were the dominant phyla in the oral microbiota, and so were in the gut microbiota. The alpha diversity of microbiota was significantly higher in the gut than in the oral cavity, and the alpha diversity of oral microbiota was higher in captive lizards than in wild conspecifics. Comparing the relative abundance of oral and gut bacteria and their gene functions, differences among different animal groups presumably resulted from human contact in artificial breeding environments and complex food processing. Differences in gene function might be related to the absolute number and/or the taxonomic abundance of oral and gut microorganisms in the wild and the water environment. This study provides not only basic information about the oral and gut microbiota of captive and wild water monitor lizards, but also an inference that feeding on frogs and aquatic products and reducing human exposure help water monitor lizards maintain a microbiota similar to that in the wild environment.

Guerrero-Sanchez S., Frias L., Saimin S., Orozco-terWengel P., Goossens B. (2023): The fast-food effect: costs of being a generalist in a human-dominated landscape. Conservation Physiology 11: coad055.
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Agricultural expansion in Southeast Asia has converted most natural landscapes into mosaics of forest interspersed with plantations, dominated by the presence of generalist species that benefit from resource predictability. Dietary shifts, however, can result in metabolic alterations and the exposure of new parasites that can impact animal fitness and population survival. Our study focuses on the Asian water monitor lizard (Varanus salvator), one of the largest predators in the Asian wetlands, as a model species to understand the health consequences of living in a human-dominated landscape in Sabah, Malaysian Borneo. We evaluated the effects of dietary diversity on the metabolism of monitor lizards and the impact on the composition of their parasite communities in an oil palm-dominated landscape. Our results showed that (1) rodent-dominated diets were associated with high levels of lipids, proteins and electrolytes, akin to a fast-food-based diet of little representativeness of the full nutritional requirements, but highly available, and (2) lizards feeding on diverse diets hosted more diverse parasite communities, however, at overall lower parasite prevalence. Furthermore, we observed that the effect of diet on lipid concentration differed depending on the size of individual home ranges, suggesting that sedentarism plays an important role in the accumulation of cholesterol and triglycerides. Parasite communities were also affected by a homogeneous dietary behaviour, as well as by habitat type. Dietary diversity had a negative effect on both parasite richness and prevalence in plantations, but not in forested areas. Our study indicates that human-dominated landscapes can pose a negative effect on generalist species and hints to the unforeseen health consequences for more vulnerable taxa using the same landscapes. Thus, it highlights the potential role of such a widely distributed generalist as model species to monitor physiological effects in the ecosystem in an oil palm-dominated landscape.

Krishnankutty S. P., Muraleedharan M., Perumal R. C., Michael S., Benny J., Balan B., Kumar P., Manazhi J., Kumar B. D., Santhosh S., Thomas G. (2019): Next-generation sequencing analysis reveals high bacterial diversity in wild venomous and non-venomous snakes from India. Journal of Venomous Animals and Toxins including Tropical Diseases 24: 41. 
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The oral cavities of snakes are replete with various types of bacterial flora. Culture-dependent studies suggest that some of the bacterial species are responsible for secondary bacterial infection associated with snakebite. A complete profile of the ophidian oral bacterial community has been unreported until now. Therefore, in the present study, we determined the complete bacterial compositions in the oral cavity of some snakes from India. Total DNA was isolated from oral swabs collected from three wild snake species (Indian Cobra, King Cobra and Indian Python). Next, the DNA was subjected to PCR amplification of microbial 16S rRNA gene using V3-region-specific primers. The amplicons were used for preparation of DNA libraries that were sequenced on an Illumina MiSeq platform. The cluster-based taxonomy analysis revealed that Proteobacteria and Actinobacteria were the most predominant phyla present in the oral cavities of snakes. This result indicates that snakes show more similarities to birds than mammals as to their oral bacterial communities. Furthermore, our study reports all the unique and common bacterial species (total: 147) found among the oral microbes of snakes studied, while the majority of commonly abundant species were pathogens or opportunistic pathogens to humans. A wide difference in ophidian oral bacterial flora suggests variation by individual, species and geographical region. The present study would provide a foundation for further research on snakes to recognize the potential drugs/antibiotics for the different infectious diseases.

Staub E., Marti H., Biondi R., Levi A., Donati M., Leonard C. A., Ley S. D., Pillonel T., Greub G., Seth-Smith H., Borel N. (2018): Novel Chlamydia species isolated from snakes are temperature-sensitive and exhibit decreased susceptibility to azithromycin. Scientific Reports 8: 5660.
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Chlamydia species have recently been recognized as emerging pathogens in snakes. However, isolation of novel snake chlamydiae is critical and their growth characteristics are largely unknown. In this study, two novel chlamydial species are described: Chlamydia serpentis and Chlamydia poikilothermis, isolated after attempts on 23 cloacal and choanal swabs from 18 PCR-positive captive snakes originating from different Swiss snake collections. Isolation success, growth curve and infectivity rates over a 48-hour time period were dependent on temperature (37 °C for C. serpentis, 28 °C for C. poikilothermis). C. serpentis and C. poikilothermis were sensitive to tetracycline and moxifloxacin during evaluation by in vitro antibiotic susceptibility assay but intermediate to resistant (2–4 μg/ml) to azithromycin. Whole genome sequencing of the isolates provided proof of the novel species status, and gives insights into the evolution of these branches of genus Chlamydia.

McKenzie J. M., Price S. J., Fleckenstein J. L., Drayer A. N., Connette G. M., Bohuski E., Lorch J. M. (2019): Field diagnostics and seasonality of Ophidiomyces ophiodiicola in wild snake populations. EcoHealth 16: 141-150.
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Snake fungal disease (SFD) is an emerging disease caused by the fungal pathogen, Ophidiomyces ophiodiicola. Clinical signs of SFD include dermal lesions, including regional and local edema, crusts, and ulcers. Snake fungal disease is widespread in the Eastern United States, yet there are limited data on how clinical signs of SFD compare with laboratory diagnostics. We compared two sampling methods for O. ophiodiicola, scale clip collection and swabbing, to evaluate whether collection method impacted the results of polymerase chain reaction (PCR). In addition, we evaluated the use of clinical signs to predict the presence of O. ophiodiicola across seasons, snake habitat affiliation (aquatic or terrestrial) and study sites. We found no significant difference in PCR results between sampling methods. Clinical signs were a strong predictor of O. ophiodiicola presence in spring and summer seasons. Snakes occupying terrestrial environments had a lower overall probability of testing positive for O. ophiodiicola compared to snakes occupying aquatic environments. Although our study indicates that both clinical signs of SFD and prevalence of O. ophiodiicola vary seasonally and based on habitat preferences of the host, our analysis suggests that clinical signs can serve as a reliable indicator of O. ophiodiicola presence, especially during spring and summer.

Walker D. M., Leys J. E., Grisnik M., Grajal-Puche A., Murray C. M., Allender M. C. (2019): Variability in snake skin microbial assemblages across spatial scales and disease states. The ISME Journal 13: 2209-2222.
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Understanding how biological patterns translate into functional processes across different scales is a central question in ecology. Within a spatial context, extent is used to describe the overall geographic area of a study, whereas grain describes the overall unit of observation. This study aimed to characterize the snake skin microbiota (grain) and to determine host–microbial assemblage–pathogen effects across spatial extents within the Southern United States. The causative agent of snake fungal disease, Ophidiomyces ophiodiicola, is a fungal pathogen threatening snake populations. We hypothesized that the skin microbial assemblage of snakes differs from its surrounding environment, by host species, spatial scale, season, and in the presence of O. ophiodiicola. We collected snake skin swabs, soil samples, and water samples across six states in the Southern United States (macroscale extent), four Tennessee ecoregions (mesoscale extent), and at multiple sites within each Tennessee ecoregion (microscale extent). These samples were subjected to DNA extraction and quantitative PCR to determine the presence/absence of O. ophiodiicola. High-throughput sequencing was also utilized to characterize the microbial communities. We concluded that the snake skin microbial assemblage was partially distinct from environmental microbial communities. Snake host species was strongly predictive of the skin microbiota at macro-, meso-, and microscale spatial extents; however, the effect was variable across geographic space and season. Lastly, the presence of the fungal pathogen O. ophiodiicola is predictive of skin microbial assemblages across macro- and meso-spatial extents, and particular bacterial taxa associate with O. ophiodiicola pathogen load. Our results highlight the importance of scale regarding wildlife host–pathogen–microbial assemblage interactions.

Pawlak A., Morka K., Bury S., Antoniewicz Z., Wzorek A., Cieniuch G., Korzeniowska-Kowal A., Cichoń M., Bugla-Płoskońska G. (2020): Cloacal gram-negative microbiota in free-living grass snake Natrix natrix from Poland. Current Microbiology 77: 2166-2171.
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Reptiles appear to be an important vector for Gram-negative pathogens, therefore, they are epidemiologically relevant. However, the composition of reptilian microbiota has been poorly recognized so far. The majority of studies concern exotic reptiles as asymptomatic carriers of Salmonella serovars. Studies of other intestinal bacteria of reptiles are rare. Only recently, the microbiota of free-living European reptiles have been investigated, however, on the basis of small samples, mainly in protected areas. Here, we aim to investigate cloacal Gram-negative microbiota of free-living Natrix natrix. Snakes (N = 45) used in the study were collected in Kraków (Poland) and its vicinity. Nineteen species of Gram-negative bacteria were isolated. The most common species were: Aeromonas hydrophila, Morganella morganii, Proteus vulgaris, Salmonella spp. The bacteria prevalent in N. natrix cloacal swabs are likely to represent the natural intestinal Gram-negative microbiota of the examined snakes. Importantly, the identified bacteria are pathogenic to humans, which clearly highlights the epidemiological potential of free-living N. natrix. The risk of infection is high for immunocompromised humans, children (under 5 years old), elderly persons, and pregnant women. Our study provides the largest dataset on intestinal Gram-negative microbiota of wild snakes. The presence of multiple human pathogens determined by us calls for the necessity of further studies on reptile-transmitted bacteria in anthropogenic environments.

Smith S. N., Colston T. J., Siler C. D. (2021): Venomous snakes reveal ecological and phylogenetic factors influencing variation in gut and oral microbiomes. Frontiers in Microbiology 12: 603.
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The gastrointestinal tract (GIT) of vertebrates contains a series of organs beginning with the mouth and ending with the anus or cloacal opening. Each organ represents a unique environment for resident microorganisms. Due to their simple digestive anatomy, snakes are good models for studying microbiome variation along the GIT. Cloacal sampling captures the majority of the microbial diversity found in the GIT of snakes—yet little is known about the oral microbiota of snakes. Most research on the snake mouth and gut microbiota are limited to studies of a single species or captive-bred individuals. It therefore remains unclear how a host’s life history, diet, or evolutionary history correlate with differences in the microbial composition within the mouths and guts of wild snakes. We sampled the mouth and gut microbial communities from three species of Asian venomous snakes and utilized 16S rRNA microbial inventories to test if host phylogenetic and ecological differences correlate with distinct microbial compositions within the two body sites. These species occupy three disparate habitat types: marine, semi-arboreal, and arboreal, our results suggest that the diversity of snake mouth and gut microbial communities correlate with differences in both host ecology and phylogeny.

Lettoof D. C., Cornelis J., Jolly C. J., Aubret F., Gagnon M. M., Hyndman T. H., Barton D. P., Bateman P. W. (2022): Metal (loid) pollution, not urbanisation nor parasites predicts low body condition in a wetland bioindicator snake. Environmental Pollution 295: 118674.
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Urban ecosystems and remnant habitat ‘islands’ therein, provide important strongholds for many wildlife species including those of conservation significance. However, the persistence of these habitats can be undermined if their structure and function are too severely disrupted. Urban wetlands, specifically, are usually degraded by a monoculture of invasive vegetation, disrupted hydrology, and chronic-contamination from a suite of anthropogenic pollutants. Top predators—as bioindicators—can be used to assess and monitor the health of these ecosystems. We measured eight health parameters (e.g., parasites, wounds and scars, tail loss and body condition) in a wetland top predator, the western tiger snake, Notechis scutatus occidentalis. For three years, snakes were sampled across four wetlands along an urban gradient. For each site, we used GIS software to measure the area of different landscapes and calculate an urbanisation–landscape score. Previously published research on snake contamination informed our calculations of a metal-pollution index for each site. We used generalised linear mixed models to assess the relationship between all health parameters and site variables. We found the metal-pollution index to have the most significant association with poor body condition. Although parasitism, tail loss and wounds differed among sites, none of these parameters influenced body condition. Additionally, the suite of health parameters suggested differing health status among sites; however, our measure of contemporary landscape urbanisation was never a significant predictor variable. Our results suggest that the health of wetland predators surrounding a rapidly growing city may be offset by higher levels of environmental pollution.

Nowakiewicz A., Ziółkowska G., Zięba P., Dziedzic B. M., Gnat S., Wójcik M., Dziedzic R., Kostruba A. (2015): Aerobic bacterial microbiota isolated from the cloaca of the European pond turtle (Emys orbicularis) in Poland. Journal of Wildlife Diseases 51: 255-259.
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We conducted a comparative analysis of the aerobic cloacal bacteria of European pond turtles (Emys orbicularis) living in their natural environment and juvenile turtles reared under controlled conditions in a breeding center. We included 130 turtles in the study. The aerobic bacteria isolated from the cloaca of the juvenile turtles were less diverse and more prevalent than the bacteria isolated from free-living adults. We isolated 17 bacterial species from juvenile captive turtles, among which the dominant species were Cellulomonas flavigena (77/96), Enterococcus faecalis (96/96), Escherichia coli (58/96), and Proteus mirabilis (41/96). From the adult, free-living turtles, we isolated 36 bacterial species, some of which are a potential threat to public health (e.g., Salmonella enterica serovars Newport, Daytona, and Braenderup; Listeria monocytogenes; Yersinia enterocolitica; Yersinia ruckeri; Klebsiella pneumoniae; Vibrio fluvialis; and Serratia marcescens), and pathogens that are etiologic agents of diseases of ectothermic animals (e.g., Aeromonas sobria, Aeromonas caviae, Hafnia alvei, Edwardsiella tarda, and Citrobacter braakii; the last two species were isolated from both groups of animals). The cloacal bacterial biota of the European pond turtle was characterized by numerous species of bacteria, and its composition varied with turtle age and environmental conditions. The small number of isolated bacteria that are potential human pathogens may indicate that the European pond turtle is of relatively minor importance as a threat to public health.

Biagi E., D’Amico F., Soverini M., Angelini V., Barone M., Turroni S., Rampelli S., Pari S., Brigidi P., Candela M. (2019): Faecal bacterial communities from Mediterranean loggerhead sea turtles (Caretta caretta). Environmental Microbiology Reports 11: 361-371.
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The loggerhead sea turtle (Caretta caretta) is the most widespread sea turtle species in the Mediterranean Sea and a relevant pollution ‘flagship species’. Here, we profiled the faecal microbiota from 29 C. caretta from a rescue centre, and explored the impact of several variables linked to both the animal itself and the environment (i.e., tank water ecosystem). We show that loggerhead turtles share more gut microbiota features with carnivorous marine mammals, than with phylogenetically close, but herbivorous, turtles, as a confirmation of the gut microbiota adaptive function to diet and environment. We also highlight a relation between the microbiota composition and the size (and consequently the age) of the turtles. Finally, we point out that the gut microbiota of sea turtles shows unexpectedly low exchange of microbes with the aquatic environment and is resilient to the stress induced by short-time captivity.

Marangi M., Carlino P., Profico C., Olivieri V., Totaro G., Furii G., Marzano G., Papini R. A. (2020): First multicenter coprological survey on helminth parasite communities of free-living loggerhead sea turtles Caretta caretta (Linnaeus, 1758) from the Adriatic Sea and Northern Ionian Sea. International Journal for Parasitology: Parasites and Wildlife 11: 207-212. 
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The prevalence of endoparasite infections in 83 free-living specimens of Caretta caretta, classified as vulnerable species, from the Adriatic Sea and Northern Ionian Sea was investigated by coprological examination. Thirty-seven (44.6%) turtles were found to be infected with helminths. The helminth infections found were: Rhytidodes gelatinosus and Sulcascaris sulcata (18.1% each), Hapalotrema mistroides (13.2%), Cymatocarpus solearis (9.6%), Eniodotrema megachondrus (7.2%), Kathlania/Tonaudia sp. (3.6%), Neospirorchis sp., Octangium sagitta and Plesiochorus cymbiformis (1.2% each). There were no significant differences in the total prevalence of helminth infections between sexes, size classes, and seasonal periods. Conversely, the prevalence of helminth infections was significantly higher (P < 0.01) in accidentally caught turtles than in stranded turtles. Highly significant differences in prevalence of helminthiases were also seen among marine sampling areas. This report provides important baseline information about the helminth fauna of free-living C. caretta in the examined geographical region. This is also the first report of O. sagitta infection in C. caretta thus broadening the host range of the parasite.

McKnight D. T., Zenger K. R., Alford R. A., Huerlimann R. (2020): Microbiome diversity and composition varies across body areas in a freshwater turtle. Microbiology 166: 440-452.
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There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft’s river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles.

Qu Y. F., Wu Y. Q., Zhao Y. T., Lin L. H., Du Y., Li P., Li H., Ji X. (2020): The invasive red-eared slider turtle is more successful than the native Chinese three-keeled pond turtle: evidence from the gut microbiota. PeerJ 8: e10271.
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The mutualistic symbiosis between the gut microbial communities (microbiota) and their host animals has attracted much attention. Many factors potentially affect the gut microbiota, which also varies among host animals. The native Chinese three-keeled pond turtle (Chinemys reevesii) and the invasive red-eared slider turtle (Trachemys scripta elegans) are two common farm-raised species in China, with the latter generally considered a more successful species. However, supporting evidence from the gut microbiota has yet to be collected. We collected feces samples from these two turtle species raised in a farm under identical conditions, and analyzed the composition and relative abundance of the gut microbes using bacterial 16S rRNA sequencing on the Roach/454 platform. The gut microbiota was mainly composed of Bacteroidetes and Firmicutes at the phylum level, and Porphyromonadaceae, Bacteroidaceae and Lachnospiraceae at the family level in both species. The relative abundance of the microbes and gene functions in the gut microbiota differed between the two species, whereas alpha or beta diversity did not. Microbes of the families Bacteroidaceae, Clostridiaceae and Lachnospiraceae were comparatively more abundant in C. reevesii, whereas those of the families Porphyromonadaceae and Fusobacteriaceae were comparatively more abundant in T. s. elegans. In both species the gut microbiota had functional roles in enhancing metabolism, genetic information processing and environmental information processing according to the Kyoto Encyclopedia of Genes and Genomes database. The potential to gain mass is greater in T. s. elegans than in C. reevesii, as revealed by the fact that the Firmicutes/Bacteroidetes ratio was lower in the former species. The percentage of human disease-related functional genes was lower in T. s. elegans than in C. reevesii, presumably suggesting an enhanced potential to colonize new habitats in the former species.

Biagi E., Musella M., Palladino G., Angelini V., Pari S., Roncari C., Scicchitano D., Rampelli S., Franzellitti S., Candela M. (2021): Impact of plastic debris on the gut microbiota of Caretta caretta from Northwestern Adriatic Sea. Frontiers in Marine Science 8: 127.
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Plastic pollution is nowadays a relevant threat for the ecological balance in marine ecosystems. Small plastic debris (PD) can enter food webs through various marine organisms, with possible consequences on their physiology and health. The loggerhead sea turtle (Caretta caretta), widespread across the whole Mediterranean Sea, is a “flagship species,” useful as indicator of the general pollution level of marine ecosystems. Ingested PD accumulate in the final section of turtles’ digestive tract before excretion. During their transit and accumulation, PD also interact with the residing microbial community, with possible feedback consequences on the host’s health. To explore the possible relationship between fecal microbial composition and PD ingestion, we collected fecal samples from 45 turtles rescued between 2017 and 2019 in the Northwestern Adriatic Sea (Italy), assessing occurrence and content of PD in the samples and in parallel the microbiome structure by 16S rRNA gene sequencing. According to our findings, almost all samples contained PD, mirroring the high level of plastic pollution in the area. We identified phylotypes associated to a high amount of PD, namely Cetobacterium somerae and other taxa, possibly responding to contamination by plastic-associated chemicals. Furthermore, putative marine pathogens were found associated to higher plastic contamination, supporting the hypothesis that PD can act as a carrier for environmental pathogenic bacteria into marine organisms. Besides confirming the role of the sea turtle as relevant flagship species for plastic pollution of the marine environment, our study paves the way to the exploration of the impact that PD ingestion can have on the microbial counterpart of large marine organisms, with potential feedback consequences on the animal and ecosystem health.

Filek K., Trotta A., Gračan R., Di Bello A., Corrente M., Bosak S. (2021): Characterization of oral and cloacal microbial communities of wild and rehabilitated loggerhead sea turtles (Caretta caretta). Animal Microbiome 3: 59.
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Microbial communities of wild animals are being increasingly investigated to provide information about the hosts’ biology and promote conservation. Loggerhead sea turtles (Caretta caretta) are a keystone species in marine ecosystems and are considered vulnerable in the IUCN Red List, which led to growing efforts in sea turtle conservation by rescue centers around the world. Understanding the microbial communities of sea turtles in the wild and how affected they are by captivity, is one of the stepping stones in improving the conservation efforts. Describing oral and cloacal microbiota of wild animals could shed light on the previously unknown aspects of sea turtle holobiont biology, ecology, and contribute to best practices for husbandry conditions. We describe the oral and cloacal microbiota of Mediterranean loggerhead sea turtles by 16S rRNA gene sequencing to compare the microbial communities of wild versus turtles in, or after, rehabilitation at the Adriatic Sea rescue centers and clinics. Our results show that the oral microbiota is more sensitive to environmental shifts than the cloacal microbiota, and that it does retain a portion of microbial taxa regardless of the shift from the wild and into rehabilitation. Additionally, Proteobacteria and Bacteroidetes dominated oral and cloacal microbiota, while Kiritimatiellaeota were abundant in cloacal samples. Unclassified reads were abundant in the aforementioned groups, which indicates high incidence of yet undiscovered bacteria of the marine reptile microbial communities. We provide the first insights into the oral microbial communities of wild and rehabilitated loggerhead sea turtles, and establish a framework for quick and non-invasive sampling of oral and cloacal microbial communities, useful for the expansion of the sample collection in wild loggerhead sea turtles. Finally, our investigation of effects of captivity on the gut-associated microbial community provides a baseline for studying the impact of husbandry conditions on turtles’ health and survival upon their return to the wild.

Sakamoto K. Q., Miyayama M., Kinoshita C., Fukuoka T., Ishihara T., Sato K. (2021): A non-invasive system to measure heart rate in hard-shelled sea turtles: Potential for field applications. Philosophical Transactions of the Royal Society B 376: 20200222.
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To measure the heart rate of unrestrained sea turtles, it has been believed that a probe must be inserted inside the body owing to the presence of the shell. However, inserting the probe is invasive and difficult to apply to animals in the field. Here, we have developed a non-invasive heart rate measurement method for some species of sea turtles. In our approach, an electrocardiogram (ECG) was performed using an animal-borne ECG recorder and two electrodes—which were electrically insulated from seawater—pasted on the carapace. Based on the measured ECG, the heartbeat signals were identified with an algorithm using a band-pass filter. We implemented this algorithm in a user-friendly program package, ECGtoHR. In experiments conducted in a water tank and in a lagoon, we successfully measured the heart rate of loggerhead, olive ridley and black turtles, but not green and hawksbill turtles. The average heart rate of turtles when resting underwater was 6.2 ± 1.9 beats min–1 and that when moving at the surface was 14.0 ± 2.4 beats min–1. Our approach is particularly suitable for endangered species such as sea turtles, and has the potential to be extended to a variety of other free-ranging species.

Díaz-Abad L., Bacco-Mannina N., Miguel Madeira F., Serrao E. A., Regalla A., Patrício A. R., Frade P. R. (2022): Red, gold and green: microbial contribution of Rhodophyta and other algae to green turtle (Chelonia mydas) gut microbiome. Microorganisms 10: 1988.
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The fitness of the endangered green sea turtle (Chelonia mydas) may be strongly affected by its gut microbiome, as microbes play important roles in host nutrition and health. This study aimed at establishing environmental microbial baselines that can be used to assess turtle health under altered future conditions. We characterized the microbiome associated with the gastrointestinal tract of green turtles from Guinea Bissau in different life stages and associated with their food items, using 16S rRNA metabarcoding. We found that the most abundant (% relative abundance) bacterial phyla across the gastrointestinal sections were Proteobacteria (68.1 ± 13.9% “amplicon sequence variants”, ASVs), Bacteroidetes (15.1 ± 10.1%) and Firmicutes (14.7 ± 21.7%). Additionally, we found the presence of two red algae bacterial indicator ASVs (the Alphaproteobacteria Brucella pinnipedialis with 75 ± 0% and a Gammaproteobacteria identified as methanotrophic endosymbiont of Bathymodiolus, with <1%) in cloacal compartments, along with six bacterial ASVs shared only between cloacal and local environmental red algae samples. We corroborate previous results demonstrating that green turtles fed on red algae (but, to a lower extent, also seagrass and brown algae), thus, acquiring microbial components that potentially aid them digest these food items. This study is a foundation for better understanding the microbial composition of sea turtle digestive tracts.

Kinoshita C., Saito A., Kawai M., Sato K., Sakamoto K. Q. (2022): A non-invasive heart rate measurement method is improved by placing the electrodes on the ventral side rather than the dorsal in loggerhead turtles. Frontiers in Physiology 13: 811947.
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Heart rate measurement is an essential method for evaluating the physiological status of air-breathing diving animals. However, owing to technical difficulties, many marine animals require an invasive approach to record an electrocardiogram (ECG) in water, limiting the application of this approach in a wide range of marine animals. Recently, a non-invasive system was reported to measure the ECG of hard-shelled sea turtles by pasting the electrodes on the dorsal side of the shell, although the ECG obtained from the moving turtle contains noise produced by muscle contraction. Here, we report that clear ECGs can be obtained by placing the electrodes on the ventral side rather than the dorsal side in loggerhead sea turtles. Using our method, clearer ECG signals were obtained with less electrical noise, even when turtles are swimming. According to the anatomical features, the electrode position on the ventral side is closer to the heart than the dorsal side, minimizing the effects of noise generated by the skeletal muscle. This new biologging technique will elucidate the functioning of the circulatory system of sea turtles during swimming and their adaptabilities to marine environments.

Kophamel S., Ward L. C., Konovalov D. A., Mendez D., Ariel E., Cassidy N., Bell I., Balastegui Martínez M. T., Munns S. L. (2022): Field‐based adipose tissue quantification in sea turtles using bioelectrical impedance spectroscopy validated with CT scans and deep learning. Ecology and Evolution 12: e9610.
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Loss of adipose tissue in vertebrate wildlife species is indicative of decreased nutritional and health status and is linked to environmental stress and diseases. Body condition indices (BCI) are commonly used in ecological studies to estimate adipose tissue mass across wildlife populations. However, these indices have poor predictive power, which poses the need for quantitative methods for improved population assessments. Here, we calibrate bioelectrical impedance spectroscopy (BIS) as an alternative approach for assessing the nutritional status of vertebrate wildlife in ecological studies. BIS is a portable technology that can estimate body composition from measurements of body impedance and is widely used in humans. BIS is a predictive technique that requires calibration using a reference body composition method. Using sea turtles as model organisms, we propose a calibration protocol using computed tomography (CT) scans, with the prediction equation being: adipose tissue mass (kg) = body mass − (−0.03 [intercept] − 0.29 * length2/resistance at 50 kHz + 1.07 * body mass − 0.11 * time after capture). CT imaging allows for the quantification of body fat. However, processing the images manually is prohibitive due to the extensive time requirement. Using a form of artificial intelligence (AI), we trained a computer model to identify and quantify nonadipose tissue from the CT images, and adipose tissue was determined by the difference in body mass. This process enabled estimating adipose tissue mass from bioelectrical impedance measurements. The predictive performance of the model was built on 2/3 samples and tested against 1/3 samples. Prediction of adipose tissue percentage had greater accuracy when including impedance parameters (mean bias = 0.11%–0.61%) as predictor variables, compared with using body mass alone (mean bias = 6.35%). Our standardized BIS protocol improves on conventional body composition assessment methods (e.g., BCI) by quantifying adipose tissue mass. The protocol can be applied to other species for the validation of BIS and to provide robust information on the nutritional and health status of wildlife, which, in turn, can be used to inform conservation decisions at the management level.

McKee R. K., Buhlmann K. A., Moore C. T., Allender M. C., Stacy N. I., Tuberville T. D. (2022): Island of misfit tortoises: waif gopher tortoise health assessment following translocation. Conservation Physiology 10: coac051.
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Translocation, the intentional movement of animals from one location to another, is a common management practice for the gopher tortoise (Gopherus polyphemus). Although the inadvertent spread of pathogens is a concern with any translocation effort, waif tortoises—individuals that have been collected illegally, injured and rehabilitated or have unknown origins—are generally excluded from translocation efforts due to heightened concerns of introducing pathogens and subsequent disease to naïve populations. However, repurposing these long-lived animals for species recovery is desirable when feasible, and introducing waif tortoises may bolster small populations facing extirpation. The objective of this study was to assess the health of waif tortoises experimentally released at an isolated preserve in Aiken County, SC, USA. Our assessments included visual examination, screening for 14 pathogens using conventional or quantitative polymerase chain reaction (qPCR) and haematological evaluation. Of the 143 individuals assessed in 2017 and 2018, most individuals (76%; n = 109 of 143) had no overt clinical evidence of disease and, when observed, clinical findings were mild. In both years, we detected two known tortoise pathogens, Mycoplasma agassizii and Mycoplasma testudineum, at a prevalence of 10.2–13.9% and 0.0–0.8%, respectively. Additionally, we found emydid Mycoplasma, a bacterium commonly found in box turtles (Terrapene spp.), in a single tortoise that showed no clinical evidence of infection. The presence of nasal discharge was an important, but imperfect, predictor of Mycoplasma spp. infection in translocated tortoises. Hemogram data were comparable with wild populations. Our study is the first comprehensive effort to assess pathogen prevalence and hemogram data of waif gopher tortoises following translocation. Although caution is warranted and pathogen screening necessary, waif tortoises may be an important resource for establishing or augmenting isolated populations when potential health risks can be managed.

Salleh M. H. M., Esa Y., Ngalimat M. S., Chen P. N. (2022): Faecal DNA metabarcoding reveals novel bacterial community patterns of critically endangered Southern River Terrapin, Batagur affinis. PeerJ 10: e12970.
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Southern River Terrapin, Batagur affinis, is a freshwater turtle listed as critically endangered on the IUCN Red List since 2000. Many studies suggest that faecal DNA metabarcoding can shield light on the host-associated microbial communities that play important roles in host health. Thus, this study aimed to characterise and compare the faecal bacterial community between captive and wild B. affinis using metabarcoding approaches. A total of seven faeces samples were collected from captive (N = 5) and wild (N = 2) adult B. affinis aseptically, crossing the East and West coast of peninsular Malaysia. The DNA was extracted from the faeces samples, and the 16S rRNA gene (V3–V4 region) was amplified using polymerase chain reaction (PCR). The amplicon was further analysed using SILVA and DADA2 pipelines. In total, 297 bacterial communities taxonomic profile (phylum to genus) were determined. Three phyla were found in high abundance in all faeces samples, namely Firmicutes (38.69%), Bacteroidetes (24.52%), and Fusobacteria (6.95%). Proteobacteria were detected in all faeces samples (39.63%), except the wild sample, KBW3. Under genus level, Cetobacterium was found as the most abundant genus (67.79%), followed by Bacteroides (24.56%) and Parabacteroides (21.78%). The uncultured genus had the highest abundance (88.51%) even though not detected in the BK31 and KBW2 samples. The potential probiotic genera (75.00%) were discovered to be more dominant in B. affinis faeces samples. Results demonstrated that the captive B. affinis faeces samples have a greater bacterial variety and richness than wild B. affinis faeces samples. This study has established a starting point for future investigation of the gut microbiota of B. affinis.

Vecchioni L., Pace A., Sucato A., Berlinghieri F., Cambera I., Visconti G., Hochscheid S., Arcuelo M., Alduina R. (2022): Unveiling the egg microbiota of the loggerhead sea turtle Caretta caretta in nesting beaches of the Mediterranean Sea. Plos One 17: e0268345.
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Microbes have central roles in the development and health of animals, being the introduction of specific microbial species a potential conservation strategy to protect animals from emerging diseases. Thus, insight into the microbiota of the species and their habitats is essential. In this manuscript, we report for the first time the bacterial composition of all the components (eggshells of hatched and unhatched eggs, internal content of unhatched eggs, intestinal content of hatchling and pipping sea turtles, and sand) of three nesting beaches of Caretta caretta along the Italian coasts of the Mediterranean Sea. The analysis of 26 amplicon samples was carried out using next-generation sequencing analysis, targeting V3–V4 regions of the bacterial 16S rRNA gene. Samples featured mainly Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, whose percentages depended on the sample type. Our results showed that, although from different sampling sites, the internal content of the unhatched eggs, intestinal content of hatchling and pipping sea turtles share the microbiota, which was yet different from that of eggshells and sand of the same nesting beach. This study suggests the maternal and environmental influence alongside a protective role of eggshells in shaping the egg microbiota of Caretta caretta sea turtles.

Walden M. A., Jania R., Kinney M. E., Devan-Song A., Drake K. K., Esque T. C., Shoemaker K. T. (2022): Computed tomography for measuring body fat reserves in the threatened Mojave desert tortoise (Gopherus agassizii). Journal of Zoo and Wildlife Medicine 53: 412-423.
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Noninvasive methods for measuring fat reserves in both captive and free-ranging animals are important for monitoring individual and population health, but chelonian anatomy and physiology present challenges to accurate measurements. Standard field-based methods for assessing body condition in Mojave desert tortoises (Gopherus agassizii) involve the qualitative body condition score, which relies on the apparent height of the temporalis muscle relative to the sagittal crest (in addition to other characteristics) and quantitative body condition indices that measure relative mass at size. However, it is unclear how these metrics relate to body fat reserves in this species. The aims of this study were to (1) describe the use of noninvasive computed tomography in measuring body fat volume of Mojave desert tortoises, (2) describe the location of fat reserves, (3) investigate relationships between fat reserves and body condition score and body condition index, and (4) explore whether relative temporalis muscle depth, measured via computed tomography, correlates with body condition score. Body condition scores were assessed for eight captive Mojave desert tortoises prior to euthanasia, and computed tomography was performed postmortem to quantify fat volume and measure temporalis muscle depth. At necropsy, the distribution of fat was documented. Fat volume calculated by computed tomography ranged from 2.83 to 145.38 cm3 (0.07–2.5% body volume). Neither qualitative body condition score nor quantitative body condition index was correlated with fat volume. Bladder content did not compromise body condition index. Body condition score was not correlated with relative temporalis muscle depth. Computed tomography is a noninvasive method for successfully identifying fat reserves and estimating total fat volume in Mojave desert tortoises. The lack of a relationship between computed tomography-determined metrics and commonly used body condition metrics indicates that computed tomography fills a critical gap in the health assessment tool kit for captive and free-ranging Mojave desert tortoises.

Capri F. C., Prazzi E., Casamento G., Gambino D., Cassata G., Alduina R. (2023): Correlation between microbial community and hatching failure in loggerhead sea turtle Caretta caretta. Microbial Ecology 86: 1923-1933.
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Microbial communities provide essential information about host ecology and could be helpful as a tool to improve species conservation efforts. However, microbes can also infect and compromise the host development process and viability. Caretta caretta is the most widespread marine turtle species in the Mediterranean basin and is the only species of sea turtle nesting along the Italian coasts. Little is known about the microbiota composition of the nest of sea turtles and its correlation with hatching failures. In this study, the microbial composition of two nests of C. caretta featuring different rates of hatching success from a nesting beach in Lampedusa (Italy) was analyzed and compared. The bacterial community was determined using culture-dependent methods and next-generation sequencing based on 16S rRNA gene metabarcoding analysis. Our results showed five dominant bacterial phyla (Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Firmicutes) and indicated different bacterial families (Pseudomonadaceae and Brucellaceae) as likely causes of hatching failures. Besides, our findings demonstrated the nests’ active role in modulating the sand’s bacterial communities. This study suggests microbiological analysis could be a valuable tool in monitoring nests to take preventive actions and reduce hatching failures.

Hancock J. M., Choma J., Mainye L., Wambi P., Stelfox M., Polyak M. M., Wambua S., Köhnk S. (2023): Using Photo-ID to document and monitor the prevalence of fibropapilloma tumours in a foraging aggregation of green turtles. Frontiers in Marine Science 10: 1217683.
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Sea turtle in-water monitoring has been conducted in Diani-Chale National Marine Reserve (DCMR) in Kenya’s South Coast since 2018, where green sea turtles (Chelonia mydas) may be found resting or foraging. Underwater surveys are conducted at a regular basis to collect data on sea turtle occurrence, abundance, and distribution, incorporating photographic identification (Photo-ID) as a capture-mark-recapture method. Photo-ID is a non-invasive, low-cost, citizen-science-friendly approach that allows researchers to obtain discrete information about individuals’ locations and health status at a given time, which is essential knowledge for spatial planning and conservation management of endangered species. When visible in the photos collected on a single individual, the occurrence of external tumours is noted, and the turtle is flagged for fibropapilloma (FP) monitoring. From July 2018 to December 2022, 2757 green turtle encounters were recorded, resulting in the identification of 571 individuals. External tumours were observed in 75 individuals. The temporal progression of the tumour’s growth was monitored through subsequent re-sightings of 47 of the affected individuals, with evidence of tumour progression and regression. Documenting the incidence of this disease on sea turtles usually involves the manipulation of captured animals, and is quite possibly under-documented in foraging grounds. Tracking the evolution of this disease using Photo-ID can be a useful, non-invasive method to understand its extent in foraging aggregations where turtles can be easily observed underwater.