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Microplastics (MPs) are particles with sizes of ≤5 mm formed when plastic materials break down. These contaminants are often found in marine environments, making it easy for sea turtles to ingest them and for their microbiome to be exposed. MPs can disrupt microbiome balance, leading to dysbiosis and making organisms more susceptible to diseases. Owing to the significance of these processes, it is crucial to dedicate research to studying the metabolic and genetic analysis of the gut microbiome in sea turtles. The objective of this study was to describe the effects of exposure to MPs on the gut microbiome of sea turtles, based on current knowledge. This review also aimed to explore the potential link between MP exposure and disease susceptibility in these animals. We show that the metabolites produced by the gut microbiome, such as short-chain fatty acids (SCFAs), polyamines, and polysaccharide A, can regulate the expression of host genes. Regulation occurs through various mechanisms, including histone acetylation, DNA methylation, and the modulation of cytokine gene expression. These processes are essential for preserving the integrity of the gut mucosa and enhancing the functionality of immune cells. Exposure to MPs disrupts the gut microbiome and alters gene expression, leading to immune system disturbances in sea turtles. This vulnerability makes turtles more susceptible to opportunistic microorganisms such as chelonid alphaherpesvirus 5 (ChAHV5), which is linked to the development of fibropapillomatosis (FP). Additionally, targeted dietary interventions or the use of live microorganisms such as probiotics can help restore microbial biodiversity and recover lost metabolic pathways. The goal of these interventions is to restore the functionality of the immune system in sea turtles undergoing rehabilitation at specialized centers. The gut microbiome plays a crucial role in sea turtle health, sparking discussions and investigations that can potentially lead to promising treatments for these animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The order Onygenales contains multiple fungal pathogens that affect free-ranging and zoo-housed reptilian species. Emydomyces testavorans, an onygenalean fungus associated with skin and shell disease, has been sporadically detected in aquatic chelonians. Because of the recent discovery of this organism, little is known about its prevalence in free-ranging chelonians. The objective of this study was to perform surveillance for E. testavorans in six free-ranging aquatic and terrestrial chelonian species in Illinois, USA: Blanding's turtles (n=437; Emydoidea blandingii), painted turtles (n=199; Chrysemys picta), common snapping turtles (n=35; Chelydra serpentina), red-eared sliders (n=62; RES; Trachemys scripta elegans), eastern box turtles (n=73; Terrapene carolina carolina) and ornate box turtles (n=29; Terrapene ornata). Combined cloacal-oral swabs (COSs) or shell (carapace and plastron surfaces) swabs were collected from 2019 to 2021 and tested for E. testavorans using quantitative PCR. The PCR detected E. testavorans in COSs of an adult male, subadult female, and juvenile male Blanding's turtle (0.6%; 95% confidence interval [CI], 0.2-1.9%) and a shell swab from an adult female RES (1.6%; 95% CI, 0-8.7%). Shell lesions consistent with E. testavorans infection were present in two of the positive Blanding's turtles. These results document the rarity of this pathogen on the landscape in Illinois. Additional studies should determine this pathogen's impact on individuals and clarify its significance for conservation efforts of Blanding's turtle, in which E. testavorans has not been reported previously., (© Wildlife Disease Association 2024.)

Background : Toll-like receptor (TLR), as an important pattern recognition receptor, is a bridge between non-specific immunity and specific immunity, and plays a vital role in the disease resistance of aquatic animals. However, the function of TLR in Pelodiscus sinensis is still unclear. Methods and Results : The sequence characteristics and homology of three TLRs (PsTLR2 , PsTLR3 and PsTLR5 ) were determined in this investigation. Their annotation and orthologies were supported by phylogenetic analysis, functional domain prediction, and sequence similarity analysis. qPCR showed that the identified TLRs were expressed in all tissues, among the high expression of PsTLR5 in the brain and liver and the high expression of PsTLR2 and PsTLR3 in the liver. PsTLR2 mRNA expression increased 6.7-fold in the liver 12 h after Aeromonas hydrophila infection, while the mRNA expression of PsTLR3 was down-regulated by 0.29 times in liver and 0.31 times in spleen. The mRNA expression of PsTLR5 was significantly up-regulated in four immune tissues, and it was up-regulated by 122.8 times in the spleen after 72 h infection. Finally, the recombinant proteins of extracellular LRR domains of these three TLRs were obtained by prokaryotic expression technology, and the binding tests were performed to discover their ability of binding pathogenic microorganisms. Microbial binding test showed that rPsTLR2, rPsTLR3 and rPsTLR5 can combine A. hydrophila , Edwardsiella tarda , Vibrio parahaemolyticus , Staphylococcus aureus , Streptococcus agalactiae and Candida albicans , while rPsTLR3 can bind A. hydrophila , E. tarda , V. parahaemolyticus and C. albicans . Conclusions : Our findings suggested that TLRs may be crucial to turtles' innate immune response against microbes.

Positive blood cultures have been identified in debilitated, stranded, and deceased green turtles ( Chelonia mydas ), suggestive of septicemia. Interpretation of these results is often difficult because multiple studies have previously identified bacteremia in clinically healthy reptiles. In this study, paired blood cultures and skin cultures obtained after aseptic preparation of the venipuncture site were collected from 50 immature free-ranging green turtles from Port Canaveral, Florida. Blood culture results were compared with health status (apparently healthy versus unhealthy, based on physical examination findings and appropriate body condition), date of collection, presence of external fibropapillomatosis, healed or unhealed injuries, and presence of barnacles. Weight, body condition score, body condition index, morphometric measures, volume of blood collected, and body temperature were compared between blood culture-positive and blood culture-negative turtles. Positive blood cultures were identified in 14% (7 of 50) of all turtles, including 15.6% (5 of 32) of apparently healthy turtles. Vibrio spp., Bacillus megaterium , Cellulomonas sp., and Staphylococcus pasteuri were isolated in blood culture from apparently healthy individuals. There was a significant association ( P = 0.048) between positive skin cultures and positive blood cultures, but isolates obtained were consistently different between paired results. There was no significant association ( P > 0.05) between blood culture results and health status, evidence of healed or unhealed injuries, external fibropapillomatosis, or presence of barnacles. Based on the results of this study, positive blood cultures suggestive of nonclinical bacteremia may be present in apparently healthy green turtles. The results of this study will aid the attending clinician in interpretation of blood culture results of apparently healthy or presumed septicemic captive and rehabilitating green turtles as part of the conservation and population recovery of this threatened species.

Aims: This study aimed to describe the bacterial microbiome associated with the carapace of three species of Galapagos giant tortoises (Chelonoidis porteri, Chelonoidis donfaustoi, and Chelonoidis vandenburghi) and determine the potential effect of the whitish lesions caused by the fungus Aphanoascella galapagosensis., Methods and Results: We used Oxford Nanopore's MinION to evaluate the external bacterial microbiome associated with the carapaces from the aforementioned species. Taxonomic assignment was carried out by Bugseq and the bacterial communities were compared between carapaces with and without lesions using a NMDS with Bray-Curtis as the dissimilarity index. We found four genera of bacteria that were ubiquitous throughout all individuals, suggesting the presence of shared taxa. The results also displayed a significant difference in the microbiome between carapaces with and without lesions, and for species-carapace interaction, but not among species., Conclusions: This study establishes a baseline of the bacterial diversity of the carapace within three Galapagos giant tortoise species, showcasing the presence of a distinctive microbial community. Furthermore, our findings suggest a significant influence of the fungus Aphanoascella galapagosensis on the bacterial populations inhabiting the carapace of these reptiles., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Humans produce 350 million metric tons of plastic waste per year, leading to microplastic pollution and widespread environmental contamination, particularly in aquatic environments. This subsequently impacts aquatic organisms in myriad ways, yet the vast majority of research is conducted in marine, rather than freshwater systems. In this study, we exposed eggs and hatchlings of the Chinese soft-shelled turtle (Pelodiscus sinensis) to 80-nm polystyrene nanoplastics (PS-NPs) and monitored the impacts on development, behavior and the gut microbiome. We demonstrate that 80-nm PS-NPs can penetrate the eggshell and move into developing embryos. This led to metabolic impairments, as evidenced by bradycardia (a decreased heart rate), which persisted until hatching. We found no evidence that nanoplastic exposure affected hatchling morphology, growth rates, or levels of boldness and exploration, yet we discuss some potential caveats here. Exposure to nanoplastics reduced the diversity and homogeneity of gut microbiota in P. sinensis, with the level of disruption correlating to the length of environmental exposure (during incubation only or post-hatching also). Thirteen core genera (with an initial abundance >1 %) shifted after nanoplastic treatment: pathogenic bacteria increased, beneficial probiotic bacteria decreased, and there was an increase in the proportion of negative correlations between bacterial genera. These changes could have profound impacts on the viability of turtles throughout their lives. Our study highlights the toxicity of environmental NPs to the embryonic development and survival of freshwater turtles. We provide insights about population trends of P. sinensis in the wild, and future directions for research., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hong Li reports financial support was provided by The National Natural Science Foundation of China. Longhui Lin reports was provided by Natural Science Foundation of Zhejiang Province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lower respiratory tract disease associated with mycoplasmal infection was detected in a free-ranging bog turtle (Glyptemys muhlenbergii) from New Jersey, USA. The presence of a mycoplasmal organism was confirmed by PCR and electron microscopy. Fluid-filled lungs were observed grossly, and there was proliferative pneumonia on histopathology., (© Wildlife Disease Association 2024.)

Research on microbial communities associated with wild animals provides a valuable reservoir of knowledge that could be used for enhancing their rehabilitation and conservation. The loggerhead sea turtle (Caretta caretta) is a globally distributed species with its Mediterranean population categorized as least concern according to the IUCN Red List of Threatened Species as a result of robust conservation efforts. In our study, we aimed to further understand their biology in relation to their associated microorganisms. We investigated epi- and endozoic bacterial and endozoic fungal communities of cloaca, oral mucosa, carapace biofilm. Samples obtained from 18 juvenile, subadult, and adult turtles as well as 8 respective enclosures, over a 3-year period, were analysed by amplicon sequencing of 16S rRNA gene and ITS2 region of nuclear ribosomal gene. Our results reveal a trend of decreasing diversity of distal gut bacterial communities with the age of turtles. Notably, Tenacibaculum species show higher relative abundance in juveniles than in adults. Differential abundances of taxa identified as Tenacibaculum, Moraxellaceae, Cardiobacteriaceae, and Campylobacter were observed in both cloacal and oral samples in addition to having distinct microbial compositions with Halioglobus taxa present only in oral samples. Fungal communities in loggerheads' cloaca were diverse and varied significantly among individuals, differing from those of tank water. Our findings expand the known microbial diversity repertoire of loggerhead turtles, highlighting interesting taxa specific to individual body sites. This study provides a comprehensive view of the loggerhead sea turtle bacterial microbiota and marks the first report of distal gut fungal communities that contributes to establishing a baseline understanding of loggerhead sea turtle holobiont., (© 2024. The Author(s).)

Introduction: Aeromonas hydrophila , a bacterium widely distributed in the natural environment, causes multiple diseases in various animals. Exploring the mechanism of the host defense against A. hydrophila can help develop efficient strategies against Aeromonas infection., Methods: Herein, we investigated the temporal influence of A. hydrophila on the Chinese soft-shelled turtle, an economically important species, at the biochemical, transcriptomic, and metabolomic levels. Plasma parameters were detected with the test kits. Transcriptome and metabolome were respectively applied to screen the differentially expressed genes and metabolites., Results: The contents or activities of these plasma parameters were significantly increased at 24 hpi and declined at 96 hpi, indicating that 24 and 96 hpi were two important time points during infection. Totals of 3121 and 274 differentially expressed genes (DEGs) from the transcriptome while 74 and 91 differentially abundant metabolites (DAMs) from the metabolome were detected at 24 and 96 hpi. The top DEGs at 24 hpi included Ccl2, Ccl3, Ccl4, Il1β, Il6, Il7, Il15, Tnf, and Tnfr1 while Zap70, Cd3g, Cd8a, Itk, Pik3r3, Cd247, Malt1, and Cd4 were the most abundant at 96 hpi. The predominant DAMs included O-phospho-L-serine, γ-Aminobutyric acid, orotate, L-tyrosine, and L-tryptophan at 24 hpi, as well as L-glutamic acid, L-arginine, glutathione, glutathione disulfide, and citric acid at 96 hpi., Discussion: The combined analysis of DEGs and DAMs revealed that tryptophan metabolism, nicotinate and nicotinamide metabolism, as well as starch and sucrose metabolism, were the most important signaling pathways at the early infective stage while tyrosine metabolism, pyrimidine metabolism, as well as alanine, aspartate and glutamate metabolism were the most crucial pathways at the later stage. In general, our results indicated that the Chinese soft-shelled turtle displays stage-specific physiological responses to resist A. hydrophila infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ji, Chen, Zhu, Hong, Liu, Wei, Zhu and Li.)

Ulcerative colitis (UC) is a recurrent intestinal disease with an increasing incidence worldwide that seriously affects the life of patients. Turtle peptide (TP) is a bioactive peptide extracted from turtles that has anti-inflammatory, antioxidant and anti-aging properties. However, studies investigating the effect of TP on the progression of UC are lacking. The aim of this study was to investigate effects and underlying mechanisms of TP and its derivative peptide GPAGPIGPV (GP-9) in alleviating UC in mice. The results showed that 500 mg/kg TP treatment significantly ameliorated colitis symptoms and oxidative stress in UC mice. TP alleviated intestinal barrier damage in UC mice by promoting mucosal repair and increasing the expression of tight junction proteins (ZO1, occludin and claudin-1). TP also modulated the composition of the gut microbiota by increasing the abundance of the beneficial bacteria Anaerotignum, Prevotellaceae_UCG-001, Alistipes, and Lachno-spiraceae_NK4A136_group and decreasing the abundance of the harmful bacteria Prevotella_9 and Parasutterella. Furthermore, we characterized the peptide composition of TP and found that GP-9 ameliorated the symptoms of dextran sodium sulfate (DSS)-induced colitis in mice by inhibiting the TLR4/NF-κB signaling pathway. In conclusion, TP and its derivative peptides ameliorated DSS-induced ulcerative colitis by inhibiting the expression of inflammatory factors and modulating the composition of the intestinal microbiota; this study provides a theoretical basis for the application of TP and its derivative peptides for their anti-inflammatory activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: Microbiome composition is increasingly considered in species reintroduction efforts and may influence survival and reproductive success. Many turtle species are threatened by anthropogenic pressures and are frequently raised in captivity for reintroduction efforts, yet little is known about turtle microbiome composition in either wild or captive settings. Here, we investigated trends in microbiome composition of captive and wild IUCN-endangered Blanding's turtles (Emydoidea blandingii)., Methods and Results: We amplified and sequenced the V4 region of the 16S rDNA locus from plastron, cloaca, and water samples of wild E. blandingii adults and two populations of captive E. blandingii juveniles being raised for headstarting. Plastron, cloaca, and water-associated microbiomes differed strongly from each other and were highly variable among captive sites and between captive and wild sites. Across plastron, cloaca, and water-associated microbial communities, microbial diversity changed over time, but not in a predictable direction between captive sites. Plastron beta diversity correlated with growth rate in captive samples, indicating that external microbiomes may correlate with individual fitness., Conclusions: Our results indicate that external and internal microbiomes vary between captive and wild turtles and may reflect differences in fitness of captive-raised individuals., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Citrobacter freundii , an opportunistic bacterial pathogen belonging to the family Enterobacteriaceae, has been sporadically reported in sea turtles. Here, the authors describe 3 unusual lesions associated with C. freundii infection in 3 loggerhead sea turtles stranded on the coast of Gran Canaria Island, Spain. It is possible that these 3 distinct lesions played a major role in the death of these turtles. The first turtle had caseous cholecystitis, a lesion not previously described in sea turtles. The second turtle had large intestinal diverticulitis, a rare condition in loggerheads. The third turtle had bilateral caseous salt gland adenitis. Histologically, numerous gram-negative bacilli were observed at the deepest edge of inflammation in all cases. Pure cultures of C. freundii were obtained from these 3 lesions. Molecular detection of C. freundii DNA in formalin-fixed paraffin-embedded samples from the lesions of the 3 turtles confirmed the microbiological isolation. These cases, in addition to expanding the limited body of knowledge on bacterial infections in sea turtles, highlight the potential pathogenic role of C. freundii in loggerhead turtles., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

As semi-aquatic species that use both terrestrial and aquatic habitats, freshwater turtles and their microbial communities are especially sensitive to the impacts of habitat disturbance. In this study, we use 16S rRNA amplicon sequencing to characterize the shell and cloacal bacterial communities of turtles in the San Francisco Bay Area. We captured western pond turtles (Actinemys/Emys marmorata) across eight sites located in urban and rural environments, along with invasive red-eared sliders (Trachemys scripta elegans). We assessed differences in western pond turtle bacterial communities diversity/composition between shell and cloacal samples and evaluated how alpha/beta diversity metrics were influenced by habitat quality. We found phylum-level bacterial taxonomic turnover in the bacterial communities of western pond turtles relative to the host tissue substrate samples. Our findings indicate that location identity elicits a high degree of lower-level (i.e. species/genus) bacterial taxonomic turnover. Further, we found that samples originating from good quality habitat had poorer shell bacterial communities but more diverse cloacal ones. The shell bacterial communities of red-eared sliders overlapped with those western pond turtles suggesting the existence of microbial dispersal between these two species. Our results add to our current understanding of turtle symbiont microbial ecology by establishing patterns of bacterial symbiont variation in an urban to rural gradient., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Sea Turtle Egg Fusariosis (STEF) is a worldwide emergent fungal disease affecting eggs and causing embryos mortality in turtle's nests such as those of Caretta caretta. It is caused by a complex of species belonging to Fusarium genus, particularly those included in the Fusarium Solani Species Complex (FSSC). During the samplings carried out in summer 2020 along the Tuscany coastlines (Italy), C. caretta eggs showed clinical signs resembling those caused by STEF. A total of 32 fungal isolates were obtained from lesioned eggs whose molecular characterization allowing identifying as belonging to FSSC / Neocosmospora spp., Fusarium oxysporum Species Complex (FOSC) / F. oxysporum and Fusarium nodosum, i.e., fungal genera and speciesincluding also well-known plant pathogens. Isolates inoculated on several plant hosts did not result in any pathogenic activity but F. nodosum causing, on wheat spikes, disease symptoms.This is the first time F. nodosum has been isolated from portions of eggs showing evident signs of fungal infection. This work represents the first report of Fusarium spp. isolated from C. caretta eggs showing lesions resembling those caused by STEF on Tuscan coast thus posing a significant concern to loggerhead sea turtle conservation also in this region., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Large-scale mortality due to Aeromonas hydrophila ( A. hydrophila ) infection has considerably decreased the yield of the Chinese pond turtle ( Mauremys reevesii ). Purslane is a naturally active substance with a wide range of pharmacological functions, but its antibacterial effect on Chinese pond turtles infected by A. hydrophila infection is still unknown. In this study, we investigated the effect of purslane on intestinal morphology, digestion activity, and microbiome of Chinese pond turtles during A. hydrophila infection. The results showed that purslane promoted epidermal neogenesis of the limbs and increased the survival and feeding rates of Chinese pond turtles during A. hydrophila infection. Histopathological observation and enzyme activity assay indicated that purslane improved the intestinal morphology and digestive enzyme (α-amylase, lipase and pepsin) activities of Chinese pond turtle during A. hydrophila infection. Microbiome analysis revealed that purslane increased the diversity of intestinal microbiota with a significant decrease in the proportion of potentially pathogenic bacteria (such as Citrobacter freundii , Eimeria praecox , and Salmonella enterica ) and an increase in the abundance of probiotics (such as uncultured Lactobacillus ). In conclusion, our study uncovers that purslane improves intestinal health to protect Chinese pond turtles against A. hydrophila infection.

The Chinese softshell turtle (CST; Pelodiscus sinensis ) is a freshwater aquaculture species of substantial economic importance that is commercially farmed across Asia, particularly in Taiwan. Although diseases caused by the Bacillus cereus group (Bcg) pose a major threat to commercial CST farming systems, information regarding its pathogenicity and genome remains limited. Here, we investigated the pathogenicity of Bcg strains isolated in a previous study and performed whole-genome sequencing. Pathogenicity analysis indicated that QF108-045 isolated from CSTs caused the highest mortality rate, and whole-genome sequencing revealed that it was an independent group distinct from other known Bcg genospecies. The average nucleotide identity compared to other known Bcg genospecies was below 95%, suggesting that QF108-045 belongs to a new genospecies, which we named Bacillus shihchuchen . Furthermore, genes annotation revealed the presence of anthrax toxins, such as edema factor and protective antigen, in QF108-045. Therefore, the biovar anthracis was assigned, and the full name of QF108-045 was Bacillus shihchuchen biovar anthracis. In addition to possessing multiple drug-resistant genes, QF108-045 demonstrated resistance to various types of antibiotics, including penicillins (amoxicillin and ampicillin), cephalosporins (ceftifour, cephalexin, and cephazolin), and polypeptides, such as vancomycin.

In this study, epibiotic bacteria on the carapace of sea turtles at three different sites in the Persian Gulf were studied. Bacterial density counted using a Scanning Electron Microscope showed that the highest (9.4 × 10 6 ± 0.8 × 10 6  cm -2 ) and lowest (5.3 × 10 6 ± 0.4 × 10 6  cm -2 ) average bacterial densities were found on the green and hawksbill sea turtles, respectively. Bacterial community analysis using Illumina 16S rRNA gene sequencing showed that Gamma- and Alpha-proteobacteria were the dominant classes on all substrates. Some genera, such as Anaerolinea, were site- and substrate-specific. In general, bacterial communities on sea turtles differed from those on the non-living substrate, stones, and exhibited lower species richness and diversity compared to the latter. Despite some similarities, the majority of bacterial communities on the two sea turtles were different. This study provides baseline information about the epibiotic bacteria of sea turtles of different species.

Stenotrophomonas maltophilia is a ubiquitous multidrug-resistant opportunistic pathogen commonly associated with nosocomial infections. The purpose of this study was to isolate and characterize extended-spectrum beta-lactamase (ESBL) producing bacteria from painted turtles (Chrysemys picta) living in the wild and captured in southeastern Wisconsin. Fecal samples from ten turtles were examined for ESBL producing bacteria after incubation on HardyCHROM™ ESBL agar. Two isolates were cultivated and identified by 16S rRNA gene sequencing and whole genome sequencing (WGS) as Stenotrophomonas sp. 9A and S. maltophilia 15A. They were multidrug-resistant, as determined by antibiotic susceptibility testing. Stenotrophomonas sp. 9A was found to produce an extended spectrum beta-lactamase (ESBL) and both isolates were found to be carbapenem-resistant. EDTA-modified carbapenem inactivation method (eCIM) and the modified carbapenem inactivation method (mCIM) tests were used to examine the carbapenemase production and the test results were negative. Through WGS several antimicrobial resistance genes were identified in S. maltophilia 15A. For example a chromosomal L1 β-lactamase gene, which is known to hydrolyze carbapenems, a L2 β-lactamase gene, genes for the efflux systems smeABC and smeDEF and the aminoglycosides resistance genes aac(6')-lz and aph(3')-llc were found. An L2 β-lactamase gene in Stenotrophomonas sp. 9A was identified through WGS., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In the United States (U.S.), pet turtles have been associated with outbreaks of salmonellosis, a serious and sometimes-fatal intestinal illness caused by Salmonella bacteria, with nearly 300,000 people being infected in some years. Children are particularly susceptible because of their propensity to put items, including small turtles, in their mouths. In 1975, a U.S. federal regulation prohibited the sale of turtles <4 inches (101.6 mm) in size, except for the purposes of export, scientific, or educational purposes. This regulation was established to reduce the incidence of salmonellosis, particularly in small children. Previous research has not evaluated the availability of turtles <4 inches in size on websites selling wildlife. We monitored 16 websites in 2021 and quantified listings of small turtles. We determined whether information on Salmonella, the 1975 federal regulation, or related state regulations were provided on the websites and determined legality of sales of small turtles by state regulations. We found that all 16 websites openly advertised and sold turtles <4 inches in size, but only half of these websites provided information about Salmonella and/or the federal regulation. These websites required buyers to confirm that they were not purchasing a turtle as a pet, thereby putting the onus on the consumer to adhere to the regulation. We documented 515 listings of turtles <4 inches in size, including 47 species and one hybrid. Our study has demonstrated that internet sales of small turtles currently represent part of the thriving online pet trade in the U.S. Enforcement of the federal regulation faces jurisdictional challenges in most states. Therefore, we recommend continued public education campaigns by public health agencies in the U.S. to help reduce the risk that pet turtle ownership presents., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Montague et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study was conducted during the 2018 nesting season at the Sugözü Beaches (Adana-Turkey) and Göksu Delta (Mersin-Turkey). Egg samples (n = 63) from loggerhead and green turtle nests (n = 43) were collected. Isolated bacteria were initially identified by phenotypic methods and then by MALDI-TOF MS. The bacterial mass spectra were analyzed using Principal Component Analysis. Bacterial isolation was performed for 55 isolates belonging to 12 genera from two major nesting sites. In Sugözü Beaches 62.2% of the bacteria species belonged to Enterobacteriaceae and in Göksu Delta 44.4% of the bacteria species belonged to Morganellaceae. Klebsiella oxytoca and Staphylococcus haemolyticus had not previously been detected in any sea turtle nests. This is the first MALDI-TOF MS study conducted for determination of bacterial variability in loggerhead turtle eggs in Turkey and serves as a reference study for the assessment of bacterial threat in sea turtle nests, enabling the establishment of suitable conservation measures and treatment processes for both sea turtles and nesting sites., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbor diverse prokaryotic and eukaryotic microbes, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides an inventory of diatoms, bacteria and diatom-associated bacteria originating from loggerhead sea turtles using both metabarcoding and culturing approaches. Amplicon sequencing of the carapace and skin samples chloroplast gene rbcL and 16S rRNA gene detected, in total, 634 diatom amplicon sequence variants (ASVs) and 3661 bacterial ASVs, indicating high diversity. Cultures of putative epizoic and non-epizoic diatoms contained 458 bacterial ASVs and their bacterial assemblages reflected those of their host. Diatom strains allowed for enrichment and isolation of bacterial families rarely observed on turtles, such as Marinobacteraceae, Alteromonadaceae and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed that related diatom genera might retain similar microbial taxa in culture, regardless of the turtle's skin or carapace source. These data provide deeper insights into the sea turtle-associated microbial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

The green sea turtle Chelonia mydas inhabit near-shore areas exposed to threatening anthropogenic activities. The granulomatous lesions in these animals may indicate infectious diseases that can be associated with environmental contamination and hazards to human health. This study aimed to characterize the granulomatous inflammation associated with bacterial and fungal infection in C. mydas off Paraná state. From September 2015 to February 2019, systematic monitoring was performed by the Santos Basin Beach Monitoring Project for sea turtles'carcasses recovery, necropsy, and cause of death diagnosis. The tissue samples were fixed in buffered formalin 10% for histochemical analysis and frozen for molecular analysis to fungi detection (Internal Transcribed Spacer region of the nuclear rDNA) and bacteria detection (16S ribosomal gene). From a total of 270 C. mydas, granulomatous lesions were observed in different organs of 63 (23.3%) individuals. The histological analysis indicated lesions in 94 organs, affecting most respiratory and digestive systems. Bacteria were identified in 25 animals, including an acid-fast bacteria detected in one animal, and fungi in 24 C. mydas. The fungi species included the genus Candida (Candida zeylanoides, n = 3), Yarrowia (Yarrowia lipolytica, n = 9; Yarrowia deformans, n = 5; and Yarrowia divulgata, n = 1), and Cladosporium anthropophilum (n = 1). No species of bacteria was identified by molecular testing. All fungi species identified are saprobic, some are important to food and medical industries, but are also pathogens of humans and other animals. Therefore, long-term monitoring of these pathogens and the C. mydas health may indicate changes in environmental quality, possible zoonotic diseases, and their effects., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: The authors have declared that no competing interests exist.

Species in the Fusarium solani species complex are fast growing, environmental saprophytic fungi. Members of this genus are filamentous fungi with a wide geographical distribution. Fusarium keratoplasticum and F. falciforme have previously been isolated from sea turtle nests and have been associated with high egg mortality rates. Skin lesions were observed in a number of stranded, post-hatchling loggerhead sea turtles (Caretta caretta) in a rehabilitation facility in South Africa. Fungal hyphae were observed in epidermal scrapes of affected turtles and were isolated. The aim of this study was to characterise the Fusarium species that were isolated from these post-hatchling loggerhead sea turtles (Caretta caretta) that washed up on beaches along the South African coastline. Three gene regions were amplified and sequenced, namely the internal transcribed spacer region (ITS), a part of the nuclear large subunit (LSU), and part of the translation elongation factor 1 α (tef1) gene region. Molecular characteristics of strains isolated during this study showed high similarity with Fusarium isolates, which have previously been associated with high egg mortality rates in loggerhead sea turtles. This is the first record of F. keratoplasticum, F. falciforme and F. crassum isolated from stranded post-hatchling loggerhead sea turtles in South Africa., (© 2022. The Author(s).)

The highly endangered European pond turtle ( Emys orbicularis ) was reintroduced in Switzerland in 2010. Up until 2019, no routine medical examinations have been carried out prior to its release or during recapture events. The aim of this study was to assess the health status of captive and free-living Emys orbicularis populations in Switzerland, taking into account the most important and frequently occurring health threats to freshwater turtles. A total of 141 European pond turtles, including captive (n = 89) and free-living (n = 52) individuals, underwent clinical examination (n = 136), choanal and cloacal swab collection for microbiology investigation (n = 140), blood sampling (n = 121), fecal examination for parasitology (n = 92), radiography (n = 84), and ultrasound (n = 46). Microbiology investigation included conventional PCR for herpesvirus, ranavirus, and Mycoplasma spp. Blood was used for the establishment of reference values for hematocrit, leukocyte count, and differential blood count as well as for biochemistry parameters tested with the VetScan VS2. An emydid Mycoplasma was detected in 40% (n = 56/140; 95%CI: 31.82-48.61%) of the turtles, including one individual with upper respiratory signs. Four animals positive for Mycoplasma arrived dead or were euthanized during the study period. Their necropsies revealed no evidence of respiratory disease. No ranavirus or herpesvirus was detected in any of the tested turtles. Two presumptively fatal infections with spirorchiid trematodes were reported during the study period. Endoparasites were detected in only 7.94% of the samples examined. This study provides comprehensive data on the current health status of the largest sample size of captive and free-living populations of Emys orbicularis ever assessed to date and serves as a baseline for future research investigations and management recommendations in this species.

The fungal pathogens Fusarium falciforme and Fusarium keratoplasticum are responsible for the sea turtle egg fusariosis (STEF) throughout main nesting areas of the world. In this study, we investigated whether eggs of the invasive alien red-eared slider turtle, Trachemys scripta, can carry these fungal pathogens. Using multilocus sequence typing of four nuclear DNA regions, we found that eggs of T. scripta naturally can carry these two Fusarium pathogenic species, as well as other Fusarium species belonging to the Fusarium solani species complex. Physiological studies on F. falciforme and F. keratoplasticum isolates revealed that their optimal growth temperature coincided with the pivotal temperature for T. scripta embryos, ca 29.5 ± 0.5 °C, providing an evidence of a potential advantageous biological property for host colonization and virulence. A host-pathogen interaction network analysis of species of the FSSC and their hosts confirmed that F. falciforme and F. keratoplasticum are generalist pathogens in a wide range of animal hosts of worldwide geographical distribution. Finally, we show that nesting areas of this invasive turtle T. scripta in the Mediterranean freshwater marshes can act as chronic reservoirs of these STEF pathogens, and this invasive species can act as a potential vector for the spread of STEF among wild native species and even to humans., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021. Published by Elsevier Ltd.)

Austwickia (Dermatophilus) chelonae is a filamentous, Gram-positive Actinobacteria in the Dermatophilaceae family. It has caused fatal granulomatous disease in diverse captive reptile species on three continents, but its presence in wild or free-ranging populations was unknown. An adult female gopher tortoise (Gopherus polyphemus) was presented euhydrated, but cachectic and infested with ticks, with two firm, encapsulated masses over the cranioventral neck and right stifle. The tortoise had moderate nonregenerative anemia and evidence of inflammation; plasma biochemistry data was within normal limits. Fine needle aspirate of the neck lesion revealed abundant necrosis and aggregates of cocci. Computed tomography delineated the masses and revealed an additional mass adjacent to the left zygomatic bone. After surgical excision, histology identified chronic granulomas with intralesional filamentous bacteria. Pan-bacterial 16S rRNA PCR and sequencing of the masses identified A. chelonae. Despite treatment with oxytetracycline and ceftazidime, the tortoise deteriorated and was euthanatized. An esophageal lesion consistent with A. chelonae was seen on postmortem examination, although it was determined that the tortoise ultimately succumbed to fungal pneumonia caused by Metarhizium robertsii, an entomopathogenic biotoxin sprayed as insect control. This case reveals A. chelonae is present in free-ranging chelonians in North America. This organism produces a toxin gene similar to diphtheria toxin, one of the most potent known biotoxins, which has not been previously identified outside the genus Corynebacterium. Novel PCR primers were designed for the toxin and rpoB genes, which were amplified and sequenced from two cases and compared with two available genomes. Selection analysis revealed that the toxin gene is under positive selection, which implies it interacts significantly with the immune system, making it a good candidate for immunodiagnostic test development., (© Wildlife Disease Association 2022.)

The identification of ticks and their associated pathogens is important for knowledge on tick-borne diseases. The objective of this study was to use morphological, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and/or molecular biology tools to identify ticks collected from turtles in north-eastern Algeria, as well as to investigate the microorganisms associated with these ticks. A total of 471 adult ticks were collected and identified morphologically as Hyalomma aegyptium, of which 248 (52.7%) were female and 223 (47.3%) were male. amongst them, 230 specimens were randomly selected for molecular and MALDI-TOF MS analysis. Molecular biology confirmed that our ticks were Hy. aegyptium. MALDI-TOF MS analysis revealed that 100% of the spectra were of excellent quality. Four spectra were selected to update our own database MALDI-TOF MS arthropod. The blind test of the 226 remaining spectra showed that all ticks were correctly identified, with scores ranging from 1.774 to 2.655 with a mean of 2.271 ± 0.16 of which, 223 (98.6%) had log score value (LSV)>1.8. Molecular biology screening showed that the ticks carried the DNA of Borrelia turcica, Rickettsia africae, Rickettsia aeschlimannii, Rickettsia sibirica mongolitimonae and with the Anaplasmataceae were close to a potentially new, undescribed Ehrlichia sp. This study confirms that MALDI-TOF MS is a reliable tool for the identification of ticks and that ticks collected from turtles in Algeria are carriers of several species of microorganisms which may be responsible for diseases in humans and animals., (Copyright © 2021. Published by Elsevier GmbH.)

Background: Neutrophil extracellular traps (NETs) represent a novel cellular mechanism of antimicrobial defense activity. Intravascular neutrophils produce extracellular web-like structures composed of chromatin, histones, and cytoplasmic granule proteins to attack and kill microbes. They may impact both pathogen and host; NETs correlate strongly with disseminated intravascular coagulation and mortality in critically ill humans. The mechanism was first discovered in human neutrophils in 2004. Presumptive heterophil extracellular traps (HETs) in a non-avian reptile species were first described in blood films of a gopher tortoise with systemic inflammation., Objective: While prior reports are limited to blood film review and in vitro studies, this descriptive case series highlights the cytological identification of presumptive HETs in nine reptile patients., Methods: Subjects included six gopher tortoises, one blood python (Python curtus ), one Burmese python ( P. bivittatus), and one desert king snake ( Lampropeltis getula splendida ). All six gopher tortoises ( Gopherus polyphemus ) had upper respiratory disease with bacterial etiology (including Helicobacter sp. and/or Mycoplasma sp.), and snakes had upper respiratory tract infection confirmed with serpentovirus (n = 2) or bacterial dermatitis (n = 1)., Results: Cytology samples with identified HETs included tissue imprints (n = 4), nasal discharge (n = 3), an oral swab (n = 1), and a fine needle aspirate of a skin lesion (n = 1). The identification of specific bacterial (n = 6) and/or viral pathogens (n = 2) was notable., Clinical Relevance: To the authors' knowledge, this is the first report of presumptive HETs recognized in reptile cytology specimens, suggesting an active cellular process in vivo in response to systemic inflammation in non-avian reptiles, and contributing to further understanding of extracellular traps in these species.

A bacterial strain designated 26B T , which had been isolated from the cloaca of a toad-headed turtle, was subjected to a comprehensive taxonomic study. Comparison of 16S rRNA gene sequences demonstrated that strain 26B T is a member of the family Neisseriaceae . Based on highest similarity values, Neisseria animaloris DSM 21642 T (95.15 %), Alysiella filiformis ATCC 15532 T (95.06 %), Uruburuella testudinis 07&#95;OD624 T (94.71 %), Uruburuella suis CCUG 47806 T (94.66 %) and Alysiella crassa DSM 2578 T (94.64 %) were identified as the closest relatives. Average nucleotide identity values based on the blast algorithm (ANIb) indicated that U. suis (76.10/76.17 %), Neisseria shayeganii 871 T (74.34/74.51 %), Stenoxybacter acetivorans (73.30/73.41 %), N. animaloris (72.98/72.80) %, A. filiformis (71.14/71.21 %) and A. crassa (70.53/71.15 %) are the next closest relatives. Like ANIb, genome-based phylogeny did not suggest the affiliation of strain 26B T with any established genus. The polyamine pattern consisted of the major compounds putrescine, 1,3-diaminopropane and spermidine and the major quinone was ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an ornithine lipid were predominant. The fatty acid profile contained predominantly C 16 : 1  ω7 c , C 12 : 0 , C 14 : 0 , C 16 : 0 and C 12 : 0 3OH. The size of the genome was 2.91 Mbp and the genomic G+C content was 54.0 mol%. Since these data do not demonstrate an unambiguous association with any established genus, we here propose the novel genus Paralysiella with the type species Paralysiella testudinis gen. nov., sp. nov. The type strain is 26B T (=CCM 9137 T =LMG 32212 T ).

Human infections caused by the bacterial pathogens transmitted from pet-turtles are becoming very common and getting more importance as the turtles are considered unsafe pet animals, mainly for children and immunocompromised people. Pet-turtles are known as the reservoir of different bacterial species as their intestinal microflora. Extrinsic stressors, such as crowding, unhygienic handling, poor water quality, polluted feeding and inadequate nutrition, can predispose pet-turtles to bacterial infections. The presence of different virulence genes leads to the virulent potential of bacteria. The virulent bacteria can cause infections in turtles and humans, if the turtle owners or shopkeepers don't practice proper sanitation while handling turtles. The aim of this review paper was to provide an overview of different bacterial species isolated from pet-turtles for awareness-raising about potential health risks related to raise pet-turtles., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The polymeric immunoglobulin receptor (pIgR) is one of the most vital components of mucosal immunity that plays a pivotal role in mediating transcytosis of polymeric immunoglobulin (pIg) on epithelial surfaces for protection against invading pathogens. Herein, we cloned the full-length cDNA of Pelodiscus sinensis pIgR, designated as P. sinensis pIgR, made of an open reading frame (ORF) of 1848 bp, molecular weight of 68.2 kDa and estimated isoelectric point of 7.00. The deduced P. sinensis pIgR sequence had a leader peptide, extracellular region containing four immunoglobulin-like domains (Ig like domains), transmembrane and intracellular regions comparable with other vertebrates. P. sinensis pIgR contained four Ig like domains that corresponded with mammalian D1, D3, D4 and D5 similar with reptile and avian Ig like domains. It had 40 potential phosphorylation sites, four putative N-glycosylation sites and several motifs resembling mammalian pIgR motifs. Phylogenetic analysis showed a close relationship between P. sinensis pIgR with avian and reptile pIgRs. P. sinensis pIgR basal levels were higher in the esophagus, small intestine and intestinnum crissum than in other organs of health turtles. Intragastric delivery of LPS and Aeromonassobria led to significant upregulation of P. sinensis pIgR in tissues of the gastrointestinal tract. A polyclonal anti- P. sinensis pIgR antibody produced in rabbit reacted with the recombinant P. sinensis pIgR protein expressed in Escherichia coli in Western blot. These studies demonstrate the existence and immune response of P. sinensis pIgR to stimulation in mucosal organs in Chinese soft-shelled turtles., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The genus Helicobacter includes spiral-shaped bacteria in the phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacteriales, that have been associated with disease in animals, including reptiles. Three wild gopher tortoise (Gopherus polyphemus) index cases presented between 2012 and 2019 with nasal discharge, lethargy, and weight loss. Cytological examination of nasal discharge from all 3 tortoises identified marked heterophilic and mild histiocytic rhinitis with abundant extracellular and phagocytized spiral shaped bacteria that stained positive with Warthin-Starry stain. Polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene revealed this to be a novel Helicobacter species. Two tortoises died despite treatment attempts, and the third was moribund and was euthanized. Histological examination of the nasal mucosa (n = 3) showed granulocytic to lymphocytic rhinitis with variable mucosal hyperplasia, erosion, and ulceration; Warthin-Starry staining highlighted the presence of spiral bacteria in the untreated tortoise. Genus-specific primers were designed, and the gyrA and groEL genes were amplified by PCR and sequenced. Phylogenetic analysis shows that this organism and other previously characterized Helicobacter from tortoises form a clade. Development and cross-validation of two qPCR diagnostic assays for the gyrA and groEL genes showed significant correlation of the results of two assays (P < 0.0001). These assays were used to survey nasal wash samples from 31 rehabilitating gopher tortoises. Mortality of tortoises significantly correlated with higher Helicobacter loads detected by qPCR (P = 0.028). Appropriate quarantine protocols for tortoises during rehabilitation should consider this organism. Upper respiratory disease in tortoises may involve complex microbial ecology; factors beyond Mycoplasmopsis (Mycoplasma) agassizii should be taken into account., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Microbial communities of animals play a role in health and disease, including immunocompromised conditions. In the northeastern United States, cold-stunning events often cause endangered Kemp's ridley turtles (Lepidochelys kempii) to become stranded on beaches in autumn. These sea turtles are admitted to rehabilitation facilities when rescued alive and are presumed immunocompromised secondary to hypothermia. To better understand the role that microbes play in the health of cold-stunned sea turtles, we characterized the oral and cloacal microbiome from Kemp's ridley turtles at multiple timepoints during rehabilitation, from admission to pre-release, by using Illumina sequencing to analyze the 16S rRNA gene. Microbial communities were distinct between body sites and among turtles that survived and those that died. We found that clinical parameters such as presence of pneumonia or values for various blood analytes did not correlate with oral or cloacal microbial community composition. We also investigated the effect of antibiotics on the microbiome during rehabilitation and prior to release and found that the type of antibiotic altered the microbial community composition, yet overall taxonomic diversity remained the same. The microbiome of cold-stunned Kemp's ridley turtles gradually changed through the course of rehabilitation with environment, antibiotics, and disease status all playing a role in those changes and ultimately the release status of the turtles., Competing Interests: The authors have declared that no competing interests exist.

Background: The emergence of tick-borne disease is increasing because of the effects of the temperature rise driven by global warming. In Turkey, 19 pathogens transmitted by ticks to humans and animals have been reported. Based on this, this study aimed to investigate tick-borne pathogens including Hepatozoon spp., Theileria spp., Babesia spp., Anaplasma spp., Borrelia spp., and Bartonella spp. in tick samples (n = 110) collected from different hosts (dogs, cats, cattle, goats, sheep, and turtles) by molecular methods., Methods: To meet this objective, ticks were identified morphologically at the genus level by microscopy; after DNA isolation, each tick sample was identified at the species level using the molecular method. Involved pathogens were then investigated by PCR method., Results: Seven different tick species were identified including Rhipicephalus sanguineus, R. turanicus, R. bursa, Hyalomma marginatum, H. anatolicum, H. aegyptium, and Haemaphysalis erinacei. Among the analyzed ticks, Hepatozoon spp., Theileria spp., Babesia spp., and Anaplasma spp. were detected at rates of 6.36%, 16.3%, 1.81%, and 6.36%, respectively while Borrelia spp. and Bartonella spp. were not detected. Hepatozoon spp. was detected in R. sanguineus ticks while Theileria spp., Babesia spp., and Anaplasma spp. were detected in R. turanicus and H. marginatum. According to the results of sequence analyses applied for pathogen positive samples, Hepatozoon canis, Theileria ovis, Babesia caballi, and Anaplasma ovis were identified., Conclusion: Theileria ovis and Anaplasma ovis were detected for the first time to our knowledge in H. marginatum and R. turanicus collected from Turkey, respectively. Also, B. caballi was detected for the first time to our knowledge in ticks in Turkey.

Background: Chinese soft-shell turtle (Pelodiscus sinensis) is an important commercial species for their high nutritional value and unique taste, but it has been a vulnerable species due to habitat loss. In this study, homologous juvenile turtles were allocated to lake, pond and paddy field to investigate the habitat effects on turtles., Results: The growth, morphology and gut microbial communities were monitored during the 4 months cultural period. It showed higher growth rate of turtles in paddy field and pond. The appearance, visceral coefficients, gut morphology and microbial communities in turtles were distinct among different habitats. The microbial community richness on Chao1 was obviously lower in initial turtle guts from greenhouses, whereas it was relative higher in turtle guts sampled from paddy fields than ponds and lake. Significant differences on dominant microbes were found among initial and subsequent samples from different habitats. Firmicutes was the most abundant phylum in the guts of turtles sampled from the greenhouse initially, while Proteobacteria was the most abundant phylum after cultivation in different habitats, followed by Bacteroidetes. The microbial composition were distinct in different habitats at 60d, and the appearance of dominant phyla and genera was more driven by sampling time than habitats at 120d. Both the sampling time and habitats affected the appearance of dominant phyla and genera during the cultivation. The functional predictions indicated that both habitat type and sampling time had significant effects on metabolic pathways, especially amino acid and carbohydrate metabolism., Conclusions: The turtles could adapt to natural lakes, artificial ponds and paddy fields. The gut microbial abundance was different among the habitats and sampling time. The species of microbes were significantly more diverse in paddy field specimens than in those from ponds and lakes. Rice-turtle coculture is a potential ecological and economic farming mode that plays important roles in wild turtle protection and food security.

Peptidoglycan recognition proteins (PGRPs), which are discovered in invertebrates and vertebrates, play an important role in antibacterial immunity. However, the function of PGRPs is largely uninvestigated in reptiles. In the present study, a short-type PGRP gene, designed as C-turtle-PGRP-S, was identified in the Chinese soft-shelled turtle, Pelodiscus sinensis. The C-turtle-PGRP-S contains a highly conserved PGRP domain and has close relationship with PGRP-S orthologues in other species according to sequence and phylogenetic analyses. C-turtle-PGRP-S gene was constitutively expressed in all detected tissues and was induced by Edwardsiella tarda. Additionally, recombinant C-turtle-PGRP-S showed PGN binding activity and antibacterial function against E. tarda. Therefore, it is suggested that the function of PGRP-S is likely to be conserved in reptile vertebrates, as observed in other vertebrates, shedding light on the evolutionary conservation of PGRPs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: In free-ranging reptile populations, bacterial, fungal, viral and parasitic pathogens may affect hosts through impairment in movements, thermoregulation, reproduction, survival, and population dynamics. The speckled dwarf tortoise (Chersobius [Homopus] signatus) is a threatened species that is mostly restricted to the Succulent Karoo biome in South Africa, and little information on pathogens of this species is available yet. We derived baseline parameters for five males and five females that were captured to genetically enhance a conservation breeding program in Europe. Upon collection of the tortoises, ticks were removed and identified. Immediately upon arrival in Europe, ocular, nasal, oral and cloacal swabs were taken for viral, bacteriological and mycological examinations. Fecal samples were collected before and 1 month after fenbendazole treatment, and analyzed for parasites. A panel of PCR, aiming to detect herpesviruses, adenoviruses and iridoviruses, was carried out., Results: Samples were negative for viruses, while bacteriological examination yielded detectable growth in 82.5% of the swabs with a mean load of 16 × 10 7  ± 61 × 10 8 colony forming units (CFU) per swab, representing 34 bacterial species. Cloacal and oral swabs yielded higher detectable growth loads than nasal and ocular swabs, but no differences between sexes were observed. Fungi and yeasts (mean load 5 × 10 3  ± 13 × 10 3 CFU/swab) were detected in 25% of the swabs. All pre-treatment fecal samples were positive for oxyurid eggs, ranging from 200 to 2400 eggs per gram of feces, whereas after the treatment a significantly reduced egg count (90-100% reduction) was found in seven out of 10 individuals. One remaining individual showed 29% reduction, and two others had increased egg counts. In five tortoises, Nycthocterus spp. and coccidian oocysts were also identified. Soft ticks were identified as Ornithodoros savignyi., Conclusions: Our baseline data from clinically healthy individuals will help future studies to interpret prevalences of microorganisms in speckled dwarf tortoise populations. The study population did not appear immediately threatened by current parasite presence.

Muscle is an important structural tissue in aquatic animals and it is susceptible to bacterial and fungal infection, which could affect flesh quality and health. In this study, Chinese soft-shelled turtles were artificially infected with two pathogens, Proteus vulgaris and Elizabethkingia meningoseptica and the effects on muscle nutritional characteristics, oxidative stress and autophagy were assayed. Upon infection, the muscle nutritional composition and muscle fiber structure were notably influenced. Meanwhile, the mRNA expression of Nrf2 was down-regulated and Keap1 up-regulated, thus resulting in a decrease in antioxidant capacity and oxidative stress. However, with N-acetylcysteine treatment, the level of oxidative stress was decreased, accompanied by significant increases in antioxidant enzyme activities and the mRNA levels of SOD, CAT, GSTCD, and GSTO1. Interestingly, there was a significant increase in autophagy in the muscle tissue after the pathogen infection, but this increase could be reduced by N-acetylcysteine treatment. Our findings suggest that muscle nutritional characteristics were dramatically changed after pathogen infection, and oxidative stress and autophagy were induced by pathogen infection. However, N-acetylcysteine treatment could compromise the process perhaps by decreasing the ROS level and regulating Nrf2-antioxidant signaling pathways.

Microbial communities associated with freshwater aquatic habitats and resident species are both critical to and indicative of ecosystem status and organismal health. External surfaces of turtle shells readily accumulate microbial growth and could carry representation of habitat-wide microbial diversity, since they are in regular contact with multiple elements of freshwater environments. Yet, microbial diversity residing on freshwater turtle shells is poorly understood. We applied 16S and 18S metabarcoding to characterize microbiota associated with external shell surfaces of 20 red-eared slider (Trachemys scripta) turtles collected from varied habitats in central and western Oklahoma, and ranging to southeast Iowa. Shell-associated microbial communities were highly diverse, with samples dominated by Bacteroidia and alpha-/gamma-proteobacteria, and ciliophoran alveolates. Alpha diversity was lower on turtle shells compared to shallow-water-associated environmental samples, likely resulting from basking-drying behavior and seasonal scute shedding, while alpha diversity was higher on carapace than plastron surfaces. Beta diversity of turtle shells was similarly differentiated from environmental samples, although sampling site was consistently a significant factor. Deinococcus-Thermus bacteria and ciliophoran alveolates were recovered with significantly higher abundance on turtle shells versus environmental samples, while bacterial taxa known to include human-pathogenic species were variably more abundant between shell and environmental samples. Microbial communities from a single, shared-site collection of the ecologically similar river cooter (P. concinna) largely overlapped with those of T. scripta. These data add to a foundation for further characterization of turtle shell microbial communities across species and habitats, with implications for freshwater habitat assessment, microbial ecology and wildlife conservation efforts., Competing Interests: The authors have declared that no competing interests exist.

Providencia rettgeri infection has occurred occasionally in aquaculture, but is rare in turtles. Here, a pathogenic P. rettgeri strain G0519 was isolated from a diseased slider turtle (Trachemys scripta) in China, and qPCR assay was established for the RTX toxin (rtxD) gene. Histopathological examination showed that many inflammatory cells were infiltrated into heart, liver and intestine, as well as the necrosis of liver, kidney and spleen. The genome consisted of one circular chromosome (4.493 Mb) and one plasmid (18.8 kb), and predicted to contain 4170 and 19 protein-coding genes, respectively. Multiple pathogenic and virulence factors (e.g., fimbria, adhesion, invasion, toxin, hemolysin, chemotaxis, secretion system), multidrug-resistant genes (e.g., ampC, per-1, oxa-1, sul1, tetR) and a novel genomic resistance island PRI519 were identified. Comparative genome analysis revealed the closest relationship was with P. rettgeri, and with P. heimbachae closer than with other Providencia spp. To our knowledge, this was first report on genomic characterization of multidrug-resistant pathogenic P. rettgeri in cultured turtles.

The expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The leatherback sea turtle (LST; Dermochelys coriacea) is highly migratory and is primarily distributed throughout tropical and subtropical regions. The LST populations found in Brazil are critically endangered. An unusual mortality event occurred between August and November 2016 with 23 LSTs stranded along a 100 km coastal segment in Iguape, Ilha Comprida and Ilha do Cardoso in southern São Paulo state. This study investigated the pathological findings and most likely causes of death of 10 LSTs. Male (n = 9) and adult (n = 9) animals predominated. All but one animal was in good body condition and all were found dead. The most prevalent gross findings were suggestive of bycatch, namely cutaneous erosions, abrasions and/or lacerations around the neck and flippers (n = 9), generalized congestion (n = 8) and pulmonary oedema (n = 6). Other prevalent gross findings were: cutaneous epibiosis by Stomatolepas elegans (n = 7); ileocecal diverticulitis (n = 7); distal oesophagitis (n = 5); and fibrinous coelomitis (n = 5). Microscopically, the most prevalent findings were: hepatic melanomacrophage centre hypertrophy or hyperplasia (n = 9); interstitial pneumonia (n = 8); multisystemic congestion (n = 6); pulmonary oedema with or without aspirated material (n = 5); adrenal coccidiosis (n = 5) with variable multiorgan involvement; and multiorgan bacterial disease (n = 5). Five animals had food ingesta (cnidarians, crustaceans) in the oesophagus or stomach; only one had evident plastic foreign bodies. Asphyxia due to entanglement in nets was the most frequently identified cause of death (n = 8); a cause of death was not identified in two animals. Our findings provide evidence of the severe negative impact of entanglement in fishing nets in LSTs, raising concerns for conservation. These findings also contribute to knowledge of the pathology of LSTs in South American populations., Competing Interests: Conflict of Interest Statement The authors declare no potential conflicts of interest with respect to the research, authorship or publication of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The microbiota is considered critical for normal vertebrate homeostasis and it may exert its effects at a local level within the gastrointestinal tract, or systemically through the production of bacterial metabolites. To date, investigations into the role that the microbiota plays in reptile physiology are rare. To address this knowledge gap, we explored the relationship between differences in microbial communities to see if they accounted for differences in haematology and biochemistry values, in different populations of nesting flatback turtles (Natator depressus). We found that microbiota composition was not correlated to any of the blood analytes we measured in flatbacks. This study is the first of its kind in reptiles and highlights the need for further investigations to determine mechanisms by which the microbiota influences the physiology and health of reptiles.

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.

Chelonians are increasingly challenged by anthropogenic threats and disease. This article summarizes recent literature and clinical experiences regarding 4 emerging infectious diseases in turtles and tortoises: ranaviruses, cryptosporidiosis, intranuclear coccodiosis of Testudines, and Emydomyces testavorans., Competing Interests: Disclosure Two of the authors (L. Adamovicz and M.C. Allender) work in a laboratory that provides diagnostic testing for reptile clinicians. This role has not influenced the structure or content of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aim of the study was to detect and genetically characterize Arcobacter butzleri in pet red-footed tortoises suspected for Campylobacter spp., using molecular techniques. A written consent from tortoise owners was obtained, after explaining the advantages of the research to tortoise owners of Grenada. Fecal samples were collected from 114 tortoises from five parishes of the country and cultured for Campylobacter spp. using selective culture techniques. A. butzleri was isolated from 4.39% of pet tortoises. Total thirteen isolates were obtained; all identified as A. butzleri by a universal and a species-specific Polymerase Chain Reaction (PCR) and direct sequencing. Genetic characterization of these isolates was performed based on Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) that generated eight different genetic fingerprints with a discriminatory power of 0.91. Campylobacter species were not detected molecularly in any of the culture-positive samples. This is the first report of infection of pet tortoises in Grenada, West Indies with A. butzleri. This study emphasizes on the risk of zoonotic transmission of A. butzleri by exotic pets, which is a serious concern for public health., Competing Interests: The authors have declared that no competing interests exist.