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The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: In 2015, a previously unrecognized intracytoplasmic erythrocytic inclusion was discovered in anemic wild-caught adult gopher tortoises (Gopherus polyphemus). Subsequently, molecular diagnostics revealed this inclusion to be a novel Anaplasma sp., Objectives: The goal of this study was to morphologically characterize these erythrocytic inclusions by light and transmission electron microscopy (TEM)., Methods: Blood samples were taken from two car-injured wild-caught gopher tortoises for the preparation of Wright-Giemsa stained smears and TEM specimens. CBC data were serially performed and morphologically examined during treatment periods., Results: Studies revealed a moderate to severe anemia with moderate regeneration as indicated by polychromasia and the presence of immature erythroid precursors. In addition, on light microscopy, one to two variably-sized round basophilic stippled paracentral erythrocytic inclusions were present per cell in both animals and involved 10%-25% of erythrocytes. TEM identified the intraerythrocytic inclusions as discrete membrane-bound cytoplasmic vacuoles (morulae) containing membrane-bound bacterial subunits that were of variable size, shape, and electron density. Serial hematologic data indicated complete remission of the infection in response to a single long-term course of doxycycline., Conclusions: The presence of a regenerative anemia in gopher tortoises from Florida revealed a newly recognized bacterial species that has morphologic characteristics similar to members of the genus Anaplasma., (© 2020 American Society for Veterinary Clinical Pathology.)

Natural antibodies (NAbs) are polyreactive, have low avidity, and are a product of B-1 cells. Evidence suggests that NAbs may play a key role in immune defense in turtles, as increased total mucosal antibodies are associated with a decreased number of extracellular intestinal parasites. However, it is unknown if this trend extends to other types of pathogens and if avidity of the NAb to the pathogen is a factor in protection. We examined the relationship between a common intracellular bacteria in turtles-Salmonella-and NAbs. Plasma and mucosal samples were taken from red-eared slider turtles. We measured levels and avidity of antibodies that bound to lipopolysaccharide (LPS), a component of Salmonella cell wall. We examined the relationship between these measures and the ability of plasma to kill Salmonella as well as infection status. Higher mucosal antibody levels were significantly associated with a decrease in likelihood of infection with Salmonella; however, plasma antibody levels were not. There was a trend for bactericidal ability of the plasma to be positively correlated with plasma antibody levels bound to LPS, but not mucosal antibody levels. Avidity was not significantly related to either killing capacity or likelihood of infection suggesting that only increased quantity and not better binding is responsible for the decreased likelihood of infection. These findings suggest that NAb regulation was sufficient to isolate the infection to the gastrointestinal tract of the turtles, allowing it to be cleared with the mucus layer. Our results add further evidence that turtles use a general, nonspecific NAb response to combat pathogens., (© 2019 Wiley Periodicals, Inc.)

Klebsiella sp. are responsible for a multitude of infectious diseases in both humans and animals. In this study, phylogenetic relationships, virulence and antimicrobial resistance gene properties of 16 Klebsiella sp. isolated from 49 pet turtles were investigated. The isolates including Klebsiella oxytoca (n = 13) and Klebsiella pneumoniae (n = 3) were identified using 16S rRNA gene sequencing and each species formed distinct clusters in the neighbour-joining phylogenetic tree. The prevalence of virulence genes including ureC (100%) and kfu (68·75%) was observed among the isolates using Polymerase chain reaction (PCR) assay. The fimH, mrkD and rmpA genes were detected in all K. pneumoniae while these were absent in every K. oxytoca isolate. In antimicrobial susceptibility testing, high resistance rates were observed against ampicillin (100%) and cephalothin (62·50%). The resistance rates against imipenem, tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid and ciprofloxacin were 12·50, 12·50, 12·50, 6·25 and 6·25% respectively. The presence of antimicrobial resistance genes such as plasmid-mediated quinolone resistance (PMQR) [qnrB (37·50%), qnrA (31·25%), qnrS (12·50%) and aac(6')-Ib-cr (12·50%)], extended-spectrum β-lactamase (ESBL) [bla CTX-M (18·75%)], β-lactamase [bla SHV-1 (18·75%)] and tetracycline resistance [tetE (12·50%)] was observed. The results revealed that pet turtle-borne Klebsiella sp. may carry different types of virulence and antimicrobial resistance genes which represents a potential threat to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: Klebsiella sp. are nonmotile Gram-negative bacteria that are found in different environments. The virulence and antimicrobial resistance properties of pet turtle-borne Klebsiella sp. have not been studied before. Phylogenetic relationships, virulence traits and antimicrobial resistance profiles of pet turtle-borne Klebsiella sp. were characterized for the first time in Korea. Multiple virulence and antimicrobial resistance genes were observed among the isolates. The occurrence of virulence and antimicrobial resistance determinants in Klebsiella sp. may represent a potential threat to public health., (© 2019 The Society for Applied Microbiology.)

The aim of this study was to investigate the occurrence and diversity of Campylobacter species in chelonians. From July 2016 to September 2017, a total of 452 individuals from a large variety of tortoises (n = 366) and turtles/terrapins (n = 86) kept in private collections and breeding centres, wildlife rescue centres, zoos, pet shops, and veterinary clinics from Northern Italy was sampled and subjected to microbiological examination. Campylobacter genus and species confirmation was performed by single and multiplex PCRs. Out of 452 samples, five (1.1%) tested positive: three for C. iguaniorum (two Testudo graeca and one Testudo hermanni), one for C. fetus subsp. testudinum (Stigmochelys pardalis) and one for C. geochelonis (Testudo hermanni). This study suggests that Campylobacter spp. are not common in chelonians, but a variety of species can be detected in these hosts, including those potentially pathogenic for humans. Further studies are needed to understand the epidemiology and the pathogenic potential for both animals and humans of reptile-associated Campylobacter spp., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Caretta caretta is threatened by many dangers in the Mediterranean basin, but most are human-related. The purposes of this research were: (i) to investigate microflora in samples from six loggerhead sea turtle nests located on the Sicilian coast and (ii) to understand microbial diversity associated with nests, with particular attention to bacteria and fungi involved in failed hatchings. During the 2016 and 2018 summers, 456 eggs and seven dead hatchling from six nests were collected. We performed bacteriological and mycological analyses on 88 egg samples and seven dead hatchlings, allowing us to isolate: Fusarium spp. (80.6%), Aeromonas hydrophila (55.6%), Aspergillus spp. (27.2%) and Citrobacter freundii (9%). Two Fusarium species were identified by microscopy and were confirmed by PCR and internal transcribed spacer sequencing. Statistical analyses showed significant differences between nests and the presence/absence of microflora, whereas no significant differences were observed between eggs and nests. This is the first report that catalogues microflora from C . caretta nests/eggs in the Mediterranean Sea and provides key information on potential pathogens that may affect hatching success. Moreover, our results suggest the need for wider investigations over extensive areas to identify other microflora, and to better understand hatching failures and mortality related to microbial contamination in this important turtle species., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Threatened and endangered green turtles (Chelonia mydas) are unique because as juveniles they recruit from pelagic to near-shore waters and shift from an omnivorous to primarily herbivorous diet (i.e. seagrass and algae). Nevertheless, when injured and ill animals are admitted to rehabilitation, animal protein (e.g. seafood) is often offered to combat poor appetite and emaciation. We examined how the fecal microbiome of juvenile green turtles changed in response to a dietary shift during rehabilitation. We collected fecal samples from January 2014 -January 2016 from turtles (N = 17) in rehabilitation at the Georgia Sea Turtle Center and used next generation sequencing to analyze bacterial community composition. Samples were collected at admission, mid-rehabilitation, and recovery, which entailed a shift from a mixed seafood-vegetable diet at admission to a primarily herbivorous diet at recovery. The dominant phyla changed over time, from primarily Firmicutes (55.0%) with less Bacteroidetes (11.4%) at admission, to primarily Bacteroidetes (38.4%) and less Firmicutes (31.8%) at recovery. While the microbiome likely shifts with the changing health status of individuals, this consistent inversion of Bacteroidetes and Firmicutes among individuals likely reflects the increased need for protein digestion, for which Bacteroidetes are important. Firmicutes are significant in metabolizing plant polysaccharides; thus, fewer Firmicutes may result in underutilization of wild diet items in released individuals. This study demonstrates the importance of transitioning rehabilitating green turtles to an herbivorous diet as soon as possible to afford them the best probability of survival., Competing Interests: Funding for this research was provided by The Coca Cola Company. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A total of 127 Amblyomma ticks (A. helvolum, A. varanense and A. geoemydae) were collected from reptiles: water monitors (Varanus salvator), Bengal monitors (Varanus bengalensis), Burmese pythons (Python bivittatus), yellow-spotted keelbacks (Xenochrophis flavipunctatus), keeled rat snakes (Ptyas carinata) and elongated tortoises (Indotestudo elongata) from nine provinces in Thailand. The presence of Borrelia spp. of the 16S rRNA, flaB, glpQ, groEL and gyrB genes was examined by conventional, semi-nested and nested PCR. Phylogenetic analyses using maximum likelihood method of housekeeping genes showed that most sequences of Borrelia spp. in these Amblyomma ticks belonged to the clade of reptile-associated (REP) borreliae. Interestingly, one Borrelia sp. in an A. geoemydae tick collected from an elongated tortoise clustered in the same clade as a Borrelia sp. detected from an A. geoemydae-infested turtle in Japan (it may belong to the same species given the identical sequences of their 16S rRNA, flaB and glpQ genes) and formed the same group with tick-borne relapsing fever (RF) borreliae of B. miyamotoi and B. theileri. Our findings are the first report on the presence of Borrelia spp. in A. helvolum and A. geoemydae ticks from reptiles in Thailand adding to the geographic distribution of Borrelia spp. in Asia., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

SYJ15 is a highly pathogenic Gram-positive Bacillus sp. with top bud spore newly isolated from dying soft shell turtle. 16SrDNA sequencing showed that it is highly homologous to B. cereus, B. thuringiensis and B. anthracis. Biochemical examinations showed that it belongs to B. cereus. To further study the new pathogen, we conducted whole-genome sequencing based on single-molecular sequencing technology from PacBio. Genome assembly analysis showed that the strain has a 5,296,886 bp chromosome, a 218,649 bp plasmid and a 5221 bp plasmid with GC content of 35.51%, 31.91% and 29.75%, respectively. The genome contains 5736 coding sequences and 6 CRISPR systems located in the chromosome as well as 11 genomic islands in the chromosome and the large plasmid. Genome function analyses were annotated by nr database, SwissProt, KEGG, COG, GO, PHI, VFDB, ARDB, Secretory_Protein and T3SS. In addition, 13 gene clusters of secondary metabolism were predicted by antiSMASH. Comparison of SYJ15 with B. subtilis, B. anthracis, B. cereus and B. thuringiensis identified 1031 core genes of the five strains and 816 genes specific to SYJ15. In addition, SYJ15 had the most common core genes with B. thuringiensis, and the least with B. subtilis. Phylogenetic analysis demonstrated that SYJ15 is between B. thuringiensis and B. cereus, suggesting that SYJ15 belongs to Bacillus cereus group. We designed a specific primer pair to distinguish SYJ15 from B. pumilus, B. licheniformis, B.subtilis, B. thuringiensis and B. cereus. In conclusion, information of SYJ15 genome will help to enhance our understanding of pathogenesis of SYJ15 and find effective treatment.

Borrelia turcica, a member of the reptile-associated Borrelia clade, is vectored by Hyalomma aegyptium. The only suggested reservoir hosts of B. turcica are tortoises of the genus Testudo. Borrelia turcica has been described to occur in several Southeastern European countries including Turkey, Romania, Bulgaria and Greece but so far nothing is known about the relationship of these populations and whether or how they are structured. Using multilocus sequence typing (MLST) on eight chromosomally located housekeeping loci (clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA) we analyzed 43 B. turcica isolates from Serres, Greece (n = 15) collected in 2017 and Izmir, Turkey (n = 28) collected in 2018. To understand their relationship a maximum-likelihood phylogenetic tree and goeBURST analysis were done based on MLST sequence data and allelic profiles, respectively. The data we generated confirmed that the samples of B. turcica investigated here were divergent from Lyme disease (LD) and relapsing fever (RF) species. Within the B. turcica clade, samples of different geographic origin (Greece, Turkey) clustered together in terminal branches; no obvious differences between the Greek and Turkish samples were noticeable. A goeBURST analysis using triple-locus variants revealed very few clonal complexes with the majority of samples appearing as singletons. Minor clonal complexes (consisting of two sequence types) comprised only Greek isolates, only Turkish isolates or both, so no pattern of clustering of isolates from the two geographical regions was observed. Interestingly, very little population structure was discerned in our study. This was surprising in view of the large geographic distance between collection sites of B. turcica and raises questions about the evolution or spatial spread of this species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

With the aim of evaluating the presence of Fusarium spp . in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles ( Caretta caretta ) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp . were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 ( Fusarium keratoplasticum ), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp . Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.

Sea turtles possess relevant characteristics to serve as sentinel species for monitoring the health of marine ecosystems, which is currently threatened. This study examined 35 loggerhead turtles from the western Mediterranean, focusing on the oral and cloacal prevalence of aerobic Gram-negative bacteria, their antibiotic resistance and the influence of several variables linked both to the animal and the environment (i.e. estimated life stage; area, season and cause of recovery; plastic ingestion). Conventional bacteriology methods led to the isolation of bacterial families commonly regarded as opportunistic pathogens (i.e. Aeromonadaceae; Enterobacteriaceae; Pseudomonadaceae; Shewanellaceae; Vibrionaceae), but pointing out sea turtles as carriers of potential zoonotic agents. The high rates of antibiotic resistance, here detected, raise important concerns on the dissemination of this phenomenon in marine environments. Moreover, several of the examined variables showed a significant influence on the prevalence of bacterial families, strengthening the role of sea turtles as mirrors of their ecosystems., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Antimicrobial resistance (AMR), encoded by plasmid-mediated AMR genes (ARGs), is an increasing global public health threat. Wildlife play a fundamental role as sentinels, reservoirs and potential vectors of ARGs. For the first time in Galapagos, we have identified and quantified the presence of ARGs in free-living giant tortoises (Chelonoidis porteri). We performed ARG analyses by quantitative PCR of faeces collected from the cloaca of 30 tortoises widely distributed across Santa Cruz Island. Validated samples (n = 28) were analysed by a panel of up to 21 different ARGs and all 28 tortoise samples were positive to one or more genes encoding resistance. Thirteen of 21 tested ARGs were present in at least one sample, and 10 tortoises (35.7%) had a multi-resistant pattern. We recommend additional research so we may more fully understand resistance patterns across taxa and geographical locations throughout the Galapagos archipelago, and the implications of ARGs for the health of wildlife, domestic animals, and humans. In this study, we found 100% of sampled giant tortoises had ARGs present in their faeces, suggesting a large-scale distribution of these genes within the archipelago., (© 2019 Blackwell Verlag GmbH.)

Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.

Dermatophilosis has been described in many animals since it was first reported in 1915. In 2010, the genus and species associated with chelonid dermatophilosis was reclassified as Austwickia chelonae. Here we discuss a series of four submissions consisting of eight juvenile African spurred tortoises (Geochelone sulcata) from a large breeding facility. Submissions began in December 2016 and continued to June 2017 and then again in November 2018. Clinical signs were originally noted in December of 2015 and consisted of facial swelling and mandibular necrosis that led to complete disarticulation of the mandibles from the skull. Affected animals were emaciated with minimal gastrointestinal contents and several had soft faeces. The facial and head lesions were caused by A. chelonae. Additionally, co-infection with Cryptosporidium ducismarci was discovered within the small intestine. This report gives information on the pathology and pathogenicity of A. chelonae and characterizes the first reported cases of cryptosporidiosis in African spurred tortoises., (Published by Elsevier Ltd.)

Background: Caretta caretta is the most abundant sea turtle species in the Mediterranean, and studies on this species have vastly expanded during recent years, including those investigating gut bacterial and parasitic communities. Members of these communities have been reported with variable prevalence and pathogenicity, mainly depending on their host and environment (e.g. lifespan, distribution, habitat, diet, health status and stressors). Indeed, many species commonly inhabiting the sea turtle gastrointestinal tract exhibit an opportunistic behaviour. This study aimed to provide baseline data on enterobacterial and parasitic composition, through bacteriological culture-based methods and the FLOTAC parasitological technique, in cloacal and faecal samples of 30 live Caretta caretta, examined upon their arrival at the Marine Turtle Research Centre (Portici, Italy)., Results: Enterobacteriaceae were isolated in 18/23 cloacal samples (78.3%), with Citrobacter and Morganella as the most common genera, followed by Proteus, Enterobacter, Providencia, and Hafnia. Parasitic elements were detected in 11/30 faecal samples (36.7%), with Enodiotrema, Rhytidodes, and Eimeria as most common genera, followed by Pachypsolus and Cymatocarpus. Additionally, Angiodyctium is reported for the first time in this host. The majority (47.8%) of sea turtles hosted exclusively Enterobacteriaceae, whereas 30.4% hosted both parasites and Enterobacteriaceae; the remaining 21.8% hosted neither of the agents., Conclusions: Bacteria and parasites evaluated in the present study are common in Mediterranean loggerhead sea turtles, with slight differences between the western and eastern basin. Although naturally present in the gastrointestinal system of free-living sea turtles, their relationship with these hosts might range from mutualism to parasitism. Indeed, members of the gut community might express their pathogenic potential in immune-compromised animals, such as those in rehabilitation facilities. Therefore, it is advisable to include in the standard work-up of rescued sea turtles a screening procedure for such opportunistic agents, in order to better evaluate the animal's health status and achieve timely intervention with appropriate treatment, thus improving rehabilitation. Furthermore, data collected from free-living sea turtles represent a starting point for investigating wild populations. However, further studies are needed to clarify the differences between sea turtle's normal gut microbiome and pathobiome.

Disease is a significant threat in the global decline of reptile species. Many aquatic reptiles live in habitats with high levels of opportunistic microbial pathogens, yet little is known about their immune system. Gut-associated lymphoid tissue is vital for protection against ingested pathogens and maintenance of normal gut microbiota. In mammals, gut mucosal immunity is well-characterized and mucosal surfaces are coated in protective antibodies. However, reptiles lack lymph nodes and Peyer's patches, which are the major sites of mammalian B cell responses. The presence or distribution of mucosal B cells in reptiles is unknown. In this study, we first set out to determine if B cells could be detected in intestinal tissues of red-eared slider turtles, Trachemys scripta. Using whole-mount immunochemistry and a primary antibody to turtle antibody light chains, we identified widely distributed B cell aggregates within the small intestine of hatchling turtles. These aggregates appeared similar to isolated lymphoid follicles (ILFs) in mammals and the frequency was much higher in distal intestinal sections than in proximal sections. To determine if these structures were inducible in the presence of microbes, we introduced an enteric Salmonella species through oral gavage. Analysis of intestinal tissues revealed that hatchlings exposed to Salmonella exhibited significantly more of these aggregates when compared with those that did not receive bacteria. These studies provide the first evidence for B cell-containing ILF-like structures in reptiles and provide novel information about gut immunity in nonmammalian vertebrates that could have important implications for ecological interactions with pathogens., (© 2019 Wiley Periodicals, Inc.)

Caretta caretta is the most common sea turtle species in the Mediterranean Sea. The species is threatened by anthropomorphic activity that causes thousands of deaths and hundreds of strandings along the Mediterranean coast. Stranded turtles are often cared for in rehabilitation centres until they recover or die. The objective of this study was to characterize the gut microbiome of nine sea turtles stranded along the Sicilian coast of the Mediterranean Sea using high-throughput sequencing analysis targeting V3-V4 regions of the bacterial 16S rRNA gene. Stool samples were collected from eight specimens hosted in the recovery centre after a few days of hospitalization (under 7) and from one hosted for many weeks (78 days). To better explore the role of bacterial communities in loggerhead sea turtles, we compared our data with published fecal microbiomes from specimens stranded along the Tuscan and Ligurian coast. Our results highlight that, despite the different origin, size and health conditions of the animals, Firmicutes, Bacteroidetes and Proteobacteria constitute the main components of the microbiota. This study widens our knowledge on the gut microbiome of sea turtles and could be helpful for the set up of rehabilitation therapies of stranded animals after recovery in specialized centres., Competing Interests: The authors have declared that no competing interests exist.

Green turtles are endangered marine herbivorous hindgut fermenters that contribute to a variety of marine ecosystems. Debilitated turtles are often rehabilitated in turtle hospitals. Since accurate diagnosis of disease is difficult, broad-spectrum antibiotics are routinely used as a general treatment, potentially causing collateral damage to the gut microbiome of the patient. Here, we evaluated the concept of the application of bacteriophage (phages) to eliminate targeted intestinal bacteria as an alternative to a broad-spectrum antibiotic (enrofloxacin) in clinically healthy, captive green turtles. Additionally, the impact of a broad-spectrum antibiotic (enrofloxacin) and phage therapy on the gut bacterial communities of green turtles was evaluated. Gut bacterial communities in faecal samples were analysed by sequencing the V1-V3 regions of the bacterial 16S rRNA. Bacteria-specific phage cocktails significantly (P < 0.05) reduced targeted Acinetobacter in phage-treated turtles during the therapy. Compared to control, no significant difference was observed in the bacterial diversity and compositions in phage-treated turtles. In contrast, bacterial diversity was significantly (P < 0.05) reduced in antibiotic-treated turtles at day 15 and throughout the trial. The alteration in the bacterial microbiota of antibiotic-treated turtles was largely due to an increase in abundance of Gram-positive Firmicutes and a concurrent decrease in Gram-negative Bacteroidetes, Proteobacteria and Verrucomicrobia. Additionally, we observed the relative abundance of several bacteria at lower taxonomic level was much less affected by phages than by antibiotics. These data offer the proof of concept of phage therapy to manipulate transient as well as indigenous bacterial flora in gut-related dysbiosis of turtles., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Turtoises are a great puzzle when it comes to their bacterial flora, the composition and structure of which are still unknown in details. Its component which has been best described so far is Salmonella spp., presumably due to the threat of reptile-associated salmonellosis in humans. This investigation tried to assess and characterize intestinal bacterial flora of imported tortoises found dead during quarantine. Most of the animals carried various serovars of Salmonella showing no antimicrobial resistance. Presence of multiresistant Escherichia coli was possibly a result of industrial breeding and high usage of antimicrobials. Thirteen bacterial species or genera like Citrobacter spp., Morganella spp., Pseudomonas spp. were identified. Their commensal character is assumed, although pathogenic potential might be verified. The results indicate global tortoise trade as a source of common and exotic bacteria or antimicrobial resistance mechanisms in new geographical areas. These dangers indicate the need for a systematic survey of exotic pets and establishment of legal requirements for reptile health conditions on breeding, trade premises and in households with such pets., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have become identified in marine ecosystem constituting a serious ecological issue. In this respect, although contamination of coastal waters and seafood, and even colonization of seabirds and fishes have been increasingly reported, molecular data are lacking to elucidate the clinical impact of ESBL producers in infected marine animals. In this study, using a genomic approach, we have analysed the genetic background of CTX-M-15-producing Enterobacter hormaechei (belonging to the international human clone ST114) and Citrobacter freundii (ST265) co-infecting a free-living green turtle (Chelonia mydas) suffering from septic arthritis, which progressed to generalized coelomitis and death. Wide resistome of these pathogens contributed to treatment failure and death of the animal., (© 2019 Blackwell Verlag GmbH.)

The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.

We evaluated cause of injury and quantified levels of three potential mycoplasmal pathogens ( Mycoplasma agassizii , Mycoplasma testudineum , and an emydid mycoplasma) in three-toed box turtles ( Terrapene carolina triunguis ) from the greater St. Louis, Missouri, US area, brought to and housed at the Wildlife Rescue Center (Ballwin, Missouri, US) in 2015 and 2016. We created a probebased quantitative PCR (qPCR) assay for the emydid mycoplasma, with a similar specificity and sensitivity as the existing qPCR assays for M. agassizii and M. testudineum . All three microbes have been implicated in the development of upper respiratory tract disease in turtles and tortoises. We assessed whether signs of respiratory disease, sex, type of trauma, or treatment (administration of antibiotics) affected the presence of pathogens. We found that the most common types of injury experienced by turtles ( n =85) were due to motor vehicles and other types of machinery, and that injuries due to motor vehicles were the most severe. We found a 61% prevalence of emydid mycoplasm a ( n =28) but M. agassizii or M. testudineum were not detected. Prevalence of disease and antibiotic treatment was too low to statistically relate to levels of mycoplasma. Sex and type of trauma were not associated with levels of emydid mycoplasma. The box turtle population we sampled did not experience signs of respiratory disease due to the fairly widespread prevalence of emydid mycoplasma. However, mycoplasmal diseases can be pathogen load-dependent. The qPCR we designed can be used to assess levels of emydid mycoplasma in other emydid species, populations, and individuals, in which there might be a positive association between the microbe and expression of respiratory disease.

Salmonella spp. are frequently shed by wildlife including turtles, but S. enterica subsp. enterica serovar Typhimurium or lesions associated with Salmonella are rare in turtles. Between 1996 and 2016, we necropsied 127 apparently healthy pelagic olive ridley turtles (Lepidochelys olivacea) that died from drowning bycatch in fisheries and 44 live or freshly dead stranded turtles from the west coast of North and Central America and Hawaii. Seven percent (9/127) of pelagic and 47% (21/44) of stranded turtles had renal granulomas associated with S. Typhimurium. Stranded animals were 12 times more likely than pelagic animals to have Salmonella-induced nephritis suggesting that Salmonella may have been a contributing cause of stranding. S. Typhimurium was the only Salmonella serovar detected in L. olivacea, and phylogenetic analysis from whole genome sequencing showed that the isolates from L. olivacea formed a single clade distinct from other S. Typhimurium. Molecular clock analysis revealed that this novel clade may have originated as recently as a few decades ago. The phylogenetic lineage leading to this group is enriched for non-synonymous changes within the genomic area of Salmonella pathogenicity island 1 suggesting that these genes are important for host adaptation.

Leptospira spp. are re-emerging zoonotic pathogens. Previous research has found that Blanding's turtles (Emydoidea blandingii) experimentally infected with Leptospira interrogans shed leptospires in their urine, suggesting that they could play a role in transmitting pathogen within an aquatic ecosystem. This study investigated whether a population of wild Blanding's turtles known to be exposed to Leptospira spp. actively shed the pathogen under natural conditions. Blood samples were collected for serologic testing and to assess the health of the turtles. Free catch urine was collected for polymerase chain reaction (PCR) testing. All turtles were seropositive for Leptospira spp. and 73.5% (25/34) of the urine samples were PCR positive. All animals appeared clinically healthy and showed no apparent signs of disease. This study confirms that wild Blanding's turtles can actively shed Leptospira spp. in their urine and suggests that they may play a role in the epidemiology of this disease in habitats in which they reside., Competing Interests: Funding for this study was received from Fluker Farms and Abaxis Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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

Development of antibiotic resistance in bacteria has challenged significantly in both veterinary and human medicine. In this study, we analyzed the potential risk of pet turtles harboring tribe Proteeae as a source of quinolone-resistant determinants, including plasmid-mediated quinolone resistance (PMQR) genes and target gene alterations in the quinolone resistance-determining region (QRDR). Antimicrobial susceptibility of 54 Proteeae isolates against ciprofloxacin, ofloxacin, levofloxacin, and nalidixic acid was examined. The PMQR genes and QRDR alterations were identified using conventional PCR assays and sequencing. Four isolates were resistant to all quinolones tested in this study. Nine isolates showed resistance to nalidixic acid and showed either intermediate resistance or susceptibility to other tested quinolones. All isolates resistant to one or more tested quinolones harbored mutations in gyrB and some also had gyrA and parC mutations. Of 54, 12 Proteeae isolates displayed the novel E466D, N440T, Q411S, and F417L mutations in gyrB . Among the PMQR genes, 41 (76%) isolates harbored the qnrD gene with the highest prevalence, whereas aac(6')Ib-cr , qnrS , qnrA , and qnrB genes were detected in 28 (52%), 9 (17.0%), 7 (13.0%), and 1 (1.9%) study isolates, respectively. The QRDR analysis of selected mutants revealed that increasing quinolone selective pressure led to a predominance of gyrA mutants. All results indicate that a healthy pet turtle can play as a potential reservoir for quinolone-resistant Proteeae, which it might cause public health risk on pet owners.

Upper respiratory tract disease (URTD) in North American tortoises (Gopherus) has been the focus of numerous laboratory and field investigations, yet the prevalence and importance of this disease remains unclear across many tortoise populations. Furthermore, much research has been focused on understanding diagnostic biomarkers of two known agents of URTD, Mycoplasma agassizii and Mycoplasma testudineum, yet the reliability and importance of these diagnostic biomarkers across populations is unclear. Gopher Tortoises (Gopherus polyphemus) have experienced significant declines and are currently protected range wide. Geographically, Alabama represents an important connection for Gopher Tortoise populations between the core and periphery of this species' distribution. Herein, we systematically sampled 197 Gopher Tortoises for URTD across seven sites in south-central and south-eastern Alabama. Plasma samples were assayed for antibodies to M. agassizii and M. testudineum; nasal lavage samples were assayed for the presence of viable pathogens as well as pathogen DNA. Lastly, animals were scored for the presence of external symptoms and nasal scarring consistent with URTD. External symptoms of URTD were present in G. polyphemus in all sites sampled in Alabama. There was no relationship between active symptoms of URTD and Mycoplasma antibodies, however the presence of URTD nasal scarring was positively related to M. agassizii antibodies (P = 0.032). For a single site that was sampled in three sequential years, seroprevalence to M. agassizii significantly varied among years (P < 0.0001). Mycoplasma agassizii DNA was isolated from four of the seven sites using quantitative PCR, yet none of the samples were culture positive for either of the pathogens. An analysis of disease status and condition indicated that there was a significant, positive relationship between the severity of URTD symptoms and relative body mass (P < 0.05). This study highlights the need for continued monitoring of disease in wild populations. Specifically, focus must be placed on identifying other likely pathogens and relevant biomarkers that may be important drivers of URTD in North American tortoises. Special consideration should be given to environmental contexts that may render wild populations more susceptible to disease., Competing Interests: The authors have declared that no competing interests exist.

The fungal order Onygenales includes many pathogens of humans and animals, and recent studies have shown some onygenalean fungi to be significant emerging pathogens of reptiles. Although many of these fungi have similar morphological features in histologic tissue sections, recent molecular analyses have revealed a genetically complex and diverse group of reptile pathogens comprising several genera, most notably Nannizziopsis , Ophidiomyces , and Paranannizziopsis Infections by members of these genera have been previously reported in a variety of reptile species, including crocodilians, lizards, snakes, and tuataras, with negative impacts on conservation efforts for some reptiles. Despite the well-documented pathogenicity of these fungi in all other extant reptile lineages, infection has not yet been reported in aquatic turtles. In this study, we report the isolation of an onygenalean fungus associated with shell lesions in freshwater aquatic turtles. The morphologic and genetic characteristics of multiple isolates ( n = 21) are described and illustrated. Based on these features and results of a multigene phylogenetic analysis, a new genus and species, Emydomyces testavorans , are proposed for these fungi isolated from turtle shell lesions., (Copyright © 2019 American Society for Microbiology.)

We investigated the prevalence of Salmonella in 227 small red-eared sliders (Trachemys scripta elegans) from 2006 to 2008. A total of 130 turtles (57.3%) tested positive for S. enterica subsp. enterica. Twenty-two serotypes including S. Montevideo, S. Newport, S. Pomona, S. Braenderup, S. Sandiego, and S. Litchfield were identified. Salmonella strains with closely related pulsed field gel electrophoresis (PFGE) patterns were isolated from several shops located in different areas from 2006 to 2008. Antimicrobial resistance was detected among strains of S. Montevideo, S. Newport, S. Braenderup, S. Sandiego, and S. Litchfield. The relatedness of antimicrobial resistance and PFGE profiles was not observed. The PFGE patterns of S. Poona strains isolated in 2006 and 2008 and the causative strains of turtle-associated salmonellosis in 2006 were identical. These results revealed a high prevalence of Salmonella enterica subsp. enterica in red-eared sliders retailed in Japan. In addition, genetically closely-related strains of turtle-associated Salmonella were repeatedly introduced into Japan over the study period and were distributed widely in Japan. These Salmonella strains present a risk of a widely disseminated outbreak of turtle-associated salmonellosis.

Competing Interests: All authors have completed and submitted the ICMJE form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Proteeae is a tribe which consists of three genera: Proteus, Providencia and Morganella. The objective of this study was to determine antimicrobial resistance profile, virulence genotype and class 1 and 2 integrons in Proteeae isolated from pet turtles and to determine the impact of antibiotic resistance on virulence and antimicrobial resistance genes. Integron-positive isolates were used to detect their gene cassette array. Sixty four Proteeae were isolated and all were resistant to macrolides (100%). Among 64 isolates 56, 52, 36 and 25 were resistant to nitrofurans, β-lactams, tetracycline and aminoglycoside respectively. Sixteen (25%) isolates were positive for intI1 while 14 (21·87%) were positive for integrase 2 (intI2). Eleven (17·18%) isolates were positive for class 1 variable region while 7 (10·93%) were positive for class 2 variable region. IMP27, a novel metallo β-lactamase gene was found in Providencia isolates. Proteus sp. were positive for every tested virulence genes and UreC gene was detected in 48·44% followed by zapA (17·19%), mrpA (17·19%) and hlyA (14·06%) genes. In this study, integron associated-antibiotic resistance genes have been identified in Proteeae isolates in a considerable range representing clear threats to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, multidrug-resistant Proteeae isolates had several antibiotic resistance genes. Integrons are important contributors to the development and dissemination of antibiotic resistance. We could detect both class 1 and 2 integrons and several gene cassette arrays in class 1 integron. The gene cassette arrays of the Class 2 integrons contained IMP27-dfrA1-aadA1-catB2-ybeA-ybgA in two isolates. To the best of our knowledge this is the first study to report detection of IMP27 in Providencia rettgeri isolates. All results indicate that healthy pet turtles act as potential reservoirs for Proteeae species with zoonotic potential., (© 2018 The Society for Applied Microbiology.)

Zoonotic transmission of Salmonella infections causes an estimated 11% of salmonellosis annually in the United States. This report describes the epidemiologic, traceback and laboratory investigations conducted in the United States as part of four multistate outbreaks of Salmonella infections linked to small turtles. Salmonella isolates indistinguishable from the outbreak strains were isolated from a total of 143 ill people in the United States, pet turtles, and pond water samples collected from turtle farm A, as well as ill people from Chile and Luxembourg. Almost half (45%) of infections occurred in children aged <5 years, underscoring the importance of the Centers for Disease Control and Prevention recommendation to keep pet turtles and other reptiles out of homes and childcare settings with young children. Although only 43% of the ill people who reported turtle exposure provided purchase information, most small turtles were purchased from flea markets or street vendors, which made it difficult to locate the vendor, trace the turtles to a farm of origin, provide education and enforce the United States federal ban on the sale and distribution of small turtles. These outbreaks highlight the importance of improving public awareness and education about the risk of Salmonella from small turtles not only in the United States but also worldwide., (© 2018 Blackwell Verlag GmbH.)

The aim of this study is to investigate the fungal diversity of green turtle nests and to examine phylogenetic relationships among these isolates. During the nesting season, samples of intra-nest sand and failed eggs were collected from 25% of the surviving nests in Sugözü Beaches, which are amongst the most important nesting beaches for endangered green turtles in the Mediterranean. Twenty-three fungi were identified by molecular techniques. Fungal isolates belonged to genera Aspergillus, Emericella, Rhizopus, Actinomucor and Apophysomyces with two undescribed species. Aspergillus variecolor, Aspergillus quadrilinieatus, Aspergillus tubingensis, Rhizopus oryzae, Actinomucor elegans and Apophysomyces variabilis were firstly detected in all sea turtle nests within this study. Our results demonstrate that 36.4% of the nests had fungal contamination. Also hatching success of the nests contaminated by fungi were significantly lower than the uncontaminated nests (P = 0.029). Also, this may represent a threat to marine turtles and a risk for the health of conservation workers. This study is the first molecular phylogenetic study associated with sea turtle nests in the eastern Mediterranean coast and contributes to the wider body of literature on fungal invasion of sea turtle nests with firstly isolated species. These findings are important for improving potential conservation measures for the nest sites.

Microsporidial spores were identified in the musculature of a loggerhead sea turtle Caretta caretta found dead on the shore in New Brunswick, Canada. Gastroenteritis was diagnosed on gross postmortem examination, with no gross abnormalities detected in the skeletal muscle. Histologically, the microsporidial spores were associated with inflammation and muscular necrosis and measured 1.1-1.7 × 2.2-3.4 µm. Spores were typically identified within sporophorous vesicles and, less often, in sporophorocysts and were weakly Gram positive, had punctate PAS staining, and were occasionally strongly acid-fast. Ultrastructural characteristics included 7-10 polar filament coils and other standard features of microsporidial spores. PCR for the microsporidial small subunit rRNA gene sequence was performed on DNA extracted from the muscle and small intestine, and the resulting amplicon was sequenced and queried against published microsporidial genomes. DNA sequences shared 98.2-99.8% sequence identity to Clade III of the Marinosporidia. This is the first report of a microsporidial infection contributing to the mortality of a sea turtle.

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

Mycoplasma spp. are known from several chelonian and other reptilian species. We determined if turtles obtained by the Turtle Rescue Team at North Carolina State University are carriers of hemotropic Mycoplasma or Bartonella spp. Spleen samples were collected at necropsy during May through July, 2014 from 53 turtles of seven species. All turtles were dead or were euthanized upon arrival due to severe traumatic injuries, or they died shortly after beginning treatment. We used PCR amplification for both bacterial genera; Bartonella spp. DNA was not amplified. Based upon sequencing of the 16S rRNA subunit, one eastern box turtle ( Terrapene carolina carolina) and one yellow-bellied slider ( Trachemys scripta scripta) were infected with Mycoplasma spp. that have genetic similarities to strains that infect other animals.

Serum from Kemp's ridley sea turtles Lepidochelys kempii and loggerhead sea turtles Caretta caretta was collected during summer in 2011, 2012, and 2013. Serum immunoglobulin Y (IgY) recognition of lysate proteins from nine bacterial species and whole bacterium-specific IgY titers to these pathogens were quantified. Serum and purified IgY recognized proteins of all bacteria, with protein recognition for some species being more pronounced than others. Circulating IgY titers against Vibrio vulnificus, V. anguillarum, Erysipelothrix rhusiopathiae, and Brevundimonas vesicularis changed over the years in Kemp's ridley sea turtles, while IgY titers against V. vulnificus, Escherichia coli, V. parahaemolyticus, B. vesicularis, and Mycobacterium marinum were different in loggerhead sea turtles. Serum lysozyme activity was constant for loggerhead sea turtles over the 3 years, while activity in Kemp's ridley sea turtles was lower in 2011 and 2012 than in 2013. Blood packed cell volume, glucose, and serum protein levels were comparable to those of healthy sea turtles in previous studies; therefore, this study provides baseline information on antibody responses in healthy wild sea turtles., (© 2017 American Fisheries Society.)

Objectives: Klebsiella spp. are regarded as major pathogens causing infections in humans and various animals. Here we report the draft genome sequence of a CTX-M-type β-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae strain CHKP0062 isolated from a Yellow-margined Box turtle., Methods: An Illumina-Solexa platform was used to sequence the genome of CHKP0062. Qualified reads were assembled de novo using Velvet. The draft genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The resistome and virulome of the strain were investigated., Results: A total of 5423 protein-coding sequences, 87 tRNAs, 24 rRNAs and 12 ncRNAs were identified in the 5 699 275-bp genome. CHKP0062 was assigned to sequence type ST2131 with the K-loci type as KL67. No virulence-associated genes were identified. However, numerous antimicrobial resistance genes were present in this strain. Plasmid contigs were assembled and revealed homology to the multidrug resistance plasmids pC15-K, pCTX-M3 and pKF3-94, with the carriage of the class A β-lactamase genes bla TEM-1b and bla CTX-M-3 ., Conclusion: The genome sequence reported in this study will be useful for comparative genomic analysis regarding the dissemination of clinically important antibiotic resistance genes among Klebsiella spp. isolated from humans and animals., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Escherichia coli pathotypes (i.e., enteropathogenic and enterotoxigenic) have been identified among the pathogens most responsible for moderate-to-severe diarrhea in low- and middle-income countries (LMICs). Pathogenic E. coli are transmitted from infected human or animal feces to new susceptible hosts via environmental reservoirs such as hands, water, and soil. Commensal E. coli , which includes nonpathogenic E. coli strains, are widely used as fecal bacteria indicator, with their presence associated with increased likelihood of enteric pathogens and/or diarrheal disease. In this study, we investigated E. coli contamination in environmental reservoirs within households ( N = 142) in high-population density communities of Harare, Zimbabwe. We further assessed the interconnectedness of the environmental compartments by investigating associations between, and household-level risk factors for, E. coli contamination. From the data we collected, the source and risk factors for E. coli contamination are not readily apparent. One notable exception is the presence of running tap water on the household plot, which is associated with significantly less E. coli contamination of drinking water, handwashing water, and hands after handwashing. In addition, E. coli levels on hands after washing are significantly associated with handwashing water contamination, hand contamination before washing, and diarrhea incidence. Finally, we observed that animal ownership increases E. coli contamination in soil, and E. coli in soil are correlated with contamination on hands before washing. This study highlights the complexity of E. coli contamination in household environments within LMICs. More, larger, studies are needed to better identify sources and exposure pathways of E. coli -and enteric pathogens generally-to identify effective interventions.

Stranded green turtles (Chelonia mydas) are often cared for in rehabilitation centers until they recover. Although the specific causal agents of diseases in stranded turtles are difficult to diagnose, we know that gut microbiota of green turtles play a vital role in health as well as a wide range of diseases. The objective of this study was to characterize and compare the gut bacterial communities between pre-hospitalization (PH) and post-rehabilitation (PR) stranded green turtles using high-throughput sequencing analysis targeting V1-V3 regions of the bacterial 16S rRNA gene. A total of eight cloacal swab samples were collected from four green turtles undergoing rehabilitation. Proteobacteria dominating in both PH and PR samples without any significant difference. Firmicutes was the second and Bacteroidetes was the third most abundant phylum in PH samples, while Bacteroidetes prevailed in PR samples, followed by Firmicutes. The predominance of the genus Bacteroides in both PH and PR samples indicates the importance of this genus in turtle gut health. At a class level, Epsilonproteobacteria was significantly (P<0.05) associated with PH samples and Deltaproteobacteria predominated (P<0.05) in PR samples. The significant abundance of Campylobacter fetus, Escherichia coli, Clostridium botulinum and Vibrio parahaemolyticus in PH samples indicate pathogenic associations with stranded green turtles with zoonotic potential. The presence of Salmonella enterica in only PR samples suggest possible acquisition of this bacteria during rehabilitation. In this study, all post-rehabilitation green turtles exhibited similar bacterial communities, irrespective of their microbial compositions at pre-hospitalization. The marked differences in the gut bacterial communities of PH and PR turtles indicate the outcome of dietary, management and environmental shift during rehabilitation. Therefore, it is important to address the process of restoring normal gut microbiota of recovered turtles prior to release back to their natural habitat., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The prevalence of Salmonella in chelonians is not known in the UK and it is not clear whether such Salmonella strains would be pathogenic for human beings. Some strains, such as members of the Arizonae subgroup, may be unable to cause anything more than very mild disease. To determine the carriage of Salmonella in pet tortoises, cloacal swabs were taken for culture. Salmonella enterica Group D was isolated from 5 of the 89 samples. All five were from the same household of seven tortoises. Salmonella isolates were shown by PCR to carry the invA and spiC genes associated with pathogenicity islands 1 and 2. Each isolate carried both genes indicating they had the genetic basis for disease and enterocyte invasion in human beings. The study indicates a low rate of asymptomatic carriage among the general population of pet tortoises. However, it does suggest that those Salmonella strains colonising the tortoise can carry Salmonellap athogenicity island (SPI)-1 and SPI-2 conferring the potential to cause disease in human beings and other animals., Competing Interests: Competing interests: None declared., (© British Veterinary Association (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

In Hawaii, glyphosate-based herbicides frequently sprayed near shorelines may be affecting non-target marine species. Glyphosate inhibits aromatic amino acid biosynthesis (shikimate pathway), and is toxic to beneficial gut bacteria in cattle and chickens. Effects of glyphosate on gut bacteria in marine herbivorous turtles were assessed in vitro. When cultures of mixed bacterial communities from gastrointestinal tracts of freshly euthanized green turtles (Chelonia mydas), were exposed for 24h to six glyphosate concentrations (plus deionized water control), bacterial density was significantly lower at glyphosate concentrations≥2.2×10 -4 gL -1 (absorbance measured at 600nm wavelength). Using a modified Kirby-Bauer disk diffusion assay, the growth of four bacterial isolates (Pantoea, Proteus, Shigella, and Staphylococcus) was significantly inhibited by glyphosate concentrations≥1.76×10 -3 gL -1 . Reduced growth or lower survival of gut bacteria in green turtles exposed to glyphosate could have adverse effects on turtle digestion and overall health., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An outbreak of Salmonella Muenchen gastroenteritis occurred in a remote coastal Aboriginal community in the Northern Territory (NT) of Australia. There were 22 people sick (attack rate 55%); 7 had laboratory confirmed S. Muenchen infection; 2 required medical evacuation and admission to the intensive care unit (ICU). We conducted a descriptive case series to investigate the outbreak. All cases ate meat from a single green turtle (Chelonia mydas). The animal's pre-death stress, improper butchering, insufficient cooking and the unsatisfactory storage of meat all likely contributed to the outbreak. Turtle meat should be prepared safely, cooked thoroughly and stored appropriately to avoid Salmonella infection., (This work is copyright. You may download, display, print and reproduce the whole or part of this work in unaltered form for your own personal use or, if you are part of an organisation, for internal use within your organisation, but only if you or your organisation do not use the reproduction for any commercial purpose and retain this copyright notice and all disclaimer notices as part of that reproduction. Apart from rights to use as permitted by the Copyright Act 1968 or allowed by this copyright notice, all other rights are reserved and you are not allowed to reproduce the whole or any part of this work in any way (electronic or otherwise) without first being given the specific written permission from the Commonwealth to do so. Requests and inquiries concerning reproduction and rights are to be sent to the Online, Services and External Relations Branch, Department of Health, GPO Box 9848, Canberra ACT 2601, or by email to copyright@health.gov.au.)

Objectives: The aim of this study was to characterise Aeromonas spp. isolated from popular species of pet turtle to assess the potential risk of pet turtles as a source of target gene alterations in the quinolone resistance-determining region (QRDR) and transferable plasmid-mediated quinolone resistance (PMQR) genes., Methods: Twenty-five isolates comprising four species, namely Aeromonas enteropelogenes, Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii, were obtained from healthy pet turtles. The antimicrobial susceptibility of the isolates to nalidixic acid, ciprofloxacin and ofloxacin was examined by minimum inhibitory concentration (MIC) determination. QRDR substitutions and PMQR genes were detected using conventional PCR assays and sequencing., Results: Although more than one-half of the isolates were resistant to nalidixic acid (14/25; 56%), most were susceptible to ciprofloxacin and ofloxacin. In QRDR substitution analysis, gyrA Ser-83→Ile substitution was predominant among A. enteropelogenes isolates, whilst two isolates of A. caviae displayed a novel Asp-95→Pro substitution. With regard to parC, Ser-80→Ile substitution was noted in all species except A. veronii. Furthermore, qnrS, qnrB and aac(6')-Ib-cr genes were detected in 68% (17/25), 8% (2/25) and 8% (2/25) of the isolates, respectively; 86% (12/14) of A. enteropelogenes isolates harboured a qnrS gene. Unexpectedly, quinolone resistance determinants were also detected in some isolates that were phenotypically susceptible to the tested quinolones., Conclusions: The current study reveals the mismatch phenomenon between quinolone resistance phenotype and genotype of turtle-borne aeromonads and suggests that susceptible isolates might be a potential risk source for storage and transmission of resistance genes., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Green turtles (Chelonia mydas) are endangered marine herbivores that break down food particles, primarily sea grasses, through microbial fermentation. However, the microbial community and its role in health and disease is still largely unexplored. In this study, we investigated and compared the fecal bacterial communities of eight wild-captured green turtles to four stranded turtles in the central Great Barrier Reef regions that include Bowen and Townsville. We used high-throughput sequencing analysis targeting the hypervariable V1-V3 regions of the bacterial 16S rRNA gene. At the phylum level, Firmicutes predominated among wild-captured green turtles, followed by Bacteroidetes and Proteobacteria. In contrast, Proteobacteria (Gammaproteobacteria) was the most significantly dominant phylum among all stranded turtles, followed by Bacteroidetes and Firmicutes. In addition, Fusobacteria was also significantly abundant in stranded turtles. No significant differences were found between the wild-captured turtles in Bowen and Townsville. At the family level, the core bacterial community consisted of 25 families that were identified in both the wild-captured and stranded green turtles, while two unique sets of 14 families each were only found in stranded or wild-captured turtles. The predominance of Bacteroides in all groups indicates the importance of these bacteria in turtle gut health. In terms of bacterial diversity and richness, wild-captured green turtles showed a higher bacterial diversity and richness compared with stranded turtles. The marked differences in the bacterial communities between wild-captured and stranded turtles suggest the possible dysbiosis in stranded turtles in addition to potential causal agents., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ticks are among the most significant human-biting ectoparasites and they play a major role in transmission of many pathogenic agents to humans. In the present study, three species of Hyalomma ticks, Hyalomma aegyptium, H. marginatum and H. excavatum, were examined for the presence of zoonotic bacteria, both male and female ticks alike. Examination of microbial diversity with tag-encoded pyrosequencing indicates that H. marginatum and H. excavatum were more diversity rich than H. aegyptium. Although numerous pathogenic and non-pathogenic bacterial genera were detected, including Acidovorax, Bacillus, Bacteroides, Bdellovibrio, Clostridium, Curvibacter, Escherichia, Flavobacterium, Limnohabitans, Paenibacillus, Ralstonia, Sarcina, Sediminibacterium, Segetibacter Stenotrophomonas and Variovorax, the predominant zoonotic bacteria represented in these ticks were genera Borrelia, Francisella, and Rickettsia. To the authors' knowledge, this work represents the first detection of Yersinia enterocolitica in the tick H. excavatum, raising questions regarding the vector competency of this tick, as well as associations of different disease representations perhaps through previously unforeseen routes of pathogen introduction. Likewise, similar questions are related to the presence of Legionella pneumophila in one H. excavatum sample.

Conservation efforts are investigating the impact of diseases within a species of interest, including prevalence and transmission and morbidity and mortality rates. However, the majority of these studies focus solely on the characteristics of a single pathogen. Recently, the role of copathogens has been reported to impact disease susceptibility and mortality. To that effect, a survey was conducted including 318 eastern box turtles ( Terrapene carolina carolina) from populations in Illinois and Tennessee in 2014 and 2015. Blood samples and oral swabs were collected for quantitative polymerase chain reaction (qPCR) of 15 different pathogens performed in a multiplex format using Fluidigm array technology. Four pathogens were found with varying qPCR prevalence: ranavirus (FV3; n = 2, 0.6%), Terrapene herpesvirus 1 (TerHV1; n = 129, 40.7%), box turtle Mycoplasma sp. (BT Myco; n = 14, 4.6%), and box turtle adenovirus (BT Adv1; n = 18, 11%). Thirteen pathogens were not identified in any sample, including Mycoplasma agassizii, M. testudineum, Salmonella enteriditis, S. typhmirium, Borrelia burgdorferi, Anaplasma phagocyophilum, tortoise intranuclear coccidia, Ambystoma tigrinum virus, Bohle iridovirus, Epizootic hematopoietic necrosis virus, and testudinid herpesvirus 2. Copathogen occurrence was rare but was observed in eight individuals with TerHV1-BT Myco detection and two animals with TerHV1-Adv1. Significant differences were observed in pathogen detection across season (TerHV1, BT Adv1, BT Myco, and TerHV1-Myco) and year (TerHV1, BT Adv1, and TerHV1-Myco). The results of this survey highlight that a single pathogen model may not adequately explain pathogen dynamics and that conservation efforts need to be aimed at detecting multiple pathogens in order to fully characterize population health.

We tested samples from 1,015 chelonians in Europe for Mycoplasma spp., herpesviruses, ranaviruses, picornaviruses, and ferlaviruses by PCR. Mycoplasma spp. were detected in 42.1% and herpesviruses were detected in 8.0% of tested chelonians. Differentiation of the herpesviruses revealed that 46.9% of the detected chelonian viruses were testudinid herpesvirus 1 (TeHV-1) and 54.3% were TeHV-3, including co-detections of TeHV-1 and -3 in 3 tortoises. TeHV-4 was detected in a leopard tortoise ( Stigmochelys pardalis), and a herpesvirus that could not be further characterized was found in a pond slider ( Trachemys scripta). Picornaviruses (topiviruses) were detected in 2.2% of the tested animals; ferlaviruses were found in 0.6%; no ranaviruses were detected in any of the animals tested. Mycoplasma spp. were detected significantly more often in Horsfield's tortoises ( Testudo horsfieldii), leopard tortoises, and Indian star tortoises ( Geochelone elegans) than in other species. Horsfield's tortoises were also significantly more often positive for TeHV-1. Mycoplasma and TeHV-1 were co-detected in 3.0%, and mycoplasma and TeHV-3 in 2.3%. The TeHV-4-positive tortoise was also positive for mycoplasma. Mycoplasma and picornaviruses were co-detected in 1.2% of the tortoises. A spur-thighed tortoise ( Testudo graeca) was positive for mycoplasma and a ferlavirus. In some cases, >2 pathogens were detected. A significant correlation between mycoplasma and herpesvirus detection was found. Of all tested animals, 47.6% were positive for at least one pathogen, demonstrating the importance of pathogen detection in captive chelonians.