Characterisation and automated quantification of induced seizure-related behaviours in Xenopus laevis tadpoles.

Epilepsy, a clinical diagnosis characterised by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient-specific treatment is vital and can be achieved by the development of rapid pre-clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re-purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure-related behaviours in X. laevis tadpoles arrayed in 24-well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4-aminopyridine (4-AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti-seizure drug valproate (VPA). Four induced seizure-like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4-AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies since X. laevis is a well-characterised and genetically tractable model organism.

Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles via lipopolysaccharide signalling.

Xenopus laevis tadpoles can regenerate functional tails, containing the spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around 1 week of age. At this stage, amputation of the tadpole's tail may either result in scarless wound healing or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six Gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli, could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

COCCIDIOSIS IN GREEN TURTLES (CHELONIA MYDAS) IN AUSTRALIA: PATHOGENESIS, SPATIAL AND TEMPORAL DISTRIBUTION, AND CLIMATE-RELATED DETERMINANTS OF DISEASE OUTBREAKS.

An epizootic of coccidiosis in free-ranging green turtles (Chelonia mydas) occurred in Australia in 1991 and the parasites were thought to be Caryospora cheloniae. Recurring outbreaks over an increased geographic range followed. We used medical records and temporal and spatial data of turtles diagnosed with coccidiosis between 1991 and 2014 to characterize the disease and factors associated with outbreaks. Most affected animals were subadults or older. Neurologic signs with intralesional cerebral coccidia were observed. Coccidia associated with inflammation and necrosis were predominantly found in the intestine, brain, kidney, and thyroid. Cases occurred in the spring and summer. Three major outbreaks (1991, 2002, and 2014) were concentrated in Port Stephens, New South Wales (NSW) and Moreton Bay, Queensland, but cases occurred as far south as Sydney, NSW. Coccidiosis cases were more likely during, or 1 mo prior to, El Niño-like events. Molecular characterization of the 18S rRNA locus of coccidia from tissues of 10 green turtles collected in 2002 and 2004 in Port Stevens and Sydney imply that they were Schellackia-like organisms. Two genotypes were identified. The Genotype 3 sequence was most common (in eight of 10 turtles), with 98.8% similarity to the 18S sequence of Schellackia orientalis. The Genotype 4 sequence was less common (in two of 10 turtles) with 99.7% similarity to the 18S sequence of the most common genotype (Genotype 1) detected in turtles from the 2014 Moreton Bay outbreak. Our study will help with the identification and management of future outbreaks and provide tools for identification of additional disease patterns in green turtles.

Caryospora-Like Coccidia Infecting Green Turtles (Chelonia mydas): An Emerging Disease With Evidence of Interoceanic Dissemination.

Protozoa morphologically consistent with Caryospora sp. are one of the few pathogens associated with episodic mass mortality events involving free-ranging sea turtles. Parasitism of green turtles (Chelonia mydas) by these coccidia and associated mortality was first reported in maricultured turtles in the Caribbean during the 1970s. Years later, epizootics affecting wild green turtles in Australia occurred in 1991 and 2014. The first clinical cases of Caryospora-like infections reported elsewhere in free-ranging turtles were from the southeastern US in 2012. Following these initial individual cases in this region, we documented an epizootic and mass mortality of green turtles along the Atlantic coast of southern Florida from November 2014 through April 2015 and continued to detect additional, sporadic cases in the southeastern US in subsequent years. No cases of coccidial disease were recorded in the southeastern US prior to 2012 despite clinical evaluation and necropsy of stranded sea turtles in this region since the 1980s, suggesting that the frequency of clinical coccidiosis has increased here. Moreover, we also recorded the first stranding associated with infection by a Caryospora-like organism in Hawai'i in 2018. To further characterize the coccidia, we sequenced part of the 18S ribosomal and mitochondrial cytochrome oxidase I genes of coccidia collected from 62 green turtles found in the southeastern US and from one green turtle found in Hawai'i. We also sequenced the ribosomal internal transcribed spacer regions from selected cases and compared all results with those obtained from Caryospora-like coccidia collected from green turtles found in Australia. Eight distinct genotypes were represented in green turtles from the southeastern US. One genotype predominated and was identical to that of coccidia collected from the green turtle found in Hawai'i. We also found a coccidian genotype in green turtles from Florida and Australia with identical 18S and mitochondrial sequences, and only slight inter-regional differences in the internal transcribed spacer 2. We found no evidence of geographical structuring based on phylogenetic analysis. Low genetic variability among the coccidia found in green turtle populations with minimal natural connectivity suggests recent interoceanic dissemination of these parasites, which could pose a risk to sea turtle populations.

Spirorchiidiasis in marine turtles: the current state of knowledge.

Blood flukes of the family Spirorchiidae are important disease agents in marine turtles. The family is near cosmopolitan in distribution. Twenty-nine marine species across 10 genera are currently recognized, but taxonomic problems remain and it is likely that more species will be discovered. Spirorchiids infect the circulatory system, where they and their eggs cause a range of inflammatory lesions. Infection is sometimes implicated in the death of the turtle. In some regions, prevalence in stranded turtles is close to 100%. Knowledge of life cycles, important for control and epidemiological studies, has proven elusive until recently, when the first intermediate host identifications were made. Recent molecular studies of eggs and adult worms indicate that a considerable level of intrageneric and intraspecific diversity exists. The characterization of this diversity is likely to be of importance in exploring parasite taxonomy and ecology, unravelling life cycles, identifying the differential pathogenicity of genotypes and species, and developing antemortem diagnostic tools, all of which are major priorities for future spirorchiid research. Diagnosis to date has been reliant on copromicroscopy or necropsy, which both have significant limitations. The current lack of reliable antemortem diagnostic options is a roadblock to determining the true prevalence and epidemiology of spirorchiidiasis and the development of effective treatment regimes.

Evidence of Diversity, Site, and Host Specificity of Sea Turtle Blood Flukes (Digenea: Schistosomatoidea: "Spirorchiidae"): A Molecular Prospecting Study.

Neospirorchis (Digenea: "Spirorchiidae") are blood flukes of sea turtles. Trematodes tentatively identified as Neospirorchis sp. infect various sites within sea turtles inhabiting waters of the southeastern United States, but efforts to obtain specimens adequate for morphologic study has proven difficult. Two genetic targets, the internal transcribed spacer region of the ribosomal RNA gene and the partial mitochondrial cytochrome c oxidase subunit I gene, were used to investigate potential diversity among parasite specimens collected from stranded sea turtles. Sequence data were obtained from 215 trematode and egg specimens collected from 92 individual free-ranging cheloniid sea turtles comprising 4 host species. Molecular analysis yielded more than 20 different genotypes. We were able to assign 1 genotype to 1 of the 2 recognized species, Neospirorchis pricei Manter and Larson, 1950 . In many examples, genotypes exhibited host and site specificity. Our findings indicate considerable diversity of parasites resembling Neospirorchis with evidence of a number of uncharacterized blood flukes that require additional study.

Molecular epidemiology and pathology of spirorchiid infection in green sea turtles (Chelonia mydas).

Spirorchiid blood fluke infections affect endangered turtle populations globally, and are reported as a common cause of mortality in Queensland green sea turtles. Both the flukes and their ova are pathogenic and can contribute to the stranding or death of their host. Of particular interest are ova-associated brain lesions, which have been associated with host neurological deficits. Accurate estimations of disease frequency and the relative effect of infection relating to different spirorchiid species are made difficult by challenges in morphological identification of adults of some genera, and a lack of species-level identifying features for ova. A new specifically designed molecular assay was used to detect and identify cryptic spirorchiids and their ova in Queensland green sea turtle tissues collected from 2011 to 2014 in order to investigate epidemiology, tissue tropisms and pathology. Eight spirorchiid genotypes were detected in 14 distinct tissues, including multiple tissues for each. We found no evidence of a characteristic pathway of the eggs to the exterior; instead the results suggest that a high proportion of eggs become lost in dead-end tissues. The most common lesions observed were granulomas affecting most organs with varying severity, followed by arteritis and thrombi in the great vessels. The number of spirorchiid types detected increased with the presence and severity of granulomatous lesions. However, compared with other organs the brain showed relatively low levels of spirorchiid diversity. An inverse relationship between host age and spirorchiid diversity was evident for the liver and kidneys, but no such relationship was evident for other organs. Molecular data in this study, the first of its kind, provides the first species-level examination of spirorchiid ova and associated pathology, and paves the way for the future development of targeted ante-mortem diagnosis of spirorchiidiasis.

Elucidation of the first definitively identified life cycle for a marine turtle blood fluke (Trematoda: Spirorchiidae) enables informed control.

Blood flukes of the family Spirorchiidae are significant pathogens of both free-ranging and captive marine turtles. Despite a significant proportion of marine turtle mortality being attributable to spirorchiid infections, details of their life cycles remain almost entirely unknown. Here we report on the molecular elucidation of the complete life cycle of a marine spirorchiid, identified as Amphiorchis sp., infecting vermetid gastropods and captive hatched neonate Caretta caretta in the Oceanogràfic Aquarium, in Valencia, Spain. Specimens of a vermetid gastropod, Thylaeodus cf. rugulosus (Monterosato, 1878), collected from the aquarium filtration system housing diseased C. caretta, were infected with sporocysts and cercariae consistent with the family Spirorchiidae. We generated rDNA sequence data [internal transcribed spacer 2 (ITS2) and partial 28S rDNA] from infections from the vermetid which were identical to sequences generated from eggs from the serosa of the intestine of neonate C. caretta, and an adult spirorchiid from the liver of a C. caretta from Florida, USA. Given the reliability of these markers in the delineation of trematode species, we consider all three stages to represent the same species and tentatively identify it as a species of Amphiorchis Price, 1934. The source of infection at the Oceanogràfic Foundation Rehabilitation Centre, Valencia, Spain, is inferred to be an adult C. caretta from the western Mediterranean being rehabilitated in the same facility. Phylogenetic analysis suggests that this Amphiorchis sp. is closely related to other spirorchiids of marine turtles (species of Carettacola Manter & Larson, 1950, Hapalotrema Looss, 1899 and Learedius Price, 1934). We discuss implications of the present findings for the control of spirorchiidiasis in captivity, for the better understanding of epidemiology in wild individuals, and the elucidation of further life cycles.

Terminal Restriction Fragment Length Polymorphism for the Identification of Spirorchiid Ova in Tissues from the Green Sea Turtle, Chelonia mydas.

Blood flukes are among the most common disease causing pathogens infecting vertebrates, including humans and some of the world's most globally endangered fauna. Spirorchiid blood flukes are parasites of marine turtles, and are associated with pathology, strandings and mortalities worldwide. Their ova embolize in tissues and incite significant inflammatory responses, however attempts to draw correlations between species and lesions are frustrated by difficulties in identifying ova beyond the genus level. In this study, a newly developed terminal restriction fragment length polymorphism (T-RFLP) method was validated as a tool for differentiating between mixed spirorchiid ova in turtle tissue. Initially, a multiplex PCR was used to differentiate between the five genera of spirorchiid flukes. Following this, PCR was performed using genus/genera-specific fluorescently tagged primer pairs and PCR products digested analysis using restriction endonucleases. Using capillary electrophoresis, this T-RFLP method could differentiate between twelve species and genotypes of spirorchiid flukes in turtles. It was applied to 151 tissue samples and successfully identified the spirorchiid species present. It was found to be more sensitive than visual diagnosis, detecting infections in 28 of 32 tissues that were negative on histology. Spirorchiids were present in 96.7% of tissues tested, with Neospirorchis genotype 2 being the most prevalent, present in 93% of samples. Mixed infections were common, being present in 60.7% of samples tested. The method described here is, to our knowledge, the first use of the T-RFLP technique on host tissues or in an animal ecology context, and describes a significant advancement in the clinical capacity to diagnose a common cause of illness in our environment. It is proven as a sensitive, specific and cost-efficient means of identifying spirorchiid flukes and ova in turtles, with the potential to contribute valuable information to epidemiological and pathological studies as well as future diagnostics for this poorly understood disease.

Ultrastructural and Molecular Characterisation of an Heterosporis-Like Microsporidian in Australian Sea Snakes (Hydrophiinae).

Four sea snakes (two Hydrophis major, one Hydrophis platurus, one Hydrophis elegans) were found washed ashore on different beaches in the Sunshine Coast region and Fraser Island in Queensland, Australia between 2007-2013. Each snake had multiple granulomas and locally extensive regions of pallor evident in the hypaxial and intercostal musculature along the body. Lesions in two individuals were also associated with vertebral and rib fractures. Histological examination revealed granulomas scattered throughout skeletal muscle, subcutaneous adipose tissue and fractured bone. These were composed of dense aggregates of microsporidian spores surrounded by a mantle of macrophages. Sequences (ssrRNA) were obtained from lesions in three sea snakes and all revealed 99% similarity with Heterosporis anguillarum from the Japanese eel (Anguillarum japonica). However, ultrastructural characteristics of the organism were not consistent with those of previous descriptions. Electron microscopic examination of skeletal muscle revealed large cysts (not xenomas) bound by walls of fibrillar material (Heterosporis-like sporophorocyst walls were not detected). The cysts contained numerous mature microsporidian spores arranged in small clusters, sometimes apparently within sporophorous vesicles. The microspores were monomorphic, oval and measured 2.5-3.0 µm by 1.6-1.8 µm. They contained isofilar polar filaments with 11 (infrequently 9-12) coils arranged in two ranks. This is the first published report of a microsporidian infection in hydrophiid sea snakes. This discovery shows microsporidia with molecular affinities to Heterosporis anguillarum but ultrastructural characters most consistent with the genus Pleistophora (but no hitherto described species). Further studies are required to determine whether the microsporidian presented here belongs to the genus Heterosporis, or to a polymorphic species group as suggested by the recognition of a robust Pleistophora/Heterosporis clade by molecular studies. The gross and histological pathology associated with these infections are described.

Molecular Characterization of Coccidia Associated with an Epizootic in Green Sea Turtles (Chelonia mydas) in South East Queensland, Australia.

In the spring of 2014, mass mortalities among wild green sea turtles occurred off the coast of south-east Queensland, Australia. The suspected causative agent was Caryospora cheloniae, an eimeriid coccidian implicated in previous epizootics. Necropsies were undertaken on a subset of 11 dead turtles, with subsequent histopathology and molecular analyses. All turtles returned positive PCR results for coccidial infection in various tissues; these included the brain, gastrointestinal tract, lung, kidney and thyroid. Granulomatous encephalitis was consistently observed, as well as enteritis and, less frequently, thyroiditis and nephritis. Sequencing and phylogenetic analyses indicated the presence of two distinct coccidian genotypes, presumably separate species-one associated with the brain, gastrointestinal tract and lung, and the second with the thyroid and kidney. Maximum likelihood and Bayesian inference analyses placed the first genotype closest to the lankesterellid genus Schellackia, rather than in the Eimeriidae, while the second was paraphyletic to the eimeriids. Presence of coccidial stages in extra-intestinal tissues of the primary host raises questions about the potential presence of intermediate or paratenic hosts within the life cycles, as well as their current placement relative to the genus Caryospora. This study represents the first genetic characterization of this emerging disease agent in green sea turtles, an endangered species, and has relevance for life-cycle elucidation and future development of diagnostics.

Molecular analysis of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 (Digenea: Spirorchiidae) reveals potential cryptic species, with comments on the validity of the genus Learedius.

Adult blood flukes of the genera Hapalotrema Looss, 1899 and Learedius Price, 1934 were collected from turtles off Queensland and the Hawaiian Islands. Specimens were identified as Hapalotrema pambanensis Mehrotra, 1973, H. synorchis Luhman, 1935, H. postorchis Rao, 1976 and Learedius learedi Price, 1934 on the basis of morphology. No major morphological differences were found between specimens from this study and previously published descriptions. DNA was also extracted and sequences obtained using custom spirorchiid-specific primers for the ITS2 and 28S rDNA regions, in order to confirm species identification and investigate phylogenetic relationships. Intraspecific genetic variation was generally low. However the ITS2 region of H. postorchis and to a lesser extent that of L. learedi showed considerable variation between specimens from the Pacific and Atlantic oceans. Further studies will be required to determine whether this variation should be considered inter- or intra-specific. Maximum likelihood phylogenetic analyses were completed for both sequenced genes. Learedius learedi was unequivocally nested among species of Hapalotrema, suggesting that the status of the genus Learedius may need to be reassessed.