Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Chapman PA

PROVIDER: S-EPMC5007010 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

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

Chapman Phoebe A PA Traub Rebecca J RJ Kyaw-Tanner Myat T MT Owen Helen H Flint Mark M Cribb Thomas H TH Mills Paul C PC

PloS one 20160831 8

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 genu ...[more]

PMID: 27579605

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Project description:After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrestAfter gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest, RNA-sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 hours (h) after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with up-regulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly down-regulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including insulin-like growth factor binding protein 1, were down-regulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported by others to be associated with hypoxia-induced embryonic arrest in diverse taxa, including budding yeast (Saccharomyces cerevisiae), the annelid Caenorhabditis elegans, and zebrafish (Danio rerio). Thus, while among oviparous species, hypoxia-induced pre-ovipositional embryonic arrest appears to be a unique adaptation of turtles, mechanisms underlying hypoxia-induced embryonic arrest per se may be highly conserved across the diverse taxa in which this mechanism operates. We report the transciptional changes in Chelonia mydas embryos incubated in either normoxia or hypoxia comapred to freshly laid eggs.

Dataset Information

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

Publications

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

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