title: 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.
authors: Veterinary-Marine Animal Research, Teaching and Investigation Unit, School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia.
authors: Phoebe A
authors: PA
authors: Chapman
authors: Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, Australia.
authors: Rebecca J
authors: RJ
authors: Traub
authors: Veterinary-Marine Animal Research, Teaching and Investigation Unit, School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia.
authors: Myat T
authors: MT
authors: Kyaw-Tanner
authors: Veterinary-Marine Animal Research, Teaching and Investigation Unit, School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia.
authors: Helen
authors: H
authors: Owen
authors: Veterinary-Marine Animal Research, Teaching and Investigation Unit, School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia.|School of Forest Resources and Conservation, University of Florida, The Florida Aquarium's Center for Conservation, Apollo Beach, Florida, United States of America.
authors: Mark
authors: M
authors: Flint
authors: School of Biological Science, The University of Queensland, St Lucia, Queensland, Australia.
authors: Thomas H
authors: TH
authors: Cribb
authors: Veterinary-Marine Animal Research, Teaching and Investigation Unit, School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia.
authors: Paul C
authors: PC
authors: Mills
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012150:Polymorphism, Restriction Fragment Length; D014200:Trematoda / Q000145:classification* / Q000235:genetics; D014201:Trematode Infections / Q000175:diagnosis* / Q000235:genetics / Q000662:veterinary; D014426:Turtles / Q000235:genetics / Q000469:parasitology*
chemical_list: D016054:DNA, Protozoan