Development of a Quantitative PCR for Rapid and Sensitive Detection of an Intranuclear Coccidian Parasite of Tortoises (TINC) and Identification of TINC in the Critically Endangered Arakan Forest Turtle (Heosemys depressa)
W. Alexander Alvarez1, DVM; Paul M. Gibbons2, DVM, MS, DABVP (Avian); Sam Rivera3, DVM, MS, DABVP (Avian); Linda L. Archer1, BS, April L. Childress1; James F.X. Wellehan Jr. 1, DVM, MS, PhD, DACZM, DACVM (Virology, Bacteriology/Mycology)
1Marine Animal Disease Laboratory, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA, 2 Behler Chelonian Center, Ojai, CA, USA, 3Zoo Atlanta, Atlanta, GA, USA
The tortoise intranuclear coccidian parasite (TINC) was first reported in radiated tortoises, Geochelone (Astrochelys) radiata, presenting with severe anorexia and lethargy.2 It has since proven to be a significant cause of disease of tortoises causing high mortality and affecting several threatened chelonian species.1 Diagnostic testing has been limited to relatively labor intensive and expensive pan-coccidial PCR and sequencing techniques with a long turnaround time. This report describes the development a quantitative PCR (real-time or qPCR) that provides a rapid, analytically specific, and economical detection of TINC. A qPCR probe targeting a specific and conserved region of TINC 18S rRNA was designed. The qPCR reaction was run on samples known to be TINC positive and the results were consistent and analytically specific. The assay was able to detect as little as 10 copies of target DNA in a sample. The development of this assay enables studies optimizing diagnostic sampling, describing geographic disease prevalence, and investigating life cycles. Testing of soil and invertebrates from enclosures of positive animals was negative and did not provide any further insights into the life cycle of the parasite. This assay was used to identify TINC in a novel host species, the critically endangered Arakan forest turtle (Heosemys depressa).
This work was funded by a 2011 ARAV Research & Conservation Grant from the Association of Reptilian & Amphibian Veterinarians.
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