DETECTION OF RANAVIRUS USING BONE MARROW HARVESTED FROM MORTALITY EVENTS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA)

Abstract

The causes of free-living chelonian mortality events are often unknown because of infrequent recovery of remains and rapid postmortem decomposition. This study describes a technique to harvest bone marrow and detect frog virus 3-like ranavirus (FV3) using quantitative polymerase chain reaction in skeletonized eastern box turtles ( Terrapene carolina carolina) ( N = 87), and assesses agreement with concurrent perimortem samples ( N = 14). FV3 was detected in bone marrow samples from 12 turtle shells (14%). Three of 14 turtles had detectable FV3 loads in both bone marrow and perimortem samples, two turtles had detectable FV3 in bone marrow only, and nine turtles tested FV3 negative in both bone marrow and concurrent perimortem samples. There was substantial agreement between FV3 testing of bone marrow and other tissues ( κ = 0.658). Harvesting bone marrow from shells is easily performed and can serve as a means for biologists and wildlife veterinarians to improve postmortem surveillance for systemically distributed pathogens, including FV3.

Investigation of multiple mortality events in eastern box turtles (Terrapene carolina carolina)

“…Remains were completely skeletonized, precluding a definitive diagnosis. However, FV3 was not detected in banked shells, lowering the likelihood of one known cause of mass mortality [ 37 ]. Almost half of the live turtles were diagnosed with a necrotizing bacterial infection (NBI) based on histopathology (N = 2), consistent culture isolates (N = 4), and compatible clinical signs (N = 5).…”

“…DNA was isolated from oral swabs, whole blood, virus isolation flasks, bone marrow, and amphibian tissue samples using a commercially available kit (QIAmp DNA Blood Mini Kit and DNAeasy kit, Qiagen, Valencia, CA, USA). Manufacturer instructions were followed with some alterations for bone marrow samples, as previously described [ 37 ]. DNA concentration and purity was assessed spectrophotometrically (NanoDrop 1000, Thermo Fisher Scientific, Waltham, MA, USA) and DNA samples were stored at -80°C.…”

Peer Review #1 of "Ranaviruses and reptiles (v0.2)"

“…Using total IgY levels as an internal control may minimise diagnostic errors resulting from seasonal variations in antibody levels. PCR based assays have been used conventionally and in quantitative real-time assays to detect reptilian ranaviruses in a number of sample types including blood, oral and cloacal swabs, and fresh and fixed tissues (Pallister et al, 2007;Allender et al, 2013a;Goodman et al, 2013, Butkus et al, 2017Leung et al, 2017;Maclaine et al, 2018). Molecular surveys of turtle populations for ranavirus have revealed that swabs and blood samples are not equally valid targets for ranavirus detection (Allender et al, 2013a).…”

“…both blood and swabs) when conducting a molecular survey for reptilian ranaviruses (a method employed in many studies). It is also possible to use bone marrow as a source of DNA for ranavirus detection from reptile carcases in which other viable tissue samples may have decayed (Butkus et al, 2017). The preferred target of ranaviral PCR assays is the major capsid protein (MCP) gene as it is highly conserved throughout the ranaviral lineage (Miller et al, 2015).…”

“…Following this, ranaviruses were predominantly isolated from box turtles (Terrapene carolina) and were identified as the aetiological agent of 'red neck disease' in the soft-shelled turtle (Pelodiscus sinensis) (Chen et al, 1999). In the last decade, several new reports of ranaviral infections in Testudines have been published (Johnson et al, 2008;Johnson et al, 2010;Belzer & Seibert, 2011;Allender, 2012;Stohr et al, 2015;Perpiñán et al, 2016;Butkus et al, 2017, Agha et al, 2017Archer et al, 2017;Adamovicz et al, 2018). Despite the increasing number of reports of infections in the Testudines, ranaviral disease in these reptiles is still likely to be underreported due to a lack of awareness, an incomplete understanding of the pathology caused by the disease, few long-term studies, and minimal population monitoring (Duffus et al, 2015).…”