Sample collection
Between January and September 2019, 23 fecal and blood samples were collected from loggerhead turtles during convalescence period in Italian rehabilitation facilities, namely Centro Recupero Animali Selvatici Il Benvenuto (RO)(n = 1) and Centro Recupero Tartarughe Marine (CRTM) “L. Cagnolaro” Centro Studi Cetacei Onlus (PE) (n = 12) for the Adriatic coastline; Centro Recupero Tartarughe Marine (CRT) of Lampedusa (AG) (n = 9) for Central Mediterranean and Stazione Zoologica Anton Dohrn (NA) (n = 1) for the Tyrrhenian coastline (Fig. 1). Blood was collected from cervical dorsal sinus of the turtles, stored in 1 ml tubes containing lithium heparin or EDTA and frozen at − 20 °C. Feces were collected from the tanks where the same turtles were individually hospitalized and the samples were stored at − 20 °C. All samples were moved to the Department of Animal Medicine, Production and Health of the University of Padova for successive analyses.
Collection of blood samples from the turtles was conducted during clinical investigations. In accordance with the National Guidelines of Italian Ministry of Health for the care and use of animals, the non-experimental clinical veterinary practice does not require permissions.
Coprological analyses
Fecal samples underwent qualitative copromicroscopic examination by means of a common concentration-flotation technique with high s.g. solution [10]. Eggs of spirorchiids were identified following keys in literature [2].
When fecal samples were positive for Hapalotrema-like eggs, DNA was extracted from frozen aliquots using PSP® Spin Stool DNA Kit (Invitek GmbH, Germany) according to manufacturer’s instructions. Amplification of the internal transcribed spacer 2 (ITS2) region of the rDNA was carried out using methods described in Stacy et al. (2010) [15]. The consensus sequences obtained were compared with the non-redundant database available in GenBank using the software BLAST [16].
Real time PCR
DNA was extracted from 400 μl whole blood with the NucleoSpin Tissue® kit (Macherey-Nagel) following manufacturer’s instructions and stored at − 20 °C.
Specific primers and probe were designed based on H. mistroides ITS2 fragment sequence (accession number: LT617052.1) from GenBank. Primer forward (5′-ACGACGCACATTTAGTCGTG-3′), reverse (5′-AAATATGCCGCACAATAGGC-3′) and fluorescent-labeled hydrolysis probe (5′-TCCTAATTTTTCCGGTGCAG-3′) were developed. For real time monitoring of the PCR signal the probe was labelled with a 5′-FAM reporter and a Black Hole Quencher at the 3′ end (Macrogen, Korea). The amplicon size was 291base pairs.
The 20 μl reactions, containing 2.5 μl of DNA, 1X QuantiNova pathogen Master Mix (Qiagen, Valencia, CA, USA), 0.8 μM of each primer and 0.25 μM of TaqMan probe, 1 μl of internal control (IC) and 1X QuantiNova IC Probe Assay and DNA-free water, were run in a Roche Light Cycler® 96 (Roche Diagnostics, Mannheim, Germany). Cycling comprised an initial heat activation at 95 °C for 2 min, 40 2-step cycling of denaturation at 95 °C for 5 s, and combined annealing-extension step at 60 °C for 30 s. Data acquisition were performed during the combined-extension step. Each assay was performed in duplicate.
Confirmation of PCR specificity and sensitivity
Primer and probe design was verified with Primer3 oligo analyser software (http://bioinfo.ut.ee/primer3-0.4). Their specificity was checked in silico by BLAST Analysis. The analytical specificity of the assay was tested on a panel of control samples including positive control DNA obtained from H. mistroides eggs (contaminating both water and blood samples) -previously isolated from splenic parenchyma of loggerhead turtles and molecularly identified (details of turtles, isolation methods and sequences accession numbers are reported in [10, 17]) and negative samples including negative blood, closely-related non target cardiovascular flukes geographically overlapped to H. mistroides in the area (i.e. Neospirorchis sp.) and bacteria (Enterobacteriaceae, Citrobacter spp., Vibrio spp., Photobacterium spp.).
Standard curves and limit of detection of the assay (LOD) were determined by using 10 2-fold dilution series of DNA from sterile water previously additioned with 100 eggs of H. mistroides. Each dilution was quantified by fluorometry (Qubit dsDNA HS assay Kit, ThermoFisher Scientific) with a limit of quantification of 10 pg/μl. The highest dilution with a positive signal in duplicate indicated the LOD.
The specificity of the assay was confirmed by end point PCR and sequencing of the PCR products. Positive controls were amplified using Platinum Taq DNA polymerase kit (Invitrogen) as described by the producer with the same couple of primers used in the real time PCR assay. Both strands of each amplicon were sequenced (Macrogen, Korea) and then compared with those present in GenBank (LT617052.1).