All the sea turtles included in this project were authorized by the Conselleria d’Agricultura, Medi Ambient, Canvi Climàtic i Desenvolupament Rural of the Regional Valencia Government in a collaborative official agreement with the Oceanogràfic Aquarium of the Ciudad de las Artes y las Ciencias of Valencia (east Spain) for rehabilitation and posterior release of animals, and for the postmortem examination of dead individuals.
In June 2014, a loggerhead nest with 131 eggs (two of them were already broken) was found in Alicante (east Spain) in the Western Mediterranean basin, an area beyond the regular nesting range for the species in the Mediterranean Sea . Given the sporadic characteristic of this nesting event, and the fact that the nest was found on a highly developed beach, the clutch was carefully removed and relocated for its protection. The clutch was divided into two egg subsets; one part (89 eggs) was relocated to a natural protected beach in the province of Valencia, and the other part (40 eggs) was kept in artificial incubation at the marine rehabilitation center of the Oceanogràfic aquarium. 102 hatchlings from both places were initially reared at the aquarium facilities under a head-starting program. 30 turtles died due to intestinal bacteria outbreak during second month of life and 15 were moved to other rescue center. The parasitic outbreak occurred 4 months after hatching. At the time of the outbreak, mean straight carapace length ± SD (95% CI) [range] was 5.91 ± 0.51 [4.6–6.84] cm and mean body weight was 43.13 ± 8.7 [21.6–57.4] g.
Sand-filtered seawater was piped through two filtration systems prior to the holding tanks. Sand filters, protein skimmers, ultraviolet light, biological filtration and ozone were used to maintain water quality. Water temperature was typically set at 25 °C (± 1) and a full light spectrum was provided by hydrargyrum quartz iodide (HQI) lightning. Neonates were kept separated from one another in plastic-mesh floating cages to prevent bite wounds and to allow close monitoring of individuals. In addition to the head started turtles, larger injured sea turtles undergoing rehabilitation were also housed at the aquarium. The turtles from the head-starting program and the animals in rehabilitation were kept in separate tanks, but shared the filtration system. Occasionally, and due to space constraints, neonates were maintained in their floating cages inside a tank where an animal being rehabilitated was also kept.
Prior to the outbreak, no preventive treatment was administered when animals were admitted from the rehabilitation center or from the head-starting program. After detecting massive copepod infestations in neonates, all the individuals were immediately treated in a tap water bath for 10 min to remove the parasite load . Additionally, three different experimental treatments were subsequently tested to remove parasites from the filtration system: (1) during week 1, formalin (Formol 40%, methyl aldehyde solution, Guinama S.L.U., Valencia, Spain) was added as follows: 0.015 ml l-1 (day 1); 0.01 ml l-1 (day 3); 0.001 ml l-1 (day 5). Ozone and ultraviolet filter was switched off during treatment and switched on again on day 7. Ten per cent of water was renewed in the system on days 1, 3 and 5. (2) During weeks 2 and 3, the filtration system was treated with chlorine (sodium hypochlorite, 150 g l-1, New Chem S.L., Alicante, Spain), which reached 0.6 ppm of free chlorine. (3) During week 4, a single dosage of 0.1 ppm of lufenuron (Program® 400, Novartis Sanidad Animal S.L., Barcelona, Spain) was applied to the system and repeated every 14 days; i.e., two additional treatments. Daily water replacement was 10% of the total system volume. To ensure that these treatments were not toxic for turtle hatchlings, three individuals were treated following the doses used in fish .
Sediment of freshwater baths of turtles was used to collect and identify parasites. Parasite specimens were collected and preserved in ethanol 70% for species identification. Complete necropsy was performed on each deceased individual within 6 h postmortem. Multiple tissues, including skin, fat, skeletal muscle, thymus, thyroid, heart, lung, liver, esophagus, stomach, intestine, spleen, pancreas, kidney, gonad, adrenal gland, salt gland and brain, were collected and fixed in 10% neutral formalin. All the tissues were routinely processed for histological examination and stained with hematoxylin and eosin (H&E) staining. To analyze gut contents of in situ B. manatorum immunohistochemical staining was undertaken for cytokeratin detection (monoclonal anti-cytokeratin, Isotype IgG1 Kappa, Clone AE1/AE3, Dako) using the avidin-biotin-peroxidase complex (ABC) method as recommended by the manufacturer. Swabs for bacteriology were taken aseptically from the coelomic cavity and the liver, lung and brain sections were frozen at −80 °C. Turtles behavior was checked there times a day as well as during feeding and treatment times. Animals were weighted once a week during the whole duration of the head-starting program.