Budd-Chiari-like pathology in dolphins | Scientific reports

Animals studied

The Canarian Marine Mammal Stranding Network has performed more than 1,200 autopsies of stranded cetaceans, including 172 striped dolphins (Stenella coeruleoalba). Of these, 4 (2%) striped dolphins were diagnosed with CLL similar or identical to those previously reported in Risso’s dolphins, common dolphins and harbor porpoises1.2. This study focused on these four striped dolphins. Biological and stranding epidemiological data for each individual are recorded in Table 1. Age category was based on total body length and gonadal development, including fetus/newborn/calf, juvenile/subadult and adult.14,15,16. Nutritional status (syn. body status) was subjectively classified as good, moderate, poor, and emaciated according to anatomical parameters such as bony prominence of spinous and transverse vertebral processes and ribs, mass of epaxial musculature, and amount fatty deposits taking into account the species and age of the animal14.15. Carcasses were categorized as very fresh, fresh, moderate autolysis, advanced autolysis, or very advanced autolysis14,15,16,17.

The authorization required for the management of stranded cetaceans has been issued by the Department of the Environment of the Government of the Canary Islands and the Spanish Ministry of the Environment. No experiments have been performed on live animals.

Pathological examinations

Necropsies followed standardized protocols14,15,16,17. Representative samples of skin, longissimus dorsi and rectus abdominis muscles, peritoneum, diaphragm, central nervous system, eye, pterygoid sac, tympanoperiotic complexes, tongue, oral mucosa, pharyngeal and laryngeal tonsils, esophagus, stomach, small and large intestine, liver, pancreas, trachea, lung, heart, aorta, kidney , ureter, bladder, urethra, lymph nodes, spleen, testis, penis, foreskin, ovary, uterus, vagina and vulva were removed and fixed in 10% neutral buffered formalin. All of these tissues were routinely processed, embedded in paraffin, and 5 μm thick sections were stained with hematoxylin and eosin (H&E) for microscopic analysis. Special histochemical techniques (sections 4–10 μm thick) to better characterize the microscopic characteristics of CLL included periodic acid Schiff, Masson’s trichrome, Picrosirius red, Perl, and Warthin-Starry.

Immunohistochemical analysis

We used a set of primary antibodies for immunohistochemical (IHC) analysis to further characterize CLL. The primary antibodies used were factor (F)-VIII (syn. Von Willebrand factor), for endothelial cells and platelets; fibrinogen, as a fibrinogen/fibrin proxy (thrombosis); pancytokeratin AE1/AE3 and cytokeratins 5, 8 and 18 for the bile duct epithelium; and vimentin as a general intermediate filament marker for mesenchymal cells. Published methodologies for IHC analyzes were followed18.19. Detailed information on the IHC methodology is recorded in Supplementary Table S2. Negative controls consisted of serial tissue sections in which primary antibodies were replaced with homologous non-immune serum. Positive controls included inner and outer striped dolphin liver with its normal constituents.

Polymerase chain reaction analyzes

During the autopsy, selected samples were taken for virological analyzes and stored frozen at -80◦C until they were processed for molecular virology tests. Approximately 0.5 g of freshly frozen tissue sample from each animal was mechanically macerated in lysis buffer and then centrifuged. DNA/RNA extraction was performed from each 300 μL macerated sample by a pressure filtration method, using a QuickGene R Mini 80 nucleic acid isolation instrument, using S-DNA Tissue Kit (QuickGene, Kurabo, Japan) according to manufacturer’s instructions with modifications: RNA carrier (Applied Biosystems, Thermo Fisher Scientific Waltham, Massachusetts, USA) was added during the lysis step20. Herpesvirus DNA was detected by conventional nested PCR using degenerate primers designed to amplify a region of the DNA polymerase gene21. Molecular detection of cetacean morbillivirus (CeMV) was performed by one or more of four different PCR methods: One-step RT-PCR of a 426 bp conserved region of the phosphoprotein (P) gene22One-step real-time RT-PCR that detects the most common CeMV strains known to circulate in the Atlantic Ocean targeting the P gene23and RT-PCR using nested primers targeting the P gene24and one-step real-time RT-PCR to detect sequences in a conserved region (192 bp) of the fusion protein gene (F)20. Molecular detection of Bartonelle spp. was also performed using a real-time PCR assay using SYBR® Green, as previously described25.

Gas sampling and analysis

Gas was collected from the LLC of cases 3 and 4 and from the intestine of case 1. To avoid contamination of atmospheric air, the livers were immersed in distilled water and the gas was collected at using an aspirometer (ES1076326) according to Bernaldo de Quirós et al.8. Gas samples were collected and stored in 5 mL additive-free glass vacutainers® (BD Vacutainer® Z. ref: 367624) and analyzed by gas chromatography within two weeks of taking the sample. Gas samples were extracted from the vacutainers and injected into the gas chromatograph (Varian 450-GC) using glass block syringes (Supelco A-2 series). Gas samples were analyzed for 15 min at 45°C and with a fixed pressure of 13.1 psi through a Varian 450-GC column and a thermal conductivity detector as well as a flame ionization detector using L helium as carrier gas8. The results were expressed as mean ± standard deviation in μmol%.

CT scan

A normal liver from an adult striped dolphin (for comparison) was scanned with a 16-slice helical CT scanner (Toshiba Astelion, Toshiba Medical System, Madrid, Spain) in the following parameters: 16 detector arrays; type of acquisition: helical; thickness: 1mm; image reconstruction interval: 0.8 mm; pitch: 1; tube rotation time: 0.75; mA 250; Kv: 120; Field of view: 512 × 512; Matrix dimensions: 1204 × 845; WW 500/WL 505. Previously the liver was dissected; the caudal vena cava and the portal veins were isolated and cannulated separately. These vessels were rinsed with tap water then a radiocontrast agent (Iomeron 300©) mixed with a saline solution was injected into both veins. The resulting DICOM images were analyzed; a soft tissue window setting (WW 500/WL 50) was applied rendering the internal volume and thus realizing a 3D model of the two venous, portal and hepatic systems.

Liver cast analysis

For a detailed assessment of hepatic vasculature, we performed a corrosion cast on a control striped dolphin (for comparison purposes) following published methodologies.26.27. At autopsy, the caudal vena cava, portal vein, hepatic artery and common bile duct were carefully dissected, isolated and rinsed with tap water. Then the liver was carefully dissected and detached from the supporting vessels while preserving the capsule and its ligaments (it was extracted with part of the diaphragm and lesser omentum). Once removed, the liver was carefully washed. Ex situ, the common bile duct, hepatic artery, portal vein and caudal vena cava were catheterized at the dorsal edge of the liver. The left branch of the caudal vena cava was clamped. Then, blue, white, red and yellow epoxy (Biodur©) was injected into the venous and arterial vessels and the common bile duct, respectively. The liver was stored at room temperature for 24 h and then at 4°C for 24 h. After that, a corrosion process was applied to the submerged liver with 10% sodium hydroxide (NaOH) solution for two weeks. Soft tissue debris was removed manually.

Bacteriological analyzes

Samples of fresh tissue (skin, muscle, lung, prescapular, pulmonary, mediastinal and mesenteric lymph nodes, liver, intestine, kidney, spleen, brain) taken routinely during autopsy, were frozen (−80°C) and selectively subjected to bacteriological analysis. These included routine culture and surface plating on routine media, for example., Columbia blood agar and preliminary identification of isolates via API® system (PLC® 20E, PLC® Rapid 20E, PLC® Staph, API® 20 Strep, API® Coryne, API® 20A). PCR targeting the 16S rRNA gene coupled with pulsed-field electrophoresis were performed on selected isolates.

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