A 4-year-old male Eurasian otter born in 2015 in the ‘Sendaviva, Natural Park of Navarra’ (42°11′31″N, 1°34′33”O) (North Eastern Spain) was transferred to the ‘Terra Natura’ wildlife park (South Eastern Spain) in 2017. The ‘Terra Natura’ wildlife park is located in the periurban surroundings of Murcia city (38°00′40″N, 1°09′54”O). The otter enclosure consisted of an area with free-access shelters and a pond containing 4 animals of this species (2 male and 2 female). No pour-on anti-sandfly insecticide was applied to the otters. In August 2019 the animal presented bilateral epistaxis, anorexia, apathy, and weight loss. The animal was sedated prior to manipulation with medetomidine (0.2 mg/kg/i.m.) and ketamine (20 mg/kg/i.m.). Whole blood was taken by venipuncture of the cephalic vein, drained into a 1 ml tube coated with ethylenediaminetetraacetic acid (EDTA) and a 1 ml tube without anticoagulant for a complete blood cell count (CBC) and biochemical analyses, respectively. The CBC was performed in a blood cell counter (ADVIA 120 Hematology System, Siemens, Italy), and the biochemical analyses were performed in an automated biochemistry analyzer (Olympus AU600 Automatic Chemistry Analyzer, Olympus Europe GmbH, Hamburg, Germany). In addition, urine was taken by Ultrasound Guided Cystocentesis for urinalysis. An echography study of the heart and abdominal organs including kidney, spleen, liver, gallbladder, pancreas, and digestive system, was performed. As the biochemical and echographic results were compatible with the clinical leishmaniosis, serological and molecular tests were performed to confirm the presence of the infection. The presence of anti-Leishmania IgG2 antibodies was evaluated in the serum of the otter by a time-resolved immunofluorometric assay (TR-IFMA) performed as described by Cantos-Barreda et al. []. In order to test that the TR-IFMA assay validated for dog serum can be applied to otter serum, a Western blot analysis was carried out. The Western blot was performed in sera from the otter with leishmaniosis-compatible signs, and from a Leishmania-seropositive dog by TR-IFMA. The sera (1:2000 dilution) were separated under reducing conditions on mini polyacrylamide gels (0.1% sodium dodecyl sulfate (SDS), 12% resolving gel, and 4% stacker gel). The resolved proteins were electrophoretically transferred to a nitrocellulose sheet (Bio-Rad, USA) and placed in a ROTI®Lumin substrate (Carl Roth, Germany) to block for 2 h at room temperature. Sheep polyclonal antibody anti-dog IgG2 (AHP498; Bio-Rad, USA) at 1:1000 dilution was used as the primary antibody, while rabbit anti-sheep IgG horseradish peroxidase (HRP)-conjugated (SAB3700702; Sigma-Aldrich, USA) at 1:1000 dilution was used as the secondary antibody to detect the bound primary antibody. Signal detection was done using a Pierce ECL2 kit (Pierce, Thermo Fisher Scientific, USA) and was digitalized in the Typhoon 9410 scanner (GE Healthcare, USA). A band of approximately 150 kDa was observed in the otter sample as well as in the canine sample (see Fig. and Additional file ). These findings corroborated that the TR-IFMA assay using sheep anti-dog IgG2 was able to detect the IgG2 of the otter. The TR-IFMA assay consisted of a non-competitive indirect method based on biotinylated K39 recombinant antigen as a capture reagent, and anti-dog IgG2 polyclonal antibody (Sheep anti-Dog IgG2, Bio-Rad, USA) Eu3 +-labelled as a detector. The test included serum from a sick Leishmania-seropositive dog as a positive control, and serum from a healthy Leishmania-seronegative dog as a negative control. The analysis was performed in a multilabel counter (VICTOR2 1420, PerkinElmer Life and Analytical Sciences, Turku, Finland). Results were expressed as Units of fluorometry for Leishmania (UFL). The cut-off was set at 22 UFL. Also, an enzyme-linked immunosorbent assay (ELISA) (Leiscan® Leishmania ELISA Test, Esteve Veterinaria, Laboratorios Dr. Esteve SA, Barcelona, Spain) detecting specific IgG antibodies against Leishmania spp. was performed, according to manufacturer’s instructions. Sera samples were diluted 1:20 and the results were expressed as sample-to-positive (S/P) ratio, calculated by optical density (OD) sample/OD low positive control. Values of S/P ratio above 1.1 were considered positives. The presence of Leishmania spp. DNA in whole blood and bone marrow was evaluated by amplification of the kinetoplast DNA sequence of Leishmania spp. using an rtPCR with primers and probes previously described []. Bone marrow was taken through fine-needle aspiration from the costochondral union, and drained into a 1 ml tube coated with EDTA. DNA was extracted using the High Pure PCR Template Preparation Kit (Roche, Germany), following the manufacturer’s instructions. The rtPCR was performed in the QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.). The internal control TaqMan Exogenous Internal Positive Control Reagents (VIC Probe) (Exo IPC) (Applied Biosystems, CA, U.S.A.) was included. The rtPCR was performed in a final volume of 20 μL, including 1× iTaq Universal Probes Supermix (Bio-Rad, CA, U.S.A), 900 nM of each primer, 200 nM of TaqMan probe, 1× Exo IPC Mix, 1× Exo IPC DNA, and 50 ng of DNA from each sample. The cycling parameters were: 50 °C for 30 s, 95 °C for 10 min, 45 cycles at 94 °C for 30 s, and 55 °C for 1 min. Each amplification run included positive and negative controls, and each measurement was accomplished in triplicate. For the Leishmania species identification, the PCR products from the positive samples were purified using the High Pure PCR Product Purification Kit (Roche, Germany) and submitted for sequencing at the Section of Molecular Biology, University of Murcia. The sequences obtained were compared to those available in GenBank. Additionally, as negative control, the same analyses were performed on a 3-year-old female Eurasian otter from the same wildlife park with no clinical signs compatible with leishmaniosis. All the results appear in Table. The occurrence of leishmaniosis was confirmed, and specific treatment was administered (allopurinol 15 mg/kg/24 h/p.o.). Treatment monitoring was performed, in November 2019, after 3 months of treatment. Bilateral nephropathy with hydronephrosis, mesenteric lymphadenomegaly, and ascites were observed. The CBC revealed decreases in white blood cells, and decreases in platelets in the Leishmania-positive otter. The serum biochemical profile for the Leishmania-positive otter showed hyperproteinemia, hyperglobulinemia, decreases of PON-1, and increases of haptoglobin and ferritin. The urinalysis revealed proteinuria and decreases in the osmolarity and specific gravity. The presence of anti-Leishmania IgG2 antibodies, and a positive result by rtPCR in terms of whole blood and bone marrow, was observed for the Leishmania-positive otter. The sequencing of positive samples confirmed the identification of L. infantum. The amplified sequence exhibited 100% similarity with a L. infantum reference sequence. In addition, oval microorganisms with an eccentric nucleus compatible with Leishmania spp. amastigotes were observed in the cytology of the spleen.