Clinical leishmaniosis in a captive Eurasian otter (Lutra lutra) in Spain: a case report

Background Captive and free-ranging wild mammals have been recognized as potential reservoirs of Leishmania infantum infection. The aim of this study was to describe the first clinical case of leishmaniosis in the Eurasian otter. Case presentation A case of clinical leishmaniosis is reported in a 4-year-old male Eurasian otter housed at a wildlife park (Murcia, South Eastern Spain). The Eurasian otter showed bilateral epistaxis, anorexia, apathy, and weight loss. A complete blood cell count and biochemical analyses revealed hyperproteinemia, hyperglobulinemia, decreases of paraoxonase-1, increases of haptoglobin and ferritin, and proteinuria. Bilateral nephropathy with hydronephrosis, mesenteric lymphadenomegaly, and ascites were also observed. L. infantum infection was confirmed by microscopy (amastigotes were detected in macrophages from spleen aspirate), molecular diagnosis (L. infantum DNA was detected by real-time polymerase chain reaction), and serology (anti-Leishmania IgG2 antibodies were detected by time-resolved immunofluorometry). The animal was treated with allopurinol for 3 months and gained weight, the epistaxis disappeared, and the ferritin concentration decreased. Conclusions This is the first report of clinical leishmaniosis in the Eurasian otter. Our results suggest that Eurasian otters are susceptible to infection with L. infantum and can develop clinical leishmaniosis in endemic areas.


Background
Leishmaniosis is a zoonotic disease caused by the protozoan parasite Leishmania infantum in the Mediterranean area, affecting a wide range of mammals, including humans [1]. Domestic dogs act as the main reservoir, and Phlebotomus perniciosus is the main vector for L. infantum in the South East of Spain, an area in which this parasite is endemic [2]. Recently, L. infantum infection has been described in mustelids such as the domestic ferret (Mustela putorius furo) in Spain [3], and free-ranging badgers (Meles meles) in Italy [4]. L. infantum infection in the Eurasian otter (Lutra lutra) was detected for the first time in wild otters from Asturias (North Western Spain) by Oleaga et al. [5]. However, as far as we are aware, no data exist about clinical cases of leishmaniosis in this mustelid, which is a "near threatened" and fully-protected species. The aim of this report was to describe the first clinical case of leishmaniosis in the Eurasian otter.
with free-access shelters and a pond containing 4 animals of this species (2 male and 2 female). No pouron anti-sandfly insecticide was applied to the otters. In August 2019 the animal presented bilateral epistaxis, anorexia, apathy, and weight loss (Fig. 1). 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. [6]. 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. 2 and Additional file 1). 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 noncompetitive 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) Eu 3 + -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 (VICTOR 2 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 [7]. 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 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 1. 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 (Fig. 3).

Discussion and conclusions
To the best of the authors' knowledge, this is the first report describing a case of clinical leishmaniosis in the Eurasian otter. This species has been reported as being susceptible to Leishmania infection, as DNA from this parasite was detected in 7 out of 10 wild healthy Eurasian otters from North Western Spain, a non-endemic area for canine leishmaniosis (CanL) [5]. Santoro et al. [9] confirmed Ehrlichia canis and Rickettsia spp. infection in the South Italian population of Eurasian otters, and Chadwick et al. [10] indicated that Toxoplasma gondii infection is common in Eurasian otters in the United Kingdom. In addition, it was reported that this species may occasionally be infected with Dirofilaria immitis [11].
The diagnosis of clinical leishmaniosis described in this report was demonstrated by serological and molecular results, similar to a previous report describing the first case of L. infantum infection in a domestic ferret (Mustela putorius furo) in Spain [3]. In dogs, the serum total levels of anti-Leishmania IgG, IgG1 and IgG2 are elevated during the active phase of Leishmania infection, the IgG2 being the predominant subclass [12,13]. At this moment, elevated levels of anti-Leishmania IgG2 in serum were found, as previously reported in dogs with clinical leishmaniosis using TR-IFMA [6]. High levels of anti-Leishmania IgG were also detected in the serum from the sick otter by the commercial ELIS A. So, both serological results (TR-IFMA and ELISA) were in accordance. The Western blot analysis showed a band of approximately 150 kDa in the otter's serum as well as in the canine serum, corresponding to the molecular weight of the IgG [14]. These findings support that the TR-IFMA using sheep anti-dog IgG2 is able to detect the IgG2 of otters. These results are in line with those of a previous study in which an indirect enzyme-linked immunoassay using anti-dog IgG detected the IgG of seven marine mammals including sea otters (Enhydra lutris) [15].
At the time of diagnosis, the otter presented bilateral epistaxis, anorexia, and apathy, all of which are clinical signs compatible with CanL [1]. The main changes detected in the analytical profile of the Leishmania-positive Eurasian otter were also in accordance with those reported in CanL: hyperproteinemia, hyperglobulinemia, decreases of PON-1, increases of haptoglobin and ferritin, and proteinuria [1,16]. After treatment, the general health condition of the animal improved, as it did not show any epistaxis, and the ferritin concentration decreased, as reported in CanL [16].
Natural infection by L. infantum in wildlife species in South-Eastern Spain was described in captive wolves and brown bears, and in free-ranging wildlife species such as foxes, rabbits and rodents [17,18]. In addition, Muñoz et al. [2] reported sand fly vector abundance in the 'Terra Natura' wildlife park. Based on our findings, we could hypothesize that Eurasian otters may act as a secondary reservoir for L. infantum infection in the Mediterranean basin. Infection in wildlife carnivores suggests that a sylvatic cycle of this pathogen may occur, being of particular importance to human and pet health [4].  Fig. 3 Cytology of spleen of the sick Eurasian otter obtained by fineneedle aspiration. Oval microorganisms with eccentric nucleus compatible with Leishmania spp. amastigotes are found into the cytoplasm of macrophages. Note the eccentric nucleus and the kinetoplast of this microorganism (arrow). The slide has been stained with Diff-quick stain and observed at ×100