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Veterinary Parasitology: Regional Studies and Reports 19 (2020) 100369 Contents lists available at ScienceDirect Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr Short Communication First report on natural infection with Leishmania infantum in a domestic T ferret (Mustela putorius furo) in Spain J. Ginera,1, A. Basurcob,1, M.M. Alcoverc, C. Rierac, R. Fisac, R.A. Lópezd,e, C. Juan-Sallésf, ⁎ M.T. Verdeb,g, A. Fernándezb,g, Andrés Yzuelg, Sergio Villanueva-Sazg,h, a Menescalia Veterinary Clinic, Ismael Merlo Actor, 5, 46020 Valencia, Spain b Animal Pathology Department, Veterinary Faculty, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain c Laboratory of Parasitology, Pharmacy Faculty, University of Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain d Department of Pathology, Faculty of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico, Ave. Universidad 3000, Circuito Exterior, Ciudad Universitaria, Coyoacan, CP 04510, Mexico City, Mexico e Aquapath. Laboratory of Veterinary Anatomic Pathology, Monte Carpatos #58, Residencial Valle Verde, CP 28610, Colima, Mexico f Noah's Path, Arquitecto Santiago Pérez Aracil 30 bajo (centro veterinario), 03203 Elche, Spain g Clinical Immunology Laboratory, Veterinary Faculty, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain h Department of Pharmacology and Physiology, Veterinary Faculty, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain A R T I C LE I N FO A B S T R A C T Keywords: A pet domestic ferret (Mustela putorius furo) with a papular lesion involving the right pinna was diagnosed with Ferret chronic pyogranulomatous dermatitis by histopathologic examination. Intralesional, intracytoplasmic oval mi- Leishmania infantum croorganisms compatible with Leishmania spp. or Histoplasma spp. were observed in macrophages and multi- Leishmaniosis nucleate giant cells. Leishmania infantum (L. infantum) infection was diagnosed by PCR, culture in Novy-MacNeal- Mustela putorius furo Nicolle medium, and immunohistochemistry. Abnormal clinicopathological results included increased alanine Dermatitis transferase, alkaline phosphatase, serum gamma glutamyl transferase and polyclonal gammpathy. Anti- Leishmania antibodies were detected by enzyme-linked immunosorbent assay, immunofluorescence antibody test and western blot using L. infantum antigen. Immunoreactivity against the 16 kDa specific L. infantum antigen fraction was observed by western blot. PCR performed in blood samples obtained from this patient after positive parasite isolation detected L. infantum DNA. To the authors' knowledge, this is the first diagnosis and isolation of L. infantum in a domestic ferret naturally infected in an endemic region (Spain) where canine and feline leish- maniosis is frequently detected. According to these findings, ferrets should be included as potential reservoir hosts of L. infantum. Future investigations should analyze the epidemiological role of ferrets in L. infantum infection including the prevalence of infection. 1. Introduction are the most important vector of canine leishmaniosis in the Iberian Peninsula: Phlebotomus perniciosus and Phlebotomus ariasi (Lucientes Leishmaniasis currently ranks second only to malaria in the World et al., 2005). Female Phlebotomus feed on a variety of vertebrate hosts, Health Organization (WHO) list of protozoan diseases that cause the including humans, livestock species, dogs, wild rabbits, hares, rodents highest human mortality. It is estimated that approximately 350 million and cats. people are under the risk of leishmaniasis infection and 12 million are Dogs are considered the main reservoir host for L. infantum but infected (Alvar et al., 2012). In southern Europe, leishmaniasis is a other animals can act as active reservoirs such as cats (Pennisi et al., zoonotic protozoan disease caused by Leishmania infantum. This parasite 2015) and rabbits (García et al., 2014). The identification of other is transmitted by phlebotomine sand flies under natural conditions domestic animals that can act as possible reservoir hosts for the parasite during feeding. Although various phlebotomine species have been im- could have a significant impact on public health. Equally, from a ve- plicated in the transmission of L. infantum in Europe, only two species terinary point of view, the identification of this disease in a new animal ⁎ Corresponding author at: Department of Pharmacology and Physiology and Clinical Immunology Laboratory, Veterinary Faculty, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain. E-mail address: svs@unizar.es (S. Villanueva-Saz). 1 These authors have contributed equally to the study. https://doi.org/10.1016/j.vprsr.2020.100369 Received 8 November 2019; Received in revised form 8 January 2020; Accepted 14 January 2020 Available online 14 January 2020 2405-9390/ © 2020 Elsevier B.V. All rights reserved. J. Giner, et al. Veterinary Parasitology: Regional Studies and Reports 19 (2020) 100369 Fig. 1. Different views of dermatologic lesions detected in this ferret (a, b, c) and full thickness biopsy performed from the ear pinna (d). a, b, c: Erythematous and oedematous papular lesion on the right ear pinna (arrowheads). A superficial ulcer is present on the tip of the ear pinna (a, b). species is deemed essential. 2.1. Histopathology and immunohistochemistry specific against L. infantum The potential role of wildlife species as reservoirs of the parasite should be taken into account as well. Among mustelids, L. infantum The skin biopsy sample was fixed in 10% formalin and embedded in infection has been identified by PCR in liver and/or spleen tissue paraffin. Four μm-thick sections were stained with hematoxylin and samples from a polecat (Mustela putorius), a European mink (Mustela eosin. Severe chronic diffuse pyogranulomatous dermatitis extending lutreola) and other wild mustelids (Meles meles, Martes foina and Martes diffusely from the superficial dermis to the subcutis was noted. martes) in the northern Iberian Peninsula (Spain) (Del Río et al., 2014). Inflammation was characterized by dense infiltrates of macrophages, None of the animals showed typical canine leishmaniosis lesions at few neutrophils, low numbers of lymphocytes and plasma cells, and post-mortem examination. rare multinucleate giant cells. The auricular cartilage was unaffected. Although no official ferret population data are available in Europe This lesion was accompanied by areas of necrosis in the dermis and (d'Ovidio et al., 2014), the domestic ferret (Mustela putorius furo) is proteinaceous edema and serocellular crusts. Macrophages and multi- nowadays a common household pet. This study describes for the first nucleate giant cells contained large numbers of oval organisms with an time naturally-occurring L. infantum infection in a ferret. eccentric nucleus and pale cytoplasm, measuring approximately 3 to 4 μm in diameter, compatible with Leishmania spp. amastigotes or Histoplasma spp. yeasts were observed (Fig. 2). To determine the pre- 2. Case report sence of Leishmania parasites in tissue section, immunohistochemistry was performed using a standard protocol with an Autostainer Link48™ A 4-year-old intact female ferret from Valencia, in the east coast of (Dako, Glostrup, Denmark) and an in-house rabbit polyclonal antibody Spain, was clinically evaluated in February 2019 because of the pre- specific for L. infantum. Blocking of endogenous peroxidase activity sence of a non-pruritic dermal lesion in the right pinna (Fig. 1). This (Dako REAL™ Peroxidase-Blocking Solution, Dako, Glostrup, Denmark) ferret was adopted at the age of two years with unknown previous was performed before sections were incubated for 30 min with the history. At the time of presentation, it lived in an apartment with other primary antiserum at room temperature (RT) (1 in 500 dilution in ferrets and had access to an outdoor terrace. It was under chronic EnVision Flex™ antibody diluent, Dako). Thereafter, sections were in- medical management with prednisolone and cyclosporine A because of cubated for 30 min at RT with Dako EnVision+ System-HRP,Rb™. The inflammatory bowel disease diagnosed one year earlier. This patient substrate used for detection was 3.3′-diaminobenzidine incubated for was also diagnosed with suppurative cholangitis six months prior to 10 min. Sections were then counterstained with hematoxylin for 8 min dermatologic presentation. (EnVision™ FLEX Hematoxylin, Dako) and covered in slides. For the On physical examination, this ferret was in good condition, active negative control, the primary antibody was replaced with non-immune and alert, normothermic and properly hydrated. Cardiac auscultation rabbit serum. A biopsy sample of the lymph node from a dog with was within normal limits. Respiratory sounds were also normal and clinical leishmaniosis was included as a positive control. The abundant there was no evidence of lymph node enlargement. The fur over the oval microorganisms present within the cytoplasm of dermal macro- back was coarse. An erythematous, edematous and non-painful papular phages stained positively (brown) (Fig. 3). lesion 5 mm in diameter was observed in the right ear pinna. Cytological evaluation from a sample taken by needle aspiration and stained with Diff-Quick revealed pyogranulomatous dermatitis; no in- 2.2. PCR fectious agents were visualized. A full thickness biopsy from the ear pinna was obtained. The presence of Leishmania spp. DNA in paraffin embedded skin biopsy was additionally evaluated by amplification of kinetoplast DNA 2 J. Giner, et al. Veterinary Parasitology: Regional Studies and Reports 19 (2020) 100369 Fig. 2. Histological sections of affected tissues with leishma- niosis in a ferret. a: Note a papular thickening of the ear (delimited with ar- rowheads) due to granulomatous inflammation. c = auricular cartilage. b: A higher magnification of this inflammatory lesion reveals infiltrates of macrophages and low numbers of lymphocytes as a well as a multinucleate giant cell (arrow). The cytoplasm of macrophages and this giant cell are ladden with oval micro- organisms with an eccentric nucleus. Hematoxylin and eosin. enzyme-linked immunosorbent assay (ELISA) using sonicated L. in- fantum antigens (MHOM/FR/78/LEM75 zymodeme MON-1). WB was performed as described by Riera et al. (1999), with some modifications. It was done on 0.1% SDS–13% polyacrylamide gel on a Mini-gel Bio RadSystem. Sera diluted at 1/50 were assayed and a protein A perox- idase conjugate (1/1000 dilution; Pierce) was used. A serum positive was considered when immunoreactivity against the 14 and/or16 kDa Leishmania antigen fraction was observed. ELISA was performed as described by Riera et al. (1999). Sera were diluted at 1/50 and a protein A peroxidase conjugate (dilution, 1/8000; Pierce) was used. The cutoff was set to 0.137 optical density units (OD) (mean + 4 standard de- viations of values from 8 ferrets with a negative result for Leishmania real-time PCR in blood). Each test included serum from a L. infantum confirmed symptomatic dog from Spain with a L. infantum isolation as positive control and serum from a healthy, non-infected dog as negative Fig. 3. Immunohistochemical staining labelling of Leishmania spp. amastigotes. control because the protein A peroxidase conjugate has been employed Note the amastigote forms labelled in brown. (For interpretation of the refer- in canine samples. Immunoreactivity from the Leishmania antigen was ences to colour in this figure legend, the reader is referred to the web version of detected by WB (band of 16 kDa) and specific antibodies against L. this article.) infantum were detected by ELISA. Medium levels of antibodies were determined with an OD result of 0.599. sequence using a quantitative polymerase chain reaction (qPCR). Each Later, other quantitative serological tests were performed including amplification was performed in triplicate, in 10 μL reaction, 15 pmol of an in-house immunofluorescence antibody test (IFAT), for detecting L. direct primer (5′-CTT TTC TGG TCC TCC GGG TAG G-3′), 15 pmol of infantum immunoglobulin G antibodies using L. infantum (strain reverse primer (5′-CCA CCC GGC CCT ATT TTA CAC CAA-3′), 50 pmol MHOM/FR/78/LEM75 zymodeme MON-1) as whole-parasite antigen of the labelled TaqMan probe (FAM-TTT TCG CAG AAC GCC CCT ACC fixed on multi-spot slides (Thermo Fisher Scientific, Waltham, CGC-TAMRA) and 2.5 μL of sample DNA. Amplification and detection Massachusetts, USA). All samples were diluted 1:20 with phosphate- were performed in the ABI Prism 7900 system (Applied Biosystems, buffered saline (PBS), and the endpoint titer of positive samples was Foster City, CA, USA.) in a two-step temperature process (94 and 55 °C) determined by preparing serial twofold dilutions of the serum starting for 45 cycles. Positive controls (DNA from L. infantum MHOM /ES /04 from the cutoff value. Immediately thereafter, 20 μL of every diluted /BCN-61) and negative controls were included in each RT-PCR analysis serum sample were applied per well. The slides were incubated for (Martín-Ezquerra et al., 2009). A positive result by PCR was obtained 30 min at 37 °C in a moist chamber. Slides were then washed twice with from the paraffin block. Additional diagnostic procedures included PBS for 5 min and once with distilled water. Following the washing detection of parasite DNA by PCR from peripheral blood sample and procedure, 20 μL of fluorescein-conjugated goat anti-ferret im- Whatman filter paper number 3 with aspirated material from the munoglobulin G (Abcam, Cambridge, United Kingdom) diluted 1:16 in perilesional excised area, serology and parasite isolation. Concerning Evans blue solution were added to each well. The slides were incubated the blood sample and Whatman filter paper results, Leishmania spp. in a moist chamber at 37 °C for an additional 30 min in the dark. The DNA was detected in both samples. washing procedure was repeated, and several drops of mounting medium were then added to the cover slips. Slides were examined under a fluorescence microscope (Leica DM750 RH; Leica 2.3. Serology Microsystems, Wetzlar, Germany) at 400 × magnification and each well was compared to the fluorescent pattern observed in the positive Anti-Leishmania antibodies were detected by western blot (WB) and 3 J. Giner, et al. Veterinary Parasitology: Regional Studies and Reports 19 (2020) 100369 and negative controls. The samples were examined by two different Gulf of Valencia on the Mediterranean Sea, a geographical area where researchers. The cutoff for this technique was set to 1:20 dilution by P. perniciosus is the most prevalent phlebotomine species detected analyzing 8 serum samples from ferrets with a negative result for (Aransay et al., 2004). Recent data about seropositivity rate of L. in- Leishmania real-time PCR in blood. The same procedure was repeated fantum in dogs in the East of Spain confirms that detection of anti- with the modification of fluorescein-conjugated goat anti-dog im- Leishmania antibodies was higher compared to the rest of the pathogens munoglobulin G (Abcam, Cambridge, United Kingdom). Due to the lack analyzed including Anaplasma spp., Ehrlichia canis, Borrelia burgdorferi of ferret positive reference serum samples, the ferret IFAT was de- and Dirofilaria immitis in this region (Miró et al., 2013). Human leish- termined by carrying out a parallel canine test run, using positive serum maniasis has also been detected in the Valencia region and a recent control (a dog from Spain with L. infantum confirmed with positive L. outbreak of cutaneous leishmaniasis was described (Roth-Damas et al., infantum culture) and negative serum control (dog from United 2017). In this sense, One Health context confirms the endemic nature Kingdom, a non-endemic area). Positive and negative controls were and the high prevalence of the infection in the European Mediterranean included on each slide. The result of the ferret IFAT was positive at regions and the potential possibility to detect the presence of L. in- 1:160 dilution with fluorescein-conjugated goat anti-ferret im- fantum infection in other animals living in the same geographical area. munoglobulin G. However, a negative result of the ferret serum sample The presence of parasite in blood samples should be considered an was obtained with fluorescein-conjugated goat anti-dog im- important factor in the risk of iatrogenic transmission of L. infantum by munoglobulin G. blood transfusion as it is reported in other species such as dogs (Tabar et al., 2008). Moreover, transmission by vectors should be taken into 2.4. In vitro isolation and cultivation account and preventive measures to avoid infection transmission from this and any other ferrets should be applied as is done in dogs (Miró For parasite isolation, Novy–MacNeal–Nicolle (NNN) medium and et al., 2017b) and cats (Brianti et al., 2017). Schneider medium supplied 100 IU/ mL penicillin and 100 μg/mL A broad range of immune responses and clinical manifestations streptomycin solution and 10% foetal calf serum was used. have been described in canine leishmaniosis. Infection in dogs may be Several drops of the aspirated material from the perilesional skin subclinical or manifested as a self-limiting disease, or a severe, and excised area were introduced into the liquid phase of the NNN and were sometimes, fatal illness. Subclinical infection is not necessarily perma- incubated in medium Schneider at 26 ± 1 °C and cultures were mi- nent and factors such as immunosuppression or concomitant diseases croscopically assessed every day. The culture was positive after three could break the equilibrium and lead to the progression of clinical days of incubation and Leishmania spp. strain isolated (Clinical disease in dogs (Solano-Gallego et al., 2009) as in cats (Pennisi et al., Immunology Laboratory, University of Zaragoza) was molecularly type 2015) and humans (Alvar et al., 2012). Although ferrets are often re- as isolated as L. infantum (Laboratory of Parasitology, University of latively resistant to the immunosuppressive effects of prednisolone, this Barcelona) and named MMST/ES/2019/ZGZ103. patient was under immune-modulating drugs therapy (prednisolone and clyclosporine) that might have caused immunosuppression and 2.5. Clinicopathological findings contributed to the clinical manifestation of Leishmania infection. The macroscopic skin lesions of canine and feline leishmaniosis can Complete blood cell count revealed hematological values within be diverse, even in the same patient. The main clinical presentations normal limits and serum biochemistry profile showed elevated serum include exfoliative, ulcerative, nodular, sterile pustular and papular alanine transferase (ALT: > 1000 U/L, reference 82–289 U/L), alkaline dermatitis, nodules at the site of parasite inoculation and onycho- phosphatase (ALKP: 239 U/L, reference 9–84 U/L), serum gamma gryphosis (Manolis et al., 2014; Pennisi et al., 2015). These lesions are glutamyl transferase (GGT: 250 U/L reference 0.2–14 UI/L), alpha-2 the result of granulomatous or pyogranulomatous inflammation that globulins (11.1 g/L; reference 4.2–8.5 g/L) and gamma-globulins (22 g/ targets different structures of the skin and of the deposition of immune L; reference 2.7–9 g/L (4.8–14.7%)) with an A/G ratio of 0.69 (re- complexes (Manolis et al., 2014). The ferret of this report presented ference 0.58–1.33). Serum protein electrophoresis revealed polyclonal with pyogranulomatous dermatitis compatible with a less common gammopathy. clinicopathological presentation of the disease (papular dermatitis or nodule at the site of parasite inoculation). In dogs, each presentation 3. Discussion may reflect a different host–parasite relationship, as in the case of the nodular and papular dermatitis, which are considered markers of high To the authors´ knowledge, this report describes the first clinical and low susceptibility to canine leishmaniosis, respectively (Manolis case of leishmaniosis in a domestic ferret (Mustela putorius furo) and is et al., 2014). the first notification of natural L. infantum infection detected by parasite Clinicopathological findings detected in this ferret included mod- culture in mustelids. The diagnosis of clinical leishmaniosis in this erately increased serum enzyme activities (ALT, ALKP and GGT) com- ferret was based on the clinical signs, pathologic findings, and con- patible with liver disorders and polyclonal hypergammaglobulinemia. firmatory L. infantum techniques, particularly PCR and culture. Inflammatory, infectious and toxic hepatic disease. Hepatic lipidosis, However, experimental infection has been described in ferrets used as and hepatic neoplasia are the most common hepatic diseases en- animal models for the study of antileishmanial compounds (White countered in this species (Huynh and Laloi, 2013). Hepatic disease et al., 1989). In dogs, the evolution and pathogenesis of natural infec- often remains subclinical, which may lead to difficulties in diagnosis. tion is highly variable and not easily comparable with experimental Canine leishmaniosis can occasionally cause hepatitis and elevation of infection. There may also be differences in the clinical course as well as liver enzymes (Rallis et al., 2005). In the ferret of this report, the cause in antibody production, among others. This situation therefore could be of ALT, ALKP and GGT elevations and if these were related to leish- extrapolable in ferrets. maniosis cannot be determined because liver biopsies were not ob- In the past decade wild animals such as lagomorphs and canids tained. other than dogs have been attributed an important role in the trans- Hypergammaglobulinemia in ferrets is associated with a variety of mission cycle of L. infantum (Miró et al., 2017a). Domestic ferrets have infections including Aleutian disease, systemic coronavirus, canine probably not played a crucial role as reservoir for L. infantum infection; distemper virus, certain neoplasia or systemic mycoses (e. g., blas- however, studies may be necessary to elucidate the epidemiological role tomycosis and coccidioidomycosis) (Melillo, 2013). In canine leishma- of domestic ferrets as household pets in this transmission cycle. niosis, serum protein electrophoresis reveals an increase of total pro- The ferret from this case report was infected in an urban environ- teins and globulins, as well as a typically polyclonal gammapathy. In ment in Valencia, on the East coast of the Iberian Peninsula fronting the this regard, the serum electrophoretogram detected in the ferret was 4 J. Giner, et al. Veterinary Parasitology: Regional Studies and Reports 19 (2020) 100369 very similar to the typical serum protein electrophoresis in dogs (Maia collar. Parasit. Vectors 10 (334), 14. and Campino, 2018) and cats (Pennisi et al., 2013) with clinical Del Río, L., Chitimia, L., Cubas, A., Victoriano, I., De la Rúa, P., Gerrikagoitia, X., Barral, M., Muñoz-García, C.I., Goyena, E., García-Martínez, D., Fisa, R., Riera, C., Murcia, L., leishmaniosis, but not specific for a single infectious disease in ferrets. Segovia, M., Berriatua, E., 2014. 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Novel areas for prevention and control of canine Leishmaniosis. Trends Parasitol. 33, 718–730. This research did not receive any specific grant from funding Pennisi, M.G., Hartmann, K., Lloret, A., Addie, D., Belák, S., Boucraut-Baralon, C., agencies in the public, commercial, or not-for-profit sectors. Egberink, H., Frymus, T., Gruffydd-Jones, T., Hosie, M.J., Lutz, H., Marsilio, F., Möstl, K., Radford, A.D., Thiry, E., Truyen, U., Horzinek, M.C., 2013. Leishmaniosis in cats: ABCD guidelines on prevention and management. J. Feline Med. Surg. 15, 638–642. Declaration of Competing Interest Pennisi, M.G., Cardoso, L., Baneth, G., Bourdeau, P., Koutinas, A., Miró, G., Oliva, G., Solano-Gallego, L., 2015. LeishVet update and recommendations on feline leishma- niosis. Parasit. Vectors 4 (8), 302. The authors have nothing to disclose. Rallis, T., Day, M.J., Saridomichelakis, M.N., Adamama-Moraitou, K.K., Papazoglou, L., Fytianou, A., Koutinas, A.F., 2005. 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