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Abstract Background Cats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries. Methods From 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities. Results Overall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis. Conclusions This study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin. Graphical Abstract

IntroductionCanine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking.MethodsIn this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA.ResultsOur results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p

Abstract Background Canine leishmaniosis (CanL) is a parasitic zoonotic disease, endemic in the Mediterranean basin including Spain. While knowledge about CanL, its management, treatment, prevention and control mounts, it remains unclear whether all clinical veterinarians follow the same international recommendations, such as those of the LeishVet group. This study was thus designed to assess recent trends in the clinical management of CanL in veterinary clinics across Spain through a questionnaire-based survey. Results were compared with those of a prior national multicenter questionnaire administered by our research team in 2005. Methods A questionnaire consisting of 28 questions about CanL was developed using Google Forms and distributed by email to 1428 veterinary clinics in Spain. Questions were designed to obtain data on common clinical signs, techniques and complementary exams used to diagnose the disease, and on its monitoring, treatment and control measures. Data were collected in a database for statistical analysis. Results Completed questionnaires were returned by 295 clinics. Compared to the situation in 2005, responses indicate that clinical signs of CanL have not changed significantly, cutaneous lesions being still the most prevalent sign observed by practitioners. Quantitative serological techniques are considered an adequate approach to diagnosis, provided their results are supported by the findings of a thorough physical exam, as well as complementary tests (complete blood count, biochemical profile, plasma protein electrophoretogram and complete urinalysis). Treatment protocols and check-ups follow international recommendations. Finally, a multimodal approach is being endorsed to adequately control CanL including preventive measures such as annual serological check-ups and the combination of repellents and vaccines. Additionally, owners are being better informed about CanL by veterinarians, which translates to the improved control of this zoonosis. Conclusions The clinical management of CanL has recently undergone significant changes owing to improvements in clinical knowledge of the disease, more unified international criteria, improved diagnostic techniques and their adequate interpretation, as well as a greater awareness of the disease transmitted to owners.

Abstract Background This report describes L. infantum infection seroprevalence in dogs in Spain through data obtained from peer-reviewed literature and a cross-sectional serological survey assessing epidemiological and habitat variables as risk factors for infection. The study also provides preliminary sand fly species distribution data and indicates factors affecting their distribution and density. Methods Three different studies were conducted in Spain: (i) a peer-reviewed literature seroprevalence survey (1985–2019); (ii) a cross-sectional serological survey (2011–2016); and (iii) a preliminary entomological survey (2013–2014). In the cross-sectional serological survey, 1739 dogs from 74 different locations including 25 Spanish provinces were tested for L. infantum by indirect immunofluorescence antibody test (IFAT) (antibody titre ≥ 1:100). Seroprevalence of L. infantum infection was analysed by province and bioclimatic zone. Statistics were used to analyse relationships between several dog- and environment-related variables and L. infantum seroprevalence. In parallel, during 2013–2014, sand flies were collected across the Iberian Peninsula and the Balearic Islands using CDC light traps to examine relationships between habitat-related factors and sand fly species densities (number of sand flies per trap per hour). Results The literature review revealed that the provinces showing the highest seroprevalence were Balearic Islands (57.1%), Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), and those showing the lowest seroprevalence were Vizcaya (0%), Cantabria (2.0%) and Álava (3.3%). In our survey, anti-Leishmania IgG antibodies were detected in 176 of the 1739 dogs rendering a seroprevalence of 10.12%. Percentage seroprevalence distributions significantly varied among bioclimatic belts. Seropositivity for L. infantum was related to size (large breed dogs versus small) and were significantly higher in younger dogs (≤ 1 years-old). In the entomological survey, 676 sand flies of five species were captured: 562 (83.13%) Phlebotomus perniciosus; 64 (9.47%) Sergentomyia minuta; 38 (5.62%) P. ariasi: 6 (0.89%) P. sergenti; and 6 (0.89%) P. papatasi. Phlebotomus perniciosus showed a greater density in the thermo-Mediterranean than in the meso-Mediterranean zone. Densities of S. minuta and P. ariasi were significantly higher in rural habitats. Conclusions This updated seroprevalence map of L. infantum infection in dogs in Spain defines non-endemic, hypoendemic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.

Abstract Background Some wild animals have been recognized as potential reservoirs of Leishmania infantum infection (e.g. carnivores, lagomorphs, rodents, etc.). Leishmania infantum was also identified infecting humans and lagomorphs (i.e. hares and rabbits) over the period of 2009–2016, with the latter acting as the main reservoirs involved in the human leishmaniosis outbreak in Madrid. Results Two cases of clinical leishmaniosis are reported in orangutans (Pongo pygmaeus pygmaeus) housed at two different centres in Madrid. The first is the case of a 36-year-old male orangutan with severe weight loss and apathy. A complete blood count and biochemical profile revealed anaemia, neutropenia, hypoalbuminaemia and elevated transaminases. Hepato-splenomegaly was also observed. Four months later, due to worsening of clinical signs (mainly bilateral epistaxis), blood and bone marrow samples were collected. Amastigotes of L. infantum were detected in macrophages from a bone marrow aspirate and by specific polymerase chain reaction. The second case was a 34-year-old female orangutan with severe weight loss and apathy and no other apparent clinical signs. A complete blood count and biochemical profile revealed anaemia, pancytopenia and hypoalbuminaemia. Splenomegaly and pericardial effusion were also observed. As leishmaniosis was included in the differential diagnosis, both blood and bone marrow samples were collected. Leishmania infantum infection was confirmed by microscopy, molecular diagnosis and serology (immunofluorescence antibody test). Both animals were treated daily with oral miltefosine for 28 days; allopurinol was also given uninterruptedly in Case 2 for at least 6 months. During follow-up, though good clinical recovery was clear, a lack of parasitological cure was confirmed molecularly in both blood and bone marrow samples from the two orangutans. In both habitats, the presence of the sand fly vector identified as Phlebotomus perniciosus was confirmed. Conclusions To our knowledge, this is the first report of L. infantum infection in great apes and in the endangered species P. p. pygmaeus. We are presently looking for L. infantum in other non-human primates living in the same peri-urban areas. If detected, we will examine the impacts of this serious disease on these critically endangered species.

Abstract Background Canine leishmaniosis (CanL) has been in the spotlight since the 2009 outbreak of human leishmaniosis in Madrid. In the framework of the Leishmaniosis Surveillance Programme set up in Madrid, this study examines Leishmania-specific seroprevalences in stray dogs for the outbreak area and rest of the Madrid region over the period spanning from the outbreak to the present (2009–2016). These data are of interest because stray dogs could be sentinels for disease surveillance in endemic areas. Since 2011, we have also been monitoring owned dogs in the outbreak area. Methods Over the study period, Leishmania infantum seroprevalence was determined in 2,123 stray dogs from the outbreak and non-outbreak areas. A serological study was also performed for owned dogs in the outbreak area: high-risk dogs such as hunting or farm dogs (n = 1,722) and pets (n = 1372). All dogs were examined and blood was collected. The variables recorded for each animal were: breed, age, sex, and clinical history indicating if the animal was healthy or clinically suspected of having any disease, and if they showed a clinical picture compatible with CanL. Results Seroprevalences of L. infantum in stray dogs were similar in the two areas examined: 4.7% (20 out of 346) in the outbreak area and 5.4% (96 out of 1,777) in the remaining Madrid region (χ 2 = 0.080, P = 0.777). A significant association was found between seroprevalence and age (z = -6.319; P

Abstract Limited data is available on feline leishmaniosis (FeL) caused by Leishmania infantum worldwide. The LeishVet group presents in this report a review of the current knowledge on FeL, the epidemiological role of the cat in L. infantum infection, clinical manifestations, and recommendations on diagnosis, treatment and monitoring, prognosis and prevention of infection, in order to standardize the management of this disease in cats. The consensus of opinions and recommendations was formulated by combining a comprehensive review of evidence-based studies and case reports, clinical experience and critical consensus discussions. While subclinical feline infections are common in areas endemic for canine leishmaniosis, clinical illness due to L. infantum in cats is rare. The prevalence rates of feline infection with L. infantum in serological or molecular-based surveys range from 0 % to more than 60 %. Cats are able to infect sand flies and, therefore, they may act as a secondary reservoir, with dogs being the primary natural reservoir. The most common clinical signs and clinicopathological abnormalities compatible with FeL include lymph node enlargement and skin lesions such as ulcerative, exfoliative, crusting or nodular dermatitis (mainly on the head or distal limbs), ocular lesions (mainly uveitis), feline chronic gingivostomatitis syndrome, mucocutaneous ulcerative or nodular lesions, hypergammaglobulinaemia and mild normocytic normochromic anaemia. Clinical illness is frequently associated with impaired immunocompetence, as in case of retroviral coinfections or immunosuppressive therapy. Diagnosis is based on serology, polymerase chain reaction (PCR), cytology, histology, immunohistochemistry (IHC) or culture. If serological testing is negative or low positive in a cat with clinical signs compatible with FeL, the diagnosis of leishmaniosis should not be excluded and additional diagnostic methods (cytology, histology with IHC, PCR, culture) should be employed. The most common treatment used is allopurinol. Meglumine antimoniate has been administered in very few reported cases. Both drugs are administered alone and most cats recover clinically after therapy. Follow-up of treated cats with routine laboratory tests, serology and PCR is essential for prevention of clinical relapses. Specific preventative measures for this infection in cats are currently not available.