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Canine leishmaniosis is a complex infection that ranges from the apparent absence of disease to a severe fatal clinical illness. Numerous epidemiological serosurveys have been carried out in Europe, although these studies often lack assessment of clinical health status. The aim of this study was to evaluate signalment, immunological and parasitological status correlated with clinicopathological findings of L. infantum -seropositive apparently healthy dogs living in endemic areas of Europe. Routine laboratory tests, endpoint in-house ELISA to quantify the anti- Leishmania antibodies, blood Leishmania quantitative PCR (qPCR) and IFN-γ ELISA were performed. Dogs were classified as healthy or sick depending on the results of routine laboratory tests. Seropositive sick dogs presented a worse clinical status that included a higher proportion of medium to high antibody levels and positive qPCR, lower IFN-γ concentration and several clinicopathological findings compared to truly healthy seropositive dogs. In conclusion, further investigation of apparently healthy L. infantum -seropositive dogs is necessary in the clinical setting, as a significant proportion of seropositive dogs that are sick with clinicopathological findings remain undetected if routine laboratory tests are not undertaken. Performing these routine laboratory tests with the combination of a thorough physical examination will improve early disease diagnosis, treatment and prognosis of clinical leishmaniosis. Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum -seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint in-house ELISA to quantify the anti- Leishmania antibodies, blood Leishmania qPCR and IFN-γ ELISA were performed. All dogs enrolled were L. infantum -seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-γ concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum -seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN-γ concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia.

Background There are several screening tools for detecting Leishmania infantum infection in dogs and various preventive measures to protect against it. Some studies have investigated them, but not many have described their current use. The aim of this study was to investigate which preventive measures and serological screening tools for L. infantum infection were employed from 2012 to 2018 in dogs from different endemic European countries. Methods A set of electronic datasheets was completed for each dog from several veterinary centres. Classification of preventive measures included: (1) repellents, (2) vaccines and (3) immunomodulators. Classification of serological tests included the: (1) direct agglutination test (DAT), (2) enzyme-linked immunosorbent assay (ELISA), (3) indirect immunofluorescence (IFI), (4) rapid tests and (5) other assays. Dogs were also classified depending on their risk of exposure and living area. Results Information from 3762 dogs was gathered. Preventive measures were applied in 91.5% of dogs and the most frequently used were repellents (86.2%) followed by vaccines (39.8%) and Leisguard® (15.3%). The different types of repellents (collar and spot-on) were used similarly. A combination of a vaccine and repellents was preferred in the high-risk group while the low-risk preferred a combination of Leisguard® and a repellent (Chi-square test: X2 = 88.41, df = 10, P Conclusions Repellents were the preferred measure, while vaccines and Leisguard® were second-line options. Some dogs were not treated by any measures, which highlights the need for dog owner education. Moreover, there seems to be a preference for rapid tests in the clinical setting to detect specific L. infantum antibodies while ELISA or IFI are less often employed. This underlines an increasing problem, as qualitative rapid tests have a variable diagnostic performance limiting the adequate diagnosis of seropositive dogs in endemic areas. Graphical abstract

The best preventive strategy for canine leishmaniasis is, to date, unknown. In the last few years, new preventive measures have become available in Europe, including vaccination against leishmaniasis and the use of domperidone. The objective of the present study was to evaluate the efficacy and safety of the different preventive measures available against leishmaniasis in client-owned dogs. A database search of medical records was carried out in 52 private veterinary practices located in endemic areas of canine leishmaniasis in Spain, Italy and Portugal. Healthy seronegative dogs were included in the study. Serology was repeated at least 6 months later, and was used to retrospectively classify dogs into healthy, infected or sick. A total of 1647 dogs were included in the study. The use of preventive measures in this population was widespread. The single most utilized measure was repellents alone in 45.7% of dogs, followed by the combination of repellents and vaccination in 23.0%, repellents and domperidone in 11.3%, vaccination alone in 4.2%, vaccination and domperidone in 2.7%, domperidone alone in 2.3%, and the combination of the three measures in 0.2% of dogs. No preventive measure was applied in 10.7% dogs. The incidence of clinical leishmaniasis in the group with no preventive treatment applied was 12.5%. In the groups where prevention was applied, the reported incidence was the following: 10.1% for the vaccination only group, 4.5% for repellents only group, 4.0% for repellents + vaccination group, and 0.5% for repellents + domperidone group. No dogs in the groups of domperidone, vaccination + domperidone, and combination of the three measures developed clinical leishmaniasis. All preventive measures resulted in a significantly lower incidence of leishmaniasis compared to not applying any measure, except for vaccination alone. The majority of preventive strategies used, with exception of vaccination alone, decreased the incidence of leishmaniasis significantly. Adverse events, mild and self-limiting in most of the cases, were reported in 5.2% of dogs and were significantly more common in dogs following vaccination. In conclusion, this is the first large-scale field study investigating the efficacy and safety of the preventive measures used routinely against leishmaniasis in client-owned dogs. Most preventive strategies used, with exception of vaccination alone, had some benefit over not applying any preventive. In this field study, the use of repellents showed a good degree of protection in dogs living in endemic areas of canine leishmaniasis. Although lower numbers of dogs are included, the use of domperidone appeared to provide additional protection. The role of vaccination and its combination with other preventive strategies needs further study.