Visceral leishmaniasis (VL) is a fatal manifestation of an infection caused by intracellular protozoa of the Leishmania genus. In New World countries, VL is classified as a zoonotic disease with domestic dogs acting as its main reservoir. Asymptomatic dogs are as competent to transmit Leishmania to the vectors as symptomatic dogs, however current diagnostic tests are limited and present low sensitivity for this important group. The development of accurate tests is fundamental to the early diagnosis, treatment, and control of canine leishmaniasis. In this study, we investigated the use of a recombinant protein (dynamin-1-like protein, Dyn-1) from L. infantum, as a potential target antigen for leishmaniasis serodiagnosis in both symptomatic and asymptomatic dogs. The antigenic performance of the protein was evaluated by means of ELISA assays using sera from symptomatic (n = 25), asymptomatic (n = 34) and non-infected dogs (n = 36) using ELISA. In addition, sera from dogs experimentally infected with Trypanosoma cruzi (n = 49) and naturally infected with Babesia sp. (n = 8) were tested to evaluate possible cross-reactivity. A crude soluble antigen (CSA) of Leishmania was used as an antigen control and K39 and K26 were used as reference antigens because they are already widely used in commercial tests. rDyn-1-based assay showed the highest sensitivity (97%) compared to the antigens K39 (88%), K26 (86%) and crude extract (95%). The highest specificity among the tests was also obtained with the protein rDyn-1 (94%), compared with the other antigens K39 (81%), K26 (87%), and crude extract (77%). This study showed that the rDyn-1 ELISA assay was able to identify 100% of asymptomatic dogs, establishing its potential as a target for the diagnosis of canine leishmaniasis.

Canine leishmaniosis (CanL) is a neglected zoonotic disease caused by Leishmania spp. Leishmania infantum is the species responsible for the zoonotic form of the disease where dogs are reservoir hosts. This study aimed to determine the seroprevalence of CanL in asymptomatic dogs in Kosovo. Blood samples were collected from 285 dogs in all seven regions in Kosovo (35-50 samples per region) from summer 2021 to spring 2022. Sera were tested using enzyme-linked immunosorbent assay (ELISA), and the presence of anti-Leishmania IgG was confirmed by an indirect fluorescent antibody test (IFAT). The true overall seroprevalence of CanL of asymptomatic dogs in Kosovo with ELISA was 4.21% (95% CI: 2.42-7.21) while with IFAT was 3.51% (95% CI: 1.92-6.34). The highest rates were found in the Prishtina region to be 8.0% (4/50) by ELISA and 6.0% (3/50) by IFAT, and in the Mitrovica region, the prevalence was 0% (0/40). There were no significant differences among the different regions, gender, age, health status, and breed. These findings highlight the presence of CanL in most regions of Kosovo and underline the veterinary relevance of clinically asymptomatic dogs infected with Leishmania.

The high prevalence of Leishmania infection was reported in dogs as the main reservoir of CanL in many locations in the old world. Detection and firmly identification of Leishmania species in asymptomatic dogs by reliable method was considered and employed. Non-invasive and non-anesthetized blood sampling in asymptomatic dogs was conducted. Nested, conventional and real-time PCR with HRM technique was performed targeting ITS-rDNA gene. 88 asymptomatic dogs were sampled from three CanL endemic provinces of Iran in 2018-2019. 23 blood samples were Leishmania positive. L. major, L. tropica and L. infantum were accurately identified for the first time with HRM targeting ITS2-microsatellite. Three samples were mixed infections. CLC software TM predictions for microsatellite ITS-rDNA were 86.93 °C: L. major, 85.76 °C: L. tropica and 86.04 °C: L. infantum. Standard strains of Leishmania species were accurately separated with almost one to 2 °C deference (L. major: 86.61 °C, L. infantum: 85.41 °C, L. tropica: 84.82 °C). Each HRM curve represents one species in a sample for helping accurate identification of Leishmania species and even mixed infection when two curves are present. Detecting parasites at primary stages in asymptomatic cases is essential using Real-time HRM. As same as mammalian Leishmania in rodents which is present at early stages and non-pathogenesis, only L. major would exist and other Leishmania disappears. This can conclude also for L. major, L. infantum and L. tropica in dogs. The role of L. major existence in canine blood should be investigated more.

Although serological assays have been widely used for the diagnosis of canine visceral leishmaniasis (CVL), they present different performances depending on the clinical profile of the dogs. This study evaluated the accuracy of serological tests, immunochromatographic (Dual Path Platform: DPP®) and enzyme-linked immunosorbent (ELISA EIE®), for CVL in relation to the detection of Leishmania DNA through real-time polymerase chain reaction (real-time PCR) in samples from symptomatic and asymptomatic dogs from a non-endemic area in the state of Rio Grande do Sul, Southern Brazil. Serum from 140 dogs (39 symptomatic and 101 asymptomatic) was tested by DPP and ELISA followed by real-time PCR. From a total of 140 samples evaluated, Leishmania DNA was detected by real-time PCR in 41.4% (58/140). Moreover, 67.2% of samples positive in real-time PCR were positive in both DPP and ELISA (39/58), showing moderate agreement between methods. In the symptomatic group, one sample non-reactive in both serological assays was positive in real-time PCR, whereas in the asymptomatic group, 17.8% non-reactive or undetermined samples in serological assays were positive in the molecular method. Leishmania DNA was not detected in 17.9% reactive samples by serological assays from the symptomatic group, and in 3.9% from asymptomatic dogs. Real-time PCR demonstrated greater homogeneity between symptomatic and asymptomatic groups compared with DPP and ELISA. The molecular method can help to establish the correct CVL diagnosis, particularly in asymptomatic dogs, avoiding undesirable euthanasia.