Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs.

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.

Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in-house ELISA.

BACKGROUND: Canine leishmaniosis caused by the protozoan Leishmania infantum is a complex infection due to its variable clinical signs and laboratory findings. Therefore, a broad range of techniques is available for diagnosis. Testing for specific antibodies in serum is the most commonly used technique, although the testing of other body fluids, such as oral transudate (OT), can be an alternative as its collection is non-invasive and testing can be performed by untrained personnel. The aim of this study was to assess and compare the detection of L. infantum-specific antibodies in paired samples of serum and OT collected from apparently healthy dogs and dogs with clinical leishmaniosis using an in-house enyzme-linked immunosorbent assay (ELISA). METHODS: Serum and OT were collected from 407 dogs, which varied in breed, sex, age, lifestyle and clinical status, by many practicing veterinarians in Spain. The main geographical areas of sampling included Barcelona (n = 110), Mallorca (n = 94), Cadiz (n = 54) and Asturias (n = 47). The majority of infected dogs were apparently healthy (89.9%) while 41 presented clinical signs and/or clinicopathological abnormalities compatible with L. infantum infection and subsequently diagnosed with leishmaniosis (10.1%). An in-house ELISA was performed to quantify the anti-Leishmania antibodies in serum and OT. RESULTS: The L. infantum infection rate determined by the in-house ELISA was 37.1% in serum samples and 32.7% in OT samples. Serum and OT ELISA results showed a positive correlation (Spearman's correlation coefficient rs = 0.6687, P < 0.0001). The percent agreement between the serum and OT ELISA results was 84%, while agreement according to Cohen's kappa statistic (κ) was substantial (0.66) when all samples were analyzed. The highest percent agreement (92.1%) between both tests was found in dogs from low endemicity regions and from sick dogs, with both groups presenting almost perfect agreement according to Cohen's κ agreement test (0.84). Few seronegative dogs (n = 23) tested positive by the OT ELISA. The agreement between serum and OT went from almost perfect to moderate when the geographical distribution and clinical status were analyzed. CONCLUSIONS: The results of this study demonstrated an almost perfect to moderate agreement between OT and serum samples tested using the in-house ELISA. These results are particularly promising in sick dogs with high antibody levels while the results seem less optimal in apparently healthy dogs with low antibody levels.

Topical Amphotericin B Semisolid Dosage Form for Cutaneous Leishmaniasis: Physicochemical Characterization, Ex Vivo Skin Permeation and Biological Activity.

Amphotericin B (AmB) is a potent antifungal successfully used intravenously to treat visceral leishmaniasis but depending on the Leishmania infecting species, it is not always recommended against cutaneous leishmaniasis (CL). To address the need for alternative topical treatments of CL, the aim of this study was to elaborate and characterize an AmB gel. The physicochemical properties, stability, rheology and in vivo tolerance were assayed. Release and permeation studies were performed on nylon membranes and human skin, respectively. Toxicity was evaluated in macrophage and keratinocyte cell lines, and the activity against promastigotes and intracellular amastigotes of Leishmania infantum was studied. The AmB gel remained stable for a period of two months, with optimal properties for topical use and no apparent toxic effect on the cell lines. High amounts of AmB were found in damaged and non-damaged skin (1230.10 ± 331.52 and 2484.57 ± 439.12 µg/g/cm2, respectively) and they were above the IC50 of AmB for amastigotes. Although there were no differences in the in vitro anti-leishmanial activity between the AmB solution and gel, the formulation resulted in a higher amount of AmB being retained in the skin, and is therefore a candidate for further studies of in vivo efficacy.