Background: Leishmania infantumis the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate.Methods: In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated.Results: The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased.Conclusion: The data indicates an increasedL. infantumresistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number ofL. infantumstrains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated.Graphical

Background and Aim: Although wild boar hunting activities and the hunting dog trade in the Atlantic Forest and Cerrado biomes of Brazil overlap both with endemic and with non-endemic areas for visceral leishmaniasis, no study to date has focused on Leishmania spp. exposure among hunting dogs and hunters. The aim of the present study was to assess the presence of Leishmania spp. antibodies in hunting dogs and hunters in different anthropized areas of two Brazilian biomes. Materials and Methods: Blood samples were collected from 170 hunting dogs and 46 hunters between October 2016 and May 2018. The presence of antibodies against Leishmania spp. in hunting dogs was screened through a dual-path platform immunochromatographic test (DPP rapid test; Bio-Manguinhos/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil) and in hunters through an rK39-based rapid immunochromatographic test. Both tests were used in accordance with Brazilian Ministry of Health recommendations. Results: Overall, although antibodies were detected through the immunochromatographic test in 3/170 (0.02%) of these female asymptomatic hunting dogs, all living in anthropized areas of the Atlantic Forest biome in South Brazil, no sample was confirmed through the enzyme-linked immunosorbent assay. All the hunters were non-reactive in the rapid immunochromatographic test. Conclusion: Our study on three suspicious hunting dogs has suggested that Leishmania (Leishmania) infantum may circulate both in endemic and non-endemic areas in Brazil. In addition, a high rate of hunting dog replacement due to death and trade may have led to less chance of infection and transmission between animals and between animals and humans, which would corroborate the outcomes reported here. Further studies should be conducted to fully establish whether hunting dogs and hunters may be used as sentinels in other areas endemic for Leishmania spp.

This research aimed to carry out an epidemiological investigation of dogs with canine leishmaniasis in an area of Rio Grande do Sul with no history of notification of the disease. Leishmaniasis is considered an endemic zoonosis in different countries. In Brazil, it occurs mainly in the Southeast and Midwest regions, having drastically increased the number of notifications in recent years. In the state of Rio Grande do Sul, there are reports of the disease in the metropolitan region of Porto Alegre and in the border region in the west of the state, as well as in the central region. The investigation was conducted in 100 dogs from the municipalities of Frederico Westphalen and region. Whole blood was collected for immunochromatographic testing (TR DPP® Canine Visceral Leishmaniasis) and lymph node aspiration. Positives were confirmed by ELISA test and indirect immunofluorescence test with total dilution or immunohistochemistry after skin biopsy. Two animals were diagnosed with visceral leishmaniasis, considered the first reported cases of the disease in the canine population in the region. Therefore, it becomes necessary to research and control the potential presence of sandflies in the area. There is a hypothesis that the animals were contaminated in other regions of the country, remaining as a reservoir until the moment of diagnosis.

Backgroud/Objectives: Canine Visceral Leishmaniasis (CVL), caused by Leishmania infantum, is a significant public health concern due to dogs serving as reservoirs for human infection. An accurate and rapid diagnostic method to distinguish symptomatic and asymptomatic CVL from healthy and vaccinated animals is essential for controlling canine and human disease. Developing innovative antibody detection techniques and exploring new antigens are essential for enhancing CVL testing efficiency. Our study focuses on a multiplex flow cytometry technique to detect Leishmania-specific antibodies in canine serum. This involved conjugating small peptides with carrier proteins or peptide tags, sequences designed to facilitate bead coupling. Methods: A peptide from the L. infantum A2 protein was coupled to beads in three forms: unconjugated, conjugated with BSA, and conjugated with a C-terminal β-alanine–lysine (x4)–cysteine TAG. This TAG was previously designed to enhance peptide solubility, improve binding efficiency, and provide functional groups for covalent attachment to the beads, ensuring stable immobilization in the multiplex assay. Results: Our results suggest that the multiplex approach shows promise as a rapid serological test for CVL, particularly with TAG-conjugated peptides, which optimize bead coupling. However, peptide/BSA conjugation revealed anti-BSA antibodies in samples from healthy and CVL dogs. Conclusions: In conclusion, our findings highlight the potential of multiplex methodologies to enhance CVL diagnostics and caution against using BSA as a bead coupling agent in serological tests for canine samples due to its impact on test specificity and sensitivity.

This research reports the clinical, histological, immunohistochemical, and molecular findings of a case of leishmaniasis by Leishmania (Viannia) in an equid in the state of Goiás, Brazil. Nodular lesions, sometimes crusty or ulcerated, were observed on the auricular pavilion, neck, and limbs of a four-year-old adult female Quarter Horse. Histomorphological findings indicated granulomatous dermatitis, revealing parasitic structures within macrophages, exhibiting an ovoid to slightly elongated morphology, measuring 2-5 µm, with a rounded nucleus and rod-shaped kinetoplast. Immunohistochemical examination showed positive staining of amastigotes of Leishmania spp. The Polymerase Chain Reaction (PCR) technique, followed by sequencing and BLASTn analysis, enabled the comparison of the percent similarity between the obtained sequences and those deposited in GenBank, which revealed a 100% of identity with sequences of the subgenus Viannia and 97.27% identity with sequences of Leishmania (Viannia) braziliensis deposited in the database. The importance of including leishmaniasis in the differential diagnosis of cutaneous nodular diseases in equids is emphasized, as well as the need for studies to elucidate the importance of these animals in the biological cycle of leishmaniasis.