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Abstract Background Leishmaniasis is not endemic in Japan, and imported cases are rare. However, there are increasing concerns regarding imported cases of cutaneous leishmaniasis from endemic countries to Japan. This report describes a case of imported cutaneous leishmaniasis that was diagnosed and treated in Japan. Case presentation A 53-year-old Pakistani man presented with skin lesions on both malleoli of his right ankle and the dorsum of the left foot. The skin lesions manifested as erythematous nodules surrounding an ulcer in the center of the lesion. The lesions of the malleoli of his right ankle each measured 3 × 3 cm, and the lesion on the top of his left foot measured 5 × 4 cm. He had been living and working in Japan but had a history of a visit to Pakistan for about 2 months in 2018. The skin lesions were biopsied. Giemsa and hematoxylin and eosin staining of biopsy samples showed amastigotes of Leishmania in macrophages, and the presence of Leishmania was confirmed by skin tissue culture. Polymerase chain reaction using biopsy specimens identified Leishmania parasites, and DNA sequence analysis revealed that the species was Leishmania tropica. The patient was treated with intravenous liposomal amphotericin B for 6 days. The erythema disappeared, and the erythematous nodules resolved within 3 weeks. Conclusion This is the first report of imported cutaneous leishmaniasis caused by L. tropica from Pakistan, and it is interesting that all three testing modalities showed positive results in this case.

Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14+ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14+ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL.

Visceral leishmaniasis (VL) is caused by parasitic protozoa of the genus Leishmania and is characterized by clinical manifestations such as fever, hepatosplenomegaly and anemia. Hemophagocytosis, the phenomenon of phagocytosis of blood cells by macrophages, is found in VL patients. In a previous study we established an experimental model of VL, reproducing anemia in mice for the first time, and identified hemophagocytosis by heavily infected macrophages in the spleen as a possible cause of anemia. However, the mechanism for parasite-induced hemophagocytosis or its role in parasite survival remained unclear. Here, we established an in vitro model of Leishmania-induced hemophagocytosis to explore the molecules involved in this process. In contrast to naïve RAW264.7 cells (mouse macrophage cell line) which did not uptake freshly isolated erythrocytes, RAW264.7 cells infected with L. donovani showed enhanced phagocytosis of erythrocytes. Additionally, for hemophagocytes found both in vitro and in vivo, the expression of signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal was suppressed by post-transcriptional control. Furthermore, the overlapped phagocytosis of erythrocytes and Leishmania parasites within a given macrophage appeared to be beneficial to the parasites; the in vitro experiments showed a higher number of parasites within macrophages that had been induced to engulf erythrocytes. Together, these results suggest that Leishmania parasites may actively induce hemophagocytosis by manipulating the expression of SIRPα in macrophages/hemophagocytes, in order to secure their parasitism.

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL.

Hemophagocytosis is a phenomenon in which macrophages phagocytose blood cells. There are reports on up-regulated hemophagocytosis in patients with infectious diseases including typhoid fever, tuberculosis, influenza and visceral leishmaniasis (VL). However, mechanisms of infection-associated hemophagocytosis remained elusive due to a lack of appropriate animal models. Here, we have established a mouse model of VL with hemophagocytosis. At 24 weeks after infection with 1 x 10(7) Leishmania donovani promastigotes, BALB/cA mice exhibited splenomegaly with an average tissue weight per body weight of 2.96%. In the tissues, 28.6% of macrophages contained phagocytosed erythrocytes. All of the hemophagocytosing macrophages were parasitized by L. donovani, and higher levels of hemophagocytosis was observed in heavily infected cells. Furthermore, more than half of these hemophagocytes had two or more macrophage-derived nuclei, whereas only 15.0% of splenic macrophages were bi- or multi-nuclear. These results suggest that direct infection by L. donovani causes hyper-activation of host macrophages to engulf blood cells. To our knowledge, this is the first report on hemophagocytosis in experimental Leishmania infections and may be useful for further understanding of the pathogenesis.

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.