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 107 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.

BackgroundIn Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear.MethodsIn this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy.ResultsLate-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi.ConclusionOur data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L. turanica to establish late-stage infections in these specific vectors of L. major suggests that the lipophosphoglycan of this species must be identical or similar to that of L. major.

Human indigenous cutaneous leishmaniasis caused by Leishmania donovani complex is endemic in Sri Lanka. We performed an entomological survey to determine the distribution of probable vector species. Sand flies were collected in districts in the dry zone, in the wet zone highlands, and in the wet zone coastal belt of Sri Lanka using CDC light traps, sticky traps and cattle-baited net traps during July, 2005. The survey was reconducted in February, 2006. Overall, 584 sand flies belonging to Phlebotomus (266 specimens, 2 species) and Sergentomyia (318 specimens, 8 species) genera were collected. A total of 266 Phlebotomus was identified as P. argentipes (258/266; 97%) and P. stantoni (8/266; 3%). The identification studies of Sergentomyia specimens showed that there are at least 8 species in Sri Lanka. Higher number of Phlebotomus sand flies (76/266) were caught in the southern part of the country compared to the other parts probably due to different ecological aspects. P. argentipes were widely distributed throughout the island whereas P. stantoni were collected only in four districts. Since P. argentipes is known to be the vector of L. donovani responsible of visceral leishmaniasis in India, this species may be incriminated as the most possible vector of human cutaneous leishmaniasis in Sri Lanka. Journal of Vector Ecology 36 (Supplement 1): S77-S86. 2011.

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.

One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection. Leishmania amazonensis-infected mice show similar ulcerative lesions. Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from either Leishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4 ؉ , CD8 ؉ , or B220 ؉ cell populations and the remaining cells were injected into Leishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4 ؉ cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4 ؉ cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.

No abstract

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.

Abstract. Elevation of serum B-cell activating factor (BAFF) is one of the characteristics of immunological disorders, including autoimmunity, but the levels of BAFF in infectious diseases have not been studied well. Here, we showed the elevation of serum BAFF in patients with visceral leishmaniasis (VL). The mean serum BAFF value in VL patients (4.65 ng/mL) was 4.3 times higher than that of healthy controls (1.08 ng/mL), and 90% of VL patients showed serum BAFF above the cutoff that was calculated as the mean + 3 SDs of the controls. This report is the first on elevation of serum BAFF during VL.Leishmaniasis is a spectrum of diseases caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) is the most severe form, generally caused by L. donovani and L. infantum. Clinical manifestations of VL include fever, anemia, and splenomegaly, and the disease is fatal if left untreated. Protective immunity against VL is associated with antigen-specific cell-mediated responses represented by lymphoproliferation and delayed-type hypersensitivity 1 and production of type 1 helper T cell (Th1) cytokines, like interferon-γ (IFN-γ) and interleukin-2 (IL-2), on antigen recall.2 In contrast, IL-10, which is associated with T-cell hyporesponsiveness, is the predominant cytokine during active VL.3 Other than the suppressed Th1 responses, immunological characteristics of active VL include strong humoral responses. In fact, high immunoglobulin G (IgG) antibodies and delayed type hypersensitivity to parasite antigens are dichotomic factors in VL, 4 and excess B-cell activation in those patients can also be presumed by the manifestation of hypergammaglobulinemia.