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1.  Evidence of Leishmania infantum Infection in Rabbits (Oryctolagus cuniculus) in a Natural Area in Madrid, Spain 
BioMed Research International  2014;2014:318254.
Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.
PMCID: PMC3958779  PMID: 24724079
2.  Erratum to 
Autophagy  2012;8(7):1163.
PMCID: PMC3429560
Lafora disease; autophagy; glycogen metabolism; laforin; malin; neurodegeneration
3.  Kinetic Analysis of Ex Vivo Human Blood Infection by Leishmania 
The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0–5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k+1) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills ∼95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k+1 for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which ∼50% and ∼25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209+ dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.
Author Summary
Leishmania infection is transmitted to mammalian hosts by phlebotomine sand flies. During the vector's bloodmeal, promastigotes are inoculated into hemorrhagic spots in the skin or are delivered into the extracellular matrix of the dermis. In the first case, blood is involved in transmission; in the second, it apparently is not. This is important, as the cellular milieu of infection can be critical for induction of the anti-parasite immune response and the subsequent course of disease. In humans, there are few comprehensive studies of the initial stages of Leishmania transmission in blood. Using blood to mimic the skin hematoma, we carried out kinetic and quantitative analyses of the reaction with serum and blood components of promastigotes from two Leishmania species with different tropism. We describe the kinetics of the promastigote reaction pathway that leads to blood infection and provide quantitative data for the cell types infected in the first five minutes of leishmaniosis transmission.
PMCID: PMC2903471  PMID: 20644618
4.  Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum 
In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement.
PMCID: PMC2193616  PMID: 11854358
Trypanosomatids; Leishmania; complement opsonization; promastigote lysis; human serum
5.  Immune Adherence–mediated Opsonophagocytosis:  The Mechanism of Leishmania Infection  
To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote–host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti–Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes. Progression of infection implies promastigote transfer from erythrocytes to acceptor blood leukocytes. After 10 min of ex vivo infection, 25% of all leukocytes contain intracellular parasites, indicating that blood cells are the early targets for the invading promastigotes. We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes. This facilitates host colonization and may represent the parasite's earliest survival strategy. In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.
PMCID: PMC1887685  PMID: 9874561
Leishmania; infection; immune adherence; natural antibodies; complement

Results 1-5 (5)