Johannes S. P. Doehl

Acquired tick resistance (ATR) is well characterized in tick‐exposed animals, compromising tick fitness through antibody‐mediated activation of basophils. Yet, anti‐tick vaccines inducing ATR have had limited success. Here, we describe a neuroimmune event preceding ATR that leads to rapid host‐mediated tick removal. Tick‐sensitized guinea pigs mechanically remove ticks within 3–6 h via an acquired neuroimmune‐induced itch response that correlates with increased dermal expression of itch‐associated genes and skin infiltration by CD3 ⁺ T cells and Iba‐1 ⁺ macrophages, independent of IgG and IgE antibodies. Inhibiting the development of acquired T cell memory by averting naive lymphocyte egress from lymph nodes with the sphingosine‐1‐phosphate receptor modulator FTY720, before tick sensitization, prevents Iba‐1 ⁺ macrophage and CD3 ⁺ T cell infiltration to the tick bite site and abrogates scratching and tick removal. This neuroimmune response is independent of Trpv1 as tick‐sensitized guinea pigs treated with the Trpv1 agonist, resiniferatoxin, remove ticks effectively. Strikingly, prior exposure to a single tick is sufficient to generate a fast and active itch‐induced tick removal (IITR) that is observed even in tick‐attached sites distant from the location of previous tick exposure. IITR represents a novel approach to tick‐borne disease prevention through early tick detection and fast removal.

Mass drug administration (MDA) is a powerful tool for malaria control, but the medicines to use, dosing, number of rounds, and potential selection of drug resistance remain open questions. Two monthly rounds of artemisinin-piperaquine (AP), each comprising two daily doses, were administered across the seven districts of Grande Comore Island. In three districts, low-dose primaquine (PMQ_LD) was also given on the first day of each monthly round. Plasmodium falciparum malaria rates, mortality, parasitemias, adverse events, and genetic markers of potential drug resistance were evaluated. Average population coverages of 80–82% were achieved with AP in four districts (registered population 258,986) and AP+PMQ_LD in three districts (83,696). The effectiveness of MDA was 96.27% (95% CI: 95.27, 97.06%; pLD districts. In comparative statistical modeling, the effectiveness of the two monthly rounds on Grande Comore Island was nearly as high as that of three monthly rounds of AP or AP+PMQ_LD in our earlier study on Anjouan Island. Surveys of pre-MDA and post-MDA samples showed no significant changes in PfK13 polymorphism rates, but low rates of PfCRT M343L and G353V raise concern as these mutations have been linked to piperaquine resistance. MDA with two monthly rounds of two daily doses of AP was highly effective on Grande Comore Island. The feasibility and lower expenses of this two-month vs. three-month regimen of AP may offer advantages for MDA programs in appropriate settings.

Aedes mosquitoes are some of the most important and globally expansive vectors of disease. Public health efforts are largely focused on prevention of human-vector contact. A range of entomological indices are used to measure risk of disease, though with conflicting results (i.e. larval or adult abundance does not always predict risk of disease). There is a growing interest in the development and use of biomarkers for exposure to mosquito saliva, including for Aedes spp , as a proxy for disease risk. In this study, we conduct a comprehensive geostatistical analysis of exposure to Aedes mosquito bites among a pediatric cohort in a peri-urban setting endemic to dengue, Zika, and chikungunya viruses. We use demographic, household, and environmental variables (the flooding index (NFI), land type, and proximity to a river) in a Bayesian geostatistical model to predict areas of exposure to Aedes aegypti bites. We found that hotspots of exposure to Ae. aegypti salivary gland extract (SGE) were relatively small (< 500m and sometimes < 250m) and stable across the two-year study period. Age was negatively associated with antibody responses to Ae. aegypti SGE. Those living in agricultural settings had lower antibody responses than those living in urban settings, whereas those living near recent surface water accumulation were more likely to have higher antibody responses. Finally, we incorporated measures of larval and adult density in our geostatistical models and found that they did not show associations with antibody responses to Ae. aegypti SGE after controlling for other covariates in the model. Our results indicate that targeted house- or neighborhood-focused interventions may be appropriate for vector control in this setting. Further, demographic and environmental factors more capably predicted exposure to Ae. aegypti mosquitoes than commonly used entomological indices. Keywords: Aedes; saliva; geostatistical; environmental; dengue fever; Zika; chikungunya

Increasing evidence suggests that the infectiousness of patients for the sand fly vector of visceral leishmaniasis is linked to parasites found in the skin. Using a murine model that supports extensive skin infection withLeishmania donovani, spatial analyses at macro-(quantitative PCR) and micro-(confocal microscopy) scales indicate that parasite distribution is markedly skewed. Mathematical models accounting for this heterogeneity demonstrate that while a patchy distribution reduces the expected number of sand flies acquiring parasites, it increases the infection load for sand flies feeding on a patch, increasing their potential for onward transmission. Models representing patchiness at both macro- and micro-scales provide the best fit with experimental sand fly feeding data, pointing to the importance of the skin parasite landscape as a predictor of host infectiousness. Our analysis highlights the skin as a critical site to consider when assessing treatment efficacy, transmission competence and the impact of visceral leishmaniasis elimination campaigns.

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150⁺ CD34^- CD48^- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4⁺ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4⁺ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ⁺CD4⁺ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4⁺ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously. Visceral leishmaniasis (VL) is a chronic often fatal disease caused by the protozoan parasites Leishmania donovani and L. infantum. Progressive disease in humans and in animal models is associated with parasite replication at systemic sites, including the bone marrow (BM) and results in significant changes in hematological profile. The mechanisms underlying hematologic dysregulation during infection are largely unknown. Using a panel of stem cell markers, we characterized murine haematopoietic stem and progenitor cells in the BM over the course of L. donovani-infection in C57BL/6 (B6) mice. Most long-term hematopoietic stem cells (LT-HSCs) in naïve mice are found in a quiescent state, representing cells with the highest degree of reconstitution potential. In contrast, during L. donovani infection, most LT-HSCs had entered cell-cycle and this correlated with a reduced potential to engraft into syngeneic recipients. HSC exhaustion and other alterations in the hematopoietic compartment did not occur in infected immunodeficient mice, but adoptive transfer of IFNγ-sufficient CD4⁺ T cells restored this phenotype. Using mixed BM chimeras, we established that IFNγ signalling and TNF signalling pathways converge at the level of BM CD4⁺ T cells, with intrinsic TNF signalling being critical for the expansion / differentiation of CD4⁺ T cells that are responsible for HSC exhaustion. Contrary to commonly held views, in the setting of experimental visceral leishmaniasis neither IFNγ nor TNF signalling in HSCs was required for their functional exhaustion. Hence, pro-inflammatory cytokines commonly associated with host protection in leishmaniasis and many other infectious diseases can also drive the development of pathogenic CD4⁺ T cells that cause long term irreversible alterations in HSC function.

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation. Millions of people around the world are at risk of infection with single-celled Leishmania parasites that cause a wide range of infectious diseases of the immune system, the leishmaniases. There is no effective vaccine for these infections while available drugs have toxic side-effects and resistance is an increasing problem. Human infection occurs through the bite of infected blood-feeding sand flies. Leishmania parasites live in the sand fly gut, where they complete a complex series of developmental changes to become infective to mammals. The parasites also modify the insect gut to promote their own transmission. Little is known about the molecular regulation of these processes. Recently, we showed that deletion of a small group of related genes from the parasite prevented completion of its development in the sand fly, suggesting a role for these sequences in transmission to the host. This study clarifies the expression pattern of these genes during parasite development and shows that the observed stalling of development is accompanied by changes in parasite-sand fly gut interactions and a loss of parasite transmission. These target genes also influence disease development in the mammalian host, confirming critical roles for their encoded proteins throughout the parasite life cycle.

Summary: Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole‐like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild‐type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.