Establishment and partial characterisation of a new cell line derived from adult tissues of the tsetse fly Glossina morsitans morsitans.

BACKGROUND: Insect cell lines play a vital role in many aspects of research on disease vectors and agricultural pests. The tsetse fly Glossina morsitans morsitans is an important vector of salivarian trypanosomes in sub-Saharan Africa and, as such, is a major constraint on human health and agricultural development in the region. METHODS: Here, we report establishment and partial characterisation of a cell line, GMA/LULS61, derived from tissues of adult female G. m. morsitans. GMA/LULS61 cells, grown at 28 °C in L-15 (Leibovitz) medium supplemented with foetal bovine serum and tryptose phosphate broth, have been taken through 23 passages to date and can be split 1:1 at 2-week intervals. Karyotyping at passage 17 revealed a predominantly haploid chromosome complement. Species origin and absence of contaminating bacteria were confirmed by PCR amplification and sequencing of fragments of the COI gene and pan-bacterial 16S rRNA gene respectively. However, PCR screening of RNA extracted from GMA/LULS61 cells confirmed presence of the recently described Glossina morsitans morsitans iflavirus and Glossina morsitans morsitans negevirus, but absence of Glossina pallipides salivary gland hypertrophy virus. GMA/LULS61 cells supported infection and growth of 6/7 different insect-derived strains of the intracellular bacterial symbiont Wolbachia. CONCLUSIONS: The GMA/LULS61 cell line has potential for application in a variety of studies investigating the biology of G. m. morsitans and its associated pathogenic and symbiotic microorganisms.

No evidence for direct thermal carryover effects on starvation tolerance in the obligate blood-feeder, Glossina morsitans morsitans.

Thermal stress during development can prime animals to cope better with similar conditions in later life. Alternatively, negative effects of thermal stress can persist across life stages and result in poorer quality adults (negative carryover effects). As mean temperatures increase due to climate change, evidence for such effects across diverse taxa is required. Using Glossina morsitans morsitans, a species of tsetse fly and vector of trypanosomiasis, we asked whether (i) adaptive developmental plasticity allows flies to survive for longer under food deprivation when pupal and adult temperatures are matched; or (ii) temperature stress during development persists into adulthood, resulting in a greater risk of death. We did not find any advantage of matched pupal and adult temperature in terms of improved starvation tolerance, and no direct negative carryover effects were observed. There was some evidence for indirect carryover effects-high pupal temperature produced flies of lower body mass, which, in turn, resulted in greater starvation risk. However, adult temperature had the largest impact on starvation tolerance by far: flies died 60% faster at 31°C than those experiencing 25°C, consequently reducing survival time from a median of 8 (interquartile range (IQR) 7-9) to 5 (IQR 5-5.25) days. This highlights differences in temperature sensitivity between life stages, as there was no direct effect of pupal temperature on starvation tolerance. Therefore, for some regions of sub-Saharan Africa, climate change may result in a higher mortality rate in emerging tsetse while they search for their first blood meal. This study reinforces existing evidence that responses to temperature are life stage specific and that plasticity may have limited capacity to buffer the effects of climate change.

Gastrointestinal parasites in captive olive baboons in a UK safari park.

From the safety inside vehicles, Knowsley Safari offers visitors a close-up encounter with captive olive baboons. As exiting vehicles may be contaminated with baboon stool, a comprehensive coprological inspection was conducted to address public health concerns. Baboon stools were obtained from vehicles, and sleeping areas, inclusive of video analysis of baboon­vehicle interactions. A purposely selected 4-day sampling period enabled comparative inspections of 2662 vehicles, with a total of 669 baboon stools examined (371 from vehicles and 298 from sleeping areas). As informed by our pilot study, front-line diagnostic methods were: QUIK-CHEK rapid diagnostic test (RDT) (Giardia and Cryptosporidium), Kato­Katz coproscopy (Trichuris) and charcoal culture (Strongyloides). Some 13.9% of vehicles were contaminated with baboon stool. Prevalence of giardiasis was 37.4% while cryptosporidiosis was <0.01%, however, an absence of faecal cysts by quality control coproscopy, alongside lower than the expected levels of Giardia-specific DNA, judged RDT results as misleading, grossly overestimating prevalence. Prevalence of trichuriasis was 48.0% and strongyloidiasis was 13.7%, a first report of Strongyloides fuelleborni in UK. We advise regular blanket administration(s) of anthelminthics to the colony, exploring pour-on formulations, thereafter, smaller-scale indicator surveys would be adequate.

Tsetse flies (Glossina morsitans morsitans) choose birthing sites guided by substrate cues with no evidence for a role of pheromones.

Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is, therefore, crucial for offspring survival. Previous laboratory studies suggested that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the physical characteristics found in the wild. We show that gravid G. m. morsitans were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. By contrast, females appear to choose larviposition sites based on environmental substrate cues. We conclude that, among the many cues that likely contribute to larviposition choice in nature, substrate features are a main determinant, while we failed to find evidence for a role of pheromones.

Improving the detection of Angiostrongylus cantonensis in the brain tissues of mammalian hosts.

Angiostrongylus cantonensis is an invasive nematode parasite that causes eosinophilic meningitis in many vertebrate hosts, including humans. This parasite is spreading rapidly through the six continents, with Europe being the final frontier. Sentinel surveillance may be a cost-effective strategy to monitor the pathogen's arrival to new geographical regions. Necropsy, followed by tissue digestion, is often used to recover helminth parasites from vertebrate host tissues, however, to detect brain parasites, this protocol is poorly utilized. Our brain digestion protocol is easily performed and 1) reduces false positivity and negativity, 2) provides accurate estimates of parasite burden and 3) helps establish a more precise prevalence. Early detection of A. cantonensis increases the efficacy of prevention, treatment, and disease control strategies for susceptible animal populations and humans.

The Trypanosoma brucei MISP family of invariant proteins is co-expressed with BARP as triple helical bundle structures on the surface of salivary gland forms, but is dispensable for parasite development within the tsetse vector.

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected tsetse flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of glycosylphosphatidylinositol (GPI)-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin.

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

Oxidative Phosphorylation Is Required for Powering Motility and Development of the Sleeping Sickness Parasite Trypanosoma brucei in the Tsetse Fly Vector.

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought.

Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi.

Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host-endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

Effects of maternal age and stress on offspring quality in a viviparous fly.

Many organisms show signs of deterioration with age in terms of survival and reproduction. We tested whether intraspecific variation in such senescence patterns can be driven by resource availability or reproductive history. We did this by manipulating nutritional stress and age at first reproduction and measuring age-dependent reproductive output in tsetse (Glossina morsitans morsitans), a viviparous fly with high maternal allocation. Across all treatments, offspring weight followed a bell-shaped curve with maternal age. Nutritionally stressed females had a higher probability of abortion and produced offspring with lower starvation tolerance. There was no evidence of an increased rate of reproductive senescence in nutritionally stressed females, or a reduced rate due to delayed mating, as measured by patterns of abortion, offspring weight or offspring starvation tolerance. Therefore, although we found evidence of reproductive senescence in tsetse, our results did not indicate that resource allocation trade-offs or costs of reproduction increase the rate of senescence.

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Big Baby, Little Mother: Tsetse Flies Are Exceptions to the Juvenile Small Size Principle.

While across the animal kingdom offspring are born smaller than their parents, notable exceptions exist. Several dipteran species belonging to the Hippoboscoidea superfamily can produce offspring larger than themselves. In this essay, the blood-feeding tsetse is focused on. It is suggested that the extreme reproductive strategy of this fly is enabled by feeding solely on highly nutritious blood, and producing larval offspring that are soft and malleable. This immense reproductive expenditure may have evolved to avoid competition with other biting flies. Tsetse also transmit blood-borne parasites that cause the fatal diseases called African trypanosomiases. It is discussed how tsetse life history and reproductive strategy profoundly influence the type of vector control interventions used to reduce fly populations. In closing, it is argued that the unusual life history of tsetse warrants their preservation in the areas where human and animal health is not threatened.

An Intranuclear Sodalis-Like Symbiont and Spiroplasma Coinfect the Carrot Psyllid, Bactericera trigonica (Hemiptera, Psylloidea).

Endosymbionts harbored inside insects play critical roles in the biology of their insect host and can influence the transmission of pathogens by insect vectors. Bactericera trigonica infests umbelliferous plants and transmits the bacterial plant pathogen Candidatus Liberibacter solanacearum (CLso), causing carrot yellows disease. To characterize the bacterial diversity of B. trigonica, as a first step, we used PCR-restriction fragment length polymorphism (PCR-RFLP) and denaturing gradient gel electrophoresis (DGGE) analyses of 16S rDNA to identify Sodalis and Spiroplasma endosymbionts. The prevalence of both symbionts in field-collected psyllid populations was determined: Sodalis was detected in 100% of field populations, while Spiroplasma was present in 82.5% of individuals. Phylogenetic analysis using 16S rDNA revealed that Sodalis infecting B. trigonica was more closely related to symbionts infecting weevils, stink bugs and tsetse flies than to those from psyllid species. Using fluorescent in situ hybridization and immunostaining, Sodalis was found to be localized inside the nuclei of the midgut cells and bacteriocytes. Spiroplasma was restricted to the cytoplasm of the midgut cells. We further show that a recently reported Bactericera trigonica densovirus (BtDNV), a densovirus infecting B. trigonica was detected in 100% of psyllids and has reduced titers inside CLso-infected psyllids by more than two-fold compared to CLso uninfected psyllids. The findings of this study will help to increase our understanding of psyllid-endosymbiont interactions.

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2-4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Cutaneous leishmaniasis and co-morbid major depressive disorder: A systematic review with burden estimates.

BACKGROUND: Major depressive disorder (MDD) associated with chronic neglected tropical diseases (NTDs) has been identified as a significant and overlooked contributor to overall disease burden. Cutaneous leishmaniasis (CL) is one of the most prevalent and stigmatising NTDs, with an incidence of around 1 million new cases of active CL infection annually. However, the characteristic residual scarring (inactive CL) following almost all cases of active CL has only recently been recognised as part of the CL disease spectrum due to its lasting psychosocial impact. METHODS AND FINDINGS: We performed a multi-language systematic review of the psychosocial impact of active and inactive CL. We estimated inactive CL (iCL) prevalence for the first time using reported WHO active CL (aCL) incidence data that were adjusted for life expectancy and underreporting. We then quantified the disability (YLD) burden of co-morbid MDD in CL using MDD disability weights at three severity levels. Overall, we identified 29 studies of CL psychological impact from 5 WHO regions, representing 11 of the 50 highest burden countries for CL. We conservatively calculated the disability burden of co-morbid MDD in CL to be 1.9 million YLDs, which equalled the overall (DALY) disease burden (assuming no excess mortality in depressed CL patients). Thus, upon inclusion of co-morbid MDD alone in both active and inactive CL, the DALY burden was seven times higher than the latest 2016 Global Burden of Disease study estimates, which notably omitted both psychological impact and inactive CL. CONCLUSIONS: Failure to include co-morbid MDD and the lasting sequelae of chronic NTDs, as exemplified by CL, leads to large underestimates of overall disease burden.

Wing length and host location in tsetse (Glossina spp.): implications for control using stationary baits.

BACKGROUND: It has been suggested that attempts to eradicate populations of tsetse (Glossina spp.) using stationary targets might fail because smaller, less mobile individuals are unlikely to be killed by the targets. If true, tsetse caught in stationary traps should be larger than those from mobile baits, which require less mobility on the part of the flies. RESULTS: Sampling tsetse in the Zambezi Valley of Zimbabwe, we found that the number of tsetse caught from stationary traps, as a percent of total numbers from traps plus a mobile vehicle, was ~5% for male G. morsitans morsitans (mean wing length 5.830 mm; 95% CI: 5.800-5.859 mm) and ~10% for females (6.334 mm; 95% CI: 6.329-6.338 mm); for G. pallidipes the figures were ~50% for males (6.830 mm; 95% CI: 6.821-6.838 mm) and ~75% for females (7.303 mm, 95% CI: 7.302-7.305 mm). As expected, flies of the smaller species (and the smaller sex) were less likely to be captured using stationary, rather than mobile sampling devices. For flies of a given sex and species the situation was more complex. Multivariable analysis showed that, for females of both species, wing lengths changed with ovarian age and the month, year and method of capture. For G. pallidipes, there were statistically significant interactions between ovarian age and capture month, year and method. For G. m. morsitans, there was only a significant interaction between ovarian age and capture month. The effect of capture method was, however, small in absolute terms: for G. pallidipes and G. m. morsitans flies caught on the mobile vehicle had wings only 0.24 and 0.48% shorter, respectively, than flies caught in stationary traps. In summary, wing length in field samples of tsetse varies with ovarian age, capture month and year and, weakly, with capture method. Suggestions that a target-based operation against G. f. fuscipes in Kenya caused a shift towards a smaller, less mobile population of tsetse, unavailable to the targets, failed to account for factors other than capture method. CONCLUSIONS: The results are consistent with the successful use of targets to eradicate populations of tsetse in Zimbabwe. Until further, more nuanced, studies are conducted, it is premature to conclude that targets alone could not, similarly, be used to eradicate G. f. fuscipes populations in Kenya.

Variant antigen repertoires in Trypanosoma congolense populations and experimental infections can be profiled from deep sequence data using universal protein motifs.

African trypanosomes are vector-borne hemoparasites of humans and animals. In the mammal, parasites evade the immune response through antigenic variation. Periodic switching of the variant surface glycoprotein (VSG) coat covering their cell surface allows sequential expansion of serologically distinct parasite clones. Trypanosome genomes contain many hundreds of VSG genes, subject to rapid changes in nucleotide sequence, copy number, and chromosomal position. Thus, analyzing, or even quantifying, VSG diversity over space and time presents an enormous challenge to conventional techniques. Indeed, previous population genomic studies have overlooked this vital aspect of pathogen biology for lack of analytical tools. Here we present a method for analyzing population-scale VSG diversity in Trypanosoma congolense from deep sequencing data. Previously, we suggested that T. congolense VSGs segregate into defined "phylotypes" that do not recombine. In our data set comprising 41 T. congolense genome sequences from across Africa, these phylotypes are universal and exhaustive. Screening sequence contigs with diagnostic protein motifs accurately quantifies relative phylotype frequencies, providing a metric of VSG diversity, called the "variant antigen profile." We applied our metric to VSG expression in the tsetse fly, showing that certain, rare VSG phylotypes may be preferentially expressed in infective, metacyclic-stage parasites. Hence, variant antigen profiling accurately and rapidly determines the T. congolense VSG gene and transcript repertoire from sequence data, without need for manual curation or highly contiguous sequences. It offers a tractable approach to measuring VSG diversity across strains and during infections, which is imperative to understanding the host-parasite interaction at population and individual scales.

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring.

BACKGROUND: As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys. METHODS AND FINDINGS: The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting. CONCLUSION: We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.

Understanding the transmission dynamics of Leishmania donovani to provide robust evidence for interventions to eliminate visceral leishmaniasis in Bihar, India.

Visceral Leishmaniasis (VL) is a neglected vector-borne disease. In India, it is transmitted to humans by Leishmania donovani-infected Phlebotomus argentipes sand flies. In 2005, VL was targeted for elimination by the governments of India, Nepal and Bangladesh by 2015. The elimination strategy consists of rapid case detection, treatment of VL cases and vector control using indoor residual spraying (IRS). However, to achieve sustained elimination of VL, an appropriate post elimination surveillance programme should be designed, and crucial knowledge gaps in vector bionomics, human infection and transmission need to be addressed. This review examines the outstanding knowledge gaps, specifically in the context of Bihar State, India.The knowledge gaps in vector bionomics that will be of immediate benefit to current control operations include better estimates of human biting rates and natural infection rates of P. argentipes, with L. donovani, and how these vary spatially, temporally and in response to IRS. The relative importance of indoor and outdoor transmission, and how P. argentipes disperse, are also unknown. With respect to human transmission it is important to use a range of diagnostic tools to distinguish individuals in endemic communities into those who: 1) are to going to progress to clinical VL, 2) are immune/refractory to infection and 3) have had past exposure to sand flies.It is crucial to keep in mind that close to elimination, and post-elimination, VL cases will become infrequent, so it is vital to define what the surveillance programme should target and how it should be designed to prevent resurgence. Therefore, a better understanding of the transmission dynamics of VL, in particular of how rates of infection in humans and sand flies vary as functions of each other, is required to guide VL elimination efforts and ensure sustained elimination in the Indian subcontinent. By collecting contemporary entomological and human data in the same geographical locations, more precise epidemiological models can be produced. The suite of data collected can also be used to inform the national programme if supplementary vector control tools, in addition to IRS, are required to address the issues of people sleeping outside.