A fine-tuned vector-parasite dialogue in tsetse's cardia determines peritrophic matrix integrity and trypanosome transmission success.

Arthropod vectors have multiple physical and immunological barriers that impede the development and transmission of parasites to new vertebrate hosts. These barriers include the peritrophic matrix (PM), a chitinous barrier that separates the blood bolus from the midgut epithelia and modulates vector-pathogens interactions. In tsetse flies, a sleeve-like PM is continuously produced by the cardia organ located at the fore- and midgut junction. African trypanosomes, Trypanosoma brucei, must bypass the PM twice; first to colonize the midgut and secondly to reach the salivary glands (SG), to complete their transmission cycle in tsetse. However, not all flies with midgut infections develop mammalian transmissible SG infections-the reasons for which are unclear. Here, we used transcriptomics, microscopy and functional genomics analyses to understand the factors that regulate parasite migration from midgut to SG. In flies with midgut infections only, parasites fail to cross the PM as they are eliminated from the cardia by reactive oxygen intermediates (ROIs)-albeit at the expense of collateral cytotoxic damage to the cardia. In flies with midgut and SG infections, expression of genes encoding components of the PM is reduced in the cardia, and structural integrity of the PM barrier is compromised. Under these circumstances trypanosomes traverse through the newly secreted and compromised PM. The process of PM attrition that enables the parasites to re-enter into the midgut lumen is apparently mediated by components of the parasites residing in the cardia. Thus, a fine-tuned dialogue between tsetse and trypanosomes at the cardia determines the outcome of PM integrity and trypanosome transmission success.

Mammalian African trypanosome VSG coat enhances tsetse's vector competence.

Tsetse flies are biological vectors of African trypanosomes, the protozoan parasites responsible for causing human and animal trypanosomiases across sub-Saharan Africa. Currently, no vaccines are available for disease prevention due to antigenic variation of the Variant Surface Glycoproteins (VSG) that coat parasites while they reside within mammalian hosts. As a result, interference with parasite development in the tsetse vector is being explored to reduce disease transmission. A major bottleneck to infection occurs as parasites attempt to colonize tsetse's midgut. One critical factor influencing this bottleneck is the fly's peritrophic matrix (PM), a semipermeable, chitinous barrier that lines the midgut. The mechanisms that enable trypanosomes to cross this barrier are currently unknown. Here, we determined that as parasites enter the tsetse's gut, VSG molecules released from trypanosomes are internalized by cells of the cardia-the tissue responsible for producing the PM. VSG internalization results in decreased expression of a tsetse microRNA (mir-275) and interferes with the Wnt-signaling pathway and the Iroquois/IRX transcription factor family. This interference reduces the function of the PM barrier and promotes parasite colonization of the gut early in the infection process. Manipulation of the insect midgut homeostasis by the mammalian parasite coat proteins is a novel function and indicates that VSG serves a dual role in trypanosome biology-that of facilitating transmission through its mammalian host and insect vector. We detail critical steps in the course of trypanosome infection establishment that can serve as novel targets to reduce the tsetse's vector competence and disease transmission.

What can a weevil teach a fly, and reciprocally? Interaction of host immune systems with endosymbionts in Glossina and Sitophilus.

The tsetse fly (Glossina genus) is the main vector of African trypanosomes, which are protozoan parasites that cause human and animal African trypanosomiases in Sub-Saharan Africa. In the frame of the IAEA/FAO program 'Enhancing Vector Refractoriness to Trypanosome Infection', in addition to the tsetse, the cereal weevil Sitophilus has been introduced as a comparative system with regards to immune interactions with endosymbionts. The cereal weevil is an agricultural pest that destroys a significant proportion of cereal stocks worldwide. Tsetse flies are associated with three symbiotic bacteria, the multifunctional obligate Wigglesworthia glossinidia, the facultative commensal Sodalis glossinidius and the parasitic Wolbachia. Cereal weevils house an obligatory nutritional symbiosis with the bacterium Sodalis pierantonius, and occasionally Wolbachia. Studying insect host-symbiont interactions is highly relevant both for understanding the evolution of symbiosis and for envisioning novel pest control strategies. In both insects, the long co-evolution between host and endosymbiont has led to a stringent integration of the host-bacteria partnership. These associations were facilitated by the development of specialized host traits, including symbiont-housing cells called bacteriocytes and specific immune features that enable both tolerance and control of the bacteria. In this review, we compare the tsetse and weevil model systems and compile the latest research findings regarding their biological and ecological similarities, how the immune system controls endosymbiont load and location, and how host-symbiont interactions impact developmental features including cuticle synthesis and immune system maturation. We focus mainly on the interactions between the obligate symbionts and their host's immune systems, a central theme in both model systems. Finally, we highlight how parallel studies on cereal weevils and tsetse flies led to mutual discoveries and stimulated research on each model, creating a pivotal example of scientific improvement through comparison between relatively distant models.

RNA interference-based antiviral immune response against the salivary gland hypertrophy virus in Glossina pallidipes.

BACKGROUND: Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded dsDNA virus that specifically infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae). GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can ultimately lead to a colony collapse. It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host's mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. Here, we investigated the involvement of RNAi during GpSGHV infections by comparing the expression of three key RNAi machinery genes, Dicer (DCR), Argonaute (AGO) and Drosha, in artificially virus injected, asymptomatic and symptomatic infected G. pallidipes flies compared to PBS injected (controls) individuals. We further assessed the impact of AGO2 knockdown on virus infection by RT-qPCR quantification of four selected GpSGHV genes, i.e. odv-e66, dnapol, maltodextrin glycosyltransferase (a tegument gene) and SGHV091 (a capsid gene). RESULTS: We show that in response to hemocoelic injections of GpSGHV into G. pallidipes flies, increased virus replication was accompanied by significant upregulation of the expression of three RNAi key genes; AGO1, AGO2 and DCR2, and a moderate increase in the expression of Drosha post injection compared to the PBS-injected controls. Furthermore, compared to asymptomatically infected individuals, symptomatic flies showed significant downregulation of AGO1, AGO2 and Drosha, but a moderate increase in the expression of DCR2. Compared to the controls, knockdown of AGO2 did not have a significant impact on virus infection in the flies as evidenced by unaltered transcript levels of the selected GpSGHV genes. CONCLUSION: The upregulation of the expression of the RNAi genes implicate involvement of this machinery in controlling GpSGHV infections and the establishment of symptomatic GpSGHV infections in Glossina. These findings provide a strategic foundation to understand GpSGHV infections and to control latent (asymptomatic) infections in Glossina spp. and thereby control SGHVs in insect production facilities.

Coevolution of hytrosaviruses and host immune responses.

BACKGROUND: Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160-174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV's specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV's asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. RESULTS: Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host's immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly's RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. CONCLUSIONS: The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive 'explorers' to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

The peritrophic matrix mediates differential infection outcomes in the tsetse fly gut following challenge with commensal, pathogenic, and parasitic microbes.

The insect gut is lined by a protective, chitinous peritrophic matrix (PM) that separates immunoreactive epithelial cells from microbes present within the luminal contents. Tsetse flies (Glossina spp.) imbibe vertebrate blood exclusively and can be exposed to foreign microorganisms during the feeding process. We used RNA interference-based reverse genetics to inhibit the production of a structurally robust PM and then observed how this procedure impacted infection outcomes after per os challenge with exogenous bacteria (Enterobacter sp. and Serratia marcescens strain Db11) and parasitic African trypanosomes. Enterobacter and Serratia proliferation was impeded in tsetse that lacked an intact PM because these flies expressed the antimicrobial peptide gene, attacin, earlier in the infection process than did their counterparts that housed a fully developed PM. After challenge with trypanosomes, attacin expression was latent in tsetse that lacked an intact PM, and these flies were thus highly susceptible to parasite infection. Our results suggest that immunodeficiency signaling pathway effectors, as opposed to reactive oxygen intermediates, serve as the first line of defense in tsetse's gut after the ingestion of exogenous microorganisms. Furthermore, tsetse's PM is not a physical impediment to infection establishment, but instead serves as a barrier that regulates the fly's ability to immunologically detect and respond to the presence of these microbes. Collectively, our findings indicate that effective insect antimicrobial responses depend largely upon the coordination of multiple host and microbe-specific developmental factors.

Trypanosome infection establishment in the tsetse fly gut is influenced by microbiome-regulated host immune barriers.

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host's physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse's resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm(Apo)) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm(Apo) adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm(Apo) flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse's PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse's larval microbiome and the integrity of the emerging adult PM gut immune barrier.

Tsetse immune system maturation requires the presence of obligate symbionts in larvae.

Beneficial microbial symbionts serve important functions within their hosts, including dietary supplementation and maintenance of immune system homeostasis. Little is known about the mechanisms that enable these bacteria to induce specific host phenotypes during development and into adulthood. Here we used the tsetse fly, Glossina morsitans, and its obligate mutualist, Wigglesworthia glossinidia, to investigate the co-evolutionary adaptations that influence the development of host physiological processes. Wigglesworthia is maternally transmitted to tsetse's intrauterine larvae through milk gland secretions. We can produce flies that lack Wigglesworthia (Gmm(Wgm-) yet retain their other symbiotic microbes. Such offspring give rise to adults that exhibit a largely normal phenotype, with the exception being that they are reproductively sterile. Our results indicate that when reared under normal environmental conditions Gmm(Wgm-) adults are also immuno-compromised and highly susceptible to hemocoelic E. coli infections while age-matched wild-type individuals are refractory. Adults that lack Wigglesworthia during larval development exhibit exceptionally compromised cellular and humoral immune responses following microbial challenge, including reduced expression of genes that encode antimicrobial peptides (cecropin and attacin), hemocyte-mediated processes (thioester-containing proteins 2 and 4 and prophenoloxidase), and signal-mediating molecules (inducible nitric oxide synthase). Furthermore, Gmm(Wgm-) adults harbor a reduced population of sessile and circulating hemocytes, a phenomenon that likely results from a significant decrease in larval expression of serpent and lozenge, both of which are associated with the process of early hemocyte differentiation. Our results demonstrate that Wigglesworthia must be present during the development of immature progeny in order for the immune system to function properly in adult tsetse. This phenomenon provides evidence of yet another important physiological adaptation that further anchors the obligate symbiosis between tsetse and Wigglesworthia.

Obligate symbionts activate immune system development in the tsetse fly.

Many insects rely on the presence of symbiotic bacteria for proper immune system function. However, the molecular mechanisms that underlie this phenomenon are poorly understood. Adult tsetse flies (Glossina spp.) house three symbiotic bacteria that are vertically transmitted from mother to offspring during this insect's unique viviparous mode of reproduction. Larval tsetse that undergo intrauterine development in the absence of their obligate mutualist, Wigglesworthia, exhibit a compromised immune system during adulthood. In this study, we characterize the immune phenotype of tsetse that develop in the absence of all of their endogenous symbiotic microbes. Aposymbiotic tsetse (Glossina morsitans morsitans [Gmm(Apo)]) present a severely compromised immune system that is characterized by the absence of phagocytic hemocytes and atypical expression of immunity-related genes. Correspondingly, these flies quickly succumb to infection with normally nonpathogenic Escherichia coli. The susceptible phenotype exhibited by Gmm(Apo) adults can be reversed when they receive hemocytes transplanted from wild-type donor flies prior to infection. Furthermore, the process of immune system development can be restored in intrauterine Gmm(Apo) larvae when their mothers are fed a diet supplemented with Wigglesworthia cell extracts. Our finding that molecular components of Wigglesworthia exhibit immunostimulatory activity within tsetse is representative of a novel evolutionary adaptation that steadfastly links an obligate symbiont with its host.

Interactions between mutualist Wigglesworthia and tsetse peptidoglycan recognition protein (PGRP-LB) influence trypanosome transmission.

Tsetse flies, the sole vectors of African trypanosomes, have coevolved with mutualistic endosymbiont Wigglesworthia glossinidiae. Elimination of Wigglesworthia renders tsetse sterile and increases their trypanosome infection susceptibility. We show that a tsetse peptidoglycan recognition protein (PGRP-LB) is crucial for symbiotic tolerance and trypanosome infection processes. Tsetse pgrp-lb is expressed in the Wigglesworthia-harboring organ (bacteriome) in the midgut, and its level of expression correlates with symbiont numbers. Adult tsetse cured of Wigglesworthia infections have significantly lower pgrp-lb levels than corresponding normal adults. RNA interference (RNAi)-mediated depletion of pgrp-lb results in the activation of the immune deficiency (IMD) signaling pathway and leads to the synthesis of antimicrobial peptides (AMPs), which decrease Wigglesworthia density. Depletion of pgrp-lb also increases the host's susceptibility to trypanosome infections. Finally, parasitized adults have significantly lower pgrp-lb levels than flies, which have successfully eliminated trypanosome infections. When both PGRP-LB and IMD immunity pathway functions are blocked, flies become unusually susceptible to parasitism. Based on the presence of conserved amidase domains, tsetse PGRP-LB may scavenge the peptidoglycan (PGN) released by Wigglesworthia and prevent the activation of symbiont-damaging host immune responses. In addition, tsetse PGRP-LB may have an anti-protozoal activity that confers parasite resistance. The symbiotic adaptations and the limited exposure of tsetse to foreign microbes may have led to the considerable differences in pgrp-lb expression and regulation noted in tsetse from that of closely related Drosophila. A dynamic interplay between Wigglesworthia and host immunity apparently is influential in tsetse's ability to transmit trypanosomes.

Antimicrobial peptide killing of African trypanosomes.

The diseases caused by trypanosomes are medically and economically devastating to the population of Sub-Saharan Africa. Parasites of the genus Trypanosoma infect both humans, causing African sleeping sickness, and livestock, causing Nagana. The development of effective treatment strategies has suffered from severe side effects of approved drugs, resistance and major difficulties in delivering drugs. Antimicrobial peptides (AMPs) are ubiquitous components of immune defence and are being rigorously pursued as novel sources of new therapeutics for a variety of pathogens. Here, we review the role of AMPs in the innate immune response of the tsetse fly to African trypanosomes, catalogue trypanocidal AMPs from diverse organisms and highlight the susceptibility of bloodstream form African trypanosomes to killing by unconventional toxic peptides.

An insect symbiosis is influenced by bacterium-specific polymorphisms in outer-membrane protein A.

Beneficial bacterial symbioses are ubiquitous in nature. However, the functional and molecular basis of host tolerance to resident symbiotic microbes, in contrast to resistance to closely related bacteria that are recognized as foreign, remain largely unknown. We used the tsetse fly (Glossina morsitans), which depends on symbiotic flora for fecundity and has limited exposure to foreign microbes, to investigate the tolerance phenomenon exhibited during symbiosis. We examined the potential role of bacterium-specific polymorphisms present in the major bacterial surface protein, outer-membrane protein A (OmpA), on host infection outcomes. Tsetse were successfully superinfected with their mutualistic facultative symbiont, Sodalis glossinidius, whereas infections with Escherichia coli K12 were lethal. In contrast, tsetse were resistant to an E. coli OmpA mutant strain, whereas recombinant Sodalis expressing E. coli OmpA became pathogenic. Profiling of tsetse immunity-related gene expression incriminated peptidoglycan recognition protein (pgrp)-lb as a determinant of the infection outcomes we observed. RNAi-induced knockdown of tsetse pgrp-lb significantly reduced host mortality after infection with otherwise lethal E. coli K12. Our results show that polymorphisms in the exposed loop domains of OmpA represent a microbial adaptation that mediates host tolerance of endogenous symbiotic bacteria.

Specific antibodies: a potential insecticide.

When tsetse flies are fed on human blood, the hemolymph of the flies contains human albumin. If the flies then ingest antibodies to human albumin, they die within a short time. The albumin fraction in their hemolymph disappears and osmoregulation is severely disturbed.

Nutritional stress affects the tsetse fly's immune gene expression.

Tsetse-transmitted trypanosomiasis poses a serious threat to human and animal health in sub-Saharan Africa. The majority of tsetse flies (Glossina spp.) in a natural population will not develop a mature infection of either Trypanosoma congolense or Trypanosoma brucei sp. because of refractoriness, a phenomenon that is affected by different factors, including the tsetse fly's immune defence. Starvation of tsetse flies significantly increases their susceptibility to the establishment of a trypanosome infection. This paper reports the effects of nutritional stress (starvation) on (a) uninduced baseline levels of gene expression of the antimicrobial peptides attacin, defensin and cecropin in the tsetse fly, and (b) levels of expression induced in response to bacterial (Escherichia coli) or trypanosomal challenge. In newly emerged, unfed tsetse flies, starvation significantly lowers baseline levels of antimicrobial peptide gene expression, especially for attacin and cecropin. In response to trypanosome challenge, only non-starved older flies showed a significant increase in antimicrobial peptide gene expression within 5 days of ingestion of a trypanosome-containing bloodmeal, especially with T. brucei bloodstream forms. These data suggest that a decreased expression of immune genes in newly hatched flies or a lack of immune responsiveness to trypanosomes in older flies, both occurring as a result of fly starvation, may be among the factors contributing to the increased susceptibility of nutritionally stressed tsetse flies to trypanosome infection.

Innate immunity in the tsetse fly (Glossina), vector of African trypanosomes.

Tsetse flies (Glossina sp.) are medically and veterinary important vectors of African trypanosomes, protozoan parasites that cause devastating diseases in humans and livestock in sub-Saharan Africa. These flies feed exclusively on vertebrate blood and harbor a limited diversity of obligate and facultative bacterial commensals. They have a well-developed innate immune system that plays a key role in protecting the fly against invading pathogens and in modulating the fly's ability to transmit African trypanosomes. In this review, we briefly summarize our current knowledge on the tsetse fly innate immune system and its interaction with the bacterial commensals and the trypanosome parasite.

Salivarian Trypanosomosis: A Review of Parasites Involved, Their Global Distribution and Their Interaction With the Innate and Adaptive Mammalian Host Immune System.

Salivarian trypanosomes are single cell extracellular parasites that cause infections in a wide range of hosts. Most pathogenic infections worldwide are caused by one of four major species of trypanosomes including (i) Trypanosoma brucei and the human infective subspecies T. b. gambiense and T. b. rhodesiense, (ii) Trypanosoma evansi and T. equiperdum, (iii) Trypanosoma congolense and (iv) Trypanosoma vivax. Infections with these parasites are marked by excessive immune dysfunction and immunopathology, both related to prolonged inflammatory host immune responses. Here we review the classification and global distribution of these parasites, highlight the adaptation of human infective trypanosomes that allow them to survive innate defense molecules unique to man, gorilla, and baboon serum and refer to the discovery of sexual reproduction of trypanosomes in the tsetse vector. With respect to the immunology of mammalian host-parasite interactions, the review highlights recent findings with respect to the B cell destruction capacity of trypanosomes and the role of T cells in the governance of infection control. Understanding infection-associated dysfunction and regulation of both these immune compartments is crucial to explain the continued failures of anti-trypanosome vaccine developments as well as the lack of any field-applicable vaccine based anti-trypanosomosis intervention strategy. Finally, the link between infection-associated inflammation and trypanosomosis induced anemia is covered in the context of both livestock and human infections.

Human/vector relationships during human African trypanosomiasis: initial screening of immunogenic salivary proteins of Glossina species.

The morbidity and mortality of vector-borne diseases is closely linked to exposure of the human host to vectors. Qualitative and quantitative evaluation of individual exposure to arthropod bites by investigation of the specific immune response to vector saliva would make it possible to monitor individuals at risk of vectorial transmission of pathogens. The objective of this study was to evaluate and compare the antibody (IgG) response to saliva from uninfected Glossina species, vectors, or non-vectors of Trypanosoma brucei gambiense by detecting immunogenic proteins in humans residing in an area endemic for human African trypanosomiasis in the Democratic Republic of Congo. Our results suggest that the immunogenic profiles observed seemed specific to the Glossina species (vector or non-vector species) and to the infectious status of exposed individuals (infected or not infected). This preliminary work tends to support the feasibility of development of an epidemiologic tool based on this antibody response to salivary proteins.

Tsetse fly saliva biases the immune response to Th2 and induces anti-vector antibodies that are a useful tool for exposure assessment.

Tsetse flies (Glossina sp.) are blood-feeding dipteran insects that transmit African trypanosomes, parasites that are responsible for human sleeping sickness and veterinary infections. Increasing attention is being paid to the effects of tsetse fly saliva deposited at the feeding site, which enables the blood-feeding process and putatively promotes parasite transmission. Here we demonstrate that saliva induces strong humoral responses against the major 43-45 kDa protein fraction (tsetse salivary gland proteins 1 and 2 - Tsal1 and Tsal2) in mice and humans and suppresses murine T and B cell responses to heterologous antigen. The saliva-induced immune response is associated with a Th2-biased cytokine profile and the production of mainly IgG1 and IgE antibody isotypes. Functionally, the antibodies raised in mice exposed to tsetse fly bites or induced after experimental saliva immunisation do not affect the fly's blood-feeding efficiency nor its survival. We propose that anti-saliva as well as anti-Tsal1/2 antibody responses can be used in epidemiological studies as a tool to analyze human exposure to tsetse flies.

Identification of a Tsal152-75 salivary synthetic peptide to monitor cattle exposure to tsetse flies.

BACKGROUND: The saliva of tsetse flies contains a cocktail of bioactive molecules inducing specific antibody responses in hosts exposed to bites. We have previously shown that an indirect-ELISA test using whole salivary extracts from Glossina morsitans submorsitans was able to discriminate between (i) cattle from tsetse infested and tsetse free areas and (ii) animals experimentally exposed to low or high numbers of tsetse flies. In the present study, our aim was to identify specific salivary synthetic peptides that could be used to develop simple immunoassays to measure cattle exposure to tsetse flies. METHODS: In a first step, 2D-electrophoresis immunoblotting, using sera from animals exposed to a variety of bloodsucking arthropods, was performed to identify specific salivary proteins recognised in cattle exposed to tsetse bites. Linear epitope prediction software and Blast analysis were then used to design synthetic peptides within the identified salivary proteins. Finally, candidate peptides were tested by indirect-ELISA on serum samples from tsetse infested and tsetse free areas, and from exposure experiments. RESULTS: The combined immunoblotting and bioinformatics analyses led to the identification of five peptides carrying putative linear epitopes within two salivary proteins: the tsetse salivary gland protein 1 (Tsal1) and the Salivary Secreted Adenosine (SSA). Of these, two were synthesised and tested further based on the absence of sequence homology with other arthropods or pathogen species. IgG responses to the Tsal152-75 synthetic peptide were shown to be specific of tsetse exposure in both naturally and experimentally exposed hosts. Nevertheless, anti-Tsal152-75 IgG responses were absent in animals exposed to high tsetse biting rates. CONCLUSIONS: These results suggest that Tsal152-75 specific antibodies represent a biomarker of low cattle exposure to tsetse fly. These results are discussed in the light of the other available tsetse saliva based-immunoassays and in the perspective of developing a simple serological tool for tsetse eradication campaigns to assess the tsetse free status or to detect tsetse reemergence in previously cleared areas.