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.

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.

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.

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.

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.

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.

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.

An immunoregulatory peptide from tsetse fly salivary glands of Glossina morsitans morsitans.

Tsetse fly Glossina morsitans morsitans is an important insect vector of African trypanosomes, which cause human African trypanosomiasis (HAT). As other hematophagous arthropods, tsetse fly relies heavily on the pharmacological propriety of their saliva to suppress host's immune reactions and get blood meal. However little information is available on immune regulators from testes fly. An immunoregulatory peptide named Gloss 2 containing amino acid sequence of QKNDTAFSCHFFEIYL SNCFNKEKYIKNYLQIM has been identified from salivary glands of the tsetse fly of G. morsitans morsitans (Diptera: Glossinidae). Gloss 2 has the ability to inhibit the secretion of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ),interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by lipopolysaccharide (LPS) in mouse splenocytes. Besides, Gloss 2 significantly suppressed the LPS-induced activation of MAPK signaling pathway through blocking phosphorylations of JNK, Erk and P38. Gloss 2 probably inhibits host inflammatory responses by inhibiting secretion of TNF-α, IFN-γ and IL-6. Considering IL-10's ability to promote humoral immune responses by enhancing class II expression B cells and inducing immunoglobulin (Ig) production, Gloss 2 may inhibit host humoral immune response by inhibiting IL-10 secretion. The immune-suppression may facilitate the blood feeding of tsetse fly and transmission of African trypanosomes to hosts.

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species.

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse's ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.

Experimental African Trypanosome Infection by Needle Passage or Natural Tsetse Fly Challenge Thwarts the Development of Collagen-Induced Arthritis in DBA/1 Prone Mice via an Impairment of Antigen Specific B Cell Autoantibody Titers.

Collagen-induced arthritis is a B cell-mediated autoimmune disease. Recently published studies have demonstrated that in some rare cases pathogens can confer protection from autoimmunity. Trypanosoma brucei parasites are tsetse fly transmitted extracellular protozoans causing sleeping sickness disease in humans and Nagana in livestock in sub-Saharan endemic areas. In the past, we demonstrated that trypanosome infections impair B cell homeostasis and abolish vaccine-induced protection against unrelated antigens. Hence, here we hypothesized that trypanosome infection can affect the onset of CIA by specifically dampening specific B-cell responses and type II collagen antibody titers in DBA/1 prone mice. We observed a substantial delay in the onset of collagen-induced arthritis in T. brucei-infected DBA/1 mice that correlates with a drastic decrease of type II collagen titers of the different IgG isotypes in the serum. Treatment of infected mice with Berenil, a trypanocidal drug, restored the development of CIA-associated clinical symptoms. Interestingly, these data were confirmed by the challenge of immunized DBA/1 prone mice with T. brucei-infected tsetse flies. Together, these results demonstrate that T. brucei infection is impairing the maintenance of the antigen specific plasma B cell pool driving the development of CIA in DBA/1 prone mice.

Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure.

BACKGROUND: Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. METHODOLOGY/PRINCIPAL FINDINGS: A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). CONCLUSION/SIGNIFICANCE: We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

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.

Insights into the trypanosome-host interactions revealed through transcriptomic analysis of parasitized tsetse fly salivary glands.

The agents of sleeping sickness disease, Trypanosoma brucei complex parasites, are transmitted to mammalian hosts through the bite of an infected tsetse. Information on tsetse-trypanosome interactions in the salivary gland (SG) tissue, and on mammalian infective metacyclic (MC) parasites present in the SG, is sparse. We performed RNA-seq analyses from uninfected and T. b. brucei infected SGs of Glossina morsitans morsitans. Comparison of the SG transcriptomes to a whole body fly transcriptome revealed that only 2.7% of the contigs are differentially expressed during SG infection, and that only 263 contigs (0.6%) are preferentially expressed in the SGs (SG-enriched). The expression of only 37 contigs (0.08%) and 27 SG-enriched contigs (10%) were suppressed in infected SG. These suppressed contigs accounted for over 55% of the SG transcriptome, and included the most abundant putative secreted proteins with anti-hemostatic functions present in saliva. In contrast, expression of putative host proteins associated with immunity, stress, cell division and tissue remodeling were enriched in infected SG suggesting that parasite infections induce host immune and stress response(s) that likely results in tissue renewal. We also performed RNA-seq analysis from mouse blood infected with the same parasite strain, and compared the transcriptome of bloodstream form (BSF) cells with that of parasites obtained from the infected SG. Over 30% of parasite transcripts are differentially regulated between the two stages, and reflect parasite adaptations to varying host nutritional and immune ecology. These differences are associated with the switch from an amino acid based metabolism in the SG to one based on glucose utilization in the blood, and with surface coat modifications that enable parasite survival in the different hosts. This study provides a foundation on the molecular aspects of the trypanosome dialogue with its tsetse and mammalian hosts, necessary for future functional investigations.

Examining the tsetse teneral phenomenon and permissiveness to trypanosome infection.

Tsetse flies are the most important vectors of African trypanosomiasis but, surprisingly, are highly refractory to trypanosome parasite infection. In populations of wild caught flies, it is rare to find mature salivarian and mouthpart parasite infection rates exceeding 1 and 15%, respectively. This inherent refractoriness persists throughout the lifespan of the fly, although extreme starvation and suboptimal environmental conditions can cause a reversion to the susceptible phenotype. The teneral phenomenon is a phenotype unique to newly emerged, previously unfed tsetse, and is evidenced by a profound susceptibility to trypanosome infection. This susceptibility persists for only a few days post-emergence and decreases with fly age and bloodmeal acquisition. Researchers investigating trypanosome-tsetse interactions routinely exploit this phenomenon by using young, unfed (teneral) flies to naturally boost trypanosome establishment and maturation rates. A suite of factors may contribute, at least in part, to this unusual parasite permissive phenotype. These include the physical maturity of midgut barriers, the activation of immunoresponsive tissues and their effector molecules, and the role of the microflora within the midgut of the newly emerged fly. However, at present, the molecular mechanisms that underpin the teneral phenomenon still remain unknown. This review will provide a historical overview of the teneral phenomenon and will examine immune-related factors that influence, and may help us better understand, this unusual phenotype.

In silico identification of a candidate synthetic peptide (Tsgf118-43) to monitor human exposure to tsetse flies in West Africa.

BACKGROUND: The analysis of humoral responses directed against the saliva of blood-sucking arthropods was shown to provide epidemiological biomarkers of human exposure to vector-borne diseases. However, the use of whole saliva as antigen presents several limitations such as problems of mass production, reproducibility and specificity. The aim of this study was to design a specific biomarker of exposure to tsetse flies based on the in silico analysis of three Glossina salivary proteins (Ada, Ag5 and Tsgf1) previously shown to be specifically recognized by plasma from exposed individuals. METHODOLOGY/PRINCIPAL FINDINGS: Synthetic peptides were designed by combining several linear epitope prediction methods and Blast analysis. The most specific peptides were then tested by indirect ELISA on a bank of 160 plasma samples from tsetse infested areas and tsetse free areas. Anti-Tsgf118-43 specific IgG levels were low in all three control populations (from rural Africa, urban Africa and Europe) and were significantly higher (p<0.0001) in the two populations exposed to tsetse flies (Guinean HAT foci, and South West Burkina Faso). A positive correlation was also found between Anti-Tsgf118-43 IgG levels and the risk of being infected by Trypanosoma brucei gambiense in the sleeping sickness foci of Guinea. CONCLUSION/SIGNIFICANCE: The Tsgf118-43 peptide is a suitable and promising candidate to develop a standardize immunoassay allowing large scale monitoring of human exposure to tsetse flies in West Africa. This could provide a new surveillance indicator for tsetse control interventions by HAT control programs.

First insights into the cattle serological response to tsetse salivary antigens: a promising direct biomarker of exposure to tsetse bites.

In the context of the Pan African Tsetse and Trypanosomiasis Eradication Campaign, the value of tsetse saliva antibodies as a biomarker of cattle exposure to tsetse flies was evaluated, as this could provide an alternative and complementary tool to conventional entomological methods. Serum immune reactivity to Glossina (G.) palpalis (p.) gambiensis, G. tachinoides and G. morsitans (m.) submorsitans whole saliva extracts (WSE) were monitored in cattle from both tsetse free and tsetse infested areas, and in cows experimentally exposed to tsetse flies and other hematophagous arthropods. In the tsetse infested area, cattle IgG responses to Glossina WSE were significantly higher during the dry season (p<0.0001) when herds are most exposed to tsetse flies and in infected animals (p=0.01) as expected in the case of a biomarker of exposure. Experimental studies further confirmed this as a quick rise of specific IgGs was observed in animals exposed to tsetse flies (within weeks), followed by a rapid clearance after exposure was stopped. In contrast to the two other tsetse species, G. m. submorsitans WSE enabled to detect exposure to all tsetse species and were associated with low level of cross-reactivity to other blood sucking arthropods. Finally, IgG responses to G. m. submorsitans salivary antigens enabled to distinguish different groups of cows according to exposure levels, thus indicating that tsetse saliva antibodies are not only indicators of tsetse exposure but also are correlated to the intensity of tsetse contacts (p=0.0031). Implementation of this new sero-epidemiological marker of cattle exposure to tsetse flies in the framework of tsetse elimination campaigns is discussed.

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.

Flying tryps: survival and maturation of trypanosomes in tsetse flies.

Survival in and colonization of the tsetse fly midgut are essential steps in the transmission of many species of African trypanosomes. In the fly, bloodstream trypanosomes transform into the procyclic stage within the gut lumen and later migrate to the ectoperitrophic space, where they multiply, establishing an infection. Progression of the parasite infection in the fly depends on factors inherent to the biology of trypanosomes, tsetse, and the bloodmeal. Flies usually eradicate infection early on with both pre-existing and inducible factors. Parasites, in contrast, respond to these stimuli by undergoing developmental changes, allowing a few to both survive and migrate within the tsetse. Here we discuss parasite and fly factors determining trypanosome colonization of the tsetse, focusing mainly on the midgut.