Vector Saliva Research Articles

Role of immune cell subsets in the establishment of vector-borne infections

Many of the pathogens responsible for diseases that result in both economic and global health burdens are transmitted by arthropod vectors in the course of a blood meal. In the past, these vectors were viewed mainly as simple delivery vehicles but the appreciation of the role that factors in the saliva of vectors play during pathogen transmission is increasing. Vector saliva proteins alter numerous physiological events in the skin; in addition, potent immunomodulatory properties are attributed to arthropod saliva. The description of specific factors responsible for these activities and their mechanisms of action have thus far remained mostly anecdotal. The National Institute of Allergy and Infectious Diseases (NIAID) sponsored a workshop in May 2012 to explore novel approaches aimed at identifying how vector saliva components affect the function of various immune cell subsets and the subsequent impact on the transmission of vector-borne pathogens. Such knowledge could guide the development of novel drugs, vaccines and other strategies to block the transmission of vector-borne pathogens. This meeting report summarizes the discussions of the gaps/challenges which represent attractive research opportunities with significant translational potential.

The transmission of Leishmania infantum chagasi by the bite of the Lutzomyia longipalpis to two different vertebrates

BackgroundSandflies are vectors of Leishmania, the causative agent of leishmaniasis in mammalian hosts, including humans. The protozoan parasite is transmitted by the sandfly bite during salivation that occurs at the moment of blood feeding. The components of vector saliva include anticlotting and vasodilatory factors that facilitate blood flow and immunomodulatory factors that inhibit wound healing and quell the immune response. Not surprisingly, these factors also play important roles in the establishment of Leishmania infection. To date, the majority of knowledge that has been generated regarding the process of Leishmania infection, including L. infantum chagasi transmission has been gathered by using intradermal or subcutaneous inoculation of purified parasites.FindingsThis study presents the establishment of a transmission model of Leishmania infantum chagasi by the bite of Lutzomyia longipalpis, the vector of American visceral leishmaniasis. The parasites were successfully transmitted by infected sandfly bites to mice and hamsters, indicating that both animals are good experimental models. The L. infantum chagasi dose that was transmitted in each single bite ranged from 10 to 10, 000 parasites, but 75% of the sandflies transmitted less than 300 parasites.ConclusionsThe strategy for initiating infection by sandfly bite of experimental animals facilitates future investigations into the complex and dynamic mechanisms of visceral leishmaniasis. It is important to elucidate the transmission mechanism of vector bites. This model represents a useful tool to study L. infantum chagasi infection transmitted by the vector.

Immunity to Protozoan Parasites

Immunity to Protozoan Parasites

Role of Leukotrienes on Protozoan and Helminth Infections

Leukotrienes (LTs), formed by the 5-lipoxygenase-(5-LO-) catalyzed oxidation of arachidonic acid, are lipid mediators that have potent proinflammatory activities. Pharmacologic or genetic inhibition of 5-LO biosynthesis in animals is associated with increased mortality and impaired clearance of bacteria, fungi, and parasites. LTs play a role in the control of helminth and protozoan infections by modulating the immune system and/or through direct cytotoxicity to parasites; however, LTs may also be associated with pathogenesis, such as in cerebral malaria and schistosomal granuloma. Interestingly, some proteins from the saliva of insect vectors that transmit protozoans and secreted protein from helminth could bind LTs and may consequently modulate the course of infection or pathogenesis. In addition, the decreased production of LTs in immunocompromised individuals might modulate the pathophysiology of helminth and protozoan infections. Herein, in this paper, we showed the immunomodulatory and pathogenic roles of LTs during the helminth and protozoan infections.

New horizon in the research on plant-virus-vector interaction

Understanding the mechanisms controlling vector-transmission of plant viruses requires integrating information from at least three different viewpoints: virus-vector interactions, plant-vector interactions and virus-plant interactions. While some of these aspects have been covered by past and present investigations, others have been bypassed completely, due either to technical bottlenecks or conceptual lacunas. Here, we highlight recent advances and needs in hitherto poorly documented aspects of vector transmission, such as characterization of the vector molecules responsible for initial viral recognition, and the role of vector saliva in inoculation and initial onset of infection in a new plant. We also propose and discuss some novel conceptual and complementary questions that are opening up fascinating new horizons in this field. We explore the possible existence of viral morphs with specific properties that facilitate acquisition by vectors, and discuss the dynamics/genetics of such viral subpopulations, which could differentiate and specialize in different host compartments.

2-DE-based proteomic investigation of the saliva of the Amazonian triatomine vectors of Chagas disease: Rhodnius brethesi and Rhodnius robustus

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

Dengue and soluble mediators of the innate immune system.

Huge emphasis has been placed on the role of the adaptive immune system in dengue pathogenesis. Yet there is increasing evidence for the importance of the innate immune system in regulating dengue infection and possibly influencing the disease. This review focuses on the interplay between the innate immune system and dengue and highlights the role of soluble immunological mediators. Type I and type II interferons of the innate immune system demonstrate non-overlapping roles in dengue infection. Furthermore, while some IFN responses to dengue are protective, others may exert disease-related effects on the host. But aside from interferons, a number of cytokines have also been implicated in dengue pathogenesis. Our expanding knowledge of cytokines indicates that these soluble mediators act upon a complicated network of events to provoke the disease. This cytokine storm is generally attributed to massive T cell activation as an outcome of secondary infection. However, there is reason to believe that innate immune response-derived cytokines also have contributory effects, especially in the context of severe cases of primary dengue infection. Another less popular but interesting perspective on dengue pathogenesis is the effect of mosquito feeding on host immune responses and viral infection. Various studies have shown that soluble factors from vector saliva have the capacity to alter immune reactions and thereby influence pathogen transmission and establishment. Hence, modulation of the innate immune system at various levels of infection is a critical component of dengue disease. In the absence of an approved drug or vaccine for dengue, soluble mediators of the innate immune system could be a strategic foothold for developing anti-viral therapeutics and improving clinical management.

Leishmania infantum proteophosphoglycans regurgitated by the bite of its natural sand fly vector, Lutzomyia longipalpis, promote parasite establishment in mouse skin and skin-distant tissues

We demonstrate that a proteophosphoglycan-rich gel secreted by Leishmania infantum inside the midgut of Lutzomyia longipalpis sand flies (promastigote secretory gel) is regurgitated along with an average dose of 500 L. infantum metacyclic promastigotes per infected bite. Using both low (10 3) and high (10 5) doses of parasites in the ears of BALB/c mice we show that the infections benefit from the presence of vector saliva and parasite gel in the skin. However, chronic infection of the spleen was only enhanced in high dose co-infections with gel. These results provide the framework for a more natural experimental model of visceral leishmaniasis.

Transcriptome Changes in <I>Culex quinquefasciatus</I> (Diptera: Culicidae) Salivary Glands During West Nile Virus Infection

Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins. Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

Background Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure.Methodology/principal findingsELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples.ConclusionOur results show the possibility of substituting Lu. longipalpis SGS for two recombinant proteins, LJM17 and LJM11, in order to probe for vector exposure in individuals residing in endemic areas.

Purification and characterization of a novel salivary antimicrobial peptide from the tick, Ixodes scapularis

A novel antimicrobial peptide was isolated from the saliva of the Lyme disease tick vector, Ixodes scapularis, henceforth designated as ISAMP ( I. scapularis Antimicrobial Peptide). ISAMP was purified using a sequential method including ultra filtration, gel filtration and reverse-phase high performance liquid chromatography. The purified peak had a molecular weight of 5.3 kDa by MALDI/TOF-MS and its amino acid sequence, determined by Edman degradation was PDxGxPxxVKAGRxPxxSI. A BLASTP search revealed that the protein is a putative 5.3 kDa secreted protein ( AAM93656) from I. scapularis. The predicted protein is composed of 69 amino acids with no conserved domain motifs. Purified ISAMP was found to have antimicrobial activities against bacteria. Gene expression studies were carried out to observe ISAMP expression in different tick tissues. RT-PCR results indicated that the gene was expressed in hemocytes, fat body and salivary gland but virtually no expression was observed in the midgut. ISAMP is only similar to other Ixodid tick proteins, thus it is a member of a unique family.

Cutaneous leishmaniasis: progress towards a vaccine

Leishmaniases are vector-borne diseases due to the protozoan parasite Leishmania . Since no prevention method is available and as current therapy is costly, often poorly tolerated and not always efficacious, the development of alternative therapies, including vaccines, constitutes the priority in the fight of Leishmania infection. This review focuses on recent advances in the development of vaccines against leishmaniasis, with emphasis on the cutaneous form. Indeed, the fact that recovery from leishmaniasis is associated with immunity against new infection provides a rational basis for the development of vaccination strategy against infection with Leishmania . Evidence from animal studies demonstrate that protection can be achieved following infection with live-attenuated Leishmania as well as through immunization with purified proteins or DNA vaccines. In addition, recent results have shown that immunization against the saliva of the insect vector could have synergistic effects with conventional vaccination. Finally, vaccination using dendritic cells was recently demonstrated as a possible tool for Leishmania vaccination.

Trypanosoma cruzi infection is enhanced by vector saliva through immunosuppressant mechanisms mediated by lysophosphatidylcholine.

Trypanosoma cruzi, the etiological agent of Chagas disease, is transmitted by bug feces deposited on human skin during a blood meal. However, parasite infection occurs through the wound produced by insect mouthparts. Saliva of the Triatominae bug Rhodnius prolixus is a source of lysophosphatidylcholine (LPC). Here, we tested the role of both triatomine saliva and LPC on parasite transmission. We show that vector saliva is a powerful inducer of cell chemotaxis. A massive number of inflammatory cells were found at the sites where LPC or saliva was inoculated into the skin of mice. LPC is a known chemoattractant for monocytes, but neutrophil recruitment induced by saliva is LPC independent. The preincubation of peritoneal macrophages with saliva or LPC increased fivefold the association of T. cruzi with these cells. Moreover, saliva and LPC block nitric oxide production by T. cruzi-exposed macrophages. The injection of saliva or LPC into mouse skin in the presence of the parasite induces an up-to-sixfold increase in blood parasitemia. Together, our data suggest that saliva of the Triatominae enhances T. cruzi transmission and that some of its biological effects are attributed to LPC. This is a demonstration that a vector-derived lysophospholipid may act as an enhancing factor of Chagas disease.

Prior Exposure to Uninfected Mosquitoes Enhances Mortality in Naturally-Transmitted West Nile Virus Infection

BackgroundThe global emergence of West Nile virus (WNV) has highlighted the importance of mosquito-borne viruses. These are inoculated in vector saliva into the vertebrate skin and circulatory system. Arthropod-borne (arbo)viruses such as WNV are transmitted to vertebrates as an infectious mosquito probes the skin for blood, depositing the virus and saliva into the skin and circulation. Growing evidence has demonstrated that arthropod, and recently mosquito, saliva can have a profound effect on pathogen transmission efficiency, pathogenesis, and disease course. A potentially important aspect of natural infections that has been ignored is that in nature vertebrates are typically exposed to the feeding of uninfected mosquitoes prior to the mosquito that transmits WNV. The possibility that pre-exposure to mosquito saliva might modulate WNV infection was explored.Principal FindingsHere we report that sensitization to mosquito saliva exacerbates viral infection. Prior exposure of mice to mosquito feeding resulted in increased mortality following WNV infection. This aggravated disease course was associated with enhanced early viral replication, increased interleukin-10 expression, and elevated influx of WNV-susceptible cell types to the inoculation site. This exacerbated disease course was mimicked by passive transfer of mosquito-sensitized serum.SignificanceThis is the first report that sensitization to arthropod saliva can exacerbate arthropod-borne infection, contrary to previous studies with parasite and bacteria infections. This research suggests that in addition to the seroreactivity of the host to virus, it is important to take into account the immune response to vector feeding.

Spit-acular Entry: Borrelia Gets Help from a Tick Salivary Protein to Move from the Mammalian Host to the Arthropod Vector

In vector-host-pathogen interactions, vector saliva plays a key role in the successful acquisition of blood and in facilitating the establishment of pathogens in mammalian hosts. In this issue of Cell Host & Microbe, Narasimhan et al. describe how a tick salivary antioxidant neutralizes reactive oxygen species at the tick-host interface and facilitates the tick vector's acquisition of Borrelia burgdorferi bacteria from an infected mammalian host.

The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors

The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC–MS/MS analysis using a LTQ–Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug's blood-feeding.

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.

Haematophagous arthropod saliva and host defense system: a tale of tear and blood

The saliva from blood-feeding arthropod vectors is enriched with molecules that display diverse functions that mediate a successful blood meal. They function not only as weapons against host's haemostatic, inflammatory and immune responses but also as important tools to pathogen establishment. Parasites, virus and bacteria taking advantage of vectors' armament have adapted to facilitate their entry in the host. Today, many salivary molecules have been identified and characterized as new targets to the development of future vaccines. Here we focus on current information on vector's saliva and the molecules responsible to modify host's hemostasis and immune response, also regarding their role in disease transmission.

Modulation of murine cellular immune responses and cytokine production by sand fly saliva

Sand flies (Diptera: Phlebotominae) are hematophagous arthropods and important vectors of Leishmania parasites. During blood feeding, both vector saliva and parasites are injected by an infected sand fly. The aim of this study was to characterize how sand fly saliva interferes with host immunity in order to clarify the mechanism underlying enhancing effect of sand fly saliva on Leishmania infection. Spleen cells from naive BALB/c mice were incubated in vitro with Phlebotomus papatasi, P. sergenti or Lutzomyia longipalpis salivary gland lysate (SGL). We studied the effect of saliva on cell proliferation and cytokine production in non‐stimulated and concanavalin A‐stimulated splenocytes. Both spontaneous and mitogen‐stimulated lymphocyte proliferation were significantly suppressed with SGLs of all three sand fly species and all SGLs doses tested (equivalent to 1/16, 1/4 and 1 gland per well). The proliferation index (a lysate‐treated/lysate‐untreated cells ratio) was significantly lower when cells were incubated with SGL and was ranging between 0.52 and 0.30 for non‐stimulated splenocytes and 0.69–0.32 for mitogen‐stimulated lymphocytes. This result indicates that saliva from different sand fly species is able to suppress host proliferative response even to the potent Leishmania‐unrelated mitogen. In parallel experiments, we analysed the effect of SGL (1 gland per well) on cytokine production. The levels of Th1 (IL‐2, IFN‐γ) and Th2 (IL‐4) cytokines were determined in cell culture supernatants by capture ELISA. Exposure to SGL alone did not affect levels of IL‐2 or IL‐4. The production of IFN‐γ was inhibited by P. papatasi SGL, whereas the SGLs of other two species were ineffective. In mitogen‐stimulated cells both Th1 cytokines were modulated; the level of IFN‐γ production was significantly suppressed and IL‐2 enhanced by SGL of all three sand fly species. The production of Th2 cytokine IL‐4 was inhibited only by L. longipalpis SGL; no changes in IL‐4 levels were noted after incubation with Phlebotomus saliva. This species‐specific immunomodulation is in agreement with our previous finding that the composition of sand fly saliva varies among species. Taken together, sand fly saliva modulate cell proliferation as well as cytokine production. These findings further increase our understanding of sand fly–host immune relationship.

Tick saliva inhibits differentiation, maturation and function of murine bone‐marrow‐derived dendritic cells

Haematophagous arthropod vectors such as mosquitoes, tsetse flies, sandflies and ticks have evolved salivary immunomodulatory factors that prevent the vertebrate host from rejecting them meanwhile enhancing pathogen transmission. As dendritic cells (DC) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC differentiation and maturation. Flow cytometry analysis revealed that the addition of saliva to bone marrow cells inhibits the differentiation of DC and decreased the population of differentiated immature DC, increasing the levels of major histocompatibility complex (MHC) class II while not altering the expression of costimulatory (CD40, CD80 and CD86) and adhesion (CD54) molecules. Furthermore, maturation of DC stimulated by lipopolysaccharide (LPS) in the presence of saliva resulted in a lower expression of costimulatory molecules, but did not alter the up-regulation of MHC class II and CD54. The lipopolysaccharide (LPS)-matured DC cultured with saliva also presented reduced production of interleukin-12, whereas interleukin-10 production was unaltered. Assessment of the function of DC cultured with tick saliva revealed them to be poor stimulators of cytokine production by antigen-specific T cells. Our data indicate a novel modulatory role for the saliva of arthropod vectors at an initial step of the immune response through the inhibition of differentiation and maturation of DC into functional antigen-presenting cells.