SAAG-4 is a novel mosquito salivary protein that programmes host CD4 T cells to express IL-4.

Mosquitoes represent the most important vector for transmitting pathogens that cause human disease. Central to pathogen transmission is the ability to divert the host immune system away from Th1 and towards Th2 responsiveness. Identification of the mosquito factor(s) critical for programming Th2 responsiveness should therefore lead to strategies to neutralize their function and thus prevent disease transmission. In the current study, we used a TCR transgenic adoptive transfer system to screen gene products present in the saliva of the mosquito Aedes aegypti for their ability to programme CD4 T cells to express the signature Th2 cytokine IL-4. The clone SAAG-4 encodes a secreted protein with a predicted size of 20 kDa whose function has previously been uncharacterized. Notably, SAAG-4 reduced host CD4 T cell expression of the signature Th1 cytokine IFN-gamma while simultaneously increasing expression of IL-4. SAAG-4 is therefore the first identified mosquito factor that can programme Th2 effector CD4 T cell differentiation.

A novel sphingomyelinase-like enzyme in Ixodes scapularis tick saliva drives host CD4 T cells to express IL-4.

Tick feeding modulates host immune responses. Tick-induced skewing of host CD4(+) T cells towards a Th2 cytokine profile facilitates transmission of tick-borne pathogens that would otherwise be neutralized by Th1 cytokines. Tick-derived factors that drive this Th2 response have not previously been characterized. In the current study, we examined an I. scapularis cDNA library prepared at 18-24 h of feeding and identified and expressed a tick gene with homology to Loxosceles spider venom proteins with sphingomyelinase activity. This I. scapularis sphingomyelinase-like (IsSMase) protein is a Mg(2+)-dependent, neutral (pH 7.4) form of sphingomyelinase. Significantly, in an in vivo TCR transgenic adoptive transfer assay IsSMase programmed host CD4(+) T cells to express the hallmark Th2 effector cytokine IL-4. IsSMase appears to directly programme host CD4 T cell IL-4 expression (as opposed to its metabolic by-products) because induced IL-4 expression was not altered when enzymatic activity was neutralized. TCR transgenic CD4 T cell proliferation (CFSE-dilution) was also significantly increased by IsSMase. Furthermore, a Th2 response is superimposed onto a virally primed Th1 response by IsSMase. Thus, IsSMase is the first identified tick molecule capable of programming host CD4(+) T cells to express IL-4.

Tick genomics: the Ixodes genome project and beyond.

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.

Tick modulation of host immunity: an important factor in pathogen transmission.

Immunological interactions at the tick host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.

Tick-induced modulation of the host immune response.

The tick-host-pathogen interface is characterised by complex immunological interactions. Host immune responses to tick infestation and infection with tick-borne pathogens involve cytokines, antibodies, complement and T lymphocyte regulatory and effector pathways. A successful host-parasite relationship is a balance between limiting the parasite by host defenses and the ability of the parasite to modulate, evade or restrict the host response. Hosts acquire immunological based resistance to tick infestation, which reduces engorgement, production of ova and viability. Salivary glands of ixodid ticks produce a complex array of immunogens and pharmacologically active molecules. Tick salivary gland derived material can modulate host cytokine, antibody and cell mediated immune responses. Both immunoregulatory and immune effector pathways of the host are suppressed. Tick feeding impairs the ability to develop a primary immune response to a thymic dependent immunogen. Lymphocytes obtained from tick infested hosts are reduced in their ability to proliferate in vitro to T lymphocyte mitogens, while responses to B lymphocyte polyclonal activators are unaltered. Normal macrophages and lymphocytes were exposed to female tick salivary gland extracts prepared daily during the course of engorgement. All extracts reduced lymphocyte responses to T cell mitogens and enhanced in vitro proliferation in the presence of a B lymphocyte mitogen. Macrophage elaboration of tumour necrosis factor alpha and interleukin-1 are significantly reduced in a differential manner. Production of interleukin-2 and interferon-gamma by T lymphocytes is reduced. Tick modulation of the host immune response could enhance the ability of the arthropod to obtain a blood meal and facilitate pathogen transmission to an immunocompetent host.

Acquired resistance to ticks: expression of resistance by C4-deficient guinea pigs.

Hartley and C4-deficient guinea pigs developed resistance to the ixodid tick. Dermacentor andersoni, after one infestation. Resistance was characterized by resistant animals of both groups allowing significantly fewer larvae (5--25%) to engorge during a second infestation than during an initial infestation (70--90%). Resistant animals in both groups developed cutaneous reactions at the site of tick attachment which were characterized by intraepidermal vesicles containing numerous basophils. In previous studies, tick-resistant Hartley guinea pigs depleted of complement by cobra venom factor were not able to express the resistance response and the skin reactions at the tick attachment sites were depleted of basophils. The use of cobra venom factor as an anti-complement probe could not distinguish the relative importance of the classical and/or alternate pathways of complement activation in the expression of tick resistance. The present study reports that C4-deficient guinea pigs, those with a total deficiency in the classical pathway of complement activation, but with an intact alternate pathway, can acquire and display tick resistance in a fashion similar to Hartley guinea pigs. This finding provides evidence that the alternate pathway of complement activation is important in the expression of tick resistance.

The induction of host resistance to tick infestation with a salivary gland antigen.

Salivary gland antigen, SGA, derived from partially engorged female Dermacentor andersoni was shown to be capable of inducing resistance to tick infestation in guinea pigs never previously exposed to ticks. Immunization regimens involved the administration of SGA by different routes and with or without the use of adjuvants. Induced resistance was expressed by significantly fewer larvae engorging, and the weight of larvae which did engorge was reduced. Salivary gland antigen has been shown to interact with the immune effector elements of animals which acquired and expressed tick resistance due to infestation. These findings demonstrated the potential for an immunologic approach to tick control.

Resistance to tick-borne Francisella tularensis by tick-sensitized rabbits: allergic klendusity.

Mammals become hypersensitive to ticks that feed upon them. That hypersensitivity was thought responsible for an observation that a large number of Francisella tularensis-infected Dermacentor variabilis failed to infect a rabbit previously exposed to ticks of that species. In a series of tests of that hypothesis, rabbits sensitized to ticks were often significantly more resistant than control animals to tick-borne tularemia. The conditions that determine the klendusity are thought to be variable and complex but the phenomenon must be of importance in the epidemiology of some arthropod-borne agents.

Modulation of host-immune responses by ticks (Acari: Ixodidae): effect of salivary gland extracts on host macrophages and lymphocyte cytokine production.

Ixodid tick infestation induces host acquired resistance, which involves immunoglobulin cell-mediated and complement-dependent effector pathways. Ticks have developed countermeasures to modulate host antiarthropod responses. Ixodid-mediated host immunomodulation results in vitro in reduced responsiveness to T-lymphocyte mitogens for cells obtained from infested hosts and impaired antibody responses to a thymic dependent antigen. Salivary gland extracts from days 0-9 of engorgement from unmated, female Dermacentor andersoni Stiles suppressed lymphocyte proliferative responses (LPS) to the T-cell mitogen Con A up to 68.4%, whereas responsiveness to E. coli LPS was enhanced. Cytokines assessed in this study included interleukin-1, IL-1, and tumor necrosis factor (TNF) alpha produced by macrophages, and interleukin-2, IL-2, and gamma interferon (IFN-G) secreted by T-lymphocytes. Salivary gland extracts prepared from tissues obtained on days 0-5 of engorgement suppressed IL-1 elaboration from 89.8% on day 0 through 37.5% on day 6. Levels of TNF were reduced from 40.7 to 94.6% throughout the course of the study. Production of IL-2 was suppressed by 14.1-31.9%, and IFN-G was reduced by 8.7-57.0%. Reduced IL-1 levels during the early phases of feeding indicated reduced host ability to activate T-lymphocytes and provide costimulatory, differentiation, and development signals for B-cells. Both IL-1 and TNF are endogenous pyrogens and activate polymorphonuclear leukocytes. Activities of TNF and IFN-G include antiviral properties and induction of expression of class I and II major histocompatibility complex molecules, which are critical components in the recognition of antigen by T-lymphocytes. The autocrine role of IL-2 in proliferation of T-lymphocytes is central to the development of immune reactivity involving T-cell regulation or effector functions or both. Reductions in cytokine levels would suppress immune responses directed toward immunogens introduced into the host during the course of tick feeding. These results indicates that immunomodulation of the host during tick feeding facilitates engorgement and pathogen transmission.

Effects of Dermacentor andersoni (Acari: Ixodidae) salivary gland extracts on Bos indicus and B. taurus lymphocytes and macrophages: in vitro cytokine elaboration and lymphocyte blastogenesis.

Cattle and laboratory animal species-acquired resistance to tick infestation has an immunological basis involving antigen presenting cells, B-lymphocytes, T-lymphocytes, and cytokines. Tick infestation has been shown to impair guinea pig antibody responses to a thymic-dependent antigen and in vitro responsiveness of lymphocytes to T-cell mitogens. Tick salivary gland extracts inhibited in vitro proliferative responses of normal murine lymphocytes to the T-cell mitogen concanavalin A (Con A) and enhanced reactivity of normal B-lymphocytes to the mitogen E. coli lipopolysaccharide (LPS). Salivary gland extracts collected daily during engorgement were shown to inhibit normal murine macrophage elaboration of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF) as well as murine T-lymphocyte production of interleukin-2 (IL-2) and interferon-gamma (IFN-G). Peripheral blood mononuclear cells collected from purebred Bos indicus and B. taurus were significantly inhibited in their in vitro responses to Con A by salivary gland extracts prepared daily from female Dermacentor andersoni stiles during the course of engorgement. Percentage of suppression of Con A responsiveness was similar for both B. indicus and B. taurus cells. The overall responsiveness of B. indicus derived T cells is significantly greater than that of similar cells from B. taurus, when mean counts per minute of methyl-tritiated-thymidine incorporation were compared for both groups. Cells of B. indicus origin were 34.5% more reactive. In vitro responsiveness of the same cell populations to LPS were significantly enhanced by the presence of tick salivary gland extracts. B. indicus lymphocyte reactivity to LPS was significantly greater (42.9%) than that of similar B. taurus cells in the absence of salivary gland extracts. B. indicus and B. taurus macrophage elaboration of IL-1 were suppressed in a similar manner by tick salivary gland extracts prepared on days 5-9 of engorgement. B. indicus macrophages produced more IL-1 than similar cells of B. taurus origin either in the presence (45.6%) or absence (43.0%) of LPS. Macrophages derived from both genetic backgrounds were significantly suppressed in their LPS induced production of TNF in the presence of tick salivary gland extracts collected on days 0-9 of engorgement. B. indicus might be able to develop more vigorous immune responses to foreign immunogens presented to the animal during tick feeding.

Murine immune responses and immunization against Polyplax serrata (Anoplura: Polyplacidae).

Mice with restricted grooming capabilities were infested with the solenophagous louse, Polyplax serrata (Burmeister). Louse burdens on Cox/Swiss and C3H/HeSN mice increased for approximately 1 mo, reaching burden/host weight ratios of 1.14 and 1.26 mg/g, respectively, followed by a steady decline. Fifty days after initial ectoparasite contact, both strains were resistant to lice. Resistance was anamnestic, lasting several months with second infestation weights reduced by 98 and 78% on Cox/Swiss and C3H/HeSN, respectively. Furthermore, mice were systemically resistant because infestations on naive body sites of resistant hosts were reduced by 59%. Host resistance was associated with the development of antilouse immune responses. After the first week of a primary infestation, the draining lymph nodes contained cells that proliferated in vitro to louse antigens. Skin responses to louse antigens were also detected: (1) delayed, (2) immediate and delayed, and (3) no significant reactivity on days 19, 34, and 54, respectively. The presence of systemic antilouse responses provided an immunologic basis for immunization against lice. Intradermal injections of soluble louse components reduced primary infestation weights by 62%. Immunized mice had immediate and delayed skin responses containing an inflammatory infiltrate 1 wk following immunization. This study, using the natural host of P. serrata, demonstrates an inducible, anamnestic immune component in louse resistance.

Characterization of an immunosuppressant protein from Dermacentor andersoni (Acari: Ixodidae) salivary glands.

A 36-kDa soluble protein was found in the salivary glands of female Dermacentor andersoni (Stiles) ticks that suppressed the in vitro proliferative response of murine splenocytes to concanavalin A (Con A). Incubating the purified protein with splenocytes reduced the incorporation of thymidine into the DNA of proliferating T-lymphocytes by more than 90% compared with cells exposed to Con A and buffer alone. The N-terminal amino acid sequence of the immunosuppressant protein was determined to be NH2-Leu-His-Lys-Ala(Asp)-Lys-Ile-Val-Lys-Leu-Thr -Glu-Glu-Ala -Arg-Lys-Tyr-Val-Gly-Arg-Xxx-Xxx-Thr-Thr-Ala-Leu-Gly-. Although the sequence exhibited a modest degree of similarity with a segment of immunoglobulin-binding protein found in several species of mammals, the mode of action of the immunosuppressant protein is unknown. This protein may play an important role in suppressing the host's acquisition of resistance to ticks.

Influence of repeated infestations with pathogen-free Ixodes scapularis (Acari: Ixodidae) on in vitro lymphocyte proliferation responses of C3H/HeN mice.

In the United States, Ixodes scapularis Say has been implicated as the vector of at least three human pathogens. Tick induced modulation of host immunity is increasingly recognized as an important factor in successful transmission or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on in vitro proliferative responses of splenic lymphocytes from C3H/HeN mice. Lymphocytes from repeatedly infested and uninfested mice were exposed to concanavalin A (Con A), Escherichia coli Castellini & Chalmers lipopolysaccharide (LPS), or I. scapularis salivary gland soluble proteins (SGSP), to determine if lymphocyte responses differed between tick-exposed and nonexposed mice. Female C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-d tick-free period between each exposure. After each infestation, tick biology parameters were measured and lymphocyte proliferative responses assessed. Acquired resistance to I. scapularis was not evident in mice subjected to tick feeding. Significant differences in the responses of lymphocytes exposed to I. scapularis SGSP were observed between infested and noninfested mice. In contrast, few differences between infested and noninfested mice were evident for lymphocytes exposed to Con A or LPS. Our results suggest that repeated exposure to I. scapularis nymphs does not affect Con A or LPS-induced proliferation of splenic lymphocytes, but significantly effects lymphocyte responses to tick salivary gland antigens.

Immunomodulation of host responses to ectoparasite infestation--an overview.

Immune reactivity stimulated by ectoparasites and other arthropods has received increased attention in recent years. It is clear that a broad range of host responses are elicited, and immune effector mechanisms appear to be active in limiting infestation with blood-feeding arthropods. Host immune responsiveness appears to be altered as a consequence of infestation by certain arthropods, and the impact of these alterations on vector-borne pathogen transmission remains to be determined. Successful techniques have been developed by a number of investigators for the artificial induction of resistance to blood-feeding arthropods. These areas are examined in this paper.

Immunological control of hematophagous arthropod vectors: utilization of novel antigens.

Immunological control of ixodid ticks requires development of a vaccine that stimulates an effective anti-tick response. However, it is important to avoid induction of intense host skin reactivity to tick feeding. Salivary-gland-derived molecules are introduced into the host during ixodid engorgement; therefore, use of these moieties might not be an optimal immunization strategy. Antigens not normally involved in acquired resistance can be used to induce anti-tick immunity. These "novel" antigens, obtained from tick gut absorptive surface, are not introduced into the host during tick feeding, but are exposed to host-immune effector elements in the blood meal, resulting in ixodid rejection, prevention of ova production and death. Anti-tick immunity is induced with microgram quantities of this ixodid gut antigen preparation. Sera obtained from immunized animals were used to identify antibody-reactive components of the resistance-inducing extract. Tick gut absorptive surface antigen glycoconjugates were identified by lectin blotting, using a series of probes with different carbohydrate specificities. The lectins, peanut and wheat germ agglutinin, and immunized host antibodies bound extract components with similar molecular weights. Solubilization and fractionization of tick gut proteins with the non-ionic detergent Triton X-114 is described.

Ixodid-host immune interaction. Identification and characterization of relevant antigens and tick-induced host immunosuppression.

Ixodid ticks are the most important vectors of pathogens to domestic and wild animals. It is established that cattle and laboratory animal species acquire resistance to tick infestation; acquired resistance has an immunological basis consisting of cell-mediated, antibody-mediated and complement-dependent effector mechanisms. Even though acquired resistance to tick feeding is expressed, host immune competence is possibly impaired during the course of tick feeding. Ixodid-induced transient immunosuppression could possibly facilitate the transmission of vector-borne pathogens and/or enhance tick feeding capabilities in the presence of a host immune response to the hematophagous arthropod. Tick tissue extracts have been used to artificially induce resistance to ixodid feeding, and this has become an area of increasing interest as a possible strategy for tick control. It is essential to have defined antigenic molecules for analysis of host responses to infestation, characterization of immunopathologic processes and for vaccine development. This report focuses on attempts to identify, characterize and isolate tick immunogens. Protein immunoblotting, utilizing sera from animals of different genetic composition and infestation patterns, was used to detect a number of tick polypeptides which are reactive with sera of infested hosts. It is clear that infestation with one ixodid species stimulates antibodies reactive with molecules derived from the sensitizing species and/or tick species in the same genus or different genera. This approach is used to identify molecules that are good candidates for use in immunization studies and for analysis of mechanisms involved in acquisition and expression of resistance to tick feeding.

Progress toward molecular characterization of ectoparasite modulation of host immunity.

Ectoparasitic arthropods and vector-borne infectious agents are global medical and veterinary public health concerns. Economic impact due to direct effects of infestation and disease transmission are significant. These problems are increased by development of arthropod resistance to insecticides/acaricides; drug resistance of vector-borne pathogens; and, lack of effective vaccines to prevent many of these diseases. There is much to be gained from understanding the complex array of immunological interactions occurring at the arthropod-host-pathogen interface. One application of that knowledge is the development of novel vaccines for the control of both ectoparasitic arthropods and the diseases they transmit. We now realize that blood-feeding arthropods are not simply flying or crawling hypodermic needles and syringes. Ectoparasitic arthropods are not passive partners in their relationships with the immune systems of their hosts. These clever invertebrates produce numerous pharmacologically active molecules that help them migrate through tissues of their hosts or to successfully obtain blood meals. Arthropod parasites stimulate a spectrum of host immune responses that could potentially impair development, reduce feeding success, or kill the ectoparasite. Not unexpectedly, arthropods have developed sophisticated arsenals of countermeasures that modulate or deviate host immune responses. Not only does arthropod modulation of host immunity facilitate survival in tissues or increase the likelihood of obtaining a blood meal, but it is increasingly recognized as a critical factor in pathogen transmission. Those countermeasures to host immune defenses are the topics of this review. Emphasis is placed on our current understanding of the molecular bases of those changes; the molecules responsible for host immunomodulation; contemporary approaches for studying these complex relationships; and, the potential for using this information to develop innovative vaccine-based control strategies.

Host immune response to northern fowl mite: immunoblot and lectin blot identification of mite antigens.

White leghorn hens were experimentally infested with northern fowl mites (Ornithonyssus sylviarum) and antibody responses to mite immunogens were monitored over 12 weeks. Mite burdens increased during the early phase of infestation and declined over the latter weeks of the study. Antigen was prepared from homogenized whole mites, which were then sonicated and extracted with non-ionic detergent. Antigen extract was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibody-reactive polypeptides were identified by immunoblotting. At the start of infestation, hens had natural, pre-existing antibodies that reacted with several mite-extract components. Individual hens had different natural antibody reactivities; however, all birds had immunoglobulins reactive with extract polypeptides of 117,000, 77,000 and 36,000 molecular weight. A variety of mite extract components reacted with hen antibodies generated in response to experimental infestation. The number of antibody-reactive mite polypeptides increased through week 8 of infestation and then decreased by week 12. Fifteen polypeptides of northern fowl mite extract were reactive with antibodies developed by the majority of infested birds. These commonly reactive polypeptides had molecular weights ranging from 40,000 to 160,000. Glycoconjugates of fractionated mite extract were identified by blotting with lectins that have different carbohydrate binding specificities. Also identified were lectins that bound extract components with the same molecular weights as those moieties complexed by immunoglobulins of infested birds.

Acquired resistance of guinea pigs to Dermacentor andersoni mediated by humoral factors.

Humoral and cell-mediated immune resistance to tick infestation has been documented in many host-parasite relationships. This study examines passive transfer of resistance to Dermacentor andersoni expressed by recipients of serum pools derived from guinea pigs that had acquired resistance through experimental infestation. Recipients of sera from high titer adult-infested animals expressed resistance evidenced by reduced engorgement weights, histological changes at tick attachment sites, and tick antigen-reactive cytophilic antibody. Recipients of lower titer sera from nymph-infested guinea pigs did not show significant reductions in engorgement weights but did exhibit inflammatory reactions at tick attachment sites similar to those of high titer sera recipients. This study, together with previous findings, suggests that immature instars of D. andersoni may not be as effective as adult ticks in stimulating sufficient humoral responses to convey biological resistance to naive recipients, and it underscores the influence the route of serum administration can have on passive transfer studies.

Acquisition and expression of resistance by Bos indicus and Bos indicus X Bos taurus calves to Amblyomma americanum infestation.

Purebred and crossbred Bos indicus calves were infested 1, 2, or 3 times with 10 female and 5 male Amblyomma americanum. Resistance was acquired by both the purebred and the crossbred calves after 1 infestation and resulted in statistically significant decreases in the percentages of females that engorged, the mean weights of engorged females, and the mean weights of egg masses. Comparisons between breeds of the percent of female ticks that engorged during the first and second infestations indicate that purebred B. indicus expressed a stronger acquired resistance to A. americanum more readily than did crossbred animals. However, calves of both genetic compositions displayed similar levels of resistance during a third exposure. All tick-exposed and control animals were skin tested with salivary gland extracts of A. americanum, A. cajennense and Dermacentor andersoni. Control, uninfested calves, did not display significant cutaneous reactivity to these extracts. All calves that had been infested had immediate, 30-min, 5-hr and delayed, 24-hr, skin reactions to Amblyomma species antigens. Reactions to D. andersoni salivary antigens in tests of both purebred and crossbred calves with acquired resistance to A. americanum suggest that Amblyomma species salivary gland antigens might have cross reactive moieties with a salivary extract prepared from D. andersoni. Peripheral blood lymphocyte in vitro responsiveness to Amblyomma species antigens was detected in purebred calves after a first, second, and third infestation, indicating the presence of cells of the immune system capable of recognizing and undergoing blast transformation in response to tick salivary components.(ABSTRACT TRUNCATED AT 250 WORDS)