Salivary Gland Lysates Research Articles

A cement protein of the tick Rhipicephalusappendiculatus, located in the secretory e cell granules of the type III salivary gland acini, induces strong antibody responses in cattle

Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding. We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalusappendiculatus. The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline. The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL[G/Y/S/F/L] tripeptide and a GSPLSGF septapeptide. Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3′ end of the RIM36 gene. Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini. An Escherichiacoli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bosindicus cattle, either experimentally infested with R. appendiculatus, or exposed to ticks in the field. The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R. appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks. The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks.

A Portuguese isolate of Borrelia lusitaniae induces disease in C3H/HeN mice

A low-passage, Portuguese isolate of Borrelia lusitaniae, strain PotiB2, was inoculated into C3H/HeN mice and disease was monitored by histopathology at 8 weeks after spirochaete challenge. Ear, heart, bladder, femoro-tibial joint, brain and spinal cord were examined. B. lusitaniae strain PotiB2 (6 of 10 mice) and B. burgdorferi sensu stricto strain N40 (9 of 10 mice) induced similar lesions in the bladder of infected mice characterised as a multifocal, lymphoid, interstitial cystitis. Moreover, both B. lusitaniae PotiB2 and B. burdorferi N40 induced lesions in the heart of infected mice. The lesions induced by B. lusitaniae PotiB2 (2 of 10 mice) were characterised as a severe, necrotising endarteritis of the aorta, with a minimal, mixed inflammatory infiltrate (neutrophils, macrophages and lymphoid cells) extending into the adjacent myocardium. In contrast, B. burgdorferi N40 induced a periarteritis of the pulmonary artery (7 of 10 mice), with no involvement of the endothelium and more extensive inflammation and subsequent necrosis of the adjacent myocardium. This infiltrate was composed entirely of mononuclear cells, predominantly mature lymphocytes and plasma cells. No lesions were noted in the joints or central nervous system with inoculation of strains N40 or PotiB2, and co-inoculation of either strain with Ixodes ricinus salivary gland lysate did not affect the resulting pathology. Serology, examined 8 weeks after inoculation, indicated a different reactivity in mice infected with B. lusitaniae PotiB2 compared with B. burgdorferi N40. Immunoblot analysis demonstrated that mice with lesions resulting from infection with B. lusitaniae PotiB2 reacted only to the flagellin protein (41 kDa) or to flagellin and OspC, whereas mice infected with B. burgdorferi N40 reacted with multiple high and low mol. wt proteins, including flagellin, p93, p39, OspA, OspB and OspC. These results indicate that B. lusitaniae PotiB2 induced pathology similar to B. burgdorferi N40 when inoculated into susceptible mice. Moreover, these results establish the first animal model of disease with B. lusitaniae. This mouse model can be used to characterise the immunopathogenesis of B. lusitaniae infection and to delineate the proteins responsible for disease induction in susceptible mice.

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan.

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.

Setting the stage for Leishmania

When a host is infected with a parasite, a symbiosis ensues that functions as a new complex organism; the infected host. The co-existence of these organisms requires integration of both physiological complexes. It has been shown that the immune system plays an important role in the fate of Leishmania in the host. White blood cells produce a cytokine context that either allows survival of the parasite or its elimination. This is dependent on the type of T-cell response activated upon infection. A T-helper 1 (Th1) response (characterized by the production of IFN-γ and IL-12) that leads to parasite killing owing to the activation of infected macrophages by IFN-γ. By contrast, a Th2 response (characterized by the production of IL- 4) allows survival of the parasite, probably because of downregulation of IFN-γ production by IL-10. Results from a mouse model indicated that salivary gland lysates of Plebotomus papatasi, the mosquito vector that transmits Leishmania major, provokes a Th2 response, which allows parasite survival (M.L. Mbow. et al., J. Immunol. 161, 5571–5577, 1998). Recently, it was shown that prior exposure of mice to bites of uninfected sandflies induced strong Th1 responsiveness with IFN-γ production, and associated resistance to Leishmania infection. These results suggest an alternative means of immunization against Leishmania infection (S. Kamhawi et al., Science 290, 1351–1354, 2000) and might have implications to the epidemiology of the disease. TS

Species-specific antigens in salivary glands of phlebotomine sandflies.

Saliva inoculated by sandfly females during feeding stimulated production of high levels of anti-saliva antibodies. To determine whether 3 species of the genus Phlebotomus have species-specific salivary antigens we performed dot-blots and immunoblots using sera from mice, hamsters and rabbits repeatedly bitten by sandflies. Important differences were found in the antigen components of the salivary gland lysates (SGL) of Phlebotomus papatasi, P. perniciosus and P. halepensis. In total 4-9 species-specific antigens were detected in each species by immunoblotting. Cross-reactivity was not detected between P. papatasi and the other species tested; in the SGL of P. papatasi sera from animals bitten by this species recognized 5-7 major antigens while sera from animals bitten by other species did not react. A weak cross-reaction was observed between P. perniciosus and P. halepensis; in SGL from P. perliciosus, the sera from rabbits and hamsters bitten by this species recognized about 8 intense bands while sera from animals bitten by P. halepensis reacted weakly with up to 4 saliva polypeptides.

Influence of lysates of the salivary glands of Lutzomyia longipalpis on the development of a Leishmania-major -like parasite in the skin of the golden hamster

Twelve years ago, some mice inoculated with Leishmania major were found to develop larger lesions, containing more amastigotes, if the inoculum used to infect them contained a lysate of salivary glands from Lutzomyia longipalpis than if no lysate was included. In the present study, outbred golden hamsters (Mesocricetus auratus) were each inoculated in a footpad with 10(4), 10(5), 10(6) or 10(7) stationary-phase promastigotes of a Leishmania-major-like parasite (MHOM/BR/71/BH49). Some of the inocula used each contained a lysate of the salivary glands from a laboratory-reared, female Lu. longipalpis. Only the hamsters inoculated with 10(7) promastigotes each developed macroscopic cutaneous lesions (all 10 co-inoculated with lysate but only two of the 10 co-inoculated with diluent). Each of the lesions developed into cutaneous nodule affecting the dermis and underlying subcutaneous tissue of the inoculated footpad, with, histologically, an intensive, diffuse and productive, inflammatory reaction. There were no apparent differences between the lesions of hamsters infected with inocula containing salivary-gland lysate and those seen in the animals infected with lysate-free inocula. Future studies will follow the histological changes at the sites of Lu. longipalpis bites.

Chrysoptin Is a Potent Glycoprotein IIb/IIIa Fibrinogen Receptor Antagonist Present in Salivary Gland Extracts of the Deerfly

Salivary gland lysates of the deerfly (genus Chrysops) contain chrysoptin, an inhibitor of ADP-induced platelet aggregation, which presumably assists the fly in obtaining a blood meal. Chrysoptin has now been isolated, and its cDNA has been cloned and expressed. Chrysoptin was purified to homogeneity using anion exchange and hydrophobic interaction chromatography and found to be a protein with a molecular mass of 65 kDa as determined by gel electrophoresis. N-terminal amino acid sequencing allowed for the synthesis of degenerate oligonucleotides that led to cloning, from salivary gland specific mRNA, of the cDNA encoding this platelet inhibitor. No RGD sites are present in the predicted sequence. A search of GenBank(TM) did not reveal significant sequence homology between chrysoptin and other proteins. The molecular mass predicted from the cDNA was 59 kDa. Predicted glycosylation and phosphorylation sites may account for this difference in molecular mass, as recombinant chrysoptin expressed in Sf21 cells had a molecular mass of 65 kDa, matching that of the natural protein. Chrysoptin functions by inhibiting the binding of fibrinogen to the fibrinogen/glycoprotein IIb/IIIa receptor on platelets with an IC(50) of 95 pmol. These results reveal that insect salivary glands are a source of fibrinogen receptor antagonists.

Phlebotomus papatasiSand Fly Salivary Gland Lysate Down-Regulates a Th1, but Up-Regulates a Th2, Response in Mice Infected withLeishmania major

AbstractA vertebrate host becomes infected with Leishmania major when the sand fly vector injects parasites into skin along with saliva. Previous studies showed that salivary gland lysate of the New World sand fly Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. However, L. major is an Old World parasite transmitted in nature by the Old World sand fly Phlebotomus papatasi. Here we examine the ability of P. papatasi salivary gland lysate to enhance infection (lesion size and parasite burden) by L. major. In addition, we examine the effects of salivary gland lysate on the immune response to L. major by monitoring the levels of cytokine mRNA from the lymph nodes draining cutaneous lesions. We found that P. papatasi salivary gland lysate dramatically exacerbated lesion development in disease-resistant CBA mice. This exacerbation of disease correlated with inhibition of the production of Th1 cytokines and associated factors (IFN-γ, IL-12, and inducible nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, whereas no changes in the levels of IL-10 and TGF-β were noted. Importantly, salivary gland lysate directly up-regulated expression of IL-4 mRNA in mice in the absence of infection with L. major.

Histologic Characterization of Experimental Cutaneous Leishmaniasis in Mice Infected with Leishmania braziliensis in the Presence or Absence of Sand Fly Vector Salivary Gland Lysate

Leishmania braziliensis is the causative agent of human cutaneous leishmaniasis in parts of the New World. In the murine model of infection, L. braziliensis does not produce severe or lasting cutaneous lesions in either BALB/c or C3H mice. However, when the parasites are injected into BALB/c mice with salivary gland lysate of the sand fly vector for the parasite, infection is significantly enhanced, as measured by lesion size, parasite burden, and the outcome of infection. Histologic examination of these cutaneous lesions showed that initially, nodular and diffuse dermal infiltrates of neutrophils, eosinophils, and histiocytes occurred in all mice. Over time, the saliva-free lesions progressed to small organized granulomas of epithelioid macrophages that contained few parasites, with eventual resolution of inflammation and mild dermal fibrosis. The saliva-associated lesions progressed to extensive, poorly organized accumulations of heavily parasitized epithelioid macrophages, with persistent neutrophils and eosinophils, and minimal fibroplasia. These results indicate that sand fly salivary gland lysate markedly modifies the inflammatory response to infection with L. braziliensis.

Heterologous Protection byLeishmania donovaniforLeishmania majorInfections in the Vervet Monkey Model of the Disease

The study was aimed at analyzing immunological cross-reactivity betweenLeishmania majorandLeishmania donovaniand possible cross-protection between the two parasite species in the vervet monkey model of the disease. Nine vervet monkeys (Cercopithecus aethiops) from the institute animal colony were sued in the study. Five of the animals had been previously infected withL. donovanibut had remained asymptomatic while the other four animals were naive and comprised the control group. Immunological responses to bothL. majorandL. donovaniantigens in the five animals with prior exposure toL. donovaniwere examined before challenge. High antibody titers to the two antigens were demonstrated in an enzyme-linked immunosorbent assay, but the antibody titers toL. donovaniwere significantly higher than those toL. major(P< 0.005). Positivein vitroperipheral blood leucocyte (PBL) proliferation toL. majorandL. donovaniantigens was also demonstrated, but there wa no significant difference in the response to the two antigens (P> 0.1). High and varying levels of interferon gamma (IFN-γ) were secreted in PBL from the five vervet monkeys when stimulated withL. majorantigen, but vervet monkey 1296 secreted marginal levels of IFN-γ. When the animals were challenged intradermally with 1 × 105virulentL. majorpromastigotes mixed with sandfly vector salivary gland lysate all four vervet monkeys in the control group developed nodules of varying sizes at the inoculation sites that eventually ulcerated. However, nodule formation and ulceration occurred at different times among these animals. The other five animals (animals with prior exposure toL. donovani) did not pick up the infection at all, but one animal from this group, vervet monkey 1296, developed a transient lesion that healed within 9 weeks, the same animal that had been shown to secrete low levels of IFN-γ. The results demonstrate high cross-reactivity betweenL. donovaniandL. majorand thatL. donovaniprotects againstL. majorinfections. This finding is important for vaccine development studies against leishmaniasis.

Maxadilan, the Vasodilator from Sand Flies, Is a Specific Pituitary Adenylate Cyclase Activating Peptide Type I Receptor Agonist

Maxadilan is a potent vasodilator peptide isolated from salivary gland lysates of the sand fly Lutzomyia longipalpis, a vector of leishmaniasis. The peptide aids the fly in obtaining blood from the skin of its vertebrate hosts but the mammalian receptor through which this insect ligand acts was unknown. We demonstrate that maxadilan is an agonist of the type I receptor for pituitary adenylate cyclase activating peptide, a neuropeptide with vascular activity. This surprising observation is a unique example of convergent evolution from a functional standpoint as these two peptides do not share significant sequence homology.

Novel anticoagulant from salivary glands of Simulium vittatum (Diptera: Simuliidae) inhibits activity of coagulation factor V.

Anticoagulant activity was detected in fractions of a reversed phase-high-performance liquid chromatography (RP-HPLC) of salivary gland lysate from Simulium vittatum Zetterstedt. Using a plasma recalcification time assay, these fractions did not inhibit factor Xa or thrombin. HPLC-purified fractions showing the anticoagulant property were pooled and examined using the activated partial thromboplastin time test conducted on normal plasma and plasmas deficient in factors V, VIII, IX, XI, and XII. The anticoagulant prolonged the clotting time of all the plasmas, except plasma deficient in factor V. The detection of antifactor V activity, together with other anticoagulants reported from Simulium spp. indicates a feeding strategy that targets enzymes in the terminal portion of the coagulation cascade.

Simulidin: a black fly ( Simulium vittatum) salivary gland protein with anti-thrombin activity

A protein purified from the salivary gland lysate of female Simulium vittatum was found to inhibit bovine α-thrombin. This protein is stable to heat, has a mass of 11,334 Da and is rich in threonine. Based on N-terminal sequencing for the first 35 amino acids, no significant sequence similarity with other proteins was detected, indicating that this salivary component may be unique in structure. Because of its source and its anti-hemostatic properties, this protein has been named simulidin.

The chemotactic effect of Phlebotomus duboscqi (diptera: psychodidae) salivary gland lysates to murine monocytes

The possibility that salivary gland lysates of Phlebotomus duboscqi are able to attract vertebrate monocytes was investigated. In vitro studies showed that salivary gland lysates of P. duboscqi, the vector of Leishmania major in Kenya, are chemotactic to mouse peritoneal monocytes. This attraction of monocytes by vector salivary gland lysates may form part of the mechanisms through which sandfly saliva ensures successful parasitization of macrophages in a susceptible host by Leishmania parasites.

Characterization of a salivary gland-specific esterase in the vector mosquito, Aedes aegypti

Esterase enzymatic activity was investigated in salivary gland lysates of adult Aedes aegypti Esterases in lysates made from female glands had higher specific activity than those in lysates from male glands towards β-naphthyl acetate but showed no difference with α-naphthyl butyrate as a substrate. Female salivary gland lysates showed no difference in activity to α- and β-forms of naphthyl acetate and no discernable activity towards α-naphthyl caprate. Both female and male salivary gland lysates exhibited phosphatase enzymatic activity but the specific activities were lower than those seen for the esterase enzymatic activity. Salivary gland esterase activity was inhibited completely by paraoxon, para-hydroxymercurobenzoate, tetraethylammonium iodide and moderately by diisopropylfluorophosphate. Eserine and phenylmethylsulfonylfluoride had no effect on enzyme activity. In a probing assay, adults of both sexes were shown to secrete esterase in saliva. Esterase activity was present in the saliva of females probing for either a sugar meal or a blood meal. Furthermore, esterase was secreted from female salivary glands in culture. Histochemical analysis of dissected salivary glands showed that the majority of the esterase enzymatic activity was in the distal-lateral lobes of the female tissues, although the proximal-lateral and medial lobes also had activity. Male salivary glands stained uniformly over all of the lobes. A salivary gland-specific esterase, designated SG-EST, appears to account for the majority of enzyme activity in the glands. SG-EST was partially purified by electroelution of an active protein from native polyacrylamide gels, and has an approximate molecular weight of 65,000 Da. In separate experiments, affinity chromatography independently identified a single 65,000 Da protein likely to be SG-EST. Native electrophoretic analysis of salivary glands revealed that, while most enzyme activity is due to SG-EST, there are two other esterases present. One of these minor moieties is present in adult tissues in addition to the salivary gland, and the other is present throughout development. Possible functions of the salivary gland esterase are discussed.

Salivary gland material from the sand fly Lutzomyia longipalpis has an inhibitory effect on macrophage function in vitro

Previous work from our laboratory demonstrated that the infectivity of the protozoan parasite Leishmania major was enhanced in mice if the infecting inoculum contained salivary gland lysates from the sand fly vector Lutzomyia longipalpis. The present study was designed to address the hypothesis that sand fly salivary gland material may function by inhibiting the host immune response. Results indicated that sand fly saliva inhibited the ability of macrophages to present leishmanial antigens to parasite-specific T cells.

Development of eosinophilia in dogs intradermically inoculated with sand fly saliva and Leishmania (Leishmania) Chagasi stationary-phase promastigotes.

Salivary gland lysates of the sand fly Lutzomia longipalpis have been shown to enhance the infectivity of Leishmania in mice. As shown herein, the simultaneous inoculation of Leishmania chagasi stationary-phase promastigotes and L. longipalpis salivary gland lysate by the intradermal route in a group of mongrel dogs induced a statistically significant eosinophilia, in relation to dogs inoculated with Leishmania or with salivary gland lysate only. These dogs had no evidence of infection, in spite of the high infectivity of the promastigotes when inoculated by the intravenous route.

Analysis of enhancing effect of sand fly saliva on Leishmania infection in mice

Salivary gland lysates of the sand fly Lutzomyia longipalpis markedly enhance the course of infection with Leishmania major in mice. Here we examine various parameters of this phenomenon. The exacerbative effect of L. longipalpis salivary gland lysates occurred in five different mouse strains; however, the character of the effect varied from one strain to another. Consistent exacerbation of infection was achieved with as little as 1/10 of a gland. The exacerbative effect applied to more than one Leishmania species and to more than one species of sand fly, since salivary gland lysates of L. longipalpis enhanced infection with L. mexicana amazonensis and salivary gland lysates of Phlebotomus papatasi enhanced infection with L. major. A synthetic rat calcitonin gene-related peptide was also found to exacerbate infection with L. major but was found to be approximately 100-fold less potent than saliva in mediating this effect. In addition, lesions induced at skin sites at which L. longipalpis had probed for a blood meal exhibited an exacerbated course of infection similar to that seen when parasites were injected with sand fly salivary gland lysates.

A mouse model of Leishmania braziliensis braziliensis infection produced by coinjection with sand fly saliva.

To development a reliable murine model of Leishmania braziliensis braziliensis infection, parasites were injected into BALB/c mice in the presence of phlebotomine sand fly salivary gland lysates, which have previously been shown to greatly increase the infectivity of L. major in mice. When injected with salivary gland lysates, L. braziliensis braziliensis produced progressively enlarging cutaneous nodules, containing many macrophages filled with Leishmania amastigotes. In contrast, L. braziliensis injected without gland extracts produced small and rapidly regressing lesions. Isoenzyme analysis, monoclonal antibodies, and the polymerase chain reaction with L. braziliensis-specific oligonucleotide primers and probes confirmed that parasites causing the lesions were L. braziliensis.

Characterization of the salivary apyrase activity of three rodent flea species

1. 1. Salivary gland lysates of the adult female fleas Oropsylla bacchi, Orchopea howardi and Xenopsylla cheopis hydrolyse ATP and ADP, but not AMP, thus characterizing the existence of a salivary apyrase activity. 2. 2. In all species Mg ++ or Ca ++ function as activators, and a pH optimum between 7 and 8 is observed. 3. 3. Salivary gland lysates of male fleas contain significantly smaller amounts of the enzyme activity than do those of female fleas. 4. 4. Immediately following a blood meal, apyrase activity and protein content of female X, cheopis salivary glands are 2–3-fold less than that of unfed fleas, indicating that salivary apyrase activity is secreted during feeding. 5. 5. It is suggested that, as in other arthopods, salivary apyrase may facilitate blood location and blood feeding by preventing ADP-induced platelet aggregation at the site of the bite.