Salivary Gland Lysates Research Articles

Human immune response to Phlebotomus sergenti salivary gland antigens in a leishmaniasis-endemic focus in Iran

ABSTRACT Salivary proteins specific antibodies have been shown to be useful biomarkers of exposure to sand fly bites. This study aimed to investigate the level, duration, and dynamics of the human immune response against the SGL of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae), and to assess the immunoreactivity of human sera with SGL components in an endemic area of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The study was carried out in 2-phase; longitudinal and cross-sectional. Sand flies were collected monthly from indoors and outdoors. In the longitudinal study, sera from healthy volunteers were collected monthly, and in the cross-sectional study, sera from healthy volunteers and patients with ACL lesion/s, were collected for immunoassay studies. The level of anti-P. sergenti saliva IgG was detected using the ELISA. Immunoreactivity of individual human sera with saliva components was also assessed by western blotting. Phlebotomus sergenti was the predominant sand fly species in the study area. The maximum and minimum percentages of IgG responses were seen in October (66%) and March (29%), respectively. Additionally, the cross-sectional study showed that 59.3% of the healthy volunteers and 80% of the patients were IgG positive. The antibody response against P. sergenti salivary gland was high during the sand fly active season and declined by the end of the activity of the vectors. Antibody response against the SGL components of P. sergenti was transient and individual-specific. Some individuals shared a strong reaction against certain individual antigens, which could be considered as vector exposure markers for further investigation. List of abbreviations ELISA: Enzyme-Linked Immunosorbent Assay; SDS PAGE: Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis; SGL: Salivary Gland Lysate; ACL: Anthroponotic Cutaneous Leishmaniasis; PBS: Phosphate Buffered Saline; BCA: Bicinchoninic Acid; PBS-T: Phosphate Buffered Saline Tween; FBS: Fetal Bovine Serum; HRP: Horseradish Peroxidase; TMB: 3,3′,5,5′-Tetramethylbenzidine; PVDF: Polyvinylidene Difluoride; SGA: Salivary Gland Antigens; OD: Optical Density; KDa: Kilodalton; VL: Visceral Leishmaniasis; CL: Cutaneous Leishmaniasis; SGs: Salivary glands

Salivary gland antigens of laboratory-bred Phlebotomus sergenti and their immunogenicity in human volunteers in laboratory condition

Objective: To investigate Phlebotomus (P.) sergenti Parrot, 1917 (Diptera: Psychodidae) salivary gland antigens and their immune response in human. Methods: Human volunteers were exposed to sand flies’ bites in the laboratory, and following each exposure the size of induration was recorded. The mean protein concentration of salivary gland lysate and specific anti-P. sergenti saliva IgG was measured. Sand fly salivary proteins were separated by SDS-PAGE and their immunoreactivity was examined by Western blotting assays. Results: Individuals exposed to P. sergenti salivary gland lysate for 8 months showed both antibody and delayed type hypersensitivity responses, although exposure for one month did not provoke any immune responses. The trend of antibody fluctuated during the exposure time and dropped by the end of antigen loading. The mean protein content was (0.36±0.08) μg in each pair salivary glands. Salivary gland lysate showed 11 to 12 major protein bands and 3 to 6 of them were immunoreactive. Conclusions: Our study showed that the salivary gland components of P. sergenti provoked both cellular and humoral immune responses in human. Furthermore, there are some mmunogenic proteins in P. sergenti saliva which could be subjected for further investigation as vector-based vaccine candidate/s against anthroponotic cutaneous leishmaniasis.

Association of Antibodies to Interferon-Inducible Protein-16 With Markers of More Severe Disease in Primary Sjögren's Syndrome.

Interferon-inducible protein-16 (IFI16) is an intracellular DNA receptor involved in innate immunity. We evaluated the frequency, phenotypic characteristics, and clinical associations of anti-IFI16 antibodies in patients with primary Sjögren's syndrome (SS), and quantitated expression levels of IFI16 in SS and control salivary gland lysates. Anti-IFI16 antibodies were assayed by enzyme-linked immunosorbent assay using sera from patients with primary SS (n = 133) and from healthy controls (n = 47). Sera from systemic lupus erythematosus (SLE) patients (n = 132) were included as disease controls. Immunoprecipitation of in vitro transcription-translated IFI16 was used to determine which portion of IFI16 the antibodies recognized. Expression of IFI16 in salivary gland lysates was quantitated by immunoblotting. Anti-IFI16 antibodies were present in the sera of 38 of 133 SS patients (29%) compared to 1 of 47 healthy controls (2.1%) (SS versus controls; P < 0.0002) and in 31 of 132 SLE controls (24%). In SS, anti-IFI16 antibodies were associated with an abnormal Schirmer's test (P = 0.003), hyperglobulinemia (P = 0.02), antinuclear antibody ≥1:320 (P = 0.01), germinal center-like structures in labial salivary gland lymphoid infiltrates (P = 0.01), and higher focus scores (3.4 versus 2.4; P = 0.005). High-titer IFI16 antibodies were directed against an epitope outside the N-terminus in 9 of 13 SS patients (69%). IFI16 was expressed in 4 of 5 (80%) of SS and 1 of 6 (17%) of control labial salivary glands. Anti-IFI16 antibodies are a prominent specificity in primary SS and are associated with markers of severe disease. IFI16 is expressed at higher levels in SS salivary glands compared to controls. These high levels in disease target tissue may contribute to the ongoing anti-IFI16 immune response.

Molecular Subsetting of Interferon Pathways in Sjögren's Syndrome.

Sjögren's syndrome (SS) is an autoimmune disease that targets the salivary and lacrimal glands. While all patients demonstrate inflammatory infiltration and abnormal secretory function in the target tissues, the disease features, pathology, and clinical course can vary. Activation of distinct inflammatory pathways may drive disease heterogeneity. The purpose of this study was to investigate whether activation of the interferon (IFN) pathway correlates with key phenotypic features. Clinical data and 1 labial salivary gland (stored frozen) were obtained from each of 82 participants (53 patients with primary SS and 29 control subjects) in the Sjögren's International Collaborative Clinical Alliance (SICCA) registry. Salivary gland lysates were immunoblotted with markers of type I or type II IFN, and patterns of IFN activity were determined by hierarchical clustering. Correlations between SS phenotypic features and IFN activity in the salivary gland were performed. A total of 58% of the SS participants had high IFN activity and differed significantly from those with low IFN activity (higher prevalence of abnormal findings on sialometry, leukopenia, hyperglobulinemia, high-titer antinuclear antibody, anti-SSA, and high focus score on labial salivary gland [LSG] biopsy). Three distinct patterns of IFN were evident: type I-predominant, type II-predominant, and type I/II mixed IFN. These groups were clinically indistinguishable except for the LSG focus score, which was highest in those with type II-predominant IFN. The SS phenotype includes distinct molecular subtypes, which are segregated by the magnitude and pattern of IFN responses. Associations between IFN pathways and disease activity suggest that IFNs are relevant therapeutic targets in SS. Patients with distinct patterns of high IFN activity are clinically similar, demonstrating that IFN-targeting therapies must be selected according to the specific pathway(s) that is active in vivo in the individual patient.

Whole Antigenic Lysates ofIxodes ricinus, but not Der-p2 Allergen-Like Protein,are Potent Inducers of Basophil Activation in Previously Tick-Exposed Human Hosts

The clinical suspicion of tick anaphylaxis is based on a history of the bite and occurs often during the warm season. Further arguments are the presence of natural hosts in the immediate environment and, eventually, the identification of the tick. The diagnosis is confirmed when immediate-type sensitization is shown by positive skin prick tests performed with specific tick extracts or the demonstration of specific IgE in vitro. In the current study, we hypothesize that hard tick-derived material contains potent inducers being able to promote basophil stimulation, which correlates with a sensitization immunological response following tick bites. To this end, biological material from two hard tick cell lines (IRE11 and IDE8 - derived from Ixodes ricinus and I.scapularis, respectively) as well as I.ricinus salivary gland and body lysates were used in a human basophil activation test (BAT) to analyse binding and cross-linking capacity of membrane-bound IgE, because basophils are one of the main effector cells of allergic reactions. Additionally, Der-p2 allergen-like gene from I.ricinus was recombinantly expressed as a 15-kDa histidine-tagged fusion protein, purified and included as a stimulus within the setup. Blood was drawn and submitted to BAT screening from a pool of 36 individuals, both bitten and who served solely as negative controls. We have found that seven subjects (19%), all of whom were at least two times tick-bitten, positively reacted to the aforementioned stimuli, whereas the reactivity level of the ones bearing single bites proved to be within the normal range. Moreover, no significant upregulation of the assessed basophil activation marker was detected in the case of Der-p2, except a faint reaction at high dosages. We conclude that at least two tick bites of the human host must occur in order to induce significant basophil activation.

Single-Cell Analysis Reveals Isotype-Specific Autoreactive B Cell Repertoires in Sjögren’s Syndrome

Microengraving is a novel technology that uses an array of microfabricated subnanoliter wells to isolate and characterize secreted proteins from larger number of single cells. This printing technique permits the capture and characterization of secreted antibodies on glass slides. Here, we profiled the antigenic repertoires of B cells reacting against salivary gland tissues in Sjögren’s syndrome (SjS), an autoimmune disease targeting the exocrine glands. Single-cell suspensions of spleen and cervical lymph node cells prepared from normal C57BL/6 and SjS-susceptible (SjSs) C57BL/6.NOD-AecAec2 mice were dispersed into subnanoliter wells (nanowells). Capture slides preincubated with mouse immunoglobulins were used for printing. Detection antibodies included fluorescence conjugated anti-IgG1, salivary gland lysates of C57BL/6 and SjSs mice. Results indicate an increase in the frequency of IgG1-secreting cells in the spleen of SjSs mice compared to C57BL/6 mice. Cells from the lymph node of SjSs mice yield higher instances of IgG1 reactive against salivary gland antigens than cells from the lymph nodes of C57BL/6 mice. These data demonstrate the isotype-specific reactivity of antibodies during the autoimmune process, and further reveals significant differences in the non-autoimmune and autoimmune antibody repertoires. These results support the generation of self-reactive B cell repertoires during the autoimmune process, at the same time, verifying that microengraving of single cells might allow for identification of novel biomarkers in SjS.

An AAV2/5 Vector Enhances Safety of Gene Transfer to the Mouse Salivary Gland

This study was designed to improve AAV-mediated gene transfer to the murine submandibular salivary glands. Our first aim was to utilize AAV pseudotype vectors, containing the genetic elements of the canonical AAV2, packaged within capsids of AAV serotypes 5, 8, and 9. Having determined that this pseudotyping increased the efficiency of gene transfer to the glands by several orders of magnitude, we next asked whether we could reduce the gene transfer inoculum of the pseudotype while still achieving gene transfer comparable with that achieved with high-dose AAV2. Having achieved gene transfer comparable with that of AAV2 using a pseudotype vector (AAV2/5) at a 100-fold lower dose, our final objective was to evaluate the implications of this lower dose on two pre-clinical parameters of vector safety. To evaluate systemic toxicity, we measured AAV vector sequestration in the liver using qPCR, and found that the 100-fold lower dose reduced the vector recovered from the liver by 300-fold. To evaluate salivary gland function, we undertook whole-proteome profiling of salivary gland lysates two weeks after vector administration and found that high-dose (5 × 109) AAV altered the expression level of ~32% of the entire salivary gland proteome, and that the lower dose (5 × 107) reduced this effect to ~7%.

Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild-caught Lutzomyia longipalpis

In order to compare the saliva effect from wild-caught and lab-reared L. longipalpis on the development of experimental cutaneous leishmaniasis, C57BL/6 mice were inoculated subcutaneously into the hind footpads with promastigotes of L. (L.) amazonensis plus salivary gland lysate from wild-caught (SGL-W) and lab-colonized (SGL-C) vectors. Lesion sizes were significantly larger in the mice infected with both saliva compared to mice infected with parasites alone; moreover, the lesions caused by parasite + SGL-C were significantly larger than the lesions caused by parasite + SGL-W. Histopathological morphometric studies regarding the acute phase of infections showed lower numbers of polymorphonuclear cells, greater numbers of mononuclear cells and parasites in SGL-C infected mice compared to SGL-W infected mice. In the chronic phase of infection, the number of mononuclear cells was lower and the number of parasites was greater in SGL-C infected mice than SGL-W infected mice. In vitro studies showed increased infection index of macrophages infected with parasites plus saliva compared to infection with parasites alone, with no difference between the saliva infection indices. SDS-PAGE gel for SGL-C and SGL-W showed differences in the composition and quantity of protein bands, determined by densitometry. These results call attention to the experimental saliva model, which shows exacerbation of infection caused by sandfly saliva.

Kinetics of Canine Antibody Response to Saliva of the Sand Fly Lutzomyia longipalpis

Zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum is transmitted from dogs to humans by sand flies and Lutzomyia longipalpis is a major vector of this disease. We studied the antibody response in dogs experimentally exposed to L. longipalpis females to characterize sand fly salivary antigens recognized by canine sera and to find out whether the level of specific anti-saliva antibodies reflects the intensity of exposure. Sera from repeatedly bitten dogs revealed up to six salivary protein bands with approximate molecular weight of 66, 55, 45, 37-39, 34, and 25 kDa in L. longipalpis salivary gland lysate. Anti-saliva immunoglobin (Ig) G and its subclasses were found to be useful markers of exposure to sand flies. Specific IgG, IgG1, and IgG2 were related to numbers of bloodfed L. longipalpis females, and increased antibody levels were detectable throughout the study, i.e. more than 6 months after the last exposure. In contrast, specific IgE response developed in some dogs only, and no correlation was observed between its level and the intensity of exposure. Screening of dog sera for specific IgG against salivary antigens of the vector is suggested as a useful epidemiological tool in VL foci. Monitoring canine antibody response to sand fly saliva also allows evaluation of the effectiveness of anti-vector campaigns.

EFFECT OF LUTZOMYIA LONGIPALPIS SALIVARY GLAND EXTRACTS ON LEUKOCYTE MIGRATION INDUCED BY LEISHMANIA MAJOR

The mechanism by which the salivary gland lysate (SGL) of Lutzomyia longipalpis enables Leishmania infection remains under investigation. One possibility is that saliva promotes cellular recruitment leading to development of skin lesions. In this study, we investigated leukocyte recruitment induced by L. major, L. major + SGL, or SGL alone into the peritoneal cavity of BALB/c mice. The administration of L. major with or without SGL induced neutrophil migration six hours after infection. Interestingly, after seven days, the BALB/c mice still had eosinophils and mononuclear cells in their peritoneal cavities. Flow cytometric analysis showed an increase in the CD4(+) CD45RB(low) T cell subset (effector or memory cells) compared with the CD4(+) CD45RB(high) subset (naive cells). Moreover, the co-injection of L. major with SGL enhanced production of interleukin-10. These results suggest that SGL can facilitate Leishmania infection by modulating leukocyte recruitment and Th2 cytokine production at the inflammatory focus.

Identification of a glycine-rich protein from the tick Rhipicephalus haemaphysaloides and evaluation of its vaccine potential against tick feeding

A cDNA coding a glycine-rich protein was identified from the Rhipicephalus haemaphysaloides tick. The cDNA named here as RH50 was 1,823 bp, including a single open reading frame (ORF) of 1,518 nucleotides. The ORF encodes a polypeptide of 506 amino acid residues with a size of 50 kDa, as calculated by a computer. The predicted amino acid sequence of RH50 showed a low homology to sequences of some known extracellular matrix-like proteins. The native protein was identified in both the fed tick salivary gland lysates and extracts of cement material using the serum against the recombinant protein. Reverse transcription polymerase chain reaction results showed that RH50 mRNA was only transcribed in partially fed tick salivary glands, not in unfed tick salivary glands or partially fed tick midgut, fat body, or ovary. The differential expression of RH50 protein in fed tick salivary glands was confirmed by immunofluorescence. The low attachment rate both in the adult and nymphal tick, and the high mortality of immature ticks (nymph) feeding on recombinant RH50-immunized rabbits were found. These results show that the RH50 protein could be a useful candidate for anti-tick vaccine development.

Modulation of murine cellular immune response and cytokine production by salivary gland lysate of three sand fly species

Saliva of sand flies (Diptera: Phlebotominae) plays an important role in transmission of Leishmania parasites by modulating host immune response. However, because of the different protein compositions of saliva, the immunomodulatory effects may vary among sand fly species. We have therefore analysed and compared the immunomodulation effects of salivary gland lysate (SGL) of three different sand flies. Spleen cells from BALB/c mice were incubated with SGL of Phlebotomus papatasi, P. sergenti or Lutzomyia longipalpis. Concanavalin A-stimulated lymphocyte proliferation was significantly suppressed with SGLs of all three sand fly species and all SGL doses tested. This result indicates that saliva from different sand fly species is able to suppress host proliferative response even to the potent mitogen. In parallel experiments, we analysed the effect of SGL on IFN-gamma, IL-2, and IL-4 production; in mitogen-stimulated cells SGLs markedly inhibited IFN-gamma production in all intervals tested (reduced up to 31%) and to a lesser degree impaired production of the other two cytokines as well. Despite some species-specific differences in the intensity of immunomodulatory effects, saliva of all sand fly species modulated cell proliferation as well as cytokine production in a similar way.

Sand fly specificity of saliva-mediated protective immunity in Leishmania amazonensis-BALB/c mouse model

Immune response of BALB/c mice to the salivary antigens of sand flies was found to vary with different species used, i.e. Phlebotomus papatasi, Phlebotomus sergenti and Lutzomyia longipalpis. Exposure of mice to bites of these sand flies elicits production of antibodies, which are largely specific to different saliva antigens previously identified as unique to the respective fly species. When immunized intradermally (i.d.) with salivary gland lysates (SGL) of L. longipalpis, BALB/c mice developed partial protective immunity against challenges in the contralateral ears with Leishmania amazonensis plus the gland lysates. Preimmunization of these mice with the lysates from the other two species was ineffective, further indicative of the specificity of saliva-mediated immune response. The partial protective immunity observed is significant, although it is not as dramatic as reported previously in a different sand fly-mouse model. There is a correlation of this immunity with a lower number of mononuclear and polymorphonuclear phagocytes at the site of parasite inoculation. Vector species-specificity of this immunity implies its elicitation by unique saliva antigen—an issue which requires attention when designing saliva-based vaccines against leishmaniasis.

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.

The mannose receptor is expressed by subsets of APC in non-lymphoid organs

BackgroundThe mannose receptor (MR) is an endocytic receptor of Mφ and endothelial cell subsets whose natural ligands include both self glycoproteins and microbial glycans. It is also expressed by immature cultured dendritic cells (DC), where it mediates high efficiency uptake of glycosylated antigens, yet its role in antigen handling in vivo is unknown. Knowledge of which APC subsets express MR will assist the design of experiments to address its immunological functions. Here the expression of MR by MHC class II positive APC in non-lymphoid organs of the mouse is described.ResultsMR positive APC were identified in several peripheral organs: skin, liver, cardiac and skeletal muscle and tongue. MR positive cells in salivary gland, thyroid and pancreas coexpressed MHC class II and the myeloid markers macrosialin and sialoadhesin, but not the dendritic cell markers CD11c or DEC-205. MR and MHC class II colocalised in confocal microscope images, implying that antigen capture may be the primary role of MR in these cells. Distinct ligands of MR were found in salivary gland and pancreas tissue lysates that are candidate physiological ligands of MR positive APC in these organs.ConclusionsThe tissue and subcellular distribution of MR suggest it is appropriately located to serve as a high efficiency antigen uptake receptor of APC.

A follow-up of Beagle dogs intradermally infected with Leishmania chagasi in the presence or absence of sand fly saliva

In this study, we compare the development of infection and/or disease in Beagle dogs intradermally infected with Leishmania chagasi, in the presence or absence of Lutzomyia longipalpis saliva, with those of intravenously infected animals. Spleen samples of all the animals inoculated with parasites had positive polymerase chain reaction tests for Leishmania DNA. Positive spleen cultures for Leishmania were detected earlier ( P≤0.018) and were more frequent (five out of the five animals) in intravenously infected animals than in the intradermally infected animals, in presence (two out of the six animals) or absence (three out of the five animals) of salivary gland lysate of L. longipalpis. Significant increase in serum antibodies against Leishmania was observed only in the intravenously infected group ( P=0.004). In addition, dogs with infection confirmed by isolation of amastigotes or detection of parasite DNA were, nevertheless, negative for anti- Leishmania antibodies up to 5 months or more after infection. Only animals of the intravenously infected group developed progressive decreases in hematocrit (Pearson r=−0.8076, P=0.0026) and hemoglobin (Pearson r=−0.8403, P=0.0012) during the infection period. No significant difference in the course of infection was observed between groups of intradermally infected animals. The data presented herein confirms that the intradermal inoculation of dogs with Leishmania produces an asymptomatic form of infection. It also fails to show an advantage in using L. longipalpis saliva as an infection-enhancing agent in experimental canine leishmaniasis.

Immunomodulatory effects of Maxadilan and Phlebotomus papatasi sand fly salivary gland lysates on human primary in vitro immune responses.

Leishmaniasis is a parasitic disease transmitted by the bite of Leishmania-infected sand flies. Here we show for the first time the ability of Maxadilan (Max), a vasodilatory peptide isolated from the sand fly Lutzomyia longipalpis, and salivary gland lysate (SGL) from Phlebotomus papatasi to decrease the secretion of Type 1 cytokines and to enhance the production of the Type 2 cytokine interleukin (IL)-6 by human peripheral blood mononuclear cells (PBMC) and monocytes. We found Max decreased the secretion of interferon (IFN)-gamma and IL-12p40 by PBMC and TNF-alpha by monocytes. SGL reduced the production of IFN-gamma by PBMC. In contrast, production of the Type 2 cytokine IL-6 was increased in Max or SGL-exposed cells. Finally, we determined that Max interacts with human cells through at least the pituitary adenylate cyclase activating polypeptide receptor. These results show that sand fly salivary gland components have an immunomodulatory effect on human cells, and this has important implications for the development of vaccines against leishmaniasis for humans.

Coinoculation of Borrelia spp. with tick salivary gland lysate enhances spirochete load in mice and is tick species-specific.

C3H/HeN mice were inoculated with 10(6) spirochetes, either Borrelia burgdorferi strain N40 or the Portuguese strain of B. lusitaniae, PotiB2. Mice receiving spirochetes coinoculated with salivary gland lysate (SGL) demonstrated significantly higher spirochete loads in target organs as measured by quantitative real-time polymerase chain reaction. This effect was tick dependent, in that Ixodes ricinus SGL specifically enhanced B. lusitaniae load, whereas I. scapularis SGL specifically increased B. burgdorferi N40 load, but did not significantly affect the dissemination of B. lusitaniae. Protein profile analysis indicated at least 5 major protein differences between I. scapularis and I. ricinus SGL, which can possibly account for this specific tick-spirochete interaction.

Characterization of the lectin from females of Phlebotomus duboscqi sand flies.

Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohydrate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d-glucosamine, d-galactosamine and d-mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.

Coinoculation of Borrelia spp. with Tick Salivary Gland Lysate Enhances Spirochete Load in Mice and Is Tick Species-Specific

Coinoculation of Borrelia spp. with Tick Salivary Gland Lysate Enhances Spirochete Load in Mice and Is Tick Species-Specific