Measurement of Recent Exposure to Phlebotomus argentipes, the Vector of Indian Visceral Leishmaniasis, by Using Human Antibody Responses to Sand Fly Saliva

Antibody (IgG) responses to the saliva of Phlebotomus argentipes were investigated using serum samples from regions of India endemic and non-endemic for visceral leishmaniasis (VL). By pre-adsorbing the sera against the saliva of the competing human-biting but non-VL vector P. papatasi, we significantly improved the specificity of a P. argentipes saliva enzyme-linked immunosorbent assay. Using this method, we observed a statistically significant correlation between antibodies to P. argenitpes saliva and the average indoor density of female sand flies. Additionally, the method was able to detect recent changes in vector exposure when sera from VL patients were assayed before, during, and after hospitalization and protected from sand fly bites under untreated bed nets. Collectively, these results highlight the utility of antibodies to P. argentipes saliva as an important tool to evaluate VL vector control programs

Hyaluronidase of Bloodsucking Insects and Its Enhancing Effect on Leishmania Infection in Mice

Hyaluronidases are enzymes degrading the extracellular matrix of vertebrates. Bloodsucking insects use them to cleave the skin of the host, enlarge the feeding lesion and acquire the blood meal. In addition, resulting fragments of extracellular matrix modulate local immune response of the host, which may positively affect transmission of vector-borne diseases, including leishmaniasis. Leishmaniases are diseases with a wide spectrum of clinical forms, from a relatively mild cutaneous affection to life-threatening visceral disease. Their causative agents, protozoans of the genus Leishmania, are transmitted by phlebotomine sand flies. Sand fly saliva was described to enhance Leishmania infection, but the information about molecules responsible for this exacerbating effect is still very limited. In the present work we demonstrated hyaluronidase activity in salivary glands of various Diptera and in fleas. In addition, we showed that hyaluronidase exacerbates Leishmania lesions in mice and propose that salivary hyaluronidase may facilitate the spread of other vector-borne microorganisms

Kinetics of Antibody Response in BALB/c and C57BL/6 Mice Bitten by <em>Phlebotomus papatasi</em>

<div><h3>Background</h3><p>Phlebotomine sand flies are blood-sucking insects transmitting <em>Leishmania</em> parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of <em>Leishmania</em> transmission. In this study, we examined the long-term kinetics and persistence of anti-<em>Phlebotomus papatasi</em> saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with <em>P. papatasi</em> salivary antigens and with the recombinant proteins.</p> <h3>Methodology/Principal Findings</h3><p>Sera of BALB/c and C57BL/6 mice experimentally bitten by <em>Phlebotomus papatasi</em> were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed <em>P. papatasi</em> females. Using western blot and mass spectrometry we identified the major <em>P. papatasi</em> antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four <em>P. papatasi</em> recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera.</p> <h3>Conclusions</h3><p>Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in <em>Phlebotomus papatasi</em>–mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of <em>Leishmania</em> transmission.</p> </div

Anti-sand fly saliva antibody response in BALB/c and C57BL/6 mice experimentally bitten by <i>Phlebotomus papatasi</i>.

<p>(A) Total protein profile, Coomassie blue-stained SDS-PAGE gel with <i>P. papatasi</i> salivary gland homogenate. (B) Western blot of <i>P. papatasi</i> salivary proteins recognized by IgG, IgG1, or IgG2b from sera of <i>P. papatasi</i>-bitten BALB/c (week 28) and C57BL/6 mice (week 5). Pre-immune sera of BALB/c and C57BL/6 mice were used as negative controls (Neg).</p

Anti-sand fly saliva antibody response in BALB/c and C57BL/6 mice bitten by <i>Phlebotomus papatasi</i>.

<p>BALB/c mice (A, C, E) and C57BL/6 mice (B, D, F) were divided into control (squares) and experimentally bitten groups (circles). Mice in the latter group were exposed to sand fly bites (arrows) in weeks 1–5 and additionally in the week 27 (only BALB/c mice). Levels of specific IgG (A, B); IgG1 (C, D); and IgG2b (E, F) were measured by ELISA at all sampling points. Full circles represent significant difference between control and bitten mice (p<0.05). Data are presented as the means ± standard errors of the means. Two independent studies were done. OD = optical density.</p

Reactivity of anti-<i>Phlebotomus papatasi</i> saliva IgG with <i>P. papatasi</i> recombinant proteins.

<p>Sera from BALB/c mice experimentally bitten by <i>P. papatasi</i> (Bitten mice) that were positive for anti-<i>P. papatasi</i> IgG (OD>cut-off: 0.19) were tested by Western blot analysis against (A) <i>P. papatasi</i> salivary gland homogenate, (B) a combination of bacterially-expressed PpSP42 and PpSP28 (Yellow-related protein AF335491 and D7-related protein AF335488, respectively), and (C) a combination of bacterially-expressed PpSP44 and PpSP30 (Yellow-related protein AF335492 and D7-related protein AF335489, respectively). The SDS-PAGE protein profiles of the <i>P. papatasi</i> salivary gland homogenate as well as the recombinant proteins were blotted and stained by Amido Black. Pre-immune sera of BALB/c mice were used as the controls (Neg).</p

Correlation between the intensity of sand fly exposure and the anti-<i>Phlebotomus papatasi</i> saliva antibodies.

<p>The correlation between the number of blood-fed sand fly females and the levels of anti-saliva antibodies in experimentally bitten BALB/c (A, C, E) and C57BL/6 (B, D) mice was performed using Spearman Rank Correlation Matrix. Positive correlation was detected in specific IgG (A); IgG1 (C); and IgG2b (E) in BALB/c mice and in specific IgG (B); and IgG1 (D) in C57BL/6 mice. OD = optical density.</p