Monitoring Leishmania infection and exposure to Phlebotomus perniciosus using minimal and non-invasive canine samples

BACKGROUND: In endemic areas of zoonotic leishmaniosis caused by L. infantum, early detection of Leishmania infection in dogs is essential to control the dissemination of the parasite to humans. The aim of this study was to evaluate the serological and/or molecular diagnostic performance of minimally and non-invasive samples (conjunctiva cells (CS) and peripheral blood (PB)) for monitoring Leishmania infection/exposure to Phlebotomus perniciosus salivary antigens in dogs at the beginning and the end of sand fly seasonal activity (May and October, respectively) and to assess associated risks factors. METHODS: A total of 208 sheltered dogs from endemic areas of leishmaniosis were screened. Leishmania DNA detection in PB on filter paper and CS was performed by nested-PCR (nPCR), while the detection of anti-Leishmania antibodies was performed using IFAT and ELISA. The exposure to P. perniciosus salivary antigens (SGH, rSP01 and rSP03B + rSP01) was measured by ELISA. RESULTS: Ninety-seven (46.6%) and 116 (55.8%) of the 208 dogs were positive to Leishmania antibodies or DNA by at least one test at the beginning and end of the sand fly season, respectively. IFAT and ELISA presented a substantial agreement in the serodiagnosis of leishmaniosis. Discrepant PB nPCR results were obtained between sampling points. Leishmania DNA was detected in CS of 72 dogs at the end of the phlebotomine season. The presence of antibodies to the parasite measured by ELISA was significantly higher in dogs presenting clinical signs compatible with leishmaniosis at both sampling points. Phlebotomus perniciosus salivary antibodies were detected in 179 (86.1%) and 198 (95.2%) of the screened dogs at the beginning and end of the phlebotomine season, respectively. CONCLUSIONS: The association between ELISA positivity and clinical signs suggests its usefulness to confirm a clinical suspicion. CS nPCR seems to be an effective and non-invasive method for assessing early exposure to the parasite. PB nPCR should not be used as the sole diagnostic tool to monitor Leishmania infection. The correlation between the levels of antibodies to P. perniciosus saliva and Leishmania antibodies suggests the use of a humoral response to sand fly salivary antigens as biomarkers of L. infantum infection.publishersversionpublishe

Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field

Recombinant antigens from Phlebotomus perniciosus saliva as markers of canine exposure to visceral leishmaniases vector

BACKGROUND: Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. METHODOLOGY/PRINCIPAL FINDINGS: The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. CONCLUSIONS/SIGNIFICANCE: Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector exposure and for estimating the risk of L. infantum transmission in dogs.This research has been funded by Instituto de Salud Carlos III ( PS09/02075 and PI1202021); Xunta de Galicia (10 PXIB 918 273PR) Fundación Mutua Madrileña. Centro de Investigacion Biomedica en Red Fisiopatología de la Obesidad y Nutrición is an ISCIII iniciative (CB06/03). CC is funded by CIBER Fisiopatología Obesidad y Nutrición (CB06/03), LLS is funded by the University Professional Development Program (FPU) of the Spanish Ministry of Education, SB-F by Xunta de Galicia. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.S

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

<div><p>Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against <i>Leishmania</i> parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from <i>Lutzomyia longipalpis</i>. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-<i>Leishmania</i> vaccines or as an antigen to measure host exposure to sand flies.</p></div

Visualization of the tunnel in 3Q6K_Llon1.

<p>The structure was visualized in PyMOL using the MOLE script for calculating the tunnel. The 3Q6K_Llon1 protein is drawn in green, the tunnel through this protein structure is in black mesh and the red-stick molecule represents serotonin.</p

Identified sand fly salivary YRPs.

<p>Identified sand fly salivary YRPs.</p

Amino acid alignment within the YRP ligand-binding sites.

<p>The alignment was created in ClustalX, and shows high conservancy in binding sites (letters in brackets below the sequences, slashes indicate amino acid substitutions in appropriate positions). Ligand binding sites are highlighted in black, three bold stars below the sequences represent the main binding sites where hydrogen bonds between amino acids and the ligand are predicted. Protein codes refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166191#pone.0166191.t001" target="_blank">Table 1</a>.</p

Sequence conservation of the amino acids lining the ligand-binding tunnel.

<p>The WebLogo server was used to visualize the sequence conservation of the amino acids lining the ligand-binding tunnel in 31 YRPs. The amino acids are colored according to their physical-chemical properties. LS and OS below the x-axis indicate the side closer to the ligand-binding site and the opposite side of the tunnel, respectively.</p

Phylogenetic tree of 33 sand fly salivary YRPs with putative glycosylation sites marked.

<p>The maximum likelihood phylogenetic tree was created in TREEPUZZLE with WAG model using quartet puzzling with 10000 puzzling steps. For rooting, the related protein (ACCN: NP650247) from <i>Drosophila melanogaster</i> (Dmel) was used. Bootstraps with support for branching are shown. The letters N, O, and C indicate putative N-, O-, and C-glycosylation sites, respectively. Protein codes refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166191#pone.0166191.t001" target="_blank">Table 1</a>.</p

A comparison of tunnels in sand fly salivary YRPs.

<p>A comparison of tunnels in sand fly salivary YRPs.</p