The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.

• MeSH
• antigeny analýza   MeSH
• hmyz - vektory parazitologie   fyziologie   MeSH
• hmyzí proteiny analýza   MeSH
• kousnutí a bodnutí hmyzem epidemiologie   etiologie   MeSH
• Leishmania infantum fyziologie   MeSH
• leishmanióza parazitologie   veterinární   MeSH
• nemoci psů epidemiologie   etiologie   MeSH
• Phlebotomus parazitologie   fyziologie   MeSH
• psi MeSH
• rekombinantní proteiny analýza   MeSH
• slinné proteiny a peptidy analýza   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Itálie epidemiologie   MeSH
• Portugalsko epidemiologie   MeSH

BACKGROUND: Canine leishmaniasis (CanL) is a zoonotic disease, caused by Leishmania infantum and transmitted by Phlebotomus perniciosus in the Mediterranean basin. Previously, an ELISA based on the P. perniciosus salivary protein SP03B was proposed as a valid tool to screen for canine exposure to sand fly bites across regions endemic for CanL. Although this approach is useful in laboratory settings, a practical tool for immediate application in the field is needed. In this study we propose the rSP03B sero-strip, the first immunochromatographic test (ICT) in the field of vector exposure able to rapidly screen dogs living in endemic areas for the presence of P. perniciosus and to aid in the evaluation of vector control programs. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was prepared using the bacterially expressed recombinant protein rSP03B as antigen. For test optimization, pre-immune sera from non-bitten laboratory-bred Beagles were used as negative controls. In order to validate the test, sera from laboratory-bred Beagles experimentally exposed to P. perniciosus bites were used as positive controls. Additionally, all samples were tested by ELISA using whole salivary gland homogenate (SGH) and the rSP03B protein as antigen. An almost perfect degree of agreement was found between the ICT and the SGH-ELISA. Furthermore, the newly proposed rSP03B sero-strip showed a sensitivity of 100% and a specificity of 86.79%. CONCLUSIONS/SIGNIFICANCE: We developed a simple and rapid ICT based on the P. perniciosus rSP03B salivary protein, able to replace the standard ELISA used in previous studies. Our rSP03B sero-strip showed to be highly sensitive and specific in the detection of antibodies (IgG) against P. perniciosus saliva. In the future, this test can be employed during large-scale epidemiological studies of CanL in the Mediterranean area to evaluate the efficacy of vector control programs.

• MeSH
• časové faktory MeSH
• chromatografie afinitní veterinární   MeSH
• ELISA metody   MeSH
• hmyz - vektory MeSH
• hmyzí proteiny MeSH
• kousnutí a bodnutí hmyzem imunologie   veterinární   MeSH
• Leishmania infantum MeSH
• nemoci psů diagnóza   parazitologie   MeSH
• Phlebotomus imunologie   MeSH
• psi MeSH
• reagenční papírky MeSH
• senzitivita a specificita MeSH
• sérologické testy veterinární   MeSH
• zoonózy MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host-vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. METHODS: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. RESULTS: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. CONCLUSIONS: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas.

• MeSH
• endemické nemoci veterinární   MeSH
• hmyz - vektory parazitologie   MeSH
• humorální imunita MeSH
• imunoglobulin G analýza   MeSH
• Leishmania infantum izolace a purifikace   MeSH
• leishmanióza krev   parazitologie   veterinární   MeSH
• longitudinální studie MeSH
• nemoci psů diagnóza   imunologie   parazitologie   MeSH
• Phlebotomus imunologie   MeSH
• protilátky protozoální krev   MeSH
• protilátky krev   MeSH
• psi imunologie   parazitologie   MeSH
• roční období MeSH
• slinné proteiny a peptidy imunologie   MeSH
• slinné žlázy chemie   parazitologie   MeSH
• sliny imunologie   mikrobiologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• psi imunologie   parazitologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Španělsko MeSH

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs. METHODOLOGY/PRINCIPAL FINDINGS: Sera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus. CONCLUSIONS: These results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.

• MeSH
• ELISA MeSH
• endemické nemoci veterinární   MeSH
• leishmanióza imunologie   veterinární   MeSH
• longitudinální studie MeSH
• nemoci psů imunologie   MeSH
• Phlebotomus imunologie   MeSH
• polymerázová řetězová reakce veterinární   MeSH
• protilátky imunologie   MeSH
• psi MeSH
• rekombinantní proteiny imunologie   MeSH
• rizikové faktory MeSH
• slinné proteiny a peptidy imunologie   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH

BACKGROUND: Canine leishmaniosis caused by Leishmania infantum is a neglected zoonosis transmitted by sand flies like Phlebotomus perniciosus. Clinical signs and disease susceptibility vary according to various factors, including host immune response and breed. In particular, Ibizan hounds appear more resistant. This immunocompetence could be attributed to a more frequent exposure to uninfected sand flies, eliciting a stronger anti-sand fly saliva antibody response. METHODS: This study aimed to investigate the prevalence of anti-P. perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in the Leishmania-endemic area of Mallorca, Spain, and to correlate these antibody levels with clinical, immunological and parasitological parameters. Anti-sand fly saliva IgG was examined in 47 Ibizan hounds and 45 dogs of other breeds using three methods: P. perniciosus whole salivary gland homogenate (SGH) ELISA; recombinant protein rSP03B ELISA; and rSP03B rapid tests (RT). Additionally, diagnostic performance was evaluated between methods. RESULTS: Results indicate significantly higher anti-SGH antibodies (P = 0.0061) and a trend for more positive SGH ELISA and RT results in Ibizan hounds compared to other breeds. General linear model analysis also found breed to be a significant factor in SGH ELISA units and a marginally significant factor in RT result. Although infection rates were similar between groups, Ibizan hounds included significantly more IFN-γ producers (P = 0.0122) and papular dermatitis cases (P < 0.0001). Older age and L. infantum seropositivity were also considered significant factors in sand fly saliva antibody levels according to at least one test. Fair agreement was found between all three tests, with the highest value between SGH and rSP03B RT. CONCLUSIONS: To our knowledge, this is the first study elaborating the relationship between anti-P. perniciosus saliva antibodies and extensive clinical data in dogs in an endemic area. Our results suggest that Ibizan hounds experience a higher frequency of exposure to sand flies and have a stronger cellular immune response to L. infantum infection than other breed dogs. Additional sampling is needed to confirm results, but anti-P. perniciosus saliva antibodies appear to negatively correlate with susceptibility to L. infantum infection and could possibly contribute to the resistance observed in Ibizan hounds.

• MeSH
• chov MeSH
• endemické nemoci MeSH
• hmyzí proteiny imunologie   MeSH
• imunoglobulin G imunologie   MeSH
• leishmanióza imunologie   veterinární   MeSH
• náchylnost k nemoci MeSH
• nemoci psů imunologie   parazitologie   MeSH
• Phlebotomus imunologie   MeSH
• psi MeSH
• slinné proteiny a peptidy imunologie   MeSH
• sliny imunologie   MeSH
• zoonózy parazitologie   přenos   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Španělsko MeSH

From July 2009 to date, a leishmaniosis outbreak has occurred in the south-west of the Madrid region (Spain) and has already accounted for more than 450 human cases in an area that comprises a population of approximately 500,000. The causative agent is Leishmania infantum and the main vector in the area is Phlebotomus perniciosus. Although canine leishmaniosis prevalence in the focus is not higher than the average in the Madrid region, a wild reservoir - the hare - has been implicated. In this study, we examined the exposure of Leishmania reservoirs in the area: dogs, hares, and wild rabbits to sand fly bites using the detection of specific IgG antibodies against P. perniciosus salivary gland homogenate or recombinant salivary proteins. Hares collected in a green park adjacent to the focus (n=59) showed positive exposure to P. perniciosus bites in comparison to hares from a non-endemic area (Czech Republic, n=18). A significant positive correlation was found between IgG response to yellow protein rSP03B and salivary gland homogenate (r=0.902) and between apyrase rSP01B and salivary gland homogenate (r=0.710). Wild rabbits captured in the study area (n=21) presented higher anti-saliva antibody levels than negative control sera and their IgG response against recombinant salivary proteins were positively correlated with salivary gland homogenate (rSP03B: r=0.710; rSP01B: r=0.666). All sera of dogs from the focus (n=34) showed higher anti-saliva IgG levels than that of non-exposed dogs. Moreover, dogs protected against sand fly bites through the use of topical insecticides and sleeping indoors showed significantly lower antibody levels than the non-protected ones. Antibody response to all three recombinant salivary proteins tested showed positive correlation with salivary gland extract (rSP03B: r=0.858; rSP01: r=0.864; and rSP01B: r=0.861). Data confirmed the exposure of hares, rabbits and dogs to P. perniciosus bites in the context of an outbreak of human leishmaniosis in Spain, highlighting their involvement in Leishmania transmission by supporting their role as potential reservoirs. This novel methodology represents a promising tool for further epidemiological studies that would help to design better strategies for the control of leishmaniosis in this area and other foci.

• MeSH
• imunoglobulin G krev   MeSH
• kousnutí a bodnutí hmyzem epidemiologie   imunologie   veterinární   MeSH
• králíci imunologie   MeSH
• leishmanióza veterinární   MeSH
• Phlebotomus imunologie   MeSH
• protilátky krev   MeSH
• psi imunologie   MeSH
• slinné proteiny a peptidy imunologie   MeSH
• zajíci imunologie   MeSH
• zvířata MeSH
• Check Tag
• králíci imunologie   MeSH
• psi imunologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Španělsko MeSH

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.

• MeSH
• ELISA MeSH
• hmyzí proteiny diagnostické užití   genetika   MeSH
• imunoblotting MeSH
• kousnutí a bodnutí hmyzem diagnóza   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• Phlebotomus imunologie   MeSH
• protilátky krev   MeSH
• psi MeSH
• rekombinantní proteiny diagnostické užití   genetika   MeSH
• sekvenční analýza DNA MeSH
• slinné proteiny a peptidy diagnostické užití   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The amine-binding properties of sand fly salivary yellow-related proteins (YRPs) were described only in Lutzomyia longipalpis sand flies. Here, we experimentally confirmed the kratagonist function of YRPs in the genus Phlebotomus. We utilized microscale thermophoresis technique to determine the amine-binding properties of YRPs in saliva of Phlebotomus perniciosus and P. orientalis, the Old-World vectors of visceral leishmaniases causative agents. Expressed and purified YRPs from three different sand fly species were tested for their interactions with various biogenic amines, including serotonin, histamine and catecholamines. Using the L. longipalpis YRP LJM11 as a control, we have demonstrated the comparability of the microscale thermophoresis method with conventional isothermal titration calorimetry described previously. By homology in silico modeling, we predicted the surface charge and both amino acids and hydrogen bonds of the amine-binding motifs to influence the binding affinities between closely related YRPs. All YRPs tested bound at least two biogenic amines, while the affinities differ both among and within species. Low affinity was observed for histamine. The salivary recombinant proteins rSP03B (P. perniciosus) and rPorASP4 (P. orientalis) showed high-affinity binding of serotonin, suggesting their capability to facilitate inhibition of the blood vessel contraction and platelet aggregation.

• MeSH
• aminy metabolismus   MeSH
• hmyzí proteiny metabolismus   MeSH
• konformace proteinů MeSH
• Phlebotomus metabolismus   MeSH
• slinné proteiny a peptidy metabolismus   MeSH
• slinné žlázy metabolismus   MeSH
• statická elektřina MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control.

• MeSH
• biologické markery krev   MeSH
• dusíkaté sloučeniny aplikace a dávkování   farmakologie   MeSH
• kousnutí a bodnutí hmyzem prevence a kontrola   MeSH
• longitudinální studie MeSH
• neonikotinoidy aplikace a dávkování   farmakologie   MeSH
• permethrin aplikace a dávkování   farmakologie   MeSH
• Phlebotomus účinky léků   MeSH
• protilátky krev   účinky léků   MeSH
• psi imunologie   MeSH
• repelenty proti hmyzu aplikace a dávkování   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• psi imunologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Španělsko MeSH

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.

• MeSH
• hmyz - vektory parazitologie   fyziologie   MeSH
• imunoanalýza metody   MeSH
• Leishmania infantum fyziologie   MeSH
• leishmanióza krev   diagnóza   parazitologie   veterinární   MeSH
• myši inbrední BALB C MeSH
• nemoci psů krev   diagnóza   parazitologie   MeSH
• Phlebotomus parazitologie   fyziologie   MeSH
• psi MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH