Latest trends in Leishmania infantum infection in dogs in Spain, Part I: mapped seroprevalence and sand fly distributions.
ABSTRACT: BACKGROUND:This report describes L. infantum infection seroprevalence in dogs in Spain through data obtained from peer-reviewed literature and a cross-sectional serological survey assessing epidemiological and habitat variables as risk factors for infection. The study also provides preliminary sand fly species distribution data and indicates factors affecting their distribution and density. METHODS:Three different studies were conducted in Spain: (i) a peer-reviewed literature seroprevalence survey (1985-2019); (ii) a cross-sectional serological survey (2011-2016); and (iii) a preliminary entomological survey (2013-2014). In the cross-sectional serological survey, 1739 dogs from 74 different locations including 25 Spanish provinces were tested for L. infantum by indirect immunofluorescence antibody test (IFAT) (antibody titre???1:100). Seroprevalence of L. infantum infection was analysed by province and bioclimatic zone. Statistics were used to analyse relationships between several dog- and environment-related variables and L. infantum seroprevalence. In parallel, during 2013-2014, sand flies were collected across the Iberian Peninsula and the Balearic Islands using CDC light traps to examine relationships between habitat-related factors and sand fly species densities (number of sand flies per trap per hour). RESULTS:The literature review revealed that the provinces showing the highest seroprevalence were Balearic Islands (57.1%), Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), and those showing the lowest seroprevalence were Vizcaya (0%), Cantabria (2.0%) and Álava (3.3%). In our survey, anti-Leishmania IgG antibodies were detected in 176 of the 1739 dogs rendering a seroprevalence of 10.12%. Percentage seroprevalence distributions significantly varied among bioclimatic belts. Seropositivity for L. infantum was related to size (large breed dogs versus small) and were significantly higher in younger dogs (??1 years-old). In the entomological survey, 676 sand flies of five species were captured: 562 (83.13%) Phlebotomus perniciosus; 64 (9.47%) Sergentomyia minuta; 38 (5.62%) P. ariasi: 6 (0.89%) P. sergenti; and 6 (0.89%) P. papatasi. Phlebotomus perniciosus showed a greater density in the thermo-Mediterranean than in the meso-Mediterranean zone. Densities of S. minuta and P. ariasi were significantly higher in rural habitats. CONCLUSIONS:This updated seroprevalence map of L. infantum infection in dogs in Spain defines non-endemic, hypoendemic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.
Project description:BACKGROUND:During last decade Lampedusa island (Italy) has been interested by a deep social change caused by the massive arrival of migrants from north Africa. The goal of this study was to evaluate current CanL burden and risk factors for Visceral Leishmaniosis (VL) on Lampedusa, actually based on very few data obtained in a previous study performed fifteen years ago. Two hundred and forty-two dogs were enrolled for the detection of Leishmania infantum infection by serology. In addition, an entomological investigation was performed to confirm the presence of Leishmania-vectors. RESULTS:Seroprevalence was of 54.13%. 223 sand flies specimens were collected. Among them, 4 species were identified: Phlebotomus perniciosus, P. papatasi, P. neglectus, Sergentomia minuta, with P. perniciosus the most abundant (67.7%; p < 0.01). CONCLUSION:The high proportion of seropositive dogs together with the presence of the most competent vector for L. infantum, P. perniciosus, demonstrate that L. infantum abundantly circulates in the island and may constitute a risk for people, particularly for hosted migrants.
Project description:An unusual increase of human leishmaniasis cases due to Leishmania infantum is occurring in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys have shown that Phlebotomus perniciosus is the only potential vector. Direct xenodiagnosis in hares (Lepus granatensis) and rabbits (Oryctolagus cuniculus) collected in the focus area proved that they can transmit parasites to colonized P. perniciosus. Isolates were characterized as L. infantum. The aim of the present work was to conduct a comprehensive study of sand flies in the outbreak area, with special emphasis on P. perniciosus.Entomological surveys were done from June to October 2012-2014 in 4 stations located close to the affected area. Twenty sticky traps (ST) and two CDC light traps (LT) were monthly placed during two consecutive days in every station. LT were replaced every morning. Sand fly infection rates were determined by dissecting females collected with LT. Molecular procedures applied to study blood meal preferences and to detect L. infantum were performed for a better understanding of the epidemiology of the outbreak.A total of 45,127 specimens belonging to 4 sand fly species were collected: P. perniciosus (75.34%), Sergentomyia minuta (24.65%), Phlebotomus sergenti (0.005%) and Phlebotomus papatasi (0.005%). No Phlebotomus ariasi were captured. From 3203 P. perniciosus female dissected, 117 were infected with flagellates (3.7%). Furthermore, 13.31% and 7.78% of blood-fed and unfed female sand flies, respectively, were found infected with L. infantum by PCR. The highest rates of infected P. perniciosus were detected at the end of the transmission periods. Regarding to blood meal preferences, hares and rabbits were preferred, although human, cat and dog blood were also found.This entomological study highlights the exceptional nature of the Leishmania outbreak occurring in southwestern Madrid, Spain. It is confirmed that P. perniciosus is the only vector in the affected area, with high densities and infection rates. Rabbits and hares were the main blood meal sources of this species. These results reinforce the need for an extensive and permanent surveillance in this region, and others of similar characteristics, in order to control the vector and regulate the populations of wild reservoirs.
Project description:An entomological survey was carried out in 2007 in two Pyrenean counties of Lleida province (north-eastern Spain), where cases of autochthonous canine leishmaniasis have been recently reported. Phlebotomus ariasi and P. perniciosus, vectors of Leishmania infantum in the Mediterranean area, were captured. The aim of the present study was to compare these phlebotomine populations with others captured in known leishmaniasis foci in Europe. Populations of these species were studied by analysing the polymorphism of seven enzymatic systems (HK, PGI, PGM, MDH, 6PGD, FUM and ACO) and compared with other specimens from endemic regions of France, Italy, Malta, Portugal and Spain captured in other campaigns, and also with previously published results. Phlebotomus ariasi was more polymorphic than P. perniciosus. Only the ACO locus had diagnostic alleles, but some other alleles show high characteristic frequencies for each species. The neighbour-joining trees separated two population groups in both species. On the basis of the isoenzyme study results, sand fly populations of the Pyrenean region in Lleida province are closely related to those of other nearby leishmaniasis endemic regions in France and Spain.
Project description:BACKGROUND: Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species. METHODOLOGY/PRINCIPAL FINDINGS: Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins. CONCLUSIONS: Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.
Project description:Leishmania parasites are transmitted by female phlebotomine sand flies that maintain the enzootic cycle by circulating between sylvatic and domestic mammals. Humans are part of this cycle as accidental hosts due to the vector's search for a source of blood. In Algeria, Human Leishmaniases (HL) are endemic and represent a serious public health problem because of their high annual incidence and their spread across the country. The aim of this study is to identify sand fly species fauna (vectors of Leishmania), determine their infection rate and identify their feeding preferences using molecular tools in a hypoendemic focus of HL located in the province of Tipaza, northern Algeria.An entomological survey using CDC light traps was conducted between July and October of 2015 in four HL affected peri-urban locations in the province of Tipaza, northern Algeria. Sand flies were identified using the morphological criteria of the genitalia for the males and spermathecae for the females. Leishmania DNA was detected in pooled female sand flies (N = 81 pools with 8-10 specimens per pool) using quantitative real-time polymerase chain reaction (qPCR) targeting two different genes: kDNA-PCR and 18S rRNA. To identify their blood meal sources, blood-fed female sand flies were analyzed by PCR-sequencing targeting the vertebrate cytochrome c oxidase I (COI) gene. A total of 4,045 sand flies were caught, of which 3,727 specimens were morphologically identified. Seven species were recorded: P. (L.) perniciosus (50.28%), P. (L.) perfiliewi (26.13%), P. (L.) longicuspis (21.92%), Sergentomyia (S.) minuta (0.85%), P. (P.) papatasi (0.42%), P. (L.) langeroni (0.32%) and P. (L.) ariasi (0.05%). Afterwards, 740 female specimens were randomly selected and divided into 81 pools and were then screened to investigate the presence of Leishmania spp. L. infantum DNA was detected in three pools, corresponding to three sand fly specimens (one each). The infection rate was 0.33% (2/600) for P. (L.) perniciosus and 2.56% (1/39) for P. (L.) perfiliewi. Analysis of the blood feeding sources (N = 88 specimens) revealed that sand flies belonging to Larroussius subgenera, mainly (71.5%) feed on small ruminants. Human blood is the second feeding source (17%), eight specimens (9%) were found to feed on equines and no domestic reservoir (dog) blood was found.The presence of human leishmaniasis cases, the high abundance of Phlebotomus (Larroussius) species which are proven or suspected vectors of L. infantum, and the detection of L. infantum DNA from its natural vectors (P. (L.) perniciosus, P. (L.) perfiliewi), in addition to the blood-feeding of positive females for L. infantum on humans blood, prove that the major elements of the epidemiological transmission cycle of L. infantum are present and indicate risk factors for an outbreak of the disease in the province of Tipaza.
Project description:<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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-? 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.<h4>Conclusions</h4>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.
Project description:In this study, the presence of Leishmania DNA and blood feeding sources in phlebotomine sand fly species commonly present in Sicily were investigated. A total of 1,866 female sand flies including 176 blood fed specimens were sampled over two seasons in five selected sites in Sicily (southern Italy). Sergentomyia minuta (n = 1,264) and Phlebotomus perniciousus (n = 594) were the most abundant species at all the sites, while three other species from the genus Phlebotomus (i.e., P. sergenti n = 4, P. perfiliewi n = 3 and P. neglectus n = 1) were only sporadically captured. Twenty-eight out of the 1,866 (1.5%) sand flies tested positive for Leishmania spp. Leishmania tarentolae DNA was identified in 26 specimens of S. minuta, while the DNA of Leishmania donovani complex was detected in a single specimen each of S. minuta and P. perniciosus. Interestingly, seven S. minuta specimens (0.4%) tested positive for reptilian Trypanosoma sp. Blood sources were successfully identified in 108 out of 176 blood fed females. Twenty-seven out of 82 blood sources identified in fed females of P. perniciosus were represented by blood of wild rabbit, S. minuta mainly fed on humans (16/25), while the sole P. sergenti fed specimen took a blood meal on rat. Other vertebrate hosts including horse, goat, pig, dog, chicken, cow, cat and donkey were recognized as blood sources for P. perniciosus and S. minuta, and, surprisingly, no reptilian blood was identified in blood-fed S. minuta specimens. Results of this study agree with the well-known role of P. perniciosus as vector of L. infantum in the western Mediterranean; also, vector feeding preferences herein described support the hypothesis on the involvement of lagomorphs as sylvatic reservoirs of Leishmania. The detection of L. donovani complex in S. minuta, together with the anthropophilic feeding-behaviour herein observed, warrants further research to clarify the capacity of this species in the transmission of pathogens to humans and other animals.
Project description: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.
Project description:Zoonotic visceral leishmaniasis is a vector-borne disease caused by Leishmania infantum in the Mediterranean Basin, where domestic dogs and wild canids are the main reservoirs. The promastigote stage replicates and develops within the gut of blood-sucking phlebotomine sand flies. Mature promastigotes are injected in the dermis of the mammalian host and differentiate into the amastigote stage within parasitophorous vacuoles of phagocytic cells. The major vector of L. infantum in Spain is Phlebotomus perniciosus. Promastigotes are routinely axenized and cultured to mimic in vitro the conditions inside the insect gut, which allows for most molecular, cellular, immunological and therapeutical studies otherwise inviable. Culture passages are known to decrease infectivity, which is restored by passage through laboratory animals. The most appropriate source of promastigotes is the gut of the vector host but isolation of the parasite is technically challenging. In fact, this option is not viable unless small samples are sufficient for downstream applications like promastigote cultures and nucleic acid amplification. In this study, in vitro infectivity and differential gene expression have been studied in cultured promastigotes at the stationary phase and in promastigotes isolated from the stomodeal valve of the sand fly P. perniciosus. About 20 ng RNA per sample could be isolated. Each sample contained L. infantum promastigotes from 20 sand flies. RNA was successfully amplified and processed for shotgun genome microarray hybridization analysis. Most differentially regulated genes are involved in regulation of gene expression, intracellular signaling, amino acid metabolism and biosynthesis of surface molecules. Interestingly, meta-analysis by hierarchical clustering supports that up-regulation of 22.4% of the differentially regulated genes is specifically enhanced by the microenvironment (i.e. sand fly gut or culture). The correlation between cultured and naturally developed promastigotes is strong but not very high (Pearson coefficient R2 = 0.727). Therefore, the influence of promastigote culturing should be evaluated case-by-case in experimentation.
Project description:Zoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe.Phlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence.Out of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta.The anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.