Density assessment and reporting for Phlebotomus perniciosus and other sand fly species in periurban residential estates in Spain.

Green periurban residential areas in Mediterranean countries have flourished in the last decades and become foci for leishmaniasis. To remedy the absence of information on vector ecology in these environments, we examined phlebotomine sand fly distribution in 29 sites in Murcia City over a 3-year period, including the plots of 20 detached houses and nine non-urbanized sites nearby. We collected 5,066 specimens from five species using "sticky" interception and light attraction traps. The relative frequency of the main Leishmania infantum vector Phlebotomus perniciosus in these traps was 32% and 63%, respectively. Sand fly density was widely variable spatially and temporally and greatest in non-urbanized sites, particularly in caves and abandoned buildings close to domestic animal holdings. Phlebotomus perniciosus density in house plots was positively correlated with those in non-urbanized sites, greatest in larger properties with extensive vegetation and non-permanently lived, but not associated to dog presence or a history of canine leishmaniasis. Within house plots, sand fly density was highest in traps closest to walls. Furthermore, the study provides a guideline for insect density assessment and reporting and is envisioned as a building block towards the development of a pan-European database for robust investigation of environmental determinants of sand fly distribution.

Longitudinal monitoring of anti-saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs.

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.

Epidemiological and genetic studies suggest a common Leishmania infantum transmission cycle in wildlife, dogs and humans associated to vector abundance in Southeast Spain.

Leishmania infantum infection was investigated in 202 wild carnivores, rodents and lagomorphs in Southeast Spain using a real-time PCR (rtPCR) in skin and organ samples, mostly spleen. Lesions compatible with leishmaniosis were not observed in any of the animals. Prevalence defined as the percentage of rtPCR-positive animals was 32% overall, and 45% in foxes (n = 69), 30% in rabbits (n = 80) and stone martens (n = 10), 19% in wood mice (n = 16), 0% in black rats (n = 10) and ranged between 0% and 100% in other minoritarian species including badgers, wild cats, wolves, raccoons, genets and hares. Most infected rabbits were rtPCR-positive in skin and not in spleen samples and the opposite was the case for foxes (p < 0.05). L. infantum prevalence was lowest in spring following months of non-exposure to phlebotomine sand fly vectors, and spatially matched recently estimated Phlebotomus perniciosus vector abundance and the prevalence of subclinical infection in dogs and humans. Prevalence increased with altitude and was greater in drier and less windy South and West compared to the coastal Southeast of the study area (p < 0.05). Genetic diversity of L. infantum from foxes, investigated by PCR-restriction fragment length polymorphisms of kinetoplast DNA, revealed B genotype in all animals, which is frequent in people and dogs in the Iberian Peninsula and Morocco. The study provides further evidence that subclinical L. infantum infection is widespread in wildlife with prevalence depending on environmental factors and that parasite tissue tropism may vary according to host species. Moreover, it suggests that sylvatic and domestic transmission cycles are closely interconnected.

Effect of trapping method on species identification of phlebotomine sandflies by MALDI-TOF MS protein profiling.

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood-feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI-TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in-house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI-TOF MS protein profiling for species identification.

Investigations of Phlebotomus perniciosus sand flies in rural Spain reveal strongly aggregated and gender-specific spatial distributions and advocate use of light-attraction traps.

The spatial and temporal distribution of Phlebotomus perniciosus (Diptera: Psychodidae) (Newstead, 1911), the sand fly vector of pathogens of public and animal health importance, was investigated in a high sand fly density rural area in Spain using light-attraction and sticky-interception traps. Traps were placed inside animal buildings and outside at increasing distance from animals. A total of 8506 sand flies were collected, 87% with light traps. Species frequency differed between trap types. The abundance of P. perniciosus decreased exponentially with increasing distance to animals and, while females were most common in the animal enclosure, males predominated in adjoining storage places. Increasing CO2 concentration had an additional positive effect on female abundance only. Both male and female density increased with rising temperature, and there was some indication that females were more active than males at higher relative humidity. The study confirms that P. perniciosus aggregates around animal premises, although male and female distributions differ and should be analysed separately to account for biological and behavioural differences. This provides further evidence that light traps offer an accurate estimation of the relative spatial and temporal abundance of P. perniciosus, conferring an added value for the study of this species and the risk of pathogen transmission.

A cross-sectional study of the impact of regular use of insecticides in dogs on Canine Leishmaniosis seroprevalence in southeast Spain.

The relationship between Canine Leishmaniosis (CanL) seroprevalence and regular use of topical insecticides was investigated in 800 pet dogs with no visible signs of CanL in Murcia, southeast Spain in 2011. Dogs were clients to 17 veterinary practices and were analyzed for Leishmania infantum antibodies in blood plasma using two commercial ELISAs (Ingezim, Ingenasa®, Spain; Leishcan, Hipra®, Spain). Owners were interviewed to gather data on dog related variables. They included date of birth, home address and frequency, duration and timing of insecticide treatments used to prevent ectoparasite infestations. The dog's residence was georeferenced and environmental data potentially associated with the dog's risk of L. infantum infection was obtained. A mixed logistic regression model was then developed to analyze the relationship between the dog's serological status and insecticidal treatment adjusted for demographic and environmental variables. Overall, CanL seroprevalence (95% confidence limits) was 18% (16-21%) including 11% in dogs not using insecticide treatments (n=60) and 19% in those using them (n=740) (p>0.05). At least 16 different insecticide products were used and 73%, 26% and 1% of dogs received 1, 2 and 3 products a year. The most frequent commercial brands used and the only ones in the market claiming anti-sandfly activity, were Scalibor collars (deltametrin 40mg/g; MSD®), Advantix pipettes (permethrin 500mg/ml and imidacloprid 100mg/ml; Bayer®) and Exspot spot-on pipettes (permethrin 715mg/ml; MSD®). Seroprevalence was 9%, 16%, 20%, 22% and 25% for dogs with Scalibor collars plus Advantix pipettes, Scalibor collars plus ExSpot pipettes, Advantix pipettes alone, Scalibor collars alone and Exspot pipettes alone, respectively. The multivariable model confirmed a significant reduction in the risk of Leishmania spp. seropositivity in dogs using the Scalibor and Advantix combination compared to those using either product alone and provided evidence of greatly increased risk of CanL in rural areas situated at 300-500m altitude and average March-July temperatures of 18.6-19°C. The study highlights the difficulty in controlling CanL infection by means of insecticide use alone and that it could be improved by using the Scalibor and Advantix combination and identifying and targeting specific geographical areas.

Comparative value of microscopy, serology and real time pcr in the diagnosis of asymptomatic canine Leishmania infantum infection

Se investigó la sensibilidad (SE) del examen citológico mediante microscopia óptica (MO) de improntas de bazo y linfonodo, de la prueba de anticuerpos ELISA (inmuno-ensayo ligado a enzima) y de la PCR (reacción en cadena de la polimerasa) a tiempo real (tr), para diagnosticar la infección asintomática por Leishmania infantum en 110 perros aparentemente sanos, del sureste de España. El porcentaje de perros positivos a MO, ELISA y PCRtr fue 2% (2/110), 27% (26/97) y 67% (39/58), respectivamente, aunque el porcentaje de PCR-positivos osciló entre 35-41% para cada tejido individualmente y 9% en sangre. La SE estimada (intervalos de confianza del 95%) de la MO en relación a la PCRtr y al ELISA fue 5% (0-12) y 8% (0-18), respectivamente. Estos resultados confirman que la mayoría de perros aparentemente sanos de una población endémica de L. infantum están infectados, que aproximadamente solo la tercera parte de éstos desarrolla anticuerpos frente al parásito y solo unos pocos tienen suficiente carga parasitaria en tejido linfoide como para ser detectada mediante MO. Consecuentemente, el grado de concordancia de la PCRtr, el ELISA y la MO en el diagnóstico de leishmaniosis canina asintomática es escaso (AU)

The sensitivity (SE) of cytological examination of spleen and lymphnode smears by optical microscopy (OM), antibody-ELISA (enzyme-linked immunosorbent assays) and real-time (rt) PCR (polymerase chain reaction), for diagnosing asymptomatic canine Leishmania infantum infection was investigated in 110 apparently healthy dogs from southeast Spain. The percentage of OM, ELISA and rtPCR positive dogs were 2% (2/110), 27% (26/97) y 67% (39/58), respectively, although the percentage of rtPCR-positive dogs were 35-41% in individual tissues and 9% in blood. The estimated SE (95% confidence interval) of OM relative to the rtPCR and ELISA tests was 5% (0-12) and 8% (0-18), respectively. Results confirm that most apparently healthy dogs from L. infantum endemic areas are infected, that approximately only one third of these infected dogs develop antibodies and that very few have parasite loads that are high enough to allow detection by OM. As a result, the degree of agreement between rtPCR, ELISA and OM for L. infantum diagnosis in subclinnically infected dogs is low (AU)

Subtyping of panic disorder patients with bradycardia.

PURPOSE: Since clinical practice suggests that panic disorder may not be a homogeneous condition, a study was carried out to test the possible existence of different groups or subgroups of panic patients. SUBJECTS AND METHODS: Thirty-two panic patients (DSM-III-R) underwent lactate challenge in our laboratory and were assessed for heart rate, blood pressure, sweating and Acute Panic Inventory. RESULTS: During the lactate challenge, patients complaining mainly of 'cardiorespiratory' symptoms (N = 12) showed tachycardia and localized sweating. Conversely, patients complaining mainly of 'pseudoneurological' symptoms (N = 16) showed bradycardia and generalized sweating. In both groups, Acute Panic Inventory scores were significantly higher during than before the panic attack, but the distribution of the scores was markedly different. DISCUSSION AND CONCLUSION: The results suggest that panic disorder may be a heterogeneous condition. Implications of these results to other phobic disorders, to Klein's false suffocation alarm theory and to the 'extended amygdala model' are discussed.