On how trap positioning affects phlebotomine sand fly density estimations.

There is a need for standardizing sand fly sampling methodology and guidance on trap positioning for quantitative sand fly studies. We investigated differences in sand fly density with 'sticky' interception and CO2 -light attraction traps, in relation to trap distance to the ground and the presence or absence of a continuous or discontinuous (wire mesh) vertical surface adjacent to the trap. The study, conducted in a dog kennel in southeast Spain, lasted 48 days and collected 692 Phlebotomus papatasi, P. perniciosus, P. ariasi and Sergentomyia minuta specimens. There were no significant differences between species with respect to trap position. Overall, density in sticky traps was highest closest to the ground and next to the continuous vertical surface, followed sequentially by traps similarly placed adjacent to the wire mesh and those hanging from a rope across the kennel yard. In contrast, density in CO2 -light traps was highest in traps hanging from the rope near the ground, followed by those next to the continuous vertical surface. The overall negative relationship between sand fly density and ground distance was not significant for CO2 -light traps next to the continuous vertical surface. Modelling also suggested that sand flies do not use the wire mesh to move vertically.

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.

A spatial ecology study in a high-diversity host community to understand blood-feeding behaviour in Phlebotomus sandfly vectors of Leishmania.

Molecular studies indicate that Phlebotomine sandflies (Diptera: Psychodidae) blood feed on many vertebrate species, of which only a few are proven parasite reservoirs. Investigating sandfly vector feeding preferences is therefore important and requires taking into account the availability and accessibility of host species. In terms of the latter, it is necessary to consider the metabolic cost to the insect of reaching the host and moving on to a suitable breeding site. The present study used statistical modelling to compare the feeding patterns of Phlebotomus perniciosus (n = 150), Phlebotomus papatasi (n = 35) and Phlebotomus ariasi (n = 7) on each of an average of 30 host species in a wildlife park in Murcia, Spain. Sandfly feeding movement costs were estimated as a function of the distance and altitude gradients saved by the insect, assuming that they displayed 'site fidelity'. Most (87%) engorged females were caught <100 m from the host on which they had fed. Although the percentage of bloodmeals was highest on fallow deer (Dama dama) (30%) and red deer (Cervus elaphus) (26%), the predicted feeding probability after considering movement cost was highest for red deer and common eland (Taurotragus oryx), and positively associated with host census. These results suggest that, under similar circumstances, sandflies prefer to feed on some host species more than on others.

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.

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.

Spatio-temporal patterns of sarcoptic mange in red deer and Iberian ibex in a multi-host natural park.

The reliance on multiple hosts to survive is what makes the management and control of multi-host infectious agents challenging. Sarcoptes scabiei causes sarcoptic mange in a wide range of mammal species with ungulates being an important host. Little is known about the role different ungulates play in sustaining endemic transmission of the disease and no study has yet to describe the long-term multi-host sarcoptic infestation dynamics in free-ranging wildlife. Here, we explore 24 years of sarcoptic mange infestation data for two Mediterranean ungulate species, red deer and Iberian ibex, living in the Sierras de Cazorla, Segura y Las Villas Natural Park of southern Spain. The temporal analysis showed a clear seasonal pattern of infestation in both ungulates with a peak in early spring and a decline throughout the summer. The spatial analysis, however, showed that caprinae rather than cervidae is the most competent host for sarcoptic mange spreading and persistence. Considering that few studies have described the spatio-temporal pattern of mange outbreaks for long periods of time, the information reported in this work aims to improve our understanding of sarcoptic mange epizootic in wild ruminant populations.

Improvements in the detection of small ruminant lentivirus infection in the blood of sheep by PCR.

The polymerase chain reaction on blood samples has been considered a complement to serological methods for the detection of small ruminant lentiviruses (SRLV) infections in sheep and goats. This is a report on the results of a study to evaluate the use of the same blood sample for the detection of infected animals by ELISA and PCR. A comparison between the results obtained by applying PCRs targeting LTR and gag sequences on blood clot, serum and peripheral blood leucocytes was made. In addition to simplifying sampling and laboratory work, the use of blood clot samples with the gag-PCR improved remarkably the detection of infected animals. Finally, this study has shown the existence of a cell-free viremia in the serum of SRLV-infected sheep.

What do we know about parasites of wildlife in high biodiversity areas with anthropogenic disturbance? The special case of Mexico.

The continual rise of anthropogenic disturbance of ecosystems has been associated with an increasing incidence of emerging diseases. The largest amount of data on emerging diseases relates to bacterial and viral pathogens, but there is a lack of parasite data, especially from wildlife. Monitoring wildlife parasitic diseases should be considered a priority, especially in high biodiversity regions with strong anthropogenic impacts, like Mexico, where the wildlife/livestock/human interface is associated with increased risk of disease transmission. Mexico belongs to the top-ten megadiverse countries and is located between two biogeographic regions. This situation makes Mexico a favourable region for the spillover of animal pathogens to human beings, causing pandemics, such as the one recently caused by influenza virus A (H1N1). The current state of knowledge of Mexican wildlife parasites is scarce and focuses mainly in Neotropical fauna. Moreover, this knowledge is heterogeneous for different parasite groups, especially concerning their pathologic effects and epidemiology. The goals of this review are to compile information on Mexican wildlife parasites and to identify knowledge gaps in order to stimulate research on pending epidemiological, public health, ecological and pathological areas, and to encourage the creation of more specialized groups from the perspective of the One-Health concept.

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.

Molecular characterization and phylogenetic study of Maedi Visna and Caprine Arthritis Encephalitis viral sequences in sheep and goats from Spain.

Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions (pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12-26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.

Flock-prevalence of border disease virus infection in Basque dairy-sheep estimated by bulk-tank milk analysis.

Bulk-tank milk (BTM) samples from 154 sheep flocks were used to estimate BDV prevalence in the Basque Country in Spain using an ELISA and a RT-PCR test. The proportion of antibody-positive flocks was 68% but varied significantly between provinces and was 93% in Araba and 54-55% in Bizkaia and Gipuzkoa. Most ELISA-positive flocks had very low antibody inhibition percentage (AIP) indicating high seroprevalence and recent BDV exposure. However, only 9% flocks were PCR-positive suggesting few infected ewes were being milked at the time of sampling. Phylogenetic analysis of the 5' NCR sequences of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study in the region, whereas the remaining isolate was closest to BDV type A. These results suggest that BDV strains in most Basque flocks have a common origin and differences in prevalence between provinces are associated to recent events affecting BDV spread such as use of communal pastures and sheep trading. The widespread distribution of BDV in the region, advocates for the implementation of BDV control strategies and highlights the potential risk of sheep as a pestivirus reservoir for other species.

Horizontal Maedi-Visna virus (MVV) infection in adult dairy-sheep raised under varying MVV-infection pressures investigated by ELISA and PCR.

A three year long experimental study was carried out to investigate horizontal MVV-infection by PCR and ELISA, in 191 one year-old latxa dairy-sheep raised in two separate groups under low and high MVV-infection pressure, respectively. Sheep originated from a previous MVV-transmission study in lambs and seroprevalence among one year-old sheep in both groups was 15% approximately. The high infection-pressure group (H-group) consisted of 147 replacement ewes that joined a milk-producing, housed dairy-flock with 42-66% MVV-seroprevalence and the low infection-pressure group (L-group) were castrated males raised in a separate shed. In contrast to results obtained when infection was investigated in lambs, the overall degree of agreement between ELISA and PCR results was very good and there was some indication that it increased further as sheep became older. MVV-prevalence did not change in the L-group and increased to 57% in three year-old sheep in the H-group (p<0.001). Random effects logistic regression confirmed seroconversion was significantly higher in the H-group compared to the L-group and was highest during the year after the sheep were introduced in the dairy flock and did not increase with age as in previous studies using less sensitive antibody assays. The evidence that horizontal transmission can be very low in spite of prolonged close contact between infected and non-infected sheep is valuable for MVV-control purposes. Furthermore it highlights the need to investigate virus excretion dynamics in infected animals and animal to animal transmission to improve our overall understanding of horizontal MVV transmission in MVV endemic populations.

PCR detection of colostrum-associated Maedi-Visna virus (MVV) infection and relationship with ELISA-antibody status in lambs.

A recent large-scale experimental study showed that bottle-feeding ovine colostrum from seropositive ewes results in high MVV-seroconversion in lambs. In contrast, relatively few lambs that naturally suckled colostrum from seropositive dams seroconverted as a result of it. Furthermore, lambs fed uninfected bovine colostrum readily seroconverted when mixed with ovine-colostrum lambs indicating that horizontal MVV transmission between lambs was efficient. MVV-infection was further investigated in the same samples using two PCR tests targeting sequences in the long-terminal repeats (LTR) and POL MVV genes. PCR-tests confirmed previous serological findings. However, the LTR-PCR was more sensitive and allowed detecting infection earlier than the other tests, including 5-8% of new-born lambs from seropositive dams, providing more evidence that prenatal MVV-infection may be more important than considered. The degree of agreement between PCR and antibody tests in individual samples was low up to 6 months of age and moderate at 10 months-old. Nine percent of lambs were always PCR-negative but seroconverted and 19% of lambs were PCR-positive at least once and did not seroconvert. However, seroconversion was associated with increasing number of times lambs were PCR-positive and ovine colostrum-fed lambs were more frequently PCR-positive than other lambs. The significance of these findings in terms of MVV-infection, epidemiology and control is discussed.

Absence of circulation of Pestivirus between wild and domestic ruminants in southern Spain.

Ruminant pestiviruses (family Flaviviridae) affect both wild and domestic ruminants worldwide, causing reproductive disorders and severe economic losses. Wild (n=1442) and domestic (n=373) ruminants from southern Spain were tested for the presence of antibodies to pestiviruses. Seropositivity was detected by both ELISA and virus neutralisation test in 1/892 (0.1 per cent) red deer, 29/125 (23.2 per cent) cattle and 17/157 (10.8 per cent) sheep. Pestivirus-specific antibodies to bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) genotypes 1, 4 and 5 were detected. Significantly higher antibody titres to BDV-4 compared with BVDV-NADL were found in one cattle farm. Results indicate that wild ruminants may act as spillover hosts from domestic ruminants, and therefore they do not represent a risk for domestic ruminants in southern Spain. The wide distribution of pestiviruses among sheep and cattle suggests that transmission between these species may occur.

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.

Diagnostic tests for small ruminant lentiviruses.

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.

Relative contribution of colostrum from Maedi-Visna virus (MVV) infected ewes to MVV-seroprevalence in lambs.

Maedi-visna virus (MVV) seroprevalence associated with consumption of colostrum from seropositive ewes was investigated in 276 housed lambs from birth to 300 days-old. At birth, lambs were allocated to five experimental groups according to the maternal MVV-serological status, source and mode of feeding colostrum (bovine or ovine and bottle fed or suckled from the dam) and type of horizontal MVV-exposure (raised with the dam or separately with other lambs). The risk of being seropositive at 300 days-old was associated with feeding ovine colostrum from seropositive ewes and increased with intake of bottle-fed ovine colostrum and was higher in lambs separated from their dams and raised with other experimental lambs compared to lambs raised with their dams. Approximately 75-87% of ELISA-positive results in lambs that had ovine colostrum was attributable to colostrum itself. However, approximately only 16% of naturally raised and 29-61% of bottle-fed ovine colostrum lambs were ELISA-positive as a result feeding ovine colostrum. These results confirm that ovine colostrum from seropositive ewes can be a major source of MVV but its overall contribution to seroprevalence in natural conditions is relatively low, and shows that horizontal MVV transmission can be an important source of infection in new-born lambs.

A retrospective clinical and epidemiological study on feline coronavirus (FCoV) in cats in Istanbul, Turkey.

The presence of antibodies to feline coronavirus (FCoV) and feline immunodeficiency virus (FIV), together with feline leukemia virus (FeLV) antigen was investigated in 169 ill household and stray cats attending a veterinary surgery in Istanbul in 2009-14. The estimated FCoV and FIV seroprevalence (95% confidence intervals) were 37% (30-45%) and 11% (6-16%), respectively and FeLV prevalence was 1% (0-3%). FCoV seroprevalence increased until 2 years of age, was highest in 2014 and among household cats living with other cats and with outdoor access, and was lower in FIV seropositive compared to seronegative cats. Symptoms typically associated with wet feline infectious peritonitis (FIP) including ascites, abdominal distention or pleural effusion, coupled in many cases with non-antibiotic responsive fever, were observed in 19% (32/169) of cats, and 75% (24/32) of these cats were FCoV seropositive. FCoV seropositivity was also associated with a high white blood cell count, high plasma globulin, low plasma albumin and low blood urea nitrogen. The percentage of FCoV seropositive and seronegative cats that died in spite of supportive veterinary treatment was 33% (21/63) and 12% (13/106), respectively. These results indicate that FCoV is widespread and has a severe clinical impact in cats from Istanbul. Moreover, the incidence of FCoV infections could be rising, and in the absence of effective vaccination cat owners need to be made aware of ways to minimize the spread of this virus.

Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: a study of environmental, demographic and social risk factors.

Recent PCR studies indicate that asymptomatic L. infantum infection is common in people in southern Europe. Understanding its spatial distribution is a requisite to evaluate the public health implications and to design disease control schemes. We investigated infection in blood samples from 657 donors in southeast Spain using PCR and antibody ELISA. They came from 19 blood centers and were interviewed about their residence, occupation, dog ownership and Leishmaniosis awareness. The percentage of PCR and ELISA positives were 8% (49/618) and 2% (13/657). Donor's residences were spatially clustered around blood donning centers and PCR prevalence was 18% in rural municipalities with 20-1330 inhabitants, 12% in those with 1467-5088 inhabitants and 3% in larger communities, and was associated with dog ownership (p<0.05). Further analysis of data from rural donors indicated that PCR status was strongly related to the climate, altitude and soil type in the donor's residence area and not to other demographic or sociologic variables. Mixed logistic regression analysis predicted PCR prevalence to be greatest in the 200-300m altitude range with a mean spring-summer (time of highest vector activity) temperature of 18.4-19.0°C. A temperature and altitude risk map was generated that will provide the basis for elaborating evidence-based vector surveillance studies.