to achieve the first objective , we conducted a cross - sectional study in the alpujarras from april through june 2006 . we then compared current leishmaniasis seroprevalence data with data from 2 cross - sectional surveys conducted in 1984 and 1991 ( 35 ) . the villages sampled for all 3 studies were similar and had been selected at random from within each of the 3 bioclimatic levels ( thermo- , meso- , and supra - mediterranean ) that comprise the inhabited zone of the alpujarras ( figure 1 ) ( 10 ) . for each level , respectively , altitudes are 0700 , 600900 , and 9001,800 m above sea level ; annual mean temperatures are 1719 , 1317 , and 815c ; and annual rainfalls are 200350 , 6001,000 , and 1,0001,600 inches . the dates for sample collection were set to coincide with organized events at which dogs were gathered ( e.g. , antirabies vaccination campaigns ) . all 3 surveys used indirect immunofluorescence for diagnosis ; protocols and positivity threshold were identical . dogs with a titer > 160 were considered positive . to determine and compare the existence or lack of statistically significant differences between present and past prevalence rates the owners of the dogs included in the cross - sectional study conducted in the alpujarras from april to june 2006 were asked to complete an epidemiologic record for each dog tested ; data on the animal and its environment were recorded for subsequent use in univariate and multivariate logistic regression analyses ( tables 1 , 2 ) . density data were included for 2 vectors , phlebotomus perniciosus and p. ariasi sandflies , captured with sticky traps in june 2006 in the same villages in which the surveys were conducted . we investigated the existence of interaction and/or confusion between variables by constructing and comparing logistic regression models . confusion was noted between the location during daytime and location at night , so location during daytime was excluded from the multivariate analysis . guard dogs ( n = 34 ) , sheepdogs ( n = 8) , stray dogs ( n = 4 ) , dogs in kennel ( n = 2 ) . * determined by multivariate logistic regression . with age as continuous variable ( 0.125 years , average 4.7 years ) , odds ratio ( or ) = 1.142 ; with weight as continuous variable ( 260 kg , average = 17.7 ) , or = 1.047 . guard dogs , sheepdogs , stray dogs , and dogs in kennels . in our 2006 survey , 57 ( 13.0% ) of 439 dogs had an antibody titer > 160 ( seroprevalence rate ) , 268 ( 61.0% ) dogs had titers 2080 , and 114 ( 26.0% ) had no titer . in terms of bioclimatic level , canine leishmaniasis seroprevalence was 20.1% at the meso - mediterranean , 13.3% at the thermo - mediterranean , and only 1.1% at the supra - mediterranean levels . statistically significant differences (  test p<0.001 ) indicate that these differences are not random . the evolution - in - time analysis ( figure 2 ) shows how over 22 years ( 19842006 ) , seroprevalence of canine leishmaniasis has progressively increased at the meso - mediterranean level , climbing from 9.2% in 1984 to 15.4% in 1991 and finally to 20.1% in 2006 ( p = 0.015 ) ; in contrast , no significant changes have taken place in global prevalence or in the other 2 bioclimatic levels studied ( 35 ; this study ) . canine leishmaniasis seroprevalence rates in the alpujarras , spain , 19842006 , by time and bioclimatic level . dogs at greatest risk for canine leishmaniasis in the disease - endemic region of the alpujarras were large dogs ( > 25 kg ) and older dogs ( > 4 years ) that worked as guard dogs or sheepdogs , slept outdoors , and lived at the thermo- or meso - mediterranean level in a village such as torvizcn , where the p. perniciosus density is > 4 sandflies / m . risk for these dogs was 54,571 greater than for dogs that were kept as pets , were small , were < 4 years of age , and slept inside a house in a village such as mecina bombarn or prtugos at the supra - mediterranean level where p. perniciosus density is < 4 sandflies / m . among the drivers of global change that have the potential to influence vector - borne diseases , climatic and nonclimatic ( socioeconomic , demographic , and environmental ) factors have been cited ( 1,2 ) . although it is not easy to attribute our findings  progressive increase in seroprevalence at the meso - mediterranean level and drop and subsequent rise at the thermo - mediterranean level ( figure 2)to 1 or more drivers , we can attempt to find some form of association in the changes that have occurred in the alpujarras throughout these 2 decades . studies on climate change in spain confirm a warming tendency ( reflected at the global level ) , which provides evidence that temperatures have been increasing for a quarter of a century ( 11,12 ) . thus , we must assume that changes in temperature , rainfall , or humidity will have equally affected the 3 bioclimatic levels researched in the alpujarras and may have influenced the spatial and temporal distribution and the seasonal dynamics of sandflies . an increase was detected in the period of p. perniciosus activity in the region ; these effects were probably more notable at the meso - mediterranean level because this is where the density of this vector species is at its highest ( 6 ; martn - snchez et al . , data ) . during the time period researched , the human population remained constant in the alpujarras ; it has , however , progressively decreased in the disperse populations and increased in population centers . unfortunately , no official figures for the canine population are available , although we were informed by staff of local town halls that in 1991 , the 615 dogs analyzed accounted for 100% of the total number of dogs registered in the villages sampled ( 3 ) . one change during the period studied was the gradual disappearance of livestock enclosures in rural population centers . ( 13,14 ) and are ideal places for sandfly blood sucking , mating , and oviposition ( 15 ) . with respect to risk factors , the increase in canine leishmaniasis seroprevalence as animal s age increases seems logical because in a leishmaniasis - endemic area , the greater the age , the longer the animal will have been exposed to sandflies and the greater the probability of having been bitten by an infected female sandfly . seroprevalence of canine leishmaniasis also increases gradually with weight , which could be attributed to the vector being more attracted to larger animals . dogs that sleep outdoors are at greater risk than those sleeping indoors because the density of the vector ( p. perniciosus ) is greater outdoors than inside a house . the association between p. perniciosus density and canine leishmaniasis seems logical , considering that p. perniciosus is the main vector species in the alpujarras . this is the only species found to have been infected in this region ( 8,9 ) , although p. ariasi is a proven vector in the nearby region of the axarquia ( 13 ) . in the alpujarras , the percentage of dogs kept as pets has increased from 42% in 1991 ( 3 ) to 59% in 2006 ( p<0.001 ) . this increase corresponds to a general increase in standard of living and , along with other factors such as the disappearance of livestock enclosures from within population centers , would have acted to reduce transmission . only the extension of the vector s activity period , which was detected at some trap sites , would have acted to increase transmission . such extension of activity may be related to the increase in temperature brought about by climate change ( 11,12 ) .