Gálvez et al. Parasites Vectors          (2020) 13:204  
https://doi.org/10.1186/s13071-020-04081-7
RESEARCH
Latest trends in Leishmania infantum 
infection in dogs in Spain, Part I: mapped 
seroprevalence and sand fly distributions
Rosa Gálvez1, Ana Montoya1, Israel Cruz2, Carlos Fernández1, Oihane Martín3, Rocío Checa1, Carmen Chicharro4, 
Silvia Migueláñez4, Valentina Marino1 and Guadalupe Miró1*
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 relation-
ships 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 Viz-
caya (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, hypoen-
demic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.
Keywords: Leishmania infantum, Canine leishmaniosis, Seroprevalence, Risk factors, Spain, Immunofluorescence 
antibody test, Phlebotomine sand fly, Bioclimatic belt
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Open Access
Parasites & Vectors
*Correspondence:  gmiro@ucm.es
1 Grupo de Investigación Epicontrol-Carnívoros, Departamento de 
Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de 
Madrid, Madrid, Spain
Full list of author information is available at the end of the article
Page 2 of 12Gálvez et al. Parasites Vectors          (2020) 13:204 
Background
Leishmaniosis caused by Leishmania infantum is a 
widespread zoonotic disease that may be transmitted 
to animals and humans by their vectors, blood-sucking 
phlebotomine sand flies [1, 2]. Other non-sand fly routes 
of L. infantum transmission include vertical and horizon-
tal routes (from blood donors, venereal transmission and 
direct dog-to-dog transmission through bites or wounds) 
[3]. In endemic areas, a population subset with subclini-
cal infection acts as a disease reservoir [3]. In Spain, CanL 
is an endemic and dynamic disease with an overall sero-
prevalence and transmission risk that vary according to 
local environmental and climatic conditions [4, 5]. In the 
Mediterranean basin, the dog is the main reservoir for L. 
infantum, and it is estimated that close to 2.5 million dogs 
are infected [6]. In Spain, CanL shows a broad seropreva-
lence range varying according to the geographical area 
[7].  In the northern provinces of the Iberian Peninsula 
traditionally classified as non-endemic, seroprevalence is 
lower [8]. Climate and environmental changes provoked 
by human activities may have caused the expansion of L. 
infantum infection in dogs towards such areas histori-
cally considered disease-free [9]. Regarding sand fly sta-
tus in Spain, Sergentomyia minuta is the most abundant 
species, followed by two vector species of L. infantum: P. 
perniciosus, which is more widespread and less affected 
by climatic conditions, and P. ariasi, which shows a pref-
erence for humid, cold areas [2].
There is great variability in how L. infantum manifests 
in a dog due to both individual factors (e.g. breed, age, 
immune status) and environmental factors (e.g. climate, 
land use) [3, 10, 11].
Control strategies should be based on local epidemio-
logical information [1]. The updated data provided here 
on the seroprevalence of CanL and on the ecology of 
sand fly vectors in Spain will be useful for the design of 
targeted control measures.
This study is Part I of a larger investigation addressing 
the situation of CanL in Spain. In Part II, we examined 
how CanL is currently managed via a multicentre ques-
tionnaire completed by veterinarians and compared the 
data obtained with a similar survey conducted in 2005.
Methods
Study area
The study area was mainland Spain and the Balearic and 
Canary Islands. Nine bioclimatic zones have been tradi-
tionally defined for the Iberian Peninsula and Balearic 
Islands [12]. Five of these areas occupying 46 × 106  ha 
were surveyed: supratemperate and mesotemperate 
within the Eurosiberian region, and supra-Mediterra-
nean, meso-Mediterranean and thermo-Mediterranean 
within the Mediterranean region. The zones not surveyed 
were the four highest regions occupying 1 × 106 ha 
(alpine and subalpine in the Eurosiberian region, and 
cryoro-Mediterranean and oro-Mediterranean in the 
Mediterranean region; mean altitudes of 2396, 1882, 
2548 and 1757 meters above sea level, respectively) 
because climatic conditions are not suitable for sand fly 
development.
Leishmania infantum seroprevalence study
Literature review
Scientific works published from 1985 to 2019 report-
ing CanL seroprevalences for mainland Spain and the 
Balearic Islands were identified. Inclusion criteria were 
seroprevalence studies conducted on randomly sampled 
dog populations in which the humoral response was 
assessed by detecting antibodies. According to the anti-
body titre cut-off established in each study, seropreva-
lence ranges were calculated for each province. These 
data were used to prepare a seroprevalence map of L. 
infantum in the dog.
Cross‑sectional serological survey
During 2011–2016, a cross-sectional seroprevalence 
study was performed in 25 Spanish provinces without 
taking into account the clinical status of dogs. Epidemio-
logical variables recorded included geographical location, 
habitat, age, sex, breed, weight, travel history and pres-
ence of clinical signs. A 5 ml blood sample was obtained 
from each dog by cephalic venipuncture and sera were 
separated and kept at − 20  °C until analysis. Serodiag-
nosis was conducted by detecting specific antibodies 
against L. infantum using the indirect immunofluores-
cence antibody test (IFAT) for anti-Leishmania-specific 
immunoglobulin G (IgG) antibodies according to stand-
ard procedures [13]. Serological analyses were conducted 
at the Pet Parasite Lab (Animal Health Department Vet-
erinary Faculty, UCM, Spain). The cut-off indicating a 
positive result was 1:100. Seroprevalence was calculated 
as the percentage of dogs testing positive for anti-L. 
infantum antibodies.
Entomological survey
Sand flies were collected from the wild using CDC light 
traps set up in the afternoon and recovered early in the 
morning. Sand flies were trapped in 2013–2014 seasons 
(from May to October). Captured sand flies were trans-
ferred to labelled 1.5  ml tubes containing 70% ethanol 
and identified to the species level. Females were cleared 
in Mark-André medium [14] and mounted on glass 
slides in Hoyerʼs medium [15]. Sand flies were identified 
according to taxonomic keys [16]. Sand fly densities were 
calculated according to the following formula: Sand fly 
density = No. of sand flies/(No. of traps × hours).
Page 3 of 12Gálvez et al. Parasites Vectors          (2020) 13:204  
Table 1 Seroprevalence of canine L. infantum infection in Spain by province based on a review of literature published from 1985 to 
2019
Province Seroprevalence (%) Seroprevalence 
range (%)
n Reference
IFAT ELISA IFAT & ELISA Commercial kit
Álava 3.3 – – – 3.3 na Sáez de Santamaría et al. [34]
Albacete – – 8.3 – 8.3 232 Benito et al. [35]
Alicante – 19.1 – – 19.1 807 Alonso et al. [36]
Almería 4.5 – – – 4.5 286 Sanchís Marín et al. [37]
Asturias 4.7 – – – 4.7 171 Miro et al. [8]
Badajoz – 7.0 – – 1.7–7.0 na Rosado et al. [38]
– – – 1.7 na Asencio et al. [39]
Balearic Is. – 1.0 – – 1.0–57.1 813 Seguí [40]
35.2 38.4 – – 353 Pujol et al. [41]
29.3 300 Solano-Gallego et al. [42]
57.1 35.7 – 52.4 121 Alcover et al. [43]
Barcelona 14.5 – – – 14.5–16.7 617 Botet et al. [44]
16.7 – – – 466 Corachan et al. [45]
Cáceres 15.0 – – – 14.0–34.2 433 Encinas Grandes et al. [46]
14.0 – – – 381 Nieto et al. [47]
– 34.2 – – 240 Rosado et al. [48]
Cádiz 31.6 31.6 98 Morales-Yuste et al. [49]
Cantabria 2.0 – – – 2.0 100 Miró et al. [8]
Castellón 5.1 – – – 5.1 118 Arnedo Pena et al. [50]
Ciudad Real 6.7 – – – 6.7 232 Benito et al. [35]
Córdoba – – 23.7 – 23.7 540 Martínez-Cruz et al. [51]
Granada 8.8 – – – 5.3–19.3 1503 Reyes Magaña et al. [52]
19.3 – – – na Reyes Magaña et al. [53]
5.3 – – – 615 Acedo-Sánchez et al. [54]
16.1 – – – 1374 Acedo-Sánchez et al. [55]
13.0 – – – 439 Martín-Sánchez et al. [56]
Girona – 19.5–24.6 – – 19.5–24.6 168 Vélez et al. [21]
Guadalajara – – 5.8 – 5.8 232 Benito et al. [35]
Huelva 6.7 – – – 6.7 702 Lepe et al. [57]
Jaén 16.0 – – – 16.1 1374 Acedo-Sánchez et al. [55]
Madrid 4.7 – – – 1.2–11.3 473 Celaya [58]
5.2 – – – 591 Amela et al. [59]
4.7 – – – 235 Castañeda et al. [60]
11.3 – – – 278 Castañeda et al. [61]
8.6 – – – 775 García Nieto et al. [62]
7.8 – – – 1803 Miró et al. [63]
8.1 – – – 1076 Gálvez et al. [20]
1.2 – – – 1372 Miró et al. [64]
Málaga 34.6 – – – 34.6 344 Morillas et al. [65]
Navarra 5.9 – – – 5.9 653 Sesma et al. [66]
Ourense 3.7 – – – 3.7–35.6 479 Amusátegui et al. [67]
35.6 – – – 101 Miró et al. [8]
Salamanca 7.0 – – – 7.0 433 Encinas Grandes et al. [46]
Sevilla 3.1 – – – 3.1 1000 Ariza-Astolfi et al. [68]
Page 4 of 12Gálvez et al. Parasites Vectors          (2020) 13:204 
Sites were geocoded by locality using ArcGis Pro v.2.3.3 
[Environmental Systems Research Institute (ESRI), Red-
lands, CA, USA].
Statistical analysis
For the cross-sectional serological survey, Chi-square 
test and Student’s t-test were used to identify significant 
associations between L. infantum seroprevalence and 
age, sex, breed, size, habitat, use given and bioclimate. 
Seroprevalence was calculated separately for every prov-
ince and bioclimatic zone. Kruskal-Wallis and Wilcoxon 
signed rank tests were used to examine relationships 
between sand fly species density (number of sand flies 
per trap per hour) and the variables bioclimatic zone, 
habitat and presence of animals (domestic, farm or wild 
fauna). Significance was set at P ≤ 0.05. All statistical tests 
were performed using the SPSS 25 package (SPSS Inc., 
Chicago, IL, USA).
Results
Forty-one scientific works reporting CanL seropreva-
lences for mainland Spain and the Balearic Islands were 
identified. The techniques used were IFI, ELISA and 
rapid tests. The review of reported seroprevalences of L. 
infantum revealed that of the 50 Spanish provinces there 
are published data for 31 of them. The provinces showing 
the highest seroprevalence were Balearic Islands (57.1%), 
Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), 
followed by Gerona (24.6%), Córdoba (23.7%), Granada 
(19.3%) and Alicante (19.1%). Provinces showing the low-
est seroprevalence were Vizcaya (0%), Cantabria (2%) and 
Álava (3.3%). Seroprevalence reported for the remaining 
provinces varied between 5–16% (Table 1 and Fig. 1).
The features of the cross-sectional seroprevalence study 
dog population are provided in Table 2. The animals sur-
veyed (n = 1739) were included into the following dog 
populations: (i) municipal animal shelter (35%, 608 stray 
dogs); (ii) hunting animal shelter (47%, 813 hunting dogs, 
2 pets, 1 guard dog and 1 shepherd dog); (iii) housed dogs 
(13.3%, 229 pets and 2 guard dogs); and (iv) farms (4.7%, 
21 shepherd dog, 11 pets, 8 hunting dogs and 1 guard 
dog). The study was conducted in 74 different locations 
in 25 Spanish provinces (see Table 3 and Fig. 2). Eleven 
new provinces without literature data were surveyed: A 
Coruña, Guipúzcoa, La Rioja, Las Palmas, Lérida, Lugo, 
Murcia, Pontevedra, Santa Cruz de Tenerife, Segovia, 
Valencia and Zamora.  
Overall, 176 of the 1739 dogs examined tested posi-
tive for L. infantum (10.1%). Significant differences were 
detected between longer deviated seroprevalence val-
ues in relation to bioclimatic belt (χ2 = 51.9968, df = 4, 
P < 0.0001): supratemperate (5.4%), mesotemperate 
(3.5%), supra-Mediterranean (13.1%), meso-Mediterra-
nean (11.9%), and thermo-Mediterranean (20.9%).
Significant differences were also detected when com-
paring seroprevalences by age group (χ2 = 21.5852, df = 5, 
P = 0.0006) and seroprevalence rates were higher in the 
younger dogs (< 1 year-old). Dog size data also revealed 
significant differences (χ2 = 12.4160, df = 2, P = 0.0020) 
and seroprevalence was higher in larger-sized dogs. Sig-
nificant differences were observed when comparing 
seroprevalences for the different habitats (χ2 = 10.5837, 
df = 2, P = 0.005) and seroprevalence was higher in rural 
habitats. No differences were observed according to sex 
Table 1 (continued)
Province Seroprevalence (%) Seroprevalence 
range (%)
n Reference
IFAT ELISA IFAT & ELISA Commercial kit
Tarragona 2.0 – – – 2.0–51.7 895 Portús et al. [69]
15.0 – – – 104 Fisa et al. [70]
10.2 – – – 902 Fisa et al. [71]
– – 10.2 – 107 Fisa et al. [72]
– – 5.7 – 2110 Fisa et al. [73]
51.7 116 Solano-Gallego et al. [42]
Toledo 8.7 – – – 8.7 232 Benito et al. [35]
Valencia – – 4.7 – 4.7 215 Benito-Hernández et al. [74]
Valladolid – – – 5.3 5.3 131 Couto et al. [75]
Vicaya 0 – – – 0 47 Miro et al. [8]
Zaragoza 8.5 – – – 8.5 1572 Castillo Hernández et al. [76]
Abbreviation: n, number of dogs surveyed; na, not applicable
Page 5 of 12Gálvez et al. Parasites Vectors          (2020) 13:204  
(χ2 = 2.4730, df = 1, P = 0.1158) or use category given to 
the dogs (χ2 = 2.7534, df = 4, P = 0.5999).
Cross-sectional survey data, together with published 
data for seroprevalence of L. infantum reveal that there 
are available data for 42 of the 50 provinces in Spain. A 
map of overall L. infantum infection seroprevalence was 
constructed (Fig.  3) by combining the data obtained 
in the literature review (Fig.  1) and the present survey 
(Fig.  2) based on seroprevalence records and the new 
areas reported here. This map provides seroprevalence 
data extrapolated to the entire country defining zones 
classified according to the seroprevalence range as: Zone 
1 (non-endemic, low risk); Zone 2 (hypoendemic, inter-
mediate risk); Zone 3 (endemic, intermediate-high risk); 
and Zone 4 (hyperendemic, high risk).
In the entomological survey conducted, fifty sites were 
sampled in 20 localities across the Mediterranean region 
(see Additional file 1: Table S1 and Fig. 4). The number of 
sites surveyed in each bioclimatic area was proportional 
to the size of the zone such that the largest bioclimatic 
belts, meso-Mediterranean and supra-Mediterranean 
were the most surveyed (22 sampling sites each) followed 
by the thermo-Mediterranean belt (6 sampling sites). A 
total of 676 specimens of five species were captured and 
further identified as follows: 562 (83.13%) P. pernicio-
sus; 64 (9.47%) S. minuta; 38 (5.62%) P. ariasi; 6 (0.89%) 
P. sergenti; and 6 (0.89%) P. papatasi. Sand fly sites were 
georeferenced and depicted as a pie chart reflecting the 
proportion of each sand fly species (Fig.  4). Traps were 
set in rural (20 sites, 60%) and periurban habitats (20 
sites, 40%).
Fig. 1 Seroprevalence of canine L. infantum infection in Spain by province based on a review of the literature published from 1985 to 2019. 
References: Miró et al. [8]; Gálvez et al. [20]; Vélez et al. [21]; Sáez de Santamaría et al. [34]; Benito et al. [35]; Alonso et al. [36]; Sanchís Marín et al. 
[37]; Rosado et al. [38]; Asencio et al. [39]; Seguí [40]; Pujol et al. [41]; Solano-Gallego et al. [42]; Alcover et al. [43]; Botet et al. [44]; Corachan et al. 
[45]; Encinas Grandes et al. [46]; Nieto et al. [47]; Rosado et al. [48]; Morales-Yuste et al. [49]; Arnedo Pena et al. [50]; Martínez-Cruz et al. [51]; Reyes 
Magaña et al. [52, 53]; Acedo Sánchez et al. [54, 55]; Martín-Sánchez et al. [56]; Lepe et al. [57]; Celaya [58]; Amela et al. [59]; Castañeda et al. [60, 61]; 
García Nieto et al. [62]; Miró et al. [63, 64]; Morillas et al. [65]; Sesma et al. [66]; Amusátegui et al. [67]; Ariza-Astolfi et al. [68]; Portús et al. [69]; Fisa et al. 
[70, 71]; Fisa et al. [72, 73]; Benito-Hernández et al. [74]; Couto et al. [75]; Castillo Hernández et al. [76]
Page 6 of 12Gálvez et al. Parasites Vectors          (2020) 13:204 
Over the 2-year study period, densities of S. minuta 
(Z = − 2.7485, P = 0.0084) and P. ariasi (Z = − 2.2811, 
P = 0.0269) were increased in rural habitats. Phleboto-
mus perniciosus showed a greater density increase in the 
thermo-Mediterranean compared with the meso-Medi-
terranean zone (Z = − 2.75663, P = 0.00584). In addition, 
the densities of the vector species (P. perniciosus and P. 
ariasi) (Z = − 2.58737, P = 0.00967) and of all phleboto-
mines (Z = − 2.70698, P = 0.00679) also rose significantly 
in the thermo-Mediterranean compared with the meso-
Mediterranean belt. New records were also observed for 
P. ariasi in Burgos, P. papatasi in Huelva and P. pernicio-
sus in Segovia.
Discussion
In a CanL endemic country like Spain with high densities 
of sand fly vectors and reservoirs, L. infantum infection 
spreads quickly amongst dog populations. The over-
all seroprevalence of L. infantum infection in the dogs 
surveyed was 10.1% (176/1739 animals), taking ≥ 1:100 
as the cut-off antibody titre. In areas where CanL is 
endemic, such a seroprevalence value of around 10% rep-
resents both dogs that develop the disease and a fraction 
of clinically healthy but persistently infected dogs [3]. In 
fact, CanL is only the tip of the iceberg in endemic areas, 
where part of the population is exposed and becomes 
infected without showing clinical evidence of disease 
[11]. In the survey, twelve provinces not sampled before 
were included. In future studies, canine L. infantum sero-
prevalence distributions need to be determined for the as 
yet unsampled provinces (in alphabetical order): Ávila, 
Burgos, Cuenca, Huesca, León, Palencia, Soria and Ter-
uel. Differences between seroprevalence distributions in 
relation to bioclimatic belt were significant. The thermo-
Mediterranean belt yielded the highest seroprevalence 
(20.9%) and may be considered of high-risk; the belts 
supra-Mediterranean (13.12%) and meso-Mediterranean 
(11.9%) of intermediate risk; and the belts supratemper-
ate (5.4%) and mesotemperate (3.5%) of low risk.
In northern Spain, where the seroprevalence was low 
compared to the rest of the Iberian Peninsula, we found 
elevated prevalence (24.3%) in Ourense [8]. Despite the 
lower seroprevalence in northern Spain, the climatic con-
ditions of the Galician province of Ourense should be 
highlighted as highly suitable for the presence of L. infan-
tum [8]. This province features exceptionally adequate 
climate conditions for the expansion of leishmaniosis and 
it belongs to the supra-Mediterranean and not the tem-
perate bioclimatic zone, like the northern region of Spain. 
Indeed, data from northern areas of Spain have already 
shown the expansion of L. infantum infection [8, 17].
According to Figs. 1 and 2, reported L. infantum sero-
prevalence in Spain ranges from 2% to 57.1% depending 
on the geographical region. Leishmania infantum sero-
prevalence variation between Spanish provinces might 
be explained by geographical differences in climate 
and seasonality associated with the bioclimatic and 
ecological requirements of the sand fly vectors [18]. In 
the European Union there is a zoonotic cutaneous and 
visceral leishmaniosis caused by L. infantum through-
out the Mediterranean region [9]. It has been proposed 
that environmental changes and global warming are 
having an impact on the geographical distribution of 
CanL infection and its vectors all over Europe [9, 18, 
Table 2 Features of the dog population in the cross-sectional 
survey of canine leishmaniosis seroprevalence (2011–2016)
Abbreviation: n, number of dogs
Variables n (%) No. positive (%)
Sex
 Female 732 (42) 65 (9)
 Male 991 (58) 111 (11)
Age group
 < 1 year 173 (11) 31 (18)
 1–2 years 473 (29) 32 (7)
 3–4 years 372 (23) 36 (10)
 5–6 years 284 (17) 35 (12)
 7–8 years 181 (11) 18 (10)
 > 9 years 154 (9) 10 (6)
Breed size
 Large (> 20 kg) 989 (60) 121 (12)
 Medium (10–19 kg) 457 (28) 34 (7)
 Small (< 10 kg) 203 (12) 12 (6)
Dog populations
 Municipal animal shelter 608 (35) 63 (10)
 Hunting animal shelter 817 (47) 79 (10)
 In the home 231 (13) 29 (13)
 Farm 83 (5) 5 (6)
Habitat
 Peri-urban 506 (29) 506 (29)
 Rural 1048 (60) 1048 (60)
 Urban 185 (11) 185 (11)
Bioclimate
 Supratemperate 93 (5) 93 (5)
 Mesotemperate 519 (30) 519 (30)
 Supra-mediterranean 526 (30) 526 (30)
 Meso-mediterranean 462 (27) 462 (27)
 Thermo-mediterranean 139 (8) 139 (8)
Use given
 Stray dog 608 (35) 63 (10)
 Hunting dog 821 (47) 80 (10)
 Pet 242 (14) 28 (12)
 Goat dog 64 (4) 4 (6)
 Guard dog 4 (4) 1 (25)
Page 7 of 12Gálvez et al. Parasites Vectors          (2020) 13:204  
19]. The map in Fig. 3 shows a snapshot of L. infantum 
seroprevalence that shows the northward emergence of 
CanL in Spain. These compendium maps of L. infantum 
seroprevalence will be useful for the implementation of 
control programmes. While it is desirable to standard-
ize the data source of the surveys upon which we cre-
ated Fig. 3, we have to take into account that they can 
present many varying factors such as dog selection pro-
cedures, serological techniques and antibody titre cut-
offs used, different periods (from 1985 up to date) and 
sample sizes, among many others.
In the present cross-sectional study, differences 
were detected in L. infantum infection seroprevalence 
according to animal age such that seroprevalences were 
significantly higher among younger dogs (< 1 year-old). 
As already reported, this could be related to an imma-
ture immune system in these dogs making them more 
vulnerable to infection in their first or second year of 
life [2, 11, 20, 21]. No significant impact of sex on infec-
tion seroprevalence was detected. In a study examining 
risk factors for canine leishmaniosis in endemic areas, 
sex did not emerge as a risk factor [22]. In the present 
cross-sectional study, seroprevalence was found to 
vary according to animal size and was higher in dogs 
weighing over 20 kg. The explanation for this could be 
that a greater weight translates to an increased risk of 
Table 3 Seroprevalence of canine L. infantum infection in Spain by province based on the cross-sectional serological survey 
conducted during 2011–2016
Abbreviation: n, number of dogs
Province Seroprevalence Localities Bioclimatic zone
n % No. positive
Álava 161 6.8 11 Arlucea, Artziniega, Madaria, Marsoño, Menoio, 
Noitegui, Quintana, Orbiso, Ollabarre, Respaldiza, 
San Román de Campezo, Sojo, Soxoguti
Mesotemperate
Asturias 110 3.6 4 Langreo, Serín Mesotemperate
Baleares (Ibiza) 40 20.0 8 Sant Andoni de Portmany, Sant Joan de Labritja Thermo-Mediterranean
Cáceres 91 19.8 18 Cañamero, Madrigal de la Vera Meso-Mediterranean
Cádiz 35 17.1 6 Los Barrios Thermo-Mediterranean
Cantabria 100 2.0 2 Torrelavega Mesotemperate
Ciudad Real 51 2 1 Puertollano Meso-Mediterranean
Coruña 32 3.1 1 La Coruña, Laracha Mesotemperate, Supratemperate
Guadalajara 81 9.9 8 El Casar, Espinosa de Henares, Torremocha de Jad-
raque
Supra-Mediterranean, meso-Mediterranean
Guipúzcoa 14 0 0 Antoñana, Izoria, Menagaray, Santurce Mesotemperate
Huelva 97 9.3 9 Valverde del Camino Meso-Mediterranean
La Rioja 66 6.1 4 Arnedo, Herce, Prejano Meso-Mediterranean
Las Palmas 104 0 0 Arguineguín Inframacaronesian
Lérida 37 8.1 3 Alcarras Supra-Mediterranean
Lugo 85 4.7 4 Lugo, Guitiriz Supratemperate
Madrid 224 10.3 23 Fuenlabrada, Móstoles, Navalcarnero, Peralejo, Serrada 
de la Fuente, Villalba, Villavicioesa, Las Zorreras
Supra-Mediterranean
Málaga 17 29.4 5 Alhaurín el Grande, Fuengirola, Mijas, Thermo-Mediterranean
Murcia 59 23.7 14 Achivel, Molina de Segura, Moratalla Meso-Mediterranean, thermo-Mediterranean
Orense 152 24.3 37 Allariz, Orense Supra-Mediterranean
Pontevedra 64 1.6 1 Vigo Mesotemperate
Santa Cruz de Tenerife 31 0 0 Tierra blanca Inframacaronesian
Segovia 14 0 0 Fuentepelayo y Cabanillas, Revenga Supra-Mediterranean
Sevilla 32 25.0 8 Castilblanco de los Arroyos Thermo-Mediterranean
Valencia 40 10.0 4 Utiel Meso-Mediterranean
Vizcaya 46 0 0 Santa Cruz, Hueto Abajo/Arriba Mesotemperate
Zamora 66 7.6 5 Arquelinos, Bretó, Pajares, Piedrahita de Castro, Reve-
nillos del Campo, Santovenia, Villarrín de Campos
Supra-Mediterranean
Zaragoza 25 4 1 Codos Supra-Mediterranean
Page 8 of 12Gálvez et al. Parasites Vectors          (2020) 13:204 
infection either because of the greater body surface sus-
ceptible to sand fly bites or because medium-sized and 
large dogs are often used for work activities and remain 
outdoors for long periods of time [20, 22]. No signifi-
cant relationship was found with factors such as habitat 
(rural, periurban or urban) or use given to the animal 
(e.g. hunting, guard, pet) of L. infantum infection. It 
should be noted that the individual immune response 
of the dog and virulence of the L. infantum isolate are 
important contributing factors [23].
The sand fly vectors of L. infantum identified here (P. 
perniciosus and P. ariasi) accounted for 88.75% of the 
total number of specimens identified. The wide distribu-
tion of these species, especially of P. perniciosus (83.13%), 
may indicate both an animal health and public health 
risk. Phlebotomus ariasi was distributed in zones of alti-
tudes belonging to the supra-Mediterranean and meso-
Mediterranean climates, as this species has a preference 
for wet and mountainous zones [9, 24–26]. Notwith-
standing, it should be underscored that six specimens 
of this species were found at an altitude of 144 m above 
the sea level, close to the coast in Carboneras (Almería), 
although in a study conducted in Hérault (France), 8 
individuals were captured at 80 metres of altitude 35 km 
from the coast [27].
Phlebotomus papatasi and P. sergenti were detected 
in the thermo-Mediterranean and meso-Mediterranean 
belts. This may be due to the fact that both species need 
environments with some aridity along with warm tem-
peratures for their development [28]. Similarly, these two 
species are epidemiologically important in other world 
zones. Thus, P. papatasi is a vector of L. major in regions 
of Africa and Asia, while P. sergenti is a vector of L. trop-
ica in North Africa, causing cutaneous leishmaniosis in 
both cases [29]. Fortunately, these two species of Leish-
mania have not been detected yet in Spain.
It should be noted that different species of sand flies 
were found here in places not described to date (P. ari-
asi in Burgos, P. papatasi in Huelva and P. perniciosus in 
Segovia). These observations reveal the lack of systematic 
surveys for the Iberian Peninsula and Balearic Islands, 
thus generating gaps in the data available or, in other 
words, a lack of representation of all the species present 
in a given zone.
According to the statistical treatment of the data, some 
variables were found to have a significant impact on the 
Fig. 2 Seroprevalence of canine L. infantum infection in Spain by province based on the cross-sectional serological survey conducted during 
2011–2016
Page 9 of 12Gálvez et al. Parasites Vectors          (2020) 13:204  
density of certain sand fly species. First, a higher den-
sity of S. minuta was observed in rural habitats. This was 
expected as this species is clearly a herpetophile [30]. 
Notwithstanding, in a study by Benito-de Martín et  al. 
[31] in Zaragoza, a greater density of this species was 
observed in periurban rather than in rural settings.
Phlebotomus ariasi was also detected at higher densi-
ties in rural habitats. The explanation for this is that this 
species is exophilic, and consequently, prefers natural 
environments and daytime resting places far from human 
settlements [30].
Finally, P. perniciosus showed greater densities in 
the thermo-Mediterranean compared with the meso-
Mediterranean belt. While this is a cosmopolitan spe-
cies that thrives in different bioclimatic zones [2, 30], its 
preferred thermo-Mediterranean region features a warm 
climate with long summers allowing for two abundance 
peaks, one in early summer and the other at the begin-
ning of autumn [2, 32]. In addition, compared with the 
meso-Mediterranean belt, the thermo-Mediterranean 
belt also showed significantly higher densities of both 
vector species (P. perniciosus and P. ariasi) and total sand 
fly species. However, this trend noted is clearly biased 
as P. perniciosus represented 83.13% of all species. Phle-
botomine vectors of L. infantum are sensitive to climate 
variations, and in the Mediterranean subregion were 
identified in the period of June-October [32]. Bioclimate 
predetermines the time needed for completion of sand 
fly development and life-cycle progression of the parasite 
within the invertebrate [33].
Fig. 3 Seroprevalence of canine L. infantum infection in Spain: compendium of Figs. 1 and 2. Zone 1 (non-endemic, low risk), Zone 2 (hypoendemic, 
intermediate risk), Zone 3 (endemic, intermediate-high risk) and Zone 4 (hyperendemic, high risk)
Page 10 of 12Gálvez et al. Parasites Vectors          (2020) 13:204 
Conclusions
This latest knowledge of seroprevalence distributions 
of canine L. infantum infection in Spain defines non-
endemic, hypoendemic, endemic and hyperendemic 
areas. As far as we are aware this is the first time to pro-
pose a CanL map for the whole territory of Spain. Along 
with the distributions of the parasiteʼs sand fly vectors, 
this information is key to approaching the control of this 
significant zoonosis. Further, these detailed maps of CanL 
in Spain including more than three decades (1985–2019) 
are an important resource for future eco-epidemiological 
analyses at the national and regional levels. Both bibliog-
raphy and survey maps combine the information needed 
for improved management of Canl and could exhibit an 
accurate prevalence predictive values. The methodology 
used to build the figure compendium can be extrapolated 
to other countries or even at the European level.
Supplementary information
Supplementary information accompanies this paper at https ://doi.
org/10.1186/s1307 1-020-04081 -7.
Additional file 1: Table S1. Detailed information about sites surveyed for 
sand flies across the Iberian Peninsula and Balearic Islands.
Abbreviations
CanL: canine leishmaniosis; IFAT: indirect immunofluorescence antibody test.
Acknowledgements
The authors wish to thank the owners of the dogs for their valuable collabora-
tion. Publication of this paper was sponsored by Bayer Animal Health in the 
framework of the 15th CVBD® World Forum Symposium.
Authors’ contributions
RG and GM conceived and coordinated the study. AM, RCH, CF, VM and OM 
participated on the completion and reporting of the serological surveys. IC, 
CCH and SM participated on the design, completion and reporting of the 
entomological survey. RG and GM were involved in the drafting of the manu-
script. All authors read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
All data generated or analysed during this study are included in this published 
article. Raw data are available from the corresponding author upon reason-
able request.
Ethics approval and consent to participate
This study was carried out in accordance with international guidelines for the 
Care and Use of Experimental Animals and Spanish Legislation (RD 53/2013).
Fig. 4 Sites surveyed for sand flies across the Iberian Peninsula and Balearic Islands shown on a bioclimatic zone map
Page 11 of 12Gálvez et al. Parasites Vectors          (2020) 13:204  
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Grupo de Investigación Epicontrol-Carnívoros, Departamento de Sanidad 
Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, 
Spain. 2 National School of Public Health, Instituto de Salud Carlos III, Madrid, 
Spain. 3 Microbiology Department, Hospital Universitario Ramón y Cajal, 
Madrid, Spain. 4 Parasitology Unit, Centro Nacional de Microbiología, Instituto 
de Salud Carlos III, Majadahonda, Spain. 
Received: 13 January 2020   Accepted: 11 April 2020
References
 1. Gálvez R, Montoya A, Fontal F, Martínez De Murguia L, Miró G. Controlling 
phlebotomine sand flies to prevent canine Leishmania infantum infection: 
a case of knowing your enemy. Res Vet Sci. 2018;121:94–103.
 2. Gálvez R, Descalzo MA, Miró G, Jiménez MI, Martín O, Dos Santos-Brandao 
F, et al. Seasonal trends and spatial relations between environmental/
meteorological factors and leishmaniosis sand fly vector abundances in 
central Spain. Acta Trop. 2010;115:95–102.
 3. Solano-Gallego L, Miró G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L, 
et al. LeishVet guidelines for the practical management of canine leish-
maniosis. Parasites Vectors. 2011;4:86.
 4. Short EE, Caminade C, Thomas BN. Climate change contribution to 
the emergence or re-emergence of parasitic diseases. Infect Dis. 
2017;10:1178633617732296.
 5. Fouque F, Reeder JC. Impact of past and on-going changes on climate 
and weather on vector-borne diseases transmission: a look at the evi-
dence. Infect Dis Poverty. 2019;8:51.
 6. Moreno J, Alvar J. Canine leishmaniasis: epidemiological risk and the 
experimental model. Trends Parasitol. 2002;18:399–405.
 7. Checa R, Gálvez R, Montoya A, Marino V, Betancor A, Fernández C, et al. 
New seroprevalence for mapping L. infantum infection and epidemiolog-
ical data affecting dogs in Spain. In: 6 World congress on leishmaniasis, 
16–20 May, Toledo, Spain; 2017. p. 1453.
 8. Miró G, Checa R, Montoya A, Hernández L, Dado D, Gálvez R. Current 
situation of Leishmania infantum infection in shelter dogs in northern 
Spain. Parasites Vectors. 2012;5:60.
 9. Ready PD. Leishmaniasis emergence in Europe. Euro Surveill. 
2010;15:19505.
 10. Miró G, Cardoso L, Pennisi MG, Oliva G, Baneth G. Canine leishmanio-
sis—new concepts and insights on an expanding zoonosis: part two. 
Trends Parasitol. 2008;24:371–7.
 11. Baneth G, Koutinas AF, Solano-Gallego L, Bourdeau P, Ferrer L. Canine 
leishmaniosis—new concepts and insights on an expanding zoonosis: 
part one. Trends Parasitol. 2008;24:324–30.
 12. Rivas-Martínez S. Pisos bioclimáticos de España. Lazaroa. 1983;5:33–43.
 13. Mancianti F, Meciani N. Specific serodiagnosis of canine leishmaniasis 
by indirect immunofluorescence, indirect hemagglutination, and 
counterimmunoelectrophoresis. Am J Vet Res. 1988;49:1409–11.
 14. Abonnenc E. Les phlébotomes de la région éthiopienne (Diptera, 
Psychodidae). Mémoires ORSTOM. 1972;55:289.
 15. Upton MS. Aqueous gum-chloral slide mounting media: an historical 
review. Bull Entomol Res. 1993;83:267–74.
 16. Gil Collado J, Morillas Marquez F, Sanchis Marin MC. Phlebotomus in 
Spain. Rev Sanid Hig Publica. 1989;63:15–34.
 17. Ballart C, Alcover MM, Picado A, Nieto J, Castillejo S, Portus M, et al. First 
survey on canine leishmaniasis in a non classical area of the disease 
in Spain (Lleida, Catalonia) based on a veterinary questionnaire and a 
cross-sectional study. Prev Vet Med. 2013;109:116–27.
 18. Reisen WK. Landscape epidemiology of vector-borne diseases. Annu 
Rev Entomol. 2010;55:461–83.
 19. Ready PD. Leishmaniasis emergence and climate change. Rev Sci Tech. 
2008;27:399–412.
 20. Gálvez R, Miró G, Descalzo MA, Nieto J, Dado D, Martín O, et al. Emerg-
ing trends in the seroprevalence of canine leishmaniosis in the Madrid 
region (central Spain). Vet Parasitol. 2010;169:327–34.
 21. Velez R, Ballart C, Domenech E, Abras A, Fernández-Arévalo A, Gómez 
SA, et al. Seroprevalence of canine Leishmania infantum infection 
in the Mediterranean region and identification of risk factors: the 
example of north-eastern and Pyrenean areas of Spain. Prev Vet Med. 
2019;162:67–75.
 22. Muniesa A, Peris A, Castillo JA, de Blas I. Variations in seroprevalences 
of canine leishmaniasis: could it be a consequence of the population 
structure? Vet Parasitol. 2016;226:5–9.
 23. Miró G, Petersen C, Cardoso L, Bourdeau P, Baneth G, Solano-Gallego 
L, et al. Novel areas for prevention and control of canine leishmaniosis. 
Trends Parasitol. 2017;33:718–30.
 24. Aransay AM, Testa JM, Morillas-Márquez F, Lucientes J, Ready PD. 
Distribution of sandfly species in relation to canine leishmaniasis 
from the Ebro Valley to Valencia, northeastern Spain. Parasitol Res. 
2004;94:416–20.
 25. Gállego M, Rioux JA, Rispail P, Guilvard E, Gállego J, Portús M, et al. Primera 
denuncia de flebotomos (Diptera, Psychodidae, Phlebotominae) en la 
provincia de Lérida (España, Cataluña). Rev Iber Parasitol. 1990;50:123–7.
 26. Lucientes Curdi J, Benito de Martín I, Castillo Hernández JA, Orcajo-Teresa 
J. Seasonal dynamics of Larroussius species in Aragon (N.E. Spain). Paras-
sitologia. 1991;33:381–6.
 27. Rioux JA, Golvan YJ, Maistre O. Presence de Phlebotomus (Larroussius) 
ariasi Tonnoir, 1921 dans Ies departements de l’Aveyron, des Bouches-
du-Rhone, du Gard., de I’HerauIt, des Pyrenees-Orientales et du Vaucluse. 
Ann Parasitol Hum Comp. 1961;36:706–7.
 28. Martínez Ortega E, Conesa Gallego E. Fenología de los flebotomos del 
subgénero Larroussius (Dip. Psychodidae, Phlebotomus) en el sureste de la 
Península Ibérica. Bol Asoc Esp Entomol. 1987;11:293–300.
 29. Ready PD. Biology of phlebotomine sand flies as vectors of disease 
agents. Annu Rev Entomol. 2013;58:227–50.
 30. Lladó Villalonga T, Rotger Sureda MJ. Estudio del flebotomo como vector 
de la leishmaniasis en la isla de Mallorca. Palma de Mallorca: Conselleria 
de Sanitat i Seguretat Social Govern Balear; 1990.
 31. Benito-De Martín MI, Lucientes-Curdi J, Orcajo-Teresa J, Castillo-Hernán-
dez JA. Seasonal dynamics of Sergentomyia minuta (Rondani, 1843) 
populations in Aragon (N.E. Spain). Parassitologia. 1991;33(Suppl.):89–97.
 32. Alten B, Maia C, Afonso MO, Campino L, Jimenez M, Gonzalez E, et al. 
Seasonal dynamics of phlebotomine sand fly species proven vectors of 
Mediterranean leishmaniasis caused by Leishmania infantum. PLoS Negl 
Trop Dis. 2016;10:e0004458.
 33. Maia C, Cardoso L. Spread of Leishmania infantum in Europe with dog 
travelling. Vet Parasitol. 2015;213:2–11.
 34. Sáez de Santamaría M, Benito A, Eraso E, Beltrán de Heredia F, Estíbalez JJ, 
Guisantes JA. Estudio seroepidemiológico de la leishmaniosis canina en 
Álava, España. Acta Parasitol Port. 1997;4:160.
 35. Benito A, Alvar, J. Estudio preliminar de la leishmaniosis en la Comunidad 
de Castilla la Mancha. In: VI Congreso Nacional de Parasitología, 25–29 
September, Cáceres, Spain; 1989; p. 194.
 36. Alonso F, Gimenez Font P, Manchon M, Ruiz de Ybanez R, Segovia M, Ber-
riatua E. Geographical variation and factors associated to seroprevalence 
of canine leishmaniosis in an endemic Mediterranean area. Zoonoses 
Public Health. 2010;57:318–28.
 37. Sanchís Marín MC, Martín Sánchez J, Amate P, Acedo Sánchez C, Miras 
N, Mostapha L, et al. Estudio epidemiológico de la leishmaniosis en la 
comarca del Campo de Níjar (Almería). Ars Pharm. 1997;38:53–61.
 38. Rosado D, Suero F, Garrudo RM, Mirón C, García Alonso M, Navarrete I, 
et al. La leishmaniosis en Zafra (Badajoz, España) del reservorio canino y 
del agente vectorial. Acta Parasitol Port. 1997;4:160.
 39. Asencio MA, Herraez O, Tenias JM, Garduno E, Huertas M, Carranza R, et al. 
Seroprevalence survey of zoonoses in Extremadura, southwestern Spain, 
2002–2003. Jpn J Infect Dis. 2015;68:106–12.
 40. Seguí MG. Estudi epidemiòlogic de la leishmaniosi a l´illa de Menorca. 
Revista de Ciencia. 1991;9:91–101.
 41. Pujol A, Cortés E, Ranz A, Vela C, Aguiló C, Martí B. Estudio de seropreva-
lencia de leishmaniosis (L. infantum) y de ehrlichiosis (E. canis) en la isla de 
Mallorca mediante técnicas inmunológicas. In: Congreso de la Asociación 
Page 12 of 12Gálvez et al. Parasites Vectors          (2020) 13:204 
de Veterinarios Especialistas en Diagnóstico de Laboratorio (AVEDILA), 
22–23 September, Palma de Mallorca, Spain; 2005. p. 9–12.
 42. Solano-Gallego L, Llull J, Osso M, Hegarty B, Breitschwerdt E. A sero-
logical study of exposure to arthropod-borne pathogens in dogs from 
northeastern Spain. Vet Res. 2006;37:231–44.
 43. Alcover MM, Ballart C, Serra T, Castells X, Scalone A, Castillejo S, et al. 
Temporal trends in canine leishmaniosis in the Balearic Islands (Spain): 
a veterinary questionnaire. Prospective canine leishmaniosis survey 
and entomological studies conducted on the Island of Minorca, 
20 years after first data were obtained. Acta Trop. 2013;128:642–51.
 44. Botet J, Serra T, Portus M, Mora R, Gallego M. Incidencia de la 
leishmaniosis en el área de Barcelona. Rev Ibérica Parasitol. 
1987;extra:51–54.
 45. Corachan M, Portus M, Filella E, Olle JE. Leishmaniasis in Catalonia. 
Preliminary study. Rev Sanid Hig Publica. 1987;61:1229–36.
 46. Encinas Grandes A, Gómez-Bautista M, Martín Novo M, Simón Martín 
F. Leishmaniasis in the province of Salamanca, Spain. Prevalence in 
dogs and seasonal dynamics of vectors. Ann Parasitol Hum Comp. 
1988;63:387–97.
 47. Nieto CG, Navarrete I, Habela M, Hernández-Rodríguez S. Seropreva-
lence of canine leishmaniasis around Cáceres, Spain. Prev Vet Med. 
1992;13:173–8.
 48. Rosado D, Arrieta E, Verdugo SG, García Alonso M, Pérez Martín E. 
Estado actual de la Leishmaniosis canina en la ciudad de Cáceres. Rev 
Exp Anim. 1995;6:93.
 49. Morales-Yuste M, Morillas-Marquez F, Díaz-Saez V, Barón-López S, 
Acedo-Sánchez C, Martín-Sánchez J. Epidemiological implications of 
the use of various methods for the diagnosis of canine leishmaniasis 
in dogs with different characteristics and in differing prevalence sce-
narios. Parasitol Res. 2012;111:155–64.
 50. Arnedo Pena A, Bellido Blasco JB, González Morán F, Arias Sánchez 
A, Calvo Mas C, Safont Adsuara L, et al. Leishmaniasis in Castellon: an 
epidemiological study of human cases, the vector and the canine 
reservoir. Rev Sanid Hig Publica. 1994;68:481–91.
 51. Martínez-Cruz MS, Martínez-Moreno A, Martínez-Moreno FJ, Martínez-
Gómez F, Hernández-Rodríguez S. Epidemiología de la leishmaniosis 
canina en Córdoba. Rev Ibérica Parasitol. 1990;50:1–7.
 52. Reyes Magaña A, Morillas-Márquez F, Valero-López A, González-Castro J, 
Benavides-Delgado I, Sanchís-Marín MC. Encuesta sobre la leishmaniosis 
canina en las comarcas naturales de la provincia de Granada (Sur de 
España). Rev Ibérica Parasitol. 1988;48:233–40.
 53. Reyes Magaña A, Morillas Márquez F, Montes Ramírez E, Sanchís Marín 
MC, Benavides Delgado I, González Castro J. La leishmaniosis en la provin-
cia de Granada: estudio de la enzootia canina. Ars Pharm. 1989;30:35–44.
 54. Acedo Sánchez C, Martín Sánchez J, Velez Bernal ID, Sanchís Marín MC, 
Louassini M, Maldonado JA, et al. Leishmaniasis eco-epidemiology in 
the Alpujarra region (Granada Province, southern Spain). Int J Parasitol. 
1996;26:303–10.
 55. Acedo Sánchez C, Morillas-Márquez F, Sanchiz-Marin MC, Martín-Sánchez 
J. Changes in antibody titres against Leishmania infantum in naturally 
infected dogs in southern Spain. Vet Parasitol. 1998;75:1–8.
 56. Martín-Sánchez J, Morales-Yuste M, Acedo-Sánchez C, Barón S, Díaz V, 
Morillas-Márquez F. Canine leishmaniasis in southeastern Spain. Emerg 
Infect Dis. 2009;15:795–8.
 57. Lepe JA, Ubeda JM, Morillas F, Guevara DC, Martín-Sánchez J, Guerrero FJ, 
Sanchos-Marín MC, Perea R. Epidemiology of leishmaniosis in the nature 
reserve of Sierra de Aracena and Picos de Aroche (Southwest Spain). Res 
Rev Parasitol. 2000;60:113–9.
 58. Celaya C. Estudio epidemiológico descriptivo de la leishmaniasis canina 
en un entorno periurbano de la ciudad de Madrid. Veterinaria en Madrid. 
1993;5:13–5.
 59. Amela C, Méndez I, Torcal JM, Medina G, Pachón I, Cañavate C, et al. 
Epidemiology of canine leishmaniasis in the Madrid region, Spain. Eur J 
Epidemiol. 1995;11:157–61.
 60. Castañeda R, Carpio I, Asensio A, Alberdi JC, García S, Domínguez T, et al. 
Estudio de seroprevalencia de la leishmaniosis canina en perros censados 
en Madrid. Consulta de Difusión Veterinaria. 1999;7:836–41.
 61. Castañeda R, García A, Miró G, Carpio I, Caballero MJ, Alberdi JC, et al. 
Estudio de seguimiento de la leishmaniosis canina en perros vagabundos 
de la Comunidad de Madrid. Pequeños Animales. 2000;25:16–27.
 62. García Nieto A, Taveira Jimenez JA, Castañeda López R, Moreno Alcalde 
S, Escacena Saínz C, Martínez Cortés M. Prevalencia de la leishmanio-
sis canina en el sistema de vigilancia en perros vagabundos de la 
Comunidad de Madrid durante el periodo 11-1996 a 4-2001. Profesión 
Veterinaria. 2002;53:56–61.
 63. Miró G, Montoya A, Mateo M, Alonso A, García S, García A, et al. A leish-
maniosis surveillance system among stray dogs in the region of Madrid: 
ten years of serodiagnosis (1996–2006). Parasitol Res. 2007;101:253–7.
 64. Miro G, Muller A, Montoya A, Checa R, Marino V, Marino E, et al. Epidemio-
logical role of dogs since the human leishmaniosis outbreak in Madrid. 
Parasites Vectors. 2017;10:209.
 65. Morillas F, Sánchez Rabasco F, Ocaña J, Martín-Sánchez J, Ocana-Wihelmi 
J, Acedo C, et al. Leishmaniosis in the focus of the Axarquia region, 
Malaga Province, southern Spain: a survey of the human, dog, and vector. 
Parasitol Res. 1996;82:569–70.
 66. Sesma B, Barricarte A. Leishmaniasis en Navarra: Revisión de actuaciones. 
Anales Sis San Navarra. 1997;20:209–16.
 67. Amusátegui I, Sainz A, Aguirre E, Tesouro MA. Seroprevalence of Leishma-
nia infantum in northwestern Spain, an area traditionally considered free 
of leishmaniasis. Ann NY Acad Sci. 2004;1026:154–7.
 68. Ariza-Astolfi C, Úbeda-Ontiveros JM, Guevara-Benítez D, Cutillas-Barrios 
C, de Rojas-Álvarez M, Reina-Mulero F. Epidemiología de la leishmanio-
sis canina en la Provincia de Sevilla (España). In: X Congreso Ibérico de 
Parasitología, 15–20 July, Madrid, Spain; 2007. p. 49.
 69. Portús M, Fisa R, Serra T, Gállego M, Mora R. Estudios seroepidemiológicos 
sobre la leishmaniosis canina en Cataluña. Med Vet. 1987;4:569–75.
 70. Fisa R, Gallego M, Portús M, Gallego J. Evolución de la leishmaniosis 
canina en zona endémica a través de su seguimiento serológico. Ciencias 
Veterinarias. 1991;4:69–76.
 71. Fisa R, Portús M, Gallego M, Valls D, Alisa MJ. El diagnóstico serológico 
de la leishmaniosis canina en la comarca del Priorat (Tarragona). Clínica 
Veterinaria de Pequeños Animales. 1992;12:231–6.
 72. Fisa R, Gallego M, Riera C, Aisa MJ, Valls D, Serra T, de Colmenares M, 
Castillejo S, Portús M. Serological diagnosis of canine leishmaniasis by 
dot-ELISA. J Vet Diagn Invest. 1997;9:50–5.
 73. Fisa R, Gállego M, Castillejo S, Aisa MJ, Serra T, Riera C, Carrio J, Gállego J, 
Portús M. Epidemiology of canine leishmaniosis in Catalonia (Spain) the 
example of the Priorat focus. Vet Parasitol. 1999;83:87–97.
 74. Benito Hernández M, Pérez-Díaz JV, Osuna-Calvet B, Domínguez MT, 
Vega-García S. The importance of a continued serovigilance in the 
control of canine leishmaniosis. A comparison of two methods of control. 
Épidemiol Santé Anima. 2004;45:87–90.
 75. Couto CG, Lorentzen L, Beall MJ, Shields J, Bertolone N, Couto JI, et al. 
Serological study of selected vector-borne diseases in shelter dogs in 
central Spain using point-of-care assays. Vector Borne Zoonotic Dis. 
2010;10:885–8.
 76. Castillo Hernández JA, Sánchez Acedo C, Gutiérrez Galindo J, Lucientes 
Curdi J, Estrada Peña A et al. Evaluación de diversas pruebas en el 
diagnóstico de la leishmaniasis canina. In: IV Congreso Nacional de Parasi-
tología. 9–13 July, Tenerife, Spain; 1985. p. 31.
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