Canine leishmaniasis in Southeastern Spain.

[FIGURE 1 OMITTED]

It has been suggested that climate change has the potential to change the transmission intensity of vector-borne diseases such as leishmaniasis, but supporting literature is lacking (1,2). Because long-term quality data on leishmaniasis caused by Leishmania infantum and its vector (3-9) are available for the Alpujarras region of southeastern Spain (Figure 1), this is an ideal area for studying changes in the prevalence of canine leishmaniasis in a changing environment. Our study objectives were to determine whether any changes had occurred in the prevalence of canine leishmaniasis over 22 years and to identify risk factors for this disease.

The Study

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 (3-5). 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 0-700, 600-900, and 900-1,800 m above sea level; annual mean temperatures are 17-19, 13-17, and 8-15[degrees]C; and annual rainfalls are 200-350, 600-1,000, and 1,000-1,600 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 [greater than or equal to]160 were considered positive. To determine and compare the existence or lack of statistically significant differences between present and past prevalence rates, we used the [chi square] or Fisher exact test.

To achieve the second objective, we conducted a survey. 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. No retrospective entomologic analysis was performed as had been done for canine leishmaniasis.

In our 2006 survey, 57 (13.0%) of 439 dogs had an antibody titer [greater than or equal to]160 (seroprevalence rate), 268 (61.0%) dogs had titers 20-80, 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 ([chi square] test p<0.001) indicate that these differences are not random. The evolution-in-time analysis (Figure 2) shows how over 22 years (1984-2006), 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 (3-5; this study).

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 Torvizcon, where the P. perniciosus density is [greater than or equal to]4 sandflies/[m.sup.2]. Risk for these dogs was 54,571x 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 Bombaron or Portugos at the supra-Mediterranean level where P. perniciosus density is <4 sandflies/[m.sup.2].

[FIGURE 2 OMITTED]

Conclusions

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; Martin-Sanchez et al., unpub. 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 [approximately equal to]100% of the total number of dogs registered in the villages sampled (3). The Alpujarras economy is based mainly on agriculture and livestock. One change during the period studied was the gradual disappearance of livestock enclosures in rural population centers. These enclosures appear to create a substantial risk for infection with Leishmania spp. (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).

The Ministerio de Educacion y Ciencia, Madrid (AGL200406909-[C.sub.0]2-02), and the Junta de Andalucia (group CVI176) provided financial assistance, and Emerging Diseases in a changing European eNvironment (EDEN; contract no. 010284, Integrated Project GOCE) awarded a PhD student grant to S.B. This publication is catalogued by the EDEN Steering Committee as EDEN0137 (www.eden-fp6project.net).

DOI: 10.3201/eid1505.080969

References

(1.) Kovats RS, Campbell-Lendrum DH, McMichael AJ, Woodward A, Cox JS. Early effects of climate change: do they include changes in vector-borne disease? Philos Trans R S[degrees]C Lond B Biol Sci. 2001;356:1057-68.

(2.) Sutherst RW. Global change and human vulnerability to vector-borne diseases. Clin Microbiol Rev. 2004;17:136-73. DOI: 10.1128/ CMR.17.1.136-173.2004

(3.) Sanchez C, Martin Sanchez J, Velez Bernal ID, Sanchis Marin 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. DOI: 10.1016/0020-7519(95)00124-7

(4.) Reyes Magana A, Morillas-Marquez F, Valero-Lopez A, Gonzalez Castro J, Benavides-Delgado I, Sanchis Marin MC. Encuesta sobre la leishmaniosis canina en las comarcas naturales de la provincia de Granada (Sur de Espana). Rev Iber Parasitol. 1988;48:233-40.

(5.) Reyes Magana A, Morillas Marquez F, Monts Ramirez E, Sanchis Marin MC, Benavides Delgado I, Gonzalez CJ. La leishmaniosis en la provincia de Granada: estudio de la enzootia canina. Ars Pharmaceutica. 1989;30:35-44.

(6.) Morillas-Marquez F, Guevara-Benitez DC, Ubeda-Ontiveros JM, Gonzalez CJ. Annual fluctuations of Phlebotomus populations (Diptera, Phlebotomidae) in the province of Grenada (Spain) [in French]. Ann Parasitol Hum Comp. 1983;58:625-32.

(7.) Alcalde Alonso M, Morillas Marquez F, Delgado V, Naranjo R. Epidemiologia de las leishmaniosis cutaneas en la provincia de Granada. Actas Dermosifiliogr. 1989;80:251-4.

(8.) Martin-Sanchez J, Guilvard E, Acedo-Sanchez C, Volf-Echeverri M, Sanchiz-Marin MC, Morillas-Marquez F. Phlebotomus perniciosus Newstead, 1911, infection by various zymodemes of the Leishmania infantum complex in the Granada province (southern Spain). Int J Parasitol. 1994;24:405-8. DOI: 10.1016/0020-7519(94)90089-2

(9.) Martin-Sanchez J, Gallego M, Baron S, Castillejo S, Morillas-Marquez F. Pool screen PCR for estimating the prevalence of Leishmania infantum infection in sandflies (Diptera: Nematocera, Phlebotomidae). Trans R S[degrees]C Trop Med Hyg. 2006;100:527-32. DOI: 10.1016/j.trstmh.2005.08.005

(10.) Rivas Martinez S, Bandullo JM, Serrada R, Allue Andrade JL, Monero del Burgo JL, Gonzalez Rebollar JL. Memoria del mapa de series de vegetacion de Espana. Madrid: Ministerio de Agricultura, Pesca y Alimentacion, ICONA; 1987. p. 31-42.

(11.) Abanades Garcia JC, Cuadrat Prats JM, de Castro Munoz de Lucas M, Fernandez Garcia F, Gallastegui Zulaica C, Garrote de Marcos L, et al. El cambio climatico en Espana. Estado de situacion. Informe para el Presidente del Gobierno elaborado por expertos en cambio climatico; 2007 [cited 2007 Dec 31]. Available from http://www.mma.es/portal/secciones/cambio_climatico

(12.) Vargas Yanez M, Garcia Martinez MC, Moya Ruiz F, Tel E, Parrilla G, Plaza F, et al. Cambio climatico en el Mediterraneo espanol. Madrid: Impresos y Revistas, Instituto Espanol de Oceanografia, Ministerio de Educacion y Ciencia; 2007. p. 32-41.

(13.) Morillas F, Sanchez Tabasco F, Ocana J, Martin-Sanchez J, OcanaWihelmi J, Acedo C, et al. Leishmaniasis in the focus of the Axarquia region, Malaga province, southern Spain: a survey of the human, dog and vector. Parasitol Res. 1996;82:269-70. DOI: 10.1007/s004360050164

(14.) Moreira ED, de Souza VMM, Sreenivasan M, Lopes NL, Barreto RB, de Carvalho LP. Peridomestic risk factors for canine leishmaniasis in urban dwellings: new findings from a prospective study in Brazil. Am J Trop Med Hyg. 2003;69:393-7.

(15.) Killick-Kendrick R. Breeding places of Phlebotomus ariasi in the Cevennes focus of leishmaniasis in the south of France. Parassitologia. 1987;29:181-91.

Joaquina Martin-Sanchez, Manuel Morales-Yuste, Carmen Acedo-Sanchez, Sergio Baron, Victoriano Diaz, and Francisco Morillas-Marquez

Author affiliations: Universidad de Granada, Granada, Spain (J. Martin-Sanchez, M. Morales-Yuste, S. Baron, V. Diaz, F. Morillas-Marquez); and Laboratorio de Analisis Veterinarios ANLAVE, Granada (C. Acedo-Sanchez)

Dr Martin-Sanchez is a professor in the Parasitology Department of the University of Granada. Her main area of research is the epidemiology of leishmaniasis.

Address for correspondence: Joaquina Martin-Sanchez, Parasitology Department, Granada University, Campus Universitario de Cartuja Granada 18.071, Spain: email: [e-mail]

Table 1. Possible factors associated with canine leishmaniasis,
southeastern Spain *

                                                % Dogs with canine
Variables                          No. dogs     leishmaniasis

Bioclimatic level                  439          13.0
Thermo-Mediterranean               210          13.3
Meso-Mediterranean                 139          20.1
Supra-Mediterranean                90           1.1

Habitat                            438          13.0
Rural                              435          13.1
Urban/peri-urban                   3            0.0

Sex                                435          12.4
Male                               253          14.2
Female                             182          9.9

Age, y                             421          13.0
[less than or equal to]<4          232          9.5
>4                                 189          18.0

Weight, kg                         405          13.6
[less than or equal to]<25         316          11.9
>25                                89           20.0

Fur length                         349          15.5
Short/ medium                      255          17.6
Long                               94           9.6

Activity                           439          13.0
Pet                                258          8.5
Hunting                            133          15.0
Other ([dagger])                   48           31.3

Location during daytime            373          15.3
House                              151          4.0
Outside                            122          28.7
In kennels                         100          16.0

Location at night                  435          13.1
Outdoors                           186          19.4
Indoors                            249          8.4

Travel away from home              438          13.0
No                                 396          12.6
Yes                                42           16.7

Clinical signs of leishmaniasis    439          13.0
No                                 413          12.3
Yes                                26           23.1

Fly protection                     375          15.2
No                                 361          15.0
Yes                                14           21.4

Phlebotomus perniciosus density    439          13.0
<4 sandflies/[m.sup.2]             303          9.9
[greater than or equal to]4        136          19.9
  sandflies/[m.sup.2]

P. ariasi density                  439          13.0
<6 sandflies/[m.sup.2]             383          10.7
[greater than or equal to]6        56           28.6
  sandflies/[m.sup.2]

Variables                          Relative     p value
                                   likelihood

Bioclimatic level                  --           0.005
Thermo-Mediterranean               Ref          --
Meso-Mediterranean                 1.640        0.092
Supra-Mediterranean                0.073        0.011

Habitat                            --           0.999
Rural                              Ref          --
Urban/peri-urban                   <0.001       --

Sex                                --           0.178
Male                               Ref
Female                             0.662

Age, y                             --           <0.001
[less than or equal to]<4          Ref
>4                                 2.094

Weight, kg                         --           0.048
[less than or equal to]<25         Ref
>25                                1.859

Fur length                         --           0.069
Short/ medium                      Ref
Long                               0.494

Activity                           --           <0.001
Pet                                Ref          --
Hunting                            1.899        0.052
Other ([dagger])                   4.876        <0.001

Location during daytime            --           <0.001
House                              Ref          --
Outside                            9.722        <0.001
In kennels                         4.603        0.002

Location at night                  --           0.001
Outdoors                           Ref
Indoors                            0.384

Travel away from home              --           0.461
No                                 Ref
Yes                                1.384

Clinical signs of leishmaniasis    --           0.122
No                                 Ref
Yes                                2.129

Fly protection                     --           0.969
No                                 Ref
Yes                                1.551

Phlebotomus perniciosus density    --           0.005
<4 sandflies/[m.sup.2]             Ref
[greater than or equal to]4        2.254
  sandflies/[m.sup.2]

P. ariasi density                  --           <0.001
<6 sandflies/[m.sup.2]             Ref
[greater than or equal to]6        3.337
  sandflies/[m.sup.2]

* Univariate analysis by logistic regression. Dogs considered
to have canine leishmaniasis were those with antibody titer
[greater than or equal to]160. We investigated the existence of
interaction and/or confusion between variables by constructing
and comparing logistic regression models. The statistical analysis
was performed using the software package SPSS 15.0 (www.spss.com).
Confusion was noted between the location during daytime and location
at night, so location during daytime was excluded from the
multivariate analysis. No interaction was detected between any pair
of independent variables. Ref, referent.

([dagger]) Guard dogs (n = 34), sheepdogs (n = 8), stray dogs (n = 4),
dogs in kennel (n = 2).

Table 2. Factors associated with canine leishmaniasis, southeastern
Spain, final model *

Variables (no. dogs)                OR (95% CI)            p value

Bioclimatic level                   --                     0.001
Thermo-Mediterranean (189)          --                     --
Meso-Mediterranean (134)            0.538 (0.196-1.476)    0.228
Supra-Mediterranean (82)            0.013 (0.001-0.126)    <0.001

Age, y                              --                     0.001
[less than or equal to]<4 (224)     --
>4 (181)                            3.223 (1.604-6.474)

Weight, kg                          --                     0.069
[less than or equal to]<25 (316)    --
>25 (89)                            1.985 (0.948-4.156)

Activity                            --                     0.00
Pet (232)                           --                     --
Hunting (129)                       2.401 (1.060-5.442)    0.036
Other (44) ([dagger])               4.831 (1.909-12.226)   0.001


Location at night                   --                     <0.001
Indoors (229)                       --
Outdoors (176)                      3.304 (1.704-6.406)

Phlebotomus perniciosus density     --                     <0.001
<4 sandflies/[m.sup.2] (277)        --
[greater than or equal to]4         7.029 (2.632-18.769)
  sandflies/[m.sup.2] (128)

* Determined by multivariate logistic regression. With age as
continuous variable (0.1-25 years, average 4.7 years), odds ratio
(OR) = 1.142; with weight as continuous variable (2-60 kg, average
= 17.7), OR = 1.047. CI, confidence interval.

([dagger]) Guard dogs, sheepdogs, stray dogs, and dogs in kennels.