Leishmania infantum in free-ranging hares , Spain , 2004-2010

Iberian hares (Lepus granatensis) were recently deemed responsible for an outbreak of human leishmaniasis affecting metropolitan Madrid, Spain. However, the reservoir potential of hares in Europe is poorly known. We report a retrospective survey on Leishmania infantum, the causal agent of zoonotic endemic leishmaniasis in the Mediterranean basin, infection status of Iberian, European (Le. europaeus) and Broom (Le. castroviejoi) hares in Spain. Spleen samples from 94 hares were tested by polymerase chain reaction. Sequencing and restriction fragment length polymorphism (RFLP) assays were performed on positive samples and RFLP patterns compared with those of strains reported in the scientific literature. DNA prevalence in hare spleen samples was 43.6% (95% confidence interval: 33.6-53.6). In all six regions studied at least one positive sample was found. RFLP revealed existence of specific hare strains of L. infantum differing from those reported in wild carnivores in Spain. The widespread presence of L. infantum in the most abundant Spanish hare species and the recent evidence of the ability of naturally infected hares to transmit the pathogen to Phlebotomus perniciosus, its main vector in the western Mediterranean, suggest that hares may have an unexpected role in the epidemiology of L. infantum in Spain.


Introduction
Diseases at the wildlife-livestock-human interface are an increasing concern for public health, animal health and animal conservation authorities worldwide [1].Also, wildlife-associated infectious diseases are at the top of human emerging diseases [2].Basic epidemiologic knowledge would constitute the foundation for targeted prevention and control measures of wildlife-associated diseases, but knowledge is scarce for many of the currently emerging threats; Leishmaniasis in Europe is a good example.Endemic Mediterranean leishmaniasis is a disease of animals and humans caused by Leishmania infantum, a protozoan causing both visceral and cutaneous zoonotic leishmaniasis in the Mediterranean basin.L. infantum has recently spread northward from Mediterranean to temperate climates in Europe (e.g.Hungary and northern Italy), apparently linked to climate change [3] but perhaps also linked to increased movements of infected hosts, mostly dogs, from endemic Mediterranean areas [4].Thus, leishmaniasis caused by L. infantum can be considered as a potentially emerging threat for central and northern European countries [4].Dogs are deemed as major reservoirs of L. infantum since they efficiently replicate the protozoan parasite and are preferred hosts for vector phlebotomine sandflies [4].Wild carnivores such as the wolf (Canis lupus), the red fox (Vulpes vulpes), the Egyptian mongoose (Herpestes ichneumon), the genet (Geneta geneta), the pine marten (Martes martes) or the Iberian lynx (Lynx pardinus) have also been implicated in the maintenance of L. infantum [5,6].Recently, naturally infected Iberian hares (Lepus granatensis) were found to efficiently allow infection of Phlebotomus perniciosus sandflies with L. infantum [7].Iberian hares were deemed as the main reservoirs of a leishmaniasis outbreak causing more than 260 human cases in the southwestern metropolitan area of Madrid since 2009 [7,8].
Three different hare species inhabit the Iberian Peninsula: (i) the Iberian hare that is present in vast areas of the Iberian Peninsula, (ii) the European brown hare (Le.europaeus) that lives in northern and northeastern Spain, and (iii) the Broom hare (Le.castroviejoi) that can be found in the Cantabrian Mountains [9].Since basic information on the relationship between L. infantum and Iberian hare species is only anecdotal [7] and Le.granatensis populations are abundant and stable [10, unpublished data], we designed a retrospective survey on samples collected from Spanish hares through wildlife disease surveillance programs.

Methods
Spleen samples from a subset of hares (n=94) collected during necropsies performed over carcasses of animals found dead or harvested by hunters in Spain from 2004 to 2010 were used in this study.Sample collection was opportunistic since they were obtained through Spanish wildlife disease surveillance programs.Hare tissues were preserved frozen at -20ºC until analysed.
According to their origin, samples were allocated to six geographic regions (listed here from north to south): Atlantic, Northern plateau, Northeast, Centre, Southern plateau and Guadalquivir river valley (Figure).
Total genomic DNA from spleen samples was extracted using a commercial kit (GenomeElute, Sigma-Aldrich, St. Louis, MO) following the manufacturer's protocol.A previously described polymerase chain reaction (PCR) targeting a 145 bp fragment present on the high copy of kDNA minicircles of L. infantum was performed on spleen samples [11].PCR products were elicited by PCR fragment size estimation in comparison with two molecular weight standards: PCR 100 pb Low Ladder and pBR 322 HaeIII Digest (Sigma-Aldrich, St. Louis, MO) after electrophoresis in 2% agarose gel.Amplicons from 23 positive hares were sequenced to confirm L. infantum infection.Moreover, a restriction fragment length polymorphism (RFLP) assay was performed on all positive samples to compare amplicon patterns with previous studies from peninsular Spain [5].For the RFLP assay, 15 µl of PCR product were digested with restriction enzymes BsiY I and Mlun NI as previously reported [12].
Basic comparison of prevalence values between sexes, species and geographic regions was performed by means of chi-squared tests.Statistical uncertainty was assessed by calculating the 95% confidence interval (CI) for each of the proportions according to the expression 95% CI. = 1.96[p(1 -p)/n] 1/2 (where "p" is the proportion in its unitary value and "n" is the sample size) and expressed in percentage.
The collected hare species were from the (i) Atlantic region: 14 1).Interestingly, the highest prevalence value was observed in hares from Central and Atlantic regions.
Sequencing was successful from nine hares and homology with L. infantum kinetoplast DNA ranged from 94% to 99%.
Twenty-two RFLP patterns were obtained from 32 hares (see Table 2).Thirteen RFLP patterns were found in thirteen individuals; six from the Atlantic region, six from Southern plateau region and one from Centre region.Eight patterns were each present in two different hares; two of the patterns were exclusively present in hares from Southern plateau region and one was only present in hares from Centre region, while five of these eight patterns were present in hares from different geographic regions − two in hares from Atlantic and Southern plateau regions respectively, one in hares from Northern plateau and Southern plateau regions, one in hares from Centre and Southern plateau regions and one in hares from Atlantic and Centre regions.Finally, one of the 22 patterns was present in three different hares: two from Southern plateau and one from Northeast region.
Nine different patterns were found in the nine positive hares (all Le. europaeus) from Atlantic region (Table 2).Five hares from Centre region (all Le. granatensis) presented four different patterns.Sixteen hares from Southern plateau (all Le. granatensis) presented 13 different patterns.RFPL patterns identified in one hare from Northeast region and one hare from Northern plateau were also present in other regions.
No similarities were found between hare RFLP patterns and those previously found in wild carnivores from continental Spain [5].

Discussion
This study shows that L. infantum is present in two of the three Spanish hare species and that specific 'hare strains' of L. infantum circulate in Spain.However, the low number of samples from Le. castroviejoi − an endangered species − prevented determining if they are exposed to L. infantum.We selected testing the presence of L. infantum DNA in spleen samples instead of detecting antibody presence because we aimed to measure the occurrence of effective infections rather than detecting exposure.The effect of possible local temporal trends in L. infantum prevalence caused by changes in vector and host population dynamics could have had an effect on prevalence rates found in this study.
In spite of sample size limitations in this study, overall L. infantum DNA prevalence in Le. granatensis and Le.europaeus seems to be above the 30% (lower limits of estimated confidence intervals).The finding of positive hares in each of the six geographic regions surveyed suggests that L. infantum is widely spread in Spanish hare populations.These findings together with the recent evidence of the ability of Ph. perniciosus to get infected through feeding on Le. granatensis [7], evidences the reservoir potential of hares for L. infantum.Infection by L. infantum in Iberian hares seems not to cause clinical disease [7] and thus L. infantum may not be of direct concern for hare conservation purposes.However, since L. infantum is an important pathogen for humans and other mammals, animal health and conservation authorities in Spain should be aware of the indirect consequences on conservation and wildlife management caused by their potential role as L. infantum reservoir.

Figure
FigureLocation of hare sampling points by geographic region,Spain, 2004Spain,  -2010 Le. europaeus and two Le.castroviejoi ; (ii) Northern plateau region: five Le.granatensis; (iii) the Northeastern region: five Le.granatensis and two Le.europaeus; (iv) Centre region: 10 Le. granatensis; (v) Southern plateau region: 54 Le. granatensis; and (vi) Guadalquivir river valley region: two Le.granatensis.Overall, 41 out of 94 Spanish hares (43.6%; 95% CI: 33.6 to 53.6) were positive for the presence of L. infantum DNA.At least one positive hare was found in each of the six geographic regions surveyed (Table1).Both Iberian and European hares tested positive for presence of L. infantum DNA by PCR.

Table 1
Prevalence of Leishmania infantum infection in hares by geographic region and species,Spain, 2004Spain,  -2010 (n=94)    (n=94) [14,15]erian and European hares are widely distributed in Spain, and their impact in the epidemiology of Mediterranean leishmaniasis deserves further research.Our findings suggest that hares have the potential to modulate the ecology of L. infantum in the near future as already evidenced in the outskirts of Madrid.The European hare inhabits vast areas of central Europe[13], constituting a potential European reservoir for L. infantum.This should be carefully considered when modeling the spread of L. infantum in those areas.Translocation of European and Iberian hares for hunting purposes between European countries is frequent e.g. between Spain and France[14,15].