Phlebotomine sand fly species distribution in Croatia and their implication in Leishmania transmission

Leishmaniasis was reported as endemic in Croatia since 1930, but it is since early 2000s that human (both visceral and cutaneous) and canine leishmaniasis (CanL) foci have been well documented from coastal and insular territories of central and southern Dalmatia (Živicnjak et al, Vet. Parasitol 2005, 131 ; 35-43 ; Sisko-Kraljevic et al, Euro Surveill 2013, 8 (29). pii=20533.201). Leishmania isolates from infected dogs were identified as belonging to Leishmania infantum zymodeme (Z) MON-1 (Gouzelou et al, ParasitVectors 2013, 6:342), the widespred agent of Mediterranean zoonotic visceral leishmaniasis. We report on a 2005–2011 phlebotomine survey performed to confirm species composition and seasonality in three central-southern counties of Dalmatia previously inquired (Sibenik-Knin, Split-Dalmatia and Dubrovnik–Neretva) (Bosnic et al, Acta Trop 2006 ; 99:42-9) ; to investigate on the current species distribution in the westernmost Istria county, including the biggest part of the Istrian peninsula, for which available information dates back 60 years (Simic and Živkovic, Arch Inst Pasteur Alger 1956 ; 383-5) ; and to search for natural Leishmania infections. Further, we performed Leishmania sp. Identification from CanL cases originating from the same counties and diagnose during 2000-2005. Sand flies collections, carried out in the frame of bluetongue disease surveillance, used blacklight suction traps employed for Culicoides monitoring. Fifteen localities in four Croatian counties were investigated. Specimens were preserved in ethanol pending morphological identification and DNA extraction. Canine Leishmania strains were identified by Multi Locus Enzyme Electophoresis. Sand flies were trapped from late May through early December. One thousand specimens were collected and seven species identified. Among Phlebotomus sand flies, P. perfiliewi was the most abundant species (54.6%), followed by P. neglectus (28.2%), P. tobbi (8.9%), P. perniciosus (5.4%), P. papatasi (0.6%) and P. mascittii (0, 1%). Sergentomyia minuta accounted for 2.2%. A difference in prevalence distribution was detected, being P. perniciosus prevalent in Istrian peninsula (49/56, 87.5%) ; P. perfiliewi in central counties (542/830, 65.3%) and P. neglectus in the southernmost county (79/114, 69.3%). A subset of 369 Larroussious females (76.2% P. perfilewi) organized in pools (1-27 specimens/pool) according to species, site and date of collection, was analysed for Leishmania DNA presence. All pools were found negative. A total of 18 canine Leishmania strains were isolated and identified as belonging to 3 zymodems of L. infantum: 15 strains belonged to ZMON-1, the commonest L. infantum zymodeme, 2 to ZMON-27 and 1 to ZMON-34, the later two being uncommon vicerotropic and dermotropic found in other Mediterranean countries. The long lasting sand flies season (May-December) and the high P. perniciosus prevalence, the main L. infantum vector, in the westernmost Istria county may represent a warning signal in re-emerging leishmaniasis. L. infantum was confirmed the CanL agent in Croatia. This study was partially funded by the FP7-UE EDENext collaborative project, Contract Number: 261504.