Several studies have shown that pre-immunization of mice with needle-injected saliva or pre-exposure to uninfected bites of Phlebotomus papatasi provided protection against infection with Leishmania major , the etiologic agent of zoonotic cutaneous leishmaniasis (ZCL).1-3 These studies were performed by using long-term laboratory colonized sand flies mainly because of the difficulty of working with wild-caught flies. A puzzling fact is that people in leishmaniasis-endemic areas succumb of ZCL despite the high frequency of uninfected bites compared with infected ones. 4 We showed in a previous study that pre-immunization of mice with salivary gland homogenate (SGH) of wild-caught P. papatasi does not confer protection against L. major co-inoculated with the same type of SGH compared with a significant protection obtained when pre-immunization and challenge were performed with SGH of long-term colonized flies. 5 It was reported that colonized and wild-caught Lutzomyia longipalpis differ in the composition and the amount of salivary proteins and these differences may account for the lower effect observed on the modulation of experimental Leishmania infection by wild-caught SGH. 6,7 Thus, these studies provide good evidence that the outcome of Leishmania infection differs significantly between colonized and wild-caught salivary gland proteins. Our aim in this study was to assess the protective effect of pre-immunization with SGH of long-term colonized P. papatasi on experimental L. major challenge co-inoculated with SGH of wild-caught P. papatasi .Phlebotomus papatasi (Tunisian strain that originated in the Governorate of Sidi Bouzid) have been reared at the Vector Ecology Laboratory of the Institut Pasteur de Tunis since 2003. 8 This colony is maintained without being supplemented periodically with wild-caught P. papatasi . Generation F 39 was used in this study. Wild sand flies were collected using CDC light traps from animal shelters located in the village of Felta (Governorate of Sidi Bouzid), a focus highly endemic for ZCL.9 Preparation of SGH from laboratory-reared and wild-caught P. papatasi was performed as described. 5 A highly virulent strain of L. major , MHOM/TN/95/GLC94, isolated from a patient in Tunisia was used in this study. 5 We used female BALB/c mice (6-8 weeks of age) bred in the animal facility of the Institut Pasteur de Tunis.Mice were immunized intradermally in the right ear with the equivalent of two pairs of salivary glands in 10 µL of phosphate-buffered saline (PBS). Two groups of 10 mice each were pre-immunized with SGH of long-term colonized female P. papatasi (F 39 ), once a week for two weeks. In the fourth week, the first [CSGH (F 39 )-L.m+CSGH (F 39 )] and the second [CSGH (F 39 )-L.m+WSGH] groups were challenged with 10 6 stationary phase L. major promastigotes in 50 µL of PBS co-inoculated subcutaneously in the right hind footpads with SGH of long-term, colonized and SGH of wild-caught, female P. papatasi , respectively. Three control groups of 10 mice each were used in this stud...