Dogs are the major reservoir of Leishmania infantum, the causative agent of human and canine visceral leishmaniasis in the Mediterranean basin. In Morocco, canine leishmaniasis (CanL) is usually believed to be widespread mainly, if not only, in the northern regions and few data are available about the situation in southern parts of the country. Here, we report the results of a preliminary, clinical, and serological study carried out in 2004–2007, in four provinces of southern Morocco. Serological analyses were processed using two different Elisa techniques, a homemade Elisa test and IDVET commercial kit, and confirmed by two different western blot (WB) tests, homemade and LDBIO commercial kits. We highlighted the presence of CanL infection in southern regions, known until then as free of the disease: 19.8% (48/243) of examined dogs displayed clinical signs compatible with CanL and the seroprevalence was particularly high, respectively, 81.8% and 87.8% by Elisa and western blot tests. Our current developed and validated homemade (Elisa and WB) tools will be cost-effective and useful for next large-scale epidemiological studies on Moroccan leishmaniasis animal reservoir. 1. Introduction Canine leishmaniasis (CanL) is a zoonotic disease caused by Leishmania infantum, a trypanosomatid protozoan, transmitted through infected sand fly (Diptera: Psychodidae) bites. CanL is endemic in the Mediterranean basin, where seroprevalence ranges between ten and 37% [1, 2] and symptoms in dogs occur in various combinations [3]. Both symptomatic and asymptomatic dogs are sources of the parasite and phlebotomine sand flies play an active role in the transmission of Leishmania to humans [4]. In northern Morocco, natural CanL was first reported in the region of Tangier [5]. Further subsequent cases have been reported in Taounate, Al Hoceima, Chefchaouen, and Ouezzane provinces [6], in Sefrou and Zouagha Moulay Yacoub [7], in Khemisset province [8], and in the province of Nador [9]. Leishmania infantum zymodeme MON-1 is the unique causative agent of CanL in the northern slopes of the Rif mountains [6] and L. infantum MON-24 was identified in dogs from the Pre-Rif area [10]. In the Rif region specifically, dog seroprevalence ranges between ten and 41% [9] and L. infantum strains are highly virulent [8, 9]. In northern slope of High Atlas Moroccan mountains, Dereure et al. [11] reported the presence of dogs infected with L. tropica (MON-102 and MON-113). Additionally, Guessous-Idrissi et al. [12] reported a case of dog with visceral leishmaniasis caused by L. tropica in
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