The current spread of zoonotic visceral leishmaniasis (ZVL) throughout arid areas of Central Tunisia is a major public health concern. The main objective of this study is to investigate whether the development of irrigation in arid bio-geographical areas in Central Tunisia have led to the establishment of a stable cycle involving sand flies of the subgenus Larroussius and Leishmania infantum, and subsequently to the emergence of ZVL. Sand flies were collected from the village of Saddaguia, a highly irrigated zone located within an arid bio-geographical area of Central Tunisia by using modified Centers for Diseases Control (CDC) light traps. Morphological keys were used to identify sand flies. Collected sand flies were pooled with up to 30 specimens per pool according to date and tested by nested Polymerase Chain Reaction (PCR) DNA sequencing from positive pools was used to identify Leishmania spp. A total of 4915 sand flies (2422 females and 2493 males) were collected from Saddaguia in September and in October 2014. Morphological identification confirmed sand flies of the subgenus Larroussius to be predominant. PCR analysis followed by DNA sequencing indicated that 15 pools were infected with L. infantum yielding an overall infection rate of 0.6%. The majority of the infected pools were of sand fly species belonging to subgenus Larroussius. Intense irrigation applied to the arid bio-geographical areas in Central Tunisia is at the origin of the development of an environment capable of sustaining important populations of sand flies of the subgenus Larroussius. This has led to the establishment of stable transmission cycles of L. infantum and subsequently to the emergence of ZVL.

Illegal waste disposal represents a risk health factor for vector-borne diseases by providing shelter for rodents and their ectoparasites. The presence of the Phlebotomus papatasi vector of Leishmania major, an etiologic agent of zoonotic cutaneous leishmaniasis (ZCL), was assessed at illegal waste sites located at the vicinity of villages in endemic areas of Central Tunisia. The study was performed over a two-year period over three nights from July to September 2017, and over three nights in September 2018. Household waste is deposited illegally forming dumpsites at the vicinity of each village and contains several rodent burrows of Psammomys obesus, the main reservoir host of L. major. Sandflies were collected from rodent burrows in the natural environment and in dumpsites using sticky traps and were identified at species level. Female sandflies were tested for the presence of L. major by PCR. Our entomological survey showed that Phlebotomus papatasi is the most abundant sandfly species associated with rodent burrows in these waste sites. The densities of P. papatasi in dumpsites are significantly higher compared to the natural environment. The minimum infection rate of P. papatasi with L. major in these illegal waste sites is not significantly different compared to the natural environment. Considering the short flight range of P. papatasi, increases in its densities, associated with burrows of P. obesus in illegal waste sites located at the edge of villages, expands the overlap of infected ZCL vectors with communities. Thus, illegal waste sites pose a high risk of spreading ZCL to neighboring home ranges. Waste management is an environmentally friendly method of controlling sandfly populations and should be included in an integrated management program for controlling ZCL in endemic countries.

Zoonotic cutaneous leishmaniasis (ZCL), endemic in Central and Southern Tunisia, is caused by Leishmania major (Kinetoplastida: Trypanosomatidae), which is transmitted by the sand fly Phlebotomus papatasi. In Tunisia, the fat sand rat Psammomys obesus and the desert jird Meriones shawi are the principal reservoir hosts of L. major. The presence of the P. papatasi vector of the L. major etiologic agent of ZCL was assessed in the vicinity of villages in endemic areas of Central Tunisia. The study was performed from September through October 2019, a period corresponding to the main peak of activity of P. papatasi. Sand flies were collected from rodent burrows located at the ecotone level, which is the transition zone between the natural environment and human settlement. Sand flies were identified to species level and tested for the presence of L. major by PCR. Our entomological survey showed that P. papatasi is the most abundant sand fly species associated with rodent burrows, and this abundance is even higher in ecotones primarily occupied by P. obesus in comparison to ecotones occupied by M. shawi. Infections with Leishmania major were detected only in P. papatasi, with an overall minimum infection rate (MIR) of 2.64%. No significant difference was observed between the MIRs in ecotones of P. obesus and of M. shawi. Incidence of ZCL in the studied areas ranged from 200 to 700 cases per 100,000 inhabitants, with a mean incidence of 385.41 per 100,000. Higher ZCL incidence was identified in ecotones of M. shawi compared to ecotones of P. obesus. ZCL cases are positively correlated with the MIRs. Considering the short flight range of P. papatasi, increases in its densities associated with burrows of P. obesus or M. shawi at the ecotone level expand the overlap of infected vectors with communities and subsequently increase ZCL incidence. Therefore, control measures should target P. papatasi populations at the ecotones.

West Nile virus (WNV) is a mosquito-borne pathogen of the Flaviviridae family that poses recurring public health threats in Tunisia, where Culex pipiens is recognized as the primary vector. Identification of circulating strains in different mosquito species is essential for targeted prevention and control. Between November 2021 and October 2022, mosquitoes were collected at four high-risk sites, and human samples were obtained through the national meningitis surveillance program. Human serum, cerebrospinal fluid (CSF), and urine samples were tested for WNV-specific IgM and IgG antibodies using ELISA, and molecular diagnosis was performed using Real-time RT-PCR (RRT-PCR). Positive samples underwent sequencing for phylogenetic characterization. Serological investigation on human serum revealed the presence of IgM and/or IgG antibodies reactive to WNV antigens, which may indicate exposure to WNV or related flaviviruses. RNA of WNV was detected in 21 mosquito pools (10.19%) belonging to Culex pipiens, Cx. perexiguus, Aedes caspius, and Ae. detritus, as well as in three human cases. Phylogenetic analysis of positive human and mosquito samples showed that all detected WNV strains belonged to sublineage 1a. The concurrent detection of WNV in vectors and humans confirms active circulation in Tunisia and underscores the role of Culex spp. Mosquitoes in transmission. Sustained multidisciplinary surveillance integrating entomological and clinical data is critical for early detection, guiding control measures, and preventing future outbreaks in humans and animals.