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.

The aac(6′)-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6′)-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6′)-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6′)-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6′)-Ib gene, which was identified as the aac(6′)-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6′)-Ib and aac(6′)-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments.

Zoonotic cutaneous leishmaniasis (ZCL) and chronic cutaneous leishmaniasis (CCL) are known to overlap in Central Tunisia. Sandflies were collected using sticky traps and CDC light-traps set in rodent burrows at the ecotones surrounding the village, in houses, and in animal shelters during July–October 2017, 2018, and 2019. A total of 17,175 sandflies were collected during the three sandfly seasons and identified morphologically to species level. Of a total of 18 sandfly species reported in Tunisia, 16 were identified in this mixed focus of ZCL and CCL. Except for the rocky mountainous areas, Phlebotomus papatasi was the most abundant sandfly species in all biotopes. In the mountainous areas, Phlebotomus sergenti is the most abundant sandfly species belonging to the genus Phlebotomus. Female sandflies were tested for the presence of Leishmania species by PCR. The overall infection prevalence of sandflies with Leishmania major and Leishmania tropica was 0.42% and 0.065%, respectively. The sequencing of PCR-amplified ITS1 products showed that L. major is the predominant species in all biotopes and transmitted mostly by P. papaptasi followed by Phlebotomus longicuspis and Sergentomyia species. Leishmania tropica was detected in Phlebotomus sergenti and in Phlebotomus longicuspis collected in bedrooms and in the ecotone of rocky mountainous areas. Our results provided strong evidence that the proximity of human settlements to biotopes of rodent reservoir hosts of L. major and of L. tropica resulted into the cocirculation of both Leishmania species leading to a mixed focus of ZCL and CCL. The epidemiology of leishmaniases in North Africa is highly complex by the high diversity of sandfly vectors and their associated Leishmania species, leading to a mixed form of cutaneous leishmaniasis. It is of major epidemiological importance to point to the risk of spillover from rural to urban areas leading to the anthroponization of cutaneous leishmaniasis. Therefore, efficient control to reduce the indoor abundance of sandfly vectors in order to reduce the incidence of leishmaniases is urgently needed.

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.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum.

Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage identity. The recognized species presents the most substantial homology with the analyzed sequence. The results of the molecular identification showed complete agreement with the morphological identifications, and Phlebotomus perfiliewi is genetically variable.