Risk Assessment of the Role of the Ecotones in the Transmission of Zoonotic Cutaneous Leishmaniasis in Central Tunisia.

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 Impact of Illegal Waste Sites on the Transmission of Zoonotic Cutaneous Leishmaniasis in Central Tunisia.

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.

Pyrethroid insecticide lambda-cyhalothrin and its metabolites induce liver injury through the activation of oxidative stress and proinflammatory gene expression in rats following acute and subchronic exposure.

Lambda-cyhalothrin (LTC) [α-cyano-3-phenoxybenzyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclo-propanecarboxylate] is a synthetic type II pyrethroid insecticide commonly used in residential and agricultural areas. The potential hepatotoxicity of pyrethroids remains unclear and could easily be assessed by measuring common clinical indicators of liver disease. To understand more about the potential risks for humans associated with LTC exposure, male adult rats were orally exposed to 6.2 and 31.1 mg/kg bw of LTC for 7, 30, 45, and 60 days. Histopathological changes and alterations of main parameters related to oxidative stress and inflammatory responses in the liver were evaluated. Further, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] in the liver tissues were identified and quantified by ultra-high-performance liquid chromatography coupled to quadripole time-of-flight mass spectrometry (UHPLC-MS-Q-ToF). Results revealed that LTC exposure significantly increased markers of hepatic oxidative stress in a time-dependent and dose-dependent manner, and this was associated with an accumulation of CFMP and 3-PBA in the liver tissues. In addition, the levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6 and IL-1ß) gene expressions were significantly increased in the liver of exposed rats compared to controls. Correlation analyses revealed that CFMP and 3-PBA metabolite levels in the liver tissues were significantly correlated with the indexes of oxidative stress, redox status, and inflammatory markers in rats exposed to lambda-cyhalothin. Overall, this study provided novel evidence that hepatic damage is likely due to increased oxidative stress and inflammation under the condition of acute and subchronic exposure to lambda-cyhalothrin and that LTC metabolites (CFMP and 3-PBA) could be used as potential biomarker in human biomonitoring studies.

Changes of Sand Fly Populations and Leishmania infantum Infection Rates in an Irrigated Village Located in Arid Central Tunisia.

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.

Irrigation in the arid regions of Tunisia impacts the abundance and apparent density of sand fly vectors of Leishmania infantum.

The distribution expansion of important human visceral leishmaniasis (HVL) and sporadic cutaneous leishmaniasis (SCL) vector species, Phlebotomus perfiliewi and P. perniciosus, throughout central Tunisia is a major public health concern. This study was designed to investigate if the expansion of irrigation influences the abundance of sand fly species potentially involved in the transmission of HVL and SCL located in arid bioclimatic regions. Geographic and remote sensing approaches were used to predict the density of visceral leishmaniasis vectors in Tunisia. Entomological investigations were performed in the governorate of Sidi Bouzid, located in the arid bioclimatic region of Tunisia. In 2012, sand flies were collected by CDC light traps located at nine irrigated and nine non-irrigated sites to determine species abundance. Eight species in two genera were collected. Among sand flies of the subgenus Larroussius, P. perfiliewi was the only species collected significantly more in irrigated areas. Trap data were then used to develop Poisson regression models to map the apparent density of important sand fly species as a function of different environmental covariates including climate and vegetation density. The density of P. perfiliewi is predicted to be moderately high in the arid regions. These results highlight that the abundance of P. perfiliewi is associated with the development of irrigated areas and suggests that the expansion of this species will continue to more arid areas of the country as irrigation sites continue to be developed in the region. The continued increase in irrigated areas in the Middle East and North Africa region deserves attention, as it is associated with the spread of L. infantum vector P. perfiliewi. Integrated vector management strategies targeting irrigation structures to reduce sand fly vector populations should be evaluated in light of these findings.

Colonization of Phlebotomus papatasi changes the effect of pre-immunization with saliva from lack of protection towards protection against experimental challenge with Leishmania major and saliva.

BACKGROUND: Sand fly saliva has been postulated as a potential vaccine or as a vaccine component within multi component vaccine against leishmaniasis. It is important to note that these studies were performed using long-term colonized Phlebotomus papatasi. The effect of sand flies colonization on the outcome of Leishmania infection is reported. RESULTS: While pre-immunization of mice with salivary gland homogenate (SGH) of long-term colonized (F5 and beyond) female Phlebotomus papatasi induced protection against Leishmania major co-inoculated with the same type of SGH, pre-immunization of mice with SGH of recently colonized (F2 and F3) female P. papatasi did not confer protection against L. major co-inoculated with the same type of SGH. Our data showed for the first time that a shift from lack of protection to protection occurs at the fourth generation (F4) during the colonization process of P. papatasi. CONCLUSION: For the development of a sand fly saliva-based vaccine, inferences based on long-term colonized populations of sand flies should be treated with caution as colonization of P. papatasi appears to modulate the outcome of L. major infection from lack of protection to protection.

Lack of protection of pre-immunization with saliva of long-term colonized Phlebotomus papatasi against experimental challenge with Leishmania major and saliva of wild-caught P. papatasi.

Immunity to saliva of Phlebotomus papatasi protects against Leishmania major infection as determined by co-inoculation of parasites with salivary gland homogenates (SGHs) of this vector. These results were obtained with long-term colonized female P. papatasi. We investigated the effect of pre-immunization with SGH of long-term colonized P. papatasi against L. major infection co-inoculated with SGH of wild-caught P. papatasi. Our results showed that pre-exposure to SGH of long-term, colonized P. papatasi do not confer protection against infection with L. major co-inoculated with SGH of wild-caught P. papatasi. These preliminary results strongly suggest that the effectiveness of a vector saliva-based vaccine derived from colonized sand fly populations may be affected by inconsistent immune response after natural exposure.

Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia.

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2%), Phlebotomus longicuspis (30.1%), Phlebotomus papatasi (12.0%), Phlebotomus perfiliewi (4.6%), Phlebotomus langeroni (0.4%) and Sergentomyia minuta (0.5%). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

Zooprophylaxis: impact of breeding rabbits around houses on reducing the indoor abundance of Phlebotomus papatasi.

Zooprophylaxis is the use of animals to deviate vectors from humans. The indoor abundance of Phlebotomus papatasi in houses with rabbit holes in the peridomestic areas are significantly lower than the indoor abundance in houses without rabbit holes in their peridomestic areas. Introduction of rabbits in artificial underground holes in peridomestic areas reduced significantly the indoor abundance of P. papatasi. Cleaning rabbit holes in peridomestic area by removing all rabbit feces induced a significant increase in the abundance of P. papatasi inside bedrooms. The ecologic niche made around houses in endemic areas by creating active rabbit holes is a major source of attractiveness of P. papatasi, and therefore it may deviate the vector from humans to rabbits. Although rabbit holes are breeding sites for P. papatasi, rabbits are not competent reservoirs for Leishmania major. Our overall findings strongly suggest that zooprophylaxis could be effective in reducing the indoor abundance of P. papatasi and subsequently may be used to control the transmission L. major in rural areas.