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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

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.

In the present study, we evaluated the effects of antibodies anti-sandfly saliva on the fecundity of Phlebotomus papatasi, vector of zoonotic cutaneous leishmaniasis in the Old World. Rabbits were repeatedly exposed to sandfly bites. Immune sera showed increased levels of anti-sandfly saliva antibody compared to the pre-exposition period. The analysis of biological parameters revealed no decline on the feeding success of females P. papatasi fed on rabbits repeatedly exposed to sandfly bites. Our results showed that anti-sandfly saliva antibodies of rabbits are not detrimental to the fitness of females P. papatasi. Thus, rabbits did not acquire resistance to sandflies following repeated exposures, and that contribute in maintaining a high density of P. papatasi. To control sandfly infestations and Leishmania transmission, more studies are needed for a better understanding of the mechanisms governing the resistance of hosts to bites of sandflies.

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.

Background The sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs. Methodology/Principal findings We carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions. Conclusions The results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa., Author summary Zoonotic visceral leishmaniasis is a neglected tropical disease caused by the parasite Leishmania infantum. In the Western Mediterranean basin, Phlebotomus perniciosus and Phlebotomus perfiliewi are the main vectors of L. infantum, and dogs are the main reservoir host of the parasite. In Northern Africa, ZVL affect mostly children less than 5 years. Understanding the chemical ecology governing the relationships between the vector, the parasite and the reservoir host is of major epidemiological importance. Previous studies based on rodent models have shown that the infection with L. infantum enhanced the attractiveness towards Lutzomyia longipaplis, the main vector of ZVL in the Americas. In this study, we reported for the first time that infected dogs are highly attractive to both male and female P. perniciosus under laboratory and filed conditions compared to uninfected dogs. Similar patterns were observed with wild populations of P. perfiliewi. Our results provided strong evidence that the parasite manipulate the reservoir hosts to enhance its transmission success by the vector.

In the Mediterranean basin, sand flies are vectors of Leishmania parasites and phleboviruses affecting humans and animals. In this study, we aimed to investigate phlebovirus and Leishmania parasites circulating in a focus of zoonotic visceral leishmaniasis (ZVL) located in a highly irrigated area within the arid Central Tunisia, known mainly to be endemic for zoonotic cutaenous leishmaniasis (ZCL) caused Leishmania major and transmitted by Phlebotomus papatasi. Sand flies were collected using CDC light traps in the village of Saddaguia, an emergent focus of ZVL located in Central Tunisia during September-October 2014, 2015, and 2016. Pools of live female sand flies were screened for phleboviruses and Leishmania by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. Dead sand flies were identified morphologically to species level. Sand flies of the subgenus Larroussius mainly Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis were predominant in this ZVL focus compared to P. papatasi. A total of 1932, 1740, and 444 sand flies were tested in 2014, 2015 and 2016, respectively. Pathogen screening performed on 4116 sand flies distributed in 148 pools revealed the presence of Leishmania infantum and Toscana virus. The minimum infection rates of sand flies with TOSV in 2014, 2015, and 2016 were 0.05%, 011%, and 0.22%, respectively. The minimum infection rates of sand flies with L. infantum in 2014, 2015, and 2016 were 0.25%, 012%, and 0.79%, respectively. No L. major was detected during the 3-years investigation in this ZVL focus. Our results showed clearly the endemic co-circulation of TOSV and L. infantum in this emergent ZVL focus. However, no co-infection of TOSV and L. infantum was detected in any of the sand fly pools investigated during the three years period. TOSV was isolated from positive pools in 2015. Phylogenetic analysis showed that the Tunisian strains of TOSV belonged to the sublineage A. Based on the present findings, our results provided strong evidence that TOSV and L. infantum are transmitted by the same predominant sand fly species of the subgenus Larroussius, and subsequently, humans and dogs could be co-infected through co-infected or successive infected bites. Our results showed clearly that the development of irrigation in arid areas contributed significantly to the establishment of stable transmission cycles of L. infantum and TOSV and subsequently to the emergence of a ZVL focus within this arid bio-geographical area characterized by the presence of multiple foci of ZCL located outside the study site. Thus, more studies are needed to better understand the impact of RNA viruses shared by vectors and reservoir hosts of L. infantum on the development of zoonotic visceral leishmaniasis.

Sand fly colonies are of major importance for experimental studies on biology, behavior, vector competence, relationship with Leishmania parasites, and vector control. This chapter is intended to provide methods and techniques used to initiate, establish, and maintain sand fly colonies. Details on collecting sand flies for colonization, colony initiation, maintenance, and experimental infection of Phlebotomus spp. with Leishmania spp. are reported.

Sand fly colonies are of major importance for experimental studies on biology, behavior, vector competence, relationship with Leishmania parasites, and vector control. This chapter is intended to provide methods and techniques used to initiate, establish, and maintain sand fly colonies. Details on collecting sand flies for colonization, colony initiation, maintenance, and experimental infection of Phlebotomus spp. with Leishmania spp. are reported.

International audience; An entomological investigation performed in 2013 covering different bio-geographical areas varying from humid in the north to the arid in the center showed that sand flies of the subgenus Larroussius including Phlebotomus perniciosus, Phlebotomus perfiliewi, and Phlebotomus longicuspis are abundant and widely distributed in Tunisia A total of 3992 collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection date were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction and sequencing Of a total of 135 pools, 23 were positive, yielding and minimum infection rate of 0.6%. Phylogenetic analysis performed using partial amino acid sequence in the polymerase gene showed that all these phleboviruses were grouped in one cluster clearly distinct from but closely related to Massilia virus and Granada virus This putative novel virus, tentatively called Saddaguia virus (SADV), is widely distributed in Tunisia Together with Toscana, Punique, and Utique viruses, SADV is the fourth recognized phlebovirus to be transmitted by sand flies in Tunisia The medical and public health interest of SADV remains to be investigated.

International audience; Dogs are the main reservoir host of Leishmania infantum, etiologic agent of human visceral leishmaniasis (HVL) and canine visceral leishmaniasis (CVL). Transmission of L. infantum to humans and dogs is mainly through the bite of infected sand flies. In the Western Mediterranean basin, Phlebotomus perniciosus is the main vector of L. infantum. However, occasional vertical transmission of L. infantum has been reported. This study investigated L. infantum vertical transmission in offspring of experimentally infected dogs. Among 14 surviving puppies from three female beagle dogs that developed CVL following an experimental infection with L. infantum, one was tested positive by indirect immunofluorescence antibody test, by PCR and by xenodiagnosis with a high parasite burden in the spleen at 14 months old. None of the remaining puppies were tested positive for L. infantum. These findings strongly suggest that infected puppies following vertical transmission can sustain infection and contribute in infecting sand flies with L. infantum. Any strategy for controlling CVL should take into consideration the vertical transmission of L. infantum.

The sand fly Phlebotomus papatasi is the main vector of Leishmania major , etiologic agent of zoonotic cutaneous leishmaniasis (ZCL), which is endemic in North Africa, the Middle East, and Asia. In North Africa, Meriones shawi is one of the two main reservoir hosts of L. major . P. papatasi populations are maintained in borrowing rodents such as M. shawi . Three fipronil-treated rodent baits were evaluated for systemic and feed through insecticidal activity against P. papatasi feeding on M. shawi . Through blood feeding bioassays, mortality rates of females P. papatasi increased with the concentration of fipronil in the rodent bait varying from 0.001% to 0.005%. In the laboratory, more than 90.0% of P. papatasi were killed within 48 h after blood feeding on the desert's jirds, M. shawi , treated up to 29 days prior with a single application of fipronil at a concentration of 0.001%, 0.0025% and 0.005%. Through larval bioassays, mortality rates of larvae that have fed on faeces of treated bait for M. shawi increase with the concentrations of fipronil. Faeces of orally-treated Meriones were significantly toxic to larvae for 5 weeks with a concentration of 0.005%. In the field, application of treated bait resulted in 80.0% reduction in the populations of P. papatasi up to 6 weeks after a single application of fipronil at a concentration of 0.005%. This is the first study to demonstrate field efficacy of fipronil-treated rodent baits for P. papatasi control and the first study to evaluate this approach in M. shawi , a principal ZCL reservoir host. These results suggest that fipronil-treated rodent baits can be used to effectively reduce the populations of P. papatasi associated with M. shawi in ZCL endemic areas.

The objective of this study was to evaluate the systemic insecticidal activity of an imidacloprid-treated rodent oral bait, against Phlebotomus papatasi Scopoli, 1786 vector of Leishmania major Yakimoff & Schokhor, 1914 (Kinetoplastida: Trypanosomatidae), etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). Shaw's gerbil Meriones shawi Duvernoy, 1842 (Rodentia, Gerbillidae) were treated with imidacloprid-treated bait (0.05%). In the laboratory, effects on adult and larval of Phlebotomus papatasi fed on treated M. shawi and on its faeces were studied. The effectiveness of this approach was tested under field conditions. In the laboratory, 100% of P. papatasi were killed within 24 hours after blood feeding on Meriones shawi treated up to four weeks prior with a single application of imidacloprid (0.05%) bait. In addition, none of the P. papatasi larvae that consumed feces from M. shawi treated with the imidacloprid bait survived to pupation. In the field, application of the imidacloprid bait resulted in a 90% reduction in the P. papatasi population up to four weeks prior with a single application of imidacloprid (0.05%) bait. This is the first study to demonstrate field efficacy of insecticide-treated rodent baits for P. papatasi control and the first study to evaluate this approach in M. shawi, a principal ZCL reservoir host. These results suggest that insecticide-treated rodent baits could be used to effectively reduce the populations of P. papatasi associated with M. shawi in ZCL endemic areas.

Background The Leishmania developmental life cycle within its sand fly vector occurs exclusively in the lumen of the insect’s digestive tract in the presence of symbiotic bacteria. The composition of the gut microbiota and the factors that influence its composition are currently poorly understood. A set of factors, including the host and its environment, may influence this composition. It has been demonstrated that the insect gut microbiota influences the development of several human pathogens, such as Plasmodium falciparum. For sand flies and Leishmania, understanding the interactions between the parasite and the microbial environment of the vector midgut can provide new tools to control Leishmania transmission. Methodology/Principal findings The midguts of female Phlebotomus perniciosus from laboratory colonies or from the field were collected during the months of July, September and October 2011 and dissected. The midguts were analyzed by culture-dependent and culture-independent methods. A total of 441 and 115 cultivable isolates were assigned to 30 and 11 phylotypes from field-collected and colonized P. perniciosus, respectively. Analysis of monthly variations in microbiota composition shows a species diversity decline in October, which is to the end of the Leishmania infantum transmission period. In parallel, a compilation and a meta-analysis of all available data concerning the microbiota of two Psychodidae genera, namely Phlebotomus and Lutzomyia, was performed and compared to P. perniciosus, data obtained herein. This integrated analysis did not reveal any substantial divergences between Old and New world sand flies with regards to the midgut bacterial phyla and genera diversity. But clearly, most bacterial species (>76%) are sparsely distributed between Phlebotominae species. Conclusion/Significance Our results pinpoint the need for a more exhaustive understanding of the bacterial richness and abundance at the species level in Phlebotominae sand flies in order to capture the role of midgut bacteria during Leishmania development and transmission. The occurrence of Bacillus subtilis in P. perniciosus and at least two other sand fly species studied so far suggests that this bacterial species is a potential candidate for paratransgenic or biolological approaches for the control of sand fly populations in order to prevent Leishmania transmission., Author summary The use of conventional microbiological methods gave us the opportunity to investigate the richness of symbiotic bacteria that inhabit the gut of P. perniciosus during its main period of activity. Our results were subsequently analyzed in the framework of what has been done on sand flies microbiota in order to validate our results and to address the question of the definition of the core bacterial microbiota of sand flies. A meta-analysis on the respective gut microbiota of Old and New World sand flies shows that the majority of bacterial species is observed only in one host whereas less than 8% are shared by more than two hosts. Our results pinpoint the need for a more exhaustive understanding of the microbiota composition and dynamic in phlebotominae, with the aim to implement new biological approaches for the control of sand fly populations in order to prevent Leishmania transmission.

La capacite du phlebotome Phlebotomus papatasi a transmettre Leishmania major, agent etiologique de la leishmaniose cutanee zoonotique (LCZ) a Meriones shawi, reservoir naturel de ce parasite, est etudiee dans les conditions de laboratoire. Les phlebotomes sont infectes apres un repas sanguin sur un M. shawi lui-meme prealablement infecte par injection sous-cutanee de L. major. P. papatasi, ainsi infecte, transmet L. major a un autre M. shawi lors d’un deuxieme repas sanguin. Deux mois apres, le deuxieme merion developpe des lesions leishmaniennes au niveau des deux oreilles sur lesquelles s’infectent de nouveaux phlebotomes. L’infectivite de M. shawi pour P. papatasi dure cinq mois, autant que la saison hivernale au Maghreb. Nous obtenons ainsi le cycle complet de la transmission de L. major de merion a merion par P. papatasi. Nos resultats montrent que les rongeurs reservoirs de parasites survivant a la periode hivernale sont la source d’infection leishmanienne la plus probable pour les phlebotomes emergents lors de la nouvelle saison estivale. Ce sont donc ces rongeurs qui joueraient le role determinant dans la persistance et la transmission de L. major entre deux saisons de transmission.

Phlebotomus papatasi (Diptera: Psychodidae) is the Old World sand fly vector of zoonotic cutaneous leishmaniasis caused by Leishmania major (Trypanosomatidae: Kinetoplastida), a debilitating and disfiguring protist parasitic disease prevalent throughout southern Mediterranean countries, the Middle East, as well as southern and eastern European countries, where it is regarded as a serious public health problem. Little is known of the mating ecology of P. papatasi, and, in particular, the role (if any) of pheromones is not known. In this laboratory- and field-based study, we have shown that a male-produced sex pheromone exists in P. papatasi. Young female P. papatasi are attracted to the headspace volatiles of small groups of males, males and females together, but not females alone. Males were not attracted to males, females, or mixed groups of males and females in the laboratory. Larger groups of males or males and females together were repellent in the laboratory study. Field experiments showed that Centers for Disease Control (CDC) light traps baited with small groups of males and females together were attractive to females, but not males. CDC traps baited with large groups of males and females together caught significantly fewer females and males than the control traps; however, the proportion of females caught compared with males overall was much higher than with CDC traps baited with small numbers of males and females. These results suggest that females may be attracted in preference to males to the vicinity of the baited traps and are highly sensitive to the concentration of male pheromone. It also suggests that P. papatasi mating behavior is fundamentally different from that of Lutzomyia longipalpis, where large mating aggregations of males and females occur.

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.

Details on the productivity and developmental times of a colony of Phlebotomus papatasi Scopoli (Diptera: Psychodidae) over 14 generations are reported and compared with findings of previous studies. The average productivity (percentage of eggs laid that were reared to adults) over six generations at 26–27 and at 29–30°C was 44.08 and 59.53%, respectively. The maximum productivity was 69.5%. The average developmental time over six generations at 26–27 and at 29–30°C was 35 and 26 d, respectively. The minimum developmental time from egg to adults was 25 d. The Tunisian strain of P. papatasi can reproduce autogenously or anautogenously, depending on the availability of a suitable bloodmeal source.

International audience; Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection.

An entomological investigation was carried out in 2014 at two sites located in Central Tunisia, one irrigated and another non-irrigated situated in arid bio-geographical areas. Sand flies of the subgenus Larroussius namely Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis are the most abundant sand fly species in the irrigated site. However, in the non-irrigated site, Phlebotomus papatasi of the Phlebotomus genus is the most abundant species. A total of 3191 sand flies were collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection date, were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction in the polymerase gene and sequenced. Of a total of 117 pools, 4 were positive, yielding a minimum infection rate of sand flies with phleboviruses of 0.12%. Phylogenetic analysis performed using partial nucleotide and amino acid sequence in the polymerase gene showed that these phleboviruses belonged to four different clusters corresponding to Toscana virus (TOSV), Saddaguia virus (SADV), Sandfly Fever Sicilian Virus (SFSV) and Utique virus (UTIV). This study provides more evidence that the abundance of P. perfiliewi is associated with the development of irrigation in arid bio-geographical areas of Central Tunisia which may have led to the emergence of phleboviruses. We report the first detection of TOSV from sand flies collected from Central Tunisia.

En Tunisie, Phlebotomus (Larroussius) perfiliewi Parrot, 1939, est soupconne d’etre implique dans la transmission de la leishmaniose cutanee sporadique (LCS). La distribution geographique de cette espece de phlebotome etait limitee au nord du pays et plus precisement dans les zones biogeographiques humides, subhumides, et semiarides. De nos jours, nous assistons a une extension recente dans la distribution geographique de P. perfiliewi et par voie de consequence de la LCS vers les zones biogeographiques arides qui sont situees au centre du pays. Cette extension est liee au developpement des systemes d’irrigations dans les zones arides, ce qui a engendre l’etablissement de nouvelles niches ecologiques favorables au developpement de P. perfiliewi.

International audience; 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.

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.

To the Editor: Toscana virus (TOSV; genus Phlebovirus, family Bunyaviridae) is transmitted by sandflies, mostly the species Phlebotomus perniciosus and P. perfiliewi (1). Initially discovered in central Italy, TOSV was recently identified in other European countries (i.e., Portugal, Spain, France, Croatia, and Turkey) (2). TOSV is a primary cause of aseptic meningitis during warm months (2). A seroprevalence study suggested that TOSV is present in Tunisia and may cause neuroinvasive infections, but definitive evidence of TOSV circulation has not been possible because it is difficult to distinguish from the antigenically related phlebovirus Punique virus (3,4); both viruses are members of the species Sandfly fever Naples virus. We investigated the prevalence of TOSV among sandflies in northern Tunisia. A total of 5,288 sandflies (3,547 females, 1,740 males) were collected during June–October 2010 by using CDC light traps (John W. Hock Company, Gainesville, FL, USA) at Utique (37°08′N, 7°74′E), a focus for visceral leishmaniasis in northern Tunisia. Sandflies were separated by sex and trapping nights and pooled with >30 specimens by pool. Pools were processed as described (4) and subjected to PCR detection of phlebovirus RNA targeting 2 genes independently (4–6) and virus isolation onto Vero cells. Of 249 pools processed, 8 strains of phleboviruses were isolated: 2 TOSV, 3 Punique virus, and 3 other phleboviruses currently being characterized. TOSV strains were obtained from 2 pools of sandflies trapped in September 2010, T152 and T166, consisting of 30 males and 30 females, respectively. These pools were positive for TOSV RNA by sequencing of 2 PCR products (201-nt and 280-nt sections in the large [L] and small [S] gene segments, respectively). Supernatant of the third passage was prepared for electron microscopy, which showed spherical and pleomorphic structures, 80–120 nm in diameter, compatible with viruses of the family Bunyaviridae. Complete genome sequencing was then done by using the Ion PGM Sequencer (Life Technologies SAS, Saint Aubin, France) (7); a total of 165,307 reads were obtained, of which 135,700 matched with the sequence of TOSV Iss.PhL3 used as reference. The viral genome of TOSV Tunisia-2010-T152 (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JX867534-JX867536","start_term":"JX867534","end_term":"JX867536","start_term_id":"469785662","end_term_id":"469785666"}}JX867534-JX867536) was composed of 12,488 nt; the complete sequence consisted of 1,869 nt, 4,215 nt, and 6,404 nt for the S, medium [M], and L RNA segments, respectively. The partial S sequence of the TOSV Tunisia-2010-T166 strain (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JX867537-JX867539","start_term":"JX867537","end_term":"JX867539","start_term_id":"469785669","end_term_id":"469785677"}}JX867537-JX867539) was identical to that of T152, but 1 synonymous mutation was observed in the partial L sequence and 1 nonsynonymous mutation in the partial M sequence (I906V). The TOSV Tunisia-2010-T152 strain was aligned with homologous sequences retrieved from the GenBank database. Genetic distances were calculated at the amino acid and nucleotide levels by using the p-distance algorithm (Technical Appendix Tables 1–3). Phylogenetic studies were performed by using the neighbor-joining method in MEGA5 (8) (Figure). The robustness of the nodes was tested by 1,000 bootstrap replications. We found that TOSV Tunisia-2010-T152 was most closely related to the prototype strain from Italy, Iss.PhL3, with nucleotide/amino acid distances of 0.031/0.052, 0.032/0.073, and 0.039/0.012 for the S, M, and L RNA sequences, respectively. Together, these genetic distances and phylogram topologies indicate that TOSV Tunisia-2010-T152 is most closely related to strains within the Italian lineage, although it may represent a distinct sublineage, more distantly related to strains belonging to the Spanish lineage (9). Figure Phylogenetic analysis of 3 segments of Toscana virus (TOSV) isolates from pools of sandflies collected in Tunisia and homologous sequences of other selected phleboviruses. A) Large segments; B) medium segments; C) small segments. Sequences are identified ... Concomitantly with virus isolation, the phenology of sandfly species was studied during May–November 2010. Sandflies were identified, and the density was calculated as described (10). Most of the sandflies belonged to the subgenus Larroussius (98.3%). P. perniciosus sandflies were the most abundant species (71.74%), followed by P. longicuspis (17.47%) and P. perfiliewi (8.82%). Other sandfly species, such as Phlebotomus (Phlebotomus) papatasi, Phlebotomus (Paraphlebotomus) sergenti, Sergentomyia minuta parotti, S. christophersi, and S. antennata were found, but these were much less abundant. The phenology of 3 main sandfly species showed 2 main peaks: 1 small peak in June and a second, larger peak during September–October (Technical Appendix Figure). In summary, of a total of 5,288 sandflies collected, 2 pools were positive for TOSV, yielding an infection rate of 0.03%. A similar infection rate was observed in Spain (0.05%) (9); however, the infection rates in Italy (0.22%) and in France (0.29%) are substantially higher (1,6). The isolation of TOSV from male and female sandflies suggests transovarial transmission in nature, as reported in Italy and Spain (1,9). In southern European countries, TOSV is mostly transmitted by P. perniciosus and P. perfiliewi sandflies (1,6,9), whereas P. perniciosus, P. longicuspis, and P. perfiliewi are the most abundant sandfly species in northern Tunisia. It is therefore probable that TOSV is transmitted by sandfly species of the subgenus Larroussius. We found that 2 phleboviruses belonging to the Sandfly fever Naples virus species, TOSV and Punique virus, are cocirculating in northern Tunisia. This finding calls for further investigation of these viruses’ potential effect on human health in this area. Technical Appendix: Genetic pairwise distances between Toscana virus strains and selected phleboviruses and phenology of sandflies collected in Utique, Governorate of Bizerte, Tunisia. Click here to view.(401K, pdf)

Background In northern Tunisia, the co-circulation of two related sand fly-borne phleboviruses, Toscana virus (TOSV) and Punique virus (PUNV) was previously demonstrated. In contrast to TOSV, a prominent human pathogen, there is no data supporting that PUNV is capable to infect and cause disease to humans. We studied the respective involvement of TOSV and PUNV in human infections in northern Tunisia through a seroprevalence study. Methods The presence of TOSV and PUNV neutralising antibodies (NT-Ab) was tested in human sera collected from 5 districts of the governorate of Bizerte, and the titres of NT-Ab were estimated by microneutralisation (MN) assay. Principal Findings A total of 1,273 sera were processed. TOSV and PUNV NT-Ab were detected in 522 (41%) and 111 sera (8.72%) respectively. TOSV seroprevalence varied from 17.2% to 59.4% depending on the district. Analysis of TOSV geometric mean titre values demonstrated a constant increase according to the age. The vast majority of sera containing NT-Ab were found to be more reactive toward TOSV than PUNV. Indeed, past infections with PUNV and TOSV were undisputable for 5 and 414 sera, respectively. Conclusions PUNV may be capable to infect humans but at a low rate. TOSV is responsible for the vast majority of human infections by sand fly-borne phleboviruses in northern Tunisia. TOSV must be considered by physician and tested in diagnostic laboratories for patients with meningitis and unexplained fever in northern Tunisia., Author Summary In northern Tunisia, two different pheboviruses are known to circulate in sand fly population, Toscana virus (TOSV) and Punique virus (PUNV). In contrast to TOSV, a prominent human pathogen, there is no data supporting that PUNV is capable to infect humans and to cause a disease. We studied the respective involvement of TOSV and PUNV in human infections in northern Tunisia through a seroprevalence study. Because TOSV and PUNV are antigenically and genetically closely related, it is difficult to distinguish between them by using broadly reactive serological tests, such as enzyme-linked immunosorbent assay (ELISA). Thus, we developed a method of microneutralisation assay using the two viruses in a comparative manner. A total of 1,273 sera were processed. We provide first evidence to support (i) that Punique virus may be capable to infect humans but at a low rate, (ii) that TOSV, the most prevalent arbovirus in Southern Europe, is responsible for the vast majority of human infections by sand fly-borne phleboviruses in northern Tunisia. Therefore, it is important to consider TOSV as an important pathogen that needs to be included in all virological diagnostic concerning patients with meningitis and unexplained febrile illness originated from Northern Tunisia.

International audience; INTRODUCTION: Sand fly saliva plays an important role in both blood feeding and outcome of Leishmania infection. A cellular immune response against a Phlebotomus papatasi salivary protein was shown to protect rodents against Leishmania major infection. In humans, P. papatasi salivary proteins induce a systemic cellular immune response as well as a specific antisaliva humoral immune response, making these salivary proteins attractive targets as markers of exposure for this Leishmania vector. Surprisingly, the repertoire of salivary proteins reported for P. papatasi-a model sand fly for Leishmania-vector-host molecular interactions-is very limited compared with other sand fly species. We hypothesize that a more comprehensive study of the transcripts present in the salivary glands of P. papatasi will provide better knowledge of the repertoire of proteins of this important vector and will aid in selection of potential immunogenic proteins for humans and of those proteins that are highly conserved between different sand fly strains. METHODS AND FINDINGS: A cDNA library from P. papatasi (Tunisian strain) salivary glands was constructed, and randomly selected transcripts were sequenced and analyzed. The most abundant transcripts encoding secreted proteins were identified and compared with previously reported sequences. Importantly, we identified salivary proteins not described before in this sand fly species. CONCLUSIONS: Comparative analysis between the salivary proteins of P. papatasi from Tunisia and Israel strains shows a high level of identity, suggesting these proteins as potential common targets for markers of vector exposure or inducers of cellular immune responses in humans for different geographic areas.

Background The sand fly Phlebotomus papatasi is an Old World vector of Leishmania major, the etiologic agent of zoonotic cutaneous leishmaniasis. This study describes the courtship behaviour of P. papatasi and compares it with that of Lutzomyia longipalpis, the New World vector of visceral leishmaniasis. Understanding the details of courtship behaviour in P. papatasi may help us to understand the role of sex pheromones in this important vector. Results P. papatasi courtship was found to start with the female touching the male, leading him to begin abdomen bending and wing flapping. Following a period of leg rubbing and facing, the male flaps his wings while approaching the female. The female then briefly flaps her wings in response, to indicate that she is willing to mate, thereby signaling the male to begin copulation. Male P. papatasi did not engage in parading behaviour, which is performed by male L. longipalpis to mark out individual territories during lekking (the establishment and maintenance of mating aggregations), or wing-flap during copulation, believed to function in the production of audio signals important to mate recognition. In P. papatasi the only predictor of mating success for males was previous copulation attempts and for females stationary wing-flapping. By contrast, male L. longipalpis mating success is predicted by male approach-flapping and semi-circling behaviour and for females stationary wing-flapping. Conclusions The results show that there are important differences between the mating behaviours of P. papatasi and L. longipalpis. Abdomen bending, which does not occur in L. longipalpis, may act in the release of sex pheromone from an as yet unidentified site in the male abdomen. In male L. longipalpis wing-flapping is believed to be associated with distribution of male pheromone. These different behaviours are likely to signify significant differences in how pheromone is used, an observation that is consistent with field and laboratory observations.

Preimmunization of mice with salivary gland homogenate (SGH) of long-term colonized (F29) female Phlebotomus papatasi Scopoli (Diptera: Psychodidae) induced protection against Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae) co-inoculated with the same type of SGH. In contrast, preimmunization of mice with SGH of wild-caught female P. papatasi did not confer protection against L. major co-inoculated with the same type of SGH. Similarly, SGH from recently colonized (F1) female P. papatasi did not protect mice against L. major. These results suggest that when developing a sand fly saliva-based vaccine, the natural vector populations should be considered.

International audience; 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.

The geographical distribution of Phlebotomus papatasi Scopoli, vector of Leishmania major Yakimoff and Schokhor (Kinetoplastida: Trypanosomatidae), the etiologic agent of zoonotic cutaneous leishmaniasis (ZCL), was assessed during September 2006 through a transect from the north to the south of Tunisia using CDC light traps. P. papatasi was found to be abundant in the arid and Saharan bioclimatic zones and rare in the humid, subhumid, and semiarid bioclimatic zones. Similarly, the highest incidence of ZCL was observed in the arid and Saharan bioclimatic zones and the lowest in the humid, subhumid, and semiarid bioclimatic zones. Our overall findings confirm the close spatial association between the abundance of P. papatasi and the incidence of ZCL.

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.