S. Cherni

Updating the Salivary Gland Transcriptome of Phlebotomus papatasi (Tunisian Strain): The Search for Sand Fly-Secreted Immunogenic Proteins for Humans

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.

Differences in the salivary effects of wild-caught versus colonized Phlebotomus papatasi (Diptera: Psychodidae) on the development of zoonotic cutaneous leishmaniasis in BALB/c mice.

ABSTRACT 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.

Co-Circulation of Toscana Virus and Punique Virus in Northern Tunisia: A Microneutralisation-Based Seroprevalence Study

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.

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.

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.

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.

Infection of sand flies collected from different bio-geographical areas of Tunisia with phleboviruses.

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 data 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.

An integrated overview of the midgut bacterial flora composition of Phlebotomus perniciosus, a vector of zoonotic visceral leishmaniasis in the Western Mediterranean Basin

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.

Laboratory and field evaluation of rodent bait treated with fipronil for feed through and systemic control of Phlebotomus papatasi.

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 48h after blood feeding on the deserts 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.

Phleboviruses associated with sand flies in arid bio-geographical areas of Central Tunisia.

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.