M. Derbali

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.

Spatial Correlation Between Phlebotomus papatasi Scopoli (Diptera: Psychodidae) and Incidence of Zoonotic Cutaneous Leishmaniasis in Tunisia

ABSTRACT 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.

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.

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.

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.

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.

Leishmania major Yakimoff et Schokhor, 1914 (Kinetoplastida : Trypanosomatidae) chez Meriones shawi Duvernoy, 1842 (Rodentia : Gerbillidae) : persistance de l’infection du mérion et de son infectivité pour le phlébotome vecteur Phlebotomus (Phlebotomus) papatasi Scopoli, 1786 (Diptera : Psychodidae)

RésuméLa capacité du phlébotome Phlebotomus papatasi à transmettre Leishmania major, agent étiologique de la leishmaniose cutanée zoonotique (LCZ) à Meriones shawi, réservoir naturel de ce parasite, est étudiée dans les conditions de laboratoire. Les phlébotomes sont infectés après un repas sanguin sur un M. shawi lui-même préalablement infecté par injection sous-cutanée de L. major. P. papatasi, ainsi infecté, transmet L. major à un autre M. shawi lors d’un deuxième repas sanguin. Deux mois après, le deuxième mérion développe des lésions leishmaniennes au niveau des deux oreilles sur lesquelles s’infectent de nouveaux phlébotomes. L’infectivité 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 mérion à mérion par P. papatasi. Nos résultats montrent que les rongeurs réservoirs de parasites survivant à la période hivernale sont la source d’infection leishmanienne la plus probable pour les phlébotomes émergents lors de la nouvelle saison estivale. Ce sont donc ces rongeurs qui joueraient le rôle déterminant dans la persistance et la transmission de L. major entre deux saisons de transmission.AbstractThe ability of the sand fly Phlebotomus papatasi to transmit Leishmania major, the etiologic agent of zoonotic cutaneous leishmaniasis, to Meriones shawi, the natural reservoir host of this parasite, was studied under laboratory conditions. Sand flies became infected with L. major after feeding on a lesion of needle-inoculated M. shawi. Moreover, P. papatasi, previously infected with L. major, transmitted the parasite to M. shawi by bite during a second bloodmeal. Two months after the blood-meal, the animal developed a lesion on its ears. Xenodiagnosis was performed on the infected animal. The infectivity of M. shawi to P. papatasi lasted for five months, period corresponding to winter season in North Africa. We have thus demonstrated the transmission of L. major by P. papatasi to M. shawi under laboratory conditions. Our results show that reservoir hosts surviving winter time are the main source of infection for P. papatasi during the following season, and subsequently they play a major role in the persistence and transmission of L. major between transmission cycles.

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

BackgroundSand 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.ResultsWhile 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.ConclusionFor 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.