W. Barhoumi

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.

Interaction between canine and human visceral leishmaniases in a holoendemic focus of Central Tunisia

Canine visceral leishmaniasis (CVL) is endemic in the Mediterranean basin. In Tunisia, CVL is spatially associated with human visceral leishmaniasis (HVL) affecting mostly children younger than 5 years old. In this study, seroprevalence of Leishmania infantum infection in dogs was assessed in highly endemic districts of the governorate of Kairouan where more than 50% of HVL cases in Tunisia were reported. An entomological investigation was also carried out in two endemic districts (Bouhajla and Haffouz) to assess sand fly fauna and infection status of sand flies with Leishmania. A total of 191 serum samples were collected from healthy dogs and tested for anti-L. infantum antibodies by indirect immunofluorescence antibody test (IFAT). Overall seroprevalence for L. infantum was 26.7% being highest among dogs in the district of Bouhajla (52.7%) and the lowest in the district of Chbika (5.2%). In dogs, seroprevalence did not differ significantly based on gender or age, with dogs younger than 1 year showing a higher seroprevalence compared to older dogs. These findings suggest strong force of infection in naïve animals in holoendemic regions leading to emerging high incidence of HVL. Concomitant to the high CVL prevalence observed in the Bouhajla district, a significantly high cumulative HVL incidence also was observed in this district. Phlebotomus perniciosus and Phlebotomus longicuspis were the most abundant sand fly species in Bouhajla and Haffouz districts. The rate of Leishmania-DNA infection in sand flies was 9.4%. This finding points to spatial correlation between the occurrence of disease in humans, a high rate of infection in dogs and a high abundance of P. pernicious and P. longicuspis. Thus, CVL is the main risk factor for transmission to humans and subsequently, it is an important parameter for controlling transmission to humans.

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.

Effet du développement des systèmes d’irrigation dans les zones arides sur l’établissement de Phlebotomus (Larroussius) perfiliewi Parrot, 1939

In Tunisia, Phlebotomus (Larroussius) perfiliewi Parrot, 1939, is highly suspected to be involved in the transmission of sporadic cutaneous leishmaniasis. The geographical distribution of this sandfly species is limited to the northern part of Tunisia and more specifically to the humid, sub-humid and semi-arid bio-geographical areas. To date, the geographical distribution of P. perfiliewi and subsequently of the sporadic cutaneous leishmaniasis extends to the arid bio-geographical areas located in central Tunisia. This extension is related to the intense development of irrigation systems in these areas, which may lead to the establishment of a new ecological niche suitable for the development of P. perfiliewi.

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.

West Nile virus in Tunisia, 2014: First isolation from mosquitoes.

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.

[Laboratory and field evaluation of an imidacloprid treated rodent oral bait for a systemic control of Phlebotomus papatasi Scopoli, 1786 (Dipetra: Psychodidae)].

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). Shaws 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.