Co-circulation of Toscana virus and Leishmania infantum in a focus of zoonotic visceral leishmaniasis from Central Tunisia.

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.

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.

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.

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 investigation on vertical transmission of Leishmania infantum in experimentally infected dogs and assessment of offspring's infectiousness potential by xenodiagnosis.

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.

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 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.

[Laboratory and field evaluation of an imidacloprid treated rodent oral bait for a systemic control of Phlebotomus papatasi Scopoli, 1786 (Dipetra: Psychodidae)]. / Évaluation au laboratoire et sur le terrain de l'imidaclopride sous forme d'appâts pour les rongeurs afin de contrôler les populations de 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). 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.

[Leishmania major Yakimoff et Schokhor, 1914 (Kinetoplastida: Trypanosomatidae) in Meriones shawi Duvernoy, 1842 (Rodentia: Gerbillidae): persistence of the infection in meriones and its infectivity for the sand fly vector (Phlebotomus) papatasi Scopoli, 1786 (Diptera: Psychodidae)]. / 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).

The 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.

[Effects of the development of irrigation systems in the arid areas on the establishment of Phlebotomus (Larroussius) perfiliewi Parrot, 1939]. / 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.

[Confirmation of the presence of Sergentomyia (Sintonius) clydei (Sinton, 1928) in Tunisia]. / Confirmation de la présence en Tunisie de Sergentomyia (Sintonius) clydei (Sinton, 1928).

An entomological investigation was performed in 2004 in the region of Rmilia, home to well-known zoonotic cutaneous leishmaniasis, located in Central Tunisia. The authors confirm the presence of Sergentomyia (Sintonius) clydei (Sinton, 1928). Thus, the sand fly fauna list of Tunisia includes 17 species.

Behavioral evidence for the presence of a sex pheromone in male Phlebotomus papatasi scopoli (Diptera: Psychodidae).

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.

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.

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.

Spatial correlation between Phlebotomus papatasi Scopoli (Diptera: Psychodidae) and incidence of zoonotic cutaneous leishmaniasis in Tunisia.

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.

Biology of Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory.

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 degrees 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 degrees 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.

Phenology of Phlebotomus papatasi (Diptera: Psychodidae) relative to the seasonal prevalence of zoonotic cutaneous leishmaniasis in central Tunisia.

The population density of Phlebotomus papatasi Scopoli (Diptera: Psychodidae), vector of Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae), the etiologic agent of zoonotic cutaneous leishmaniasis (ZCL), was assessed May-November 2005 in central Tunisia by using sticky traps. The densities of P. papatasi were found to peak in early spring and again in the autumn. The lowest densities were observed in August. Prevalence of ZCL in the governorate of Sidi Bouzid peaks in December, 3 to 3.5 mo after the fall sand fly population peak, suggesting a close temporal association with the abundance of P. papatasi.