First detection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Culicoides spp. (Diptera: Ceratopogonidae)

Background Culicoides (Diptera: Ceratopogonidae) species are known to be the vectors of Bluetongue virus and African Horses Sickness virus (AHSV) in different areas of the world. Nevertheless, other researchers have hypothesized that these arthropods could be involved in the transmission of other pathogens such as Schmallenberg virus, Plasmodium and Leishmania parasites. Identification of the Culicoides’ potential vector competence is crucial in understanding the worldwide Culicoides/Leishmania life cycle. Findings Blood fed and parous females of biting midges Culicoides spp. were collected between 2009 and 2010 in Central Tunisia. DNA was extracted from individual blood fed Culicoides and used as a template in a genus-specific PCR. Leishmania DNA was detected in 14 Culicoides imicola specimens and one Culicoides circumscriptus. In a second step, parasite identification was performed based on a single copy Topo-isomerase II gene specific amplification and sequencing. Leishmania infantum was identified in two infected Culicoides spp. Conclusion This is the first report of Leishmania DNA detection from naturally infected wild caught Culicoides spp. Our finding supports the assumption that Culicoides spp. are a potential vector for L. infantum.

Biting midges of the genus Culicoides are known to be vectors of a wide range of pathogens, most notably arboviruses [1]. In early research on Leishmania transmission, Culicoides have been suspected to be vectors of Leishmania donovani in India [2]. However, since this date and until the beginning of the 20th century, no data were published concerning the potential involvement of biting midges in Leishmania transmission. Between 2004 and 2010, some researchers reported the natural infection of Culicoides biting midges by Herpetomonas and Sergeia kinetoplastid protozoa [3][4][5].
In 2011 Dougall et al. [6] reported the natural infection of Forcypomyia day-feeding midges with Leishmania using both molecular approach and microscopic detection of promastigotes in their gut. Recently, Seblova [7] have experimentally proved the susceptibility of reared Culicoides nubeculosus to infection with Leishmania infantum. All these previous studies have highlighted the potential role of midges in the Leishmania transmission.
Moreover, other studies have demonstrated the infection of domestic dogs (reservoirs of L. infantum) with Blue-Tongue Virus (BTV) in Morocco [8]. This implies that Culicoides can feed on canine hosts and thereby transmission of L. infantum by this arthropod genus is possible.
In our study, we emphasize the likelihood of Leishmania transmission via Culicoides.
Insects were collected between 2009 and 2010 in Central Tunisia, using two light traps types: home-made CDC (Center of Disease Control, Atlanta, USA) miniature and OVI (Onderstepoort Veterinary Institute) traps in rural areas. Traps were set before sunset and collected the next morning. All insects were collected in a beaker filled with 70% ethanol. Sampled insects were carried to the laboratory and Culicoides were separated from other insect genera. Females Culicoides specimen were divided into engorged (i.e. insects with a full or partial blood meal), parous and unfed midges. For each female specimen, wings and genitals were mounted for morphological identification using Chaker's key [9].
Total DNA from each individual, blood fed or parous, biting midge abdomen and head, was extracted and PCR was performed to detect Leishmania DNA.
In the first step, DNA from all specimens (blood fed and parous) was tested using genus-specific PCR primers targeting a part of small subunit rRNA gene for the detection of Leishmania spp. infection according to the protocol of Spanakos et al. [10]. In the second step, DNA from positive specimens was re-analyzed by a second set of primers targeting a Topo-isomerase II single copy gene. PCR products from this last PCR were sequenced for identification of Leishmania to the species level according to the protocol of Haouas et al. [11].
In total, 259 biting midges of Culicoides (blood fed, n = 189; parous, n = 70) were tested for Leishmania spp. infection. Morphological identification showed that these tested midges belonged to the following species: Culicoides imicola (n = 196), C. jumineri (n = 35), C. cataneii (n = 3), C. paolae (n = 10), C. newsteadi (n = 10), C. circumscriptus (n = 3) and C. sp. (unidentified Culicoides) (n = 2). Among these collected samples, 15 Culicoides specimens were positive for Leishmania spp. DNA using genus-specific PCR primers. To confirm this result all positive PCR products were sequenced and sequences were blasted using Blastn algorithm against the "non-redundant" GenBank sequence database. These positive specimens belonged to C. imicola species (n = 14) and C. circumscriptus species (n = 1). All of them were engorged females and no parous female was positive for Leishmania spp. In the 15 positive specimens only two were positive for Leishmania Topo-isomerase II gene primer set. They correspond to C. imicola (n = 1) and C. circumscriptus (n = 1). The sequencing of these Topo-isomerase II positive PCR products confirmed the presence of L. infantum DNA in the abdomens of both Culicoides species. It is the first case in the world of Leishmania DNA detection from wild caught biting midges Culicoides. This preliminary finding highlights the potential role of this insect in Leishmania transmission. Our result corroborates the findings of Seblova et al. who have succeeded to experimentally infect reared C. nubeculosus with L. infantum [7].
It is noteworthy that even phlebotomine sand flies captured in the same area as biting midges were positive for Leishmania DNA in their mid gut. Among them three Phlebotomus perniciosus specimen were infected with L. infantum [12]. This result indicates that L. infantum could be transmitted by more than one arthropod genus. This assumption is supported by the studies of Coutinho et al. who have reported the presence of L. infantum DNA and promastigote forms in Rhipicephalus sanguineus (tick) and Ctenocephalides felis felis (fleas) respectively [13,14].
Despite infected biting midges are blood fed the hypothesis of contaminated blood meals could be excluded. Indeed, infected engorged females were also analyzed to identify their blood meal origin according to the protocol of Haouas et al. [11] and Homo sapiens, Capra hircus and Gallus gallus hosts were identified (unpublished data). These hosts are not known to be reservoirs of Leishmania in Tunisia. The presence of L. infantum DNA in Culicoides specimens fed on animals that are not regarded as reservoirs of the parasite may indicate that these female Culicoides would have taken their first blood meal from Leishmania reservoirs (the dog in the case of L. infantum in Tunisia). Then, these females would have taken a second blood meal (just before being captured) from uninfected hosts (human, goats or chicken).
Our preliminary findings raise important questions to solve in future epidemiological studies on the Leishmania life cycle. Therefore, to confirm the vector role of Culicoides in Leishmania transmission, studies should be accompanied by direct microscopic observations to confirm Leishmania development and survival.

Conclusion
Our study reports for the first time the detection of Leishmania DNA in the abdomens of wild caught Culicoides spp. Nevertheless, further studies such as the isolation of the parasite and its iso-enzymatic identification are mandatory to confirm this preliminary result.