Darine Slama

Transmission of Visceral Leishmaniasis in a Previously Non-Endemic Region of Tunisia: Detection of Leishmania Dna in Phlebotomus perniciosus

ABSTRACT: Visceral leishmaniasis (VL) has been endemic in northern Tunisia and has occurred sporadically in the center of Tunisia. Recently, there have been several cases from areas known to be free of VL. We report in this work all human and canine cases of VL recorded between 2003 and 2011 and an entomological study of phlebotomine fauna in a previously non-endemic region. Sixty-three cases of VL were diagnosed and identified as L. infantum using several different methods. Eight species of 179 sand flies were caught and identified by both morphological and molecular methods. Two genera were present, Phlebotomus and Sergentomya, with an abundance of the subgenus Phlebotomus (Larrousius) spp., a classic vector of VL in Tunisia. Moreover, Leishmania DNA was detected in seven unfed Phlebotomus pernicousus and L. infantum was identified in three of them. This result confirms the establishment of a transmission cycle of VL in the studied region by the coexistence of infected vectors with infected hosts.

Blood meal analysis of culicoides (Diptera: ceratopogonidae) in central Tunisia.

To evaluate the host preferences of Culicoides species (Diptera: Ceratopogonidae) in Central Tunisia, we identified the source of blood meals of field collected specimens by sequencing of the cytochrome b (cyt b) mitochondrial locus and Prepronociceptine single copy nuclear gene. The study includes the most common and abundant livestock associated species of biting midges in Tunisia: C. imicola, C. jumineri, C. newsteadi, C. paolae, C. cataneii, C. circumscriptus, C. kingi, C. pseudojumineri, C. submaritimus, C. langeroni, C. jumineri var and some unidentified C. species. Analysis of cyt b PCR products from 182 field collected blood-engorged females’ midges revealed that 92% of them fed solely on mammalian species, 1.6% on birds, 2.4% on insects and 0.8% on reptiles. The blast results identified the blood origin of biting midges to the species level with exact or nearly exact matches (≥98%). The results confirm the presence of several Culicoides species, including proven vectors in Central Tunisia. Blood meal analyses show that these species will indeed feed on bigger mammals, thereby highlighting the risk that these viruses will be able to spread in Tunisia.

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

BackgroundCulicoides (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.FindingsBlood 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.ConclusionThis 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.

Prevalence of Toxoplasmosis in Sheep, Goats and Cattle in Southern Tunisia

The prevalence of Toxoplasma gondii infection in ruminants (sheep, goats, and cattle) in Southern Tunisia is largely unknown. Antibodies and DNA to T. gondii were determined in serum and apex of the heart samples of 261 animals (204 sheep, 32 goats and 25 cattle) using the modified agglutination technique test (MAT) and the PCR respectively. Antibodies to T. gondii (MAT, 1:20) were found in 40.2% (95% CI: 33.4%, 47.2%), 34.5% (95% CI: 19.1%, 53.2%) and 12% (95% CI: 03.15%, 32.13%) in sheep, goats and cattle respectively. Seroprevalence significantly (p 3 years) and females were significantly (p<0.05) more infected than younger and male animals respectively. The seroprevalence was highest in sheep and specifically in the Barbarine breed. The presence of Toxoplasma DNA was detected in 11 of 96 samples (11.5%). The prevalence observed in the present study indicates a widespread exposure to T. gondii in South Tunisia. Results indicate, for the first time in Tunisia, that infected sheep and goats represent a potential source of T. gondii infection for humans in particular in the South of the country (Gafsa area).

Molecular phylogeny of 42 species of Culicoides (Diptera, Ceratopogonidae) from three continents

The genus Culicoides includes vectors of important animal diseases such as bluetongue and Schmallenberg virus (BTV and SBV). This genus includes 1300 species classified in 32 subgenera and 38 unclassified species. However, the phylogenetic relationships between different subgenera of Culicoides have never been studied. Phylogenetic analyses of 42 species belonging to 12 subgenera and 8 ungrouped species of genus Culicoides from Ecuador, France, Gabon, Madagascar and Tunisia were carried out using two molecular markers (28S rDNA D1 and D2 domains and COI mtDNA). Sequences were subjected to non-probabilistic (maximum parsimony) and probabilistic (Bayesian inference (BI)) approaches. The subgenera Monoculicoides, Culicoides, Haematomyidium, Hoffmania, Remmia and Avaritia (including the main vectors of bluetongue disease) were monophyletic, whereas the subgenus Oecacta was paraphyletic. Our study validates the subgenus Remmia (= Schultzei group) as a valid subgenus, outside of the subgenus Oecacta. In Europe, Culicoides obsoletus, Culicoides scoticus and Culicoides chiopterus should be part of the Obsoletus complex whereas Culicoides dewulfi should be excluded from this complex. Our study suggests that the current Culicoides classification needs to be revisited with modern tools.

Association between host species choice and morphological characters of main sensory structures of Culicoides in the Palaeartic region

Culicoides (Diptera: Ceratopogonidae) serve as vectors of several mammalian and avian diseases, including bluetongue, Schmallenberg, African horse sickness, avian malaria and Oropouche. Host preference investigations are necessary to assess the transmission routes of vector-borne diseases and to inform mitigation strategies. A recent study examining the main sensory structures (palps and antennae) of Culicoides species suggests that they be classified as ornithophilic or mammalophilic according to their feeding habits. We analyzed Culicoides host preferences according to the literature and carried out a multiple correspondence analysis linking these preferences with morphological data. Seven out of 12 variables were found to be reliable predictors of host preference in Culicoides species: Antenna Flagellomer-Sensilla Coeloconica-Number: (7–10) and (11–13); Antenna Flagellomer-Sensilla Coeloconica IV–X: presence; Palpus-size: wide and/or narrow opening and shallow pit; Palpus-Shape: strongly swollen; Antenna-Short sensilla trichodea-distal part segment IV to X-Number: 2 seta each. Our results demonstrate that the presence of sensilla coeloconica and the maxillary palpus can be used to separate ornithophilic and mammalophilic or ornithophilic/mammalophilic species.

Clinical Presentation of Cutaneous Leishmaniasis caused by Leishmania major.

Background/Aims: The diagnosis of cutaneous leishmaniasis (CL) is based on the microscopic detection of amastigote, isolation of the parasite, or the detection of Leishmania DNA. Nevertheless, since these techniques are time consuming and not usually available in many endemic countries, the diagnosis remains clinical. Consequently, such disease may be overlooked because of its similarity to other skin diseases. The aim of this study is to describe the clinical polymorphism of CL caused by Leishmaniamajor.Methods: A cross-sectional survey was carried out on 166 patients. Diagnoses were made by both microscopic examination of stained tissue-scraping smears and PCR. The Leishmania species was identified by restriction enzyme analysis of the ribosomal internal transcribed spacer 1 region. The clinical polymorphism was analyzed only for patients with a positive diagnosis for CL and L. major as the identified species. Results and Conclusion: Of the 166 patients, 75 patients fit the inclusion criteria. Twelve different types of CL caused by L. major were defined. The most common type was the ulcero-crusted form followed by the papulonodular form and the impetigenous form. The ulcerated, mucocutaneous, lupoid, and sporotricoid forms were less common. The eczematiform, erysipeloid, verrucous, psoriasiform, and pseudotumoral types were represented by a single case. Zoonotic CL caused by L. major can simulate many other skin diseases, which may lead to a significant spread of this disease and increases in morbidity and drug resistance. This large polymorphism may be the result of a complex association between the genetics of the parasite and the immune response of the host.

Culicoides spp. (Diptera: Ceratopogonidae) in Tunisia

Culicoides is a genus of biting midges in the family Ceratopogonidae. The female midges require blood meals for egg production. There are over 1000 species in the genus, which is divided into many subgenera. Several species are known to be vector of many diseases and parasites, which can affect animals. As vectors of viruses, Culicoides species are of the higher veterinary importance. More than 75 arboviruses, belonging to Bunyaviridae, Reoviridae and Rabdoviridae families, were isolated from different Culicoides species. In Mediterranean region, the principal vector of Bluetongue virus is represented by Culicoides imicola, and also other European Culicoides biting midges are implicated in virus transmission. Despite the virulence of these species and his colonisation in Tunisia, they are still considered as neglected area due to the rarity or the absence of programmes to control these biting midges. Thus, the available data on species composition, dominant species, breeding sites and host preferences are urgently needed to better understand these biting midges and to develop reliable tools to prevent the spread of other diseases that threaten human and animal life.

Novel Method for Identifying the Culicoides (Diptera: Ceratopogonidae) Larvae Stage IV

Background: Many entomological investigations of Culicoides have used the adult specimens as samples for their studies. This is because immature stages are poorly known in most Culicoides species. The lack of correct identification systems of Culicoides larvae has been one of the major obstacles to understanding the field ecology of Culicoides species. Materials and methods: Culicoides larvae stage was extracted from mud. Larvae stage 2 and 3 were placed in Petri dishes to continue their development to stage IV after pupation. Larvae stage IV showing the same morphology was divided into 2 groups. First group was grown up to reach the adult stage. Second group of larvae stage IV was used for molecular identification. Thus, DNA was extracted and used as a template for a genus-specific PCR. PCR products were then sequenced and analyzed. Results: This seems to be the first report identifying Culicoides larvae stage IV by molecular and morphological techniques. By using this method, the sequence of Internal Transcribed Spacer 1 of two Culicoides larvae: C. circumscriptus and C. Puncticollis are identified for the first time.