The initial detection of Toscana virus in phlebotomine sandflies from Turkey.

Toscana virus (TOSV) is a prominent arthropod-borne viral agent of human central nervous system infections occurring in the Mediterranean region. The main transmission route to susceptible individuals involves sandflies as vectors. Despite several reports revealing widespread TOSV activity in Turkey, vectors remained unidentified. A sandfly field survey was carried out in five provinces in Central, Southeast and Mediterranean Anatolia in 2017 to identify TOSV and related sandfly-borne phleboviruses and Leishmania parasites, with evidence for circulation in the region. A total of 7136 sandfly specimens, collected via standard methods, were evaluated in 163 pools. TOSV was detected in 11 pools (6.7%), comprising Phlebotomus major sensu lato, Sergentomyia dentata and Phlebotomus papatasi species. TOSV partial L and S segment sequences were characterized, that phylogenetically clustered with local and global genotype A strains. An amino acid substitution outside the conserved motifs of the viral polymerase, also present in previous TOSV sequences in endemic regions, was observed. Leishmania tropica was detected in a single pool of Ph. sergentii (0.6%). This is the first report of TOSV in sandflies from Turkey, and this study further provides evidence for additional sandfly species with the potential to transmit TOSV.

Sandfly surveillance and investigation of Leishmania spp. DNA in sandflies in Kosovo.

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo - Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. The presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance.

Investigations of Phlebotomus perniciosus sand flies in rural Spain reveal strongly aggregated and gender-specific spatial distributions and advocate use of light-attraction traps.

The spatial and temporal distribution of Phlebotomus perniciosus (Diptera: Psychodidae) (Newstead, 1911), the sand fly vector of pathogens of public and animal health importance, was investigated in a high sand fly density rural area in Spain using light-attraction and sticky-interception traps. Traps were placed inside animal buildings and outside at increasing distance from animals. A total of 8506 sand flies were collected, 87% with light traps. Species frequency differed between trap types. The abundance of P. perniciosus decreased exponentially with increasing distance to animals and, while females were most common in the animal enclosure, males predominated in adjoining storage places. Increasing CO2 concentration had an additional positive effect on female abundance only. Both male and female density increased with rising temperature, and there was some indication that females were more active than males at higher relative humidity. The study confirms that P. perniciosus aggregates around animal premises, although male and female distributions differ and should be analysed separately to account for biological and behavioural differences. This provides further evidence that light traps offer an accurate estimation of the relative spatial and temporal abundance of P. perniciosus, conferring an added value for the study of this species and the risk of pathogen transmission.

Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

Ongoing activity of Toscana virus genotype A and West Nile virus lineage 1 strains in Turkey: a clinical and field survey.

Toscana virus (TOSV), West Nile virus (WNV) and tickborne encephalitis virus (TBEV) are among major viral pathogens causing febrile disease and meningitis/encephalitis. The impact of these viruses was investigated at a referral centre in Ankara Province, Central Anatolia in 2012, where previous reports suggested virus circulation but with scarce information on clinical cases and vector activity. Serum and/or cerebrospinal fluid samples from 94 individuals were evaluated, in addition to field-collected arthropod specimens that included 767 sandflies and 239 mosquitoes. Viral nucleic acids in clinical samples and arthropods were sought via specific and generic nested/real-time PCRs, and antibody responses in clinical samples were investigated via commercial indirect immunofluorescence tests (IIFTs) and virus neutralization. A WNV antigen assay was also employed for mosquitoes. WNV neuroinvasive disease has been identified in a 63-year-old male via RNA detection, and the WNV strain was characterized as lineage 1. TOSV infections were diagnosed in six individuals (6.3%) via RNA or IgM detection. Partial sequences in a 23-year-old female, presented with fever and transient pancytopenia, were characterized as TOSV genotype A. Febrile disease with arthralgia and/or peripheral cranial nerve involvement was noted in cases with TOSV infections. Previous WNV and TOSV exposures have been observed in 5.3% and 2.1% of the subjects, respectively. No confirmed TBEV exposure could be identified. Morphological identification of the field-collected mosquitoes revealed Culex pipiens sensu lato (74.4%), Anopheles maculipennis (20.9%), An. claviger (2.1%) and others. Sandfly species were determined as Phlebotomus papatasi (36.2%), P. halepensis (27.3%), P. major s. l. (19.3%), P. sergenti (8.9%), P. perfiliewi (4.4%), P. simici (2.6%) and others. Viral infections in arthropods could not be demonstrated. TOSV genotype A and WNV lineage 1 activity have been demonstrated as well as serologically proven exposure in patients. Presence of sandfly and mosquito species capable of virus transmission has also been revealed.

Emergence and co-infections of West Nile virus and Toscana virus in Eastern Thrace, Turkey.

The objective of this study was to identify the impact of West Nile virus (WNV) and Toscana virus (TOSV) in febrile diseases of unknown aetiology in Eastern Thrace, Turkey; this study was conducted during August-October 2012, and included 18 clinical cases and 296 blood donors for local serosurveillance. Antibodies were determined via commercial assays and further tested for specificity via neutralization assays (NA). Viral RNAs were sought via specific and/or generic primers. WNV infections were diagnosed in seven patients (38.8%), detected via RNA+IgM in four, RNA in one and IgM and low avidity IgG in two cases. The most common symptom was fever (>38°C), followed by headache, malaise/fatigue, myalgia/arthralgia, muscle stiffness/lower back pain, anorexia, nausea/vomiting, diarrhoea, supraorbital/retrobulbar pain and abdominal pain. Neurological symptoms were noted in one individual. WNV strains in RNA-detectable patients were characterized as lineage 1. TOSV RNA or IgM were identified in two individuals with confirmed WNV infections and in one patient without evidence of WNV exposure. The clinical and laboratory findings in individuals with WNV/TOSV co-infection were comparable to those in WNV-induced disease. The TOSV strain in the patient with detectable viral RNA was characterized as genotype A. In local blood donors, seroreactivity for specific WNV and TOSV immunoglobulins was observed in 1.7% (5/296) and 14.4% (26/180), respectively. These findings indicate the emergence of WNV and TOSV-associated diseases in Eastern Thrace. WNV/TOSV co-infections were documented for the first time.

Intraspecific variability of natural populations of Phlebotomus sergenti, the main vector of Leishmania tropica.

An intraspecific study of Phlebotomus sergenti was performed on populations from Turkey, Syria, Israel, and Uzbekistan by four different approaches: geometric morphometrics, RAPD analysis, internal transcribed spacer 2 (ITS2) sequencing (nuclear marker), and cytochrome B sequencing (mitochondrial marker). In RAPD analysis, distinct clades were formed in accordance with the geographical origin of the specimens. There was no distinct grouping according to place of origin within the Turkish samples from various localities in south-eastern Anatolia, which suggests a gene flow between populations separated spatially by the Amanos mountains, a mountain range of a considerable altitude. The results of ITS2 rDNA sequencing complied with the previously published intraspecific division of P. sergenti into two branches, northeastern and southwestern. However, mtDNA haplotypes formed three lineages with specimens from Turkey and Israel, sharing a common clade. A previously postulated hypothesis about a complex of sibling species within P. sergenti is therefore questionable. Cytochrome B seems to be a more discriminative marker for intraspecific variability assessment.

Molecular evidence for similarity between Anopheles hyrcanus (Diptera: Culicidae) and Anopheles pseudopictus (Diptera: Culicidae), sympatric potential vectors of malaria in France.

Malaria was a former public health problem in the Camargue, southeastern France, where members of the Hyrcanus group were recently described as the main malaria potential vectors. However, the systematic status in this group, which includes at least two sympatric sibling species, Anopheles hyrcanus (Pallas) and Anopheles pseudopictus Grassi as well as a morphologically intermediate form in the Camargue, is unclear. Indeed, both species have been alternatively considered as separated or synonymous species. We examined sequence variation of the internal transcribed spacer (ITS) 2 and domain-3 (D3) of 28S ribosomal DNA and the cytochrome oxidase subunit I and II (COI and COII) genes of mitochondrial DNA of the Hyrcanus group mosquitoes from the Camargue and Turkey to infer the taxonomic status of the members of this group. DNA sequence analysis of ITS2 and D3 showed no difference between either species or geographical origin (mean pairwise genetic distances d = 0.000-0.003). The COI and COII sequences between French specimens also were nearly identical (d = 0.001-0.002), whereas French and Turkish Anopheles were genetically distinct (d = 0.009-0.014). The distinction between populations of the two areas, supported, respectively, by four and five fixed mutations, attested the differentiation by the distance. Finally, the high degree of genetic similarity, despite morphological differences between An. hyrcanus, An. pseudopictus, and an intermediate form, suggests that these three taxa may belong to a single species in the Camargue.

A comparison of the intraspecific variability of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae).

Phlebotomus sergenti populations from different areas of the Mediterranean basin are known to exhibit high intraspecific variability. Previous studies of ITS2 revealed the presence of two branches that may represent sibling species. To corroborate this finding by other tools, two colonies of P. sergenti originating from Turkey and Israel, each belonging to a different ITS2 branch, were compared by three different methods: geometric morphometric analysis of wing shape, RAPD (random amplified polymorphic DNA), and cross-mating study. For geometric morphometric analysis, two-dimensional Cartesian coordinates of 16 landmarks from the wings were digitized and analyzed. Significant shape differences were found between colonies but not between sexes within each colony. RAPD results formed two distinctive clades corresponding to the origin of the colony but also showed heterogenity among members of both colonies. In cross-mating studies, viable hybrid F1 and F2 progeny were obtained when both Turkish males/Israeli females and Israeli males/Turkish females were crossed. F1 progeny was included in RAPD analysis and these hybrids formed a distinctive clade with an intermediate position between the two parental clades. No significant differences were found in egg production of crossed sand flies. The cross-mating study showed that there is no reproductive barrier between P. sergenti from different geographical areas. On the other hand, RAPD and geometric morphometric analysis revealed a significant difference between colonies and confirmed the suitability of previous ITS2 analysis for discrimination among sand fly populations. Further development of molecular markers should resolve a possible existence of sibling species within Phlebotomus sergenti.

Variability in natural populations of Anopheles sacharovi (Diptera: Culicidae) from southeast Anatolia, revealed by morphometric and allozymic analyses.

Four populations of Anopheles sacharovi Favre occurring in different ecological subregions at altitudes between 353-1,126 m in the Sanliurfa Province of southeast Turkey were compared using morphometric and allozyme analyses. Four allozyme loci were assessed for genetic differentiation among samples from four localities. The similarity phenogram obtained from the allozyme data showed that populations at Birecik and Sandi branched as a separate group from the Pamuklu and Gedik populations. The Gedik population at the highest altitude (1,126 m) was clustered as a separate branch when linear measurements of 63 morphological characteristics were examined. The UPGMA phenogram also showed that Pamuklu and Sandi formed a cluster while Birecik and Gedik formed separate groups.

Altitudinal variation in morphometric and molecular characteristics of Phlebotomus papatasi populations.

Four populations of the phlebotomine sandfly Phlebotomus (Phlebotomus) papatasi (Scopoli) (Diptera: Psychodidae), in different ecoregions at altitudes between 368 and 1117 m in the Sanliurfa Province of Turkey, were compared using morphometric and isoenzyme analyses. A similarity phenogram obtained from allozyme data showed that heterozygosity was extremely low, particularly for the alleles which were found to be completely fixed in populations at Hamdun (HMD) and Alitas (ALT). Populations at Akcakale (AKL) and ALT branched as a separate group from populations at Hayatiharrani (HHR) and HMD. The ALT population at the highest altitude (1117 m), and the HHR population (488 m) were clustered distinctly when linear measurements of 46 morphological characteristics were examined. A UPGMA (unweighted pair-group method using arithmetic averages) phenogram also showed that ALT and HHR clustered separately, whereas AKL and HMD formed another group.

Comparison of nocturnal activities of mosquitoes (Diptera: Culicidae) sampled by New Jersey light traps and CO2 traps in Belek, Turkey.

Temporal and seasonal variations in the nocturnal activities of adult females of six locally common mosquitoes, Aedes cretinus, Ochlerotatus caspius, Oc. dorsalis, Culex pipiens, Cx. tritaeniorhynchus and Culiseta annulata were studied at Belek, Turkey, using New Jersey light traps and CO2 charged olfactory traps. In addition to assessing the relative value of these traps as sampling devices capable of measuring activity at different periods of the night, these studies also examined the seasonal contribution of each species to the phenology of the overall mosquito population. While statistical results showed that differences in activation time preferences were found to be not significant for each species, the nocturnal activity profiles of Cx. tritaeniorhynchus, Ae. cretinus and Oc. caspius were definitely differed from those of other species. Ae. cretinus was a common species with a rate of 26.1% at 18:00-20:00, whereas the adult rate of Cx. tritaeniorhynchus was determined to be the lowest (11.4%) at that time interval. Significant differences were found at the time intervals 18:00-20:00, 22:00-24:00 and 02:00-04:00 in the general bias of mosquito populations among different months. While the nocturnal patterns of Cx. tritaeniorhynchus and Ae. cretinus were found to be bimodal, it was monomodal for Oc. caspius and Cu. annulata, unimodal for Cx. pipiens and Ae. dorsalis. While 69.6% of females captured by traps throughout the study period were found to be unfed, 24.8% were fed, 4.1% were half-gravid and 1.4% were gravid. Statistical results of trap preferences of Aedes species were found to be significant (P < 0.05). Oc. caspius and Ae. dorsalis preferred New Jersey light traps to CO2 traps. Light trap and CO2 trap collections can be influenced by design features including the amount of contrast between the light sources and surroundings. These features were found to be highly significant factors for the biting activity Cx. pipiens and Cx. tritaeniorhynchus.

Evaluation of protective efficacy of K-OTAB impregnated bednets for cutaneous leishmaniasis control in Southeast Anatolia-Turkey.

Large-scale field trials were performed in an endemic focus of cutaneous leishmaniasis in both urban and rural settlements of Sanliurfa City, SE Anatolia, Turkey, to evaluate the efficacy of insecticide impregnated bednets. An intervention field trial promoting the use of K-OTAB (deltamethrin-tablet formulation) impregnated bednets by the local inhabitants of five quarters for cutaneous leishmaniasis was performed between July 2000 and July 2001. The aim was to examine the monthly and annual efficacy of such bednets in controlling cutaneous leishmaniasis and to compare effect of impregnated bednets (IB) with non-impregnated bednets (NIB). Results showed significant (P < 0.05) reduction in cutaneous leishmaniasis incidence in the intervention areas from 1.87 % to 0.035% in Yenice and from 2.3% to 1.32% in Suruç B. In contrast, cutaneous leishmaniasis incidence increased in the untreated control areas from 1.03% to 1.38% in Suruç A. The annual incidence of the second control area for cutaneous leishmaniasis experiments showed almost no change at all(1.1% in July 2000; 1.02% in July 2001). Similar to results of control areas, cutaneous leishmaniasis incidence rates showed an increase despite the bednets in the Suruç C (1.22% to 1.47%) where NIBs were distributed.

Effect of insecticide-treated bednets for malaria control in Southeast Anatolia-Turkey.

Deltamethrin is one of the most effective insecticides for vector control, already widely used for bednet impregnation to control malaria. To evaluate the efficacy of deltamethrin-impregnated bednets in malaria control and in reducing the biting nuisance caused by Anopheles sacharovi, field trials were carried out in an endemic area of malaria in the surrounding rural settlements of Sanliurfa City, SE Anatolia, Turkey. Preliminary studies commenced in August 1999 with pre-intervention house-to-house surveys to identify villages outside of Sanliurfa City with high malaria incidence, to collect socio-economic, epidemiological and entomological data, and to determine physical properties of the study areas. An intervention field trial promoting the use of K-OTAB (deltamethrin-tablet formulation) impregnated bednets by local inhabitants of four villages was performed between July 2000 and July 2001. Its aim was to examine the monthly and annual efficacy of such bednets in controlling malaria and to compare the effect of impregnated bednets (IB) with non-impregnated bednets (NIB). The experimental design consisted of four villages. Gedik was selected as the intervention area using IBs, Orgulu served as the control area, and in Persiverek and Sandi NIBs were implemented. All 1,406 inhabitants of the 146 households were recruited for the study. Results showed significant (P < 0.05) reduction in malaria incidence in Gedik from 8.29% in the pre-treatment year to 1.57% in the post-treatment year. In contrast, malaria incidence slightly increased in Orgulu from 6.55% to 7.58%. Similar results were obtained from the other two villages where NIBs were used; malaria incidence rates increased from 2.16% to 6.77% (Persiverek) and from 1.9% to 9.8% (Sandi). Entomological surveys, employing different techniques, were carried out randomly at selected collection sites within the intervention and control settlements every month from June 2000 to June 2001 to determine the fluctuation of seasonal population sizes and compare the monthly density of malaria vectors between intervention and control areas.

Species composition and seasonal dynamics of mosquitoes in the Belek region of Turkey.

The species composition and seasonal population dynamics of mosquitoes trapped in the Belek Region of Turkey are described. Eight traps (four New Jersey light traps and four CO2 baited traps) were activated weekly at four different sampling sites (wooded area, open field, village, and road edge). Between May and December, 1997, 4,542 specimens representing seven species (Culex pipiens, Culex tritaeniorhynchus, Aedes caspius, Aedes cretinus, Aedes dorsalis, Aedes vexans, and Culiseta annulata) were collected. The most abundant species was Cx. pipiens (26.7% of total catch) followed by Cx. tritaeniorhynchus (23.8%), Ae. caspius (23.4%), Ae. cretinus (10.7%), Ae. dorsalis (8.7%), Cs. annulata (6.2%), and Ae. vexans (0.1%). This is the first record of Ae. cretinus in the area since 1984. The species was captured throughout the study period, although it was most abundant in late summer. Culex tritaeniorhynchus reached its highest density in August, while Cx. pipiens was found in high numbers throughout most of the study period.