Seroepidemiological and entomological survey in a new focus of zoonotic visceral leishmaniasis in Kars province, Northeastern Turkey

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Seroepidemiological and entomological survey in a new focus of zoonotic visceral leishmaniasis in Kars province, Northeastern Turkey Barıs¸ Sari a , M. Emin Limoncu b , I. Cuneyt Balcioglu c , Adnan Aldemir d , Gencay Taskin Tasci a , Yunus Kilic¸ a , Seray Toz e , Berna Demirci a , Samiye Demir f , Ozge Erisoz Kasap g , M. Kirami Olgen h , Yusuf Ozbel e,∗ a

Kafkas University, Faculty of Veterinary Medicine, Department of Parasitology, Kars, Turkey Celal Bayar University, Vocational School of Health Services, Manisa, Turkey c Celal Bayar University, Faculty of Medicine, Department of Parasitology, Manisa, Turkey d Kafkas University, Faculty of Science, Department of Biology, Kars, Turkey e Ege University, Faculty of Medicine, Department of Parasitology, I˙ zmir, Turkey f Ege University, Faculty of Science, Department of Biology, I˙ zmir, Turkey g Hacettepe University, Faculty of Science, Department of Ecology, Ankara, Turkey h Ege University, Department of Geography, Izmir, Turkey b

a r t i c l e

i n f o

Article history: Received 22 November 2014 Received in revised form 17 January 2015 Accepted 18 February 2015 Keywords: Leishmaniasis Visceral Sand flies Kars Turkey

a b s t r a c t Visceral leishmaniasis (VL) has now been recorded from 38 provinces of Turkey. Twentyone VL cases were reported within six years from settlements located in most northeastern Turkey and we therefore aimed to carry out an entomological and seroepidemiological survey in this new focus for clarifying risk factors. Blood samples from 290 children and 165 dogs were collected. Sera samples were investigated for anti-Leishmania antibodies using indirect fluorescent antibody test. Sand fly collection for determining the fauna and seasonal activity was performed in all settlements by CDC light traps between June and September 2006. Although no seropositive child was detected during the survey the overall seroprevalence rate of canine leishmaniasis was found as 7.2%. A total of 4154 sand flies were collected and 10 species of genus Phlebotomus were identified belonging to Adlerius, Larroussius, Paraphlebotomus and Phlebotomus subgenera. Among them Phlebotomus kandelakii s.l. (55.44%), Phlebotomus balcanicus (12.62%) and Phlebotomus neglectus (4.40%) was detected as probable vector species for this new focus. The poor sanitation, very high population size of sand flies, probably because of very short season, no control measures for sand flies as well as dogs, and presence of microclimate suitable for sand flies were considered as main risk factors in the area. © 2015 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Ege University, Faculty of Medicine, Department of Parasitology, Bornova, I˙ zmir, Turkey. Tel.: +90 232 390 4724; fax: +90 232 388 1347. E-mail address: [email protected] (Y. Ozbel). http://dx.doi.org/10.1016/j.vetpar.2015.02.021 0304-4017/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Sari, B., et al., Seroepidemiological and entomological survey in a new focus of zoonotic visceral leishmaniasis in Kars province, Northeastern Turkey. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.02.021

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1. Introduction Leishmania protozoan parasites, transmitted by the bite of Phlebotomine sand flies (Diptera: Psychodidae), cause visceral, cutaneous or mucocutaneous leishmaniasis. Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and if not treated, can potentially be a lethal disease. Zoonotic VL caused by Leishmania infantum is endemic in all countries bordering the Mediterranean Basin WHO (2010). L. infantum is responsible for human VL and canine leishmaniasis (CanL), while Leishmania tropica is mainly responsible for cutaneous leishmaniasis (CL) in Turkey. So far, all Leishmania strains isolated from VL patients were identified as L. infantum MON1 or from dogs as L. infantum MON-1 and MON-98 in Turkey (Özbel et al., 2000; Ok et al., 2002) based on multi-site DNA polymorphism analyses. In addition to this, two strains isolated from two VL patients were identified as “Leishmania major variant” four differing from Friedlin strain of Leishmania major in the sequences of N-acetylglucosamine-1-phosphotransferase (NAGT) genes with four bases substitutions (Akman et al., 2000). Recently L. major and L. donovani were also reported as causative agents of CL in East Mediterranean Region of Turkey (Koltas et al., 2014). South and Southeast Anatolia regions are endemic for CL, while human VL and CanL have been mainly seen along the Aegean, Mediterranean coasts and Central Anatolia Region (Ok et al., 2002). Dogs are recognized as the main reservoir hosts of zoonotic VL and the prevalence of CanL in Mediterranean countries varies from 1.1% to 48.4% (Dujardin et al., 2008). Sand flies belonging to subgenera Larroussius and Adlerius are proven or suspected vectors of Leishmania in the Mediterranean Basin and in some Eurasian countries (Killick-Kendrick, 1990; Sadlova et al., 2003). In Turkey, the first epidemiological study for CanL showed a seroprevalence rate of 1.6% among 1150 dogs from Ege and Mediterranean Regions in 1981 (WHO, 1993). In the epidemiological studies carried out in 22 different provinces between 1993 and 2006 in Turkey, the average prevalence of CanL was reported as 15.7% (changing between 1.45% and 27.5%) (Dujardin et al., 2008). Zoonotic VL has been reported from all geographical regions of Turkey with low numbers. The total official VL cases are between 30 and 40 per year (Dujardin et al., 2008; MoH records) but these numbers are believed to be underestimated even the disease has compulsory notification in Turkey. Seroepidemiological studies were previously carried out in Turkey in different endemic regions for VL (Özensoy Töz et al., 1998 and Özbel et al., 2000). Visceral leishmaniasis cases have reported from the eastern part of the country by Büyükavcı et al. in 2005 and the authors were reviewed 21 cases of childhood VL reported between 1996 and 2002 from eastern Turkey, 60% of whom were from Kagizman town and its villages. In all 21 patients, the disease was diagnosed by demonstrating the amastigote forms of Leishmania in bone marrow aspiration smears. We therefore aimed to carry out entomological and seroepidemiological survey on human and dogs in Kagizman town and the villages where the VL patients were diagnosed for finding

risk factors in this geographical area located in high altitude and most northeastern part of Turkey. 2. Materials and methods 2.1. Study area Kagizman town (40 09 N; 43 08 E) belongs to Kars province and is located in most northeastern part of Turkey. The town is divided into 62 villages covering an area of 1972 km2 . Eleven and 10 VL cases were reported from Kagizman town and its 5 rural villages (Yukarikaraguney, Gunindi, Karabag, Kuloglu and Karakus) between 1996 and 2002, respectively. These five villages and Kagizman town, representing active foci of VL, were selected as study sites. A village (Kotek) where no VL cases reported so far was also included in the study as control area only for sand fly seasonal activity survey. Physical examination and all samplings were done between July and September 2006. The geographical features and other information about the sampling sites were given in Table 1. All of the villages are in high altitude (>1.300 m asl) and located near the Aras River that also crosses through Kagizman town and all settlements have similar ecological aspects. The climate is classified as a humid continental and meadows and grasslands are the dominant flora. Kagizman town has a temperate climate compared to other districts of Kars; the minimum mean temperature is ∼2.6 ◦ C in January and the maximum mean temperature is ∼23.1 ◦ C in July; the average annual rainfall is 425.5 mm. The major economic activity is cattle/sheep breeding. People are also engaged in orcharding and vegetable gardening. 2.2. Physical examination and sampling 2.2.1. Human A total of 290 children aged between 2 and 14 years old were randomly selected (Table 1). After receiving the written consent form from their parents, children underwent a physical examination for detection of clinical symptoms by authorized physicians. Five ml of venous blood sample was collected to the blood tube and centrifuged for obtaining sera. 2.2.2. Dogs A total of 165 household dogs from five villages were also selected randomly (Table 1). Dog sampling could not be performed in the districts of Kagizman town because of the all dogs were stray dogs. Prior to sample collection, the dogs were examined for clinical signs of CanL including weight loss, skin lesions, hair loss, local or generalized lymphadenopathy, epistaxis, onychogryphosis and kerato-conjunctivitis. Peripheral blood was taken from a femoral or brachial vein in 5 ml blood tubes and centrifuged for obtaining sera. Popliteal lymph node aspirates were obtained only from 6 dogs using 21 G needles and used for Giemsa-stained smears and parasite culture. Parasitological examination was regarded as positive on the basis of smear and/or culture positivity. Dog owners provided the consent form for sample collection from dogs.

Please cite this article in press as: Sari, B., et al., Seroepidemiological and entomological survey in a new focus of zoonotic visceral leishmaniasis in Kars province, Northeastern Turkey. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.02.021

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144 290 165

0

21 20.090 645 –

15

– –

1341 43 00 49 E 40 13 10 N

Total

–

10 10 21 11 8 0 60 65 103 62 27 40 22 28 48 1 4 1 2 2 1065 800 800 795 1000 245 110 100 70 120 20 10 17 17 30 43 15 37 E 43 09 50 E 43 01 15 E 42 57 23 E 43 23 17 E 40 12 27 N 40 14 00 N 40 09 50 N 40 05 33 N 40 10 35 N

1422 1565 1837 1329 1778

62 – – 11 23.012 – 0 43 07 15 E 40 08 22 N

Kagizman town (7 districts) Villages Gunindi Karabag Karakus Kuloglu Yukarikaraguney Control village Kotek

1419

Distance from Kagizman (km) Altitude (m) Long Lat

Table 1 The geographical characteristics and sampling information in the study area.

Number of houses

Population (2009 census)

No of previous VL Cases

Dogs sampled

Children sampled

No. of light traps set up

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2.2.3. Serological test Standard serodiagnostic procedures for human (de Korte et al., 1990) and dog (Abranches et al., 1991) were followed. The immunofluorescence antibody test (IFAT) was performed using promastigotes from local L. infantum MON-1 (MHOM/TR/05/EP126) stocks obtained by mass cultivation in RPMI–1640 containing 10% FCS. Briefly, promastigotes were washed eight times using phosphate buffered saline (PBS) by centrifugation and then re-suspended at a concentration of 2 × 106 ml−1 in saline. Ten ␮l of this suspension was transferred per spot onto multispot IFAT slides. After air-drying, the slides were stored at −20 ◦ C until further use. Twofold serial dilutions (1:16 to 1:16,384) of the human and dog sera in PBS were dispensed onto the antigencoated wells. Following an incubation at 37 ◦ C for 30 min, slides were washed and stained with FITC-labeled antihuman IgG conjugate (BioMerieux 75692) for human sera and FITC-labeled anti-dog IgG conjugate (Sigma, A9042) for dog sera. Slides were covered and examined under a fluorescence microscope. Titers ≥1:128 were scored as seropositive for both sera samples (Özbel et al., 2000). Samples with an antibody titer at 1:64 dilution were scored as borderline positive although the seroprevalence rates were calculated from only seropositives.

2.2.4. Sand fly collection All sand fly sampling was carried out, in five villages and seven districts belonging to Kagizman town from June to September 2006, using CDC miniature light traps operated overnight inside and/or outside of the houses and animal barns. The sand fly activity work was carried out only in Kagizman town and the control village, Kotek. Because of the very short summer season in this geographical region, the collections were made once every month in Kagizman town (between June and September – twice in July) and Kotek village (between July and September). The maximum and minimum temperature and humidity values were measured using thermo-hygrometer accompanying each light trap. The houses and animal sheds were usually made of brick, mud, wood and/or stone. Almost all villagers raise cattle and sheep and they have at least one dog. In sand fly sampling period, a total of 144 light traps were set, one or two light traps in each location. They were operated between 18:00 PM and 09:00 AM hours. The insect catch was checked each morning and sand flies were sorted and kept in 96% ethanol until identification. The collected sand flies were mounted on permanent microscope slides for species identification, which was carried out according to the keys by Lewis (1982), Artemiev (1980), Artemiev and Neronov (1984) and descriptions by Léger et al. (1983). The species identification was made by examining the morphology of male genitalia [aedeagus, coxite, style, spines on style, basal lobe (for Paraphlebotomus), length of penis pump, antennal formula] and shape of female spermathecae [number of segments, spermathecal duct] and pharynx [pharyngeal teeth and armature], and antennal formula.

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Table 2 Leishmaniasis seroprevalence by IFAT. Number and results of sampled dogs according to the five rural villages. Villages

Dogs sampled

Gunindi Karabag Karakus Kuloglu Yukarikaraguney Total

Seropositive dogs, N (%)

27 40 22 28 48

3 (11.1) 4 (10) 0 5 (17.8) 0

165

Borderline (1:64) dogs, N

Male/Female

2 5 1 1 5

12

26/1 35/5 22/0 28/0 41/7

14

2.2.5. Ethical consideration The study was approved by the Local Human Research Ethical Committee of the School of Medicine, Celal Bayar University, Manisa, Turkey (April 28, 2006; No: 0046). Animal part of the study was approved by the Local Animal Care and Ethics Committee of the School of Medicine, Ege University, Izmir, Turkey. The instructions and policies of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No: 85–23, revised 1996) were applied in the study.

152/13

Clinical symptoms POS (%) 5 (18.51) 5 (12.50) 1 (4.54) 5 (17.85) 9 (18.75) 25

clinical symptom and were polysymptomatic, respectively. On the other hand, at least one of the clinical symptoms was also detected in 13 (8.49%) out of 153 seronegative dogs. The presence of Leishmania amastigotes was confirmed by microscopy in one (58.33%) of 6 lymph node aspirates of which found to be seropositive (Table 2). It is also noted that the dogs are always used for guarding and kept outside of the houses by their owners in this region.

3.2. Sand flies 3. Results A total of 4.154 sand flies were collected using 144 light traps and 10 species of genus Phlebotomus were determined in the study area; P. balcanicus Theodor, Phlebotomus halepensis Theodor, P. kandelakii s.l. Shchurenkova, Phlebotomus neglectus Tonnoir, Phlebotomus galilaeus Theodor, Phlebotomus caucasicus Marzinowski, Phlebotomus jacusieli Theodor, Phlebotomus sergenti Parrot, Phlebotomus alexandri Sinton, Phlebotomus papatasi Scopoli. Some female specimens belonging to Adlerius subgenus could not be identified morphologically and they were noted as “unidentified Adlerius spp.” (Table 3). In overall collection, one Larroussius species, P. kandelakii s.l. (55.38%) and one Adlerius species P. balcanicus (12.61%), were most abundant species and found in all sampling sites and days (Figs. 1 and 2). The average female/male ratio was 0.70 (1093/1543) for all specimens. In periods of high sand fly activity, the maximum and

3.1. Human and dogs A total of 138 (47.5%) male and 152 (52.5%) female children were included in the study. None of the children was found to be seropositive although hepatosplenomegaly was detected in 12 (4.1%) children during physical examination. These children were then transferred to state hospital for further analysis. During the serological survey, we could reach 12 of the 21 children with previous history of VL and their antibody titers were ≤1:64. Overall, 12 of 165 dogs (7.27%) were found seropositive by IFA Test. Although no seropositive dog was detected in two villages, Karakus and Yukarikaraguney, the seropositivity rates of CanL were found to be between 10% and 17.8% in other three villages. Ten (83.33%) and two (16.66%) out of 12 seropositive dogs showed at least one

Table 3 The number and percentage of collected sand flies from each locality in study area. Sand fly species

Kagizman town

Villages Günindi

Adlerius Unidentified Adlerius spp. P. balcanicus P. halepensis Larroussius P. kandelakii s.l. P. neglectus P. galilaeus Paraphlebotomus P. caucasicus P. jacusieli P. sergenti P. alexandri Phlebotomus P. papatasi Total a b

198 439 84 1629b 8 1 1 29 – 2

Karaba˘g

Kulo˘glu

Y. Karagüney

Köteka

Total, N (%)

5 – 7

167 42 102

44 21 5

5 – 4

10 22 40b

429 (10.34) 524 (12.62) 242 (5.83)

32b 1 –

129 172b 0

490b – –

– – –

23 2 2

2303 (55.44) 183 (4.40) 3 (0.07)

12 – – –

18 1 20 –

20 – – 11

– 4 – –

– – – –

245

1

15

61

–

30

2636

58

666

652

13

129

51 (1.22) 34 (0.82) 20 (0.48) 13 (0.31) 352 (8.47) 4154

No VL patient was reported. Dominant species in the study site.

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Fig. 1. (a) Geographical location of study area, Kagizman town, in Turkey; and the density and distribution of (b) P. kandelakii s.l., (c) P. neglectus and (d) P. balcanicus.

minimum temperature and humidity ranged between 19.5 ◦ C and 33.4 ◦ C, and 26% and 82%, respectively. The dominant species according to the villages/districts are; (i) P. kandelakii s.l. in five districts of Kagizman town center (75.01%; 1597/2129), Kuloglu (75.15%; 490/652) and Gunindi (55.17%; 32/58) villages; (ii) P. balcanicus in two districts of Kagizman town center (47.14%; 239/507); (iii) P. neglectus only in Karabag village (25.67%; 172/670) and (iv) P. halepensis in Yukarikaraguney (30.76%; 4/13) and in control village, Kotek (31.00%; 40/129) (Table 3). 3.2.1. Seasonal activity Fig. 2 shows the changes in the seasonal activity of sand flies collected in the districts of Kagizman town. It is determined that eight species of Phlebotomus are active between June and August but, only three species, P. kandelakii s.l., P. balcanicus and P. halepensis are present during all transmission season. P. kandelakii s.l. has a sharp increase in mid of July (81.63%). Only two sand fly specimen was found in September. Fig. 2 shows the changes in the seasonal activity of four Phlebotomus species in Kagizman town and control village, Kotek. In Kotek village, P. halepensis and P. balcanicus have peaks in July and they decreased sharply in August and September. P. papatasi is predominant in August while the activity of other species decreased. More male specimens

than females were captured in June and July but we have observed an increase in the total number of female specimens toward end of the season, in August. Seasonal activity results according to the sex of total collected sand fly specimens and suspected vector species, P. kandelakii s.l., within Kagizman town were given in Table 4.

4. Discussion Information and knowledge on ecology and epidemiology of leishmaniasis is very important for effective and sustainable control of the disease (Rassi et al., 2005). Molyneux (1998) emphasizes the importance of stratification of epidemiological patterns in order to assess priorities and to design focused control strategies (Ashford, 1999). The present study was designed to understand the epidemiological specifications of leishmaniasis occurred in Kagizman town where new endemic site for VL is located. We also aimed to clarify risk factors in this geographical area located in high altitude and most northeastern part of Turkey. Serological screening of dogs by IFA Test showed that the disease is circulating among dogs probably via two species of sand flies belonging to Larroussius subgenus, P. kandelakii s.l. and P. neglectus. Here, we reported the results of first seroepidemiological and entomological study of a

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Fig. 2. Seasonal activity of four dominant Phlebotomus species in Kagizman town and control village, Kötek.

new emerging focus of human and canine VL in northeastern part of Turkey. Large-scale seroepidemiological screenings are more efficient to obtain data about seroprevalence of leishmaniasis among human. In the most recent study carried out in Tbilisi, Georgia very close to present study area, 4.250 children were screened for human VL by DAT and rK39 dipstick test and overall seroprevalence was reported as 7.3% (Giorgobiani et al., 2011). Several seroepidemiological studies related to leishmaniasis carried out in Turkey reported different seroprevalence rates of asymptomatic children by IFAT, 2.6% in northwestern part (Dogan et al., 2008) and 0.16% in the North Part of Central Anatolia (Ertabaklar et al., 2005). In a study performed in western part of Turkey, Western blotting were found to

be more reliable test among other diagnostic methods for detecting asymptomatic infections in endemic areas (Sakru et al., 2007). In the present study, we could not find any seropositive children by IFAT, probably because of the small sampling size. Dogs represent a significant risk factor for human VL as indicated by Gavgani et al. (2002). The villages included in the study are close to each other geographically (Fig. 1) and had similar vegetation. Previously, 4, 1 and 2 VL patients were officially reported in Karabag, Gunindi and Kuloglu villages, respectively (Büyükavcı et al., 2005). The seropositivity ratios were 10%, 11.1% and 17.8% among dogs in Karabag, Gunindi and Kuloglu villages, respectively. It is also noted that there are 2 and 1 VL patients in Yukarikaraguney and Karakus villages where no seropositive dogs

Table 4 Seasonal activity results according to sex of total collected sand fly specimens and suspected vector species, P. kandelakii, within Kagizman town. Months June P. kandelakii s.l. Male (%) Female (%) Sex ratio (F:M)

105 (56.75) 80 (43.25) 0.76

Total All sand flies Male (%) Female (%) Sex ratio (F:M)

185

Total

725

435 (60) 290 (40) 0.66

July 737 (54.75) 609 (45.25) 0.82 1346 981 (59.46) 669 (40.54) 0.68 1650

August 36 (31.58) 78 (68.42) 2.16

September

Total

0 0 0

878 767 0

114

0

1645

125 (48.27) 134 (51.73) 1.07

2 0 2

1543 1093 0.70

259

0

2636

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were found. Although similar findings were obtained or indicated in previous studies (Costa et al., 2002; Barao et al., 2007; Giorgobiani et al., 2011), no correlation was observed between number of previously reported VL patients and CanL seroprevalence in these villages (p = 0.703, ANOVA). The overall CanL seroprevalence rate was recorded as 7.2% and reported first time from this new endemic focus and it is one of the most important findings obtained in the present study. Several studies showed no specific correlation between seropositivity and gender or breed of the dogs (Mohebali et al., 2005; Papadopoulou et al., 2005). However, in our survey, we could not evaluate the relationship between seropositivity and gender or breed of dogs, because of the majority (92.7%) of them were male (people living in the region would like to keep male dogs) and all dogs were mix breed. P. kandelakii s.l. was found to be predominant sand fly species in Kagizman town (61.79%) and two villages, Kuloglu (75.15%) and Gunindi (55.17%). This species seems to play a role as vector of human and canine visceral leishmaniasis in the area (Table 3). In previous studies, natural infection of P. kandelakii and Phlebotomus perfiliewi with Leishmania promastigotes were demonstrated in Ardabil province, the most important focus of VL in northwestern Iran (Rassi et al., 1997). The vectorial status of P. kandelakii have proved by providing evidences of high anthropophily and showing the natural infection of Leishmania infantum In the Meshkinshahr district of Ardabil province (Rassi et al., 1997) and Shirvan district (Rassi et al., 2012) of Iran. These three endemic areas are very close to present study area. A study carried out in Tbilisi-Georgia a geographically closest area to our study area where an active focus of VL is (Giorgobiani et al., 2012), reports the presence of five species of Phlebotomus, P. balcanicus, P. halepensis; of the subgenus Adlerius; P. kandelakii, P. wenyoni of the subgenus Larroussius; P. sergenti of the subgenus Paraphlebotomus, with dominancy of P. kandelakii (33.5%) and P. balcanicus (18.9%). These authors also investigated the presence of Leishmania parasites by direct dissection method and found 12 (10 P. kandelakii; 2 P. balcanicus) infected among 659 female sand flies (Giorgobiani et al., 2012). P. balcanicus, the most abundant species in Van (Deger and Yaman, 2005), a neighboring city of Kars, was the second most abundant species in Kagizman town (Table 3). P. balcanicus together with P. kandelakii were reported as suspected vectors of VL in Armenia (Dergacheva and Ogenasyan, 1987). In our study area, we recorded 10 Phlebotomus species and determined the dominancy of P. kandelakii s.l. and P. balcanicus but we could not check female sand flies for parasites. Unlike their results, we could not found P. wenyoni but detected six more other species. P. neglectus, the proven vector of zoonotic VL in Greece (Léger et al., 1988; Chaniotis et al., 2000), was found to be predominant only in Karabag village. This species is the suspected vector in Yugoslavia (Ivovic´ et al., 2004), Albania (Velo et al., 2005), Croatia (Bosnic´ et al., 2006) and Italy (Maroli et al., 2008). P. neglectus is associated with VL transmission in western and central parts of Turkey (Tok et al., 2009; Ertabaklar et al., 2005). The species identification was also confirmed in a later study by molecular analysis based on mitochondrial cytochrome b (Cyt b) and nuclear

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elongation factor1 (EF 1-␣) genes (Kasap et al., 2013). The finding about the presence of P. neglectus in northeastern part of Turkey can be a milestone for the movement of this species from east to west. Azizi et al. (2006) reported P. alexandri infected with L. infantum in Fars province, southern Iran. P. caucasicus is a suspected vector of zoonotic CL in Central Iran (Rassi et al., 2004) which was found naturally infected with Leishmania promastigotes. In our study area, both sand fly species were present in low number. In a recent study, it is reported that Phlebotomus transcaucasicus was determined as vector species of L. infantum in northwestern Iran (Rassi et al., 2009). This species was not found during our entomological survey. However, three male specimens of P. galilaeus a phylogenetically related species is present in two villages although with very low numbers among all sand flies collected. The identification of the subspecies of P. perfiliewi group members is not easy (Depaquit et al., 2013) but our specimens were morphologically compared with the specimens from northern Cyprus where well known area for P. galilaeus. P. halepensis was caught in all villages and Kagizman town but only was predominant in the control village, Kotek. Although P. halepensis has widespread occurrence from Turkey to Israel and Jordan, across Transcaucasia to Iran and southern Turkmenistan and its distribution coincides with both visceral (L. infantum) and cutaneous (L. major, L. tropica) leishmaniases, the studies related to its vector statuses are very limited (Sadlova et al., 2003). In this study, the seasonality of the sand fly fauna in Kagizman town was also examined. Sand flies were active from mid of June to mid of August. Only one activity peak were noted in July that corresponds to the relatively high temperature and humidity. We also observed very similar results for suspected vector species, P. kandelakii s.l., within Kagizman town (Table 4). The seasonal activity of the most probable vector, P. kandelakii s.l. was higher in July (Fig. 2). However in Kotek where no VL cases are reported, P. kandelakii s.l. was not as active as P. halepensis or P. papatasi (Fig. 3). The findings related to sand fly seasonal dynamics recorded in Tbilisi, Georgia (Giorgobiani et al., 2012), are supported by our findings about exceptionally short season starting mid of June, peaking in July, dramatic decreasing in August and then zero in early September. The population size was very much higher in July than the other months. The observed increased captures in July probably depend on short suitable season for growing of sand flies. The activity of all species is also very high in July, declined markedly in August and sand flies were not collected in September, except two specimens. We also notified that this sharp decrease was the parallel of the sharp declining in the maximum temperature from 33.4 ◦ C to 16.3 ◦ C. The findings in the present study show the presence of several potential vectors of VL in the study area. As for the vector competence of the sand flies identified, P. kandelakii s.l., P. balcanicus and P. neglectus may all play a role in the transmission of Leishmania parasites in this new VL foci. P. galilaeus, P. caucasicus and P. alexandri had low density in the study area and they are unlikely to have a role in VL

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transmission, but complementary studies are suggested to determine the proven vector(s). Here, we reported the leishmaniasis infection rates in dog population and the distribution of suspected sand fly vector species in the northeastern part of Turkey for the first time. When we take into account present and previous studies together, it is confirmed that eco-epidemiological studies covering all aspects of leishmaniasis are necessary to establish “identity card” of each endemic area containing all these information. This can provide us a standardization to compare the endemic sites in all over the world as well as necessary differences for implementing or improving effective control methods according to the site. The risk factors detected in the area were; poor sanitary conditions and very high population size of sand flies probably because of very short season (July–August), no control measures against sand flies as well as to protect dogs, even though the area has high altitude, a microclimate is present especially in big town, Kagizman, and habitat suitability for transmitting the parasite. Conflicts of interest The authors declare there are no conflicts of interest. Acknowledgement This study is supported by Scientific Research Project Coordination Office of Celal Bayar University with the Project No: SH-2006-024. References Abranches, P., Silva-Pereira, M.C.D., Conceiao-Silva, F.M., Santos-Gomes, G.M., Janz, J.G., 1991. Canine leishmaniasis: pathological and ecological factors influencing transmission of infection. J. Parasitol. 77, 557–561. Akman, L., Aksu, H.A.Z., Wang, R.-Q., Ozensoy, S., Ozbel, Y., Alkan, M.Z., Ozcel, M.A., Culha, G., Ozcan, K., Uzun, S., Memisoglu, H.R., Chang, K.P., 2000. Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region. J. Eukaryot. Microbiol. 47, 545–554. Artemiev, M.M., 1980. A revision of sandflies of the subgenus Adlerius (Diptera, Phlebotominae, Phlebotomus). Zool. Zhurnal 59, 1177–1192 (in Russian; English translation edited by R. Killick-Kendrick and available on request to the corresponding author). Artemiev, M.M., Neronov, V.M., 1984. Distribution and Ecology of Sandflies of the Old World (Genus: Phlebotomus). Institute of Evolution, Morphology and Animal Ecology, USSR, Moscow, pp. 1–208. Ashford, R.W., 1999. Cutaneous leishmaniasis: strategies for prevention. Clin. Dermatol. 17, 327–332. Azizi, K., Rassi, Y., Javadian, E., Motazedian, M.H., Rafizadeh, S., Yaghoobi Ershadi, M.R., Mohebali, M., 2006. Phlebotomus (Paraphlebotomus) alexandri: a probable vector of Leishmania infantum in Iran. Ann. Trop. Med. Parasitol. 100, 63–68. Barao, S.C., Camargo-Neves, V.L.F., Resende, R.M., Da Silva, L.J., 2007. Human asymptomatic infection in visceral Leishmaniasis: a seroprevalence study in an urban area of low endemicity. Preliminary results. Am. J. Trop. Med. Hyg. 77, 1051–1053. ´ S., Gradoni, L., Khoury, C., Maroli, M., 2006. A review of leishBosnic, maniasis in Dalmatia (Croatia) and results from recent surveys on phlebotomine sandflies in three southern countries. Acta Trop. 99, 42–49. Büyükavcı, M., Karacan, M., Tan, H., Akda˘g, R., 2005. Visceral childhood leishmaniasis in Ka˘gızman, eastern Turkey: a new endemic area. Ann. Trop. Paediatr. 25, 41–43. Chaniotis, B., Spyridaki, I., Scoulika, E., Antoniou, M., 2000. Colonization of Phlebotomus neglectus (Diptera: Psychodidae), the major vector of visceral leishmaniasis in Greece. J. Med. Entomol. 37, 346–348.

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