Introduction Leishmaniasis is a group of zoonotic infections caused by protozoan parasites of the genus Leishmania; an estimated persons are

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1 Sero-epidemological Survey on Canine Visceral Leishmaniasis and the Distribution of Sandfly Vectors in Northwestern Turkey: Prevention Strategies for Childhood Visceral Leishmaniasis by Nihal Dogan, a Yusuf Ozbel, b Seray Ozensoy Toz, b Ener Cagri Dinleyici, c and Ozcan Bor c a Department of Microbiology, Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey b Department of Parasitology, Ege University Faculty of Medicine, Bornova, Izmir, Turkey c Department of Pediatrics, Eskisehir Osmangazi University Faculty of Medicine, Eskisehir, Turkey Summary Visceral leishmaniasis (VL) caused by Leishmania infantum, is an endemic disease in Aegean and Mediterranean Regions among humans and dogs. In this study, a sero-epidemiological survey for VL and cutaneous leishmaniasis (CL), which both are sporadically reported in the region, were carried out in the villages of Eskisehir, Afyon, and Bilecik cities. The study was designed according to the location of the sporadic cases of VL and CL, and blood samples of 111 dogs were randomly collected. Lymph node aspiration samples were taken from dogs that have popliteal lymphadenopathy. Sand flies were also collected using CDC light traps in the several localities. The sera samples were screened using IFAT, ELISA, rk39 ELISA and dip-stick tests for anti-leishmania antibodies. A total of 15 (13.51 per cent) dogs out of 111 were found to be seropositive by at least one of the tests. The seropositivity ratios among dogs were found to be 27.5 per cent (8/29), 9.09 per cent (4/44) and 7.8 per cent (3/38) in Afyon, Bilecik and Eskisehir cities respectively. Leishmania amastigotes were detected in 4 of the 14 lymph node aspiration samples (eight seronegative, six seropositive), and all of them were seropositive dogs. One year later, two of the dogs were found to be dead and the other two were severely ill. Among the 179 collected Phlebotomus specimens from, Phlebotomus major was found to be abundant (35.7 per cent) and the other species were P. simici (28.5 per cent), P. similis (34.7 per cent) and P. alexandri (1.1 per cent). In the study area, canine VL is more spread than human VL. Because dogs are playing an important role for VL in Mediterranean Basin, and development of appropriate control measures will be necessary for childhood VL. Introduction Leishmaniasis is a group of zoonotic infections caused by protozoan parasites of the genus Leishmania; an estimated persons are Acknowledgements We thank the Health Directorate of Eskisehir, Ku tahya, Afyon and Bilecik cities for helping us in the field to collect blood samples from children and dogs. Special thanks go to Dr Metin Korkmaz and Dr Nermin Sakru from Ege University Medical School Department of Parasitology for helping us in laboratory work. This work was supported by the grant (No: 2000/4) from Research Support Department of Osmangazi University. Correspondence: Dr Ener Cagri Dinleyici, Department of Pediatrics, Eskisehir Osmangazi University Faculty of Medicine, TR-26480, Eskisehir, Turkey. 5timboothtr@yahoo.com4. affected per year, worldwide. In recent years there have been exciting advances in diagnosis, treatment and prevention of leishmaniasis [1, 2]. Two type of leishmaniasis among human has been shown in Turkey; anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica and visceral leishmaniasis (VL) caused by Leishmania infantum [3, 4]. The VL caused by L. infantum is zoonotic and domestic dogs act as principal reservoir host and transmission is by the bite of a phlebotomine sandfly [1, 2]. Canine visceral leishmaniasis (CVL) is a widespread disease in all Mediterranean countries, with an average prevalence of 5 8 per cent in the total dog population, exceeding 30 per cent in some areas [5, 6]. The knowledge about the epidemiology is limited in Turkey [7]. As in humans, the disease elicits strong humoral immune response in infected dogs and in recent ß The Author [2005]. Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 212 doi: /tropej/fmi102 Advance Access Published on 15 November 2005

2 Fig. 1. Map of Turkey showing the study area. years, canine serology has been frequently used not only to confirm patient infections but also to detect latent infections. Different diagnostic methods are used for Leishmania mass screening surveys but dipstick test as a rapid, sensitive, and specific diagnostic test would be extremely valuable in mass screening and intervention campaigns [8]. Extensively genotyping studies also showed that all isolates obtained from human and dogs belong to the same group, as Leishmania infantum MON1 [9]. Epidemiological surveys on leishmaniasis are basic research to understand the situation and to set up control strategies in the endemic regions. The present field survey was planned after seven childhood VL and four CL coming from the study region. In this study, we are reporting that epidemiological situation (incidence of CVL with different diagnostic procedures including dip-stick test and analysis of distribution of vector Phlebotomus species) of leishmaniasis in the new focus for childhood VL. Materials and Methods Study area and sampling Totally seven different localities, each have one human VL case which were reported between 1997 and 2000 years and confirmed parasitologically by bone marrow aspiration., were chosen for the present study (Fig. 1). The altitudes of the villages included in this study varied from 120 to 1500 meters above sea level. The main occupation of the villagers was farming, animal and silkworm breeding. In Afyon city, people are mainly working in marble factories. The ACL cases only were reported from Iscehisar Table 1 The seropositivity ratio according to the cities City Dogs sampled n Seropositive n (%) Seronegative n (%) Afyon 29 8 (27.5) 21 (62.5) Bilecik 44 4 (9) 40 (91) Eskisehir 38 3 (7.9) 35 (92.1) Total (13.5) 96 (86.5) town of Afyon city. Dogs sampled in the villages had owners and were used for hunting and/or guarding. Dogs were kept outside the houses. In total, 111 dog blood samples were collected from three villages in Bilecik city; one village in Kutahya city, two towns in Afyon city and one district located in the center of Eskisehir city (Table 1). The sera were separated, transported to the laboratory and stored at 20 C until use. Serological tests IFAT: IFAT was carried out using promastigotes from local L. infantum stocks (MHOM/TR/95/EP14) obtained by mass culturing in RPMI-1640 containing 10 per cent FCS. Promastigotes were harvested and washed eight times in phosphate-buffered saline (PBS). The methodology for human serodiagnosis has been described previously. Titers 1:128 were considered as seropositive [5]. rk39 ELISA: Microtiter plates were coated overnight with 20 ng of rk39/well (2 mg antigen in 10 ml of 100 mm bicarbonate buffer, ph 9.4 at 4 C). Plates were blocked with 100 mg of casein buffer (ph 7.4) Journal of Tropical Pediatrics Vol. 52, No

3 Table 2 Clinical and laboratory findings of seropositive dogs No Dog Code City/Village Age/Sex Smear IFAT rk39 ELISA Whole ELISA Intersept Symptoms 1 BIL-D02 Bilecik/Kayabali 10/M Neg 256 POS POS Neg LAP 2 BIL-D03 1/F Neg 128 POS POS POS LAP 3 BIL-D5* Bilecik/Kure 9/M POS 2048 POS POS POS Weakness, fever, hair depilation, skin lesions 4 BIL-D21 10/M Nd 1024 nd POS POS LAP 5 AFY-D01 Afyon/Iscehisar 6/M POS 2048 POS POS POS LAP 6 AFY-D03 8/M POS 512 POS POS POS LAP 7 AFY-D09 3/M Nd 256 POS POS POS LAP 8 AFY-D10 1/M POS 4096 POS POS POS Weakness, skin lesions, hair depilation, LAP,onycogryphosis 9 AFY-D12 2/M Nd 512 POS POS POS Normal 10 AFY-D21 9/M Nd 256 POS POS POS Normal 11 AFY-D23 Nd Nd 256 POS POS POS Normal 12 AFY-D28 Nd Nd 256 POS POS POS Normal 13 ESK-D16 Eskisehir/Center 2/M Nd 128 POS POS POS Normal 14 ESK-D17 4/M Nd 256 POS POS Neg Normal 15 ESK-D19 4/M Nd 256 POS POS POS Normal *Successfully cultivation; nd: Not done. for 1 h and incubated with 100 ml of sera diluted in casein buffer (1:400) for 1 h at 37 C. The methodology for human serodiagnosis has been described previously. Values greater than the mean of negative controls plus three standard deviations were considered positive [4]. Whole ELISA: Leishmania infantum promastigotes (MHOM/TR/95/EP14- MON1) were solubilized in SDS (1:100) and were diluted in 100 mm bicarbonate buffer, ph 9.4. One hundred ml per well were used to coat 96-well microtiter plates (Linbro) 1 h at room temperature. The methodology for human serodiagnosis has been described previously. Values greater then the mean of negative controls plus three standard deviations were considered as positive [4]. Dip-stick test (Intersep Leishmania Strip Test): This is a rapid chromatographic immunoassay for the qualitative detection of antibodies against Leishmania in serum or plasma (Intersep Leishmania Test, Berkshire, UK). The methodology for human serodiagnosis has been described previously. Lymph node aspiration: Popliteal lymph node puncture was performed by a veterinarian, aseptically to dogs having popliteal lymphadenopathy. Tissue aspirates were used for culturing on NNN medium and also stained with Giemsa and examined by microscopy. Sand fly collection Sand flies were collected by CDC light traps. Traps were placed in and near the houses of past VL patients. On each catching night, three light traps were set in each of the selected houses. Flies were immersed in 70 per cent ethanol and mounted in chloralhydrate medium for later identification. Species-identification was done mainly according to the keys and descriptions presented by Killick- Kendrick [11]. Results The seropositivity ratios among dogs were found to be 27.5 per cent (8/29), 9.09 per cent (4/44) and 7.89 per cent (3/38) in Afyon, Bilecik and Eskisehir cities respectively. The all information about seropositive dogs was shown in Table 2. The concordance between IFAT and whole or rk39 ELISA tests results was 100 per cent, excluding one dog rk39 ELISA could not be applied. Dipstick test was positive 13 out of 15 dogs. Lymph node aspiration was performed for 14 (8 seronegative, 6 seropositive) dogs having lymphadenopathy. Only four coming from seropositive dogs out of 14 lymph aspiration samples were found to be positive. These four cases were also positive with four serologic examinations. Clinical symptoms (weakness, skin ulcer, scaling, depilation, onychogriphosis, fever, lymphadenopathy, fatigue, conjunctivitis) were observed in two smear-positive dogs. Leishmania parasites were isolated from one dog (BIL-D5) having clinical symptoms in Bilecik city. One year later, two of the dogs were found to have died and the other two were severely ill. 214 Journal of Tropical Pediatrics Vol. 52, No. 3

4 Table 3 Phlebotomus species collected in two regions Subgenus Species Bilecik Iscehisar Total Female Male Total% Female Male Total% Female Male Total% Adlerius P. simici % % % Paraphlebotomus P. sergenti % % % P. alexandri % Larroussius P. major % % % Total A total of 166 and 13 Phlebotomus specimens were collected. Four Phlebotomus species identified belonging to Paraphlebotomus (Phlebotomus sergenti, P. alexandri), Larroussius (P. major) and Adlerius (P. simici) subgenus. Three species were found to be abundant equally, except P. alexandri in Bilecik while only one species, P. major, was abundant in Afyon (Table 3). Discussion The changes in seropositivity among dogs in a wide range ( per cent) has shown that CVL is an important factor in the protection of public health [5 7]. The percentage of seropositivity of our study is in accordance with previous studies performed other regions recorded human VL cases in Turkey [4 9]. In a detailed screening carried out in the localities where human VL cases have been found in western Turkey, the mean seropositivity rate among dog populations was 5.3 per cent (range 3.6 per cent 25 per cent) [7]. Since a high proportion of infected dogs are asymptomatic, serodiagnosis is essential in evaluating the prevalence of Leishmania infections in large dog populations [12]. However, serodiagnosis may not detect early infections and may also underestimate prevalence of infection [13]. The clinical symptoms were observed in two dogs among seropositive dogs (13.3 per cent) in our study. Similar observations were made in other regions in Turkey and Mediterranean countries [5, 7, 14, 15]. In dogs, clinical symptoms of the disease are included skin lesions, such as exfoliative dermatitis, local or generalized lymphadenopathy, weight loss, epistaxis, diarrhea and renal failure. Lymphadenopathy was the most frequently encountered symptom like our study [16]. The gold standard for diagnosis of VL is the detection of parasites in tissue smears especially spleen aspirates [17]. However difficulties in obtaining and examining tissue mean that serological methods are increasingly being used. Ozensoy et al. [4] reported that 18 of 494 dogs were positive by rk39-elisa and that sensitivity and specificity were reported to be 93 per cent and 100 per cent, respectively. Reithinger et al. [8] reported that ELISA was 100 per cent sensitive in detecting culturepositive dogs and 84 per cent sensitive for the detection of parasitologically-confirmed field dogs. The sensitivity of a diagnostic test can change with the course of infection and that these dogs appear to have developed an immune response that controls infection. The sensitivity and specificity of ELISA depend on the type of antigen used and changes to the standard experimental protocol. In this study, the concordance between IFAT and whole or rk39 ELISA tests results was 100 per cent, excluding one dog rk39 ELISA could not be applied. The rapid test used in the present study has per cent (13/15) concordance with other serological tests. Rapid strip test is used to detect antibody to rk39 and is both sensitive ( per cent) and specific ( per cent), whereas these diagnostic procedures were less sensitive in Europe and Africa than India [1]. With dipsticks, a large number of samples can be processed quickly and with minimum effort. Compared to microscopy, ELISA or PCR, the technological expertise (i.e., training of personnel) necessary to perform the dipstick tests is minimal, as is the requirement for specialized laboratory equipment. Another advantage of dipstick tests is that patients (in our case, dog owners) can see the results for themselves, which will contribute to a better working relationship between local communities and people carrying out the surveys and would increase compliance rates. From an epidemiological point of view, a dipstick test allows interventions to be implemented in situ. The outcome should be to significantly reduce the mean duration of infection Journal of Tropical Pediatrics Vol. 52, No

5 of dogs that have become infected, thereby significantly enhancing the impact of the intervention on the basic reproductive number. Four Phlebotomus species identified P. sergenti, P. alexandri, P. major and P. simici during our study in the two different regions. Previous surveys have identified 17 species of sandflies which are potential vectors, P. papatasi, P. sergenti, P. neglectus, P. syriacus, P. perfilievi being the most common within the Aegean and Mediterranean regions [18, 19]. In this field study, same Phlebotomus species previously reported were found, but interestingly, three species of Phlebotomus identified in the regions were showed almost same abundance [18, 20]. Many reports confirm the importance of Larroussius spp. in the transmission of L. infantum and show that more than one species of this subgenus may transmit in the same place [11]. Spraying houses with insecticide is the most widely used intervention for controlling sand flies in endemic areas [21]. Where sand flies are endophagic and most active when people are asleep, bed nets provide considerable protection [22]. In endemic areas, a dog-culling program is used for prevention of VL, but determining of infection in dogs takes a long time. Also the study region is not endemic area and people were sleep inside, deltamethrin-treated collars may provide dogs with long term protection against sand fly bites. Field trials on CVL in Iran showed that providing all dogs in a community with collars also protects children against L. infantum infection [2]. Childhood VL may be fatal despite a new diagnostic method and treatment modalities. We have to point out that the potential risk of high CVL prevalence, while only sporadic, human VL cases have been reported in these places. We conclude that prevention of childhood VL may be possible with the some prevention strategies, as follows: To organize several courses on the situation of leishmaniasis to the physicians and the veterinarians working in the region. To report new cases to the local Health Directories in the city. To treat all new cases immediately and to follow the patients after therapy. To give basic knowledge about vector Phlebotomus and personnel protection methods. To perform residual insecticide spraying of houses and also of animal shelters by municipalities. To whitewash the walls of all animal barns to prolong and increase the effect of insecticide. To diagnose new canine leishmaniasis cases and treat, or remove, immediately. To use insecticide-impregnated dog collars to protect dogs against Phlebotomus. References 1. Davies CR, Kaye P, Craft SL, et al. Leishmaniasis: new approaches to disease control. BMJ 2003;326: Gavgani AS, Hodjati MH, Mohite H, et al. 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