Microsoft Word - 2- Dr yaghoobi RTL.doc Iranian J Arthropod-Borne Dis, 2010, 4(1): 17–23 MR Yaghoobi-Ershadi et al.: Sand fly Surveillance within … 17 Original Article Sand fly Surveillance within an Emerging Epidemic Focus of Cutaneous Leishmaniasis in Southeastern Iran *MR Yaghoobi-Ershadi1, M Hakimiparizi1, AR Zahraei-Ramazani2, H Abdoli2, AA Akhavan1, M Aghasi3, MH Arandian2, AA Ranjbar4 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Iran 2Isfahan Training and Health Research Center, National Institute of Health Research, Tehran University of Medical Sciences, Iran 3School of Public Health, Kerman University of Medical Sciences, Iran 4Control of Communicable Disease Unit, Department of Public Health, Rafsanjan University of Medical Sciences, Iran (Received 19 Dec 2009; accepted 10 Apr 2010) Abstract Background: Cutaneous leishmaniasis due to Leishmania major has become a hot topic in Iran. The objective of this study was to determine some ecological aspects of sand flies in the study area. Methods: Sand flies were collected biweekly from indoors and outdoors fixed places in the selected villages, using 30 sticky paper traps from the beginning to the end of the active season of 2006 in Kerman Province, south of Iran. The flies were mounted and identified. Some blood fed and gravid female sand flies of rodent burrows and indoors were dissected and examined microscopically for natural promastigote infection of Leishmania parasite during Au- gust to September. Results: In total, 2439 specimens comprising 8 species (3 Phlebotomus and 5 Sergentomyia) were identified. The most common sand fly was P. papatasi and represented 87.1% of sand flies from indoors and 57.2% from outdoors. The activity of the species extended from April to end October. There are two peaks in the density curve of this spe- cies, one in June and the second in August. Natural promastigote infection was found in P. papatasi (12.7%). Conclusion: Phlebotomus papatasi is considered as a probable vector among gerbils and to humans with a high percentage of promastigote infection in this new focus of cutaneous leishmaniasis. The Bahraman area which until recently was unknown as an endemic area seems now to represent a focus of zoonotic cutaneous leishmaniasis trans- mission in Iran. Keywords: Sand fly, Phlebotomus papatasi, Ecology, Leishmania major, Iran Introduction Phlebotomine sand flies are of wide- spread importance in the transmission of Leish- mania pathogens in Iran. Increasing knowledge of sand flies is a cornerstone for establishing control measures to prevent leishmaniasis. Cu- taneous and visceral leishmaniasis are endemic in Iran and remain to be a growing health threat to community, development and the en- vironment in our country (Nadim et al. 1994). In the last two decades, the reported cases especially that of cutaneous leishmaniasis due to Leishmania major (CLM) have increased, and they were reported in areas that previ- ously were known to be non- endemic by the public health services (Yaghoobi- Ershadi et al. 2001, 2004). This ignored form of ne- glected diseases is common in many rural ar- eas of 17 out of the 30 provinces in Iran. About *Corresponding author: Prof Mohammad-Reza Yaghoobi- Ershadi, E-mail: yaghoobi.reza@gmail.com Iranian J Arthropod-Borne Dis, 2010, 4(1): 17–23 MR Yaghoobi-Ershadi et al.: Sand fly Surveillance within … 18 80% of leishmaniasis cases reported in the country are of the CLM form. Based on the reports of Control of Communicable Disease Unit, Rafsanjan Health Center, southern Iran, cutaneous leishmani- asis has not been presented in the area until 2002. Since 2004 the reported cases of cu- taneous leishmaniasis have suddenly increased in the study area. During 2003-2004, a total of 257 cases were officially reported all by passive case detection, most of them were from Bahraman District (Rafsanjan Health Center, unpublished data) 70 km from Raf- sanjan County but this is probably a large underestimate. A preliminary survey showed that Rhombomys opimus was the main reser- voir host and zoonotic cutaneous leishmani- asis is caused by L. major (School of Public Health, Tehran University of Medical Sciences, unpublished data). Bahraman rural district (Noogh) of Raf- sanjan County is an important agricultural axis from the point of view of growing pistachio in Iran so in depth entomological study must be carried out for preparation of control pro- gram to prevent the spread of disease to the neighboring villages and counties. The objective of this study was to de- termine the species richness and relative abun- dance of sand flies, their distribution, monthly prevalence, number of generations and leish- manial infection of sand flies in the area. Materials and Methods Study site The investigation was carried out from 10 April to 11 October 2006 in 5 villages, Javadieh, Aliabad, Ahmadabad, Najmabad and Daghogh, in the rural district of Bahraman, Raf- sanjan County, Kerman Province, in the southeast of the Islamic Republic of Iran. Rafsanjan County (29º 45´ N, 54º 50´- 56º 45´ E) is situated in the south east of Iran, 100 km northwest of Kerman at an altitude of 1460 m above sea level. It has a desert climate, very hot and dry in summer, cold and dry in winter. In 2006, the maximum mean monthly temperature was 31.9º C (July) and the minimum was 5.6º C (January). The total annual rainfall was 32.6 mm, with a monthly minimum of 0.2 mm (July and August) and maximum of 17.4 mm (January). The mini- mum mean monthly relative humidity was 14% in August and the maximum was 61% in March (Rafsanjan Meteorological Office, per- sonal communication) Sand flies sampling and monitoring Sand flies were collected biweekly from fixed sites indoors (bedrooms, sitting rooms, toilets and stables) and outdoors (rodent bur- rows), using 30 sticky traps (castor oil-coated white papers 20cm×30cm) from the begin- ning to the end of active season (early April- mid October). Collected sand flies were stored in 70% ethanol. For species identifi- cation, sand flies were mounted in Puri's me- dium, produced at the medical entomology department (Smart et al. 1965) and identified after 24 h using the keys of Theodor and Mesghali (Theodor and Mesghali, 1964), then they were mounted and segregated by sex. Detection of natural leptomonad infection in sand flies and their age determination Sand fly collections were made during August to September in the selected villages using sticky traps around rodent burrows and aspirator from human dwellings. Collections were transferred to the laboratory at Rafsan- jan health center for dissection. Blood fed fe- males collected indoors were kept alive for 3-4 days to allow blood digestion and then dissected. All fed, gravid, semi-gravid and some unfed females collected from rodent bur- rows were dissected in a fresh drop of sterile saline (9/1000) and examined microscopically for natural promastigote infection in the ali- mentary canal. The physiological age of each Iranian J Arthropod-Borne Dis, 2010, 4(1): 17–23 MR Yaghoobi-Ershadi et al.: Sand fly Surveillance within … 19 female was determined by the presence or absence of granules in the accessory glands (Foster et al. 1970). All female sand flies were identified by the morphology of the pharyn- geal armature and of the spermatheca (Theo- dor and Mesghali 1964). Results In this entomological survey, a total of 2439 adult sand flies (2152 from rodent bur- rows and 287 from indoor resting places) were collected and identified. They comprised 8 phlebotomine species with three Phleboto- mus spp. and five Sergentomyia spp. (Table 1). The following five spices were found in- doors: P. papatasi (87.1%); P. sergenti (0.7%); S. clydei (5.2%); S. sintoni (1.4%) and S. baghdadis (5.6%). In the rodent burrows: P. papatasi (57.2%); P. sergenti (0.2%); P. mongolensis (0.05%); S.clydei (22.7%); S. tiberiadis (0.05%); S. sintoni (13.3%); S. dentata (0.1%) and S. baghdadis (6.4%). The most prevalent species of indoors were P. papatasi, S. clydei and S. baghdadis and in case of outdoors they were P. pa- patasi, S. clydei, S. sintoni and S. baghdadis respectively. While other species were less frequent. The number and relative abundance of these species are given in Table 1. Phlebotomus papatasi started to ap- pear in late April and disappeared in mid October in rodent burrows. There are two peaks in the density curve of this species, one in early June and the second in early Au- gust (Fig. 1) while, the first peak of activity of the species was at the end of May and the second in mid of July in indoors. The de- crease in Sand fly density in mid October was most probably due to the cold weather and rains. There are also two peaks in the density curve of other species in rodent burrows in the area, one in late May or early June and the second in middle of August (Fig. 2). The sex ratio, i.e., number of males per 100 females of P. papatasi was 525 and 126 in indoors and rodent burrows respectively by the trapping method, sticky trap. P. pa- patasi (165), S. clydei (8), S. baghdadis (2) and S. sintoni (2) were collected in the vi- cinity of rodent burrows and were dissected. The results of these dissections showed that P. papatasi (12.7%) was infected with pro- mastigotes. In three cases a large number of promasitgotes were observed in the gut as well as in the head of P. papatasi. In September 2006, six P. papatasi were collected from indoor places and dissected for the presence of promastigotes. All were negative (Table 2). 0 50 100 150 200 250 9 Apr. 23 Apr. 6-M ay 20-M ay 29-M ay 10-Jun 26-Jun 11-Jul 29-Jul 14 Aug. 29 Agu. 15 Sept. 27 Sept. 11 Oct. Month,2006 no .o fS an d fli es pe r 30 Tr ap s P.papatasi outdoors P.papatasi indoors Fig. 1. Monthly prevalence of P. papatasi in indoors and outdoors, Bahreman area, Rafsanjan County, 2006 Iranian J Arthropod-Borne Dis, 2010, 4(1): 17–23 MR Yaghoobi-Ershadi et al.: Sand fly Surveillance within … 20 0 20 40 60 80 100 120 140 9 A pr . 23 Ap r. 6- Ma y 20 -M ay 29 -M ay 10 -Ju n 26 -Ju n 11 -Ju l 29 -Ju l 14 Au g. 29 Ag u. 15 Se pt . 27 Se pt . 11 Oc t. Day of sand fly collection no .o fs an d fli es pe r 30 tr ap s S.clydei S.baghdadis S.sintoni Fig. 2. Monthly prevalence of S. sintoni, S. baghdadis and S. clydei in Bahreman area, Rafsanjan County (rodent burrows), 2006 Table 1. Richness, number, and relative abundance of sand fly species collected from indoors and outdoors in Bah- raman rural district, Rafsanjan County, Kerman Province, Iran, 2006 Table 2. Natural promastigote infection in sand flies collected from rodent burrows, Bahraman rural district, Rafsanjan County, Kerman Province, Iran, summer 2006 Site Species No. of sandflies dissected Age group No. of sandflies with promastigotes % infected N P ? T G O H Outdoors P. papatasi 165 13 141 11 21 18 3 3 12.7 S. clydie 8 1 6 1 0 0 0 0 0 S. baghdadis 2 0 2 0 0 0 0 0 0 S. sintoni 2 0 1 1 0 0 0 0 0 Indoors P. papatasi 6 0 5 1 0 0 0 0 0 T, total; G, gut; O, oesophagus; H, head; P, parous; N, nuliparous;? age not known* *Some females that oviposited all of their eggs had accessory glands devoid of identifiable granules so recognizing parous sand flies from nuliparous ones was impossible. Species Collection site Richness No. and (%) of sand flies P .papatasi P .sergenti P . m ongolensis S.clydei S.tiberiadis S.sintoni S.dentata S.baghdadis No. 250 2 0 15 0 4 0 16 Indoors 5 % 87.1 0.7 0 5.2 0 1.4 0 5.6 Outdoors 8 No. 1230 4 1 490 1 286 2 139 % 57.2 0.2 0.05 22.7 0.05 13.3 0.1 6.4 Iranian J Arthropod-Borne Dis, 2010, 4(1): 17–23 MR Yaghoobi-Ershadi et al.: Sand fly Surveillance within … 21 Discussion In the rural district of Bahraman, Ker- man Province, cutaneous leishmaniasis due to L. major is a serious and increasing public health problem (Rafsanjan Health Center 2005, Kerman University of Medical Sciences, un- published data). This paper presents the first report of sand flies in this focus. Eight spe- cies (three of Phlebotomus and five of Ser- gertomyia) were collected and identified. P. papatasi, a proven vector of L. major in Iran (Yaghoobi-Ershadi et al. 1995), is the most abundant species in Bahraman, specially in in- doors. Phlebotomus sergenti accounts for 0.7% and 0.2% of sand flies collected from indoors and outdoors respectively and was found only in some villages in the region. This species has never been implicated in the transmis- sion of L. major in Iran. Only one male of P. mongolensis (0.05%) was collected from ro- dent burrows. With regard to its low fre- quency, it seems that the role of females of this species in the circulation of L. major among the rodents is doubtful in this part of Iran. Phlebotomus papatasi has two peak activities in indoors and outdoors and it re- presents that the spices has two generations in the study area. In routine collections by sticky traps in indoors and rodent burrows, males of P. pa- patasi predominate. The same results were also obtained on S. baghdadis and S. clydei in rodent burrows. In case of S. sintoni col- lected from outdoors, females were predomi- nated. These results conformed to what was happened in other parts of the country such as Isfahan, Badrood, Natanz, Nikabad, Arde- stan, Abardezh of Varamin, Sabzvar, Garm- sar and so forth (Yahgoobi- Ershadi and Javadian 1997,Yaghoobi-Ershadi et al. 2001, 2004, 2003, Akhavan et al. 2007). It should be mentioned that the parameter, sex ratio varies greatly according to the trapping method and among species of Genus Phlebotomus and Sergentomyia (Schlein et al.1989). The estimation of the age structure by dissection of the 165 females of P. papatasi from rodent burrows during the period of ob- servation, showed an overall parous: nulipa- rous ratio of 141:13. It should be mentioned that about 6.7% of the females of this spe- cies that would have oviposited all of their eggs had accessory glands devoid of identifi- able granules so recognizing parous sand flies from nuliparous ones were impossible. Leishmanial infection rates of P. pa- patasi from rodent borrows of the other CLM foci in Iran (i.e. Abardezh, Ahwaz, Dezful, Isferayen, Lotfabad, Shush, Turkmen- Sahra, Isfaham and Badrood) ranged from 0.2% to 15.6% during 1967-2001 (Mesghali et al. 1967,Javadian et al. 1976, Nadim et al. 1994, Yaghoobi-Ershadi and Akhavan 1999). In the present study, 12.7% of the vec- tor species were infected. Comparison of the present findings with those obtained from Iran and other countries (Nadim et al. 1994, Jan- ini et al. 1995) showed high natural infection rates with promastigotes from rodent burrows in Rafsanjan of southern Iran. Based upon these entomological data, P. papatasi is con- sidered the most likely vector of CL in Raf- sanjan. However, further studies are neces- sary to isolate Leishmania strains from P. papatasi and to characterize the genotype of Rafsanjan sand flies, taking into account cur- rent findings about the multi- locus microsa- tellite typing which has been employed to in- fer the population structure of P. papatasi in provinces of Khorasan, Khuzestan, Baluchis- tan, Azerbaijan, Isfahan, Fars and Yazd of Iran (Hamarsheh et al. 2009). In conclusion, based on this survey, Bahraman rural district seems to be a typical focus of P. papatasi and L. major. It should be added to the list of CL foci due to L. ma- jor, so it is necessary to propose a new update map of the distribution of these foci in Iran. Iranian J Arthropod-Borne Dis, 2010, 4(1): 17–23 MR Yaghoobi-Ershadi et al.: Sand fly Surveillance within … 22 Acknowledgments We are grateful to DM Gooya, Direc- tor General, Center of Communicable Dis- eases Control and Dr A Mozaffari from Raf- sanjan University of Medical Sciences for their close collaboration. Our appreciation is also offered to M Alizadeh, K Mohammadi Poor and M Mozaffari from Rafsanjan Health Center for helping us in carrying out this program. This study was financially supported by Na- tional Institute of Health Research, Academic Pivot for Education and Research, Tehran University of Medical Sciences ( Project No: 241.68.1) and partly by Tehran University of Medical Sciences (Grant number 3215-63-02- 85 to Dr MR Yaghoobi-Ershadi). The authors declare that they have no conflicts of interest. 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