Nocturnal activities and host preferences of Phlebotomus orientalis in extra-domestic habitats of Kafta-Humera lowlands, Kala-azar endemic, Northwest Ethiopia
© Lemma et al.; licensee BioMed Central. 2014
Received: 28 September 2014
Accepted: 6 December 2014
Published: 17 December 2014
Phlebotomus orientalis feeds on a variety of wild and domestic animals and transmits Leishmania donovani from hitherto unknown reservoir hosts to humans in extra-domestic habitats in the Metema - Humera lowlands. The aim of this study was to determine the nocturnal activities of P. orientalis and its preferred blood meal hosts.
Collections of Phlebotomus orientalis were made by using CDC light traps to determine the density as P. orientalis/hour CDC trap and preference to rodents by using Turner’s traps in agricultural fields, animal shelters and thickets of Acacia seyal in Baeker site-1 and Gelanzeraf site-2. The blood meal sources were detected by Reverse Line Blot (RLB) of cytochrome b polymerase chain reaction (PCR) amplification in August, 2012 from collections of sand flies in thickets of A. seyal (March 2011) and dense mixed forest (July 2011) in Baeker site 1. RLB PCR involved first amplification of animal specific sequences of cytochrome b using PCR techniques. Then the amplified sequence was hybridized with 11 species-specific probes for domestic animals adsorbed on nitrocellulose membrane for calorimetric color detection.
A total of 6,083 P. orientalis (2,702 males and 3,381 females) were collected at hourly intervals using 22 CDC traps from January to May 2013. The peak activities of P. orientalis were at 1.00 a.m (134.0 ± 7.21) near animal shelters, 3.00 a.m (66.33 ± 46.40) in agricultural fields and 21:00 pm (40.6 ± 30.06) in thickets of A. seyal. This species was not attracted to the different species of rodents in trials carried out in March and April 2013. RLB PCR identified 7 human (28%), 9 mixed (human and cattle) (36%) and 2 cattle (8%) blood meals while 7 were unknown (28%).
Female P. orientalis can bite humans in extra-domestic habitats of Kafta-Humera lowlands at any hour of the night with peak biting after midnight.
Of the total half a million kala-azar (Visceral Leishmaniasis; VL) cases in the world, 90% occur in Bangladish, India, Nepal, Sudan, Ethiopia and Brazil . In Sudan and northwest Ethiopia, kala-azar is transmitted by P. orientalis- and it claims the lives of thousands of people -. Phlebotomus orientalis is found to exhibit anthropophilic behavior and it transmits VL from man to man and/or to dog and vice versa ,. The dog was considered as an intermediate host between a possible sylvatic cycle and the anthroponotic cycle . The large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages in Sudan might also indicate a possible human reservoir and anthroponotic transmission . Phlebotomus orientalis was also reported to be exo-phagic/exophilic (zoonotic VL) -. Zoonotic transmission of VL in east Africa was also suggested based on evidence of VL outbreaks among people that camped in uninhabited areas of eastern and former southern Sudan in addition to isolation of L. donovani from wild animals in the uninhabited Dinder National Park -. Experimental study on host preferences of P. orientalis and the isolation of L. donovani in east Africa have indicated that Canis familiaris (dogs), Herpeistes ichneumon (mongoose), Genetta genetta (genet cat), Acomys spp. and Arvicanthis spp. as preferred hosts for source of blood meals ,,,. The result of P. orientalis from human bait also showed humans to be preferred hosts ,-. Attraction of P. orientalis towards cattle, sheep, goat, mule, donkey and horse has not been defined in east Africa as a whole, even though high Leishmania sero-prevalence rate for these domestic animals has been reported in eastern Sudan  and northwest Ethiopia .
In the Metema-Humera lowlands, seasonal labour migrants who stay in agricultural fields for weeding and harvest of sesame, sorghum and cotton are exposed to Zoonotic kala-azar infections from June to October . In these areas, the nocturnal activities (dieal periodicity) and the peak biting hours of P. orientalis were not studied. Adequate knowledge on these aspects helps to reduce kala-azar incidence by avoiding contact with sand flies at that particular period. Information on the host preferences of this vector could also give important clues in identification of reservoir hosts of kala-azar. Thus the aim of this study was to elucidate the nocturnal activities and host preferences of P. orientalis in Kafta – Humera lowlands.
Kafta-Humera, Welkait and Tsegede are three districts in the Western Zone of Tigray region and the vast land at an altitude of around 600 m above sea level, which is endemic to kala-azar. Humera town (14°17’N, 036°39’E; altitude 637 m) is the capital of Kafta-Humera district and it is the sit for the administrative centers of the district and the Zone. It is bordered to Eritrea in the north and Sudan in the west. It is located 180 km west of Shirao town (Tahatay Adiyabo district of Tigray region) and 253 km northwest of Gondar town (Amhara region). Kafta-Humera district has a total population of 92,167(47,909 men and 44,258 women) and covers an area of 4, 542.33 square km . In the district small towns such as Rawyan (14°17′ N, 36°37′E), May Kadra (14°08′ N, 36°34′ E), Baeker (14°00′ N; 36°61′E) and Adebay (14°17′N, 36°38′E) are surrounded by uniform agricultural fields that are occasionally interrupted by thickets of Acacia seyal in depressions. All the dense mixed forests were converted into agricultural fields except in the Kafta Shiraro Park and some rocky outcrops. The part of the dense mixed forest in Baeker is used for grazing of domestic animals especially during the rainy season when all arable land is covered with crops. The common trees and shrubs in Kafta-Humera areas are A. seyal, A. mellifera, Balanites aegyptiaca, Terminalia spp., Boswellia papyrifera, Ficus sycomorus, Sclerocarya birrea, Zizypus spp., Dalbergia melanoxylon, Boscia angustifolia, Sterculia africana, Adansonia digitata, Dichrostachys cineria and Syzgium guineese.
Goats and sheep are browsing and grazing in agricultural fields and sparse thickets of A. seyal including the dense mixed forest (Part of Kafta-Shirao park in Baeker at about 7 km from Site 1) during the day and shelter near the camps at night unlike cattle which grazed at night during dry season (October - June). In agricultural rainy season, however, animals are guarded in the dense mixed forest and rocky uncultivated areas. Similar points on agricultural fields, animal shelter and sparse thickets of A. seyal were selected for the repeated sand fly collections during studies of nocturnal activities and host preference towards rodents. The sampling points on agricultural fields and animal shelter were separated by about 400 m with the huts located between them in both Site 1 and 2. The sampling point for animal shelter was about 2 m from where the goats and sheep sheltered in both sites.
The vegetations in Metema-Humera lowlands on black cracking soil were described as Acacia seyal - Balanites aegyptiaca forest and Argeissus - Combretum savannah woodlands , which have been transformed into big agricultural fields. During transformation of forest into fields, all trees except Balanites aegyptiaca were cleared in order to prevent gully erosion. Thus, agricultural fields in these areas have this tree species at about 25 m intervals (Figure 1). The soil in the study sites was black soil (eutric vertisols)  also reported to be associated with P. orientalis and kala-azar in Ethiopia and adjacent Eritrea and Sudan ,,-. It is thought that such soil is hygroscopic and swells during the rainy season, then loses water and shrinks quickly during the dry season resulting in very deep cracks, creating essential microhabitats for the vector, P. orientalis,. Vertisol at shallow depths (45 cm) near Balanites aegyptiaca with relatively cool temperature, stable humidity and enhanced soil decomposition rate, were reported as suitable breeding site for sand fly larvae compared to open fields with seasonal 5–6°C temperature and 38.07% (average) relative humidity fluctuations . The annual mean maximum temperature in Baeker and Humera towns vary from 29.10 to 41.2°C while the monthly mean minimum range from 13.50 to 25.40°C with November to May being the dry season that is characterized by high mean maximum temperatures (35.7 – 40.83°C), lack of heavy rain and cracking of black soil. March, April and May were the hottest months with mean maximum temperatures ranging from 38.9 to 40.83°C. The rainy season started in May and extended up to October with the highest rainfall occurred in August (Mean ± SD: 199.97 ± 126.53). Average annual rainfall received by the area from 2011 to 2013 was 791 mm .
Nocturnal activities of Phlebotomus orientalis
A total of 22 CDC light traps were deployed on agricultural fields (n = 11), animal shelters (n = 3) and thickets of sparse A. seyal (n = 8), during January – May, 2013, to collect sand flies at one hourly intervals by changing sand fly cages before they were kept in separate tubes containing 96% ethanol for subsequent species identification. After Sergentomyia spp. were separated, Phlebotomus spp. were identified by using appropriate keys - following dissection and mounting of the specimens using Hoyer’s medium.
Host preference of Phlebotomus orientalis using small rodents in Turner and Hoogstraal’s box traps
In order to evaluate the host preference of P. orientalis towards rodents, locally made box traps based on the design of Turner & Hoogstraal  were used. A total of 30 rodents (2 Rattus spp., 18 Arvicanthis spp., 4 Tatera spp., and 6 Acomys spp.) in 30 box traps were used as baits in Site 1 and 2 agriculture fields ( n = 10), animal shelter (n = 10) and thickets of A. seyal (n = 10) for a total of 10 days in March and April, 2013.
Blood meal analysis of Phlebotomus orientalis using Reverse Line Blot (RLB) of cytochrome b PCR product
Sand fly collection and preservation
Any freshly blood-fed female P. orientalis encountered during previous sand fly collection from thickets of A. seyal (March, 2011) and dense mixed forest (July, 2011) in Baeker, was preserved for molecular identification of animal species that served as source of blood for this vector using Reverse Line Blot (RLB) of cytochrome b PCR product. For this purpose, the head of each fed female was mounted on the slides by Hoyer’s medium for later species identification. The remaining body (thorax and abdomen) was individually placed in empty sterile eppendorf tube with silica gel grains and cotton pads inside, bearing a corresponding label with the mounted specimen on the slide. They were stored at −20°C until molecular blood meal analysis was conducted in August 2012 in the Department of Microbiology & Molecular Genetics, Hadassah Medical School, Hebrew University, Israel.
The thorax and abdomen of freshly-fed P. orientalis from each tube with silica gel were placed on filter and were transferred to test tube for digestion of tissues using lysis buffer (200 μl) and proteinase K (10 μl). Each Phlebotomus orientalis was homogenized using sterile wooden sticks and incubated in water bath (65°C) for 2 hours. Phenol extraction of DNA was made by adding 180 μl phenol before centrifugation at maximum speed (1,400 r.p.m) for 2.5 minutes. For ethanol DNA extraction, the aqueous part (150 μl) was transferred into a test tube containing sodium chloride solution (8 μl) before 400 μl cold (−20°C) ethanol was added and placed in refrigerator (−20°C) for 2 hours and cold centrifugation at maximum speed (1400 r.p.m) for 10 minute that precipitated DNA. Finally, pure DNA was obtained by removing the ethanol and suspending in 50 μl double distilled water.
This study described a blood meal identification approach based on PCR amplification of the mitochondrial cytochrome b gene (cyt b) followed by RLB analysis as already described ,. RLB is a highly reproducible technique in which species-specific oligonucleotides (probes) are covalently linked to nylon membranes through the formation of amide bonds between the carboxyl group present on the membrane and amino-linked oligonucleotides which hybridizes with Biotinylated PCR products of the mitochondrial cyt b gene to reveal color that will be detected by colorimetric or enhanced chemiluminescent (ECL) systems. The species-specific oligonucleotide probes designed by Abassi et al.  for human (ATG CAC TAC TCA CCA GAC GC), cattle (ATT ATG GGT CTT ACA CTT T), sheep (TCC TAT TTG CGA CAA TAG CTT CCT), goat (ATA CAT ATC GGA CGA GGT CTA), camel (CGT TGG AAT TGT TTT ATT), donkey (CTA CTT TTC ACA GTT TAG CTA CA), dog (CAG ATT CTA ACA GGT TTA ), mouse (TGG AGT ACT TCT ACT GTT CGC AGT), rat (CAG TCA CCC ACA TCT GC), chicken (CAT CCG GAA TCT CCA C) and avian (TAC ACA GCA GAC AC) were used which adsorbed on the 3–4 mm strips of membrane in the following order from the top to bottom: Human, donkey, cattle, sheep, goat, camel, dog, mouse, rat, chicken and avian. One probe was found effective in identification of any avian species (36).
Polymerase chain reaction
To make 25 μl of final volume for PCR reaction, 20 μl master mix (1 μl forward primer, 1 μl backward primer and 18 ddH2O) and 5 μl DNA sample were mixed. PCR amplification of the mitochondrial cytochrome b gene was made using primers Cyto1 (5'-CCA TCA AAC ATC TCA GCA TGA TGA AA-3') and Cyto2 (5'- CCC CTC AGA ATG ATA TTT GTC CTC-3') before gel electrophoresis at 120 V in 1× TAE buffer in 1.5% agarose gels. The DNA fragments were visualized by UV light for determination of the sizes. The PCR product left from gel electrophoresis (18 μl) was used for RLB hybridization.
Species-specific probes from the 11 species were bounded on 5.5 × 15 cm biodyne C membrane (Gelman, USA). The species-specific probes were adsorbed on the membrane so that they would identify the corresponding PCR product indicating the species of animals P. orientalis fed. The membrane was first incubated with 0.1 M HCl and 10% EDAC solution before the bounding of probes and was later cut into strips of 3–4 mm width for the process of sample hybridization.
The nocturnal activities of P. oreintalis during 12 hours of the night were estimated from mean number P. orientalis/hour/CDC. All statistical analyses were performed using the Statistical Package of Social Science (SPSS), version 16. Comparisons of nocturnal activities during the night hours were analyzed using one-way analysis of variance (ANOVA). P-values less than 0.05 for mean number P. orientalis/hour/CDC were considered as significantly different.
Population dynamics of Phlebotomus orientalis
Host preference of Phlebotomus orientalis towards rodents using Turner and Hoogstraal’s traps
No P. orientalis was collected in rodent baited Turner Box’s trap. Only 5 Sergentomyia species (S. schwetzi) were collected from 30 Turner’s box trap deployed for 10 days in the agricultural fields, animal shelters and thickets of A. seyal in both sites.
Host preference study of Phlebotomus orientalis based on molecular analysis of blood meals
So far no effective VL control measures have been conducted in Sudan and northwest Ethiopia, mainly due to the sylvatic nature of the habitats of adult P. orientalis and the lack of enough knowledge on transmission dynamics of L. donovani between unknown reservoir hosts and humans . Studies on nocturnal and feeding activities of adult P. orientalis could give an insight about the behavior of this vector which is an essential component to design kala-azar control strategies. The results on nocturnal activities from hourly CDC and molecular blood meal analysis showed a risk of P. orientalis bite throughout the night as already reported . Use of bed net by labour migrants involved in agricultural activities in the study areas is required.
The fact that the highest mean value of P. orientalis collected near animal shelters might indicate that goats and sheep were also sources of blood meals. The role of goats and sheep in the epidemiology of kala-azar is a subject of further study. Only one serological study in eastern Sudan indicated 8.5% L. donovani infection rate in goats using DAT . In Kenya, P. duboscqi and P. martini were reported to be attracted to goats . Recently, RLB of cytochrome b PCR products of blood meal collected from P. orientalis in domestic areas in Kafta-Humera district showed goats and sheep as a source of blood meal for this vector in addition to cattle (Yared et al., personal communication). The mean landing rate on human bait near goat and sheep shelters was almost twice the rates in the agricultural fields. Thus, goats and sheep might have zooprophylactic effect in preventing humans from kala-azar as, e.g. cattle . This study has also shown that cattle to be important source of blood for P. orientalis in extra-domestic habitats where 72% of this species was found to have human and/or cattle blood-meal source. Mixed (human and cattle) blood meal was found to be very high (36%) which might indicate that P. orientalis had interrupted feeding on both hosts. Interruptions might have occurred due to inherent behavior of the female fly or due to the physical disturbance by the human or cattle hosts during the probing of the fly. Previous study has shown that cattle serve as the main source of blood for P. orientalis in domestic areas . The reason why more cattle (92%) served as source of blood than human (2.2%) in domestic and predomestic areas using Enzyme Linked Immuno Sorbent Assay (ELISA)  in the area of Metema compared with this study is difficult to explain, but might need further investigation.
The bands of DNA fragments (cyt. b) on agarose gel (28%), that were not captured by the 11 probes used, may represent wild animals in the area. The probes that were not used include wild rodents and small carnivores. The existences of small carnivores such as genet cats and mongoose were confirmed during our study on reservoirs in these areas especially in the dense mixed forest (unpublished data). Either rodents or small carnivores or both could be the unknown source of blood for P. orientalis. Attractiveness towards mongoose (63.9 ± 12.1/CDC/night), genet cat (17.4 ± 3.72/CDC/night) and Nile rat (2.6 ± 0.56/CDC/night) compared to the control (0.4 ± 0.16/CDC/night) were reported in eastern Sudan . No P. orientalis or other sandfllies were attracted to the rodents from all Turner and Hoogstraal box traps in this study. Similar study using CDC or sticky traps is required before reaching a conclusion.
The results of this study were in complete agreement with other observations , showing night-long human bite of P. orientalis. The nocturnal activities and human biting behavior was described as wave-like to show non-uniformity of biting rate at different hours of the night . Such wave-like biting was due to the effect of wind. There were different trends of winds according to the observations during this study. Sometimes it began at 19:00 pm and ended at 21:00 p.m. or started at 21:00 p.m. and came down at 23:00 or 24:00 p.m. In few instances strong wind persisted until 24:00 p.m. Having understood this, long study period was used as the probability of unsuccessful sand fly collection was high.
Phlebotomus orientalis could bite humans at different hours of the night and there is a risk of kala-azar infection in agricultural fields, thickets of A. seyal and in the camps. It is also attracted to domestic animals in the extra-domestic habitats for blood meal. Phlebotomus orientalis is most probably catholic in its feeding habits and having wide range of mammals as blood meal sources depending on the available host in space and time. Whether these animals are potential carriers of Leishmania causing Kala-azar needs further investigation.
Despite several attempts made to investigate the reservoir host of L. donovani in Sudan and northwest Ethiopia, further study, with more emphasis on wild animals in extra-domestic animals, are required.
We would like to thank the Bill and Melinda Gates Foundation Global Health Program [Grant number OPPGH5336] and Gondar University for funding this research. We would also thank Abel Haile, for technical assistance and for sand fly sampling, and Shewangizaw Sime (driver) and all who assisted us during our field activities in Metema – Humera low lands. Our thanks also go to Tigray regional state and western Tigray zone administration for their unreserved co-operation during the execution of this research.
- Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, Alvar J, Boelaert M: Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?. Nat Rev Microbiol. 2007, 5: 873-82. 10.1038/nrmicro1748.View ArticlePubMedGoogle Scholar
- Hoogstraal H, Heyneman D: Leishmaniasis in the Sudan Republic. 30. Final epidemiological report. Am J Trop Med Hyg. 1969, 18: 1091-1210.Google Scholar
- Gemetchu T, Zerihune A, Assefa G, Lemma A: Observations on the sandfly (Phlebotomidae) fauna of Setit-Humera (Northwestern Ethiopia). Ethiop Med J. 1975, 13: 41-51.Google Scholar
- Elnaiem DA, Hassan HK, Ward RD: Phlebotomine sandflies in a focus of visceral leishmaniasis in a border area of eastern Sudan. Ann. Trop. Med. Parasitol. 1997, 91: 307-318. 10.1080/00034989761157.View ArticlePubMedGoogle Scholar
- Seaman J, Mercer J, Sondorp E: The epidemic of visceral Leishmaniasis in Western Upper Nile, southern Sudan: course and impact from 1984–1994. Int J Epidemiol. 1996, 25: 862-871. 10.1093/ije/25.4.862.View ArticlePubMedGoogle Scholar
- Hailu A, Gebre-Michael T, Berhe N, Balkew M: Leishmaniasis. In Epidemiology and Ecology of Health and Disease in Ethiopia Shama Books. Edited by Berhane Y, Hailemariam D, Kloos H. Addis Ababa: 2006:615–634.Google Scholar
- Ritmeijer K, Dejenie A, Assefa Y, Hundie B, Mesure J: A comparison of miltefosine and sodium stibogluconate for treatment of visceral leishmaniasis in an Ethiopian population with high prevalence of HIV infection. Clin Infect Dis. 2006, 43: 357-364. 10.1086/505217.View ArticlePubMedGoogle Scholar
- Lambert M, Dereure J, El-Sail SH, Bucheton B, Dessein A, Boni M, Feugier E, Dedet JP: The sandfy fauna in the visceral leishmaniasis focus of Gedaref, in the Atbara-river area of eastern Sudan. Ann Trop Med Parasitol. 2002, 96: 631-636. 10.1179/000349802125001474.View ArticlePubMedGoogle Scholar
- Dereure J, El-Safi H, Bucheton B, Boni M, Kheir M, Davoust B, Pratlong F, Feugier E, Lambert M, Dessein A, Dedet J: Visceral leishmaniasis in eastern Sudan: parasite identification in humans and dogs; host-parasite relationships. Microbes Infect. 2003, 5: 1103-1108. 10.1016/j.micinf.2003.07.003.View ArticlePubMedGoogle Scholar
- Zijlstra EE, El-Hassan AM: Leishmaniasis in Sudan. Visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2001, 95: S27-S58. 10.1016/S0035-9203(01)90218-4.View ArticlePubMedGoogle Scholar
- Schorscher J, Goris M: Incrimination of Phlebotomus (Larroussius) orientalis as a vector of visceral leishmania sis in Western Upper Nile province, southern Sudan. Trans R Soc Trop Med Hyg. 1992, 86: 623-627. 10.1016/0035-9203(92)90154-5.View ArticleGoogle Scholar
- Elnaiem A, Conners S, Thmoson M, Hassan M, Hassan H, Aboud A, Ashford W: Environmental determinants of the distribution of Phlebotomusorientalis in Sudan. Ann Trop Med Parasitol. 1998, 92: 877-887. 10.1080/00034989858925.View ArticlePubMedGoogle Scholar
- Hassan M, Osman OF, El-Raba'a FM, Schallig HD, Elnaiem DE: Role of the domestic dog as a reservoir host of Leishmania donovani in eastern Sudan. Parasit Vectors. 2009, 2: 26-10.1186/1756-3305-2-26.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirk R: Studies in leishmaniasis in the Anglo-Egyptian Sudan. XII. Attempts to find a reservoir host. Trans Roy Soc Trop Med Hyg. 1956, 50: 169-177. 10.1016/0035-9203(56)90079-7.View ArticlePubMedGoogle Scholar
- Ibrahim ME, Lambson B, Yousif AO, Deifalla NS, Alnaiem DA, Ismail A, Yousif H, Ghalib W, Khalil AG, Kadaro A, Barker DC, El Hassan AM: Kala-azar in a high transmission focus: an ethnic and geographic dimension. Am J Trop Med Hyg. 1999, 61: 941-944.PubMedGoogle Scholar
- Elnaiem DA, Hassan MM, Maingon R, Nureldin GH, Mekawi AM, Miles M, Ward RD: The Egyptian mongoose, Herpestes ichneumon, is a possible reservoir host of visceral leishmaniasis in eastern Sudan. Parasitology. 2001, 122: 531-536. 10.1017/S0031182001007594.View ArticlePubMedGoogle Scholar
- Haile T, Lemma A: Isolation of Leishmania parasites from Arvicanthis Ethiopia. Trans Roy Soc Trop. Med Hyg. 1977, 71: 180-181. 10.1016/0035-9203(77)90092-X.View ArticlePubMedGoogle Scholar
- Mutinga MJ, Ngoka JM, Schnur LF, Chance ML: The isolation and identification of leishmanial parasites from domestic dogs in Machakos District of Kenya, and possible role of dogs reservoirs of kala-azar in East Africa. Ann Trop Med Parasitol. 1980, 74: 139-144.PubMedGoogle Scholar
- Ashford RW, Hutchinson MP, Bray RS: Kala-azar in Ethiopia: pidemiological studies in a highland valley. Ethiop Med J. 1973, 11: 259-264.PubMedGoogle Scholar
- Hailu A, Balkew M, Berhe N, Meredith E, Gemetchu T: Is Phlebotomus (Larroussius) orientalis a vector of visceral leishmaniasis in south-west Ethiopia?. Acta Trop. 1995, 60: 15-20. 10.1016/0001-706X(95)00093-T.View ArticlePubMedGoogle Scholar
- Kolaczinski H, Reithinger R, Dagemlidet T, Worku A, Ocheng K, Kabatereine K, Brooker S: Risk factors of visceral leishmaniasisin East Africa: a case–control study in Pokot territory of Kenya and Uganda. Inter J Epidemiol. 2008, 8: 1-9.Google Scholar
- Mukhtar AH, Sharief SH, El S, Harith AE, Higazz TB, Adam AM, Sulieman H: Detection of antibodies to Leishmania donovaniin animals in a kala-azar endemic region in eastern Sudan: a preliminary report. Trans R Soc Trop Med Hyg. 2000, 94: 33-36. 10.1016/S0035-9203(00)90429-2.View ArticlePubMedGoogle Scholar
- Gebre-Michael T, Balkew M, Berhe N, Hailu A, Mekonnen Y: Further Metema (north-west Ethiopia) with observations on their natural blood meal sources. Parasit Vectors. 2010, 3: 6-10.1186/1756-3305-3-6.PubMed CentralView ArticlePubMedGoogle Scholar
- WHO Expert Committee: Studies on Leishmaniasis vectors/Reservoirs and their control. The Old World, Tropical Africa. Geneva: World Health organization Technical Report Series; 1981:1–164.Google Scholar
- Tigray Region report: Livelihood Profile Tigray Region, Ethiopia. Humera Sesame and Sorghum Livelihood Zone. Tigray Regional report. Mekele, Ethiopia: 2007:1–13.Google Scholar
- Gemetchu T: The distribution of sandflies (Diptera, Psychodidae, Phlebotominae) in north-west Ethiopia. Sinet Ethiop J Sci. 1983, 6: 65-73.Google Scholar
- Gebre-Michael T, Malone JB, Balkew M, Ali A, Berhe N, Hailu A, Herzi AA: Mapping the potential distribution of Phlebotomus martini and P. orientalis (Diptera: Psychodidae), vectors of kala-azar in East Africa by use of geographic information systems. Acta Trop. 2004, 90: 73-86. 10.1016/j.actatropica.2003.09.021.View ArticlePubMedGoogle Scholar
- Fuller GK, Lemma A, Haile T, Gemeda N: Kala-azar in Ethiopia: survey of south-west Ethiopia. The leishmanin skin test and epidemiological studies. Ann Trop Med Parasitol. 1979, 73: 417-431.PubMedGoogle Scholar
- Thomson MC, Elnaiem DA, Ashford RW, Connor SJ: Towards a kala-azar risk map for Sudan: mapping the potential distribution of Phlebotomus orientalis using digital data of environmental variables. Trop Med Int Health. 1999, 4: 105-113. 10.1046/j.1365-3156.1999.00368.x.View ArticlePubMedGoogle Scholar
- Moncaz A, Kirstein O, Gebresellassie A, Lemma W, Yared S, Gebre-Michael T, Hailu A, Shenker M, Warburg A: Characterization of breeding sites of Phlebotomus orientalis – The vector of visceral leishmaniasis in northwestern Ethiopia. Acta Trop. 2014, 139: 5-14. 10.1016/j.actatropica.2014.06.013.View ArticlePubMedGoogle Scholar
- Lemma W, Tekie H, Balkew M, Gebre-Michael T, Warburg A, Hailu A: Population dynamics and habitat preferences of Phlebotomus orientalis in extra-domestic habitats of Kafta Humera lowlands – kala azar endemic areas in Northwest Ethiopia. Parasit Vectors. 2014, 7: 359-10.1186/1756-3305-7-359.PubMed CentralView ArticlePubMedGoogle Scholar
- Abonnenc E, Minter DM: Bilingual keys for the identification of the sandflies of the Ethiopian region. Cah O R S T O M, ser. Entomol Med. 1965, 5: 3-24.Google Scholar
- Quate LW: Leishmaniasis in the Sudan Republic: Phlebotomus sandflies of the Paloich area in the Sudan (Diptera: Psychodidae). J Med Entomol. 1964, 1: 213-268.View ArticlePubMedGoogle Scholar
- Lewis J: A taxonomic review of the genus Phlebotomus (Diptera:Psychodidae). Bull Brit Mus (Nat Hist) (Ent). 1982, 45: 121-209.Google Scholar
- Turner ER, Hoogstraal H: Leishmaniasis in the Sudan Republic. 23. Sandflies (Phlebotomus) attracted to rodent baited traps (Diptera: Psychodidae). J Med Entomol. 1965, 2: 137-139.View ArticlePubMedGoogle Scholar
- Abbasi I, Cunio R, Warburg A: Identification of blood meals imbibed by Phlebotomine sand flies using cytochrome b PCR and reverse line blotting. Vec Borne Zoo Dis. 2008, 2: 280-285.Google Scholar
- Garlapati RB, Abbasi I, Warburg A, Poché D, Poché R: Identification of bloodmeals in wild caught blood fed Phlebotomus argentipes (Diptera: Psychodidae) using cytochrome b PCR and Reverse Line Blotting in Bihar, India. J Med Entomol. 2012, 49: 515-521. 10.1603/ME11115.View ArticlePubMedGoogle Scholar
- Elnaiem DA: Ecology and control of the sand fly vectors of Leishmania donovani in East Africa, with special emphasis on Phlebotomus orientalis. J Vector Ecol. 2011, 36: S23-S30. 10.1111/j.1948-7134.2011.00109.x.View ArticlePubMedGoogle Scholar
- Mutinga MJ, Kyai FM, Kamau C, Omogo DM: Epidemiology of leishmaniasis in Kenya- host preference studies using various types of animal baits at animal burrows in Marigat, Baringo District. Insect Sci Appl. 1986, 7: 191-197.Google Scholar
- Hoogstraal H, Dietlein DR, Hevneman D: Leishmaniasis in the Sudan Republic: 4. Preliminary observations on man-biting sand flies (Psychodidae: Phlebotoraus) in certain Upper Nile endemic areas. Trans Roy Soc Trop Med Hyg. 1962, 56: 411-422. 10.1016/0035-9203(62)90016-0.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.