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Population dynamics and habitat preferences of Phlebotomus orientalis in extra-domestic habitats of Kafta Humera lowlands – kala azar endemic areas in Northwest Ethiopia
© Lemma et al.; licensee BioMed Central Ltd. 2014
Received: 17 June 2014
Accepted: 18 July 2014
Published: 6 August 2014
Kafta Humera lowlands are endemic for kala-azar (visceral leishmaniasis). These lowlands are characterized by black clay soil which is used for growing sesame, sorghum and cotton for commercial purposes.
The aim of this study was to determine seasonal dynamics and habitat preferences of Phlebotomus orientalis, the vector of kala-azar, in extra-domestic habitats of Kafta Humera lowlands.
CDC-light Trap [CDC-LT] and Sticky paper Trap [ST] were used to collect sand flies from different habitats before species identification by their morphological characteristics using appropriate keys. Data summarized and analyzed included: species, sex, density, habitats, type of trap used and date (month).
A total of 389,207 sand flies using CDC-LT (n = 955) and ST (n = 5551) were collected from May 17, 2011 to June 6, 2012. The highest Mean Monthly Density (MMD) of P. orientalis trapped by CDC-LT was found in thickets of Acacia seyal in March (64.11 ± 75.87). The corresponding highest MMD of P. orientalis trapped by STs was found in April (58.69 ± 85.20) in agricultural field. No P. orientalis were caught in September using CDC traps and July-October using sticky traps. The overall MMD of P. orientalis trapped by CDC-LT was 15. 78 ± 28.93 (n = 320) in agricultural field, 19.37 ± 36.42 (n = 255) in thickets of A. seyal, and 3.81 ± 6.45 (n = 380) in dense mixed forest. Similar habitats in different localities did not show statistically significant difference for the MMD of P. orientalis trapped by CDC-LT (p = 0.117) and ST (p = 0.134).
Agricultural fields and thickets of A. seyal habitats, which exhibit extensive soil cracks and fissures, as opposed to dense mixed forests, serve as preferred breeding sites for P. orientalis.
Visceral leishmaniasis (VL) or kala-azar is vector borne disease that is almost fatal if left untreated [1, 2]. More than 90% of VL cases in the world occur in Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil . Phlebotomus martini and Phlebotomus orientalis are the two principal vectors of kala-azar in East Africa [3, 4]. Phlebotomus martini is associated with Macrotermes termite mounds in southern Ethiopia  while P. orientalis is associated with Acacia seyal - Balanites aegyptiaca forest and deeply cracking soil in eastern Sudan and northwestern Ethiopia [6–18]. P.orientalis is believed to depend on the black cracking soil as day resting and breeding site in dry season [7, 8]. Elnaiem et al.  considered termite mound as principal day resting site for P. orientalis compared to tree holes, crevices, soil cracks and chicken coops in eastern Sudan. The breeding site of P. orientalis has not been identified beyond doubt as attempts to find larvae were not successful . Further, the sugar feeding habits and diapauses of this species have not been described [7, 8]. The reservoir hosts of zoonotic VL (ZVL) in Eastern Africa Region are not known. Due to lack of knowledge on zoonotic transmission cycles and the ecology of the vector, little has been achieved in control of VL in the region. A. seyal – B. aegyptiaca forest was reported to have the highest P. orientalis population in Sudan [7, 12, 13, 15–17]. Elnaiem et al.,  described P. orientalis as forest species with special preference to dense A. seyal than B. aegyptiaca, Combretum kordofanum, Hyphaena or Zizipus trees. The smooth surface of A. seyal, however, lacks cracks, fissures or cavities to serve as resting site during wet rainy season as oppose to Balanites sp. or other trees in the forest .
Metema–Humera lowlands in northwest Ethiopia are endemic for kala-azar and accounted 60% of all VL cases in Ethiopia . Matema–Humera lowlands are, geographically, an extension of eastern Sudan [8, 15, 17] and have similar rainfall pattern and vegetation [7, 8]. The most kala-azar affected part of Matema–Humera lowlands is the Kafta-Humera district with the annual incidence that range from 1000 to 2000 cases, with higher prevalence (>80%) in labour migrants from Amhara and Tigray highland areas compared to the permanent residents in the area . World Health organization report on leishmaniasis in tropical Africa  indicated that 45.6% of Humera population involved in farm activities were positive for leishmanin skin test compared to 8.3% in non-farmers (urban and farm-owning population) with annual sero-conversion rate of 7% and less than 1% respectively. Kala azar infections in labour migrants from non-endemic highland areas of Tigray and Amhara regions were addressed to the travel history to Kafta-Humera [21, 22]. Almost all (156/157 or 99.4%) kala azar cases in the labour migrant visiting Kafta Humera during June – October rainy season were aged from 15 to 49 compared to 68.9% (104/151) in permanent residents for the same age group (22) indicating this age group, that involved in agricultural activities in the extra-domestic habitats, as high risk group. Kala azar is the most important public health problem and cause high mortality and morbidity rate, especially among the young adult working forces, and has serious impact on the socio-economy of Kafta-Humera district. While treatment of kala-azar patients saves lives, it does not stop the disease from becoming a public health threat. Prevention of kala-azar transmission by vector control requires in depth understanding of the biology and ecology of sand fly vectors. Study on population dynamics and habitat preferences of P. orientalis are among the first steps in vector management to control kala-azar. Thus, the aim of this study was to describe the seasonal dynamics of P. orientalis and habitat preferences in extra-domestic habitats of the Kafta Humera lowlands.
Study sites and habitats
Information about mean average annual rainfall, mean maximum and minimum temperature of Humera and Baeker towns during January 2011 to December 2013 were obtained from Ethiopian National Meteorology agency to describe the climate of the study area.
Sand flies collection and identification
At least 12 CDC - light traps/month (Model512, Hock and Co.,USA) were used to collect sand flies from agricultural fields, thickets of A. seyal and dense mixed forests at the four localities (sampling sites). Similarly, 20 – 388 sticky traps/month were used to collect sand flies from the three habitats. CDC light traps were set at 6 p.m, hanged at about 0.5 meter above ground level, and left overnight till 6 a.m. Sesame oiled sticky traps were randomly placed horizontally on the ground at about 5 m interval. Sand flies from sticky traps were collected in 95% alcohol before transferring to saline containing detergent for washing, sorting and counting Sergentomyia and Phlebotomus spp. Similarly, sand flies collected using CDC traps were sorted and counted. Sand flies were dissected in saline and mounted in Hoyer’s medium, after the head is separated and turned upside down before placing cover slip. The last segment of the abdomen was also removed for visualizing the spermathecae in female sand flies. Species identification was carried out using the appropriate keys [7, 24, 25].
Study on habitat preference and population dynamics (Bionomics)
The sand fly species, sex, habitat, numbers, date and type of trap used were documented. Mean Monthly Density (MMD) of trapped sand flies was determined by total counts divided by number of traps used. Comparisons of P. orientalis MMD in different habitats were used for study of habitat preference of this vector. Similarly, MMD of P. orientalis at different months were used to determine the population dynamics. Resting sites of P. orientalis in the dense mixed forest during rainy season were determined by comparing the result of P. orientalis cached using the sticky traps placed on the ground and the emergence traps deployed on the tree trunks. Hand used torch light battery was also used for making observations of sand flies resting sites.
The density of sand flies calculated as average numbers of male and female sand flies per trap per day and the results were entered into Statistical Package of Social Sciences (SPSS) version 16 for analysis of the data using descriptive statistics (Mean ± SD), analysis of variance (ANOVA) and Post hoc Tukey Honestly Significant Difference (HSD) tests so that seasonal dynamics and habitat preferences of P. orientalis could be studied. P-values less than 0.05 were considered as statistically significant difference.
Climate: temperature and rainfall
Phlebotomus orientalis density
Total sand flies collected from the agricultural fields, thickets of A. seyal and dense mixed forest using CDC and sticky traps from May 17, 2011 to June 6, 2012 G.C
CDC (No. traps (n) = 955)
Sticky (n = 5551)
Sergentomyia s pp
The overall MMD of P. orientalis (female + male) trapped by CDC-LT was 15. 78 ± 28.93 (n = 320) in agricultural field, 19.57 ± 36.42(n = 255) in thickets of A. seyal, and 3.81 ± 6.45 (n = 380) in dense mixed forest. For STs, the overall MMD of P. orientalis was 14.76 ± 38.78 (n = 2378) in agricultural field, 11.45 ± 15.56 (n = 1500) in the thickets of A. seyal and 0.95 ± 2.16 (n = 1168) in dense mixed forest. ANOVA result has showed statistically significant mean difference (p = 0.000) for different habitats. However, similar habitats in different localities did not show statistically significant difference for the MMD of P. orientalis trapped by CDC-LT (p = 0.117) and ST (p = 0.134). During the August rains, habitat with more protection such as dense mixed forest, harbored more number of P. orientalis/ CDC (1.04 ± 1.2) compared to agricultural fields (0.24 ± 0.42) and thickets of A. seyal (0.2 ± 0.44). Sticky traps placed on the ground in July and August did not collect sand flies when compared to emergence traps deployed on the tree trunks in the dense mixed forests. A total of 44 (32 female, 12 male) P. orientalis and 8115 Sergentomyia sp. (3736 female, 4379 male) were collected using 77 emergence traps.
P. orientalis abundance in different sampling sites in relation to specific habitats, i.e., agricultural fields, thickets of A. seyal and mixed forest using CDC traps
Thickets of A. seyal
Dense mixed forest
Adebay extra domestic site 1
Baeker extra domestic site 2
Gelanzeraf extra domestic site 4
Rawyan extra domestic site 3
Three decades ago, the vegetations of Metema – Humera lowlands were described as A. seyal – B. aegyptiaca forest and Argeissus - Combretum savannah woodlands  which have now been converted mainly into big mechanized agricultural fields, especially in Kafta Humera district. Absence of A. seyal – B. aegyptiaca forests and termite mounds in the study areas in this district gave advantage to analyze fewer habitats such as agricultural fields and thickets of A. seyal in addition to dense mixed forests in the periphery of Kafta - Shiraro National Park. These habitats are distinctly separated from each other as opposed to habitats studied previously that had no demarcation between agricultural fields and the different types of forests [7, 14, 16].
In the highlands of Belessa valley in Ethiopia, the population of P. orientalis was reported to show no significant variation from September (rainy season) to April (dry season) . In contrast, in Sudan, the numbers of P. orientalis captured using STs were reported to be few in the early dry season (January and February) and increased significantly in March until it reached peak number in April. The population of P. orientalis declined in May and June when rain commenced [7, 8]. Similarity in the result between numbers of P. orientalis/trap in Sudan and this study could be due to similar ecology shared between eastern Sudan and northwestern Ethiopia along the border areas [8, 16]. During the study of sand flies in the Dinder National Park next to Ethiopian border, P. orientalis showed a slight peak from December 1994 to February 1995 and then dropped in March – May, and then peaked suddenly in June 1995 in thickets of A. seyal. In this study, however, P. orientalis was reached peak mean numbers in March and April with sudden dropped in May (Figure 4). The effect of wind in affecting sand fly collection in May and June should not be under estimated. The trend of seasonal dynamics of P. orientalis in dense mixed forest did not fully match the patterns in agricultural field and thickets of A. seyal (Figure 4). Generally, MMD in dense mixed forest was low compared to other habitats, but it showed an increase during dry season as other habitats until July. In July, MMD reached its peak value unlike other habitats where P. orientalis population declined. These results might be enough for the conclusion of dense mixed forest not to be the breeding site for P. orientalis. The seasonal changes in P. orientalis population in dense mixed forest might be due to the inter-habitat shift of this vector. During rain and wind stress season (May-June), P. orientalis from neighboring agricultural fields and thickets of A. seyal might have shifted to dense mixed forests where cracks and burrows in tree trunks serve them protected. The cooler temperature inside the forest, which has prevented soil crack formation, might have played a significant role for dense forests not to act as breeding site.
Due to the fact that most previous studies [7, 8, 14] have considered P. orientalis as forest species and owing to the lack of comparisons among different types of forests (thickets of A. seyal, A. seyal – B. aegyptiaca and dense mixed forests), the exact habitats of P. orientalis were not identified. The overall mean collections of P. orientalis using sticky traps from the three different habitats such as 11.50/sticky traps (n = 2418) from agriculture fields, 12.17/sticky ( n = 1500) from thickets of A. seyal and 1.08/sticky (n = 1633) from dense mixed forest in Kafta-Humera lowlands could be compared with similar study in Sudan . The overall result of mean P. orientalis/ sticky from the different forests in the Paloich areas in South Sudan 0.195/sticky trap (n = 100)  was similar with the result obtained from dense mixed forest in Kafta Humera districts. These results were lower than the overall results obtained from agricultural fields and thickets of A. seyal and might also suggest dense forests not to be breeding sites for P. orientalis. Habitats with cracks during dry season due to exposure to the heat of the sun such as agricultural fields and less dense thickets of A. seyal might be a breeding sites of P. orientalis. Males of P. orientalis collected from thickets of A. seyal and agricultural fields were found with unrotated genitalia (data not shown) indicating these habitats as places where this vector emerged from larvae. A mere absence of P. orientalis in CDC light traps set in forest habitats of Paloich area (Sudan) could not have been exclusively due to the failure of P. orientalis not being attracted to light as already described . The absence of cracking type black soil or other breeding sites of P. orientalis were decisive. Dense forests could be devoid of soil cracks as already reported  and unsuitable for P. orientalis breeding. Habitats of P. orientalis were reported to have persistently lower normalized difference vegetation index (NDVI) value during the dry season and experienced more extreme dry and wet seasons than P. orientalis negative sites . Our field observations in northwest Ethiopia concur with this. Typically, agricultural fields and thickets of A. seyal (sparse or thin), where P. orientalis was caught in abundance appeared to experience full exposure of sun during dry season whereas being covered with vegetation during rainy season. Agricultural fields and sparse or thin thickets of A. seyal, including A. seyal - B. aegyptiaca woodlands, could be a target for future control of kala-azar in the northwest Ethiopia and Sudan.
Less dense thickets of A. seyal and agricultural fields in Kafta Humera lowlands, which are characterized by deep black cracking soil in dry season, are breeding and resting habitats for P. orientalis, compared to dense mixed forests where P. orientalis shelters temporarily during rainy season. Kala-azar infections in Kafta Humera lowlands might have been related with the visits or permanent settlement on the agricultural fields where tickets of A seyal often found in narrow depressions around the agricultural fields.
Further studies on man – P. orientalis contact and parasite isolations from the vectors, human and reservoir hosts in extra-domestic habitats will show the exact areas where labour migrants are exposed to infection of kala-azar.
We would like to thank the Bill and Melinda Gates Foundation Global Health Program [Grant number OPPGH5336] and University of Gondar for funding this research. We would also thank Abel Haile, for technical assistance and sand fly collection, and Shewangizaw Sime (driver) and all the staffs in Humera who assisted us during our field and laboratory activities in Kafta 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.
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