INVESTIGATION OF LEISHMANIASIS VECTORS IN EL-KADOBA VILLAGE, WHITE NILE STATE, SUDAN

Visceral leishmaniasis (VL; kala-azar) is one of the most important parasitic tropical diseases in Sudan and the Sudan is considered to be one of the most important foci in the world. The visceral leishmaniasis has been described in Sudan since the beginning of the twentieth century. In Sudan, VL is caused by Leishmania donovani complex: MON 18, MON 30 and MON 82 zymodemes and The proven vector is Phlebotomus (Larroussius) orientalis, in this study a survey was carried out to identify the principal vector of VL based on Leishmania infection, morphological characters and to determine some ecological aspects of the sand flies prevalent in the area . Ten species of sand flies were recorded, three Phlebotomus species and seven Sergentomyia species of these sandflies, P. rodhaini was collected only from Acacia seyal/Balanities aegyptiaca at the Island whereas S. hunti was collected from the Acacia nilotica forest only of the total collections, P. orientalis representing 3.80% (248 specimens) of the total collection and P. papatasi were 3.11% of the collection. Sand flies identification was done under a binocular microscope at 40x (magnification). The main features used for sand flies identification were the sperm theca of the female, the termination of the male and the pharyngeal and the ciboria toothed structures of both sexes. According to results of this study we recommended the following: more studies are needed in the future to determine the transmission season, and infection rates of Leishmania parasites in human and the animal host in this area. Annually Entomological surveys must be done to determine density of Sand fly Vectors and Encourage the use of personal protection tools (ITNs), repellents, and improving of houses to avoid bite of sand fly.


Introduction
The Leishmaniasis is wide spread group of diseases worldwide. The disease is caused by obligatory, intracellular, kinetoplastid protozoan parasites of the genus Leishmania, which are transmitted by sandflies as flagellated promastigotes. Depending on the Leishmania species and the immune response of the host, the disease manifests itself in a variety of clinical forms; cutaneous (CL), mucocutaneous (MCL) and visceral leishmaniasis (VL).
Leishmaniasis is endemic in 88 countries on four continents: Africa, Europe, Asia, Central and Latin America (WHO, 1998). It is estimated that approximately 12 million people are currently infected and 350 million are at risk of acquiring leishmaniasis in 88 countries. The annual incidence rate is estimated to be 1.5-2 million cases of cutaneous leishmaniasis and 500,000 cases of visceral leishmaniasis (WHO, 2006). Visceral leishmaniasis (VL; kala-azar) is the most severe form of the disease, which is fatal if untreated with an almost 100% mortality rate. The disease is characterized by irregular fever, loss of weight, splenomegaly, hepatomegaly and/or lymphadenopathy and anaemia.
Visceral leishmaniasis is caused by L. donovani on the Indian subcontinent and in East Africa, by L. infantum in the Mediterranean region and by L. chagasi, in the New World (Desjeux, 1991). Ninety percent of VL cases occur in India Brazil, and Sudan (WHO, 2006).
Cutaneous leishmaniasis (oriental sore) is an endemic disease in northern Africa and western and central Asia. The disease produces skin lesions, on the face, arms and legs, causing serious disability and permanent scars (WHO, 2006). The disease is caused by L. major, L. tropica and L. aethiopica in the Old World whereas, in the New World is caused by L. maxicana and L. braziliensis complexes.
Ninety percent of all cases of CL occur in Afghanistan, Brazil, Iran, Peru, Saudi Arabia and Syria, with 1-1.5 million new cases reported annually world-wide (WHO, 1996).
It is less common, chronic form of disease in evolution and especially difficult to treat. The disease produces lesions resembling leprosy, which do not heal spontaneously, due to deficiency of the immune response (Desjeux, 1996;WHO, 1998). DCL is due to L. aethiopica and L. amazonensis (Desjeux, 1996).
Muco-cutaneous leishmaniasis (espundia) produces disfiguring lesions to the face, destroying the mucous membranes of the nose, mouth and throat (WHO, 1998;2006). The disease is mostly caused by L. braziliensis, L. panamensis and L. guyanensis in the New World, but mucosal lesions have been reported in the Old World due to L. donovani, L. major and L. infantum in immunosupresed patients (Desjeux, 1996).  Sudan is considered to be one of the areas of leishmaniasis in the world where recent epidemics had occurred with high incidence rates. The three clinical forms of leishmaniasis occur in Sudan e.g. VL including PKDL, CL and MCL (Fig. 1).
Sudan is considered to be one of the most important foci of VL in the world (Zijlstra and El-  In Sudan, the VL is thought to be a zoonotic disease with some probable anthroponosis in epidemic situations (Desjeux, 1991). The idea that VL is a zoonotic disease was strongly accepted due to repeated outbreaks of VL in people camping in uninhabited woodlands of southern and eastern Sudan (Kirk, 1956). Furthermore, the finding of infected P. orientalis in the uninhabited Dinder National Park (DNP) strongly suggest the presence of a reservoir other than man

2.1.Study Area
El-Kadaba village is located on western bank of the White Nile River (White Nile State) (32 o 14′ E, 14o 72′ N), it distances about 200 km south of Khartoum (capital of Sudan). The area is flat and covered by an alluvium of silt clay soil of the two types, black cracking clay soil and sand deposited by the river. Climate of it resembles a semi desert region therefore it is part of the arid climate with three distinct seasons; winter (November-February), summer (March-June) and autumn (July-Octobor) with an estimated annual rainfall of <400 mm.

2.2.Sample Size
In this study about 6527 sandflies were collected from diferent habitats during all the period of survey, during period of survey, about 52 females of sandflies were dissected to investigate about Leishmania promastigotes. These dissected species are: 35 flies were P. orientalis, 16 were P. papatasi and one female was P. rodhaini.

Light Traps
The light traps used to collect nocturnally active sandflies were CDC miniature light traps (Model 512, John Hock Co., Gainesville, Florida, USA), connected to 6volts rechargeable batteries and sandflies cages (Plate 2). The light traps were generally hanged at 30 cm above the ground level at outdoor and indoor sites, between 18:00-06:00 HR. Captured sandflies were separated from other insects and preserved in 70% alcohol for identification.
Plate 1: CDC miniature traps used for sandflies collection during July 2008

Sticky Paper Traps
The sticky paper traps were xerox paper sheets (15  21cm 2 ) coated on both sides with diesel oil or caster oil and fixed vertically on sticks held at 15 cm above the ground (Plate 3) and left overnight from 18:00-06:00 HR and collected by the next morning. Sandflies found stuck on the oiled papers were removed using small brushes, washed in dilute detergent and preserved in 70% alcohol for identification.
Plate 2: Sticky paper traps used for sandflies collection during July 2008

Data Analysis
Obtained Data was analyzed by a computer through the program (SPSS) and excel program. Then presented in tables and figures.

Results
Ten species of sandflies were recorded, three Phlebotomus species and seven Sergentomyia species namely: Phlebotomus papatasi, P. orientalis, P. rodhaini Sergentomyia clydei, S. schwetzi, S. antennatus, S. squamipleuris, S. africana, S. sintonious and S. hunti.  The above table shows that species of Sergentomyia are found with high numbers more than other genus and S. squamipleuris was the highest density in both habitats. The above table shows that P. orientalis are indicated statistical significant according to P. value (less than 0.05) and female of P. orientalis more than male in all habitats except in forest.   Figure 5: The above figure shows that the abundance of female of Phlebotomus orientalis in forest is higher than male while male of Phlebotomus orientalis in grass is higher than female

Discussion
The study found that sand flies collected from different habitats at El-Kadaba village and its surrounding during July 2008. Using one-way ANOVA, the P. orientalis were collected significantly in higher numbers from the island (Acacia/Balanities thicket) (5.50± 0.98), than the village The current study revealed that numbers of Phlebotomus sand flies dissected in El-Kadaba village and its surrounding habitats were not Leishmania promastigotes carrier , after dissected about 52 females of sand flies during period of survey, these dissected species are : 35 flies were P. orientalis, 16 were P. papatasi and one female was P. rodhaini. None of all these species were found infected with Leishmania promastigotes.
The morphological features of P. orientalis collected during this study were similar to those of the corresponding species reported by Kirk and Lewis (1951) and Quate (1964). The record of P. orientalis vector in the northern Sudan was a unique phlebotominae finding in this area. The presence of this sandfly vector which typically inhabits savannah environments in northern

Conclusion
1) The sandflies species found in the area consists of three Phlebotomus and seven Sergentomyia species which are namely P. orientalis, P. papatasi, P. rodhaini, Sergentomyia clydei, S. schwetzi, S. antennatus, S. squamipleuris, S. africana, S. sintanious and S. hunti. 2) S. squamipleuris, S. clydei and S. schwetzi were the most dominant species in the area. 3) P. orientalis were collected from all habitats in the area. 4) P. rodhaini was collected only from the woodland of Acacia seyal/Balanities aegyptiaca in the Island near El-Kadaba village. 5) P. orientalis was absent from indoor sites of El-Kadaba village. 6) P. orientalis, is the only proved sand fly vector of VL, was collected from study area. 7) All females of sand fly vectors that dissected were not infected by Leishmania promastigotes.

Recommendations
According to results of this study it is recommended that. : Annually Entomological surveys must be done to determine density of Sand fly Vectors by seasons and to determine the transmission season, and infection rates of Leishmania parasites in human and the animal host in this area , Encourage the use of personal protection tools (ITNs), repellents, and improving of houses to avoid bite of sand flies, and Control of Leishmaniasis procedures must be directed towards reservoirs, mass treatment of patients, and Vectors.