Studies On Sandflies, Transmission And Reservoir Host Of Visceral Leishmaniasis In Eastern Sudan

Abstract

The study described in this thesis has two components (1) Field work component conducted during June 1999 to July 2003 in Dinder National Park (DNP) and some villages along rivers Rahad and Atbara areas in eastern Sudan (2) Laboratory component performed at: Centre for Applied Parasitology and Entomology, Keele University, U.K, Faculty of Science and Institute of Endemic Diseases, University of Khartoum and National Malaria, Schistosomiasis and Leishmaniasis Administration, Federal Ministry of Health, Sudan. In this study entomological investigations and studies on the reservoir hosts of visceral leishmaniasis were conducted. The entomological investigations on the prevalence of P. orientalis in villages of river Rahad area and Atbara river area included transmission of the parasites and the infection rates of Phlebotomus orientalis with Leishmania donovani in villages and woodland of DNP. Sandflies were collected using light traps set at outdoors sites between 18:00- 06:00 hrs. The results of this work indicated that the vector was prevalent in all villages investigated in the rivers Rahad area and Atbara. The infectivity of the vector was investigated using the Polymerase Chain Reaction (PCR). The study showed the presence of infected flies in villages in river Rahad area with an average infection rate of 1.6% of L. donovani in P. orientalis. However, infection rates of the parasites in the vector in woodland of the Dinder National Park (DNP) were done by both PCR and direct microscopy. The results showed an active focus of zoonotic transmission of L. donovani in the uninhabited site of DNP; this was demonstrated by finding the parasite in 3.4% (7 out of 184) and 3.2% (5 out of 157) of flies collected in March 1998 and May 1999, respectively. Attempt of establishment a functional colony of P. orientalis was conducted during June-September 2003. Females P. orientalis were collected from DNP using light traps and human bait techniques. The flies were transported to the insectary of the National Malaria, Schistosomiasis and Leishmaniasis Administration, Ministry of Health, Khartoum. The flies were maintained under the laboratory condition at temperature 25 oC and humidity of 75-90%. Loss in different stages and their developmental time were investigated. Significantly, more loss was observed in the first instars larva than other stages (28.3 ± 5.66; P < 001). The whole life cycle of P. orientalis from oviposition to emergence of adult was found ranging between 48-60 days. Host preference study based on attraction of P. orientalis and other sandfly species to different animals was conducted in UmKura’a Warden Camp. Inverted light traps with bulb were hanged above animal cage between 18:00-06:00 hrs to collect sandflies attracted to different animal traps. The results showed clear preference of the P. orientalis to dogs (228.8 ± 53.5) compared to mongoose (Herpestes ichneumon) (63.88 ± 12.08), genet (Genetta genetta) (17.4 ± 3.72), and Nile rat (A. niloticus) (2.6 ± 0.56) (P<0.001). However, all sandfly species collected were found attracted to the dog baited traps (P<0.001) except S. bdfordi and S. antennata. Investigations for incrimination of a possible reservoir host (s) of Leishmania were conducted in two zoonotic foci of visceral leishmaniasis (1) Dinder National Park (DNP) and (2) Peri-domestic habitats of adjacent villages of eastern Sudan. The study included investigation of wild animals. Different traps were used for capturing animals including, animal cages, large spring traps (for carnivores), and multi-live trap, locally made faraneep (rats) and active chasing (for ground squirrels). Samples from spleen, liver and skin were taken from 250 and 254 during 1999 and 2000 respectively. Samples were kept in Isopropanosl for subsequent PCR and on glass slides for microscopy. Animals were captured, in March-November 1999 and April-June 2000 and examined for L. donovani infection using microscopy and two sensitive Polymerase Chain Reaction (PCR) systems. Infections of L. donovani were detected in 2 out of 14 Egyptian mongooses (Herpestes ichneumon), 1 out of 168 Arvicanthis niloticus and 1 out of 8 Mastomys natalensis. Samples from 68 other wild animals captured from the study area were all negative for Leishmania infection. The role of the domestic dogs (Canis familiaris) for the transmission of VL was investigated in the River Rahad area. Cross-sectional surveys were done during May 2002 to investigate the infection rates of Leishmania parasites in dog populations. A longitudinal survey was done to study the variation in the seroprevalence of VL infection among the dog populations in the area using the Direct Agglutination Test (DAT) during three different transmission seasons; November 2000, May 2001 and May 2002. For the infection rates study blood samples and lymph node aspirates were taken on filter papers Whattman No 3 for serology and subsequent PCR. Lymph node aspirates were prepared on glass slides for microscopy. The blood samples (n= 87) and lymph nodes (n= 36) collected during May 2002 were subjected to PCR. However, blood samples collected during the May 2002 were subjected to both DAT and FAST tests. The presence of anti- Leishmania anti-body for the season May 2002 was tested by freeze-dried dog-DAT (KIT, Amsterdam). The seasonal seroprevalence were tested using DAT antigen prepared in Khartoum. The results revealed one positive blood samples out of 20 samples tested by PCR, whereas, lymph node aspirates samples showed negative results. The results of the serological investigations were found to be 6.9% for DAT and 51.7% for FAST test for the blood samples collected during May 2002. The results of the serology tests showed high exposure of the dogs to L. donovani. The microscopic examination showed no parasites in the lymph node aspirates. The seasonal seroprevalence studies revealed no significant differences in DAT positivity between the seasons of November 2000 (7.1%), May 2001(7.1%) and May 2002 (9.1%) (χ2=0.259; P<0.878). The result of a socioeconomic questionnaire showed that the Masaleet people compared to other tribes mostly possessed dogs.