Host preference and human blood index of Phlebotomus orientalis, an exophilic sand fly vector of visceral leishmaniasis in eastern Sudan.

Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani, transmitted by Phlebotomus orientalis, is a serious systemic disease that causes high morbidity and mortality rates in Sudan and other parts of East Africa and the world. Despite progress in understanding the epidemiology of the disease in East Africa, little is known about the host preference of P. orientalis in kala azar endemic villages of Sudan, which have some of the highest VL incidence rates in the world. The present study used host choice experiments and blood-meal identification approaches to determine the host preference of P. orientalis in kala azar endemic villages in Gedarif state, eastern Sudan. In the host choice experiment, tent traps were used to compare the attractiveness of cows, donkeys, sheep and goats for host-seeking P. orientalis. In the blood-meal identification study, blood-fed P. orientalis females, captured inside houses and peri-domestic habitats, were subjected to molecular typing using cytochrome b gene (cyt b) amplification and sequence analysis. Cows and donkeys were the most attractive to blood-seeking P. orientalis, followed by goats. Similarly, the blood-meal analysis of P. orientalis showed that the vector preferentially feeds on cows, followed by donkeys, humans and goats. The human blood index of P. orientalis was 19.4% (42/216), indicating a high zoophilic habit of the vector, both inside and outside the houses. Although the order of host preference varied by location, it was clear that cows are the most preferred host of P. orientalis in the area. Results are discussed in relation to the role of domestic/livestock animals in VL zoopotentiation and zooprophylaxis. Inference is made on the potential impact of insecticide treatment of cows in control of the vector and the transmission of VL in Sudan and other parts of East Africa.

Infection of Leishmania donovani in Phlebotomus orientalis Sand Flies at Different Microhabitats of a Kala-Azar Endemic Village in Eastern Sudan.

A study was carried out to compare the infection rates of Leishmania donovani in Phlebotomus orientalis sandflies at different microhabitats of a VL endemic village in Gedarif state, Sudan. DNA extracts of 1078 P. orientalis sand fly females sampled by CDC light traps from indoor, outdoor, peri-domestic, and sylvatic sites, in three transmission seasons, March-June 2016-18, in Helat-Belo village, were subjected to independent PCR amplifications targeting Leishmania kDNA and the cpb gene followed by ITS1 region sequencing. Leishmania kDNA was detected in 1.4% of the 1078 P. orientalis females captured in the area. Two of these specimens showed a characteristic 741 bp band of L. donovani after cpb gene amplification. The DNA sequence of the ITS1 region of the parasites matched the ITS1 L. donovani genotype F. There were no signficant differences between rates of infection of L. donovani in P. orientalis captured at different sites. Blood meals found in infected flies origninated from human (5 specimens), cattle (4 specimens) and donkey (2 specimens). The finding of fresh cow and donkey blood in the infected flies suggests the possible role of these animals in the zoopotentiation and/or zooprophylaxis against VL. The study provides important information for VL transmission models and control programs in East Africa.

Genetic Structure of Phlebotomus orientalis (Diptera: Psychodidae) in Leishmaniasis Endemic Foci of Sudan.

BACKGROUND AND OBJECTIVE: Visceral leishmaniasis (VL) remains a major concern in many parts of Sudan. The disease is transmitted by Phlebotomus orientalis. The objective of this study was to determine genetic structure of Phlebotomus orientalis population from 5 geographical regions in Sudan. MATERIALS AND METHODS: A total of 194 individual sand flies were collected from 5 geographic regions in Sudan. The field collected sand flies were analyzed by Random Amplified Polymorphic DNA (RAPD) using 30 primers. RESULTS: Eight hundred and 65 bands from 4 RAPD primers were analyzed for genetic variation. A higher level of intrapopulational variability was detected in populations of P. orientalis from eastern Sudan compared to those populations from central and northern Sudan. Diagnostic bands were detected in populations of P. orientalis central Sudan. Hieratical clustering analysis showed clear clustering into 2 main populations with 1 population subdivided into 4 subpopulations. However, these populations did not show any correlation with their geographical origins. Furthermore, the low genetic differentiation among subpopulations was supported by fixation index (FST) estimated by analysis of molecular variance (AMOVA). CONCLUSION: It is concluded that the populations of P. orientalis from the selected areas in Sudan have a low genetic differentiation. However, assessment of genetic structure of P. orientalis populations is important for understanding the patterns of transmission of VL in different endemic areas.

Understanding sand fly sampling methods: sticky traps are attraction-based and not interceptive sampling tools of Phlebotomus orientalis.

BACKGROUND: Sticky traps are generally viewed as interceptive sand fly sampling methods; although no previous experimental evidence has supported this assumption. In this study, we tested this assumption experimentally for Phlebotomus orientalis, the principal vector of visceral leishmaniasis in East Africa, and propose an explanation for the highly male-biased collection of sticky traps. METHODS: A number of field experiments were carried out in March-June 2016-2019, in Gedarif state, eastern Sudan. In the first experiment, we compared numbers of P. orientalis caught on sticky traps made of black, red, transparent, white, yellow, green and blue A4 size papers set simultaneously at different lunar light conditions. In the second and third experiments, we compared numbers of P. orientalis captured on sticky traps placed side-by-side horizontally or vertically on the ground, or horizontally on a 15 cm height stool. We also witnessed mating behaviour of sand flies following their landing on un-sticky papers placed on the ground. RESULTS: Phlebotomus orientalis showed significant attraction to white, yellow and transparent traps, with negligible numbers caught on the black and the red traps. Similarly, significantly higher numbers of P. orientalis were attracted to the horizontal traps, resulting in an 8-fold increase in sand fly trapping efficacy as compared to the vertical traps. Placing the traps on the stools resulted in significant reduction in this attraction. In contrast to the sticky traps that captured only very few females; we found that when male sand flies land on un-sticky white paper they successfully lure females and copulate with them. CONCLUSIONS: We demonstrate that, for P. orientalis, sticky traps are more attractant-based than interception-based sampling tools. Further, our findings support the notion that males of this sand fly species likely utilize the bright surface of the trap papers to perform mating rituals that attract the females for copulation. However, pre-mature death in the sticky oil hampers the completion of these rituals, and thus results in failure to attract the females. These findings inform our understanding of P. orientalis behaviour and have important implications for optimization of sticky trap design for vector surveillance purposes.

Amine-binding properties of salivary yellow-related proteins in phlebotomine sand flies.

The amine-binding properties of sand fly salivary yellow-related proteins (YRPs) were described only in Lutzomyia longipalpis sand flies. Here, we experimentally confirmed the kratagonist function of YRPs in the genus Phlebotomus. We utilized microscale thermophoresis technique to determine the amine-binding properties of YRPs in saliva of Phlebotomus perniciosus and P. orientalis, the Old-World vectors of visceral leishmaniases causative agents. Expressed and purified YRPs from three different sand fly species were tested for their interactions with various biogenic amines, including serotonin, histamine and catecholamines. Using the L. longipalpis YRP LJM11 as a control, we have demonstrated the comparability of the microscale thermophoresis method with conventional isothermal titration calorimetry described previously. By homology in silico modeling, we predicted the surface charge and both amino acids and hydrogen bonds of the amine-binding motifs to influence the binding affinities between closely related YRPs. All YRPs tested bound at least two biogenic amines, while the affinities differ both among and within species. Low affinity was observed for histamine. The salivary recombinant proteins rSP03B (P. perniciosus) and rPorASP4 (P. orientalis) showed high-affinity binding of serotonin, suggesting their capability to facilitate inhibition of the blood vessel contraction and platelet aggregation.

Habitat preference and seasonal dynamics of Phlebotomus orientalis in urban and semi-urban areas of kala-azar endemic district of Kafta Humera, northwest Ethiopia.

Visceral leishmaniasis is a significant public health problem in northwest Ethiopia, particularly in Kafta Humera district. The study was designed to determine the species composition and population dynamics of sand flies in five villages representing urban and semi-urban areas of Kafta Humera district namely, Setit Humera, Mykadra, Rawyan, Bereket and Adebay. Sand flies were collected for two to three nights monthly from May 2011 to April 2012 using CDC light-traps and sticky traps. Traps were placed in villages, at periphery of villages and farm fields. Sticky traps were also used for sampling indoor active sand flies. In total, 13,097 sand fly specimens of eight Phlebotomus species and 91,949 Sergentomyia specimens were collected. Among the Phlebotomus, P. orientalis was the predominant species (58.1%) followed by P. papatasi (29.6%), P. lesleyae (5.6%), P. bergeroti (3.8%), P. duboscqi (2.1%), P. alexandri (0.4%), P. heischi (0.2%) and P. rodhaini (0.2%). Significantly, higher number of P. orientalis was caught in Adebay village and the least in Setit Humera town. Seasonal abundance of P. orientalis increased during the dry season (January-May) and dropped drastically in the wet season (late June-September). Significant positive correlation was found between monthly abundance of P. orientalis and the monthly average air and surface soil temperature, while a negative correlation was found with monthly average rainfall and relative humidity. Higher number of P. orientalis was collected outdoors, especially in the farm fields followed by periphery of villages. Thus, various observations strongly suggested P. orientalis to be the principal vector in the study areas, where farm lands and periphery of villages were identified as the most risky habitats, whereas the indoors were the least ones. Appropriate control methods should be designed and implemented according to the knowledge of P. orientalis habitat preferences and seasonal dynamics in the district.

Comparative study on the nocturnal activity of phlebotomine sand flies in a highland and lowland foci of visceral leishmaniasis in north-western Ethiopia with special reference to Phlebotomus orientalis.

BACKGROUND: Leishmania donovani, the causative agent of visceral leishmaniasis (VL), is most probably vectored by Phlebotomus orientalis in north-western Ethiopia. The aim of this study was to determine and compare the nocturnal activity patterns of Ph. orientalis in VL endemic foci of Libo-Kemkem (highland) and Metema (lowland) districts of north-western Ethiopia. METHODS: Sampling of sand flies was conducted bimonthly from January-May 2012 in the highland and from March-June 2013 in the lowland. Sand flies were sampled using two CDC light traps placed in compounds occupied by both cattle and humans. Sampling of sand flies started at 18:00 h and ended at 06:00 h. Every hour, a cage was replaced by another cage. RESULTS: In total, 9479 nocturnally active sand flies were collected from both study areas. Six Phlebotomus species (Ph. orientalis, Ph. duboscqi, Ph. papatasi, Ph. bergeroti, Ph. rodhaini and Ph. martini) and several Sergentomyia spp. were identified. In both areas, of the six Phlebotomus spp., Ph. orientalis was the preponderate. In the highland, the hourly activity pattern of Ph. orientalis females was higher before midnight with a peak in density between 22:00-23:00 h, whereas in the lowland after midnight between 03:00-04:00 h. CONCLUSIONS: The present study showed that Ph. orientalis females exhibited different nocturnal activity patterns with a peak in the early part of the night in the highland and in the latter part of the night in the lowland areas. As the risk of acquiring L. donovani infections vary in the two areas, appropriate control strategies should be developed according to the activity of Ph. orientalis in the respective areas.

Preliminary study on investigation of zoonotic visceral leishmaniasis in endemic foci of Ethiopia by detecting Leishmania infections in rodents.

OBJECTIVE: To investigate the zoonotic visceral leishmaniasis (ZVL) by identification of the most probable reservoir hosts using parasite isolation and analysis of a possible transmission dynamics of the disease in extra-domestic agricultural fields and rural villages. METHODS: Rodents were collected from selected study sites in kala-azar endemic areas based on information for localities of kala-azar cases for screening of Leishmania infections using parasitological, serological and polymerase chain reaction (PCR) from March, 2013 to January, 2014. Ketamine (Clorketam Veterinary) was used to anaesthesize the rodents according the prescribed dosage (average 2 mg/kg for intra-venous route). The blood obtained using sterile needle was dropped into sterile filter paper and allowed to air dry before sealing in plastic bags. The tissues from liver, spleen and skin were macerated in Locke's solution before transferring them into NNN medium. Blood and touch smears of liver, spleen, skin and bone marrow were prepared for fixing using methanol and staining by Giemsa stain for microscopy. These tissues were also used for DNA extractions and PCR amplification of Leishmania infection. RESULTS: A total of 335 rodents (13 species) were analyzed by sampling internal organs. The infection rate by PCR was 11.1% (6/54) for Arvicanthis nilothicus compared to 17.6% (3/17) and 12.5% (2/16) for Acomys cahirinus and Tarera (G) robustus respectively. Almost all the infections were found from bone marrow samples (8/48 or 16.7%) compared with 1/91 (1.1%) liver, 2/87 (2.2%) spleen and 0/87 (0%) skin. In all study sites with past human VL cases, rodents and proved vectors shared similar habitats. CONCLUSIONS: Leishmania donovani might circulate among different species of rodents in kala-azar endemic lowlands and valleys of Ethiopia by Phlebotomus orientalis and Phlebotomus martini. Detailed studies to substantiate the preliminary data on the possible role of these rodents are urgently needed.

A review of visceral leishmaniasis during the conflict in South Sudan and the consequences for East African countries.

BACKGROUND: Visceral leishmaniasis (VL), caused predominantly by Leishmania donovani and transmitted by both Phlebotomus orientalis and Phlebotomus martini, is highly endemic in East Africa where approximately 30 thousands VL cases are reported annually. The largest numbers of cases are found in Sudan - where Phlebotomus orientalis proliferate in Acacia forests especially on Sudan's eastern border with Ethiopia, followed by South Sudan, Ethiopia, Somalia, Kenya and Uganda. Long-standing civil war and unrest is a dominant determinant of VL in East African countries. Here we attempt to identify the correlation between VL epidemics and civil unrest. OBJECTIVE AND METHODOLOGY: In this review, literature published between 1955 and 2016 have been gathered from MSF, UNICEF, OCHA, UNHCR, PubMed and Google Scholar to analyse the correlation between conflict and human suffering from VL, which is especially apparent in South Sudan. FINDINGS: Waves of forced migration as a consequence of civil wars between 1983 and 2005 have resulted in massive and lethal epidemics in southern Sudan. Following a comprehensive peace agreement, but especially with increased allocation of resources for disease treatment and prevention in 2011, cases of VL declined reaching the lowest levels after South Sudan declared independence. However, in the latest epidemic that began in 2014 after the onset of a civil war in South Sudan, more than 1.5 million displaced refugees have migrated internally to states highly endemic for VL, while 800,000 have fled to neighboring countries. CONCLUSION: We find a strong relationship between civil unrest and VL epidemics which tend to occur among immunologically naïve migrants entering VL-endemic areas and when Leishmania-infected individuals migrate to new areas and establish additional foci of disease. Further complicating factors in East Africa's VL epidemics include severe lack of access to diagnosis and treatment, HIV/AIDS co-infection, food insecurity and malnutrition. Moreover, cases of post-kala-azar dermal leishmaniasis (PKDL) can serve as important reservoirs of anthroponotic Leishmania parasites.

Characterization of breeding sites of Phlebotomus orientalis - the vector of visceral leishmaniasis in northwestern Ethiopia.

We studied breeding sites of Phlebotomus orientalis (Diptera: Psychodidae) the vector of visceral leishmaniasis in northern Ethiopia. Although numbers were rather small, 165 sand flies were captured emerging from vertisol cracks. The most productive breeding sites were cracked vertisols, dry river banks and close to trees. No sand flies were caught emerging from sandy clay loam soils in peri-domestic habitats but a few were captured emerging from gaps in a stone wall. Abiotic parameters in vertisols close to trees and in open field from which P. orientalis had emerged, were compared. Soil pH was slightly alkaline and salinity was low. Organic matter contents were similar in both sites. Temperatures and RH remained relatively stable near trees from the end of the rainy season through mid dry season, yet fluctuated markedly at the shallower depth in the open field. During the rainy season, cracks in the soil were sealed resulting in significant lowering of the oxygen concentrations near the tree. Gravimetric water content of soil near trees was lower than open field at shallow depth but similar deeper down. We conclude that ambient conditions suitable for sand fly larvae at shallow depths (45cm) are restricted to areas close to trees. However, deeper in vertisols (90cm) suitable conditions are apparently maintained throughout the dry season even in open fallow fields.

Sandflies (Diptera: Psychodidae) in a focus of visceral leishmaniasis in White Nile, Sudan

Visceral leishmaniasis (VL) has been known to occur since the 1980s on the western bank of the White Nile River (Central Sudan), 150 km south of Khartoum, and has resulted in high mortality. The most recent outbreak of the disease in this area began in 2006. Entomological surveys were carried out during May 2008, June 2010 and May and July 2011 in the White Nile area. Sandflies were collected using Centers for Disease Control light traps and sticky oil traps in the village of Kadaba and the nearby woodland. Phlebotomus females were dissected for the presence of Leishmania promastigotes. A total of 17,387 sandflies, including six species of Phlebotomus and 10 species of Sergentomyia, were identified. The Phlebotomus species recorded were Phlebotomus orientalis, Phlebotomus papatasi, Phlebotomus bergeroti, Phlebotomus duboscqi, Phlebotomus rodhaini and Phlebotomus saevus. P. orientalis was collected in both habitats. The relative abundance of P. orientalis in the woodland habitat was higher than that recorded in the village habitat. In the woodland habitat, there was a notable increase in the relative abundance of P. orientalis during the surveys conducted in 2008 and 2010 compared to 2011. None of the 311 P. orientalis females dissected were infected with Leishmania promastigotes, although relatively high parous rates were recorded in both habitats. Based on the distribution of P. orientalis recorded in this study, this species is the most likely vector of VL in the endemic focus in the White Nile area. Further investigation is required to elucidate the seasonal abundance and distribution of the vector, as well as the transmission season of VL in both habitats so that appropriate control strategies for the vector can be designed.