Epidemiology and Transmission of Lymphatic Filariasis in Southern Sudan

BY: ELTAYA HASSAN AWAD ELKARAM B. Sc. (General) Zoology Faculty of Science Omdurman Islamic University Qualifying M.Sc Zoology University of Khartoum

A thesis Submitted in Fulfillment of the Requirements of the Master Degree in A Thesis Submitted in Fulfillment of the Requirements of the Zoology

Master Degree in Zoology Department of Zoology

Faculty of Science

Department of Zoology Faculty of Science

University of Khartoum ******************************************************* May 2007

ABSTRACT

This study on Lymphatic Filariasis and its vectors has been carried out at Bahr El Jebal State (Juba and Terkaka towns) between the periods August 2004 – September 2004 and April 2005 – June 2005 . Mosquitoes , the vectors of this disease , were collected using Pyrethrum spray (Knock down ). This insect survey showed that Anopheles gambiae s.s. is the common species in Juba area but in Terkaka Culex quinquefasciatus is the common species . All insect components namely head , thorax and abdomen were dissected in search of the parasites. The result revealed that the parasite , causing Elephantiasis (chronic filariasis ) is Wuchereria bancrofti. From the total number of mosquitoes collected (2729) only 81 individuals were found infected : 60 Anopheles and 21 Culex The overall rate of the mosquitoes which carried the first , second and third stages of the parasite was 2.9 %, but the rate of mosquitoes which carried the infective stage (L3) reached its maximum at Rajaf west (4.2 %) . The observations showed that the disease is prevalent in Juba and Terkaka . Males were more affected by the disease than females specially in the villages. A similar visit was also made to Blue Nile State being a second area where this disease prevailed .

الخالصة

تمت هذه الدراسة لمرض الفالريا الليمفية و العائل الناقل له بوالية بحر الجبل )مدينتي جوب ـا

وتركاكا( في الفترة من أغسطس 2004 – سبتمبر 2004 و ابريل 2005 – يونيو 2005 .

تم جمع البعوض ) العائل الناقل ( لهذا المرض بواسطة الرش ال ـرذاذي لمبي ـد الب ـايريثرم . اوضح

المسح الحشري لهذه الد ارسة ان انوفيليس قامبيا س .س. اكثر االنواع شيوعا في منطقة جوب ـا اما في

تركاكا فبعوض كيولكس كوينكيفاسكياتس هو اكثر االنواع شيوعا .

للبحث عن الطفيل تم التشريح الجزاء جسم البعوضة الثالث ـة ، الـرأس ، ال ـصدر وال ـبطن واظهرت

النتائج بأن الطفيل المسبب لمرض داء الفيل هو يوشيريريا بانكروفتي .

من العدد الكلي للبعوض )2729( الذي تم جمعه وجد ان 81 بعوض ـة فقـط مـصابة : 60 انوفيليس و

21 كيوليكس .

المعدل للبعوض المصاب الذي يحمل االطوار االول , الثاني والثال ـث للطفي ـل 9.2% لك ـن

المعدل للبعوض الذي يحمل الطور الثالث المعدي )ل3( وصل اقصاه ف ـي منطقـة الرج ـاف غـرب

. )%2.4(

اوضحت المشاهدة بأن المرض يتفشى في جوبا وتركاكا . الرجال اكثر اصابة بالمرض م ـن

النساء و خاصة في القرى .

تضمنت هذه الد ارسة زيارة مشابهة لوالية النيل االزرق كمنطقة ثانيه يتفشى بها هذا المرض.

Chapter One

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION Lymphatic Filariasis (LF) is a major cause of clinical morbidity and an impediment to socioeconomic development affecting some 80 countries in the tropics and subtropics (Farid et. al., 2003). And it puts at risk more than a billion people. Over 120 million people have already been affected by it, over 40 million of them are seriously incapacitated with the disease. One third of this number is in Africa and most of the remainder are in South Asia (particularly India), the Pacific and America. In tropical and subtropical areas where Lymphatic Filariasis is wellestablished, the prevalence of infection is continuing to increase. A primary cause of this increase is the rapid and unplanned growth of cities, which creates numerous breeding sites for the mosquitoes that transmit the disease (WHO, 2000). The causative agent of Lymphatic Filariasis in a nematode which belong to the following systematic status : Kingdom: Animalia. Phylum: Nematoda. Class: Secerneutea. Subclass: Spiruria. Order: Spirurida. Suborder: Spirurina. Family: Filariidae. Subfamily : Filarioidea Genus: Wuchereria

Species:bancrofti.( www.keepbair alive, com/ para. Html).2006

Lymphatic Filariasis is evidently endemic in Sudan; this is based on previous published (Satti and Abd El-Nur,1974) and unpublished data of scattered spot surveys and hospital records (lymphoedema or/hydrocele). Of the 26 Sudanese States, 12 states are considered LF endemic areas (Farid et. al., 2003). These include the 10 Southern States in addition to South Dar-Fur and Blue Nile States. The total population of these states is around 8 millions and depending on Rajaf and Radom studies, 3 millions are at risk of having the disease, two thirds of them are in Southern State. A report of Ministry of Health stated that between 1948 to 1968 some 7957 cases were recorded although details and parasitological verification of these cases were not presented. Lymphatic Filariasis is a parasitic disease that is spread by mosquitoes (Anopheles, Culex, Aedes and Mansonia), caused by three nematode worms of the Family Filariidae. These are Wuchereria bancrofti, Brugia malayi and Brugia timori. There are fifteen species of filarial worms recorded from mammals and which can develop in mosquitoes. They include four species and Wuchereria bancrofti is one of them (Nelson, 1959). The latter is responsible for 90% of worldwide infections with 9% caused by Brugia malayi in southeast and eastern Asia and only 1% is reserved for Brugia timori in the Pacific Region (Michael and Bundy, 1997). Lymphatic Filariasis or Bancroftian disease depends on two hosts: humans and several species of mosquitoes which ingest microfilaria when they bite a person. These microfilariae undergo a process of transformation in mosquitoes to become infective larvae. The microfilariae mature into adult worms which can live for several years in man lymphatic system and produce millions of microfilariae. The latter circulate in the peripheral blood stream, usually at night .The worms lodge in the lymphtic system – net work of nodes and vessels that

regulate the delicate fluid balance between the tissue and blood and are essential for fighting infection - causing stagnation of the lymph and swelling (Evans, 2003). In endemic areas a proportion of infected persons develops no clinical symptoms although microfilariae can be found in their blood (microfilaraemia). Such persons can remain asymptomatic for several years or progress to acute and chronic filariasis (Monica , 1999). When the diagnosis of the disease becomes difficult by the use of classic ways (because the parasite is nocturnal in periodicity) the immunochromatogra - phic diagnostic test (ICT) is the suitable way at any time (WHO, 2000). Two primary biological characteristics of bancroftian parasites render transmission of infection insufficient: the lack of any animal reservoir host and no amplification of the parasite within the mosquito vector (Elsetouhy and Ramzy, 2003). It is impossible for a bite by a mosquito carrying a single infective larva to cause microfilariae (Hairston and DeMeillon, 1968), perhaps paradoxically, the efficiency of Lymphatic Filariasis transmission is actually quite low. Generally, a person needs to be exposed to many mosquito bites for a period of months or years before becoming infected. In contrast malaria can be caused by just one bite from a malaria-carrying mosquito. The microfilaria are not injected when the mosquito bites, they must migrate through the puncture site (Evans, 2003). WHO decided that Lymphatic Filariasis should be eliminated as a public health problem. Elimination of lymphatic filariasis means a reduction of the disease incidence close to zero as a result of deliberate efforts requiring continued and coordinated activities (WHO, 2002). 1.2 Objectives: Apparently Lymphatic Filariasis is transmitted by several genera or species of mosquitoes. And all these genera do exist in the Sudan. But

work or surveys about transmission vector have never been practiced in this country. All the efforts of combating this disease were directed towards treatment of ailing individuals but no control strategies were directed towards the vectors. Therefore, the objectives of this study are to: 1. Confirm existence of the disease and determine the genera and species of the transmission vector. 2. Identify the species of filarial worms responsible for this diseas.

1.3 Mosquitoes: Mosquitoes are insects belonging to the Order Diptera; they have a worldwide distribution being found in the tropics, in the temperate zones and in the Arctic Circle. They have been found breeding not only in underground tunnels but also in deep mines (Gordon and Lavoipierre, 1962). The term “Mosquitoes” or “Musketa” is a Spanish or Portuguese word meaning “little fly” while British people call mosquitoes “Gants”, for any two winged insect which bites and sucks the blood of man (Floore, 1999). Modern writers used the name Culex to refer to mosquitoes but this tern is now retained as a mosquito genus. Among the other important genera are Aedes and Anopheles. These three genera belong to the Phylum: Arthropoda, Class: Insecta, Subclass: Pterygota, Order: Diptera, Suborder: Nematocera, Family: Culicidae, Subfamily: Culicinae. The latter is divided into three tribes: the Anophelini (the Anophelines), Culicini (the Culicines) and the Toxorhychitini or elephant mosquitoes (Gordon and Lavoipierre, 1962). 1.3.1 What are mosquitoes: Culicine mosquitoes are small long legged organisms. They may be distinguished from all other Nematocerous flies by:

1. Proboscis with palps which are held straight and rigid, and which are not pendulous. 2. Scales are present on the body , the legs and the wings. The colouration of the mosquito is dependent on the colour of the scales. 3. Wing venation in which the third vein is straight and lies between two forked veins (Gordon and Lavoipierre, 1962). Mosquitoes are the most important single group of insects with regard to public health. They are remarkable and fully cosmopolitan group with 3.000 species distributed throughout the world (WHO, 1972). Since mosquitoes live in close association with man, travelling with and feeding on him, but existing for the most part as free-living insects, they are in particular well adapted to harbour and transmit agents of disease. The Subfamily Culicinae contains many genera of mosquitoes, of which the medically important ones are Culex, Aedes, Mansonia, Sabeines, Haemagogos and Anopheles (WHO, 1972). 1.3.2 Status of mosquitoes in the Sudan: Survey of mosquito fauna of the Sudan revealed that there are about 76 species of the two genera, Aedes 31, and Culex 45 (Lewis, 1943, 1945, 1953, 1955; El-Rayah, 1976). Many species of Anopheline mosquitoes are found in the Sudan,about (31) species have been identified (Nugud, et.al. 1997). The most dominant species of culicine mosquitoes in Sudan is Culex quinquefasciaus and others ( Abu Groon , 1988).

1.4 The genus Culex: Members of the genus Culex are found both in temperate and tropical zones throughout the world. Several species occur over very large geographical areas and the group which forms Culex pipiens complex is cosmopolitan.

The genus Culex was early shown to be of medical importance, when Manson in 1877 discovered the developing stages of Wuchereria bancrofti in a Culex mosquito. These mosquitoes are now recognised to be not only important transmitters of filariasis but also vectors of several diseases e.g mosquito-borne encephalitis (Gordon and Lavoipierre, 1962).

1.4.1 Culex quinquefasciatus in urban and rural areas: Although Culex quinquefasciatus occurs mainly in urban areas, it is also present in rural areas. A brief description of comparative studies on Culex quinquefasciatus in urban and rural areas is summarized below: a. Mosquito density: Culex quinquefasciatus is usually much more numerous in urban areas than in rural areas. As quoted by Singh (1965), the highest density of Culex quinquefasciatus per man-hour in an urban area (Gaya) was 495 and in rural Delhi, only 3-7. In Djakarta, the monthly average total collection of this mosquito in three houses was from 150 to 456 (Chow et. al., 1959).

b. Susceptibility of the mosquito to the urban and rural strains of W. bancrofti: Singh (1965) stated that the difference in infection rate of mosquitoes in urban and rural areas seems to be due to mosquito longevity. The mosquito density would have some effect on this

(Chow, et. al. 1959).

1.4.2 History of Culex quinquefasciatus Say in the Sudan:

The old name of this mosquito was Culex pipiens fatigans Wiedmann. The mosquito was recognized to be Subspecies of Culex. pipiens, which hybridized with it to a significant extent in nature. In the Sudan there is a little opportunity for hybridization (Lewis, 1956). In vast extent of the Sudan it had been only known from a comparatively small area near the coast where it might have been introduced by shipping. It seems probable that Culex pipiens fatigans has acquired its wide tropical

distribution largely by association with man, but it had apparently failed to penetrate far inland across the desert by rail. It may have reached Kassala by road from Eritrea. Additional localities were Gebeit Gold Mine, Halaib, Tokar and Port Sudan (Lewis, 1956).

1.4.3 Breeding site of Culex: Culex mosquitoes breed in quiet waters of almost all kinds from artificial containers and catchment basins of drainage systems to large bodies of permanent water. Water in which there is much organic material, including sewage, is often favoured (WHO, 1972).

1.4.4 Classification of Culex quinquefasciatus: Kingdom: Animalia. Phylum: Arthropoda. Subphylum: Hexapoda. Class: Insecta. Subclass: Pterygota. Order: Diptera. Suborder: Nematocera. Family: Culicidae. Subfamily: Culicinae. Tribe: Culicini. Genus: Culex. . Species: Culex quinquefasciatus Say (Gordon and Lavoipierre, 1962).

1.5 The genus Anopheles: Members of the genus Anopheles have a worldwide distribution. More than 200 species and varieties have been described. The genus Anopheles was so named in 1818, but it was not until some 80 years later that the particular association of Anophelines with the transmission of mammalian malaria (in contrast to the part played by Culicine mosquitoes in the

transmission of avian malaria) was demonstrated (Gordon and Lavoipierre, 1962). Anopheline mosquitoes are predominantly important vectors of malaria to man in some parts of the tropics they are also important vectors of filariasis. They are not known to transmit infections other than these. No species of anopheline have been shown to be vectors of viruses.

1.5.1 Species and distribution of Anopheles mosquitoes in Sudan: About 31 Anopheles species have been identified in Sudan but only few of them are known to be malaria vectors (Nugud et. al.,1997). Anopheles gambiae has been identified only from southern Sudan (Petrarca et. al., 1986). Anopheles arabiensis has been identified from eastern Sudan in Kassala Province (Lewis 1956, 1959), Khashm El-Girba area (Haridi, 1972a) Gedaref State (Karrar, 1996, Abd El-Rahim, 1998), New Halfa Town (Hiemeidan, 2000) and Northern Sudan (Dukeen, 1981), Western Sudan, Kordofan area (El-Mamoun, 1993; El-Tayeb, 2001). An. Arabiensis was also reported from Khartoum State by Omer (1968), Abu Groon (1988); El-Khawam (1989), Ali (1992), El-Imam (1997), El-Sayed (1998), and Nurein (2000). In the Gezira area it was reported by El-Rayah (1976) and El-Safi (1992). Anopheles pharoensis co-exists with Anopheles arabiensis in Sudan particularly in irrigated and swampy areas (Akood, 1980). Anopheles rufipes was reported from ElRahad area, North Kordofan State, mid west of Sudan by El-Tayeb (2001).

1.5.2 Anopheles gambiae:

Anopheles gambiae is not a single species, but a complex of species. The latter look alike morphologically, they are actually different species, which have been identified cytologically. Anopheles gambiae is complex divided into seven separate species, and they are identified by examination of their chromosomes. These different species are sexually isolated. They are morphologically identical but intrinsically they are different. In the Sudan we have only two species out of these seven ones mentioned, they are Anopheles gambiae and Anopheles arabiensis. These two species have also certain geographical distribution. Anopheles gambiae is equatorial in distribution, while Anopheles arabiensis, has adapted itself to the dry and arid zone. It is found in the Sahara, and the Savana and other arid areas of Africa (so Anopheles gambiae is equatorial and Anopheles arabiensis is in the centre of the Sudan). Anopheles arabiensis is a rural species. This is a very important fact in relation to malaria transmission. Anopheles arabiensis does not usually breed in any type of polluted water; it breeds in clean water, which is exposed to sunlight, and pure air (Habib, 1982).

1.5.3 Breeding sites of Anopheles: Most anopheline mosquitoes breed in static fresh water pools; each species being with particular ecological conditions (White, 1982). Anopheles arabiensis is adapted to change its breeding sites according to the change in the environment (El-Imam, 1997). Anopheles gambiae normally breeds in shallow, sunlit puddles, completely or partially exposed to direct sunlight (Muirhead-Thompson, 1951). In Sudan Anopheles arabiensis was found to utilize different types of breeding sites, such as pools made by the Nile water, rain pools during the rainy season, irrigation canals, seepage from water pipes during the dry season and

water storage tanks (El-Imam, 1997). (Dukeen ,1981) reported breeding sites of Anopheles arabiensis in underground waters (wells).

1.5.4 Seasonal abundance and density of Anopheles: In general terms seasonal changes in Anopheles gambiae population tend to follow the seasonal pattern of rainfall. Thus in Savanna zones with a single rain season numbers start to rise explosively soon after the first rain falls, reaching a peak in middle of the rains and declining steadily thereafter as water levels become stabilized and vegetation and predators become established (Walton, 1947; Adam and Grjebine, 1956; Draper and Smith, 1957). The exact pattern seems to vary from one district to another (Gillies and DeMeillon, 1968). Thus Hamon et. al. (1956) recorded that the seasonal rise begins in some areas ahead of the rains in the middle of the dry season, owing to the partial drying and pooling of swamps and rice fields. In equatorial zones with two seasons An.gambiae population responds with two annual peaks (Mouchet, 1962; Gillies, 1954c). At Isiolo in northern Kenya, Heish (1947) found that the peak production followed the short rains (October-December) rather than long rains (March-June). In certain situations, the seasonal relationship may be reversed. Thus in some of the large rivers in Cameroon, pooling on sand bank in the dry season leads to a population peak when the river is low .During the rains these are swept and the numbers of the species become low (Mouchet, 1962).

1.5.5 Classification of Anopheles gambiae: Kingdom: Animalia. Phylum: Arthropoda. Subphylum: Hexapoda. Class: Insecta. Subclass: Pterygota. Order: Diptera.

Suborder: Nematocera. Family: Culicidae. Subfamily: Culicinae. Tribe: Anophilini. Genus: Anopheles. Species: Anopheles gambiae (Gordon and Lavoipierre, 1962). 1.6 What is Lymphatic Filariasis: Lymphatic Filariasis or elephantiasis is one of humanity’s oldest and most debilitating diseases but few people have heard of it, let alone know any thing about it. It does not make the headlines and it does not kill outright, but it causes permanent disability silently destroying people’s lives. The facts are intimidating. Lymphatic Filariasis is a parasitic disease that has spread by mosquitoes. A-thread-like worm causes the disease which depends on two hosts: humans and several species of mosquito (Evans, 2003). 1.6.1 History of the disease: In 1860 Otto Wuchereria demonstrated the presence of filaria, or filaria larvae in urine. In 1871 Timoth Lewis discovered the presence of microfilaria in peripheral blood, later in 1876, Joseph Bancrofti discovered the adult form. Finally in 1878, Patrick Manson observed the development of Wuchereria bancrofti in mosquitoes (www.worldhistory .com/wink/F/Filariasis.htm.4October2005). Lymphatic Filariasis has been a worldwide scourge of civilization for thousands of years. Depicted on the pharaonic murals of Egypt and in the ancient medical texts of China, India, Japan and Persia, elephantiasis and hydrocele were first associated with parasitic filarial worms and their mosquito vectors in the late 19th century by French, English and Australian physicians working with patients from Cuba, Brazil, China and India (WHO, 1996). Elephantiasis have been known since antiquity; the

ancient Greek and Roman writers likened the thickened and fissured skin of infected persons to that of the elephant (Schmidt and Roberts, 1977). 1.6.2 Cause of filariasis: The thread like, parasitic filarial worms Wuchereria bancrofti and Brugia malayi that cause Lymphatic Filariasis live almost exclusively in humans. These worms lodge in the lymphatic system. This network of nodes and vessels maintains the delicate fluid balance between the tissues and blood. They are an essential component for the body’s immune defence system. Worms live for 4-6 years, producing millions of immature microfilariae (minute larvae) that circulate in the blood (WHO, 2000). 1.6.3 Vectors of Lymphatic Filariasis: The disease is transmitted by mosquitoes that bite infected humans and pick up the microfilariae that develop, inside the mosquito, into the infected stage in a process that usually takes 7-21 days. The larvae then migrate to the mosquitoes’ biting mouthparts, ready to enter the punctured skin following the mosquito bite, thus completing the cycle (WHO, 2000)

1.7 Vectors of W. bancrofti in the world: The impact of permethrin-impregnated bed nets on the resting and feeding behaviour of mosquito vectors of Wuchereria bancrofti was studied in six pairs of villages (treated and untreated) before and after intervention. The study villages were in Kwala District, near the coast of Kenya, where bancroftian filariasis is highly endemic, transmitted by a combination of both Anopheline and Culicine mosquito vectors (Bogh and Pedersen, 1998). Several species of anthropophilic mosquitoes were collected at night in Monrovia, Liberia, and dissected to ascertain whether they were carrying the filarial larvae of Wuchereria bancrofti. Of the mosquitoes caught,

55.8% were Culex fatigans (Culex quinuefasciatus), 32.8% were Culex antennatus and 8.1% were A. gambiae . The developing filarial stages were found in some mosquitoes, but mature larvae were only encountered in delayed dissection. Offspring of Culex quinuefasciatus, Culex antennatus, Culex poicilipes, Anopheles gambiae, Anopheles melas, Anopheles ziemanni, Aedes aegypti and Mansonia uniformis were reared to the adult stage in the laboratory and fed blood containing microfilariae of the rural filarial strain to see if they were capable of transmitting Wuchereria bancrofti. In all mosquito species, except Mansonia uniformis, the microfilaria developed and matured (Kuhlow, 1984). The common house mosquito, Culex quinuefasciatus Say is the vector of human filariasis due to Wuchereria bancrofti in Sri Lanka.(Samarawickrema et al 1980 & Abdulcader 1966). Filariasis is a major public health in India. It is estimated that about 122 million people inhabit the area in which Wuchereria bancrofti is endemic. In this area of endemicity, about five million people have one or more manifestations of these disease and about 13 million people have microfilariae in their blood .The mosquito vector is Culex quinuefasciatus. (Pattanayak , 1966). Filariasis caused by Wuchererai bancrofti has been known to exist in Burma for a long time. It appeared to be on the increase, and caused public concern particularly after World War II. The vector has been ascertained as being Culex pipiens fatigans (Hayashi, 1966).

1.8 Diagnosis: Until very recently, diagnosing Lymphatic Filariasis had been extremely difficult since the parasites had to be detected microscopically in the blood. And in most parts of the world, the parasites have a “nocturnal periodicity” that restricts their appearance in the preiferal circulation to only hours around midnight. The new development of a very sensitive and very specific simple “ICT card test” to detect circulating parasite antigens without the need for laboratory facilities and using only finger-prick blood droplets, taken anytime of the day, has completely transformed the approach to diagnosis. With this and other new diagnostic tools, it will be possible both to improve our understanding of where the infection actually occurs and to monitor more easily the effectiveness of treatment and control programmes (WHO, 2000).

1.8.1 Identification of ICT Card Test: ICT (immunochromatographic testing) is a rapid card-based test. It is highly sensitive and specific, with excellent negative predictive values but low positive predictive values as it detects antigenaemia in microfilaraemic individuals (Reda, 1997). 1.9 Status of Lymphatic Filariasis: Elephantiasis is a complication of advanced Lymphatic Filariasis. It is seen as coarse thickening, hardening, and cracking of the skin overlying enlarged fibrosed tissues. The legs are more commonly affected than the arms and in W. bancrofti endemic areas, the thigh also is often involved. Grossly enlarged limbs make walking difficult. Secondary bacterial and fungal infections of the skin can occur. Elephantiasis is more commonly seen in filariasis endemic areas of Africa, China, India and the Pacific region (Monica, 1999).

In brugian filariasis, symptoms of infection develop more rapidly and children are often infected. Elephantiasis occurs less frequently and tends to involve only the lower limbs. In male adults, the scrotum and spermatic cord are not usually affected and hydrocele is rare (Monica, 1999). 1.10 Asymptomatic Lymphatic Filariasis: In endemic areas a proportion of infected persons develop no clinical symptoms although microfilariae can be found in their blood (microfilaraemia). Such persons can remain asymptomatic for several years or progress to acute and chronic filariasis (Monica, 1999). 1.10.1 Acute Lymphatic Filariasis: In the acute form there are recurrent attacks of fever (filarial fever) with painful inflammation of lymph nodes (lymphadenitis) and lymph ducts (lymphangitis). The lymphatics involved are those of the limbs, genital organs (especially spermatic cord) and breasts (Plate 1a). In bancroftian filariasis, the lymph glands in the groin and lymphatics of male genitalia are frequently affected. Inflammation of the spermatic cord and repeated attacks can lead to blockage of the spermatic lymph vessels, leading to accumulation of fluid in the scrotal sac, which becomes distended (hydrocele). In brugian filariasis, the affected lymph nodes are mostly situated in the inguinal and axillary regions with inflammation of distal lymphatics. Acute attacks can last for several days and are usually accompanied by a rash and eosinophilia. Damage to the lymphatics leads to thickening and oedema (lymph fluid in surrounding tissues). Infections with subperiodic W. bancrofti and B. timori are associated with ulcers which form along the inflamed lymph vessels (Monica, 1999).

1.10.2 Chronic Lymphatic Filariasis: This is characterized by hydrocele (Plate 1b); lymphodema and elephantiasis of limbs (Plates 1c & 1d). Hydrocele is common in bancroftian filariasis. Microfilariae are rarely found in the blood of patients with hydrocele or elephantiasis but can be found occasionally in hydrocele fluid (Monica, 1999).

Symptomatic Lymphatic Filariasis

*After : maven.smith.edu/~sawlab/fgn/pnb/wuchban.html 01/01/1996

Plate ( 1 a ): Lymphatic Filariasis of breast

Plate ( 1 b ): Lymphatic Filariasis of hydrocele Symptomatic Lymphatic Filariasis *After: maven.smith.edu/~sawlab/fgn/pnb/wuchban.html 01/01/1996

Plate ( 1 c ): Lymphatic Filariasis of arm

Plate ( 1 d ): Lymphatic Filariasis of leg and hydrocele

1.11 Distribution of the disease in the world: Human Lymphatic Filariasis occurs in humid tropical foci in Africa, America, Asia and numerous islands in the Pacific Ocean. Local epidemiology of the disease is strongly influenced by the behaviour and ecology of the various species of mosquito vectors, so that seven principal epidemiological zones of Bancroftian filariasis distribution are based on regional variations of the vectors. Brugian filariasis is restricted to parts of Southeast Asia and Australasia (Map 1). Bancroftian filariasis in urban situations is becoming increasingly prevalent in warm countries due to transmission by Culex quinquefasciatus ( Culex pipiens fatigans) (Map 2) which breed prolifically in polluted water. As these mosquitoes have developed wide spread resistance to insecticides (WHO, 1986) it is preferable to control them by environmental management and source reduction (WHO, 1982). 1.12 Lymphatic Filariasis endemic in countries in the Eastern Mediterranean Regions: It is estimated that approximately 0.4 million LF infected individuals, representing <1% of the LF global burden, live in countries of the Eastern Mediterranean Regions (Michael and Bundy, 1997). The disease is known to be focally endemic in 3 countries: Egypt, Sudan and the Republic of Yemen, whereas the LF situation in Djibouti, Oman, Pakistan, Saudi Arabia and Somalia is currently uncertain. However, clinical cases have been reported in Oman, Pakistan, Saudi Arabia and Somalia. In the countries of the region, LF is entirely transmitted by Culex mosquitoes in mostly rural and semi-urban areas (EL-Setouhy and Ramzy, 2003). Funds to support LF elimination activities in the countries of the Eastern Mediterranean Region have been available through several

Map (1) : Distribution of Wuchereria bancrofti in the world *After: WHO(1986)

Map (2) : Distribution of bancroftian filariasis in urban vectors *After: WHO(1986)

funding agencies. In particular, the Arab Fund for Economic and Social Development, located in Kuwait, donated a generous contribution aimed at helping countries to start LF elimination (EL-Setouhy and Ramzy, 2003).

1.13 Egypt and Wuchereria bancrofti: The population of Egypt (administratively consisting of 26 governorates) currently amounts to about 68 million, of which more than 60% reside in the densely populated governorates of the Nile Delta. In Egypt, nocturnally periodic Lymphatic Filariasis caused by Wuchereria bancrofti infection has been endemic in rural areas for a long time (Southgate, 1979). Culex pipiens is the main vector mosquito and is extremely abundant throughout the country. The disease has a focal distribution, causing a major public health problem in 6 governorates in upper Egypt. However, due to a sustained control measures by the Ministry of Health and Population (MOHP), most endemic villages have low infection prevalence rates and intensities (Weil et. al., 1999). 1.14 Filariasis in urban areas after World War II: In some large cities, C. p. fatigans breeding increased enormously, particularly after World War II. For example, in Rangoon the sewage system was destroyed by bombing and other means, with the result that polluted material was seen to collect in the open drains, which are ideal for C. p. fatigans ’ breeding. Urban filariasis has, therefore, occurred. The same situation appears to have occurred in the Manila area. Although it seems that filariasis transmission has not been noted. (Singh, 1965), classified an area where C. p. fatigans density is comparatively high but with no infection as a potential risk of filariasis transmission. Abdulcader (1965) stated that in Ceylon bancroftian filariasis became a problem only after the war. Before the war, rural filariasis (B. malayi)

was most apparent, while urban filariasis (W. bancrofti) was confined to two coastal towns Calle and Metara, in the southern part of the country (Chow, 1966).

1.15 History of Lymphatic Filariasis in Sudan: In reviewing the incidence of filariasis in Sudan Kirk (1957) summarized the information given in the reports of Archibald (1914), in Geneina Town of Darfour. In 1930s, unidentified microfilariae in massive lymph-scrotum were found. Woodman and Bokhary (1941) also reported prevelance of filariasis in the Sudan. Wuchereria bancrofti has not been incriminated as a cause except in a small area in the Nuba Mountains in Kordofan Province. In (1944) Mahdi discovered the disease in the area around Kadogli in the Nuba Mountains where occasional cases of elephantiasis were known to exist (Abdalla, 1974). In 1966 Kirk, carried out a complete randomized survey of Kadogli Town where Mohy Eldin had described his cases.(Satti and Abd- El- Nur, 1974). Reconnaissance survey was carried out in February (1968), when several cases were encountered, , many with hydrocele and elephantiasis at Farbronga and Habeila. In June of the same year a survey was conducted in the same areas (Satti and Abd-El- Nur, 1974). All those results from surveys were taken from blood samples. Insects were observed at those areas (Nuba Mountains and South-Western Darfur). The main features of this collection were the absence of Culex fatigans and the abundance of the malaria vectors, Anopheles gambiae and A. funestus, and of the yellow fever vector Aedes aegypti, Hawking (1957) stated that “the chief vectors of Wuchereria bancrofti in Africa seem to be Anopheles gambiae and A. funestus. Culex fatigans has been found to be a poor vector”(Satti and Abd- El- Nur,1974).

There were some cases reported at Yabus Bridge village and Zalingi of the Sudan (Map 3a).(Abdalla, 1974). The current presence of Lymphatic Filariasis in Sudan includes Kassala, Gadaref, Sennar, Blue Nile, Upper Nile and Bahr Eljebel, Eastern Equatoria, South Kordofan, White Nile and Khartoum states (Map 3b and Table 1) (Federal Ministry of Health (2005). Unless epidemiological surveys are been done, and other states can not be considered free of Lymphatic Filariasis. Mapping of Lymphatic Filariasis in Sudan is currently hampered because of the conflict in parts of the country. Therefore there are certain areas that can not be accessed or reached for epidemiological surveys (EL-Setouhy and Ramzy, 2003). 1.16 Bancroftian filariasis and other filarial diseases in Sudan in 1970’s: Bancroftian filariasis has not been reported before from western Sudan and the discovery of one case from Zalingi area in Darfur State indicated the need for surveys to determine the prevalence of infection in that Province (Abdalla , 1974). In the east in the region along the Ethiopian border, the presence of the disease has long been suspected (Kirk, 1957). The identification of microfilariae of Wuchereria bancrofti in cases of elephantiasis and hydrocele in Yabus area proved the presence of the disease in the eastern region. Considering that the previously known focus of bancroftian filariasis in Kadogli area in Central Sudan, the discovery of new cases in Zalingi area in the west and in Yabus area in the east of the country indicated that the disease is patchy in distribution but may be more widespread than is realized.

Map ( 3 a ) : Distribution of Wuchereria bancrofti in the Sudan in the past *After: Abdalla (1974)

Map ( 3 b ) : Status of Lymphatic Filarasis in Sudan * After: Federal Ministry of Health (2005).

Table (1) . ICT Prevalence of Lymphatic Filariasis in certain states

State surveyed No. of sites examined Highest Prevalence

Kassala 4 14% (n. 102)

Gadaref 5 22% (n. 50)

Sennar 3 1% (n. 83)

Blue Nile 4 51% (n. 100)

Upper Nile 8 28% (n. 50)

Bahr El-Jebel 2 42% (n. 50)

Eastern Equatoria 5 54% (n. 50)

South Kordofan 9 52% (n. 50)

White Nile 4 10% (n. 100)

Khartoum 7 20% (n. 100)

* After: Federal Ministry of Health (2005).

Bryant (1935) failed to demonstrate Wuchereria bancrofti in cases of hydrocele and elephantiasis heavily infected with Onchocerca volvulus and accordingly he included these conditions among the clinical manifestations of onchocerciasis in the Province. Subsequent observations by other workers (Cruickshark, 1936; Kirk et. al., 1959; Haseeb et. al., 1962) were in agreement with his opinion. According to Augustinne (1957) the association of scrotal elephantiasis, hydrocele and lymphoid reactions with onchocerciasis, as reported by various investigators in Africa, is characteristic of the African type of the disease. In Bahr El- Ghazal Province Onchocerca volvulus may well be the cause of the clinical conditions but the discovery of the co-existence of bancroftian filariasis and onchocerciasis in Yabus leads one to suspect the possible presence of Wuchereria bancrofti infections in some parts where onchocerciasis existed. Loa loa was also suggested as a possible cause of elephantiasis and hydrocele (Cruickshark, 1936). These clinical conditions are common in loasis endemic region in western Equatoria Province. Woodman and Bokhary (1941) discovered 12 people infected with Wuchereria bancrofti but failed to demonstrate the infection in cases of elephantiasis, hydrocele, lymphocele and lymphodenopathy. They were of the opinion that the conditions were manifestations of Loa loa infection (Woodman and Bokhary, 1941; Woodman, 1949). This conclusion is different from the experience of workers in other countries and condition examination of such cases may show microfilariae of Wuchereria bancrofti in addition to usual Loa loa infection.

1.17 Cases of filariasis in the Sudan (historical) : In the 70s, Abdalla (1974) identified the microfilariae of Wuchereria bancrofti in 5 cases of elephantiasis and hydrocele; the patients were from two regions of the country other than Nuba Mountains. In one of these areas in addition to infection with Wuchereria bancrofti, people were found infected with Onchocerca volvulus (Abdalla, 1974).

1.17.1 Case reports:  Case No 1: Man 27 years old from Tour Village near Zalingi in Darfur Province. He was admitted to Khartoum Civil Hospital with hydrocele of the scrotum and microfilariae of Wuchereria bancrofti were found in his blood. They were present in blood smears taken during both day and night, but the blood microfilaria count, using Khott’s concentration method, showed that they were approximately 100 per ml at 23 hours, and only 20 per ml at 11 hours, indicating subperiodicity with nocturnal peak. Cases No. 2, 3, 4 and 5 were seen in southern Blue Nile Province in the area around Yabus River near the Ethiopian border. In all 4 cases the microfilaria of Wuchereria bancrofti were found in skin shavings but no subcutaneous nodules were present.  Case No 2: Female, 16 years old, with elephantiasis of the left leg.

 Case No 3: Female, 20 year old with elephantiasis of the left leg.  Case No 4: Male, about 35 years of age. He had bilateral hydroceles, enlarged inguinal lymph nodes, and thickened skin of the legs. Microfilariae of Wuchereria bancrofti were present in blood smears.  Case No 5: Male, about 45 years of age with elephantiasis of scrotum. Microfilariae of Wuchereria bancrofti were found in his blood.

1.17.2 Survey at Yabus Bridge: At Yabus Bridge Village, where cases No. 2, 3 and 4 were discovered, 20 adults of both sexes, about 15 to 50 years old, were examined for evidence of filariasis and onchocerciasis. Blood smears and skin shaving showed that, out of the 20 individuals 2 were infected with Wuchereria bancrofti which were found in blood smear taken between 22 and 23 hr. Smears were also taken between 10 and 11 hr. and except in 2 cases, these smears did not show the presence of microfilariae. Microfilarial counts were not done but it was apparent from microscopical examination of the smear from those two cases that the microfilariae were fewer in day than in night blood (Abdalla, 1974). The clinical manifestations of filariasis were not detected and the skin changes of onchocerciasis were not seen but, of the 6 individuals who showed microfilariae of Onchocerca volvulus, 2 had subcutaneous nodules over the ribs and hips. In addition, the group of people who showed both infections included a man, about 40 years old, who had one subcutaneous nodule on each side of the chest. He was blind, although his eyes appeared normal and the expression of his face was characteristic of “Sudan blindness” due to onchocercal retinochoroditis. His face as described by Bryant (1935) possessed “that vacant gazing expression so characteristic of the blind”. 1.18 Filarial worms: Fifteen species of filarial worms from mammals are known to develop in mosquitoes; these include four species of Wuchereria:  Wuchereria bancrofti.  Wuchereria malayi.  Wuchereria pahangi.  Wuchereria patei.

Six species of Dirofilaria, three of Setaria and two of Dipetalonema ,and one species of bird filaria Aproctoides, four species of Foleyella from frogs and Conispiculum and Oswaldofilaria from lizards all these undergo larval development in mosquitoes. There seems to be no doubt that many more will be found to be transmitted by mosquitoes in nature. It is therefore rather surprising to find only four references to filarial larvae of animals having been seen in mosquitoes during human filariasis surveys. Poynton and Hodking (1938) and Halcrow (1954) found a few larvae thought to be Dirofiaria immitis. Raghavan et. al. (1952) found larvae of Dirofilaria repens and Carter (1948) found some very long infective larvae, which he suggested to have originated from water-buffaloes (Nelson, 1959).

1.19 Wuchereria bancrofti : 1.19.1 Biology of Wuchereria bancrofti: Adult Wuchereria bancrofti live in the major lymphatic ducts of humans tightly coiled into nodular masses. They are normally found in the afferent lymph channels near the major lymph glands in the lower half of the body. Rarely, they invade a vein. The females are ovoviviparous, producing thousands of larvae known as microfilariae, which are not differentiated as normal first-stage larvae and are sometimes considered advanced embryos (Schmidt and Roberts, 1977). The microfilariae are released into the surrounding lymph by the female. Some may wander into the adjacent tissues but most are swept into the blood via the thoracic duct. Throughout much of the geographical distribution of the parasite, there is a marked periodicity of microfilariae in the peripheral blood. That is they can be demonstrated at certain times of the day, while at other times, they virtually disappear from the peripheral circulation.In the nocturnal rhythm maximum number may

usually be found between 10 pm and 2 am. For this reason, night-feeding mosquitoes are the primary vectors of Wuchereria in areas where microfilarial with nocturnal periodicity occurs where . During the day, the microfilariae are concentrated in blood vessels of the deep tissues of the body, predominantly in the pulmonary vessels proximal to the pulmonary arterioles. The causes of the periodicity remain obscure, but they apparently do not involve daily release of a new generation of progency by the adult female. Stimuli, such as arterial oxygen tension and body temperature, are probably involved (Schmidt and Roberts, 1977).

1.19.2 Morphology of Wuchereria bancrofti: The adult worms live in the lymph glands or ducts, often in inextricable tangles. The female is 65 to 100 mm long and only 0.25 mm in diameter (Plate 2a); the male is about 40 mm long and 0.1 mm in diameter (Plate 2b).The body tapers to a fine head slightly swollen at the end, with a simple pore as a mouth. The oesophagus is partly muscular and partly glandular with the vulva opening a little behind its middle. The males have the tail coiled like the tendril of a vine, with numerous pairs of papillae; there is one long and one short spicule (Chandler and Read, 1961).

1.19.3 Lifecycle of Wuchereria bancrofti: During a blood meal, an infected mosquito introduces third stage filarial larvae onto the skin of the human host, where they penetrate into the bite wound (Plate 3). They benetrate into lymphatics and develop into adults . Adults produce sheathed microfilariae . In the tropics they have nocturnal periodicity i.e. they appear in the peripheral circulation chiefly between 10 pm and 4 am except in the South Pacific. The microfilariae migrate and move actively in lymph and blood channels (www.dpdcdc.gov/dpdx.May 2004.22=19=10).

A mosquito usually ingests the microfilariae during a blood meal. Ultimately, the microfilariae lose their sheath in the first 2 to 6 hours in the insect’s stomach, after which they penetrate the gut of the host and reach the thoracic muscles. The first cuticular moult occurs about 2 days later (Schmidt and Roberts, 1977). In the muscles they become characteristically sausage-shaped and short, now most of the organ systems are present. Within 2 weeks the second moult takes place. The larva is now an elongate, slender filariform third stage, and development ceases. The filariform larvae are infective to definitive host. They migrate through out the haemocoel, eventually reaching the labium, or proboscis sheath, from which they escape when the mosquito is feeding .(Fig. 1)

1.20 Microfilariae of Wuchereria bancrofti: Microfilariae are not differentiated as normal first-stage larvae and are sometimes considered advanced embryos. The microfilariae of W. bancrofti retain the egg membrane as a “sheath” (not to be confused with the sheath of some third-stage strongyle larvae, which is the second-stage cuticle).

1.20.1 Sheath of Microfilariae: The sheath is rather delicate and close-fitting but can be detected where it projects at the anterior and posterior ends of the microfilariae. The location of these and the presence or absences of a sheath are used to identify the several species of microfilariae found in humans (Schmidt, and Roberts, 1977).

Plate ( 2 a ) : Female of Wuchereria bancrofti *After: maven.smith.edu/~sawlab/fgn/pnb/wuchban.html 01/01/1996

Plate ( 2 b ) : Male of Wuchereria bancrofti *After: maven.smith.edu/~sawlab/fgn/pnb/wuchban.html 01/01/1996

Plate ( 3 ) : This microphoto shows infective larvae of filarial worms emerging from a mosquito's proboscis *After : WHO, (1979)

Fig ( 1 ) : Life cycle of Wuchereria bancrofti *After: www.dpd.cdc.gov/dpdx.may2004.22:19:10

1.20.2 Main features of microfilariae of W. bancrofti in blood: The microfilariae of W. bancrofti are sheathed, their body is gently curved, and the tail is tapered to a point, the nuclei can be visualized individually and do not extend to the tip of the tail ,they have a lower number of nuclei and they measure 4-10 cms in length (Fig. 1ı) (Monica, 1999). This indicates a more precocious arrest of cellular divisions.

Fig ( 1ı ) : Microfilariae of Wuchereria bancrofti *After: Monica (1999)

1.20.3 Periodicity of microfilariae: As already indicated, the microfilariae of various species exhibit periodicity to a varying extent and this phenomenon has been investigated extensively. this phenomenon is well marked of Wuchereria bancrofti and other filarial speices. The various theories put forward to explain periodicity have been comprehensively reviewed by Ansari (1970). Hawking (1967). Four main periodicity patterns were recognized (Smyth, 1996). : a. Wuchereria bancrofti with microfilariae reaching a peak about midnight transmitted by night-biting mosquitoes. b. Dirofilaria immitis in whose microfilariae are present all the 24 hours transmitted by night-biting mosquitoes. c. Loa loa in which microfilaria are numerous at midday but almost absent at night, transmitted by a day-biting fly. d. Wuchereria bancrofti (so called pacific type) a type morphologically indistinguishable from type (a) above but in which the microfilariae are always present, being more numerous in the afternoon than at night transmitted by day-biting mosquitoes. Hawking (1967) points out that the behaviour of microfilariae appears to be adapted to promote transmission by arranging the maximum number of microfilariae in the peripheral blood at times when the arthropod vector is likely to bite. There has been much speculation and experiments to determine the reason for the periodicity of the parasite of Wuchereria bancrofti. One theory was that the larvae are concentrated in internal organs during the day, and circulate in the blood at night to synchranize with their nightbiting mosquito transmitters, chiefly Culex quinquefasciatus (Culex pipiens fatigans) and certain species of Anopheles. Another theory was that the embryos are born at a certain time each day, and are then

distributed in the host within the next 24 hours (Chandler and Read, 1961).

1.21 The identification of infective filarial larvae in mosquitoes:

Faust (1949) recorded 73 species of mosquitoes as possible vectors of Wuchereria bancrofti. The incrimination of many of these is doubtful, being based on the assumption that the larvae found in “wild” mosquitoes are of human origin. Many workers have in fact assumed that it is not possible to distinguish the infective larvae of the human parasites from those of animals flarial worms. During the 1950’s investigations on filariasis in Kenya revealed that little was known about the identification of filarial larvae in mosquitoes. The World Health Organization Expert Committee on filariasis (1957) recognized this gap in our knowledge and recommended that further research was needed in the differentiation of filarial larvae in their arthropod hosts (Nelson, 1959). Although detailed laboratory studies have been carried out on the development of several species in mosquitoes, very few attempts have been made to apply this information on epidemiological investigations. Lyengar (1957) has reviewed some of this work and reproduced the illustrations of the infective larvae of Wuchereria bancrofti made by Lebredo (1905) and Kobayashi (1940) and those of Wuchereria bancrofti made by Feng (1936). He also illustrated the infective larvae of D. immitis. In the Island of Pate, off the North East Kenya Coast, several larvae with a quite distinctive morphology were found in mosquitoes in and around the village of Faza. In this localized area, seven species of filarial worms were present in the very limited domestic and wild animal

populations. The human population had a high infection rate with Wuchereria bancrofti. An attempt was made to identify the filarial larvae in the wild mosquuiotes by comparing them with a collection of known infective larvae obtained by feeding mosquitoes on animals known to be infected with a particular species. In this way a “reference collection” of infective larvae was compiled and it was possible to identify with confidence several of the local species (Nelson, 1959). 1.21.1 Description of filarial larvae: In this respect Nelson (1959) said, “In this paper no attempt has been made to describe the larvae in great detail, only those characters which were found useful in differentiation have been stressed”. He added, “This paper is not concerned with the identification of first and second stage larvae but only with the third stage or infective larvae. It has been shown by Newton and Pratt (1945) and Wharton( 1957) that third stage larvae from abdomen move rapidly to the proboscis when the mosquito starts feeding. Third stage larvae are regarded as infective whether they occur in the proboscis or else where in the mosquito host. These larvae have a well developed cuticle. They are usually very active, they have lost the skin of the second ecdysis and there is no sign of an anal plug (Nelson, 1959). The specific length of the larvae is an important character for differentiation of the infective larvae, e.g. Dinofilaria spp. With an average of less than 1,000 µ, can be immediately distinguished from Wuchereria spp. and Setaria with an average of more than 1,400 µ (Nelson, 1959). Various factors affect the length of the larvae: if they are dissected out several days after they have reached maturity they are usually longer than larvae removed on the day following the second ecdysis. In mosquitoes with a very heavy infection physical crowding results in some of the larvae being shorter than normal. Another important factor affecting the

length is the species of mosquito host. Kartman (1953) has shown that the larvae of D. immitis are longer in C. quinquefasciatus than in Ae. aegypti. The position of the anus is an important feature in differentiation: the anal aperture is much nearer the caudal extremity in Dinofilaria than in Wuchereria and the position of the anus in Setoria is intermediate. This feature together with the differences in shape of the larvae between the anus and the extremity have been used by Wharton (1957) to differentiate W. malayi from D. immitis. He calculated what he has called the “Anal ratio” which is the distance from the anus to the caudal extremity divided by the breadth of the larva half way between those two points. This ratio is useful if only fragments of larvae are available for examination or if the terminal anatomy is not very clear. The “Anal ratio” in Dinofilaria average approximately 2, in Wuchereria the ratio is nearer 4. The most useful character for differentiation of the larvae is the shape of the caudal extremity. With very little experience it is possible to recognise even with the low power objective, the cigar shaped end of Dinofilaria, which clearly distinguishes it from Wuchereria, which has a much more definite narrowing between the anus and extremity. Most species of infective larvae have three caudal papillae with the dorsal one usually the most prominent. The other two are in a lateral or ventral position. The number, shape and size of these papillae are of importance in differentiation. The papillae are often very small and can only be seen by differential focusing under high magnification (Nelson, 1959).

1.21.2 Identification of species (Wuchereria bancrofti L3 stage, infective stage): Host: man, Developement in mosquitoes was first demonstrated by Manson (1878). Infective larvae have been examined from C. fatigans, A. gambiae and A. funestus and measured as follows:  Length: 1170 µ-1575 µ.  Breadth: 18 µ-32 µ.  Anus to caudal extremity: 56 µ-72 µ. The three bubble-like caudal papillae on the rounded extremity of the infective larvae of Wuchereria bancrofti are quite characteristic. They are large, equal in size, nearly spherical and at times, they appear to be pedunculated (Fig. 2). All three papillae are not always obvious, their relative prominence depends up on the position in which the larvae are mounted. There is a slight narrowing of the larva of Wuchereria bancrofti between the anus and rounded extremity, which gives the posterior end of the larva the appearance of a knobbed club.

Fig ( 2 ): Caudal end of infective larvae of W.b *After : Manson (1878)

1.22 The presence of filarial larvae in mosquitoes: At least 30 species of filarial worms are known to complete their development in mosquitoes. Of these only two, B. malayi and W. bancrofti, are infective to man. The problem of differentiating filarial larvae in mosquito depends on the parasites prevalent in domestic and wild animals in any particular area. Before any species of mosquito can be incriminated as a vector of either B. malayi or W. bancrofti, the stage of development of the larvae in mosquito must be identified. Four larval stages are found in mosquitoes: 1. The microfifilaria: which may be found in the stomach or in other parts of the insect soon after a blood meal. 2. The first-stage larva: a short sluggish sausage stage which in Brugia and Wuchereria is confined to the thoracic muscles, but which in other species occurs in malpighian tubules or fat bodies. 3. The second-stage larva: a long sausage stage with a developing intestinal canal. In Brugia and Wuchereria it is confined to the thorax and is relatively inactive. 4. The third-stage larva: the final stage of development in mosquito. The larva at this stage is very active and can be found in any part of the insect, not just in the head or proboscis. Only this stage should be referred to as infective. In studies on transmission, it is the third-stage infective form that requires special attention. Simple morphological characteristics, such as length, breadth, position of the anus, and shape of the caudal extremity, can be used to separate Wuchereria and Brugia from almost all infective forms in animals that have been described. However, it is not possible to separate the different species of Brugia, one from another except by injecting them into susceptible animals and wait for their full development (WHO, 1967). 45

1.23 Mortality of infected mosquito by larvae of Wuchereria bancrofti: Studies done before by several workers have shown that the ingestion of large numbers of microfilariae brings about high mortality in mosquitoes during parasite development. In the South Pacific region, Rosen (1955) observed high mortalities in Ae. polynesiensis when fed on microfilariae densities of over 9000 per ml. Hairston and DeMeillon (1968) re- examined the data of Rosen and reported that the average success of larvae through the mosquito phase of the lifecycle was very much higher for intermediate densities than for high densities. Bryan and Southgate (1976) found that the parasite loads (they studied) did not increase the daily mortality of the vectors. Symes (1960) observed that high mortality became apparent when feeding was carried out with microfilariae counts exceeding 5500 per ml. Fluctuation in temperature often caused high mortalities. 1.24 Epidemiology: Many mosquito vectors of Wuchereria have a preference for human blood and often breed near human habitation. At least 77 species and subspecies of mosquitoes in the genera Anopheles, Aedes, Culex and Mansonia are known as intermediate hosts for Wuchereria bancrofti in areas where the periodic strain of Wuchereria is found. The mosquito vectors are primarily night feeders. The species of mosquito serving as vectors in a particular area seem to depend more on coincidence (which species feeds when the larvae within the definitive host are available) rather than on physiological determinatnts of host specificity. Nevertheless, the periodicity has a practical epidemiological significance because this fact determines which mosquito species must be controlled and, consequently, what control measures must be applied.

Suitable breeding sites for mosquitoes are abound in tropical areas. Some sites are difficult or impossible to control such as tree holes, hollows at the bases of palm fronds and so-on while others can be controlled with a degree of effort. Hollow coconuts, killed while still green by rats gnawing holes in them , fill with rain water and become havens for developing mosquito larvae. These can be collected and burned. Even dugout canals that are unused for a few days can partially be filled with rain water and become mosquito nurseries. Conditions for transmission of Wuchereria vary from locality to locality and from country to country. The epidemiologist must consider each case independently within the framework of the biology of the vector and host, putting in mind the economic and technical resources that are available to best advantage (Schmidt and Roberts, 1977). 1.25 When do mosquitoes become infective ? : The inefficiency of transmission of Wuchereria bancrofti from Culex quinuefasciatus to the human host based on observations in Ragoon, Hairston and DeMeillon (1968) implied that the probability was low for infective mosquitoes harbouring one, two or three larvae to produce patient infections in the human host. Thus for the human host to be microfilariae positive both sexes of the parasite must be present and mated. It is impossible for a bite by a mosquito carrying a single infective larva to cause microfilariae. Half the mosquitoes carrying two larvae and one-quarter of those carrying three larvae cannot cause microfilariae on the assumption of an equal sex ratio and independent distribution of the two sexes among mosquitoes. The studies of DeMeillon, Hayashi and Sebastian (1967) suggest that only a proportion of the infective larvae leave the mosquito host at one feeding. Thus, as suggested by Gubler and Battacharya (1974) the human population will receive a single larva or no larvae from bites of these infected mosquitoes. Patient infections would

therefore not often occur from a single bite. The people would be repeatedly infected with low or sub minimal doses of infective larvae. Gubler and Battacharya (1974) suggested that instead of producing patient infection such exposure would likely induce an immune response by the host. 1.26 Filariasis-sex and ethnic group: There are nevertheless obvious differences according to sex as well as ethnic groups. The blood survey carried out in the Kemmendine area (Rangoon) shows that the prevalence rates among males are 6.4%, 12.7% and 4.7% for Burmese, Indians and Chinese respectively. On the other hand, the rates for females are 3.0%, 6.5% and 2.9% respectively in these ethnic groups (p < 0.05). This clearly indicates that the rates in males are always higher than those in females in all ethnic groups. The average rate in males, which is twice the female rate, was 6.7%. The average rate for both sexes is 10.0% for Indians, 4.6%, for Burmese and 3.9% for Chinese. (Hayashi, 1966).

1.27 Relationship between age and Wuchereria bancrofti: The age distribution of the microfilariaemia rate is given in (Table 2 ). Generally speaking, there are two distinct peaks in the distribution pattern. The first peak appears in the 15-19 years age-group and the second around 50 years of age. This suggests that there have been at least two relatively important endemics in the past. The picture for the younger age-group, i.e. up to around 25 years, directly reflects the situation brought about by a recent endemics in the area. It must also be noted that the prevalence among Indians is higher than in the other ethnic groups for

Table ( 2 ):Age distribution of the microfilariaemia rate by sex and by ethnic group in Kemmendine, Rangoon

Age Burmese India Chinese Combined Total Male Female Male Female Male Female Male Female No. Pos% exam

0-4 0.0 0.0 0.0 3.8 11.1 0.0 0.3 0.3 675 0.3 5-9 1.5 2.4 12.5 2.3 0.0 6.3 2.1 2.5 1512 2.2 10-14 5.2 3.9 10.3 12.9 3.8 0.0 5.4 4.1 1560 4.8 15-19 8.5 6.8 16.7 16.7 12.5 0.0 9.0 7.1 1253 8.1 20-24 8.4 3.8 14.7 2.9 10.0 0.0 8.8 3.7 1190 6.3 25-29 8.2 2.2 21.4 16.0 0.0 7.7 8.7 2.9 1100 5.6 30-34 7.2 2.5 16.0 3.7 0.0 0.0 7.6 2.5 841 4.9 35-39 7.9 1.5 15.6 0.0 0.0 0.0 8.4 1.4 811 4.6 40-44 10.0 1.8 9.1 5.6 10.0 8.3 10.0 2.2 663 5.7 45-49 12.2 2.6 19.0 0.0 10.0 0.0 12.7 2.4 555 6.5 50-54 10.3 4.2 15.0 6.3 0.0 25.0 10.4 4.6 449 7.3 55-59 6.2 0.6 11.1 0.0 0.0 0.0 6.1 0.6 315 3.2 60-64 2.0 0.0 14.3 33.3 0.0 0.0 3.4 0.7 253 2.0 65-69 4.3 4.8 0.0 0.0 0.0 0.0 3.6 4.4 123 4.1 70 1.8 5.3 0.0 0.0 0.0 0.0 1.6 5.1 141 3.5 Total 6.4 3.0 12.7 6.5 4.7 2.9 6.7 3.1 4.9 No.exam. 5055.0 5425.0 361.0 292.0 170.0 137.0 5587.0 5854.0 11441

*After: Hayashi (1966)

almost all age-groups, and that the prevalence among males is higher at particularly all ages (Hayashi, 1966). 1.28 Immunity to Lymphatic Filariasis: Relatively little is known about protective immunity in human filariasis, and there is little direct evidence that such immunity exists. The present study was prompted by the observation that filariasis prevalence rates increase rapidly with age in teenage children in Egypt (Ramzy et. al. 1994). Another study revealed that the intensity of infection in villages in the Nile Delta tended to decrease with age (Farid et. al.1997) (also Weil GJ and other, unpublished data). Relatively few longitudinal studies of filariasis have been conducted, and fewer still have used sensitive diagnostic methods. Data were collected by(Weil , et. al. 1996) for 1,853 subjects (914 males and 939 females) in 362 households that were scattered in all areas of the 5 study villages they found that prevalence rates for microfilaremia, antigenemia, and antifilarial antibody varied significantly by village, however antibody rates being higher than antigenemia rates, which were generally higher than microfilaremia (Table 3). Prevalence rates for disease, levels of microfilaremia, antigenemia, and antifilarial antibody were significantly lower in children 10-19 years of age than in older subjects. Disease prevalence rates increase with age. In contrast, age prevalence curves for microfilaremia, antifilarial antibody and antigenemia have convex patterns (Fig. 3). No significant gender differences were observed in filarial antigen, microfilaria or antibody prevalence rates. Positive filarial antigen test results were present in 88.7% of those with microfilaremia and 5.5% of microfilaremic subject with no clinical evidence of filariasis (endemic normals).

Table ( 3 ) : Perevalence rates of filariasis in different Egyptian villages

Village n Clinical Micro- Anti- Antibody disease filaremia genemia

ELKOISAM 812 1.4 10.8 15.0 44.6

KAFRSAAD 244 0.8 9.0 8.2 27.9

KAFRTAHORIA 116 0.9 6.0 12.9 34.5

KAFR- 373 0.3 2.1 3.8 10.5 ELSHORAFA

TAHORIA 308 1.6 9.1 12.0 35.1

Total 1.853 1.1 7.8 11.2 33.3

* After: Weil and Ramzy et .al,(1996)

Fig ( 3 ) : Relationship between Microfilaremia, antigenenmia, antibody of filariasis * After: Weil and Ramzy et .al,(1996)

1.29 Economic and social impact: Because of its prevalence often in remote rural areas, on the one hand, and in disfavoured peri-urban and urban areas, on the other, Lymphatic Filaraisis is primarily a disease of the poor. In recent years, Lymphatic Filariasis has steadily increased because of the expansion of slum areas and poverty, especially in Africa and the Indian sub-continent. As many filariasis patients are physically incapacitated, it is also a disease that prevents patients from having a normal working life. The fight to eliminate Lymphatic Filariasis disease is also a fight against poverty. Lymphatic Filariasis exerts a heavy social burden that is especially severe because of the specific attributes of the disease, particularly since chronic complications are often hidden and are considered shameful. For men, genital damage is a severe handicap leading to physical limitations and social stigmatization. For women, shame and taboos are also associated with the disease. When effected by lymphodema, they are considered undesirable and when their lower limbs and genital parts are enlarged they are severely stigmatized; marriage, which is in many situations an essential source of security, is often impossible (WHO, 2000). 1.30 Treatment of filariasis: In communities where filariasis is endemic the primary goal of treating the affected community is to eliminate microfilariae from the blood of infected individuals. For treating the individual both Albendazole and Diethylcarbmizen (DEC) have been shown to be effective in killing the adult-stage filarial parasite (necessary for complete cure of infection), but ideal treatment regimens still need to be defined (WHO, 2000).

1.31 The WHO strategy for the elimination Lymphatic Filariasis : Two primary biological characteristics of bancroftian parasites render transmission of infection insufficient: the lack of any animal reservoir host and being anematode there is no amplification of the parasite within the mosquito vector. Moreover, a new concept of disease elimination has resulted from recent fundamental developments in diagnostic methods and therapies, including improved diagnosis by the immunochromatographic diagnostic test (ICT), and antigen capture card format that detects filarial antigens in whole blood taken during daytime (EL Setouhy and Ramzy, 2003). Furthermore, it is now recognized that the annual single-dose regimen of new microfilaricidal (and to a lesser extent macrofilaricidal) drug treatment combinations (albendazole+DEC or albendazole + ivermectin) is safe for community-wide control programmes aiming to interrupt transmission of infection. These combinations impressively reduce mf by 99% for a full year (Ismail, 1996). Such developments prompted the World Health Assembly (WHA) to pass a resolution in 1997 calling for “elimination of Lymphatic Filariasis as a public health problem”. Consequently, WHO, (2002) developed a new strategy and the global programme for elimination of Lymphatic Filariasis was initiated with the aim of eliminating Lymphatic Filariasis as a public health problem by the year 2020. The programme has two principal goals: 1. To interrupt transmission of the infection, the essential strategy is to treat the entire at-risk population for a period long enough to ensure that microfilariae levels in the blood remain below those necessary to sustain transmission by mosquitoes. For the yearly single-dose, 2- day regimens are advocated (albendazole 400mg + ivermectin 200mcg/kg) in areas where onchocerciasis is co-endemic with

bancroftian filariasis (WHO, 2002), this period has been estimated to be 4-6 years, corresponding to the reproductive lifespan of the parasite. 2. To alleviate the suffering caused by the disease. The new strategy is aimed at management of the consequences of Lymphatic Filariasis infection, particularly the lymphoedema (swelling of upper and/or lower limbs), elephantiasis and lymphoceles (scrotal swellings) and operated cases of lymphoceles or hydroceles. It is now evident that excessive local hygiene supplemented with antibiotics to inhibit bacterial super-infection can prevent or even reverse the lymphoedema and elephantiasis consequential to filarial infection (El- Setouhy and Ramzy, 2003).

Chapter Two

MATERIALS AND METHODS

2.1 Study area: This study was carried out during the period August-September 2004 at Juba area in Southern Sudan, and also during April - June 2005 again at, Juba and Terkaka. The study area lies between the longitudes 30° 20¯ and 31° 45¯E and the latitudes 4° 40¯and 5° 00NJuba is about 1200 km south of Khartoum and Terkaka is about 200 km north from Juba .(Wilson ,1963). 2.1.1 Topography: The topography or land of Juba is slightly undulating and is gently sloping northwards, and it is marked with distinct isolated hills such as Logwek (Jebel Rajaf West) Longwi (Jebel Rajaf East).Both hills are located to the south of the town and west of the River Nile.Korok (Jebel Kujur) is located west of the town; it is the nearest area to the town, while Nyarkonyi (Jebel Lado) is located further north of the town and Bilinyang hills are further to the east of the town. Within these surrounding hills the city of Juba is situated. At the basin of these hills the area is dissected again by a number of tributary streams (Khors) such as Lilio and Markunon to the south of the town and east of the Nile, Dorodo and Wolliang to the south of the town and west of the Nile (Lejulugor, 2003). 2.1.2 Drainage: The river Nile is the main catchment through which water drains to the North. The Luri and Kit Rivers and most of the small streams as tributaries of the Nile do dry up during the dry season. However, the ground water is available at the depth of 18 meters. This turns to rise up during the rainy season (Lejulugor, 2003).

2.1.3 Climate: The climate is dry monsoon with 5-6 humid months though rainfall has recently become unreliable and it varies between 700-1300 mm per annum. 2.1.3.1 Temperature: The mean annual average temperature is about 27.5 degree C´ in December. The temperatures therefore are uniformly high so that weathering and crops growth are never slowed down by low temperature (Rainfall Annual Report 1996/97). The southern part is cooler than the north. The dry seasons are shorter than the long and cool wet season. (Lejulugor, 2003). 2.1.3.2 Rainfall: The rain begins in April through to November with a dry spell in June and the peak period occurs between May and August. The rainfall often occurs in small showers with occasional storms and the number of days with more than one(1)mm rainfall are about 95 days per annum. This means rainless period can occur even during the wet season" the maximum intensity of rainfall is only 70-90 mm per annum ". (Lejulugor, 2003). 2.1.4 Soils: The soils of the area are alluvial and colluvial along the banks of the Nile. The textures range from light sandy looms, and loamy sands to heavy clays. These are good soils for the production of a range of crops, which include cereals, vegetables, pulses and fruits. (Lejulugor, 2003).

2.1.5 Vegetation: The vegetation in Juba town area is open savannah grassland with relatively tall grasses and big scattered trees and thickets along the rivers , streams and any water courses. Grass is often burnt around Juba town in dry season and as such the trees are indiscriminately cut down to provide land for cultivation ,grazing , building , firewood and charcoal making . This causes desertification and environmental hazards in the area up to perimeters of 15-30 miles around Juba town . 2.1.6 Human population: The human population in Juba and Terkaka area is of Nilotic African tribes (Baria tribe) in Juba and (Mondari tribe) in Terkaka.They live in small villages (Plate 4) and most of them work as farmers and fishermen and still others have a vegetate life. In the countryside inhabitants are infiltrated in hamlets surrounded by an area of cultivation separated by an area of natural or secondary vegetation. The watering points are rivers, wells and scattered ponds. Often these places represent the main man and /or animal fly contacts (The Federal republic of the Sudan Bahr El Jebel State. Ministry of Agriculture and Animal WealthJuba. The preliminary report 1994). 2.1.6.1 Population practices: Most of the population in the study area are agro-pastoralists keeping animals (cattle/goats) and practicing agricultural activities in small scale or back yard farming, or fishermen. 2.1.6.1.1 The Agricultural sector: Most people are either employed or self employed in agriculture. Most of this is done by traditional hand tools. Before the war most of the food was produced in the rural areas, and urban centres were supplied from those areas. There were no food deficiency syndromes.

Plate (4) : Types of houses in Juba and Terkaka

And no food was imported into the state but the war since 1953 destabilized the farming community so much that little food is produced locally, mostly in and round town under government control. As a result people began to depend mostly on relief food hand outs .

2.1.6.1.2 The fishing sub sector: Fish are abundant in the Nile River and its tribularies, in the various ponds created by the Nile and the Terkaka Lakes. In fact 95% of the fish come from Terkaka province. The remainder comes from Bahar EL-Jebel and Kajokaji province. The normal annual average harvest is 4,000 metric tons (The Federal republic of the Sudan Bahr El Jebel state. Ministry of Agriculture and Animal Wealth-Juba. The preliminary report 1994). 2.1.7 Poverty status: Prices of food items in Juba have always been on the rise and it has been difficult for the various state governments to bring them down by control (laws or orders). The reasons for the price rise are: • Food is insufficient, it is provided by air flight into the state from outside.

• The supply has always been little compare to the high demand. The acute lack of governments funds led to insufficient quantities of vaccine, drugs and logistic facilities for fast movement (The Federal republic of the Sudan Bahr El Jebel state. Ministry of Agriculture and Animal Wealth-Juba. The preliminary report 1994). 2.1.8 Transport and communication: Juba town except for the islands is well supplied with a network of all weather accessible roads the year round. The town is also connected to the National capital Khartoum by air and river communication as well as tele- communication links. Radio Juba plays a key role in dissemination of news of varied nature including education and agricultural information to the farming communication. Juba town is also connected to East Africa and the Republic of Congo (Former Zaire) by both air and road, although 60

currently the intensification of the civil war has rendered transport by road difficult if not impossible. The only link to the neighbouring East Africa countries is by air. (Lejulugor, 2003). 2.2 Endemic areas in Juba: Two trips to Juba were carried out during the period of August to September 2004, and to Juba town and Terkaka area during the period from April to June 2005, to collect vector samples and to look for patients of the disease (Lymphatic Filariasis) at the centre and also rural areas (Plates 5a & 5b & 5c ).

The study area was divided into five regions : 1. Juba zone consisting of (Juba Nubari, Ghabat, Jebel Nuga, Tamarjia Line, Kator (out skirts). 2. Gondokro Island. 3. Monki. 4. Rajaf west and Rajaf east, Lologo ( villages Lateral of Juba). 5. Terkaka (200 km from Juba).(map 4) All the areas were known to be affected by Lymphatic Filariasis, according to surveys of Ministry of health using ICT card.(personal Communication) Trip to Terkaka is by boat (about 5 hours with the current and about 10 hours against the current. (Plate 6).

Plate (5 a) : In Gabat area

Plate (5 b) : In Monki area

Plate (5c) : In Terkaka

Map (4) : The study area

Plate (6) : Return Trip from Terkaka

Methods 2.2.1 Entomological survey: 2.2.1.1 Detection of breeding sites: Detection of mosquito breeding sites in and around Juba town and Terkaka took place during the study period (August 2004 and April 2005) . All possible mosquito sites in endemic areas were visited including the surrounding farms . wells , and mountains . 2.2.1.2 Collection of adult Mosquitoes: One method namely spraying was used for collection of adult mosquitoes resting inside houses. Indoor spray collection (pyrethrum spray collection or knock down collection) method was carried out early in the morning , usually between 6-9 pm. Collection sites were selected randomly taking in consideration that each hut is occupied by a number of persons and is near to one or more of the mosquito breeding sites. People were first requested to leave the hut and then the whole floor surfaces as well as beds and any other areas were completely covered from wall to wall with white cloth sheets. All windows, doors, eves and other openings through which mosquitoes could otherwise escape were firmly closed. The hut was filled with mist of 0.2% solution of pyrethrum diluted in kerosene using spray- pumps or spray guns (Plate 7a) (spray can) of minimum capacity. And also swingfog (Plate 7b) machine (with 4 batteries and one liter benzene), and flit guns were used (Plate 7c). After spraying, the hut was kept closed for 10 minutes and then opened to collect mosquitoes knocked down on the sheets using a torchlight (Plate 8). Dead mosquitoes were carefully collected and placed gently into plain containers containing Modified Carnoy’s fixative (ethanol + Glacial acetic acid 3:1 respectively . + Glycerin ) at 20°c. Samples were carefully transported to the laboratory for examination in Khartoum.

Plate (7a) : Hand pump

Plate (7 b) : Swing fog

Plate (7 c) : Flit gun

Plate (8) : Collection of mosquitoes

2.2.1.3 Identification of adult mosquitoes: Collection sites at Juba and Terkaka were divided into two groups according to their distance from the river or pools. The number of mosquitoes collected from each hut was recorded and all mosquitoes collected from each group were dissected. In the laboratory of National Malaria Administiration at Khartoum records were made of adult female mosquitoes collected. All adult Anopheline and Culicine mosquitoes collected were identified and dissected apart (head, thorax and abdomen) and then examined microscopically for detection of parasites . All adult Anopheline and Culicine mosquitoes collected were identified to species level using the morphologic identification key prepared by Gillies and De-Meillon (1968) and Abd El-Nur and Nugud (1994). 2.3 Dissection of adult mosquitoes: After identification of mosquitoes as regards their genera and species, samples were dissected according to Gordon & Lavoipierre (1962). The legs and wings were removed, laid in a drop of saline on a slide which was then placed on the stage of adissecting microscope. The head, thorax, and abdomen were separated and transferred to separate drops of saline. The severed head was steadied with one needle, and , with the aid of another very fine needle, the labium was split from the base to the tip. The thorax, abdomen and the remainder of the head were then teased up and examined as individual fresh preparations under the low powers of a compound microscope (x10, x40) to observe filarial species. The staining of slides containing the parasite was done and after 24hr later stain were , washed by lcohol and cleared in berlyes fluid and covered. The preparations were re-examined carefully using a compound microscope at about (10-40-or 100) magnification.

Infective mosquitoes were defined as those containing L3 larvae in any of the body segments. Mosquitoes carrying microfilaria, L1, L2, or L3 larvae were defined as infected. The numbers of stages of all Wuchereria bancrofti larvae were recorded for each body part. Third stage (L3) larvae were examined carefully under high power of binocular microscope for the three caudal papillae characteristic of Wuchereria bancrofti. 2.4 Blood smears: Blood smears were very difficult to obtain from the population.. Blood samples were randomly taken from ten students at the camp of (Ezat EL Sudan No.9 ), in addition to seven people at Jebel Nuga, one of them suffering from the disease , all the sample were taken after 10 pm. The smears were stained in Giemsa stain according to conventional methods . 2.5 Visit to the Blue Nile state:

The Blue Nile state is regarded as an epidemic area of the Lymphatic Filariais disease. In Baow locality of this state about 100 adult mosquitoes were captured and dissected. 2.6 The visit to Egypt: In the Sudan the filarial worms were identified as Wuchereria bancrofti. But my supervisor and other interested workers advised me to visit Egypt. This idea was enhanced and encouraged by some Egyptian parasitologists who visited the Sudan at that time (2005). The visit materialized in March 2006, and extended for 21 days during which I gained a lot of information about filariasis (Plate 9a & 9b). I visited several research centres concerning Filariasis, Malaria and Leishmaniasis. These visits were arranged by the Egyptian Ministry of Health. The importance of the visits lies in the following:

1. I could make the necessary comparisons between my findings and the Egyptian key-records. 2. I had the opportunity to read most of the literature concerning filariasis especially that of Wuchereria bancrofti. I was lucky enough to read the very important paper of Nelson (1959) which is missing in the Sudan. 3. I could also managed to witness the identification of the worm through the usage of PCR. 4. I was invited to visit Naval American Medical Research Unit (NAMRU), and make use of its library. 5. I was provided with many slides and papers pertaining to my research. 6. Needless to say that I have created friendly relations and contacts with many Egyptian experts in this filed.

Plate (9a) : Project Manger (Prof. Adel Gad ) of Filarial program in Cairo

Plate (9 b) : Team of Filarial programme in Cairo

Chapter Three

RESULTS 3 .1 Breeding sites in Juba and Terkaka : Some regions at or in the areas of Juba and Terkaka like (Ghabat , Temarjia line, Gondokro island and Lolgo area) are characterized by their poor and inadequate drainage and sanitation. Pools isolated from the main river were polluted and rendered unhealthy for drinking. These bodies are suitable as breeding sites for mosquitoes particularly Culex quinquefasciatus .

3.2 Anopheline and Culicine mosquito fauna of the study area: The anopheline mosquito in the study areas was An. gambiae s.s . A total of 2013 adult females was collected ( 931 from Juba zone, 180 from Gondokro island, 95 from Monki, 739 from Lateral area of Juba - Rajaf west, Rajaf east and Lologo) and 68 from Terkaka. The culicine mosquito was Culex quinquefasciatus . A total of 716 insects was collected , 281 from Juba zone, 120 from Gondokro island, 5 from Monki, 162 from Lateral area of Juba, 148 from Terkaka. (Tables and Figurs 4a, 4b, 4c, 4d, and 4e).

3.3 Identification of different stages of Wuchereria bancrofti: The Wuchereria bancrofti larvae dissected out of the different parts of the body (head, thorax, and abdomen) of both Anopheles gambia s.s and Culex quinquefasciatus mosquitoes were identified according to Schmidt & Roberts,1977 and Nelson,1959.  First stage larva (L1): sausage – like stage .(Plate 10a)

 Second stage larva (L2) : J - shaped stage .(Plate 10b)

 Third stage larva (L3): infective larva .this is characterised by the three large cow teat-like knobs on the caudal extremity .(Plates 10c ı , 10 c п )  Microfilaria embryos: long and the tail is tapered to a point.(Plates 10 d ı ,10 d п).Some dissected mosquitoes consisted of sheathed microfilariae, this must have been captured immediately after feeding (Plate 10 d ш). 3.4 Notes on larval length of Wuchereria bancrofti L3 Larvae found in the heads, thorax and abdomen of mosquitoes collected from Juba and Terkaka: According to measurement of Nelson,(1959) the length of L3 larvae of Wuchereria bancrofti ranged between 1170-1575 µ and breadth 18-32 µ. In this study the length of L3 in the head was 1288 µ and breadth was 29 µ; in the thorax the length was 995 µ, and the breadth was 17.2 µ and in the abdomen the length was 1265 µ and the breadth was 23 µ. 3.5 Notes on length of Wuchereria bancrofti microfilaria found in the mosquito and human blood: In this study the avarge length of microfilaria in the human blood smear was approximately 287 µ this measurement is similar to length of microfilaria in the mosquito’s abdomen before the development to stage one (L1) which is 250 µ according to (Lardeux & Cheffort 2002). 3.6 Results of blood smears: No parasites were seen in students blood, although they came from endemic areas. Of the seven people of Jebel Nuga only one has microfilaria (mf) in his blood (Plate 11). Appendix (1) , (Plate 1) shows microfilaria from www.dpd.cdc. gov/dpdx.may2004.22:19:10 .

Mosquito fauna of the study area Table (4a) : Endemic areas Culex Anopheles Total Number of quinquefasciatus gambiae Juba zone Spices Ghabat 197 501 680 Juba Nubari 0 0 0 Kator 5 45 50 Tamarjia line 62 20 82 Jebel Nuga 35 365 400 Gross Total 281 931 1212

Mosquito fauna of the study area Table (4b): Endemic area Culex Anopheles Total Number of quinquefasciatus gambiae species

Gondokro island 120 180 300

Figer 4b

Mosquito fauna of the study area Table (4c): Endemic area Culex Anopheles Total Number of quinquefasciatus gambiae species

Monki 5 95 100

Fig 4c

Mosquito fauna of the study area Table (4d): Endemic areas Culex Anopheles Total Number of quinquefasciatus gambiae species

Rajaf west 99 304 403 Rajaf east 50 240 290 Lologo 13 195 208

Fig 4d

Mosquito fauna of the study area Table (4e): Endemic area Culex Anopheles Total Number of quinquefasciatus gambiae species

Terkaka 148 68 216

Fig 4e

Plate ( 10 a ) : Sausage stage L1 of Wuchereria bancrofti

Plate ( 10 b ) : Second stage L2 of Wuchereria bancrofti

Plate ( 10 c ı ) : Third stage L3 (infective stage ) of Wuchereria bancrofti

Plate ( 10 c п) : Third stage L3 ( caudal extremity Cow teat– like knobs ) of Wuchereria bancrofti

Plate ( 10 d ı ) : Microflaria of Wuchereria bancrofti in mosquitoes

Plate ( 10 d п ) : Tapered tail of Microflaria of Wuchereria bancrofti

Plate ( 10 d ш ) : Sheathed Microflaria of Wuchereria bancrofti in mosquito

Plate ( 11 ) : Microflaria of Wuchereria bancrofti in human blood

3.7 Infection rate: General infection rate of Wuchereria bancrofti in mosquitoes (mosquitoes with LI, L2, L3 and microfilaria) in the study area is shown in Tables and Figures (5a, 5b,5c,5d, and 5e). At Juba zone (Ghabat, Juba Nubari Tamarjia line, Jebel Nuga and Kator) an overall rate 2.5% (Culex quinquefasciatus 1.7%, Anopheles gambiae 2.7%), Gondokro island the overall infection rate was about 1% (Culex 1.6%, Anopheles 0.5%),in Monki (0%) no infection was detected, in Rajaf west the overall rate was 7.6% (Culex 2.02%, Anopheles 9.5%), and in Terkaka the overall infection rate was 7.4% (Culex 8.1%, Anopheles 5.8%). The grand infection rate of Culex and Anopheles at all areas (Juba zone, Lateral of Juba and Terkaka) was 3% (Culex 2.9%, Anopheles 2.9%). 3.8 The Proportion of Mosquitoes harbouring L3 in any part of the body: The infectivity rate defined as the proportion of moquitoe harbring L3 in any part of its body (Tables and Figures 6a,6b,6c,6d, and 6e), show the proportion of infective mosquitoes found in all areas.In the highest endemic area Rajaf west the infectivity rate was 4.2% (Culex 0%, Anopheles 5.5%) in Terkaka 4.1% (Culex 4.7%, Anopheles 2.9%) in Juba zone 2.3% (Culex 2.1%, Anopheles 1.4%). In the highest endemic area in Juba zone, Ghabat recorded 2.5% (Culex 2.7%, Anopheles 2.5%). Gondokro island recorded 0.3% (Culex 0.8%, Anopheles 0%). While in Monki all mosquitoes were negetive.

3.9 Location of Wuchereria bancrofti L3 infection in heads, thorax and abdomen of Culex quinquefasciatus and

Anopheles gambiae in Juba area and Terkaka: Infective forms were encountered in the head, thorax and abdomen of mosquitoes . Out of 2729 mosquitoes 50 were infected (41 in Juba and 9 in

Terkeka) , (Tables 7a,7b,7c,7d,7e) show the location of infective larva in the body of vector, in Ghabat there were 18 mosquitoes (13 An. (3 in the head , 3 in the thorax and 7 in the abdomen) , 5 Culex in the abdomen ) .Tamarjia Line there were 2 mosquitoes (1 Culex in the abdomen , and 1 An. in the abdomen also). In Jebel Nuga there were 3 An. In the abdomen. In the Rajaf west there were 17 An. (2 in the haed , 5 in the thorax and 10 in the abdomen). In the Terkaka the were 9 mosquitoes (7 Culex (6 in the thorax and 1 in the abdomen) , 2 An. In the abdomen ).

Prevalence rate of Mosquitoes infection by larvae of

W.bancrofti (Culex quinquefasciatus & Anopheles gambiae) Table (5a): Endemic Infected Culex Anopheles Overall Culex An. areas mosquitoes quinquefasciatus gambiae infectio quin. Gambiae Juba n rate (%) (%) zone (%)

Ghabat 23 4 19 3.3 2.2 3.7 (n= 680) (n= 179) (n= 501) Juba 0 0 0 0 0 0 Nubari

Kator 0 0 0 0 0 0 Tamarjia 2 1 1 2.4 1.6 5 (n= 82) (n= 62) (n= 20) Jebel 6 0 6 1.5 0 1.6 Nuga (n= 400) (n= 35) (n= 365) Gross 31 5 26 2.5 1.7 2.7 Total (n= (n= 281) (n= 931) 1212)

Prevalence rate of Mosquitoes infection by larvae of W.bancrofti (Culex quinquefasciatus & Anopheles gambiae) Fig 5a

Juba Nubari Tamarj ia Line

Juba Nubari Tamarjia Line

Prevalence rate of Mosquitoes infection by larvae of

W.bancrofti (Culex quinquefasciatus & Anopheles gambiae)

Table (5b): Endemic infected Culex Anopheles Overall Culex An. area mosquitoes quinquefasciatus gambiae Infection quin. gambiae rate (%) (%) (%) Gondokro 3 2 1 1 1.6 0.5 island

Table (5c): Endemic infected Culex Anopheles Overall Culex An. area mosquitoes quinquefasciatus gambiae Infection quin. gambiae rate (%) (%) (%) Monki 0 0 0 0 0 0

Fig 5c

Table (5d): Endemic infected Culex Anopheles Overall Culex An. areas mosquitoes quinquefasciatus gambiae Infection quin. gambiae rate (%) (%) (%)

Rajaf 31 2 29 7.6 2.02 9.5 west

Rajaf 0 0 0 0 0 0 east

Lologo 0 0 0 0 0 0

Fig 5d

Table (5e): Endemic infected Culex Anopheles Overall Culex An. areas mosquitoes quinquefasciatus gambiae Infection quin. gambiae rate (%) (%) (%) Terkaka 16 12 4 7.4 8.1 5.8

Fig 5e

The Proportion of Mosquitoes harbouring L3 :(Culex quinquefasciatus & Anopheles gambiae): Table (6a): Endemic No. of L3 Culex Anopheles overall Culex quin. An. areas quinquefasciatus gambiae Infectivity % gambiae rate % Juba zone % Ghabat 18 5 13 2.5 2.7 2.5 Juba 0 0 0 0 0 0 Nubari

Kator 0 0 0 0 0 0 Tamarjia 2 1 1 2.4 1.6 5 Jebel Nuga 3 0 3 0.7 0 0.8 Gross 23 6 17 1.8 2.1 1.8 Total

The Proportion of Mosquitoes harbouring L3 :(Culex quinquefasciatus & Anopheles gambiae):

Fig. 6a

The Proportion of Mosquitoes harbouring L3 :(Culex quinquefasciatus & Anopheles gambiae): Table (6b): Endemic No. of L3 Culex Anopheles Overall Culex An. gambiae areas quinquefasciatus gambiae Infectivity quin.% % rate %

Gondokro 1 1 0 0.3 0.8 0 island

Fig 6b

The Proportion of Mosquitoes harbouring L3 :(Culex quinquefasciatus & Anopheles gambiae): Table (6c): Endemic No. of L3 Culex Anopheles Overall Culex An. areas quinquefasciatus gambiae Infectivity quin.% gambiae % rate %

Monki 0 0 0 0 0 0

Fig 6c

The Proportion of Mosquitoes harbouring L3 : (Culex quinquefasciatus & Anopheles gambiae): Table (6d): Endemic No. of L3 Culex Anopheles Overall Culex An. areas quinquefasciatus gambiae Infectivity quin.% gambiae % rate %

Rajaf west 17 0 17 4.2 0 5.5 Rajaf east 0 0 0 0 0 0 Lologo 0 0 0 0 0 0

Fig 6d

The Proportion of Mosquitoes harbouring L3 : (Culex quinquefasciatus & Anopheles gambiae): Table (6e): Endemic No. of L3 Culex Anopheles Overall Culex An. areas quinquefasciatus gambiae Infectivity quin.% gambiae % rate %

Terkaka 9 7 2 4.1 4.7 2.9

Fig 6e

Location of Wuchereria bancrofti L3 infection in (head , Thorax and Abdomen) of Culex quinquefasciatus and Anopheles gambiae in Juba area and Terkaka: Table (7a): Endemic areas Location Of L3

Head Thorax Abdomen

Juba zone

Ghabat 3An. 3 An. 5 Culex, 7 An.

Juba Nubari 0 0 0

Kator 0 0 0

Tamarjia line 0 0 1 Culex, 1 An.

Jebel Nuga 0 0 3 An.

Gross Total 3 3 17

Table (7b): Endemic areas Location Of L3

Head Thorax Abdomen Gondokro island 0 0 1

Table (7c): Endemic areas Location Of L3

Head Thorax Abdomen Monki 0 0 0

Location of Wuchereria bancrofti L3 infection in (head , Thorax and Abdomen) of Culex quinquefasciatus and Anopheles gambiae in Juba area and Terkaka:

Table (7d): Endemic areas Location Of L3

Head Thorax Abdomen

Rajaf west 2 An. 5 An. 10 An.

Rajaf east 0 0 0

Logo 0 0 0

Table (7e): Endemic areas Location Of L3

Head Thorax Abdomen

Terkaka 0 6 Culex 1 Culex, 2 An.

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3.10 Epidemiological surveys : Assessment of an epidemiological survey of this disease is summarised in : 1- Most of the population in the study area are agro-pastoralists keeping animals (cattle / goats) and practicing agricultural activities in small farming . 2- Apparently mosquitoes are the main vectors of the disease of filariasis. 3- Rains are very frequent : when it rains people sleep indoors . Mosquitoes then invade their dwellings . They have no nets to protect them against mosquito bites. Males were more affected than females probably because of the nature of their daily practice which made them more exposed to mosquito bites than women . On the other hand women may use some traditional methods for protection e.g smoking , oils . Hence it appears that men are more affected by the disease than women . 4- No statistical information about the prevelance of the disease is available due to absence of hospital records . (Plates 12a , 12b ,12c ,12d) show patients in some region of the study area . Appendix (2) (Plates 2a , 2b) show people infected with filariasis worldwide.

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Plate ( 12 a ) : Patient in Terkaka (man)

Plate ( 12 b ) : Patient in Rajaf west (woman)

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Plate ( 12 c ) : Patient in Monki (man)

Plate ( 12 d ) : Patient in Terkaka (man)

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CHAPTER FOUR DISCUSSION

Mosquitoes are the most important single group of insects with regards to public health. It is a remarkably adaptable and fully cosmopolitan group, distributed throughout the world. Sudan is very rich in its mosquito fauna. During recent years enormous transformations have taken place in the Sudan environment. Many agricultural schemes have been introduced e.g. Managil extention for cultivation of cotton. Many forests have been removed and replaced by cultivation of crops. These agricultural practices led to usage or application of pesticides to combat agricultural pests. On the other hand extension of towns or urbanization led to the propagation of urban mosquitoes like Culex quinquefasciatus which is the most dominant species in Sudan (Abu Groon, 1988). The total number of the three genera (Anopheles, Culex and Aedes) is 107 species. Salman(2000) reported 31 species of Anopheles only three or four of them are of medical importance. The remainder 76 consist of Aedes, Culex and Mansonia. Mosquitoes are the main vectors of disease the Lymphatic Filariasis (WHO, 1972). Different species of mosquitoes participate in this according to localities. Culex quinquefasciatus is largely responsible for transmission of Wuchereria bancrofti in the world. Zaroug (1984) mentioned that Culex quinquefasciatus in Sudan does not transmit any disease, but it causes unbearable nuisance to people by its irritating bites. Results of this study showed that the density of Anopheles gambiae is higher in all areas of Juba except Tamarjia line. In contrast to this in Terkaka area numbers of Culex quinquefasciatus are higher than Juba areas.

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In Sudan no previous work was done to reveal the transmission vector of the Lymphatic Filariasis .In all previous studies only blood smears were examined in addition to ICT test. But no knowledge of which genera or species of mosquitoes transmitted the disease. Satti and Abdel Nur in their paper entitled: Bancroftain filariasis in the Sudan (1974) said “insects of medical importance were collected in both areas (Nuba mountains and South-western Darfur). The main features of this collection were the absence of Culex fatigans and the abundance of the malaria vectors An. gambiae and An. funestus and of the yellow fever vector Aedes aegypti”. Hawking (1957) stated that “the chief vectors of Wuchereria bancrofti in Africa seem to be An. gambiae and An. funestus. Cx. fatigans has been found to be a poor vector”. The study revealed that Anopheles gambiae and Culex quinquefasciatus were the vectors of human filariasis due to Wuchereria bancrofti in southern Sudan (Juba & Terkaka).In other countries including Srilanka (Gratz 1974) and East Africa(White ,1971a) Culex pipiens fatigans ( quinquefasciatus ) is the sole vector of the disease. In Monrovia, Liberia Culex quinquefasciatus was incriminated carrying the filarial larvae of Wuchereria bancrofti (Kuhlow, 1984). Also Anopheles gambiae transmits the parasite of Wuchereria bancrofti in some areas in the world e.g. in Tanzania coast (Frances and Bushrod 1981). In some localities Anopheles gambiae and Culex quinquefasciatus serve together as vectors of Lymphatic Filariasis while in other localities there were different genera and different species in the same country e.g. Malaya. Chow (1966) mentioned “urban filariasis is mainly transmitted by Culex pipiens fatigans (quinquefasciatus). However, in certain rural area in Malaya, the main vector of Wuchereria bancrofti is Anopheles whartoni (Reid, 1963).

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Hairstone and De Meillon (1968) implied that the probability was low for infective mosquitoes harbouring one, two or three larvae to produce patient infections in the human host. Lymphatic Filariasis (LF) is a severely debilitating and stigmatizing disease . In its most obvious manifestations LF causes elephantiasis ,an abnormal enlargement of the limbs (legs or arms) and of the genitals (scrotum, breasts and vulva) (W.H.O 2001) Bancroftian filariasis which is the true picture of Lymphatic Filariasis, is a major public health problem in the tropics with more than 100 million people infected in 73 countries (Michael et al,1996). Wuchereria bancrofti is the most well documented and wide spread parasite causing Lymphatic Filariasis . It is more common to find elephantiasis in patients affected with it and it characteristically includes symptoms associated with genitalia or chyluria in heavily infected patients (www.pdp.cdc.gov/dpd/dpdx/HTML/Filariasis asp?2006). Wuchereria bancrofti infects man only, no animal reservoir (WHO, 1967 ). Wuchereria bancrofti was a very wide spread parasite; it was a periodic form but of focal distribution in the humid tropical zone. It is transmitted by night-biting mosquitoes. It has disappeared from the main land of Australia, from North America, and from some of the islands in the Caribbean, but it is invading new areas in the growing towns of Asia, Africa and South America. The diurnal sub periodic form of Wuchereria bancrofti is restricted to South pacific area, where it is transmitted mainly by day biting mosquito.The periodic form is absent from this area the sub periodic form was predominantly rural (WHO, 1967). In the Polynesia where no Anopheles occur, Wuchereria bancrofti form are sub periodic, being adapted to uptake and transmission by the

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Aedes (stegomyia) scutellaris group which bites during daytime and evening (Macodonald, 1976). In a few forest situations of Southeast Asia there are also sub periodic strains of Wuchereria bancrofti transmitted by the Aedes (finglaya) niveus group, which may indicate the possible origins of the Polynesian sub periodic strain(Samarawickrema et al 1979). The efficiency of LF transmission is actually quite low. Generally, a person needs to be exposed to many mosquito bites over a period of a months or years before becoming infected (Evans , 2003). In Sudan the prevalence of the disease is very high in most states till now, that is natural because of the rapid and unplanned growth of cities which creates numerous breeding sites for mosquitoes whose density is the major determining factor of filarial transmission . Ghabat area, Jebel Nuga Tamarjia line and Kator are parts of Juba centre (outskirts) while, Rajaf east, Rajaf west. Monki, Lologo are villages which are far from Juba. In all these areas in addition to Terkaka and Gondokro island there is prevalence of the disease and infected mosquitoes. This indicates that filariasis is clearly an urban and rural disease in Juba and Terkaka.This is confirmed earlier by WHO,( 2002) who published that filariasis is prevalent in remote rural areas and in disfavoured peri-urban and urban areas. This was also confirmed by Samarawickrema, (1980) who said “Culex quinquefasciatus, the vector of Wuchereria bancrofti is common and found in all urban areas of Srilanka. But the disase in rural areas is more frequent than in urban ones. So LF is a disease of the poor”. Due to rapid urbanization without proper sanitation or use of insecticides, Culex quinquefasciatus, has increased enormously in many towns and cities, particularly in the developing countries. Comparatively

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speaking, urban filariasis is much difficult to control than rural filariasis (Chow, 1966). In this study Sausage shaped first stage larvae L1 existed in mosquito’s thorax. According to WHO (1967), second stage larvae (L2) were very rare in the mosquito. In this study only one insect carried this stage. In this study the third stage L3 infective larvae to man occurred in head , thorax and abdomen of mosquitoes. This is similar to the findings of Newton & Pratt (1945) and Wharton (1957) who stated that L3 occur in the proboscis or elsewhere in the mosquito host. It was also mentioned that the larvae at third stage are very active and can be found in any part of the insects not just in the head or proboscis (WHO, 1967). The three bubble like caudal papillae on the round extremity of the infective larvae are not all seen in this study. Lyengar, (1956) emphasized that the size of the papillae in illustration shows three large cow teat-like knobs on the caudal extremity. Also Manson (1878) said “all three papillae are not always obvious, their relative prominence depends upon the position in which the larvae are mounted. In this study the length of L3 is about 1288µ and bredth 29µ according to Nelson, (1959)the length of L3 ranged between 11701575µ, breadth between 18µ-32µ. In this study, the infection rate of mosquitoes in Southern Sudan (Juba and Terkaka) was 3% , (Culex quinquefasciatus 0.8%, Anopheles gambiae 3.4%). In Terkaka the infection rate was 7.4%. (Culex quinquefasciatus 8.1% and An. gambiae 5.8% ~ 6%). These results are similar to the findings of David, et. al . (2003) who illustrated that the prevalence of mosquito infection with Wuchereria bancrofti ranged from

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0%-3.5% by dissection . The infection rate in Terkaka is 7.4% this is natural, according to Laurence (1963) who stated that dissection method at Vellor gave an infection rate of 15%.

Out of 2729 there were 50 mosquitoes which carried the infective stage 41 from Juba , and 9 from Terkaka , only five larvae of L3 in the head .Infectivity rate in all endemic areas was 1.8% (Culex 0.9% , Anopheles 1.6%).This is similar to the investigation of Kuhlow and Zielke , (1978) that An. gambiae in Savannah and forest region of Liberia ( from 16 An. gambiae the infectivity rate 1.1% ). Rajaf west account for the highest infectivity rate as regards Anopheles (out of 17 Anopheles gambia the infectivity rate was 5.5% ). In Terkaka the infectivity rate was higher than in other areas of Juba as regards Culex quinquefasciatus (4.7% probably of the high density of Culex in Terkaka). Ghabat area participates with Terkaka in this aspect. Its infectivity rate was 2.7%. There are some places in the study areas like Kator and Lologo with high density of mosquitoes but no infected people or mosquitoes were met with . Dissection is a very effective and very laborious method for monitoring infection prevalence in vector population, (Burkot,& Ichimori,2002 ) . It is the suitable way to confirm the presence of (W.b ) through its characters particularly L3 , third stage. Through keen observations in Blue Nile, Juba and Terkaka, and using tests of ICT card,it appears that males are more affected than females. This is also recorded in the Kemmendine area of Rangoon where Hayashi (1966) mentioned that the microfilaria positive rate in males is almost twice as high as in females. Also observations revealed that leg’s elephantiasis is higher than arm’s. This is similar to the study done in 109

Kenya by Wijers & Kinyajui (1977) who stated that affected persons of Wuchereria bancrofti in Jaribuni (Kenya) were twelve men and eleven women all with leg elephantiasis. Three of the men also had arm filariasis and one woman had arm and breast filariasis. In Blue Nile, Juba, and Terkaka no patient suffered from arm, or breast elephantiasis. Genital organs as regards to females were not seen, but there were some cases of hydrocele observed in Blue Nile . The age distribution of the microfilariaemia rate is between 15-19 years as stated by Hayashi (1966) in Rangoon, Burma. But in Kenya Wijers & Kinyajui, (1977) mentioned that the youngest Mambrui child with a microfilariaemia was a boy aged three years and in Jaribuni it was a baby just twelve month old. In Sudan there were cases of children filariasis as stated by Ministry of health (2006, personal communication). Confirmation of identification of the species of filarial worms and their stages was done in Egypt. Now according to results of this study and surveys done before (in the past and present), clinical data and present surveys by ICT card (Ministry of health), Lymphatic Filariasis is prevalent in Sudan and puts most of populations of the South at risk. That is in addition to South Darfour and Blue Nile states. The disease is also suspected to be present in other central and eastern states. In some tribes in Juba the disease (elephantiasis) particularly that which affect feet is considered a curse on the person. The increase of the disease is a natural result of lack of treatment since the 1944 when the disease was identified. No efforts were made to eliminate the disease. Globally the infection of lymphatic disease has been recognized as the second leading cause of permanent and long term disability, with the

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deforming, mutilating disease of the limbs and genitals resulting not only in physical crippling but also in serious psycho social crippling. In addition to WHO there are many resources which can contribute to eliminate Lf disease like: World Bank, the Government of Sudan, African union, Atomic Energy Agency, Ministry of Health and Tropical Disease Research (TDR) .Appendix (3) . There must be two major objectives to eliminate Lymphatic filariasis : 1- To stop the spread of filariasis infection in all endemic states. 2- To alleviate and prevent the suffering and disability of affected people By 2020 we hope that LF will be a disease of the past, existing only in medical textbooks.

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Chapter five

Conclusions And Recommendations 5.1 Basic information of filariasis 1. Lymphatic Filariasis may occur in childhood. There was a baby just 12 years old infected, appendix (4) (Plate 3). 2. Lymphatic Filariasis is not a Killer disease .But silently destroying people's lives. 3. The microfilaria microfilariae live from 4 – 6 years circulating in the blood . 4. Acute Lymphatic Filariasis attacks can last for several days and are usually accompanied by a rash and eosinophilia. Damage to the lymphatic circulation or system leads to thickening and eventual blockage of lymphatic vessels with lymphoedema (lymph fluid in surrounding tissues). 5. Chronic Lymphatic Filariasis is characterized by hydrocele, lymphodema and elephantiasis. 6. Hydrocele is common in bancroftian filariasis. 7. Elephantiasis is a complication of advanced Lymphatic Filariasis it is seen as a coarse thickening, hardening, and cracking of the skin overlying enlarged fibrotic tissue. Appendix (4) (Plate 4). 8. The legs are more commonly affected than the arms. 9. Lymphatic Filariasis is diagnosed by :-  Finding and identifying the microfilariae of Wuchereria bancrofti in blood collected at correct time.  Occasionally detecting the microfilaria of Wuchereria bancrofti in hydrocele fluid or in the urine of patients with chyluria.  ICT card at day time. 112

10. Treatment should be once in a year. Administration is by a dose of anti-filarial drugs, or daily use of DEC fortified cooking salt. 5.2 Conclusions : The present research work was carried out to study the epidemiology and transmission of lymphatic filariasis at Southern Sudan (Juba & Terkaka) the work was done in the period August 2004 to September 2004 and April 2005 to June 2005. The findings obtained can be summarized in the following points : 1. The most dominant Anopheline mosquito in the two areas (Juba & Terkaka) was Anopheles gambiae s.s. and the most dominant culicine one was Culex quinqefasciatus. 2. In Terkaka Culex quinqefasciatus was dominant compared to Anopheles gambiae s.s. 3. In Juba and Terkaka the filarial disease is caused by Wuchereria bancrofti, transmitted by Anopheles gambiae, and Culex quinqefasciatus. 4. Stages of Wuchereria bancrofti (L1, L2, L3) in addition to microfilariae were detected in both species . 5. The infection rate of Anopheles gambiae with Wuchereria bancrofti was higher compared to Culex quinqefasciatus in Juba area. 6. The infectivity rate of Wuchereria bancrofti in Culex quinqefasciatus was significantly higher in Terkaka than in Juba. 7. Microfilariae in the blood were detected in only one patient (out of 17 persons ) in Juba. 8. Through observations and results of surveys by (ICT) card the percentage of the disease in Sudan is high. 9. Males were more affected than females.

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10. No genitalia cases were recorded in Juba or Terkaka . In the Blue Nile state more than one case of hydrocele were seen . 11. Much information has been obtained from Egypt : a. Definition of worms and confirmation of identification. b. Identification of stages (L1, L2, L3) and microfilaria. c. vector of Wuchereria bancrofti in Egypt wasCulex pipiens.

5.3 Recommendations: 1. The government and competent authorities should have declared the disease of Lymphatic Filariasis as a serious endemic disease just like malaria, schistosmiasis, leishmaniasis and provide the drugs an attempt to control the disease . 2. There must be regular medical compaigns against the disease in all affected states. 3. Reports of disease spread should be regularly submitted to the WHO to take its responsibility for distribution of ICT cards and drugs. 4. Sensitive test cards (ICT) should be available in laboratories so that patients could undergo medical test during the day and medical workers should be trained how to use these cards in all states of Sudan. 5. Training should be directed to clinical lymphological techniques to permit early detection of lymphatic changes. 6. Ministry of Higher Education & WHO should encourage universities to teach the elements of basic and clinical lymphology, so as to enable graduates to diagnose lymphoedema by inspection and palpation in absence of ICT cards .

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7. The use of simple noninvasive (e.g. inspection, palpation and skin- fold measurement) is recommended in order to identify individuals with abnormal lymph absorption syndromes in endemic communities. 8. Examination of environmental factors acting during the prenatal and neonatal periods, with long-term follow-up when appropriate, should be encouraged, such as:  Cellular and antigen studies on cord blood.  Study the scope of immune tolerance developing to filarial antigens in infants of infected mothers. 9. Ministry of Health should do its best to fight the transmission vectors by using effective insecticides-specially during rainy season in different areas of the Sudan. Medical advice for patients of chronic stage and hopeless cases is essential to minimize their pain. Patients must suppress growth of bacterial and fungi in wounds caused by the disease. 10. Due to the differences in biology of the various vector species preliminary studies of the vector’s bionomics and insecticide susceptibility and response are necessary in order to find out the most feasible and practical vector control method suitable to local epidemiological and entomological conditions. 11. Usage of insecticides in houses must be intensified. 12. Remote areas in eastern, western and southern Sudan must not be a hindrance for research . 13. Encouragement of researchers in this respect is needed . 14. It is advised that people should sleep inside nets. 15. Strategy for achieving eradication of Lymphatic Filarasis includes :

1- Treatment of all populations at risk . 2- Dissemination of Health Education to raise awareness in patients and to promote the benefits of intensive simple hygiene activities on affected body parts. 115

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APPENDIXES Appendix (1)

Plate (1): Microfilaria from the blood *After: www.dpd.cdc.gov/dpdx.may2004.22:19:10

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Appendix (2) Photographs of people infected with Filariasis (distribution by the organizers of Filariasis seminars . Khartoum ,2003)

Plate ( 2 a ): Lymphatic Filariasis of leg (male)

Plate ( 2 b ): Lymphatic Filariasis of leg (female)

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Appendix (3)

The Carter Center Lymphatic Filariasis Elimination Program Lymphatic filariasis, a parasitic disease transmitted by mosquitoes, is a leading cause of permanent and long-term disability. In its severest form, lymphatic filariasis leads to elephantiasis — a crippling condition in which limbs or other parts of the body are grotesquely swollen or enlarged. In communities endemic with lymphatic filariasis, as many as 10 percent of women can be affected with swollen limbs and 50 percent of men can suffer from mutilating genital disease. These conditions have a devastating effect on the quality of life of those affected, impacting them not only physically but also emotionally and economically.

In a collaborative effort, The Carter Center and its partners are working to show that the transmission of lymphatic filariasis can be interrupted on a large scale in Nigeria with mass community drug treatment and health education. In 2005, more than 3 million people were treated to prevent lymphatic filariasis in the two Nigerian states of Plateau and Nasarawa — a remarkable 92 percent of the eligible population.

Eliminating Lymphatic Filariasis : Lymphatic filariasis is one of six diseases the Carter Center's International Task Force for Disease Eradication determined could be eradicated using current tools and technologies (Read the ITFDE summary [PDF]). In 1997, the World Health Assembly, the directing body of the World Health Organization, called for the global elimination of lymphatic filariasis as a public health problem . .

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The strategy for elimination is based on treating everyone eligible to take the medicine living in an affected community with a dose of two drugs: ivermectin (Mectizan®, donated by Merck & Co., Inc.) and albendazole (donated by GlaxoSmithKline) in sub- Saharan Africa or diethylcarbamazine and albendazole elsewhere. Integrating Treatment : The Carter Center's Lymphatic Filariasis Elimination Program is based on the same community education and drug distribution system as the Center's River Blindness Program. In fact, the drug Mectizan, which is used in combination with albendazole to prevent transmission of lymphatic filariasis, is the same drug used worldwide to prevent transmission of river blindness. After years of success distributing Mectizan for river blindness in Nigeria, the Center began a pilot project to determine the feasibility of adding albendazole to the treatment regimen in communities where both river blindness and lymphatic filariasis are endemic. Partnering for Support : Elimination of lymphatic filariasis is only possible through collaboration with multiple organizations and agencies. Carter Center partners include the Nigeria Ministry of Health and the ministries of health in Plateau and Nasarawa states, as well as the Emory University Lymphatic Filariasis Support Center, the Centers for Disease Control and Prevention, the Bill & Melinda Gates Foundation, Merck, and GlaxoSmithKline. Most of all, through committed community action, Nigerians themselves are freeing their families and friends from the misery of lymphatic filariasis,www.cartercenter.org/health/lf/index.html (2007) .

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Appendix (4)

Photographs of people infected with Filariasis (distribution by the organizers of Filariasis seminars . Khartoum ,2003)

Plate ( 3 ): Lymphatic Filariasis of Hydrocele (boy) *After : Federal Ministry of Health (2003)

Plate ( 4 ): Elephantiasis acute and abnormal case – the most extreme form of Lymphatic Filariasis *After :Http://www.cartercenter.org/health/lf/index.html (2007) .

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