CHARACTERIZATION OF CATTLE FILARIAL PARASITE MORPHOLOGICALLY SIMILAR TO WUCHERERIA BANCROFT! (NEMATODA: FILAROIDAE) IN SOUTHERN GHANA BY HELENA BAIDOO (B.Sc. Hons.) (10097418) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT FOR THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY IN ZOOLOGY (APPLIED PARASITOLOGY) SEPTEMBER, 2003 University of Ghana http://ugspace.ug.edu.gh 4 5 2 6 . ?3>- ^ Wltcv University of Ghana http://ugspace.ug.edu.gh DECLARATION This thesis is the result of research work undertaken by Helena Baidoo under the supervision of the following names listed below and all references stated have been duly acknowledged. HELENA BAIDOO (Student, 10097418) (Supervisor) (Supervisor) (Supervisor) University of Ghana http://ugspace.ug.edu.gh DEDICATION TO ALL THE MEMBERS OF THE BAIDOO FAMILY University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS I wish to express my deepest appreciation to my supervisors, Dr. Daniel Boakye, Prof. M D. Wilson both of the Parasitology Unit of the Noguchi Memorial Institute for Medical Research (NMIMR) and Prof. D. Edoh of the Zoology Department, University of Ghana for their excellent guidance, advice and profound contribution throughout this work. I am so much grateful to Prof. D. Ofori-Adjei, Director of the NMIMR for allowing me to use the facilities of the Institute. Very special thanks to Mr. Sampson Otoo (Technician) of the Parasitology Unit for the major role he played both in my fieldwork and laboratory analysis. I really cannot thank him enough for the assistance given throughout and most especially when I needed him most and for his teaching skills. I also want to thank Mr. Jonas Asigbee (Chief Technician) who was very instrumental in the planning of my field trips right from the beginning of the work. I am also indebted to major field workers including Dr. Akumiah (Veterinary Doctor) and Mr. Ibrahim Issah, both of the Ministry of Food and Agriculture Unit at Winneba for their immense support during my fieldwork. I really appreciate the warm reception and tremendous help given me by Mr. Hammond, Mr. Amoako and all the Fulani herdsmen. In fact without granting me the permission to use their animals for research this work would not have been carried out. I am also grateful to Dr. Okyere Darko of the Veterinary University of Ghana http://ugspace.ug.edu.gh Unit at Takoradi, for his goodwill and support and linking me up with research personnel, Mr. Okine, Uncle Ebo etc at Axim and for the various contacts he made on my behalf. I also appreciate the moral and spiritual support of my special friends, Beatrice and Grace for being there for me all the time. Special thanks also go to friends in the Parasitology Unit most especially Mrs. Anita Ghansah, Mr. Charles Brown, Ms Naiki Puplampu, Mrs. Ogoe, Mrs. Cofie, and my roommate Ms Nkem Okoye. The encouragement and friendly support given me by my course mates, Helena Nartey, Fred Aboagye-Antwi, Victoria Atsorebo, Isaac Aboagye and my lecturer Dr. Bimi is very much appreciated. My heartfelt thanks and gratitude go to the entire members of my family, especially to my Mom and Dad for their support and encouragement throughout the course of my study. Above all I want to thank the almighty God for seeing me through another phase of my life. This project was supported by WHO Special Programme for Research Training in Tropical Diseases (TDR) grant (M8/181/4/b,316) to Helena Baidoo. IV University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION.................................................................................................................. i DEDICATION.................................................................................................................... ii ACKNOWLEDGEMENTS_______________________________________________ in TABLE OF CONTENTS................................................................................................... v UST OF ILLUSTRATIONS.................................................................................................x LIST OF TABLES..............................................................................................................xi LIST OF APPENDICES...................................................................................................xii LIST OF ABBREVIATIONS.......................................................................................... xiii ABSTRACT...................................................................................................................... jcv CHAPTER ONE................................................................................................................ 1 GENERAL INTRODUCTION.__________________________________________ 1 1.1 Introduction......................___ ........................................................................... 1 1.2 Rationale and Aims of the Study.........................................................................5 1.2.1 General objective..........................................................................................5 1.2.2 Specific objectives........................................................................................6 CHAPTER TWO................................................................................................................ 7 LITERATURE REVIEW..............................................................................................7 2.1 Filarial Parasites______ —............__.............__ ......._.......___......._.......... 7 2.1.1 Human filarial diseases .............................................................................8 University of Ghana http://ugspace.ug.edu.gh 2.1.1.1 Lymphatic filariasis...............................................................................8 2 1.1.2 Onchocerciasis.......................................................................................9 2.1.1.3 Loasis.................................................................................................... 9 2.1.1.4 Other filarial infections........................................................................10 2.1.2 Zoonotic filarial infections...................................................................... 13 2.1.2.1 Setaria species.....................................................................................15 22 Filarial Nocturnal Periodicity .....................................................................——18 23 The Vectors of Human Filariasis........................................................................22 2.3.1 Identification of Culexpipiens complex......................................................24 2.3.2 Identification of the An. gambiae and An. Junestus complex.................... 25 2.4 Trasmission Dynamics in Filariasis................................................................28 2.4.1 Factors that influence microfilaria prevalence and density........................31 2£ The Global Burden of Lymphatic Filariasis...................................................... 32 2.6 Socio-economic Impact of Filariasis.................................................................33 2.7 Diagnosis of lymphatic filariasis.......................................................................35 2.7.1 Direct detection of microfilariae................................................................ 35 2.7.2 Concentration methods for microfilariae....................................................37 2.7.3 The Diethylcarbamazine (DEC) Provocation Test.....................................39 2.7.4 Detection of filarial antigen........................................................................39 2.7.5 Filarial antibody assay............................................................................... 41 2.7.6 PCR assays for filarial detection in vectors............................................... 43 2.8 Control of lymphatic filariasis...........................................................................46 2.8.1 Vector control.......................................................................................... 46 University of Ghana http://ugspace.ug.edu.gh 2.8.1.2 Insecticide treated/untreated bed-nets ............................................... 48 2.8.2 Chemotherapy.............................................................................................50 2.8.2.1 Mass drug treatment with DEC.......................................................... 51 2.8.2.2 Use of Ivermectin................................................................................ 52 2.8.2.3 Albendazole......................................................................................... 54 2.8.2.4 Combination therapy with DEC and Ivermectin................................. 54 CHAPTER THREE.......................................................................................................... 56 MATERIALS AND METHODS________________________________________ 56 3.1 Study Sites 3.2.1 Screening of cattle for filarial infections.................................................... 59 3.2.1.1 Collection of cattle blood for molecular studies..................................60 3.2.2 Determination of microfilaraemic density..................................................60 3.2.3 Morphometric studies of cattle blood microfilariae................................. 61 33 Entomological Studies...................................................................................... 62 3.3.1 Mosquito surveys...................................................................................... 62 3.3.2 Dissection of mosquitoes for filarial infections..........................................62 3.4 Molecular Studies.......................................................................................... 65 3.4.1 Chemical and Reagents ...........................................................................65 3 .4.2 Molecular identification of filarial worms................................................ 65 3.4.2.1 Extraction of filarial DNA...................................................................65 34.2.2 PCR identification of cattle filariae.................................................. 66 3.4.3 Molecular identification of Anopheles gambiae s.l.................................... 66 3.4.3.1 DNA extraction of mosquitoes using Bender buffer protocol............ 66 vii University of Ghana http://ugspace.ug.edu.gh 3.4.32 Species identification of Anopheles gambiae s.L using PCR...............67 3.4.4 PCR identification of Anopheles junestus group........................................ 68 3.4.5 Analysis of PCR product (Agarose gel electrophoresis)............................ 68 3.4.6 Estimation of size of PCR product............................................................. 69 CHAPTER FOUR.......................................................................................................... 70 RESULTS.................................................................................................................... 70 4.1 Study Population ____ ...____ ......................................................................... 70 42 Microfilaraemic density and observed frequencies.... ......—............------------- 70 43 Morphometric/Morphological Description of Cattle Filariae ................. — 72 4.3.1 Sheathed microfilaria..................................................................................73 4.3.2 Unsheathed microfilaria..............................................................................73 4.3.3 Comparison of cattle filariae with Wuchereria bancrofti...........................74 4.3.4 Comparison with Setaria species................................................................74 4.4 Mosquito Surveys ............................................................................................ 77 4.5 Molecular Identification of Cattle Filariae and Anopheles Mosquitoes 79 CHAPTER FIVE............................................................................................................ 84 DISCUSSIONS AND CONCLUSION___________________________________ 84 REFERENCES................................................................................................................. 87 APPENDICES................................................................................................................122 APPENDIX 1.............................................................................................................122 APPENDIX n............................................................................................................ 124 APPENDIX m........................................................................................................... 126 viii University of Ghana http://ugspace.ug.edu.gh APPENDIX IV...........................................................................................................130 APPENDIX V............................................................................................................ 145 APPENDIX VI...........................................................................................................146 ix University of Ghana http://ugspace.ug.edu.gh LIST OF ILLUSTRATIONS Figure 2.1 Figure 2.2 Figure 3.1 Figure 3.2 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Microfilaria of Wuchereria bancrofti Elephantiasis of the leg (WHO image library) A map of Ghana showing the study sites, Winneba, Axim, Somanya, and Accra, the Capital City The front (top) and rear (bottom) view of mosquito net erected over cattle with flap open for mosquito entry The microfilariae densities of the cattle studied at Winneba Unsheathed microfilaria (mi) observed in cattle blood after staining with Giemsa (xlOOO) Sheathed microfilaria (mf) observed in cattle blood after staining with Giemsa (xlOOO) Proportion of various species of mosquitoes collected off cattle Agarose gel electrophoregram of the amplified PCR An. jimestus s.s DNA fragment Agarose gel electrophoregram of the amplified An .gambiae s.s DNA fragment Agarose gel electrophoregram of PCR product amplified from rDNA products of W. bancrofti and cattle filarial parasite Infective stage of (L3) of filarial worm found in culex mosquito University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 3.1 Table 32 Table 33 Table 4.1 Table 4.2 Constituents of PCR reaction mix for Wuchereria bancrofti Typical reaction component for a 25(0.1 PCR reaction for An. gambiae identification Standard reaction component for a 25|_il PCR reaction for identification An. fiinestus The sex and age distribution of cattle proportions at the three sampling sites Microfilaria intensities during the 24-hour collection period xi University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES APPENDIX I APPENDIX H APPENDIX m APPENDIX IV APPENDIX V APPENDIX VI Chemicals and Reagents DNeasy protocol for DNA purification from animal tissues Morphometric measurements of cattle filarial Results of microscopic examination and demographic features (Field data) Formulas Constituents of 25|j.l PCR reaction mix for the different reactions University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS CDC Centre for Disease Control and Prevention, Atlanta, USA DPD Division of Parasitic Diseases DEC Diethylcarbamazine citrate WHO World Health Organization ADL Adenolymphangitis ELISA Enzyme-linked immunosorbent assay ICT Immunochromatographic whole blood test PCR Polymerase chain reaction bp base pair dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dTTP deoxythymidine triphosphate dGTP deoxyguanosine triphosphate EDTA Disodium ethylene diamine tetraacetate.2H20 pH Hydrogen-ion exponent RNA Ribonucleic acid RNase Ribonuclease rDNA Ribosomal DNA rpm Revolution per minute SddH20 Sterile double distilled water S.S. sensu stricto University of Ghana http://ugspace.ug.edu.gh TAE Tris-acetate EDTA TE Tris EDTA Tm Melting temperature Tris 2-amino-2-hydroxyl-1,3 -propanediol TBE Tris-Borate-EDTA Buffer uL Microlitre XIV University of Ghana http://ugspace.ug.edu.gh ABSTRACT The monitoring of the current global strategy for the elimination of lymphatic filariasis (LF) can be confounded if other parasites indistinguishable from Wuchereria bancrofti also occur in endemic areas. An incidental examination of cattle blood revealed microfilariae that were morphologically similar to W. bancrofti. This study was therefore conducted to characterize these cattle filarial parasite. A total of 284 cattle from Somanya, Winneba and Axim were screened randomly for filarial parasites using the traditional blood smear technique. Blood was collected from the positive cattle at 4-hour intervals over a 24-hour period to determine microfilarial intensity and periodicity, and for molecular studies.Six hundred and ninety mosquitoes were collected off the positive cattle from 00.00 - 04.00am by an aspirator into paper cups and sent to the laboratory. The mosquitoes were morphologically identified and dissected for filarial infections. DNA extracted from the microfilariae (mf) and infective stages (L3s) of the parasites were subjected to PCR analysis using W. bancrofti primers (NV1 and NV2). The prevalence of filarial infections were 3.5% (5/141) and 6% (2/33) at Winneba and Somanya respectively and negative for 110 cattle blood screened at Axim. An overall prevalence of filarial infections among cattle for the three study sites was found to be 2.4% (7/286). The geometric mean densities among infected cattle were found to vary between 4.6-20 microfilariae/1 OOul of blood and trend suggestive of subperiodicity. The two morphological types of microfilariae (sheathed and unsheathed) were observed and both were morphometrically similar, but different from W. bancrofti suggesting that they were not the latter species but rather Setaria species. One distinctive feature about the xv University of Ghana http://ugspace.ug.edu.gh microfilaria was the presence of a prominent inner korper, which is absent in W. bancrofti. Also both cattle filarial parasites and the infective larvae were negative by PCR. Thus indicating that they are not W. bancrofti. Among the 690 mosquitoes collected off cattie, 612 (88.7%) were Culex, 29 (4.93%), Anopheles, 43 (6.23%) Mansonia and 1 (0.15%) Aedes. Two infective stages of filarial worms (L3) were found in Culex mosquitoes. These results seem to indicate that the presence of these cattle filarial parasite in LF endemic areas may not confound the monitoring of LF intervention programmes. xvi University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE GENERAL INTRODUCTION 1.1 Introduction Lymphatic filariasis also known as elephantiasis is a debilitating disease, which result from infection with vector-bome tissue dwelling nematodes called filariae. It is ai important public health and socio-economic problem worldwide. Both male and femali are equally susceptible to infection but due to different local, cultural, and social worl practices as well as exposure to insect vectors, one sex may be more exposed to infectioi than the other. All ages are susceptible and potentially microfilaremic as stated by Cool and Saunders, 1996. Microfilaremia rate increases with age through childhood and earl; adulthood, though clinical manifestations may be inapparent in endemic areas Manifestation of acute and chronic filariasis usually occurs after years of repeated anc intense exposure to infected vectors. * The disease is rarely fatal but the consequences of infection can cause significan personal and socio-economic hardships for those who are infected. Morbidity of humar filariasis is due mainly to the host reaction to microfilaria or to developing adult worms in different areas of the body (Evans etal., 1993). 1 University of Ghana http://ugspace.ug.edu.gh The World Health Organization (WHO) ranks lymphatic filariasis as the second leading cause of permanent disability worldwide. Currently 120 million people are infected worldwide, out of which 103 million (90%) of these infections are caused by infections with Wuchereria bancrofti and 13 million (about 10%) either by Brugia malayi or Brugia timori. (Michael et al., 1996). More than 1.1 billion people (20%) of the world’s population live in areas where they are at risk of infection. About half of the world’s burden of lymphatic filariasis is transmitted by Cx. qumquefasiciatus in India. This and other man-biting mosquitoes belonging to the Culex pipiens complex are responsible for most or all of the bancoftian filariasis transmission in Asian countries, Indonesia, Egypt, urban East Africa and the Americas (Subramanian et al., 1997). In the Papuan sub-region and tropical Africa including Ghana, Anopheles mosquitoes transmit lymphatic filariasis (Appawu et al., 2001). Surveys in Ghana have indicated that bancroftian filariasis is present in most parts of the country, with considerable regional variations in prevalence (Dunyo et al., 1996). Studies along the coast of Ghana have revealed endemic foci along the west coast of the country with an overall microfilaria (mf) prevalence of 9-25% and microfilarial intensities of 321- 1172 mf/tnl of blood. Hydrocele affected 8.5-27% whilst elephantiasis of the limbs affected 8.5-27% of populations (Dunyo et al., 1996). Hunter (1992) observed that a number of communities in Ghana with filariasis had water impoundment, such as small dams provided for agricultural purposes For example in Okyereko, an irrigation project community in the Central Region of Ghana where the prevalence of microfilaraemia was 2 University of Ghana http://ugspace.ug.edu.gh 26.4%, and hydrocele and elephantiasis were 13.8% and 1.4% respectively (Dzodzomenyo etal., 1999). Fortunately, several advancements in the drug therapy regimen for community-wide control programmes have been made. Several studies have shown 1hat simultaneous treatment with albendazole and the antifilarial drug ivermectin enhanced the suppression of microfilaiae (Ottesen et al., 1990; Dreyer et al., 1995; Bockarie et al., 1997). It has also been reported that the disease can be eliminated worldwide with the use of mass chemotherapy of the affected communities (Kazura et al. 1993; Ottesen & Ramachandran 1995; Bockarie et al., 1998). The co-administration of DEC with either ivermectin or albendazole, which is a single dose therapy, has been shown to reduce blood microfilaria levels effectively and to the same long-lasting degree as tihe previously recommended 12- day treatment course of DEC. This strategy for controlling the disease has been adopted by the WHO for its programme on the global elimination of lymphatic filariasis. For it’s implementation to be successful there will be the need for a very effective monitoring and evaluation system because many control programmes have failed as a result of premature cessation of control activities probably resulting from ineffective monitoring (Ottesen etal., 1997). Effective monitoring depends on correct identification of parasites in human and insect hosts, (vectors) and also an in-depth knowledge of vector movement and distribution is essential. These entomological parameters need to be built up within any intervention 3 University of Ghana http://ugspace.ug.edu.gh programme because information on these parameters gives indication of the situation at any point in time. Determination of pre-intervention and post intervention entomological parameters of disease transmission have been used for monitoring intervention programmes against insect-bome diseases. A classical example is the monitoring of onchocerciasis control (Yameogo et ah, 1999). This approach however, could be misleading if there are other filarial parasites indistinguishable from those implicated as causative agent of the disease. This situation could become more confusing if the same vectors transmit the disease. An example is the occurrence of the cattle filarial Onchocerca ochengi found in the members of the Simulium damnosum complex. This is a situation that could affect the accurate assessment and monitoring of oncho cerciasis control (Toe et al, 1998). The programme to eliminate lymphatic filariasis is an initiative with a bold objective, which is to eliminate LF as a public health problem by the year 2020 (WHO, 2000). This elimination programme has been planned and has already started in many African countries including Ghana. The institution of a monitoring strategy based on vector transmission indices has been planned and a molecular methodology is being developed (Boakye etal., 2001). 4 University of Ghana http://ugspace.ug.edu.gh 1.2 Rationale and Aims of the Study An incidental examination of catde blood revealed some filarial parasites that on morphological examination showed some similarity to W. bancrofti. A polymerase chain reaction (PCR) conducted using the W. bancrofti Primers, NV1 and NV2 (Ramzy et al., 1997) on the DNA of pooled parasite from the cattle also gave a product of the same size as W. bancrofti (M. D. Wilson, pers. Comm.). These observations indicated that the presence of these worms could pose a limitation to the use of entomological parameters for monitoring lymphatic filariasis intervention especially if the vectors are found to be members of the Anopheles gambiae complex and that these parasites are widely distributed in areas endemic for bancroftian filariasis. There is therefore the urgent need to properly characterize these worms and, determine their distribution and identify the possible vectors. 1.2.1 General objective The project seeks to use both morphological and molecular methods to identify cattle filarial parasites morphologically similar to Wuchereria bancrofti. The vectors of these parasites will also be determined in order to establish how important they will be in the monitoring of lymphatic filariasis intervention in Ghana. 5 University of Ghana http://ugspace.ug.edu.gh 1.2.2 Specific objectives The specific objectives of the study are as follows: 1. To examine cattle blood for the presence of filarial parasites 2. To determine if these parasites exhibited any form of periodicity as observed in Wuchereria bancrofii 3. To use morphological methods to identify these cattle parasites 4. To apply molecular methods developed for specific identification of W bancrofii on the cattle parasites 5. To determine the prevalence of blood filarial infection in cattle 6. To determine if these cattle parasites are also transmitted by members of Anopheles gambiae complex 6 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1 Filarial Parasites Filarial parasites (filaroids) are long hair-like tissue-dwelling nematodes. All except the Guinea worm Dracunculus medinensis (which uses a copepod) employ arthropods as intermediate hosts. All filaroids have a similar life cycle that includes an obligatory maturation stage in a blood-sucking insect or copepod, and a reproductive stage in the tissues or blood of a definitive host (McMahon and Simonsen, 1996). Adult male and female worms live in the lymphatic, skin, or other tissues. Microfilariae (Fig 2.1) are produced by the female worm, which either circulate in the blood or invade the skin, and are ingested by the vector. Larval development but not multiplication occurs within the muscles of the vector. The infective stage L3 migrates to the proboscis and is transmitted to the new host during feeding. Unlike malaria, the infective stage is not directly injected into the skin of the new host. They are deposited onto the skin whilst the mosquito is feeding and find their own way through the skin, usually via the puncture made by the mosquito. Filaroids have been found in all classes of vertebrates except fish and are especially common in birds (McMahon and Simonsen, 1996). All but one of the species that cause human disease (the Guinea worm) belongs to the Family Onchocercidae. University of Ghana http://ugspace.ug.edu.gh 2.1.1 Human filarial diseases Of the hundreds of described filarial parasites, only 8 species cause natural infections in humans (Nissen, 2001). Human filarial parasites may be classified according to the habitat of the adult worms in the vertebral host. As such there are three groups of filarial parasites. These are the cutaneous group (Loa loa, Onchocerca volvulus and Mansonella streptocerca); lymphatic group (Wuchereria bancrofii, Brugia malayi and Brugia timori) and ihe body cavily group (Mansonella perstans and Mansonella ozzardi). Parasites of the cutaneous and lymphatic groups are of the most significant clinical interest (Nissen, 2001). 2.1.1.1 Lymphatic filariasis Wuchereria bancrofii, Brugia malayi and Brugia timori are mosquito-bome and cause lymphatic filariasis. In many people's mind lymphatic filariasis is synonymous with "elephantiasis" characterized by the enlargement of scrotum or limbs (Figure 2.2). In most endemic areas elephantiasis only occurs in a small proportion of the people suffering from lymphatic filariasis. Bancroftian filariasis, caused by W. bancrofii is responsible for 90% of lymphatic filariasis cases and is found throughout the tropics and in some sub-tropical areas. Brugia malayi is confined to Southeast and Eastern Asia and is transmitted by mosquitoes including Mansonia, Anopheles and Aedes (WHO, 1997). Brugia timori is found only in Timor and its adjacent islands (McMahon & Simonsen 1996) and is transmitted by Anopheles mosquitoes (WHO, 1997). In West Africa the vectors of W. bancrofii are An. gambiae and An. Junestus whilst in east and Central Africa, it is Culex mosquitoes (Subramanian et al., 1997). University of Ghana http://ugspace.ug.edu.gh 2.1.1.2 Onchocerciasis Onchocerciasis is a debilitating disease affecting 20-40 million people in 34 countries of Sub-Saharan Africa, the Arabian Peninsula and South America. It is transmitted by Simulid black fly. Microfilariae invade the skin and give rise to dermatitis, premature aging of the skin and skin atrophy. Development of the adult worm leads to nodule formation. Microfilariae invade the eye and cause an inflammatory reaction that can lead to blindness (Duke, 1990). In recent years, the prospects for its control have been improved with the development of community directed treatment with ivermectin, which is a safe effective and affordable drug. However encephalopathies with occasional fatal outcomes have been reported following its use to treat persons coinfected with Onchocerca volvulus and Loa loa (Boussinesq and Gardon, 1997; Chippaux et at, 1996; Gardon et al., 1997). These adverse outcomes are an obstacle to the sustainability of large-scale ivermectin treatment for the control of onchocerciasis and lymphatic filariasis in areas of Africa where there is a potential for coendemicity with L. loa (Boussinesq and Gardon, 1997). 2.1.13 Loasis Loasis occurs in the forested areas of West and Central Africa particularly in Nigeria and Cameroon. It is caused by Loa loa (sometimes called the African eye worm), which is transmitted to humans by the daytime-biting tabanid flies. Loasis very rarely causes serious complication and the adult worms generally go unnoticed unless they pass through the bridge of the nose or the conjunctiva of the eye. The disease is characterized by a range of clinical manifestation including "Calabar swellings", occurring commonly 9 University of Ghana http://ugspace.ug.edu.gh in the hotter months, pruritis and eye inflammation (Pinder et al., 1988). A localized, sometimes painful, swelling also occurs when the worm dies. It is the most common filaroid infecting travellers from non-endemic areas (Pinder et al, 1988). 2.1.1.4 Other filarial infections Mansonella perstans which occurs in Africa, Central and South America, Mansonella streptocerca (Africa) and Mansonella ozzardi (Central and South America) are all transmitted by Culicoid midges. Most infections are asymptomatic but in the worse cases symptoms similar to O. volvulus may occur. This is especially relevant in Brazil where M. ozzardi and O. volvulus are sympatric in parts of the Amazonia oncherciasis focus. Also occasionally symptoms, which look very much like loasis, may occur (WHO, 1997). Attempts at controlling mansonelliasis have been few, but a recent report indicates that ivermectin may be effective as a microfilaricide. Other filariae also known to infect man includes Wuchereria kalimantari, B. arbuta, B. beaveri, B. guyanensis and Dipetalonema sprenti. 10 University of Ghana http://ugspace.ug.edu.gh Fig 2.1: Microfilaria of Wuchereria bancrofti, A=Head space, B=Inner body, C=nerve ring and D= Sheath 11 University of Ghana http://ugspace.ug.edu.gh Fig 22: Elephantiasis of the leg (WHO filariasis image library) 12 University of Ghana http://ugspace.ug.edu.gh 2.1.2 Zoonotic filarial infections Human infections with various known and unknown animal filarial parasites have regularly been reported throughout the tropical and subtropical parts of the world including the southwestern United States of America (Nelson, 1965; Orihel and Eberhand, 1998). There could be a wide range of animal parasites that can infect man as well as their animal host. These include; Dirofilaria immitis, D. repens, D. tenuis, D. spectans, D. striata, Setaria equina mdMeningonema peruzzi. Dirofilaria immitis is the most important filaroid parasite of domestic animals endemic in tropical and subtropical and warm temperate regions of the world (including the south western United States) which occasionally infects man (Rodrigues-Silva et al., 1995). Adult worms reside in the right ventricle and pulmonary artery of the dog while the unsheathed microfilariae circulate in the blood. Once ingested by the appropriate mosquito vector, the microfilariae undergo development into infective larvae, which can be transmitted to both dogs and humans. However the worms never reach full maturity in humans. Most cases are asymptomatic and the worm becomes calcified in the lung resulting in a "coin lesion" which may be mistaken for carcinoma or tuberculosis. There are more than 200 cases of human pulmonary dirofilariasis reported throughout the world (Schneider et al., 1986). Buckley (1958) demonstrated that man could suffer serious complications when inoculated with Brugia species from animals. Thereafter furnished experimental proof by Danaraj (1956) suggested that animal filariae were responsible for tropical pulmonary eosinophilia quite common on the Kenya coast. 13 University of Ghana http://ugspace.ug.edu.gh Another dog filaroid, Dirofilaria repens can cause subcutaneous nodules, peri-orbital lesions, coin lesions in the lungs, and breast lumps in humans and rare eases have been reported throughout the world (Pampiglione et al, 1995). High levels of antifilarial IgG, IgE and IgM antibodies are present in patients with dirofilariasis (Simon el al., 1997) but there can be high background level of dirofilaria-associated antibody in communities which are in close contact with infected dogs and the prevalence of IX immitis antibodies in humans is closely related to the number of infected dogs in the community (Welch and Dobson, 1974). At least six cases of human filarial dermatitis have been attributed to Brugia beaveri (Routh and Ebowmik, 1993), a filarial species whose natural host is the Raccoon and nine cases of lymph node infection by an unidentified Brugia species (Baird et al, 1986) possibly from the domestic cat. Animal filaroids seldom seen to develop and become sexually mature and produce a microfilaraemia in man but it does occur from time to time (Orihel and Eberhard, 1998). Greene etal. (1978) found circulating microfilariae in a patient from Alabama who was suffering from Lupus erythematosis. Although the species could not be identified with certainly, the most likely species appeared to be Mansonella interstitium, which is a filaroid of squirrels. There has also been report of microfilaraemia and eosinophilia in a _ 70-year-old Greek man who had never been in an area endemic for human filarial parasites and was presumably infected with an animal species (Petrocheilou et al., 1998). 14 University of Ghana http://ugspace.ug.edu.gh 2.1.2.1 Setaria species Setaria species are widely distributed throughout the world and many species of the adult have been described. More than 200 species of filariae have been described in literature; the life cycle of only 38 have been studied. Nelson (1962) stated that 17 species have been found in Africa alone. Mosquitoes are suitable intermediate host of 23 out of the 38 species and these include filariae from lizards, frogs, birds, bats, carnivores, herbivores and primates. Setaria species are parasitic in the peritoneal cavity of sheep, cattle, the ox, deer and horses They are also known to be the major causative agents of epizootic cererobrospinal nematodiasis in sheep raised in Korea since 1939 (Kimura & Niimi, 1939,1940,1941). Adult Setaria digitata is commonly found free with the abdominal cavity of several ungulates, which include cattle, sheep and horses in the Far East and Asia. In Korea, two species S, digitata and S. marshalli have been reported to parasitize cattle (Rhee et al., 1994). Prevalence of Setaria spp among cattle raised in Korea ranges from 5 to seventy percent (Lshii et al., 1953; Paick etal., 1976; Rhee etal., 1994 Moon and Kang, 2000; Mohanty et al., 2000). In one particular study, a total of 110 cattle were examined in a bancroftian filariasis endemic area to determine the prevalence of infection of the bovine filarial parasite Setaria digitata. About 12.5% of cattle examined were found to harbour both adult worms in the peritoneum nd microfilariae (mf) in circulation. Seventy percent of the cattle were amicrofilaraemic but harboured adult worms. A third group of cattle (16.5%) were free of detectable mf and adult worms. The presence of adult worms and/or 15 University of Ghana http://ugspace.ug.edu.gh mf did not influence the antibody levels of the 4-antigen preparation of S. digitata (Mohanty etal, 2000). Although the adult worms of most Setaria species in the abdominal cavity are mostly harmless to cattle serious pathogenic results could occur in animals such as sheep, goats and horses in which larvae can migrate erratically into the central nervous system (Innes & Shoho, 1952, 1953). The ectopic parasitism of S. digitata has been reported in the eye of a horse (Jemelka, 1976). Except for one report on S. digitata in the cavity of a cow (Nair et al, 1993), heterotropic parasitism of cattle with Setaria species is rarely known. A case of a single-eyed blindness of two cattle in Korea and the isolation of a female S. digitata has been reported (Sung-Shik et al., 2001). The worm removed was 5.6 cm long and was identified as female S. digitata after the light and electron microscopic morphological description of Rhee et al. (1994). Setaria cervi (Setaria labiato-papillosa) is a cosmopolitan nematode parasite of cattle which is used to assess the efficacy of potential antifilarial agents, and bears close similarity to human filarial parasite Wuchereria bancroft (Singhal et al., 1969, 1972). Work done by Heisch et al. (1959) revealed that out of 200 cows examined; 7 had microfilariae in blood films, 5 of which were infections due to Setaria cervi; the microfilariae were also similar to Setaria equina. Although in the survey only 5 cows were found with microfilariae in blood films, adult Setaria cervi were commonly seen in the peritoneal cavities of cattle slaughtered at the abattoir in Mombassa. Microfilarial densities have always been very low in the cattle examined in Kenya. This has made 16 University of Ghana http://ugspace.ug.edu.gh feeding experiments with mosquitoes very difficult This problem has been overcome by implanting living adult setaria in the peritoneal cavities of monkeys, using technique developed by Williams (1955). * Zoonotic Setaria infections in human is rare, however Panaitescu et al. (1999) have reported four cases of human filariasis due to Setaria labiatopapillosa in Bucharest, Romania. The vector insect however could not be specified. These are the first reported cases of human infection with Setaria labiatopapillosa. 17 University of Ghana http://ugspace.ug.edu.gh 2.2 Filarial Nocturnal Periodicity Periodicity is a well known phenomenon which occurs with many filaroid worms, and various hypotheses put forward to explain periodicity have been comprehensively reviewed (Oishi, 1959; Hawking, 1967; Kamine, 1972; Masuya, 1976). According to the periodic pattern of microfilariae (mf) in the human host’s peripheral blood, lymphatic filariasis caused by Wuchereria bancrofti may be separated into three forms: (Aikat, 1977) a nocturnal periodic form, widely found in tropical and subtropical zones in Africa, Asia and Latin America, in which microfilarial densities peak close to midnight; (Dreyer et al., 1996) a non-periodic or diurnal, sub-periodic form, prevalent in the islands of the South Pacific, in which maximum densities of mf occur around 16.30 hours; and (Gatika et al., 1994) a nocturnal, sub-periodic form, with a focal distribution in western Thailand, which is characterized by a peak in microflarial density at around 20.30 hours (Harinasuta et al., 1970 a,b; WHO, 1992). A mathematical method for the analysis of microfilarial counts in peripheral blood during a 24-h cycle was developed by Sasa and Tanaka (1972), who believed that the circadian variation followed a simple harmonic wave. Aikat and Das (1977) devised another method, based on simple trigonometry, to calculate at which time densities of mf in the peripheral blood peaked and to calculate a periodicity index. The data available from surveys of periodicity among the mf of human parasites from various regions of the world were analyzed by Tanaka (1981), using both of these methods. Although the two 18 University of Ghana http://ugspace.ug.edu.gh methods gave the same results, Tanaka (1981) considered that the trigonometric method of Aikat and Das (1977) was the easier to perform. The method proposed by Aikat and Das (1977) have been used in various studies to establish the periodic pattern of W. bancrofti mf in the peripheral blood of subjects worldwide and also to determined appropriate time for sampling. For instance in the Brazilian city of Maceio, in Alagoas state, a study was conducted by Rocha and Fontes (1998) to establish the periodicity of W. bancrofti. In the study it was found that although all the subjects had a detectable microfilaraemia from 23:00 hours to 06.00 hours, no mf could be detected in most (71.4%) of the smears prepared from samples collected at 15.00 hours. Samples collected during 15.00 hours, contained 170 times fewer mf7nl than those collected at 01.00 hours when microfilaraemia were generally most intense. Therefore for diagnosis of bancroftian filariasis in this area, blood samples should collected between 22.00 and 3.00 hours, when microfilarial counts will be at least 90% the peak counts. This study confirmed other studies that have been done with regards to periodic patterns of W. bancrofti (Dreyer et al, 1996; Simonsen etal., 1997). Not much work has been done on animals in terms of periodicity but there have been a few reported studies on dogs, cattle and ungulates in general. Dirofilaria immitis is probably the best known filarial in the laboratory throughout the world. As such it has been used widely for the study of microfilaria periodicity in animals. It produces microfilariae that circulate in the peripheral bloodstream as well as the blood of all other parts of canine body. There is a tendency towards microfilarial periodicity in a day 19 University of Ghana http://ugspace.ug.edu.gh (circadian rhythm) as well as seasonal periodicity showing a summit in summer throughout a year (Oishi, 1959). This appears to vary in different countries. Thus Tarplee and Bradley (1982) found maximal numbers at midnight in the USA; Euzeby and Laine (1951) found the lowest numbers at 08:00 hr and the greatest at 20:00 hr in France; Webber and Hawking (1955) found minimum parasitaemia at 06:00 hr and maximum at 18:00 hr in a Chineese strain of D. immitis in England. Moreover, several investigators in Japan have referred to the periodicity of D. immitis as being lowgrade nocturnal (Masuya, 1976), and a distinct nocturnal: maximal numbers were found at 24 hr and minimal numbers at 10:00 hr and the number of maximum was 6.5 times of minimal count from 28 dogs naturally infected with D. immitis (Oishi 1959). On the contrary, Angus (1981) reported that there are both a distinct diurnal (16:00 hr) and lowgrade nocturnal (from 24:00 to 01: 00 hr) peal® in the periodicity of D. immitis microfiariae in cephalic venous blood of dogs in South Queensland. There were diurnal periodicity- maximum microfilarial counts of D. immitis were found at 11:00 hr and minimal at 22:00 hr in a dog from Tanzania (Matola, 1991). Moreover the microfilaraemia in a dog infected with D. immitis was diumally subperiodic with maximum microfilariae numbers between 12:00 and 16:00 hr (Grieve and Lamia, 1983) and Schnelle and Young (1944) observed minimum microfilaraemia at 11:00 hr and maximum at 16:30 hr in the USA A recent study by Rhee et al. (1998) in Korea determined the periodicity of D. immitis at two-hour intervals for 72 consecutive hours in 10 naturally infected war dogs aged 3-9 years. This study was done to facilitate harvesting of the microfilariae for possible use as an animal model and to elucidate 20 University of Ghana http://ugspace.ug.edu.gh further periodicity of the microfilaria depending on geographic location. Although the periodicity had been observed as being low-grade nocturnal, maximal microfilaria, counts were found at 21:00 h and minimal at 11:00 hr, giving rise to an evident peak in fluctuation of the larval counts. Studies by Nelson et al. (1962) revealed that Setaria species just like Mansonella species are normally aperiodic and as such the microfilariae circulate in the peripheral blood throughout a 24-hr period without significant changes in their numbers. University of Ghana http://ugspace.ug.edu.gh 2.3 The Vectors of Human Filariasis A wide range of mosquitoes acts as vectors for filariasis. Depending on geographic region, human filariasis can be transmitted by mosquito species belonging to the genera Culex, Aedes, Anopheles, Mansonia, Psorophora and Coquillettidia. Culex quinquefasciatus, which is the most important species among the Culex pipiens complex, is the principal vector of Bancroftian filariasis in areas where Wuchereria bancrofti has nocturnal periodicity, but in other places, other species of mosquitoes may serve as insect host of the parasite (White 1989, WHO 1992). In Brazil, Cousey et al. (1945), Rachou et al. (1956) incriminated species of Aedes and Anopheles as hosts of secondary importance. In Maceio, capital state of Alagoas, northerwest Brazil, Deane etal. (1953) and Fontes et al. (1994) established that Cx. spp is the most important insect host of W. bancrofti. The Cx. pipiens complex consists of several named species including; Cx. pipiens s.s., Cx quinquefasciatus, Cx. austratralicus and Cx. globocoxitus (Savage & Miller, 1995). These species differ mainly in their behaviour, biology and morphologically, in minor details. The shape of the male genitalia, shape of larval siphon and number of the first siphonal hair are mostly used as distinguishing features. Within the Cx. pipiens complex, Cx quinquefasciatus is the most important vector species of Wuchereria bancrofti in Asia and parts of both urban and semi-urban East Africa (White, 1971, Subramanian et al., 1997) but it is considered an unimportant LF vector in West Africa (Jayasekera et ah, 1980, Zielke and Chlebowsky, 1980, Dzozomenyo etal., 1999, Appawu etal., 2001). 22 University of Ghana http://ugspace.ug.edu.gh Aedes egypti is frequently found in human habitations in Maceio and it was considered necessary to determine if this species plays a role in the transmission of W. bancrofti. Aedes polynesiensis and Aedes samoanus are the most important vectors in the pacific area. The former breeds in crab holes and tree holes making it a very difficult mosquito to control with conventional methods. They are day-biting mosquitoes thus accounting for the diurnal periodicity of filariasis in this region. Aedes poecilius, a night-biting mosquito is a major vector in the Philippines. Mansonia species are an important vector of B. malayi, and sometimes W. bancrofti, in areas where there are extensive areas of aquatic plants. Anopheles species are important vectors of W. bancrofti in parts of Africa and Southern Asia and in Papua New Guinea. The Anopheles gambiae complex is considered as the most important LF vector in Ghana (Dzozomenyo etal., 1999, Appawu etal, 2001). An. gambiae si is a group of six morphologically indistinguishable yet genetically and behaviorally distinct mosquito species that vary dramatically in their importance as vectors of various diseases including LF in Africa (Coluzzi et al, 1979). Anopheles, gambiae sensu stricto Giles, An. arabiensis Patton, and An. quadriannulatus, are freshwater species; saltwater species are An. merus, and An. melas, An. bwambae, is the only mineral water species (White, 1985). The species are morphologically indistinguishable. Anopheles funestus Giles is the second most important LF vector in Ghana (Dunyo et al, 1996; Dzodzomenyo et al., 1999; Appawu et al, 2001). Anopheles, funestus is also a member of a species complex comprising at least nine members, the adults of which are 23 University of Ghana http://ugspace.ug.edu.gh not easily distinguished on the basis of morphological characteristics (Gillies & De Meillon 1968; Gillies & Coetzee, 1987) although some species may be distinguished using larval characteristics. The members of this complex are An. funestus s.s., An. vaneedeni Gillies and Coetzee, An. parensis Gillies, An. arum Sobti, An. conjusus Evans and Leeson, An. rivulomm Leeson, An. juscivenosus Leeson, An. leesoni Evans, and An. brucei Service. Traditionally, the only other method for distinguishing members of the An.Junestus group has been by chromosomal inversion karyotypes (Green, 1982). More recently, however, single-strand conformation polymorphism (SSCP) analysis has been used to identify four member of this group (Koekemoer et al., 1999). Of the nine species in the complex, An. funestus s.s. has the widest distribution. This mosquito is also highly anthropophilic (Gillies and De Meillon, 1968). 2.3.1 Identification of Culexpipiens complex Adults of the Cx. pipiens complex are light brown mosquitoes that lack distinctive markings on the proboscis and legs, and are not rapidly separated from other Culex (Culex) mosquitoes. Adult females of the complex are usually identified by the presence of distinctive, basal, pale abdominal bands. Abdominal bands are broadly rounded medially and distinctly constricted sublaterally before joining large, lateral scale patches. Male adults of Cx. pipiens and Cx. quinquefasciatus, and with less precision Cx. pipiens- quinquefasciatus hybrids, can be identified by use of the DV/D ratio of the genitalia (Savage and Miller, 1995). Larvae of the Cx. pipiens complex can be identified by the presence of a moderately long siphon that has 6-13 pecten teeth locate on the basal 1/3, and 4-branched siphonal tufts, one of which is laterally and out-of-alignment with the 24 University of Ghana http://ugspace.ug.edu.gh other three; and double-branched lateral setae on abdominal segments IE-IV (Savage & Miller, 1995). The shape of the siphon and number of branches on setae I on abdominal segment 3-4 can be used to characterised Cx. pipeins and Cx. quinquefasciatus. However, no reliable means of specific larval identification is available in areas where hybrids may occur. Recent molecular studies have led to the development of species specific polymerase chain reaction (PCR) primers that can be used in a PCR “cocktail” to identify Cx. salinarius, Cx. restuans, and the Cx. pipiens complex (Crabtree et al., 1995). Unfortunately, variation between Cx. pipiens s.s. and Cx. quinquefasciatus was insufficient to develop diagnostic primers for these taxa. Later, Crabtree et al. (1997) used genomic subtractive hybridization to identify a region of nucleic acid heterology between the genomes of the two latter species. 2.3.2 Identification of the An. gambiae and An.funestus complex After the discovery of the existence of the sibling species in An. gambiae group in 1956 by crossing experiments (White, 1974), several methods have been exploited to try and develop a consistent and easy to use identification method. At first, hybridisation experiments were the only possibility, but naturally this is far too laborious and moreover inconvenient because live mosquitoes are needed. The morphology of the species was studied, but this can only distinguish between some of the species and even then there is a lot of overlap (White, 1974). Gillies and De Meillon, (1968) and Gillies and Coetzee, (1987) provides established identification techniques, by using morphological characters, 25 University of Ghana http://ugspace.ug.edu.gh for the anopheline mosquitoes. An. gambiae adult females are identified by their smooth palps with 3 pale bands on the 3rd 4th and 5* segments; the wing field is pale with yellowish or creamy markings and has pale fairly long costal spots. The femora, tibia and 1st tarsal segment speckled to a variable degree. The abdomen is pale brown and hairy with scales on the 8th tergite and scales on the cerci. Adult female Anopheles funestus are identified by the observation of three pale bands on the 2nd, 3rd and 5th segments of the palps. The dark wings also bears characteristic pale scales, with the costa having four pale spots usually shorter than the intervening dark areas. The abdomen is dark brown and lack scales including the cerci, while the legs are usually dark with a small apical white spot on the tibia. Identification by cytotaxonomy was and perhaps still the most reliable method to use. Species are identified on the basis of polytene chromosome banding patterns. The X chromosome differentiates between An. gambiae, An. arabiensis and An. quadricmnulatus; the two autosomal chromosomes more subtly distinguish the other species (White, 1974). The use of isoenzymes of the enzyme octanol dehydrogenase for species identification (Miles, 1978, 1979) has the advantage that it is not stage or sex-specific. It can however only be applied to freshly caught or frozen specimens. Moreover, there are no single or sets of isoenzymes that are uniquely associated with the different species of complex; there is always some overlap (Collins etal., 1988). Other investigative techniques that 26 University of Ghana http://ugspace.ug.edu.gh were brought to practice at a large scale include the use of cuticular hydrocarbons (Carlson & Service, 1980) and of sex chromosome heterochromatin (Gatti et al., 1982). In 1987 two DNA probe-based identification methods were independently developed. A major advantage of these techniques is that they can be applied to frozen, dried, alcohol and isopropanol preserved specimens, because of the stability of DNA molecules. There have been several improvements on these techniques (Hill et al., 1991a, 1991b). The latest identification method that was developed makes use of a technique called the polymerase chain reaction (PCR). PCR involves the amplification of unique sequences from a mixture of sequences via the use of two primers (Saiki et al, 1985, 1988). Each primer anneals to a complementary piece of DNS, which triggers the binding of a DNA polymerase that then copies the segment. By this procedure, it is possible to synthesize many copies of a chosen piece of DNA in the presence of vast excesses of other DNA sequences (Kocher et ah, 1989). The PCR-based assay also differentiates between all complex members except An. bwambae (Paskewitz and Collins, 1990; Collins et ah, 1990; Scott e/ ah, 1993). 27 University of Ghana http://ugspace.ug.edu.gh 2.4 Trasmission Dynamics in Filariasis Several scientists have extensively reviewed the host-parasite relationship and the transmission dynamics of filariasis. Two modelling frameworks lymfasim (Plaisier etal., 1998) and Epifil (Chan et al., 1998) are based on the life history of the parasite, its transmission, the unmuno-regulatory role of the host immune system, the development of disease and the impact of control measures. In general, the mosquito infection rate, that is the number of mosquitoes that contain microfilariae after a blood meal and the number of microfilariae ingested per mosquito, increases with increasing parasitaemia. Although there is no multiplication in the vector, the number of infective larvae is around six times greater than that expected by microfilariae density (Brito et al., 1998). Bockarie et al. (1997) has shown that the annual infective biting rate and the annual transmission potential show a positive correlation with microfilariae rate, microfilariae density and prevalence of leg oedema suggesting that transmission intensity is a major determinant of patent infection and morbidity. Kazura et al. (1997) studied the relationship between annual transmission potential (ATP) and disease status in 1666 individuals from five similar but distinct W. bancrojti-inkcted communities from a highly endemic area of Papua New Guinea. Annual transmission potentials and microfilariae prevalence (MF) in these villages were: 2,344 (MF 94%); 1,338 (MF 82%); 279 (MF 56%); 179 (MF 66%) and 31 (MF 52%) respectively. In all villages the prevalence of leg odema was highly positively related to the ATP (correlation coefficient r=0.89 and probability p=0.04). The incidence of acute 28 University of Ghana http://ugspace.ug.edu.gh filarial attacks is also related to the transmission intensity. Gyapong et al. (1996) found that the incidence of acute filarial attacks were reduced in the dry season when the transmission potential was at its lowest. The concept of “facilitation” - that the proportion of microfilariae that develop increases as the number of microfilariae ingested increases (WHO, 1992) is an important consideration when the possibility of eradication of filariasis is being considered. In theory, it should be possible to reach a point where there are insufficient circulating microfilariae in the population to support transmission (WHO, 1992). It is important to note however, that persons with microfilariae densities as low as 3/ ml can still infect mosquitoes and that residual low-density microfilaraemics after mass treatment programs have the potential to cause rapid resurgence of filariasis (Lowrie etal, 1989; Southgate, 1992). Beckett (1973) found that a heavy uptake of microfilariae from the blood can cause life-threatening damage to the internal organs of the mosquito during larval development but this finding is not supported by the recent work of Brito et al. (1998). The feeding of mosquitoes on individuals with medium or low microfilaria] densities may therefore enhance transmission potential especially if the main vector is a culicine rather than an anopheline (Webber, 1981). The reasons for this difference, termed “limitation” is discussed by Bryan et al. (1990), Webber and Southgate (1981); Southgate and Bryan (1992) who suggest, that in contrast to culicines, anopheline mosquitoes have a well developed pharyngeal armature which damages microfilariae when they are ingested. If the number of ingested microfilariae is low there may be insufficient viable microfilariae 29 University of Ghana http://ugspace.ug.edu.gh to infect the mosquito. Loss of microfilariae also occurs in fluid expelled from the anus of anopheline but not culicines (Bryan & Southgate, 1988a,b). The concepts of facilitation and limitation and their role in filariasis transmission has been critically reviewed by Wada et al. (1995) who concluded that “there was no clear evidence to support the existence of facilitation and limitation-based unstable equilibrium in relation to microfilariae prevalence and density below which filariasis would spontaneously disappear even when the vector was Anopheles. Instead, the existence of a critical level of man/mosquito contacts for the disappearance of filariasis was suggested”. The microfilariae density in animals varies considerably from one place to another. Unfortunately much work has not been done on animals to determine prevalence of filarial infections in Ghana. However, prevalence of Setaria species among cattle raised in Korea ranges from 5 to 70% (Moon and Kang, 2000). Work done at the coastal part of Kenya revealed that out of the 200 cows examined; 7 had microfilariae in blood films. 5 of these infections were with Setaria labiato-papillosa (,Setaria cervi); the microfilariae were similar also to S. equina. Although in the survey only 5 cows were found with microfilariae in blood films, adult S. labiato-papillosa were commonly seen in the peritoneal cavities of cattle slaughtered at the abattoir in Mombassa. Microfilarial densities have always been very low in the cattle examined in Kenya. This has made feeding experiments with mosquitoes very difficult. This problem has been overcome by implanting living adult Setaria in the peritoneal cavities of monkeys, using the technique 30 University of Ghana http://ugspace.ug.edu.gh described by Williams (1955). Adequate microfilarial densities have been maintained for several weeks and complete development of S. labiato-papiHosa have been seen in Aedes aegypti fed on the infected monkeys. 2.4.1 Factors that influence microfilaria prevalence and density A spontaneous decrease in microfilaraemia prevalence can occur in the absence of vector control or mass chemotherapy. In a study in Benin the prevalence of microfilaraemia decreased from 9.4% to 0.48% over a 10-year period. The prevalence of people with chronic pathology remained the same. The changes could not be explained by environmental or sociological changes in the region or even by changes in the demographics of the study population (Myung etal., 1998). Microfilariae density is also subject to considerable daily variation but this is unrelated to lunar phase lactation, or the menstrual cycle (Nathan et al., 1982). Fasting during the month of Ramadan has been shown to reverse nocturnal periodicity (Nathan etal., 1982). The fecund life span of W. bancrofti has been calculated by various means and methods and has been variously estimated from 5 to 15 years. A recent study by Vanamail et al. (1996) suggests that the life span is at the lower end of previous estimates - around five years. 31 University of Ghana http://ugspace.ug.edu.gh 2.5 The Global Burden of Lymphatic Filariasis Current global estimates suggest that around 80 countries are endemic for lymphatic filariasis. Of the three parasites causing LF, Wuchereria bancrofti accounts for over 90% of the global burden. Brugia malayi is limited in distribution to Asia, and Brugia timori to a few islands in Indonesia. It has been estimated that 1.1 billion people living in areas endemic for this disease are exposed to the risk of infection, and that there are about 120 million cases with either disease or infection (microfilaria carriers). Almost half (49.2%) of the 120 million estimated cases are in the (WHO, 2001) South-East Asia Region (India alone accounts for about 40% of the global cases) and another 34.1% of cases are in the African region; the rest are in the western Pacific (16.1%), eastern Mediterranean (0.3%) and Americas (0.3%). The 120 million cases of LF include 83.63 million cases of microfilaria carriers, 16.02 million cases of lymphoedema, and 26.79 million cases of hydrocele; this clearly shows that the burden of genital manifestations in filariasis in terms of hydrocele is greater than that due to lymphoedema. Of the 120 million LF cases in the world, although Asia accounts for the majorily of infection (because of the large populationsliving in endemic areas), the prevalence of W. bancrofti is higher in Africa (8.97%) than in Asia (2.25%). However, this is expected to be an underestimate as the proportion of endemic population who are amicrofilaraemic but have evidence of infection in terms of a positive antigen test (as many as an additional 18%of endemic populations)was not included, so the actual number of infected or diseased people could be much higher. 32 University of Ghana http://ugspace.ug.edu.gh 2.6 Socio-economic Impact of Filariasis It is necessary to understand the social and economic impact of diseases in order to set priorities within the health system. This requires information on the types of symptom that are experienced; their prevalence, frequency and duration; and the nature of the associated social and economic costs. Much of this information is not available for lymphatic filariasis, and in its absence it is not surprising that the disease is not given higher priority in much of the endemic world (Evans et al, 1993) According to Gyapong etal. (1996a) a major hurdle for procuring funding for control and research has been the lack of information regarding the social and economic impact of the disease. The estimated socio-economic impact of filariasis varies widely from study to study and documentary evidence of loss of production and income is difficult to find. The current global estimate of the disability associated with lymphatic filariasis is 850,000 disability-adjusted life years lost (Gyapong et al, 1996). This is probably a serious under-estimate given the lack of recent prevalence information for many of the endemic countries. There is very strong evidence that late stage chronic disease with its accompanying disability reduces productive capacity as suggested by Evans et al. (1993), but the economic impact is reduced somewhat by the fact that most people with advanced chronic pathology are beyond their most productive years (Wijers, 1977). The impact on some communities can be severe. Gyapong et al. (1996) found that because of chronic filariasis 4.1% of the productive female labour force and 20% of the productive male labour force were disabled by between 10 and 60% and 20% people with advanced 33 University of Ghana http://ugspace.ug.edu.gh 2.7 Diagnosis of lymphatic filariasis Efficient diagnosis of W. bancrofti infection is especially important as control programmes move towards the new strategy of community treatment and repeated, annual mass therapy with single-dose DEC or combination regimens (Ottesen and Ramachandran, 1995). Several methods are available for diagnosis of lymphatic filariasis infections. These include; direct and concentrated techniques, Antigen detection (ELISA) microfilaria membrane filtration, DNA detection (PCR), antibody serology, and, ICT card test (WHO, 1994). 2.7.1 Direct detection of microfilariae In areas where microfilariae exhibit nocturnal periodicity blood should be taken within two hours either side of midnight when the highest density of microfilariae is expected to occur. Where they show diurnal periodicity blood should be taken two hours either side of midday. Simonsen et al. (1997) have devised a method to adjust for the effect of sampling time on microfilariae density. Using their technique it is possible to predict what the microfilariae density would be at midnight in blood collected at probably, 2200 hours. The simplest technique for direct diagnosis of microfilaraemia is a Giemsa-stained thick blood film of capillary blood collected by finger-prick (Schultz, 1988). If a measured amount of blood is used (such as 40- 60 (101, the number of microfilariae per ml can be 35 University of Ghana http://ugspace.ug.edu.gh calculated (Moulia-Pelat et al, 1992). A modification (Southgate 1974), which allows easier counting of microfilariae, is to expel 60jj,l of blood onto the slide so it forms three even linear strips. The dried films are de-haemoglobinised in buffered water before staining with Giemsa stain. The disadvantage of thick films, like other direct methods, is that they underestimate the prevalence of microfilaraemia if microfilariae densities are low because theoretical detection limit for such procedures is between 15 and 50 microfilariae per ml (Panicker et al., 1991). Even if 60pl of blood is used subjects with less than 60 microfilariae per ml will not be reliably detected. Another problem is loss of microfilariae from the film during processing especially if anticoagulated blood is used. Southgate (1973) observed a loss of up to 51% and Denham et al (1971) a loss of between 10% and 40%. Loss of microfilariae is minimised if un-anticoagulated blood is used and the films are dried over night at room temperature (Partono and Idris, 1977). Youssef et al (1995) have shown that applying a thin film of agar to the thick film before staining greatly reduces the loss of microfilariae. The thick film does lack sensitivity when microfilariae density is low but is still a useful and cheap technique for survey work where other more sensitive techniques are too expensive (Moulia-Pelat etal., 1992) or when it is difficult for cultural or other reasons to obtain venous blood. It has also been suggested that more microfilariae are present in capillary blood than venous blood and that the use of capillary blood may be an advantage when microfilariae densities are low (Eberhard et al, 1988). There is no question, however, that direct 36 University of Ghana http://ugspace.ug.edu.gh techniques often fail to identify patients with low parasite densities and concentration techniques should be used wherever possible (Weller et al., 1982). Acridine orange staining and fluorescence microscopy has been used as an alternative to Giemsa staining and various combinations of stains can be used to demonstrate the internal structure of microfilariae (Lowrence and Simpson, 1969). Counting chamber techniques using various diluents have been used for counting microfilariae by several investigators (Denham, 1979; Southgate, 1973) and, although not as sensitive as concentration techniques, are a reasonable compromise if 0.1ml of blood is used (McMahon et al., 1979). 2.7.2 Concentration methods for microfilariae The most widely used is the method developed by Knott (1935). For this method one ml of blood is added to 9ml of a 1% formalin solution in normal saline. After red cell lysis is complete the mixture is centrifuged and the deposit examined for microfilariae. Because the formalin preserves the microfilariae, Knott's tests can be set up in the field and processed later in the laboratory. The theoretical detection limit is one microfilaria per ml. The accuracy of the Knott's test and its ease of use is compromised when blood is processed from individuals with excessive amounts of plasma gamma globulins, a common finding in tropical populations. The formalin precipitates the protein and makes the examination of the deposit very difficult. The method has been improved by Melrose et al. (2000) who added a small Triton X-100 to the diluent, which dissolved most of the proteinaceous deposit thus enhancing the visibility of the microfilariae. If a citrate- 37 University of Ghana http://ugspace.ug.edu.gh microfilariae and other blood parasites in diluted, saponin-lysed blood and it is claimed to be 7.5 times more sensitive than a thick film (Petithory et al, 1997). 2.7.3 The Diethylcarbamazine (DEC) Provocation Test If 1.5 to 2 mg of DEC per kg of body weight is given during the daytime, microfilariae are "provoked" into leaving the lungs and entering the peripheral circulation where they can be detected by any of the techniques above (WHO, 1987). The use of this test should be discouraged as it has a lower sensitivity than night blood collection and mns the risk of also provoking a severe reaction (see below) especially in areas where W. bancrofti occurs with O. volvulus or L. loa (WHO, 1987). The other antifilarial drugs ivermectin and albendazole do not induce microfilariae to enter the blood during the day (Dunyo et 2.7.4 Detection of filarial antigen t Filarial antigenaemia is associated with active filarial infection and several assays for filarial antigen using both polyclonal and monoclonal antibodies raised against various antigens have been developed (Harinath, 1986; Lai et al., 1987; Weil and Liftis, 1987; Weil et al, 1987; Zheng et al, 1987; Cheirmaraj et al, 1990). The first commercial assay Trop Bio Og4C3 Antigen Test (Trop Bio Pty Ltd, Townsville, Australia) is based on the assay developed by More and Copeman (1990; 1991). The monoclonal antibody is raised against Onchocerca gibsoni antigen and shows very strong specificity for W. bancrofti antigen. Since it detects antigen from both adult worms and microfilariae the al, 1999). -ST /P 39 University of Ghana http://ugspace.ug.edu.gh Og4C3 assay will detect amicrofilaraemic and microfilaraemic infections (More and Copeman, 1990) and is a very good marker of active filarial infection with adult worms (Chanteau et al., 1994a,b). Unlike microfilaraemia, antigen levels show no significant nocturnal or diurnal variation and blood can be taken at any time of day or night (Moulia- Pelat etal., 1993). Blood samples taken onto filter paper strips can be used for the Og4C3 assay and Lalitha et al. (1998) and Itoh et al. (1998) found that the capillary blood collected on filter paper and serum gave comparable results. By contrast, Gyapong et al. (1998) found that the sensitivity of the filter paper test to be significantly inferior with only 50.3% positive with both tests. More and Copeman (1990) showed no cross-reaction in the Og4C3 test with Brugia species or other helminths but there is a recent report by Rocha et al. (1996) of a person from a "non endemic" filarial area who tested positive with the Og4C3 test. This individual was found to be parasitized by Hymenolepsis nana. Whether this represents a true cross-reaction or not is debatable since the same individual also showed a positive immunoblot with crude B. malayi antigen. The reported diagnostic sensitivity of the Og4C3 assay for W. bancrofti varies from 73% (Chanteau et al., 1994a) to 100% (Lammie et al., 1994). The variation in sensitivity might be partially explained by the variation in the amount of blood used for the detection of microfilariae, which varied from 20(j.l to 1ml. A more recent study by Rocha et al. (1996) suggests that the sensitivity of the Og4C3 assay may be reduced when microfilariae density is very low. At microfilarial densities of <1, 1 to 30, and >30 the sensitivity was 72.2, 97.6 and 100% 40 University of Ghana http://ugspace.ug.edu.gh respectively and the log OD of the assay was strongly associated with microfilariae density. The ICT antigen test (ICT Diagnostics, Sydney, Australia) is rapid immuno- chromatographic technique using specific monoclonal and polyclonal antibodies. It utilises capillary or venous blood and is simple enough for field use by technicians and requires a minimum training (Weil etal., 1997). The efficacy of filarial antigen tests has been reviewed by Nguyen etal. (1999) and Phantana etal. (1999). Phantana etal. (1999) also compared the ICT to thick blood films and membrane filtration and obtained the following results; Sensitivity 100%, specificity 96.3%, predictive value positive 70.7% and predictive value negative 100%. Pani et al. (2000) however, found it less sensitive than the filtration test for detecting low-level microfilaraemia citing an 88.3% positive predictive value when compared with the latter test. 2.7.5 Filarial antibody assay Lack of readily obtainable adult worms has made it very difficult to prepare W. bancrofii antigen for immuno-diagnosis but the recent introduction of ultrasonic detect (Amaral et al., 1994; Suresh et al., 1997) should make the task easier. The only animals that can be infected with W. bancrofii are the leaf monkeys Presbytis cristata (Palmieri etal., 1982; Rajasekariah et al., 1986) and P. melalophos (Sucharit et al., 1982) and these animals are expensive and difficult to maintain in captivity (Tranke et al., 1987). Wuchereria bancrofii microfilariae can be maintained in culture or isolated from blood (El Bassiouny et al., 1993) and used to prepare antigen for antibody studies. The viability of separated 41 University of Ghana http://ugspace.ug.edu.gh or cultured microfilariae can be ascertained by means of a tetrazolium formazan assay (Mukheijee era/., 1997). Fortunately, there is marked cross reactivity between filaroid species (Tandon et al., 1981) and wide range of other crude filarial parasites antigens have been utilised for filarial antibody detection: Dirofilaria immitis (Ata et al, 1993); Setaria cervi (Almeida etal, 1990); Setaria digitata (Dissanayake and Ismail, 1980); B. malayi (Ottesen etal, 1985). There is a down side to this cross reactivity - antifilarial antibodies cannot be used to distinguish between filaroid species (Rajasekariah etal., 1986). Various methods can be used to detect filarial antibody: Complement fixation, indirect haemagglutination, gel diffusion, immunoelectrophoresis, counter current immunoelectrophoresis, indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA) have all be used (Ambroise-Thomas, 1980). Currently, almost all filarial antibody studies use ELISA. Cross reactivity limits the usefulness of IgG antibody in filariasis diagnosis but they may be of some value in communities where parasites other than W. bancrofti are absent or rare (Chanteau et al, 1991) and have been used to diagnose occult filariasis in Indian children (Chaturvedi et al, 1995). Humans are not able to synthesize anti-phosphocholine or anti-carbohydrate antigen IgG4 (Maizels et al, 1987; Lai et al., 1991) therefore the filarial IgG4 antibodies assay greatly increases specificity and enhances the diagnostic ability of the test (Lai and Ottesen, 1988). Chanteau et al (1995); Terhell et al. (1996) and Mahanty et al. (1994) have shown that antifilarial IgG4 is a good index of the intensity and duration of filarial exposure in 42 University of Ghana http://ugspace.ug.edu.gh endemic populations, and (Maizels et al., 1995) found that the level of IgG4 antibody correlates with microfilariae counts. Filter paper collection techniques can be used to detect filarial IgG4 antibody (Chanteau etal., 1991; Terhill etal., 1996). IgG4 antibodies are extremely useful for the detection of Brugia species because they will not be detected by the current antigen tests since they are specific for W. bancrofti (Rahmah et al., 1998). There appears to be very little literature on the usefulness of anti-filarial IgM antibody for filariasis diagnosis but Ata et al. (1993) found it to be more specific than IgG. It should be borne in mind that sero-positivity does not always indicate active filarial infection. The antibody may have been raised by exposure to infective larvae without an adult worm being present or the antibody may persist after parasites have been cleared and although the sensitivity of antibody tests is high, the specificity and hence the predicative value, is low (Chanteau et al., 1991). 2.7.6 PCR assays for filarial detection in vectors Molecular detection of filarial DNA in humans or mosquitoes can best be done with polymerase chain reaction (PCR) assays to detect specific DNA sequences. A repeat sequence (188 bp) designated Sspl, identified from a W. bancrofti genomic library was found to characterize Wuchereria (Zhong et al., 1996) as distinct from Brugia and other filariae. A PCR assay developed to amplify this Sspl family of repeated DNA elements using specific primers (NV-1 and NV-2). This assay was shown to be sensitive enough to detect 0.1 picogram of W. bancrofti genomic DNA, representing less than 1% of one infective larval DNA within its mosquito host (Chanteau etal, 1994). 43 University of Ghana http://ugspace.ug.edu.gh Recently, several research groups in African countries started using this sensitive PCR mosquito-pool screening approach in preference to the classical method of dissecting mosquitoes to determine their filarial infection and transmission potential. Also in India, scientist at VCRC have devised simpler, quicker and less expensive PCR assay procedure for screening mosquitoes to detect W. bancrofii (VCRC unpublished data; Ottesen etal., 1997). The PCR approach to detect W. bancrofii can be used for diagnosis of infection in the human host and more importantly, detection of filarial DNA in the mosquito vector (Farid et al., 2001). A reliable, sensitive, specific and faster immunochromatographic (ICT) card format for rapid diagnosis of W. bancrofii active infection in available (Weil etal., 1997), but is more costly ($1.50/test). Thus, compa tests or mosquito dissection, the PCR technique is economical, can be applied to humans and has more practical value for xenomonitoring by detection of filarial DNA in mass screening of mosquito vectors. The PCR method has advantage of detecting a single worm in a pool of wild-caught mosquitoes (Ramzy et al., 1997), being cost effective for field application. Thus, screening of pools for wild caught mosquitoes by PCR, a non- invasive means, could be used for identifying endemic regions, but would be particularly useful for monitoring transmission in areas where mosquito infection rates are very low, especially during active control programmes. In applying this assay to field collected samples, experiences of the DNA diagnostics laboratory of the Onchocerciasis Control Programme (OCP) in West Africa may prove to humans is also a 93 red to ICT card \ 44 University of Ghana http://ugspace.ug.edu.gh be a helpful model in overcoming this obstacle. For the past nine years, the OCP’s laboratory has been using a PCR assay based on the amplification of a 150bp repeat sequence family to monitor infection in the Simulium damnosum complex vectors of African onchocerciasis. Initially the OCP laboratory’s efforts were primarily directed toward identifying individual parasites dissected by the OCP’s field teams, but the laboratory has been successfully applying a pool screening approach to monitor infection in the vector population (Laurent et al, 1999). Similarly, the Onchocerciasis Elimination Programme of the Americas (OEPA) has been applying this technology to monitor transmission since 1999 (Toe et al, 1999). PCR assays are not quantitative therefore it is impossible to determine if a positive pool contains one or more than one infected mosquito. However, it is possible to state that pools that produce negative results do not contain any infected mosquitoes. This observation has been used to develop a method to calculate the prevalence of infection in the vector population, based upon the simple computer program called Poolscreen (Katholi et al, 1995). In addition to the development of practical ways to employ the PCR detection techniques in specific field situations, additional research is needed to develop and apply similar PCR assays specific for B. malayi and B. timori (without cross­ reactions to other enzootic Brugia species) for xenomonitoring prevalence via vector. Also, for measuring transmission potential and monitoring the impact of transmission control, specific PCR assays that detect only L3 infective larvae would be useful. 45 University of Ghana http://ugspace.ug.edu.gh 2.8 Control of lymphatic filariasis Rapid population growth and inadequate sanitation in urban and rural areas have resulted in enhancement of transmission of W. bancrofti and expansion of its distribution. On the other hand, due to the long-term control efforts by some countries, there is evidence of reduction in the prevalence of the disease. The control strategy based on vector control and treatment of microfilaria carriers, practiced in the region till recently had a limited effect since it lacked the necessary political commitment and financial support. Recently there have been significant developments in regards to filariasis control based primarily on mass chemotherapy supplement by case management and vector control. These measures constitute the substance of the revised strategy for control of lymphatic filariasis (Rafei, 1999-2000). 2.8.1 Vector control A variety of means for controlling the vectors of filariasis are available today. These include Bacillus sphaericus, a toxin-producing bacterium: polystyrene beads, which help to limit the breeding of mosquitoes in certain situations; insecticide-impregnated bednets and curtains, which limit host/vector contact; indoor spraying of long-lasting pyrethroid insecticide, especially effective for adult Mansonia and Culex; and community participation in integrated vector management. All these methods help to decrease vector numbers and transmission, but exactly how and when the tools are cost-effective and useful in large scale-scale control programmes for lymphatic filariasis have not yet been clearly defined (Webber, 1991). 46 University of Ghana http://ugspace.ug.edu.gh Vector control have a part to play and can be very successful in situations where malaria and filariasis have common vectors. In the Solomon Islands for instance, where both malaria and filariasis are transmitted by Anopheles farauti and Anopheles koliensis, the prevalence of microfilaraemia was reduced from around 15% to <2% by vector control earned out during a malaria campaign (Webber, 1977, 1991). Vector control however does take a long time to become effective. For instance, Schuurkamp et al. (1987) estimated that it would take 11 years to reduce the prevalence of microfilaraemia to <2% in the Tabubil area of the Western Province of Papua New Guinea using vector control alone. Such prolonged campaigns are very labour intensive and costly and the development of insecticide resistance makes them become less and less effective. Data collected during a 5-year vector control program in Pondicherry, India, found that vector control alone might have little impact on the overall age-prevalence of infection even when sustained for long periods (Srividya etal., 1996). Toilet pits and the like are prime breeding grounds for Culex quinquefasiattus (Nwoke et al., 1993) and the use of polystyrene beads can be a very effective control mechanism for this and other Culex species. Using this approach Maxwell et al. (1989) reduced the numbers of mosquitoes entering houses by about 97%. Maxwell et al. (1999) found that although the treatment of excess pools and pit toilets with polystyrene beads could reduce mosquito populations in homes, the cost of achieving this could not be justified by its impact on filariasis alone, but the reduction in nuisance biting might increase the community support for other control programs. 47 University of Ghana http://ugspace.ug.edu.gh 2.8.1.2 Insecticide treated/untreated bed-nets The impact of bednets on the transmission of the parasites causing lymphatic filariasis has received less attention than their impact on the transmission of malaria parasites. Burkot et al. (1990) showed that use of untreated bednets reduced transmission of W. bancrofii by Anopheles punctulatus in Papua New Guinea by 70%. Although the nets had no apparent effect on the size of the vector population, the human blood index was reduced from 96% to 90% for indoor resting mosquitoes, and from 84% to 46% for outdoor resting, while the proportion of blood meals taken in dogs increased. Recently, Bockarie et al. (2002) demonstrated that long-term use of untreated bednets significantly reduced the prevalence of both human infections with W. bancrofii and symptomatic bancroftian filariasis, in an area of Papuan New Guinea where An. farauti was the vector. The prevalence of microfilaraemia, antigenaemia and hydrocoele among the bednet users were 27% and 70% lower, respectively, than those observed in the individual who were not using bednets. The probable impact of insecticide-treated materials on lymphatic filariasis has mainly to be inferred, indirectly from knowledge on their effects on the various mosquito species that act as vectors of the filarial. It is known that most human filariasis is caused by W. bancrofii transmitted by Anopheles spp and Culex quinquefasciatus (White, 1989; WHO, 1994). Insecticide-treated material generally has a substantial killing effect on anopheline mosquitoes as documented in the studies on malaria. In contrast, Curtis et al. (1996) demonstrated that pyretheroid-impregnated bednets kill very few Cx. quinquefasciatus, although they do reduce this species’ success in feeding (to a greater extent than 48 University of Ghana http://ugspace.ug.edu.gh impregnated eave and wall curtains). Repellency could be part of the explanation for the low mortality of Cx. quinquefasciatus, but Curtis et al. (1996) noted that: ‘Presumably this mainly associated with the general tolerance of Cx. quinquefasciatus to tarsal contact with insecticides. It is possible that Culex are more readily irritated than Anopheles and inclined to leave the pyrethroid-treated surfaces. Bogh et al. (1998) studied the impact of pyrethrum-impregnated bednets on the resting and feeding behaviour of filarial vectors on the Kenya coast. The introduction of the impregnated bednets reduced the numbers of indoor-resting An. gambiae si and An. fimestus by 95.6% and 98.1% respectively but as expected caused no significant change in the number of Cx. quinquefasciatus collected indoors. Despite the feet that pyrethrim- impregnated bdnets do not kill Cx. quinquefasciatus, these results indicate that the impregnated bednets are likely to reduce W. bancrofti transmission by An. gambiae, An. fimestus Mid Cx. quinquefasciatus efficiently. In a more recent investigation Qinones et al. (2000) also detected changes in mosquito feeding behaviour a consequence of the introduction of pyrethrim-impregnated bednets. The effect of pyrethrum-impregnated bednets on the transmission of W. bancrofti was investigated directly for the first time by Pedersen and Mukoko (2000). In the study it was observed that the overall mosquito density in the village with pyrethrin-impregnated bednets, reduced by 22.6% in the post- intervention year. The transmission of W. bancrofti was drastically reduced in the studied villages. 49 University of Ghana http://ugspace.ug.edu.gh 2.8.2 Chemotherapy There are essentially two regimes used to control filarial infections using chemotherapy: selective and mass treatment. In selective therapy individuals are examined for the presence of disease and those found to be infected are treated. There are major problems associated with that approach. If microfilaraemia is used as the indicator of infection many infected people will be overlooked since not all those who have the disease are microfilaraemic. People with very low microfilarial densities may not be detected and it has been shown that such people are capable of infecting mosquitoes and causing a resurgence of disease (Lowrie et al., 1989). This approach has however worked in many countries including Japan. The use of antigen testing will overcome these problems but there is a problem with the logistics involved with screening all members of the community. If selective chemotherapy is used the lost infections are balanced by new infections to produce a dynamic equilibrium and there needs to be continual reassessment of the filarial status of the community to identify the newly infected. Woolhouse etal. (1997) by studying the transmission potential of a number of diseases, including helminth parasites, found that, typically, 20% of the infected host population contributes at least 80% of the net transmission potential and that control programs which fail to cover all of this core group, a problem with selective treatment programs, will be less effective in reducing levels of infection in the group as a whole. Using mass chemotherapy j-egardless of parasite status therefore overcome problems associated with selective therapy. 50 University of Ghana http://ugspace.ug.edu.gh Mass treatment aims to treat all members of the endemic community at the same time and will therefore treat pre-patent as well as patent infections. In other words the community becomes the focus of the control program rather than the individual. Originally antimony and arsenic based drugs and naphthalene sulfonic acid (Suramin) were used to treat filariasis but with limited success. These compounds had a moderate amount of anti- filarial activity and were very toxic. Suramin has been shown to be moderately active against adult W bancrofii and Brugia phahangi (Howells et al., 1983) but it has no effect on microfilariae and will therefore not interrupt the transmission cycle in the short term. It is also more toxic than other antifilarial drugs and is not widely used today. Levamisole has been shown to have limited activity against filarial parasites (Rogers and Denham 1976; McMahon 1979). 2.8.2.1 Mass drug treatment with DEC Diethylcarbamazine (DEC) has been available for treatment of filarial disease for almost 50 years and is till used today. It destroys larval worms and even some of the adult worms but has drawbacks if used in heavy infections when the destruction of large numbers of worms may cause side effects. Current practice is a single annual dose of 300 mg of DEC for adults and 150 mg for children (often combined with Ivermectin, and sometimes Albendazole). This simplifies the treatment, increases compliance and can be easily accommodated into existing primary health care networks without over-burdening them (Wijers, 1984). Low dose DEC has been shown to significantly reduce the prevalence and density of microfilariae in treated communities and reduce the prevalence of chronic pathology (Partono etal., 1984, 1989). Panicker etal. (1991) found that there 51 University of Ghana http://ugspace.ug.edu.gh was a reduction in microfilariae prevalence of 74.9% in the annual treatments and 90% in the biannual treatments. The authors also found that attacks of filarial fever and incidence of recent oedema cases were also significantly reduced after DEC treatment. A trial in Tanzania achieved microfilariae clearance rates of 92% (Meyrowitsch et al., 1996). Meyrowitsch and Simonsen (1998) have shown that the beneficial effects of DEC treatment persist for at least 4 years and although microfilaraemia does recur in some patients, especially those who had high microfilaria densities before treatment, the level of microfilaraemia does not reach the pretreatment levels. Khan et al. (1998) found that 5 years after a dose of 72 mg/kg of DEC given over a 21 day period, 51% of subjects were stil amicrofilaraemic, 36.4% had densities less than pre-treatment levels and 11.8% had increased microfilariae counts. In Samoa, 3 annual treatments of DEC 6 mg/kg, achieved an estimated 80% reduction of microfilaria in the population and lowered the annual transmission potential from 2.18 to 0.67 (Kimura et al., 1992). Mataiki et al. (1998) obtained similar results with a five-year annual treatment program in Fiji. 2.8.2.2 Use of Ivermectin Ivermectin has been used very successfully for the treatment of onchocerciasis for a number of years (Goa et al., 1991; Townson etal., 1994) and a single annual dose of 400 Hg/kg, either alone or in combination with Diethylcarbamazine, has proved to be very effective producing long-term suppression of microfilaraemia in Bancroftian lymphatic filariasis in a number of countries (Zheng et al., 1991a,b; Cartel et al., 1992) and is equally effective against brugian filariasis (Shenoy et al., 1993). Nquyen et al. (1996) found that twice yearly doses of 100 fig/kg of Ivermectin did not reduce the prevalence of 52 University of Ghana http://ugspace.ug.edu.gh microfilaraemia, but when the dose was increased to 400 ng/kg the prevalence dropped from 21% to 7% and the microfilariae density to 0.5% of its initial value. Plaisier et al. (1999) found that Ivermectin is very effective at reducing the microfilariae load and at a does of 400 jig/kg a single treatment irreversibly reduces the microfilariae of the adult parasite by at least 65%. Zheng etal. (1991) found that Ivermectin was especially useful in the treatment of filarial relapses after DEC treatment Like DEC, Ivermectin treatment does give rise to adverse side effects. Cao etal. (1997) describes them as mild and “flu like”, generally mild, and well tolerated by patients. Mild reactions were also noted by Zheng etal. (1991) who also found that local flare-up of acute filariasis was less likely with Ivermectin than with DEC. The severity of the reaction was strongly associated with the pre-treatment microfilariae density but independent of the dose (Cao et al., 1997). Weil et al. (1991) stated that local adverse reactions such as nodule formation, lymphangitis and epididymitis are not seen with Ivermectin therapy because these reactions are caused by the death of adult filarial worms and Ivermectin is not a macrofilaricide. As with DEC, the macrofilariacidal action of Ivermectin is debatable. Ismail etal. (1996) found that multiple, high dose Ivermectin treatment (12 fortnightly doses of 400 jig/kg) does have macrofilaricidal activity but the results are neither predictable nor consistent. Eberhard et al. (1997) reported that although filarial antigen fell after treatment, in no case did it fall to zero, even in individuals who remained amicrofilaraemic for several years after treatment suggesting that some adult worms survived. By contrast however, Dreyer et al. (1995b; 1996) could find no evidence of macrofilariacidal activity. In their 53 University of Ghana http://ugspace.ug.edu.gh study 15 men who had living, adult W. bancrofti detected by ultrasound were treated with 400-|ig/kg-body weight of Ivermectin at 2-week intervals for 6 months (total dose 4.8 mg/kg). Microfilaraemia was rapidly suppressed but no changes in the motility or location of the adult worm were detected. In another study Dreyer et al. (1995b) removed live adult worms from several patients 8 months after being treated with 400 Hg/kg of Ivermectin. 2.8.23 Albendazole Albendazole has been used for the treatment of intestinal helminths for a number of years but it has only recently been tried as an anti-filarial. Addiss et al. (1997) have shown that at a stated dose of 400 mg per person it is an effective microfilaricide. It is even more effective when combined with DEC or Ivermectin. The problem of adverse reactions with Albendazole is no better or worse than with DEC or Ivermectin. The potential for Albendazole to be used as a macrofilaricide for the treatment of individual patients is regarded by Ottesen et al. (1999) as one of the most important questions in filarial research. 2.8.2.4 Combination therapy with DEC and Ivermectin A combination of DEC and Ivermectin has proven to be very effective in providing rapid and long-term clearance of microfilariae. This is the current global strategy for controlling the disease. Nicolas et al. (1997) found DEC and Ivermectin to be more effective than either drug alone for clearing circulating filarial antigen in both amicrofilaraemic and microfilaraemic subjects. Addiss et al. (1997) also showed that a 54 University of Ghana http://ugspace.ug.edu.gh combination of 200-400 pg/kg of Ivermectin plus 400 mg of albendazole is more effective in clearing microfilariae than either drug alone. Ismail et al. (1998) studied the effects of Albendazole, DEC, and Ivermectin alone and in combination and showed that although all were well tolerated and effective macrofilaricide, a single dose of a combination of 600 mg of Albendazole and 400 (ig/kg of Ivermectin was the most effective. Decreasing levels of filarial antigens after treatment suggested that all four regimes have significant macrofilaricidal activity. The most effective, however, was a combination of 600 mg of Albendazole with 6 mg/kg of DEC. With this combination, filarial antigen levels decreased by 77% fifteen months after therapy. 55 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.1 Study Sites The study was conducted in two districts that are known from a 1994 national survey to be endemic for filariasis and one non-endemic district (Gyapong et al, 1994). These are namely Winneba, Axim and Yilo Krobo districts all located in the southern sector of Ghana (Fig 3.1). Depending on the number of cattle Kraal that were found in the districts, villages were randomly selected at random within each one for cattle blood sampling and examination. Mosquitoes were also collected off the microfilaraemic cattle screened. Winneba is located at the southern sector of Ghana, about three kilometres off the main Accra-Cape Coast road. The vegetation is coastal savanna with stretches of mangrove swamps. This area receives the least amount of rain in Ghana (Dickson and Benneh, 1988). The annual rainfall is between 74 and 89 centimetres. Relative humidity is however, high throughout the year and thus compensates for the scanty annual rainfall. The inhabitants are mainly fishermen and farmers and grow crops like cassava, plantain and other vegetables. There are 6 kraals in the area and the number of cattle per site ranges from 40-150. Three kraals located at Muni lagoon, animal husbandly and Mpotah were chosen randomly for screening and collection of mosquitoes. 56 University of Ghana http://ugspace.ug.edu.gh Axim is also located in the south-western part of Ghana. This is the wettest climatic district in Ghana. Mean annual rainfall is above 190 centimetres and, and on the average there is no month that receives less than 2.5 centimetres of rain. The highest mean monthly temperature of about 30°C occurs between March and April and the lowest of about 26°C in August. Average monthly relative humidity ranges between 75-80% during the two rainy seasons. Six sampling sites namely Asaasetre, Teleko Bokazo, Nkroful Secondary School, Kanbule, Arokpogwe and Police Quarters with varying number of cattle were chosen because of scarcity of cattle in Axim. Yilo-krobo District of Ghana is located at the southern part of Somanya District. This site, which is non-endemic for filariasis, was included in the study because an incidental examination of catde blood revealed some filarial parasites that were morphologically similar to Wuchereria. Yilo-krobo district has a mean annual temperature of about 26oC, Rainfall in the area is seasonal with two peaks occurring in June and September. The area has wide stretches of savanna or grassland which cattle owners use as grazing fields. The vegetation type is that of savanna woodland. 57 University of Ghana http://ugspace.ug.edu.gh Fig 3.1: A map of Ghana showing the study sites, Yilo-Krobo, Winneba (Awutu Efutu District), Axim ( Nzema East) and Accra, the Capital City. 58 University of Ghana http://ugspace.ug.edu.gh These studies were undertaken to help identity and classify the filarial parasites that were found in the cattle blood. The cattle were screened using traditional staining techniques for filarial parasites. Microfilariaemic density was determined at regular intervals of time over a 24 hr period. Blood was also collected into heparinized tubes for molecular studies. Moiphometric measurements were taken and the values obtained were compared to those of W. bancrofti and Setaria species. 3.2.1 Screening of cattle for filarial infections Screening was carried out between the hours of 06-08 hrs in the morning and 1700-1900 hrs in the evening at ihe three study sites. The cattle were randomly selected after which they were tagged and information on age, sex and type of breed were recorded. The ear lobe of each cattle was cleaned with a cotton wool ball soaked in alcohol. It was then pricked with a lancet and the blood was allowed to ooze freely. The blood was then drawn into a sterile calibrated capillary tube and later transferred onto a microscope slide and smeared uniformly avoiding bubble formation. The slides were air-dried after which they were immersed in tap water until the haemoglobin leached out of the smear. This took about 3-5 minutes. The slides were placed horizontally and the smear allowed to dry at room temperature. After this the blood films were stained for 25 minutes in a 1:20 dilution of Giemsa stain of pH of 6.8-7.2. The films were then washed for 3-5 minutes under running tap water. They were then dried in a vertical position. The stained blood films were then observed under low and high power to detect the presence of microfilaria. 3.2 Parasitological Studies 59 University of Ghana http://ugspace.ug.edu.gh 3.2.1.1 Collection of cattle blood for molecular studies About 10ml of blood was obtained from the positive animals by bleeding from the jugular vein using 10ml syringes and needles. This was done on a four hourly interval for 24 hours at the various sites where infections were detected. The blood was stored in heparinized tubes. They were then kept cold in an icebox in the filed and transported to the laboratory where they were preserved at 4°C until ready to be used. lOfxl of blood was also collected using capillary tube into tubes containing 1ml of 3% acetic acids. 3.2.2 Determination of microfilaraemic density The counting chamber technique for determining microfilariae concentration (mf) in the blood was used. A 3% acetic acid was prepared and 990|il aliquoted into tubes. lOOjxl capillary tubes were then used to draw blood from the ear lope and then drained into the tubes containing the 3% acetic acid and thoroughly mixed. The content of each tube was transferred to a clean counting chamber and examined under the microscope at high power xlOO magnification. The microfilariae were counted and the density expressed as mf7100|xl of blood. This was repeated over a 24-hour period at 4 hour intervals to investigate the variations in blood microfilarae density. The total counts of microfilariae in each tube were used to calculate the geometric mean using formular given in appendix 60 University of Ghana http://ugspace.ug.edu.gh 3.2.3 Morphometric studies of cattle blood microfilariae The thick blood films prepared in the field were used for morphometricc studies. Microfilariae, at lOOx magnification were drawn onto A4 paper using camera lucida. One centimetre on a ruler was first traced on the paper. Then the outline of each microfilaria was drawn using a pencil. The total length, width, the length of the inner korper and headspace of each microfilaria were measured from the paper using a thread. In all about 40 sheathed microfilaria and 5 of the unsheathed were drawn. The values obtained which were in centimetres were converted into micrometers by dividing by 103 taking into account the magnification at which the drawings were made. Comparative statistical analyses were also carried out. 61 University of Ghana http://ugspace.ug.edu.gh 3.3 Entomological Studies Mosquitoes were collected as part of the study to determine the vectors of the cattle filarial parasites. Specially made mosquito nets were erected at the various sampling sites. They were sorted out in the laboratory and dissected for all stages of filarial parasites. 3.3.1 Mosquito surveys The cattle that were infected were isolated and confined in a mosquito net, which had an opening at one side to allow entry of mosquitoes (Fig 3.2). Resting mosquitoes on nets or those coming to take blood meal were then collected by aspiration between the hours of 00-04hr. Mosquitoes were also obtained from rooms of microfilaraemic individuals living close to the sampling site using pyrethrum spray catch. The mosquitoes were kept in paper cups and sent to the laboratory where they were sorted out according to species using the criteria of Grilles and de Meillon (1968). 3.3.2 Dissection of mosquitoes for filarial infections The legs and the wings of each female mosquito were first removed and then placed in a drop of normal saline on a slide. The head, thorax and the abdomen were separated under approximately 20X magnification, and transferred to separate drops of saline. Using fine needle, the labium was separated from the other parts of the proboscis. The infective filarial worms if present normally emerge from the labium into the saline. The remainder 62 University of Ghana http://ugspace.ug.edu.gh of the head, thorax and the abdomen were dissected separately at XI0 of the dissecting microscope and examined for infections with all filarial stages (LI, L2 and L3). Any filarial-like parasites were recorded and the positive slides as well as a few negatives were placed in racks and kept at - 4°C for the subsequent molecular studies. 63 University of Ghana http://ugspace.ug.edu.gh Fig 3.2: The frout (top) and rear (bottom) view of the mosquito net erected over cattle, with flap open for mosquito entry 64 University of Ghana http://ugspace.ug.edu.gh 3.4 Molecular Studies 3.4.1 Chemical and Reagents The various chemicals and reagents used were prepared as outlined in Appendix I 3.4.2 Molecular identification of filarial worms One technique that has been found to be highly sensitive and specific in identifying Wuchereria bancrofti using parasite DNA is the polymerase chain reaction (PCR) which has been developed to amplify a family of repeated DNA element, the 188bp SspJ repeat, specific for Wuchereria genus. The blood that was obtained from the jugular vein was washed thoroughly and used for DNA extraction followed by PCR using the appropriate primers. 3.4.2.1 Extraction of filarial DNA Aliquots of 100-200^1 of blood were transferred into 1.5ml eppendorf tubes. Approximately 1ml sterile distilled water was added, vortexed and centrifuged at 14,000 rpm for 10 minutes and the supernatant decanted. This washing process was repeated several times until the haem component of the blood was completely washed off. The worms (which are pelleted) were then stored in iso-propanol at -20°C until ready to be used for DNA extraction. The parasite’s genomic DNA was extracted using the DNeasy Tissue Kit (QIAGEN Inc., USA). The modified protocol for extraction of DNA from animal tissue was followed (see Appendix II) for details. 65 University of Ghana http://ugspace.ug.edu.gh 3A.2.2 PCR identification of cattle filariae The PCR method using the previously published oligonucleotide primer sequences NV-1 (5 ’ -CGT GATGGC ATC AAAGT AGCG-3 ’) and NV-2 (5’- CCCTCACTTACCATAAGACAAC-3’) for identification of W. bancrofti was used (Ramzy et al., 1997). The composition of the PCR reaction mix is shown in Table 3.1 of appendix VII. The contents of each tube was mixed thoroughly, centrifuged briefly and overlaid with 25jli1 of mineral oil to minimize evaporation and refluxing. A positive control containing known W. bancrofti DNA and a negative control without DNA were also included for each reaction. The PCR cycling conditions used were an initial denaturation at 94oC for 3 minutes, followed by 35 cycles of denaturation at 94oC for 1 minute, annealing at 55oC for 1 minute and extension at 72oC for 2 minutes and a final cycle of 94oC for 1 minute, annealing at 55oC for 1 minute and extension at 72oC for 10 minutes. 3.4.3 Molecular identification of Anopheles gambiae s.l The Anopheles gambiae were first morphologically identified. The genomic DNA from carcass of dissected An. gambiae s.l. mosquitoes was used as template for the PCR method for the identification of the member species (Scott et al., 1993). 3.4.3.1 DNA extraction of mosquitoes using Bender buffer protocol Genomic DNA was extracted from Anopheles mosquitoes using bender buffer (0.1 M NaCl, 0.2M Sucrose, 0.1M Tris-HCl, 0.05M EDTA pH 8.0 and 0.5% SDS) extraction protocol proposed by Collins et al. (1987). Each mosquito was triturated in a 100(0.1 66 University of Ghana http://ugspace.ug.edu.gh Bender buffer in a 1.5ml eppendorf tube and incubated at 650C for 30 minutes. 125|j,l of phenol was added to homogenate. The mixture was then vortexed, and spun at 14,000 rpm for 10 minutes. The supernatant was transferred into a fresh tube and 250^1 of equal volume of phenol/chloroform was then added, vortexed and spun at 14,000rpm for another 10 minutes. This process was repeated depending on how impure the mixture is. The supernatant was then transferred into a new tube and 250^.1 of pre-chilled absolute ethanol and 1 Ojal of 8M-potassium acetate was added to the mixture and kept at or at - 40oC for just one hour or overnight. The mixture was spun at 10,000rpm for 10 minutes after which the supernatant was poured off. The pellet was then rinsed with 200nl of 70% ethanol, spun at 100,000rpm for 5 minutes and the supernatant was poured off. The pellet was allowed to dry for about an hour and redissolved in 25|il of TE RNase and kept on ice for 1 hour. Two microlitres was used for PCR. 3.4.3.2 Species identification of Anopheles gambiae si using PCR PCR method for the identification of mosquitoes of the An. gambiae complex published by Scott et al. (1993) was used. The sequence details of the named oligonucleotide primers are as follows; UN (GTG TGC CCC TTC CTC GAT GT) GA (CTG GTT TGG TCG GCA CGT TT), ME (TGA CCA ACC CAC TCC CTT GA), AR (CTG GTT TGG TCG GCA CGT TT) and QD (CAG ACC AAG ATG GTT AGT AT). The UN anneals to the same position on the rDNA sequences of all five species and GA anneals specifically to An. gambiae s.s., ME to both An. merus and melas, AR to An. arabiensis, and QD to An. quadriannulalus. The expected sizes of the PCR products are 390, 315, 153 and 464/466 respectively. The amplification was carried out using a PTC 100 thermal cycler 67 University of Ghana http://ugspace.ug.edu.gh (MJ Research Inc., USA) using the cycling conditions: 94oC for 2 minutes, 30 cycles of (94oC for 30 s, 50oC for 30 s, 72oC for 30 s). A typical mix for a 25 pi reaction is given in Table 3.2 of Appendix VI. 3.4.4 PCR identification of Anopheles funestus group This reaction was performed using protocol developed by Koekemoer etal. (2002) which is a species-specific PCR assay able to rapidly identify five of the most commonly found members of the An. funestus group: An. funestus, An. rivulorum, An. leesoni, An. parensis andAn. vaneedeni. Two sets of primers, 3DA and 3DB were used and their sequences are GAC CCG TCT TGA AAC ACG GA and TCG GAA GGA ACC AGC TAC TA respectively. The following PCR cycling conditions were used, initial denaturation at 94oC for 3minutes, 30 cycles of (94oC at 30s, 400C for 30s, 72s), and 1 cycle of 94oC for 30s, 40oC for 30s and 72oC for lmin. Details of a 25 pi reactions mix for the reaction are found in Table 3 .3 of Appendix VI. 3.4.5 Analysis of PCR product (Agarose gel electrophoresis) Electrophoresis buffer (lxTAE) was prepared, (see appendix I). Agarose was first weighed into beaker and dissolved in specific volume of the buffer already prepared making a 2% gel. The slurry was heated in a microwave oven until the agarose was completely dissolved. The solution was cooled under tap water and ethidium bromide (0.5|ig/ml) was added and poured into the gel tray with the well-forming comb. This was allowed to cool and set. The comb was carefully removed and the gel was transferred and 68 University of Ghana http://ugspace.ug.edu.gh positioned in the chamber with just enough electrophoresis buffer solution to submerge it. The PCR products, mixed with orange G loading buffer were carefully loaded A DNA molecular weight ladder (Sigma, USA) was run along side to enable the estimation of the size of the PCR products. The gel was run at a voltage of 100-120V until the orange G tracking dye had migrated to give distance. 3.4.6 Estimation of size of PCR product The gels were visualized under UV using a UV transilluminator (UPA, USA), at short wavelength and photographed using a Polaroid model IBI 46400 (Polaroid Inc., USA) fitted with an orange filter and a Polaroid type 667 film. The size of the PCR products was estimated by comparison with the mobility of marker of known DNA molecular sizes. The expected length for W bancrofti is 188bp. The expected lengths of Anopheles gambiae and Anopheles funestus are 390bp and 400bp respectively. 69 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1 Study Population A total of 284 cattle 33 from Somanya, 141 from Winneba and 110 from Axim were studied. The mean ages of cattle were 3.8 years (range 2-25 years) for Somanya, 5.6 years (ranges 3 months-9 years) for Winneba and 6.3 years (range= 2-25 years) for Axim. The age distribution is shown in Table 4.1 and it shows that most (63%) of the cattle at Winneba were between 6-10 years, 59.4% were between 2-5 years at Axim and were about equal of the age groups of the cattle screened at Somanya. Females formed 79.4% of tile cattle from Winneba, 84.7% at Axim and 75 .8% at Somanya. West African Short Horn formed majority of the breed types (66% for Winneba; 87.4% for Axim and 100% for Somanya). Details of demographic characteristics of the cattle are given in Appendix V 4.2 Microfilaraemic density and observed frequencies The prevalence rates of filarial infections were 3.5% (5/141) and 6% (2/33) at Winneba and Somanya respectively, and zero for 110 cattle blood screened at Axim. The cattle found to be positive were of the West African Short Horn breed type and were above 10 years of age. The geometric mean densities among infected cattle were found to vary 70 University of Ghana http://ugspace.ug.edu.gh between 4.6-20 microfilariae/100(il of blood (Table 4.2 of appendix VI). Maximal microfilarial densities of 26 microfilariae/100jj.1 of blood at 2:00 pm and minimal of 6 microfilariae/1 00p.l of blood at 2:00 am on the average were recorded. The curve is suggestive of a diurnal subperiodic. This feature distinguishes the cattle filariae from W. bancrofti which exhibits a nocturnal form of periodicity. Table 4.1: The sex and age distributions of cattle populations at the three study sites Site • # Sex Age Categories #F 10y Winneba 141 29 112 7 45 89 0 (20.6%) (79.4%) (4.9%) (32%) (63.1%) Somanya 33 8 25 10 12 11 0 (24.2%) (75.8%) (30.3%) (36.3%) (33.3%) Axim 110 16 94 0 66 34 10 (14.4%) (84.7%) (59.4%) (30.6%) (9.9%) Total 284 53 231 17 123 134 10 71 University of Ghana http://ugspace.ug.edu.gh M f i nt en sit ie s ( m f/l O O ul ) Time (hrs) Figure 4.1: The microfilaria densities of the five cattle studied at Winneba 72 University of Ghana http://ugspace.ug.edu.gh 4.3 Morphometric/Morphological Description of Cattle Filariae 4.3.1 Sheathed microfilaria A total of five sheathed microfilariae were studied. Their sheaths stained pink in Giemsa (Fig 4.3). The length and width measured ranged between 152-182fim (mean=162 ± 12.2) and 6-7jun (mean=7.4 ± 0.9) respectively. The headspace measured between 6- 8[m (mean=6.8 ± 0.8) whilst the inner korper was between 25-38p,m (mean=32.4 ± 5.3). The nucleus is large and located at the end of the column. The column of the nucleus is very compact and it starts out multiple and also stains deeply in Giemsa. The tail is anucleated and tapered to a round tip. The inner body is very conspicuous and stains pink in Giemsa. 4.3.2 Unsheathed microfilaria A total of 40 unsheathed microfilariae were studied. These were morphologically similar to the sheathed ones except that they lacked sheath (Fig 4.2). The morphometric measurements such as width, headspace and inner korper were quite similar to those of the sheathed except for the length, which was found to be between 128-200p,m (mean=164 ± 15.6). The column of the nucleus is also compact just like the sheathed microfilariae and start out multiple and stain intensely in Giemsa. There is also a large nucleus at the end of the column. The tail also tapers to a round tip and is anucleated. The inner body stained with Giemsa and was veiy conspicuous. 73 University of Ghana http://ugspace.ug.edu.gh 4.3.3 Comparison of cattle filariae with Wuchereria banerofti The two cattle filarial worms were compared with published characteristics of W. banerofti (WHO, 1997). There were similarities as well as differences in their morphology. Differences were very evident in their lengths, which is much longer in W. banerofti, (244-296pm) than the cattle filarial (128-200pm). The sheath of W. banerofti does not stain in Giemsa at all but that of the cattle filariae stained pink in Giemsa. The nucleus of the cattle filarial is very compact and stained intensely in Giemsa whilst that of W bancrofii is dispersed and does not stain in Giemsa. The presence of a prominent inner koiper in the cattle filariae however distinguishes it from W. bancrofii, which does not possess an inner koiper. Similarities were found in features such as the presence of sheath, a short headspace and length of width. Also the tails in both filariae are anucleated tapering to a rounded posterior end. 4.3.4 Comparison with Setaria species The cattle filarial worms were compared to the general description published for Setaria worms (Nelson et al., 1962; Omri et al., 1978). Morphometries of the cattle microfilaria especially of the length of the microfilaria (128-200pm) falls within the range of Setaria species (122-365[xm). The width of the cattle filarial however was slightly wider (4-8(xm) than Setaria species (4-6jxm), but the difference was not significant. There was also striking similarity in terms of features such as sheath and inner body, which stained intensely in both cases as well as the tapering of the tail. 74 University of Ghana http://ugspace.ug.edu.gh Fig 4.2: Unsheathed microfilaria (mf) observed in cattle blood after staining with Giemsa (X1000). A = Head Space, B = Compact nuclei, C = Inner korper D= tail end. 75 University of Ghana http://ugspace.ug.edu.gh Fig 4.3: Sheathed microfilaria (mf) from cattle blood after staining with Giemsa (XlOOO). A = Head space, B=Compact nuclei, C = Inner korper and D = Sheath 76 University of Ghana http://ugspace.ug.edu.gh 4.4 Mosquito Surveys A total number of 690 mosquitoes were collected off cattle at Winneba and Culex species was the most abundant species forming 88.7% (612/690). Mansonia species was 6.2% (43/690), Anopheles species was 4.9% (29/690) and the least was Aedes, which was 0.15% (1/690) [Fig 4.3], Two infective stages of filarial worms (L3) were found and both occurred in Culex mosquitoes thus an infective rate of 0.3% (2/612). There were some 4 microfilariae (mfs) in some of the Culex mosquitoes dissected. In total there were 3 Lis (first stage of the filarial parasite), 1 L2s (second stage) and 2 L3s (third/infective stages) thus giving infection rate in the mosquitoes as 0.9%. There were 42 mosquitoes caught by pyrethrum spray catch and these were mostly of the Anopheles species comprising of 7 Anopheles gambiae and 35 Anopheles funestus. None of the Anopheles mosquitoes was found to have the infective stage of the W. bancrofti. However there were 2L2’s and 1 microfilaria in one of the Anopheles gambiae (Fig 4.8). 77 University of Ghana http://ugspace.ug.edu.gh 88.7% Fig 4.4: Proportion of various species of mosquitoes collected off cattle at Winneba (n=690) 78 University of Ghana http://ugspace.ug.edu.gh 4.5 Molecular Identification of Cattle Filariae and Anopheles Mosquitoes A total of 30 catttle filariae from blood were amplified using W. bancrofti primers. None of these parasites was positive by PCR. Two infective stages (Fig 4.8) and the other stages together with ihe microfilariae that were were found in the dissected mosquitoes were also amplified by the set of primers. Of the Anopheles species there were 7 Anopheles funestus (Fig 4.5) confirmed and 3 Anopheles gambiae (Fig 4.6) and the rest were negative by PCR. None of the parasites was positive by PCR (Fig 4.7). 79 University of Ghana http://ugspace.ug.edu.gh M 1 2 3 4 5 6 7 Fig 4.5: Agarose gel electrophoregram (2%) of the amplified Anopheles funestus s.s. DNA fragment of band size 400bp observed under UV light after staining with ethidiom bromide. Lane 1= lOObp DNA molecular marker, lanes 1-7 — Anopheles funestus (Mosquitoes collected off cattle). 80 University of Ghana http://ugspace.ug.edu.gh M 1 2 3 4 Fig 4.6: Agarose gel electrophoregram (2%) of the amplified Anopheles gambiae s. s. DNA fragment of band size 390bp observed under UV light after staining with ethidium bromide. (Mosquitoes collected of cattle). Lane l=100bp marker, lane 2 = negative control and Lanes 3-5 = Anopheles gambiae s.s. 81 University of Ghana http://ugspace.ug.edu.gh M 1 2 3 4 5 6 7 Fig 4.7: Agarose gel electrophoregram (2%) of PCR products amplified rDNA products in Wuchereria bancrofti microfilariae, cattie filarial parasites and mosquitoes. The reactions were performed using oligonucleotide primers NV-1 and NV-2 designed to amplify a family of the DNA elements, the 188 base pair (bp) Sspl repeat, specific for the genus Wuchereria. Lane M = 100 base pair marker, lane 1 = Positive control, lane 2 = Negative control and lane 3-7 = Filarial worm from catde 82 University of Ghana http://ugspace.ug.edu.gh Fig 4.8: Infective stage (L3) of the filarial worm found in Culex mosquito (xlO). A= Head space, B= Inner korper and C= Compact nuclei. 83 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSIONS AND CONCLUSION The ultimate aim of the present study was to use morphological and molecular techniques to characterize a filarial parasite reported in cattle that shared some morphological similarities with the human filarial parasite, Wuchereria bancrofti. Morphometries of the cattle filarial worms made it possible to classify it under the genus Setaria. Comparison of the cattle filarial worm to W. bancrofti using published identification keys (WHO, 1997) revealed some differences as well as similarities. One distinctive feature about the cattle filarial worm is the presence of a very prominent inner korper in 70% of the worms identified. This feature is not very distinct in W. bancrofti. Also the compact large nuclei distinguished it from Wuchereria which has dispersed nuclei. The differences in morphometric measurements of the sheathed and unsheathed microfilariae were found not to be significant which suggest that they probably belong to the same Genus if not the same species. Even though Setaria species are sheathed, only 5 of the cattle filariae were sheathed. This could have been due to loses during slide preservation and preparation since some lose sheath were found on slides. The results supports what has been reported earlier by Nelson etal. (1962) that Setaria spp from cattle, sheep, the ox, deer and horses, stain intensely with Giemsa stain same as W. bancrofti and B. malayi. The similarity is evident in features such as the short headspace, tail that is devoid of nucleus and lengths. It is therefore not out of place to University of Ghana http://ugspace.ug.edu.gh find that there was no amplification using microfilariae and L3 DNA since the primers were specifically designed for W. bancrofti DNA sequences. The PCR results confirm that the cattle filarial worms were not W. bancrofti. The periodicity of a given species or geographic variant is especially useful in determining the best time of day to collect blood samples for examination. Several studies have confirmed the nocturnal periodicity of W. bancrofti worldwide (Sasa, 1976; WHO, 1992; Dreyer et al, 1996; Simonsen et al., 1997). Normally, in Africa, where night-biting mosquitoes transmit nocturnal forms of the parasite, the highest intensities in bancroftian filariasis, the mf periodicity in the peripheral blood occur at night and few or none during the day. In several foci of Southeast Asia and tie Pacific but not in Africa, W. bancrofti and B. malayi are known to be subperiodic meaning that the microfilariae are detectable in the peripheral blood at any time. So far studies have established Setaria species to be aperiodic (Nelson et al 1962; Omri et al, 1978). The present study however does not support these findings which seem to indicate subperiodicity. However more studies are needed before any firm conclusion can be drawn, because of the small sample size and limited period of this study. About half of the world’s burden of lymphatic filariasis is transmitted by Cx. quinquefasciatus in India. This and other man-biting forms of Culexpipiens complex are responsible for most or all of the bancroftian filariasis transmission in Asia countries, Indonesia, Egypt, urban East Africa and the Americas. Globally, the majority of W. bancrofti is transmitted by Cx. quinquefasciatus. Anopheles mosquitoes are known to 85 University of Ghana http://ugspace.ug.edu.gh transmit lymphatic filariasis in rural areas of tropical Africa and the Papua sub-regions. In Ghana, members of the An. gambiae complex and An. funestus are the known vectors of the disease (Gyapong et al., 1994; Dzozomenyo et al., 1999; Appawu et al., 2001). From the present study Culex species was found to be the most dominant species (89%), with an infective rate of 0.3%. This species has however been found to be unimportant in the transmission of lymphatic filariasis in Ghana. It is therefore difficult to understand why the same mosquito species is a possible vector of cattle filariae and not of W. banerofti. The An. gambiae and An. junestus caught off cattle did not harbour any of the infective stages of the parasites and most importantly formed the minority. This implies that there could not be cross infections from human to cattle since different vectors are responsible for transmission. It can be concluded that the cattle filarial cannot be Wuchereria banerofti. This is based on the morphological, molecular and periodicity studies conducted. The two forms of the cattle filariae were found to be similar morphologioally and the absence of sheath could have resulted from loss during processing. Features of the cattle filarial agree with general features of Setaria species. Culex species has been implicated by the present study as the vector of the cattle filariae. In Ghana it is known to be refractory to W. banerofti. These findings suggest that entomological parameters can still be used for monitoring LF intervention programmes without any problem most especially in our study area. However further studies need to be conducted especially in other LF endemic areas for confirmation. 86 University of Ghana http://ugspace.ug.edu.gh REFERENCES ADDISS, D. G., BEACH, M. J., STREIT, T. G., LUTWICK, S., LECONTE, F., LAFONTANT, J. G, HIGHTOWER A W, LAMMIE, P. J. (1997) Randomized placebo-controlled comparison of ivermectin and albendazole alone and in combination for Wuchereria bancrofti microfilaraemia in Haitian children. Lancet 350 (9083): 1036. AIKAT, T. K, & DAS, M. (1977). A modified statistical method for analysis of periodicity of microfilaria. 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(1991). Can Anopheline transmitted filariasis be eliminated? J. Trop. Med. Hyg. 94 (4): 241-4. WEBBER, W. A. F. & HAWKING, F. (1955). Experimental maintenance of Dirofilaria repens and D.immitis in dogs. Exp. Parasitol. 4: 143-164. WEIL, G. J. & LIFTIS, F. (1987). Identification and partial characterization of a parasite antigen in sera from human infected with Wuchereria bancrofti. J. Immunol. 138:3035-3041. 117 University of Ghana http://ugspace.ug.edu.gh WEIL, G. J., JAIN, D. C., SANTHANAM, S., MALHORTRA, A., KUMAR, H„ SETHUMADHAVAN, K. V. P., LUFTIS F. & GHOSH, T. K. (1991). A monoclonal antibody-based enzyme immunoassay for detecting parasite antigenemia in bancroftian filariasis. J. Infect. Dis. 156: 350-355. WEIL, G. J., LAMMIE, P. J. & WEISS, N. (1997). The ICT Filariasis Test: a rapid format antigen test for diagnosis of bancroftian filariasis. Parasitology Today 13: 401-404. WELCH, J. S. & DOBSON, C. (1974). 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Lymphatic filariasis, In Geographical Distribution of Arthropod- borne Diseases and their Principal Vectors, World Health Organization, Vector Biology and Control Division, WHO/VBC/89.967. pp. 23-34. WIJERS, D. J. (1977). Bancroftian filariasis in Kenya. Prevalence study among adult males in the Coast Province. Ann. Trop. Med. Hyg. Parasitol. 71 (3): 313-31. WILLIAMS, H. E. (1955). Studies on the bovine filarial Setaria cervi (Rudolphi, 1819). Parasitology 45: 1-2, 56-62. WOOLHOUSE, M. E., FERGUSON, N. M., DONNELLY, C. A. & ANDERSEN, R. M. (1997). The epidemiology of BSE in cattle herds in Great Britain II. Model construction and analysis of transmission dynamics. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352 (1355): 803-38. WORLD HEALTH ORGANIZATION (1987). Control of Lymphatic filariasis. A manual for health personnels. WHO, Geneva, pp 89 WORLD HEALTH ORGANIZATION (1992). Lymphatic ffilariasis: the disease and its control. Fifth report of the WHO Expert Committee on Filariasis. Geneva: World Health Organization, Technical Report Series No. 821. WORLD HEALTH ORGANIZATION (1994). Lymphatic filariasis infections and disease: control strategies. Report of a consultative meeting held at the University Sains Malaysia, Penang, Malaysia. Geneva: World Health Organization, mimeographed document TDR/CTD/Fil/PENANG/94.1. WORLD HEALTH ORGANIZATION (1995). WHO Tech. Rep. Ser. 852: 1-104 WORLD HEALTH ORGANIZATION (1997). Bench aids for the diagnosis of filarial imfections. Geneva. 119 University of Ghana http://ugspace.ug.edu.gh WORLD HEALTH ORGANIZATION (2000). Eliminate Filariasis: Attack Poverty. The Global Alliance to Eliminate Lymphatic Filariasis. Proceedings of the First Meeting, Santiago de Compostela, Spain. Document WHO/ CDS/ CEE/ 2000.5. 41pp YAMEOGO, L., TOE L., HOUGARD, J. M., BOATIN, B. A. & UNNASCH, T. R. (1999). Pool screening polymerase chain reaction for estimating the prevalence of Onchocerca volvulus infection in Simulium damnosum sensu lato: results of a field trial in an area subject to successful vector control. Am. J. Trop. Med. Hyg. 60:124-128. YOUSSEF, F. G., ANASSANEIN, S. H. & CUMMINGS, C. E. (1995). A modified staining method to detect Wuchereria bancrofti microfilariae in thick smear preparations. Ann. Trop. Med. Parasitol. 89 (1): 93-4. ZHENG, H. J. ET AL. (1987). Parasite antigens in sera and urine of patients with bancroftian and brugian filariasis detected by sandwich ELIZA with monoclonal antibodies. Am. J. trop. Med. Hyg 36: 684-688 ZHENG, H. J., PIESSWENS, W. F., TAO, Z. H., CHENG, W. F., WANG, S. H., CHENG, S. H., YE, Y. M., LUO, L. F., CHEN, X. R. & GAN, G. B. (1991). Efficacy of ivermectin for control of microfilaremia recurring after treatment with diethylcarbamazine. I. Clinical and parasitologic obcervations. Am. J. Trop. Med. Hyg. 45 (2): 168-74. ZHONG, M., MCCARTHY, J., BIERWART, L., LIZOTTE-W ANIEWS KI, M., CHANTEAU, S., NUTMAN, T. B., OTTESEN, E. & WILLIAMS, S. A. (1996). A polymerase chain reaction assay for detection of the parasite 120 University of Ghana http://ugspace.ug.edu.gh Wuchereria banerofti in human blood samples. Am.. J.Trop. Med.Hyg. 54: 357- 363. 121 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX I CHEMICALS AND REAGENTS Preparation of Giemsa solution Preparation of500ml of stock solution Reagents Needed: Giemsa Powder 3.8g Glycerol 250ml Methanol 250ml Procedure: The methanol was measured and added to a measured amount of Giemsa powder in a dry brown bottle. Glycerol was measured out and added to the stain preparation. The mixture was heated to 50°C for up to two hours, and mixed at intervals. Preparation of 2% Giemsa solution 49ml of buffered water, pH 7.1 was added to 1ml of Giemsa stain and mixed gently. Preparation of Electrophoresis buffer Electrophoresis buffer, Tris-acetate (TAE), concentrated stock solution; 242g of Tris base was dissolved in 27.1ml of glacial acetic acid and 100ml of0.5M ofEDTA (pH 8.0), and the volume was made up to 1000ml with distilled water. The 50x solution was dispersed into aliquots and stored at room temperature. 122 University of Ghana http://ugspace.ug.edu.gh Preparation of Ethidium bromide Ethidiim bromide, lOmg/ml: O.lg of ethidium bromide was added to 10ml of water and stirred on a magnetic stirrer for several hours to ensure that the dye has dissolved. The container was wrapped with an aluminium foil and stored at room temperature. Gloves were worn when working with the solution that contained this dye because it is carcinogenic. Preparation of Orange G 5x orange G: 20% (w/v) Ficol, 25mM EDTA, 2.5% (w/v) orange G: 2.5g of Ficol and 0.25g of orange G were dissolved in a few ml of sterile distilled water, then 500ul of 5M EDTA was added and the volume made up to 10ml with distilled water. • lOObp ladder molecular weight marker (Sigma-P1473): the marker contains 10 bands ranging from lOOObp in exact lOObp increments. This marker was prepared according to manufacturer recommendations. 123 University of Ghana http://ugspace.ug.edu.gh APPENDIX II DNeasy Protocol for DNA purification from animal tissues 1. Cut up to 25mg tissue (up to 1 Omg spleen) into small pieces, place in a 1. 5ml microcentrifuge tube, and add 180ul Buffer ATL. 2. Add 20ul proteinase k, mix by vortexing, and incubate at 55°C until the tissue is completely lysed. Vortex occasionally during incubation to disperse the sample, or place in a shaking water bath or on a rocking platform. Optional: RNase treatment of the sample. Add 4ul of RNase A (lOOmg/ml), mix by vortexing, and incubate fir 2minutes at room temperature. 3. Vortex for 1 Ssec. Add lOOul Buffer AL to the sample, mix thoroughly by vortexing, and incubate at 75°C for lOminutes. 4. Add 200ul ethanol (96-100%) to the sample, and mix thoroughly by vortexing. 5. Pipet the mixture from step 4 into the DNeasy mini column sitting in a 2-ml collection tube (provided). Centrifuge at 8000rpm for 1 minute. Discard flow­ through and collection tube. 6. Place the DNeasy mini column in a new 2-ml collection tube (provided), add 500p.l buffer AW1 and centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube. 7. Place the DNeasy mini column in a 2ml collection tube (provided), add 500ul buffer AW2 and centrifuge for 3 minutes at full speed to dry the DNeasy membrane. Discard flow through and collection tube. 124 University of Ghana http://ugspace.ug.edu.gh 8. Place the DNeasy mini column in a clean 1. 5ml or 2ml micro centrifuge tube (not provided), and pipette 200ul Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 minute, and then centrifuge for 1 minute at 8000rpm to elute. 9. Repeat elution as described in STEP 8. 125 University of Ghana http://ugspace.ug.edu.gh APPENDIX III MORPHOMETIC MEASUREMENTS & ANALYSIS UNSHEATHED MICROFILARIAL (MORPHOMETRICS) # Length (fim) Width (|um) Head space(jim) Inner cupa(jj.m) 1 167 8 8 26 2 157 7 7 35 3 167 7 8 35 4 146 8 6 22 5 164 8 7 27 6 151 6 7.5 30 7 144 6 8 33 8 128 7 7 32 9 159 6 6 33 10 144 6 6 35 11 159 7 7 33 12 158 7 7.5 31 126 University of Ghana http://ugspace.ug.edu.gh 29 29 32 28 32 28 27 35 25 25 30 25 22 23 24 30 22 25 39 151 5 8 141 6 6.5 156 6 8 152 7 7 149 6 6 155 5 7 178 8.7 8.5 172 8.7 8 168 8.7 6.8 168 8.4 7.5 196 7 8 149 8.7 7 172 7.4 6 173 8.7 8 169 7.4 6 166 8 8 155 8 7 180 8.7 8.5 173 8 7 127 University of Ghana http://ugspace.ug.edu.gh 32 200 7 8 36 33 185 8 7.5 37 34 160 8 8 38 35 190 7 7.5 33 36 183 8 7.3 28 37 170 8 6 35 38 188 8 7 33 39 168 8 6 34 40 160 6 5 32 Mean 164.275 7.31 7.1525 30.2 St Dev 15.62212025 1.05946285 0.85033553 4.686040371 128 University of Ghana http://ugspace.ug.edu.gh SHEATHED MICROFILARIAL (Morphometries) # Length (jim) Width (|im) Head space(jim) Inner cupa(jxm) 1 160 6 7 32 2 152 8 7 30 3 153 8 8 25 4 165 8 6 38 5 182 7 6 37 Mean 162.4 7.4 6.8 32.4 St Dev 12.17784874 0.89442719 0.836660027 5.319774431 129 University of Ghana http://ugspace.ug.edu.gh RESULTS OF MICROSCOPIC EXAMINATION AND DEMOGRAPHIC FEATURES (FIELD DATA) APPENDIX IV Screening At Somanya ID # Age Sex Breed Result 1 5m F WASH NEG 2 5m M WASH NEG 3 7m F WASH NEG 4 7m F WASH NEG 5 iy F WASH NEG 6 3y F WASH NEG 7 ly F WASH NEG 8 7m M WASH NEG 9 6m F WASH NEG 10 3m F WASH NEG 11 ly M WASH NEG 12 ly M WASH NEG 13 7y M WASH NEG 14 7y F WASH NEG 130 University of Ghana http://ugspace.ug.edu.gh 15 3m F WASH NEG 16 3m F WASH NEG 17 3y F WASH NEG 18 7m F WASH NEG 19 3y M WASH POS 20 2y F WASH NEG 21 3y M WASH NEG 22 3y M WASH NEG 23 10y F WASH NEG 24 lOy F WASH NEG 25 6y F WASH NEG 26 10Y F WASH NEG 27 lOy F WASH NEG 28 lOy F WASH NEG 29 lOy F WASH NEG 30 3y F WASH NEG 31 lOy F WASH NEG 32 32y F WASH NEG 33 vy F WASH POS 131 University of Ghana http://ugspace.ug.edu.gh Screening at Winneba (Muni lagoon ID# Age Sex Breed Result 139 6y M ZEBU NEG 165 5y F ZEBU NEG 101 6y F WASH NEG 105 5y F WASH NEG 129 4y F WASH NEG 136 6y F ZEBU NEG 159 7y F WASH NEG 102 8y F WASH NEG 122 7y F ZEBU NEG 147 6y F WASH NEG 112 8y F ZEBU NEG 164 7y M ZEBU NEG 107 3y F ZEBU NEG 135 6y F WASH NEG 154 8y F WASH NEG 103 8y F WASH NEG 145 8y M WASH NEG 178 iy F WASH NEG 132 University of Ghana http://ugspace.ug.edu.gh 182 8y F WASH NEG 163 By F WASH NEG 115 9y F ZEBU NEG 142 8y F WASH NEG 169 lOy F WASH POS 148 9y F ZEBU NEG 167 6y F WASH NEG 179 8y F WASH NEG 18 5y F ZEBU NEG 151 7y M WASH NEG 6 2y F WASH NEG 8 2y F ZEBU NEG 166 5y F WASH NEG 150 7y F ZEBU NEG 118 7y F WASH NEG 10 2y M ZEBU NEG 5 3y F WASH NEG 114 8y F ZEBU NEG 102 6y F WASH NEG 168 3y M WASH NEG 103 4y M WASH NEG 21 3y M WASH NEG University of Ghana http://ugspace.ug.edu.gh 17 3y M ZEBU NEG 171 6y F ZEBU NEG 9 3y M ZEBU NEG Screening at animal husbandary 2131 4y F WASH NEG 2132 6y F WASH POS 2133 5y F WASH NEG 2135 6y F WASH NEG 2136 7y F WASH NEG 2137 5y M WASH NEG 2138 8y F WASH NEG 2139 7y F WASH NEG 2140 8y F WASH NEG 2121 5y F WASH NEG 2122 8y F WASH NEG 2123 6m F WASH NEG University of Ghana http://ugspace.ug.edu.gh 2124 8y F WASH NEG 2125 8y F WASH NEG 314 9y F WASH NEG 2126 6y F WASH NEG 2127 2y M WASH NEG 2128 3y M WASH NEG 2129 7y F WASH NEG 2130 6y F WASH NEG 2111 8y F WASH NEG 2112 3m F WASH NEG 2113 8y . F WASH NEG 2114 6y F WASH NEG 2115 3y M WASH NEG 2116 4y M WASH NEG 2117 4y F WASH NEG 2118 3y F WASH NEG 2119 3y M WASH NEG 2120 6y M WASH NEG 2142 5y F WASH POS 2143 3y M WASH NEG 2144 2y M WASH NEG 2145 9m F WASH NEG University of Ghana http://ugspace.ug.edu.gh 2146 iy M WASH NEG 2147 ly M WASH NEG 2148 6m F WASH NEG 2150 6y F WASH NEG 2110 3y F WASH NEG 2109 2m M WASH NEG 2108 3m M WASH NEG 2107 3m F WASH NEG 1681 4y F ZEBU NEG 1682 6y F ZEBU NEG 1683 6y F ZEBU NEG 1684 6y F ZEBU NEG 1685 1 5y M WASH NEG 1686 6y F WASH NEG 1687 3y F WASH NEG 1688 6y F ZEBU NEG 1689 6y F WASH NEG 1690 5y M ZEBU NEG 1671 4y F ZEBU NEG 1672 4y M ZEBU NEG 1673 8y F ZEBU NEG 1674 1.5y M WASH NEG University of Ghana http://ugspace.ug.edu.gh 1675 5y M WASH NEG Screening at Mpotah 1641 8y F ZEBU NEG 1642 8y F ZEBU NEG 1643 8y F WASH NEG 1644 7y F WASH NEG 1645 4y F WASH POS 1646 7y F WASH NEG 1647 8y F ZEBU NEG 1648 7y F WASH NEG 1649 8y F ZEBU NEG 1650 8y F ZEBU NEG 1651 8y F ZEBU POS 1652 5y F ZEBU NEG 1653 10y F ZEBU NEG 1654 5y F WASH NEG 1655 8y F ZEBU NEG 1656 8y F ZEBU NEG ' 1657 7y F WASH NEG 1658 4y M ZEBU NEG 1659 8y F ZEBU NEG 137 University of Ghana http://ugspace.ug.edu.gh 1660 1661 1662 1663 1664 1665 1666 1667 1668 1670 1676 1677 1678 1679 1680 2101 2102 2104 201 202 203 8y F ZEBU NEG 7y F WASH NEG 8y F ZEBU NEG 8y F ZEBU NEG 8y F WASH NEG 8y F WASH NEG 7y F WASH NEG 6y F ZEBU NEG 8y F ZEBU NEG 6y F ZEBU NEG By F WASH NEG 8y F ZEBU NEG 6y F WASH NEG 8y F WASH NEG 8y F WASH NEG 8y F WASH NEG 8y F ZEBU NEG 7y F WASH NEG 8y F ZEBU NEG 7y F ZEBU NEG 6y F WASH NEG 138 University of Ghana http://ugspace.ug.edu.gh Screening at Axim (Asaasetre) ID# Age Sex Breed Result 204 lOy F WASH NEG 205 4y F WASH NEG 206 5y F WASH NEG 207 5y F WASH NEG 208 4y F WASH NEG 209 6y F WASH NEG 210 12y F WASH NEG 231 20y F WASH NEG 232 3y F WASH NEG 233 2y F WASH NEG 234 3y F WASH NEG 235 5y F WASH NEG 236 6y F WASH NEG 237 4y F WASH NEG 238 25y F WASH NEG 239 23 y F WASH NEG 240 24y F WASH NEG 211 3y F WASH NEG 212 8y F WASH NEG 139 University of Ghana http://ugspace.ug.edu.gh 213 24y F WASH NEG 214 15y F WASH NEG 215 17y F WASH NEG 216 30y F WASH NEG 111 14y F WASH NEG 218 4y M WASH NEG 219 8y F WASH NEG 220 3y F WASH NEG 221 2y F WASH NEG 222 ly F WASH NEG 223 3y F WASH NEG 224 3y F WASH NEG 225 8y F WASH NEG 226 5y F WASH NEG 227 2y F WASH NEG 228 2y F WASH NEG 229 ly F WASH NEG 230 2y M WASH NEG 140 University of Ghana http://ugspace.ug.edu.gh Screening at Teleko Bokazo 241 9y F WASH NEG 242 8y F WASH NEG 243 7y F WASH NEG 244 7.5y F WASH NEG 245 6y F WASH NEG 246 6y F WASH NEG 247 8y F WASH NEG 248 ly F WASH NEG 249 3y F WASH NEG 250 4y F WASH NEG 191 3y F WASH NEG 192 3y F WASH NEG 193 lOy F WASH NEG 194 ly F WASH NEG 195 3y F WASH NEG 107 4y F WASH NEG 108 6.5y F WASH NEG 109 6y F WASH NEG 110 5y F WASH NEG 130 3y F WASH NEG 121 4y M WASH NEG 141 University of Ghana http://ugspace.ug.edu.gh Screening at Nkroful Sec Sch 200 7y M WASH NEG 199 5y F WASH NEG 198 4y F WASH NEG 197 5y M WASH NEG 196 6y F WASH NEG 101 7y F WASH NEG 241 9y M WASH NEG 102 8y F WASH NEG 103 7y M WASH NEG 104 6y F WASH NEG 105 5y F WASH NEG 106 2y M WASH NEG Screening at Kanbule 122 4y F WASH NEG 123 4y F WASH NEG 124 3y M WASH NEG 125 3y M WASH NEG 126 3y F WASH NEG 127 5y M WASH NEG 128 5y F WASH NEG 142 University of Ghana http://ugspace.ug.edu.gh 129 4y F WASH NEG 131 6y F WASH NEG 132 5y F WASH NEG 133 8y F WASH NEG 134 8y F WASH NEG 135 5y F WASH NEG Screening at Arokpogwe 181 5y M WASH NEG 182 8y M WASH NEG 190 8y M WASH NEG 183 5y F WASH NEG 184 5y F WASH NEG 185 5y F WASH NEG 186 4y F WASH NEG 187 6y F WASH NEG 188 5y F WASH NEG 189 8y F WASH NEG 141 6y F WASH NEG 142 3y F WASH NEG 143 5y F WASH NEG 144 3y F WASH NEG 143 University of Ghana http://ugspace.ug.edu.gh 145 3y F WASH NEG Screening at Police Quarters 111 3y F WASH NEG 112 7y F WASH NEG 113 3y F WASH NEG 114 4y F WASH NEG 115 3y F WASH NEG 116 4y M WASH NEG 117 5y F WASH NEG 118 4y F WASH NEG 119 3y F WASH NEG 120 4y F WASH NEG 171 2y F WASH NEG 172 6y M WASH NEG 173 4y M WASH NEG 144 University of Ghana http://ugspace.ug.edu.gh APPENDIX V FORMULAS 1. Geometric Mean: Geometric Mean = antilog {X log X+l} - 1 2. Prevalence: Number of cattle infected X 100 Total Number screened 3. Infective rate: Number of cattle infectedX 100 Total cattle dissected 145 University of Ghana http://ugspace.ug.edu.gh APPENDIX VI Constituents of a 25CCH1 PCR reaction mix Table 3.1: Constituents of PCR reaction mix for W. banerofti Reagent Volume Final concentration Sterile distilled water To make up 25 (xl lOx PCR buffer lx 50mM MgCb 2.5mM DON1V of each of dATP, 0.5^1 each 200[jM each dCTP, dGTP, dTTP RO1SVYFG1 primer 0.5 (0.1 200 nM 20nM NV-2 primer 0.5 (j.1 200 nM 5U Taq polymerase 0.125)0.1 O0ORA,LN1PD DNA template A CCRT Final Volume 25(0.1 146 University of Ghana http://ugspace.ug.edu.gh Table 32: Typical reaction components of a 25pJ PCR reaction for An. gambiae identification H20 To complete final volume of 25JJ.1 Final conc 10X PCR reaction buffer 2.5p;l IX dNTPs 2.510.1 20mM UN Primer l.Op.1 12.5ng GA, AR, ME, QD 1.0^1 each RON1V 0.6 U Taq polymerase (5U/p.l) O.lul 0.625 2-2.5 mM each dATP, dCTP, dGTP, dTTP Table 33: Typical reaction components for a 25pl reaction for identification An. funestus H20 To complete final volume of 25jil Final Conc 10X PCR reaction buffer containing 2.5|j.l IX 25 mM MgCl l.Ojal dNIP solution 2.5|xl Primers, D3 A, D3B l.Ofxl 20^iM 0.6 U Taq polymerase (5,L110D Q 0.1 nl 0.625 2-2.5 mMeach dATP, dCTP, dGTP, dTTP 20mM 147 University of Ghana http://ugspace.ug.edu.gh Table 42: Microfilaria intensities during the 24-hour collection period ID# Age Sex Mf intensities (mf/lOOpl) at the hours of examination 6:00am 10:00am 2:00pm 6:00pm 10:00pm 1o oCN 169 lOyrs F 22 18 16 7 13 16 2142 5yrs M 29 11 7 11 12 4 2132 6yrs F 21 92 0 60 34 88 1651 8yrs F 4 5 5 0 2 22 1645 4yrs F 0 3 1 0 3 1 Geometric Mean 20 18.4 4.6 7.6 11.6 15.5 148 University of Ghana http://ugspace.ug.edu.gh