Snails' Population Dynamics and Their Parasitic Infections with Trematode in Barakat Canal, Gezira Scheme 2011

Snails' Population Dynamics and their Parasitic Infections with Trematode in Barakat Canal, Gezira Scheme 2011

By Arwa Osman Yousif Ibrahim B.Sc (Honours) in Science (Zoology), University of Khartoum (2007)

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Medical Entomology and Vector Control Blue Nile National Institute for Communicable Diseases University of Gezira

Main Supervisor: Dr. Bakri Yousif Mohammed Nour Co-Supervisor: Dr. Azzam Abdalaal Afifi

July, 2012

Snails' Population Dynamics and their Parasitic Infections with Trematode in Barakat Canal, Gezira Scheme 2011

Arwa Osman Yousif Ibrahim

Supervision Committee:

Supervisor Dr. Bakri Yousif Mohammed Nour ……………. Co-Supervisor Dr. Azzam Abd Alaal Afifi …………….

Snails' Population Dynamics and their Parasitic Infections with Trematode in Barakat Canal, Gezira Scheme 2011

Arwa Osman Yousif Ibrahim

Examination committee:

Name Position Signature Dr. Bakri Yousif Mohammed Nour Chairman ……………. Prof. Souad Mohamed Suliman External examiner ……………. Dr. Mohammed H.Zeinelabdin Hamza Internal Examiner …………….

Date of Examination: 17/7/2012

Snails' Population Dynamics and their Parasitic Infections with Trematode in Barakat Canal, Gezira Scheme 2011

By Arwa Osman Yousif Ibrahim

Supervision committee:

Main Supervisor: Dr. Bakri Yousif Nour …………………………. Co-Supervisor: Dr. Azzam Abd Alaal Afifi …………………………

Date of Examination…………….

DEDICATION

To the soul of my grandfather

To everyone who believed in me

To everyone who was there when I was in need

To everyone who supported, helped and stood beside me

To all of you, my immense appreciation

Acknowledgements

I would like to express my deep gratitude to my main supervisor Dr. Bakri nour and Co-supervisor Dr. Azzam Afifi for their valuable advices and comments throughout this study.

My immense gratitude is also due to prof. Samira Hamid Dean of BNNICD institute.

I would like to thank all the Blue Nile Institute staff especially the Laboratory staff Safia Siddig, Amel Hassan, Mostafa Mohielden, Asia Elhag, Sally Widaa, Gasim Mostafa, Midhat Modather, Howida Amin .

Thanks to University of Gezira staff Dr. Mona Elhag department of agricultural Engineering, for her infinite help, Dr. Hala Aloub Department of Crop Protection for helping in plant identification and the staff of the Soil Department for their help in PH measurements.

My gratitude also extends to Ms. Nidal Ahmed department of Zoology, University of Khartoum for her huge help in snail and cercariae identification.

My thanks due to Dr. Ehab Farah and Dr. Siddig Essa for their assist in statistical analysis.

At last I want to thanks everyone I might forgot who helped me and encouraged me, to all of you, I extend my deep gratitude.

Snails' Population Dynamics and their Parasitic Infections with Trematode in Barakat Canal, Gezira Scheme 2011

Arwa Osman Yousif Ibrahim

Msc in medical entomology and vector control (July 2012)

Blue Nile Institute for communicable disease

University of Gezira ABSTRACT

Freshwater snails are the intermediate host for the most of trematode worms which are responsible for a number of disease conditions in humans and many other vertebrates. Across sectional study was conducted in Barakat canal (June- July 2011) to explore the present snail species and their relation with trematode infection, with observation on environmental factors affecting them. Three surveys at different environmental conditions were conducted, first after canal re-filling, the second after canal clearing and the third after vegetation re-grow. A total of 1540 freshwater snails were collected from the study site with overall species density Cleopatra builmoides

(43%), Lymnaea natalensis (17.9%), Bulinus truncatus (14%), Biomphalaria pfeifferi

(13%), Melanoides tuberculata (7%), Lanistus carinatus (5%), and Bulinus forskalii

(0.1%). The overall density in the three surveys was (27%), (37%), and (36%) respectively. Snails were screened for trematode cercariae under artificial light, six of the seven species were shed different trematode cercariae with infection rate in the three surveys 15%, 52%, and 79% respectively. Six cercariae morphotypes were shed

Longi-furcate apharengeate Monostome cercaria (LPM), three types of

Xiphidiocercariae, Echinostomecercariae, Plurolophocercouscercariae, Longi-furcate pharengeate Distome cercaria LPD and two types of Furcocercouscercariae. A list of recommendations were made, since the planning of snail control reassures and evaluate their impact, knowledge of their ecology, population trends and dynamism are essential requirement towards understanding disease transmission and control.

دراسة على القواقع في ترعة بركات )مشروع الجزيرة( ومعدل اصابتها بالديدان المثقبة

أروى عثمان يوسف إبراهيم

أطروحه لنيل درجة الماجستير في الحشرت الطبية ومكافحة نواقل االمراض)يوليو 2012(

مهعد النيل االزرق القومي لالمراض السارية

جامعة الجزيرة

الملخص

تعتبر القواقع من اهم العوائل الوسيطة للديدان المفلطحة المسببة للكثير من االمراض لالنسان و الفقاريات عامة, اجريت دراسة مقطعية في ترعة بركات في الفترة من يونيو- يوليو 2011 بهدف معرفة انواع القواقع الموجودة ودرجة اصابتها بالديدان المفلطحة مع دراسة العوامل البيئية المحيطة. اجريت ثالثة مسوحات فى ظروف مختلفة, المسح االول تم بعد فتح الترعة , والثاني بعد تنظيف الترعه من الحشائش بينما اجري الثالث بعد نمو الحشائش المزالة. جمع حوالى 1540 قوقع لسبعة انواع مختلفة كليوباترا بوليمويدس )46%(, ليمنيا نتالينسيس )17.9%(, بولينس ترنكاتس )14%(, بايومفالريا بفيفيراي )13%(, ميالنويدس تيوبركيوالتا )7%(, النيستس كارينيتس )5%( وبولينس فورسكالي )0.1%(. بينما كانت الكثافة الكلية للقواقع في كل مسح 26%, 37% و36% على التوالي. لمعرفة و حساب معدل اصابة القواقع عرضت لمصدر ضوء لتحفيز خروج السركاريا, ستة من انواع القواقع اخرجت انواع مختلفة من السركاريا لونجيفوركات ا فارينجايات مونوستوم, ثالثة انواع زيفيديوسركاريا, ايكينوسركاريا, بلورولوفوسيركس, نوعين من الفركوسيركس و لونجيفوركات فارانجيات دايستوم سركاريا بمعدل اصابة كلي فى المسوحات الثالث 15%, 52% و79% على التوالي.هذه الدراسة قد تشكل اضافه مهمة للمعرفة الديدان المفلطحة في السودان وبمحاولة تعميمها فالمعلومات المتحصل عليها بهدف التخطيط السليم لمكافحة القواقع و معرفة العوامل المؤثرة عليها تؤدي لمكافحة الديدان المفلطحة بطريقة غير مباشرة.

LIST OF CONTENTS Contents Page DEDICATION iii ACKNOWLEDGEMENTS iv ABSTRACT v ARABIC ABSTRACT vi LIST OF CONTENTS vii LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF PLATES xvi LIST OF APPENDIXES xvii CHAPTER ONE 1.1 Introduction 1 1.2 Justification of the study 3 1.3 General and specific objectives 3 1.4 Study design 3 CHAPTER TWO : LITERATURE REVIEW 4-29

CHAPTER THREE: MATERIALS AND METHODS 30-33

CHAPTER FOUR : RESULT 34-64

CHAPTER FIVE

DESCUSSION 65-75

RECOMMENDATIONS 76

REFRENCES 77- 100

APPENDEXES 101- 105

LIST OF TABLES

Table (1):Animal schistosomes which can infect human accidentally 12

Table (2): The observed turbidity during the study period in Barakat 46 canal in the three surveys. Table (3): The observed velocity during the study period in Barakat 47 canal in the three surveys. Table (4): The observed Vegetation density during the study period 48 in Barakat canal in the three surveys. Table (5): the reported vegetation types in Barakat canal during the 49 study period Table (6): The natural infection rate of the collected snail species 52 with trematode cercariae in the three surveys (June-July 2011) Table (7): The Vivax LPM type and its specific infection rate to snail 54 species in the three surveys (June-July 2011) in Barakat canal Table (8): The Armate Xiphidiocercariae type and its specific 56 infection rate to snail species in the three surveys (June-July 2011) in Barakat canal Table (9): The undescribed Xiphidiocercariae type 1 and its specific 57 infection rate to snail species in the three surveys (June-July 2011) in Barakat canal Table (10): The Virgulate Xiphidiocercariae type and its specific 58 infection rate to snail species in the three surveys (June-July 2011) in Barakat canal Table (11):The Echinostome type and its specific infection rate to 59 snail species in the three surveys (June-July 2011) in Barakat canal Table (12): The Paraplurolophocercous type and its specific 60 infection rate to snail species in the three surveys (June-July 2011) in Barakat canal Table (13): The furcocercous cercariae type 3 and its specific 61 infection rate to snail species in the three surveys (June-July 2011) in Barakat canal Table (14): The Strigea LPD cercariae type and its specific infection 62 rate to snail species in the three surveys (June-July 2011) in Brakat canal Table (15): The furcocercouscercariae type 2 and its specific 63 infection rate to snail species in the three surveys 2011) .

LIST OF FIGUERS

Figure (1): The overall percentage of snail species density in the three surveys 34 during June-July 2011 in Barakat canal Figure (2): Snail Species density in the three surveys in Barakat canal during 35 June-July 2011 Figure (3): The snail species density in survey (1) after canal opening in 40 Barakat canal (June-July 2011). Figure (4): The snail species density in survey (2) after vegetation clearing in 41 Barakat canal (June-July 2011). Figure (5): The snail species density in survey (3) after vegetation growth in 42 Barakat canal (June-July 2011) Figure (6): Average of water temperature in the three surveys conducted in 43 Barakat canal (June-July 2011). Figure (7): Average of water depth in the three surveys conducted in Barakat 44 area during June-July 2011 Figure (8): Average of water PH in the three surveys conducted in Barakat 45 canal (June-July 2011). Figure (9): The overall percentage of snail species infection rate in the three 50 surveys during June-July 2011 in Barakat canal

Figure (10): The rate of cercariae types' shed by the collected snail species in 45 the three surveys in Barakat canal June-July 2011 Figure (11): The density of cercariae types in the three surveys in Barakat 53 canal 2011

LIST OF PLATES

Plate (1): The selected study area (Barakat canal) 31

Plate (2): Cleopatra bulimoides 36

Plate (3): Lymnaea natalensis 36

Plate (4): Bulinus truncates 37

Plate (5): Biomphlaria pfeifferi 37

Plate (6): Melanoides tuberculate 38

Plate (7): Lanistus carinatus 38

Plate (8): Bulinus forskalii 39

Plate (9, 10 and11): Longifurcate-Phayrngeate Monostome cercariae 55 LPM

Plate (12): ArmateXiphidiocercaria 56

Plate (13):Xiphidiocercaria type1 shed by Biomphalaria pfeifferi and 57 Cleopatra bulimoides

Plate (14): VirgulateXiphidiocercaria 58

Plate (15): Echinostomecercaria 59

Plate (16 and17):Paraplurolophocercous cercariae 60

Plate (18): Un-described Furcocercouscercaria type 3 61

Plate (19):Longifurcate-Pharyngeate distome cercariae (LPD) 62

Plate (20): Un-described Furcocercouscercaria type 2 63

LIST OF APPENDICES

Appendix (I): The percentage of the collected snail species density in 101 the three surveys during June-July 2011 in Barakat canal Appendix (II): The percentage of snail species density in the three 102 surveys during June-July 2011 in Barakat canal

Appendix (III): The density of shed cercariae types in the three 103 surveys in Barakat canal June-July 2011 Appendix (IV): The correlation between surveys and water 104 temperature in the study area Appendix (V): The correlation between surveys and water PH in the 104 study area Appendix (VI): The correlation between surveys and water depth in 105 the study area

CHAPTER ONE

INTRODUCTION

Freshwater snails are the first intermediate host for most of trematode worms. About 350 snail species are estimated to be of possible medical or veterinary importance. The Planorbidae, Physidae, Lymnaeidae, and Ancylidae are in the Order Basommatophara represent one of the most important families of freshwater snails, they have a wide distribution. There are approximately 40 recognized genera of planorbids that are found in all continents except Antarctica, in almost any freshwater lakes, pools, or streams. In habitats ranging from tropical rainforests to desert oaises, and they are significant both medically and economically as intermediate hosts for digenetic trematode worms infecting human and domestic animals throughout the tropics. The other family is Prosobranchs which is widely distributed in central and eastern Africa, the common are genera Bellamya, Pila, lanistus, Gabbia, Cleopatra and Melanoides .All snail species prefer stable conditions with fairly dense vegetation and the environmental requirements of the various species are broadly similar. They prefer slower running water, gradual Changes in the water level, little turbidity, some organic pollution, partial shade and optimal water temperature, under these conditions, aquatic vegetation and algae, which are the main sources of nutrition for aquatic snails, flourish creating favorable habitats for intermediate snail hosts presence. The densities of snails differ highly among sites, and their variability within irrigated areas is related to the canal type, distance of sites from the canal passage. The variability is also due to water stagnation, water depth, shading, the density and composition of aquatic vegetation. Digenetic trematodes are responsible for a number of disease conditions in humans and many other vertebrates. They have a heteroxenous life

14 cycle with freshwater snail as their first intermediate host. The adult stages are found in different vertebrate definitive hosts including amphibians, fishes, reptiles, birds and mammals. Disease characteristics of fluke infections vary with the parasite species and the site or organ of infection and are linked with the life cycle. The distributions of freshwater snails correlate with the occurrence of different trematode taxa in a particular region. 1.2- Justification of the study:

The irrigation canals in the Gezira scheme provide flourish environment for water-borne diseases transmitted by fresh water snails, mainly schistosomiasis which is endemic in Gezira state threatening both human and animals. Other trematode diseases such as cercerial dermatitis, fascioliasis, and paramphistomiasis which have a huge economic effect on livestock and human.

1.3-General objective:

Study snail species of medically and veterinary importance and their natural infection with trematode worms in Barakat canal .

1.4- Specific objectives:-

1- To determine the present snail species in the area. 2- To determine and estimate the prevalence of natural infection rate among snails population. 3- To observe environmental factors affecting snails.

1.5- Study design:

Cross-sectional study was conducted in Barakat canal to collect medical and veterinary snail intermediate hosts. Three surveys had been done in canals around an endemic area. Snails were collected using scooping method. The snails were screened in the laboratory for cercariae and their prevalence of natural infection was estimated. Also the ecological parameter which affect the presence of Schistosome intermediate host and other snails which act as human and animal hosts in the canals was observed,e.g water turbidity, pH, vegetation type and water velocity.

CHAPTER TWO

LITERATURE REVIEW

Digenic trematodes pose a real threat on human and animal populations, as their different orders contain many parasitic infectious families. Trematode adults live in the gut and its accessory organs; lungs, bladder, gills and buccal cavity. The life cycle of digenetic trematode involve a wide range of larval stages differ in their physiological requirements, feeding process and morphology (Erasmus, 1972). As the parasites are mostly host specific, higher heterogeneity of the host promotes higher heterogeneity of the parasites (Hechinger and Lafferty, 2005). Similarly, higher snail diversity leads to higher trematode diversity (Smyth, 1962; Poulin, 2005). Trematode infection usually results in parasitic castration of the snail host (Esch and Fernandez, 1994), reduce the egg production or increase snail mortality rates (Lafferty, 1993).which may have important effects on gastropod population biology and life history (Brown et al., 1988; Sorensen and Minchella, 2001) and could contribute to the regulation of host populations at high prevalence (Lie and Ow- Yang, 1973; Brown et al. 1988). Prevalence of trematode infection generally increases with snail size and age. Several different factors, such as parasitic alteration of growth rates, changes in survivorship and increased exposure to infection with age may contribute to this pattern (Brown et al. 1988; Sorensen and Minchella, 2001). There is considerable intra-specific variation in parasite prevalence in snail hosts (Kuris and Lafferty, 1994). However, geographical patterns of variation in trematode prevalence, and how these relate to variation in snail growth rates and life history, remain unknown.

2.1- The general Trematode life cycle:

The digenens exhibit many variation of their life cycle pattern. The typical life cycle consist of molluscan primary host in which the multiplication occurs, an intermediate host, and vertebrate final host. The primary hosts get infected by free-swimming miracidium penetration; or ingestion of full developed miracidium inside an egg. Inside the mollusk miracidium develop into sporocyst or in some species redia which they give massive numbers of the infective stage cercariae which search for the final host depending on sensory and environmental cues. After penetration the host they encyst to give mata-cercariae either on vegetation, substrates, or second intermediate host. Meta-cercriae develop into adults who are specialized for maintaining their position in a microhabitat, with the provision of physiological and immunological favorable conditions to develop their egg and release it (Erasmus, 1972).

2.2- Family Schistosomatidae:

Schistosomes are helminth of the Family:Schistosomatidae .They belong to phylum Platyhelminthes, sub-phylum Cercomeridae, Class Trematoda, Sub-class Digenoidae, Super-order Anepitheliocystidia, Order Strigeatida, Super-family Schistosomatidae (Jordan and Webbe,1993).They alternate generations with a sexual phase in the definitive mammalian hosts, and sexual phase in intermediate snail hosts. They have life span of many years and daily produce large numbers of eggs, which may traverse the gut and bladder tissues in their way to the lumen of execratory organs (Ahmed, 2006).

2.3- Family Echinostomatidae:

Echinostome cercariae may infect fish or snail as secondary intermediate host and birds or mammals including man as definitive host. They are

18 recognized by the double row of spines on the circumoral collar. The size, number and arrangement of these spines are important for the taxonomy of the group. Echinostoma revolutum is a common species of the genus Echinostoma, are widespread and relatively abundant; not extremely host specific, Eggs hatch in water and miracidia penetrate the first intermediate host which typically are snails (Physa, Lymnaea, Helisoma), The metacercaria often are found with molluscs, planaria, fish and tadpoles. Definitive hosts pick up these parasites from ingesting any of these hosts and humans will become infected if they eat raw muscels or snails (Soulsby, 1982).

2.4- Family Paramphistomatidae:

They parasitize fishes, amphibians, reptiles, birds and mammals. In domestic animals a large number of them have been isolated from the rumen and reticulums of ruminants, some species occur in the large intestine of ruminants, pigs, and mammals (Soulsby, 1982).

2.5- Family Fasciolidae:

Family Fasciolidae belong to Order Echinostomida, Sub-order Echinostomata. It includes several veterinary and medically important parasites. The family has five genera Fasciola, Fascioloides, Fasciolopsis, Parafasciolopsis, and Protofasciola (Olson et al., 2003).Flukes of the family are localized in liver, gall bladder, and the intestine. The life cycle of fasciolid flukes includes one intermediate host which belong to Family Lymnaeidae (Jurasik and Dubinsky, 1993).

2.6- Family paragonomidae:

Paragonimids are lung flukes of mammals including humans. Snails and crustaceans act as the first and second intermediate hosts respectively. The economical species occur in Asia, Africa and the Americas, mostly

19 in tropical or subtropical areas, more than half of all nominal species occur in East Asia, especially China. A few species are found in temperate regions of North America and Northern Asia. Only two genera are recognized, Paragonimus and Euparagonimus, but the taxonomy of the family remains confused (Blair, et al. (1999). Paragonimids are restricted to two superfamilies of caenogastropods. Despite these differences, some species of Paragonimus exhibit remarkably low levels of host specificity, with different populations utilizing snails of different families.

2.7- Family Amphistomatidae:

The prevalence of amphistomes in domestic ruminants in Africa, particularly cattle, is high (Dinnik, 1964). In Africa, Calicophoron microbothrium is one of the most common species occurring in cattle, sheep and goats (Dinnik and Dinnik, 1962; Dinnik, 1964; Horak, 1971). The adult trematodes found in the rumen and reticulum, are not usually associated with clinical disease (Rolfe and Boray, 1987).

2.8- Cercariae of trematode worms:

Cercariae are the infective stage of human, released in massive numbers from their snail host. Cercarial output is directly influenced by the temperature which increases the emergence from the snail and accelerates of cercarial production within the snail host. Under high temperature conditions some cercariae encyst within snails (Poulin, 2005). The daily cycles of cercarial emergence are recognized as an adaptive mechanism to enhance parasite transmission. Several hypotheses have been proposed to explain the functional significance of these daily cycles (Shostak and Esch, 1990).The most accepted hypothesis suggests that cercarial emergence is timed to coincide with the presence of the next host,

20 particularly for those cercarial species in which the target hosts do not regularly cohabit with the molluscan host producing the cercariae (Combes et al., 1994).The pattern of emergence in fresh water system varies according to circadian rhythm (one emergence peak /24hrs) or ultradian rhythm (two emergence/24hrs) to enhance the probability of successful transmission (Pages J.R, Theron.A, 1990, Fingerut, et al., 2003). The important factors affecting these rhythms types are photo- period and thermo-period. Many trematodes show a marked increase in cercariae shedding with increased temperature (Fried et al., 2002; Fredensborg et al., 2005). There is no correlation between snail species and the rhythmatic emergence of cercariae (Niemman and Lewis, 1990). In Sudan the schistosome emergence peaks vary over an interval of twelve hours from one scheme to another, between 6:00am to 6:00pm depending on Schistosome population considered (Ahmed, 2002). The emergence correlates with the host activity either diurnal like in human schistosomiasis, nocturnal like S. rhodaini which infect rodents, or emergence at dusk or down like S. margrebowiei (Pitchford and Dutoit, 1976). 2.9- Snail water-borne diseases:

The fresh water snails harbor and transmit various types of trematode worms causing illness for mammals either man or animal, such as schistosomiasis, fascioliasis, paragonimiasis and clonorchiasis (Stricland G.T, 2000), also cercarial dermatitis, and Amphistomosis with great concern.

2.9.1- Schistosomiasis:

Schistosomiasis is one of the major health problems in Sudan. The disease is endemic in all irrigation agricultural schemes (El Tash, 2000; Ahmed et al., 2002, Ahmed, 2005). In almost all region of the country

21 and in recent years it was increased in distribution and prevalence as a result of progressive expansion in water resources development and population movement (Blue Nile health project Annual Report, 1981). 2.9.1.1- Epidemiology of schistosomiasis:

Schistosomiasis is a chronic, parasitic disease caused by trematode worms of the genus schistosoma. Three of the five recognized species groups of Schistosoma rely on snails of the family Planorbidae to complete their life cycles and each species group requires a specific planorbid genus. Five species of Schistosoma; S.haematobium (Bilharz,1852),S.mansoni (Sambon,1907),S.japonicum (Katsurada,1904), S.intercalatum (Fisher,1934)and S.mekongi(Voge et al.,1978) and new discovered Malaysian species(Greer et al.,1980) infect humans causing disease as they reside in their abdominal veins (Ross ; Bartly et al., 2002). Most intermediate hosts of human Schistosoma parasites belong to three genera, Biomphalaria, Bulinus and Oncomelania there is great deal of parasite strain specificity for snail specie (WHO, 1997). In Africa and the Americas, snails of the genus Biomphalaria are intermediate hosts of S. mansoni, Snails of the genus Bulinus are the intermediate hosts of S. haematobium in Africa and the Eastern Mediterranean, as well as of S. intercalatum in Africa. In south-east Asia, Oncomelania serves as the intermediate host of S. japonicum, and Tricula as the intermediate host of S. mekongi (WHO, 1997). More than 207 million people are infected worldwide, with an estimated 700 million people at risk in 74 endemic countries, 85% live in Africa (King, 2009), in Africa, the mortality due to urinary schistosomiasis are estimated to be 150,000 per year, and the number of people dying of intestinal schistosomiasis all estimated to be 130,000 per year (Fenwick, 2003). Although mortality due to schistosomiasis is low compared to millions of cases, the disease has huge impact on public health and

22 socio-economic development (WHO, 2006).The disease is prevalent in tropical and sub-tropical areas, especially in poor communities without access to safe drinking water and adequate sanitation, refugee movements and migration to urban areas are introducing the disease to new areas, also increasing population size and the corresponding needs for power and water result in development schemes and environmental modifications which also lead to increase transmission. 2.9.1.2- Mode of transmission:

Schistosomiasis transmission takes place where the ecologies of the schistosome parasite, the aquatic snail intermediate host, and the human definitive host converge in space and time in surface waters. Climate and the distribution of surface waters suitable for snail intermediate hosts and the free-swimming parasite are crucial in macro geographic distribution of schistosomiasis worldwide (Kloos and David, 2002). Micro geographic variations in the physical environment, human settlement patterns, the distribution of freshwater bodies and the intensity of exposure and contaminative contact by humans and the prevalence of the pathogenic worms and host snails largely determine the prevalence of infection within endemic areas and communities (Kloos and David, 2002). The disease transmission depends on excretory contamination of snail habitats and direct contact with infective water. This ecological relationship makes schistosomiasis a disease that is closely linked to rural water resources development, population increase, inadequate sanitation and lack of effective medical treatment (Kloos and Thompson 1979). 2.9.1.3- Distribution of Schistosomiasis intermediate hosts in Sudan:

Several surveys were conducted in different part of the Sudan studying snail distribution in all endemic areas. The distribution of Bulinus forskalii in the Blue Nile and spring of Nuba mountain in south Kordofan state, Biomphalaria alexandria and B.pfeirfferi in the Blue Nile, B.biossyi

23 and B.pfeirfferi in the White Nile was reported(ref).In Barber region in North Sudan Biomphalaria species were recorded (Archibald, 1933). In Gezira state both species Bulinus and Biomphalaria were reported by (Stephenson, 1947). And then Greany (1952) described Bulinus truncatus, B.forskalii and B.looses, and Biomphalaria africanus, B.alexandria in different canals of Gezira scheme. Manjing, (1978) reported B. pfeirfferi in all Gezira canals since the irrigation system in Gezira-Managil scheme provide favorable conditions for snail breeding and schistosomiasis transmission (Madsen et al, 1988). Hilali, (1992) described Biomphalaria pfeirfferi in Managil agricultural scheme, and he found the same speices with Bulinus forskalii in Khartoum state (Hilali, 1995).

2.9.2- Animal Schistosomiasis:

Animal schistosomes can be found in mammals, birds and reptiles mainly crocodiles; the life cycle is identical to human schistosome (Horák and Kolár∨ová, 2001). Water snails infected by the first larvae (miracidia) serve as obligatory intermediate hosts in which asexual multiplication occurs, and then they release the infective stage (cercariae) which then penetrate the skin of vertebrates and develop into adults. Sometimes, a non-specific host might be infected, although it might be light infection but the repeated infections can cause significant losses due to long-term effects on animal growth and productivity, as well as increasing their susceptibility to other parasitic or bacterial diseases (De Bont and Vercruysse, 1998). Epizootiological surveys conducted in the White Nile State of the Sudan showed that 70-90% of cattle in irrigated areas were infected with S. bovis (Majid et al., 1980).

Schistosomiasis is posing a real threat on ruminants, mainly cattles in Africa and Asia, where it is estimated that at least 165 million animals are

infected (De Bont, and Vercruysse, 1997). Out of the 10 species reported to naturally infect cattle only Schistosoma mattheei and Schistosoma bovis have received particular attention, mainly because of their recognized veterinary significance (Taylor, 1987). Another species of importance in animals is Schistosoma japonicum because of its zoonotic aspect of transmission.

Table (1): Animal schistosomes which can infect human accidentally:

Parasite Host Snail host

Austrobilharzia terrigalensis Birds Genus littorina

Schistosoma bovis Cattle, sheep, goats Bulinus sp.

Schistosoma mattheei Cattle and sheep Bulinus sp.

Trichobilharzia ocellata Birds Lymnae sp.

Trichobilharzia stagnicolae Birds Lymnae sp.

2.10.3- Cercarial dermatitis:

It was known about 80 years ago throughout the world, the cercariae of schistosomes of animals, especially those from birds, cause dermatitis when they penetrate the skin of people in contact with waters of natural habitats (Verbrugge et al., 2004). Although it die in the skin, recent work suggests that this is not always the case, and that some parasites from such infections may persist and cause neurological problems in humans (Hradkova and Horak, 2002). There is strong evidence that outbreaks of dermatitis occur continually around the world, both in marine and freshwater habitats (Larsen et al., 2004). For the well-known genera (e.g. Trichobilharzia), it is thought that the restricted specificity towards the

25 intermediate snail host is the key limiting factor in explaining disease distribution (Blair, and Islam, 1983). The identification of bird schistosomes causing dermatitis remains a complicated matter; for many of the avian schistosomes the life cycles and participating intermediate as well as final hosts are not known (Horák and Kolár∨ová, 2001). It is often not possible to distinguish cercariae of different species within one genus, and to be sure which parasite is responsible for the dermatitis. Laboratory maintenance of the life cycle with subsequent recovery and identification of adult worms and, sometimes, characterization of other developmental stages is necessary (Blair and Islam, 1983; Hradkova and Horak, 2002). 2.9.4- Paragonimiasis:

An anthropozoonosis disease essentially confined to lungs. It is due to a parasite belonging to Paragonimus genus, out of the forty species discovered in the world, and about ten are pathogen in humans (Blair et al., 1999). Two hundred million people would be exposed to this disease and twenty million persons would be infected all over the world (Toscano, et al., 1995). Most human cases have been reported on the Asian continent. In Africa, endemic foci were reported in about ten countries, mainly around the Guinean Gulf and in the central part of the continent. Two species of Paragonimus are described in Africa (Voelker, and Vogel, 1965) and other two species were suspected (Cabaret, et al., 1999). Compared to the second intermediate hosts (crustaceans), little information on snails the first intermediate hosts in the life-cycle of African Paragonimus is available. The first reports concerned with the prosobranch Potadoma freethii (Thiaridae) which was found infected by Paragonimus spp in cameroon (Vogel and Crewe, 1965; Sam-Abbenyi, , 1985) and Nigeria (Sachs, and Cumberlidge, 1989) , another species was a thiarid and was named as Melania spp in Nigeria (Arene, et al., 1998).

2.9.5- Fasciolosis:

Considered as an important helminthic disease caused by two trematode Fasciola hepatica and Fasciola gigantica, snail of the family Lymnidae act as the intermediate host, this disease belongs to the plant-borne trematode zoonosis in Europe, the Americas and Oceania only F. hepatica is a concern, but the distributions of both species overlap in many areas of Africa and Asia (Mas-Coma et al., 2005). The definitive host range is very broad and includes many herbivorous mammals, including humans, the life cycle includes freshwater snail as an intermediate host of the parasite (Torgerson and Claxton, 1999). Recently, worldwide losses in animal productivity due to fasciolosis were conservatively estimated at over 3.2 billion $ (Spithill, et al., 1999). In addition, fasciolosis is now recognized as an emerging human disease, the (WHO) has estimated that 2.4 million people are infected with Fasciola, and a further 180 million are at risk of infection (Anonymus, 1995). The distribution of the species is limited to the tropics and has been recorded in Africa, the Middle East, Eastern Europe and south and eastern Asia (Torgerson and Claxton, (1999). Human fasciolosis has been reported from countries in Europe, America, Asia, Africa and Oceania, many studies carried out in recent years have shown human fasciolosis to be an important public health problem (Chen and Mott, 1990). The incidence of human cases has been increasing in 51 countries of the five continents. In Africa, human cases of fasciolosis, except in northern parts, have not been frequently reported; the highest prevalence was recorded in Egypt where the disease is distributed in communities living in the Nile delta (Mas-Coma et al., 2005).

Human F. hepatica infection is determined by the presence of the intermediate snail hosts, domestic herbivorous animals, climatic

27 conditions and the dietary habits of man (Chen and Mott, 1990). Sheep, goats and cattle are considered the predominant animal reservoir, while other animals can be infected, they are usually not very important for human disease transmission. Humans are infected by ingestion of aquatic plants that contain the infected metacercariae. In natural and constructed water reservoirs, infection with Fasciola gigantica was documented and the disease is of considerable veterinary and economic importance in Sudanese livestock, heavily infected animals suffer substantial reduction in growth rate and production inefficiency, and cause economic losses due to partial liver rejections in abattoirs, in addition, costly annual treatment of animals with fasciolicides is becoming ineffective because the parasite has now shown some degree of resistance (Goreish and Musa, 2005).

2.9.6- Amphistomosis:

The disease is caused when heavy infection with immature flukes results in acute gastroenteritis with high morbidity and mortality, particularly in young animals (Dinnik, 1964; Horak, 1971; Rolfe and Boray, 1987; Brown, 1994). Amphistomosis in domestic ruminants results in serious economic loss to the wool, meat and milk industries (Horak, 1967). The distribution of amphistomes is determined by the distribution of their snail intermediate hosts (Dinnik, 1964). Bulinus truncatus and Bulinus tropicus are the intermediate snail hosts of C. microbothrium in northern and southern Africa respectively (Chingwena et al., 2002).The epidemiology and seasonal pattern of infection depends on the species of definitive and intermediate host (Rolfe et al. 1991), the system of management and grazing habits of the cattle (Horak, 1967; Boray, 1969), the biological potential of the snail hosts (Swart and Reinecke, 1962a,b ;Dinnik, 1964; Horak, 1971), the potential of the flukes to infect intermediate and definitive hosts (Dinnik and Dinnik, 1954; Dinnik,

1964; Horak, 1967), the topography of the snail habitats and the climate (Rolfe et al. 1991).Natural infection of Biom. pfeifferi snails by C. sukari has also been reported in Kenya and Zambia (Dinnik, 1965).

2.10- Fresh water snail species:

Only the most important species will mentioned according to their relation with trematodes worms:

2.10.1- Biomphalaria species:

Biomphalaria species is freshwater snails of the family planorbidae they are distributed all over the Sub-Saharan area, recently they found in Madagascar, probably due to human activity, spatial distribution, recruitment, and migration of Biomphalaria pfeifferi is highly influenced by the rainy/dry periods of the season (Charbonell et al., 2002). It has a small migration distance and this through active dispersal, against or following water flow, long distance migration is possible through other animals, like birds, or through human activities (Charbonnel et al., 2002). All known African species are susceptible to S.mansoni, most of them have been found naturally infected in the field, they classified in to four groups; Pfeifferi, Sudanica, Choanomphela and Alexandrina group. Infection of Biomphalaria pfeifferi with amphistome cercariae are new records for Zimbabwe (Chingwena, et al., 2002), Amphistome cercariae, furcocercous cercariae types, Echinostomes and Xiphidiocercariae were isolated from Biomphalaria species in Africa (Frandsen and Christensen, 1984).

2.10.2- Bulinus species:

The genus Bulinus is naturally divided into two different subgenera: Physopsis and Bulinus spp. which also includes the old subgenus

Pyrgophysa (Mandhle-Barth, 1962). They divided to Africanus group, Reticulates group, Forsklii group, and Truncatus group which all contain the susceptible species to S.haematobium and intercalatum. Researchers reported various cercariae species in Bulinus species; the main intermediate host for Amphistomes was B. tropicus. Infections in B. globosus, B. forskalii with amphistome cercariae are new records for Zimbabwe (Chingwena, et al.,2002 ).in Spain Schistosoma bovis reported from Bulinus truncatus and B. wrighti (Mouahid and Théron, 1986). Echinstome, Xiphidiocercariae reported in Africa (Frandsen and Christensen, 1984).In Sudan Ibrahim, (2007) reported Xiphidiocercariae, Echinstomecercariae and Amphistome types from Bulinus species.

2.10.3- Lymnaea species: Family Lymnaeidae, are fresh water snail with a thin dextral right opening shell preferred to live in stagnant habitat in slow streams with heavy vegetation, they act as the first intermediate host of animal schistosomes throughout the world (Ferete et al., 2005), also the most important and widespread intermediate host of Fasciola hepatica in Africa, Asia, Europe and north America is Lymnae trunculata (Soliman, 2008).various studies indicated that Lymnaeidae were infected with larvae fasciolid species (De kock et al ,2003;Soliman,2008; Barraggan- Saenz et al ,2009) and trematodes such as Paramphistomum (Dryffuss et al.,2004) ,Holostom (Klockars et al.,1928) ,Trichobilharzia , Diplostomidae , Plagiorchiidae , schistosomatidae , Azygiidae, Notocotyidae and Strigeidae (Faltynkova and Hass,2006).C.T by

2.10.4- Melanoides species: Belong to Order Sorbeococh, Family Thiaridae M. tuberculata (Muller, 1774), is now distributed in all Neotropical region. It was reported for the first time in Brazil in 1984 (Vaz et al., 1986), Introduced in some areas as

30 a competitor of the native snail Biomphalaria glabrata, which is a host of

Schistosoma mansoni (Pointier, 2001; Pointier,.et al 1993) and it Reduces the number of (Schistosoma mansoni) snail hosts present in the community. M. tuberculata is known to be the intermediate host of several species of the eye fluke which has agymnocephlous type of cercariae. In Venezuela, Diaz et al. (2002, 2008) described a gymnocephalous cercaria of Philophthalmus gralli which utilizes M. tuberculata as intermediate host. M.tuberculata Has been recorded for the first time as the first intermediate host of Echinochasmus japonicus and its natural infection rate was found to be 1.1% (Cheng and Fang, 1989), in Iran Farhanak et al., (2005) also reported Echinostomecercariae From Melanoides tuberculata.These snails also collected from ponds around the Ninh Binh province, Vietnam settlements and were infected with cercariae of Clonorchis scinensis, the causative agent of human clonorchiasis, at a rate of 13.3% (Kino et al., 1998). Furthermore M.tuberculata-associated larval stages of Philophthalmus have been recorded from Jordan (Dimitrov et al., 2000). Xiphidiocercariae are described from M. tuberculata which collected from Giza and Qualiobyia (Wanas et al., 1993). M. tuberculata populations can harbor viable infections and infect birds by cercaria which could cause cercarial dermatitis in human (Barber and Caira, 1995). 2.10.5- Cleopatra species: Cleopatra species (Troshel, 1857) are a genus of freshwater snails with an operculum; belong to the super-family Cerithoidea, family Paludomidae within the subfamily Cleoptrinae. The habitat of species in this genus includes slow-running freshwater stream, Cleopatra Serve as an intermediate host for Prohemistomum vivax which infect dogs, cats and kittens in Egypt and occasionally infect man (Wykoff et al., 1965).

2.11.6- Lanistus species: They belong to order Architaenioglossa, family Ampullariidae, this species is known from Egypt, Ethiopia, Uganda and Kenya. They are probably found throughout the Nile, including Lake Nasser (Ibrahim et al., 1999).They occur in southern Sudan, southeast Ethiopia and Uebi and Giuba rivers in south Somalia (Brown, 1994).They live in standing and slow flowing fresh water with rich vegetation, and have been incriminated for nematode rat- worms (Angiostrongylus cantonensis in Egypt (Yousif ; Ibrahim, 1978). In Kenya Lanistus purpureus was found infected with trematode cercariae (Kariuki, et al, 2004), and also there was a record of non-furcocercous cercariae of Lanistus carinatus. Lanistus carinatus is considered as a biological agent against Bilharzia hosts. 2.11- Snail's life cycle:

The hermaphroditic or parthenogenetic reproductive habits of many snails favor their successful introduction into new locations (Jarne and Stadler, 1995). In all species of snails eggs are laid at intervals in batches of 5–40, each batch surrounded by a mass of jelly-like material. The young snails hatch after 6–8 days and reach maturity in 4–7 weeks, depending on the species and environmental conditions, temperature and food availability are among the most important limiting factors, a snail lives more than year and lays up to 1000 eggs during its life (WHO, 1997).

2.12- Snail's ecology: In most areas, seasonal changes in rainfall, water level and temperature cause marked fluctuations in snail population densities and transmission rates. Snail habitats include almost all types of freshwater bodies ranging from small temporary ponds and streams to large lakes and rivers. Within each habitat, snail has focal distribution and their density is varying seasonally. In general, the aquatic snail hosts of schistosomes occur in

32 shallow water near the shores of lakes, ponds, marshes, streams and irrigation channels (Malek, 1985), they are most common in waters with organic matters such as faeces and urine, Plants serve as substrates for feeding and oviposition as well as providing protection from high water velocities and predators. Snail intermediate hosts of Schistosoma haematobium and S. mansoni are tolerant to physical and chemical conditions, sunlight and relatively high water temperatures affect the snail populations because bacterial decomposition processes may result in reduced oxygen tension. In the irrigation schemes, stream velocities in canals are considered a crucial issue in relation to schistosomiasis transmission (Boelee ; Madsen, 2006). Presence and density of snails differ highly among sites, and their variability within irrigated areas, in most instances, is related to the canal type, distance of sites from the canal off-take, and the composition and density of aquatic vegetation (Appleton 1978; Madsen 1996a; Khallaayoune et al. 1998). A major part of this variability, however, is also due to specific local conditions such as temporary water stagnation, water depth, shading, and density and composition of aquatic vegetation, all these can be influenced by human Interventions, like cleaning and maintenance works, removing the aquatic macrophytes, the application of pesticides and temporary drying out of canals and structures (Boelee ; Madsen, 2006). 2.13- Environmental factors affecting snails' population:

Limited ecological studies on actual or suspected vectors of bilharziasis have been undertaken by Cridland (1957, 1958) in Uganda, Webbe and Msangi (1958) and Webbe (1960) in Tanganyika. , McClelland (1956) and Teesdale and Nelson (1958) in Kenya, and Malek (1958) and Madsen (1985, 1988) in Sudan. Environmental factors affecting snails are include physical, chemical, and biological factors, each of these factors have been studied to evaluate their effect on snail population:

2.13.1- Physical factors:

2.13.1.1- Temperature:

All schistosome's intermediate hosts are tolerating themselves to the temperature variation of their habitat, the effect of the annual range of temperature upon the whole breeding season affect snail reproduction and their generation over the year. The optimum temperature for mollusks between 22°C and 26°C with an average 18°C per year and the snail ability for partly dehydration during hibernation or aestivation make them able to withstand extremes of temperature (WHO, 1956). In southern and central Sudan the schistosomiasis hosts can withstand seasonal and diurnal temperature (Malek, 1958). Malek (1958) suggested that temperature was not a limiting factor in the distribution of bilharziasis vectors in the Sudan although he concluded that temperature does influence their rate of reproduction. Gaud (1958) found that temperature was important but not the only factor influencing the seasonal rhythms of snails. 2.13.1.2- Sun light:

The light intensity has an indirect effect on snails by increasing the microflora (food source) and high dissolved-oxygen content, as it was proved by (Babiker, 1985), in Sudan when turbidity decreased seasonally, snail growth increased by the indirect effect of sun light which is flourished the aquatic plants. In Gezira irrigated area the majority of canals are almost without shade so snails are concentrating on the aquatic weeds and grass on edges, although they need light, some species like Bulinus truncatus can breed in complete darkness for five months under laboratory condition (Malek, 1958).

2.13.1.3- Water current velocity:

The water movement is important as it is motivating oxygenation of the habitat; schistosome intermediate hosts prefer stagnant or slow running water. Larger species and older snails are most affected by current than small species and younger snails. Some species do colonize in fast running water and that probably referred to the different geographical races show different resistance to the current, observations conclude that Biomphalaria pfeifferri are less able to withstand rapid flowing water than Bulinus trancatus, that explain the only record of Biomphalaria of the Blue Nile was by Archibald (1933), and it hasn't been reported since then. Biomphalaria ruppellii, Biomphalaria sudanica and Bulinus ugandae have been reported from slowly flowing White Nile upstream in Jebel Awllya dam (Malek, 1950). High water velocity after heavy rains may sweep away snails food-microflora- (Marti, 1986).

2.13.1.4- Turbidity:

The effect of turbidity is difficult to assess, short periods of high turbidity, such as occur during flooding, have no adverse effect upon snails, however, prolonged turbidity will probably affect the growth of algae on which snails are apparently dependent for their proper development (De Mellion et al, 1958).According to Welch, (1952) all natural waters show some degree of turbidity, resulting from rich plankton growth and organic matters suspension. According to Madsen (1990) turbidity alone doesn't affect snail population, although Myer- Lessen et al., (1994) proofed that clear water is essential for the presence of fresh water snails, where adult and juvenile are not affected by turbidity, the egg masses become unviable because of slit decomposition.

2.13.1.5-Water depth:

Schistosomiasis intermediate hosts are occurring in shallow water near shores, amphibious species occur on moist soil, water plants and surfaces near water (WHO, 1997). Under natural condition it is rare to find snails in depth exceeding 1.5-2 meters, which mainly associated with food and shelter availability near the surfaces. Experiments with Bulinus trancatus showed that they exist for periods of time at a depth of 10 meters, snail species which live near the bottom or in deeper water are of less importance for schistosomiasis transmission (WHO, 1956).

2.13.2- Chemical factors:

2.13.2.1- Salinity:

It refer to the total salt content in fresh waters and it can be measured by the sodium chloride proportion which is low in most inland waters, the total amount of dissolved solids or the total salinity of the water is less importance than the proportions of the constituent salts. Different snail species have different degree of salt tolerance thus Bulinus truncatus is live in less than 4000 parts per million of salt in the water. Calcium is required for the shell formation is taken mainly with food, since its concentration in water is very low many researchers found that the variation which occur in natural habitat without effect on snail development and life cycle although its affect algae composition (Madsen and Christensen, 1992). Copper, barium, nickel and zinc salts are toxic to snails causing distress or even death, but sudden rises in chloride content even if it is happen for long intervals of time may be lethal for eggs or young snails (WHO, 1956). According to Levesque et al., (1978) the egg laying is decline when the concentration of NaCl, MgCl2, and KCl belw specific level.

2.13.2.2- Hydrogen-ion concentration (pH):

The intermediate schistosomiasis hosts are tolerant for wide range of pH between 4.8 to 9.8 within this variation there is no effect on snails density (Malek, 1958; Madsen, 1985). pH is affected by different environmental factors such as vegetation, chemical composition of water, sun light, carbon dioxide content, and substratum matters (WHO, 1956).

2.13.2.3- Dissolved oxygen:

Pulmonates snail have lungs enable them to use atmospheric oxygen for respiration when they come to the surfaces, and they use their mantles for respiration when they are submerged. Snails depend more on oxygen dissolved in water than atmospheric oxygen but low oxygen concentration reduce movement, feeding and reproduction, in Sudan the dissolved oxygen averaged between 4.7 to 7 p.p.m (Malek, 1958).

2.13.2.4- Pollution:

Water contamination causes by decaying organic matters of animal or vegetation origin, urine and feces excreta, or even industrial wastes; such polluted habitat is favorable for intermediate hosts, decaying organic matters in canal waters have strong relation with snail occurrence, Chemical pollution from discharged industrial wastes has lethal effect on snail population (WHO, 1956). Pollution has an indirect effect on vegetation by reducing the oxygen supply which alters the nature of the bottom substratum. Although polluted water may harbor bilharziasis actual and potential hosts as it reported in the White Nile bank at kosti (Malek,1950),many perennial streams between Kalkuting and Niertiti, In Jabel Marra western Sudan Biomphalaria pfeifferi and Bulinus spp. were found in heavily polluted water with human and animal excrement, but the water has well flow (Malek, 1950 ).

2.13.3- Biological factors:

The effect of fauna and flora upon snail's population is very powerful, the relationships of snails with animals include commensalism, symbionts, predation and parasitism, while the associated plants are aquatic weeds and microflora. Many vertebrates and invertebrates attack snails; also ecto-parasites and endo-parasites, fungi, viruses, bacteria, digenic trematodes and leeches.

Coleoptera of the family Dytiscidae and the dragon fly nymphs prey on aquatic snail egg masses (Taylor, 1894; cooke, 1913), some snail species act as competitors or predators like Marisa cornuariet , pila spp. ,Lanistus spp., also fishes like Gambusia affinis , Lebistes reteculatus can prey on snails just like some birds species such as open-bill stork (Anastomus lamelligerus) ,wood rail (Himantornis haematopus) ,the fin foot (Podica senegalensis) ,and certain species of ducks (Pteronetta hartlaubi) (WHO, 1956; Malek, 1958).

2.13.3.1- Aquatic weeds:

The aquatic fauna may be free-flouting, totally emerged or sub-merged; broad leaf plants provide suitable surface for egg deposition and protect snails of sun light. In canals of Gezira irrigated area Andrews in 1945 reported Spirogyra decina and S.maxima as flouting plants while the important plants near the banks were Cyperus, Vossia, Phragmites and Echinochloa. In the Gezira irrigation scheme in Sudan Hilali et al. (1985) found that snails were often associated with aquatic macrophytes. A similar association was observed by Madsen et al. (1988). 2.13.4- Seasonal and climatic changes:

Rain fall cycles affect snail life cycle by altering water level which has a reflection on population size ,humidity , temperature and all of this elements are collaborated tgether leading in snail population fluctuation

(WHO,1956). Canals of the Gezira scheme support snails where there is a diurnal rise or fall of 20-30 cm, and Malek (1958) points out that small gradual change of water level do not seem to disturb snails.

2.14- Snails control measures:

Snail control is an important preventive measure in an integrated approach to control schistosomiasis transmission (Madsen and Christensen, 1992; Sturrock, 2001).The major method used for snails control in fresh water habitat regarding snail-water borne disease to reduce exposure to trematode cercariae are molluscicide, environmental measures, and bio-agent (WHO, 1993), this is also applicable for areas where the transmission is focal and the water amount is small (Madsen, 1992). Snails control play a significant role in morbidity control programmes for maintaining low level morbidity achieved by chemotherapy (Christensen et al., 1987). Alternative approaches include not only modified techniques and strategies for molluscicide application, but also environmental control measures and biological interventions to reduce the application of Niclosamide and to provide more long-lasting effects.

2.14.1- Chemical control: The most effective method of achieving significant reductions in snail population density is the use of molluscicides such as niclosamide, to terminate aquatic snails, focal molluscicide application, which is implemented seasonally according to the transmission pattern, is the most recommended approach to snail control (Klumpp and Chu, 1987), but for its passive environmental impact, makes it necessary to develop new approaches to snail control. This is more important in areas where irrigation water is used intensively for fishing, livestock, domestic purposes and even drinking (Yoder, 1983; van der Hoek et al., 1999,

2001, 2002; Boelee and Laamrani 2004; Nguyen-Khoa et al., 2005). The beneficial applied molluscicide has to be highly specific for snails, non- toxic to fauna and flora, remain effective for long period at low concentration and easy to apply in the field. In Gezira state they started with copper sulphate in 1969, but they substitute it due to its negative effect on fishes, aquatic vegetation and vertebrate fauna, the alternative was Sodium Pentachlorophorate which is also found to be unsuitable, so they come-up with Niclosamide which the only effective choice till now with the great concern upon snail resistance (WHO, 1998) against Niclosamide. Researchers concentrated on molluscicide of plant origin to avoid negative environmental impact and sucssesful triles were accomplished allover the world.

2.14.2- Environmental control: According to Bradley and Webbe (1978) the benefits of environmental control is its persistent effect without continuous re-application. Basically rely on habitat modification by acceleration of water current, fluctuate water levels, vegetation clearance and intermittent irrigation periods to make it unfavorable for snail reproduction and occurrence, the removal of vegetation manually or mechanically diminished the food resources for snails to a higher degree (Jordan and Webbe., 1993). In addition, as vegetation once removed takes a long time to re-grow, it had a direct impact in minimizing snail populations. Also, aquatic snails attached to the vegetation will be removed (Allam, 2000).The pond filling, drainage water level fluctuation in irrigation canals are effective in fresh water snail control (Madsen and Christensen, 1992), as well as stream straightening, Deeping of marginal areas of irrigation canals, and increasing the water velocity (WHO, 1993). In the Rahad irrigation scheme in the Sudan Meyer-Lassen (1992) observed the disappearance of Bulinus truncatus after the removal of aquatic plants. In contrast, Hilali et

40 al., (1985) reported less impressive results from the Gezira irrigation scheme, where mechanical weed removal alone did not lead to a significant reduction of the snail population, because of rapid re- colonization of the canals by weeds. Environmental measures should be considered in the preparation phase of new irrigation projects (Madsen, 1990), such impressive outcome of snail reduction was accomplished in Japan (WHO, 1991), china (Beijing, 1998), Morocco (Boelee and Laamarni, 2004). 2.14.3- Biological control: Friendly-environmental approach has to develop after the negative result of molluscicide by usng predators and competitors organisms against snails. The use of Marisa cornuarietis in Puerto Rico and Pomacea haustrum in Brazil, also Barbosa (1973) reported that introduction of Biom. straminea into habitat of Biom. glabrata eliminate them after three years. Introduction of predators in snail habitat responsible for high mortality of young Lymnae elodes (Eisenberg, 1970; Hopkins, 1973). Fishes also are used as bio-control agent in the countries of eastern mediterinain region, Somalia and Sudan. WHO (2003) lists 34 fish species, Tilapia melanopleura, Astronotus ocellatus (Feitosa and Milward, 1986), aquatic birds like ducks (Michelson, 1957), chelonian (Coelho et al., 1975) are also recorded as snail predators. Dragon fly nymphs, giant water beetles, are serving as snail predators, in laboratory observations leeches, ostracods crustacean found to be snail predator (Guimar et al., 1983). There are only few examples of successful biological control of schistosome intermediate hosts, and they have been applied only on limited scale (Pointier and Giboda 1999). The focus of biological control has been on the introduction of a non-susceptible snail species that may act as competitors of the intermediate hosts (Madsen, 1990, 1996b). One promising component of biological control is the introduction of parasite

41 resistant snails into endemic areas to replace the resident susceptible snails and avoid the often destructive changes to the local ecosystem that accompany other methods of snail control (Sturrock, 2001). 2.14.5- Integrated snail control measures:

This approach is define by the WHO as a rational decision-making process for the optimal use of resources for vector control, based on that the combination between environmental, bio-gents and chemical approaches has to give an exceptional result in snail elimination (McCullough and Mott, 1983) such progression was achieved in Philippines (Santos, 1984), Egypt (Mobarak, 1982), Brazil (Katz et al., 1980) and China (Chu et al., 1981).

CHAPTER THREE

MATERIALS AND METHODS

3.1- Study area:

Gezira state is one of the most agricultural states in Sudan. It lies between the Blue Nile and the White Nile rivers. It is bounded by Khartoum state in the north, Gedarif state in the east, White Nile state in the west and Sennar state in the south.It lies in the rich savanna region between latitude 13°-15.2° N and longitude 32.5°-34° E. The area has hot dry summer from April to June with an average temperature 32°C and relative humidity 20%, and the cool dry winter from December to march with average temperature 22°C and relative humidity 30%. The rainy season start in the late June and end in October.

3.2- The selected canal:

This study was conducted in Barakat area, the selected canal is a minor canal, and known site of infected communities with schistosomiasis .the water contact of both human and animals was observed during the study, one section with 200 meter length is divided into ten sites each of it measured 20 meter was selected to conduct the study.

Plate (4): The selected study area (Barakat canal)

3.3- Malacological survey:-

3.3.1- Snails sampling:

Three surveys were conducted from June-July (2011) in the selected section of the canal .In each site snails were collected by scooping method using flat wire-mesh of metal frame (40X30cm) supporting a mesh of 1.5 micro-size attached to an iron handle of 1.5meter long as described by (Amin, 1972), by taking 10-15 dips/site, start with the edge then scraping the bottom and vegetation. The collected samples were put in jars containing water of the canal and transported to BNNICD laboratory for sorting out and identification. Samples of the emerged, sub-emerged and floating vegetation was taken and transported to the Faculty of Agriculture, Department of crop protection, University of Gezira for identification.

3.4- Ecological factors monitoring: The main ecological factors influencing snail populations were monitored on the three surveys, measuring water temperature, water depth, water pH and observation of water turbidity, and velocity. Also the presence of predators, vegetation density and types were recorded. 3.4.1- Water temperature: The temperature was measured per site using mercury thermometer (0- 50°C) by immersing it for 15 minute under the water surface. 3.4.2- Water depth: A two meters wooden stick was immersed twice in the water to measure the depth, and then the average was calculated. 3.4.3- Water turbidity: It was observed at the canal and determined as low, medium or high turbid water according to the colour. 3.4.4- Water velocity: It was observed and recorded as stagnant, slow, medium, and fast running current. 3.4.5- Water pH: A few volume of the water was taken from each site in separate labeled bottle then transported to the soil laboratory of Gezira University to measurement using pH-meter. 3.4.6- Vegetation types and density: The density of plants was observed and reported as sparse where there is little amount, low, medium and thick, and then the vegetation samples were taken from the canal and transported to the University Of Gezira, Department of crop protection for species identification. 3.4.7- Presence of predators, animals and birds: Predators species presence was recorded, and the animals (cows, goats, others) and birds in contact with the canal at the collection time were recorded.

3.5- Laboratory work: 3.5.1- Snails identification: The collected snails identified to the genus and species level, guided by Mandle-Barth (1962) key to the identification of East and Central African Fresh water snail of medical and veterinary importance based on morphological features only.

3.5.2 - Natural infection of the snails (Snail screening for cercaria):

The collected identified snails were examined for trematode cercariae presence, 3-7 snails of the same species were put in 5ml tap water in glass bottles, and then exposed to artificial light for 3-5 hours (Webbe and Sturrock, 1964), the presence of trematode cercaria was confirmed under dissecting microscope and some iodine drops were added to kill and stain cercariae for identification. The cercariae were preserved in 5%formaldhyde and 70%ethanol for identification following the Danish Bilharzia laboratory key developed by Frandsen and Christensen (1984).

3.6 - Data analysis:

The ANOVA test was applied to investigate the significance relationship between snail species density in each survey, and the correlation test used to signify the relation of density and environmental factors.

CHAPTER FOUR

RESULTS

4.1- Identification of snails: Various snail species were found in the canal during the study period, with total 1540 in the three surveys and their overall prevalence was Cleopatra bulimoides 663 (43%), lymnaea natalensis 271 (17.9%), Bulinus truncates 211 (14%), Biomphalaria pfeifferi 206 (13%), Melanoides tuberculata 104 (7%), Lanistus carinatus 82 (5%), and Bulinus forskalii 3 (0.1%).

50 45 40 35 30 25

20 Density Density % 15 Density % 10 5 0

Snail species

Figure (1): The overall density of snail species in the three surveys in

Barakat canal during June-July 2011

c* 60

b* Survey 1 % 40 Survey 2 a* 30 Survey 3

a* a Density

20 a,b b b* b* b* a a. 10 a* b* a b a a* a a a 0

Snail species

Figure (2): Snail Species density in the three surveysin Barakat canal during June-July 2011

*= significance difference P>0.05

4.1.1- Cleopatra bulimoides : plate (2)

The results Figure (2) revealed that the density at first was the lowest (25%), which statistically differ from surveys 2 (40%) and 3 (60%) at

(F=10.570, df (2,27); P <0.05).

Plate (2): Cleopatra bulimoides (Olivier, 1804)

4.1.2- lymnaea natalensis: plate (3)

The density in survey 1 (33%) and survey 2 (15%) showed no significant differences (Figure 2), while there is such difference between survey 1 and survey 3 (9%) at (F=4.505, df (2,27) ; P<0.05).

Plate (3): Lymnaea natalensis (Krauss, 1848)

4.1.3- Bulinus truncatus: plate (4)

Statistical analysis showed a significant difference (F= 9.734, df (2, 27); P <0.05) between density in survey 1 (8%) compared to survey 2 (16%) and survey 3 (15%), (Figure 2).

Plate (4): Bulinus truncatus (Audouin, 1827)

4.1.4-Biomphalaria pfeifferi: plate (5)

The results in (Figure 2) showed that there is no significant variation between survey 1 (26%), surveys 2 (9%) and 3 (8.9%) respectively, at

(F=2.743), df (2,27); P>0.05).

Plate (5): Biomphalaria pfeifferi (Krauss, 1848)

4.1.5-Melanoides tuberculata: plate (6)

There was no significant difference (P> 0.05) between the third survey which showed the lowest density (2%) and the first one (4%) (Figure 2), while there is a difference between their density and the second survey

(12%) at (F=7.341,df (2,27) ; P<0.05).

Plate (6): Melanoides tuberculata (Muller, 1774)

4.1.6-Lanistus carinatus: plate (7)

Although the density of this snail in the three surveys wasn't vary too much and it was arranged as 3%, 7%, and 5% (Figure 2), but statistical analysis revealed a significant difference at (F=3.609, df (2,27); P<0.05).

Plate (7): Lanistus carinatus (Olivier, 1804)

4.1.7-Bulinus forskalii: plate (8)

This species had the lowest density compared to other species, not found in the first survey and had low density in the others (0.01% and 1%) (Figure2). The statistical analysis revealed there is no significant difference between all surveys at (F=0.600, df (2,27) ; P>0.05).

Plate (8): Bulinus forskalii (Ehrenberg, 1831)

4.2- Snail density in relation to the different ecological conditions in the three surveys:

4.2.1- Survey (1):

Result in Figure (3) showed that the highest density was recorded for Lymnaea nataleinsis and decreased to the Lanistus carinatus, with no record for Bulinus forskalii, with overall density 414 (27%) which is the lowest compared to other surveys P>0.05.

35 30 25 20

15 Density Density % 10 5 0

Snail species

Figure (3): The snail species density in survey (1) after canal re-filling in Barakat canal (June-July 2011).

4.2.2- Survey (2):

The highest density was for Cleopatra bulimoides and the lowest was for Bulinus forskalii with a harmonize abundance for the seven species Figure (4), survey 2 also reported the highest density among the three surveys 575 (37%).

45 40 35 30 25

20 Density % Density 15 10 5 0

Snail species

Figure (4): The snail species density in survey (2) after vegetation clearing in Barakat canal (June-July 2011).

4.2.3- Survey (3):

The density of Cleopatra bulimoides was the highest in survey three with low density of Melanoides tuberculata, Lymnaea nataleinsis and Bulinus forskalii Figure (5).The overall survey density 551 (36%).

30 Density % Density 20

Snail species

Figure (5): The snail species density in survey (3) after vegetation growth in Barakat canal (June-July 2011).

4.3- Environmental factors effect:

4.3.1- Water temperature: Figure (6) reveal that the water temperature in survey 1 ranged between (25 -27 °C) with average 26.4 °C, in survey 2 the range between (26.5-26 °C) with average 26.1 °C, while in the third survey the measured temperature was 27 °C in all sites. Statistical analysis revealed that there is no correlation between water temperature and snail density (r = 0.052) with no significance difference P>0.05.

27.2 27 26.8 26.6 26.4

Average 26.2 Temp 26 25.8 25.6 Survey 1 Survey 2 Survey 3

Figure (6): Average of water temperature in the three surveys conducted in Barakat canal (June-July 2011).

4.3.2- Water depth:

It fluctuated during the three surveys, it was found at first survey (50cm), while at survey 2 (43cm), and at the last survey (42.3cm), Figure (7), statistically there is a negative correlation (r = -0.904) with significance P< 0.01 between the depth and total density.

Average 44 water depth 42

38 survey1 survey2 survey3

Figure (7): Average of water depth in the three surveys conducted in

Barakat area during June-July 2011

4.3.3-Water PH:

The water pH varied during the three surveys, although it showed a degree of alkalinity. The pH during survey 1 ranged between (8.2-7.7) with average (7.9), in survey 2 the range was (7.4-7.7) with average (7.59), whereas at the third survey the range was between (7.3-7.9) with average (7.68) (Figure 8). The statistical analysis proved there is a significant moderate negative correlation between pH and total snail density when (r = -0.531, P>0.001).

7.9

7.8

7.7

Average PH 7.6

7.5

7.4 survey1 survey2 survey3

Figure (8): Average of water pH in the three surveys conducted in

Barakat canal (June-July 2011).

4.3.4- Water turbidity:

The observation in (Table 2) revealed that the turbidity had noticeable variation. During survey (1) the water was almost clear, very turbid at survey (2) after canal cleaning and moderate turbid at survey (3).

Table (2): The observed Turbidity during the study period in

Barakat canal in the three surveys.

Site Water turbidity N0. Survey 1 Survey 2 Survey 3 1 Low Mid High 2 High(Algae) High High 3 Low High High 4 Mid High Mid 5 Mid High Mid 6 High(Algae) High Mid 7 High(Algae) High Mid 8 Mid High Mid 9 Mid High Mid 10 Mid High Mid

4.3.5- Water velocity:

At the three surveys the water current wasn’t run very fast but it was ranged between low flowing and medium (Table 3).

Table (3): The observed velocity during the study period in Barakat canal in the three surveys.

Site Water velocity N0. Survey 1 Survey 2 Survey 3 1 Stagnant Slow Mid 2 Stagnant Slow Mid 3 Stagnant Slow Mid 4 Stagnant Slow Mid 5 Stagnant Slow Mid 6 Slow Slow Stagnant 7 Slow Slow Mid 8 Slow Slow Stagnant 9 Stagnant Slow Mid 10 Stagnant Slow Mid

4.3.6- Vegetation density and vegetation type:

The result in Table (4) illustrated the vegetation density which showed a clear variation in surveys and sites ranging from sparse, medium to thick vegetation. The dominant plant species found in the study period are illustrated in Table (5) showed only the scientific and common names of the observed plant, pistia stratiotes, Alternanthera philoxeroides, Pediastrum boryanum, Cynodon dactylon, Ipomoea hildebrandtii, Potamogeton crispus, Ocimum basilicum, Ziziphus spinachristi, Calotropis procera, Acacia nilotica, and Azaridechta indica.

Table (4): The observed Vegetation density during the study period in Barakat canal in the three surveys.

Site Vegetation density N0. Survey 1 Survey 2 Survey 3 1 Thick Sparse Sparse 2 Thick Sparse Mid 3 Thick Sparse Thick 4 Thick Sparse Mid 5 Thick Sparse Mid 6 Sparse Mid Mid 7 Thick Mid Sparse 8 Thick Mid Sparse 9 Mid Mid Sparse 10 Mid Mid Sparse

Table (5): Types of vegetation in Barakat canal during the study period

Scientific name Common name Growth status Cynodon dactylon Bermuda grass Sub-emerged Ipomoea hildebrandtii The Nile Ipomoea at the edge Acacia nilotica Arabic gum at the edge Ziziphus spina-christi The christs thorn jujube at the edge Ocimum basilicum Basil bee at the edge Calotropis procera Rubber bush at the edge Pistia stratiotes Water lettuce Floating Alternanthera philoxeroides Alligator weed Floating Potamogeton crispus Curlyleaf pond weed Emerged Azaridechta indica Neem at the edge Pediastrum boryanum Algae floating

4.3.7- Presence of predators, animals and birds:

The most predators species found were water beetles, dragon fly nymphs, fishes, water bugs. The predator's density was lower at the third survey than the others. While the dominant animal species contact with the canal are sheep, goats, cows, and dogs with a record of reptile at the third survey. The observed birds were black horn, doves, and sparrows.

4.4- Snails and their natural infection rates:

Figure (9) illustrates the snail infection rates with some trematode cercariae in the three surveys. The overall natural infection rate of snail species was 49.1% and arranged as: Cleopatra bulimoides (65%), Bulinus truncatus (47%), lymnaea natalensis (43%), Melanoides tuberculata (42%), Biomphalaria pfeifferi (30%), Lanistus carinatus (9%), and Bulinus forskalii (0%).

30 infection rate%

Infectionrate % 20

Snail species

Figure (9): The overall infection rate of snail species in the three surveys during June-July 2011 in Barakat canal

4.4.1-The snail species specific infection rates: The variation in the snails infection rate in the three surveys is illustrated in Table (6) which demonstrated the infection rate in survey 1, survey 2, and in survey 3, the overall infection rate in each survey was 15%, 52%, and 79% respectively.

Table (6): The natural infection rate of the collected snail species with Trematode cercariae in the three surveys (June-July 2011)

Surveys Snail species Examined Infected Infection NO. snails snails rate% 1 C. bulimoides 88 28 32a* 2 C. bulimoides 125 84 67b* 3 C. bulimoides 122 106 87 b Total 335 218 65

1 B. truncatus 33 1 3a* 2 B. truncatus 63 28 44b* 3 B. truncatus 66 47 71b Total 162 76 47

1 Ly. natalensis 7 1 14a 2 Ly. natalensis 22 3 14 a* 3 Ly. natalensis 20 18 90 b Total 49 22 45

1 M. tuberculata 23 1 4 a* 2 M. tuberculata 62 31 50b 3 M. tuberculata 10 8 80 a* Total 95 40 42

1 Bio. pfeifferi 107 7 7 a 2 Bio. pfeifferi 34 19 56 a 3 Bio. pfeifferi 43 29 67 a Total 184 55 30

1 L. carinatus 1 1 100 a 2 L. carinatus 10 0 0.0 a 3 L. carinatus 0 0 0.0 a Total 11 1 9

1 B.forskalii 0 0 0.0 2 B.forskalii 1 0 0.0 3 B.forskalii 1 0 0.0 Total 2 0 0.0

*≡ significant difference at P<0.05

The total of examined snails = 838 The total of infected snails =412 The overall snail infection rate = (412/838) x100 =49.1%

4.5- Cercariae type and their specific infection rate among collected snails:

A total of nine different types of cercariae belonging to four morphotypes were shed by the snails Figure (10) is arranged as: Longi-furcate apharengeate Monostome cercaria LPM (45.5%), ArmateXiphidiocrcariae (19%), Type1Xiphidiocercariae (12%), VirgulateXiphidiocercariae (11.5%), Echinostomecercariae (8%), Plurolophocercouscercariae (3.5%), Type3Furcocercouscercariae (0.31%), Longi-furcate pharengeate Distome cercaria LPD (0.1%) and Type2 Furcocercouscercariae (0.09%).

50 45 40 35 30 25

20 Density% 15 10 5 0

Figure (10): The density of cercariae types in the three surveys conducted in Barakat canal (June-July2011).

4.5.1- The identified Cercariae types and their specific rate:

4.5.1.1- Longifurcate-Pharyngeat Monostome cercariae (LPM):

It belong to species of the family Cyathocotylidae (intestinal parasite of birds, mammals and reptiles), also called vivax cercariae. They have longifurcate tail, oral sucker is present (plates 9, 10, and 11). They develop in sporocyst, encyst in fish. These cercariae where found in the three surveys, with total density 1104 (45.5%) and cercariae rate 3.06 (Table 7), shed by Bulinus truncatus, Cleopatra bulimoides, Lymnaea natalensis, Lanistus carinatus and Melanoides tuberculata.

Table (7): The Vivax LPM type and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Longi-furcate Cleopatra bulimoides 207 723 aphyrngeate Biomphalaria pfeifferi 45 63 Monostome LPM Melanoides tuberculata 33 106 (vivax) Bulinus truncatus 53 45 Lymnaea natalensis 22 147 Lanistus carinatus 1 20 Total 362 1104

Plates (9, 10 and 11): Longifurcate-pharyngeate Monostome (LPM)

4.5.1.2-Armate Xiphidiocercariae:

Belong to family Plagiorchiidae (intestinal parasite of all vertebrates). They have un-forked tail, stylet is present (Plate 12). It develops in sporocyst, encyst in vertebrates –amphibians and reptiles. This type was shed by Bulinus truncatus, Cleopatra bulimoides and Melanoides tuberculata, their total density was 465 (19%) and cercariae rate 31 (Table 8).

Plate (12): Armate Xiphidiocercaria

Table (8): The Armate Xiphidiocercariae type and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Armate Cleopatra bulimoides 3 150 Xiphidiocercariae Biomphalaria pfeifferi 6 300 Melanoides tuberculata 6 15 Total 15 465

4.5.1.3-Un-described Xiphidiocercariae type (1):

It was shed by Biomphalaria pfeifferi and Cleopatra bulimoides, with density 300 (12%) and cercariae rate 50, (Table 9), they have a bubble capsule encounter the head and un-forked tail (Plate 13).

Plate (13): Un-described Xiphidiocercaria type (1)

Table (9): The undescribed Xiphidiocercariae type 1 and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Type (1) Cleopatra bulimoides 3 150 Xiphidiocercariae Biomphalaria pfeifferi 3 150 Total 6 300

4.5.1.4-Virgulate Xiphidiocercariae:

Tail without dorsoventral finfolds, the virgulate organ is present in the region of oral sucker; ventral sucker is smaller than oral sucker (Plate 14). They belong to family Lecithodendriidae which parasitize birds, amphibians and bats. This type was shed by Bulinus truncatus, with density 278 (11.5%) and cercariae rate 15.44 (Table 10).

Plate (14): Virgulate Xiphidiocercariae

Table (10): The Virgulate Xiphidiocercariae type and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Virgulate Bulinus truncatus 18 278 Xiphidiocercariae

4.5.1.5-Echinostome cercariae:

It belongs to family Echinostomatidae (parasite of birds). It have un- forked tail, ventral sucker on mid ventral surface of the body, no stylet, no eye spots, presence of spine collar around oral sucker (plate 15). It develops in redia, encyst in fish and amphibians. Shed by Bulinus truncatus and Melanoides tuberculata, with density 183 (8%) and cercariae rate 22.88 (Table 11).

Plate (15): Echinostomecercariae

Table (11): The Echinostome type and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Echinostomecercariae Melanoides tuberculata 1 170 Bulinus truncatus 7 13 Total 8 183

4.5.1.6-Pleurolophocercus cercariae:

They belong to family Heterophyidae which parasitized birds and mammals. The tail un-forked with well developed finfolds, ventral sucker is absent; eye spots present (Plates 16 and 17). They encyst in fish and amphibians' this type was shed by Melanoides tuberculata and Bulinus truncatus, with density 84 (3.5%) and cercariae rate 4.42 (Table 12).

Plates (16, 17): Pluerolophocercous cercariae

Table (12): The Plurolophocercous type and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Plurolophocercous Melanoides tuberculata 7 64 Bulinus truncatus 12 20 Total 19 84

4.5.1.7- Un-described forked tail cercariae (furcocercous) type (3):

It was shed by Biomphalaria pfeifferi, with density 7 (0.31%) and cerariae rate 1.17 (Table 13); they belong to furcated types with a forked tail (plate 18).

Plate (18): Un-described furcocercous cercariae type (3)

Table (13): The furcocercous cercariae type 3 and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Type (3) furcocercous Biomphalaria pfeifferi 6 7

4.5.1.8- Longifurcate-Pharyngeate distome cercariae (LPD):

The tail is longifurcate, no body and furcal finfolds, pharynx is present (Plate 19), called Strigida cercaria. it encyst in snails, tadpoles, reptiles and fish.produced by the families Strigeidae and Diplostomatidae which parsitised birds and mammals. Shed by Bulinus truncatus with density 3 (0.1%) and cercariae rate 3 (Table 14).

Plate (19): Longifurcate-Pharyngeate distome cercariae LPD Strigea

Table (14): The Strigea LPD cercariae type and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Longifurcate- Bulinus truncatus 1 3 Pharyngeate distome cercariae LPD(Strigea)

4.5.1.9- Un-described furcocercous cercariae type (2):

It belong to the furcocercouscercariae types (plate 20), it was shed only in survey three by Cleopatra bulimoides, with density 1 (0.09%) and cercariae rate 0.2 (Table 15).

Plate (20): Un-described furcocercous cercariae type (2)

Table (15): The furcocercouscercariae type 2 and its specific infection rate of snail species in the three surveys (June-July 2011) in Barakat canal:

Cercariae types Snail species No.of No. of infected cercariae snails Type (2) furcocercous Cleopatra bulimoides 5 1

4.5.2- The density of cercariae types in the three surveys:

The result in (Figure 11) demonstrate the density of each reported cercaria types during the study, such as the LPM type which appeared in the three surveys, 14%, 48%, and 38% respectively, the Armate Xiphidiocercariae reported only in the first 32% and second surveys 68%. The un-described Xiphidiocercariae type1 was only recorded in the first survey 100% the same as the virgulate Xiphidiocercariae which was reported in the third survey only with density 100%. In the third survey Echinostomcercariae appeared with density 100%. Plurolophocercous type reported in survey 1 (76%) and 2 (24%).At the third survey the furcocercous type 2 and type 3 were recorded with density 100% for each. The Strigae cercariae which appeared in survey 1 had density (100%).

120

100

60 survey 1 survey 2 40 survey 3

Figure (12): The density of cercariae types in the three surveys in Barakat canal 2011

CHAPTER FIVE

DISCUSSION

The agricultural sector development in Sudan has a huge role in provision of ideal habitat for snail breeding and snail-water borne diseases progression. Water-associated diseases are debilitating and seriously reduce productivity of agricultural communities in the Sudan (El Tash, 2005). Studies on larval trematode infection in freshwater snails in Africa are modest in number to provide information about, snail-borne diseases, larval trematode fauna and the potential transmission sites. The international health community recapitulated that there is a serious gap of information in biomedical and epidemiological aspects of water borne diseases vectors information, causative agents and disease determinants, to evaluate the trematode effect on snail populations (Ibrahim, 2007).

Human changes to freshwater habitats, such as the construction of small impoundments, have been implicated in facilitating snail populations and creating environments conducive to trematode infection (Johnson, et al., 2002). This study was conducted in Barakat canal during the hot season (April- July). Three surveys (two in June and one in July) were carried; each survey has its unique ecological condition. Before the first survey the canal was dry for approximately three months, so snail sampling was started after three weeks of the canal re-fill, the habitat was enriched with floating, sub-emerged, emerged vegetation and small bushes around the canal. At the second survey the canal was cleaned by scrapping all the vegetation except a few sub-emerged plants (Cynodon dactylon), so the canal was almost clear of plants and highly turbid, where at the last one

78 the canal re-flourished with vegetation but in low density compared to the first one and the water was almost turbid.

The collected snail species during the study period were identified to the species level and the fluctuation in their overall density was Cleopatra bulimoides (43%), lymnaea natalensis (17.9%), Bulinus truncatus (14%), Biomphalaria pfeifferi (13%), Melanoides tuberculata (7%), Lanistus carinatus (5%), and Bulinus forskalii (0.1%), after a period of drought only tolerant snails that are capable of withstanding desiccation and aestivate are re-appear when rain is fall or canal opening, also intermittent irrigation may dry out snails regularly and arrest their reproduction, this is likely to create a negative effect on the snail population density as well. Intermittent irrigation can promote the passive transport of snails, as these may be attached to loose debris when irrigation resumes (Boelee et al.,1997), so that explain why the lowest snail density was in the first survey (27 %). The low density (0.1%) of Bulinus forskalii might refer to aestivatation, although this species has widespread but sporadic distribution in every type of habitat. It is depending on rainfall to increase in density, this finding are similar to those reported by Cridland (1957) in Uganda and Teesdale (1962) in Kenya when they reported the occurrences of B. forskalii after rain fall. Environmental factors effect on freshwater pulmonates have been studied by a few researchers in Africa, the chemical, physical, and biological factors determine the species diversity and snail density to be found in a particular water body at a given period of time, the ecological conditions for transmission of schistosomiasis in favorable snail habitats can vary considerably from site to site and area to area, even within short distances (Pesigan, 1958).Various species react similarly to the same environmental influence, their ecological requirements being qualitatively similar but

79 quantitatively different, this applies to most African intermediate hosts of bilharziasis, and it is very difficult to identify and estimate the importance of an individual environmental factor whether physical, chemical or biological-when all may be mutually affecting one another and their combined effect influencing a certain species as recorded by (Webbe, 1962) in Tanganyika.

The present study only emphasized on part of the environmental factors such as water depth, water temperature, water pH, turbidity, velocity, vegetation density and type. The water temperature was varied among surveys with no correlation to snail density (r = 0.059 ,P>0.05), it had narrow fixed range between 25°C- 27°C in each survey/ site and that is the optimum temperature for mollusks which is between 25°C- 26°C, in such case the density couldn't get affected, that contract what reported by Malek (1958); Gaud (1958) and Teesdale (1962) which investigations shows that in small water bodies, a constant high temperature can reduce the population, and this through the reduction of dissolved oxygen and volume of the water. Several studies have revealed facts about pH as an important and limiting factor of the freshwater pulmonate snails distribution (Madsen, 1985; Teesdale, 1962; Malek, 1958) according to them the pH itself is not a limiting factor, but its correlation with other more important factors, as the availability to substratum, sunlight, and carbon dioxide has more influence on the distribution (WHO, 1956), in South Africa De Mellion et al, (1958) reported PH ranged 7.0 - 8.2 which contracted this study, pH has a significant negative correlation with the snail density (r = -0.531, P>0.001), in all surveys the water was alkaline with PH range 7.3-8.2 and that may related to organic pollution, due to the increase in phosphate and ammonium salts as reported by (Feema, 1981). The water depth has no effect on snails except for those places where a sudden marked rise or fall resulted in the flushing out of the

80 habitat, there appeared to be little correlation between water fluctuations and snail populations Teesdale (1962) and Malek (1958) points out that small gradual changes of water level do not seem to disturb snails in the canals of the Gezira scheme where there is a diurnal rise or fall of 20-30 cm, and that contract this study findings where there is anegative but significant correlation between water depth and snails density (r = -0.904, P>0.01). Although the minor canals like Barakat canal may appear homogeneous there is marked variation in physical and chemical conditions of the water, this variation related to the density and composition of the aquatic plants (Madsen, 1988). The snail density in the second survey was the highest (37%) despite of the vegetation removal, Bulinus truncatus density was (16%) in the second survey and that contract with Hilali et al. (1985) who reported high snail density in Gezira irrigation scheme, where mechanical weed removal alone did not lead to a significant reduction of the snail population, because of rapid re-colonization of the canals by weeds and intensified cleaning of hydraulic structures led to a short-term reduction in density and snail populations rapidly re-colonized the habitats (Laamrani et al.2000), in contrast, Meyer-Lassen (1992) observed the disappearance of B. truncatus after the removal of aquatic plants in the Rahad irrigation scheme. Frequently, very high densities of snails can be found at the starting point of low-order canals (Boelee et al.1997), this could be due to snails being washed into such canals and their subsequent upstream migration, in the study period the canal current was stagnant to slow run so the density wasn't affected , although in the second survey canal cleaning and sediment removal reduce resistance and could increase water flow velocity (Oomen et al.1990), Often, there is a relationship between water flow velocities and the location of breeding sites in an irrigation system,

81 more snails are found at sites with low-flow velocities (Boelee; Madsen, 2006). As reviewed in the literature (Williams and Hunter, 1967) reported that all species prefer thick plants, in contrast of that the observed snail density in different sites when some species get their maximum density in sparse vegetation sites and the same species entirely disappeared in sites with medium or thick vegetation density like Ly.nataleinsis, Bio.Pfeifferi, C.bulimoides and M.tuberculata, that proved Watson (1950) observation that water plants are desirable but not an essential feature of the habitat controlling snails density. The observed plants during the study period were, pistia stratiotes, Alternanthera philoxeroides, Cynodon dactylon, Ipomoea hildebrandtii, Potamogeton crispus, Ocimum basilicum, Ziziphus spinachristi, Calotropis procera, Acacia nilotica, and Azaridechta indica (Table ,5), according to Malek, (1958) the occurrence of pistia stratiotes was not reported in Gezira canal but only in the White and Blue Nile, that contrast with the current study. The revelation conclusion by many researchers including (Brown, 1980) when they conducted behavioral studies on snails' population showed that they are capable of escaping un-favorable conditions during the day, so habitat measurements do not necessarily reflect the actual conditions experienced by the snails. A total of 838 examined snails 412 were found naturally infected with trematode cercariae (49.1%) arranged as Cleopatra bulimoides (65%), Bulinus truncatus (47%), lymnaea natalensis (43%), Melanoides tuberculata (42%), Biomphalaria pfeifferi (30%), Lanistus carinatus (9%), and Bulinus forskalii (0%), a high infectivity was observed in the third survey where 79% of the snails were infected and that could be refer to the high cercariae diversity as six morphotypes were isolated in this survey which done after the re-growing of vegetation, and that attracted livestock and birds, so the canal contaminated with fecal matters

82 inducing the trematode transmission, also in the third survey each snail species shows its highest rate where the temperature was 27°C and probably the temperature is the reason, according to (Poulin, 2005) Cercarial output is directly influenced by the temperature due to both stimulating effect of temperature increasing the emergence from the snail and the acceleration of cercarial production within the snail host, the combined effect of the two will increase the cercariae release in great numbers. in contrast the low infection rate in the first survey 15% contributed with snail density and cercariae diversity due to drought since there was no host contact, and the absence of final hosts led to the absence of trematodes, many studies showed consistent relationship between the density and heterogeneity of the snail populations with the trematode density and heterogeneity (Hechinger and Lafferty, 2005) which match the finding in this study both in density and heterogeneity of trematode. The effectiveness of exposure by a shedding snail depends upon numbers of factors, such as the amount of time spent in a given area, number of cercariae shed, water current, vegetation, and the number and distribution of suitable intermediate hosts (Campell, 1973 and Hyman, 1967). On the other hand the probability of a snail becoming infected increases with age and is dependent upon the time spent in the aquatic environment and the ability to inoculate by miracidia, even though, snail populations grant higher trematode diversity, prevalence and intensity of infection are the major determinant factors of the rate of transmission of digenetic trematodes from the snail-intermediate host to the next host in their life cycle (Margolis et al., 1982; Anderson and May, 1991). Other trematode larvae found in Barakat canal are longifurcate- pharyngeate Monostome LPM (45.5%), Armate Xiphidiocercariae (19%), , type (1) Xiphidiocercariae (12%), Virgiulate Xiphidiocercariae (11.5%) Echinostome cercariae (8%), pluerolophocercous cercariae (3.5%),

83 type(3) furcocercous cercariae (0.31%), Longi-furcate pharengeate Distome cercariae LPD (0.1%), and type (2) furcocercous cercariae (0.09), such variation and diversity of digenean fauna is dependent on the presence of final and intermediate host (Gardner and Campbell, 1992). However, the cercariae types distribution is depend on the presence of definitive host and the adult parasites but not on the presence of snail intermediate host (Upthala, A. et al., 2010). The dominant cercariae type shed was LPM and it is the only type shed by Lymnaea and Lanistus species, also shed by Melanoides, Cleopatra, Bulinus and Biomphalaria species beside other cercariae types. The record of cercariae co-infection in the current study appeared in Cleopatra bulimoides, lymnaea natalensis, Bulinus truncatus, Biomphalaria pfeifferi, Melanoides tuberculata match many researchers finding, according to (Lafferity et al., 1994) the inter-species antagonism is the explanation for the scarcity of the double infections. Sousa (1992) reported that double infection could be more pathogenic compared to single infection. Echinostomecercariae were found in Melanoides tuberculata that match Cheng and Fang (1989) finding when they reported Echinostome cercariae (Echinochasmum japonicum), there presence in Bulinus truncatus (Frandsen and Christensen, 1984), (Lie et al., 1965, 1966, 1967) shows that Echinostome cercariae are antagonistic to the development of larvae of other trematode species in the same host, this contrast this study because some members of Melanoides tuberculata harbor two types of cercariae the same as Bulinus truncatus which harbor also Plurolophocercous beside the Echinostome. Cleopatra bulimoides recorded as a host of LPM (vivax) cercariae only, but in this study three types were reported beside LPM which are type 2 furcocercous, type 1 Xiphidiocercous, and Armate Xiphidiocercous and this species also found by Ibrahim, (2007) in Khartoum state, also Xiphidiocercariae found in this study was shed by Cleopatra bulimoides,

Biomphalaria pfeifferi and Melanoides tuberculata, that reported by Ibrahim, (2007) in sudan. Similarly other four xiphidiocercariae were described by Yousif & Ibrahim (1979) from Cleopatra bulimoides, other xiphidiocercariae from M. tuberculata, were procured in other countries e.g Thailand by Dechruksa et al., (2007) and Ukong et al. (2007). As for pleurolophocercous cercariae which shed by M. tuberculata and Bulinus truncatus, that match Bogea et al (2005) in Brazil, Velasquez et al (2006) in Columbia, Ukong et al (2007) and Dechruksa et al (2007) in Thailand and Skov et al (2009) in Vietnam when M. tuberculata liberated this type, in Egypt four cercariae types xiphidiocercariae, furcocercous cercariae and pleurolophocercous were reported by Yousif et al., (2010) from M. tuberculata. During the entire study period the Bulinus truncatus and Biomphalaria pfeifferi snails shed only Xiphidiocercariae, longifurcate-pharyngeate Monostome, Echinostome, and pluerolophocercous cercariae, even they were screened for three weeks about four times, they didn’t shed the identified human cercariae species although the Bio. pfeifferi shed undescribed furcocercous cercaria, this finding could relate to the low prevalence of infection following exposure to low doses of miracidia in natural habitat, snail–schistosome combinations suggests that such mismatches regularly occur in the field, and can play an important role in diminishing the number of patent infections achieved, although intermediate hosts can survive long periods of continuous desiccation, intermittent irrigation may dry out snails regularly and arrest their reproduction (Boelee and Madsen, 2006). Many workers including Archibald (1933), Barlow & Abdel Azim (1945), Gerber (1952), Olivier (1956a, 1956b) and Shiff (1960) have made observations on the reaction of molluscan hosts of bilharziasis to drought, the capacity of snails to survive out of water for weeks or even months has important consequences in relation to the epidemiology of

85 bilharziasis and to control measures. The temperature may also be responsible for the non-infectivity of Bulinus because infection with schistosome renders snail less tolerant to desiccation hence, only those snails which have better adaptability are capable of reactivating from aestivation bearing schistosome infection (Marti, et al., 1985). Other reason for the non-infectivity of Bulinus species their infection with Echinostomecercariae, based on some researchers opinion in the Gezira irrigation system of Sudan, Echinostomecercariae have been noted to be abundant in local B. truncatus populations, a factor which may to some extent be responsible for the uneven distribution and low prevalence of S. haematobium in this region (Amin, personal communication cited by McCullough, 1981), since the Echinostome are more pathogenic for snails than other trematodes, it invade their reproductive organs and digestive glands causing castreation or even death (WHO, 1956), so some how they could of use as biocontrol agents against snails populations, it has to be noted that study of the trematode fauna in endemic areas may reveal the existence of certain species that could be manipulated to achieve biological control of snail-transmitted diseases (Loker et al., 1981), the same situation might applied on their infection with Xiphidiocercariae as what reported by Paperna (1967) who noted that if Bulinus truncatus rohlfsi previously infected with xiphidiocercariae were considerably less likely to develop S.haematobium infections. In addition, the prolonged drought may have extreme long-term impacts on snail populations and cercariae shedding in the study area, This in turn, may result in changes in human infection patterns and the age distribution of schistosomiasis (El Kholy et al., 1989), conversely Webbe and Msangi (1958) recorded that Bulinus nasutus shed mammalian-type cercariae only 21 days after a pond, which had been dry for five months, was refilled by rainfall; and they concluded that in view of the short period which elapsed between the refilling of the pool and the shedding

86 of mature cercariae by the snails, the snail may possibly have contained immature infections, infected snails did not appear to survive for long periods so the survivors after a period of drought are not infected before their aestivation or they are in early ages to help cercariae maturation. Clearly, the trematodes mainly schistosomes pose a real threat on humans and animals life, some of this families found in the current study and identified as furcocercous cercariae types, the need for understanding veterinary schistosome species, not only in relation to disease management in animals, but also to reduce transmission to humans from animal reservoirs, which carry the potential to create new hybrid species. Human Fasciolosis can no longer be considered as a secondary zoonotic disease but must be considered a major human parasitic disease (Sam- Abbenyi, 1985). Although during the study period Lymnaea nataleinsis shed only furcated cercariae type (LPM), and no fasciola cercariae (Amphistome) were librated even with animal contact (cows, goats and sheep), that donot mean there is no threat upon human since the fasciolosis high prevalence in humans are not necessarily related to areas where fascioliasis is a great veterinary problem and humans can contract fascioliasis when they ingest infective metacercariae by eating raw aquatic plants (and, in some cases, terrestrial plants, such as lettuce, irrigated with contaminated water), drinking contaminated water, using utensils washed in contaminated water or eating raw liver infected with immature flukes ( Mas-Coma, 2005). As a final point, higher snail diversity leads to higher trematode diversity, apart from that, conditions influencing the proliferation of snail population will inevitably enhance the existence of trematode parasites in their intermediate host, and in some riverine habitats provide physicochemical conditions that favor the proliferation of freshwater snail fauna. For this reason, environmental conditions in different habitats, in different climatic zones may support the trematode life cycle differently

(Upthalla et al., 2010). It is important to notice that spatial variation in infection can easily arise as a consequence of the distribution of the second intermediate and final hosts and / or habitat characteristics which affect the risk of infection (Curtis and Hurd, 1983; Frandsen and Esch, 1991; Sousa, 1994). In conclusion, the small scale study do not represent the effect of the environmental factors that may inhibit snail survival or development perfectly because it needs long period of time to assess the effect and come with a proper conclusion. Further investigations of environmental factors especially the chemical water composition, e.g., measurements of specific dissolved ions, need to be carried out at all canals of Gezira state as a start and could be expanded all over endemic areas, since the planning of snail control reassures and evaluate their impact, knowledge of their ecology, population trends and dynamism are essential requirement towards understanding disease transmission and control. Hopefully, this study adds more information to the trematode fauna in Sudan.

5.1-Recommendations:

 Further studies needs to be established to understand the parasite biodiversity since the trematode transmitted diseases are of great concern affecting human and animals worldwide and could be a suspected danger in Sudan.

 Large scale study of environmental factors has to be made to provide a base line data to maintain a rational, cost effectiveness and applicable snail control measure.

 In order to understand and fight the parasitic diseases, reported cercariae types need more identification to the species level to study the characteristics of the adult worms and their host parasite relationship.

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Appendixes:

Appendix (І): The percentage of the collected snail species density in the three surveys during June-July 2011 in Barakat canal

Snail Species Survey (1) Survey (2) Survey (3) Total density

Cleopatra bulimoides 103(25%) 230(40%) 330(60%) 663(43%) lymnaea natalensis 135(33%) 86(15%) 50(9%) 271(17.5%)

Bulinus truncatus 33(8%) 94(16%) 84(15%) 211(14%)

Biomphalaria pfeifferi 107(26%) 50(9%) 49(8.9%) 206(13%)

Melanoides tuberculata 24(4%) 70(12%) 10(2%) 104(7%)

Lanistus carinatus 12(3%) 43(7%) 27(5%) 82(5%)

Bulinus forskalii 0(0%) 2(0.01%) 1(1%) 3(0.1%)

Total 414(27%) 575(37%) 551(36%) 1540

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50 C.bulimoides L.nataleins 40 B.truncatus 30 Density % Density M. tuberculate

20 Bio-pfeifferi La.carinatus 10 B.forskalii 0 Survey 1 Survey 2 Survey 3 Snail species

Appendix (IІ): The percentage of snail species density in the three surveys during June-July 2011 in Barakat canal

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Appendix (ІII): The density of shed cercariae types in the three surveys in Barakat canal June-July 2011

Type of cercaria Survey 1 Survey 2 Survey 3 Total

Vivax cercariae (LPM) 159(14%) 529(48%) 417(38%) 1104(45.5%)

ArmateXiphidiocercaria 150(32%) 315(68%) 0(0%) 465(19%)

Type1Xiphidiocercaria 300(100%) 0(0%) 0(0%) 300(12%)

VirgulateXiphidiocercaria 0(0%) 0(0%) 278(100%) 278(11.5%)

Echinostomecercaria 0(0%) 0(0%) 183(100%) 183(8%)

Paraplurolophocercous 0(0%) 64(76%) 20(24%) 84(3.5%)

Type3 Furcocercous 0(0%) 0(0%) 7(100%) 7(0.31%)

Stergiea cercariae (LPD) 3(100%) 0(0%) 0(0%) 3(0.1%)

Type 2 Furcocercous 0(0%) 0(0%) 1(100%) 1(0.09%)

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Appendix (IV): The correlation between surveys and water temperature in the study area

Correlations Water density temp density Pearson 1 .052 Correlation Sig. (2-tailed) .786 N 30 30 Water Pearson .052 1 temp Correlation Sig. (2-tailed) .786 N 30 30

No significance difference

Appendix (V): The correlation between surveys and water pH in the study area

Correlations Water survey PH survey Pearson 1 -.531** Correlation Sig. (2-tailed) .003 N 30 30 Water Pearson -.531** 1 PH Correlation Sig. (2-tailed) .003 N 30 30

**. Correlation is significant at the 0.01 level (2-tailed).

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Appendix (VI): The correlation between surveys and water depth in the study area

Correlations water survey Depth survey Pearson 1 -.904** Correlation Sig. (2-tailed) .000 N 30 30 water Pearson -.904** 1 Depth Correlation Sig. (2-tailed) .000 N 30 30

**. Correlation is significant at the 0.01 level (2-tailed).

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