Establishment Studies of the Life Cycle of Raillietina Cesticillus, Choanotaenia Infundibulum and Hymenolepis Carioca

Establishment Studies of the life cycle of Raillietina cesticillus, Choanotaenia infundibulum and Hymenolepis carioca.

Hanan Dafalla Mohammed Ahmed B.V.Sc., 1989, University of Khartoum

Supervisor: Dr. Suzan Faysal Ali

A thesis submitted to the University of Khartoum in partial fulfillment of the requirements for the degree of Master of Veterinary Science

Department of Parasitology Faculty of Veterinary Medicine University of Khartoum

May 2003

Dedication

To soul of whom, I missed very much, to my brothers and sisters

ACKNOWLEDGEMENTS

I thank and praise, the merciful, the beneficent, the Almighty Allah for his guidance throughout the period of the study. My appreciation and unlimited gratitude to Prof. Elsayed Elsidig Elowni, my first supervisor for his sincere, valuable discussion, suggestions and criticism during the practical part of this study. I wish to express my indebtedness and sincere thankfulness to my current supervisor Dr. Suzan Faysal Ali for her keen guidance, valuable assistance and continuous encouragement. I acknowledge, with gratitude, much help received from Dr. Shawgi Mohamed Hassan Head, Department of Parasitology, Faculty of Veterinary Medicine, University of Khartoum. I greatly appreciate the technical assistance of Mr. Hassan Elfaki Eltayeb. Thanks are also extended to the technicians, laboratory assistants and laborers of Parasitology Department. I wish to express my sincere indebtedness to Prof. Faysal Awad, Dr. Hassan Ali Bakhiet and Dr. Awad Mahgoub of Animal Resources Research Corporation, Ministry of Science and Technology, for their continuous encouragement, generous help and support. I would like to appreciate the valuable assistance of Dr. Musa, A. M. Ahmed, Dr. Fathi, M. A. Elrabaa and Dr. Iltigani Allam in identifying the insects' specimens. My special thanks will also be extended to Dr.Yahia, M. M. for offering me T. castaneum specimens. I am indebted to Dr.Mubark Elsidig, Mrs. Eatimad Awad and Mr. Mohammed Damona for their help in insects photographing. I do appreciate the unlimited help of Dr. Abdulwahab H. A. and Dr. Abdulbagi, M. A. in statistical analysis.

Deepest thanks to my colleague Kamal, H. Ali for his continuous help advice and assistance in computer work. Indeed, I wish to thank my friends and colleagues for their help and encouragement. Animal Resources Research Corporation, Ministry of Science and Technology, supported this work. My thanks are due again and forever to Almighty Allah who helped me to finish this work.

TABLE OF CONTENTS

PAGES Dedication……………………………………………………….. i

Acknowledgements………………………………………………. ii Table of contents…………………………………….…………... iv

List of Tables ………………………………………………….. xiii

List of Plates …………………………………………………….. xv Abstract ………………………………..………………………… x

Abstract………………………………………………………….. xii

INTRODCTION 1

List of abbreviations …………………………………… 3

CHAPTER ONE: LITERATURE REVIEW 4

1.1. Poultry tapeworms……………………………….………… 4 1.2.Tapeworms taxonomy and morphology……………………… 4 1.2.1. Raillietina cesticillus………………………………….. 4 1.2.2. Choanotaenia infundibulum…………………………… 5

1.2.3. Hymenolepis carioca………………………….……….. 5

1.3. The life cycle of poultry tapeworms…………………….….. 6

1.3.1. The life history and biology of Raillietina cesticillus.... 7

1.3.2. The life cycle of Choanotaenia infundibulum….. 10

1.3.3. The life history of Hymenolepis carioca…………….. 11

1.4. Prevalence of Raillietina cesticillus, Choanotaenia infundibulum and.Hymenolepis 1 carioca……………….. 2 1.5. Pathogenicity of poultry tapeworms……………………….. 14

1.5.1.Pathogenicity of Raillietina cesticillus…………… 14

1.5.2. Pathogenicity of Choanotaenia infundibulum……… 14

1.5.3. Pathogenicity of Hymenolepis carioca……………. 15

1.6. Alphitobius diaperinus……………………………………... 15

1.6.1. Alphitobius diaperinus as an intermediate

Host for cestodes and nematodes of poultry…………. 15

1.6.2. The economic importance of Alphitobius diaperinus. 16

1.7. Tribolium castaneum………………………………………… 17

1.7.1. Life cycle…………………………………….……… 17

1.7.2. Experimental infection of the meal beetles Tribolium

Castaneum and T. confusum with tapeworms….. 18

1.8. Musca domestica………………………………………..…. 19

1.8.1. Life cycle. ………………………………………….. 19

1.8.2. Musca domestica and disease transmission…………. 20

1.9. Family Histeridae-Hister beetles…………………………..… 21

1.10. Family Anthicidae- ant-like flower beetles…………..…….. 22

1.11. Diagnosis and identification of poultry tapeworms………… 22

1.12. Control of poultry tapeworms………………………………. 22

1.12.1. Control of the intermediate host…………………… 22

1.12.1.1. Changing management practice…… 23

1.12.1.2. The use of insecticides…………… 23

1.12.1.3. The use of drugs………………….. 23

1.12.1.4. Biological control…….……………. 24

1.12.1.5. Mechanical barrier……….………… 24

1.12.2. Treatment of infected chickens…………………. 24

CHAPTER TWO: MATERIALS AND METHODS 26

2.1. Collection of chicken tapeworms…………………………. 26

2.2. Preparation of infective material……………………..…….. 27

2.3. Experimental insects………………………..……………….. 27

2.3.1. Tribolium castaneum……………………….……….. 27

2.3.1.1. Rearing and maintenance……………….…. 27

2.3.1.2. Formation of infected insect colonies…….. 28

2.3.1.3. Cysticercoids recovery…………….…..…... 28

2.3.2. Alphitobius diaperinus……………………………….. 29

2.3.2.1. Rearing and maintenance…………....…….. 29

2.3.2.2. Formation of infected insect colonies……… 29

2.3.3. Musca domestica……………………………...………. 30

2.3.3.1. Rearing and maintenance…………………... 30

2.3.3.2. Exposure to infection……………..……... 30

2.3.3.2.1. Embedding tapeworm gravid

segments in sterile chicken manure …. 31

2.3.3.2.2. Maceration of the gravid segments. 31

2.4. Experimental chickens………………….…………..……… 32

2.4.1.Rearing and housing………..…………….………... 32

2.4.2. Administration of the infection……………….……. 32

2.4.3. Recovery of the mature tapeworms….……………... 32

2.5. Search for natural intermediate hosts for chicken tapeworms.. 33

2.5.1. Collection of data……………………….…….……. 33

2.5.2. Collection of samples……………………………… 33

2.5.3. Cysticercoids recovery……………..….………….... 33

2.5.4. Identification of potential insects………….….….… 34

2.6. Experimental design…………………………..………..….... 34

2.6.1. Experimental infection of Tribolium castaneum with

different species of cestodes………………..……….. 34

2.6.2. Experimental infection of Alphitobius diaperinus….

with different species of cestodes…………….…… 34

2.6.3. Experimental infection of Musca domestica

with different species of cestodes……………..….. 37

2.6.4. Experimental infection of White leghorn chicks

with different species of cestodes…………….…… 36

2.6.4.1.Design of Experiment 1……………….…… 36

2.6.4.2.Design of Experiment 2………..…….….… 36

2.6.4.3.Design of Experiment 3…..……………….. 36

2.6.4.4.Design of Experiment 4………..………..… 39

2.7. Statistical analysis……………………………………...….…. 39

CHAPTER THREE: RESULTS 40

3.1. Establishment of some tapeworm species in adult

Tribolium castaneum………….……………..……..….… 40

3.2. Establishment of R. cesticillus in adult T. castaneum……...... 40

3.3. Establishment of C. infundibulum in adult T. castaneum… 46

3.4. Establishment of H. carioca in adult T. castaneum……....… 46

3.5. Variability in infection between R. cesticillus,

C. infundibulum and H.carioca………..……...……….…. 48

3.6. Effect of C. infundibulum infecting adult T. castaneum 48

on the mortality rate (%)……………………………...….. 40

3.7. Effect of the size of infective dose on C. infundibulum

established in adult T. castaneum……………………….. 50

3.8. Effect of the size of infective doses on H. carioca

established in adult T. castaneum……………………… 50

3.9. Effect of submerging gravid segments of R. cesticillus for

five hours, in water on infection intensity, infection rate and

abundance of infection………………………..………… 50

3.10. Establishment of some tapeworm species on A. diaperinus 52

3.11. Effect of starving A. diaperinus on establishment

of C. infundibum…………….………………….…… 52

3.12. Establishment of tapeworm infection in M. domestica larvae 55

3.13. Establishment of tapeworm infection

in the definitive host…………………………….……….. 56

3.13.1. Establishment of R. cesticillus

in white leghorn chicks………..……….………… 56

3.13.2. Establishment of H. carioca

in white leghorn chicks………………………... 59

3.13.3. Establishment of C. infundibulum

in white leghorn chicks…………..……………… 59

3.13.3.1. Using cysticercoids established

in T. castaneum………………………… 59

3.13.3.2. Using cysticercoids established in

A. diaperinus……………..……………… 59

3.14. Naturally infected intermediate hosts……………………... 59

CHAPTER FOUR: DISCUSSION 67 REFERENCES…………………………………………... 78

APPENDICES…………………………………………… 91

LIST OF TABLES

Page 1 Adult Tribolium castaneum exposed to different cestode species, at different starvation periods (days), time of dissection post infection (days) and dose of infection.……………………………………………… 34 2 Alphitobius diaperinus exposed to different cestode species, at different starvation periods (days), time of dissection post infection (days) and dose of infection... 35 3 Musca domestica exposed to different cestode species, at different starvation periods (days), time of dissection post infection (days) and dose of infection... 37 4 White leghorn chicks exposed to infection with different metacestodes, with different age and slaughtered 15 days post infection (d.p.i) ………………………… 38 5 Establishment of some tapeworm species in three invertebrate intermediate hosts…………………… 41 6 Mean infection intensity, infection rate (%) and abundance of infection of three cestode species infecting T. castaneum under laboratory condition…………… 45 7 Effect of C. infundibulum infecting adult T castaneum on mortality rate (%) under laboratory condition… 49 8 Effect of size of infective doses of C. infundibulum and H. caioca (established in adult T. castaneum) on infection intensity, infection rate and abundance of infection…………………………………………….. 51

9 Effect of submerging, for five hours, gravid segments of R. cesticillus in water on infection intensity, infection rate and abundance of infection……….…... 53 10 Effect of starving A. diaperinus on C. infundibulum, establishment……………………………………….… 54 11 Infectivity of R. cesticillus and H. carioca to white leghorn chicks…………………………………… 58 12 Infectivity of C. infundibulum to white leghorn chicks. 60 13 Prevalence of C. infundibulum and R. cesticillus in naturally infected insects collected in Shambat and Elhalfaya Localities 2000/2001……………………. 63 14 Appendix (8): Mean square values showed infection intensity, infection rate and abundance of the infection of three cestodes infecting adult T. castaneum……... 92 15 Appendix (9): Mean square values showed infection intensity, infection rate and abundance of the infection of C.infundibulum, infecting adult A. diaperinus and starved for different periods………………………… 92

LIST OF PLATES

1 Mature Raiteillina cesticillus cysticercoid from adult Tribolium castaneum 21d.p.i with deep notch at the broader end (a) and wide rostellar hook-circlet (b).x 25. 42 2 Mature twins' cysticercoids of Choanotaenia infundibulum from adult Tribolium castaneum 20d.p.i, with capsule enclosing eccentric postioned larvae (a) and convoluted mucus-like mass (b). x 10………………………………… 43 3 Mature Hymenoplis carioca cysticercoid from adult Tribolium castaneum 20d.p.i with unarmed sucker larva .x 25…………………………………………………… 44 4 Mature Choanotaenia infundibulum cysticercoid from adult Tribolium castaneum (6-9) d.p.i with capsule, carcareous corpuscles are clearly seen (a). x 10………….. 47 5 Mature Choanotaenia infundibulum cysticercoid from adult Tribolium castaneum (15-16) d.p.i with capsule, carcareous corpuscles are clearly seen (a). x 10………….. 47 6 Scolex of Railietina cesticillus, showing broad rostellum (a) and unarmed suckers (b). x 25………………………… 57 7 Scolex of Choanotaenia infundibulum, showing pointed distinctive rostellum with a single raw of slender hooks (a) and unarmed elongated suckers (b).x 4……………………. 61 8 Ant-beetle, Anthicus formicarius, collected from shambat, natuarlly infected with C. infundibulum. x 12…………….. 65 9 Hypocalccus praecox, collected from shambat, natuarlly

infected with R. cesticillus. x 12…………………………. 66 10 Carcinops troglodytes collected from shambat, natuarlly infected with R. cesticillus. x 12…………………………. 66

ABSTRACT

Establishment of the life cycle of three cestodes Raillietina cesticillus, Choanotaenia infundibulum and Hymenolepis carioca was studied in three insects that act as intermediate hosts, namely Tribolium castaneum, Alphitobius diaperinus and Musca domestica and in white Leghorn chicks, the definitive host. Insects were experimentally infected with gravid segments of the three cestodes. White Leghorn chicks were reared in insect- proof cages and experimentally infected with cysticercoids previously established in adult T. castaneum and A. diaperinus. In a field study, 1205 different insects collected in Shambat and Elhalfaya in Khartuom North, were dissected to determine their natural infection with poultry cestodes. Raillietina cesticillus, C. infundibulum and H. carioca established in adult T. castaneum whereas C. infundibulum was the only tapeworm that established in adult and larvae of A. diaperinus. Nevertheless, R. cesticillus and C. infundibulum did not established in M. domestica larvae. R. cesticillus had the highest infectivity to T. castaneum compared to the other tapeworms. Raillietina cesticillus, C. infundibulum and H. carioca established in white Leghorn chicks. Age of the cysticercoid and type of intermediate host in addition to the procedure of administering the infection affected their establishment. Search for natural intermediate hosts for poultry cestodes in the field revealed the presence of four species of beetles carrying metacestode infection. Two of the beetles, Anthicus formicarius and A. diaperinus, were

found infected with C. infundibulum whereas the other two, Hypocalcculus praecox and Carcinops troglodytes, were found harbouring R. cesticillus cysticercoids. No metacestodal infection was found in adult T. castaneum or M. domestica larvae. This is the first record of A. formicarius, H. praecox and C. troglodytes as intermediate hosts for C. infundibulum and R. cesticillus in the Sudan. It is concluded that the present findings contribute to the understanding of the properties of some experimental models e.g. C. infundibulum in A. diaperinus, R. cesticillus in T. castaneum and H. carioca in T. castaneum that can be used in several fundamental investigations (chemotherapy, immunology etc.). Immune mechanism of the insect may be an essential factor that reduces the infectivity of C. infundibulum cysticercoids developed in A. diaperinus. The nature of this reaction requires further investigations. It is recommended that for the purpose of control of these cestodes it is essential to manage their potential intermediate host(s).

ﻤـﻠﺨﺹ ﺍﻷﻁﺭﻭﺤـــﺔ

ﺃﺠﺭﻴـﺕ ﻫـﺫﻩ ﺍﻟﺩﺭﺍﺴـﺔ ﻟﺘﻭﻁـﻴﺩ ﺜﻼﺜﺔ ﺩﻴﺩﺍﻥ ﺸﺭﻴﻁﻴﺔ ﻫﻰ ﺩﻭﺩﺓ ﺍﻟﺭﻴﻠﻴﺘﻴﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ ، ﻭﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨـﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟـﻴﻡ ﻭﺍﻟﻬﻴﻤﺎﻨﻭﻟـﺒﺱ ﻜﺎﺭﻴﻭﻜـﺎ ﻓﻲ ﺍﻟﻌﻭﺍﺌل ﺍﻟﻭﺴﻴﻁﺔ : ﺍﻟﺘﺭﺍﻴﺒﻭﻟﻴﻡ ﻜﺎﺴﺘﺎﻨﻴﻡ ، ﺍﻻﻟﻔﻴﺘﻭﺒـﻴﺹ ﺩﻴﺒﺭﻴﻨﺹ ﻭﺍﻟﺫﺒﺎﺒﺔ ﺍﻟﻤﻨﺯﻟﻴﺔ ، ﺜﻡ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻨﻬﺎﺌﻲ ﺍﻟﺩﻭﺍﺠﻥ ﺒﺎﺴ ﺘﺨﺩﺍﻡ ﺼﻐﺎﺭ ﻓﺼﻴﻠﺔ ﺍﻟﻭﺍﻴﺕ ﻟﻴﻕ ﻫﻭﺭﻥ. ﺍﻟﻌﻭﺍﺌل ﺍﻟﻭﺴﻴﻁﺔ ﺘﻤﺕ ﺇﺼﺎﺒﺘﻬﺎ ﺘﺠﺭﻴﺒﻴﺎ ﺒﺎﻟﻘﻁﻊ ﺍﻟﻨﺎﻀﺠﺔ ﻟﻬﺫﻩ ﺍﻟﺩﻴﺩﺍﻥ ﻭ ﺘﻤﺕ ﺘﺭﺒﻴﺔ ﺼﻐﺎﺭ ﺍﻟﺩﻭﺍﺠـﻥ ﻓـﻲ ﻅـﺭﻭﻑ ﺘﻀﻤﻥ ﻋﺩﻡ ﺇﺼﺎﺒﺘﻬﺎ ﺒﺄﻱ ﻤﻥ ﺍﻟﺤﺸﺭﺍﺕ ﺍﻟﻨﺎﻗﻠﺔ ﻟﻠﺩﻴﺩﺍﻥ ﺍﻟﺸﺭﻴﻁﻴﺔ ﻭﻤﻥ ﺜﻡ ﺘﺠـﺭﻴﻌﻬﺎ ﺒﻴﺭﻗﺎﺕ ﺍﻟﺩﻴﺩﺍﻥ ﺍﻟﺸﺭﻴﻁﻴﺔ ﻭﺍﻟﺘﻲ ﺴﺒﻕ ﻨﻤﻭﻫﺎ ﻓﻲ ﺍﻟﻌﺎﺌﻠﻴﻥ ﺍﻟﻭﺴﻴﻁﻴﻥ : ﺍﻟﺘﺭﺍﻴﺒﻭﻟﻴﻡ ﻜﺎﺴﺘﺎﻨﻴﻡ ﻭﺍﻷﻟﻔﻴﺘﻭﺒـﻴﺹ ﺩﻴﺒﺭﻴﻨﺹ ، ﺒﺎﻹﻀﺎﻓﺔ ﺇﻟﻰ ﺍﻟﻌﻤل ﺍﻟﻤﻴﺩﺍﻨﻰ ﻭﺍﻟﺫﻱ ﺘﻡ ﻤﻥ ﺨﻼﻟﻪ ﺠﻤﻊ 1205 ﻋﻴﻨﺔ ﺤﺸـﺭﺍﺕ ﻤـﻥ ﻤـﺯﺍﺭﻉ ﺘﻘﻊ ﺒﻤﻨﻁﻘﺘﻲ ﺸﻤﺒﺎﺕ ﻭﺍﻟﺤﻠﻔﺎﻴﺎ ﺒﺎﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻱ ﻟﻤﻌﺭﻓﺔ ﺇﺼﺎﺒﺘﻬﺎ ﺒﺎﻟﺩﻴﺩﺍﻥ ﺍﻟﺸﺭﻴﻁﻴﺔ. ﺘﻭﻁـﺩﺕ ﻫـﺫﻩ ﺍﻟﺩﻴـﺩﺍﻥ ﺍﻟـﺜﻼﺜﺔ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻭﺴﻴﻁ ﺍﻟﺘﺭ ﺍﻴﺒﻭﻟﻴﻡ ﻜﺎﺴﺘﺎﻨﻴﻡ ﻭ ﺘﻭﻁﺩﺕ ﺩﻭﺩﺓ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨـﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟـﻴﻡ ﻓـﻲ ﺍﻟﻌـﺎﺌل ﺍﻟﻭﺴـﻴﻁ ﺍﻷﻟﻔﻴﺘﻭﺒﻴﺹ ﺩﻴﺒﺭﻴﻨﺹ ﻭﻟﻡ ﺘﺘﻭﻁﺩ ﺩﻭﺩﺓ ﺍﻟﺭﻴﻠﻴﺘﻴﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ ﺃﻭ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟﻴﻡ ﻓﻲ ﻴﺭﻗﺎﺕ ﺍﻟﺫﺒﺎﺒﺔ ﺍﻟﻤﻨﺯﻟﻴﺔ . ﺩﻭﺩﺓ ﺍﻟﺭﻴﻠﻴﺘﻴﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ ﺃﻅﻬﺭﺕ ﺃﻜـﺒﺭ ﻗـﺩﺭ ﻤﻥ ﺍﻟﺘﻭﻁﻴﺩ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻭﺴﻴﻁ ﺍﻟﺘﺭﺍﻴﺒ ﻭﻟﻴﻡ ﻜﺎﺴﺘﺎﻨﻴﻡ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﺩﻴﺩﺍﻥ ﺍﻷﺨﺭﻯ . ﺘﻭﻁﺩﺕ ﺍﻟﺩﻴـﺩﺍﻥ ﺍﻟﺜﻼﺜﺔ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻨﻬﺎﺌﻲ ﻭﻗﺩ ﻭﺠﺩ ﺃﻥ ﻋﻤﺭ ﻴﺭﻗﺎﺕ ﺍﻟﺩﻴﺩﺍﻥ ﻭﻨﻭﻉ ﺍﻟﻌﺎﺌل ﺍﻟﻭﺴﻴﻁ ﻭﻁﺭﻴﻘﺔ ﺘﺠﺭﻴﻊ ﻫﺫﻩ ﺍﻟﻴﺭﻗﺎﺕ ﻟﻪ ﺍﺜﺭ ﻓﻲ ﺘﻭﻁﻴﺩ ﺍﻟﺩﻴﺩﺍﻥ ﻓﻲ ﺍﻟﻌﻭﺍﺌل ﺍﻟﻭﺴﻴﻁﺔ ﺍﻟﻤﻌﻨﻴﺔ. ﺃﻅﻬﺭ ﺍﻟﺒﺤﺙ ﺍﻟﻤﻴﺩﺍﻨﻲ ﻭﺠﻭﺩ ﻋﻭﺍﺌل ﻭﺴﻴﻁﺔ ﻤﺼﺎﺒﺔ ﺒﺎﻟﺩﻴﺩﺍ ﻥ ﺍﻟﺸﺭﻴﻁﻴﺔ ﻭﻫﻰ ﺃﺭﺒﻌﺔ ﺃﺠﻨﺎﺱ ﻤـﻥ ﺍﻟﺨﻨﺎﻓﺱ ﻭﺠﺩﺙ ﻨﺎﻗﻠﺔ ﻟﺩﻭﺩﺘﻲ ﺍﻟﺭﻴﻠﻴﺘﻴﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ ﻭ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟﻴﻡ ﻭﻫﻲ ﺍﻷﻨﺴﻴﻜﺹ ﻓﻭﺭﻤﻴﻜﺎﺭﻴﺱ ﻭﺍﻷﻟﻔﻴﺘﻭﺒﻴﺹ ﺩﻴﺒﺭﻴﻨﺹ ﻴﺤﻤﻼﻥ ﺩﻭﺩﺓ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟﻴﻡ ﻭﻭﺠﺩ ﺃﻥ ﺍﻟﻬﺎﻴﺒﻭﻜﺎﻟﻜﺎﺱ ﺒـﻴﺭﻜﻭﺱ ﻭﺍﻟﻜﺎﺭﺴـﻴﻨﻭﺏ ﺘﺭﻭﻗﻠﻭﺩﺍﻴﺕ ﻴﺤﻤﻼﻥ ﺩﻭﺩﺓ ﺍﻟﺭﻴﻠﻴﺘﻴ ﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ ﺒﻴﻨﻤﺎ ﺤﺸﺭﺓ ﺍﻟﺘﺭﺍﻴﺒﻭﻟﻴﻡ ﺍﻟﺒﺎﻟﻐﺔ ﻭﻴﺭﻗﺎﺕ ﺍﻟﺫﺒﺎﺒﺔ ﺍﻟﻤﻨﺯﻟﻴﺔ ﻻ ﻴﺤﻤﻼﻥ ﺃﻱ ﻨﻭﻉ ﻤﻥ ﺍﻹﺼﺎﺒﺔ ﺒﻴﺭﻗﺎﺕ ﺍﻟﺩﻴﺩﺍﻥ ﺍﻟﺸﺭﻴﻁﻴﺔ. ﺍﻟﺠﺩﻴـﺭ ﺒﺎﻟﺫﻜـﺭ ﺃﻥ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺴﺠﻠﺕ ﻷﻭل ﻤﺭﺓ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ ﺃﻥ ﺍﻷﺠﻨﺎﺱ ﺍﻷﻨﺴﻴﻜﺹ ﻓﻭﺭﻤـﻴﻜﺎﺭﻴﺱ ﻭ ﺍﻟﻬﺎﻴـﺒﻭﻜﺎﻟﻜﺎﺱ ﺒﻴﺭﻜﻭﺱ ﻭﺍﻟﻜﺎﺭﺴﻴﻨﻭﺏ ﺘﺭﻭﻗﻠﻭﺩﺍﻴﺕ ﺘﻌﻤل ﻜﻭﺴﺎﺌﻁ ﺘﻨﻘل ﺩﻭﺩﺘﻲ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟﻴﻡ ﻭﺍﻟﺭﻴﻠﻴﺘﻴﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ.

ﻤﻥ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺘﺴﺘﻨﺘﺞ ﺇﻤﻜﺎﻨﻴﺔ ﺍﺴﺘﺨﺩﺍﻡ ﺩﻭﺭﺓ ﺤﻴﺎﺓ ﺩﻭﺩﺓ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟﻴﻡ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻭﺴـﻴﻁ ﺍﻷﻟﻔﻴﺘﻭﺒـﻴﺹ ﺩﻴﺒﺭﻴـﻨﺹ ؛ ﺍﻟﺭﻴﻠﻴﺘﻴﻨﺎ ﺴﺴﺘﻴﺴﻠﺹ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻭﺴﻴﻁ ﺍﻟﺘﺭﺍﻴﺒﻭﻟﻴﻡ ﻜﺎﺴﺘﺎﻨﻴﻡ ﻭﺍﻟﻬﻴﻤﺎﻨﻭﻟﺒﺱ ﻜﺎ ﺭﻴﻭﻜﺎ ﻓﻲ ﺍﻟﻌﺎﺌل ﺍﻟﻭﺴﻴﻁ ﺍﻟﺘﺭﺍﻴﺒﻭﻟﻴﻡ ﻜﺎﺴﺘﺎﻨﻴﻡ ﻜﻨﻤﺎﺫﺝ ﺘﺴﺘﺨﺩﻡ ﻓﻲ ﺍﻟﺩﺭﺍﺴﺎﺕ ﺍﻟﺒﺤﺜﻴﺔ ﺍﻷﺴﺎﺴـﻴﺔ ﻤـﺜل ﻋﻠﻡ ﺍﻷﺩﻭﻴﺔ ﻭﻋﻠﻡ ﺍﻟﻤﻨﺎﻋﺔ ﻭﻏﻴﺭﻫﺎﹰ . ﺭﺒﻤﺎ ﻴﻜﻭﻥ ﺍﻟﺠﻬﺎﺯ ﺍﻟﻤﻨﺎﻋﻲ ﻋﺎﻤﻼ ﺃﺴﺎﺴﻴﺎ ﻓﻲ ﺘﻘﻠـﻴل ﺇﺼﺎﺒﺔ ﺤﺸﺭﺓ ﺍﻷﻟﻔﻴﺘﻭﺒﻴﺹ ﺩﻴﺒﺭﻴﻨﺹ ﺒﺩﻭﺩﺓ ﺍﻟﻘﻭﺍﻨﻭﺘﻴﻨﻴﺎ ﺃﻨﻔﻨﺩﺒﻴﻭﻟﻴﻡ ﻭﻫﺫﺍ ﻴﺤﺘﺎﺝ ﺇﻟﻰ ﻤﺯﻴﺩ ﻤﻥ ﺍﻟﺒﺤـﺙ . ﻭﺘﻭﺼﻲ ﺍﻟﺩﺭﺍﺴﺔ ﻋﻠﻰ ﺍﻟﺴﻴﻁﺭﺓ ﻋﻠﻰ ﺍﻟﺩﻴﺩﺍﻥ ﺍﻟﺸﺭﻴﻁﻴﺔ ﺒﻤﻜﺎﻓﺤﺔ ﺍﻟﻌﻭﺍﺌل ﺍﻟﻭﺴﻴﻁﺔ ﺍﻟﻨﺎﻗﻠﺔ ﻟﻬﺫﹾﻩ ﺍﻟﺩﻴﺩﺍﻥ.

INTRODUCTION

Tapeworms, along with their close relatives, the acanthocephalans, are more and better adapted to parasitism than any other group of metazoan parasites (Read and Simons, 1963). The cestodes form a group of worms, which with a few exceptions exhibit two striking morphological features; they possess an elongated tape-like body and lack an alimentary canal. Adult tapeworms occur only in tubular habitat, usually the alimentary canal, but occasionally in the bile or pancreatic ducts. On the other hand, larval habitat shows a wide range of variation, and larvae can be found in almost any organ of both vertebrate and invertebrate hosts, although most larvae show a predilection for a particular site (Smyth, 1996). Chickens comprise one of the definitive hosts for cestodes, and as the result they suffer so much effect such as stunted growth, lowering egg production and bring about emaciation and death (Kaushir and Deorani, 1971). The mere presence of Raillietina cesticillus was found to be associated with an increase in death rate in infected birds due to intestinal obstruction (Dutt, 1961). Reduction in productivity is also attributed to tapeworms; Enigk and Sticinsky (1959b) found that egg production was markedly reduced in chickens infected with Choanotaenia infundibulum, Hymenolepis carioca, Raillietina cesticillus and Davaenia proglottina. Several reports showed the importance of tapeworms intermediate hosts as vehicles of other pathogenic agents e.g Alphitobius diaperinus, which is considered to be an intermediate host for Choanotaenia infundibulum (Elowni, 1979), plays an important role in transmitting some species of protozoa (Apuya et al., 1994), bacteria, i.e. species of Salmonella (McAllister et al., 1994; Davies and Wary 1995), and viruses, e.g. Merck's

Disease virus (Loomis, 1984). It is worth mentioning that eggs of chicken tapeworms are also found to be another vehicle of infectious disease agents; Lee et al., (1974) isolated certain strains of avian reovirus and avian adenovirus from the eggs of Raillietina tetragona. Whether these viruses multiply within the larvae inside the intermediate host or not and their role in the epidemiology of infectious disease, needs further investigation (Elowni, 1984). Relatively very little work has been done in the Sudan investigating the way the cestodes take to continue their cycles, and even though, the local intermediate hosts despite their importance when constructing a control program, stand upon local information. The present study is an attempt to investigate the establishment of the life cycle of three chicken cestodes, namely Raillietina cesticillus, Choanotaenia infundibulum and Hymenolepis carioca, in three invertebrate hosts, namely Tribolium castaneum, Alphitobius diaperinus and Musca domestica and the definitive host the chicken. In addition, search for potential intermediate hosts responsible for maintaining this cycle.

1.1 List of abbreviations °C degree celsius Cm centimetre CV Coefficiet of variation DMRT Duncan's Multiple Range Test d.p.i. days post infection Kg kilogram Log Logrithm µm micrometer PPM Part Per Million

CHAPTER ONE LITERATURE REVIEW

1.1. Poultry tapeworms Over 1400 species of tapeworms have been described from wild and domestic birds (Reid, 1984, 1995). Of 193 genera belonging to 17 families that have been identified from birds (Reid, 1984), as many as 45 species of tapeworms, belonging to 10 different genera have been reported to parasitize domestic fowl (Elowni, 1977). Several species of chicken tapeworms for example Raillietina cesticillus, Raillietina tetragona and Raillietina echinobothrida and Hymenolepis carioca have been shown to be cosmopolitan (Elowni, 1977). Cotugnia digonopora and Raillietina cohni, R. tetragona, R. cesticillus, R. echinbothrida, Amoebotaenia cuneata, Hymenolepis cantaniana, H. carioca, H. villosa, Davainea proglonttina, Metroliasthes lucida have been reported from different parts of Africa (Sawada, 1954; Permin et al., 1997; Poulsen, et al., 2000; Eshetu, 2001). In the Sudan, R. cesticillus, R. tetragona, R. echinobothrida, C. infundibulum, C. digonopora, Amoebotaenia cuneata and H. carioca have been reported from local and foreign breeds of chicken from different parts of the country (Abdel-Malik, 1959; Eisa, et al., 1976, Elowni, 1977; Saad et al., 1989 and Ali, 1994).

1.2. Tapeworms taxonomy and morphology 1.2.1. Raillietina cesticillus Family: Davaineidae (Fuhrmann, 1907) Genus: Raillietina (Fuhrmann, 1891) Species: Raillietina cesticillus (Molin, 1858) It is 4 cm, rarely 15 cm long. It has a large scolex with a wide rostellum armed with 400-500 small hooks. The suckers are inconspicuous and unarmed. The eggs, 75-88 µm in diameter, occur singly in egg capsules (Soulsby, 1982).

1.2.2. Choanotaenia infundibulum Family: Dipylidiidae Wardle, Mc Leod and Radinovski, 1974 Genus: Choanotaenia Railliet, 1896 Species: Choanotaenia infundibulum (Bloch, 1779) It may reach 23 cm in length and the segments are markedly wider posteriorly than anteriorly, giving the worm a characteristic shape. The rostellum is armed with 16-20 slender hooks. Genital pores alternate regularly. The uterus is sac-like. However, the proglottids leave the body before they are completely gravid and there is doubt as to whether the uterus is replaced by egg capsules. The eggs have distinct elongate filaments (Soulsby, 1982).

1.2.3. Hymenolepis carioca Family: Hymenolepididae Railliet and Henry, 1909 Genus: Hymenolepis Weinland, 1858 Species: Hymenolepis carioca de Magalhaes, 1898 The adult is slender and up to 8 cm long (Soulsby, 1982). The suckers unarmed; rostellar sac present; rostellum rudimentary; three testes usually in straight raw, genital pores unilateral, located anterior to the middle of the proglottid margin; an inner membrane enveloping the oncosphere elongated into a football shape with granular deposits at poles, embryonal hooks 10-12 µm (Reid, 1984).

1.3. The life cycle of poultry tapeworms Poultry tapeworms with known life history require intermediate hosts for completion of the life cycle. Investigations have shown invariably that intermediate hosts of tapeworms have been invertebrates, such as beetles, flies, ants, snails, slugs, crustaceans etc. The type of intermediate host that serves a particular tapeworm in its successful transference, depends on the habitat of the avian host, e.g. terrestrial birds (chickens, turkey, guinea fowl, etc.), intermediate host must lead a terrestrial life or at least amphibious (Wehr, 1972). Intermediate hosts may swallow individual eggs in the faecal mass or devour the entire proglottid after being attracted by odour or movement (Reid, 1984). Following ingestion, it hatches in the digestive tract and the liberated oncosphere penetrates the intestinal wall, and enter the body cavity. Hatching and penetration involves breast like movement of the six hooks and secretion from the penetration glands as the parasite invades the body cavity (haemocoel) of the intermediate host (Reid, 1984). The hexacanth embryo develops into a white, bladder-like spherical body known

as cysticercoid in the next several weeks. The cysticercoid usually remains alive in the invertebrate intermediate host and infective to the bird host for many months, e.g. Raillietina cesticillus retains its infectivity for five and half months (Elowni, 1984). In a few life cycles, the cysticercoids survive ingestion by a second intermediate or auxiliary host (Reid, 1984). Mechanical or chemical actions within the gut of the definitive host free the cysticercoid. The scolex evagenates, and attaches to the intestinal wall. The cyst wall degenerates and is lost, while the strobila proliferates from the neck region to form a new tapeworm. Most tapeworms require 2-3 weeks prepatent period in the bird to mature and release the first proglottids in the faeces (Reid, 1984).

1.3.1. The life history and biology of Raillietina cesticillus (Molin, 1858) Many authors have reported the life history of this cestode. Guberlet (1916) and Joyeux (1920) tried to infect houseflies, Musca domestica, larvae and adults, but no larval forms of the cestode were recovered. On the other hand Ackert (1918, 1936) reported recovery of adult R. cesticillus following feeding young chickens adult stage of houseflies. Workers, such as Gram (1928), found cysticercoids of R. cesticillus in the beetles Anisotarsus agilis and Choeridium histeriodes. Since that time, numerous beetles in which the cysticercoids developed have been found. The detailed description of the life cycle and biology of this worm has been reported by Reid et al. (1938). Several species of beetles were tested as possible intermediate hosts of Raillietina cesticillus, two genera and twelve species of ground beetles (Carabidae), which previously have not been reported can act as intermediate hosts for this worm. The time required for the cyst to develop to maturity 14-16 days, the numbers of cysticercoids per

beetle ranged from 1 to 626, which appeared to be correlated to the number of proglottids fed. Considerable variations in the size of cysticercoid from different beetles were noted. Dutt et al. (1961) traced the pathway of oncosphere until its final location in the body cavity of the invertebrate intermediate host. They found that the oncosphere burrowing through the enteric caeca of the midgut. Elowni (1977) detected the ocnosphere in the body cavity in Tribolium castaneum 18 hours following infection and the embryonal hooks showed no activity, suggesting that, penetration had occurred sometimes earlier. The time of development of the cysticercoid was found to be 12-30 days (Dutt et al., 1961), 14 days (Voge, 1961) and 15 days (Elowni, 1977), depending upon the incubation temperature. Dutt et al. (1961) found four species to be natural hosts for R. cesticillus, namely Anthicus confucii, Opatroides vicinus, Dermestes maculatus, and Hister orientalis. Elowni (1977) reported the infectivity of R. cesticillus to the beetle Tribolium castaneum. From a total number of 165 beetles fed on gravid segments of R. cesticillus, 75% developed the infection with a mean number of cysts 5.21 and a range of 1-27. The total number of gravid segments offered to the starved beetles had no significant effect on total number of cysts recovered. Su-XZ and Lin-YG (1985) found Tribolium ferrugineum served as an intermediate host of R. cesticillus, incidence of cysticercoids (17.4%), the number per beetle 1-13, and at 16-20˚C the cestode took 28 days to develop in the intermediate host and fully developed cysticercoids were obtained within 31-34 days. The second step of the life cycle is the development of the cestode in the definitive host. Reid et al. (1938) fed chickens with cysticercoids, previously established in ground beetles. Gravid segment appeared in chicken droppings 13 days following infection, though freed gravid

segments were found in the intestine 11 days post-infection. Most of the gravid proglottids were voided by the chickens in the afternoon and evenings. The periodicity in shedding segments is thought to be associated with feeding and digestion in the chicken and with absorption and assimilation in the tapeworm (Reid et al., 1938 and Dutt et al., 1961). Gravid proglottids were found to be motile in the intestine, and retained their motility whenever remained warm. This motility is not affected by heat, light or gravity (Reid et al., 1938). The prepatency was found to be 13-16 days (Dutt et al., 1961), 12 days (Gray, 1972), 13.5-21.5 days with a mean of 17.64 days (Elowni, 1977) and 15 days post infection (Su-XZ and Lin- YG, 1985). The age of cysticercoid has a significant effect on the infectivity of R. cesticillus to young chicken. Four months old cysticercoids of R. cesticillus retained infectivity which gradually decreased until completely lost seven months (Elowni, 1984). The habitat of the adult tapeworm R. cesticillus is the lumen of the upper third of the chicken intestine. The scolices are embedded in the mucosa about 5 cm below the entrance of the bile and pancreatic ducts or short distance above the duodenal loop or as far back as Meckel's diverticulum (Reid et al., 1938). Adult worms were recovered in the intestines of White Leghorn chicks attaching to the mucosa 1-17 cm posterior to the level at which the bile duct joins the intestine (Elowni, 1977). Gray (1972 a, b) studied the patency phase of R. cesticillus. A posterior migration of the young cestodes during the first 7 days of infection was confirming those of Foster and Daugherty (1959). A progressive destrobilisation occurring 70 days after infection. No regeneration occurred and the scolices were eventually eliminated. There are marked differences in

the rate of destrobilisation in the two host sexes (destrobilisation proceeded more rapidly in the female than in the male). There was an effect of the host age on the course of infection. Males became resistant to cestodes with increasing age and this resistance is manifested by an increased rate of destrobilisation and consequent loss of scolices, and in decreased rate of growth of the worm. The cestodes distributed over a great length of intestine in old than in young birds. In female hosts, age resistance develops more rapidly than in male until the birds were 84 days old and from this age onwards the manifestation of resistance declined.

1.3.2. The life cycle of Choanotaenia infundibulum (Bloch, 1779) The life history of C. infundibulum has been known since 1889 when Grassi and Rovelli in Italy described from the housefly, Musca domestica, cysticercoids which they believed to be those of this species. In Germany, Soloviov (1911) fed a young hen 15-20 specimens of Musca domestica daily during the summer. By late fall, the hen was passing segments of C. infundibulum, whereas three control birds were negative. In the United States, Guberlet (1916) fed segments of C. infundibulum to 88 flies, Musca domestica, most of which died within a few days. One out of six flies that lived for 12 days contained 5 cysticercoids of this cestode. In another experiment, Guberlet fed each of 3 chicks 50 Musca domestica caught around chicken roosts. Three weeks later, postmortem examination showed that one chick had 6 specimens of C. infundibulum, one had one specimen of this tapeworm, and the remaining chicks as well as 3 controls were negative. Guberlet tried to infect Musca domestica maggots with C. infundibulum; the results were negative.

In France, Joyeux (1920) infected a beetle, Geotrupes syvaticus, with C. infundibulum, but he did not state that he succeeded in infecting chicken by feeding them infected beetles. In the United States, Jones (1930) found a beetle, Cratacanthus dubius, containing cysticercoids of C. infundibulum, but she did not infect a chicken by feeding the infected beetle. Horsfall and Jones (1973) demonstrated the life cycle of C. infundibulum in the beetle Aphodius granarius and the grasshopper, Melanoplus femurrubrum. In these hosts, the development of the cysticercoids to the infective stage took 48 days and 17-20 days at 60-75˚F and 75-90˚F in case of Aphodius granarius and Melanoplus femurrubrum, respectively. Chickens passed gravid segments of C. infundibulum 13-15 days after feeding with the infective cysts obtained from the above mentioned two hosts. In addition, another 7 species of beetles were recorded for the first time as intermediate hosts for C. infundibulum, one of them was naturally infected. In India, Dutt and Sinha (1961) found two species of beetles, Anthicus confucii and Opatroides vicinus harbouring the cysticercoids of this cestode in nature. On an experimental study, 15 species of beetles including the two mentioned above and 15 species of grasshopper were found to take the infection. It, generally, took 5-7 days for the larvae to develop in the intermediate host. Mature worms were obtained in 8 days after infection. In USSR, Dushkin (1970) described briefly the life cycle of C. infundibulum. The intermediate hosts are beetles of the genera Amara and Calanthus. Cysticeroids develop in these at 17-20˚C in about 28-32 days and fowls infect themselves by eating the beetles. The tapeworms become sexually mature about 17 days after the cysticercoids have been ingested. In the Sudan, Hamad (1987) while studying the pathogenicity of C. infundibulum in White Leghorn chicks, showed that the beetles Alphitobius diaperinus and Tribolium castaneum had

acquired high infection with a maximum of 120 cysticercoids per beetle, and the infection rate in chickens was 1-2%.

1.3.3. The life history of Hymenolepis carioca (de Magalhaes, 1898) Guberlet (1919) observed that chickens became infected with this tapeworm after they had been fed stable flies caught around poultry yards. It has been demonstrated by Jones (1929), Gram and Jones (1929) that dung beetles act as intermediate hosts. Horsfall (1938) successfully grew cysticercoids of this species in Tribolium castaneum and T. confusum. When flour beetles containing cysticercoids of H. carioca were fed to young chickens, the latter became infected with the adults of this worm. Cysticercoids develop in beetles to a stage, which is infective for chickens within approximately 3 weeks. Development of the adult worm in the chicken to the time when gravid segments are passed requires 2-4 weeks (Wher, 1972). Reid (1984) enumerated 26 species belonging to 9 families of beetles and one species of termites as experimental or natural intermediate hosts; dung and ground beetles are the most common source of infection.

1.4. Prevalence of Raillietina cesticillus, Choanotaenia infundibulum and Hymenolepis carioca Wilson et al. (1994) conducted a 12-months survey to determine the prevalence of poultry helminth in Northwest Arkansas commercial broiler chickens. A total of 3542 intestinal tracts from market- ready broilers were collected from two commercial broiler companies (A and B). R. cesticillus was found in 27.2% of company A farms and 69.2% of company B farms.

In 1997, in Morogoro region, Tanzania, Permin et al. conducted a cross sectional study on the prevalence of helminths in rural scavenging poultry on 600 adult chickens selected randomly from 6 villages during wet and dry seasons. All chickens were infected with one or several species of helminths, the average being 4.8 ± 1.7 helminths per chicken during the wet season and 5.1 ± 1.8 during the dry season. A total of 29 different species were shown in the trachea or gastrointestinal tract. Raillietina cesticillus, 8.7% (wet season), 2.7% (dry season); Choanotaenia infundibulum 0.0% (wet season), 3.7% (dry season) and Hymenolepis carioca 9% (wet season), 18% (dry season); in addition to other species of cestodes and nematodes. In Denmark, Permin et al. (1999) performed cross-sectional prevalence study in Danish poultry system in 268 adult chickens selected randomly from 16 farms from October 1994 to October 1995. Raillietina cesticillus and Choanotaenia infundibulum were found in battery cages, but not in the back yard or deep-litter systems in which nematodes were recovered. Another cross-sectional study for gastrointestinal helminths and haemoparasites was conducted by Poulsen et al. (2000) in chickens kept in extensive system in Ghana, West Africa. Chickens were found 100% infected. R. Cesticillus and C. infundibulum comprised 12% and 13%, respectively. An over dispersed distribution was seen for most of the helminth species. In Botswana, Kgatleng district Oodi, 13 adult indigenous chickens, were examined for helminth parasites. Two species of nematodes, Ascaridia galli and Hetrakis gallinarum and species of cestodes, genus Raillietina were recovered. Ascaridia galli occurs concurrently with Raillietina spp. (Mushi, et al., 2000).

In four districts in Amhara region, Ethiopia, A survey was done in October 1998 to August 1999 by Eshetu et al. (2001), the prevalence rate for R. cesticillus was 5.62% and 4.49% for C. infundibulum with highest prevalence rate being observed in low land areas.

1.5. Pathogenicity of poultry tapeworms Generally, the domestic fowl, Gallus gallus domesticus, as one of the more frequent hosts of cestodes, suffer a considerable damage; tapeworms sap its energy, cause stunted growth, lower egg production, bring about emaciation and not infrequently death (Elowni, 1977).

1.5.1. Pathogenecity of Raillietina cesticillus The pathogenic effects of this cestode are manifested by retardation in growth, reduction in egg laying, diarrhoea and general weakness. In heavy infection the bowl may be occluded and the normal movement of the intestinal content greatly disturbed. Nervous symptoms such as twisting of the head and neck in an abnormal position and paralysis of one or two legs (Dutt, 1961; Reid, 1963; Barger et al., 1969; Mathur and Pande, 1969; Kaushik and Deorani, 1971; Soulsby, 1978; Ackert and Case, 1981 and Bhowmik and Sinha, 1983). However, Botero and Reid (1969) considered R. cesticillus as non-pathogenic and this view is based on extensive control experiments with broilers and layers on optimal nutritional diets.

1.5.2. Pathogenicity of Choanotaenia infundibulum Different pathogenic effects were produced by C. infundibulum such as loss of appetite, drooping, thrust, anaemia, emaciation and reduction in egg production. In severe infections death may occur (Swada, 1955; Dutt,

1961; Reid, 1963; Barger et al, 1969; Nadakal et al., 1971; Hofstad, 1978; Soulsby, 1978; Allam, 1981; Bhowmik and Sinha, 1983).

1.5.3. Pathogenicity of Hymenolepis carioca Hymenolepis carioca is considered to be the least pathogenic cestode. Yet, in heavy infections, clinical signs such as enteritis, diarrhoea, anaemia and paleness of comb and wattles, in addition to thickening of the wall of the intestine with the presence of haemorrhagic exudates (Barger et al., 1969; Kaushik and Deorani, 1971; Soulsby, 1978; Allam, 1981; and Bhowmik and Sinha, 1983).

1.6. Alphitobius diaperinus, Panzer (Tenebrionidae) Alphitobius diaperinus is a cosmopolitan mycophagus beetle, which feeds on mouldy and damaged food products. It is an important beetle in the grain and poultry industries (Schroeckenstein et al., 1988). According to Elrabaa (1967) this species was commonly found in pigeon houses in association with pigeon cotes and bat roosts. Elowni (1979) showed the occurrence of this beetle in two poultry farms in Khartoum area with high prevalence of poultry cestodes. The life cycle of the lesser meal beetle averages 70 days, of which 48 days are spent as a larva, with temperature and diet affecting the life length (Loomis, 1984).

1.6.1. Alphitobius diaperinus as an intermediate host for cestodes and nematodes of poultry Elowni (1979) first reported the role of Alphitobius diaperinus as intermediate host of poultry tapeworms in two farms in Khartoum, Sudan,

with a prevalence rate of 11% and 54%. In intensive search done in quail breeding in Japan, by Uchida et al. (1984), Alphitobius diaperinus was not found to harbour Choanotaenia infundibulum. Similarly, in a survey done in cage layer houses in Brazil (Avancini and Ueta, 1990) did not find larval stages of C. infundibulum or even other parasites in this beetle. However, Moya et al. (1977) described A. diaperinus as an intermediate host for the nematode Sublura suctoria in Japan.

1.6.2. The economic importance of Alphitobius diaperinus Adults and larvae of A. diaperinus were found to be infected with Gregarines, a protozoan infection commonly encountered in broiler and turkey production in south-eastern, north-eastern and central Piedmont regions of North Carolina, USA, with high level of infection in larval stages in specific smallest larvae (Apuya et al., 1994). McAllister et al. (1994) showed the reservoir competence of the lesser mealworm, A. diaperinus, for Salmonella typhimurium relative to broiler chicken production. Salmonella typhimurium was isolated from faeces, and from both external and internal body of adults and larvae, after exposure to the microorganism. Salmonella positive cloacal swaps were obtained from one-day old broiler chicks within 24 hours after eating one infected lesser worm adult and larvae. In addition, A. diaperinus was proved to carry Salmonella enteritidis in poultry (Davies and Wary, 1995). Another species of Salmonella was also isolated from this beetle, S. Mbandak, from cage layer houses (Olsen and Hammack, 2000) together with other salmonellae isolated from housefly, Musca domestica, and dump fly, Hydrotaea. Castrillo et al. (1998) isolated 24 strains of Beauveria bassiana

(Deuteromyotina: hyhpomycetes) form the darkling beetle A. diaperinus from poultry houses in North Carolina and West Virginia, USA. Loomis (1984) and Jordan (1990) implicated adult A. diaperinus in transmission of Marek's Disease carrying the virus for several weeks. Despins and Axtell, (1994, 1995) showed in experimental work, that broiler chicks and turkey poults restricted to feed on A. diaperinus larvae, exhibited reduction in growth. They even did not compensate for reduced weight after returned to starter feed, and consumed larvae diet in the presence of their normal feed. Loomis (1984) reported that lesser meal worm beetle, A. diaperinus may invade Styrofoam insulation used in enclosed poultry houses and cause severe damage, particularly to ceiling areas requiring extensive repair. Individuals exposed to the beetle A. diaperinus as a result of their occupations, developed symptoms of asthma, rhinitis, conjunctivitis, urticaria and angioedema (work related symptoms). In addition, they developed IgE mediated sensitivity to A. diaperinus antigen (Schroeckenstein et al., 1988).

1.7. Tribolium castaneum (Coleoptera, Tenebrionidae) The flour beetles are cosmopolitan insects that have been associated with grain stored by man at least since early Egyptian times. In nature, they live under bark where they were semi-predators feeding on both living and dead materials. Today they are found in stored grains of many kinds and in products manufactured from grains (Robert, 1985)

1.7.1. Life cycle Each female may deposit from 400-500 white eggs among feed particles. The number of the larval instars varies between 5 and 12. The time required to develop from egg to adult depends upon the moisture and temperature. Under optimum conditions of enviroment and food the developmental period is approximately 30 days (Robert, 1962).

1.7.2. Experimental infection of the meal beetles Tribolium castaneum and T. confusum with tapeworms Horsfall (1938) experimentally infected adult meal beetles, Tribolium castaneum and T. confusum, for the first time, with three cestodes, Raillietina cesticillus, Choanotaenia infundibulum and Hymenolepis carioca, the infectivity percent was 82, 28 and 17 respectively. Cysts developed in these beetles were used for experimental infection of chicken, the establishment percent was 100 for H. carioca and 83 for R. cesticillus, the least one was C. infundibulum, 33. Two groups of chickens of different ages were fed with suspected R. cesticillus infected beetle, younger chickens contain higher number of R. cesticillus (40 and 37) than older ones (26 and 25). Luttermoser (1940) exposed adult and larvae of T. castaneum to infection with R. cesticillus, R. echinobothrida and H. carioca. Adult meal beetle contained cysticercoids of both H. carioca and R. cesticillus; in addition, cysticercoids of R. cesticillus were also recovered from T. castaneum larvae, whereas neither larvae nor adult beetles became infected with cysticercoids of R. echinobothrida. Adult meal beetles were found to be more suitable intermediate hosts than the larvae, Since 80.3% of the adults and only 70.2% of the larvae contain cysticercoids and higher mean number

of cysts (11.5) in the adult than in larvae (6.7). The process of metamorphosis of the beetle did not destroy the cysticercoids since the cysts were found in pupae and young adult beetles that contracted the infection as larvae. Meal beetle Tribolium confusum was used to study the high temperature stress upon cysticercoids. It was found that high temperature stress (38.5oC) applied during the development of H. cestilli and R. cesticillus in the beetle T. confusum, showed the presence of sensitive period coinciding in both species with the period of maximal growth and differentiation. Elowni (1977) studied the development of R. cesticillus in the beetle T. castaneum, in addition the beetle was exposed to experimental infection with different chicken cestodes including the C. infundibulum, H. carioca, Cotugnia digonopora and R. tetragona, the last three cestodes showed negative results.

1.8. Musca domestica (Linnaeus, 1758) Musca domestica, the common housefly, has a cosmopolitan distribution and is important as a mechanical carrier of various infectious agents including viruses, bacteria and protozoa. It also acts as an intermediate host for a number of helminths (Soulsby, 1982).

1.8.1. Life cycle The housefly lays 100-150 eggs at a time and a total of about 1000. Fresh horse manure is preferred, but the fly will also develop in faeces of other animals and man as well as in all sorts of decaying organic matter and refuse. The eggs are 1 mm long, elongate and creamy-white in colour and

the dorsal surface has two curved, rib-like thickenings. The larva hatches in 12-24 hours and grows into a maggot 10-12 mm long in 3-7 days depending on enviromental temperature, and the pupa remains in the larval skin, which turns brown and becomes rigid to form the puparium. The full-grown larva leaves the material in which it has developed to pupate in the ground. The pupal stage lasts 3-26 days depending on the temperature. Fertilisation and oviposition takes place a few days after emergence of the fly and the whole cycle may be completed in about 8 days at 33-35˚C, so that a number of generations develop in one summer (Soulsby, 1982).

1.8.2. Musca domestica and disease transmission Houseflies have been found to harbour about 100 different pathogens and charged with transmitting 65 of them (Greenberg, 1965). The pathogens recovered from this fly range from viruses to helminths. It includes the viruses of poliomyelitis, infectious hepatitis and Newcastle disease virus; bacteria associated with cholera (Vibrio), enteric infections caused by species of Salmonella and Shigella, pathogenic Escherichia coli, haemolytic streptococci, Staphylococcus aureus, agents of trachoma, bacterial conjunctivitis, diphtheria, tuberculosis, and leprosy and yaws. In addition, flies can carry the cysts of protozoa, including those of Entamoeba histolytica, which causes amoebic dysentery and the eggs of the thread worm, Trichuris trichiura, the hookworm, Ancylostoma duodenale Heterakis gallinarum and other nematodes and cestodes (Loomis, 1984; Kettle, 2000). The housefly is a biological vector and intermediate host of certain cestodes of poultry, e.g. Choanotaenia infundibulum and Raillietina cesticillus (Soulsby, 1982). The fly is also encountered in the transmission of

habronemiasis in horses (Kettle, 2000). Fly larvae have only a tenuous association with myiasis, the invasion of living tissue with dipterous larvae. When they occur in an advanced stage of myiasis in sheep, they do not feed on living tissues, but on the exudates and matted wool (Pont, 1973). Milushev (1978) showed the tendency of Musca domestica to transmit the oocysts of Eimeria tenella, both sporulated and non-sporulated. No sporulation occurred inside the gut of the fly, but the oocysts remain viable and retain sporulation capacity after their excretion. In addition, chicks were infected with coccidida when fed flies infected with oocysts.

1.9. Family Histeridae-Hister beetles Hister beetles are small (0.5-10 mm in length) broadly oval beetles that are usually shining black in colour; the elytra are cut-off square at the apex, exposing one or two apical abdominal segments. Hister beetles are generally found in or near decaying organic matter such as dung, fungi and carrion, but are apparently predaceous on other small insects living in these materials. Some species, which are very flat, occur under the loose park of stumps or logs; a few live in nests of ants or termites. A few species are elongate and cylindrical, these live in galleries of wood-boring insects (Borror et al., 1974). The two genera Hypocacculus and Carcinops, belonging to this family, are very wide spread genera of predatory Histeridae associated with birds' nests, but typically found in animal and in particular poultry housing. The beetles breed in the dung and feed on the eggs and larvae of flies in particular Muscidae (Roger, 2001). Carcinops spp. i.e. Carcinops pumlio are often used as biological control agents of housefly (Diptera, muscidae) eggs and larvae, however the

beetles are found to be a competent reservoir of Salmonella entertidis (Gray et al., 1999). Other Carcinops species such as Carcinops 14 striatus has been proven to be a natural intermediate host for the chicken tapeworm Hymenolepis carioca (Jones, 1929).

1.10. Family Anthicidae ant-like flower beetles These beetles are 2-6 mm in length and somewhat ant-like in appearance, with the head deflexed and strongly constricted behind eyes and with pronotum oval. Anthicids generally occur in flowers and foliage, some occur under stone and logs and in debris, and a few occur on sand dunes (Borrer et al., 1974). The species Anthicus formicarius, is almost cosmopolitan and is known to occur in Africa (Roger, 2001).

1.11. Diagnosis and identification of poultry tapeworms Diagnosis of poultry cestodiasis usually is made at necropsy. A postmortem examination of a representative number of the flock is the most satisfactory means of diagnosis since not only may the burden of parasites be assessed, but also the species may be determined (Soulsby, 1965). Since species identification is most accomplished by examining the scolex and any distinctive attached hooks, considerable effort should be spent in its recovery. The general length and width of strobila may also provide a diagnostic feature (Reid, 1984). Distinctive diagnostic characteristics may also be recognised by examining the eggs of chicken tapeworms found free in the intestinal contents or recovered by teasing from gravid proglottids (Reid, 1959, 1984). Presence of tapeworms and their identification may also be accomplished by examining discharged eggs, gravid proglottids or whole worm from live birds.

1.12. Control of poultry cestodes This can be accomplished through: 1.12.1. Control of the intermediate host 1.12.1.1. Changing management practice Since a delicate ecological balance is required to establish tapeworm infection in both definitive and secondary host, a change in management practices may provide a relatively an easy method of parasite control. Poultry management should stress sanitary measures to capitalize on the advantage of modern poultry system (Reid, 1984). Arthropod parasite problems will be minimized by thorough cleaning of houses between batches of birds, whole flock replacement rather than partial culling and replacement, smooth house construction and mesh to keep out wild birds and keeping manure too dry for fly breeding.

1.12.1.2. The use of insecticides This is also beneficial in controlling the arthropod intermediate hosts. These compounds must be accepted by the enviromental protection agency (EPA) as causing no hazardous residues in eggs, meat or edible poultry products (Loomis, 1984). The type of insecticide used is selected in accordance to type of insects and their habitat.

1.12.1.3. The use of drugs Ivermectin incorporated in feed of broiler chicken at a level of 2 PPM for 5 weeks was found to be efficient in eliminating lesser meal worm larvae; no Ivermectin residues were found in the livers of treated chickens even without withdrawal period. However, the feeding of Ivermectin may reduce body weight gain and feed efficiency of older ages (Miller, 1990).

1.12.1.4. Biological control Predators of insects' eggs and larvae are one of the controlling tools, e.g. the mite Macrocheles muscae domesticae used alone or in combination with other larvicides is effective against Musca domestica larvae. Beetles such as Carcinops pumilo is used in fly control strategy, which involves its movement between poultry houses to facilitate its establishment. However, this beetle was found to harbour Salmonella enteritids so its movement between poultry facilities should be considered (Gray, 1999). Also Bacillus thuringenesis (Berliner) was found effective against fly larvae and harmless to the mite Macrocheles muscae domesticae (Wicht et al., 1970).

1.12.1.5. Mechanical barrier This consists of bands of polyethylene terrepthalate resins to prevent climbing by lesser mealworm Alphitobius diaperinus larvae in poultry houses. Attaching to wooden post latex caulk adhesive staples were 100% effective (Geden and Carlson, 2001).

1.12.2. Treatment of infected chickens A large number of drugs have been recommended for treatment of cestodiasis in poultry with variable results. These include: Niclosamide (Yomesan; Bayer; Leverkusen) twenty mg/kg/day of the drug is effective against Raillietina cesticillus (Boisvenue and Hendrix, 1965). The compound is 100% effective against R. tetragona at a dose rate of 125-250 mg/bird weight 1-1.5kg (Ioda and Singh, 1975). The use of levamisole at 40 mg and Niclosamide at 200 mg/head enhances the cestodal action of the latter drug (Hadykto-Mv; Kulik-OM, 1991).

Dichlorophene: the use of the drug at a dose rate of 1.25 mg/kg body weight at repeated doses was 100% effective against Raillietina species (Prasanna et al., 1974). Hexachlorophene: this drug was found to be 100% effective against Raillietina cesticillus at a dose range of 26-50 mg/kg body weight (Keer, 1948). Its combination with other compounds such as phenothiazine 500 mg or nicotine sulphate 50 mg, at the rate of 50 mg has highly effective results against tapeworms, particularly Raillietina spp. (Hungerford, 1955). Bunamidine: a single dose of 50-mg/kg-body weight was found non- significant against single and mixed infection of R. cesticillus, R. tetragona, and R. echinobothrida. The drug gave an efficacy of 100% against R. cesticillus when administered at 50-mg/ kg body weight twice with 48 hours interval in doses. At 100 mg/kg body weight, the drug gave a significant result against R. tetragona and R. cesticillus (Gogoi and Chaudhuri, 1982). Tin compounds: tin sulphate and tin phosphate at a dose of 1g/kg body weight were effective against R. cesticillus and Amoebotaenia sphenoides with varying percentage, but they were 100% effective against Hymenolepis Carioca (Prasanna et al., 1974). Dibutyl tin dilaurate given as capsule or incorporated in feed was effective against R. cesticillus and R. tetragona with 90% and frequently 100% (Edgar, 1956). Mebendazole: this broad spectrum anthelmintic is very effective against Raillietina species at a dose rate of 50, 25 and 10 mg/kg by weight (Matta, 1980)

CHAPTER TWO MATERIALS AND METHODS

2.1. Collection of chicken tapeworms Tapeworms were collected from viscera of different breeds of chicken, including White leghorn (culled from large scale poultry farms), Baladi, Fayoumi and their crosses (reared at rural areas around Khartoum North Province); slaughtered at Khartoum North Slabs located at Khartoum North Central Market. The first specimens of viscera collected were wrapped in plastic bags, but due to occurrence of quick putrefaction, the rest of the samples were brought to the laboratory in paper bags. The collected viscera were washed with running tap water to remove clotted blood, feathers and other debris, if present. Following the method adopted by Elowni (1977), the entire viscera were freed from their mesenteries and divided into parts: duodenum refers to the parts of the intestine that begins at the exit from the gizzard and terminates at the level of the bile and pancreatic ducts' common papilla. The ileum follows the duodenum and terminates where the two caeca join: the vestige of the yolk sac (Meckel's diverticulum) is a common anatomical feature half-way down this piece of intestine and divides it into an upper and lower ileum. Both upper and lower ilea are then equally divided into anterior and posterior parts, before the intestine was examined for tapeworms. The large intestine starts from the point where the two caeca join and terminates at the cloaca. Each of these segments was placed in a shallow tray with a black background. Using a pair of stainless steel blunt edges scissors, the intestines were slit opened longitudinally. The scolices were freed from the mucosae

by cutting underneath the attachment point when robust species such as Raillietina tetragona and Cotugnia digonopora were encountered or scraped with glass slide; with Hymenolepis carioca, a delicate form, the mucosa was teased with dissecting needles under a dissecting microscope. Worms were then collected in glass petri dishes togather with gravid proglottids found free in the intestines.

2.2. Preparation of infective material Looking for mature eggs (infective material) the last segments of the collected tapeworm, were separated from their strobillae, placed on glass slide and cut on its (their corners) corner with sharp dissecting needle to determine the maturation of their eggs. Gravid proglottids, found free in the intestine or attaching to their strobillae, were shredded on filter paper using sharp dissecting needle and fed immediately to the prepared insects.

2.3. Experimental insects 2.3.1. Tribolium castaneum 2.3.1.1. Rearing and maintenance About 100 adult beetles, Tribolium castaneum, were obtained from Food Research Centre, Shambat. It was then maintained in the laboratory in an incubator (Gallenhamp, England) at a temperature ranging between 30- 33ºC, in wide-mouthed glass jars (20x14 cm) containing wheat flour (Sayga Wheat Flour Mills, Khartoum North) and brewer's yeast at rate of 5%. The culture jar was closed with muslin cloth, kept in place by rubber band. Grumble papers were introduced into the jars to provide walking and pupation sites. Labelling was made on the jar side indicating the origin of insects and the date of culturing. Culture jar was left inside the incubator for

two weeks and then sieved with a 25-mesh sieve to remove the adult insects into another medium. When the newly adult beetles started to emerge, the culture was left for another week, then shifted to new culture.

2.3.1.2. Formation of infected insect colonies Adult T. castaneum, 1-2 weeks old were distributed in glass jars (8.5x5 cm), 10 beetles in each, tightly closed with muslin cloths and rubber bands and then starved for several days (1-6) days. The starved beetles were then placed in labelled petri dishes and fed en masse freshly prepared infective material (see 2.2), and incubated for over night. In some cases, beetles were repeatedly exposed to infection, especially when Choanotaenia infundibulum and Hymenolepis carioca were involved. Wheat flour togather with brewer's yeast, were offered to the beetles on the next day, then the arthropods were kept for varying periods following infection before they were examined for cysticercoids.

2.3.1.3. Cysticercoids recovery Beetles were killed by decapitation and placed in either physiological saline or tap water in glass petri dishes. The chitinous exoskeleton was torn into small pieces under stereoscopic dissecting microscope. Each piece was agitated with the dissecting needle to dislodge any adhering cysticercoids. The recovered cysts were then studied and counted to evaluate the establishment of different cestodes and they might either be used to infect the definitive host, i.e. chicken, or preserved in one of these solutions: 4%, 5% formaline (Appendix 1, 2) or Zenker's fluid (Appendix 3). The infection rate, the abundance of infection and intensity of infection were then determined according to Buscher and Otim (1986). The infection rate was

expressed as percentage of beetles infected / number of beetles examined; the abundance of infection as the mean number of cysticercoids recovered / beetles examined and the intensity of infection as the mean number of cysticercoids recovered / infected beetles. The mortality rate was sometimes determined for insects as percentage of dead insects /number of insects examined.

2.3.2. Alphitobius diaperinus 2.3.2.1. Rearing and maintenance From Shambat area and during July 2000, about 50 adult Alphitobius diaperinus beetles were collected in plastic bags from highly infested poultry houses. Alphitobius diaperinus was then maintained in the laboratory in wide-mouthed glass jars (20x14 cm), containing insect free mash and sterile poultry manure in equal quantities. The jars were tightly closed with muslin cloths and rubber bands. Moist filter paper was placed in the jar to raise the humidity and to induce fungal growth (Elowni, 1977). Sterilization was done in an oven (Memmert-854 Schwatbach, West Germany) at 140ºC for two hours (Robert and O'sulaivan, 1950). One week later, the emerging larvae were transferred to another jar (20x14 cm) containing the same media.

2.3.2.2. Formation of infected insect colonies Adults and larvae of the beetle Alphitobius diaperinus, were distributed in glass jars (8.5x5 cm), 10-15 beetles in each, tightly closed with muslin cloths and then starved for 1-7 days. Moist filter papers were placed inside these jars. In some cases sterile dampened sand was used as media during the starvation period.

Adults and larvae of this beetle were exposed to parasitic infection in the same way as for adult Tribolium castaneum. Dissection of adults and larvae was carried out 16-20 days post infection as done before for the previously mentioned beetle.

2.3.3. Musca domestica 2.3.3.1. Rearing and maintenance Musca domestica, common house flies, were caught from a small hut, situated closely adjacent to the dairy cattle pens, near Faculty of Veterinary Medicine, University of Khartoum, using 20-cm diameter hand net. The captured flies were introduced into 45-cm square wooden wire cage through a circular opening situated into the frontal side, which was covered with a piece of cloth to prevent the flies from getting out. The cage with the flies was then placed on the top of a laboratory stool, whose legs were immersed to about 10cm in a detergent, e.g. powder soap, to prevent the climbing ants to gain access to the cage. Cubes of sugar and sufficient amount of water was placed inside the cage. To facilitate oviposition, a mixture of 80 grams grass meal, 10 grams brewer's yeast and 10 grams powdered milk, made in pasty consistency was placed inside the cage in a shallow plastic tray.

3.3.3. Exposure to infection Two methods were used: 3.3.3.1. Embedding tapeworm gravid segments in sterile chicken manure Following the method adopted by Alictata and Chang (1939), glass jars (8.5x5 cm) were half filled with sterile chicken manure and then water was added. Gravid segments of Choanotaenia infundibulum or Raillietina

cesticillus were then embedded in the moist manure. Ten larvae of Muscua domastica; first, second and third instars per each jar, were placed with the segments and incubated overnight. Whenever, pupae appeared, they were transferred to other jars with slightly dampened sand and covered with muslin cloths and rubber bands. A piece of cotton daily soaked in sweetened milk was placed on the top of each jar as feeding for the emerging flies. Seventeen to twenty days post infection, flies were either trapped in plastic bags and incubated in the fridge for 1-2 hours or drowned. Flies and pupae were dissected in the same manner as the previously mentioned insects.

3.3.3.2. Maceration of the gravid segments First, second and third stage larvae of Musca domestica were picked up with sharp forceps and collected in glass petri dishes half-filled with tap water and washed thoroughly in order to remove any adherent material. Twenty larvae were then distributed in each glass jar (8.5x5 cm) or petri dish with a piece of cotton soaked in tap water, to provide moisture and hiding sites, as they are known to react negatively to light (Loomis, 1984) and allowed to starve for 2-3 days. Ten starved larvae were placed in each labelled petri dish with filter paper containing the infective material (see 2,2) and another piece of filter paper was used as covering for the larvae. They were incubated for 3-4 hours, then the larvae were moved to other jars with sterile chicken manure and incubated for varying periods and dissected as the above mentioned ones. Some larvae were transferred to petri dishes or glass jars with mixture of grass meal, milk powder and brewer's yeast.

2.4. Experimental chickens 2.4.1. Rearing and housing A total of 66, 1-day old White leghorn chicks were purchased from commercial poultry farm and reared in insect proof cages, and fed on balanced ration and watered ad lib.

2.4.2. Administration of the infection Cysticercoids obtained from experimentally infected beetles were fed to 1-7 days old chicks in tap water, using gelatin capsules or Pasteur pipettes. The required numbers of the mature larvae were removed with the Pasteur pipette; to which a rubber sucker was attached to one end, shortly after recovery. Cysticercoids were then introduced directly into the pharynx of the bird or put inside the gelatin capsule, which was then given quickly to the chick to avoid disintegration of the gelatin capsule before administration.

2.4.3. Recovery of the mature tapeworms Two weeks after administration of the tapeworms' larvae the chicks were sacrificed with a sharp knife. The entire viscera of the bird were exposed, the intestines were taken out, freed from their mesenteries and longitudinally opened with a pair of stainless blunt edges scissors, on a shallow tray with a black background, and the attaching scolices of the worms were located. Recovered worms were collected in petri dishes and washed several times in physiological saline to remove adhering mucus and debris and then fixed in Rouda bush (Appendix 4), then preserved in 70% alcohol (Appendix 5), or 10% formal saline (Appendix 6), and were further identified microscopically.

2.5. Search for natural intermediate hosts for chicken tapeworms 2.5.1. Collection of data Numerous specimens of miscellaneous arthropods were collected from poultry houses at Shambat and Elhalfaya localities during 2000/2001. Poultry facility from which the arthropods were collected, adopting deep litter system for production of table eggs; except for one house in which two batteries were arranged side by side.

2.5.2. Collection of samples Alphitobius diaperinus (321 samples), Tribolium castaneum (333 samples), and unidentified insects (326 samples); together with 44 Musca domestica larvae; were hand-caught from manure, inside the houses and also from outside, where a lot of manure was scattered. Portion of manure was taken to the laboratory and arthropods sorting was done. These insects might either be found in the most humid area i.e., underneath the drinkers or semi- dry one, i.e. in the bottom or under the laying jars. Cans half-filled with water were embedded in the ground, to trap the creeping and jumping insects. Also light trap was placed inside the pen for collection purpose.

2.5.3. Cysticercoids recovery Collected arthropods were dissected in the laboratory in the same manner as for experimental insects (see section 2.3.1.3). Recovered cysts preserved in Zenker's fluid (appendix 3), 4% or 5% formalin (Appendix 1,2).

2.5.4. Identification of potential insects Collected insects exhibiting potentiality to natural parasitic infection were preserved in screw-capped bottles, containing 5% glycerin alcohol

(Appendix 7). The area from which the samples were collected, time of collection, the habitat and the initials of the collector were labelled. Preliminary identification was done to the family level, then the specimens were sent to a reference laboratory, The Natural History Museum, London, United Kingdom for confirmation.

2.6. Experimental design 2.6.1. Experimental infection of Tribolium castaneum with different species of cestodes Adult Tribolium castaneum starved for varying periods were exposed to experimental infection with different species of cestodes: Raillietina cesticillus, Choanotaenia infundibulum, Hymenolepis carioca and Cotugnia digonopora at different infection doses, then the beetles were dissected after different incubation periods (Table 1).

2.6.2. Experimental infection of Alphitobius diaperinus with different species of cestodes Adults and larvae of Alphitobius diaperinus were starved from 1-7 days and fed on gravid segments of different cestodes and dissected after 16-20 days (Table 2). 2.6.3. Experimental infection of Musca domestica with different cestode species Musca domestica maggots were exposed to cestodal infection of R. cesticillus and C. infundibulum, following 2-3 days starvation periods (Table 3).

Table 1: Adults Tribolium castaneum exposed to different cestode species, at different starvation periods (days), time of dissection post infection (days) and dose of infection (number of segments). Experi- cestode No. of No.of Starvation No. of Time of ment species beetles beetles/ period segments/ dissection No. group (days) group (days) 1 R. cesticillus 20 10 2 2 21 C. 36 12 1 4 20 infundibulum H. carioca 30 10 5 10 20 C. digonopora 20 10 6 5 20 2 R. cesticillus 60 10 4 2 19 C. 60 10 4 2 19 infundibulum H. carioca 60 10 4 2 19 Control 60 10 4 2 19 3 C. 60 10 4 2 60 infundibulum Control 60 10 4 2 60 4 H. carioca 30 10 6 100 12-13 H. carioca 30 10 6 35 15 5 C. 60 10 6 10 6-9 infundibulum C. 60 10 6 15 15-16 infundibulum 6 R. cesticillus 40 10 2 5 21 R. cesticillus 40 10 2 5 21

Table 2: Alphitobius diaperinus exposed to different cestode species, at different starvation periods (days), time of dissection post infection (days) and dose of infection (number of segments). Exper- Cestode No. of No. of Starvation No. of Time of iment species beetles beetles/ period segments dissection No. group (days) (days) 1 R. cesticillus 30** 10 6 4 21 C. infundibulum 20** 10 5 4 16 H. carioca 36** 12 5 10 23 C. digonopora 30** 10 4 5 30 2 C. infundibulum 60*** 15 7 20 20 3 C. infundibulum 60*** 10 4 2 20 C. infundibulum 60*** 10 6 2 20 4 C. infundibulum 36* 12 0 2 20 C. infundibulum 36* 12 4 2 20 * Larvae (2-4 mm). ** Larvae (1 cm). *** Adults.

Table 3: Musca domestica larvae exposed to different cestode species, at different starvation periods (days), time of dissection post infection (days) and dose of infection (number of segments). Experi- Cestode No. of No. of Starvation No. of Time of ment species larvae larvae/ period segments/ dissection No. group (days) group (days) 1 C. 200 10 2-3 5* 15-20 infundibulum C. 120 10 2-3 10-20** 15-20 infundibulum 2 R. cesticillus 140 10 2-3 5* 15-20 R. cesticillus 80 10 2-3 10-15** 15-20 * Segments were shredded on filter papers. ** Segments were embedded in chicken manure.

2.6.4. Experimental infection of White leghorn chicks with different cestode species 2.6.4.1. Design of experiment 1 Ten White leghorn chicks, 2-days-old, were fed, 100, 135-days-old Raillietina cesticillus cysticercoids using Pasteur pipette. These cysts were previously established in T. castaneum adult beetles. Ten chicks were used as non-infected control. Infected and control chicks were sacrificed 2 weeks following infection (Table 4).

2.6.4.2. Design of experiment 2 A total of 20, 2-days-old White leghorn chicks were fed a total of 200, 20-days-old C. infundibulum cysticercoids, previously established in adult Alphitobius diaperinus beetles. Ten chicks received the infection using Pasteur pipette and the other 10 chicks by gelatin capsules. All chicks were sacrificed 2 weeks post infection (Table 4).

2.6.4.3. Design of experiment 3 A total of 20, 7-days-old White leghorn chicks were fed cysticercoids of C. infundibulum in gelatin capsules. These cysts were previously established in adult T. castaneum. Ten chicks were fed 100 cysticercoids of C. infundibulum, 6-9-days-old infection; all chicks were sacrificed 15days post infection (Table 4).

Table 4:White leghorn chicks exposed to infection with different cestode larvae of different age, and slaughtered 15 days post infection. Experi- Cestode No. of Age of No. of Time of ment species chicks cysticercoids cysticercoids/ slaughter No. (days) chick (days) 1 R. cesticillus 10 135 10* 15 Control 10 0 10* 15 2 C. 10 6-9 10** 15 infundibulum C. 10 15-16 10** 15 infundibulum 3 C. 10 20 10* 15 infundibulum C. 10 20 10** 15 infundibulum 4 H. carioca 3 12-13 8,6,6** 15 H. carioca 3 15 3,2,2** 15 * Cysticercoids administered by Pasteur pipette. ** Cysticercoids administered by gelatin capsules. All chicks were not starved before infection. Age of cysticercoids measured from the time the beetles were exposed to infection.

2.6.4.4. Design of experiment 4 Six, 5-days-old White leghorn chicks were fed, using gelatin capsules, cysticercoids of Hymenolepis carioca. Three chicks were fed 12-13-days-old cysts, whereas the remaining 3 chicks, 15-days-old cyst. All chicks were sacrificed 2 weeks post infection (Table 4).

2.7. Statistical analysis Experiments were analyzed using Student's‘t’test, F-test or correlation. Percentage data were transformed using arcsine or √ log 10(x+1) + 0.5.

CHAPTER THREE RESULTS

3.1. Establishment of some cestode species in adult Tribolium castaneum Adults Tribolium castaneum starved for varying periods and fed different doses of infective materials of Raillietina cesticillus, Choanotaenia infundibulum, Hymenolepis carioca and Cotugnia digonopora, was found only refractory to C. digonopora. The adult beetles showed variations in their susceptibilities to the three former cestodes; being more susceptible to R. cesticillus (45%), then C. infundibulum (25%) the least was H. carioca (3.3%). Infection intensity also varied between the recovered cestodes. R. cesticillus had the highest intensity (5.6) and the highest infection range (1- 16), (Table 5). Different developmental stages were recovered from infected beetles, including immature and mature larvae. Mature cysts of R. cesticillus (Plate 1), showed deep notch at the broader end and characteristic wide circlet rostellar hooks; mature cysts of C. infundibulum with capsules, enclosing eccentric positioned larvae and convoluted mucus-like mass (Plate 2), and mature unarmed sucker larva of H. carioca (Plate 3).

3.2. Establishment of R. cesticillus in adult T. castaneum Raillietina cesticillus fed to adult T. castaneum (2 gravid segments per 10 beetles) starved for 4 days, was successfully established (67.8%), with infection intensity of 6.07, abundance of infection 4.1 and infection range 1- 32 cysts per beetle (Table 6).

Table 5: Establishment of some cestode species in three invertebrate intermediate hosts. Species Cestode Infection Infection Abundance Range Of species intensity rate Of of invertebrate (%) infection infection host T. castaneum R. cesticillus 5.6 45 2.6 1-16 Adults C. 2.1 25 0.5 1-6 infundibulum H. carioca 3 3.3 0.1 3 C. 0.0 0.0 0.0 0.0 digonopora A. diaperinus C. 3.22 23.3 0.8 1-20 adults infundibulum A. diaperinus R. cesticillus 0.0 0.0 0.0 larvae C. 3 4.5 0.14 3 infundibulum H. carioca 0.0 0.0 0.0 0.0 C. 0.0 0.0 0.0 0.0 digonopora M. domestica R. cesticillus 0.0 0.0 0.0 0.0 larvae C. 0.0 0.0 0.0 0.0 infundibulum

Plate 1: Mature Raiteillina cesticllus cysticercoid from adult Tribolium castaneum 21d.p.i with deep notch at the broader end (a) and wide rostellar hook-circlet (b). x 25.

Plate 2: Mature twins' cysticercoids of Choanotaenia infundibulum from adult Tribolium castaneum 20d.p.i, with capsule enclosing eccentric postioned larvae (a) and convoluted mucus-like mass (b).x 10.

Plate 3: Mature Hymenolepis carioca cysticercoid from adult Tribolium castaneum 20d.p.i with unarmed sucker larva.x 25.

Table 6: Means infection intensity, infection rate and abundance of infection of three cestode species infecting adult T.castaneum under laboratory conditions. Cestode Infection Infection Abundance Infection species intensity Rate Of range (%) infection R. cesticillus 6.07 a 67.8 a 4.1 a 1-32 (56.2) C. infundibulum 1.75 b 19.3 b 0.4 b 1-5 (26.9) H. carioca 0.0 c 0.0 c 0.0 c 0.0 (0.01) (0.01) (0.01) Control 0.0 c 0.0 c 0.0 c 0.0 (0.01) (0.01) (0.01) ±SE 0.13 0.39 0.09 *Means followed by the same letter in each column are not significantly different at 0.05 level of probability according to Duncan's Multiple Range Test (DMRT). **Arcsine transformed values are in parentheses. SE = Standard Error

No significant correlation was found between infection rate and infection intensity (r=0.540), in addition no significant correlation was also observed between infection rate and abundance of infection (r=0.094). Mortality rate was 1.67%.

3.3. Establishment of C. infundibulum in adult T. castaneum Adult T. castaneum starved for 4 days and fed (2 gravid segments per 10 beetles) contracted the infection with C. infundibulum (19.3%), the mean infection intensity 1.75, abundance of infection 0.4 and infection range 1-5 (Table 6). No significant correlation was observed between infection rate and infection intensity (r=0.146), also no significant correlation between infection rate and abundance of the infection (r=0.432). Mortality rate was 10.56% (19d.p.i.) without significant correlation with infection rate (r=0.794). Mature cysts of C. infundibulum with or without capsules were recovered from both dead and alive beetles from 7-19 d.p.i (Plate 4,5). These cysts were evaginated in tap water after their dissection from recently dead beetles; evagination took about 5-45 minutes post dissection.

3.4 Establishment of H. carioca in adult T. castaneum Tribolium castaneum, adult beetles (starved for 4 days and fed 2 gravid segments per 10 beetles) were not found positive to H. carioca upon dissection 19d.p.i. (Table 6).

Plate 4: Mature Choanotaenia infundibulum cysticercoid from adult Tribolium castaneum (6-9) d.p.i with capsule, carcareous corpuscles are clearly seen (a). x 10.

Plate 5: Mature Choanotaenia infundibulum cysticercoid from adult Tribolium castaneum (15-16) d.p.i with capsule, carcareous corpuscles are clearly seen (a).x 10.

3.5. Variability in infection between R. cesticillus, C. infundibulum and H. carioca Adult T. castaneum was found susceptible to R. cesticillus (67.8%) compared to C. infundibulum (19.3%) and H. carioca (0.0%) at this level of infective dose (2 gravid segments per 10 beetles), (Table 6). The analysis of variance (ANOVA) on infection intensity, infection rate and abundance of infection showed a significant difference between these three cestodes (p<0.01), (Table 6, Appendix 8). R. cesticillus had a higher infection intensity (p<0.05), higher infection rate (p<0.05) and also higher abundance of infection (p<0.05) compared to the other cestodes and the control. Also, C. infundibulum had a higher infection rate (p <0.05), higher infection intensity (p <0.05) and higher abundance of infection compared to H. carioca and the control. On the other hand, no significant difference was found between the control and H. carioca.

3.6. Effect of C. infundibulum infecting adult T. castaneum on the mortality rate (%). Adult T. castaneum (starved for 4 days and fed 2 gravid segments of C. infundibulum) dissected 1-60-d.p.i showed 18.3%, mortality rate that started from the first week and continued two months following infection. Significant difference between the infected beetles and the control was noticed in the second week (p<0.05), (Table 7). In addition highly significant correlation (r=1.14) was present between infection rate and mortality rate.

Table 7: Effect of C. infundibulum infecting adult T. castaneum under Laboratory conditions, on mortality rate (%). Treatment Mortality rate (%) per week 1 2 3 4 5 6 7 8 9 C. infundibulum 1.7 a 8.3 a 3.3 a 1.7 a 2.1 a 1.7 a 0.0 a 0.0 a 2.1 a (1.1) (2.4) (1.3) (1.1) (2.7) (1.1) (0.7) (0.7) (2.7) Control 0.0 a 0.0 b 1.9 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a (0.7) (0.7) (4.1) (0.7) (0.7) (0.7) (0.7) (0.7) (0.7) ±SE 0.29 0.55 2.46 0.29 1.39 0.29 0.0 0.0 1.39 Means followed by the same letter in each column are not significantly different at 0.05 level of probability based on t- test. Analysis is based on transformed value √log10 (x+1)+0.5. SE=standard error

3.7. Effect of the size of infective dose (number of gravid segments) on C. infundibulum established in adult T. castaneum Adult T. castaneum starved for 6 days and exposed to different sizes of infective dose (10 for 2 consecutive days) and (15 for 3 consecutive days) showed significant difference (p <0.01) on infection intensity, infection rate and abundance of infection, (Table 8). Difference was also observed in infection range.

3.8. Effect of the size of infective doses (number of gravid segments) on H. carioca established in adult T. castaneum. Adult T. castaneum starved for six days, fed 100 gravid segments per 10 beetles and another group of beetles fed 35 gravid segments per 10 beetles, both groups successfully contracted H. carioca infection. No significant difference between the two sizes of the infective dose on either infection rate, infection intensity and abundance of infection was detected, (Table 8).

3.9. Effect of submerging gravid segments of R. cesticillus for five hours, in water on infection intensity, infection rate and abundance of infection. Adult T. castaneum (10 beetles) fed five gravid segments of R. cesticillus, immediately after recovery from adult worm, established R. cesticillus compared to the other group fed five gravid segments of the worm submerged in water for five hours. Significant differences in infection intensity (p<0.05), infection rate (p<0.01), and abundance of infection (p<0.01) between the two groups was observed (Table 9).

Table 8: Effect of size of infective doses (number of gravid segments) of C. infundibulum and H. carioca (established in adult T. castaneum)on infection intensity, infection rate and abundance of infection. Number of gravid Infection Infection Abundance Infection segments intensity rate of infection range (%) C. infundibulum 10 1.32 35.74 0.6 1-6 (37.2) 15 2.94* * 63.33* * 1.8 * * 1-13 (54.63) ±SE 0.13 3.49 0.13 H. carioca 35 1.07 ns 20.4 ns 0.27 ns 1-2 (27.1) 100 1.67 ns 26.7 ns 0.47 ns 1-5 (30.8) ±SE 0.47 3.62 0.05 * *P <0.01, ns = not significant. Arcsine transformed values are in parentheses. SE = Standard Error.

Table 9: Effect of submerging, for five hours, gravid segments of R. cesticillus in water on infection intensity, infection rate and abundance of infection. Treatments Infection Infection Abundance Infection intensity rate of range (%) infection Gravid segments 7.34* 68.6** 5.2 ** 1-32 not submerged (58.5) Gravid segments 0.01 0.0 0.01 0.0 submerged (0.01) ±SE 1.52 3.35 0.28 *P < 0.05, **P < 0.01 Arcsine transformed values are in parentheses. SE = Standard Error

3.10. Establishment of some metacestodes in A. diaperinus Alphitobius diaperinus larvae (measuring approximately 1 cm long) starved for varying periods and fed different sizes of tapeworm segments, allowed the establishment of C. infundibulum in contrast to the other cestodes (Table 5). Two fully developed cysts with capsules surrounding mucoid-like cells and 1 free moving larva was recovered 16 d.p.i from the newly hatched adult beetle. Nevertheless, no cestode larvae of R. cesticillus, H. carioca or C. digonopora were recovered during dissection of larvae, pupae or newly hatched adult beetles. In addition, A. diaperinus adult beetles were also found susceptible to C. infunlibulum when exposed to this cestode (Table 5).

3.11. Effect of starving Alphitobius diaperinus (larvae and adult) on establishment of C. infundibulum. Adult Alphitobius diaperinus (10 beetles) starved for 4 and 6 days and fed 2 gravid segments differed in establishing C. infundibulum. The analysis of variance (ANOVA) on infection intensity, infection rate and abundance of infection showed a significant difference between these two starvation periods (p <0.01), (Table 10,appendix 9). Only fully developed cysts with or without capsule were recovered 20 d.p.i. No early developmental stages were observed. On the other hand, A. diaperinus larvae starved for 4 days and fed C. infundibulum (2 gravid segments per 12 larvae) contracted the parasitic infection of C. infundibulum compared to non-starved group given the same infective dose. Significant difference in infection rate, infection intensity and abundance of infection (p <0.01) was noticed (Table, 10).

Table 10: Effect of starving A. diaperinus on C. infundibulum establishment. Starvation Infection Infection Abundance Range of periods intensity rate (%) of infection infection (days) (A) 4 4.5 b 13.3 b 1.2 a 1-27 (21.2) (A) 6 5.88 a 15.3 a 0.9 b 1-12 (24.2) ±SE 0.21 0.40 0.04

(B) 0 0.01 0.0 0.01 0 (0.01) (B) 4 6.11* 43.5** 2.3** 1-14 (42.7) ±SE 1.11 2.86 0.13 Means followed by the same letter in each column are not significantly different at 0.05 level of probability according to DMRT. * P < 0.05, ** P < 0.01, SE = Standard Error. Arcsine transformed values are in parentheses. A=adults, B=Larvae.

3.12. Establishment of metacestode infection in Musca domestica larvae All larval stages, 1st, 2nd and 3rd of Musca domestica that were exposed to either C. infundibulum or R. cesticillus failed to establish these cestodes (Table 5). Gravid segments offered to the flies larvae inside the moist poultry manure, disappeared after a few hours, suggesting consumption by these larvae. Moreover, larvae allowed to feed on gravid segments shredded on moist filter paper, consumed all infective material in less than 4 hours and started seeking additional feed. Incubating M. domestica larvae in moist cotton piece during starvation period seemed to be satisfactory method for preparing them for parasitic infection. Also placing larvae between two pieces of filter paper during feeding time was successful in contrast to the other method used i.e. red light placed over the petri dish with M. domestica larvae, was not successful in keeping larvae on feeding, they still moved around until they reached the rim of the petri dish. In addition starving the larvae for more than two days was not suitable as high deaths were observed among larvae even after feeding on gravid segments and transference to another medium with moist sterile poultry manure. Moreover, transferring previously infected larvae to a medium composed of wheat bran, milk powder and brewer's yeast (freshly prepared, moist media) was not suitable, as most larvae escaped. Drowning or freezing of adult flies allowed strained flies to be dissected and examined for parasitic infection, yet drowning was found to be more effective, as frozen flies soon recovered and began flying. It was also observed during this study the presence of unidentified species of ants predating on M. domestica larvae.

3.13. Establishment of tapeworm infection in the definitive host 3.13.1. Establishment of Raillietina cesticillus in White Leghorn chicks Raillietina cesticillus cysticercoids (135-days-old), previously established in adult Tribolium castaneum, and given by means of gelatin capsules, to non starved chicks (10 cysts/chick), were recovered 15 days post infection as mature worms (Plate 6), attaching to intestinal mucosae, 14-26 cm posterior to the level where pancreatic and bile ducts join. R. cesticillus was significantly established (p <0.05) (Table 11).

3.13.2. Establishment of Hymenolepis carioca in White Leghorn chicks Hymenolepis carioca, cysticercoids, established in adult T. castaneum, and used to infect White Leghorn chicks in two age groups, i.e. 12-13 days old and 15 days-old, showed variations in establishment. 12-13-days old cyst failed to establish, when dissecting chicks 15-days post infection, whereas 15-days cysts were significantly established (p <0.05) (Table 11).

3.13.3. Establishment of Choanotaenia infundibulum in White Leghorn chicks 3.13.3.1. Using cysticercoids established in T. castaneum Choanotaenia infundibulum cysticercoids (6-9 days-old) and (15- 16days-old), previously established in adult Tribolium castaneum, and given to less than one week-old White Leghorn chicks, using gelatin capsules, were not recovered 15-days post-infection (Table 12).

Table 11: Infectivity of R. cesticillus and H. carioca to white leghorn chicks. Species of Age of No. Cysticercoids/ Infectivity Infection cestodes Cysticeroids chick (%) range (days) R. cesticillus 135 10 5** (20.5) 1-2 Control 0 0 0.0 0 ±SE 1.45 H. carioca 12-13 8,6,6 0.0 (0.0) 0 H. carioca 15 3,2,2 38.9* (43.5) 1-2 ±SE 12.6 * P < 0.05, ** P < 0.01, SE = Standard Error Arcsine transformed values are in parentheses.

Plate 6: Scolex of Railietina cesticillus, showing broad rostellum (a), wide rostellar hook circlet (b) and unarmed suckers(c). x 25.

Table 12: Infectivity of C. infundibulum to white leghorn chicks. species of Age of No.Cysticercoids/ Infectivity Infection invertebrate Cysticeroids chick (%) range host (days) T. 6-9 10 0 0 castaneum 15-16 10 0 0 A. 20* 10 0 0 diaperinus adult A.diaperinus 20** 10 1 1 adult

* Cysticeroids were administered by Pasteur pipette. ** Cysticeroids were administered by gelatin capsules.

3.13.3.2. Using cysticercoids established in Alphitobius diaperinus Cysticercoids of Choanotaenia infundibulum (20-days-old), recovered from experimentally infected adult A. diaperinus, and administered to white leghorn chicks, by means of gelatin capsules, were established (1%) (Plate 7). However, cysticercoids administered to the other group of chicks, using Pasteur pipette, were not established (Table 12).

3.14. Naturally infected intermediate hosts Searching for natural intermediate hosts of chicken tapeworms at Shambat and Elhalfaya localities revealed the abundance of miscellaneous arthropods inhabiting poultry houses. Chicken litter was highly infested with different types of insects, in particular badly constructed houses, and heaps of manure around them. Inside these houses, the presence of humidity may favour the presence of insects. A total number of 1205 different insects were dissected to determine whether they harboured tapeworm cysticercoids. From a total number of 60 A. diaperinus, adult beetles collected from Shambat Locality, 17 (28.33%) were found harbouring cysticercoids of C. infundibulum. The range of infection was 1-119 cysticercoids per beetle and 18.59 infection intensity (Table 13). Of these 60 A. diaperinus beetles, 50 were collected from deep litter and the 10 from battery-cage showed no parasitic infection. Another group of 261 beetles of the same species, comprises (195) adult, 24 pupae and 42 larvae, that were collected from poultry houses in Elhalfaya locality, within which 46 (23.59%) adults, 2 (4.76%) larvae and 1 (4.17%) pupa, were infected with cysticercoids of C. infundibulum. Infection intensity was 12.5 in adult beetles and 1 for the other developmental stages, the range of infection for adult beetles 1-209 and 1 for both pupae and larvae

Plate 7: Scolex of Choanotaenia infundibulum, showing pointed distinctive rostellum with a single raw of slender hooks (a) and unarmed elongated suckers (b). x 4.

Table (13): Prevalence of C. infundibulum and R. cesticillus in naturally infected beetles collected at Shambat and Elhalfaya Localities in 000/2001. Locality Species of Total no. of Cestode species Total no. of Total no. of Infection Infectio Infec beetles beetles dissected recover-ed infected cysticercoids intensity n rate tion beetles recovered (%) range

Shambat A. diaperinus 60 C. infundibulum 17 316 18.59 28.33 1-119 A.formicarius 119 6 45 7.5 5.04 2-22 H. praecox 40 R. cesticillus 4 45 11.5 10 3-36 C. troglodytes 40 R. cesticillus 9 86 9.56 22.5 1-28 Elhalfa-ya A.diaperinus 195* C. infundibulum 46 575 12.5 33.59 1-207 24** 1 1 1 4.17 1 42*** 2 1 1 4.76 1 * Beetles were collected as adult. ** Beetles were collected as pupae. ***Beetles were collected as larvae.

(Table 13). In this locality (Elhalfaya), adult beetles were collected, in two separate sampling, first sample, 98, of which 21 (21.44%) were found infected with C. infundibulum, 24.095 infection intensity and range of infection 1-209. The other group (97) that was collected after application of some sanitary measures gave 25 infected beetles (25.77%), infection intensity (2.76) and range of infection 1-8. No early developmental stages were recovered, and almost all cysts were found mature, with or without capsules. Some cysticercoids showed variation in sizes, and appeared dark in colour and surrounded by black capsules. The family Anthicidae was also found to be the natural reservoir of the chicken cestode C. infundibulum (Plate 8). A total number of 119 adult Anthicus formicarius beetles, were dissected for determination of cestode infection. Anumber of 6 (5.04%) positive beetles to C. infundibulum released 45 cysticercoids, with 7.5 infection intensity and 1-22 range of infection (Table 13). Infection intensity and infection rate of C. infundibulum in Anthicus formicarius greatly differed in the two management practices. In the deep litter, 4(20%) adult A. formicarius were infected with infection intensity (9.75) and range of infection (3-22). Nevertheless, in the battery-cage, only 2(2.02%) beetles were found infected with infection intensity (3) and (2-4) range of infection, which indicates the high prevalence of cestode infection in deep litter. A total number of 80 adult beetles belonging to the family Histeridae, were collected from poultry houses at Shambat locality. Hypocalccus praecox was examined for the presence of parasitic infection. Four beetles (10%) were found positive to R. cesticillus, with 11.25 infection intensity

Plate 8: Ant-beetle, Anthicus formicarius, collected from shambat, natuarlly infected with C. infundibulum. x 12

And infection range between 3-36 cysticercoids per beetle (Table 13, Plate 9). The other species Carcionops troglodytes was also found harbouring R. cesticillus cysticercoids. Nine specimens (22.5%) were infected with 86 total number of cysticercoids, with (9.95) infection intensity and 1-28 range of infection (Table 13, Plate 10). From a total number of 333 adult Tribolium castaneum and 44 Musca domestica larvae, collected from Shambat locality, no parasitic infection was observed during their dissection.

Plate 9: Hypocalccus praecox, collected from shambat, natuarlly infected with R. cesticillus.x12

Plate 10: Carcionops troglodytes collected from shambat, harbouring R. cesticillus in the nature.x12

CHAPTER FOUR DISCUSSION

In this study, the beetle Tribolium castaneum was found to be susceptible to infection with three species of cestodes viz Raillietina cesticillus, Choanotaenia infundibulum, and Hymenolepis carioca following experimemtal feeding of gravid segments. Mature cysticercoids of these parasites were recovered following dissection of this insect 20days following infection. This result confirm those obtained by Horsfall (1938), Luttermoser (1940) and Elowni (1977), although Elowni, (1977) found that T. castaneum is refractoy to experimental H. carioca infection. Infections with these parasites in T. castaneum differed with regards to infection rate; infection intensity with R. cesticillus appears to be the most successful in parasitism. Such differences in infection rates and infection intensities, however, may be attributed to differences to the number of segments from different tapeworms offered to the similler number of beetles, differences in starvation periods prior to exposure of beetles to infection as it is not possible to judge the exact number of onconspheres present in individual gravid segment of different tapeworm species fed to grantee comparable infection doses. In this respect, starvation of beetles, though expected to reduced feeding discimination, thus reducing variability in the size of infective dose, nevertheless, T. castaneum in this study appeared to have a feeding preference to R. cesticillus graivd segments as compared with shredded segments of either C. infundibulum or H. carioca. Such observation confirmed that of Horsfall, (1938). Another observation that T. castaneum is more suitable intermediate host to R. cesticillus as compared with other species of poultry tapeworms is that no mortality was recorded in

beetles infected with this tapeworm in contrast to C. infundibulum which mediated total losses in all infected beetles group and within a period of two months after infection. The absence of correlation between infection rate and infection intensity and also infection rate and abundance of infection in both worms, R. cesticillus and C. infundibulum infecting adult T. castaneum, agreed with those findings obtained by Yan and Norman (1995). Results showed that there was significant correlation between infection rate and mortality rate 60.d.p.i in C. infundibulum infecting T. castaneum, and the absence of such correlation between these variables 19 d.p.i. This suggests that persistence of the infection in the beetles after day 19 of infection is the crucial factor mediating mortality and marking this species of beetle as a compartively unsuitable host for this cestode. Such mortality may be a failure of the defense mechanism of this insect to overcome the parasitic infection and/or the successfulness of the parasite to evade the host defense mechanism that has been reported in some parasites (Lacki, 1976) or to depletion of insect nutrient reserves by mature cysticercoids which had developed as early as 6 d. p.i.The present study did not investigate the effect of age and sex of beetles on the survival of tapeworms in this intermediate T. castaneum host. However, Elowni 1984 noticed that infectivity of R. cesticillus cysticercoids from T. confusum declined as the insect ages and by 7 month in the intermediate host such cysticercoids became uninfective to chikens. Results showed that infectivity of R. cesticillus to T. castaneum adversely affected with prior immersion of gravid segments in tapwater possibly because of a change in osmolarity mediating premature hatching of oncospheres before the eggs were ingested by the insect. Generally, inside

the intermediate host, normal hatching process begins by partial destruction of the covering membranes, that includes the egg shell and the cytoplasmic membrane by the insect's mouth parts and the rest of the hatching process is completed in the intestine (Miller, 1961; Lethbridge, 1971). Oncospheres liberated before ingestion by the beetle are almost uninfective. Other factors may be that tapwater, with some salinity, may reduce the palatability of proglottids to the beetle and hence reduce the size of infective dose. Our results indicated that adults and larvae of A. diaperinus were successfully reared, maintained and experimentally infected with C. infundibulum. The larvae were found to be refractory to R. cestcillus, C. digonopora and H. carioca, these findings agreed with those obtained by the luttermoser (1940), Voge, et al (1964), Lethbridge (1971), and Rau (1979), who infected coleopteran larvae with cestodes. However, they disagreed with Moya et al, 1977, who assigned this beetle as only intermediate host for the nematode Subulura suctoria and Uchida et al, 1984 and avancini and Ueta, 1990, who deny the helminths transmitting capacity of A. diaperinus. Prior starvation was found to be an important factor in the establishment of C. infundibulum in A. diaperinus, as non starved larvae failed to acquire infection. Beetles starved for 6 days showed significantly higher infection rate than beetles starved for 4 days. This may be due to the effect of starvation in reducing feeding discrimination (Elowni, 1977) and hence increasing feeding ability. Mature and fully developed cysts with or without capsules, were only recovered from experimentally infected adults and larvae of A. diaperinus 16-20 d.p.i, this indicates the suitability of this beetle as an intermediate host for this tapeworm species. Moreover, cysticercoids were obtained from pupae and newly hatched adults that contracted the infection in larval stage

indicating that, the process of metamorphosis did not affect the development of the cysticercoids. Asimilar result has been reported by Luttermoser (1940) for R. cesticillus. The present study indicated that, Musca domestica larvae, are not suitable host for either C. infundibulum or R. cesticillus despite the two methods applied to ascertain ingestion of viable eggs. This result agreed with those obtained by Guberlet (1919), and Joyeux (1929), who reported negative results from attempts to parasitize these larvae with either C. infundibulum or R. cesticillus. Several factors may contribute to this non- suitability, such as the prior need for disruption of egg shell by specific forms of mouth parts (Reid, 1951; Miller 1961; Lethbridge, 1971), anatomy of the intestinal wall to allow oncospheral peneration (Calentine et al., 1970; Lethbridge, 1971) and physiological or immunological enviroments within the invertebrate to allow or prevent parasite development (Salt, 1963; Götz and Vey, 1974; Vey and Götz, 1975). Which one of these factors may attribute to failure of M. domestica larvae to establish these cestodes under study needs further investigations. Our results confirmed that R. cesticillus cysticercoids at 135-days of age were infective. They were significantly established in white leghorn chicks. This finding was in agreement with those obtained by Elowni (1984). The findings in this work illustrated that 15-days old cyst of H. carioca were infective to young white leghorn chicks. This result agreed with Horsfall (1938), as T. castaneum is a suitable intermediate host for developing H. carioca cysticercoids to the infective forms, yet Horsfall (1938), reported (32-39) days-old cysts to be infective to the definitive host. In contrast to Reid (1984), in this study, (12-13) days old cysticercoids of H. Carioca were not established in the final host, and this may be due to their

non physiological maturation despite their complelete morphological maturation. The findings of this study confirmed that the administration procedure of cestodal larvae affected their establishment in their final hosts. Cysticercoids of C. infundibulum, 20-days old, were only established when administered by means of gelatin capsules instead of Pasteur pipette. The same result was obtained by Elowni (1984), when comparing the efficiency of the two methods in the establishment of R. cesticillus in the definitive host. However, he found that, the significant differences between these methods are clearly observed when the final host is subjected to starvation before infection. However, twenty-days old cysticercoids of C. infundibulum, developed in adult A. daiperinus, were infective to white leghorn chicks despite their low infectivity. This result agreed with Hamad (1987), yet he did not mention the age of these cysts. The recovered cysts resembled those cysts obtained 14 d.p.i (Elowni, 1977), which suggested that maturation of C. infundibulum cysticercoids might begin at/or before 14 d.p.i and then gradually lost their infectivity to the definitive host. Other findings could support this suggestion, concerning R. cesticillus. Voge (1961) and Elowni (1984), demonstrated that, in T. confusum incubated at 30ºc, the cysticercoids of this worm, became infective 14 d.p.i, reach the peak maturity at 30d.p.i, and gradually lost their infectivity until they completely become non infective after 7month p.i. The reason for this reduction in the infectivity might be due to effective insect defense mechanism that was stimulated by the presence of the parasite (Salt 1961, Ubelakie et al, 1970), with a consequent adverse effect on the parasite that resulted in loss of infectivity. A second reason might be due to factors concerned with activation and excystment in the alimentary tract of the final

host. Rothman (1959), when investigating the effect of the different constituents of the digestive juice, on the activation and excystment of selected tapeworms including H. diminuta, he found that young age (less than 30 days) cysticercoids of this worm, readily excysted at 37ºC without addition of trypsin. However, the presence of such substance, enhanced the excystment of the older age cysts causing weakening of the cysts and setting the trapped larvae. The present results indicated that, (6-9) and (15-16) days old cysticercoids of C. infundibulum established in T. castaneum were not infective to white leghorn chicks, even though these cysts appeared completely formed and readily evaginated in tap water or physiological saline after their release from the insect body cavity. They might resembled those obtained from both grasshopper (Dicromorpha viridis and Melanoplus ferurrubrum) and the beetles (Stenocellus debilipes, Stenocellus conjuctus, Aliphitophagus bifasciatus, Apocellus sphaericollis, Ataenius cognataus, Aphodius spp., and Aphodius granarius) by Horsfall et al, 1937; that failed to infect the primary hosts, despite their apparent complete development and evagination in tapwater. These cysts might attain morphological maturation but they still need additional time to be physiologically mature and hence established in the definitive host. However, cysts aging (32-39) days old, which developed in the same host has been established in the final host (Horsfall, 1938). The present results confirmed that, A. diaperinus as a natural intermediate host for C. infundibulum that agreed with that obtained by Elowni (1979). The infection rate of C. infundibulum in the collection areas (Shambat and Elhalfaya) was 28.33% and 23.59% respectively compared to 11% in (Kuku) and 54.8% (Shambat) recorded by Elowni (1979).

Cysticercoids of C. infundibulum were detected in all beetle’s developmental stages, with highest infection rate being observed in the adult stage. A similer observation was reported by Elowni (1979). Wide range of infection was observed in this limited survey (1-209) in Elhalfaya and (1-119) Shambat; and high average number of cyst per infected beetle (18.59) in Shambat and (12.5) in Elhalfaya, compared to (1-69) and (1-61), infection range, and mean cyst per each beetle (9.54) and (8.33) in Kuku and Shambat, respectively. In this survey adult A. diaperinus naturally infected with C. infundibulum, showed evidence of immune reaction against C. infundibulum cysticercoids, that appeared as a black material surrounding and covering these cysts that may be developed by this host in response to the presence of the parasite. Such reaction have been reported to occur commonly in coleoptera (Salt, 1963; Ġotz and Vey (1974); Vey and Ġotz (1975), yet further investigation, concerning this matter should be done. Three new intermediate hosts for poultry cestode were reported in this study for the first time in the Sudan. The family Histeridae to which Hypocacclus praecox and Carcinops troglodyte, belong to, are responsible for transmitting R. cesticillus. Reid (1984) reported that Carcinops pumilo acts as intermediate host for this cestode. Jones (1929) detected Hymenolepis carioca cysticercoids in Hister (Carcinops 14 striatus). Thus, this family may be responsible for circulating more than one tapeworm in this country. Anthicus formicarius was found harbouring cysticercoids of C. infundibulum with higher infection rate in the deep-litter. Dutt, et al. (1961), found the same genus to be an intermediate host for both R. cesticillus and C. infundibulum. The importance of these coleopteran beetles in the maintenance of cestoidosis should be further evaluated.

Tribolium castaenum was not found to be a natural intermediate host for any of poultry cestode, this result coincided with that obtained by Reid (1964) and Elowni (1977). In addition, Musca domastica, larvae were not found carrying any cestode. No records of poultry tapeworm infection in flies larvae have been found, other than that reported by Guberlet (1919) and Avancini (1990), claiming Stomoxys calcitrans to be an intermediate host for H. carioca. The results presented here in stemming from either experimental procedures or natural infections address two crucial points of practical implications: a) Tapeworms of poultry are common in the Sudan (Abdel-Malik, 1959, Eisa, et al., 1976, Elowni, 1977, Saad et al., 1989 and Ali, 1994). Their control requires both treatment of the definitive host and elimination of the intermediate host. As each specific species of tapeworm is transmitted by specific species of intermediate host, e.g. beetles, ants, flies, grasshoppers etc, the positive identification of both the causative parasite and its potential intermediate host(s) is an indispensable matter. The present findings contribute to achieving parasite control more objectively. They even report new findings. b) Chichen tapewoms have been used as experimental modeles in several fundamental investigations (e.g. chemontherapy, immunology etc). The present findings contribute to the understanding of the properties of the se modeles e.g C.infundibulum in A.diaperinus, R.cesticillus in T.castaenum and H.carioca in T. castaenum.

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APPENDICES

1 4% Formalin Formalin, 33% 4.0ml Distilled water 96.0ml 2 5% Formalin Formalin, 33% 5.0ml Distilled water 95.0ml 3 Zenker's fluid Sodium sulphate 1.0gm Mercuricchloride 5.0gm Potasium dichromate 2.5gm Distilled water 100.0ml 4 Rouda bush Ethyl alcohol, 95% 240.0ml Formalin, 33% 50.0ml Glycerine 100.0ml Glacial acetic acid 50.0ml Distilled water 460.0ml 5 70% Alchohol Alchohol 70.0ml Distilled water 30.0ml 6 10% Formal saline Formalin, 33% 10.0ml 10.0ml Normal saline 90.0ml 90.0ml 7 5% glycerine Alchohol Glycerine 5.0ml 5.0ml Alchohol 95.0ml 95.0ml

Table 14: Appendix (8): Mean square values showed infection intensity, infection rate and abundance of the infection of three cestodes infecting adult T. castaneum under laboratory condition Source df Infection Infection Abundance of intensity Rate of variation (%) infection Cestodes 3 49.06** 4304.50** 24.02** Error 20 0.11 0.90 0.05 CV(%) 16.93 4.57 19.86 **P<0.01 Degree of freedom =df

Table 15: Appendix (9):Mean square values showed infection intensity, infection rate and abundance of the infection of C. infundibulum, infecting adult A. diaperinus (under laboratory condition) and starved for different periods Source df Infection Infection Abundance of intensity Rate of variation (%) infection Starvation 1 5.69 27.0** 24.02** period Error 10 0.27 1.0 0.05 CV(%) 10.01 4.40 19.86 **P<0.01 Degree of freedom =df