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Studies on Fowl Spirochetosis in Khartoum State

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Studies on Fowl Spirochetosis in Khartoum State View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by KhartoumSpace Studies on fowl spirochetosis in Khartoum state By Iman Mohammed El Nasri Hamza B.V.Sc University of Khartoum 1985 M.Sc University of Khartoum 1997 A Thesis Submitted to the University of Khartoum in fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) Department of Microbiology Faculty of Veterinary Medicine University of Khartoum August 2008 TABLE OF CONTENTS List of tables……………………………………………………………vii List of figures……………………………………………………….......ix Dedication………………………………………………………………xi Preface………………………………………………………………….xii Acknowledgment………………………………………………………xiii English summary……………………………………………….. ……..xiv Arabic summary……………………………………………………..…xiii Introduction………………………………………………………....….xiv Chapter one: Review of literature 1.1Spirochetosis……………………………………..….……… 1 1.2 The disease in Sudan……………………………………...………….1 1.3 Causative agent……………………………………….…….…….….2 1.4 Pathogenesis………………………………………………………….7 1.5 Borrelial genetic………………………………………………….…..7 1.6 Cultivation of Borrelia anserina ……………………..……………..8 1.7 Serotypes…………………………..………………………………...9 1.8 Incubation period………………………………………….………10 1.9 Clinical signs……………………..……………………………..... 11 1.10 Morbidity and mortality……………………………..……………12 1.11 Post mortem lesions……………………………………………….12 1.11.1 Slpeens ………………………………………………..12 1.11.2 Livers ……………………………………………………14 1.11.3 kidneys………………………………………………..…..15 1.11.4 Intestines …………………...……………………………15 1.11.5 Lungs ……………………………………………………15 1.11.6 Brain…………………………………………… ………. 16 1.12 Hematological changes…………….…………………..…………16 1.13 Transmission……………………… ………………….………… 16 1.14 Methods of diagnosis……………………………………………..20 1.14.1 Serology……………………..………………..…………..20 i 1.14.1.1 Agglutination test…………………..……….…………..21 1.14.1.2 Spirochete immobilization test………..….……………..21 1.14.1.3 Agar gel precipitation test.……………..……………….22 1.14.1.4Direct fluorescent antibody test…………..……………..22 1.15 Control of disease…………………………………………………23 1.15.1 Vaccine……………………………………………………23 1.16 Preservation of Borrelia anserina…………………………………25 Chapter two: Materials and methods 2.1 Experimental birds……………………………..……….. ..............28 2.2 Field isolates……………………………….………….……….….28 2.3 Embryonated eggs… …………………………….……………….29 2.4 Housing of chicken…………………………………………….…..29 2.5 Preparation of reagents……………………………………………31 2.5.1 sodium chloride………………………………..…………..31 2.5.2 Anticoagulant………………………………….……………31 2.5.2.1 Ethylenediamintetraacetic acid EDTA…………….….31 2.5.2.2 Sodium citrate 2%………………………………….…31 2.5.2.3 Sodium citrate 1%………….………………..…….…31 2.5.2.4 Citrate saline…………………………………..……...31 2.5.3 Phosphate buffer………………………….…………………..32 2.5.4 Preparation of stains……….............................……………...32 2.5.4.1 Giemsa solution…………........................………………32 2.5.4.2 Crystal violet…………………………………………... 32 2.5.4.3 Carbol fuchsin……………….…………………….… ... 32 2.5.5 Methods of staining…………………………………………..33 2.5.5.1 Giemsa staining……………………………………..…...33 2.5.5.2 Grams staining……………………….……………...…..33 2.5.5.3 Crystal violet staining…………………..………………33 2.6 Preparation of blood smears………………………….……..... ..33 ii 2.6.1 Stained smears………………………………………………33 2.6.2 Wet smears…………………………………………………..33 2.7 Sterilization……………………….……..……………...….……... .33 2.7.1 Glassware equipment…………………………..….……….…..33 2.7.2 Dimethyl sulphoxide…………………………………………...34 2.7.3 Glycerol……………………….……………….……….……..34 2.7.4 Sodium citrate…………………..……………….….….………34 2.8 Preparation of infected materials………………….………..….….34 2.8.1 Infected blood………………………….………………………34 2.8.2 Infected serum………………………………….………..……34 2.8.3 Infected blood clot………………..………………………......35 2.8.4 Preparation of infected plasma……………………….……….35 2.8.5 Infected ticks………………………………………..…………35 2.8.6 Tick homogenate………………………………………………35 2.8.7 Intestinal content………………………………………………36 2.9 Pathogenicity of Borrelia anserina…………………..………… ... 36 2.9.1 Experimental infection of chicks……………………..……… 37 2.9.2 Experimental infection of growers….………………………….40 2.9.3 Experimental infection of adult chicken…….………...………..41 2.9.4 Experimental infection by tick feeding………………………..42 2.10 Propagation and serial passage of Borrelia anserina Field isolates……………………………………………………….42 2.10.1 Embryonated eggs………………………………..…………..43 2.10.1 Yolk sac route…………………………………...………....43 2.10.2 Chorioallantoic membrane route……………….………......44 2.10.3 Allantoic sac route ………………………………………....45 2.10 4 Harvest...................................................................................45 2.10.2 Propagation of Borrelia anserina in day- old- chicks ........... 46 2.10.3 Propagation of Borrelia anserina in 6- 8 month old birds……47 iii 2.11 Infectivity of Borrelia anserina in chicken after passage in chick embryo………………………..………………………... 47 2.12 Preservation of Borrelia anserina…………………..…………... 48 2.12.1 Serum………………………………….…………..………. 48 2.12.2 Citrated blood………………………….…………..………. 49 2.12.3 Plasma……………………………..………..….…..……… 50 2.12.4 Buffy coat………………………………….……..…..…… 50 2.12.5 Yolk and chorioallantic fluid…………….…………...……. 50 2.12.6. Organs…………………………………………..…………. .51 2.13 Experimental infection of local breeds……………….……….. .51 2.14 Experimental infection of pigeons…………………………… ..51 2.15 Cultivation of Borrelia anserina in bacteriological media…………………………………………………..…… …...52 2.16 Oral infection using tick homogenate…………………….……… 52 2.17 Comparative studies on spleen weights………………….………. 52 2.18 Passage of Borrelia anserina field isolate……………….……… .52 2.19 Serology……………………………………………..……..……. .53 2.19.1 Preparation of agglutination antigen………….……...……….53 2.19.2 Preparation of immobilization antigen………..….…...……….54 2.19.3 Perparation of hyperimmune sera…………..……..…….…….54 2.19.4 Preparation of convalescent serum…….……………...………54 2.19.5 Plate agglutination test………………. .………….....……… .55 2.19.6 Tube agglutination test…………….… ……………..……….55 2.19.7 Immobilization lysis test……….…… ………………………56 2.20 Cross protection study of the four field isolates in susceptible chicken…………………. ………..…….……….. 56 Chapter three:Results 3.1 Incubation period………............................................….…………. 59 3.1.1 Soba1 isolate………...............................………….………….. 62 iv 3.1.2 Butri isolate……………………................................................ 65 3.1.3 Alkadaro isolate…………...................................……….......... 65 3.1.4.Taiba isolate.............................................................................. .65 3.2 Clinical signs.................................................................................... 68 3.3 Post mortem lesions......................................................................... 73 3.4 Histopathological changes.............................................................. 73 3.5 Spirochetemia.................................................................................. 76 3.5.1 Spirochetemia in chicks.......................................................... 76 3.5.2 Spirochetemia in growers........................................................ 78 3.5.3 Spirochetemia in adults........................................................... 78 3.6 Propagation of Borrelia anserina.................................................... 81 3.6.1 Propagation in chicks embryos.................................................81 3.6.2 Propagation of Borreila anserina in one -day old chicks........................................................................................89 3.6.3 .Propagation of Borrelia anserina in 6 –8 month old birds.....89 3.6.4 Growth of Borrelia anserina in chicken after passage in chick embryo………………………………………… ………91 3.7 Comparative study on spleen weight……………………………...91 3.8 Tick feeding…………………………………………….….……..94 3.9 Experimental infection in local chicken breed………….………100 3.10 Experimental infection in pigeons…………………………… 100 3.11 Preservation of Borrelia anserina………..……………………..100 3.11.1 Liquid nitrogen……………………………………….…….100 3.11.1.1 Infected serum…………………………………….…… 100 3.11.1.2 Infected citrated blood……………….…………….……..102 3 .11.1.3 Infected plasma…………………………………..………102 3.11.1.4 Infected embryonic materials…………………………….102 3.11.1.5 Infected organs…………………………………..…..……102 3.11.2Cold preservation ……………………….….………….……..105 v 3.11.2.1 Infected serum……………………………..……………. 105 3.11.2.2 Infected citrate blood……………………………….……105 3.11.2.3 Infected plasma and buffy coat…………………….…….105 3.11.2.4 Infected embryonic materials…………….………...…… 105 3.11.2.5 Infected tissues……………………………..……..….….. 105 3.12 Serological tests…..…………………………….….…….…… 107 3.12.1 Plate agglutination test…………………………..….……. 107 3.12.2 Tube agglutination test…………………….……...………107 3.12.3 Spirochete immobilization test…………………………….108 3.13 Cross protection tests…………………………………………..109 Chapter four:Discussion ……………………… … ……. 113 Conclusions and Recommendations ………………………. ……. 127 References…………………………………..…………………….. 128 List of appendices vi List of Tables 1 Grading of Borrelia anserina………………………….…….…….. 13 2 Borrelia anserina field isolates………………………………..…... 30 3 Doses of infected materials given via different routes in chicks…... 38 4 Doses of infected materials given via different routes in grower…. 38 5 Doses of infected materials given via various routes in adult…….. 39 6 Cross infection studies in chicken..……………………….............. 58 7 Incubation period in days in chicken after experimental infection with the four field isolate using different inoculum through various routes……………………………………………………….…….60 8 Incubation period in days ( means of 10 birds ± S D ) i n
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