臺灣博碩士論文加值系統

球蟲症是種嚴重影響家禽產業的寄生蟲疾病。許多抗球蟲藥都被使用來預防球蟲症的發生，然而過度使用抗球蟲藥會使得球蟲產生抗藥性。本研究主要目的是評估Sulfaclozine(0.5g/L)與Toltrazuril(0.025g/L)這兩種球蟲藥對於台灣地區的E. tenella與E. acervulina野外株的抗藥性影響。本實驗的陰性對照組乃純化自活蟲疫苗的兩株藥敏型球蟲純株(E. acervulina-V與E. tenella-V)，均保存在廠商的實驗室中未經任何藥物處理。野外型球蟲純株(E. acervulina-W與E. tenalla-W)則分離自本研究中的送檢雞場。前期試驗的結果顯示E. acervulina-W(106顆/雞)與E. tenella-W(1.5x104顆/雞)，分別可以達到病變指數2到3。因此選定這兩種劑量當作標準攻毒量。實驗設計將14日齡大白肉雞(N=70)隨機分成七組(n=10隻/組)，其中包括一組陰性控制組(沒感染沒治療組；UUC組)與兩組陽性對照組組(有感染沒治療組；IUC)，與四組感染後治療組(每個田間組與疫苗組均使用Sulfaclozine與Toltrazuril治療)。結果顯示兩種抗球蟲藥在疫苗株組均有較高的抗球蟲效力，這可能是因為E. acervulina-W與E. tenalla-W對Sulfaclozine與Toltrazuril這兩種球蟲藥產生抗藥性所導致。此外相較於野外株組的雞

隻，疫苗株組的雞隻在使用Toltrazuril治療之後有較高的平均增重率，但是使用Sulfaclozine治療的組別沒有如此的結果。
關鍵字：球蟲抗藥性、E. acervulina、E. tenalla、Sulfaclozine、Toltrazuril

Coccidiosis is a parasitic disease that major affects many susceptible livestock especially poultry. A large number of prophylactic coccidiostats were administered as medicated feed to control chicken coccidiosis. However, the extensive use of these drugs has resulted in the development of drug resistance by Eimeria spp. The purpose of this study was to evaluate the drug resistance of two coccidiocides, Sulfaclozine (0.5g/L) and Toltrazuril (0.025g/L), to E. acervulina and E. tenella wild strain in Taiwan. Two known drug susceptible strains (E. acervulina-V and E. tenella-V) obtained from a live vaccine were used as negative control, because they were always maintained in the manufactory’s laboratory without any drug treatment. The wild strains (E. acervulina-W and E. tenella-W) were isolated from a chicken farm in this study. A preliminary trial to determine the inoculation dose showed that E. acervulina-W (106 oocysts/bird) and E. tenella-W (1.5×104 oocysts/bird) could cause the gross lesions up to scores between 2 and 3. Therefore, these two dosages were selected as the standard for infection. Fourteen days old broiler (n=70) were randomly divided into seven groups (n=10 birds/group) including one negative control (uninfected untreated control, UUC), two positive control (infected untreated control, IUC), and
four infected treated groups (each vaccine strain group or field strain group treated with Toltrazuril and Sulfaclozine). The results showed that both Toltrazuril and Sulfaclozine have higher anticoccidial index in vaccine strain groups than in field strain groups. It was suggested that the field strain E. acervulina and E. tenella have higher drug resistance to Toltrazuril and Sulfaclozine than vaccine strains. Additionally, compared to chickens in field strain group, chickens in vaccine strain group have higher mean weight gain after intake Toltrazuril, but no such findings in the groups consumed Sulfaclozine.
Key words: coccidiosis drug resistance, E. acervulina, E. tenella, Sulfaclozine, Toltrazuril.

Table of Contents
中文摘要 (Chinese) I
Abstract (English） III
Acknowledgements V
Tables of Contain VI
List of Tables VIII
List of Figures IX
1. Introduction 1
2. Literature review 3
2.1. Classification and taxonomy 3
2.2. Etiology 3
2.4. Life cycle 5
2.5. Transmission 6
2.6. Susceptibility to environmental factors 7
2.7. Epidemiology 7
2.8. Economic impact 7
2.8. Diagnostic methods 8
2.8.1. Clinical findings 8
2.8.2. Microscopic examination 9
2.8.3. Gross Lesions 9
2.8.4. Polymerase chain reaction (PCR) 11
2.9. Treatment, prevention and control coccidiosis 11
2.9.1. Treatment 11
2.9.2. Prevention and control 12
2.9.2.1. Biosecurity 12
2.9.2.2. Vaccination 12
3. Materials and methods 15
3.1. Parasite 15
3.1.1. Wild strains of Eimeria 15
3.1.2. Vaccine strains of Eimeria 16
3.1.3. Isolation and identification of Eimeria spp. 16
3.1.3.1. Morphology 16
3.1.3.2. Isolation of Eimeria wild strains 16
3.1.3.3. Isolation of Eimeria vaccine strains 17
3.1.4. Detection of infection site 17
3.1.5. Genomic DNA extraction 17
3.1.6. PCR amplification and gel electrophoresis 18
3.1.7. Cloning and nucleotide sequencing of the ITS-2 rDNA 19
3.1.8. Pathogen Index (PI) 19
3.2. Anticoccidia drugs 20
3.3. Experimental design 20
3.4. Birds, feed and housing 22
3.5. Propagation of Eimeria 22
3.6. Drug resistance trials 23
3.7. Statistical analysis 24
4. Results 25
4.1. Morphology and measurement 25
4.2. Isolation of Eimeria spp. 25
4.3. Site of infection 26
4.4. PCR amplification of Eimeria ITS-2 26
4.5. Cloning and nucleotide sequencing of the ITS-2 rDNA 27
4.6. Pathogen index 27
4.7. Oocysts per gram (OPG) of feces 27
4.8. Average weight gains 28
4.9. Lesion scoring 28
4.10. Drug resistance 29
5. Discussion 30
6. References 51
Annexes 58
Biosketch of Author 62
Appendix 63

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