VPM Faculty meeting July 23, 2015

Size: px

Start display at page:

Download "VPM Faculty meeting July 23, 2015"

冈沛易
5 years ago
Views:

1 Diagnostic Puzzles: Putting All The Pieces Together 诊断拼图 : 将所有图片拼在一起 6 th Leman China Swine Conference 第六届李曼中国养猪会 Nanjing, China 中国南京 Jerry Torrison 杰瑞托瑞森, DVM, PhD 兽医学博士, Dipl. ACVPM Director 主管, Veterinary Diagnostic Laboratory 兽医诊断实验室 Professor 教授, Veterinary Population Medicine 兽医人口医学

3 Getting the Whole Picture 了解全面情况

4 Getting the Whole Picture 了解全面情况 Clinical Signs 临床症状 Farm History 农场历史 Medications 用药 Neighbour Farms 临近的农场 Feed 饲料 Pathology 病理学 Genetics 品种 Bacteriology 细菌学 Virology 病毒学 Mortality 死亡率 Weather 天气

RIC-PCR earson correlation (Opt:0.50%) [0.0%-2.0%] [13.4%-79.8%] ERIC-PCR BOX-PCR 80 85 90 95 100. Bacterial isolate. NA. Lung. CLI, ERY(I), PEN, SPE (I), SPD (I), TYL. Lung, liver. NA. Lung. CLO (I), CLI, ERY, OXYT, PEN, SPE, SPD, SPT, TYL.

Lung, lymph node, pericardium. CLI, ERY (I), OXYT, PEN, SPE, SPD (I), TIL, TYL. Lung, pleura. CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. Lung. CLI, ERY(I), PEN, SPE, SPT (I), TYL. Tonsil.

Lung 5. AMP (I), CLI, ERY, PEN, SPE, SPD, SPT, TYL. Liver 5. NA. Joint 8. NA. Lung, liver. AMP (I), CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. Lung. CLI, ERY (I), PEN, SPE, SPT, TIA (I), TYL.

CLI, ERY (I), OXYT, PEN, SPE, SPD, SPT, TIA (I), TIA, TIL, TYL. Lung, liver. CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. Lung, pleura. AMP (I), CLI, ERY (I), PEN, SPE, SPD, SPT, TYL. Lung, liver. AMP (I), CLI, ERY, PEN, SPE, SPT, SPD, TIA, TYL.

Lung, pleura. CLI, ERY (I), OXYT, PEN, SPE, SPT, TIL, TYL. Lung, lymph node, spleen. CLI, ERY (I), PEN, SPE, SPT, TYL. Lung, liver. AMP (I), CLI, ERY, NEO, PEN, SPE, SPD, SPT (I), TIA (I), TYL. Lung. CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL.

00% 0.25 >16 CLSI* Breakpoint Ceftiofur 97.65% 0.5 0.5 2 Chlortetracycline 62.35% 8 >8 Enrofloxaxin 100.00% 0.12 0.12 Florfenicol 97.65% 0.5 0.5 2 Gentamicin 17.65% 8 >8 Oxytetracycline 21.

5 RIC-PCR earson correlation (Opt:0.50%) [0.0%-2.0%] [13.4%-79.8%] ERIC-PCR BOX-PCR Bacterial isolate. NA. Lung. CLI, ERY(I), PEN, SPE (I), SPD (I), TYL. Lung, liver. NA. Lung. CLO (I), CLI, ERY, OXYT, PEN, SPE, SPD, SPT, TYL. Lung, pleura. AMP (I), CLI, ERY, NEO, PEN, SPE, SPD, SPT, TYL. Lung, pleura, spleen. AMP (I), CLI, ERY (I), PEN, SPE, SPD, SPT (I), TYL. Lung. CLI, ERY (I), PEN, SPE, SPT (I), TYL. Lung, lymph node, pericardium. CLI, ERY (I), OXYT, PEN, SPE, SPD (I), TIL, TYL. Lung, pleura. CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. Lung. CLI, ERY(I), PEN, SPE, SPT (I), TYL. Tonsil. CLI, ERY(I), PEN, SPE, SPT, TYL. Tonsil. CLI, ERY(I), PEN, SPE (I), SPT (I), TYL. Tonsil. CLI, ERY(I), PEN, SPE (I), SPT (I), TYL. Lung. CLI, ERY(I), PEN, SPE, SPT (I), TYL. Lung. CLI, ERY(I), PEN, SPE, SPT (I), TYL. Lung. CLI, ERY(I), PEN, SPE, SPT (I), TYL. Lung. CLI, ERY(I), PEN, SPE (I), SPD, SPT,TYL. Lung 5. AMP (I), CLI, ERY, PEN, SPE, SPD, SPT, TYL. Liver 5. NA. Joint 8. NA. Lung, liver. AMP (I), CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. Lung. CLI, ERY (I), PEN, SPE, SPT, TIA (I), TYL.. Bacterial isolate. NA. Bacterial isolate. NA. Lung, pleura, liver. AMP (I), CLI, ERY (I), OXYT, PEN, SPE, SPD, SPT (I), TIL, TYL. Lung, liver. CLI, ERY (I), OXYT, PEN, SPE, SPD, SPT, TIA (I), TIA, TIL, TYL. Lung, liver. CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. Lung, pleura. AMP (I), CLI, ERY (I), PEN, SPE, SPD, SPT, TYL. Lung, liver. AMP (I), CLI, ERY, PEN, SPE, SPT, SPD, TIA, TYL. Pleura, pericardium, lung. CLI, ERY (I), OXYT, SPE, SPT (I), TIL, TYL. Lung, pleura, liver. CLO (I), CLI, ERY (I), OXYT, PEN, SPE, SPT (I), TIA, TYL. Lung, pleura, liver. CLI, ERY (I), PEN, SPE, SPD, SPT, TYL. Lung, pleura. CLI, ERY (I), OXYT, PEN, SPE, SPT, TIL, TYL. Lung, lymph node, spleen. CLI, ERY (I), PEN, SPE, SPT, TYL. Lung, liver. AMP (I), CLI, ERY, NEO, PEN, SPE, SPD, SPT (I), TIA (I), TYL. Lung. CLI, ERY (I), OXYT, PEN, SPE, SPT, TYL. NA. NA. Lung, liver, skin.. Lung, pleura. PT ( ATCC.. NA.. Lung. EN ATCC.. ATCC... Actinobacillus AT MVDL Susceptibility Data Actinobacillus pleuropneumoniae 2008/2009 % Susceptible 2009 MIC MIC90 Ampicillin 80.00% 0.25 >16 CLSI* Breakpoint Ceftiofur 97.65% Chlortetracycline 62.35% 8 >8 Enrofloxaxin % Florfenicol 97.65% Gentamicin 17.65% 8 >8 Oxytetracycline 21.62% >8 >8 Penicillin N/A 0.5 >8 Spectinomycin 12.94% Tetracycline N/A Tiamulin 87.06% Tilmicosin 74.12% Trimeth./Sulphamethoxa zole N/A 2 2 Tulathromicin (Draxxin) 82.43% 16 16

6 A puzzle is an act of fitting separate pieces together as a test of knowledge, ingenuity, skill and patience. 拼图是测试知识, 独创性, 技能和耐性把分别的部分拼凑在一起的一项活动

7 Jigsaw puzzles assemble information to complete the picture Tangram puzzles use shapes to make changing pictures 拼图游戏 : 收集信息来完成七巧板游戏利用形状来制造持续会变的图片

8 Start with a question 从问问题开始 : 1.What is causing disease or death in my pigs? 是什么在导致我的猪生病或死亡? 2.Is a specific disease agent present or absent? 有特定的病原存在或缺失吗? 3.When should I vaccinate or treat my pigs? 我该什么时候给我的猪接种疫苗或者进行治疗

9 Minnesota VDL Pathogen Discovery 明尼苏达兽医诊断实验室对于病原的发现 Molecular Development 分子学开发 Molecular Diagnostics 分子诊断 Virology 病毒学 Pathogen Discovery 病原发现 Pathology 病理学 & Histology 组织学 Serology 血清学 Bacteriology 细菌学 Electron Microscopy 电子微镜学

10 Minnesota VDL Pathogen Discovery 明尼苏达兽医诊断实验室对于病原的发现 Molecular Development 分子学开发 Molecular Diagnostics 分子诊断 Virology 病毒学 Pathogen Discovery 病原发现 Pathology 病理学 & Histology 组织学 Serology 血清学 Bacteriology 细菌学 Electron Microscopy 电子微镜

11 PCV1 PCV2 PCV3

12 PCV Background 圆环病毒背景 Porcine circoviruses 猪圆环病毒 Small non-enveloped single stranded DNA virus 小的未被包围单链 DNA 病毒 Infect primarily macrophages, but also other cells 主要感染巨噬细胞, 但也有其它细胞 Widespread in swine populations 在猪群里非常普遍

13 History 历史 PCV Background PCV 背景 Picornavirus-like particles (1974): non-pathogenic contaminants of cell line PK-15 与小核糖核酸病毒相似的粒子 (1974): 细胞系 PK-15 的非病原污染物 PCV (1982): new virus, small, icosahedric, non-enveloped, singlestranded DNA 猪圆环病毒 (1982): 新型病毒, 小的, 二十面体, 未被包围, 单链 DNA PMWS (1996): First case of Postweaning Multisystemic Wasting Syndrome (PMWS). Associated with PCV. PMWS (1996): 第一例断奶仔猪多系统衰竭综合征, 和 PCV 有关联

14 PCV Types 猪圆环病毒类型 PCV1 1970s: Non-pathogenic cell culture contaminant 无病原细胞培养污染物 PCV2 1990s o Diverse range of clinical syndromes (PCVAD) 临床综合征的多种范围 ( 猪圆环病毒相关疾病 ) o Postweaning multisystemic wasting syndrome (PMWS) 断奶仔猪多系统衰竭综合征 - Europe 欧洲 ( ) - North and South America 北美和南美 ( ) o Subtypes 亚型 : PCV2a, PCV2b, PCV2c, PCV2d, PCV2e PCV o PDNS, abortions and perivascular inflammation (myocarditis) o 猪皮炎肾病综合征, 流产, 血管周炎 ( 心肌炎 )

15 PCV2-associated diseases PCV2 关联疾病 Madson et al, 2016

16 PCV2-associated diseases PCV2 关联疾病 Clinical signs 临床症状 Late nursery-early finishing (8-12 weeks) 保育后期 - 育肥早期 (8-12 周 ) 5-30% morbidity (5-30% 发病率 ) Failure to thrive, paleness 生长慢, 苍白 Sometimes: dyspnea, jaundice, diarrhea 有时 : 黄疸腹泻 Secondary bacterial infections unresponsive to antibiotics 继发细菌感染, 抗生素无效

17 PCV2-associated diseases PCV2 关联疾病

18 PCV2-associated diseases PCV2 关联疾病 Photos: Steve Henry

19 Mortality curve example from PCVD herd 猪圆环病毒病猪群死亡率曲线图范例 2% Percent risk of death by week 按周计算的死亡风险率 1% Expected mortality rate 预期死亡率 0% Dr. Steve Henry, 2006 Week in finish barn 育肥舍周数

20 PCV2-associated diseases Gross lesions 组织病变 PCV2 关联疾病 Enlarged lymph nodes 淋巴结肿大 Non-collapsed lungs 未衰竭的肺 White-spotted kidneys 布满白斑的肾脏 Yellowish liver 微黄的肝脏

21 PCV2 Histopathology PCV2 组织病理学 Microscopic lesions 显微病变 Lymphocytic depletion (lymph node, spleen, ileum) 淋巴细胞性损害 ( 淋巴结, 脾脏, 回肠 ) Intracytoplasmic inclusion bodies within macrophages 有巨噬细胞的胞质内包涵体 Multinucleated giant cells 多核巨细胞 Non-lymphoid tissues 非淋巴系组织 Interstitial pneumonia 间质肺炎 Nephritis 肾炎 Hepatitis 肝炎

22 PCV2 Histopathology PCV2 组织病理学 Inclusion bodies 包涵体 Multinucleated giant cells 多核巨细胞

23 PCV2- Immunohistochemistry PCV2 免疫组织化学

24 Porcine Dermatitis and Nephropathy Syndrome (PDNS) 猪皮肤炎和肾病综合征

25 PCV2 Abortion PCV2 流产 Brunborg et al, 2007 Madson, 2009

26 PCV2-associated diseases PCV2 关联疾病 Coinfections 同时感染 Parvovirus 细小病毒 PRRSv 猪繁殖呼吸系统综合征 Bacterial infection (including S. enterica serotype Choleraesuis) 细菌感染 ( 包括沙门氏菌血清型 ) Genetic variation 基因变异 (ORF 2 羊痘疮 2) PCV2a less likely to cause disease than PCV2b PCV2a 与 PCV2b 相比不太可能会导致疾病 PCV2a + PCV2 b and mpcv2b = more virulence PCV2a + PCV2 b 和 mpcv2b 毒性更强

27 Frequency pattern of PCV2 ORF2 sequencing at the Minnesota VDL: 年至 2015 年明尼苏达兽医诊断实验室 PCV2 ORF2 序列的发生规律

28 Finding new strains of PCV2 寻找 PCV2 新的新菌种

29 Traditional sequencing: Sanger sequencing 传统测序 :Sanger 测序法 Developed by Sanger et al. in 年由 Sanger 等人研发 Uses primers: you need to know what you are looking for 利用引子 : 你需要知道你要找的对象 We use it mainly to characterize viral strains (PRRSV, SIV, ) 主要用于定性病毒株 ( 蓝耳, 流感 )

Traditional sequencing: Sanger sequencing 传统测序 :Sanger 测序法 By Estevezj - Own work, CC BY-SA 3.0, https://commons.

curid=23264166 CCGCGGGCTGTTGCTCGCAATTGCTTTCTTTGT GGTGTATCGTGCCGTTCTGTTTTGCTGTGCTCG

30 Traditional sequencing: Sanger sequencing 传统测序 :Sanger 测序法 By Estevezj - Own work, CC BY-SA 3.0, CCGCGGGCTGTTGCTCGCAATTGCTTTCTTTGT GGTGTATCGTGCCGTTCTGTTTTGCTGTGCTCG CCAACGCCAGCAACGACAGCAGCTCCCATCTA CAGCTGATTTACAACTTGACGCTATGTGAGCTG AATGGCACAGATTGGCTAGCTAACAAATTTGAT TGGGCAGTGGAGAGTTTTGTCATCTTTCCCGTT TTGACTCACATTGTCTCCTATGGTGCCCTCACT ACCAGCCATTTCCTTGACACAGTCGCTTTAGTC ACTGTGTCTACCGCCGGGTTTGTTCACGGGCG

Sample Types 样本类型 NGS Processing 下一代基因测序处理过程 Tissue 组织, Tissue Homogenate 组织匀质 Feces 粪便, Fecal Swab 粪便拭子 Serum 血清, Oral Fluids 口腔液体 Isolates 分离体 (viral,

31 Sample Types 样本类型 NGS Processing 下一代基因测序处理过程 Tissue 组织, Tissue Homogenate 组织匀质 Feces 粪便, Fecal Swab 粪便拭子 Serum 血清, Oral Fluids 口腔液体 Isolates 分离体 (viral, bacterial 病毒, 细菌 ) PCR-amplicons PCR 扩增子 Virus/bacteria-specific Nucleic Acid Isolation 病毒 / 细菌特异的核酸分离 Library Prep 数据库预备 Sequencing 测序 MiSeq, PE 250 bp HiSeq, PE 250 bp

32 Next Generation Sequencing 下一代基因测序 Millions of reads ( bp) 成千上万的读数 Overlapping reads 重叠的读数 Aligned sequence 排好的序列

33 Next Generation Sequencing 下一代基因测序

35 PCV3-associated diseases PCV3 关联疾病 2016 Palinski et al J. Virol. doi: /jvi Phan et al 2016 Virology Journal

36 Outline Red = PCV3 (ISH) 红色 =PCV3 Perivascular myocarditis (HE) 血管周心肌炎 Top differentials 最大差异 - PRRSv - PCV2

37 Outline Perivascular myocarditis 血管周心肌炎 Co-infection 共同感染 PCV2 and PCV3 Green 绿色 = PCV2 Red 红色 = PCV3

38 Outline Severe myocarditis 严重心肌炎 Red 红色 = PCV3

39 Outline Interstitial pneumonia 间质性肺炎 Red 红色 = PCV3

40 Outline Broncho-associated Lymphoid Tissue 与支气管相关淋巴系组织 Red 红色 = PCV3 Interstitial pneumonia 间质性肺炎 Red 红色 = PCV3

41 Outline Spleen white pulp 脾脏白浆 No clear evidence of lymphoid depletion 没有明显淋巴细胞性损害的证据 (Red 红色 =PCV3)

42 Outline Lymph node lymphoid follicle 淋巴结淋巴系卵泡 No evidence of lymphoid depletion 没有淋巴系损害的证据 (Red=PCV3)

43 Outline Lymph node lymphoid follicle 淋巴结淋巴系泡 No evidence of lymphoid depletion 没有淋巴系损害的证据 Co-infection 共同感染 PCV2-PCV3 Green 绿色 = PCV2 and Red 红色 =PCV3

由原位杂交发现组织里的 PCV3 Development of a PCR for PCV3 因为 PCV3 发育的 PCR retrospective testing 追溯测试 : PCV3 in 27% of VDL

44 PCV3 Unusual clinical presentation or unusual pathology: viral myocarditis/vasculitis 不正常的临场表现或者不寻常的病理 : 病毒性心肌炎 / 血管炎 New Porcine Circovirus identified by NGS (PCV3) 由下一代基因序列测定法确认新的猪圆环病毒 Presence of PCV3 in tissues by ISH 由原位杂交发现组织里的 PCV3 Development of a PCR for PCV3 因为 PCV3 发育的 PCR retrospective testing 追溯测试 : PCV3 in 27% of VDL cases 27%VDL 案例里发现 PCV3 Experimental inoculation? 试验接种? Virus isolation attempts unsuccessful so far 目前为止尝试病毒分离都不成功

45 Diagnostic Tools 诊断工具 Clinical history 临床历史 Histopathology 组织病理学 Virology 病毒学 Immunohistochemistry 免疫组织化学 Electron microscopy 电子微镜学 PCR 聚合酶链式反应 Next Generation Sequencing 下一代基因序列测定 In situ hybridization 原位杂交

46 Diagnostic Tools: Electron Microscopy 诊断工具 : 电子微镜学

47 Diagnostic Tools: In Situ Hybridization 诊断工具 : 原位杂交

48 Diagnostic Tools Histopathology 诊断工具组织病理学

49 History of pathogen discovery at the Minnesota VDL 明尼苏达兽医诊断实验室发现病原历史 PRRSV 蓝耳病病毒 (1991) Avian metapneumovirus 禽偏肺病毒 (1998) Rotavirus B and C 轮状病毒 B 和 C (2009) Brachyspira hampsonii 汉普森短螺旋菌 (2011) PEDV 流行性腹泻病毒 (2013) PDCoV 猪德尔塔冠状病毒 (2014) Senecavirus 猪塞内加谷病毒 (2015) Porcine circovirus type 3 猪圆环病毒 3 型 (2016) Other avian and fish viruses 其它鸟类及鱼类病毒

50 Minnesota VDL Pathogen Discovery 明尼苏达兽医诊断实验室对于病原的发现 Molecular Development 分子学开发 Molecular Diagnostics 分子诊断 Virology 病毒学 Pathogen Discovery 病原发现 Pathology 病理学 & Histology 组织学 Serology 血清学 Bacteriology 细菌学 Electron Microscopy 电子微镜

51 PCV1 PCV2 PCV3

<4D F736F F D203135A1A1CEE2BAA3B3ACA1A1A3A8CEACA3A92E646F63>

<4D F736F F D203135A1A1CEE2BAA3B3ACA1A1A3A8CEACA3A92E646F63> ( ) 2014 10 40 5 Journal of Hunan Agricultural University (Natural Sciences), Oct. 2014, 40(5):525 529 DOI:10.13331/j.cnki.jhau.2014.05.013 http://www.hunau.net/qks 育肥猪血清中猪细小病毒 16WS 株的分离与鉴定 ( 410128) *