6th Leman China Swine Conference Nanjing, China Tailored Diagnostic Strategies: Diagnostic Investigations and Monitoring Strategies 针对目标的诊断策略：诊断调查和监测策略 Jerry Torrison DVM, PhD & Fabio Vannucci DVM, PhD 杰瑞·托瑞森 兽医博士和法比欧·费洛奇 兽医博士 PRRS incidence & prevalence TTS (time to stability) 到稳 蓝耳病发生率和患病率 定的时间 Outbreak Investigation with virus sequences over time and Antimicrobial 耐药性 space 通过病毒序列在时间和空间的分布 resistance 进行疫情调查 State IL 伊 State MN 明 利诺伊州 尼苏达州 System 1 System 3 系统1 系统3 EWMA by State 按州看EWMA EWMA by State SUMMER夏季 SPRING 春季 • 场点数 平均周数 Physiology 生理 Serum calcium, phosphorous and globulin concentrations 血清钙， 磷和球蛋白浓度 12 10 8 6 4 serum mineralserum concentration,mg/dl 2 0 0 5 10 15 20 25 30 sow number Calcium Phos Globulin OutlineDiagnostic Investigation of Reproductive Disease 对于繁殖疾病的诊断性调查 • Attempts to get away from the frustration: basics and challenges • 尝试远离挫折麻烦：基础和挑战 • Sampling strategies: fetal infections versus maternal illness • 取样策略：胎儿感染与母体感染相较 • Differentials: what to expect (?) • 差异：该期待什么（？） • Still got nothing: bang your head against the wall (sow crate) • 仍然什么都没有：拿头撞墙（母猪栏） • Applying new technologies (NGS): don’t look too close • 适用新的技术（下一代基因测序）：不要看得太仔细 • What does it mean? • 那是什么意思？ • Case study: PCV3-associated abortion and myocarditis • 案例：和猪圆环病毒3型有关的流产和心肌炎 AttemptsOutline to get away from the frustration 尝试远离麻烦 • Understanding the basics 懂得最基础的 - Abortion = loss of progesterone from corpus luteum - 流产=因为黄体素的关系损失孕酮 - Disruption of the endocrine equilibrium of the pregnancy - 孕期内分泌失调 Abortions 流产 Return to service (RTS) 再配种 Stillbirths 死胎 木乃化 Resorption Mummification Days 0 10-16 35 65 112-116 125 skeletal Mating Implantation Fetus begins mineralization Farrowing 分娩 配种 着床 developing Organogenesis Immunity 胎儿免疫开始发育 骨骼矿质化，器官形成 AttemptsOutline to get away from the frustration • Understanding the challenges 了解挑战 - Same diagnostic questions: Who, When and How (?) – different thought process - 一些诊断上的问题：谁，何时，如何？- 不同的思考过程 Fetal infection 胎儿感染 Pathogenesis of History of the the potential farm and the pathogens clinical problem causing abortion 史以及 床 农场历 临 潜在病原体的发病 问题 机理导致流产 Maternal illness 母体疾病 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • History of the farm 农场历史 - Reproductive performance 繁殖能力 - Stage of gestation 妊娠阶段 - Sow illness: subpopulation (?) - 母猪的病：亚种群？ - Diagnostic submissions 诊断提交 Reproductive Disease: Diagnostic Investigations 繁殖疾Outline病：诊断性调查 • Sampling strategies 取样策略 - Acute abortions versus recurring abortion in different stages may require multiple sampling over time 急性流产与在不同阶段反复流产可能需要随时间多次取样 - Likely not to sample/submit the first affected sow or abortions 不太可能提交或从 第一头被感染母猪或第一次流产取样 - Serology 血清学 Sows o Identifying introduction of new pathogen – acute, convalescent, pen/herd mates 确定 母猪 新病原体的进入-急性的，恢复期的，圈/群里的同伴 Fetus o Monitoring for status for a specific pathogen 监测某一个具体病原体的状态 >65 days Pathogen-specific: no reliable Se/Sp 特定病原：没有可靠的Se/Sp 大于65天 o 的胎儿 o Total IgG/IgM: in-utero antigen exposure 总IgG/IgM：在子宫内对抗原的暴露 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Sampling strategies (Tissues) 取样策略（组织） Reproductive Disease: Diagnostic Investigations 繁殖疾Outline病：诊断性调查 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Sampling strategies (Tissues) 取样策略（组织） ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Sampling strategies 取样策略 - Fetal infections 胎儿感染 o In general > day 14 post-infection o 基本上 感染后 > 14天 o Sow: seroconversion and no systemic infection (viremia or bacteremia) o 母猪：血清转化并且无系统性感染（病毒血症或菌血症） o Fetus and/or placenta: pathogen detection (not equally distributed!) o 胎儿以及/或胎盘：发现病原（不是均匀分布的！） o 4-6 fetuses/litter from at least 3 litters (assuming fetal infection 50%, litter of 12) o 至少3窝里的没窝4至6个胎儿（假设胎儿感染率是50%，每窝12只） Outcomes 结果: Reduced litters and abortions o 减少的窝数和流产数 Stillbirths 死胎 Resorption – RTS 再吸收 Mummification 木乃伊化 Days 0 10-16 35 65 112-116 125 着床 免疫 天数 Mating 配种 Implantation Bone mineralization Immunity Farrowing 分娩 骨矿化 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Sampling strategies 取样策略 - Maternal illness (acute phase of the disease) 母体疾病（疾病的急性期） o Sow: may not have seroconverted – opportunity for pathogen detection o 母猪：可能还没有血清转化 – 发现病原的机会 o Fetus and/or placenta: primary pathogen may not be present (but send it out!) o 胎儿以及/或胎盘：主要病原可能还未呈现（但是把送出去！） ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Gross lesions 组织病变 ReproductiveOutline Disease: Diagnostic Investigations 生殖疾病：诊断性调查 Bacterial and fungal abortion • Gross lesions 肉眼病变 细菌及真菌堕胎 Usually clustering of affected animals 通常感染动物 Viral abortion • 聚集在一起 病毒堕胎 • Variable: size and immune status of the herd 变数： 群大小和免疫状态 • Primary, secondary/opportunistic 主要，次要/机会 主义的 • Fetal icterus 胎儿黄疸 • Splenomegaly 脾肿大 • Placental hemorrhage and necrosis 胎盘性产前出血 和坏死 • Culture 培养 - Placenta, lung, stomach contents 胎盘，肺，胃内容 Elizabeth Macarthur Agricultural Institute, NSW, Australia 物 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 Bacterial and fungal abortion • Micro lesions 微小病灶 细菌和真菌堕胎 Viral abortion 病毒堕胎 • Suppurative (usually) • Nonsuppurative or Lymphocytic – hint* 非化脓 • 化脓性的（通常） 性或淋巴细胞性 – 提示* - Pneumonia 肺炎 - Encephalitis 脑炎 - Placentitis 胎盘炎 - Myocarditis 心肌炎 (Brucella suis) - Vasculitis 血管炎 （猪布鲁氏杆菌） (Chlamydia sp) （衣原体） ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 Infections of the fetus 胎儿感染 Maternal illness 母体疾病 PRRSv 蓝耳病病毒 PRRSv 蓝耳病病毒 CSF 猪瘟 CSF 猪瘟 Leptospira 细螺旋体 Leptospira 细螺旋体 PRV 伪狂犬病毒 Influenza virus A 甲型流感病 毒 PCV2 猪圆环病毒2型 Zearalenone 毒素 PPV (1 and 2) 猪细小病毒（1和2） Erysipelas 丹毒 BVD 牛病毒性腹泻 EMCV 脑心肌炎病毒 Teschovirus, Enterovirus 猪肠病毒，肠 病毒 Opportunistic bacterial infections 机会 性细菌感染 Brucella suis (placenta)*布鲁氏杆菌 （胎盘） Chlamydia sp (placenta)*衣原体（胎盘） ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Zearalenone (toxin-associated abortion) 毒素（毒素相关联堕胎） - Fusarium roseum: estrogenic mycotoxin 粉红廉孢： 动情素性霉菌毒素 Dietary level 日粮水平 Clinical findings 临床发现 Prepuberal gilts 发情前小 母猪 (ppm) Vulvovaginitis and Prolapses 外阴阴道炎和下垂 Cycling gilts/sows 循环后 1-3 Anestrous and Pseudopregnancy 休情及假怀孕 备/母猪 3-10 Reduce litter due to early embryonic death 由于早 Pregnant sows 怀孕母猪 >30 期胚胎死亡造成幼崽数减少 - Diagnosis 诊断： - Contaminated feed no longer available - 拿不到已经污染的饲料 - Clinical and transient 临床和暂时的 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 减压 在此撞头 说明： 1. 找个坚硬的表面 2. 按照圆圈里的说明去做 3. 重复第二项直到失去知觉 4. 如果失去知觉， 停止减压 活动 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Next Generation Sequencing (NGS) – pathogen/emerging variant discovery • 下一代基因测序 – 病原/发新发变异的发现 DNA/RNA extraction DNA/RNA 提取 Whole sequencing of clinical sample 临床 样本的完全测序 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • Next Generation Sequencing (NGS) – pathogen/emerging variant discovery • 下一代基因测序 – 病原/新变异的发现 ~ <1% potential sequences of interest 小 于1%的重要的潜在序列 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 • NGS current challenges 下一代基因测序法当前挑战 - Data analysis and interpretation: “normal “ virome within the sample - 数据分析和解读：样本里“正常的”病毒组 - Potential biases on sample prep: DNA virus, RNA virus, bacteria 准备样本时潜在的 偏差：DNA病毒，RNA病毒，细菌 - Turnaround: >3weeks 时间：大于3周 - Cost: $ 300/sample 花费：一个样本300美金 - Lack of correlation with lesions or morphological changes in the tissues 与组织病变 和形态改变缺乏相关性 o Combine with other techniques for detection of the potential pathogen in the tissues 与 其它技术合并来发现组织里的潜在病原 Immunohistochemistry (IHC)免疫组织化学 In situ hybridization (ISH) 原位杂交法 Antibody-based assay 以抗体为基础的检测 Sequence-based assay 以序列为基础的检测 ReproductiveOutline Disease: Diagnostic Investigations 繁殖疾病：诊断性调查 Immunohistochemistry (IHC) 免疫组织化学 In situ hybridization (ISH) 原位杂交法 Antibody-based assay 以抗体为基础的化 Sequence-based assay 以序列为基础的化验 验 CaseOutline study: PCV3-associated reproductive failure 案例：猪圆环病毒3型关联的繁殖失败 • Chronic/recurrent diagnostic investigation (abortions and post- weaning wasting) • 慢性/反复诊断性调查（流产及断奶后消瘦） • Findings 发现: - Fetus 胎儿: viral epicarditis 病毒心外膜炎 - Nursery 保育: viral myocarditis, vasculitis + bacterial infections 病 毒心肌炎，血管炎+细菌感染 • NGS 下一代基因测序 - ~50 seqs – pcv-like (SFBeef and PorkNW2/USA/2009) - ~1600 seqs – pcv-like (SFBeef and PorkNW2/USA/2009) - ~80 seqs – pcv-like (SFBeef and PorkNW2/USA/2009) - Background (?): Astrovirus 4, Rota, hepacivirus, PCMV CaseOutline study: PCV3-associated reproductive failure 案例：猪圆环病毒3型关联的繁殖失败 • In situ hybridization: probes PCV-like and Astrovirus 4 原位杂交法：探查 猪圆环病毒相似体及星状病毒4 - Astrovirus 4: negative 星状病毒4：阴性 - PCV-like (PCV3): positive 猪圆环病毒相似体（3型）：阳性 CaseOutline study: PCV3-associated reproductive failure 案例：猪圆环病毒3型关联的繁殖失败 CaseOutline study: PCV3-associated reproductive failure 案例：猪圆环病毒3型关联的繁殖失败 • Going forward 进一步 - Commonly found in the US swine population 对于美国猪群很常见 o Recombinant ELISA 重组酶联免疫吸附: 55% positive 55% 阳性 (Palinski et al 2017) – n=83 o Retrospective PCR screening by UMN-VDL 明大兽医诊断实验室进行的聚合酶链反应回顾检查 (Miller et al, unpublished) – n=477 130 PCV3+ (~27%) 99 PCV2+ (~21%) 43 PCV2/3+ (~9%) o Ongoing 进行中: Retrospective PCR screening by UMN-VDL明大兽医诊断实验室进行的聚合酶 链反应追溯检查 (Zhen et al, unpublished) – n=74 15 PCV3+ (20%) CaseOutline study: PCV3-associated reproductive failure 案例研究：猪圆环病毒3型关联的繁殖失败 • PCV2 versus PCV3 猪圆环病毒2型与3型比较 - Genetic 遗传 o Capsid proteins: amino acid sequence with 37% identity o 衣壳蛋白：氨基酸序列37%相似 - Clinical (nonspecific): can be indistinguishable - 临床（非特异）：可能无法分辨 o Abortion, PDNS, post-weaning wasting o 流产，猪皮炎肾病综合征，断奶后消瘦 - Diagnosis strategies: PCV2-like 专断策略：与2型相似 o PCV3: lymphoid depletion not a remarkable feature o 3型：淋巴损耗并不是一个突出特征 o Co-infection may occur 协同感染可能会发生 CaseOutline study: PCV3-associated reproductive failure 案例：猪圆环病毒3型关联的繁殖失败 • PCV3: is this real (?) 猪圆环病毒3型：这是真的吗？ - Koch’s postulate – no (yet) Koch的假定 – 还不确定 - Vaccine cross-protection – seems unlikely - 疫苗的交叉保护 – 看上去有可能.