Differentiating Neonatal Diarrhea 鉴别新生猪腹泻 Darin Madson, DVM, PhD, DACVP 达林 麦迪逊博士 Iowa State University

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1 Differentiating Neonatal Diarrhea 鉴别新生猪腹泻 Darin Madson, DVM, PhD, DACVP 达林 麦迪逊博士 Iowa State University

2 General concepts 一般概念 Swine microflora is vast = ~10 14 bacteria 猪的菌群很多 = ~10 14 细菌 New-born pigs = sterile gastrointestinal tract 新生猪 = 无菌消化道 At birth 出生时 Race for bacterial colonization 细菌快速定殖 Overgrowth is common 超速生长很常见 Environment and immunity play a role 环境和免疫力扮演着一定的角色 Neonatal diarrhea is: Often infectious and associated with management issues 新生儿腹泻 : 经常是有传染性的并于管理问题有关

3 Management Influences 管理的影响 Transmission 传播 Gestation 怀孕 Stalls vs. pens vs. open lots 畜舍, 猪栏与开放场地 Parity distribution 同等分布 Gilt acclimation 小母猪适应 Housing design 猪舍设计 Sanitation 卫生 Flooring material 铺地材料 Room management 房间安排 pig flow 猪流动 Dam immunity 母畜免疫 Gestation 怀孕 Stalls vs. pens vs. open lots 畜舍, 猪栏与开放场地 Parity distribution 同等分布 Vaccination feed back 免疫 - 反馈 Sow milking 母猪喂奶 Feeding practices 喂养 Sow comfort 母猪舒适 Pig suckling 猪哺乳

4 Primary infectious causes 主要传染起因 Clostridium difficile 难辨梭菌 Clostridium perfringens type A A 型产气荚膜梭菌 Clostridium perfringens type C C 型产气荚膜梭菌 Coccidiosis (Isospora suis) 球虫 Colibacillosis (enterotoxigenic E. coli; ETEC) 大肠杆菌 Rotavirus 轮状病毒 TGEV (coronavirus) 猪传染性胃肠炎 Combined infections are common 合并感染很常见

5 Mechanisms of diarrhea 腹泻的机制 Moeser et al. 2007

6 TGEV 传染性胃肠炎 Rotavirus 轮状病毒 (A, B, C) E coli 大肠杆菌 Coccidiosis 球虫 C perf. type C C 型产气荚膜梭菌 C 型产气荚膜梭菌 C perf. type A C difficile A 型产气荚膜梭菌 难辨梭菌 Weeks of Age 年龄 ( 周 )

7 Clostridium difficile 难辨梭菌 Common intensive management systems 常见 集约化管理系统 Gilt and sow litters affected 影响小母猪和母猪产仔 All piglets colonized by 48 hrs. 所有猪仔 48 小时后分群 1 7 days of age 于 1-7 天感染疾病 Watery to pasty diarrhea 水状到糊状腹泻 Dysbacterosis overgrowth of C. difficile secretion of TcdA and TcdB (cytotoxins) 菌群失调 - 梭菌过度生长 分泌 TcdA 和 TcdB ( 细胞毒素 ) Antibiotics use? 使用抗生素? Effects of crated gestation/sanitation on flora/immunity? 猪舍妊娠 / 卫生 / 免疫的影响

8 Clostridium difficile 难辨梭菌 Mesocolonic edema 水肿 Fibrinonecrotic colitis 结肠炎

9 Clostridium perfringens type A A 型产气荚膜梭菌 Pasty creamy - watery diarrhea 糊状 - 乳状 - 水状腹泻 1 7 days of age1-7 天时腹泻 Poor response to antibiotics 对抗生素反映欠佳 High morbidity, low mortality 高发病率, 低死亡率 Offending strains are β 2 toxin positive 侵害病株 β2 毒素阳性 Common flora of sows and pigs 母猪和猪常见的菌群 Most strains β 2 toxin negative 多数菌株 β2 毒素阴性 No gross lesions 无肉眼可见的病变

10 Clostridium perfringens type C C 型产气荚膜梭菌 Acute form: 1-7 days-of-age 急性 :1-7 天 Subacute form: 7-14 days-of-age 亚急性 :7-14 天 Segmental in small intestine 节段性小肠 Acute without maternal immunity 急性 - 没有母体的免疫力 Bloody diarrhea 血性腹泻 High mortality 死亡率高 Subacute partially protective maternal immunity 亚急性 - 部分保护的母体免疫力 Watery diarrhea, wasting 水状腹泻 消瘦 High mortality 死亡率高

11 Clostridium perfringens type C C 型产气荚膜梭菌 Hemorrhage, emphysema and fibrinous mucosal membrane 出血, 肺气肿和纤维素黏膜

12 Coccidiosis Isospora suis 球虫病 Predominantly 5 14 days-of-age 主要在 5-14 天时感染 fewer up to 5 weeks-of-age 较少 5 周以上 White pasty-to-watery scour 白色糊状到水状的腹泻 Disease is dose-related 疾病是剂量相关的 Environmental contamination by oocysts 卵囊引起的环境污染 Oocyts are environmentally resistant 卵母细胞对环境有抗性 Farrowing crate flooring type and sanitation are critical 产仔舍地板的类型和卫生是关键

13 Coccidiosis Isospora suis 球虫病

14 Enterotoxigenic E. coli (ETEC) 肠毒素大肠杆菌 ( 大肠杆菌 ) Most often in pigs less than a week old 最常出现在一周以下的小猪中 Predispose by chilling, poor consumption of colostrum/milk 易由着凉, 初乳和奶不足引起 Non-hemolytic & hemolytic strains 非溶血和溶血性的菌株 Bacteria attach to small intestinal mucosa 细菌附着于小肠黏膜 Bacterial fimbria; F4 (K88), F5 (K99), F6 (987P), F41 细菌菌毛 ; Bacteria secrete enterotoxins that stimulate secretion by intestinal mucosa 细菌分泌, 刺激肠粘膜分泌的肠毒素 Heat liable (LT) 热不稳定 Heat stable (Sta, Stb) 热稳定

15 Enterotoxigenic E. coli (ETEC) 肠毒素大肠杆菌 ( 大肠杆菌 )

16 Rotavirus 轮状病毒 Three antigenic groups 三种抗原组 Groups A, B & C A 组,B 组和 C 组 Disease 疾病 Virus replicates in villus enterocytes variable villus atrophy (more severe in younger animals) 病毒在绒毛细胞复制 不同程度绒毛萎缩 ( 年幼的动物更为严重 ) 1day to 8 weeks-of-age 1 天至 8 周 Most common: 2-3 weeks-of-age (or post-weaning) 最普遍 :2-3 周 ( 或断奶后 ) Degree of maternal/mucosal immunity determines disease 母体 / 粘膜的免疫程度决定疾病 Little: Watery diarrhea with undigested milk 很少 : 水状腹泻, 带未消化奶 Some: Creamy diarrhea ( milk scours ) 一些 : 乳状腹泻 ( 奶腹泻 ) Much: Subclinical 严重 : 亚临床

17 Rotavirus 轮状病毒 Normal 正常 Thin walled intestines 薄壁肠

18 TGEV 猪传染性胃肠炎 1 serotype of TGEV 1 血清型猪传染性胃肠炎病毒 Replicates in villus enterocytes villus atrophy 在肠绒毛细胞复制 绒毛萎缩 Maternal/mucosal immunity determines disease 母体 / 粘膜的免疫程度决定疾病 Little/none: Epizootic All ages affected 很少 / 没有 : 流行性所有年龄 Moderate: Enzootic 1-3 weeks of age 中度 : 地方性 1-3 周 Much: Protection 重度 : 保护 Epizootics more common in cold months 在寒冷时更易流行

19 TGEV 猪传染性胃肠炎 Severe villous atrophy 严重肠绒毛萎缩 )

20 Questions? 问题? Thank you for listening 感谢听讲

21 Diagnosing Neonatal Diarrhea ( 新生猪腹泻诊断 ) APPENDIX ( 附录 )

22 General Concepts Diagnosis Viral Diseases 病毒性疾病 Virus is present a short time 病毒出现时间短 Select acute pigs for testing (1 st 24 hours of diarrhea) 选择急性感染的猪做测试 ( 腹泻 24 小时内 ) Bacterial Diseases 细菌性疾病 一般概念 - 诊断 Therapeutic antibiotics interferes with testing 治疗抗生素干扰测试 Select untreated pigs for testing 选择未经治疗的猪测试 Intestinal mucosa autolyzes rapidly 肠粘膜迅速自溶 Select only euthanized pigs for testing 只选择 安乐死 的猪测试 Fix intestinal segments within 15 minutes of death 死亡的 15 分钟内固定肠段 Lesions are often segmental in intestine 病变往往是节段性的 View many segments at each level 查看同一级别多个节段 Collect multiple segments of each level 收集同一级别多个节段

23 Diagnostic Approach 诊断方法 Acutely affected untreated pigs 未经治疗急性感染的猪 Fecal samples (cecal contents) chilled 粪便样本 ( 盲肠内容物 ) 冷却 Fresh (chilled) and fixed (fix within 15 min. of death) 新鲜 ( 冷藏 ) 并固定 ( 在死亡 15 分钟内 ) Jejunum 空肠 Ileum 回肠 Spiral colon (tied and frozen for C. difficile) 螺旋结肠 ( 捆绑并冻结用来测难辨梭菌 ) Mesenteric lymph nodes 肠系膜淋巴结 Liver 肝 Convalescent serum (TGEV) 恢复期血清 (TGEV)

24 Clostridium sp. Diagnosis C. perfringens type C C 型产气荚膜梭菌 梭状芽孢杆菌诊断 Typical gross and microscopic lesions 典型的可见和微观病变 Culture and identification 培养和鉴定 Typing is by detection of toxin genes by PCR 通过 PCR 检测毒素基因来测基因型 C. perfringens type A A 型产气荚膜梭菌 Microscopic lesions (few) 微观病变 ( 较少 ) Anaerobic bacterial culture 厌氧细菌培养 Elimination of other causes of diarrhea 消除其他原因引起的腹泻 Diagnosis by exclusion 通过排除诊断 C. Difficile 梭状芽孢杆菌 Typical gross and microscopic lesions 典型的可见和微观病变 Colonic contents 结肠内容物 Positive culture 阳性培养 Quantitative toxin assay 定量毒素分析

25 Coccidiosis Diagnosis 球虫病的诊断 Lesions 病变 Most severe in the distal small intestine 远端小肠中最严重 Gross 肉眼可见 : Fibrinonecrotic enteritis 纤维坏死肠炎 Microscopic 微观 : Atrophic and fibrinonecrotic enteritis + coccidia 萎缩性胃炎和纤维坏死肠炎 + 球虫 Diagnosis (confirm presence of I. suis) 诊断 ( 确认 I. suis 存在 ) Histopathology 病理组织学 Fecal examination 粪便检查 Will not work for acute cases 不适用于急性病例

26 Enterotoxigenic E. coli Diagnosis 肠毒素大肠杆菌诊断 Sample untreated acute pigs 取样未经治疗的急性感染猪 Distal jejunum and ileum 远端空肠和回肠 Typical gross and microscopic lesions 典型的肉眼可见和微观病变 Identify virulent E. coli 识别毒大肠杆菌 Mucosal impression smears 粘膜压制涂片 Bacterial culture 细菌培养 Isolate predominant E. coli 提取主要的大肠杆菌 Identify virulence attributes 找出致病属性 PCR

27 Rotavirus Diagnosis 轮状病毒的诊断 Acutely affected pigs (day 1 of diarrhea) 急性感染猪 ( 腹泻第一天 ) Fecal samples 粪便样本 EM (will not distinguish A, B or C 不能区分 A,B 和 C) 电镜 PAGE PCR Ag ELISA (commercially available for only A 市场有售仅检测 A) Frozen or fixed jejunum and ileum (fix in 10% NBF within 15 minutes of euthanasia) 新鲜冷冻或固定的空肠和回肠 ( 处死后的 15 分钟内在 10% 的 NBF 中固定 ) Histopathology, IHC 病理组织学, 免疫组化 FA 免疫荧光

28 TGEV Diagnosis TGEV 诊断 Acutely affected pigs (day 1 of diarrhea) 急性感染猪 ( 腹泻第一天 ) Fecal samples 粪便样本 EM 电镜 PCR Fresh chilled or fixed jejunum and ileum 新鲜冷冻或固定的空肠和回肠 Typical lesions 典型病变 FA 免疫荧光 IHC 免疫组化 Serologic testing of convalescent sera 恢复血清的血清学检测 SN (will not differentiate TGEV and PRCV 不能区分 TGEV 和 PRCV) Blocking ELISA to differentiate from PRCV (use sera with > 1:40 SN titer) 阻断 ELISA 来区分 PRCV( 使用 SN 滴度 >1:40 的血清 )

29 TGEV Clinical Disease TGEV 临床疾病 Epizootic 流行性 Nearly all litters all pigs affected 几乎所有胎次 - 所有猪都被影响影响 Severe watery diarrhea and vomiting 严重水样腹泻和呕吐 Nearly 100% mortality 近 100% 的死亡率 Enzootic 地方性 Much litter/litter variation 更多胎次与胎次之间的不同 1-3 weeks-of-age most common 1-3 周最常见 Watery flocculent diarrhea +/- vomiting 水样腹泻 +/- 呕吐 Variable mortality 不同死亡率