Leman China Swine Conference 2012 Overview of Enteric Disease in the Growing Pig 生长猪的肠道疾病 Connie Gebhart, PhD gebha001@umn.edu Pospischil, A. et al. 2002. SHAP
Diarrheal Diseases 腹泻疾病 Nursery age 保育期 E. coli, enterotoxigenic 大肠杆菌, 产肠毒素 Rotavirus 轮状病毒 TGE 猪传染性胃肠炎 Coccidiosis 球虫病 Grow/finish age 育肥期 Salmonellosis 沙门氏菌病 Swine dysentery 猪痢疾 Colonic spirochetosis 结肠螺旋体病 Proliferative enteropathy 增生性肠病 TGE, PED, HBS
Diarrheal Disease Agents in Growing Pigs 生长猪的腹泻病原 WEEKS OF AGE 周龄 E. coli Rotavirus Coronavirus (TGE) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Salmonella L. intracellularis B. hyodysenteriae B. pilosicoli Trichuris suis typical age distribution 年龄分布 peak incidence 高发期
Post-weaning E. coli 断奶后大肠杆菌 Clinical signs: 临床表现 Occur days to weeks after arrival in nursery 到保育室数天至数周后出现 Pathogenic, toxigenic E.coli up to 14 weeks of age 产毒素, 致病性大肠杆菌出现年龄可至 14 周 Liquid, yellow diarrhea, ill-thrift, death (edema disease) 液体, 黄痢, 进食不良, 死亡 ( 水肿病 ) Pathogenesis caused by fimbrialbased colonization / toxinmediated secretory diarrhea 由菌毛群落 / 毒素调节的分泌性腹泻的发病机制 Virulence factor genes 毒性因子基因 Fimbrial 菌毛 Toxins 毒素 Adhesins 粘附素 F4 (K88) F5 (K99) F6 (987P) F18 F41 LT STa STb Stx1 STx2e EAST1 AIDA-I Paa Eae
Post-weaning E. coli 断奶后大肠杆菌 Gross: reddened intestinal mucosa, empty GI tract; mesenteric lymph nodes usually not enlarged 可见 : 变红的肠黏膜, 空胃肠道, 肠系膜淋巴结通常不增大 Edema Disease (F18 E.coli) 水肿病 Secretes shiga-like exotoxin 分泌 shiga 样外毒素 Affects endothelial cells=edema 影响内皮细胞 = 水肿 Pittman JS. Enteritis in grower-finisher pigs caused by F18-positive Escherichia coli. J Swine Health Prod. 2010;18(2):81 86.
Rotavirus 轮状病毒 RNA virus; double-stranded RNA 双链病毒 Groups A,B,C 分 A,B,C 组, Group A most common A 组最常见 Clinical signs: 临床表象 Watery to pasty diarrhea, dehydration 水样到糊状腹泻, 脱水 Pathogenesis 发病机理 Destroys intestinal villous enterocytes 破坏肠绒毛肠 Villous atrophy malabsorption diarrhea dehydration 绒毛萎缩吸收不良腹泻脱水
Rotavirus 轮状病毒 Pale intestines with fluid ingesta 苍白, 带流食的肠道 Histologically: 组织学 : Moderate villus atrophy 轻度的绒毛萎缩 Enterocyte vacuolization and necrosis 肠上皮细胞空化和坏死 Targets the mature enterocytes 针对成熟的肠细胞 Group A Rotavirus, IHC A 组轮状病毒, 免疫组化
Coronavirus 冠状病毒 Causes TGE (Transmissable Gastroenteritis) and PED (Porcine Epidemic Diarrhea) 引发肠胃炎和猪流行性腹泻 Clinical signs: profuse watery diarrhea, rapid dehydration, vomiting, death 临床症状 : 大量水泻, 迅速脱水, 呕吐, 死亡 Malabsorptive diarrhea from villus damage 绒毛损伤导致吸收不良性腹泻, Malabsorption diarrhea dehydration 吸收不良 腹泻 脱水
Coronavirus 冠状病毒 Thin walled, pale intestine (fetid odor) 薄壁, 苍白的肠道 ( 臭味 ) Histologically 组织学 Severe villous atrophy 严重的绒毛萎缩 PED resembles TGE except: PED 和 TGE 很像, 除了 : Longer incubation period 潜伏期更长 Lower mortality 死亡率较低 Slower spread 缓慢蔓延 Older pigs more lethargic 老猪更无活力 Normal Atrophied
Salmonella 沙门氏菌 S.typhimurium= enteric form 鼠伤寒 = 肠道形式 Serogroup B4,5 pathogens usually- Typhimurium, Derby, Agona, Heidelberg 通常是血清 B4,5 病原体 - 伤寒, 德比, 阿戈纳, 海德堡 S. choleraesuis= systemic form 猪霍乱 = 系统形式 Serogroup C 血清型 C Clinical signs and lesions: 临床征兆和损伤 Enteric form: usually only diarrhea, enlarged mesenteric lymph nodes, button ulcers in large intestine 肠道形式 : 通常只有腹泻, 肠系膜淋巴结肿大, 大肠有 钮状溃疡 Septicemic form: cyanosis, splenomegaly /hepatomegaly, pneumonia 败血性形式 : 苍白, 脾肿大 / 肝肿大, 肺炎
Salmonella 沙门氏菌 Enteric form: Most infections are subclinical; watery diarrhea, feces with fibrin, blood flecks 肠道形式 : 大多数感染为亚临床型, 水样腹泻, 粪便中带纤维蛋白, 血斑点 Button ulcers in colon, inflammation 结肠 钮状溃疡, 发炎 Septicemic form: Pneumonia a common feature, coughing 败血形式 : 肺炎是共同特点, 咳嗽 Tonsils and lungs are sites of invasion 扁桃体和肺是入侵点 Cyanotic extremities 青紫四肢
Brachyspira 短螺旋体属 Swine Dysentery 猪痢疾 Strongly beta-hemolytic: 强 β- 溶血性 B. hyodysenteriae primary etiologic agent 猪痢疾密螺旋体 - 主要病原体 B. hampsonii brachyspiral colitis in N.A. 结肠炎 B. suanatina provisional species isolated in Denmark and Sweden (swine and mallards) 现行种是在丹麦和瑞典 ( 猪和野鸭种 ) 分离出 Porcine Intestinal Spirochetosis 猪小肠螺旋体病 Weakly beta-hemolytic: 弱 β 溶血性 B. pilosicoli primary etiologic agent 主要病原体 B. innocens, B. murdochii, B. intermedia considered commensal B. innocens, B. murdochii, B. intermedia 被认为是共生的
B. hyodysenteriae 猪痢疾密螺旋体 Clinical signs: mucoid and/or bloody diarrhea, weight loss, lethargy 临床症状 : 粘液性和 / 或出血性腹泻, 消瘦, 嗜睡 Lesions in large intestine 大肠病变 Colitis with fibrinous exudate 结肠炎纤维素性渗出物 Histologically: 组织学 Goblet cell hyperplasia, superficial mucosal inflammation 杯状细胞增生, 表面粘膜炎症 May see large spirochetes in mucosa 粘膜中可见大螺旋菌
Prevalence of Swine Dysentery Worldwide 世界范围 猪痢疾的流行 Global economic significance 全球经济影响 North America 北美 Common disease in the 1970 s and 1980 s 20 世纪七八十年代的常见病 Identification by phenotypic characteristics 由表型特征识别 Associated with classic dysentery signs 与典型痢疾症状相关 More sporadic cases in 2000 s 在 2000 s 有较多散发病例 Often mucoid diarrhea, colitis 通常出现粘液样腹泻, 结肠炎 Brachyspira a re-emerging issue? 短螺旋体属是个新出现的问题? Culture may be more sensitive/universal 培养可能会更加敏感 / 有共性 Novel Brachyspira species cultured 培养出新种类
B. pilosicoli 螺旋体 Porcine intestinal spirochetosis 猪肠道螺旋体病 Clinical signs: pasty diarrhea, ill-thrift, gaunt, tucked up abdomen 临床症状 : 糊状腹泻, 进食不佳, 憔悴, 腹部卷起 Grossly, milder catarrhal lesions in mucosa 肉眼可见, 温和的鼻粘膜病变 Histologically: 组织学 Attachment of bacteria in colon 结肠中有附带细菌 Hampson et al. 2000. Vet. Microbiol. 73:76-84.
Novel Brachyspira species 新短螺旋体种 B. hyodysenteriae and B. pilosicoli identified by specific PCR 由特异性 PCR 识别猪痢疾密螺旋体和肠道螺旋体 Novel Brachyspiras not detected by routine PCR 无法由常规 PCR 识别的新型短螺旋体属 Identified by culturing and then nox gene sequencing 通过培养和 nox 基因测序鉴定 Often signs not classic dysentery, but chronic diarrhea 通常情况下表现不是典型痢疾, 而是慢性腹泻 Pathogen-specific PCR tests may miss other/new pathogens 病原体的特异 PCR 检测可能会错过其他的 / 新的病原体 B. murdochii often only isolate found: commensal? B. murdochii 往往是唯一发现的菌株 E.U. reports suggest B. murdochii may contribute to chronic diarrhea 欧盟报告显示 B. murdochii 可能导致慢性腹泻 Etiology may be multifactorial 病因可能是多重因素
Brachyspira species 螺旋体属种 2010-2011 at U of MN VDL Novel Strongly Hemolytic (NSH) Brachyspiras 新型强血溶性螺旋体 10% of Brachyspira isolates 10% 的螺旋体菌株 Genetically distinct from B.hyodysenteriae 遗传学上与 B.hyodysenteriae 不同 All from suspected enteric disease (8 with bloody diarrhea) 都源于疑似肠道疾病 (8 个带血腹泻 )
Brachyspira Identification 短螺旋菌鉴定 nox Sequencing nox 测序 Species identification of isolates by PCR and sequencing of nox (NADH oxidase) 通过 PCR 和 nox 测序进行种属鉴定 Forward Primer Targets conserved regions and amplifies variable region 针对保守区域, 放大可变区 Broad-taxon amplification with species-specific discrimination 用种特异区分进行宽群放大 PCR product sequenced and compared to NCBI database 对 PCR 产物测序并与 NCBI 数据库比较 PCR Product Reverse Primer Conserved 保护区 Variable Region 变量区 Conserved
Condensed nox Gene Tree 简要 nox 物基因树 Entire nox gene sequence of NSH Brachyspiras 所有 nox 基因序列 Sequences cluster together 序列聚集在一起 Represent 2 clades 代表 2 个分支 Clusters with B. murdochii 与 B murdochii 群集 Little similarity to B. hyodysenteriae 与猪痢疾密螺旋体很少相似 NSH Clade I NSH Clade II
16S Phylogenetic Analysis 16S 系统进化分析 16S rrna sequencing is the standard 16S rrna 基因测序是标准 Results consistent with nox analysis 与 nox 分析结果一致 Novel Brachyspira sequences still cluster together; 2 groups 新型螺旋体序列仍聚集在一起,2 组 Cluster nearest B. murdochii 与最近的 B murdochii 聚集 NSH Clade I NSH Clade II
Phenotypic Characterization of Brachyspira 短螺旋体属表型特征 Isolate 菌株 Phenotypic profile* 表型情况 Tests 测试 Hemolysis Spot indole Hippurate a-galactosidase a-glucosidase b-glucosidase B. hyodysenteriae ATCC 27164 210011 Strong + - - + + B. pilosicoli ATCC 51139 101100 Weak - + + - - B. murdochii ATCC 51284 100011 Weak - - - + + NSH Brachyspira provisionally named B. hampsonii and contains Clades I and II 被命名为 B. hampsonii 的新型强血溶性短螺旋体属并包含 I 和 II 分枝 NSH Brachyspira 200000 Strong - - - - - *Phenotypic profile = 0: negative reaction/no hemolysis; 1: positive reaction/weakly hemolytic ; 2: strongly hemolytic.
Significance of B. hampsonii B.hampsonii 的重要性 Preliminary epidemiology (retrospective) 初步流行病学 ( 追溯 ) Strong relationship between isolation of NSH Brachyspira and diarrhea, especially bloody diarrhea NSH 短螺旋体属菌株和腹泻, 特别是出血性腹泻之间的紧密联系 Reported Condition 报告的情况 Novel isolate 新菌株 Non-novel non-typable 非新型非分型 Odds Ratio 比例 C.I. No Diarrhea 无腹泻 0 23 - - Diarrhea 腹泻 19 37 24.4 1.4-424.3 Bloody Diarrhea 带水腹泻 8 1 34.4 3.9-306.1 Experimental reproduction of Brachyspira colitis: 短螺旋菌结肠炎的再生实验
B. hampsonii Pathogenesis B. hampsonii 发病机理 Pathogenesis confirmed by experimental reproduction in pigs 发病机理在再生实验中被证实 Groups (n=4) 组 Unchallenged control 对照 Results 结果 no lesions 无病变 B. murdochii mild colitis 轻度 B. hampsonii Clade I moderate colitis 一般 B. hampsonii Clade II moderate colitis 一般 B. hyodysenteriae moderate colitis 一般 Both B.hampsonii Clades I and II produced mucodiarrhea with gross and histological lesions B.hampsonii 分支 I 和 II 都产生了肉眼可见有组织病变的黏膜腹泻
Proliferative Enteropathy (PE) 增生性肠病 (PE) Worldwide economic impact in pigs 对世界养猪生产经济影响 Increasingly reported in other species 越来越多的在其它种中出现 Horse, hamster, ferret, fox, deer, ratites, rabbit, monkey, etc 马, 鼠, 雪貂, 狐狸, 鹿, 平胸鸟, 兔, 猴等 Mimics and/or predisposes pigs to other diseases 与猪的其他疾病相似 : PCV2, Salmonellosis, swine dysentery, colibacillosis, colonic spirochetosis, hemorrhagic bowel syndrome PCV2, 沙门氏菌病, 猪痢疾, 大肠杆菌病, 结肠螺旋体病, 出血性肠易激综合征
Characteristics of PE in Pigs 猪的 PE 特征 PHE (proliferative hemorrhagic enteropathy) PHE( 增生性出血性肠病 ) Acute form; 4-12 months 急性型 ;4-12 个月 Older pigs, bloody diarrhea, death 老龄猪, 血性腹泻, 死亡 PIA (porcine intestinal adenomatosis) PIA( 猪小肠腺瘤 ) Chronic form; 6-20 weeks 慢性型 ;6-20 周 Young growing pigs, diarrhea, poor growth, weight variation 年幼生长猪, 腹泻, 生长发育不良, 体重变化 Subclinical 亚临床 (difficult-to-diagnose form of PIA) ( 难以诊断的 PIA) No diarrhea, but performance/growth rate is impacted 没有腹泻, 但产能 / 增长率受影响 Reduced feed intake and loss of homogeneity 食量下降和生长不均匀
Equine PE 马 PE Prevalence/awareness increasing 流行性 / 意识提高 Outbreaks commonly reported 发病通常被发现 Also a production problem 也是一个 生产 问题 Recovered foals sell for 63% less 治愈的马驹售价降低 63% Poor-doer, colic, peripheral edema, hypoproteinemia* 表现差, 绞痛, 血管神经性水肿, 低蛋白血症 * Source of infection in horses? 马的传染源? Probably not pigs 不是猪 Pig parallels to equine PE 猪与马的 PE 相似
Pig Parallels to Equine PE Both affect rapid growing breeds/genetics 都影响快速增长的品种 Both occur after transport to new facilities 都在转移至新地点后发生 - Lawsonia is ubiquitous/stress initiates disease? 内劳森普遍存在 / 压力引发疾病? - Endemic in some environments? 在某些环境中流行吗? 猪与马的 PE 相似 Like most production animal students, Doreen breezes through the chapter on Equine Medicine. The Far Side
Epidemiology of PE VNTR Typing PE 流行病学 VNTR 分型 Lawsonia isolates from diverse animal species and geographies are phenotypically/antigenically similar 来自不同的物种和地域的内劳森菌株表型 / 抗原相似 No difference between ostrich, hamster, pig or horse isolates 鸵鸟, 仓鼠, 猪或马的菌株之间无差异 No difference between E.U. and U.S. isolates (temporal or geographic) 欧盟和美国的菌株无区别 ( 时间和地域 ) VNTR molecular typing technique detects minor genetic differences VNTR 分子组型技术检测到轻微的遗传差异
Epidemiology of PE VNTR Typing PE 流行病学 VNTR 分型 Isolates within an outbreak have IDENTICAL profiles 同一病发内的菌株具有相同的特征 Form of PE does not affect VNTR profile PE 形式不影响 VNTR 特征 Multiple temporal samples identical within site 站内多时间样本相同 Isolates between outbreaks have DIFFERENT profiles 病发之间的菌株具有不同特征 Differences between E.U and U.S. outbreaks are no greater than between Iowa and Minnesota outbreaks 欧盟和美国病发之间的差异不大于爱荷华州和明尼苏达州之间病发的差异 Isolates from pigs vs other animal species show MARKEDLY DIFFERENT profiles 猪与其他物种菌株表现明显不同的特征 VS
VNTR Typing Pig site IV 2 3 1 Pig site III 2 3 1 Pig site II 2 3 1 15540D (Denmark) Pig site I 2 3 1 VPB 1-4 963/93 (U.K.) PHE00-MN1 Zoo pig Mini pony Horse Ostrich Ferret Spider monkey VTRN 分型 Pig 猪 Non-pig 其它
Host Adaptation of Lawsonia 内劳森的宿主适应 Evaluated the susceptibilities of pigs and horses to porcine and equine Lawsonia isolates 评估猪和马对猪和马的内劳森菌株的易感性 Species-specific infection=similar lesions, shedding and serologic responses 种属特异性感染 = 类似的病变, 脱落和血清反应 Cross-species infection=significantly different responses 跨种感染 = 显著不同的反应 Host susceptibilities driven by species of origin 由物种起源引起的宿主的易感性