Mutations in the Salmonella enterica serovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis Delta crp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis Delta crp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis Delta crp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis Delta crp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis Delta crp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis

Pathogenesis of Actinobacillus Pleuropneumoniae (I)Exotoxin Induced Apoptosis in Procine Alveolar Macrophage

胸膜肺炎放線桿菌是引起豬隻纖維素性、出血性、壞死性、胸膜肺炎的病原，世界各國均有本病之發生，造成養豬業界極大的經濟損失。 胸膜肺炎放線桿菌產生多種毒性因子，其中外毒素Apx 被證實為造成疾病的主要毒性因子。胸膜肺炎放線桿菌依其血清型不同，生成二至三種外毒素(ApxI-IV)，這些外毒素具有不等程度之溶血性及急性細胞毒殺效果。這些外毒素除造成溶血，亦傷害肺臟內皮細胞及第一線防衛細胞-肺泡巨噬細胞。近年有研究指出ApxIII 可誘發豬肺泡巨噬細胞之凋亡，雖ApxIII 誘發豬肺泡巨噬細胞凋亡之訊息傳導路徑仍待釐清，但此研究顯示肺泡巨噬細胞凋亡可能為本病致病機制之一；至於ApxI 或ApxII是否誘發肺泡巨噬細胞凋亡，至今則仍無相關研究。相較於其他Apx 毒素，ApxI之溶血性與細胞毒性均屬最高，為極重要毒性因子，故本計劃擬利用胸膜肺炎放線桿菌第十血清型僅生成ApxI 之特性，製備具高生物活性之ApxI，探討此毒素是否在in vivo 及in vitro 下誘發豬肺泡巨噬細胞凋亡，釐清相關訊息傳導路徑，包括毒素接受體及下游訊息分子之確認，藉此深入瞭解ApxI 在胸膜肺炎放線桿菌致病機制扮演之角色，以利本病防治策略之擬定

Actinobacillus Pleuropneumoniae Exotoxin ApxI Induces the Expression of Proinflammatory Cytokines in Porcine Alveolar Macrophages

胸膜肺炎放線桿菌為猪隻重要呼吸道病原，引起纖維素性、出血性、壞死性胸膜肺炎，世界各國均有本病發生，往往造成養猪業極大經濟損失。胸膜肺炎放線桿菌產生多種毒性因子，其中外毒素Apx 為造成疾病的主要毒性因子。胸膜肺炎放線桿菌依其血清型不同，生成二至三種外毒素(ApxI-IV)，這些外毒素具有不等程度之溶血性及急性細胞毒殺效果。相較於其他Apx 毒素，ApxI 溶血性與細胞毒性均屬最高，為極重要毒性因子，過去本實驗室已證明ApxI 除上述生物活性，尚可誘發猪肺泡巨噬細胞之凋亡，所涉及訊息傳導路徑包括MAPK 成員p38 及JNK 之活化，導致內源性與外源性凋亡路徑活化，進而誘發猪肺泡巨噬細胞凋亡。另利用特異抗體阻斷細胞表面分子CD18 活性，ApxI 所誘發之凋亡亦被抑制，顯示CD18 可能為ApxI 接受體或參與ApxI 誘發之凋亡，並推測此為ApxI 傷害宿主細胞機制之一。至於ApxI 是否尚有其它生物活性如誘發肺泡巨噬細胞前炎症細胞素生成，至今仍無相關研究，故本計劃擬探討: (1) ApxI 是否誘發猪肺泡巨噬細胞表現前炎症細胞素IL-1β、IL-8 及TNF-α；(2)確認ApxI 細胞接受體；(3)釐清相關訊息傳導路徑，包括p38、JNK、NF-κB 在ApxI誘發猪肺泡巨噬細胞前炎症細胞素表現中所扮演的角色及各訊息分子間之交互關係；藉此瞭解ApxI對宿主細胞之生物活性及毒素在胸膜肺炎放線桿菌致病機制扮演之角色，以利本病防治策略之擬定。Actinobacillus pleuropneumoniae (APP), the causative agent of porcine fibrinous, hemorrhagic,necrotizing pleuropneumonia, has resulted in great economic loss in swine industry worldwide. APPproduces a variety of virulence factors to facilitate its infection. Among various virulence factors, theexotoxins of A. pleuropneumoniae (Apx) are the major virulence factors contributing to the pathogenesis ofthis disease. There are four types of Apx toxins (ApxI-IV) exerting varied degrees of hemolytic and cytotoxicactivities. In comparison with other Apx toxins, ApxI elicits its most significant effects on hemolysis andcytolysis. Previously, we have demonstrated that ApxI induces typical apoptosis in porcine alveolarmacrophages (PAM) at a concentration- and time-dependent manner. The activation of MAPK p38 and JNKby ApxI leads to subsequent involvement of both intrinsic and extrinsic apoptotic pathways. Inhibition ofeither MAPK or caspase activity protected PAM from ApxI-mediated apoptosis. Furthermore, application ofan antibody specifically blocking a cell surface molecule CD18, spared PAM from apoptosis by ApxI,suggesting that CD18 may be a potential receptor of ApxI or may have a role in the apoptotic effect of ApxI.However, up to now whether ApxI confers effects additional to cytolysis and apoptosis on PAM remainsunclear. Therefore, the objectives of proposed study are to: (1) investigate whether ApxI induces theexpression of proinflammatory cytokines IL-1β, IL-8 and TNF-α in PAM; (2) identify the receptor of ApxIon PAM; and (3) delineate the underlying signaling mechanisms involved in the proinflammatory cytokineexpression, including the roles of p38, JNK, and NF-κB, and the correlation between these signalingmolecules. Through the elucidation of ApxI effects on PAM, it would further our understanding toward thepathogenesis of APP and provide useful information in the development of proper strategy for disease controlin the future

Pathogenesis of Actinobacillus Pleuropneumoniae-Exotoxin Induced Apoptosis in Procine Alveolar Macrophage (I)

胸膜肺炎放線桿菌是引起豬隻纖維素性、出血性、壞死性、胸膜肺炎的病原，世界各國均有本病之發生，造成養豬業界極大的經濟損失。胸膜肺炎放線桿菌產生多種毒性因子，其中外毒素Apx 被證實為造成疾病的主要毒性因子。胸膜肺炎放線桿菌依其血清型不同，生成二至三種外毒素(ApxI-IV)，這些外毒素具有不等程度之溶血性及急性細胞毒殺效果。這些外毒素除造成溶血，亦傷害肺臟內皮細胞及第一線防衛細胞-肺泡巨噬細胞。近年有研究指出ApxIII 可誘發豬肺泡巨噬細胞之凋亡，雖ApxIII 誘發豬肺泡巨噬細胞凋亡之訊息傳導路徑仍待釐清，但此研究顯示肺泡巨噬細胞凋亡可能為本病致病機制之一；至於ApxI 或ApxII是否誘發肺泡巨噬細胞凋亡，至今則仍無相關研究。相較於其他Apx 毒素，ApxI之溶血性與細胞毒性均屬最高，為極重要毒性因子，故本計劃擬利用胸膜肺炎放線桿菌第十血清型僅生成ApxI 之特性，製備具高生物活性之ApxI，探討此毒素是否在in vivo 及in vitro 下誘發豬肺泡巨噬細胞凋亡，釐清相關訊息傳導路徑，包括毒素接受體及下游訊息分子之確認，藉此深入瞭解ApxI 在胸膜肺炎放線桿菌致病機制扮演之角色，以利本病防治策略之擬定。Actinobacillus pleuropneumoniae, the causative agent of porcine fibrinohemorrhagicnecrotizing pleuropneumonia, has resulted in great economic loss inswine industry worldwide. A. pleuropneumoniae produces a variety of virulencefactors to facilitate its infection. Among various virulence factors, the exotoxins Apxof A. pleuropneumoniae have been characterized as the major virulence factorscontributing to the pathogenesis of this disease. A. pleuropneumoniae toxins (ApxI-IV) possess varied degrees of hemolytic and cytolytic activities. Not only do theycause hemolysis, damage pulmonary epithelial cells, but also impair the first line ofdefense cells in lung, the alveolar macrophages. It has been demonstrated that ApxIIIinduces apoptosis in alveolar macrophages via a caspase-dependent pathway.Although the underlying signaling events remain unclear, this finding suggests thatapoptosis of alveolar macrophages may be one of the mechanisms by which A.pleuropneumoniae causes disease. Notably, whether other Apx such as ApxI andApxII contribute to apoptosis in porcine alveolar macrophages resulting in pulmonarydamage is currently unknown. The fact that ApxI possesses the highest hemolytic andcytotoxic activity than other Apx, indicate its important role in the pathogenesis ofthis disease. Therefore, the objectives of proposed project are to investigate whether A.pleuropneumoniae serotype 10-derived ApxI induces apoptosis in porcine alveolarmacrophages both in vivo and in vitro, and to delineate the underlying signalingmechanisms involved in this process, including the identification of receptor for ApxIand downstream signaling molecules. Elucidating the role of ApxI in the pathogenesisof APP would further our understanding toward the pathogenesis of this disease andprovide useful information in development of proper strategy for disease control

Development of monoclonal antibodies against bovine viral diarrhea virus

本試驗的目的為製備牛病毒性下痢病毒（BVDV）之單源抗體，以期利用於本病之診斷 及其他有關BVDV之研究。本試驗係以純化之BVDV為抗原免疫Balb／C 小白鼠, 取其脾 臟細胞與NS-1骨髓瘤細胞以PEG-1500進行融合反應, 經HAT 培養液和ELISA 法篩選出 出72株強陽性融合瘤, 取其中10株以限數稀釋法進行單株化, 最後獲得四株穩定分泌 抗體之融合瘤細胞株, 分別命名為42E1A、14C4F、35E5B、22B8B。 為確定這些單源抗體之特異性, 本試驗配合酵素連結免疫轉印技術, 中和反應測試等 血清學方法予以定性分析試驗結果得知這些細胞株所分泌之單源抗體分子亞型均屬Ig G1, 其中14C4F 及35E5B 二株單源抗體具有中和能力; 42E1A 所作用之抗原決定區位 於85Kd及78Kd之病毒蛋白上, 35E5B 及22B8B 則可抗56Kd之病毒蛋白, 而14C4F 會抗 何病毒蛋白則無法藉酵素連結免疫轉印技術測知。 此外, 應用這些單源抗體於免疫過氧化氫 測試法, 可使感染BVDV之MDBK細胞呈現棕 色反應; 應用之於墨點酵素檢測技術時, 42E1A、35E5B及22B8B三株的敏感性皆達 PFU 的病毒量, 此較具敏感性之三株可供臨床診斷之用

Molecular Characterization and Antigenicity Study of Actinobacillus pleuropneumoniae Outer Membrane Protein (II)

豬胸膜肺炎放線桿菌(APP)為豬隻急性且致死性的病原菌，為一具有莢膜、無鞭毛、無運動性且不形成芽胞之革蘭氏陰性球桿菌，常造成豬隻出血性、壞死性、纖維素性之胸膜肺炎，由於豬隻在發病前常無臨床症狀之呈現，且罹病豬隻常是體型較大之肥育後期豬隻，因此APP一直都是養豬國家重大經濟損失的原因之一。目前已知APP的致病因子除Apx毒素外，其外膜蛋白(outer membrane proteins)亦是重要之致病因子。因此本計畫將持續豬胸膜肺炎放線桿菌之蒐集，並以multiplex PCR鑑定菌株之血清型別；分析以分子選殖、表現之重組外膜蛋白Oml其抗原性；並進行小鼠及豬隻動物試驗，以評估重組外膜蛋白在豬胸膜肺炎防治上之可行性。Actinobacillus pleuropneumoniae (APP) is a gram-negative, capsulated, short rod-shaped, and non-mobile bacterium that causes acute and fatal pleuropneumonia in swine. Affected pigs often developed hemorrhagic, necrotizing, fibrinous pleuropneumonia. The fact that APP occurs most often in fattening pigs that succumbed to sudden death without prior symptoms has caused great economic losses in swine industry. It is well known that there are many virulence factors of APP. In addition to Actinobacillus pleuropneumoniae toxins (Apx), the outer membrane proteins also played an important role in the pathogenesis of this disease. Therefore, the purpose of this project is to continuously collect APP strains from fields and to identify the serotype of the collected isolates using multiplex PCR. Moreover, the antigenicity of recombinant outer membrane protein Oml will be analyzed and its potential in disease control will be further evaluated both in mice and in target animal pigs

Establishment of Disease Inspection System on Notifiable Fish Diseases

[宣詩玲老師]台灣於近年來加入世界貿易組織後，國際間活魚及魚卵之貿易漸為頻繁，每年約由國外進口一百萬個以上的魚卵，並進口大量之冷藏或冷凍魚體，因此實施嚴格之檢疫工作，方可避免國際間魚類重大傳染病之傳播。本計畫擬利用先前計畫所開發之分子生物學診斷方法，配合並參考OIE所制訂之標準作業流程，來檢驗國內及國外進口相關水產品中之魚類重大傳染性病原。本年度計畫中，已成功建立一從”採樣”至”樣本檢驗”之標準作業流程，並建立基本之檢疫結果資料庫，以供查詢之用。檢驗結果方面，所蒐集之進百餘個進口鮭魚及台灣本土鱒魚、香魚樣本中，目前並無檢驗出包括infectious hematopoietic necrosis virus (IHNV),Viral Hemorrhagic Septicaemia (VHS)與bacterial kidney disease (BKD)可疑之樣本，但持續之監控仍持續進行中。另外，有鑑於國際間之魚貨貿易頻繁，為增加檢疫之多樣性，本單位亦積極建立其它OIE列表之魚類重大傳染性疾病之陽性對照質體，而本計畫中亦將利用長鏈引子合成之技術，進行部分infectious pancreaticnecrosis virus (IPNV)質體之構築，期望未來對於檢疫工作有所幫助。[張文發老師]為我國建立輸入甲殼類重要疾病之監控系統。1.完成樣品採集地點之選定及採樣方式與時間2.完成採樣樣本之DNA與RNA萃取3.完成樣本之PCR與RT-PCR之檢測4.建立監控結果之資料5.研究成果除參與人員可獲得有關甲殼類疾病監控與分子生物學技術之良好訓練外，尚具學術及應用價值。6.依相同模式，可用於其他進口動物重要傳染性疾病與我國出口水產品之監控，有助我國防疫檢疫技術之提升。7.長期對進口水產品進行監控，建立資料，有助於檢疫單位對外國重要疾病疫情之瞭解與掌控，並增加對為諮商談判之有效籌碼。[宣詩玲老師]The international trade of aquatic animal products between Taiwan and other countries becomes more and more frequent since the participation of the World Trade Organization(WTO). However, large quantity of imported fishery products may raise the risk for distribution of highly transmissible aquatic animal disease pathogens into Taiwan. Therefore, more strict inspection should be carried out to avoid the transmission of aquatic pathogens to Taiwan. In this project, we have applied the inspection techniques established in the past few years of COA projects to examine the collected samples from importers. Also, we have established the Standard Operating Procedures (SOP) to standardize the procedures from "sampling" to "laboratory inspections" for examining the aquatic animal diseases and set up a database for future analysis. The inspection results demonstrated that among more than 100 samples, all samples were negative for infectious hematopoietic necorsis virus (IHNV), viral hemorrhagic stpticaemia virus (VHSV), and bacterial kidney disease (BKD). While the inspection was still on going, we have also tried to construct a positive control for another OIE listed disease, infectious pancreatic necrosis virus (IPNV). In this project, by using long primer synthesis technique, a positive control for IPNV gene will be cloned and incorporated into multiplex RT-PCR system to increase the diversity of the inspection. With the approaches described above, we hope we can provide some information about current situation of OIE listed aquatic animal disease pathogens in Taiwan.[張文發老師]The import/export of aquatic animals is a significant trade sector in Taiwan. It is therefore important to prevent the accidental import/export of any aquatic disease causing agents that might threaten this industry. We propose to strengthen and integrate technologies to better support aquaculture quarantine policy on the import/export of live crustaceans. To improve flexibility in the event of new or emerging diseases

Jejunojejunal intussusception and colonic impaction in a 12-day-old orphan foal

A 12-day-old 14.4-kg, female foal of Australian Miniature Pony breed was presented at the National Chung Hsing University Veterinary Medicine Teaching Hospital. Taiwan, with a history of weakness of 2 days duration. The mare died of unknown cause on the previous day. Abnormal findings during physical examination included an estimated dehydration of 3 to 5%, dark red oral mucous membranse, elevated heart, and respiratory rates, stiffened feces, and absence of intestinal motility on ausculation. Continual nursing care was followed by intravenous fluid therapy and nonsteroidal anti-inflammatory drug and antibiotic treatment. The foal's condition deteriorated rapidly, and feeding was refused. Subsequently, she developed signs of pyrexia and panting. In spite of oxygen supplement, she died 18 hours after admission. Postmortem examination revealed the presence of a jejunojejunal intussusception, 20 cm in length, a torsion of jejunum proximal to the intussusception also was noted. An ulcerative lesion, 3 cm in diameter, was found on the mucosa of duodenum. Large amounts of sand, coat hair, and hay mantling in bloody discharge were observed in a colonic impaction. That it was an acute case was evident by the involvement of intussusception and ulceration, indicating desquamation of the necrotic epithelial cells with edema and distinct infiltration of neutrophils. These results suggested that a practical management strategy for nursing an orphan foal should be performed to avoid a similar case in the future

Suppurative meningitis in a 7-day-old Formosan sambar deer (Cervus unicolor swinhoei) caused by Escherichia coli

This article describes the clinical and pathological features of an orphan 7-day-old, male Formosan sambar fawn that was hospitalized for treatment of weakness. The fawn had been deprived of colostrum and developed suppurative meningitis that was attributed to Escherichia coli