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

豬假性狂犬病gE基因缺損與萎縮性鼻炎重組次單位PMT毒素雙價疫苗之研發及免疫效益評估

Pseudorabies (PR) is a highly contagious and fatal viral disease in swine. The clinical symptoms and mortality depend on the age of affected swine. However, PR infection is more often to become imperceptible in growing to finishing pigs but will induce high mortality in suckling and weaning piglets when antibody titer is impairment. For controlling this disease, vaccination of the pregnant sows can mount good protective maternal antibody to prevent suckling and weaning piglets from PR infection. In addition, Progressive atrophic rhinitis (PAR) is an important respiratory disease and causes the snout deformation, turbinate atrophy, septum deviation, and growth retardation in swine. PAR can affect all ages of swine but piglets are most susceptible. Both diseases and the following complication of porcine respiratory disease complex (PRDC) will induce tremendously economic loss on production in a pig farm. Multivalent vaccine is a good strategy for immunization and can be applied to reduce the stress and the cost of management during vaccination. The purpose of this project is to develop the bivalent of gE(-) PR/PAR of recombinant subunit PMT vaccine, and test its efficacy and safety in mice, rabbits, swine and field trials in order to control PR and PAR for decreasing the diseases outbreak in pig farms.豬假性狂犬病(Pseudorabies; PR)為高傳染性及高致死性的疾病,特別若爆發於哺乳仔豬及離乳幼豬之時期,感染後的死亡率甚至可高達100%.此外,豬進行性萎縮性鼻炎(progressive atrophic rhinitis; PAR)為豬隻重要呼吸道疾病,感染豬隻之鼻黏膜會急速壞死且造成鼻甲骨萎縮,導致呼吸系統第一道防禦機制嚴重受損,因而其他病原十分容易長驅直入,為豬隻罹患呼吸道綜合感染症(procine respiratory disease complex; PRDC)之主因.本計畫為預防此二種疾病發生,除可減少因PR或AR本身所造成之直接經濟損失外,亦可降低所繼發各種PRDC的發生,並大幅降低豬隻因呼吸道疾病死亡的損失及因治療或預防添加藥物所需的成本.豬假性狂犬病與萎縮性鼻炎具有相同的免疫適期,均需藉由免疫懷孕母豬產生的移行抗體以保護仔豬免於早期之感染.本計畫的主要目的即先以小白鼠進行免疫攻毒之安全性試驗及田間母豬隻免疫試驗,進行PR與PAR雙價疫苗免疫效力的評估.而最終則是開發新一代的gE基因缺損PR與PAR重組次單位PMT毒素雙價疫苗,並藉由本實驗能成功移轉高安全、高效率及成本低廉並具競爭之PR與PAR雙價疫苗,以減少養豬業之經濟損失

Establishment of a Recombinant Subunit Pasteurella Multocida Toxin Based Multivalent Vaccine Platform

豬萎縮性鼻炎(AR)為豬隻之重要疾病，其主要致病因子為巴斯德桿菌所產生的PMT毒素，可導致豬隻鼻甲介骨萎縮，並波及骨骼發育使得豬隻有生長遲緩情形。本計劃為新提計畫，其目的是要建立一重組巴氏桿菌毒素多價疫苗平台，來簡化先前所研究上市之AR次單位疫苗抗原成份，使得該疫苗平台可以納入其他抗原來做為多價疫苗。本計劃將採用超音波、French pressure或噴射等擊碎方式，來將細菌的細胞壁打破，以得到半純化之重組PMT抗原，而後將利用半純化之抗原進行各種組合，混和適當佐劑後，於小鼠及豬隻進行安全性及效力試驗，來了解AR次單位疫苗最基本而有效之抗原成份，期望在未來建立該次單位疫苗之平台後，將有助於其他有效抗原成分之納入，來發展可保護豬隻對抗多重疾病之多價疫苗。Pasteurella multocida is characterized as the primary causative agent of progressive atrophic rhinitis (PAR) in swine. The P. multocida toxin (PMT) has been identified as the major virulent factor to induce nasal turbinate atrophy and growth retardation of the piglets. Vaccination is known to be an effective way to prevent PAR in pigs. In our previous study, it was revealed that a vaccine composed of several recombinant subunit PMTs (rsPMT) and bacterins induced protective efficacy in swine. To facilitate future development of multivalent vaccine in control of multiple diseases in swine, the aim of this project is to establish an rsPMT-based multivalent vaccine platform. By disrupting the bacterial cell wall via sonication, French pressure cell press or jet-spray, the semi-purified rsPMT will be collected, characterized and formulated into different combinations. Thereafter, these prototype vaccines will be subjected to safety and efficacy tests in mice and pigs to determine the minimal effective composition of the rsPMT-PAR vaccine. By this way, it may serve as the base for incorporation of other effective immunogen(s) in order to develop an effective multivalent vaccine for swine in the future

The Development of Recombinant Subunit Vaccine against Swine Progressive Atrophic Rhinitis

豬萎縮性鼻炎為感染豬隻之重要細菌性疾病,而PAR之主要病原曾引起諸多的爭議,但目前已被證實PAR的主要致病因子為P. multocida toxin (PMT),且只有產毒能力之D型與A型P. multocida才能導致臨床PAR的產生.而D型P. multocida產生的PMT毒素,除能誘導鼻甲介骨萎縮的病變外,並且會波及全身性骨骼發育而使生長遲緩.但由於D型P. multocida的細胞毒素萃取過程繁複、耗時且成本相當高,而所萃取之細胞毒素又有不活化完全時其安全性之慮.本實驗室曾對PAR作一系列之研究,同時為得到高產量與安全性之PMT毒素,因此以基因工程技術研發次單位PMT毒素疫苗,並已證實所研發之基因工程疫苗單獨或經混合不活化A型P. multocida與B. bronchiseptica,對豬隻皆具有良好的保護能力,亦可測得具保護性之中和抗體力價.本病於台灣豬場之感染率高達75%,而目前國外進口之商品化PAR疫苗因生產PMT類毒素步驟繁瑣,且病原在自然培養狀況下毒素產量又極低,故PMT類毒素疫苗價格十分高昂,若疫苗中只添加不活化之菌體以取代類毒素時,其免疫效力又極端不足,導致農民使用PAR疫苗意願低落.本實驗計畫的目的即應用所研發完成之PAR重組PMT次單位疫苗,進行生產過程之監控與重組蛋白特性之評估,也同時針對生產的抗原進行質與量的監測,並評估新一代PAR重組次單位PMT疫苗,於田間豬場在母豬與仔豬的免疫保護效力與抗體消長之情形,期望藉由本實驗能成功移轉高效能且具國際經濟競爭性的重組次單位PAR疫苗,以貢獻國內養豬業者.The toxin-producing strains of Pasteurella multocida (both type D and A) are characterized to be the major causative agent of progressive atrophic rhinitis (PAR) in swine. The P. multocida toxin (PMT) with a calculated molecular weight of 146 kDa has also been identified to cause the atrophy of nasal turbinate and skeletal bones. PAR can affect all ages of swine but piglets are most susceptible. The economic lose due to PAR or following the complication of porcine respiratory disease complex (PRDC) will become tremendously in swine production. Vaccine is a good strategy for immunization and can be applied to reduce the cost and lost of management. The purpose of this project is to provide the tested recombinant subunit PMT protein for immunogenic control and for vaccine trial in field in order to prevent the economic loss in pig farm