腸炎沙門氏菌感染雞隻細胞之生理反應

Abstract

腸炎沙門氏菌感染症為重要的公共衛生議題之一，受感染的雞隻為重要的宿主，並且透過禽類相關食品傳染至人類。雖然腸炎沙門氏菌不易造成雞隻全身性的感染症狀，但是腸炎沙門氏菌毒力株可以在雞隻腸道增殖，入侵到卵巢顆粒細胞以及造成肝臟持續性的感染，進而引起產蛋率下降。而雞隻的產蛋機制受到許多器官共同調控，因此本研究設計一連串相關的試驗，藉此找出蛋雞感染腸炎沙門氏菌後造成產蛋率下降的可能機制。首先雞卵巢顆粒細胞為腸炎沙門氏菌入侵卵巢時所偏好的目標細胞，而該細胞參與調控濾泡生長、濾泡閉鎖以及排卵。此研究分析從不同發育程度濾泡所分離出的顆粒細胞在感染腸炎沙門氏菌毒力株(ATCC17025)後，對於類固醇生成及相關基因調控的影響。另外，於排序濾泡及排序前濾泡所分離出的顆粒細胞分別添加黃體生成激素(luteinizing hormone，LH)及促濾泡激素(follicle-stimulating hormone，FSH)，以模擬雞隻體內賀爾蒙調控。從實驗結果發現，LH receptor、FSH receptor、steroidogenic acute regulatory proteins、P450 side-chain cleavage以及3beta-Hydroxysteroid dehydrogenase的mRNA表現量下降。顆粒細胞的黃體激素產量也因為腸炎沙門氏菌感染而顯著地減少。推知顆粒細胞對於LH及FSH的刺激反應以及其類固醇生成途徑可能會被腸炎沙門氏菌感染而抑制。從toll-like receptor 15 (TLR15)以及B-cell lymphoma 2 (Bcl-2)的mRNA表現量推知，當受到腸炎沙門氏菌感染時，F1濾泡傾向於提高免疫反應以去除病原，然而其餘發育程度較低的濾泡則選擇以細胞凋零及濾泡閉鎖來避免病原於卵巢感染，進而造成垂直感染。因此，我們推測腸炎沙門氏菌感染能夠減緩類固醇生成以及抑制濾泡生長進而可能會延長排卵間距，造成產蛋率下降。 另一方面，肝臟是調控脂質代謝的主要器官，負責生成和組裝卵黃所需要的脂質。因此同樣使用腸炎沙門氏菌感染LMH-2A肝臟細胞株，並且添加β-estradiol模擬肝臟受到腸炎沙門氏菌感染時，對於肝臟脂質代謝以及卵黃脂蛋白合成的影響。從實驗結果發現LMH-2A肝臟細胞株內的大部分與脂質生成及脂質運送相關基因的mRNA表現量，包括sterol regulatory element binding protein 1、acetyl-CoA carboxylase、fatty-acid synthase、long-chain-fatty-acid-CoA ligase 1、peroxisome proliferator-activated receptor-γ、and very-low-density lipoproteins (VLDL) II，都顯著地上升。另外estrogen receptor alpha 的mRNA表現量下降則顯示卵黃脂質的形成可能會受到腸炎沙門氏菌感染的抑制。隨著腸炎沙門氏菌的感染時程，胞內ApoB蛋白堆積增加，胞外VLDL分泌量減少而LMH-2A內脂質堆積逐漸增加，都顯示LMH-2A運送出的脂質也會被腸炎沙門氏菌感染所抑制。因此我們推測腸炎沙門氏菌感染肝臟可能會增加脂質合成並且降低脂質運輸作用。這些都可能進一步造成肝臟過量的脂質堆積，並且減緩濾泡的卵黃堆積，進而降低濾泡生長速度。 從以上研究結果得知，腸炎沙門氏菌感染造成雞隻產蛋率下降可能與降低濾泡類固醇生成作用以及改變肝臟脂質代謝與堆積有關。

Salmonella Enteritidis (SE) is one of important public health concerns and infected chickens serve as reservoir which potentially transmits to humans through food. Although SE seldom causes systemic infection in chickens, virulent SE strains can colonize in intestines, invade ovarian follicular cells, and lead a persistent infection of the liver. Infection by SE causes decreased egg production in laying hens clinically. The egg production in laying hens are orchestrated by several organs; therefore, serial experiments were performed to elucidate the possible mechanisms of the decreased egg production in laying hens infected with SE. First, the chicken granulosa cells (cGCs) of ovary is the preferred site for SE infection and it is involved in follicular growth, atresia, and ovulation. The alteration of target genes and steroidogenesis in cGCs at distinct stages of follicular maturity were studied. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were applied to the cGCs isolated from hierarchical and prehierarchical follicles, respectively, to imitate the effects of gonadotropin during in vivo SE infection. Attenuated responses to LH and FSH as well as retardant progesterone production due to down-regulated the mRNA expressions of LH receptor, FSH receptor, steroidogenic acute regulatory proteins, P450 side-chain cleavage, and 3beta-Hydroxysteroid dehydrogenase were observed. The progesterone production in cGCs was also significantly reduced during the infection. Moreover, there was a tendency toward pathogen elimination in F1 follicles by induction of a strong immune response and cell apoptosis in smaller follicles to avoid bacterial transovarian infection. It is our speculation that slowed steroidogenesis and impeded follicular growth may result in an increased ovulation interval, and further decreased egg production during SE infection. Secondly, the liver is the primary organ for lipid metabolism in chickens and the site for production and assembly of main components in yolk. We proceeded with a time-course experiment using LMH-2A cells that were infected with SE and co-incubated with β-estradiol to evaluate if SE infection affected lipid metabolism and subsequently changed lipoprotein formation of egg yolk. The results indicated that lipid accumulation significantly increased in infected LMH-2A cells, and the mRNA expressions of lipid transportation and most lipogenetic genes including sterol regulatory element binding protein 1, acetyl-CoA carboxylase, fatty-acid synthase, long-chain-fatty-acid-CoA ligase 1, peroxisome proliferator-activated receptor-γ, and very-low-density lipoproteins (VLDL) II were significantly up-regulated. Moreover, declined lipid transportation of hepatocytes was evidenced by the down-regulation of estrogen receptor alpha which promotes VLDLy formation, increased intra-cellular accumulation of ApoB protein, and decreased cellular excretion of VLDL protein. SE infection probably elevated lipid synthesis and reduce lipid transportation in the chicken liver. These changes of cGCs and LMH-2A may lead excessive lipid accumulation in liver and slower lipoprotein deposition in yolk. Collectively, our study explained that reduced egg production in SE infected laying hens may be associated with the decreased steroidogenesis and the progressive changes in chicken liver lipid metabolism.