畜牧兽医学报  2019, Vol. 50 Issue (6): 1123-1134. DOI: 10.11843/j.issn.0366-6964.2019.06.002    PDF    
鸡坏死性肠炎生物性防控研究进展
阚刘刚, 刘艳, 吴媛媛, 范文晓, 王忠     
中国农业大学动物科技学院动物营养学国家重点实验室, 北京 100193
摘要:鸡坏死性肠炎(necrotic enteritis,NE)是现代化肉鸡集约养殖生产中最为突出的肠道疾病之一,给家禽产业带来了巨大的经济损失。随着世界范围内的立法限制、禁用抗生素生长促进剂,鸡坏死性肠炎的发病率在全球范围内呈上升趋势,这对家禽的生产和健康产生严重威胁。因此,迫切需要新的技术和新的产品代替抗生素来预防和控制鸡坏死性肠炎。作者着眼于NE的危害和疫苗、噬菌体、卵黄抗体、植物提取物、酸化剂、益生菌、低聚糖与微生物多糖等生物性防控进展,综述了目前有效预防和控制鸡坏死性肠炎的生物性措施的最新研究情况,既为鸡坏死性肠炎生物性防控提供技术参考,也为其深入研究提供了理论思考。
关键词    坏死性肠炎    生物防控    
Advances in Biological Prevention and Control of Avian Necrotic Enteritis
KAN Liugang, LIU Yan, WU Yuanyuan, FAN Wenxiao, WANG Zhong     
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Abstract: Avian necrotic enteritis (NE) is one of the most prominent intestinal diseases in broiler modern intensive production, which has brought huge economic losses to the poultry industry. With the worldwide restrictions and the bans on antibiotic growth promoters, the incidence of chicken necrotic enteritis is on the rise globally, which poses a serious threat to poultry production and health. Therefore, there is an urgent need for new technologies and products to replace antibiotics to prevent and control avian necrotic enteritis. This article reviews the advances in biological measurements for the prevention and control of chicken necrotic enteritis. It provides a technical reference for the biological prevention and control of chicken necrotic enteritis, and provides theoretical considerations for its in-depth research.
Key words: chicken     necrotic enteritis     biological prevention and control    

鸡坏死性肠炎(necrotic enteritis, NE)是一种发病机制复杂的肠道疾病,主要由产气荚膜梭菌感染引起,除此之外还包括一系列的诱发因素。据估计仅在美国,每年因鸡坏死性肠炎造成的损失就超过60亿美元[1]。除此之外,产气荚膜梭菌还能随着食物链进入人体内,威胁公众健康[2],因此及时防治鸡NE十分重要。

1 鸡坏死性肠炎的危害

产气荚膜梭菌是NE的主要致病因子,它是一种严格厌氧的革兰阳性菌,可形成孢子,它属于鸡肠道内的常在菌(浓度约为每克食糜102~105CFU),正常情况下不会诱导NE发生,是一种典型的条件致病菌。而当存在鸡舍饲养环境卫生条件差、垫料潮湿,鸡群感染艾美耳球虫、免疫抑制性疾病(如马立克病、传染性法氏囊病、鸡网状内皮细胞增生症、禽白血病等),或鸡群饲养密度过大(30只·m-2),鸡舍温度过高(热应激),或鸡群饲喂高动物蛋白日粮,或高非淀粉多糖(NSP)的饲料原料(如小麦、大麦等),或饲喂霉菌毒素污染的饲料等诱发因素的情况时,均可破坏肠道正常的微生物区系平衡、促进肠道产气荚膜梭菌大量繁殖且分泌毒素、诱发肠道出现炎症反应,进而导致肠道内环境稳态及黏膜屏障完整性被破坏,最终形成坏死性肠炎[3-6]。已知产气荚膜梭菌可产生的毒素有16种(α、β、γ、ε、ι、θ等),近年来又发现了一些新毒素(NetB、TpeL)。按原来的分类方法(根据分泌毒素不同进行分类)可将产气荚膜梭菌分为A、B、C、D、E 5种血清型[7]。事实上,并非所有的产气荚膜梭菌菌株都能够诱导坏死性肠炎。Parish[8]首先报道了A型产气荚膜梭菌可引起鸡坏死性肠炎,随后的研究发现,NE主要由A型产气荚膜梭菌引起,而少数可由C型菌株诱发[9],这表明具有特异性毒力因子的产气荚膜梭菌能对家禽致病。一直以来,人们认为α毒素是产气荚膜梭菌的主要毒力因子,可水解磷脂酰胆碱和鞘磷脂,破坏细胞膜结构[10]。但新的研究表明单纯的NetB毒素也可诱发NE[11]。此外,产气荚膜梭菌分泌的细菌素、胶原蛋白酶及其黏附力等也在NE发生过程中发挥重要作用[12-14]

研究发现鸡NE通常发生于仔鸡出生后2~4周[15],根据发病症状可分为急性临床坏死性肠炎和亚临床坏死性肠炎,且这两种情况下,家禽肠黏膜结构都会受到破坏,严重影响动物生产性能[16-17]。在急性临床病例中,鸡群通常毫无预兆地急性发病,发病1~2 h内开始出现死亡,排红褐色乃至黑褐色煤焦油样稀便,死亡率通常为2%~10%,也可高达50%[18];而亚临床型坏死性肠炎肉鸡并不表现出明显的临床症状,通常不会出现死亡或死亡率很低,但肠道黏膜的慢性损伤及炎症反应会导致营养物质消化吸收不良,表现为采食量下降,肉鸡增重和饲料转化效率降低[19]。由于亚临床坏死性肠炎鸡群通常没有任何临床疾病迹象,难以监测发现,因而鸡群不能及时得到治疗,虽不会出现高的死亡率,但其造成的经济损失更为严重。

鸡坏死性肠炎病灶可见于十二指肠、空肠和回肠,有时盲肠也会出现病变,通常表现为肠壁薄、易碎、充满气体,在发生严重感染时,肝、肾等器官也发现病灶[2]。对坏死性肠炎鸡的病理观察表明,轻微病变时肠道黏膜表面覆盖一层松散粘连的纤维蛋白,病变加重时肠道出现坏死、溃疡并伴随出血点,坏死点周围的纤维蛋白结痂,病变最严重时肠黏膜出现弥漫性坏死,坏死组织和炎症细胞碎片紧密黏附于坏死区域,形成典型的坏死性肠炎。观察肠道病变组织切片发现,肠绒毛融合变短,肠腔中出现细胞碎片,且肠腔、肠黏膜和固有层中存在大量杆状细菌,肠固有层存在强烈的炎症反应,固有层充血,伴随大量异嗜白细胞和淋巴细胞浸润[10, 20-22]。了解机体发生疾病时的这些内在变化将有利于我们发展对坏死性肠炎的整体认知,对筛选出针对该疾病的干预措施起着十分重要的作用。

2 NE生物性防控研究进展

尽管药物性饲料添加剂(如杆菌肽锌、乙酰水杨酸杆菌肽、恩拉霉素、维吉尼亚霉素、阿维霉素、黄霉素、泰乐菌素、金霉素等)用于控制NE发挥着较大作用,但是抗生素耐药性和食品动物抗生素残留等问题频发,促使越来越多的研究人员开始专注于探索预防动物疾病的替代策略。这些预防性措施可直接靶向病原菌,控制病原菌的定植、增殖或干扰病原菌毒力因子和致病因子的表达;或者提高动物的免疫力,增强动物对病原菌或疾病的抵抗力。笔者将重点介绍疫苗、噬菌体、特异性抗体、植物提取物、有机酸、益生菌、低聚糖与微生物多糖等生物学策略对产气荚膜梭菌诱导的鸡坏死性肠炎的预防和控制效果的最新研究进展。

2.1 疫苗

针对鸡NE的疫苗主要有产气荚膜梭菌活菌疫苗、灭活疫苗、类毒素疫苗和重组蛋白疫苗等。Mishra和Smyth[23]研究了非毒性NetB阳性产气荚膜梭菌作为口服活疫苗在肉鸡上预防NE的效果,以霍乱毒素为佐剂,在肉鸡2和12日龄进行疫苗接种,在23日龄进行毒性产气荚膜梭菌攻毒,分别在21和28日龄进行采样检测。21日龄结果显示非毒性NetB阳性产气荚膜梭菌接种可提高肉鸡血清IgY水平和黏膜IgA水平,而在产气荚膜梭菌攻毒后(28日龄结果),非毒性NetB阳性产气荚膜梭菌接种肉鸡血清IgY水平和黏膜IgA水平也显著高于攻毒未接种肉鸡组,且肠道病变记分也显著降低,但肉鸡体重无显著差异。该结果表明非毒性NetB阳性产气荚膜梭菌疫苗可提高机体免疫力,保护肉鸡抵抗产气荚膜梭菌感染。也有学者研究母体免疫对其后代疾病抵抗力的影响。Crouch等[24]给肉种鸡肌内注射A型产气荚膜梭菌α-类毒素疫苗,研究其后代仔鸡的NE抵抗力。疫苗接种后种鸡体内α毒素抗体滴度显著增加,并可随着种蛋转移到它们的后代,降低了后代仔鸡的NE病变及死亡率,该结果表明α毒素可作为肉鸡预防NE的关键性保护免疫原。Keyburn等[25]给肉种鸡皮下注射重组NetB毒素,发现肉种鸡血清中NetB特异性IgY抗体水平升高,同样种蛋和后代仔鸡中NetB特异性IgY抗体水平也升高;之后对后代仔鸡进行产气荚膜梭菌攻毒试验,发现来自免疫接种母鸡的后代NE病变显著低于对照组。此外,Jiang等[26]用重组弱毒沙门菌疫苗(表达α毒素C-端片段和NetB毒素)接种肉鸡后产生了良好的免疫保护效果,作用效果优于只表达一种抗原的重组沙门菌疫苗(表达α毒素C-端片段或NetB毒素)。而da Costa等[27]也发现α毒素抗原和NetB毒素抗原联合使用效果更显著,他们的试验结果表明α毒素C-端片段(CPA 247-370)和NetB的无毒变体(NetB W262A)联合接种肉鸡后,肉鸡对NE的抵抗力高于毒素单独接种的效果。这不仅说明α毒素和NetB毒素在免疫接种时具有协同作用,它们在产气荚膜梭菌感染过程中可能也存在协同诱导NE的作用。杜吉革等[28]的研究表明,产气荚膜梭菌重组ε毒素突变体无毒力且保留了良好的免疫原性,为D型产气荚膜梭菌病新型疫苗的研制提供了重要的依据。

产气荚膜梭菌的蛋白质也可作为疫苗的候选物,包括丙酮酸:铁氧还蛋白氧化还原酶、延伸因子-Tu、果糖-二磷酸-醛缩酶和假想蛋白等[29-30]。肉鸡皮下注射重组蛋白丙酮酸:铁氧还蛋白氧化还原酶或延伸因子-Tu,可降低NE肉鸡肠道病变记分和空肠IL-8的mRNA表达,丙酮酸:铁氧还蛋白氧化还原酶还可增加NE肉鸡的体增重[29]。此外,Tsiouris等[22]发现抗球虫的弱毒疫苗(Paracox-5)可降低NE肉鸡肠道病变记分,对NE感染有保护作用。

2.2 噬菌体

噬菌体是细菌的天然病毒,包括温和性噬菌体和裂解性噬菌体,其中裂解性噬菌体会破坏宿主细菌的DNA,在宿主菌内进行复制,然后溶解宿主细菌并释放大量子噬菌体,达到抗菌的效果。Miller等[31]研究了从环境中分离的对产气荚膜梭菌具有破坏作用的多价噬菌体“鸡尾酒”INT-401,发现不同给药方式(灌胃、饮水和饲料,每只鸡2.5×109 PFU·d-1)处理5 d都可显著降低NE肉鸡死亡率,并提高肉鸡体重和饲料转化效率。另外,Simmons等[32]克隆了梭菌属噬菌体phiCP39O和phiCP26F的裂解酶,在大肠杆菌内重组表达,斑点和浊度分析发现其对产气荚膜梭菌具有裂解作用。

2.3 卵黄抗体

蛋鸡经疫苗免疫后,免疫系统受抗原刺激产生免疫球蛋白。在鸡蛋形成过程中特异性免疫球蛋白可在鸡蛋中沉积,形成卵黄抗体(immunoglobulin of yolk,IgY)。IgY在控制动物肠道疾病的研究中发挥了良好的效果[33-34]。但其在鸡坏死性肠炎模型中的作用效果研究较少。我们知道产气荚膜梭菌的毒素种类繁多,准确定位其关键抗原的特异性抗体可能是该研究的难点之一,需要更多的研究工作来挖掘IgY在鸡坏死性肠炎疾病中的应用潜力。

2.4 植物提取物

众所周知,植物广泛地应用于传统医学[35]。此外,植物提取物(也称为植物源抗生素)具有抗菌、抗炎、抗氧化和抗寄生虫活性[36-38],探究其在动物营养方面的作用也已成为一个热门的研究方向。植物提取物的生物活性成分主要为次级代谢产物,如萜类化合物、酚类、糖苷类和生物碱,以醇类、醛类、酮类、酯类、醚类和内酯类的形式存在[39]。近年来的研究报告指出使用植物提取物或衍生的植物源化合物作为家禽饲料添加剂可能对其健康和生产性能产生积极影响[37, 40]。植物的抗菌化学物质大致可分为几种,如单宁(多酚)、精油、油性树脂和生物碱等[41]。科学研究表明,单宁对家禽的营养生理和动物健康都具有显著的作用[42]。Tosi等[43]发现添加栗子单宁提取物显著降低了球虫+产气荚膜梭菌攻毒模型肉鸡的肠道病变及肠道产气荚膜梭菌数量。单宁的抗菌活性可能与它们的生化特性有关,包括对酶的络合作用和“铁剥夺”,进而抑制病原菌的代谢[44-45]。最近的一项研究报道栗子提取物改善了乳酸菌过胃的耐受性以及对低pH和胆汁盐的耐受性,表明单宁酸还可与益生菌联合使用,增强肠道健康[46]。精油(essential oil,EOs)是植物中不直接参与植物正常生长、发育或繁殖的次级代谢产物[47],认为它们参与植物防御,其中大多数可能具有抗菌特性[48]。EOs的抗菌活性与其疏水性等化学特性有关,疏水性使其能够与细菌细胞膜脂质相互作用,从而干扰细菌的代谢、细胞壁和细胞膜的通透性、导致细菌细胞的关键分子和离子大量泄漏。已有研究探讨了EOs对NE肉鸡的控制效果[49-50]。Yin等[51]添加120 mg·kg-1混合精油(含25%百里香酚、25%香芹酚)显著降低了NE肉鸡死亡率及肠道病变,降低了致病菌毒力因子(VF0073-ClpE、VF0124-LPS、VF0350-BSH)的表达,并增加了NE肉鸡回肠卷曲乳杆菌、Lactobacillus agilis的相对丰度,而降低了唾液乳杆菌及约氏乳杆菌的相对丰度。这些研究表明EOs对产气荚膜梭菌诱导的NE的影响可能与它对病原菌的直接抑菌作用和调节肠道菌群的间接作用相关。Sun等[52]也发现补充EOs(有效物质百里香酚和香芹酚)增加了产气荚膜梭菌感染肉鸡的肠道绒毛高度(villus height, VH)和VH与隐窝深度(crypt depth, CD)的比值,并降低血浆内毒素水平,降低肠道细胞通透性和sIgA分泌量。有研究总结了EOs可能的抗菌作用模式,主要有细胞壁降解、细胞质膜损伤、膜蛋白损伤、细胞内容物渗漏、细胞质凝固和质子动力耗竭等[53]。另外,植物油性树脂和肉桂醛也对NE具有保护作用,Kim等[54]发现饲料添加8 mg·kg-1的辣椒和姜黄的油性树脂混合物可显著改善NE肉鸡的体增重及肠道病变,并增加了NE肉鸡回肠乳酸杆菌的相对丰度,而肉桂醛(600 mg·kg-1)能提高NE肉鸡抗氧化能力,下调空肠促凋亡基因的表达[55]

另外一些成分复杂、含有多种生物活性物质的植物源添加剂,也具有良好的动物营养作用。如厚朴树皮提取物(0.33或0.56 mg·kg-1)能增加NE感染肉鸡采食量、体增重及平均体重,并显著降低饲料转化率(feed conversion rate,FCR)和肠道病变记分,并提高空肠、肝、脾及胸肌的抗氧化基因的表达[过氧化氢酶(catalase,CAT)、血红素氧化酶1(heme oxygenase-1,HMOX1)及超氧化物歧化酶(superoxide dismutase,SOD)]及肝和脾黄曲霉毒素B1醛还原酶的表达[56]。而Lee等[57]发现,与NE对照相比,饲料添加1%或3%宽叶韭根粉(含有黄酮类和酚类物质)显著增加肉鸡体增重,降低肠道病变及粪便排出球虫卵囊数,降低了NE引起的空肠IL-8、IL-17及诱导型一氧化氮合酶基因表达的升高,增加空肠紧密连接分子JAM2、occludin及Mucin-2的表达。此外,Kang等[58]发现添加β-胡萝卜素氧化物(2、4、6 mg·kg-1)能显著增加NE感染肉鸡的平均体重(感染后7和14 d),并显著降低肠道病变及产气荚膜梭菌的定植量。上述研究结果表明了植物提取物(或植物源添加剂)对控制家禽NE的积极效果,但多数植物提取物的潜在作用机制尚不清楚,还需要大量研究进行探索与确定,这将有利于植物提取物的广泛应用。

2.5 酸化剂

脂肪酸的抗菌活性由来已久[59],其中,月桂酸对产气荚膜梭菌有较高的抗菌活性[60],它可直接抑制或杀灭肠道中的产气荚膜梭菌,从而降低家禽NE发病率。Timbermont等[61]的研究发现,与阳性对照组相比,增加日粮中月桂酸和丁酸的浓度(188、150 mg·kg-1)显著降低了NE肉鸡数量,增强了肉鸡对NE的抵抗力,只增加日粮中丁酸浓度时,发现丁酸以剂量依赖的方式增强肉鸡对NE的抵抗力。此外,Song等[62]的研究发现,NE肉鸡日粮中添加包被丁酸钠(800 mg·kg-1)可显著提高肉鸡的生产性能,上调空肠紧密连接蛋白claudin-1、claudin-4、ZO-1和occludin等的表达量,并降低盲肠中产气荚膜梭菌和大肠杆菌的数量,对肉鸡坏死性肠炎有较好的防治效果。Eshak等[63]也证明丁酸钠对NE肉鸡具有保护效果。而Jerzsele等[64]发现了与之矛盾的结果,他们的试验结果表明包被丁酸钠(1.5 g·kg-1)对NE肉鸡无益生效果。丁酸盐对产气荚膜梭菌无直接抑制作用,但它具有抗炎、增加宿主肠道黏液和抗菌肽分泌、维持肠绒毛结构等功能[65-68]。推测日粮中丁酸添加的浓度可能是其发挥效果的限制因素之一。因此,丁酸在控制肉鸡NE时出现的矛盾结果可能是其添加剂量不同造成的,此外,实验动物种类或者环境因素的不同也会对试验结果产生巨大影响。

2.6 益生菌

益生菌被定义为“改善肠道微生物平衡对宿主发挥有益影响的活性微生物添加剂”[69]。事实上,益生菌可直接与宿主相互作用以增强宿主免疫力、促进肠道发育、刺激代谢,从而降低机会性病原体感染的风险。益生菌也可以产生具有抗菌活性的分子,如细菌素、抗菌肽,可以靶向特定的病原体,抑制病原体的增殖或致病毒素的产生,甚至直接杀死病原体[70]。除此之外,一些益生菌还可改变肠道pH,与病原菌竞争黏附位点及营养素,从而抑制病原菌在宿主体内的定植。研究发现许多微生物分离株都对产气荚膜梭菌具有抗菌活性,它们大多属于芽胞杆菌属和乳酸杆菌属,此外一些丁酸梭菌和酵母菌也具有抗产气荚膜梭菌效果。

2.6.1 益生芽胞杆菌

多种芽胞杆菌菌株对产气荚膜梭菌具有抗菌活性。Barbosa等[71]的研究表明,源自鸡粪便的200多株芽胞杆菌中的枯草芽胞杆菌、地衣芽胞杆菌和短小芽胞杆菌等具有拮抗产气荚膜梭菌的活性,并发现细菌素和抗菌肽是其发挥抗菌作用主要活性物质[72-74]。最新的研究报道主要集中于枯草芽胞杆菌和地衣芽胞杆菌。Tactacan等[75]发现,日粮中添加枯草芽胞杆菌孢子(QST 713,1×106CFU·g-1)可显著提高NE肉鸡体重,降低饲料转化率,肠道病变记分和死亡率也显著降低。Jayaraman等[76]的试验结果表明,添加枯草芽胞杆菌PB6(500 g·t-1)可显著降低NE肉鸡的FCR及肠道病变记分,此外,肠道产气荚膜梭菌数量也显著降低,肠道VH及VH/CD显著上升,保证了绒毛结构完整。研究发现枯草芽胞杆菌和布拉酵母菌共同饲喂肉鸡后,肉鸡法氏囊和胸腺等免疫器官相对质量增加,空肠IgA阳性细胞数量显著增加,小肠中IL-6、TNF-α、转化生长因子-β(transformation growth factor -β,TGF-β)及sIgA的分泌量上升,此外,小肠杯状细胞数量和空肠Occludin、Claudin-2及Claudin-3的mRNA表达也显著升高,表明枯草芽胞杆菌具有刺激免疫,提高机体免疫水平,维持肠道屏障功能等效果[77]。肠道微生物菌群平衡是健康肠道必不可少的条件之一,肠道的微生物群形成了复杂的生态群落,通过其代谢物质、免疫调节功能以及与宿主间的相互作用影响宿主的正常生理活动和对疾病的易感性,肠道微生物区系与动物机体健康紧密相关[78]。研究发现NE感染严重破坏了肉鸡肠道的微生物菌群结构,而添加地衣芽胞杆菌H2可改善NE肉鸡肠道微生物区系的紊乱,使微生物菌群结构恢复到与健康肉鸡相似的水平[79-80]。虽然具体的作用机制仍不清楚,但推测该功能地衣芽胞杆菌是发挥其益生作用的重要因素。而一项关于地衣芽胞杆菌的研究发现,地衣芽胞杆菌H2(1 g·kg-1)可降低NE肉鸡血清葡萄糖含量,而提高高密度脂蛋白胆固醇的含量,并上调NE肉鸡肝中脂肪氧化分解相关基因的表达[81]。此外,Wu等[82]发现产乳酸和细菌素的凝结芽胞杆菌也对NE肉鸡生产性能具有改善作用,并显著降低了肠道病变记分和盲肠及肝中产气荚膜梭菌数量。

2.6.2 乳酸杆菌

乳酸杆菌也具有良好的抗菌活性,对控制肉鸡NE起着积极作用[83]。研究发现日粮中添加约氏乳杆菌B15(1×106CFU·g-1)增加了亚临床坏死性肠炎(SNE)肉鸡的采食量、体重及日增重,降低了其饲料转化率;提高了肠道胰岛素样生长因子-1和表皮生产因子的水平,促进损伤肠道恢复,减轻了回肠上皮细胞脱落、固有层充血等绒毛结构损伤;回肠固有层中IgA+ B细胞的数量升高,约氏乳杆菌BS15显著增加了血清及肠道中IgA、IgG和IFN-γ的水平及外周血和回肠固有层中CD3+CD4+T细胞百分率;提高了宿主抗氧化酶(SOD、CAT)的活性并降低了SNE肉鸡回肠凋亡基因(casepase3)的表达[84-85];约氏乳杆菌BS15还通过改变肝脂质代谢减少肝脂肪积累并改变鸡肉中脂肪酸组成[86-87]。此外,约氏乳杆菌BS15可调整肠道菌群,促进SNE肉鸡肠道的发育[88]。但遗憾的是约氏乳杆菌BS15仅有预防SNE的作用,而无治疗作用。乳酸杆菌的抗菌作用通常归因于细菌素的分泌或有机酸的产生。饲料补充嗜酸乳杆菌可降低产气荚膜梭菌感染肉鸡的肠道病变记分、促炎细胞因子mRNA表达和血清内毒素含量[89],而提高肠道中有益菌的相对丰度,并减少了肠道中病原菌的相对丰度[90]。Cao等[91]报道将发酵乳杆菌1.2029添加到雏鸡饲料中可刺激宿主免疫系统,提高细胞因子表达水平,降低产气荚膜梭菌引起的肠道病变和炎症发生率。然而,该研究并未检测肉鸡肠道中产气荚膜梭菌数量的变化。Layton等[92]测试了益生菌产品FloraMax-B11(由11种乳酸菌和灭活酵母组成)的功效,发现FloraMax-B11可降低巨型艾美耳球虫、鼠伤寒沙门菌和产气荚膜梭菌感染小鸡的肠道病变程度和产气荚膜梭菌数量降低。

2.6.3 其他菌株

益生肠球菌可产生细菌素,称为肠球菌素,其对革兰阳性菌和革兰阴性菌具有良好的抗菌活性[93]。肉仔鸡从孵化当天开始饲喂屎肠球菌,到28 d后发现肉鸡肠道产气荚膜梭菌及其他病原体数量减少,并且乳酸菌(乳酸杆菌和双歧杆菌)数量增加[94]。Klose等[95]发现多种肠球菌都对产气荚膜梭菌具有拮抗活性,这可能归因于酸和过氧化氢的产生。此外,Eeckhaut等[96]发现在健康肉鸡饲料中添加Butyricicoccus pullicaecorum(B.p)可降低饲料转化率;对肠道微生物菌群分析发现,B.p对肠道微生物α多样性无显著影响,但降低了肠道有害菌的相对丰度,当B.p添加到NE肉鸡日粮中时,显著降低了肠道病变发生率。此外,丁酸梭菌MIYAIRI 588也可降低肉鸡坏死性肠炎的发生率和严重程度[97]。这些研究表明一些丁酸产生菌也对NE肉鸡具有保护作用。

大量研究表明益生菌对宿主健康具有改善作用。但近年来,也有一些文章发现益生菌的作用并非想象的那么强大。益生菌发挥其益生作用的方式之一就是平衡肠道微生物菌群,但有文章报道益生菌对抗生素造成的肠道菌群失衡的恢复作用远远比不上移植自体粪便微生物组的效果[98],且益生菌对幼龄儿童的肠道菌群结构及多样性无显著影响[99]。此外,Zmora等[100]也发现益生菌对宿主黏膜菌群结构及肠道转录组的影响较为短暂,且具有个体差异性,提示益生菌并不是完全普遍适用的,并具有菌株特异性。因此,开发个性化益生菌产品将会是益生菌的研究发展方向之一。

2.7 低聚糖与微生物多糖

益生元是刺激宿主共生菌群并增强宿主内益生菌功效的一类添加剂,主要是难消化的低聚糖[101]。其中甘露寡糖(mannan oligosaccharides, MOS)是微生物来源的主要益生元,MOS是酵母细胞壁的组分,可与动物消化道壁上的各种受体[102]和致病菌膜上的受体相结合[103]。此外,甘露聚糖-蛋白质结合物可与动物免疫系统相互作用,增强机体免疫系统活性[104]。Yitbarek等[105]研究MOS对产气荚膜梭菌感染肉鸡的饲喂效果,发现饲料添加2 g·kg-1 MOS上调了TLR-2b、TLR-4、IL-12p35和IFN-γ的基因表达,激活了先天免疫,增强肉鸡的免疫功能,但对肉鸡的生产性能无显著改善作用,而Fowler等[106]的结果则表示MOS可以改善产气荚膜梭菌感染肉鸡的总体生产性能。酵母葡聚糖(glucans)属于多糖的一种,是酵母细胞壁三维结构的一部分,具有维持酵母细胞壁强度和吸收毒素的能力[107]。Glucans在动物营养中的主要作用是吸附霉菌毒素。Tian等[108]添加200 mg·kg-1酵母β-glucans到NE肉鸡饲料中,发现空肠中产气荚膜梭菌特异性抗体水平提高,VH及CD增加,肠道病变及产气荚膜梭菌定植量降低,但同样对NE肉鸡的生产性能无显著影响。另一项关于酵母细胞壁(富含甘露寡糖和β-glucans)的研究发现,酵母细胞壁显著增加了NE感染肉鸡日增重、降低了饲料转化率,但对肠道病变记分无显著影响,此外,肠道产气荚膜梭菌数量降低,而乳酸杆菌和双歧杆菌的数量增加[109]。Abudabos和Yehia[110]测试了一种商业酵母提取物添加剂Saf-Mannan(有效物质为甘露寡糖)保护肉鸡抵抗NE感染的能力。试验在小鸡孵化后开始饲喂0.05% Saf-Mannan,16日龄进行产气荚膜梭菌攻毒,并在小鸡30日龄时进行相关指标测定。结果显示Saf-Mannan添加组的肉鸡整体肠道状态良好,VH得到改善,肉鸡生产性能有所提高,此外,与阳性对照组相比,Saf-Mannan处理的肉鸡小肠中产气荚膜梭菌数量显著减少。

2.8 其他物质

除了上述研究的抗生素替代物之外,外源酶、某些微量元素和某些氨基酸等也具有缓解肉鸡NE造成的肠道损伤等作用。Liu等[111]在小麦日粮中添加木聚糖酶(5 500 U·kg-1),发现产气荚膜梭菌感染肉鸡的肠道损伤得到改善,空肠VH/CD比值增加,并可降低血清内毒素水平,上调回肠occludin的基因表达,且由产气荚膜梭菌感染导致的小肠细胞凋亡指数显著升高也得到了改善。饲料中小麦由于富含NSP是NE的诱发因素之一,而添加木聚糖后能降低消化液黏度,提高消化液通过率,改善营养物质的消化吸收,从而减少小肠内病原菌的数量,这可能是木聚糖酶改善小麦日粮NE肉鸡健康的机制之一[112-113]。此外,Liu等[114]发现饲料中添加溶菌酶(40 mg·kg-1)也可降低产气荚膜梭菌感染肉鸡肠道病变记分及回肠产气荚膜梭菌定植量。而Xu等[115]采用艾美耳球虫+产气荚膜梭菌感染建立肉鸡NE模型,评估有机硒添加量(0.25、0.5、1 mg·kg-1)对NE肉鸡的影响,发现添加量为0.5 mg·kg-1时,有机硒能显著增加NE肉鸡体重及NetB毒素抗体水平,并减轻肠道病变,此外,对肠道和脾组织细胞因子mRNA的表达具有上调作用。

Star等[116]在肉鸡NE疾病模型中将苏氨酸:赖氨酸的比例从0.63提高到0.67,发现肉鸡体增重高于对照组,但对NE发病率及肠道病变没有显著改善作用。Xue等[117]报道肉鸡饲料中添加10 g·kg-1谷氨酰胺(glutamine, Gln)能增加0~24日龄健康肉鸡采食量和体增重,降低饲料转化率,降低健康肉鸡肠道CD,提高VH:CD的比值,而对NE肉鸡生产性能无显著影响,但能降低肠道CD及空肠和回肠的病变记分。推断Gln对NE肉鸡肠道的保护作用可能与Gln的供能作用有关[118],在肠道发生损伤时,Gln分解供能促进肠细胞快速增殖更新,维持肠道功能。也有研究报道,肉鸡饲料中添加L-精氨酸(1.87%)可减轻NE肉鸡肠道损伤,提高肠道occludin及claudin-2的表达,上调空肠NOD1及IL-10的表达,提高空肠sIgA的分泌量,降低回肠及盲肠中产气荚膜梭菌的定植量[119]。L-精氨酸(0.3%)改善肠道NE肉鸡肠道损伤,缓解了产气荚膜梭菌感染造成的菌群失衡[120],这可能与肠道巨噬细胞利用精氨酸生成杀菌信号分子NO有关。

尽管某些营养素具有缓解NE感染的效果,然而从病原菌诱发肠道疾病的致病机制分析,NE感染导致肠道损伤、消化道酶活性和微生物区系的变化,影响了机体对饲料中大分子营养物质的消化和吸收,导致肠道细胞对营养素摄取的量不足,此时补充肠道上皮细胞可直接利用的单体氨基酸或其他营养素可改善肠道的营养饥饿状态或抗氧化状态,这可能是营养素改善肠道屏障结构和功能的原因。但营养素并无直接预防和治疗病原菌感染导致的肠道疾病,故应正确区分营养素和非营养性功能添加剂在细菌性肠道疾病预防中的作用。

3 总结与展望

随着抗生素耐药菌株的出现,抗生素的使用受到限制,多种细菌性疾病的发病率逐渐上升,鸡坏死性肠炎就是其中之一。科研工作者开始研究抗生素替代物以期降低鸡坏死性肠炎带来的危害。笔者综述了现阶段鸡坏死性肠炎的生物性防控手段及其作用效果,为鸡坏死性肠炎生物性预防提供了参考资料,但同时现阶段多数的研究都还停留在对鸡坏死性肠炎控制效果的表观研究上,缺乏对坏死性肠炎发病机制及新型干预手段作用机制的深层次研究。理解这些深层次机制问题是我们开发新型干预手段,控制坏死性肠炎的科学关键点。这方面的研究还有待深入。

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