Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 7: 4387 - 4401

DOI: https://doi.org/10.12418/CJAN2023.408

RESEARCH PAPER

Effects of Benzoic Acid on Growth Performance, Immune Function and Intestinal Health of Broilers Coinfected with Eimeria and Clostridium perfringens

  • FAN Zheng , 1 ,
  • LIU Yuanbin 1 ,
  • ZHANG Qiaochu 1 ,
  • SONG Yaru 1 ,
  • CHU Licui 2 ,
  • HOU Danxi 2 ,
  • ZUO Jianjun , 1, * ,
  • WANG Weiwei , 1, *
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  • 1 College of Animal Science, South China Agricultural University, Guangzhou 510000, China
  • 2 DSM (China) Co., Ltd., Shanghai 200137, China
* ZUO Jianjun, professor, E-mail: ;
WANG Weiwei, associate professor, E-mail:

Received date: 2023-01-16

  Online published: 2023-07-11

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Abstract

This experiment was conducted to investigate the effects of benzoic acid on the growth performance, immune function and intestinal health of broilers coinfected with Eimeria and Clostridium perfringens. A total of 240 one-day-old 817 female broilers were randomly divided into 3 groups with 8 replicates per group and 10 broilers per replicate. Broilers in control group (CON group) and model group (CP group) were fed a basal diet, and broilers in benzoic acid group (BA group) were fed the basal diet supplemented with 1 000 mg/kg benzoic acid. Broilers in the CP group and BA group were infected with Eimeria at 12 days of age and Clostridium perfringens at 16 to 19 days of age. The experiment period was 28 days. The results were as follows: 1) compared with the CON group, coinfection with Eimeria and Clostridium perfringens significantly decreased the average final body weight (FBW) and average daily gain (ADG) of broilers aged 1 to 21 days and 1 to 28 days, and significantly decreased the average daily feed intake (ADFI) of broilers aged 22 to 28 days and 1 to 28 days (P<0.05); significantly increased liver index and bursa of Fabricius index of broilers at 21 days of age (P<0.05); significantly increased the serum interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) contents of broilers at 21 days of age and serum interleukin-6 (IL-6) content at 28 days of age (P<0.05); significantly decreased the serum immunoglobulin A (IgA) content of broilers at 21 days of age (P<0.05); significantly decreased the ratio of villus height to crypt depth (VH/CD) of jejunum of broilers at 21 days of age and ileum of broilers at 28 days of age (P<0.05), and significantly increased the crypt depth (CD) of jejunum and ileum of broilers at 28 days of age (P<0.05); significantly up-regulated the mRNA relative expression levels of IL-1β in jejunum and interleukin-8 (IL-8) in ileum of broilers at 21 days of age (P<0.05); significantly down-regulated the mRNA relative expression levels of zonula occluden-1 (ZO-1) and mucin-2 (MUC-2) in jejunum of broilers at 21 days of age and the mRNA relative expression levels of ZO-1 in jejunum, Occludin and ZO-1 in ileum of broilers at 28 days of age (P<0.05). 2) Compared with the CP group, the supplementation of benzoic acid significantly alleviated the decrease of FBW, ADG and ADFI of broilers caused by Eimeria and Clostridium perfringens coinfection (P<0.05); significantly decreased the liver index and bursa of Fabricius index of infected broilers at 21 days of age (P<0.05); significantly decreased the serum TNF-α content of infected broilers at 21 days of age (P<0.05); significantly increased the serum immunoglobulin G (IgG) and immunoglobulin M (IgM) contents of infected broilers at 21 and 28 days of age (P<0.05); significantly increased the VH/CD of jejunum and ileum and villus height (VH) of ileum of infected broilers at 21 days of age and jejunum VH/CD of infected broilers at 28 days of age (P<0.05); significantly decreased the jejunum CD of infected broilers at 28 days of age (P<0.05); significantly decreased the mRNA relative expression levels of IL-8 and TNF-α in ileum of infected broilers at 21 days of age and the mRNA relative expression levels of IL-8 in ileum of infected broilers at 28 days of age (P<0.05); significantly increased the jejunal Occludin mRNA relative expression level of infected broilers at 28 days of age (P<0.05). In conclusion, dietary supplementation of 1 000 mg/kg benzoic acid can effectively relieve intestinal inflammation and intestinal barrier damage caused by Eimeria and Clostridium perfringens coinfection on broilers aged 1 to 28 days, enhance the immune function, improve the intestinal tissue morphology, maintain the intestinal health, and then improve the growth performance of broilers.

Key words： benzoic acid; Eimeria; Clostridium perfringens; broilers; growth performance; immune function; intestinal health

Cite this article

FAN Zheng , LIU Yuanbin , ZHANG Qiaochu , SONG Yaru , CHU Licui , HOU Danxi , ZUO Jianjun , WANG Weiwei . Effects of Benzoic Acid on Growth Performance, Immune Function and Intestinal Health of Broilers Coinfected with Eimeria and Clostridium perfringens[J]. Chinese Journal of Animal Nutrition, 2023 , 35(7) : 4387 -4401 . DOI: 10.12418/CJAN2023.408

饲料端“禁抗”后,畜禽疾病频发,其中禽坏死性肠炎(necrotic enteritis,NE)尤为突出[1]。NE又称肠毒血症,是一种主要由A型产气荚膜梭菌(Clostridium perfringens,CP)引起的肠道疾病,在生产中常与球虫病相伴或相继发生[2]。家禽感染NE后,食欲减退,肠道出血、溃疡、坏死,腹泻率提高,生长性能降低,急性NE感染还可导致鸡群突然大量死亡。NE给全球养殖业带来了巨大的经济损失[3],据统计,年经济损失高达20亿美元[4]。因此,为了遏制禽NE的病发率,兼顾抗生素杀菌抑菌和促生长两大特性,寻求绿色安全的饲用抗生素替代品迫在眉睫。研究表明,短链有机酸能够作用于畜禽小肠后端和大肠,增强肠道屏障功能和免疫功能,并在消化道后端创造弱酸性环境,抑制病源微生物的定植,调控肠道微生态平衡,提高养分消化率和生长性能,且无污染、无残留,应用前景广阔[5⇓-7]。苯甲酸作为最简单的芳香酸,具备短链有机酸的功能。目前苯甲酸在正常饲养肉鸡上的应用研究已经较多,但关于苯甲酸在预防球虫和产气荚膜梭菌联合感染肉鸡上的应用研究较少。因此,本研究旨在探究苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡生长性能、免疫功能及肠道健康相关指标的影响,为白羽肉鸡生产中缓解NE感染提供一种新的营养策略。

1 材料与方法

1.1 试验材料

试验所采用的苯甲酸(含量≥99.5%)由上海某公司提供。鸡球虫病三价活疫苗(堆型、巨型、柔嫩艾美尔球虫混合株)购自佛山某生物技术有限公司。产气荚膜梭菌(编号:ATCC 13124,主要产生α毒素)购自广东省微生物菌种保藏中心。

1.2 试验设计

试验选用体况正常、健康的1日龄817雌性白羽肉仔鸡240只,随机分为3组,分别为对照组(CON组)、模型组(CP组)和苯甲酸组(BA组),每组8个重复,每个重复10只,各组试验鸡初始体重差异不显著(P>0.05)。CON组和CP组肉仔鸡饲喂基础饲粮;BA组肉仔鸡饲喂基础饲粮+1 000 mg/kg苯甲酸。CP组和BA组肉仔鸡在12日龄时每只灌服0.5 mL混合型球虫卵液(含2×104个混合虫卵),再于16~19日龄期间每天灌服1 mL产气荚膜梭菌菌液(活菌数为1×109 CFU/mL);CON组肉鸡同期灌服等量无菌的生理盐水或庖肉培养基。试验期为28 d。

1.3 基础饲粮

试验采用玉米-豆粕型基础饲粮,基础饲粮根据我国《肉鸡饲养标准》(NY/T 33—2004)配制,其组成及营养水平见表1。
表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of basal diets (DM basis) %

项目
Items		 含量Content
1~21日龄1 to 21 days of age 22~28日龄22 to 28 days of age
原料Ingredients
玉米Corn 55.63 61.03
豆粕Soybean meal 37.63 31.88
豆油Soybean oil 2.37 3.13
石粉Limestone 1.27 1.21
食盐NaCl 0.35 0.35
磷酸氢钙CaHPO4 1.75 1.49
氯化胆碱Choline chloride 0.20 0.20
DL-蛋氨酸DL-Met 0.20 0.12
L-赖氨酸盐酸盐L-Lys·HCl 0.02 0.01
预混料Premix1) 0.58 0.58
合计Total 100.00 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.14 12.56
粗蛋白质CP 21.61 19.48
钙Ca 1.11 0.99
总磷TP 0.78 0.70
有效磷AP 0.38 0.33
赖氨酸Lys 1.16 1.01
蛋氨酸Met 0.52 0.42
蛋氨酸+半胱氨酸Met+Cys 0.88 0.75
色氨酸Trp 0.25 0.22
苏氨酸Thr 0.81 0.73

1)预混料为每千克饲粮提供The premix provided the following per kg of diets:VA 9 500 IU,VB1 2 mg,VB2 6 mg,VB6 4 mg,VB12 0.025 mg,VD3 62.5 μg,VE 30 IU,VK3 2.65 mg,生物素 biotin 0.032 5 mg,叶酸 folic acid 1.25 mg,D-泛酸 D-pantothenic acid 12 mg,烟酸 nicotinic acid 50 mg,Cu (as copper sulfate) 8 mg,Fe (as ferrous sulfate) 80 mg,Mn (as manganese sulfate) 100 mg,Zn (as zinc sulfate) 75 mg,I (as potassium iodide) 0.35 mg,Se (as sodium selenite) 0.15 mg。

2)营养水平均为计算值。Nutrient levels were calculated values.

1.4 饲养管理

饲养试验于广东省畜牧研究所家禽代谢室进行。各组肉鸡饲养环境和管理条件保持一致且符合817白羽肉鸡饲养要求,按照所在试验场地的常规管理规程和免疫程序进行饲养管理。肉鸡饲养期间,自由采食和饮水。饲养前3 d保持24 h光照,之后每天光照时间减少2 h,直至每天光照时间恒定为16 h。试验鸡除每周称重1次外,在12日龄和16~19日龄攻毒前禁食12 h,攻毒当天进行称重。于21和28日龄进行屠宰取样。

1.5 屠宰取样

分别于20和27日龄21:00,从每个重复中挑选1只体重接近该重复平均体重的肉鸡,每组8只,共计24只,禁食12 h后,颈静脉采血10 mL,静置30 min后,于3 000 r/min离心10 min,分离上层血清,于-30 ℃保存。将采血后的肉鸡进行解剖,分离器官及肠道,称重免疫器官,收集空肠、回肠组织置于2 mL冻存管中,液氮速冻。取十二指肠、空肠和回肠中段组织2 cm左右,在磷酸盐缓冲液(PBS)中清洗食糜后,置于4%多聚甲醛溶液中固定。

1.6 指标测定

1.6.1 生长性能

每周对试验鸡进行空腹称重,计算1~21日龄、22~28日龄肉鸡的平均末重(FBW)、平均日采食量(ADFI)、平均日增重(ADG)和料重比(F/G)。

1.6.2 免疫器官指数

屠宰前对每只待屠宰试验鸡进行称重,记录活体重,取样后去除肝脏、脾脏、胸腺和法氏囊周边脂肪,称重并记录,计算器官指数,计算公式为:
器官指数=器官重(g)/活体重(kg)。

1.6.3 血清免疫指标

采用酶联免疫吸附测定(ELISA)法测定21、28日龄肉鸡血清中免疫球蛋白A(IgA)、免疫球蛋白G(IgG)、免疫球蛋白M(IgM)含量(试剂盒购自上海酶联生物科技有限公司)和血清中细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)含量(试剂盒购自武汉华美生物工程有限公司)。

1.6.4 肠道组织形态

将固定好的肠道组织样用于制作苏木精-伊红(HE)染色石蜡切片,并将切片置于显微镜40倍下观察,测量绒毛高度(VH)、隐窝深度(CD),并计算二者的比值(VH/CD)。

1.6.5 肠道病理损伤评分

肉鸡肠道损伤评分标准参照张贝贝[8]的文章,评分标准如下:0分,无明显损伤;0.5分,小肠的浆膜面和肠系膜严重充血;1分,肠壁变薄、变脆、有红色出血点(≥5个);2分,肠腔内有气体,肠壁出现针尖样坏死或溃疡点;3分,肠腔内充满气体,肠壁出现成片状坏死或溃疡;4分,肠腔内有大量气体,出现弥漫性坏死。

1.6.6 肠道屏障及炎症相关基因表达

采用组织总RNA抽提试剂盒[EZ-10,生工生物工程(上海)股份有限公司]提取空肠、回肠组织总RNA,提取完毕后使用NanoDrop 2000核酸蛋白测定仪测定RNA的浓度与纯度,通过1%琼脂糖凝胶电泳确定RNA完整性。确认提取的总RNA可用于后续试验后,采用逆转录PCR试剂盒(南京诺唯赞生物科技股份有限公司)对提取的组织总RNA进行反转录制备cDNA,然后使用实时荧光PCR仪及SYBR Green染料体系(南京诺唯赞生物科技股份有限公司)对反转录得到的cDNA进行PCR。以β-肌动蛋白(β-actin)作为内参基因,采用2-ΔΔCt法来计算各目的基因的mRNA相对表达量。
内参基因β-actin,肠道炎症相关基因IL-1β、IL-6、IL-8、IL-10、TNF-α,肠道屏障相关基因闭合蛋白-1(Claudin-1)、闭锁蛋白(Occludin)、闭合小环蛋白-1(ZO-1)、黏蛋白-2(MUC-2)引物序列根据NCBI中GenBank提供的基因序列,使用软件Prime Premier 5.0进行设计,各基因特异性上、下游引物见表2。试验所用引物均由北京擎科生物科技有限公司合成。
表2 PCR特异性引物序列

Table 2 Specific primer sequences for PCR

项目
Items		 引物序列
Primer sequence (5'—3')		 产物长度
Product length/bp
β-肌动蛋白β-actin F:TGCCTGCAGAAGAAGCCTCG
R:GACGGGCTCAAAAACCTCCT		 204
白细胞介素-1β IL-1β F:TGACCAAACTGCTGCGGAGGC
R:TCGAAGGACTGTGAGCGGGTGT		 157
白细胞介素-6 IL-6 F:CAAGAAGTTCACCGTGTGCG
R:GGAGAGCTTCGTCAGGCATT		 133
白细胞介素-8 IL-8 F:TTGGAAGCCACTTCAGTCAGAC
R:GGAGCAGGAGGAATTACCAGTT		 120
白细胞介素-10 IL-10 F:GCTGCCAAGCCCTGTTGCT
R:TCCCGTTCTCATCCATCTTCTCG		 441
肿瘤坏死因子-α TNF-α F:GAGCAGGGCTGACACGGAT
R:CAGGCACAAAAGAGCTGATGG		 152
闭合蛋白-1 Claudin-1 F:ACTACAATGCCGCTGTCCTG
R:TCTTTCCAGTGGCGATACCTAC		 172
闭锁蛋白Occludin F:TTCGTCATGCTCATCGCCTC
R:TCCACGGTGCAGTAGTGGTA		 158
闭合小环蛋白-1 ZO-1 F:CTTCAGGTGTTTCTCTTCCTCCTC
R:CTGTGGTTTCATGGCTGGATC		 131
黏蛋白-2 MUC-2 F:CATTCAACGAGGAGAGCTGC
R:TTCCTTGCAGCAGGAACAAC		 248

1.7 数据处理与分析

试验数据经过Excel 2021进行初步处理后,采用SPSS 25.0统计软件进行单因素方差分析(one-way ANOVA),方差齐性时使用Duncan氏法多重比较检验组间差异的显著性,方差不齐时使用Welch检验与Games-Howell检验判断差异的显著性,试验数据用平均值和均值标准误(SEM)表示,P<0.05为差异显著,0.05≤P≤0.10表示有显著趋势。

2 结果与分析

2.1 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡生长性能的影响

由表3可知,1~21日龄时,CP组肉鸡FBW和ADG与CON组相比显著降低(P<0.05),BA组肉鸡生长性能与CON组和CP组差异不显著(P>0.05);22~28日龄时,CON组和BA组肉鸡ADFI显著高于CP组(P<0.05),BA组肉鸡ADG恢复到CON组水平(P>0.05);从整个试验期(1~28日龄)来看,CON组和BA组肉鸡FBW、ADFI均显著高于CP组(P<0.05),且BA组肉鸡ADG显著高于CP组(P<0.05)。
表3 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡生长性能的影响

Table 3 Effects of benzoic acid on growth performance of broilers coinfected with Eimeria and Clostridium perfringens

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
1~21日龄1 to 21 days of age
平均初重IBW/g 42.69 42.68 42.62 0.02 0.153
平均末重FBW/g 368.01a 354.53b 356.10ab 2.36 0.017
平均日采食量ADFI/g 24.95 24.21 24.85 0.20 0.276
平均日增重ADG/g 15.49a 14.85b 14.93ab 0.11 0.017
料重比F/G 1.63 1.63 1.67 0.01 0.302
22~28日龄22 to 28 days of age
平均日采食量ADFI/g 65.12a 59.24b 64.23a 1.08 0.048
平均日增重ADG/g 33.54 31.95 34.58 0.53 0.129
料重比F/G 1.89 1.85 1.87 0.09 0.750
1~28日龄1 to 28 days of age
平均末重FBW/g 601.58a 587.06b 604.00a 3.05 0.028
平均日采食量ADFI/g 35.03a 33.91b 35.03a 0.21 0.025
平均日增重ADG/g 19.76ab 19.44b 20.05a 0.10 0.025
料重比F/G 1.73 1.74 1.76 0.01 0.223

同行数据肩标无字母或相同字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下表同。

In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05). The same as below.

2.2 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡免疫器官指数的影响

由表4可知,21日龄时,CP组肉鸡肝脏指数和法氏囊指数显著高于CON组和BA组(P<0.05)。28日龄时,各组间免疫器官指数差异不显著(P>0.05)。
表4 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡免疫器官指数的影响

Table 4 Effects of benzoic acid on immune organ indexes of broilers coinfected with Eimeria and Clostridium perfringens g/kg

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
21日龄21 days of age
肝脏指数Liver index 24.52b 26.84a 24.79b 0.36 0.007
脾脏指数Spleen index 1.04 1.15 1.16 0.04 0.423
法氏囊指数Bursal of Fabricius index 3.13b 4.26a 3.16b 0.16 0.001
胸腺指数Thymus index 4.01 3.99 3.84 0.11 0.825
28日龄28 days of age
肝脏指数Liver index 23.41 24.12 22.93 0.25 0.137
脾脏指数Spleen index 1.30 1.24 1.20 0.03 0.140
法氏囊指数Bursal of Fabricius index 2.78 2.65 2.52 0.07 0.304
胸腺指数Thymus index 5.32 4.95 5.12 0.16 0.683

2.3 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡血清细胞因子含量的影响

由表5可知,21日龄时,与CON组相比,CP组肉鸡血清IL-1β、TNF-α含量显著提高(P<0.05),血清IL-6含量有提高的趋势(P=0.092);与CP组相比,BA组肉鸡血清TNF-α含量显著降低(P<0.05),血清IL-6含量有降低的趋势(P=0.092)。28日龄时,CP组肉鸡血清IL-6含量显著高于CON组和BA组(P<0.05),各组间肉鸡血清IL-1β、IL-10和TNF-α含量差异不显著(P>0.05)。
表5 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡血清细胞因子含量的影响

Table 5 Effects of benzoic acid on serum cytokine contents of broilers coinfected with Eimeria and Clostridium perfringens pg/mL

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
21日龄21 days of age
白细胞介素-1β IL-1β 0.027b 0.042a 0.037ab 0.002 0.003
白细胞介素-6 IL-6 43.28 64.45 47.73 3.97 0.092
白细胞介素-10 IL-10 0.53b 0.91ab 1.09a 0.10 0.037
肿瘤坏死因子-α TNF-α 0.23b 0.37a 0.25b 0.03 0.040
28日龄28 days of age
白细胞介素-1β IL-1β 0.046 0.053 0.036 0.005 0.410
白细胞介素-6 IL-6 14.09b 18.87a 13.71b 0.93 0.027
白细胞介素-10 IL-10 1.02 0.91 0.90 0.05 0.505
肿瘤坏死因子-α TNF-α 0.009 0.012 0.009 0.001 0.337

2.4 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡血清免疫球蛋白含量的影响

由表6可知,21日龄时,与CON组相比,CP组和BA组肉鸡血清IgA含量显著降低(P<0.05);与CP组相比,BA组肉鸡血清IgG、IgM含量显著提高(P<0.05)。28日龄时,与CON组相比,CP组和BA组肉鸡血清IgM含量显著提高(P<0.05);与CP组相比,BA组肉鸡血清IgG含量显著提高(P<0.05)。
表6 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡血清免疫球蛋白含量的影响

Table 6 Effects of benzoic acid on serum immunoglobulin contents of broilers coinfected with Eimeria and Clostridium perfringensμg/mL

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
21日龄21 days of age
免疫球蛋白A IgA 170.67a 86.94b 62.57b 15.29 <0.001
免疫球蛋白G IgG 1 516.82b 1 563.02b 2 105.06a 104.32 0.017
免疫球蛋白M IgM 294.97b 291.58b 437.87a 39.29 0.006
28日龄28 days of age
免疫球蛋白A IgA 252.93 209.41 219.31 16.50 0.575
免疫球蛋白G IgG 1 544.61b 1 964.50b 2 424.41a 121.33 0.003
免疫球蛋白M IgM 164.86b 235.11a 232.61a 12.58 0.015

2.5 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道组织形态的影响

由表7可知,21日龄时,与CON组相比,CP组肉鸡空肠VH/CD显著降低(P<0.05);与CP组相比,BA组肉鸡空肠、回肠VH/CD和回肠VH均显著提高(P<0.05)。28日龄时,与CON组相比,CP组肉鸡空肠和回肠CD显著提高(P<0.05),回肠VH/CD显著降低(P<0.05);与CP组相比,BA组肉鸡空肠CD显著降低(P<0.05),空肠VH/CD显著提高(P<0.05)。
表7 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道组织形态的影响

Table 7 Effects of benzoic acid on intestinal morphology of broilers coinfected with Eimeria and Clostridium perfringens

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
21日龄21 days of age
绒毛高度VH/μm 1 131.64 1 045.85 1 041.78 23.66 0.257
空肠Jejunum 隐窝深度CD/μm 174.30 183.01 177.94 4.52 0.786
绒毛高度/隐窝深度VH/CD 6.94a 5.26b 6.36a 0.24 0.003
绒毛高度VH/μm 798.23b 713.26b 896.44a 24.16 0.002
回肠Ileum 隐窝深度CD/μm 205.29 193.59 184.64 8.40 0.640
绒毛高度/隐窝深度VH/CD 3.96b 3.75b 4.84a 0.19 0.041
28日龄28 days of age
绒毛高度VH/μm 1 043.69 1 090.53 1 154.17 33.71 0.437
空肠Jejunum 隐窝深度CD/μm 159.65b 217.44a 177.55b 10.83 0.018
绒毛高度/隐窝深度VH/CD 6.69ab 5.29b 7.54a 0.39 0.046
绒毛高度VH/μm 798.11 879.38 865.41 20.03 0.218
回肠Ileum 隐窝深度CD/μm 156.01b 235.93a 169.70ab 12.62 0.049
绒毛高度/隐窝深度VH/CD 5.65a 4.50b 5.13ab 0.19 0.036

2.6 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道病理损伤评分的影响

由表8可知,21日龄时,与CON组相比,CP组肉鸡回肠病理损伤评分显著提高(P<0.05)。28日龄时,与CON组相比,CP组肉鸡空肠病理损伤评分显著提高(P<0.05);与CP组相比,BA组肉鸡空肠病理损伤评分显著降低(P<0.05)。
表8 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道病理损伤评分的影响

Table 8 Effects of benzoic acid on intestinal pathological damage score of broilers coinfected with Eimeria and Clostridium perfringens

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
21日龄21 days of age
空肠Jejunum 0.31 0.88 0.56 0.12 0.156
回肠Ileum 0.06b 0.94a 0.56ab 0.13 0.014
28日龄28 days of age
空肠Jejunum 0.38b 1.19a 0.75b 0.10 0.002
回肠Ileum 0.50 0.94 0.75 0.09 0.138

2.7 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道炎症因子相关基因表达的影响

由表9可知,21日龄时,与CON组相比,CP组肉鸡空肠IL-1β、回肠IL-8 mRNA相对表达量显著上调(P<0.05),空肠IL-6(P=0.090)、回肠IL-1β(P=0.097)、回肠IL-6(P=0.086)mRNA相对表达量有上调趋势;与CP组相比,BA组肉鸡回肠IL-8、TNF-α mRNA相对表达量显著下调(P<0.05),回肠IL-1β mRNA相对表达量有下调趋势(P=0.097)。28日龄时,与CON组相比,CP组肉鸡回肠IL-10 mRNA相对表达量显著降低(P<0.05);与CP组相比,BA组肉鸡回肠IL-8 mRNA相对表达量显著降低(P<0.05);空肠炎症因子相关基因mRNA相对表达量各组间均无显著差异(P>0.05)。
表9 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道炎症因子相关基因表达的影响

Table 9 Effects of benzoic acid on the expression of genes related to intestinal inflammatory factors of broilers coinfected with Eimeria and Clostridium perfringens

项目
Items		 组别Groups SEM P值
P-value
CON CP BA
21日龄21 days of age
白细胞介素-1β IL-1β 1.00b 1.87a 1.21ab 0.17 0.007
白细胞介素-6 IL-6 1.00 2.35 1.66 0.26 0.090
空肠Jejunum 白细胞介素-8 IL-8 1.00 0.98 1.13 0.11 0.825
白细胞介素-10 IL-10 1.00 2.64 3.51 0.46 0.060
肿瘤坏死因子-α TNF-α 1.00 1.77 1.51 0.18 0.220
白细胞介素-1β IL-1β 1.00 1.44 1.02 0.10 0.097
白细胞介素-6 IL-6 1.00 2.00 1.44 0.19 0.086
回肠Ileum 白细胞介素-8 IL-8 1.00b 1.72a 0.91b 0.15 0.023
白细胞介素-10 IL-10 1.00 1.35 1.23 0.19 0.752
肿瘤坏死因子-α TNF-α 1.00a 0.99a 0.90b 0.02 0.008
28日龄28 days of age
白细胞介素-1β IL-1β 1.00 0.98 1.17 0.08 0.711
白细胞介素-6 IL-6 1.00 1.06 1.61 0.13 0.077
空肠Jejunum 白细胞介素-8 IL-8 1.00 0.92 1.04 0.08 0.837
白细胞介素-10 IL-10 1.00 2.27 1.55 0.35 0.379
肿瘤坏死因子-α TNF-α 1.00 1.37 1.15 0.10 0.354
白细胞介素-1β IL-1β 1.00 1.10 0.93 0.05 0.409
白细胞介素-6 IL-6 1.00 1.10 0.89 0.09 0.659
回肠Ileum 白细胞介素-8 IL-8 1.00a 0.98a 0.51b 0.08 0.002
白细胞介素-10 IL-10 1.00a 0.66b 0.44b 0.08 0.008
肿瘤坏死因子-α TNF-α 1.00 1.33 1.03 0.08 0.201

2.8 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道屏障相关基因表达的影响

由表10可知,21日龄时,与CON组相比,CP组肉鸡空肠ZO-1、MUC-2 mRNA相对表达量显著下调(P<0.05),空肠Claudin-1 mRNA相对表达量有下调趋势(P=0.086);与CP组相比,BA组肉鸡各肠道屏障相关基因mRNA相对表达量无显著变化(P>0.05)。28日龄时,与CON组相比,CP组肉鸡空肠ZO-1、回肠Occludin和ZO-1 mRNA相对表达量显著降低(P<0.05),回肠MUC-2 mRNA相对表达量有降低趋势(P=0.070);与CP组相比,BA组空肠Occludin mRNA相对表达量显著提高(P<0.05)。
表10 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道屏障相关基因表达的影响

Table 10 Effects of benzoic acid on expression of genes related to intestinal barrier of broilers coinfected with Eimeria and Clostridium perfringens

项目
Items		 组别Groups SE P值
P-value
CON CP BA
21日龄21 days of age
闭合蛋白-1 Claudin-1 1.00 0.69 0.66 0.07 0.086
空肠Jejunum 闭锁蛋白Occludin 1.00 0.87 1.16 0.06 0.117
闭合小环蛋白-1 ZO-1 1.00a 0.51b 0.59b 0.08 0.034
黏蛋白-2 MUC-2 1.00a 0.34b 0.49b 0.10 0.001
闭合蛋白1 Claudin 1 1.00 1.16 0.90 0.08 0.485
回肠Ileum 闭锁蛋白Occludin 1.00 0.88 0.78 0.05 0.178
闭合小环蛋白-1 ZO-1 1.00 0.87 0.95 0.03 0.250
黏蛋白-2 MUC-2 1.00 0.69 0.71 0.07 0.154
28日龄28 days of age
闭合蛋白-1 Claudin-1 1.00 1.43 1.21 0.14 0.512
空肠Jejunum 闭锁蛋白Occludin 1.00b 1.32b 1.47a 0.08 0.041
闭合小环蛋白-1 ZO-1 1.00a 0.86b 0.87b 0.03 0.042
黏蛋白-2 MUC-2 1.00 0.82 0.74 0.08 0.441
闭合蛋白-1 Claudin-1 1.00 0.84 0.79 0.08 0.630
回肠Ileum 闭锁蛋白Occludin 1.00a 0.62b 0.78b 0.08 0.007
闭合小环蛋白-1 ZO-1 1.00a 0.59b 0.66b 0.05 0.001
黏蛋白-2 MUC-2 1.00 0.72 0.64 0.07 0.070

3 讨论

3.1 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡生长性能的影响

大量研究表明,球虫或产气荚膜梭菌感染显著降低肉鸡的体重、ADG和ADFI,提高F/G[9⇓⇓-12]。本研究也发现,球虫和产气荚膜梭菌联合感染后,肉鸡各阶段FBW、ADG、ADFI均有不同程度降低。与前人研究[9⇓⇓-12]相比较,虽然各试验所使用的产气荚膜梭菌菌株和感染模型存在差异,但是均证实了同一结论,即产气荚膜梭菌感染可降低肉鸡的生长性能。球虫感染引起的肠道黏膜损伤是产气荚膜梭菌引起禽类NE发生的主要诱导因子之一,预防球虫病也是降低NE发病率的有效办法。Yan等[13]报道,饲粮中添加包被苯甲酸可以缓解球虫攻毒带来的肉鸡生长性能下降。黄灵杰等[9]研究也表明,饲粮中添加苯甲酸对球虫攻毒肉鸡体重有提高的趋势。本试验中,在球虫和产气荚膜梭菌联合感染肉鸡的同时在饲粮中补充苯甲酸,肉鸡生长性能得到显著的提升,基本恢复至CON组水平,说明在球虫和产气荚膜梭菌联合感染条件下,饲粮中添加适量苯甲酸对球虫和产气荚膜梭菌联合感染造成的肉鸡生长性能下降有一定的恢复效果。推测其原因可能是苯甲酸进入肠道后,在肠道内定点释放,创造弱酸性环境,抑制多种病原体定植,改善肠道健康,减少病原微生物与宿主的营养竞争,增强营养物质吸收,从而提高生长性能[9,14]。
另外,本研究依旧按照817白羽肉鸡生长阶段(1~21日龄、22日龄后)来统计生长性能数据,前人试验中也有采用类似的方式来划分数据采集阶段[15],但这存在一定的局限性。在本研究中,肉鸡于12日龄预先感染球虫,再于16~19日龄感染产气荚膜梭菌,二者均为诱导肉鸡NE的条件,可合并为一个整体的感染阶段。按照常规感染/攻毒试验的阶段划分规范,本研究应该划分为3个试验阶段,即感染前(1~11日龄)、感染期间(12~21日龄)和感染后的恢复阶段(22~28日龄),并按这3个阶段来采集生长性能数据,以确认正式开展细菌感染/攻毒前各组试验鸡生长性能没有统计学差异,这样处理可能更为合理和规范。

3.2 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡免疫功能的影响

机体免疫功能对于维护机体健康至关重要,而免疫器官的发育状态直观反映了免疫功能的强弱,在一定范围内,免疫器官相对重量越大,免疫水平越高[16],但感染状态下,免疫器官相对重量的增加往往是器官肿大的表现。胸腺、脾脏和法氏囊为家禽的主要免疫器官,肝脏在维持机体代谢平衡的同时也具备一定的免疫功能[17]。本试验发现球虫和产气荚膜梭菌联合感染显著提高了21日龄鸡肝脏指数和法氏囊指数,表现为感染早期肝脏与法氏囊肿大,机体受产气荚膜梭菌感染水平较高。这与孙亚楠[18]、Hussein等[19]的研究结果相似,说明球虫和产气荚膜梭菌感染早期机体处于抗感染的高免疫水平状态。而饲粮中添加苯甲酸后,肉鸡感染早期的肝脏与法氏囊的肿大被缓解,表明机体受感染程度降低。推测其原因:一方面在于苯甲酸抑制梭菌生长,另一方面可能是由于苯甲酸进入消化道后,影响了产气荚膜梭菌通过肠道向肝脏中的转移,降低了机体的感染程度,进而缓解了肝脏的肿大现象。
球虫和产气荚膜梭菌感染可引发机体炎症反应,释放炎性因子。谢涛[20]研究发现,肉鸡感染球虫后血清中白细胞介素-2(IL-2)的分泌水平提高,进一步介导细胞免疫反应,引起肉鸡的肠道损伤,而饲粮中添加酸化剂能够缓解肠道损伤。孙秋娟等[21]试验结果显示,28日龄感染产气荚膜梭菌的肉仔鸡血清IL-1β含量提高,炎性因子IL-1β被证明可诱导发热反应,激活细胞因子网络。本试验中,球虫和产气荚膜梭菌联合感染后,肉鸡21日龄时血清IL-1β和TNF-α含量显著提高,表明机体炎症水平提高,而在感染的同时补充苯甲酸,肉鸡血清IL-1β、IL-6和TNF-α含量降低,IL-10含量提高,与前人研究结果[20⇓-22]基本一致,表明苯甲酸可以更好地调动机体抗炎反应,削弱球虫和产气荚膜梭菌感染带来的炎症反应强度。
免疫球蛋白是体液免疫应答中最主要的免疫分子,其最突出的生物学特性是能够特异性地与相应抗原结合,消除侵入体内的异物。免疫球蛋白含量的升高可以体现出机体免疫水平的增强。薛敬洁等[23]研究发现,球虫感染降低了罗曼公雏鸡血清IgA、IgG和IgM含量。黄灵杰等[9]也报道,球虫攻毒降低了肉鸡42日龄时血清IgM含量,在补充苯甲酸后,肉鸡21日龄时血清IgM含量有所提高。本试验中,球虫和产气荚膜梭菌联合感染后,肉鸡21日龄时血清IgA含量显著下降,补充苯甲酸后,肉鸡血清IgG和IgM含量显著提高。与前人研究结果[9,23]相似,表明饲粮中添加苯甲酸可在一定程度上缓解球虫和产气荚膜梭菌联合感染导致的体液免疫功能降低。

3.3 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道组织形态及肠道病理损伤评分的影响

肠道作为机体营养物质吸收的主要部位,在维持机体健康和提高生长性能上发挥了重要作用。肠黏膜形态结构组成了肠道的机械屏障,能够避免肠道内细菌或抗原物质穿过肠黏膜进入其他组织,是肠道屏障功能的结构基础。肠道VH、CD、VH/CD是评价肠道消化吸收能力的重要指标[24]。肠道VH增加会促进肠道对营养物质的吸收,CD增加则对应肠上皮组织分泌消化液的能力减弱,VH/CD在一定程度上可以反映肠道吸收功能的强弱,比值越高表示消化吸收能力越强[25-26]。Cao等[27]研究表明,产气荚膜梭菌感染肉鸡的肠道损伤评分显著提高。本研究结果与上述研究结果基本一致,且本研究表明饲粮中补充苯甲酸可降低肠道病理损伤评分。Li等[28]研究发现,产气荚膜梭菌感染会导致肉鸡肠道VH和VH/CD显著降低。刘阳等[29]研究也表明产气荚膜梭菌感染显著增加了肉鸡十二指肠CD,显著降低了十二指肠和回肠VH和VH/CD。本试验中,球虫和产气荚膜梭菌联合感染显著降低肉鸡21日龄时空肠VH/CD和28日龄时回肠VH/CD,显著提高肉鸡28日龄时空肠和回肠CD,与前人研究结果[28-29]基本一致,但损伤的肠段略有差异,可能与球虫和产气荚膜梭菌感染的方式及菌株类型不妥有关,但也基本说明球虫和产气荚膜梭菌感染降低了肉鸡肠道吸收能力。补充苯甲酸后,肉鸡21日龄时空肠、回肠VH/CD和回肠VH得到显著提高,28日龄时空肠VH/CD和空肠CD分别得到的显著提高与降低。这表明添加苯甲酸后,因EM/CP感染而降低的肉鸡肠道吸收能力得到了一定程度上的恢复。其原因可能是在较高水平苯甲酸的添加下,肠道pH降低,增加机体细胞分裂,使得VH增加,VH/CD提高。另外,有机酸也被证明可以通过促进消化功能的变化和改变肠道微生物的数量来改善肠道形态[30]。

3.4 苯甲酸对球虫和产气荚膜梭菌联合感染肉鸡肠道炎症及屏障相关基因表达的影响

肠黏膜通过发挥免疫功能抵御产气荚膜梭菌在肠道中的增殖,其激活主要表现为炎症因子的释放。IL-1β、IL-6为促炎细胞因子,在机体的炎症反应中发挥作用,机体受到病原微生物入侵,会刺激其释放[31]。IL-8可诱导细胞形态发生改变,释放中性粒细胞,增强免疫细胞的抗菌能力。TNF-α是调控机体免疫反应、炎症反应和细胞凋亡的关键因子[32]。IL-10能够拮抗炎性因子,缓解炎症反应,是一种抗炎细胞因子[33]。病原菌入侵时,会激活免疫反应,促炎细胞因子被释放,但促炎细胞因子过度释放会引发代谢异常和炎症反应,导致机体受到损伤。郭双双[34]研究表明,产气荚膜梭菌感染上调了肠道IL-1β和γ-干扰素(INF-γ)mRNA的表达。Oh等[35]研究发现,NE感染显著上调了鸡肠道IL-1β和IL-10 mRNA的表达。Collier等[36]也观察到,鸡感染NE后,肠道中IL-10 mRNA的表达量显著增加。Hong等[37]研究显示,产气荚膜梭菌感染肉仔鸡,其肠道α-干扰素(IFN-α)、IFN-γ、IL-1β等基因的mRNA表达被抑制,而IL-8、IL-10、IL-15等基因的mRNA表达被上调。这些研究均表明NE感染能诱导宿主基因参与肠黏膜先天性免疫。本试验中,球虫和产气荚膜梭菌联合感染诱导了肉鸡的肠道炎症,导致21日龄肉鸡空肠或回肠的促炎细胞因子IL-1β、IL-6、IL-8、TNF-α和抗炎细胞因子IL-10 mRNA相对表达量均增加,在28日龄这些现象得到缓解。Abd El-Hack等[38]研究发现,相较于产气荚膜梭菌单独感染,球虫和产气荚膜梭菌联合感染后IFN-α、IFN-γ和IL-1β的mRNA表达均被下调,与本研究结果类似。而在球虫和产气荚膜梭菌感染的同时补充苯甲酸,可以观察到21日龄肉鸡肠道促炎细胞因子IL-1β、IL-6、IL-8、TNF-α的mRNA相对表达量下调,虽未完全恢复至CON组水平,但相较于CP组已经有了明显的降低,说明饲粮中补充苯甲酸可以缓解因球虫和产气荚膜梭菌感染所导致的炎症反应,预防或缓解NE带来的损伤。这与Timbermont等[39]的观点相似,推测原因可能是苯甲酸进入后肠部位后,进入有害菌细胞,打破该细胞内酸碱平衡,降低呼吸相关酶的活性,最终杀死有害菌,降低有害菌对肠道的侵害与损伤,缓解肠道损伤,从而下调肠道炎症相关基因的表达[40]。
动物的肠道屏障功能能有效抵御病原菌入侵,保障机体健康。肠道机械屏障主要由固有层、肠上皮紧密连接和黏液层组成,在维持肠道健康方面发挥着重要作用。紧密连接主要由Occludin、闭合蛋白(Claudins)和闭合小环蛋白(ZO)构成[41]。其中Claudin-1和Occludin负责形成肠道屏障[42];ZO是紧密连接支持结构的基础,具有连接跨膜蛋白与细胞骨架、维持上皮极性等功能[43]。MUC-2作为小肠黏液层中最主要的黏蛋白成分,可阻碍病原体及毒素等的入侵[44-45]。夏亿[46]研究显示,产气荚膜梭菌感染导致黏液中MUC-2含量减少,肠道屏障功能减弱。张贝贝[8]研究发现,球虫和产气荚膜梭菌联合感染下调了肉鸡肠道Claudin-1和Occludin的mRNA相对表达量。曹力等[47]研究发现,肉鸡感染产气荚膜梭菌后,肠道Occludin的mRNA相对表达量下调,并伴随着肠上皮紧密连接的通透性增加。本研究中,球虫和产气荚膜梭菌联合感染2 d后,肉鸡21日龄时空肠Claudin-1、ZO-1、MUC-2和回肠Occludin、MUC-2的mRNA相对表达量被下调,表明肉鸡小肠机械屏障受到破坏,直至感染9 d后(28日龄)小肠机械屏障仍旧存在一定程度的损伤。有报道称酸化剂可以通过调控肠道免疫功能抑制炎症反应,进而维持肠道屏障功能[48]。谢涛[20]研究也发现,饲粮中添加复合酸化剂可以缓解肉鸡感染球虫后空肠紧密连接蛋白Occludin、Claudin-1、闭合小环蛋白-2(ZO-2)和MUC-2表达的下调。本试验中。肉鸡在球虫和产气荚膜梭菌联合感染的同时在饲粮中补充苯甲酸,一定程度上缓解了感染早期(21日龄)肉鸡肠道屏障相关基因表达的下调,在28日龄时,相较与CP组,BA组肠道屏障相关基因的表达存在一定优势,表明饲粮中添加苯甲酸能在一定程度上通过调节肠道黏蛋白和黏膜紧密连接蛋白相关基因的表达,改善肠道屏障紧密连接结构和功能的完整性,缓解球虫和产气荚膜梭菌联合感染导致的肠道损伤,提高肉鸡的生长性能。推测其原因可能是苯甲酸通过抑制促炎细胞因子的释放来改善肠道屏障的完整性[40],因为细胞因子TNF-α、IL-1β、IL-10被证明能够通过调节肠道紧密连接蛋白的表达来调节肠道通透性[49]。

4 结论

饲粮中添加1 000 mg/kg苯甲酸能有效缓解球虫和产气荚膜梭菌联合感染对1~28日龄肉鸡造成的肠道炎症及肠道屏障损伤,增强免疫功能,改善肠道组织形态,维护肠道健康,从而提高生长性能。

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