腹泻仔猪肠道菌群结构特征及肠道炎症因子、腺苷酸环化酶和鸟苷酸环化酶mRNA表达变化研究

doi:10.3969/j.issn.1004-1524.20231249

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (11): 2465-2475.DOI: 10.3969/j.issn.1004-1524.20231249

腹泻仔猪肠道菌群结构特征及肠道炎症因子、腺苷酸环化酶和鸟苷酸环化酶mRNA表达变化研究

吴兴凤¹^,²(), 章啸君³, 朱江², 余敏洁², 徐娥¹, 马灵燕², 肖英平²^,^*()

1.贵州大学动物科学学院,高原山地动物遗传育种与繁殖教育部重点实验室,贵州贵阳 550025
2.浙江省农业科学院农产品质量安全与营养研究所,省部共建农产品质量安全与危害因子防控国家重点实验室,浙江杭州 310021
3.金华市农业科学研究院畜牧兽医与水产研究所,浙江金华 321017

收稿日期:2023-11-03 出版日期:2024-11-25 发布日期:2024-11-27
作者简介:吴兴凤(1998—),女,布依族,贵州都匀人,硕士研究生,研究方向为动物营养与饲料科学。E-mail:wxf18826@163.com
通讯作者: *肖英平,E-mail:xiaoyp@zaas.ac.cn
基金资助:
金华市农业科技重点项目(2021-2-010);国家自然科学基金(31402083);国家自然科学基金(32372907)

Study on structural characteristics of intestinal microflora and expression changes of intestinal inflammatory factors, adenylate and guanylate cyclase mRNA in diarrheal piglets

WU Xingfeng¹^,²(), ZHANG Xiaojun³, ZHU Jiang², YU Minjie², XU E¹, MA Lingyan², XIAO Yingping²^,^*()

1. Key Laboratory of Genetics, Breeding and Reproduction of Plateau Mountain Animals, Ministry of Education, School of Animal Science, Guizhou University, Guiyang 550025, China
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Institute of Animal Husbandry, Veterinary Medicine and Fisheries, Jinhua Academy of Agricultural Sciences, Jinhua 321017, Zhejiang, China

Received:2023-11-03 Online:2024-11-25 Published:2024-11-27

摘要/Abstract

摘要：

选取同批次的21日龄仔猪,在断奶3 d后采集55份健康仔猪和37份腹泻仔猪的粪便样本,测定短链脂肪酸含量,采用高通量测序技术基于16S rRNA基因分析菌群结构,并分别选择6头腹泻和健康仔猪屠宰采集结肠黏膜样品对炎症因子、腺苷酸环化酶和鸟苷酸环化酶的mRNA表达水平进行检测。结果显示,与健康仔猪相比,腹泻仔猪粪便菌群的香农指数、Chao1指数显著(P<0.05)降低;在门水平上,变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)的相对丰度显著提高,拟杆菌门(Bacteroidota)的相对丰度显著降低;在属水平上,大肠埃希菌-志贺菌属(Escherichia-Shigella)、乳酸杆菌属(Lactobacillus)和拟普雷沃氏菌属(Alloprevotella)的相对丰度显著升高,瘤胃球菌属(Ruminococcus)、瘤胃球菌科_uncultured(Ruminococcaceae_uncultured)、普雷沃氏菌科_NK3B31(Prevotellaceae_NK3B31_group)、理研菌科RC9属(Rikenellaceae_RC9_gut_group)、UCG-010_norank和狭义梭菌属_1(Clostridium_sensu_stricto_1)的相对丰度显著降低。菌群功能预测结果显示,与健康仔猪相比,腹泻仔猪中粪便菌群的糖酵解/糖异生、D-谷氨酰胺和D-谷氨酸代谢、D-精氨酸代谢通路的相对丰度显著升高,细菌趋化性,以及缬氨酸、亮氨酸和异亮氨酸的生物合成(valine,leucine and isoleucine biosynthesis)通路的相对丰度显著降低。腹泻仔猪粪便中的乙酸、丙酸和丁酸含量较健康仔猪显著降低,且乙酸含量与理研菌科RC9属、UCG-010_norank和瘤胃球菌科_uncultured的相对丰度呈显著正相关,丁酸含量与罗氏菌属(Roseburia)、不动杆菌属(Agathobacter)和狭义梭菌属_1的相对丰度呈显著正相关。与健康仔猪相比,腹泻仔猪肠道中促炎因子——肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的mRNA表达水平显著升高,抗炎因子——白细胞介素-10(IL-10)的mRNA表达水平显著降低(P<0.05),腺苷酸环化酶mRNA的表达水平显著升高。由此可见,断奶健康仔猪与腹泻仔猪的肠道菌群结构与功能存在显著差异,断奶后仔猪肠道中致病菌增加、有益菌减少和短链脂肪酸含量降低,以及促炎因子和腺苷酸环化酶mRNA水平的升高,导致仔猪发生腹泻。

关键词: 断奶仔猪, 腹泻, 肠道菌群, 腺苷酸环化酶, 鸟苷酸环化酶

Abstract:

In the present study, the same batch of 21-day-old piglets were selected. Fecal samples of 55 healthy piglets and 37 diarrhea piglets were collected after 3 days of weaning. The content of short-chain fatty acids was determined, and the microbial community structure was analyzed by high-throughput sequencing of 16S rRNA gene. Six diarrheal and healthy piglets were selected and slaughtered to collect colonic mucosa samples to detect the mRNA expression levels of inflammatory factors, adenylate cyclase and guanylate cyclase. It was shown that the Shannon index and Chao1 index of microbial community in diarrheal weaned piglets were significantly (P<0.05) decreased compared with those in the healthy group. At the phylum level, the relative abundance of Proteobacteria and Firmicutes in the microbial community of diarrhea weaned piglets was significantly increased, yet the relative abundance of Bacteroidota was significantly decreased compared with those of the healthy pigles. At the genus level, the relative abundance of Escherichia-Shigella, Lactobacillus and Alloprevotella was significantly increased, yet the relative abundance of Ruminococcus, Ruminococcaceae_uncultured, Prevotellaceae_NK3B31_group, Rikenellaceae_RC9_gut_group, UCG-010_norank and Clostridium_sensu_stricto_1 was significantly decreased than those of the healthy piglets. The prediction of microbial function showed that the relative abundance of glycolysis/gluconeogenesis, D-glutamine and D-glutamate metabolism, and D-alanine metabolism pathways in the microbial community of diarrheal piglets was significantly increased compared with the healthy group, while the relative abundance of bacterial chemotaxis and valine, leucine and isoleucine biosynthesis pathways was significantly reduced. The contents of acetic acid, propionic acid and butyric acid in the intestine of diarrhea piglets were significantly lowered than those in the healthy piglets. The relative abundance of Rikenellaceae_RC9_gut_group, Ruminococcaceae_uncultured and UCG-010_norank flora was significantly positively correlated with the content of acetic acid, and the relative abundance of Roseburia, Agathobacter and Clostridium_sensu_stricto_1 was significantly positively correlated with the content of butyric acid. The mRNA levels of pro-inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in the intestine of diarrhea piglets were significantly increased compared with those in the healthy piglets, while the mRNA level of anti-inflammatory factor interleukin-10 (IL-10) was significantly decreased, yet the mRNA level of adenylate cyclase was significantly increased. In conclusion, there were significant differences in the structure and function of intestinal microflora between weaned healthy piglets and diarrheal piglets. After weaning, the increase of relative abundance of pathogenic bacteria, the decrease of relative abundance of beneficial bacteria, the decrease of short-chain fatty acid content in the intestinal tract of piglets, and the increase of pro-inflammatory factors and adenylate cyclase mRNA levels led to diarrhea in piglets.

Key words: weaned piglet, diarrhea, intestinal microflora, adenylate cyclase, guanylate cyclase

中图分类号:

S858.28

吴兴凤, 章啸君, 朱江, 余敏洁, 徐娥, 马灵燕, 肖英平. 腹泻仔猪肠道菌群结构特征及肠道炎症因子、腺苷酸环化酶和鸟苷酸环化酶mRNA表达变化研究[J]. 浙江农业学报, 2024, 36(11): 2465-2475.

WU Xingfeng, ZHANG Xiaojun, ZHU Jiang, YU Minjie, XU E, MA Lingyan, XIAO Yingping. Study on structural characteristics of intestinal microflora and expression changes of intestinal inflammatory factors, adenylate and guanylate cyclase mRNA in diarrheal piglets[J]. Acta Agriculturae Zhejiangensis, 2024, 36(11): 2465-2475.

图/表 11

表1 实时定量PCR引物序列

Table 1 Primer sequences for real time quantitative PCR

基因名称 Gene name	引物序列 Primer sequence
TNF-α	F:5'-CTGTAGGTTGCTCCCACCTG-3' R:5'-CCAGTAGGGCGGTTACAGAC-3'
IL-2	F:5'-CCCTTGCACTCATGGCAAAC-3' R:5'-ACAGCAGTTACTGTCTCATCATCA-3'
IL-8	F:5'-ACTTCCAAACTGGCTGTTGC-3' R:5'-TGCTGTTGTTGTTGCTTCTCA-3'
IL-10	F:5'-CCAGCTCAGCACTGCTCTAT-3' R:5'-TCATCTTCATCGTCATGTAGGCT-3'
AC	F:5'-GTCCACGTGCTGAAGAGTCA-3' R:5'-GTAGACACTCCCCAAAGGGC-3'
GC	F:5'-AACCGCAGATGAGAAGGAGC-3' R:5'-CGTCGGATCAAGGTGTCCAT-3'
GAPDH	F:5'-TCGGAGTGAACGGATTTGGC-3' R:5'-TGACAAGCTTCCCGTTCTCC-3'

表2 仔猪粪便测序结果

Table 2 Sequencing results of fecal samples of piglets

样品 Sample	OTU数量 OTU number	香农指数 Shannon index	辛普森指数 Simpson index	Chao1指数 Chao1 index
健康仔猪Healthy piglets	53 170±577	9.02±0.13 a	0.020±0.001 b	14 091±532 a
腹泻仔猪Diarrheal piglets	53 012±648	7.39±0.20 b	0.040±0.004 a	8 632±640 b

图1 健康仔猪与腹泻仔猪肠道菌群结构的主坐标分析(PCoA) H,健康仔猪;D,腹泻仔猪。下同。

Fig.1 Principal co-ordinates analysis (PCoA) of microbial community structure in healthy and diarrheal piglets H, Healthy piglets; D, Diarrheal piglets. The same as below.

图2 健康仔猪与腹泻仔猪肠道的菌群结构 Firmicutes,厚壁菌门;Bacteroidota,拟杆菌门;Proteobacteria,变形菌门;Fusobacteriota,梭杆菌门;Actinobacteriota,放线菌门;Lactobacillus,乳酸杆菌属;Prevotella_9,普雷沃氏菌属_9;Clostridium_sensu_stricto_1,狭义梭菌属_1;Prevotella:普雷沃氏菌属;Prevotellaceae_NK3B31_group,普雷沃氏菌科_NK3B31;Alloprevotella,拟普雷沃氏菌属;Prevotella_7,普雷沃氏菌属_7;Rikenellaceae_RC9_gut_group,理研菌科RC9属;Others,其他。

Fig.2 Microbial community structure in healthy and diarrheal piglets

图3 健康仔猪和腹泻仔猪肠道差异菌属热图

Fig.3 Heat map of differential genera in healthy and diarrheal piglets

图4 健康仔猪(左)与腹泻仔猪(右)肠道菌群网络互作分析图中节点的大小与肠道菌群丰度呈正相关;红色和绿色连接线分别代表正相关和负相关;菌群间的连接线越多,表示菌群间的关系越密切。

Fig.4 Correlation network of microbial community in healthy (left) and diarrheal (right) piglets The size of the nodes in the figure is positively correlated with the abundance of the microbiota. The red and green lines represent positive and negative correlation, respectively. The more the connection lines between the microbiota, the closer the relationship between the microbiota.

图5 健康仔猪与腹泻仔猪肠道菌群的功能预测差异分析

Fig.5 Difference analysis of microbial function prediction in healthy and diarrheal piglets

表3 仔猪粪便中的短链脂肪酸含量

Table 3 Short-chain fatty acids (SCFAs) content in feces of piglets mg·g-1

样品 Sample	乙酸含量 Acetic acid content	丙酸含量 Propionic acid content	异丁酸含量 Isobutyric acid content	丁酸含量 Butyric acid content	异戊酸含量 Isovaleric acid content	戊酸含量 Valeric acid content	总短链脂肪酸 Total SCFAs content
健康仔猪	5.83±0.17 a	2.75±0.14 a	0.25±0.04 a	2.00±0.13 a	0.23±0.03 a	0.48±0.08 a	11.54±0.41 a
Healthy piglets
腹泻仔猪	4.80±0.39 b	2.09±0.24 b	0.21±0.03 a	1.43±0.22 b	0.07±0.03 b	0.45±0.06 a	9.05±0.84 b
Diarrhea piglets

图6 健康仔猪与腹泻仔猪肠道差异菌属与短链脂肪酸含量的相关性分析

Fig.6 Correlation analysis within differential microbiota and short-chain fatty acids content in healthy and diarrheal piglets

图7 健康仔猪与腹泻仔猪肠道中炎症因子的表达水平 TNF-α,肿瘤坏死因子-α;IL-2,白细胞介素-2;IL-8,白细胞介素-8;IL-10,白细胞介素-10。标“*”的表示差异显著(P<0.05)。下同。

Fig.7 Expression level of inflammatory factors in intestinal tract of healthy and diarrheal piglets TNF-α, Tumor necrosis factor-α; IL-2, Interleukin-2; IL-8, Interleukin-8; IL-10, Interleukin-10. “*” indicate significant difference at P<0.05.

图8 健康仔猪与腹泻仔猪肠道中腺苷酸环化酶(AC)和鸟苷酸环化酶(GC)的表达水平

Fig.8 Expression levels of adenylate cyclase (AC) and guanylate cyclase (GC) in intestinal tract of healthy and diarrheal piglets

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腹泻仔猪肠道菌群结构特征及肠道炎症因子、腺苷酸环化酶和鸟苷酸环化酶mRNA表达变化研究

Study on structural characteristics of intestinal microflora and expression changes of intestinal inflammatory factors, adenylate and guanylate cyclase mRNA in diarrheal piglets

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