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

doi:10.3969/j.issn.1004-1524.20231249

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (11): 2465-2475.DOI: 10.3969/j.issn.1004-1524.20231249

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S858.28

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.

Figures/Tables 11

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'

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

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.

Fig.2 Microbial community structure in healthy and diarrheal piglets

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

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.

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

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

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

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.

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

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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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