Supplementary file 1_Elevated butyric acid and histamine in feces and serum as an indicator of onset of necrotic enteritis in broiler chickens.docx

BackgroundClostridium perfringens (CP) induced necrotic enteritis (NE) is an economically significant intestinal disease of broiler chickens. Identifying potential biological markers during the development of NE might facilitate early disease control measures. Therefore, the current study aimed to identify the metabolites and metabolic pathways changes associated with the onset of NE in serum and feces of CP-infected broiler chickens.
MethodologyThe protein content of the feed was abruptly altered from 20% to 28% using a well-established NE model before challenging the birds with CP. Then, we performed a targeted, fully quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) -based assay for analyzing the metabolomics profile of serum, feces, and jejunal contents in NE birds. The data were analyzed to understand the trend of metabolite distribution, relationships between metabolites and pathway impacts.
ResultsBirds with NE showed metabolic variations including lipids, amino acids, and organic acids, across all the biological samples analyzed. This variation was higher in serum samples (310/597 metabolites, 51.92%), compared to fecal (182/608 metabolites, 29.93%), and jejunal samples (125/607 metabolites, 20.59%). A robust statistical analysis of these metabolites identified 19 common metabolites, including butyric acid and histamine. Pathway analysis identified that six of them were enriched in key pathways, like tricarboxylic acid cycle (TCA cycle) (citric acid and cis-aconitic acid), glyoxylate and dicarboxylate metabolism (citric acid and cis-aconitic acid), arginine-proline metabolism (spermine and creatinine), butanoate metabolism (butyric acid), and histidine metabolism (histamine). These pathways were related to energy synthesis, nitrogen metabolism, and immune response in NE birds.
ConclusionThis study highlights metabolic differences in birds with NE and underscores butyric acid and histamine as potential early biomarkers for NE diagnosis. The upregulation of these metabolites across serum, jejunal and fecal samples reflects their local and systemic impacts on the disease. These biomarkers play key roles in several NE hallmark features, including gut barrier disruption, dysbiosis of microbes and tissue injury through immune system activation, and systemic inflammation. Future studies need to validate our findings across field conditions and different predisposing factors.

背景：产气荚膜梭菌（Clostridium perfringens, CP）诱导的坏死性肠炎（necrotic enteritis, NE）是一种对肉鸡产业具有重要经济影响的肠道疾病。鉴定坏死性肠炎发病过程中的潜在生物标志物，有助于制定早期疾病防控措施。因此，本研究旨在鉴定产气荚膜梭菌感染肉鸡的血清与粪便中，与坏死性肠炎发病相关的代谢物及代谢通路变化。

方法：本研究采用已成熟的坏死性肠炎造模模型，先将饲料蛋白含量从20%骤升至28%，随后以产气荚膜梭菌攻毒肉鸡。随后，采用基于液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）的靶向全定量检测方法，分析坏死性肠炎模型肉鸡的血清、粪便及空肠内容物的代谢组谱。对检测数据进行分析，以明确代谢物分布趋势、代谢物间关联及通路影响效应。

结果：经检测的所有生物样本中，坏死性肠炎模型肉鸡均出现脂质、氨基酸及有机酸类代谢物的显著变化。相较于粪便样本（182/608种代谢物，占比29.93%）与空肠样本（125/607种代谢物，占比20.59%），血清样本的代谢物变化幅度更高（310/597种代谢物，占比51.92%）。对上述代谢物开展严谨的统计学分析，共筛选出19种共有差异代谢物，其中包括丁酸与组胺。通路富集分析显示，其中6种差异代谢物参与了关键通路的富集，包括三羧酸循环（tricarboxylic acid cycle, TCA循环）（柠檬酸、顺乌头酸）、乙醛酸与二羧酸代谢（柠檬酸、顺乌头酸）、精氨酸-脯氨酸代谢（精胺、肌酐）、丁酸代谢（丁酸）以及组氨酸代谢（组胺）。上述通路与坏死性肠炎模型肉鸡的能量合成、氮代谢及免疫应答密切相关。

结论：本研究揭示了坏死性肠炎模型肉鸡的代谢差异，并明确丁酸与组胺可作为坏死性肠炎早期诊断的潜在生物标志物。这些代谢物在血清、空肠及粪便样本中均呈现上调表达，反映了其对疾病的局部与全身影响。这些生物标志物在坏死性肠炎的多个核心特征中发挥关键作用，包括肠道屏障破坏、微生物群失调、免疫激活介导的组织损伤以及全身炎症反应。未来研究需在实际养殖场景与不同易感因素下验证本研究结果。