Hassan Heba M, Fadel Mai A, Soliman Mohamed A
Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center ARC, Dokki, Giza, Egypt.
Pharmacology and Pyrogen Unit, Department of Chemistry, Toxicology and Food Deficiency, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, Egypt.
Vet World. 2020 Nov;13(11):2338-2345. doi: 10.14202/vetworld.2020.2338-2345. Epub 2020 Nov 5.
Lipopolysaccharide (LPS) is an integral part of the outer cell membrane complex of Gram-negative bacteria. It plays an important role in the induction and stimulation of the immune system. Various LPS purification protocols have been developed. However, analysis of their efficacy is limited by contamination during downstream applications or the public health hazard of LPS. The aim of this study was to evaluate a modified method for extracting LPS as well as assess the purity of the extracted LPS by high-performance liquid chromatography (HPLC) analysis. Further, we evaluated its immunopotentiating function by measuring the relative RNA expression levels of splenic immune-related genes such as interleukin 1β (IL-1β) and interferon-γ (IFN-γ), after intramuscular injection of increasing concentrations of the extracted LPS in specific pathogen-free (SPF) chick.
Isolation, identification, and serotyping of Typhimurium were performed using chicken flocks. We then performed molecular typing of isolates using conventional polymerase chain reaction (PCR). A new protocol for purification of LPS from isolate (. Typhimurium) was conducted. HPLC analysis of the extracted LPS in the current study was compared to existing methods. An study was performed to evaluate the ability of LPS to induce an immune response by measuring relative IFN-γ and IL-1β gene expression after injecting increasing concentrations of the extracted LPS into SPF chicks.
Isolation and serotyping revealed that was of the serovar Typhimurium. Confirmation was conducted by molecular typing through conventional PCR. Fractionation of the LPS extract by HPLC revealed a high degree of purity comparable with standard commercial LPS. These results demonstrate the high purity of extracted LPS based on our modified method using propanol and sodium hydroxide mixture. Intramuscular injection of the extracted LPS in 22 day-old SPF chicks, compared to the negative control, revealed significant upregulation of IFN-γ and slight downregulation of IL-1β.
The new modified method can be used for high purity LPS extraction and demonstrates effective immunopotentiating activity.
脂多糖（LPS）是革兰氏阴性菌外细胞膜复合物的一个组成部分。它在免疫系统的诱导和刺激中发挥重要作用。已经开发了各种LPS纯化方案。然而，它们的功效分析受到下游应用过程中的污染或LPS对公共健康危害的限制。本研究的目的是评估一种改良的LPS提取方法，并通过高效液相色谱（HPLC）分析评估提取的LPS的纯度。此外，在特定病原体-free（SPF）雏鸡中肌肉注射浓度递增的提取LPS后，通过测量脾脏免疫相关基因如白细胞介素1β（IL-1β）和干扰素-γ（IFN-γ）的相对RNA表达水平，我们评估了其免疫增强功能。
使用鸡群进行鼠伤寒沙门氏菌的分离、鉴定和血清分型。然后我们使用常规聚合酶链反应（PCR）对分离株进行分子分型。进行了一种从分离株（鼠伤寒沙门氏菌）纯化LPS的新方案。将本研究中提取的LPS的HPLC分析与现有方法进行比较。进行了一项研究，通过在向SPF雏鸡注射浓度递增的提取LPS后测量相对IFN-γ和IL-1β基因表达，评估LPS诱导免疫反应的能力。
分离和血清分型显示为鼠伤寒血清型。通过常规PCR进行分子分型予以确认。通过HPLC对LPS提取物进行分级分离显示出与标准商业LPS相当的高纯度。这些结果证明了基于我们使用丙醇和氢氧化钠混合物的改良方法提取的LPS具有高纯度。与阴性对照相比，在22日龄SPF雏鸡中肌肉注射提取的LPS显示IFN-γ显著上调，IL-1β略有下调。
新的改良方法可用于高纯度LPS提取，并显示出有效的免疫增强活性。
