BioVector® 胞内劳森菌标准菌株说明书

BioVector® Lawsonia intracellularis Standard Strain Manual

第一部分 中文说明

一 产品基本信息与遗传学背景

• 菌株名称：胞内劳森菌（Lawsonia intracellularis）。

• 物种分类：细菌界（Bacteria），变形菌门（Pseudomonadota），$\delta$-变形菌纲（Deltaproteobacteria），脱硫振子菌目（Desulfovibrionales），脱硫振子菌科（Desulfovibrionaceae），劳森菌属（Lawsonia）。

• 核心生物学与生理学特征：

  • 专性胞内寄生菌（Obligate Intracellular Bacterium）：胞内劳森菌是一种革兰氏阴性（Gram-negative）、弯曲或微弯的微需氧弯曲杆菌。该菌具有极度苛刻的寄生特性，在宿主细胞外完全无法生长和繁殖。在体内，它特异性地寄生在哺乳动物（尤其是猪和马）的小肠（回肠）隐窝上皮细胞的细胞质内。

  • 增殖性肠病（PPE）的唯一病原体：它是引发猪增殖性肠病（Porcine Proliferative Enteropathy, PPE，俗称“回肠炎”）的特异性病原体，临床表现为肠壁上皮细胞恶性增生、增厚，导致严重的出血性或坏死性肠炎。

• 来源历史：通常分离自患有急性或慢性回肠炎的病猪回肠黏膜病变组织。通过将其接种于体外培养的大鼠小肠上皮细胞（IEC-18）或人类结肠癌细胞（McCoy细胞）中，在微需氧条件下进行特殊的分离与传代维持。

• 生物安全级别：2级（BSL-2）。涉及活菌扩增及受感染细胞系的操作，必须在二级生物安全柜内严格按照厌氧/微需氧胞内菌规范执行。

二 核心科研价值与转化医学应用

胞内劳森菌是兽医学、比较医学及肠道黏膜免疫学研究中公认极难培养但危害巨大的标杆性病原体：

1. 猪回肠炎发病机制与肠道增生研究：用于深入探究该菌如何通过特定分泌系统（如三型分泌系统 T3SS 或相关效应蛋白）注入宿主细胞，如何激活 Wnt/beta-catenin 等信号通路进而阻断小肠隐窝上皮细胞的分化，并异常驱动其进行不受控制的恶性增殖。

2. 兽用疫苗与抗生素药物筛选（Vaccine & Drug Screening）：是评价针对 PPE 的新型灭活疫苗、亚单位疫苗及活疫苗效能的关键抗原株；同时用于体外筛选对胞内菌敏感的抗生素（如泰乐菌素、泰万菌素、渥尼妙林等）的胞内杀菌动力学。

3. 分子诊断质控物（Diagnostics & Quality Control）：作为官方标准的参考品，广泛用于开发和校准检测猪/马回肠炎的常规 PCR、实时荧光定量 qPCR 试剂盒、原位杂交（ISH）探针以及血清学 ELISA 诊断芯片。

三 实验室菌株复苏、细胞协同培养与冷冻保存标准步骤

由于胞内劳森菌是专性胞内寄生菌，严禁将其接种于任何普通细菌培养基（如血平板、LB肉汤等）上。复苏和传代必须依赖活的宿主细胞系。

1. 宿主细胞准备与微需氧环境配置

• 推荐宿主细胞：大鼠小肠上皮细胞（IEC-18, ATCC CRL-1589）或 McCoy 细胞（ATCC CRL-1696）。

• 细胞培养基：含 10% 优质胎牛血清（FBS）的高糖 DMEM 或 McCoy's 5A 培养基。

• 核心微需氧参数（关键）：胞内劳森菌的生长对氧气浓度极度敏感。混合培养必须置于特殊的微需氧环境（通常为：8.0% - 10.0% $O_2$、8.0% - 10.0% $CO_2$，其余为 $N_2$）中进行，普通 5% $CO_2$ 三气孵箱或完全严格厌氧环境均会导致细菌死亡。

2. 冻存菌种的复苏与细胞接种（Thawing & Inoculation）

1. 提前一天将 IEC-18 或 McCoy 细胞接种于 T25 培养瓶中，使其在复苏当天达到约 30% - 50% 的低融合度（此时细胞处于旺盛分裂期，最有利于劳森菌的入侵与体内繁殖）。

2. 从液氮中取出胞内劳森菌的裂解冻存管，立即投入 37 摄氏度水浴中急速融化。

3. 消毒冻存管外部，移入生物安全柜。将菌液吸出，直接加入到长有低融合度宿主细胞的培养瓶中。

4. 迅速转入 37 摄氏度专用微需氧孵箱中孵育 3 至 5 小时（或过夜），以利于细菌充分吸附并侵入细胞。

5. 次日轻轻吸除含有残余冻存液的旧培养基，更换为新鲜的细胞培养基，重新送回微需氧孵箱中连续培养 5 至 7 天。

3. 菌株传代与细菌释放（Passaging & Harvesting）

胞内劳森菌在胞内过度繁殖会导致宿主细胞裂解并释放。日常传代需要人工辅助将其从宿主细胞中释放出来：

• 细胞裂解释放法：

  1. 当培养至第 6-7 天时，吸除旧培养基。加入无菌去离子水或低渗 KCl 溶液（0.1%），在 37 摄氏度下孵育 10 分钟使宿主细胞溶胀。

  2. 使用移液管对瓶壁进行剧烈吹打，或者通过无菌注射器针头（如 21G 针头）来回抽吸 3-5 次，依靠机械剪切力彻底破碎宿主细胞，释放出胞内的劳森菌。

  3. 将裂解液在 500 × g 离心 3 分钟以沉淀未破碎的细胞碎片；收集含有细菌的上清液。

• 接种传代：将收集到的细菌上清液，按 1:3 至 1:5 的比例直接加到新准备好的低融合度（30%）宿主细胞平板/瓶中，继续置于微需氧环境培养。

4. 菌株长期冷冻保存（Cryopreservation）

• 冻存保护液：常规细胞冻存液（90% FBS + 10% DMSO）或者特殊的蔗糖-磷酸盐-谷氨酸缓冲液（SPG）补充 10% DMSO。

• 冻存操作：

  1. 收集感染率极高（细胞内充满密密麻麻菌体）的混合培养物。

  2. 可选择直接消化收集带有细菌的完整宿主细胞，或者通过上述裂解法收集游离菌体。

  3. 加入冻存液重悬后，迅速分装入冻存管，立即投入标准程序降温盒中。

  4. 置于 -80 摄氏度过夜后，次日必须迅速转移至 -196 摄氏度液氮罐 中进行长期冷冻保存。

Part 2 English Section

I General Information and Genetic Architecture

• Organism Name: Lawsonia intracellularis Reference Strain.

• Taxonomic Classification: Domain Bacteria, Phylum Pseudomonadota, Class Deltaproteobacteria, Order Desulfovibrionales, Family Desulfovibrionaceae, Genus Lawsonia, Species Lawsonia intracellularis.

• Core Biological and Physiological Profiles:

  • Obligate Intracellular Bacterium: Lawsonia intracellularis is a Gram-negative, curved, or microaerophilic rod-shaped bacterium. This pathogen possesses a unique physiological property: it is completely unable to replicate outside of a supportive eukaryotic host cell. In vivo, it specifically targets and colonizes the cytoplasm of actively dividing intestinal (ileal) crypt epithelial cells of mammalian species, primarily pigs and horses.

  • Etiological Agent of PPE: It stands as the definitive causative pathogen of Porcine Proliferative Enteropathy (PPE, commonly referred to as ileitis). PPE is characterized by abnormal hyperplastic proliferation of infected enterocytes, resulting in a marked thickening of the intestinal wall, causing severe hemorrhagic or necrotic enteritis.

• Source Isolation Provenance: Typically recovered from the diseased ileal mucosa mucosa harvested from pigs exhibiting acute or chronic porcine proliferative enteropathy. Cultivation and maintenance are achieved by co-culturing the bacterium inside rat small intestinal epithelial cell matrices (IEC-18) or human colon cancer lines (McCoy cells) under custom microaerophilic conditions.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) parameters. Any workflows involving active cellular infection matrices require certified Class II biosafety containment frameworks and microaerophilic atmospheric gas systems.

II Strategic Research Value and Translational Fields

Lawsonia intracellularis is universally recognized as one of the most fastidious yet economically devastating pathogens within veterinary medicine and mucosal gastroenterology:

1. Mechanisms of Proliferative Enteropathy & Cell-Cycle Subversion: Deployed to map how its dedicated secretion apparatus (such as Type III Secretion Systems [T3SS] or related effector loops) alters eukaryotic homeostatic machinery, specifically up-regulating pathways like Wnt/beta-catenin to arrest normal enterocyte differentiation and force host cell hyperproliferation.

2. Veterinary Vaccine Development and Antimicrobial Profiling: Serves as the index target for verifying the structural potency and protective parameters of novel inactivated, subunit, or modified live vaccines targeting PPE. It is also used to benchmark the intracellular bactericidal kinetics of targeted macrolides and pleuromutilins (e.g., tylosin, tylvalosin, valnemulin).

3. Molecular Diagnostic Standard Verification: Acts as the official positive metrology baseline to optimize and validate clinical qualitative/quantitative PCR assays (qPCR), in vitro immunohistochemistry setups, in situ hybridization (ISH) diagnostic probes, and serological enzyme-linked immunosorbent assay (ELISA) monitoring kits.

III Thawing, Cell Co-Culture, Passaging, and Cryopreservation Routines

Because Lawsonia intracellularis is an obligate intracellular pathogen, never plate this bacterium onto standard cell-free agar formulas (such as blood agar, nutrient agar, or LB broth). Revitalization requires viable eukaryotic host cellular matrices.

1. Host Cell Configuration and Ambient Microaerophilic Parameters

• Recommended Host Cell Lines: Rat intestinal epithelial cells (IEC-18, ATCC CRL-1589) or McCoy cells (ATCC CRL-1696).

• Cultivation Media: High-glucose DMEM or McCoy's 5A matrix, supplemented with 10% premium Fetal Bovine Serum (FBS).

• Critical Atmospheric Gas Balance (Mandatory): Proliferation dictates narrow microaerophilic ranges. Co-cultures must be incubated within specialized microaerophilic environments (optimized at 8.0%–10.0% $O_2$, 8.0%–10.0% $CO_2$, with the balance configured as $N_2$). Standard 5% $CO_2$ incubators or absolute anaerobic configurations will rapidly cause irreversible loss of bacterial viability.

2. Cryovial Thawing and Cellular Infection Protocol

1. One day prior to thawing, seed the target host cells (IEC-18 or McCoy) into standard T25 flasks to establish a low confluency of approximately 30%–50% on the day of infection. (Dividing enterocytes provide the optimal targets for bacterial endocytosis and intracellular division).

2. Retrieve the Lawsonia intracellularis frozen lysate from liquid nitrogen storage and submerge it instantly into a 37 degree Celsius water bath. Thaw rapidly within 1 minute.

3. Swab the vial with 70% ethanol, transit to a biosafety cabinet, and transfer the thawed bacterial suspension directly into the flask containing the low-confluency host cell layer.

4. Promptly return the infected flask to the 37 degree Celsius microaerophilic incubator for 3 to 5 hours (or overnight) to optimize bacterial attachment and cell entry kinetics.

5. The following day, gently aspirate the media containing residual cryoprotectant elements, replenish with fresh cell culture media, and return to the microaerophilic workspace for 5 to 7 days of continuous incubation.

3. Subculturing and Mechanical Bacterial Release

Overwhelming intracellular bacterial loads eventually lead to spontaneous cell lysis. To achieve efficient passaging, manual harvesting is required to extract intact bacteria from intact cells:

• Hypotonic Mechanical Lysis Protocol:

  1. At day 6–7 post-infection, aspirate the spent growth medium. Introduce sterile deionized water or a hypotonic KCl solution (0.1%) and incubate at 37 degree Celsius for 10 minutes to induce host cell swelling.

  2. Aggressively pipette the fluid against the flask baseline or pass the slurry back and forth through a sterile 21-gauge syringe needle 3–5 times. The resulting mechanical shear forces disrupt the weakened host membranes, releasing intact bacteria.

  3. Centrifuge the crude lysate at 500 × g for 3 minutes to sediment large cellular debris; carefully collect the bacteria-rich supernatant fluid.

• Inoculation Routine: Blend the collected bacterial supernatant directly into fresh flasks pre-seeded with 30% confluent host cells, and store under microaerophilic parameters.

4. Cryopreservation Protocol

• Freezing Medium Matrix: Standard cell cryopreservation matrix (90% FBS + 10% DMSO) or a specialized sucrose-phosphate-glutamate (SPG) transport buffer fortified with 10% analytical-grade DMSO.

• Freezing Routine: Harvest heavily infected cultures showing peak intracellular densities. Researchers can freeze down the intact host cells carrying the bacteria, or execute the hypotonic mechanical lysis routine detailed above to freeze isolated bacterial fractions. Resuspend the pellet in freezing media, aliquot into cryovials, and place immediately inside an isopropyl alcohol controlled-rate freezing box. Keep at -80 degree Celsius overnight, and shift into the liquid nitrogen vapor phase (-196 degree Celsius) the next day for long-term preservation.

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