这项工作得到了中国国家重点研究开发计划 (2017YFD0500102) 的资助;中国国家自然科学基金 [31572544];兽医病因生物学国家重点实验室 [SKLVEB2016KFKT005];上海农业应用技术发展项目, 中国 [G2016060201]。

小鼠感染实验由中国江苏省实验动物监测委员会批准, 并在南京农业大学实验动物中心 (许可证编号: SYXK (Su) 2017-0007) 上进行。
1. 细菌的制备
注:猪血清2型毒株 P1/7 是从患病的猪与脑膜炎19分离出来的。P1/7 菌株生长在托德-休伊特汤 (THB, 公式每公升 THB: 心脏输液, 3.1 克; neopeptone, 20.0 克; 葡萄糖, 2.0 克; 氯化钠, 2.0 克; 磷酸二钠, 0.4 克; 碳酸钠, 2.5 克) 和电镀在托德-威特特琼脂 (临屋区, 公式每公升心脏输液, 3.1 克;neopeptone, 20.0 克;葡萄糖, 2.0 克;氯化钠, 2.0 克;磷酸二钠, 0.4 克;碳酸钠, 2.5 克;琼脂, 15.0 克) 在37°c 和 5% CO2。
<ol><li>应变 P1/7 浮游细胞的采集<ol>
<li>收集5毫升浮游细胞从中对数阶段培养 (OD600= 0.6), 离心机为3分钟在 8000 x g, 然后洗涤3次与 PBS。</li>
		<li>并用重悬的细胞与5毫升25% 甘油在 THB, 整除成5管, 然后存储在-80 摄氏度。</li>
	</ol>
</li>
	<li>应变 P1/7 生物膜状态细胞的采集<ol>
<li>采取20毫升隔夜文化和增加180毫升新鲜的 THB;将稀释过的文化分成10圆的培养板, 然后把盘子放在37摄氏度的孵化器中, 5% CO2为 24 h。</li>
		<li>孵化后, 轻轻摇动盘子, 并用重悬不粘在盘子上的细菌, 然后用吸入法丢弃上清。 注意: 轻轻摇动盘子, 避免 resuspending 沉淀物。</li>
		<li>加入5毫升的 PBS, 以收获生物膜状态细胞, 完全并用重悬沉积物, 然后将样品转移到新管上。</li>
		<li>油脂实验生物膜状态细胞从最后一步以以下参量:60 W, 4 个周期, 5 s 在和十年代。</li>
		<li>离心机为3分钟在 8000 x g 和存放上清在-80 °c。 注: 上清可能含有生物膜成分, 可用于在感染前并用重悬生物膜细菌, 如动物实验所述 (步骤 3)。</li>
		<li>在 THB 中并用重悬10毫升25% 甘油的沉淀物, 然后将生物膜状态细胞储存在-80 摄氏度。</li>
	</ol>
</li>
</ol>2. 扫描电镜 (SEM) 分析
<ol><li>用4% 多聚甲醛 12-24 小时在4摄氏度固定收集的生物膜状态细胞和浮游细胞。</li>
	<li>用蒸馏水快速冲洗样品, 并以乙醇 (25%、50%、70%、90% 和 100%) 的浓度增加为30分钟, 在每个溶液中依次脱水样品。</li>
	<li>将干试样粘附在带有双面胶带的金属夹上, 最后将其涂在带有金和钯的蒸发器中。</li>
	<li>用扫描电子显微镜观察样品, 使用以下参数: EHT (超高压) = 10 伏;WD (工作距离) = 7.0 或8.0 毫米;放大 = 5000 x;信号 A = SE1。</li>
</ol>3. 动物实验
<ol><li>在本研究中使用 SPF 6 周的雌性 BALB/c 小鼠。通过颅内 subarachnoidal 感染的所有小鼠, 其注射部位位于 bregma 3.5 毫米延髓。Chiavolini 和al17清楚地描述了注射部位的立体定向坐标。<ol>
<li>病理学分析, 感染两组小鼠 (每组5只小鼠), 使用浮游细胞或生物膜状态细胞, 剂量为 3 x 107菌落形成单位 (CFU), 另有5只老鼠注射 PBS 作为控制。</li>
		<li>为检测 TLR2 和细胞因子在脑中的 mRNA 表达, 在 3 x 107 CFU 中使用浮游细胞或生物膜状态细胞感染两组小鼠 (每组6只小鼠)。</li>
		<li>通过电镀连续稀释来确定活菌的数量。</li>
		<li>弄死所有小鼠在12小时后感染后进行进一步的研究。 注: 在感染前必须清除在培养基中的甘油。从-80 °c 恢复的浮游细胞和生物膜状态细胞被洗涤3次与 PBS。然后, 浮游细胞悬浮与 PBS, 稀释到适当剂量的感染;生物膜状态细胞与储存的上清液悬浮 (从步 1.2.5) 和稀释到适当剂量的感染。感染量不应超过50µL。</li>
	</ol>
</li>
	<li>脑组织中 TLR2 和细胞因子 mRNA 表达的检测<ol>
<li>使用 rna 提取试剂盒从脑组织中提取总 RNA。<ol>
<li>对于每只老鼠, 使用全脑组织提取 RNA。把整个脑组织分成5管。服用多达100毫克的脑组织, 并添加1毫升的裂解液, 每管含有株溶藻基质提供的试剂盒。</li>
			<li>在6.0 的设置中, 在一个均质机上处理管, 然后离心管在 12000 x g 和4°c 为5分钟。</li>
			<li>离心后, 将上相转移到一个新的离心管。</li>
			<li>在室温下孵化转移样品5分钟;添加300µL 的氯仿, 漩涡十年代, 然后孵化5分钟室温。</li>
			<li>离心管在 12000 x g 和4°c 5 分钟转移上部阶段到新的管子。</li>
			<li>将500µL 的冷绝对乙醇添加到管中, 然后将其反转5次, 然后将样品贮存在-20 摄氏度, 至少为1小时。</li>
			<li>离心管在 12000 x g 和4°c 为15分钟和去除上清。用500µL 冷75% 乙醇在 RNase 的 H2O 上清洗颗粒。</li>
			<li>除去乙醇, 空气干燥的颗粒在室温下5分钟, 并并用重悬 RNA 在100µL RNase 无2O。</li>
			<li>在室温下孵化 rna 5 分钟, 用 rna bioanalyzer 测定 rna 浓度。</li>
		</ol>
</li>
		<li>进行 cDNA 合成, 包括消除基因组 DNA (gDNA), 使用 thermocycler 与反向转录 (RT) 试剂盒。<ol>
<li>将1µg 的 RNA 添加到含有2µL 5x gDNA 消除缓冲液的管中, 1 µL gDNA 消除酶和 RNase 的 H2O, 直到体积达到10µL. 在2摄氏度孵育42分钟。</li>
			<li>添加10µL 的主混合 (1 µL 的 rt 酶混合 I, 1 µL 的 rt 底漆混合, 4 µL 5x 缓冲器 2, 4 µL RNase 免费 H2O) 到反应解决方案从最后一步, 然后轻轻混合。立即进行 RT 反应:37 °c 为15分钟, 然后85°c 5 s。</li>
			<li>使用 SYBR qPCR 试剂盒实时 pcr 机进行定量实时 pcr (RT qPCR) 分析。添加10µL 的2x 酶, 0.8 µL 的前底漆, 0.8 µL 的反向底漆, 0.4 µL 50x 火箭参考染料 II, 2 µL 的 cDNA 模板, 和 RNase 免费 H2O, 直到总容量20µL。</li>
			<li>使用以下热参数运行每个样本: 三十年代在94°c, 跟随40个周期 5 s 在95°c 和三十四年代在60°c, 以十五年代的最后阶段在95°c, 1 分钟在60°c, 十五年代在95摄氏度。qPCR 的底漆列于表 1。管家基因B2m和18s rRNA被用作内部控制。</li>
		</ol>
</li>
		<li>根据 2-ΔΔCt方法计算相对褶皱变化。</li>
	</ol>
</li>
	<li>
组织学切片观察
	<ol>
<li>24 h 固定10% 福尔马林后, 将固定的脑组织重建成适当大小的组织块, 在 2-3 毫米。</li>
		<li>把纸巾放在嵌入的卡带里, 浸泡在一个新的固定液中, 用自动真空脱水脱水。</li>
		<li>取出固定组织, 并在每种溶液中依次脱水以增加乙醇 (70%、80%、95%、95% 和 100%) 的浓度。</li>
		<li>将该样品与二甲苯 Transparentize 15 分钟, 将其浸在液体石蜡中60°c 90 分钟, 然后用嵌入机嵌入。</li>
		<li>将石蜡块嵌入组织切成3µm 片, 在45摄氏度的水上平铺切片, 然后在空气中晾干。</li>
		<li>在60摄氏度孵育3小时的切片, 用 haematoxylin 和伊红 (他) 方法染色, 并用中性胶封住。</li>
		<li>用光学显微镜观察大脑的组织病理学变化。采用四点分级系统评价组织学变化。使用未配对的t检验进行统计分析。 充血/出血:缺席, 评分 0;小面积的局灶充血/出血, 评分 1;大面积的局灶充血/出血, 或多处小面积的局灶充血/出血, 评分 2;多达三个大焦距充血/出血部位, 得分 3;弥漫性充血/出血, 评分4。 坏死:缺席, 评分 0;病灶坏死, 评分 1;弥漫性发生 1-10 坏死细胞, 评分 2;汇合坏死灶, 评分 3;广泛的汇合坏死, 评分4。 炎症细胞浸润:缺席, 评分 0;少数和焦点渗透, 得分 1;多焦浸润或渗透形成聚集, 得分 2;最多三集, 分数 3;超过四集, 分数4。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

这里描述的颅内 subarachnoidal 感染途径与其他感染途径相比有明显的优势。它允许调查人员研究宿主-细菌相互作用和细菌成分对宿主免疫应答的影响直接在大脑, 这模仿细菌进入中枢神经系统。因此, 这种感染途径可以扩展, 以调查其他细菌引起的脑膜炎机制。此外, 还可用于检测药物对细菌性脑膜炎的疗效。
为了获得良好的结果, 使用这个模型, 以下关键步骤是明确的。生物膜的形成需要在实验条件下进行检验。本研究采用扫描电镜分析。其他方法也可用于检测生物膜的形成, 如组织培养板法和管法21。感染前一天, 浮游细胞和生物膜状态细胞的整除数需要从-80 摄氏度解冻, 以确定 CFU。第二天, 细菌应稀释到适当剂量根据 CFU 的感染。模拟感染的控制组注射 PBS 需要包括在内, 由模拟感染的对照组的小鼠在感染实验期间不应表现出任何症状。不同菌株可能具有不同的传染性剂量, 因此强烈建议进行初步实验以确定适当的感染剂量。在本研究中, 根据我们的初步实验, 对 3 x 107 CFU 的感染进行了剂量分析。这一剂量的生物膜细菌能够诱导5只小鼠48小时感染后的严重脑膜炎和随后死亡的初步实验。
猪血脑或血脑脊液屏障的破坏是造成脑膜炎22、23、24的重要一步。颅内 subarachnoidal 感染的途径不适用于评价猪的能力, 以此来突破这些障碍。其他感染途径, 如 ip, 静脉注射, 或 i.n., 可用于实现这一目标。
在这里, 我们首先表明, S. 猪的生物膜有助于诱导脑膜炎, 利用颅内 subarachnoidal 感染途径。这种感染模型不仅有助于进一步了解猪链球菌脑膜炎的发病机制, 而且适用于研究其他细菌引起的脑膜炎的致病机理和新药对细菌性脑膜炎的疗效。

猪链球菌(猪) 是全世界猪的主要细菌病原体, 造成严重疾病, 包括脑膜炎、肺炎、败血症、心内膜炎和关节炎1。它也是一个新兴的人畜共患病的特工。到目前为止, 据报道, 九型血清型可能导致人类感染, 包括血清型 2, 4, 5, 9, 14, 16, 21, 24, 312,3,4。在人类和猪中, 脑膜炎是感染猪的主要临床征象之一。在越南和泰国,美国猪是5成人脑膜炎的主要病因。微生物生物膜是粘在一起的微生物, 并集中在界面上;它们对细菌的毒力、不同环境下的生存和耐药性5至关重要。生物膜通常被一个细胞外基质所包围, 通常含有多糖、蛋白质和 DNA6。后者能够诱发宿主炎症反应和细胞因子的产生7。在以前的研究中, 生物膜的形成已被用于链球菌脑膜炎。生物膜有助于罗非鱼鱼模型中的链球菌脑膜炎链球菌, 生物膜的形成已经通过腹腔内接种8在体内的脑组织和脑膜表面周围显露出来。在脑膜炎期间,肺炎链球菌处于生物膜状状态, 而这种生物膜状态下的细菌更有效地诱发了老鼠感染模型9中的脑膜炎。此外, 在我们以前的研究中, 在小鼠脑中与猪的相关的生物膜状态通过生存分析导致细菌毒力10。然而, 直接证据的生物膜参与的猪链球菌脑膜炎需要进一步的调查。
采用腹腔 (ip)11、鼻腔 (i.n.)12、静脉注射 (静脉注射)13和 intracisternal (中) 感染14条的方法, 研制出了猪鼠感染动物模型.15,16. 然而, 感染的 ip、i.n. 和静脉注射途径不适合于直接在脑部研究在脑膜炎中猪的表面成分的作用。这些包括生物膜胞外基质。虽然在美国猪感染中使用了内源接种, 但是这些文件中没有描述精确的注射部位。与此相反, 颅内 subarachnoidal 接种的注射部位的立体定向坐标在前一项研究中已经明确描述过17。这允许容易地承认接种点和更加简单的实验性协议。此外, 颅内 subarachnoidal 感染的途径, 模拟细菌进入中枢神经系统从鼻窦或中耳17, 中耳和脑膜炎的关系, 由猪.已经证明了马德森et al18。此外, 通过应用颅内 subarachnoidal 感染的小鼠, 我们已经证明了猪的小 RNA rss04 导致脑膜炎在我们以前的研究10。
在本研究中, 采用颅内 subarachnoidal 感染途径对小鼠进行了研究, 探讨了生物膜在猪链球菌脑膜炎中的作用。通过这种感染途径, 小鼠感染了浮游细胞或猪的生物膜状态细胞。生物膜对小鼠脑组织中 TLR2 和细胞因子表达的组织病理学分析清楚地表明,猪的生物膜对脑膜炎有促进作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td height="46">Todd Hewitt Broth(THB)</td>
			<td style="width:225px;">Becton, Dickinson and Company</td>
			<td style="width:115px;">DF0492078</td>
			<td style="width:304px;">Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td style="width:225px;">DSBIO</td>
			<td style="width:115px;">16C0050</td>
			<td style="width:304px;">Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:184px;">Milli-Q Reference Water Purification System</td>
			<td style="width:225px;">Merck KGaA</td>
			<td style="width:115px;">Z00QSVCUS</td>
			<td style="width:304px;">Without Dnase/ Rnase</td>
		</tr>
		<tr height="21">
			<td>NaCl</td>
			<td style="width:225px;">Tianjin Kemiou Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10019318</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>Na2HPO3</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009012-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>KCl</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009017-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="20">
			<td>KH2PO4</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009019-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="24">
			<td>KOH</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009014-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="42">
			<td>Glycerol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10010618</td>
			<td style="width:304px;">Diluted with equal volumu of purified water, autoclave at 121&#176; for 15 min</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">4% paraformaldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">80096675</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">25% Glutaraldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">30092436</td>
			<td style="width:304px;">10-fold diluted with purified water for fixation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ethanol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10009218</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10006818</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spctrophotometre</td>
			<td style="width:225px;">DeNovix Inc.</td>
			<td style="width:115px;">DS-11+</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ultrasound cell crusher</td>
			<td style="width:225px;">NingBo Scientz Biotechnology Co.,Ltd</td>
			<td style="width:115px;">JY96-IIN</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Centrifuge</td>
			<td style="width:225px;">Hitachi Koki Co., Ltd</td>
			<td style="width:115px;">CT15RE</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Refrigerator</td>
			<td style="width:225px;">Aucma Co., Ltd</td>
			<td style="width:115px;">DW-86L500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Scanning electron microscope</td>
			<td>Zeiss</td>
			<td style="width:115px;">EVO-LS10</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastRNA Pro Green Kit</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">#6045-050</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep-24 Instrument</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">116005500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Instrument for PCR</td>
			<td style="width:225px;">SensoQuest GmbH</td>
			<td style="width:115px;">1124310110</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantStudio 6 Flex</td>
			<td style="width:225px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">4485689</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">SYBR Premix Ex Taq II</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR820A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">PrimeScript RT reagent kit with gDNA Eraser</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR047A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Fully Enclosed Tissue Processor</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ASP200S</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Heated Paraffin Embedding Module</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">EG1150H</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Semi-Automated Rotary Microtome</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">RM2245</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Water bath for paraffin sections</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">HI1210</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Autostainer XL</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ST5010</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Agilent 2100</td>
			<td style="width:225px;">Agilent Technologies</td>
			<td style="width:115px;">G2939A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Optical microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:115px;">BX51</td>
			<td style="width:304px;"></td>
		</tr>
	</tbody>
</table>

intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model

猪链球菌不仅是全球猪的主要细菌病原体, 而且是一种新兴的人畜共患病剂。在人类和猪中, 脑膜炎是感染猪的主要表现。适当的感染模型是了解病原体致病机制的基本工具。研究了小鼠感染的几种途径, 探讨了猪感染的发病机制。然而, 腹腔, 鼻腔和静脉感染的途径不适合研究猪的表面成分在脑膜炎直接在脑中的作用, 如生物膜胞外基质。虽然 intracisternal 接种已被用于猪的感染, 但精确注射部位尚未描述。本文用小鼠模型描述了颅内 subarachnoidal 感染的途径, 探讨了生物膜在猪链球菌脑膜炎中的作用。通过位于 bregma 3.5 毫米延髓的注射部位, 将猪的浮游细胞或生物膜状态细胞直接注入小鼠蛛网膜下腔。用生物膜状态细胞注射小鼠脑组织中 TLR2 和细胞因子的病理学分析和 mRNA 表达明显表明,猪的生物膜在猪的脑膜炎中起着决定性的作用。这种感染途径明显优于其他感染途径, 允许研究宿主-细菌相互作用。此外, 它允许细菌成分对宿主免疫应答的影响直接在大脑被评估, 并模仿细菌进入中枢神经系统。这种感染途径可以扩展, 以调查其他细菌引起的脑膜炎机制。此外, 还可用于检测药物对细菌性脑膜炎的疗效。

在实验条件下对生物膜的形成进行了 SEM 分析。如图 1所示, 浮游细胞 (图 1a) 与生物膜状态细胞之间的生物膜形成有显著差异 (图 1B)。SEM 分析表明, 生物膜细菌在团簇和多层, 它们被包裹在细胞外基质, 而浮游细菌的密度更低, 主要分散单独。
生物膜胞外基质的成分, 如 DNA, 能够诱发宿主炎症反应和细胞因子的产生。由于 TLR2 和细胞因子 CCL2、IL-6 和 TNF α参与了与脑膜炎有关的脑炎症反应, 根据先前的研究10,11,20, 这些基因的 mRNA 表达比较在体内的老鼠感染的浮游细胞和那些感染的生物膜状态细胞。这样做是为了探讨生物膜对小鼠脑组织炎症反应的影响。如图 2所示, 在12小时感染后, 生物膜状态细胞与浮游细胞相比, TLR2、CCL2、IL-6 和肿瘤坏死因子α的表达明显较高。
感染生物膜细菌的老鼠比那些感染浮游细菌的小鼠表现出更严重的脑膜炎症状 (僵硬的姿势、共济失调或惊厥)。猪P1/7 损伤小鼠脑组织的组织学检查显示, 脑膜、大脑皮层、心室或间脑的充血/出血、坏死和炎症细胞浸润 (图 3).与感染浮游细菌的小鼠相比 (图 3E H), 在 12 H 感染后, 在感染生物膜细菌的小鼠脑组织中观察到更严重的病理变化 (图 3A D),包括大面积的充血/出血, 坏死, 或强烈的炎症细胞浸润。表 2记录了感染浮游和生物膜细菌的小鼠脑部的总病理变化。没有观察到由 PBS 注射的小鼠的病理改变和神经系统症状。这些数据清楚地表明,猪的生物膜有助于诱导脑膜炎的发生。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57658/57658fig1.jpg"> 图 1:应变 P1/7 浮游细胞和生物膜状态细胞的 SEM 图像.在 THB 培养基中培养细菌。SEM 分析表明, 浮游细胞 (A) 密度低得多, 主要分散, 而生物膜细菌 (B) 聚集在团簇和多层, 被包裹在细胞外基质中。<a href="/files/ftp_upload/57658/57658fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57658/57658fig2.jpg"> 图 2:生物膜激活 CCL2、IL-6、TNF α和 TLR2 在体内的小鼠脑组织中的 mRNA 表达 .每组六只 BALB 小鼠通过颅内 subarachnoidal 感染途径注射, 3 x 107 CFU 浮游细胞或生物膜状态细胞。小鼠在感染后12小时被安乐死。结果显示, 与浮游细胞感染小鼠相比, 生物膜状态细胞感染小鼠 mRNA 表达的褶皱变化。(A) 基因B2m被用作参考基因;(B) 基因18s rRNA被用作参考基因。对非配对t检验用于统计分析。** p ≤0.01, 和 p ≤0.05。<a href="/files/ftp_upload/57658/57658fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57658/57658fig3.jpg"> 图 3:感染 P1/7 浮游细菌和生物膜状态细菌的小鼠脑组织的组织学分析.由感染生物膜状态细菌的小鼠的脑样本;来自感染浮游细菌的老鼠的脑标本。a、B、E 和 F: 脑膜和大脑皮层;C 和 G: 心室;D 和 H: 间脑。Δ, 充血/出血;-坏死;0: 炎症细胞浸润。放大倍数 = 200X;刻度条 = 100 µm.<a href="/files/ftp_upload/57658/57658fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>底漆名称</td>
			<td>序列 (5 '-3 ')</td>
			<td>基因符号</td>
		</tr>
<tr>
<td>IL-6 转发</td>
			<td>CTTCCATCCAGTTGCCTTCT</td>
			<td>Il6</td>
		</tr>
<tr>
<td>IL-6 反转</td>
			<td>CTCCGACTTGTGAAGTGGTATAG</td>
			<td>Il6</td>
		</tr>
<tr>
<td>TLR2 转发</td>
			<td>CACTATCCGGAGGTTGCATATC</td>
			<td>Tlr2</td>
		</tr>
<tr>
<td>TLR2 反转</td>
			<td>GGAAGACCTTGCTGTTCTCTAC</td>
			<td>Tlr2</td>
		</tr>
<tr>
<td>CCL2 转发</td>
			<td>CTCACCTGCTGCTACTCATTC</td>
			<td>Ccl2</td>
		</tr>
<tr>
<td>CCL2 反转</td>
			<td>ACTACAGCTTCTTTGGGACAC</td>
			<td>Ccl2</td>
		</tr>
<tr>
<td>TNF-α向前</td>
			<td>TTGTCTACTCCCAGGTTCTCT</td>
			<td>Tnf</td>
		</tr>
<tr>
<td>TNF-α反转</td>
			<td>GAGGTTGACTTTCTCCTGGTATG</td>
			<td>Tnf</td>
		</tr>
<tr>
<td>β2m 转发</td>
			<td>GGTCTTTCTGGTGCTTGTCT</td>
			<td>B2m</td>
		</tr>
<tr>
<td>β2m 反转</td>
			<td>TATGTTCGGCTTCCCATTCTC</td>
			<td>B2m</td>
		</tr>
<tr>
<td>18s 前进</td>
			<td>GTAACCCGTTGAACCCCATT</td>
			<td>18s rRNA</td>
		</tr>
<tr>
<td>18s 反向</td>
			<td>CCATCCAATCGGTAGTAGCG</td>
			<td>18s rRNA</td>
		</tr>
</tbody></table>表 1:qPCR 的底漆.
<img alt="Table 1" src="/files/ftp_upload/57658/57658tbl2.jpg"> 表 2: 感染的小鼠脑组织病理学变化美国猪 P1/7 菌株.四点评分系统用于评估组织学变化, 如协议科3.3.7 所述。总分被计算为不同病理学变化的分数之和。平均值被计算为小鼠的总得分/数量。对非配对t检验用于统计分析。** ≤0.01。

Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

本文描述了小鼠颅内 subarachnoidal 感染的途径, 研究了生物膜在猪链球菌脑膜炎中的作用。这种感染模型也适用于研究其他细菌性脑膜炎的发病机制和新药对细菌性脑膜炎的疗效。

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

7.2K 次观看.  Nanjing Agricultural University. 本文描述了小鼠颅内 subarachnoidal 感染的途径, 研究了生物膜在猪链球菌脑膜炎中的作用。这种感染模型也适用于研究其他细菌性脑膜炎的发病机制和新药对细菌性脑膜炎的疗效。

视频: Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...