The presence of infectious extracellular Francisella tularensis subsp. novicida in murine plasma after pulmonary challenge.
The presence of infectious extracellular Francisella tularensis subsp. novicida in murine plasma after pulmonary challenge.
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存在传染性细胞外土拉弗朗西斯菌亚种。
DOI：
10.1007/s10096-007-0434-x
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
Arulanandam,BP
中科院分区：
文献类型：
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作者：
Yu,J-J;Raulie,EK;Murthy,AK;Guentzel,MN;Klose,KE;Arulanandam,BP
图拉氏方济氏菌是一种革兰氏阴性的兼性胞内细菌，是人畜共患病图拉热症的病原体[1]。由于其低感染剂量和高死亡率，图拉氏丝虫被认为是一种潜在的生物武器[2]。图拉氏丝孢霉可分为几个亚种，包括与人类疾病相关的亚种：图拉氏丝孢霉亚种。Tularensis(A型)和图拉氏亚种(F.tularensis subsp.Holarctica(B型)和F.novicida和F.mediasiatica[3]。然而，我们关于方济氏菌的致病机制和对感染的免疫反应的大部分知识都来自于对图拉氏原虫和新氏原藻的研究[4-6]。这两种微生物在人类身上都得到了减毒，而在老鼠身上却保持了毒力。众所周知，方济各氏菌可以进入宿主细胞并在宿主细胞内复制，而破译巨噬细胞内生长的机制一直是一个强有力的研究重点。然而，人们对弗朗西塞氏菌的胞外相知之甚少。图拉氏丝虫是从受感染的动物和人类的血液中培养出来的，尽管报告的病例很少[7，8]。具体地说，Long等人[8]证明，静脉感染72小时后从小鼠制备的血浆中可以恢复LVS。这些细胞外细菌在血液中的LV总量中所占比例不到1%。然而，Forestal等人最近的一项研究[9]表明，经皮内或鼻腔感染的小鼠血浆中有更高比例(50%)的胞外弗朗西斯杆菌(A型Schu S4和LVS)。在这里，我们独立地证实，在肺图拉热症小鼠模型中，经鼻内新城疫霉菌攻击后，血浆中存在胞外杂交菌[6]。此外，我们证明了血浆中的胞外杂交菌具有高度的传染性，这一阶段可能有助于在肺部感染后将细菌从肺迅速传播到肝脏。新城疫杆菌U112株与图拉氏F菌A和B型[10]具有高度的抗原性和遗传相似性，并且对LD50约为10个集落形成单位(CFU)的小鼠具有高毒力[6]。在这项研究中，我们监测了新城疫霉菌在小鼠体内的复制和传播。细菌在37℃添加0.1%半胱氨酸的胰酶大豆发酵液(TSB)中生长。用胰酶大豆琼脂(TSA)连续稀释法测定接种物的浓度。雌性BALB/c小鼠(6-8周龄，来自英国巴尔港杰克逊实验室)接受400 CFU(40 LD50)的刺激。所有的动物护理和实验程序都遵循德克萨斯大学圣安东尼奥分校的机构动物护理和使用委员会的指导方针进行。肺和肝脏的细菌载量通过TSA上的组织匀浆平板进行评估。在肺攻击的24小时内，在肺中观察到大量的新城疫霉菌，约1×107CFU/g组织，48h增加到1×108CFU/g组织，然后在72小时下降到1×106CFU/g组织。在感染小鼠的肝脏中，细菌负荷逐渐增加，从24小时的100CFU/g组织上升到72小时的约1×107CFU/g组织(图1a)。这些结果表明，肺部暴露后，新城疫霉菌从肺部扩散到次级器官。为了确定迅速扩散到次级器官的可能机制，分别在24、48和72小时从眼眶神经丛采集血液。
Francisella tularensis is a gram-negative, facultative intracellular bacterium and is the causative agent of the zoonotic disease tularemia [1]. F. tularensis has been considered a potential biological weapon due to its low infectious dose and high mortality rate [2]. F. tularensis can be classified into several subspecies, including those relevant to human disease: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B); F. novicida and F. mediasiatica [3]. However, most of our knowledge about the pathogenesis of Francisella and the immune responses to the infection have come from studies of F. tularensis LVS (derived from holartica) and F. novicida [4–6]. Both organisms are attenuated in humans, while retaining virulence in mice. It is well established that Francisella enters and replicates within host cells, and a strong research focus has been on deciphering the mechanisms for intramacrophage growth. However, little is known about the extracellular phase of Francisella. F. tularensis has been cultured from blood of infected animals and humans, although the reported cases have been rare [7, 8]. Specifically, Long et al.[8] demonstrated that LVS could be recovered from plasma prepared from mice 72 h after intravenous infection. These extracellular bacteria accounted for less than 1% of total LVS within the blood. However, a recent study by Forestal et al.[9] indicated a much higher percentage (> 50%) of extracelluar Francisella (type A SCHU S4 and LVS) within plasma of mice infected intradermally or intranasally. Here, independently, we confirm the presence of extracellular Francisella in plasma after intranasal F. novicida challenge in a murine model of pulmonary tularemia [6]. Moreover, we demonstrate that the extracellular Francisella in plasma is highly infectious and that this phase may contribute to the rapid dissemination of the bacterium from lungs to the liver after pulmonary infection.F. novicida strain U112 shares a high degree of antigenic and genetic similarity with F. tularensis type A and B [10] and is highly virulent in mice with a LD50 of approximately 10 colony forming units (CFU) by the intranasal (in) route [6]. In this study, we monitored the replication and dissemination of F. novicida in mice. Bacteria were grown at 37 C in Trypticase Soy Broth (TSB) supplemented with 0.1% cysteine. The concentrations of the bacterial inocula were determined by serial dilution plating on Trypticase Soy Agar (TSA). Female BALB/c mice (6–8 weeks old, obtained from the Jackson Laboratory, Bar Harbor, ME) were challenged in with 400 CFU (40 LD50). All animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines of the University of Texas at San Antonio. Bacterial loads in the lungs and livers were assessed by plating tissue homogenates on TSA. Within 24 h of pulmonary challenge, a large number of F. novicida, approximately 1× 107 CFU/g tissue, was observed in the lungs, which increased to 1× 108 CFU/g tissue by 48 h and then decreased to 1× 106 CFU/g tissue by 72 h. In the livers of infected mice, there was a progressive increase in bacterial load with levels rising from 100 CFU/g tissue at 24 h to approximately 1× 107 CFU/g tissue by 72 h (Fig. 1 a). These results demonstrate dissemination of F. novicida from the lungs to the secondary organs after pulmonary exposure. To determine a probable mechanism for the rapid spread to the secondary organs, blood was collected from the orbital plexus at 24, 48 and 72 h