类鼻疽伯克霍尔德菌经鼻感染BALB/c小鼠模型的构建与评价

目的 构建类鼻疽伯克霍尔德菌（Burkholderia pseudomallei, BP）经鼻感染BALB/c小鼠动物模型,为后续类鼻疽菌的毒力研究和急性类鼻疽的致病机制研究提供可靠的动物模型。方法 采用经鼻主动吸入的感染途径,通过大体解剖、组织病理和组织匀浆计数菌落等方法观察类鼻疽伯克霍尔德菌感染小鼠的病理生理反应、脏器病理损伤和细菌定植情况,分析急性类鼻疽感染小鼠模型的生物学特征,并比较致病性类鼻疽临床株与非致病性类鼻疽泰国株（伯克霍尔德菌）感染BALB/c小鼠的不同表现。结果 急性类鼻疽菌经鼻感染模型中,BALB/c小鼠死亡多集中在感染后第3到第5天,3×10⁵~3×10⁶ CFU可作为急性类鼻疽BALB/c小鼠病死模型的合适攻毒剂量,而半数致死量约在3×10⁴~3×10⁵ CFU。大体解剖和组织HE染色均可见肺脏、脾脏和肝脏组织中形成脓肿或坏死病灶,其中在脾脏最明显,并与攻毒剂量呈正相关。类鼻疽菌感染的小鼠血液、肺脏、脾脏及肝脏中均发现类鼻疽菌定植,且定植量与组织特异性有关,血液中分离到的活菌浓度最高Log₂对数值：（10.28±0.34） CFU/mL],定植总量最高的脏器是肺脏Log₂对数值：（7.54±2.11） CFU]。虽然类鼻疽伯克霍尔德菌与伯克霍尔德菌株在细胞水平上的生物学效应类似（多核巨细胞形成、胞内增殖等）,但是对BALB/c小鼠的毒性差异显著。伯克霍尔德菌即使在高剂量（8×10⁷ CFU）时对小鼠仍是非致死性的,而且无法在小鼠脏器中有效定植。结论 成功构建了经鼻吸入性感染BALB/c小鼠的急性类鼻疽动物模型,明确了类鼻疽菌造成的组织病理损伤特点,发现了类鼻疽伯克霍尔德菌与伯克霍尔德菌株在动物水平上的显著生物学差异。

Establishment and evaluation of a BALB/c mouse model of Burkholderia pseudomallei via nasal infection

Objective To establish an animal model of BALB/c mice infected with Burkholderia pseudomallei through the nose (inhalation route), provides a reliable animal model for the follow-up studies on the virulence of melioidosis and the pathogenesis of acute melioidosis. Methods The experiment was carried out through infecting with Burkholderia pseudomallei through the nose (inhalation route). The pathophysiological response, visceral pathological damage and bacterial colonization of the mice infected with Burkholderia pseudomallei were observed by gross anatomy, histopathology and tissue homogenate count, and the biological characteristics of the mouse model of acute melioidosis were analyzed accordingly. Then we compared the physiological responses in BALB/c mice between the Burkholderia pseudomallei and non-pathogenic Burkholderia thailandensis. Results In the model of acute nasal infection with Burkholderia thailandensis, most death happened between the 3rd to 5th day after infection, 3×10⁵-3×10⁶ CFU was the suitable dose for acute fatal melioidosis model of BALB/c mice, and the medium lethal dose was about 3×10⁴-3×10⁵ CFU. Both gross anatomy and tissue HE staining showed that abscesses or necrosis were found in the lung, spleen and liver, especially in the spleen and lung, which was positively correlated with the challenge dose. Viable bacteria was isolated from the blood, lung, spleen and liver of Burkholderia pseudomallei-infected mice, and the bacteria account colonization was related to tissue specificity. The concentration of live bacteria isolated from in the blood was the highest Log₂ value: (10.28±0.34) CFU/mL], and the organ with the maximum quantity of bacteria was the lung Log₂ value: (7.54±2.11) CFU/total organ]. It has been reported that the biological effects of Burkholderia pseudomallei and its homologous non-pathogenic Burkholderia thailandensis were similar at the cellular level, like multi-nuclear giant cell formation and active intracellular replication, while it is still unclarrified in the differences of virulence in mice. In this study, it was proved that Burkholderia thailandensis was not fatal to mice even at a high dose (8×10⁷CFU), or detected from mice infected with it via nasal. Conclusion We successfully established a reliable BALB/c mouse model (acute lethal model) of melioidosis via nasal infection, described its biological characteristics, and identified the different biological responses between Burkholderia pseudomallei and its homologous non-pathogenic Burkholderia thailandensis in mice.