The presence of infectious extracellular Francisella tularensis subsp. novicida in murine plasma after pulmonary challenge

Abstract

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 (i.n.) 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 i.n. 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 1×10 CFU/g tissue, was observed in the lungs, which increased to 1×10 CFU/g tissue by 48 h and then decreased to 1×10 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 1×10 CFU/g tissue by 72 h (Fig. 1a). 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 Eur J Clin Microbiol Infect Dis (2008) 27:323–325 DOI 10.1007/s10096-007-0434-x