Mouse Models of Aerosol-Acquired Tularemia Caused by Francisella tularensis Types A and B

After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.Abbreviations: LVS, live vaccine strain; SUBLN, submandibular lymph node; MESLN, mesenteric lymph nodeTularemia is an infectious disease caused by the gram-negative bacterium Francisella tularensis. Clinical disease was first seen in Japan in 1837, when people who ate meat from rabbits developed fever, chills, and glandular tumors.²⁷ In the United States, the disease was first described in 1911 by Dr Edward Francis, who observed a plague-like illness in ground squirrels. In 1914, the first human case in the United States was described in a butcher.⁴⁸ Dr Francis, for whom the microorganism was named, characterized the clinical signs, symptoms, and the mode of transmission after infection.Tularemia is one of the most pathogenic diseases known, causing disease after inoculation or inhalation of as few as 10 microorganisms.²⁷ The microorganism is infectious by many routes, including ingestion of contaminated food or water, through microabrasions or bites from various arthropods, or inhalation of the infectious agent. The species F. tularensis includes several subspecies including tularensis, holarctica, novidia, and mediaasiatica. The type A F. tularensis subspecies tularensis is highly virulent in humans and is the dominant species in North America. Type B F. tularensis subspecies holarctica (also called palearctica) is usually found in European countries and is less virulent.⁴⁵ Humans infected with either subspecies can develop an acute febrile illness.F. tularensis is a facultative intracellular pathogen and multiplies in macrophages. Target organs are associated with the reticuloendothelial system and include lymph nodes, lungs, spleen, liver, and kidneys. In human pneumonic tularemia, infection progresses systemically from the lungs to other organs and causes pathologic changes, primarily to the liver and spleen.^9,11,19,38 Humans infected by aerosol exposure also show hemorrhagic inflammation of the airways, which can progress to bronchopneumonia.⁴³ Alveolar spaces become filled with a mononuclear cell infiltrate. Pleuritis with adhesions and effusion and hilar lymphadenopathy are commonly found.³³ The fatality rate for untreated pneumonic infections is approximately 30% to 60% for type A F. tularensis and approximately 10% for infections with type B F. tularensis.⁴² Because of its high infectivity, morbidity, and mortality, this microorganism is classified as a Category A Select Agent. F. tularensis can cause natural infections in a variety of animals, including rabbits, cats, prairie dogs, voles, raccoons, squirrels, rats, lemmings, wild mice, and other species.^{1,3,4,12,30,35,40,49} Although the source of infection can be inapparent, outdoor-housed rhesus macaques can acquire tularemia.^15,37Animal models using virulent (for humans) strains are essential for the development of efficacious vaccines and evaluation of antimicrobials against pathogenic diseases affecting humans. Due to the virulence of this microorganism, most experimental infections in rodent model systems use the Live Vaccine Strain (LVS) to minimize the risk to laboratory workers,¹⁷ although many recent studies use the more virulent type B and type A strains. LVS can be lethal to mice but is attenuated in humans. LVS is lethal in naïve mice and causes pathologic reactions similar to those seen with virulent strains in humans.⁸ However, using an LVS model of infection is not without potential problems. First, dose lethality varies widely, depending on the route of LVS infection. Naïve mice are most sensitive to intraperitoneal challenge, followed by intravenous, intranasal, aerosol, and intradermal challenge. Furthermore, the LD₅₀ for LVS administered by aerosol is greater than 10³ microorganisms, whereas the LD₅₀ for virulent F. tularensis strains is less than 10 microorganisms.⁸To better understand the pathogenesis of this important Category A pathogen, we determined the virulence and investigated the histopathogenesis of type A and type B F. tularensis in mice challenged by small-particle aerosol. We compared the histopathogenesis of infection over a period of 3 wk in BALB/c and C57BL/6 mice challenged with 1468 microorganisms or 100 microorganisms of F. tularensis SCHU S4 (type A). Type B (strain 425) infection was similarly investigated in BALB/c mice challenged with 102 microorganisms. Bacterial burdens in spleens, livers, lungs, and blood were determined.