Use of a Safe, Reproducible, and Rapid Aerosol Delivery Method to Study Infection by Burkholderia pseudomallei and Burkholderia mallei in Mice

Abstract

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 102, 103 and 104 organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 103 and 104 B. pseudomallei cells, animals infected with 102 organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those seen in human infections.

Characterization of an autotransporter adhesin protein shared by Burkholderia mallei and Burkholderia pseudomallei

“…The model entails the use of a Microsprayer® to deliver bacteria directly into the murine lungs [67]. The device generates aerosol particles from the tip of a bent, 23-gauge nebulizing tube attached to a high-pressure stainless steel syringe that contains bacteria.…”

Antibodies against In Vivo -Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei

“…Specific-pathogen-free (SPF) female BALB/c and C57BL/6 mice were purchased from Envigo and allowed to acclimate for at least 1 week prior to use. After administration of anesthetic, the mice were inoculated intratracheally using a Microsprayer nebulizing device (PennCentury) as previously reported (77,93,144). The infected animals were monitored daily, food and water were provided ad libitum, and humane endpoints were strictly observed.…”

“…For BALB/c mice challenged with WT B. mallei ATCC 23344 and B. pseudomallei 1026b, inoculating doses of approximately 8,000 (10 LD 50 ) and 25,000 (5 LD 50 ) CFU were used, respectively. These values were previously established while developing the Microsprayer aerosol delivery platform (144). For BALB/c mice challenged with WT B. pseudomallei K96243, an inoculating dose of ϳ300 CFU was used.…”

“…The serum samples from mice that survived aerosol infection with B. mallei ATCC 23344 are described elsewhere (144). The serum samples from horses experimentally infected with B. mallei ATCC 23344 were obtained in the context of a separate study (unpublished data) (NIH-NIAID award U01AI077764).…”

Vaccines for the Prevention of Melioidosis and Glanders

“…Manifestation of symptoms depends greatly on the route of infection, with the most severe occurring due to the inhalational form of infection [9]. Acute infection with B. pseudomallei can lead to symptoms of severe pneumonia and rapid septicemia.…”