The US Centers for Disease Control and Prevention lists Brucella as a potential bioterrorism threat requiring enhanced diagnostic capacity and surveillance (http://emergency.cdc.gov/bioterrorism/). Successful treatment and management of patients after exposure to biological threat agents depends on accurate and timely diagnosis, but many biothreat agents present with similar, vague clinical signs – commonly referred to as ‘flu-like illness’. Diagnosis of brucellosis is notoriously challenging, especially early in infection, and definitive diagnosis may require invasive methods, e.g. bone marrow biopsy. We studied the pathogenesis of Brucella suis aerosol infection in rhesus macaques in an effort to guide the diagnostic algorithm in case of possible intentional exposure of humans. Rhesus proved to be an excellent model for human brucellosis; the data showed that PCR DNA amplification testing of non-invasive diagnostic samples has the potential to definitively detect a point-source outbreak immediately and for several days after exposure.

b After a relatively short untreated interval, pneumonic plague has a mortality approaching 100%. We employed a murine model of aerosol challenge with Yersinia pestis to investigate the early course of pneumonic plague in the lung, blood, and spleen. We fit a mathematical model to all data simultaneously. The model fit to the data was acceptable. The number of organisms in the lung at baseline was estimated to be 135 (median) or 1,184 (mean) CFU/g. The doubling time was estimated as 1.5 to 1.7 h. Between 1 and 12 h postexposure, counts declined, but they then increased by 24 h, a finding hypothesized to be due to innate immunity. The model predicted that innate immunity declined with a half-time of 3 to 3.8 h. The threshold for bacteremia was 6.4 ؋ 10 4 to 1.52 ؋ 10 6 CFU/g. By 42 to 48 h, stationary phase was obtained. Lung bacterial burdens exceeded 10 log CFU/g. Obviating early defenses allows for rapid amplification of Y. pestis in bacteremia, making the rapid course with high mortality understandable.

Aerosolized Burkholderia pseudomallei, the causative agent of melioidosis, can infect many species of mammals (including humans), causing rapid, severe pneumonia with high mortality. Diagnosis in humans is challenging, as few organisms can be detected in blood or other noninvasive samples. Although it cannot be said that the model is established, studies to date indicate that rhesus macaques may represent a good model of human melioidosis. This is supported by the results of this study. The early progression of meliodosis in the rhesus macaque was studied in an effort to better understand the disease and the application of rapid diagnostic methods. Results indicate that a PCR analysis of key diagnostic samples such as nasal swabs, throat swabs, tracheo bronchial lymph node aspirates and broncho-alveolar lavage may be a useful component of a rapid diagnostic algorithm in case of aerosol exposure.

Bacillus anthracis is considered a likely agent to be used as a bioweapon, and the use of a strain resistant to the first-line antimicrobial treatments is a concern. We determined treatment efficacies against a ciprofloxacin-resistant strain of B. anthracis (Cip r Ames) in a murine inhalational anthrax model. Ten groups of 46 BALB/c mice were exposed by inhalation to 7 to 35 times the 50% lethal dose (LD 50 ) of B. anthracis Cip r Ames spores. Commencing at 36 h postexposure, groups were administered intraperitoneal doses of sterile water for injections (SWI) and ciprofloxacin alone (control groups), or ciprofloxacin combined with two antimicrobials, including meropenem-linezolid, meropenem-clindamycin, meropenem-rifampin, meropenemdoxycycline, penicillin-linezolid, penicillin-doxycycline, rifampin-linezolid, and rifampinclindamycin, at appropriate dosing intervals (6 or 12 h) for the respective antibiotics. Ten mice per group were treated for 14 days and observed until day 28. The remaining animals were euthanized every 6 to 12 h, and blood, lungs, and spleens were collected for lethal factor (LF) and/or bacterial load determinations. All combination groups showed significant survival over the SWI and ciprofloxacin controls: meropenem-linezolid (P ϭ 0.004), meropenem-clindamycin (P ϭ 0.005), meropenem-rifampin (P ϭ 0.012), meropenem-doxycycline (P ϭ 0.032), penicillindoxycycline (P ϭ 0.012), penicillin-linezolid (P ϭ 0.026), rifampin-linezolid (P ϭ 0.001), and rifampin-clindamycin (P ϭ 0.032). In controls, blood, lung, and spleen bacterial counts increased to terminal endpoints. In combination treatment groups, blood and spleen bacterial counts showed low/no colonies after 24-h treatments. The LF fell below the detection limits for all combination groups yet remained elevated in control groups. Combinations with linezolid had the greatest inhibitory effect on mean LF levels.

The objective of this study was to evaluate the natural history and pathogenesis of Francisella tularensis in a murine model of inhalational tularemia with the SchuS4 strain. Before the efficacy of antimicrobials could be assessed in this model, further model development was required to determine the optimal time to start therapy. This study helped define the time course of infection after aerosol challenge by quantifying the presence of bacteria in lung, blood, and spleen at multiple harvest points. In this study, mice were infected via a targeted inhaled dose of 100 50% lethal doses (LD 50 s) (LD 50 ‫؍‬ 300 CFU) of F. tularensis by whole-body aerosol. At 1, 24, 36, 48, 60, 72, 75, 78, 81, 84, 87, and 90 h postchallenge, groups of 15 animals were sacrificed and blood, lung, and splenic tissue samples were harvested, homogenized, plated, and incubated to evaluate the bacterial load in those tissues. It was determined that of the 3 sample types harvested, splenic tissue provided the most consistent bacterial counts, which steadily increased with the progressing infection. Further, it was determined that lung samples from all (15/15) animals were positive for infection at 75 h postaerosolization and that 14/15 animals had positive splenic tissue counts. Bacterial levels in blood were not predictive of treatment initiation. For future therapeutic evaluation studies in this model using F. tularensis (SchuS4), it was determined that therapy should be initiated at 75 h postchallenge and validated by spleen involvement.

Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases.

The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis , Burkholderia pseudomallei , B. mallei , Yersinia pestis , and Bacillus anthracis . We validated methanol treatment for B. pseudomallei . These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories.