SARS-CoV-2 Aerosol and Intranasal Exposure Models in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the worldwide COVID-19 pandemic. Animal models are extremely helpful for testing vaccines and therapeutics and for dissecting the viral and host factors that contribute to disease severity and transmissibility. Here, we report the assessment and comparison of intranasal and small particle (~3 µm) aerosol SARS-CoV-2 exposure in ferrets. The primary endpoints for analysis were clinical signs of disease, recovery of the virus in the upper respiratory tract, and the severity of damage within the respiratory tract. This work demonstrated that ferrets were productively infected with SARS-CoV-2 following either intranasal or small particle aerosol exposure. SARS-CoV-2 infection of ferrets resulted in an asymptomatic disease course following either intranasal or small particle aerosol exposure, with no clinical signs, significant weight loss, or fever. In both aerosol and intranasal ferret models, SARS-CoV-2 replication, viral genomes, and viral antigens were detected within the upper respiratory tract, with little to no viral material detected in the lungs. The ferrets exhibited a specific IgG immune response to the SARS-CoV-2 full spike protein. Mild pathological findings included inflammation, necrosis, and edema within nasal turbinates, which correlated to positive immunohistochemical staining for the SARS-CoV-2 virus. Environmental sampling was performed following intranasal exposure of ferrets, and SARS-CoV-2 genomic material was detected on the feeders and nesting areas from days 2-10 post-exposure. We conclude that both intranasal and small particle aerosol ferret models displayed measurable parameters that could be utilized for future studies, including transmission studies and testing SARS-CoV-2 vaccines and therapeutics.

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1ß, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1ß at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.

Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues.

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of "inactivated" Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.

Pulmonary Delivery of Ceftazidime for the Treatment of Melioidosis in a Murine Model.

Burkholderia pseudomallei, the etiological agent responsible for melioidosis, exhibits a great public health toll in its endemic regions. The elevation of B. pseudomallei to a Tier I select agent underscores the urgent need for effective therapeutics and preventatives. The current treatment regimen for melioidosis is suboptimal, requiring an intensive phase of intravenous antibiotic followed by months of oral antibiotics. Inhaled antibiotics are a promising avenue to pursue for pulmonary diseases, including melioidosis, since this mode of delivery mimics the likely exposure route and can provide high drug doses directly to the infected tissue. Ceftazidime was delivered via a nose-only system to BALB/c mice challenged with B. pseudomallei. Mice treated with nebulized ceftazidime became symptomatic but survived until study end, which was comparable to those treated intraperitoneally. Upon necropsy, bacteria remained within the spleens of the majority of the experimental animals. The effectiveness of nebulized ceftazidime warrants additional studies to improve the treatment regimen and to test as a prophylactic therapy against B. pseudomallei.

Formulation and Characterization of Nanocluster Ceftazidime for the Treatment of Acute Pulmonary Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. The disease is responsible for a high proportion of human pneumonia and fatal bacteremia in the endemic areas of the world and is highly resistant to most commonly available antibiotics. Studies have shown that prophylactic antibiotic treatment, when administered 24 h following bacterial challenge, can prevent infection in a murine model. Prophylactic treatment against this disease using a pulmonary antibiotic formulation has not previously been examined, but may reduce the number of treatments required, allow for the delivery of higher doses, eliminate the need for intravenous administration, and help to minimize systemic side effects. Ceftazidime was formulated as a dry powder aerosol suitable for pulmonary delivery using previously developed NanoCluster dry powder technology. Pharmacokinetics of aerosolized ceftazidime was analyzed in a mouse model. This study demonstrates that ceftazidime can be formulated using NanoCluster technology as a dry powder aerosol suitable for pulmonary delivery to humans. We have also demonstrated the retention of nebulized ceftazidime in mouse lungs for up to 6 h after exposure. The results indicate that this treatment may be useful as a prophylactic treatment against melioidosis. Future work will examine the efficacy of this treatment against B. pseudomallei aerosol challenge.