Dose-dependant acute or subacute disease caused by Burkholderia pseudomallei strain NCTC 13392 in a BALB/c aerosol model of infection.

AIMS: The goal of this study was to examine, for the first time, the virulence and pathogenicity of aerosolized Burkholderia pseudomallei, strain NCTC 13392, in BALB/c mice in order to develop an animal model for testing novel medical countermeasures (MCMs) for the treatment of human acute and subacute (a disease state between acute and chronic) melioidosis. METHODS AND RESULTS: BALB/c mice were exposed to varying doses of aerosolized bacteria. Acute disease was seen in animals exposed to a very-high dose (≥103  CFU per animal) and death occurred 3-4 days postchallenge (pc). Bacteria were detected in the lungs, liver, kidney and spleen. In contrast, animals exposed to a low dose (<10 CFU per animal) survived to the end of the study (day 30 pc) but developed weight loss, a bacterial tissue burden and increasing clinical signs of infection from day 20 pc onwards, mimicking a subacute form of the disease. Pathological changes in the tissues mirrored these findings. CONCLUSIONS: This proof of concept study has shown that B. pseudomallei strain NCTC 13392 is virulent and pathogenic in BALB/c mice, when delivered by aerosol. By varying the doses of aerosolized bacteria it was possible to mimic characteristics of both human acute and subacute melioidosis, at the same time, within the same study. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia pseudomallei, the aetiological agent of melioidosis, causes a serious and often fatal disease in humans and animals. Novel MCMs are urgently needed for both public health and biodefense purposes. The present model provides a useful tool for the assessment and evaluation of new MCMs (e.g. therapeutics and vaccines) and offers the potential for testing new treatments for both subacute to chronic and acute melioidosis prior to human clinical trials.

Sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus.

UNLABELLED: To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10(5) PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE: Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

Method for assessing IFN-γ responses in guinea pigs during TB vaccine trials.

AIMS: We sought to develop a new method that enables the assessment of the immune response of guinea pigs during TB vaccine evaluation studies, without the need to cull or anaesthetize animals. METHOD AND RESULTS: Guinea pigs were vaccinated with five different formulations of oral BCG. One week prior to challenge with Mycobacterium bovis, blood (50-200 µl) was taken from the ears of vaccinated subjects. Host RNA was isolated and amplified following antigenic restimulation of PBMCs for 24 h with 30 µg of bovine PPD. The up- or down-regulation of γ-interferon (IFN-γ), a key cytokine involved in protection against tuberculosis, was assessed using real-time PCR. The relative expression of prechallenge IFN-γ mRNA in the vaccinated groups (n=5) correlated (P<0·001) with protection against M. bovis challenge. CONCLUSION: We have demonstrated that it is possible to take blood samples and track IFN-γ responses in guinea pigs that then go on to be exposed to M. bovis, thus providing prechallenge vaccine uptake information. SIGNIFICANCE AND IMPACT OF THE STUDY: This methodology will also be applicable for tracking the immune responses of vaccinated guinea pigs over time that then go on to be challenged with M. tuberculosis during human TB vaccine evaluation studies.

The expression of ferritin, lactoferrin, transferrin receptor and solute carrier family 11A1 in the host response to BCG-vaccination and Mycobacterium tuberculosis challenge.

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P≤0.05). In addition, lactoferrin (P≤0.002), transferrin receptor (P≤0.05) and solute carrier family 11A1 (P≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

Disinfection of feline calicivirus (a surrogate for Norovirus) in wastewaters.

AIMS: To compare the inactivation of feline calicivirus (FCV) (a surrogate for Norovirus, NV) with the reduction of a bacterial water quality indicator (Escherichia coli), a human enteric virus (poliovirus) and a viral indicator (MS2, FRNA bacteriophage), following the disinfection of wastewaters. METHODS AND RESULTS: Bench-scale disinfection experiments used wastewater (sterilized by gamma-irradiation) seeded with laboratory-cultured organisms. Seeded primary effluent was treated with different doses of applied free chlorine (8, 16 and 30 mg l(-1)). FCV and E. coli were easily inactivated by >4 log10, within 5 min with a dose of 30 mg l(-1) of applied chlorine. Poliovirus was more resistant and a reduction of 2.85 log10 was seen after 30 min, MS2 was the most resistant organism (1 log10 inactivation). In further experiments seeded secondary effluent was treated with different doses of u.v. irradiation. To achieve a 4-log10 reduction of E. coli, FCV, poliovirus and MS2 doses of 5.32, 19.04, 27.51 and 62.50 mW s cm(-2), respectively, were required. CONCLUSIONS: Feline calicivirus and E. coli seeded in primary wastewater were very susceptible to chlorination compared with poliovirus and MS2. In contrast, FCV seeded in secondary wastewater was more resistant to u.v. irradiation than E. coli but more sensitive than poliovirus and MS2. SIGNIFICANCE AND IMPACT OF THE STUDY: FRNA phage was more resistant to inactivation than all the viruses tested. This suggests FRNA phage would be a useful and conservative indicator of virus inactivation following disinfection of wastewaters with chlorination or u.v. irradiation.

Intranasal bacille Calmette-Guerin (BCG) vaccine dosage needs balancing between protection and lung pathology.

Intranasal vaccination may offer practical benefits and better protection against respiratory infections, including tuberculosis. In this paper, we investigated the persistence of the Mycobacterium bovis-strain bacille Calmette-Guerin (BCG) Pasteur, lung granuloma formation and protection against pathogenic tuberculous challenge in mice. A pronounced BCG dose-dependent granulomatous infiltration of the lungs was observed following intranasal, but not after subcutaneous, vaccination. Corresponding doses of BCG, over a 100-fold range, imparted similar protection against H37Rv challenge when comparing the intranasal and subcutaneous vaccination routes. Interestingly, a BCG dose-dependent reduction of the H37Rv challenge infection was observed in the lungs, but not in the spleens, following both intranasal and subcutaneous vaccination. In the light of the observed concurrence between the extent of granuloma formation and the level of protection of the lungs, we conclude that intranasal vaccination leading to best protective efficacy needs to be balanced with an acceptable safety margin avoiding undue pathology in the lungs.

Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains.

Different types of microcarriers were assessed for the large-scale culture of influenza virus in the Madin-Darby canine kidney (MDCK) cells. Both porous and solid carriers were examined. A higher titre of influenza A/PR8/34 virus was recovered from cultures using solid (1.3x10(9) PFU per ml) rather than porous carriers (4.0x10(8) PFU per ml). High titres of virus (1.0x10(9) PFU per ml) were also obtained from roller bottle cultures of MDCK cells and the traditional culture technique using embryonated hens' eggs (3.9x10(9) PFU per ml). We found that solid carriers composed of dextran with a positive charge are the most suitable carriers for the large-scale growth of influenza A virus in MDCK cells using serum-free media.