An Assessment of Germicidal Ultraviolet Treatment Cabinets and Carousels Using a Bacteriophage Surface Challenge.

Introduction: Modern germicidal ultraviolet C (UVC) equipment can deliver automated UV disinfection treatment by predetermined or self-monitoring cycle. Limited information exists about the performance of such UV systems for treating SARS-CoV-2 and other viral contaminants on surfaces. Published studies differ in their approaches due to the absence of an approved test method. Methods: The ability of germicidal UVC irradiation systems to disinfect surfaces at room and cabinet scale was assessed. Test carriers, seeded with bacteriophage Phi6, were irradiated following a new standard test method. Powered air-purifying respirator equipment was then used to introduce a more demanding challenge. Results: Treatments of seeded carriers using UVC cabinets gave Phi6 log reductions up to 4.58 logs, with little difference between systems. Subsequent treatments, with carriers located on respirator ensembles, were similar, despite shadowing effects. Differences existed for various combinations of cabinet and carrier location. The Phi6 log reduction range was slightly wider for carousel systems, with the most exposed carrier positions giving the greatest Phi6 reductions for seeded respirators. Discussion: Cabinets demonstrated similar performance despite different technical specifications, with maximum observed Phi6 reduction indicating a measurable level of efficacy. There was a more obvious difference in performance between the two carousels, where one delivered an almost twofold higher UVC dose than the other, the most likely explanation for observed performance differences. Conclusion: UVC cabinets and carousels demonstrated Phi6 reductions that could augment routine cleaning measures for reusable respirators. In real-world scenarios, germicidal UVC devices could therefore potentially offer benefits for reducing contact transmission from infectious viruses.

Room-Based Assessment of Mobile Air Cleaning Devices Using a Bioaerosol Challenge.

Introduction: The widespread transmission of the SARS-CoV-2 virus has increased scientific and societal interest in air cleaning technologies, and their potential to mitigate the airborne spread of microorganisms. Here we evaluate room scale use of five mobile air cleaning devices. Methods: A selection of air cleaners, containing high efficiency filtration, was tested using an airborne bacteriophage challenge. Assessments of bioaerosol removal efficacy were undertaken using a decay measurement approach over 3 h, with air cleaner performance compared with bioaerosol decay rate without an air cleaner in the sealed test room. Evidence of chemical by-product emission was also checked, as were total particle counts. Results: Bioaerosol reduction, exceeding natural decay, was observed for all air cleaners. Reductions ranged between devices from <2-log per m3 room air for the least effective, to a >5-log reduction for the most efficacious systems. One system generated detectable ozone within the sealed test room, but ozone was undetectable when the system was run in a normally ventilated room. Total particulate air removal trends aligned with measured airborne bacteriophage decline. Discussion: Air cleaner performance differed, and this could relate to individual air cleaner flow specifications as well as test room conditions, such as air mixing during testing. However, measurable reductions in bioaerosols, beyond natural airborne decay rate, were observed. Conclusion: Under the described test conditions, air cleaners containing high efficiency filtration significantly reduced bioaerosol levels. The best performing air cleaners could be investigated further with improved assay sensitivity, to enable measurement of lower residual levels of bioaerosols.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

The immune response in bovine primary dermal fibroblasts is influenced by Interleukin 8 promoter haplotype and vitamin D.

Interleukin-8 (IL-8) is a potent inflammatory chemokine, and two gene promoter haplotypes have been previously reported to segregate in cattle populations. Our earlier work showed how these divergent IL8 genotypes influence IL-8 expression and other immune response parameters at a systemic level. Here we extend that work to characterise the influence of haplotype on the local immune response - in primary bovine dermal fibroblasts. Furthermore, we also investigated how this response is modulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Significant induction of IL8 expression was observed in cells from both haplotypes at 3 and 24 h post-stimulation with the TLR1/2 ligand, Pam3CSK4 and with the TLR4 ligand, LPS. IL8 expression was elevated in response to both LPS and Pam3CSK4 in fibroblasts carrying the IL8-h1 haplotype and this result was supported by significantly enhanced IL-8 protein secretion. Gene expression profiles for other known fibroblast immune mediators (SAA3 and CCL20) did not show significant differences between haplotypes but NOS2 gene expression was significantly elevated in response to vitamin D, even above the level detected in response to both TLR ligands. In conclusion, this work has demonstrated that the IL-8 response of dermal fibroblasts is dependent on IL8 haplotype and that the immune response profile in these cells is significantly differentially regulated by 1,25(OH)2D3. Fibroblasts have important immune response capacity and their function in driving inflammatory responses (including iNOS) is underappreciated. Understanding the relationship between cattle genotype and immune function is critically important for uncovering sustainable solutions for animal disease.

1,25(OH)D vitamin D promotes NOS2 expression in response to bacterial and viral PAMPs in primary bovine salivary gland fibroblasts.

OBJECTIVES: The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs). METHODS: Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18 h before stimulation with lipopolysaccharide (LPS µg/ml), lipoteichoic acid (LTA µg/ml) or polyinosinic:polycytidylic acid (poly I:C-20 µg/ml) PAMPs for 6 h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR). RESULTS: RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, ß-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.

A preclinical ultrasound method for the assessment of vascular disease progression in murine models.

INTRODUCTION: The efficacy of preclinical ultrasound at providing a quantitative assessment of mouse models of vascular disease is relatively unknown. In this study, preclinical ultrasound was used in combination with a semi-automatic image processing method to track arterial distension alterations in mouse models of abdominal aortic aneurysm and atherosclerosis. METHODS: Longitudinal B-mode ultrasound images of the abdominal aorta were acquired using a preclinical ultrasound scanner. Arterial distension was assessed using a semi-automatic image processing algorithm to track vessel wall motion over the cardiac cycle. A standard, manual analysis method was applied for comparison. RESULTS: Mean arterial distension was significantly lower in abdominal aortic aneurysm mice between day 0 and day 7 post-onset of disease (p < 0.01) and between day 0 and day 14 (p < 0.001), while no difference was observed in sham control mice. Manual analysis detected a significant decrease (p < 0.05) between day 0 and day 14 only. Atherosclerotic mice showed alterations in arterial distension relating to genetic modification and diet. Arterial distension was significantly lower (p < 0.05) in Ldlr-/- (++/--) mice fed high-fat western diet when compared with both wild type (++/++) mice and Ldlr-/- (++/--) mice fed chow diet. The manual method did not detect a significant difference between these groups. CONCLUSIONS: Arterial distension can be used as an early marker for the detection of arterial disease in murine models. The semi-automatic analysis method provided increased sensitivity to differences between experimental groups when compared to the manual analysis method.