A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources |
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| Affiliation: | 1. Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China;2. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, China;3. Department of Animal Science, Cornell University, Ithaca, NY 14853, USA;1. Food Lipid Biomarkers and Health Group, Institute of Food Science Research (CIAL, CSIC-UAM), Madrid, Spain;2. IMDEA-Food, CEI UAM, Madrid, Spain;3. VALORNUT Research Group, Department of Nutrition and Food Science, Faculty of Pharmacy, Complutense University of Madrid, Spain;4. Cogni-UNED Research Group, Faculty of Psychology, UNED, Madrid, Spain;5. Department of Molecular Medicine, University of Padova, Padova, Italy;1. State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 210029, China;2. Department of Immunology, Nanjing Medical University, Nanjing, 210029, China;1. Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistraße 52, Hamburg 20251, Germany;2. Université Bordeaux, ISPED, Centre INSERM U1219, Inria, SISTM, F-33000, Bordeaux, France;3. CHU de Bordeaux, pôle de santé publique, F-33000, Bordeaux, France;4. Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Martinistraße 52, Hamburg 20251, Germany;5. Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Martinistraße 52, Hamburg 20251, Germany;1. Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile;2. Departamento de Ingeniería Química, Universidad de la Frontera, Temuco, Chile;3. Centro de Excelencia en Investigación Biotecnológica Aplicada al Medio Ambiente (CIBAMA-BIOREN), Universidad de la Frontera, Temuco, Chile;4. Planta de Procesos Industriales Microbiológicos (PROIMI-CONICET), Avenida Belgrano y Pasaje Caseros, 4000 Tucumán, Argentina |
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| Abstract: | ![]()
An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 108 cfu/ml into a suspension of Ped-2E9 (106/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test, Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 μm filter. |
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