A 6-HOUR IN-VITRO VIRULENCE ASSAY FOR LISTERIA-MONOCYTOGENES USING MYELOMA AND HYBRIDOMA CELLS FROM MURINE AND HUMAN SOURCES

被引:38
作者
BHUNIA, AK
STEELE, PJ
WESTBROOK, DG
BLY, LA
MALONEY, TP
JOHNSON, MG
机构
[1] Department of Food Science, University of Arkansas Biotechnology Center, University of Arkansas, Fayetteville, AR 72703
[2] Plizer Pharmaceuticals Central Research Division, Groton, CT 06333, Eastern Point Rd.
关键词
LISTERIA MONOCYTOGENES; MURINE MYELOMA; MURINE HYBRIDOMA; HUMAN HYBRIDOMA; VIRULENCE ASSAY; LACTATE DEHYDROGENASE ASSAY;
D O I
10.1006/mpat.1994.1011
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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. © 1994 Academic Press.
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页码:99 / 110
页数:12
相关论文
共 25 条
[1]   MONOCLONAL ANTIBODY-COLONY IMMUNOBLOT METHOD SPECIFIC FOR ISOLATION OF PEDIOCOCCUS-ACIDILACTICI FROM FOODS AND CORRELATION WITH PEDIOCIN (BACTERIOCIN) PRODUCTION [J].
BHUNIA, AK ;
JOHNSON, MG .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1992, 58 (07) :2315-2320
[2]   MONOCLONAL-ANTIBODY SPECIFIC FOR LISTERIA-MONOCYTOGENES ASSOCIATED WITH A 66-KILODALTON CELL-SURFACE ANTIGEN [J].
BHUNIA, AK ;
JOHNSON, MG .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1992, 58 (06) :1924-1929
[3]  
DELCORRAL F, 1990, J FOOD PROTECT, V53, P1003, DOI 10.4315/0362-028X-53.12.1003
[4]   FEEDING TRIALS OF LISTERIA-MONOCYTOGENES WITH A NONHUMAN PRIMATE MODEL [J].
FARBER, JM ;
COATES, EDF ;
BEAUSOLEIL, N ;
FOURNIER, J .
JOURNAL OF CLINICAL MICROBIOLOGY, 1991, 29 (11) :2606-2608
[5]   INVITRO MODEL OF PENETRATION AND INTRACELLULAR GROWTH OF LISTERIA-MONOCYTOGENES IN THE HUMAN ENTEROCYTE-LIKE CELL-LINE CACO-2 [J].
GAILLARD, JL ;
BERCHE, P ;
MOUNIER, J ;
RICHARD, S ;
SANSONETTI, P .
INFECTION AND IMMUNITY, 1987, 55 (11) :2822-2829
[6]   TRANSPOSON MUTAGENESIS AS A TOOL TO STUDY THE ROLE OF HEMOLYSIN IN THE VIRULENCE OF LISTERIA-MONOCYTOGENES [J].
GAILLARD, JL ;
BERCHE, P ;
SANSONETTI, P .
INFECTION AND IMMUNITY, 1986, 52 (01) :50-55
[7]   ENTRY OF L-MONOCYTOGENES INTO CELLS IS MEDIATED BY INTERNALIN, A REPEAT PROTEIN REMINISCENT OF SURFACE-ANTIGENS FROM GRAM-POSITIVE COCCI [J].
GAILLARD, JL ;
BERCHE, P ;
FREHEL, C ;
GOUIN, E ;
COSSART, P .
CELL, 1991, 65 (07) :1127-1141
[8]   NONHEMOLYTIC LISTERIA-MONOCYTOGENES MUTANTS THAT ARE ALSO NONINVASIVE FOR MAMMALIAN-CELLS IN CULTURE - EVIDENCE FOR COORDINATE REGULATION OF VIRULENCE [J].
KATHARIOU, S ;
PINE, L ;
GEORGE, V ;
CARLONE, GM ;
HOLLOWAY, BP .
INFECTION AND IMMUNITY, 1990, 58 (12) :3988-3995
[9]   LISTERIA BRAIN-STEM ENCEPHALITIS - 2 OWN CASES AND LITERATURE-REVIEW [J].
KOHLER, J ;
WINKLER, T ;
WAKHLOO, AK .
INFECTION, 1991, 19 (01) :36-40
[10]   HEMOLYSIN SUPPORTS SURVIVAL BUT NOT ENTRY OF THE INTRACELLULAR BACTERIUM LISTERIA-MONOCYTOGENES [J].
KUHN, M ;
KATHARIOU, S ;
GOEBEL, W .
INFECTION AND IMMUNITY, 1988, 56 (01) :79-82