ABSTRACT
Yersiniae are gram-negative bacilli belonging to the family of Enterobacteriaceae. Three species are recognized as human and animal pathogens: Yersinia pestis, the agent of plague, Y. pseudotuberculosis,, and Y enterocolitica.lThe latter can provoke enteritis, mesenteric adenitis, and sometimes septicemia in humans. Such infections may be followed by reactive arthritis or erythema nodosum. 2 The species is heterogeneous and has been subdivided in five biotypes characterized by different metabolic activity patterns. 3 Besides, each biotype includes a number of serotypes with specific antigens, 4 among which there are polysaccharidic antigens referred to as antigens “O”, flagellar or “H” antigens, and a few “surface” or “envelope” antigens corresponding, at least in some strains, to a fimbrial structure 5 (Figure 1). The study of these antigens is of interest since only some of the antigenic profiles have so far been characteristic of potentially pathogenic strains. 6 Furthermore, essential virulence determinants of yersiniae are located on plasmids of about 70 kilobases in size. 7 , 8 The plasmids of the three species are structurally and functionally related. 9 , 11 Several phenotypic properties, depending on temperature, are specified by these virulence plasmids. 12 One of them is the secretion of large amounts of proteins after incubation at 37°C in a Ca++ deficient medium. 13 , 14 Since these proteins were initially detected in the outer membrane ,they were called YOPs for Yersinia Outer Membrane Proteins. 15 As shown by reaction with polyclonal antisera and monoclonal antibodies (MAbs), the YOPs of the different species are immunologically related. 15 , 17 A number of observations have suggested that these proteins are indeed involved in the pathogenic process of Yersinia. 18 , 19 MAbs could be a powerful means to study their structure and role in pathogenesis. In order to obtain hybridomas secreting MAbs against these antigens, rats have been immunized with Y. enterocolitica. For this experiment, the strain chosen was W22703(pGC 565). It is a strain of the 0:9 serotype, known to produce YOPs. 14 The antigen was prepared as described in reference 14. The bacteria were first incubated overnight at 28°C in brain-heart infusion (BHI) containing 25 μg/ml kanamycine. With 250 μl of this preculture, we seeded 4 times 10 ml of BHI added with 20 mM sodium oxalate, 20 mM MgCl2, 20 mM glucose, and 25 μg/ml kanamycin. Incubation was performed at 28°C for 2 h, then at 37°C for 4 h. After centrifugation, the cells were washed and resuspended in saline at a density of 3 × 109 bacteria per milliliter according to the optical density at 600 nm.
