Two techniques were used to isolate outer membrane proteins from Campylobacter jejuni, EDTA-lysozyme extraction and sodium-N-lauroylsarcosinate (Sarkosyl) solubilization. The protein profiles of the two preparations were similar, with a few additional bands in the EDTA-lysozyme preparations. The major outer membrane protein was 43,000 (43K) daltons, and there were 8 to 10 minor bands ranging from 92K to 14K daltons. There was no difference in the protein profile of a strain causing an infection (strain 17) and the resulting stool isolate (strain 17J). Sera collected before the infection and during the acute and convalescent stages were used with Western blotting and immunoautoradiographic techniques to determine the antigenicity of outer membrane proteins. A number of antigenic proteins were detected before the infection by their reaction with preinfection serum (61K, 51K, 43K, 40K, 34K, and 31K daltons), and three additional bands appeared during the infection when acute and convalescent sera were used (92K, 56K, and 19K daltons). Furthermore, an area of the gel at less than 14.4K daltons that did not stain with Coomassie brilliant blue became visible in the immune blots when the convalescent serum was used.
Bacteroides fragilis caused severe, nonhemorrhagic, watery diarrhea when 109 CFU of a porcine or human isolate was administered orogastrically to 3-day-old rabbits. The bacterium colonized the intestinal tract with a predilection for the large intestine (109 CFU/g of cecal contents). Diarrhea occurred at an average of 4.6 days postinoculation, and 84% of rabbits were dead or moribund at an average of 8.8 days postinoculation. The disease was characterized by watery diarrhea and dehydration. Severe histologic lesions including inflammation, exfoliation of epithelial cells, and crypt hyperplasia were observed throughout the colon. There was no indication of bacteremia or of bacterial adherence to or invasion of intestinal epithelial cells. Rabbits inoculated with nonenterotoxigenic B. fragilis were colonized with B. fragilis but did not develop clinical disease or intestinal lesions. While the pathogenesis of this disease is undefined, clinical signs of disease and histologic changes were consistent with a mechanism of net secretion of fluid into the small intestine and decreased absorption of fluid from the large intestine. Enteric disease caused by enterotoxigenic B. fragilis in infant rabbits was similar to naturally occurring enteric disease associated with the bacterium in humans and livestock. This study established that enterotoxigenic B. fragilis is enteropathogenic in intact infant rabbits.
Fifty Campylobacterjejuni isolates, including 29 from humans associated with an outbreak of enteritis, 20 from cattle, and 1 from a milk source, were serotyped on the basis of extractable thermostable antigens and examined by bacterial chromosomal restriction endonuclease digest analysis. Serotyping showed specific differences between the human isolates and the milk isolates, but each of these generally, although not consistently, reacted with 4 of the 42 C. jejuni typing antisera. Restriction patterns of all of the human isolates and some of the cattle isolates were indistinguishable, confirming the suspected link between the cattle and the human outbreak. The single milk isolate had a restriction pattern unlike those of the human isolates, and therefore its involvement in the transmission of infection from the cattle to the humans could not be confirmed.
Forty Campylobacter jejuni and 17 Campylobacter coli strains that constitute the set of reference strains for our serotyping scheme were each examined for the presence of plasmid DNA. Agarose gel electrophoresis of alkaline-extracted DNA showed the occurrence of 29 bands in 11 C. jejuni strains and 40 bands in C. coli strains. Plasmids ranged in size from 1.6 to 70 megadaltons. Most strains that carried plasmids had between 2 and 6 of them; however, one strain had 14 plasmids, and two strains contained only 1 plasmid each. Repeated electrophoresis demonstrated that all plasmid profiles were stable. A different plasmid profile was seen for each of the 19 plasmid-carrying strains, but it was clear that plasmids of the same or similar molecular weight could be found in different strains. On the basis of these findings, we are persuaded that plasmid profiles determined by a rapid procedure for DNA extraction will play a significant role in resolving complexities among strains that are difficult to serotype and could be useful in epidemiological studies in which the implicated isolates are plasmid bearers.
Lipopolysaccharides (LPS) were extracted from eight strains of Campylobacterjejuni and purified by enzyme treatment to remove traces of RNA, DNA, and protein. This material was used to sensitize sheep erythrocytes for the passive hemagglutination assay that is presently used to serotype C. jejuni. The results confirmed that the thermostable antigen typing scheme is based on LPS (0) antigens. The LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining was found to consist of a series of slow migrating bands which could not be elinminated by treatment with NaOH, urea, or EDTA. However, the use of LPS double labeled with 14C and 32p yielded evidence that the bands of high molecular weight were indeed aggregations of low-molecular-weight LPS molecules.Campylobacter jejuni and Campylobacter coli have become recognized as major causes of human bacterial enteritis (40, 41) and interest in the epidemiology of infections due to these organisms has led to the development of serotyping systems. Schemes that differentiate strains through differences in the specificities of surface antigens (13), thermolabile antigens (21), and thermostable antigens (19, 35) have been described. Currently, attention is being directed towards characterizing the biological and biochemical properties of the antigens to seek insight into the basis for the extensive serological heterogeneity noted to occur among strains of these species. At the outset, the thermostable antigens were thought to be lipopolysaccharide (LPS), the somatic 0 antigens common to gram-negative species (34), and investigations at the molecular level that support this view have recently been forthcoming (22,(27)(28)(29). Although the LPS of C. jejuni and C. coli appear similar to LPS of the Enterobacteriaceae in that both have considerable serological heterogeneity, it appears that, structurally, LPS in C. jejuni and C. coli may be quite unique. In the present report, enzyme-purified LPS was used to study the serological specificity of the 0 antigen by passive (indirect) hemagglutination (PHA) and to characterize the structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Evidence is provided to show that the thermostable antigen typing scheme is based on 0 antigens composed of LPS which have structural features different from the LPS of other gram-negative bacterial species. MATERIALS AND METHODSBacterial strains and cultural conditions. The strains of C. jejuni used in this study are listed in Table 1. Also, the smooth strain of Escherichia coli O:111B4 and the Ra (SL3749) and Rc (SL3748) mutants of Salmonella typhimurium were used.Stock cultures were maintained at -70°C in 15% glycerol-I% proteose peptone no. 3 (Difco Laboratories, Detroit, Mich.) and, when required, strains were thawed, plated on brucella agar (Difco), and incubated for 48 h under
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