Detection of Salmonella enterotoxin using rabbit ileal loops.

The presence of an enterotoxin produced by Salmonella in broth culture has been demonstrated by using the rabbit ileal loop model. Response by the animal to enterotoxin in sterile culture supernatant fluids is enhanced when the intestinal lumen is washed with a mucolytic agent prior to the administration of toxin. Fluid secretion is untreated intestinal loops was also observed if enterotoxin was administered with a live, invasive Salmonella strain which did not evoke a secretory response. A limited survey of Salmonella isolated for clinical and food sources indicated the common occurrence of enterotoxin production, and stock cultures maintained the ability to produce the toxin. The host-adapted species which were tested varied in their ability to produce enterotoxin.     

Detection of antibodies in Brugia pahangi-infected cats by counter immunoelectrophoresis, indirect fluorescent antibody test and enzyme-linked immunosorbent assay

In cats infected with Brugia pahangi, antibodies first appeared against the larvae (L3), then against the adults (L5) and the microfilariae (mf). Homologous antigens were better than antigens prepared from heterologous species (Dirofilaria immitis, Dipetalonema viteae, Litomosoides carinii and Onchocerca gutturosa) in detecting antibodies to B. pahangi in the infected cats by indirect fluorescent antibody test (IFAT). Metabolic products of L5, but not L3 or mf, of B. pahangi were antigenic and were used in the enzyme-linked immunosorbent assay (ELISA) for detection of antibodies. Using various homologous antigens, IFAT was found to be more sensitive than counter immunoelectrophoresis and ELISA in the detection of antibodies in the infected cats. The best antigen was cryosections of L3, with a positivity rate of 81%. However, using L3, L5 and mf antigens in IFAT, a total positivity of 97% was obtained.     

Identification of Salmonella enterotoxin by coagglutination reaction

It has been shown that the intensity of enterotoxin production by various strains of Salmonella is different (it ranges from + to +). Thus, in addition to adhesive properties and skin permeability factors, the ability of various Salmonella strains to produce enterotoxin is one of the pathogenic factors of these microorganisms. Further biological characteristics of the infective agents will promote the detection of epidemiologically significant types of microorganisms during outbreaks of toxicoinfections.     

Study of the antigenic structure of Salmonella by means of immunoelectrophoresis]

The method of immunoelectrophoretic analysis was applied to the study of the antigenic structure of 180 freshly-isolated and laboratory cultures of salmonellae belonging to 29 serological types. A complicated set of specific and common antigens of bacteria was revealed in direct and cross experiments with the use of homologous and heterogenous antisera; immunophoregrams were drawn for each of the serological type under study. General regularities in immunophoretic, diffuse and serological characteristics of the individual antigens of salmonellae were established. It was shown that different serological types of salmonellae possessed surface K antigens along with the O and H antigens. On immunophoregrams K-antigens of salmonellae were revealed in the form of a bright precipitation arch in the central or the anode zone; O antigens gave a characteristic two-component precipitation line in the cathode zone of the phoregram.     

Characterization and cloning of enterotoxin genes of Salmonella typhimurium

Five of fifty five strains of Salmonella typhimurium of human origin was hybridized with both the LT-A and LT-B gene of Escherichia coli. The remarkably erythromatous and indurated response on rabbit skin and significant elongation of Chinese Hamster Ovary (CHO) cells indicated the production of enterotoxin of these isolates. The Salmonella enterotoxin is heat-labile and is not a secretory product. The LT gene of E. coli was used to analyze the chromosome and plasmid DNA from Salmonella typhimurium strains for toxin gene sequences. Southern blot analysis demonstrated that the toxin gene was located on the plasmid but not on the chromosome. Restriction enzymes BamHI, EcoRI, HindIII and PstI were used to analyze the DNA isolated from salmonella strains Nos.22, 52, 55 and 59. Three DNA fragments with size of 5.2 Kb of strain 22, 5.0 Kb of strain 52 and 8.6 Kb of strain 59 were identified as containing the enterotoxin gene. Plasmid pUC19 was used as the vector to clone these DNA fragments in E. coli. The rabbit skin permeability test indicated that Salmonella enterotoxin could be synthesized at readily detectable levels in these transformed E. coli.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

Detection of Candida albicans mannan by immunodiffusion,counterimmunoelectrophoresis, and enzyme-linked immunoassay

Anti-mannan was produced in rabbits after peptidoglucomannan in adjuvant was injected. The antiserum was used to detect mannan by immunodiffusion and counterimmunoelectrophoresis (CIE) in gel and by sandwich enzyme-linked immunosorbent assay (ELISA). The antiserum detected lower concentrations of mannan of serotype A than of serotype B. Except in CIE, the reactions were more pronounced at 4°C than at higher temperatures. CIE detected 0.8 g/ml mannan A or 12.5 /ml mannan B. Sandwich ELISA detected 3 ng/ml mannan A or 10⁵ ng/ml mannan B. Mannan was not detected in the serum of patients or rabbits with candidiasis.Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education and Welfare.     

Detection of acyl-homoserine lactones by Escherichia and Salmonella

Escherichia and Salmonella do not synthesize quorum-sensing signaling molecules of the N-acyl-l-homoserine lactone (AHL) type but they can detect AHLs produced by other species of bacteria. AHLs are present in the bovine rumen but not in the remainder of the gastrointestinal tract. Enterohemorrhagic E. coli (EHEC) responds to AHLs extracted from the bovine rumen. Salmonella fails to detect AHLs in the gastrointestinal tracts of pathogen-free mice or pigs, suggesting that AHLs are not present. However, Salmonella does detect the AHL production of Yersinia enterocolitica in mouse Peyer's patches. In response to AHLs, EHEC represses flagellar genes and the LEE pathogenicity island while it activates the acid fitness island, whereas Salmonella activates the rck operon and a gene, srgE, encoding a putative Type III secreted effector.     

Purification of Salmonella stanley enterotoxin and its immunology and dermatotoxicity.

Isolation of an enterotoxic factor from cell-free-culture-supernatant of S. stanley was achieved to homogeneity using salt precipitation, dialysis and molecular seive chromatography through Sephadex G-100 and G-200 columns. The purified enterotoxic factor yielded a single protein band on polyacrylamide gel electrophoresis, induced antibodies in the rabbit and showed single band on agar gel precipitation. It induced fluid accumulation in the rabbit ligated ileal loop (RLIL) and was neutralized by the homologous antiserum. Antigenically it was not related to cholera toxin but with enterotoxin of other Salmonella serotypes. It also exerted dermatotoxic effect in the rabbit skin causing marked central necrosis with peripheral erythema.