[The influence of viscosity on adhesion and invasion of Salmonella strains in an in vitro model]. / Untersuchungen zum Einfluss der Viskosität auf Adhäsion und Invasion bei Salmonellen im in-vitro-Modell.

The influence of viscosity on the adhesion and invasion behaviour of Salmonella strains of different origin was investigated using an in-vitro-model. These processes seem to be strain-dependent. Compared to the controls, the number of internalized Salmonella was elevated. This increase was a result of the greater number of Salmonella which adhered to Caco-2-cells and was detected only for strains from organs of calves having died from salmonellosis. The average motility of these strains was determined to be 1.6 +/- 0.5 mm/h. A possible association between adhesion ability and motility was discussed.

Studies of the presence of the virulence factors, adhesion, invasion, intracellular multiplication and toxin formation in salmonellas of different origin.

Salmonellas of different origin were classified into two groups (11 strains of common serovars which had been isolated from organs of calves having died from salmonellosis and 18 strains belonging to rare serovars which showed uncommon metabolic characteristics and had been isolated from spices and spiced foods). The strains were examined with regard to different virulence parameters. All salmonellas investigated possessed the genetic information on invasion (invA) and toxin formation (stn). They adhered equally well to epithelial cells, could penetrate into these and survive and multiply inside the cells. The formation of toxic substances could be detected in all strains after co-cultivation with epithelial cells in the CHO-K1 test. Significant differences between the groups of strains could be demonstrated only for the invasion of epithelial cell monolayers. Since adhesion, invasion and the ability of intracellular survival and multiplication as well as toxin formation constitute virulence parameters of salmonellas, it must be assumed that also the Salmonella serotypes studied which have been rarely observed epidemiologically constitute a risk for humans.

Detection of the induction of Salmonella enterotoxin gene expression by contact with epithelial cells with RT-PCR.

All strains of Salmonella enterica investigated were found to carry the Salmonella enterotoxin gene (stn) as determined by PCR and hybridization studies. However, when using CHO-K1 cells for testing the toxicity of the strains, not all strains showed a toxic effect (cell elongation) on the cells or did so only at a low level. The cultivation of Salmonella in contact with epithelial cells (IEC-6) led to an increase in the production of toxin. The stn gene expression was detectable with the help of the RT-PCR after 3 h of incubation. The RNA of the strains was isolated, transcribed into cDNA (with MMLV-reverse transcriptase) and amplified using PCR. The PCR products were separated electrophoretically using a polyacrylamide gel and detected by silver staining.

[In vitro studies of adhesion and invasion by Salmonella strains of bovine epithelial cells]. / In-vitro-Untersuchungen zu Adhäsions- und Invasionseigenschaften von Salmonella-Stämmen bovinen Ursprungs.

24 Salmonella strains were divided into three groups according to the circumstances of isolation. The adhesion and invasion abilities of the strains were determined using two permanent cell lines (IEC-6, VERO) and an epithelial cell line from the small intestine of a calf fetus (pKD). Strains of different groups showed no differences in their ability to adhere to the cells tested. Significant differences were found for the invasion ability. Strains isolated from organs of calves suffering from salmonellosis showed a significant higher invasiveness for permanent cell lines and a considerable higher invasiveness for pKD cells than strains of the other groups.

Cultivation of Salmonella in contact with epithelial cells.

An in vitro cultivation model for Salmonella having contact to epithelial cells was developed, which led to an increase in the production of toxic substances. The toxin assay on CHO-K1 cells was used for the determination of the toxic activities. Salmonella strains cultivated in contact with a monolayer of the intestinal cell line IEC-6 produced considerably more toxin than Salmonella strains cultivated on VERO cells. The toxin formed was heat-labile.

[The specificity of antibodies reacting with Salmonella antigens in ELISA]. / Untersuchung zur Spezifität der im ELISA mit Salmonellaantigen reagierenden Antikörper.

Among cattle sera from herds without a history of salmonellosis and without vaccination against salmonellas, we sometimes found samples which reacted positively in the ELISA with salmonella antigen. Contrary to antibodies caused by a proved salmonella infection, these antibodies could be eliminated by a whole-cell lysate of E. coli. In the colostra of cows which had received a salmonella live vaccine orally in the first days of life but were raised and kept without known exposition to salmonellas, we also found antibodies which reacted in the salmonella ELISA. They could not be eliminated by E. coli lysate.

Course of infection and humoral immune reaction in calves infected orally with different Salmonella serovars.

5 groups of five calves each were orally infected with the salmonella serovar S. agona, S. dublin, S. enteritidis, S. infantis or S. typhimurium. The course of infection and the humoral immune response against 5 antigen preparations of the homologous and the 4 heterologous salmonella serovars were monitored in each group. Antibodies against two different protein antigens and against two differently prepared LPS antigens were determined by ELISA and the level of H-agglutinin was assessed by micro agglutination test. All 5 serovars proved to be virulent for calves. The specificity of antibodies produced following the infection are not always consistent with the Kauffmann-White scheme. There was a relation of age and strain for infection on immune response, whereas the severity of infection did no correlate with an increase of antibody production. The serological tests used were good for investigation of a defined host-parasite-interaction but not satisfactory for serovar specific diagnosis of salmonella infection in cattle.

[The toxin-producing ability of salmonella from bovine test material]. / Untersuchungen zum Toxinbildungsvermögen von Salmonellen aus bovinem Untersuchungsmaterial.

Cell-free supernatants of 40 Salmonella strains of different serovars were tested for the existence of enterotoxic substances in the rabbit skin permeability factor test, rabbit ileal loop test, tissue culture assays (CHO K1, RTG-2), and baby mouse test. There were differences in the test results between the strains even within the same serovar. Correlation coefficients between the results of different toxin tests were low. It is therefore improbable that the "enterotoxic activity" of Salmonella is caused by only one toxic substance. The activities revealed in the different test systems could not be related to virulence or the epizootiological behaviour of the strains. The in vitro occurrence of the virulence factor "enterotoxin production" is lower for Salmonella than for E. coli and V. cholerae.

[Detection of enterotoxin production in bovine Salmonella isolates]. / Nachweis der Enterotoxinbildung bei bovinen Salmonella-Isolaten.

54 Salmonella-strains (7 serovars) of bovine origin isolated from faecal samples, rectal swabs as well as from organs of emergency-slaughtered or dead animals were tested for enterotoxin production (heat-labile and heat-stable) in rabbit-ileal-loop-assay (RILT), skin-permeability-factor-test (HPT), CHO- and Y1-cell-culture-assay and in baby-mouse-test (BMT). The cell-free supernatants (CFS) were used in the tests. The Y1-cells did not respond to the Salmonella toxins. While the RILT was suitable, the CHO-cell-assay proved to be the most sensitive and easy-to-handle system. The results confirm the frequent occurrence of the biological property of enterotoxin production within the species Salmonella enterica. Therefore, this property is in our opinion not useful as an epizootological marker for salmonellae.

[The occurrence of cytotonic and cytotoxic enterotoxins in Salmonella]. / Das Vorkommen von zytotonischen und zytotoxischen Enterotoxinen bei Salmonellen.

A survey is given on the presence and the detection of entero- and cytotoxin production of Salmonella. Their role in the pathogenesis of Salmonellosis has not yet been clarified completely. The standardization of the terms of toxins according place and modus of action is proposed. Cytotonic and cytotoxic enterotoxins were demonstrated in Salmonella strains. Toxins, like the Verotoxin of E. coli, acting in enterocytes as well as in other cells of organisms have not been detected in Salmonella strains. For detection cell culture assays are preferred, DNA-hybridization will be of importance in the near future.

Relation between Ca2+-ATPase and endogenous calmodulin of human erythrocyte membranes.

Short incubation of erythrocyte membranes with oleic acid releases Ca2+-independently bound endogenous calmodulin together with a minor fraction of membrane-associated proteins without destruction of the membranes. The released endogenous calmodulin is similar if not identical to cytosolic calmodulin reversibly bound to ghosts in a Ca2+-dependent manner. The release of endogenous calmodulin proceeds without affecting the activity of Ca2+-ATPase when ghosts are incubated with oleic acid in the presence of Ca2+ plus ATP and thereafter freed from oleic acid by washings with serum albumin. Kinetic parameters of Ca2+-ATPase of ghosts with and without endogenous calmodulin are identical as are amounts of exogenous calmodulin bound to these ghosts. Thus, endogenous calmodulin does not function as an essential part of Ca2+-ATPase.

Ca/EGTA solutions: comparison between measured and calculated free calcium ion concentrations in the micromolar range.

We prepared a series of Ca/EGTA solutions and compared the pCa2+free values obtained by calculation and measurement with an ion sensitive electrode. Two possible reasons for the discrepancies between these values were studied: (1) the effect of various association constants used for the calculation of pCa2+free, and (2) the influence of deviations of the calcium total concentration in the Ca/EGTA solutions from the values used for the calculation. The results obtained showed that the main reason for differences between calculated and measured pCa2+free values was the nonequimolarity of both stock solutions CaCl2 and EGTA. A linear dependence of measured electrode potentials with Nernstian behaviour on the calculated pCa2+free in the Ca/EGTA solutions has been obtained with carefully equalized stock solutions.