In-vitro-Untersuchungen zur Adhäsivität, Invasivität und intrazellulären Persistenz von Salmonella-enterica-Impfstämmen

Abstract

Vaccination against Salmonellosis is mostly based on Salmonella live vaccine strains. In most cases it is well documentated, that the vaccines are effective and harmless, but the underlying mechanisms of attenuation resist often speculative. In order to investigate the causes of attenuation, three Salmonella live vaccine strains for use in animals (S.Typhimurium "ZOOSALORAL H", S.Dublin "BOVISALORAL" and S.Gallinarum 9R "NOBILIS") were examined for their adhesive, invasive and intracellular capabilities in a cell culture modell. Virulent wild type strains S.Typhimurium "MOSKAU" (parent strain of "ZOOSALORAL H"), S.Dublin 442/012 (parent strain of "BOVISALORAL") and S.Gallinarum 1910/93 were used for pairwise comparison. Host cells were chick embryonal fibroblasts (HEF), fetal calf lung cells (FKL), and cells from a human laryngeal carcinoma cell line (HEp-2). Bacterial adhesion patterns were assessed by light microscopy. An invasion assay (coincubation of bacteria and host cells for 3 h to 7 days) was used for the quantification of adhesion, invasion and intracellular survival.

All vaccine and wild type strains adhered to host cell monolayers with a diffuse adherence pattern, invaded into host cells, and could be recovered from the intracellular environment for at least 2 days after inoculation. However, beside this very similar behaviour on the qualitative level there were significant quantitative differences depending on the strain, the host cell and the parameter tested:
The vaccine strain "ZOOSALORAL H" was less able to survive in host cells than its parent strain. The number of intracellular bacteria of "ZOOSALORAL H" declined to 3 - 18 % within the first 24 h post inoculation. During the same period the numbers of intracellular wild type bacteria increased to 218 - 612 %.
The vaccine strain "BOVISALORAL" significantly less effectively adhered to HEF and FKL cells and less effectively invaded into FKL cells than its parent strain. After 24 h of coincubation numbers of intracellular bacteria of "BOVISALORAL" reached only 30 % (FKL) or 20 % (HEF) of those yielded by S.Dublin 442/012.
The S.Gallinarum vaccine strain 9R "NOBILIS" adhered significantly less effectively to all host cells than the wild type strain of S.Gallinarum that was used for comparison. No significant correlation was detected between the recognized host species adaption of a Salmonella serotype and the behaviour of the Salmonella vaccine and wild type strains in the various cell culture systems.

The results suggest, that the attenuation of these Salmonella vaccine strains is rather caused by downregulation of adhesion, invasion, and intracellular survival capabilities than due to a complete loss of these properties. Thus, the reduced virulence of "ZOOSALORAL H" may be determined by a reduced intracellular survival in the phagosomes of non-professional host phagocytes. Similarily, an adhesion defect may contribute to the avirulent phenotype of S.Dublin strain "BOVISALORAL". Results also suggest that the attenuated phenotype of S.Gallinarum strain 9R "NOBILIS" may not be determined by impaired adhesion, invasion or intracellular survival. Furthermore, the host species adaption of certain Salmonella serovars seems not to be caused by a host species specific interaction between the bacterial cells and the non-professional phagoycytes.