Abstract

Seven cases of progressive osteomyelitis associated with Salmonella enterica spp. arizonae were identified in a colony of ridgenose rattlesnakes (Crotalus willardi) at the Knoxville Zoological Gardens.² The Salmonella arizonae serotype 56:Z4,Z23 was consistently isolated from all bony lesions but was isolated only once from cloacal swabs. Twelve other S. arizonae serotypes were isolated from cloacal swabs but not from the bony lesions. The present study was initiated to see if it was possible to use cell culture invasion assays to compare the pathogenicity of the 56:Z4,Z23 serotype with four of the intestinal isolates. Our hypothesis was that an in vitro assay could differentiate the pathogenic 56:Z4,Z23 serotype from the intestinal isolates. Two cell lines, human HeLa cells and viper heart cells, were used in the invasion assays. Multiwell plates with a confluent monolayer of cells were incubated with each of the five S. arizonae serotypes. After incubation, the cells were treated with antibiotics, rinsed, and then lysed to release any bacteria which had invaded the cells during the incubation period.^1,3 The results of the HeLa cell invasion assay supported our initial hypothesis. The number of colony forming units recovered was significantly higher (p<0.05) for the 56:Z4,Z23 serotype compared to the intestinal isolates. The results of the viper heart assay varied from this pattern. One intestinal isolate, serotype 48:I-Z, was recovered in significantly higher (p<0.05) numbers than the 56:Z4,Z23 serotype.

Based on these results, the HeLa cell invasion assay appears to be able to differentiate between the pathogenic 56:Z4,Z23 serotype and the apparent commensal intestinal serotypes. Evaluation of the invasion assay with other known pathogenic and non-pathogenic Salmonella sp. seems warranted. The cause of failure of the viper heart cell assay to differentiate between the 56:Z4,Z23 and other Arizona serotypes is unclear. The two cell lines are grown and maintained at different ambient temperatures, which may contribute to the observed differences. Viper heart cells have not, to our knowledge, been previously used for invasion assays and intrinsic cell properties are another potential cause for the observed results. Development and investigation of other reptile cell cultures, such as snake macrophages, might prove more useful as an in vitro test for differentiating between these Salmonella arizonae serotypes.

Literature Cited

1.  Mills SD, Finlay BB. Comparison of Salmonella typhimurium invasion, interacellular growth and localization in cultured human epithelial cells. Microbiol Pathol. 1994;17:409–423.

2.  Ramsay EC, Daniel GB, Bemis DA, Tryon BW. Salmonella arizonae osteomyelitis in a colony of Arizona ridge-nosed rattlesnakes (Crotalus w. willardi). In: Proceedings of the American Association of Zoo Veterinarians. 1996:218–219.

3.  Yabuuchi E, Ikedo M, Ezaki T. Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells. Microbiol Immunol. 1986;30:1213–1224.