An investigation of Erysipelothrix rhusiopathiae isolates affecting marine mammals and the cloning, expression and evaluation of a putative recombinant 66 kD immunoprotective surface protein

Robert Charles Osgood

Abstract

Erysipelothrix rhusiopathiae is a nonmotile, Gram positive, non-sporeforming rod found widely in nature. It is known to cause chronic or acute erysipelas in swine and erysipeloid in humans. In cetaceans the organism is capable of causing diamondback type cutaneous lesions or an acute fatal form of septicemia. Several environmental isolates of Erysipelothirix rhusiopathiae implicated in the death or disease of marine mammals were acquired from various marine mammal facilities and compared to each and to known ATCC isolates of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum using microbiological, immunological and molecular biological techniques. Microbiological analysis included carbohydrate fermentations, hydrogen sulfide production, sodium citrate utilization as well as the employment of several staining procedures and morphological studies. The identity of the isolates was confirmed using previously published polymerase chain reaction (PCR) primers that amplify a Erysipelothrix rhusiopathiae specific 399 base pair 16s rRNA DNA sequence, and a 384 base pair 16S rRNA DNA sequence specific only to Erysipelothrix tonsillarum . The recombinant form of the 66kD surface protein was obtained from the cloned and expressed coding sequence of The U.S. Navy Space and Naval Warfare System Center (SPAWARSYSTENS) isolate of Erysipelothrix rhusiopathiae , acquired from The U.S. Navy Marine Mammal Program. The species specific PCR, the immunological and the microbiological testing confirmed the identity of the environmental isolates as Erysipelothrix rhusiopathiae and revealed some differences between the environmental strains themselves as well as some differences between the environmental strains and the ATCC reference isolates of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum . Western blot analysis further illustrated within-group and between-group differences. Although some differences were expected, enough variation between the two groups and within the groups suggests and agrees with the present notion that isolates of the genus Erysipelothrix may be diverse enough at this point to add other species members to the genus. The His-tagged recombinant form of the SpaA protein was produced and its performance was analyzed in an ELISA. This recombinant form of the protein did not function in the ELISA as well as a non-recombinant extracted form of the protein. The failure of this form of the protein may possibly be due to interference of the His tag, improper folding of the protein or possibly destruction of continuous or conformational epitopes on the recombinant protein during its purification.