Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.

Addition of monoclonal antibodies specific for Rickettsia akari to the rickettsial diagnostic panel.

Monoclonal antibodies were produced from mice infected with Rickettsia akari (the etiologic agent of rickettsialpox) and evaluated for specificity in indirect fluorescent-antibody tests with 23 different rickettsial antigens. Of the nine antibodies that were evaluated, two were specific for R. akari and four reacted with R. akari and all other spotted fever group rickettsiae. The remaining three antibodies reacted with some, but not all, members of the spotted fever group. None of the antibodies reacted with typhus, scrub typhus, trench fever, or Q fever rickettsiae. Adding these antibodies to the list of available diagnostic reagents will facilitate identification of rickettsial diseases, particularly those caused by members of the spotted fever group, where the clinical presentations are similar and the etiologic agents are closely related antigenically.

Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts.

Ethyl acetate as a substitute for diethyl ether in the formalin-ether sedimentation technique.

Ethyl acetate appears to be a satisfactory subsitute solvent for diethyl ether in the Formalin-ether sedimentation technique. In comparative studies, concentration of organisms with ethyl acetate was equal to or greater than that with diethyl ether. No distortion or alteration of morphology was observed with eigher solvent, and preparations were comparable in appearance and ease of examination. In addition, ethyl acetate is less flammable and less hazardous to use than diethyl ether.