Mitchell Carl

Detection of Rickettsia tsutsugamushi by gene amplification using polymerase chain reaction techniques.

Scrub typhus is commonly undiagnosed in endemic areas due, in part, to dependence on retrospective serodiagnosis. Since the etiologic agent, R. tsutsugamushi, will not grow in cell-free systems, a rapid direct-agent detection system such as provided by polymerase chain reaction (PCR) methodology is needed. Genes coding for the variable 56-kDa antigen of R. tsutsugamushi were amplified through 35 cycles using 20-mer oligonucleotide primers and Taq polymerase. Amplification of 1-ng samples of DNA extracted from purified prototype R. tsutsugamushi Karp, Gilliam, and Kato strains was detected by direct visual inspection of the electrophoresed, ethidium bromide-stained, specific bands. Specificity of the PCR was shown when PCR amplification of various non-scrub typhus rickettsial DNAs was unsuccessful. R. tsutsugamushi DNA extracted from the blood of infected mice could be PCR amplified and the 1477-base pair product detected by either direct visualization or by specific hybridization with amplified non-radioactive digoxigenin-11-dUTP-labeled Karp 56-kDa DNA probe.

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of rickettsia typhi and Rickettsia prowazekii

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.

Detection of rickettsiae in arthropod vectors by DNA amplification using the polymerase chain reaction.

Abstract : Polymerase chain reaction (PCR) amplification of DNA was used to detect Rickettsia rickettsii and R. typhi in experimentally infected adult Dermacentor variabilis ticks and Xenopsylla cheopis fleas, respectively. A primer pair derived from the 17-kDa-antigen gene sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base (bp) fragment of the genome of the rickettsiae. The specific PCR-amplified product in extracts of individual infected fleas or ticks was detected readily on ethidium bromide-stained agarose gels. The amplified 434-bp sequence was not detected in uninfected controls. The PCR procedure provides a rapid, sensitive, and highly specific assay for detection of rickettsial infection in arthropod vectors. Keywords: Antigen detection, Indirect immunofluorescence, Rickettsia rickettsii, oligonucleotide primers.

The Importance of the Crystalline Surface Layer Protein Antigens of Rickettsiae in T-cell Immunity

Studies in animal models have demonstrated that solid immunity to typhus rickettsiae is dependent on immune T cells. In addition, the surface protein antigen (SPA) of typhus rickettsiae has been shown to be an effective immunogen, protecting vaccinated animals against subsequent challenge with virulent organisms. In the present studies we describe three classes of human lymphocytes which are capable of lysing cells infected with typhus rickettsiae. The first class is CD3,8-positive and is capable of specially lysing both HLA-matched and mismatched targets infected with typhus rickettsiae. Since this cytotoxic effector can be generated with IL-2 as well as with SPA it appears to be a lymphokine-activated killer (LAK). The second class of lymphocytes is CD3,4-positive and is capable of producing gamma interferon in response to the SPA of typhus rickettsiae. Gamma interferon in turn can cause the lysis of cells infected with typhus rickettsiae as well as inhibit intracellular rickettsial growth. A third cytotoxic effector which is CD3,4-positive and which is capable of lysing only HLA-matched targets infected with typhus rickettsiae was generated with a sonicated antigen, and its fine antigenic specificity is not known at present. We conclude that the SPA is an immunologically important protein for the human host and represents an outstanding candidate for a subunit vaccine against typhus.

Diagnosis of acute typhus infection using the polymerase chain reaction.

We used the polymerase chain reaction (PCR) for the diagnosis of an acute rickettsial infection. A primer pair derived from the 17-kDa antigen sequence of Rickettsia rickettsii amplified specifically a 434-bp DNA fragment from the genome of Rocky Mountain spotted fever and endemic and epidemic typhus. The assay was able to detect as few as 30 rickettsiae. Detection of PCR-amplified DNA with a digoxigenin-labeled DNA probe confirmed an acute human infection with Rickettsia prowazekii.