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Chapter 61 : The Rickettsiaceae, Anaplasmataceae, Bartonellaceae, and Coxiellaceae
The order Rickettsiales consists of the family Rickettsiaceae and the family Anaplasmataceae. The family Rickettsiaceae contains the genus Rickettsia and the genus Orientia. Members of the family Rickettsiaceae are short rods or coccobacilli, and members of the family Anaplasmataceae are small pleomorphic cocci. The family Bartonellaceae consists of two genera, one of which, Bartonella, includes human pathogens belonging to the α2-group proteobacteria. Currently, the principal techniques used for the serodiagnosis of rickettsial diseases are probe-based immunoassays. Immunoprobe-based tests include the indirect fluorescence assay (IFA) and its micromodification, the micro-IF (MIF) test. The MIF test is still considered as the gold standard. Other immunoprobe tests include immunoperoxidase assays (IPAs); enzyme-linked immunosorbent assays (ELISAs), in which the antigens are adsorbed either onto the well of a microtiter plate or onto nitrocellulose in a dot blot or slot blot configuration; and immunoblot assays (IBAs). IFAs and IPAs require the whole bacterium as the antigen. The remaining immunoprobe assays use either highly purified rickettsiae or an extract. The development of real-time PCR-based diagnosis and rapid PCR-based diagnostic methods has been the focus of much attention in many research laboratories. Recently, a protein-based method for the detection of the small-cell variant protein A (ScvA) plus other protein markers comprising a Coxiella fingerprint via matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry has been developed.
Taxonomy of human pathogens of the order Rickettsiales, with representative species from each genus and bacteria from genera that were formerly in the order Rickettsiales and were kept in this chapter for historical purposes.
Correlation between IFA titers and ODs for ELISA with EK-rP44 as the antigen. Each circle represents one sample. The rho value calculated by Spearman’s rank correlation was 0.740 (P <0.001; n = 181). (Reprinted with permission from reference 44 .)
(A) Dot blot immunoassay of HGE patient sera with different IFA testing titers by use of 0.5 μg of affinity-purified recombinant P44 (rP44) antigen. The sera were diluted 1:1,000. (B) Layout of experiment. (Reprinted with permission from reference 51 .)
Western immunoblot analysis of anti-HGE (presently, anti-HGA) sera using purified HGE (presently, HGA) agent isolate 13 and rP44 antigen. The sera used for this study included a horse anti-HGE-agent serum; five convalescent-phase serum samples, from patients 2, 3, 11, 13, and 16; five serum samples collected at different times of illness over a 2-year period from patient 21, who was suspected of having persistent infection or reinfection; and negative control serum (IFA titer, <1:20). Samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis consisted of 10 μg of purified whole-cell preparations of HGE agent strain 13, affinity-purified rP44, HL-60 cells, and E. coli BL21(DE30)/pLysS. These proteins were transferred to a nitrocellulose sheet and incubated with a 1:1,000 dilution of antisera. The number at the bottom of each panel represents the IFA test titer of the serum sample. The numbers on the left indicate molecular masses in kilodaltons based on broad-range prestained standards (Bio-Rad) (adapted and reprinted with permission from reference 51 ).
Representative types of pathogenic rickettsiae in humans
Examples of PCR primer sets that allow for identification and differentiation of rickettsial species via RFLP analysis
Primers used for PCR