Greub. 1470), (CRIB-18), and (CRIB-01). strain BL was used to grow (ATCC 50802). Purified bacteria were injected intravenously to 6-week-old BALB/c mice (two mice per antigen). After four immunizations, blood was collected, and sera were obtained by centrifugation at 5,200 for 5 DCC-2618 min at 4C. Twofold-diluted sera were tested by immunofluorescence for antibody reactivity against purified antigens. Fluorescein isothiocyanate-coupled anti-mouse immunoglobulin antibody (Bio-Rad, Rheinach, Switzerland) was used as a secondary antibody. Phosphate-buffered saline-milk and uninfected axenic (ATCC 30010) were used as unfavorable controls. The IgG/IgM Micro-IF test kit (ANILabsystems, Vantaa, Finland) was used to test the antibody reactivity of mice sera against antigens. Mice responded DCC-2618 properly to injected antigens. Very high titers of 1/32,768 and 1/16,384 were obtained after immunization with and strain Hall’s coccus, respectively. Comparable results were obtained for each duplicate sera, with titers varying by less than one dilution. Sera elicited against any of all and strains. In contrast, no or little cross-reactivity was detected between distantly related was detected when we tested sera elicited with any strain Hall’s coccus that reacted with (titer of 1/128). A tree inferred from DCC-2618 SDIs was built by using the neighbor-joining option of the PHYLIP software (10). 16S rRNA sequences were used to infer another neighbor-joining tree (observe Fig. ?Fig.1A1A for the GenBank accession figures), using Kimura-corrected p-distance and the complete deletion option of the MEGA software (18). Both SDI and 16S rRNA-based trees were rooted using as an outgroup. The phyletic tree based on the SDI was congruent with the tree inferred from 16S rRNA encoding gene sequences (Fig. ?(Fig.1A).1A). This suggests that cross-reactivity between users of the order is usually proportional to the level of relatedness between each species, i.e., to the likeliness of common epitopes. Open in a separate windows FIG. 1. (A) Congruence of the phylogenetic tree based on the 16S rRNA encoding gene (left panel) and the phyletic tree based on the SDI (right panel). Both trees were built by using the neighbor-joining method and were outrooted with strain Hall’s coccus, “type”:”entrez-nucleotide”,”attrs”:”text”:”AF366365″,”term_id”:”14030551″,”term_text”:”AF366365″AF366365; and strain BN9, “type”:”entrez-nucleotide”,”attrs”:”text”:”Y07556″,”term_id”:”1491675″,”term_text”:”Y07556″Y07556. (B) Western blot pattern of each strain BN9; C and D, strain Hall’s coccus; E and F, strain BN9strain Hall’s coccusstrain BN91,024 (0)1,024 (4)128 (1)32 (10) 32 (10) 32 (11) 32 (17) 32 (17)strain Hall’s coccus1,024 (4)16,384 (0)512 (3)64 (12) 32 (12)32 (12) 32 (19)64 (19)experienced immunogenic proteins of approximately 130, 70, 43, and 30 kDa. For strain BN9, a protein of approximately 70 kDa exhibited the strongest immunogenicity. For of about 70, 53, 45, and 30 kDa exhibited the highest immunogenicity. For possess immunogenic proteins of 130, 89, 43, and 25 kDa (Fig. ?(Fig.1B).1B). Earlier, Western blot has been used to confirm immunofluorescence results in two studies investigating the role of that exhibited a strong reactivity against proteins of about 70, 40, and 28 kDa (11). Here, we also recognized a 70-kDa parachlamydial immunogenic protein. In the second study, a 64-kDa protein exhibited the strongest immunogenicity (4, 25). Since DCC-2618 serology was not discriminative at the species level for as a new agent of pneumonia (2, 11, 21) should be interpreted as a hint for a role of either or spp. showed a high seroprevalence. The absence of cross-reactivity between spp., spp. (Table ?(Table1),1), and all three tested in our study support the idea that humans are frequently exposed to spp. or cross-reacting related species. Whether spp. cause respiratory tract diseases remains to be exhibited since Kumar et al. (17) and Johnsen et al. (15) did not confirm early studies by Lieberman et al. (19, 20). Discrepant results might be due to some cross-reactivity of the enzyme-linked immunosorbent assay used by Lieberman et al. with that exhibit Rabbit Polyclonal to SEPT1 common immunogenic proteins. In conclusion, we have explained the level of cross-reactivity and the molecular excess weight of immunogenic proteins of spp. Appl. Environ. Microbiol. 63:115-121. [PMC free article] [PubMed] [Google Scholar].