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At the 10th international Chlamydia conference in June 2002 [see: Antalya report], Poppert and colleagues presented their work on the use of fluorescence in situ hybridization (FISH) probes for the characterization of the Chlamydiales. The images presented were strikingly beautiful and chlamydiae.com is pleased to present them here.  Full details of the oligonucleotide probe sequences used are on the web at Probebase.net [Loy et al., 2003] (use the search term 'chlamydia')  and are outlined here:

Table 1: Oligonucleotide probe sequences specific for the Chlamydiales and formamide concentration in the hybridization buffer required for specific fluorescence in situ hybridization.

This work has now been published  [Poppert et al., 2002]. The authors developed a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes [Fig 4] was demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells [Figs 6 - 12], including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis [Fig 12]. FISH reliably stained chlamydial inclusions after 12 hours growth in vitro [Fig 7]. The method was found to be highly specific, reliably permitting  the detection of a wide range of chlamydiae in a single step [Poppert et al., 2002].

Figure 1. Title and authors. Presentation © Poppert et al., 2002 and shown here with the consent of the authors.	Figure 2. Outline of the FISH methodology.	Figure 3. Applications of FISH in medical microbiology.	Figure 4. Diagram of the coverage of the hierarchic probe sets used. The probes are described on probebase.net

Figure 5. The probebase.net web site.	Figure 6. Illustration of how simultaneous binding of three differently coloured probe sets may be demonstrated. Here the three probes together appear white.	Figure 7. Early inclusions of C. pneumoniae detected 12 hours post infection, showing the ability of the method to resolve single chlamydial particles.	Figure 8. C. pneumoniae reticulate bodies in immature inclusions, 24 hours post infection. These reticulate bodies are clearly larger than the elementary bodies seen in Fig 7.
Figure 9. Mature inclusions of C. pneumoniae 72 hours post infection. The electron micrograph (left) shows that the inclusion is packed with infectious elementary bodies whose release completes the developmental cycle.	Figure 10. Combined FISH (blue and red) and immunofluorescence (green). The DNA intercalating dye DAPI has been used to mark DNA with blue fluorescence.	Figure 11. Double infection of cells with C. pneumoniae and C. trachomatis showing the use of Chlamydiaceae family - specific blue probe 574 in conjunction with C. trachomatis species - specific probe 623 (green) and C. pneumoniae species - specific probe 214 (red). Note in these figures a variety of fluorochromes may readily be used with each probe.	Figure 12. Double infection of HeLa 229 cells with C. pneumoniae and C. trachomatis. Hela cells are stained red with a eukaryotic cell probe EUK. C. pneumoniae, labelled with blue probe 214, is clearly distinguished from C. trachomatis which is stained with green probe 623. Note the double infected cell at the centre of the field
Figure 13. Parachlamydia sp. UWE25, a fresh water parachlamydia, in its Acanthamoeba host. The Parachlamydia react with both the red and blue probes; the amoeba are stained with a green eukaryotic cell specific probe, and the bottom right hand picture is an overlay of all three probes.	Figure 14. The advantages of FISH for the in situ identification of the chlamydiales.	Figure 15. The authors and their institutions.

Poppert et al., 2000b suggest that FISH probably exceeds the sensitivity of culture-based methods for chlamydial detection, but is not as sensitive as nucleic acid amplification-based methods. Since their FISH primers are targeted against rRNA they believe that FISH, unlike classic amplification based methods, primarily detects only intact and viable chlamydiae. This requires confirmation. They considered the high specificity of FISH, its ability to encompass a wide range of chlamydiales and the fact that it can be used on tissue directly makes it particularly appropriate for the investigation of blood vessels for chlamydophila-like organisms. Chae et al., 1997 have also described the use of in situ hybridization for the detection of C. suis.

[MEW April 2003].

References

Chae, C., Cheon, D. S., Kwon, D., Kim, O., Kim, B., Suh, J., Rogers, D. G., Everett, K. D. & Andersen, A. A. (1997). In situ hybridization for the detection and localization of swine Chlamydia trachomatis. Veterinary Pathology 36, 133 - 137.

Loy, A., Horn, M., Wagner, M. (2003). probeBase - an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res., 31(1), [In press]. See: probeBase

Poppert, S., Essig, A., Marre, R., Wagner, M. & Horn, M. (2002). Detection and differentiation of chlamydiae by fluorescence in situ hybridization. Applied and Environmental Microbiology 68, 4081 - 4089.

Poppert, S., Essig, A., Marre, R., Wagner, M. & Horn, M. (2002b). A comprehensive oligonucleotide probe set for the identification of chlamydiae by fluorescence in situ hybridization (FISH). pp 29 - 32 In: Proceedings of the 10th international symposium on human chlamydial infections. International Chlamydia Symposium San Francisco ISBN 0-9664383-1-0

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