Paraffin sections from gill tissues.

<p>The sections of gill tissue from <i>Clarias gariepinus</i> have been processed for <i>in situ</i> hybridization or stained with HES. A) Primary filament showing dark-blue stained <i>Candidatus</i> Actinochlamydia clariae inclusions, stained with antisense DIG-labelled RNA-probe against <i>Ca.</i> A. clariae 16S rRNA. Cells with inclusions are particularly frequent at the filament tip. Bar = 100.0 µm. B) Same primary filament stained with a sense probe, demonstrating absence of staining in the inclusions (examples indicated by arrows). Bar = 100.0 µm. C) HES stained section of the same filament tip. Bar = 100.0 µm. D) Magnification of an IHC stained inclusion, where the actiniae are discernible (arrowhead). Bar 10.0 = µm.</p

Semi-thin sections of infected primary lamellas from African sharptooth catfish.

<p>Pictures of semi-thin sections of the primary gill lamellas from African sharptooth catfish infected with <i>Candidatus</i> Actinochlamydia clariae. Arrows point to cysts of variable sizes. The majority of the cysts are located towards the apical part of the primary lamellas. A) Bar = 60.0 µm. B) Bar = 30.0 µm.</p

Section through inclusion with IBs.

<p>Sections through inclusions containing mainly IBs. The IBs have condensed nucleoids, but the rest of the content of the bacteria is not condensed. A) Bar = 5.0 µm. B) Bar = 0.5 µm. C) Bar = 0.5 µm.</p

Degenerating epitheliocysts.

<p>Pictures of degenerating epitheliocysts from <i>Clarias gariepinus</i> that contains inclusion with RBs (RB) and IBs (IB). The host cell cytoplasm loose the normal electron density and the mitochondria increase in size and become spherical (asterisks). The morphology of the tubular extensions from the inclusion changes from the normal irregular, star-shape (in transverse section), to round tubules (arrows). A) Bar = 5.0 µm. B) Bar = 1.0 µm. C) Bar = 0.5 µm.</p

Morphology of tubular extensions.

<p>These two figures show the tubules from the inclusion. Picture A) shows transverse sections of the tubules close to the inclusion membrane. The tubules are irregular, star-shaped, with electron lucent material close to the inclusion membrane. Bar = 1.0 µm. B) shows a section through a neighbouring cells showing that the tubules, containing electron dense material, are extending into this cell. Cell nucleus (Nu). Bar = 1.0 µm.</p

Phylogenetic tree.

<p>The phylogenetic tree shows the relationship between <i>Candidatus</i> Actinochlamydia clariae from African sharptooth catfish and selected members of other families within the order Chlamydiales. The best-fitting nucleotide substitution model was used during maximum likelihood analysis and the tree was bootstrapped (50 000 quartet puzzling steps) in TREE_PUZZLE. The scale bar shows the number of nucleotide substitutions as a proportion of branch lengths.</p

Nucleotide similarity of <i>Ca</i>. Actinochlamydia clariae (Accession nos: JQ480299, JQ480300, JQ480301) compared to other members of the order Chlamydiales.

<p>Nucleotide similarity of <i>Ca</i>. Actinochlamydia clariae (Accession nos: JQ480299, JQ480300, JQ480301) compared to other members of the order Chlamydiales.</p

Section through inclusion with EBs.

<p>Section through a large inclusion from the gills of <i>Clarias gariepinus</i> containing mainly EBs. A) The cyst is beginning to open to the gill surface (arrow), and the host cell cytosol shows signs of degeneration. A neighbouring cell contains an inclusion with RBs only (asterisk). Bar = 5.0 µm. B) Section through EB showing the cap area with associated protein structures (arrow). Tangential section through the cap area (ring) showing the hexagonal arrangement of the proteins. Bar = 0.2 µm. C) Section through free EBs showing the smooth cap areas (arrows) with proteins, an electron dense core (nucleoid), and condensed cytoplasm consisting mainly of ribosomes. Bar = 0.2 µm.</p

Cysts of <i>Candidatus</i> Actinochlamydia clariae.

<p>High magnification pictures of cysts containing <i>Ca</i>. A. clariae. Figures A) and B) show medium size cysts with tubules/channels radiating from the inclusion giving the cysts its distinct morphology. A) and B) Bars = 30.0 µm. B) A tangential section of the cyst clearly shows that the projections are tubules/channels (ring). C) This figure shows a large cyst, about 30 µm in diameter, containing mostly EBs (asterisk). C) Bar = 30.0 µm.</p