Table 1.
PCR-RFLP primers and fragment sizes for differentiation of C. abortus 1B vaccine strain from wild-type strains.
Fig 1.
Placentas infected with vaccine-type and wild-type C. abortus strains, and negative control.
(A) Placenta vt-P1: showing the oedema and thickening of the whole placenta covering and the typical dark red or grey-whitish colouration of the cotyledons and partially covered with a cream-coloured exudate on the intercotyledonary areas. (B) Placenta wt-P1: showing the oedema and thickening of the whole placenta partially covered with a cream-coloured exudate on the intercotyledonary area and dark red and grey cotyledons. (C) Placenta Neg-P1: showing red colour of the cotyledons, and thin and clear and translucent intercotyledonary membranes.
Fig 2.
PCR-RFLP analysis of C. abortus-positive placental samples.
Restriction enzyme digestion patterns for vt-P1 (lanes 4, 11 and 18) and vt-P2 (lanes 5, 12 and 19), wt-P1 (6, 13 and 20) and wt-P2 (7, 14 and 21), control wt C. abortus strains S26/3 (1,8 and 15) and AB7 (2, 9 and 16) and control C. abortus 1B vaccine strain (3, 10 and 17) of PCR fragments (see Materials and methods and Table 1) with enzymes SfcI (lanes 1–7), HaeIII (lanes 8–14) and Sau3AI (lanes 15–21).
Fig 3.
Histopathological changes in the placentas of sheep infected with C. abortus vaccine-type strains.
(A, C and E) Placenta vt-P1: showing (A) necrosuppurative placentitis and (C) inflammatory infiltration forming a compact layer consisting primarily of large numbers of leucocytes in the epithelium of the cotyledon (arrow) attached to superficial amorphous necrosuppurative material; and (E) mixed infiltration, primarily of PMN, in different stages of degeneration, and abundant necrotic debris. (B, D and F) Placenta vt-P2: showing (B) necrosuppurative placentitis and (D) extensive compact layer of leucocytes in the trophoblast layer and basement membrane (arrow); and (F) a heavy and compact suppurative infiltration. Black outlined areas shown in top images indicates areas expanded in images located immediately below. (Scale bar: A: 5 mm; B: 10 mm; C: 500μm; D: 1 mm; E, F: 50μm).
Fig 4.
Histopathological changes in the placental blood vessels of sheep infected with C. abortus vaccine-type and wild-type strains.
(A-C) Placenta vt-P1: showing (A) necrosuppurative vasculitis, with severe mixed inflammatory infiltration (black arrow) and partially occlusive thrombosis (*); (B) partially occlusive fibrinoid thrombosis in multiple vessels (*) with revascularisation, and ischemic necrosis of the trophoblast cells (N); (C) severe fibrinoid necrotising arteritis displaying a dense band of amorphous and intensely eosinophilic material (orange arrow); (D) Placenta vt-P2: showing severe occlusive thrombosis, with neovascularization, mineralization of the thrombi (*). (E) Placenta wt-P1: showing severe fibrinoid necrotising mural arteritis (orange arrow), multiple foci of thrombosis (*), severe perivascular inflammatory infiltration (black arrow) and ischemic necrosis of the trophoblast cells (N). (F) Placenta wt-P2, severe necrotising arteritis, showing mural degeneration (orange arrow), mixed infiltration (black arrow) and ischemic necrosis of the surrounding tissue (N). Haematoxylin and eosin. (Scale bar: A, B: 100μm; C, D, E: 500μm; F: 250μm,).
Fig 5.
Trophoblast cells showing intracytoplasmic inclusion bodies of C. abortus vt- and wt-strains in sheep placentas.
Placentas vt-P1 (A-D) and wt-P1 (E-H) were stained by HE for histological examination (A and C for vt-P1; E and G for wt-P1) and chlamydial organisms labelled by IHC using genus-specific anti-LPS mAb 13/4 and counterstained with haemotoxylin (B and D for vt-P1; F and H for wt-P1). Note the presence of cytoplasmic chlamydial inclusion bodies in trophoblast cells indicated by arrows. (Scale bar: A, B, E, F: 50 μm; C, D, G, H: 25 μm).
Fig 6.
Histopathological changes in the placentas of sheep infected with C. abortus wild-type strains.
(A, C and E) Placenta wt-P1: note (A) necrosuppurative placentitis, (C) extensive, severe, suppurative infiltration of the trophoblast cells, necrotic material (N) in the luminal face of the layer and multiple haemorrhages in the basal tissue. (E) wt-P1: suppurative infiltration and necrotic debris in the trophoblast cells; (B, D and F) Placenta wt-P2, (B) necrosuppurative placentitis (D) fibrinoid necrosis of the trophoblast cells (N) with foci of mineralization (orange arrow) and suppurative infiltration in forming a dense layer of leucocytes in the basement membrane (black arrow), (F) suppurative infiltration containing PMN in different degrees of degeneration and necrotic debris. Black outlined areas in images represents areas expanded in images located in their immediate below (Scale bar: A: 10 mm; B: 5 mm; C, D: 500μm; E, F: 50μm).
Fig 7.
Immunohistochemical detection of chlamydial antigen in ovine placentas.
Both vt- and wt-placentas were labelled by IHC using genus-specific anti-LPS mAb 13/4 and counterstained with haematoxylin. (A and B) Placenta vt-P1: showing (A) diffuse positive labelling in the cotyledonary trophoblast layer, with (B) intense and abundant labelling of the trophoblastic cells. (C and D) Placenta vt-P2: showing (C) positive labelling of the trophoblast layer, with (D) the intense multifocal to coalescent labelling of the trophoblast cells. (E and F) Placenta wt-P1: showing (E) intense multifocal to coalescent immunolabelling (F) with suppurative infiltration in the trophoblast layer. (G and H) Placenta wt-P2: diffuse moderate labelling of the trophoblast cells, showing (H) intense labelling of the individual mononuclear cell surrounding by abundant cellular debris. The outlined black squares in the images on the left indicate the magnified area shown in the images on their immediate right. IHC anti-chlamydia mAb 13/4. (Scale bar. A, C, E and G: 500μm; B, D, F, and H: 50μm).