Formalin-fixed and paraffin-embedded placenta specimens were analyzed by using histopathologic and immunohistochemical techniques. Hematoxylin and eosin–stained histologic sections of all placenta specimens (n = 235) were examined for the type and degree of placentitis or vasculitis. Paraffin-embedded sections of specimens positive for Chlamydia-like organisms by 16S rRNA PCR (n = 43) were analyzed for Parachlamydia spp. and Waddlia by using specific mouse polyclonal antibodies as described (10). Optimization experiments for immunohistochemical analysis were performed by using infected amebal and infected HEp-2 cell pellets. Briefly, Acanthamoeba castellanii cultures were infected with P. acanthamoebae strain Hall coccus and W. chondrophila strain ATCC 1470. HEp-2 cell monolayers were infected with Cp. abortus strain S26/3. Uninfected cells were used as negative controls. Amebal and cell pellets were prepared as described (11). Optimization of the immunohistochemical protocol for experimentally infected amebal pellets showed the species specificity of mouse antibodies to P. acanthamoebae and W. chondrophila. We did not observe cross-reactivity of both antibodies with Cp. abortus–infected HEp-2 cell pellet (data not shown).

To test placental specimens, we used mouse polyclonal antibody against P. acanthamoebae and W. chondrophila at dilutions of 1:1,000 and 1:2,000, respectively. Antigen detection was performed with the ChemMate Detection Kit (Dako, Glostrup, Denmark) according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinated in xylene and rehydrated through graded ethanol to water. Antigen was detected by using repeated microwave heating (750 W for 10 min) in citrate buffer, pH 6.0 (Target Retrieval Solution, Dako). Specimens (slides) and primary antibodies were incubated for 1 hour. Negative and positive controls of each section were included as described (9).

Histopathologic lesions such as purulent or necrotizing placentitis were observed in 149 (63.4%) of 235 specimens. Placentitis was observed in 5 of 8 specimens positive for P. acanthamoebae, and vasculitis was observed in 1 of 8 specimens (Table). Positive antigen labeling was observed in 6 of 8 specimens for Parachlamydia spp., but antigen labeling was negative in all specimens for Waddlia (Table). The Figure, panel A shows positive immunohistochemical labeling in 1 of these specimens. Among the 35 placentas positive by PCR for Chlamydia-like organisms other than P. acanthamoebae, 28 (82.3%) showed obvious purulent or necrotizing placentitis by histologic analysis. Four of the 28 specimens with placentitis also had vasculitis. A total of 24 (68.6%) of 35 specimens were positive when tested with antibody against P. acanthamoebae, and all 35 specimens were negative when tested with antibody against W. chondrophila.

Two placental specimens positive for Parachlamydia spp. by immunohistochemical analysis and 16S rRNA PCR were further investigated by transmission electron microscopy for ultrastructural evidence of Chlamydia-like organisms. Briefly, placental tissue specimens were fixed with glutaraldehyde and osmium tetroxide and embedded in Epon resin. Ultrathin sections (80 nm) were mounted on gold grids (Merck Eurolab, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Fluka, Buchs, Switzerland) and lead citrate (lead nitrate and tri-natrium dehydrate, Merck Eurolab), and analyzed with a Philips (Eindhoven, the Netherlands) CM10 electron microscope. Both placentas showed Chlamydia-like structures (Figure, panel B).

To our knowledge, this is the first description of Parachlamydia spp. in bovine abortion. The organism was detected by PCR (9) and within placental lesions by immunohistochemical analysis by using an antibody specific for Parachlamydia spp. and electron microscopy. All specimens were negative for Waddlia by immunohistochemical analysis. Isolation of Parachlamydia spp. from aborted bovines is necessary to confirm that this agent causes bovine abortion. Parachlamydia spp. may be involved in lower respiratory tract infections in humans (5,6) and may replicate within both pneumocytes (13) and human macrophages (14). Thus, caution should be taken when handling bovine abortion material because of the potential zoonotic risk.