trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs. It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species (Banks et al., 1970; Peterson et al., 1990; Girjes et
al., 1993; Donati et al., selleckchem 1996, 2006, 2009). The detection of these antibodies could be useful in the diagnosis of mixed infections or in the detection of immunogenic antigens as vaccine candidates. A previous study (Donati et al., 2009) reported a strong in vitro neutralizing activity to Chlamydia suis in 80% of selleck pig sera that, due to the presence of high microimmunofluorescence (MIF) titres, suggested C. suis infection. A close relationship
between C. suis and Chlamydia trachomatis has already been reported in relation to the ompA DNA sequence similarity (Kaltenboeck et al., 1997), together with morphology and other features, such as the production of glycogen in cell culture (Rogers et al., 1996) and the sensitivity to cathelicidins (Donati et al., 2007). In view of these features, in the present study, we evaluated the neutralizing activity against D–K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs. A total of 17 MIF chlamydia-positive selected sera were tested: 11 sera collected from C. suis-infected pigs showing C. suis neutralizing activity and six sera from patients infected with D, E, F, G, H and K C. trachomatis serovars, respectively. As a negative control, 10 human and 10 pig MIF chlamydia-negative sera were used. Before performing the neutralization assay, human and pig sera were diluted at a MIF titre of 128 to C. trachomatis and C.
suis, respectively, to obtain a uniform Obatoclax Mesylate (GX15-070) antibody concentration. Italian urogenital C. trachomatis isolates D–K (Donati et al., 2009), and the Italian C. suis isolate 7MS06 (Donati et al., 2007) were grown in LLC-MK2 cells and EBs were purified by sucrose density-gradient ultracentrifugation using the method of Fukushi & Hirai (1988). In addition, purified EBs of the reference strains Chlamydia muridarum Nigg, Chlamydophila pneumoniae IOL-207, Chlamydophila psittaci 6BC and the Italian Chlamydophila felis FEIS-M isolate were used to check the species-specificity of neutralizing antibodies in the human and pig sera. EB preparations were titrated to contain 4 × 105 inclusion-forming units (IFU) mL−1 and stored frozen in 0.25 M sucrose–10 mM potassium phosphate–5 mM glutamic acid, pH 7.4 (SPG), at −70 °C. As a source of complement, aliquots of fresh rabbit serum were stored at −70 °C and used in the neutralization assay at a 5% final concentration.