subsp. with subsp. SC, in which it was recognized postinfection for significantly longer than standard serological test reactions. subsp. small-colony type (SC) is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious disease which represents a major danger to raising cattle, particularly in Africa. CBPP is also a problem in other parts of the world, including some European countries, where the disease all of a sudden reemerged 2 decades ago. CBPP is definitely a disease of major economic concern in the affected countries, not only due to the morbidity and mortality but also due to restrictions on cattle trade imposed by international regulations. Hence, control of the disease is definitely a priority for countries in which it is endemic, in order to eradicate the disease as quickly as possible after outbreaks and to avoid its distributing, as well as for countries which are free of CBPP, in order to keep that status. The main problem in eradication is the frequent event of subacute or asymptomatic infections and the persistence of chronic service providers after the medical phase. Serological analysis is the most important diagnostic tool for the control of CBPP, but it MRS 2578 is definitely significantly hampered from the relatively low level of sensitivity and specificity MRS 2578 of the methods. The match MRS 2578 fixation test (CFT), which is currently the established and most widely used serodiagnostic test, offers been shown to be relatively sensitive in the acute phase of the disease, but it levels off rather quickly and is insensitive 3 months after illness (1, 29). In contrast to CFT, immunoglobulin G (IgG) and IgA reactions to many antigens of subsp. SC are prolonged for several weeks, as demonstrated by immunoblot analysis of sera and bronchial-lavage samples which were sequentially collected from experimentally contact-infected cattle (1). In addition to problems with level of sensitivity, the specificity of current serological checks is definitely reduced due to cross-reactions with additional closely related users of the cluster, which can lead to false-positive results (11, 14, 28, 33). By using a competitive enzyme-linked immunosorbent assay based on a monoclonal antibody which specifically identified a yet-uncharacterized approximately 80-kDa antigen of subsp. SC, the specificity of serodiagnosis of CBPP could be significantly improved (21). It is therefore important to characterize specific antigens of subsp. SC. A few antigens of subsp. SC have been characterized, including the lipoproteins LppA (12) and LppB (35). The major lipoprotein, LppA, was shown to belong to a family of lipoproteins which is definitely created in all users of the cluster. LppA was recently found to contain only a few epitopes, which are specific to subsp. SC (15, 25). LppB is present only in strains belonging to the African cluster of subsp. SC and not in the Western cluster (35). In order to develop more efficient serodiagnostic tools and to study the molecular mechanism of virulence which distinguishes the highly pathogenic subsp. SC from your additional significantly less pathogenic or nonpathogenic users of the cluster, it is essential to acquire fundamental molecular knowledge about those factors which discriminate subsp. SC from additional closely related mycoplasmas. In the Rabbit Polyclonal to MRPS33 present study, we have analyzed the genetic, antigenic, and biochemical properties of a newly recognized lipoprotein, LppQ, which is definitely specific to subsp. SC and which induces an early immune response in cattle with CBPP which persists long after other immune responses. MATERIALS AND METHODS Strains and growth conditions. strains used in this study are outlined in Table ?Table1.1. They were cultured in standard medium at 37C (5) until stationary growth phase. The cells were harvested by centrifugation at 13,000 for 20 min, washed three times in TES buffer (10 mM Tris-HCl, 1 mM EDTA, 0.8% NaCl, pH 8.0), and then resuspended in TES buffer to a concentration of approximately 109 ml?1. For gene cloning, strains XL1-blue MRF ((Tn(Tetr)]; XLOLR (([F Tn(Tetr)] r Su? (Stratagene, La Jolla, Calif.); and BL21(DE3) (F? (T7-RNA-(lysogenic) strains were cultivated in Luria-Bertani broth at 37C in an orbital shaker-incubator (31). Antibiotics (ampicillin, 100 g/ml; kanamycin, 50 g/ml; or tetracycline, 50 g/ml) were added.