A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.

Cross-reactivity between antibodies to different human coronaviruses (HCoVs) has not been systematically studied. By use of Western blot analysis, indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA), antigenic cross-reactivity between severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and 2 HCoVs (229E and OC43) was demonstrated in immunized animals and human serum. In 5 of 11 and 10 of 11 patients with SARS, paired serum samples showed a > or =4-fold increase in antibody titers against HCoV-229E and HCoV-OC43, respectively, by IFA. Overall, serum samples from convalescent patients who had SARS had a 1-way cross-reactivity with the 2 known HCoVs. Antigens of SARS-CoV and HCoV-OC43 were more cross-reactive than were those of SARS-CoV and HCoV-229E.

Porphyromonas gingivalis is a bacterial pathogen associated with chronic periodontitis that results in destruction of the tooth's supporting tissues. The major virulence determinants of P. gingivalis are its cell surface Arg-and Lys-specific cysteine proteinases, RgpA/B and Kgp. Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in saliva and gingival crevicular fluid, is believed to play an important role in innate immunity. In this study, bovine milk LF displayed proteinase inhibitory activity against P. gingivalis whole cells, significantly inhibiting both Arg-and Lys-specific proteolytic activities. LF inhibited the Argspecific activity of purified RgpB, which lacks adhesin domains, and also inhibited the same activity of the RgpA/Kgp proteinaseadhesin complexes in a time-dependent manner, with a first-order inactivation rate constant (k inact ) of 0.023 min ؊1 and an inhibitor affinity constant (K I ) of 5.02 M. LF inhibited P. gingivalis biofilm formation by >80% at concentrations above 0.625 M. LF was relatively resistant to hydrolysis by P. gingivalis cells but was cleaved into two major polypeptides (53 and 33 kDa) at R 284 to S 285 , as determined by in-source decay mass spectrometry; however, these polypeptides remained associated with each other and retained inhibitory activity. The biofilm inhibitory activity of LF against P. gingivalis was not attributed to direct antibacterial activity, as LF displayed little growth inhibitory activity against planktonic cells. As the known RgpA/B and Kgp inhibitor N-␣-p-tosyl-L-lysine chloromethylketone also inhibited P. gingivalis biofilm formation, the antibiofilm effect of LF may at least in part be attributable to its antiproteinase activity.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibodies to Histophilus somni exopolysaccharide (EPS), which is created during biofilm formation. When an index value of 0.268 was used, the sensitivity of the assay for infected calves was 90.5% at 3 weeks postinfection, but the number of positive animals increased by week 4. The specificity of the assay for healthy calves was 92.5%. The EPS ELISA may aid in identifying calves with H. somni diseases.

is capable of intracellular survival within professional phagocytic cells, but the mechanism of survival is not understood. The Fic motif within the direct repeat (DR1)/DR2 domains of the IbpA fibrillary network protein of is cytotoxic to epithelial and phagocytic cells, which may interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and Fic to resistance to host defenses, strains and mutants that lacked all or a region of (including the DR1/DR2 regions) were tested for survival in bovine monocytic cells and for serum susceptibility. An mutant lacking IbpA, but not the DR1/DR2 region within , was more susceptible to killing by antiserum than the parent, indicating that the entire protein was associated with serum resistance. strains expressing IbpA replicated in bovine monocytes for at least 72 h and were toxic for these cells. Virulent strain 2336 mutants lacking the entire gene or both DR1 and DR2 were not toxic to the monocytes but still survived within the monocytes for at least 72 h. Monitoring of intracellular trafficking of with monoclonal antibodies to phagosomal markers indicated that the early phagosomal marker early endosome antigen 1 colocalized with all isolates tested, but only strains that could survive intracellularly did not colocalize with the late lysosomal marker lysosome-associated membrane protein 2 and prevented the acidification of phagosomes. These results indicated that virulent isolates of were capable of surviving within phagocytic cells through interference in phagosome-lysosome maturation. Therefore, may be considered a permissive intracellular pathogen.

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S-ribosylhomocysteinase (LuxS) is required for the synthesis of the autoinducer-2 (AI-2) quorum-sensing signalling molecule in many Gram-negative bacteria. The bovine (and ovine) opportunistic pathogen Histophilus somni contains a luxS, and forms a biofilm containing an exopolysaccharide (EPS) in the matrix. Since biofilm formation is regulated by quorum sensing in many bacteria, the role of luxS in H. somni virulence and biofilm formation was investigated. Although culture supernatants from H. somni were ineffective at inducing bioluminescence in the Vibrio harveyi reporter strain BB170, H. somni luxS complemented the biosynthesis of AI-2 in the luxS-deficient Escherichia coli strain DH5α. H. somni strain 2336 luxS was inactivated by tranposon mutagenesis. Comparison of RNA exression profiles revealed that many genes were significantly differentially expressed in the luxS mutant compared to the wildtype, whether the bacteria were grown planktonically or in a biofilm. Furthermore, the luxS mutant had a truncated and asialylated lipooligosaccharide (LOS), and was substantially more serum-sensitive than the wildtype. Not surprisingly, the luxS mutant was attenuated in a mouse model for H. somni virulence, and some of the altered phenotypes were partially restored after the mutation was complemented with a functional luxS. However, no major differences were observed between the wildtype and the luxS mutant in regard to outer membrane protein profiles, biofilm formation, EPS production, or intracellular survival. These results indicate that luxS plays a role in H. somni virulence in the context of LOS biosynthesis, but not biofilm formation or other phenotypic properties examined.

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