Protein and antigen profiles of Leptospira interrogans serovar Hardjo

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.

An investigation of the therapeutic value of vaccinia-immune IgG in a mouse pneumonia model

Vaccinia-immune globulin (VIG) was used to treat severe complications of smallpox vaccination, but its use was controversial because it resolved disease in only some clinical cases. VIG is a pool of hyperimmune sera collected from individuals with a high neutralizing titre against the intracellular mature form (IMV) of vaccinia virus (VACV), but activity against the extracellular enveloped form (EEV) was often not considered. Here, the efficacy of anti-VACV antibodies (Abs) in protecting mice from intranasal infection with the VACV strain Western Reserve (WR) was evaluated. Mice were immunized passively with hyperimmune rabbit Abs (IgG) generated against inactivated IMV or produced following infection by VACV; subsequently, animals were challenged with VACV WR. The results demonstrated that: (i) good protection requires Abs to EEV in addition to IMV; (ii) Abs were effective when given before or up to 4 days after infection; and (iii) protection of mice from VACV WR correlated with a reduction of virus replication in lungs, but not in brain. In agreement with studies conducted before smallpox was eradicated and recent studies using EEV antigens for immunization, this study reiterates the importance of anti-EEV Abs in protecting against orthopoxvirus infection and illustrates the need to evaluate both anti-IMV and anti-EEV neutralizing Abs in VIG.

Phage Lytic Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia

ABSTRACT Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are badly needed. We report the use of Cpl-1, the lytic enzyme of a pneumococcal bacteriophage, as an intravenous therapy for pneumococcal bacteremia in a mouse model. A 2,000-μg dose of Cpl-1 reduced pneumococcal titers from a median of log10 4.70 CFU/ml to undetectable levels (<log10 2.00 CFU/ml) within 15 min. This dose given 1 h after intravenous infection led to 100% survival at 48 h, compared to the 20% survival of buffer-treated controls. In advanced bacteremia, treatment with two doses at 5 and 10 h still resulted in significantly longer survival (P < 0.0001) and a hazard ratio of 0.29 (95% confidence interval, 0.04 to 0.35). The enzyme is immunogenic, but the treatment efficacy was not significantly diminished after previous intravenous exposure of mice and hyperimmune rabbit serum did not neutralize the activity. Cpl-1 is also very effective as a topical nasal treatment against colonization by S. pneumoniae. In vitro, the enzyme is active against many serotypes of S. pneumoniae, independent of their penicillin resistance, and it is very specific for this species. Bacteriophage enzymes are unusual but extremely effective antimicrobials and represent a new weapon against infections with resistant bacteria.

Partial characterization and time course analysis of Hymenolepis diminuta coproantigens

An analysis of Hymenolepis diminuta specific antigens in infected rat faeces was carried out. Using a capture type antibody sandwich ELISA assay based on a hyperimmune rabbit anti-worm somatic antisera it was demonstrated that, although antigen was present in faeces before patency, the onset of egg production led to a sharp increase in the levels of parasite antigen in the faeces. Levels of antigen in host faeces were independent of worm burden. Parasite eggs did not contribute significantly to faecal antigen levels. Western blot analysis indicated a number of highly specific antigens at around Mr 69,000, Mr 37,000, Mr 50,000 and Mr 27,000 with a low molecular weight smear at between Mr 30,000 and Mr 34,000 present in the faeces of H. diminuta infected rats. Some cross reaction occurred with an antigen of around Mr 66,000 in the faeces of non H. diminuta infected rodents. Antibody activity against this antigen was removed by affinity adsorption of the antibody solution against normal rat faeces.

Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans

SUMMARYThree ELISA assays, based on hyperimmune rabbit serum raised against adult cestode somatic antigen, were applied in this study for the detection ofTaenia- andEchinococcus-specific antigens in host faeces. The first assay, using an antiserum againstTaenia pisiformisantigen extract, was used in a time-course ofT. pisiformisexperimental infection in dogs. The assay was shown to be considerably more sensitive than microscopical detection of eggs in faeces. Antigen was present in faeces before patency and antigen levels were independent ofT. pisiformisegg output. The second assay, involving a test for human taeniasis based on antibodies againstT. solium, was applied in two field studies carried out in China and Guatemala. The test was highly specific, no false positive reactions occurred with human faecal samples and the test was capable of diagnosing individuals who would not have been detected by coproscopy or treatment to recover the tapeworm. A third assay was designed forE. granulosusand demonstrated 87·5% sensitivity and 96·5% specificity with samples from naturally and experimentally infected dogs withEchinococcusorTaeniainfections. In both the humanTaeniaand canineEchinococcusstudies antigen could be detected in faecal samples from infected hosts stored in 5% formalin for 6 months. Further refinements to these tests for field application are discussed.

The serological relationship betweenYersinia enterocoliticaO9 andEscherichia coliO157 using sera from patients with yersiniosis and haemolytic uraemic syndrome

SUMMARYSera from patients with yersiniosis. shown to contain antibodies toYersinia enterocoliticaO9; and sera from patients with haemolytic uraemic syndrome (HUS) caused byEscherichia coliO157, were used to investigate serological cross-reactions betweenY. enterocoliticaO9 andE. coliO157. Lipopolysaccharide (LPS) was isolated from strains ofY. enterocoliticaO9 andE. coliO157 and reacted with sera by immunoblotting and ELISA. Sera from patients with HUS contained antibodies to the LPS ofE. coliO157 only; 80% of sera from patients with yersiniosis contained antibodies to the LPS ofY. enterocoliticaO9 andE. coliO157. This one-way cross-reaction was also detected using hyperimmune rabbit antisera.

Analysis of lipopolysaccharide antigens ofTreponema hyodysenteriae

SUMMARYLipopolysaccharide (LPS) extracts obtained fromTreponema hyodysenteriaeof serogroups A, B, D and E, and fromT. innocenswere examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), silver-staining, and immunoblotting with hyperimmune rabbit sera. All organisms possessed multiple LPS bands, but their position and number differed.Immunoblotting of LPS with grouping sera identified three or four major antigenic LPS components in the 10-42 kDa range in all organisms: these components were largely specific to each type-organism of a serogroup, and presumably represented group antigens. Although some minor cross-reactivity occurred between LPS from organisms in the different groups, this was insufficient to merit changes to the current LPS serogrouping system for T. hyodysenteriae. Besides this LPS ‘complex’, other higher-molecular-weight material which appeared to be a common component of the treponemes examined was present in low concentrations. Organisms with different serotypes within a serogroup apparently possessed common LPS bands, but also had unique LPS bands which may account for their serotype specificity. One ‘untypable’ organism lacked group-specific LPS and was thought to be a mutant of a group B organism. The loss of serogroup LPS by the isolate suggested that this material is an external component of the cell wall. The availability of an atypical organism lacking LPS components may facilitate further studies on the pathogenesis of swine dysentery.