The serological response to Verocytotoxigenic Escherichia coli in patients with haemolytic uraemic syndrome

AIMS: To screen sera from 80 patients with clinical haemolytic uraemic syndrome (HUS) and serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157, for antibodies to Verocytotoxin-producing Escherichia coli (VTEC) belonging to serogroups O5, O26, O104, O111, O128, O145, O153 and O165. METHODS AND RESULTS: Sera were screened by an LPS-based ELISA and SDS-PAGE/immunoblotting. None of the 80 sera contained antibodies binding to long-chain LPS of any of the LPS types employed; however, nine sera contained antibodies binding to R3 LPS-core epitopes. CONCLUSIONS: The presence of patients' serum antibodies to the LPS of E. coli O157, in the absence of antibodies to the LPS of a range of other VTEC, demonstrated that cases of HUS may be caused by strains of O157 VTEC alone and that concurrent infection with multiple strains of VTEC is not a prerequisite for cases of HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibodies to long-chain LPS of VTEC other than O157 were not detected, and so there was no evidence of infection with VTEC belonging to more than one serogroup. The results of immunoassays such as ELISAs and micro-agglutinations must take into consideration antibodies binding to R3 epitopes located on LPS-core.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Human antibody responses to R3-core epitopes on the lipopolysaccharide of Escherichia coli O157

AIMS: To establish the incidence of serum antibodies binding to the R3-core lipopolysaccharide (LPS) of verocytotoxin-producing Escherichia coli (VTEC) O157, in patients with serum antibodies to E. coli O157 LPS, and to characterize the class(es) of antibodies binding to epitopes on the R3-core. METHODS AND RESULTS: SDS-PAGE profiles of LPS prepared from VTEC O157 were used in combination with immunoblotting to detect and characterize serum antibodies binding to the R3-core LPS of VTEC O157. Of 417 sera, referred to the Laboratory of Enteric Pathogens (LEP) for routine O157 serology and found to have serum antibodies to long-chain VTEC O157 LPS, 31 had antibodies binding to the R3-core of VTEC O157 LPS. The majority of the 31 sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. Patients who did not develop haemolytic uraemic syndrome (HUS) produced antibodies of the IgM class to R3-core and IgG-class antibodies to long-chain LPS more frequently than patients with HUS. CONCLUSIONS: Only 7.4% of sera received by the LEP, and shown to have antibodies to VTEC O157 LPS, contained antibodies binding to the R3-core of VTEC LPS. Most sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. SIGNIFICANCE AND IMPACT OF THE STUDY: Patients infected with VTEC O157 produced antibodies binding to the R3-core epitopes of VTEC O157 LPS only rarely, and these antibodies are unlikely to interfere with the serodiagnosis of infections caused by these organisms.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.     

Use of immunological adsorption in a heterologous immunoenzyme analytical system for detecting specific antibodies to serogroup B and D1 Salmonella lipopolysaccharides in healthy donors and in persons with a history of salmonellosis

The sera obtained from healthy donors and from subjects with a history of Salmonella infections (B, C1, D1 and E1) of medium severity were studied. In the initial testing in the heterologous enzyme immunoassay the levels of antibodies to LPS, groups B and D1, were somewhat higher in the subjects with a history of the infections than in healthy donors. After a single adsorption with homologous LPS the levelling of differences between the two groups was observed. After two adsorptions with heterologous LPS the level of the reaction in both the groups increased, and the differences between these groups augmented. The adsorption of sera with heterologous LPS increased the diagnostic sensitivity of the reaction only to 14-23% in the determination of the serogroup of the causative agent. An increase in the antibody level after adsorption with heterologous LPS was supposedly due to the additional detection of group-specific antibodies with low avidity, which appeared as the result of the elimination of cross-reacting highly avid antibodies, as well as non-immunoglobulin molecules complementary to LPS.     

Comparison of usefulness of lipopolysaccharides extracted by phenol and trichloroacetic acid from Salmonella Typhimurium and Enteritidis for serodiagnosis of salmonelosis

Salmonella Typhimurium and Salmonella Enteritidis are the two predominant serogroups, responsible for about 80% of all human cases of salmonelosis in Poland. Therefore we compared the usefulness of lipopolysaccharides antigens extracted by phenol (Westphal method) and trichloroacetic acid (Boivine method) from Salmonella Typhimurium and Enteritidis in ELISA method for the determination of antibodies. We used one home - made LPS antigen and two others commercially available antigens from SIGMA - Aldrich. Our study showed that the presence of antibodies was found in 35 (74.5%) sera from 47 samples from patients with suspected salmonelosis. There was no significant statistical differences of frequency of appearance of antibodies to all three Salmonella antigens in sera from patients with salmonelosis and in sera from control group. This study showed that all three antigens are useful for determination of IgA, IgG, IgM antibodies for Salmonella serogroup B and D in routine serological diagnosis of salmonelosis. However, it should be considered possibility of cross-reaction between LPS antigen of Salmonella and antibodies to Yersinia enterocolitica which could be correlated with similarity between somatic antigens of these two pathogens.     

Development and evaluation of a rapid multianalyte particle-based flow cytometric assay for the quantification of meningococcal serogroup B-specific IgM antibodies in sera for nonculture case confirmation

Serogroup-specific antibody has been shown to be present in the sera of patients recovering from meningococcal disease, and thus the detection of such antibodies may aid in the confirmation of disease. There are currently no standard methods for measuring meningococcal serogroup B-specific antibody in sera. Here, we report the development of a microsphere-based immunoassay which utilizes colominic acid from Escherichia coli 07:K1 (L):NM to detect immunoglobulin M directed against serogroup B polysaccharide. The serogroup B assay was incorporated into a multiplex assay which also detects serogroup-specific immunoglobulin M for meningococcal serogroups A, C, Y and W-135. Using the method of cross-standardization, serogroup B-specific immunoglobulin M concentrations were assigned to the standard serum CDC 1992. The assay is able to detect increases in specific immunoglobulin M concentrations from acute to convalescent phase serum from serogroup B cases, and can be utilized in conjunction with the previously developed tetraplex immunoglobulin G detection assay for serogroups A, C, Y and W-135.     

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.     

Evaluation of usefulness of the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to lipopolysaccharides of VTEC strains in patients suspected for Escherichia coli O104:H4 infection in Poland

The aim of the study was to investigate the presence of antibodies to lipopolysaccharides obtained by modified Boivin's method from E. coli serotype O104:H4 and O26, O103, O111, O121, O145, O157 in sera of 7 patients with acute diarrhea, suspected in clinical investigation for infection caused by E. coli O104:H4. Additionally, to determine the cut-off levels, the 75 sera from blood donors were tested. The high level of antibodies to LPS E. coli O104 was diagnosed in three patients from family outbreak caused by E. coli serotype O104:H4. In one of those patients, 7-years boy with HUS, we observed also a significant decrease of level of IgA, IgG and IgM antibodies in serum sample obtained in chronic phase of the disease. Furthermore, we showed that two others patients with clinical evidence of VTEC infection, not connected with this family outbreak, had a high level of antibodies to E. coli of other serotypes: one to O157 and one to O103. We did not observe presence of antibodies to LPS from E. coli O26, O111, O121 i O145 in the sera of tested patients. In conclusion, we confirmed that ELISA based on lipopolysaccharides obtained from different serotypes of E. coli may be helpful in laboratory diagnosis of infection caused by VTEC in humans.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona, were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona. In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Abstract Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona , were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona . In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Escherichia coli O157 serology: false-positive ELISA results caused by human antibodies binding to bovine serum albumin

An analysis of farm workers and rural dwellers for serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157 detected sera with antibodies binding to bovine serum albumin (BSA) by ELISA. These antibodies were not specific for BSA when examined by immunoblotting, and the ELISA values were reduced to a background level when plates were blocked with normal rabbit serum.     

California serogroup and Powassan virus infection of cats

One hundred and seventy five sera from cats in Ontario, Canada, were tested for hemagglutination inhibition (HI) antibodies to three arboviruses; namely, Powassan (POW) of the Flavivirus serogroup, and Snowshoe hare (SSH) and Jamestown Canyon (JC) viruses of the California (CAL) serogroup. All sera were negative for antibodies to POW virus. Twelve cats possessed CAL serogroup antibodies including 3 with antibodies to SSH alone, 6 with antibodies to JC alone, and 3 with antibodies to both SSH and JC antigens. POW virus was inoculated into seven cats, one intracerebrally and six intravenously. Neurologic signs were not detected in any of the cats. Histologic lesions of a nonsuppurative encephalitis and encephalomyelitis were observed in the intracerebrally inoculated cat and in one of the intravenously inoculated cats, respectively. POW virus was not isolated from the brain or spinal cord of either of these two cats. HI antibodies were detected in the sera of all inoculated animals. HI antibodies were not detected in the CSF of any animal.     

Homologous and heterologous antibody responses to lipopolysaccharide after enterohemorrhagic Escherichia coli infection

To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli(EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme-linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.     

Isolation and characterization of a lipopolysaccharide mutant of Legionella pneumophila

A mutant unable to bind a monoclonal antibody (mAb 1E6) directed against serogroup 1 lipopolysaccharide (LPS) was isolated from L. pneumophila strain Philadelphia-1. SDS-PAGE analysis of isolated LPS from the mutant and wild type revealed that there were no obvious structural differences between the two LPS. The results from Western-blot experiments showed that the mutant LPS was unable to bind mAb 1E6 but retained the ability to bind polyclonal serogroup 1 antibodies. Loss of the LPS epitope recognized by mAb 1E6 did not alter the ability of the mutant to multiply in human monocyte-like U937 cells. Also, the mutant, like wild type, was resistant to killing by normal human serum. These results show that a minor change in the antigenic composition of serogroup 1 LPS has no effect on the virulence properties of strain Philadelphia-1. Additionally, this mutant may be useful for molecular genetic analysis of serogroup 1 LPS biosynthesis and assembly.     

Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structural elucidation of the O36-antigen polysaccharide

The O-specific polysaccharide (OPS) isolated from the lipopolysaccharide of Proteus mirabilis O36 was found to have a pentasaccharide repeating unit of the following structure: -->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAc6Ac-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. The structure is unique among Proteus OPS, which is in agreement with the classification of this strain into a separate Proteus O-serogroup. Remarkably, the P. mirabilis O36-polysaccharide has the same structure as the OPS of Escherichia coli O153, except that the latter is devoid of O-acetyl groups. The cross-reaction of anti-O36 antibodies with the O-part of E. coli O153 lipopolysaccharide is observed. In the present study, two steps of serotyping Proteus strains are proposed: screening of dry mass with enzyme-linked immunosorbent assay and immunoblot with the crude lipopolysaccharides. This method allowed serotyping of 99 P. mirabilis strains infecting the human urinary tract. Three strains were classified into serogroup O36. The migration pattern of these lipopolysaccharides fraction with long O-specific PSs was similar to the standard laboratory P. mirabilis O36 (Prk 62/57) lipopolysaccharide. The relatively low number of clinical strains belonging to serogroup O36 did not correspond to the presence of anti-P. mirabilis O36 antibodies in the blood donors' sera. Twenty-five percent of tested sera contained a statistically significant elevated level of antibodies reacting with thermostable surface antigens of P. mirabilis O36. The presence and amount of antibodies correlated with Thr399Ile TLR4 polymorphism types (P=0.044).     

Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

Detection of porin antigen in serum for early diagnosis of mouse infections with Salmonella typhimurium

Abstract The monoclonal antibodies to porin, an outer membrane protein isolated from Salmonella typhimurium and sandwich enzyme linked immunosorbent assay (ELISA) has made possible the detection of porin from sera of S. typhimurium -infected mice. The specificity of the monoclonal antibodies was ascertained based on their cross-reactivity with porins isolated from S. typhi, Shigella flexneri and Escherichia coli and lipopolysaccharide (LPS) of S. typhimurium and E. coli . Serum samples were found to be positive for porin as early as 3 days after intravenous and 5 days after oral infection. In addition, a positive correlation was observed between the bacterial load and the concentration of porin detected in the sera. On the other hand, analysis of sera for anti-porin antibody showed diametrically opposite time kinetics with antigenaemia. These results indicate that porin accumulates in the serum of infected mice much earlier than the appearance of antibodies to porin. Thus detection of porin holds promise for early diagnosis of typhoid.     

Level of IgG antibodies to bacteria of 12 species in the blood sera of rabbits, immunized with preparations obtained from Neisseria meningitidis, grown under the stress conditions

The blood sera of rabbits, immunized with preparations obtained from N. meningitidis of serogroups A, B or C, cultivated under the stress conditions, were studied. These sera were found to contain IgG antibodies not only to N. meningitidis antigens, but also to the bacterial antigens of 12 species. The sera of rabbits, immunized with meningococcal preparation of serogroup A, were found to have the elevated levels of IgG antibodies, in comparison with the control, to the antigens of 3 other bacterial species; the blood sera of rabbits, immunized with meningococcal preparation of serogroup B, were found the elevated levels of IgG antibodies to the antigens of 11 other bacterial species; and the blood sera of rabbits, immunized with meningococcal preparation of serogroup C, to the antigens of 9 other bacterial species. The study of serogroup B meningococci, used as an example, revealed the influence of the growth phase of the culture on the content of cross-reacting antigens. Their greatest amount was determined at the stationary phase when the stressor effect on the culture reached its maximum and their least amount, at the exponential phase when the stressor effect on the culture was minimal. It was, therefore, found to be expedient to obtain immunodiagnostic and test systems from N. meningitidis cultures, grown to middle of the exponential phase of growth.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.