Serogroup-specific Antigen Research Articles

Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels.

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.

A serologicalsurveillance of bluetongue disease in sheep and goats in Iraq by using acompetitive ELISA Technique

In this study, a sero-survillance on ovine and caprine serum samples from all over Iraqexcept the governorates of Iraqi Kurdistan region was conducted. It aimed for the detection ofspecific antibodies directed against VP7 protein - the sero-group specific antigen of bluetongue virus – by using a competitive ELISA technique.The results showed that among a totalof 3277 serum samples, 1436 samples were found to be positive forming 43.82% of totalsamples, while 1687 samples were negative with 51.48 % and 154 sample were suspiciousforming 4.69 %. The highest positive serum samples were found in Al Basrah Province(southern of Iraq) with 60.41% and in Al Anbar Province (Western of Iraq) with 61%, whilethe lowest of positive samples were found in Nineva (Northern of Iraq) with 22.35%.The results also showed a closely ratio between positive percentage of goats and sheepsamples with the positive goats serum samples ( in five provinces ) were 45 of 114 samplesrepresenting 39.47 %,while the positive sheep serum samples ( in fifteen provinces) were1391 of 3163 samples representing43.97%. Our study is complementary to the previousworks held in this field and it represents the first field serological survey for bluetongueinsheep and goats in Iraq.

Complete Genome Characterisation of a Novel 26th Bluetongue Virus Serotype from Kuwait

Bluetongue virus is the “type” species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing “bluetongue” (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen “VP7” showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein “VP2” identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other “eastern” or “western” BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.

A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes

Since 1998, six distinct serotypes of Bluetongue virus (BTV) have invaded Southern and Central Europe, persisting in some regions for up to 6 years and resulting in the deaths of >1.8 million sheep. Rapid and reliable methods of virus detection and identification play an essential part in our fight against bluetongue disease (BT). We have therefore developed and evaluated a duplex, one-step RT-PCR assay that detects genome segment 7 (encoding the major serogroup (virus-species) specific antigen and outer-core-protein VP7) from any of the 24 BTV serotypes. Although Seg-7 is highly conserved, there are sequence differences in the near terminal regions that identify two distinct phylogenetic groups. Two sets of primers (targeting Seg-7 terminal regions of viruses from these two groups) were included in a duplex RT-PCR assay system. Assay sensitivity was evaluated using tissue culture derived virus, infected vector insects and clinical samples (blood and other tissues). The assay reliably amplified Seg-7 from any of the BTV strains tested, including isolates of the 24 BTV serotypes and isolates from different geographic origins. No cross-reactions were detected with members of closely related Orbivirus species ( African horsesickness virus (AHSV), Epizootic haemorrhagic disease virus (EHDV), Equine encephalosis virus (EEV) and Palyam virus (PALV)).

Expression and antigenic characterization of the major core protein VP7 of Chuzan virus, a member of the Palyam serogroup orbiviruses

The Palyam serogroup-specific antigen, VP7, of Chuzan virus strain K-47 was expressed in insect cells by a recombinant baculovirus. The expressed protein appeared as a single band of 38 kDa corresponding to the predicted molecular mass of Chuzan virus VP7 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In immunoprecipitation analysis, the recombinant VP7 was not only recognized by all polyclonal antibodies against the Palyam serogroup viruses (PALV) tested in this study, but also by antisera to bluetongue virus (BTV) serotype 1, epizootic haemorrhagic disease virus (EHDV) serotypes 1 and 2. However, in Western immunoblot assay, no positive signals were observed between this protein and these antisera, even in the homologous reaction using antiserum to Chuzan virus. These findings demonstrate that the common antigenic determinants on the VP7 proteins of Chuzan virus and the other PALV serotypes are mainly conformational and that the proteins share some epitopes with those of BTV and EHDV beyond the serogroup. No cross-reactivities were detected between Chuzan virus VP7 and antisera to BTV and EHDV in agar gel immunodiffusion (AGID) and indirect ELISA tests, indicating that the recombinant VP7 is useful as a diagnostic reagent for serological tests of congenital abnormalities of cattle caused by PALV.

A particle counting immunoassay for the direct detection of Clostridium difficile serogroup specific antigen in faecal specimens.

The potential of a particle counting immunoassay (PACIA) for the direct detection of Clostridium difficile serogroup G specific antigen in faecal specimens was evaluated. F(ab')2 fragments from a rabbit anti-serogroup G antiserum were covalently coupled to carboxylated latex beads. This reagent was mixed with acid extracts of faecal specimens and the reaction was assayed with an optical counter which discriminated unagglutinated from agglutinated latex particles. Culture for C. difficile, faecal cytotoxin detection, PACIA and serogrouping of C. difficile isolates were performed on 249 stools. Of the 71 culture-negative specimens, none gave a positive result in the cytotoxin assay or in PACIA. Faecal cytotoxin was detected in 100 of the 178 culture-positive specimens. PACIA was positive for 63 of the 71 faecal specimens that yielded serogroup G C. difficile on culture. PACIA gave negative results for all other culture-positive stools tested with one exception, from which a serogroup A7 C. difficile strain was isolated. PACIA detection of serogroup G antigen in faecal specimens showed a sensitivity of 88.7%, a specificity of 99.7%, a predictive value of a positive culture with a serogroup G strain of 98.4%, and a predictive value for specimens that were culture-negative for a serogroup G strain of 95.6%. The results indicate that PACIA with specific antiserum is a rapid and reliable method for detecting serogroup specific antigens of C. difficile in faecal specimens. Clinical applications of the method are discussed.

Further evidence that genotypically closely related strains of Legionella pneumophila can express different serogroup specific antigens.

The relationship between serogroup and genotype of Legionella pneumophila strains was investigated by restriction fragment length polymorphism (RFLP) typing with a previously standardised method. Of the 51 RFLP types identified, 19 comprised strains of more than one serogroup. Several RFLP types included strains of five or more serogroups. To determine if sharing the same RFLP type indicates that strains are genotypically indistinguishable or merely that they are superficially similar, 31 strains were selected for further analysis with an extended range of restriction endonucleases and nucleic acid probes. In some cases, strains of a particular RFLP type were indistinguishable, while in others the restriction fragment patterns showed minor differences. It is possible that in the latter case the strains are diverging representatives of a parent clone. We conclude that analysis of restriction fragment patterns, either probed or unprobed, provides a more accurate measure of the ancestral relationship between strains than can be obtained with serological methods.

Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis.

Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38-40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38-40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.

Detection of Antigen Determinants and Peptide Mapping of the Membrane Proteins of Erwinia chrysanthemi Pathovars.

Erwinia chrysanthemi strains isolated from various hosts were sero logically grouped with three antisera prepared against E. chrysanthemi strains Ku8601 isolated from pear, R8 from rice and 511-3 from corn. The strains isolated from carnation, pear, potato, onion, taro and welsh onion reacted with anti-Ku8601-serum, which were classified as serogroup I. Rice and corn strains respectively reacted with anti-R8 and anti-511-3 sera, showing high specificity which were classified as serogroup II and serogroup III. Membrane proteins of the bacterial strains belonging to each serogroup were separated with SDS-PAGE and immunoblotted on nitrocellulose sheet to determine antigenic proteins. The strains of serogroup I commonly possessed antigenic polypeptides of 38.5 and 5.9kd that were considered to be serogroup-specific antigens. While the strains of serogroup II possessed antigenic polypeptides of 41.7 and 11.2kd and those of serogroup III possessed 41.7, 40.3, 21.4 and 10.2kd, respectively. Close relationship was observed between serological property and host of origin only in rice and corn strains.

Heat stability of protective antigen of Leptospira interrogans serovar lai

Protective antigen (PAg; glycolipid antigen; molecular size, 23 to 30 kilodaltons), the serogroup-specific antigen partially purified from leptospiral cells, is one of the most important protective antigens. The heat stability of PAg was compared with that of whole-cell (WC) antigen by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, protective activity, opsonin-inducing activity, agglutinating antibody-inducing activity, and an inhibition test in an enzyme-linked immunosorbent assay. A band of 23 to 30 kilodaltons of PAg, which was seen in untreated PAg and WC, shifted to a position with a molecular size of ca. 20 kilodaltons after heat treatment of PAg at 80 degrees C for 30 min and WC at 100 degrees C for 30 min. In the enzyme-linked immunosorbent assay inhibition test with monoclonal antibody LW2 and a sonicated antigen of WC, the inhibition rate of PAg and WC to sonicated WC was reduced by heat treatment at 80 degrees C for 30 min and at 100 degrees C for 30 min, respectively. Agglutinating antibody-inducing activities and opsonin-inducing activities of PAg and WC in mice were reduced by heat treatment under the same conditions; these activities were assayed by a microscopic agglutination test and by chemical luminescence response in serum from immunized mice, respectively. Protective activity of heated PAg and heated WC in cyclophosphamide-pretreated mice agreed with the results of immunogenicity in mice. These results indicate that the Leptospira PAg is one of the important protective antigens and is altered by heat treatment at 80 degrees C. Furthermore, the immunogenicity and antigenicity of the PAg present in WC are more stable than that of the extracted PAg, and the coexistence of other cellular components with PAg might protect and stabilize PAg from the heat treatment.

Immunological Reactivity and Passive Protective Activity of Monoclonal Antibodies against Protective Antigen (PAg) of Leptospira interrogans Serovar lai

Monoclonal antibody (MAb) AG1 against the protective antigen (PAg) was produced and characterized. It had been extracted from Leptospira interrogans serovar lai by the chloroform-methanol-water method and was of glycolipid nature (23-30Kd). The fact that the PAg was a serogroup-specific antigen was shown by MAb AG1, because MAb AG1 agglutinated serovars of serogroup Icterohaemorrhagiae. Purified MAb LW2 and LW3 which are agglutinating antibodies of serovar lai and AG1 passively protected hamsters from leptospiral infection. Induction of the reactive oxygen intermediates by MAbs from peritoneal exudate macrophages of mice were observed in the chemical luminescence assay and the MAbs reacted with the PAg in the dot enzyme-linked immunosorbent assay. However, MAb LW4a against the genus-specific antigen present in the sub-surface of leptospiral cells did not show protective and reactive-oxygen-inducing activities; they reacted with the non-protective glycolipid antigen of low molecular weight (Fr I, 10-15Kd) in the dot enzyme-linked immunosorbent assay. These results indicated that anti-PAg antibody exhibited opsonic activity against Leptospira and the production of reactive oxygens by macrophages led to leptospiricidal action as one of the defence mechanisms of the host against leptospiral infection. However, the antibody against the genus-specific glycolipid antigen may not be important for protection against leptospiral infection.

Antigenic Homogeneity among Legionella pneumophila Serogroups 1 to 6 Evaluated by Crossed Immunoelectrophoresis

The objective of this study was to investigate the antigenic profiles of Legionella pneumophila serogroups 1 to 6 and antigenic relatedness among individual serogroups. A reference precipitate pattern which had 71 anodic- and 6 cathodic-migrating antigens was established with a pooled antigen preparation of sonicated L. pneumophila cells of serogroups 1 to 6 against purified homologous rabbit antibody. Sixteen antigens made heat stable by boiling for 10 min were found, of which one could be demonstrated as being the common antigen of gram-negative bacteria and another could be shown as being the serogroup-specific antigen of L. pneumophila. Precipitin patterns similar in overall appearance and precipitin numbers were demonstrated for each serogroup by testing antigen preparations of individual serogroups against reference system antibody. Crossed-line experiments revealed < 5 non-cross-reacting antigens among serogroups, with resulting matching coefficients (ratio of the number of cross-reactive to total antigens) all above 0.91. The matching coefficients found when testing antigen preparations from L. pneumophila serogroups 7 and 8 were 0.93 and 0.99, respectively. By contrast, the matching coefficients of Tatlockia micdadei, Fluoribacter bozemanae, Fluoribacter dumoffii, and Fluoribacter gormanii was 0.39, 0.45, 0.39, and 0.41, respectively. Comparison of precipitate patterns and experimental results between the previously described monovalent L. pneumophila serogroup 1 system and the polyvalent crossed immunoelectrophoresis system with L. pneumophila serogroups 1 to 6 substantiated the antigenic homogencity of L. pneumophila and the significant difference in antigenic composition between this taxon and other members of the family Legionellaceae. In addition, reproducibility of the antigenic profile of L. pneumophila by crossed immunoelectrophoresis was demonstrated.

Electrophoretic and serological characterization of the lipopolysaccharides of Legionella pneumophila.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteinase-K-digested cell lysates to provide preliminary data on the LPS chemotypes for 20 strains of Legionella pneumophila (serogroups 1 to 8). The profiles of all strains except Chicago 2 (serogroup 6) were similar in the number, spacing, and size distribution of the bands visualized on silver-stained gels and were indicative of smooth LPS. However, compared with the bands from Salmonella minnesota smooth LPS, their banding pattern was much tighter, with three to four legionella bands for every salmonella band. The proteinase K digest of Chicago 2 was unique in that only two widely separated silver-stained bands were seen. LPS profiles of 10 serogroup 1 strains were identical, and the profile of Knoxville 1 was not altered by extensive in vitro passage. We used immunoblotting to investigate the serological specificities of the LPSs. When a rabbit antiserum prepared against a serogroup 1 strain was used to probe nitrocellulose sheets that bound LPS from strains belonging to eight different serogroups, it recognized only the LPS from the homologous serogroup. Similar results were observed with serogroup 2, 4, and 6 antisera. Our data indicate that L. pneumophila has a smooth-type LPS with an unusual banding pattern and that it is a serogroup-specific antigen.

Production of monoclonal antibodies to Legionella pneumophila serogroups 1 and 6.

To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, we have produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common antigen. The second antibody, LP-I-81, was specific for serogroup 1. This antibody was able to agglutinate bacterial cells belonging to the serogroup 1 reference strains. Philadelphia and Knoxville. Microagglutination assays of environmental and clinical isolates revealed a subgroup of serogroup 1 environmental isolates which were not agglutinated by LP-I-81. This subset of isolates was segregated to certain buildings in the medical complex. Immunodiffusion studies showed identity between the LP-I-81 antigen and the serogroup-specific antigen of serogroup 1 organisms. This antigen could be absorbed out of the serogroup 1 organism extract with LP-I-81-coated Staphylococcus aureus, leaving the serogroup-common antigens. Three hybridomas were produced to serogroup 6. All three produced antibodies which were serogroup 6 specific and agglutinated serogroup 6 bacteria.

Antigens and Toxic Components of Legionella in Pathogenesis and Immunity

Legionellae are opportunistic facultative intracellular pathogens with several antigens and toxic components which may function synergistically in contribution to pathogenesis and immunity. The serogroup specific antigens are associated with endotoxic activities of the bacteria and are potent adjuvants in inducing hypersensitivity and potentiating antibody responses to various antigens. Two protein toxins interfere with oxidative metabolism of host cells and are potential inhibitors of intracellular bactericidal activities. Tissue damage in the host appears to be caused by lysis of infected cells with release of toxic substances of host and bacterial origins. In host defense, the serogroup antigens provide primarily group-specific immunity, and the acid soluble protein toxin induces cross-protection among different serogroups. The combined effects of antibodies to these antigens in promoting phagocytosis and abrogating toxicity would enhance intracellular killing of the invading bacteria. The serogroup antigen complexes may also function as a potent adjuvant to modulate host responses to the invading bacteria.

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.

Three years' experience of serodiagnosis of Legionella pneumophila infections.

During a three-year-period, serum samples from 209 patients with acute pneumonia were tested for antibodies to Legionella pneumophila, using an indirect micro-immunofluorescent antibody test performed with heat-killed serogroup specific antigens. Low antibody levels, defined by titres ranging from 32 to 128, were demonstrated in 73 of the patients. Seroconversion indicating current L. pneumophila, Serogroup 1, infection was documented in 3 previously healthy individuals. High IgM antibody titres suggested such an infection in 3 other patients. Thirteen patients showed either static or single antibody titres of 256 or greater. Only 7 of these patients had antibodies detectable with formalin-inactivated L. pneumophila, Serogroup 1, antigen. The high frequency of stationary antibody titres to heat-inactivated L. pneumophila antigen found in the present study suggests that formalin-inactivated organisms may be a superior antigen for the serodiagnosis of current Legionnaires' disease.

Immunological and biochemical relationships among flagella isolated from Legionella pneumophila serogroups 1, 2, and 3.

Flagella were isolated from virulent Legionella pneumophila serogroups 1, 2, and 3. Antiserum made against purified serogroups 1 flagellin agglutinated live, flagellated serogroups 1, 2, and 3 but not heat-killed or nonflagellated bacteria. A single line of identity was seen in immunodiffusion slides between the flagella isolated from the three serogroups and antibody to flagellin isolated from serogroups 1, 2, and 3. Indirect immunoperoxidase staining showed that antibody to flagellin isolated from serogroup 1 organisms reacted with flagella on serogroup 1, 2, and 3 bacteria. Indirect immunoperoxidase staining was also showed that antibody to flagellin isolated from serogroup 1 L. pneumophila did not react with the serogroup-specific cell surface antigen, thus demonstrating that the flagella- and the serogroup-specific antigen are separate antigens. The amino acid content of the flagella from the three serogroups was essentially the same, with aspartate, glutamate, alanine, and threonine comprising 41% of the total. Thirty-five percent of the amino acids were hydrophobic, and there were not detectable amounts of cysteine, tryptophan, or tyrosine.

Detection of hypersensitivity toLegionella pneumophila in guinea pigs by skin test

Cross-reacting antigens of three serogroups ofLegionella pneumophila differed serologically and immunologically from the serogroup-specific antigens. Intradermal injection of cross-reacting antigens into sensitized guinea pigs evoked skin hypersensitivity. Animals were sensitized either by injection of inactivatedL. pneumophila in adjuvant or by infection with live organisms. Skin reactions were measurable about 2–4 h after injection and continued to increase in intensity for the first 24 h, followed by a gradual decline over the next 48 h. Histological examination of skin reactions taken from test sites at 48 h revealed infiltration of mononuclear cells in and about the small subcutaneous blood vessels and throughout the dermis, compatible with a delayed-type reaction. The overall appearance and time course of the reaction resembled a combination of immediate and delayed types of hypersensitivity. Each cross-reacting antigen of the three serogroups evoked skin reactions in animals which had been sensitized to any of those serogroups, but was not reactive in nonsensitized animals. These observations indicate the possibility of detecting present or past infection ofL. pneumophila by skin tests.

Serospecificity and opsonic activity of antisera to Legionella pneumophila.

A high-molecular-weight surface component (F-1 fraction) has been isolated from the four serogroups of Legionella pneumophila. Antibody raised against live organisms was found by microagglutination assay to be specific for the homologous serogroup. Agglutinating activity of antiserum was markedly diminished after absorption with the homologous, but not heterologous, F-1 fraction. In addition, it was found that L. pneumophila organisms were not interiorized by rat alveolar macrophages or mouse peritoneal macrophages in the absence of antiserum, whereas homologous antiserum effectively opsonized the organisms. The opsonizing activity of serogroup-specific antisera was eliminated by absorption of the antisera with the homologous, but not heterologous, F-1 fraction. These data indicate that the serogroup-specific antigen of L. pneumophila resides in the F-1 fraction and that antibody to the F-1 fraction is required for phagocytosis of L. pneumophila by mammalian phagocytes.