Broadly reacting precipitating and agglutinating antigen of leptospirae.

A saprophytic Leptospira biflexa strain of equine origin was found which cross-reacts with immune rabbit antisera to 14 pathogenic Leptospira interrogans serotypes. Sera from goats experimentally inoculated with the saprophyte showed multiple low-level cross-agglutination reactions against a battery of live L. interrogans serotypes. Sonically treated and saline-extracted suspensions of the L. biflexa strain and serotypes canicola, icterohaemorrhagiae, and pomona yielded a common precipitating protein antigen that was detected by immunodiffusion and immunoelectrophoresis with all of the antileptospiral sera examined. In cross-absorption and gel diffusion tests, the precipitinogen from each of the strains was shown to be identical. Formaldehyde treatments and heating at 100 degree C suggest that the cellular location of the common antigen is either somatic or subsurface, and Sephadex G-200 gel filtration enabled the isolation of the active fraction of the L. biflexa antigen. Monoprecipitin sera against the common antigen of L. biflexa were produced by immunizing rabbits with specific precipitates in agar. In gel diffusion and immunoelectrophoresis tests the antisera with each of the soluble antigens developed a single precipitin formation, and the antisera agglutinated formolized and heated whole-cell suspensions of serotypes canicola, icterohaemorrhagiae, and pomona at low dilutions. The soluble L. biflexa antigen was evaluated as an immunogen and in passive immunity tests for protection against death and kidney infection in hamsters. No cross-protection occurred when the hamsters were challenged with virulent leptospires. In contrast, the animals vaccinated or administered hamster immune serum before challenge died earlier than the control animals.     

Detection of leptospiral antibodies in animal sera by means of fractionated antigenic extracts

Sodium dodecyl sulphate extracts of the reference strains Mus 127, Castellón 3 and Arborea of the Ballum agglutinogenic serogroup of Leptospira interrogans (the species of pathogenic leptospira), of strain Patoc 1 of the saprophytic species of L. biflexa, and of strain 3055 of illini serotype, the sole representative of L. illini, were each fractionated by ultracentrifugation in a sucrose density gradient into 10 fractions. The fractions were tested by complement fixation and immunodiffusion against the sera of animals during the process of immunisation and during the course of naturally occurring infections. The fractions could be divided into three main pools of serological activity: pool I (fractions 1, 2 and 3), pool II (fractions 4, 5 and 6), pool III (fractions 7, 8 and 9). Pool I was species/genus specific; fraction 1 tended to be species specific while fraction 2 reacted with antisera to all strains whether pathogenic or saprophytic. Pool II was serogroup specific and reacted only with antisera to members of the same serogroup. Pool III was serotype (serovar) specific and revealed the identity of the infecting strain at an early stage of infection.     

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.     

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.     

First isolation of bacteriophages for a spirochaete: Potential genetic tools for Leptospira

Three bacteriophages of the saprophytic aquicole bacterium Leptospira biflexa were isolated from sewage waters from the outskirts of Paris, France. These phages do not infect representative strains of the pathogenic species Leptospira interrogans, and their host range is restricted to serovar patoc of the saprophytic species. The phages were found to be lytic and no lysogenic state could be demonstrated. Electron micrographs showed that the phages were morphologically identical and had polyhedral heads and contractile tails. Their genomes were sensitive to restriction enzymes and consisted of double-stranded DNA. Pulsed-field agarose gel electrophoresis indicated that their genomes were linear: 60 kb for LE1 and 50 kb for LE3 and LE4.     

Sero-Epidemiological Study on Equine Influenza in Japan

A serological survey was conducted on horse sera collected for 7 years just before the first outbreak of equine influenza (EI) infection in Japan in 1971. No antibodies against the A/Equi-1/Prague/56 (equi-1) and A/Equi-2/Miami/63 (equi-2) strains of EI virus were detected in any of the sera of 452 native horses when employing hemagglutination inhibition (HI) and complement fixation (CF) tests against viral (V) antigen. On the contrary, of the 80 imported horses, 48 (60.0%) had HI titers of 1:8 or higher against equi-1 and 23 (28.8%) against equi-2. In the CF-V test 42.6% of the horses showed titers of 1:4 or higher against equi-1 antigen and 42.9% against equi-2 antigen. However, all the test sera of the native and imported horses were negative (less than 1:4) in CF tests against soluble human influenza antigen. Epidemiological analysis was carried out to clarify the relationship between the history and the presence of serum antibody against EI viruses in individual imported horses.     

Antibodies to a Novel Leptospiral Protein,LruC, in the Eye Fluids and Sera of Horses with Leptospira-Associated Uveitis

Screening of an expression library of Leptospira interrogans with eye fluids from uveitic horses resulted in identification of a novel protein, LruC. LruC is located in the inner leaflet of the leptospiral outer membrane, and an lruC gene was detected in all tested pathogenic L. interrogans strains. LruC-specific antibody levels were significantly higher in eye fluids and sera of uveitic horses than healthy horses. These findings suggest that LruC may play a role in equine leptospiral uveitis.     

Enzymatic degradation of H2O2 by Leptospira.

The enzymes responsible for reducing H2O2 were surveyed in 49 strains of Leptospira by using semiquantitative assays for catalase and peroxidase. The survey revealed a differential distribution of catalase and peroxidase activities between the two leptospiral complexes. The pathogenic Leptospira interrogans strains gave strong catalase and weak or negative peroxidase reactions. Conversely, the nonpathogenic Leptospira biflexa strains gave strong peroxidase and negative or weak catalase reactions. An intermediate group of four L. biflexa strains, which were isolated from mammals, fell into the high peroxidase, low or negative catalase group. One water isolate, H-23, gave strong reactions for both enzymes and was examined for virulence and in vitro growth parameters. Results indicate metabolic differences between pathogens and water forms in their abilities to reduce H2O2.     

Recent vesicular stomatitis virus infection detected by immunoglobulin M antibody capture enzyme-linked immunosorbent assay.

We developed an enzyme-linked immunosorbent assay (ELISA) that was capable of detecting immunoglobulin M (IgM) antibody to vesicular stomatitis virus (VSV) in the sera of experimentally and naturally infected cattle and horses. The detection of IgM in the sera of these animals permitted an estimate of the recency of infection by VSV serotype New Jersey. A VSV serotype New Jersey epizootic strain isolated from a horse and passed once in an Aedes albopictus cell line was used to infect a horse and a calf. Sera from these animals were used to standardize the ELISA. This assay was used to test sera from cattle and horses involved in the 1982 VSV epizootic. Comparative antibody titrations were performed by three systems: the serum-dilution plaque-reduction neutralization, complement fixation, and indirect immunofluorescent tests. The antibody titers by neutralization and the ELISA were comparable for the period that IgM was present; when IgM ELISA titers diminished, the neutralization titers remained high. The complement fixation and indirect immunofluorescent antibody titers followed closely the IgM pattern determined by the ELISA. The capture IgM ELISA is applicable for the rapid detection of IgM antibody to VSV in cattle and horses and is a useful assay of recent infection.     

Comparative measurement of equine influenza virus antibodies in horse sera by single radial hemolysis, neutralization, and hemagglutination inhibition tests.

Single radial hemolysis (SRH), neutralization (NT), and hemagglutination inhibition (HI) tests were carried out on sera from horses immunized against the Prague and Miami strains of equine influenza virus. The HI and NT tests demonstrated good sensitivity; the sensitivity of the SRH test was somewhat lower. The NT titers of individual sera were correlated very closely with the HI titers, although the NT titers were higher. SRH zone diameters of individual sera also showed significant correlation with the NT and NI titers. The SRH test appears to be suitable for large-scale serological surveys and offers the advantages of rapidity and simplicity.     

Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera

This paper describes the production and purification of a group-specific recombinant protein VP7 of African horse sickness virus serotype 3 (AHSV-3) and validation of an I-ELISA for the detection of IgG-antibodies to VP7 in horse sera. Baculovirus-expressed VP7 crystals were purified from infected insect cells. Analytical accuracy of the I-ELISA was examined using sera (n = 38) from an experimentally infected horse, from foals born to vaccinated mares, from guinea-pigs immunized with nine serotypes of AHSV, and from sera of animals infected with other orbiviruses. Compared to traditional serological assays, the I-ELISA was more sensitive in detection of the earliest immunological response in an infected horse and declining levels of maternal immunity in foals. Antibodies to all nine serotypes of AHSV could be detected. Cross-reactivity to related orbiviruses was not observed. Diagnostic accuracy of the I-ELISA was assessed by testing sera from vaccinated horses (n = 358) residing in AHS-enzootic areas and from unvaccinated horses (n = 481) residing in an AHS-free area. Sera were categorised as positive or negative for antibodies to AHSV using virus neutralisation tests. The TG-ROC analysis was used for the selection of the cut-off value. At a cut-off of 11.9 of the high positive control serum (percentage positivity), the I-ELISA specificity was 100%, sensitivity 99.4%, and the Jouden index was 0.99.     

Interactions of virulent and avirulent leptospires with primary cultures of renal epithelial cells

A primary culture system for the cells of mouse renal-tubular epithelium was established and used to observe the adhesion of leptospires. Virulent strains of serovars copenhageni and ballum attached themselves to epithelial cells within 3 h of infection whereas an avirulent variant of serovar copenhageni did not adhere to epithelial cells at all within the experimental period of 24 h. The saprophytic Leptospira biflexa serovar patoc became attached non-specifically to inert glass surfaces as well as to the cells. The adhesion of leptospires to epithelial cells was not inhibited by homologous antibody.     

Serological comparison among various strains of equine infectious anemia virus

Summary The serological relationship between 8 strains of equine infectious anemia (EIA) virus was investigated by means of complement fixation and neutralization tests. All strains had a common complement fixing antigen, although there were some differences in the intensity of CF reaction in certain antigen-antiserum combinations.Cross neutralization tests revealed that all strains were only neutralized by homologous antisera. The respective antibody titers recorded ranged from 32 to 512. When horses were infected with the virus strains, neutralizing antibodies could be detected as early as 32 to 87 days after inoculation and maximum titers were demonstrable after 42 to 148 days. Of 34 horses infected with 6 strains of EIA virus, 33 horses had neutralizing antibody when they were tested 52 to 127 days after inoculation. Antibodies were only produced against the virus strains inoculated, and not against heterologous strains.     

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses.     

LruA and LruB, novel lipoproteins of pathogenic Leptospira interrogans associated with equine recurrent uveitis

Recurrent uveitis as a sequela to Leptospira infection is the most common infectious cause of blindness and impaired vision of horses worldwide. Leptospiral proteins expressed during prolonged survival in the eyes of horses with lesions of chronic uveitis were identified by screening a phage library of Leptospira interrogans DNA fragments with eye fluids from uveitic horses. Inserts of reactive phages encoded several known leptospiral proteins and two novel putative lipoproteins, LruA and LruB. LruA was intrinsically labeled during incubation of L. interrogans in medium containing [14C]palmitic acid, confirming that it is a lipoprotein. lruA and lruB were detected by Southern blotting in infectious Leptospira interrogans but not in nonpathogenic Leptospira biflexa. Fractionation data from cultured Leptospira indicate that LruA and LruB are localized in the inner membrane. Uveitic eye fluids contained significantly higher levels of immunoglobulin A (IgA) and IgG specific for each protein than did companion sera, indicating strong local antibody responses. Moreover, LruA- and LruB-specific antisera reacted with equine ocular components, suggesting an immunopathogenic role in leptospiral uveitis.     

Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains.

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D stain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two- to fourfold differences were observed between (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns, Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferon-treated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.     

Evaluation of commercial antisera for serotyping heat-labile antigens of Campylobacter jejuni and Campylobacter coli.

Commercial antisera for serotyping 22 heat-labile antigens of Campylobacter jejuni and Campylobacter coli were evaluated by using 66 isolates from human and nonhuman sources. Test results were compared with results of tests using antisera produced at the Centers for Disease Control (CDC), Atlanta, Ga. All strains (three isolates of each of the 22 serotypes) were typeable with the CDC antisera. Of 66 test strains, 39 (59%) were typed as the same serotype with both sets of antisera. Twenty-four strains (36%), including two heat-labile serotype reference strains, were nonreactive with the commercial antisera, and three strains (4.5%) were typed as serotypes different from those obtained with CDC antisera. Five of the 22 commercial antisera correctly serotyped all homologous strains. Our study indicated that two polyvalent antiserum pools, 7 unabsorbed antisera, and 16 absorbed monovalent antisera are weak and need modification to enhance their antibody titers. Further studies are necessary to explain the antigenic change to a different serotype in three strains.     

Lipopolysaccharide serotyping of Neisseria meningitidis by hemagglutination inhibition.

The method of hemagglutination inhibition was used to investigate the antigenic diversity of lipopolysaccharide (LPS) from Neisseria meningitidis and to develop a serotyping systems based on this antigen. The system uses outer membrane complex prepared by a simple extraction procedure to inhibit homologous hemagglutination reactions involving sheep erythrocytes sensitized with purified LPS and rabbit antiserum raised to whole meningococci. Antisera with specificity for eight different LPS determinants were used as typing sera to serotype a cross section of 67 meningococcal strains. Only two strains (both group A) were not typable with the eight sera, and most strains had more than one type. Comparison of LPS type and bactericidal serotype suggests that the LPS and protein serotypes are independent serological markers.     

Hemagglutination by several strains of equine infectious anemia virus

Summary Six strains of equine infectious anemia (EIA) virus propagated in equine leukocyte cultures were found to agglutinate horse erythrocytes. Concentrated virus material containing about 20 units of complement fixation (CF) titer showed hemagglutinating (HA) titers ranging from 4 to 8 units. The HA activity remained stable after ether treatment and was reduced by trypsin, formaldehyde and KIO₄. Cesium chloride equilibrium density gradient centrifugation revealed two populations of hemagglutinin, one in the density range of 1.15–1.16 g/ml coinciding with a peak of CF antigen and the other at round 1.27 g/ml. However, after the antigen was treated with ether, hemagglutinin showed a single peak at about 1.27 g/ml. Hemagglutinin receptors on the erythrocytes were inactivated by trypsin and formaldehyde but their activity was enhanced by neuraminidase. Hemagglutination was inhibited by sera from horses infected with homologous strain for EIA virus. The hemagglutinin showed immunological properties similar to those of the hemagglutinin of guinea pig erythrocytes as reported in our previous paper.With 2 Figures     

LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species

Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.