Production of a cell wall-specific monoclonal antibody to Fibrobacter succinogenes.

A monoclonal antibody (mAb) was raised against Fibrobacter succinogenes and produced after fusion as ascites in BALB/c mice. An ELISA was used to test for specificity and sensitivity of the mAb to detect F. succinogenes. The mAb BD1 was tested for sensitivity and cross-reactivity in detecting F. succinogenes with ELISA. The lower limits for F. succinogenes detection in pure and mixed culture-using mAb BD1 with ELISA was 10(5) cells ml-1. Twenty-six other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA but none was detected. Electron micrographs of F. succinogenes cells with immunogold labelling showed that the mAb BD1 reacted exclusively with cell wall epitopes but not intracellular material, as confirmed by ELISA.

Immune recognition of Ornithodoros tick (Acari: Argasidae) salivary antigens by anti-Psoroptes cuniculi antibodies.

Rabbits infested with either Ornithodoros sp. ticks or Psoroptes cuniculi mites were assayed for anti-tick antibody by enzyme-linked immunosorbent assay. Titration of rabbit serum against Ornithodoros sp. salivary gland extract (SGE) demonstrated both mite- and tick-infested animals to have elevated anti-tick antibody titers. Western blot analysis demonstrated the anti-mite and anti-tick antisera to contain antibodies with affinities for both common and unique subsets of Ornithodoros SGE proteins.

Isolation and characterization of mouse monoclonal antibodies reactive with soft tick (Acari: Argasidae) salivary antigens of high molecular weight.

The purpose of this study was to immunologically characterize soft tick salivary antigens. BALB/c mice hyperimmunized with salivary gland extract prepared from Ornithodoros talaje (Guérin-Méneville) were observed to develop high titers of antitick salivary antigen antibodies. Subsequent fusion of splenic lymphocytes from the hyperimmunized mice with SP-2/0-AG14 myeloma cells resulted in the production of 10 antitick IgM-producing hybridoma clones. Partial characterization of the respective tick antigens by gel filtration and SDS-PAGE demonstrated all 10 monoclonal antitick antibodies to be reactive with a salivary gland extract fraction containing proteins 50-110 kDa in molecular weight. Cross-reactivity assays and electrophoretic comparison of salivary gland extract specimens demonstrated similar proteins in several ixodid tick genera and species.

Detection and quantification of Ornithodoros-specific anti-tick antibody by competitive inhibition ELISA.

The objective of this study was to develop a highly specific enzyme-linked immunosorbent assay (ELISA) for the serological detection of anti-Ornithodoros tick antibodies in animals. Affinity-purified rabbit anti-Ornithodoros IgG antibodies were employed in indirect competitive inhibition ELISA assays designed to measure the anti-Ornithodoros antibody titers in other animal species using the domestic goat (Capra hircus) as a large animal model. Repeated infestation of goats with Ornithodoros coriaceus was found to elicit the formation of antibodies capable of inhibiting the binding of the Ornithodoros-specific rabbit IgG. Western blot analysis of goat and rabbit anti-tick antisera demonstrated both animal species to respond immunologically to a set of 9 major protein bands in O. coriaceus salivary gland extracts. The results of these experiments demonstrate that a history of animal exposure to O. coriaceus may be detected serologically by competitive inhibition ELISA.

Evidence of common and genus-specific epitopes on Ornithodoros spp. Tick (Acari: Argasidae) salivary proteins.

New Zealand White rabbits were repeatedly infested with Ornithodoros turicata (Duges), Ornithodoros talaje (Guérin-Méneville), and Ornithodoros coriaceus (Koch) at 2-wk intervals. Blood samples were taken from each animal 10 d after each infestation and the titer of anti-tick antibody was determined by enzyme-linked immunosorbent assay. Subsequent cross-reactivity studies demonstrated that the antitick antisera nonspecifically bound to salivary gland extract proteins prepared from several other tick genera and species' Amblyomma maculatum (Koch), Dermacentor andersoni (Stiles), Dermacentor variabilis (Say), and Ornithodoros moubata (Murray). Absorption of the antisera against an immobilized extract of A. maculatum substantially increased specificity at the genus level. Western blots of electrophoretically separated Ornithodoros salivary gland extract samples were used to further compare the specificity of absorbed and nonabsorbed antitick antisera. The blots demonstrated that many of the Ornithodoros salivary gland extract proteins bear genus specific epitopes. Some differences were noted among the Ornithodoros species examined with respect to the degree of antigenic relatedness with the ixodid ticks.

Detection of Salmonella enteritidis in eggs and chicken with enzyme-linked immunosorbent assay.

An ELISA previously developed for the rapid detection of Salmonella enteritidis (SE) in environmental samples was modified and applied to food samples. A sandwich ELISA was designed that employs affinity-purified rabbit polyclonal antibodies for the capture stage and highly specific monoclonal antibodies for the detection stage. Thirty-nine species of bacteria other than SE, including 32 Salmonella species, were included in cross-reactivity testing with ELISA. Results showed no reactivity with any species tested besides SE. Salmonella enteritidis was added to homogenized food samples (chicken skin, meat, and eggs) to test ELISA sensitivity. The lower limit for ELISA detection of SE was 10(4) cells/mL for pure cultures and in 10% meat (wt/vol), 10(5) cells/mL in 10% skin (wt/vol), and 10(7) cells/mL in 10% eggs (wt/vol). Salmonella enteritidis detection with ELISA was confirmed with the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) method. Results were obtained within 24 h for ELISA method compared to 96 h for the BAM procedure. Results show that sensitivity of ELISA can vary with the type of food tested for detection of SE.

Development and application of a monoclonal antibody against Thiothrix spp.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.

An enzyme-linked immunosorbent assay (ELISA) for detection of Clostridium aldrichii in anaerobiodigesters.

Monoclonal antibodies (Mabs) against Clostridium aldrichii were prepared by in vivo and in vitro immunization with whole cells and produced after fusion as ascites in BALB/c mice. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Mabs to detect Cl. aldrichii. The lower limit for Cl. aldrichii detection in pure and mixed culture with ELISA was 10(5) cells ml-1. Twenty other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA, but none was detected. The ELISA was used for detection of Cl. aldrichii over a 16-month period in five mesophilic continuously-stirred tank reactors (CSTR) with wood, glucose, sludge or sorghum as substrates. The population of Cl. aldrichii in the poplar wood anaerobic digester effluent was 10(6)-10(7) cells ml-1 over that time. These numbers were confirmed by anaerobic microbiological methods. Results from the ELISA technique were obtained in 36 h vs 3 weeks for culture methods. It is concluded that the ELISA is a useful, time-saving method for identification, detection and quantification of Cl. aldrichii in axenic, mixed culture, and in complex undefined cultures such as those found in anaerobic digesters.

Detection of Salmonella enteritidis in environmental samples by monoclonal antibody-based ELISA.

We have developed a enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (ASCII) for the detection of Salmonella enteritidis in environmental samples. ELISA was used to test for sensitivity and specificity of ASCII. 38 other species of bacteria, including 31 Salmonella species were included in cross-reactivity testing with ELISA. ASCII showed no reactivity with any other species tested. ASCII was found to be an IgG1 specific for S. enteritidis lipopolysaccharide (LPS). The lower limits for S. enteritidis detection was 10(5) cells/ml for pure cultures and in 10% sludge (w/v). Environmental samples (raw wastewater, wastewater effluents, mixed liquor and aerobically digested sludge) were obtained twice from five sites and ELISA tested for the presence of S. enteritidis. ELISA results compared to the American Public Health Association (APHA) method of Salmonella detection were not significantly different (P greater than 0.05). The ELISA took 24 h for completion compared to 96-120 h for the APHA procedure. Results demonstrate the reliability of the ELISA and, more importantly, provides a rapid means of detection of S. enteritidis in environmental samples.

Antibody responses of laboratory mice to sequential feedings by two species of argasid ticks (Acari: Argasidae).

The host antibody response of CD1 mice resulting from sequential exposures to two species of Ornithodoros ticks was isotyped (ELISA) in an effort to determine the mechanism for the development of acquired resistance. In addition, salivary gland proteins from two argasid and one ixodid species were examined (RIA) for cross-reactivity. Significant antibody responses, detectable for at least 90 d after last exposure, were shown to occur. Isotyping showed that the initial response was of the IgM class with a secondary class switch to the IgG1 subclass occurring. Evidence that cross-reactive proteins exist between argasid and ixodid ticks is also discussed.

Floc Formation by Azospirillum lipoferum Grown on Poly-beta-Hydroxybutyrate.

Azospirillum lipoferum RG6xx was grown under conditions similar to those resulting in encystment of Azotobacter spp. A. lipoferum produced cells of uniform shape when grown on nitrogen-free beta-hydroxybutyrate agar. Cells accumulated poly-beta-hydroxybutyrate and often grew as chains or filaments that eventually lost motility and formed capsules. Within 1 week, vegetative A. lipoferum inocula were converted into microflocs arising from filaments or chains. Cells within microflocs were pleomorphic, contained much poly-beta-hydroxybutyrate, and were encapsulated. Some cells had a cystlike morphology. Up to 57% of the dry weight of encapsulated flocs was poly-beta-hydroxybutyrate, whereas vegetative cells grown in broth with combined nitrogen had only 3% of their dry weight as poly-beta-hydroxybutyrate. Neither encapsulated cells in flocs nor nonencapsulated vegetative cells were significantly desiccation resistant. Under starvation conditions (9 days) only 25% of encapsulated cells remained viable, whereas vegetative cells multiplied severalfold. In short-term germination experiments with encapsulated flocs, nitrate, ammonium, and soil extract promoted formation of motile vegetative cells. Most cells in treatments lacking combined nitrogen eventually depleted their visible poly-beta-hydroxybutyrate reserves without germinating. The remaining cells retained the reserve polymer and underwent size reduction.