Strain-specific Monoclonal Antibodies Research Articles

Immunomagnetic separation for isolation and enrichment of Lacticaseibacillus paracasei strain Shirota from human feces

Probiotics are live microorganisms that, when consumed in adequate amounts, confer health benefits on the host. The beneficial effects of probiotics are believed to be exerted when the microbes reach the intestine alive and engage in metabolic activity. The number of ingested probiotics that are able to reach the intestine is much fewer than that of the commensal intestinal bacteria. This makes it difficult to characterize the physiological state of probiotics in vivo owing to interference by other bacteria. Here, we established and evaluated an immunomagnetic separation (IMS)-based approach using a strain-specific monoclonal antibody to isolate Lacticaseibacillus paracasei strain Shirota (LcS) from human feces. Six healthy adults ingested a bottle of LcS-containing commercially available fermented milk each day for 4 consecutive days, and feces were collected after the final ingestion. Our approach successfully yielded a collection of more than 70 % of LcS from fecal samples in all subjects. Furthermore, the proportion of LcS among total bacteria, which initially stood at approximately 1 % before IMS, surged to over 70 % after IMS. This substantial increase signifies a concentration of LcS several-fold higher than the original fecal ratio. We conclude that LcS can be separated and enriched from human feces with acceptable level of efficiency and specificity by using our IMS approach.

Human germinal centres engage memory and naive B cells after influenza vaccination.

SummaryInfluenza viruses remain a major public health threat. Seasonal influenza vaccination in humans primarily stimulates pre-existing memory B cells, leading to a transient wave of circulating antibody-secreting plasmablasts1–3. This recall response contributes to “original antigenic sin,” the selective boosting of antibody specificities from prior exposures to influenza virus antigens4. It remains unclear whether such vaccination can also induce germinal centre (GC) reactions in the draining lymph node (LN) where diversification and maturation of recruited B cells can occur5. Here we used ultrasound-guided fine needle aspiration to serially sample the draining LNs and investigate the dynamics and specificity of GC B cell responses after influenza vaccination in humans. We show that influenza vaccine-binding GC B cells can be detected as early as 1 week after vaccination. In 3 out of 8 participants, we detected vaccine-binding GC B cells up to 9 weeks after vaccination. Between 12% and 88% of the responding GC B cell clones overlapped with those detected among early circulating plasmablasts. These shared B cell clones had high frequencies of somatic hypermutation (SHM) and encoded broadly cross-reactive monoclonal antibodies (mAbs). In contrast, vaccine-induced B cell clones detected only in the GC compartment exhibited significantly lower SHM frequencies and predominantly encoded strain-specific mAbs, suggesting a naïve B cell origin. Electron microscopy-based epitope mapping revealed that some of these strain-specific mAbs recognized epitopes that were not targeted by the early plasmablast response. Our results indicate that influenza virus vaccination of humans can elicit a GC reaction to which B cell clones targeting novel epitopes are more likely to be recruited, thereby broadening the spectrum of vaccine-induced protective antibodies against this rapidly mutating pathogen.

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab')2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.

Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus

To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.

Strain-Specific Monoclonal Antibodies to the E2 Protein of Classical Swine Fever Virus, Paderborn Strain

Monoclonal antibodies (MAbs) against the E2 protein of classical swine fever virus (CSFV) are useful for diagnosis and strain characterization. A purified, baculovirus-expressed CSFV E2 protein from the Paderborn strain was formulated with a saponin adjuvant and successfully used to induce an antigen-specific immune response in mice. After cell fusion a panel, designated F92G, of 12 mouse hybridomas (5-2, 11-1, 14-1, 25-2, 28-2, 31-1, 34-1, 35-2, 37-3, 38-2, 39-1, 41-1) producing CSFV-E2 specific MAbs were selected based on their Ig subclass and secretion level (μg IgG/mL). Nine IgG 1/k, two IgG 2b/k, and one IgG 2a/k MAbs were further characterized using immunoperoxidase reactivity, ELISA, and Western blot analysis. Immunoglobulin concentration-dependent immunoperoxidase and ELISA reactivity was observed for some of the MAbs with certain antigens. In general there were several reactivity patterns exhibited by the MAbs, with CSFV strains representing different genetic subgroups (by immunoperoxidase staining) and recombinant antigens (by ELISA). It was interesting to note that in some cases the strain-specific reactivity of a MAb was dependent on the test, thereby providing a clue regarding the nature of the binding site.

Analysis of the Epitope Structure of Plum pox virus Coat Protein

Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

First Identification of an Unusual Recombinant Potato virus Y Strain in Potato in Mexico.

Potato virus Y (PVY) has been reported in potato crops in Mexico (3), with tobacco necrotic variants found in the central State of Mexico (4). Nevertheless, many individual states are currently declared PVY free and distribution of individual strains of PVY in potato in different states of Mexico and in different solanaceous crops had not yet been studied. A limited field PVY survey was conducted on potato in the State of Chihuahua in August 2009. More than 900 random potato leaf samples were collected from cvs. Snowden, Atlantic, FL1867, Felsina, Fianna, Gigant, and Alpha. Seven were found to be PVY-positive and had been collected from cvs. Fianna, Snowden, and FL1867. The PVY status of the collected samples was initially determined with the PVY-specific Immunostrips (Bioreba, Reinach, Switzerland) and by double-antibody sandwich-ELISA using the polyclonal PVY detection kit (Agdia, Elkhart, IN). To determine the strain specificity of these PVY isolates following ELISA tests, the infected original samples were inoculated onto tobacco plants at the four-leaf stage and symptom appearance and development were observed for 8 weeks side-by-side with control isolates PB-Oz (PVYO), N4 (PVYNTN), and Mont (PVYN) (1), followed by the standard PVY strain typing by reverse transcription (RT)-PCR (2). Only one of the PVY-positive samples, originally from symptomless potato cv. Fianna, induced systemic PVY infection in tobacco by producing stunting, mosaic, and vein clearing. No systemic vein necrosis, characteristic of isolates Mont and N4, was observed in Nicotiana tabacum cvs. Burley, Xanthi, or Samsun after inoculation with this isolate during all 8 weeks of observation. This isolate, PVY-M3, was typed as a PVY recombinant by RT-PCR, with two recombinant junctions characteristic of European PVYNTN strains (2). It was further analyzed by triple-antibody sandwich-ELISA using four PVYO and PVYN strain-specific monoclonal antibodies. Monoclonals 1F5 (Agdia) and SASA-N (Scottish Agriculture Science Agency [SASA], Edinburgh) reacted to this isolate and identified PVY-M3 serologically as PVYN serotype, characteristic of other PVYNTN recombinants. Monoclonals MAb2 (Agdia) and SASA-O (SASA), specific to PVYO and PVYC strains, did not react to PVY-M3. Taken together, the combination of biological, serological, and molecular characteristics define this recombinant isolate from Mexico as belonging to the same PVY strain group represented by the isolate PVY-L26 (1). To our knowledge, this is the first report of such an unusual PVYNTN recombinant strain from Mexico. Presence of this isolate, with no vein necrotic symptoms induced on tobacco and with PVYNTN genome, will necessitate development of new detection methods for the seed potato industry in Mexico.

First Report of Plum pox virus Recombinant Strain on Prunus spp. in Canada.

In July of 2008, during the Canadian Plum pox virus (PPV) eradication survey, three Prunus spp. trees (B-5, B-6, and C-1) in a home owner's yard in Grimsby, ON, Canada were found to be infected with PPV by triple antibody sandwich (TAS)-ELISA using the 5B generic monoclonal detecting antibody (1) and reverse transcription (RT)-PCR using the well-validated universal primer set P1/P2. All three trees were grafted on an unknown plum rootstock cultivar. Tree B-5 contained grafts of unknown peach (P. persica) and plum (P. domestica) cultivars, tree B-6 contained the same peach graft as tree B-5 and an unknown apricot (P. armeniaca) cultivar, and tree C-1 was grafted with the same plum cultivar as tree B-5. Strain typing was done by TAS-ELISA using strain-specific monoclonal antibodies for D, M, C, and EA strains. Positive results were obtained with the M-specific test. Strain typing by RT-PCR also was done using primers specific for D, M, W, and recombinant (Rec) strains. Positive results were obtained with the M and Rec primers (4). The 605-bp fragment generated by the PPV Rec primers, which spans the recombination site, was cloned and sequenced. The nucleotide sequences obtained from B-5, B-6, and C-1 are 99% identical to each other and approximately 98 and 99% identical to the PPV Rec isolates BOR-3 and J4c, respectively. Correspondingly, percentage identities are approximately 90% for PPV M, 84% for PPV isolate D-Fan (a typical Canadian D isolate), and 69% for PPV W. The deduced amino acid sequence of B-5, B-6, and C-1 are 98 to 99% identical to each other, 99% identical to the PPV Rec isolates BOR-3 and J4c, 92% identical to isolates of PPV M, and only 84% identical to the typical Canadian D isolate PPV Fan. The P3-6K1 genomic region was amplified using primers that generate a 836-bp fragment (2). This region was 97 to 98% identical to the PPV Rec isolates BOR-3 and J4c, 96 to 97% identical to isolates of PPV D, but only 86% identical to isolates of PPV M. The data above confirm that the PPV isolates B-5, B-6, and C-1 belong to the strain PPV Rec (3). To our knowledge, this is the first report of PPV Rec in North America, and together with PPV D and W, it represents the third PPV strain found on this continent. An intensive survey of all Prunus spp. within a 1.5-km radius area surrounding the home owner's property failed to reveal any additional PPV-positive plants. The three positive plants were removed.

The complete nucleotide sequence of the Plum pox virus El Amar isolate

) is the causal agentof the economically important sharka disease of stone fruit trees. To date, sixstrains of PPV have been recognized (PPV-M, -D, -Rec, -EA, -C, and -W),differing by their serological and molecular properties [1], the PPV-M, -D, and-Rec strains being the most common and widespread. The minor EA (El Amar)strain consists of divergent isolates, so far geographically limited to Egypt. TheoriginalElAmarisolatewaspartiallysequencedintheearly1990s[4]andwaslaterused to produce strain-specific monoclonal antibodies [3]. Recently, the completenucleotide sequence of an isolate belonging the EA strain and deriving from theoriginal isolate was reported [2]; (AM157175). We report here the completedfull-length sequence of the original ElAmar isolate kept in the virus collection ofINRA Bordeaux.This new sequence shows 3.8% divergence from theAM157175sequence, providing evidence for a higher variability within the PPV-EA strainthan in other PPV strains.

Sporadic distribution of Plum pox virus M strain in natural sources in the Czech Republic

The presence and distribution of M strain of Plum pox virus (PPV-M) were investigated in natural hosts of Sharka, plums, myrobalans and blackthorns in the Czech Republic. Leaves or flowers of trees were evaluated for the presence of PPV by specific polyclonal antibodies at first. PPV infected samples were investigated for the presence of PPV-M by strain specific monoclonal antibodies. 102 PPV isolates from plum, 81 from myrobalan and 25 from blackthorn were typed. PPV-M was detected in six plum trees, six myrobalan trees and in one shrub of blackthorn. Sporadic incidence of PPV-M was proved in all investigated areas of the Czech Republic. Molecular and serological typing of different PPV strains in natural hosts, plum, apricot, and peach orchards was proposed to realize in Central Europe.  

PRESENCE OF A RECOMBINANT ISOLATE OF PLUM POX VIRUS IN APULIA

During spring 2003, a small focus of infection by Plum pox virus (PPV) that involved four trees was discovered in an apricot orchard in the Province of Brindisi (Apulia, southern Italy). Virus isolates from all these trees were serologically typed by DASI-ELISA using four strain-specific monoclonal antibodies (Mabs). One isolate (PPV-BR) was also characterized by RFLP analysis of PCR products derived from two genome regions, i.e. coat protein (CP) and P3-6K1. PPV-BR proved to be a recombinant as it typed as PPV-M by strain-specific Mabs and RFLP analysis of the CP gene, but as PPVD by analysis of the P3-6K1 gene. Sequence analysis of the (Cter)NIb-(Nter)CP region of this recombinant showed that the recombination breakpoint was located in the C terminus of the NIb gene, as has been reported for other recognized PPV recombinants. This represents the first record of a recombinant isolate of PPV from Italy.

Colonization of pepper roots by a plant growth promoting Pseudomonas fluorescens strain

The effect of a Pseudomonas fluorescens strain (Aur 6) isolated from Lupinus hispanicus on pepper seedlings (Capsicum annum cv. Roxy) was studied. This strain had already shown its capacity as a plant growth promoting bacteria (PGPB) on seedlings of Lupinus albus, Pinus pinea and Quercus ilex. Strain Aur 6 significantly enhanced all biometric parameters measured: fresh weight, height, neck root diameter and slender index (height/neck root diameter). The PGPB effects could be related to auxin and siderophore production, as strain Aur 6 produced substances of both classes in pure culture. For a further monitoring of Aur 6 in the rhizosphere, strain-specific monoclonal antibodies were developed. Using the strain-specific it could be proofed that Aur 6 is an effective and persistent colonizer of pepper roots, but it is not able to behave as an endophyte.

Adsorption and anchoring of Azospirillum strains to roots of wheat seedlings

Recent microscopic evidence acquired using strain-specific monoclonal antibodies and specific gene probes confirms earlier claims that some strains of Azospirillum lipoferum and A. brasilense, but not others, are capable of infecting the interior of wheat roots. The present study was performed to determine whether this strain specificity in the infection of the interior of wheat roots was apparent in the first 24 h of adsorption (`anchoring') of Azospirillum cells to the root surface. Strains of A. brasilense, originally isolated from surface-sterilised wheat roots (Sp 245, Sp 107) or with a proven ability to infect the interior of wheat roots (Sp 245), showed no greater ability to anchor to the roots than other Azospirillum strains isolated from the wheat rhizosphere (Sp 246) or from the rhizosphere or rhizosphere soil of other gramineae (Sp 7, Cd, S 82). The SEM images showed that at the root tip the Azospirillum cells were principally located in cracks between epidermal cells. In the root hair zone the bacteria were more numerous but again principally located in the depressions between epidermal cells. In all zones of the roots mucilage was present, and near the tip this appeared to have been partially digested, forming `halos' around the bacteria and revealing fibril-like strands attached to the bacteria. Subsequent studies were conducted using a technique originally developed for investigating competition of rhizobia for adsorption sites on legume roots. In the adaptation of this technique it was found that the presence of any significant concentration of Ca++ in the incubation medium reduced bacterial adsorption, as did concentrations of (PO4)3- above 50 mM. The influence of the pH of the incubation medium on the adsorption of ten different strains of Azospirillum showed, that with one exception, strains isolated from the roots or rhizosphere of wheat showed optimum adsorption at pH 6.0, and all other strains pH 7.0. Apart from this effect of pH no differences in adsorption were detected between strains with a proven capacity to infect wheat roots and those unable to do so. However, strains varied in their capability to compete for adsorption sites, there being a tendency for strains with a proven capacity to invade the internal tissues of wheat roots to be more competitive for adsorption sites.

Characterization of a strain-specific monoclonal antibody to hepatitis delta virus antigen

Sequences of the hepatitis delta virus (HDV) vary to different degrees among isolates. A monoclonal antibody, designated as HP6A1, against the antigen of HDV (HDAg) has been characterized for its specificity. HP6A1 bound to HDAg of isolate 25 (genotype I) that was used for immunization, but not to others of both genotypes I and II. The epitope recognized by HP6A1 was then determined by a phage library displaying various heptapeptides. A consensus peptide deduced has the best match with that of residues 4–10 of HDAg (isolate 25). To confirm the phage mapping result, Escherichia coli recombinant proteins containing different lengths and various segments of HDAg (isolate 25) were constructed. The shortest HDAg segment contained in the fusion protein that reacted with HP6A1 was residues 1–10. When this peptide was added to the N-terminus of a heterologous protein engineered for eucaryotic expression, the fusion protein was detected by HP6A1. It is concluded that HP6A1 recognizes an epitope located at the N-terminus of HDAg (isolate 25). Since viruses of quasi-species exist in natural infections, a question of how different viral strains interact in vivo remains to be explored. The highly specific MAb opens a possibility to examine the fate of one strain in the presence of other related species in a cell transfection system.

Symptomless Spread of Blight-inducing Strains of Xanthomonas campestris pv. campestris on Cabbage Seedlings in Misted Seedbeds

Xanthomonas campestris pv. campestris induces two types of symptoms, namely, black rot and blight. Black rot symptoms are V-shaped lesions and black veins on the leaf, and blight symptoms are sudden collapse of interveinal tissues following the lack of veinal necrosis at early stages of infection. These two symptoms can occur simultaneously. However, the tendency to induce either symptom type is strain-dependent. Six strains were evaluated for their rate and pattern of spread in misted seedbeds by using strain-specific monoclonal antibodies and miniplate enrichment/ELISA. Data on pathogen incidence was defined as the presence of the pathogen in or on plants rather than visual symptoms. The results indicated that blight-inducing strains spread to more seedlings than black rot-inducing strains. The high incidences of blight-inducing strains in experimental plots were associated with non-randomness of spatial pattern of pathogen spread, indicating that high incidence is primarily due to the spread from adjacent plants by leaf contact and water splash. Most ELISA-positive seedlings were symptomless, indicating that the sensitivity of the system used in this study was adequate for detection of latent or epiphytic spread.

Isolation and characterization of a transfectant influenza B virus altered in RNA segment 6.

This report describes the successful generation of an influenza B transfectant virus altered in RNA segment 6, which encodes the neuraminidase (NA) protein. The procedure for selection of the transfectant virus relies on the use of strain-specific anti-NA monoclonal antibodies to inhibit growth of the helper virus within the system. A transfectant virus has been engineered which has a coding change in the NA protein. This change resulted in attenuated growth in vitro that could be rescued by addition of exogenous bacterial NA. The mutant virus-associated NA activity was unstable as a result of the engineered changes. The ability to genetically manipulate influenza B virus segment 6 will allow us to assess the function of both NA and the small protein NB, also coded from this RNA, within the context of the virus infectious cycle.

Quantification of Within-Field Spread of Soybean Mosaic Virus in Soybean Using Strain-Specific Monoclonal Antibodies

ABSTRACT Strain-specific monoclonal antibodies were used to follow the temporal increase and spatial spread of soybean mosaic virus (SMV) strain G-5 released from a point source. The use of strain-specific monoclonal antibodies allowed discrimination of within-field temporal and spatial spread of SMV strain G-5 from non-G-5 SMV isolates that originated from exogenous field sources. SMV isolates originating from exogenous sources have potential to alter the temporal and spatial pattern of within-field virus spread, which could potentially affect the choice of models used to quantify within-field pathogen spread. Analysis of SMV epidemics in field-plot experiments indicated that the logistic model was the most appropriate model to describe and compare the temporal spread of SMV among years. On the basis of ordinary runs analyses, within-field spread of SMV strain G-5 was random in 1991 and 1994, but was mostly aggregated in 1992 and 1993. Non-G-5 SMV isolates arising from exogenous sources displayed a random spatial pattern over time. This is the first study in which pathogen incidence (detection of SMV using strain-specific monoclonal antibodies) as opposed to disease incidence (based on symptoms) was employed to monitor and model SMV spread in time and space.

Characterization of V3 Loop-Pseudomonas Exotoxin Chimeras: CANDIDATE VACCINES FOR HUMAN IMMUNODEFICIENCY VIRUS-1

To develop a candidate vaccine for human immunodeficiency virus, type 1 (HIV-1), chimeric proteins were constructed by inserting sequences derived from the V3 loop of gp120 into a nontoxic form of Pseudomonas exotoxin (PE). Inserts of 14 or 26 amino acids, constrained by a disulfide bond, were introduced between domains II and III of PE. V3 loop-toxin proteins expressed in Escherichia coli and corresponding to either MN (subtype B) or Thai (subtype E) strains, were recognized by strain-specific monoclonal anti-gp120 antibodies. When loop sequences were introduced into an enzymatically active form of the toxin, there was no loss of toxin-mediated cell killing, suggesting that these sequences were co-transported to the cytosol. Sera from rabbits injected with nontoxic PE-V3 loop chimeras were reactive for strain-specific gp120s in Western blots, immunocapture assays, enzyme-linked immunosorbent assays, and neutralized HIV-1 infectivity. Since toxin vectors were designed to receive oligonucleotide duplexes encoding any V3 loop sequence, this approach should allow for the production of V3 loop-toxin chimeras corresponding to multiple HIV isolates.

Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems.

Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plusssee) in the spring of 1990. The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718. These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plusssee by the direct analysis of low-molecular-weight RNAs from bacterioplankton. We developed one MAb each for the bacterial isolates PX54 and PU7718 that did not show any cross-reactivity with other bacterial strains by immunofluorescence microscopy. Each MAb recognized the general lipopolysaccharide fraction of the homologous strain. These MAbs were tested successfully for their ability to be used for the in situ detection and counting of bacteria in lake water by immunofluorescence microscopy. During the spring of 1993, A. hydrophila PU7718 showed a depth distribution in Lake Plusssee with a pronounced maximum abundance at 6 m, whereas Comamonas acidovorans PX54 showed a depth distribution with a maximum abundance at the surface. The application of these MAbs to the freshwater samples enabled us to determine the cell morphologies and microhabitats of these strains within their natural environment. The presence of as many as 8,000 cells of these strains per ml in their original habitats 3 years after their initial isolation demonstrated the persistence of individual strains of heterotrophic bacteria over long time spans in pelagic habitats.

Molecular microbial ecology approaches applied to diazotrophs associated with non-legumes

Species- and genus-specific oligonucleotide probes, as well as monoclonal antibodies and polyclonal antisera, were used to study the taxonomic identity and ecology of diazotrophic bacteria associated with non-legumes. Species-specific oligonucleotide probes directed against variable regions of the ribosomal rRNAs of five Azospirillum spp., Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Acetobacter diazotrophicus and new Burkholderia spp. isolates, as well as genus-specific Azospirillum and Azoarcus probes, have been developed and applied to prove the identification of new isolates. In addition, different approaches to identify bacteria on the basis of restriction fragment length polymorphism (RFLP) and PCR-fingerprinting techniques allow the characterization of the diversity of populations even at strain level. On the other hand, strain-specific monoclonal antibodies directed against different Azospirillum brasilense strains enabled us to localize and quantify inoculant bacteria in different parts of the rhizosphere. In addition, antibodies were used successfully to enrich and (re)isolate specific bacteria from different sources. The application of confocal laser scanning microscopy offers high resolution images of diazotrophic bacteria specifically labelled with fluorescent probes in plant tissues.