Bacterial populations on the surfaces of organic and conventionally grown almond drupes.

AIMS: To compare the bacterial populations on organically and conventionally grown almond drupes before and after hull split. METHODS AND RESULTS: We constructed 16S rRNA gene libraries, containing approx. 3000 sequences each, from the bacteria from organically and conventionally grown drupes before and after hull split. We observed that before hull split both conventionally and organically grown drupes were colonized by relatively few types of bacteria that were mostly common phyllosphere-associated Proteobacteria. However, the organically grown drupes contained significantly more Alphaproteobacteria and the conventionally grown drupes contained significantly more Gammaproteobacteria. The conventionally grown drupes also contained significantly more sequences associated with the phylum Actinobacteria. After hull split, we observed a significant increase in bacterial diversity, with many newly appearing sequences that were not normally associated with the phyllosphere. CONCLUSIONS: Organic and conventional growing methodologies influence the types of bacteria on almond drupes and hull split results in a burst of microbial diversification. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of organic produce is increasing due to consumer preferences, but it was unknown how this methodology affects the bacterial populations on almond drupes. This is the first study to compare the bacterial populations of organically and conventionally grown almond drupes.

Bacterial population structure and dynamics during the development of almond drupes.

AIMS: To describe the bacterial populations and their dynamics during the development of almond drupes. METHODS AND RESULTS: We examined 16S rRNA gene libraries derived from the bacterial populations on almond drupes at three stages of development: (i) when the drupes were full sized, but before embryo development, (ii) when the drupe hulls first began to split and (iii) when the drupes were fully mature, but before harvesting. Our data revealed that the immature drupes were colonized by relatively few types of bacteria, belonging mostly to common phyllosphere-associated bacteria within the genera Pseudomonas, Pantoea, Methylobacterium and Sphingomonas. However, after the hulls first began to split, the level of bacterial diversity increased and continued to do so until the drupes were fully mature. At the last sampling period, we observed several sequences belonging to bacteria that are not usually associated with the phyllosphere, including some identical to Salmonella enterica. CONCLUSIONS: The bacterial populations on almond drupes before hull split were composed of relatively few types, most of which were commonly associated with the phyllosphere. However, after hull split, the level of microbial diversity increased, which was mostly due to increased levels of bacteria that are not normally associated with the phyllosphere, including Salm. enterica. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the bacterial populations associated with almond drupes and their dynamics during development. Of specific significance is the observation that Salm. enterica was present on the drupes just prior to harvesting, which may represent a critical control point.

Effects of sodium bisulfate on the bacterial population structure of dairy cow waste.

AIMS: To determine the effects of sodium bisulfate (SBS) on the bacterial populations in cattle waste. METHODS AND RESULTS: We applied SBS at 0, 60, 70 or 100 kg week(-1) to cattle waste as it accumulated on the floors of four cattle pens, housing eight cattle each. We observed significant pH decreases in all of the treated wastes on day one; however, the 60 kg week(-1) treatment returned to control levels by day four, while the others remained significantly lower. Heterotrophic plate counts of the waste revealed that all treatments reduced the bacterial populations in the wastes on day one; however, all returned to control levels by day four. The 16S rRNA gene libraries derived from the wastes revealed significant reductions in sequences associated with the phyla Bacteroidetes and Firmicutes and increases in the Proteobacteria, Actinobacteria and Spirochaetes on day one, but resembled the control by day seven. Sequences associated with Escherichia coli increased significantly after SBS application, but became undetectable by day seven. CONCLUSIONS: SBS application significantly alters the bacterial population structure of waste during the first few days of application, but the populations return to almost normal after 7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of SBS to animal waste can reduce emissions; however, biosecurity precautions must be rigorously maintained during the initial application to ensure that pathogenic E. coli is not released into the environment.

Identification of the binding site of two monoclonal antibodies to human protamine.

We have previously developed a number of monoclonal antibodies (Mabs) that bind to protamine. One of these antibodies, Hup1N, binds to human protamine 1 but not to protamine 2. In contrast, Mab HupA binds both protamine 1 and protamine 2. The epitopes for these two Mabs were observed to overlap, and were localized to the evolutionarily conservative amino-terminal region of protamine 1. This assignment is based on antibody binding to protamine from different species in which the protamine sequence is known, as well as analysis of antibody binding to synthetic peptides and synthetic peptides with specific amino acid substitutions.

Characterization of monoclonal anti-furosemide antibodies and molecular modeling studies of cross-reactive compounds.

Four mouse monoclonal antibodies directed against furosemide have been isolated and characterized. The cross-reactivity of the antibodies with eight compounds which are structurally and/or functionally related to furosemide was determined using a competition ELISA. All of the compounds, including furosemide, were then modeled using molecular mechanical and quantum mechanical methods in an attempt to correlate antibody binding with the conformational and electronic properties of the molecules. The results of these experiments demonstrated that all of the cross-reactivity observed could be readily explained using these techniques. Furthermore, these results should allow for more accurate prediction of unexpected cross-reactivities with these antibodies when they are used in immunoassays for determination of furosemide.

Characterization of monoclonal antibodies to aflatoxin M1 and molecular modeling studies of related aflatoxins.

Aflatoxin M1 (AFM1) and seven structural analogs were used to investigate the correlation between antibody binding and the conformational and electronic properties of these molecules. Mice were immunized with AFM1-BSA and hybridomas secreting anti-AFM1 antibodies were isolated and characterized. The cross-reactivities of seven structurally similar aflatoxins were determined by competition enzyme-linked immunosorbent assay (cELISA). In an effort to correlate antibody binding with three-dimensional properties of the analogs, all of the aflatoxins (and the immunogen) were modeled and global energy minima were determined using molecular, mechanical and quantum mechanical methods. The results demonstrate that, for these molecules, loss of optimum structure and introduction of steric hindrance in the portion of the molecule that would fit into the antibody binding site are more important to binding than simply loss of a determinant group. Molecular computational techniques can give reasons for the wide variation in IC50 values observed between structural analogs and can be used as a tool for determining which conformational and electronic properties of molecules are most important for antibody binding.

Sequences of cDNAs encoding immunoglobulin heavy- and light-chain variable regions from two anti-dioxin monoclonal antibodies.

We report the cDNA cloning of the gamma- and kappa-chain-encoding genes for two mouse monoclonal antibodies (mAb) which recognize dioxins. The nucleotide sequences encoding the variable regions of these mAb were also determined. Although the mAb have similar dioxin-binding characteristics, their deduced variable region amino-acid sequences are very different.

One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography.

A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or IgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.

Increased responsiveness to intravenous lipopolysaccharide challenge in steers grazing endophyte-infected tall fescue compared with steers grazing endophyte-free tall fescue.

Fescue toxicosis in cattle occurs as a result of consumption of ergot alkaloids in endophyte-infected (E+, Neotyphodium coenophialum) tall fescue (Festuca arundinacea). The condition is characterized by pyrexia, decreased weight gains, rough hair coats, and decreased calving rates. The objective of this experiment was to investigate whether steers grazing E+ fescue have altered host response to lipopolysaccharide (endotoxin, LPS) challenge compared with steers grazing endophyte-free (E-) fescue. Angus steers (n=8) had continuously grazed either E+ (n=4) or E- (n=4) tall fescue grass for 8 months prior to the experiment. The E+ steers had lower body weight, depressed average daily gain, and decreased basal serum prolactin compared with the E- steers prior to LPS administration. Each steer received a single bolus i.v. injection of LPS (0.2 microgram/kg body weight; Escherichia coli; 026:B6) dissolved in sterile saline, and blood was serially collected every 30 min for 4 h and at 24 h post LPS administration. LPS increased serum tumor necrosis factor-alpha (TNF-alpha), cortisol, and haptoglobin but decreased plasma glucose and IGF-I. Importantly, however, TNF-alpha, cortisol, and IGF-I responses to LPS were greater in E+ compared with E- steers. These results indicated that animals grazing E+ fescue had altered integrated metabolic host response compared with animals grazing E- fescue. Potentially, combined exposure to E+ fescue and a bacterial LPS could have greater deleterious effects on the animal compared with exposure to only one of the two and would likely lead to increased catabolism.

Monoclonal antibodies for dioxin: antibody characterization and assay development.

A set of 5 anti-dioxin monoclonal antibodies (mAbs), named DD-1, DD-3, DD-4, DD-5 and DD-6, have been isolated. In order to evaluate the ability of these mAbs to recognize various kinds of polychlorinated dibenzodioxins and dibenzofurans, a competition enzyme-linked immunosorbtion assay (ELISA) was developed. All 5 antibodies recognize tetrachloro- and pentachloro-dibenzodioxins and -dibenzofurans. They fail to bind either non-chlorinated, mono-, hexa-, or octa-chlorinated dibenzodioxins, nor do they recognize non-chlorinated, octachloro- or 1,2,3,4,8,9-hexachloro-dibenzofurans. Chlorine substitution on both rings appears necessary for antibody recognition. In the course of our experiments, 3 of the mAbs did not recognize any of the polychlorinated biphenyls (PCBs) tested, while 2 mAbs (DD-1 and DD-6) weakly recognized the 3,3',4,4'-tetrachloro congener. DD-4 and DD-5 are the most specific of the antibodies for the dibenzodioxin and dibenzofuran structure. They do not recognize any of a panel of chlorinated phenols, benzenes, or pesticides. Significantly, these antibodies do not react with PCBs, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, trichlorophenol, or 2,4,5-trichlorophenoxyacetic acid (the latter is weakly recognized by DD-6), any or all of which might be present in large quantities in some dioxin-contaminated samples. Finally, the competition ELISA is able to easily detect 0.5 ng of the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin. It should thus prove useful as an environmental screen for contamination.

Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Murine monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of selected probiotic bacteria present in a continuous-flow competitive exclusion culture known to be effective at reducing chicken cecal and crop colonization by Salmonella typhimurium. Veillonella, Enterococcus avium and S. typhimurium were grown anaerobically in batch culture of Viande Levure broth in pure culture and mixed culture. The mixed cultures produced significantly more acetate and propionate than any of the pure cultures with acetate and propionate being the predominant volatile fatty acids. The association in mixed culture resulted in a significant increase in cell numbers compared to the respective pure cultures. The ELISA was capable of detecting 10(4) cells per ml of the bacteria. The plots of cell numbers determined by the ELISA versus direct plating increased in accordance with increases in cell numbers with r2 values of 0.950, 0.922 and 0.940 for the pure culture incubations and 0.901, 0.924 and 0.905 in the mixed culture incubation for E. avium, S. typhimurium and Veillonella, respectively. The results indicate that the monoclonal antibodies can be used to quantitatively assay individual probiotic bacterial species grown in a mixed culture incubation.

Efficacy of Salmonella enteritidis-immune lymphokines on horizontal transmission of S. arizonae in turkeys and S. gallinarum in chickens.

Salmonella arizonae (SA) and S. gallinarum (SG) are of economic importance to international poultry production because of their pathogenesis in young poultry during the first week after hatching. Previous studies from our laboratory have shown immune lymphokines (ILK) produced by S. enteritidis (SE)-immunized chickens provide protection against SE organ invasion in day-old chickens and turkey poults. Previous studies have also demonstrated that SG organ invasion was significantly decreased by administration of ILK to broiler chicks. The objective of the present study was to determine the effect of ILK on the incidence of horizontal transmission of SA in turkey poults and of SG in broiler chicks. The effect of ILK administration on horizontal transmission of SA in poults and SG in chicks was assessed in a seeder/contact model. Seeders were challenged with the appropriate bacterium (SA turkeys, SG chicks), contacts were either untreated or administered ILK. Seeders and contacts cohabited within an experimental group throughout the experiment. Mortality and organ invasion as a result of horizontal transmission were determined. There were no significant differences in mortality between non-treated and ILK-treated contact poults. In contrast, SG was extremely pathogenic to young broiler chicks. Non-treated contact chicks had a mortality rate of approximately 68% whereas significant (P < 0.05) reduction in mortality was demonstrated in the contact chicks treated with ILK (15%). Horizontal transmission, as determined by organ invasion, of SA to contact turkey poults and SG to contact broiler chicks was also significantly (P <0.05) decreased by immunoprophylactic administration of ILK. Bacterial recovery of SA from the liver/spleen and the cecal tonsil in contact poults and SG from contact chicks treated with ILK was dramatically reduced when compared to non-treated contact poults and chicks. Our results strongly suggest the immunoprophylactic administration of SE-immune lymphokines to young turkey poults and broiler chicks significantly reduces the horizontal transmission of Salmonella in poultry. These results suggest the possibilities of using a non-vaccine immunologically-based preventative strategy against Salmonella in poultry.

Production and characterization of a monoclonal antibody against bovine haptoglobin and its use in an ELISA.

Haptoglobin (Hp) is the major acute phase reactant found in cattle. As such, it is an excellent indicator of early disease processes and could be used as a marker for pre-clinical illness in cattle. The production of monoclonal antibodies (mAbs) directed against bovine Hp and bovine hemoglobin is described. The anti-haptoglobin mAbs (Hap1, Hap2, Hap3) and the anti-bovine hemoglobin (Hb) mAb (BoHem1) were characterized and tested for cross-reactivity by means of enzyme-linked immunosorbent assay (ELISA) and immunoblotting analyses. Additionally, the development of an ELISA based on an anti-haptoglobin mAb is discussed.

Validation of immunoassays for bovine haptoglobin.

Haptoglobin (Hp) is an acute phase protein. The plasma concentration of Hp increases rapidly following tissue damage associated with infection and inflammation. Thus Hp levels could be used as a screening test for organic disease, an objective index of disease activity and response to therapy, or as a sign of microbial infection. Recently, an enzyme-linked immunosorbent assay for bovine Hp was described. We have now developed three different immunoassay formats for bovine Hp and report on their validation and relative value to the diagnosis of bovine disease. Hp levels measured using these three immunoassays were compared and contrasted with results obtained for Hp estimation as measured by the increase in the protection of peroxidase activity against acid inactivation following binding with bovine haemoglobin. The quantitative Hp immunoassays evaluated in the present study are simple, rapid, inexpensive, reproducible, and well suited for both field and laboratory use.

Detection of salmonella in poultry using a silicon chip-based biosensor.

Salmonella typhimurium was detected to levels as low as 119 CFUs using the Threshold Immunoassay System. This immunoassay system utilizes solution-based binding of the biotin and fluorescein labeled antibodies to salmonella, followed by filtration-capture of the immunocomplex on a biotin-coated nitrocellulose membrane. Lastly, an anti-fluorescein urease conjugate is bound to the immunocomplex. Detection of the bound immunocomplex is made possible via the silicon chip-based light-addressable potentiometric sensor. In the presence of the urea, urease converts the substrate to ammonia and CO2 and this results in a pH change at the silicon surface. The resultant pH change is monitored with time and the signal output is reported in microV s(-1). An experiment whereby chicken carcass washings were fortified with salmonella showed a recovery of 90%, indicating that the technique can be used to test for salmonella under these conditions. Precautions must be used with this instrument as sample debris will affect sample flow through the membrane and hence the signal output.

Immunosorbents coupled on-line with liquid chromatography for the determination of fluoroquinolones in chicken liver.

Four fluoroquinolones were analyzed in fortified chicken liver using an automated, on-line immunoaffinity extraction method. The fluoroquinolones were extracted from the liver matrix using an immunoaffinity capture column containing anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix components had been washed away, the captured fluoroquinolones were automatically eluted directly onto a reversed phase column. Liquid chromatographic analyses were performed by isocratic elution using 2% acetic acid/acetonitrile (85:15) as the mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Overall recoveries from fortified liver samples (20, 50, and 100 ng/g) ranged between 85.7 and 93.5% with standard deviations of <5%. The limit of quantification for each fluoroquinolone was 1 ng/mL. The limits of detection, based on a signal-to-noise ratio of 5:1, were 0.47, 0.32, 0.87, and 0.53 ng/mL for ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin, respectively.

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 1. Development and characterization of monoclonal antibodies and molecular modeling studies of antibody recognition.

Sulfonamide antibiotics are used to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Current residue methods for the analysis of sulfonamides in animal-based food products include bioassays, chromatographic methods (HPLC, GLC), and immunoassays. Most immunoassays have employed highly specific polyclonal antibodies. In this paper, we describe the isolation of monoclonal antibodies against sulfadimethoxine (SDM) that vary in their sensitivities and cross-reactivities against a large number of sulfonamides. The most sensitive monoclonal antibody, designated SDM-18, exhibits an IC(50) value for SDM of 1.53 ppb. Another monoclonal antibody, designated SDM-44, exhibits IC(50) values for six sulfonamides well below the established threshold level of 100 ppb for animal tissues. Molecular modeling studies of the cross-reactive drugs suggest that, depending on the monoclonal antibody, both steric and electronic features govern antibody binding. Due to the diversity of these monoclonal antibodies, it should be possible to design both compound- and class-specific monoclonal antibody-based immunoassays.

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 2. Evaluation of rapid extraction methods and implications for the analysis of incurred residues in chicken liver tissue.

Several rapid extraction methods were evaluated for use with a monoclonal antibody-based competitive inhibition ELISA (cELISA) to detect sulfadimethoxine (SDM) in chicken liver tissue. These methods included extraction of the samples with (1) aqueous buffer with or without ultrafiltration, (2) acetonitrile/water, (3) methanol/water, or (4) acetone. The organic extraction methods were evaluated with or without solvent evaporation prior to dilution into assay buffer for the cELISA. The aqueous-based extraction methods were compatible with the cELISA. However, of the organic extraction methods, only the acetone liver extract with solvent evaporation prior to analysis was compatible with the cELISA. The cELISA method coupled to aqueous- or acetone-based sample extraction as well as an HPLC method was evaluated for the analysis of chicken liver tissues fortified with SDM at levels from 0.2 to 0.025 ppm. Mean SDM recoveries for the HPLC method and for the cELISA method using samples prepared by aqueous extraction, aqueous extraction and ultrafiltration, or acetone extraction, evaporation, and reconstitution were 68.9, 95.7, 60.1, and 52.5%, respectively. For the analysis of samples obtained from an SDM incurred residue study, HPLC and cELISA analysis of the same organic extract gave results that were highly correlated (R(2) = 0.976; p < 0.0001). However, results obtained from the analysis of aqueous extracts by cELISA did not correlate well with those obtained by HPLC (R(2) = 0.61, p > 0. 0006). This was attributed to the coextraction of cross-reactive SDM-related residues that were not quantified by the HPLC method. The presence of these residues should be considered during data interpretation when ELISA methods coupled with rapid aqueous extraction of samples are used in SDM residue monitoring programs.

Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella typhimurium DT104 in rumen contents in vitro.

Escherichia coli O157:H7 and Salmonella Typhimurium DT104 are important foodborne pathogens affecting the beef and dairy industries and strategies are sought to rid these organisms from cattle at slaughter. Both pathogens possess respiratory nitrate reductase that also reduces chlorate to the lethal chlorite ion. Because most anaerobes lack respiratory nitrate reductase, we hypothesized that chlorate may selectively kill E. coli O157:H7 and Salmonella Typhimurium DT104 but not potentially beneficial anaerobes. In support of this hypothesis, we found that concentrations of E. coli O157:H7 and Salmonella Typhimurium DT104 were reduced from approximately 1,000,000 colony forming units (CFU) to below our level of detection (< or = 10 CFU) following in vitro incubation (24 h) in buffered ruminal contents (pH 6.8) containing 5 mM added chlorate. In contrast, chlorate had little effect on the most probable number (mean +/- SD) of total culturable anaerobes (ranging from 9.9 +/- 0.72 to 10.7 +/- 0.01 log10 cells/ml). Thus, chlorate was bactericidal to E. coli O157:H7 and Salmonella Typhimurium DT104 but not to potentially beneficial bacteria. The bactericidal effect of chlorate was concentration dependent (less at 1.25 mM) and markedly affected by pH (more bactericidal at pH 6.8 than pH 5.6).

Inhibition of in vitro Salmonella Typhimurium colonization in porcine cecal bacteria continuous-flow competitive exclusion culturest.

Continuous-flow (CF) chemostate cultures were used as models to determine the potential usefulness of undefined porcine cecal bacteria as competitive exclusion (CE) cultures against colonization by Salmonella Typhimurium. One culture, pCF1, was derived from cecal bacteria of an animal maintained on antibiotic-free feed, while the other culture, pCF4, was derived from cecal bacteria of an animal maintained on feed containing chlortetracycline. The effectiveness against a chlortetracycline-resistant Salmonella Typhimurium was examined in CF cultures maintained in the absence (pCF1 and pCF4) and presence (cpCFl and cpCF4) of chlortetracycline. CF cultures were inoculated with each of 10(2), 10(4), and 10(6) Salmonella Typhimurium CFU/ml. Chemostat inocula of 10(2) Salmonella CFU/ml resulted in no Salmonella Typhimurium being detected at 2 and 3 days postinoculation in pCF1 and pCF4, respectively, and after 2 days in both cpCF1 and cpCF4. Inoculations of 10(4) Salmonella Typhimurium CFU/ml resulted in clearance from pCF1 and pCF4 within 4 days and within 3 days from cpCF1 and cpCF4. Following inoculation with 10(6) CFU/ml, no Salmonella Typhimurium were detected in all CF cultures by 6 days postinoculation. The results indicated that in vitro CF cultures of porcine cecal bacteria were able to inhibit the growth of Salmonella Typhimurium. The ability to limit Salmonella Typhimurium growth was not restricted by prior exposure of the cecal bacteria to the feed additive chlortetracycline. The present study demonstrates the potential application of CF cultures as models to aid in the identification of CE cultures against salmonellosis in pigs.