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.

Survival of Escherichia coli O157:H7 in wastewater from dairy lagoons.

AIM: To determine the survival of Escherichia coli O157:H7 in dairy wastewater from on-site holding lagoons equipped with or without circulating aerators. METHODS AND RESULTS: Survival was monitored in dairy lagoon microcosms equipped with or without scale-size circulators. Both laboratory strains of E. coli O157:H7 and an isolate of E. coli H7 from wastewater had poor survival rates and none proliferated in water from waste lagoons with or without circulators. Furthermore, the decline of E. coli O157:H7 was not enhanced in those microcosms equipped with circulators. Strain variation in survival was observed in both circulated and settling waters. The decline rate of E. coli O157:H7 Odwalla strain increased proportionately with the inoculum load. Escherichia coli failed to establish itself in wastewater even after four sequential inoculations simulating continuous faecal input into the lagoon. The native aerobic bacteria survived longer with a decimal reduction time of 21.3 days vs either introduced or native E. coli, which declined rapidly with decimal reduction time of 0.5-9.4 days. CONCLUSIONS: Escherichia coli O157:H7 failed to establish and proliferate in dairy wastewater microcosms equipped with or without circulating aerators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study furthers our knowledge of pathogen survival in wastewater, and suggests that proper management of wastewater before its use in irrigation is essential to reduce pathogen transfer to crops.

Fluorescent marker for the detection of crop and upper gastrointestinal leakage in poultry processing plants.

Previous published research has identified the crop as a source of Salmonella and Campylobacter contamination for broiler carcasses and reported that broiler crops are 86 times more likely to rupture than ceca during commercial processing. Presently, we evaluated leakage of crop and upper gastrointestinal contents from broilers using a fluorescent marker at commercial processing plants. Broilers were orally gavaged with a fluorescent marker paste (corn meal-fluorescein dye-agar) within 30 min of live hang. Carcasses were collected at several points during processing and were examined for upper gastrointestinal leakage using long-wavelength black light. This survey indicated that 67% of the total broiler carcasses were positive for the marker at the rehang station following head and shank removal. Crops were mechanically removed from 61% of the carcasses prior to the cropper, and visual online examination indicated leakage of crop contents following crop removal by the pack puller. Examination of the carcasses prior to the cropper detected the marker in the following regions: neck (50.5% positive), thoracic inlet (69.7% positive), thoracic cavity (35.4% positive), and abdominal cavity (34.3% positive). Immediately prior to chill immersion, 53.2% of the carcasses contained some degree of visually identifiable marker contamination, as follows: neck (41.5% positive), thoracic inlet (45.2% positive), thoracic cavity (26.2% positive), and abdominal cavity (30.2% positive). These results suggest that this fluorescent marker technique may serve as a useful tool for rapid identification of potential changes, which could reduce the incidence of crop rupture and contamination of carcasses at processing.

Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine.

Lymphokine (ILK) secreted from concanavalin A-stimulated T cells from Salmonella Enteritidis-immune chickens is an undefined mixture of proteins that confers protection against Salmonella infectivity when administered to day-old chicks. It has previously been shown that polyclonal antibodies raised against human granulocyte colony-stimulating factor (GCSF) can neutralize the heterophil activation that is responsible for ILK's protective effect. Western blot analysis of ILK probed with anti-GCSF antibodies detects a prominent protein of mass 33 kDa. We have sequenced the first 20 amino acids of this protein and found it to be identical to residues 24 to 43 of P33, a 326-amino acid protein of unknown function encoded by the chicken mim-1 gene. The primary structure of P33 consists of two 140-residue imperfect repeats that are each homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have expressed mim-1 in Escherichia coli and demonstrated in vitro that recombinant P33 is chemotactic for heterophils, the avian equivalent of mammalian neutrophils. We have also constructed a derivative of P33 that consists of residues 33 to 165 (P33[33-165]), the first repeat sequence of P33 that is homologous to LECT2. P33(33-165) is chemotactic for heterophils both in vitro and in vivo, inducing an influx of heterophils into the peritoneum in a response similar to that observed with ILK. These results suggest that P33 functions as a chemotactic factor in chickens and that it plays an active role in ILK-mediated protection against Salmonella infection.

Role of Campylobacter jejuni potential virulence genes in cecal colonization.

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.

Genotypic variation among arcobacter isolates from a farrow-to-finish swine facility.

Arcobacter spp. were isolated from nursing sows and developing pigs on three farms of a farrow-to-finish swine operation and market-age pigs at slaughter. Isolates were identified by polymerase chain reaction and genotypic fragment patterns were examined by pulsed-field gel electrophoresis (PFGE). Incidences of Arcobacter-positive samples increased progressively as the pigs aged, resulting in all of the pens at the end of the growth cycle in the finishing barn containing Arcobacter-positive feces. However, only 10 of 350 cecal samples from slaughtered pigs were positive. There was little similarity between genotypic patterns for Arcobacter collected from the three farms. The level of genotypic variation revealed by PFGE suggested that pigs in this farrow-to-finish operation were colonized by multiple Arcobacter parent genotypes that may have undergone genomic rearrangement, common to members of Campylobacteraceae, during successive passages through the animals. Additionally, the level of genotypic diversity seen among Arcobacter isolates from farms of a single farrow-to-finish swine operation suggests an important role for genotypic phenotyping as a source identification and monitoring tool during outbreaks.

Determination of fluoroquinolones in serum using an on-line clean-up column coupled to high-performance immunoaffinity-reversed-phase liquid chromatography.

A simple, rapid and reliable method for the simultaneous analysis of the fluoroquinolones ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin in bovine serum has been developed. Upon injection of serum samples, an on-line protein G-linked column was employed to automatically remove serum components that otherwise would interfere with analyses. A high-performance immunoaffinity chromatography (HPIAC) column containing covalently bound anti-sarafloxacin antibodies was then used to capture the fluoroquinolones while allowing the remainder of the serum components to elute to waste. After binding to the HPIAC column, the fluoroquinolones were eluted directly onto a reversed-phase (RP) column for final separation of the compounds prior to fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. Due to use of a clean-up column in tandem with a highly selective HPIAC column, the only off-line sample preparation required was dilution (10-fold) in phosphate buffered saline (PBS) and passage of the samples through a 0.2-microm filter to remove particulate matter prior to injection. No significant interferences from the sample matrix were observed, indicating good selectivity with the HPIAC column. The method yielded high recoveries from fortified bovine serum that were >95% for all four fluoroquinolones with good reproducibility (C.V. values <7.0%). The on-line, automated method described here provides a simple, sensitive and specific assay for multiresidue detection of fluoroquinolones in serum.

Cecal volatile fatty acids and broiler chick susceptibility to Salmonella typhimurium colonization as affected by aflatoxins and T-2 toxin.

Four experiments were conducted using day-of-hatch, mixed-sex broiler chicks to evaluate the effects of aflatoxins and T-2 toxin on cecal volatile fatty acids (VFA) and the susceptibility to Salmonella colonization. All chicks in these experiments were challenged orally with 10(4) cfu of Salmonella typhimurium (ST) on Day 3. In Experiments 1 and 2, chicks were fed diets containing 0, 2.5, or 7.5 mg aflatoxins/kg of diet and were allowed to develop their microflora naturally. In Experiment 3, all chicks were orally gavaged on the day of hatch with a competitive exclusion (CE) culture (PREEMPT) and were fed diets containing 0, 2.5, or 7.5 mg T-2 toxin/kg. In Experiment 4, the chicks were fed diets containing 0, 7.5, or 15.0 mg T-2 toxin/kg and one-half of the chicks were orally gavaged on the day of hatch with the CE culture. In Experiments 1 and 2, with the exception of increased total VFA at 5 d in chicks fed the 7.5 mg T-2 aflatoxins/kg diet, there were no treatment effects on cecal propionic acid, total VFA, or incidence or severity of ST colonization. In Experiment 3, the only alteration in concentration of cecal propionic acid or total VFA was a significant reduction in total VFA at 5 d in chicks fed the 2.5 mg T-2 toxin/kg diet. No significant treatment differences were observed for numbers of Salmonella cecal culture-positive chicks or for numbers of ST in the cecal contents. In Experiment 4, with minor exceptions, the chicks treated with the CE culture had higher cecal concentrations of propionic acid and were less susceptible to Salmonella colonization than the non-CE-treated chicks. In the non-CE-treated chicks, T-2 toxin had no effect on any of the parameters, and 85 to 90% of the chicks were Salmonella cecal culture-positive. In the CE-treated chicks, there was a decrease in propionic acid concentration at 3 and 11 d and an increase in susceptibility to Salmonella colonization of the chicks fed the 15.0 mg T-2 toxin/kg diet. These results indicate that cecal concentrations of VFA can be affected by toxins, such as high concentrations of T-2 toxin, and that resistance to Salmonella colonization may be reduced. Further research is necessary to determine the biological significance of these changes.

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.

Anti-inflammatory effects of ergotamine in steers.

The objective of this experiment was to investigate whether the ergot alkaloid, ergotamine (ET), an alkaloid used to model fescue toxicosis in cattle, modifies the response of cattle to endotoxin (LPS) challenge. Steers (n = 16) were divided into the following treatment groups: control (C), ergotamine (ET), endotoxin (LPS), and ET + LPS. ET and ET + LPS groups received a single bolus intravenous injection of ET (40 microg. kg. body wt(-1)), whereas C and LPS steers received a single bolus injection of sterile vehicle. Thirty minutes after ET/vehicle administration, a single bolus intravenous injection of LPS (0.2 microg. kg. body wt(-1)) was given. Blood was collected at various time points for 48 hr post. Endotoxin increased rectal temperature (RT) and the circulating levels of tumor necrosis factor-alpha (TNF-alpha), cortisol, haptoglobin (Hp), thromboxane B(2) (TXB(2)). The circulating Hp, TNF-alpha, and TXB(2) increases were blunted by pretreatment with ET compared with ET + LPS. Ergotamine by itself increased circulating cortisol and RT, whereas it decreased serum prolactin (PRL). Therefore, whereas administration of LPS at 0.2 microg/kg to steers resulted in an expected response, the combination of ET + LPS attenuated major effects of LPS alone. Thus, acute administration of ET appeared to be anti-inflammatory as it decreased the inflammatory response to LPS, an effect likely driven at least in part by the ET-caused cortisol increase.

Salmonella enteritidis hilA gene fusion response after incubation in spent media from either S. enteritidis or a poultry Lactobacillus strain.

The purpose of this study was to determine if growth of a poultry probiotic lactobacilli strain can influence S. enteritidis virulence expression by measuring the response of a hilA-lacZY transcriptional fusion. beta-galactosidase activity was not detected when S. enteritidis was incubated in Lactobacillus-spent medium (24 h growth, pH 4.1, 50.4 mM lactate) but was detectable in spent medium from 4 h growth cultures of Lactobacillus sp. (final OD of 0.213, pH 5.7, 12 mM lactate) when pH and lactate were adjusted to that of the 24 h-pH 4 spent media levels. Adjusting the pH of the 24 h spent medium from 4 to 6, resulted in a measurable beta-galactosidase activity that was significantly higher than expression in LB broth. When S. enteritidis was grown in Salmonella-spent media (24 h growth, pH 4.2, 78 mM acetate), hilA expression was increased 4-fold over expression in the LB broth.

Separation and quantification of two fluoroquinolones in serum by on-line high-performance immunoaffinity chromatography.

To demonstrate that two structurally similar chemicals can be extracted from a complex matrix and then separated from each other on the basis of their relative affinities for an antibody, an automated column-switching system was used, incorporating on-line, high-performance immunoaffinity chromatography (HPIAC). A high-affinity monoclonal antibody (Mab Sara-95) against the fluoroquinolone sarafloxacin was covalently cross-linked to a protein G column and used to capture fluoroquinolones in fortified serum samples. Interference from matrix components adhering nonspecifically to the column was minimized by the insertion of a protein G cleanup column between the injection port and the Mab Sara-95 derivatized HPIAC column. Upon injection, serum samples containing the fluoroquinolones passed through both columns. The cleanup column detained serum components, that otherwise would bind nonspecifically to the HPIAC column, but allowed the fluoroquinolones to pass through unhindered to the HPIAC column. The fluoroquinolones were then eluted from the HPIAC column according to their relative affinities for the antibody, and individual peaks were monitored using fluorescence detection. By using an on-line cleanup column in tandem with an HPIAC column, the fluoroquinolones could be separated from the serum matrix and then separated from each other on the basis of their affinity for Mab Sara-95 without the use of organic solvents or reversed-phase liquid chromatography (RPLC). This method demonstrates true immunoaffinity separation of structurally related compounds in a complex matrix.

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).

Expression of the hilA Salmonella typhimurium gene in a poultry Salm. enteritidis isolate in response to lactate and nutrients.

Pathogens express virulence genes in response to the combination of environmental conditions present in the host environment. The crop is the first gastrointestinal environment encountered in birds. However, feed withdrawal alters the crop environment resulting in an increased pH, and decreased concentrations of lactate, glucose and amino acids compared with unmoulted birds. Salmonella enteritidis infections increase significantly in hens that have been force moulted by feed withdrawal. The present study examined the effects of pH, carbohydrate sources, amino acids and lactate on expression of Salm. enteritidis virulence by measuring expression of hilA. The hilA gene encodes a transcriptional activator that regulates expression of Salmonella virulence genes in response to environmental stimuli. HilA expression was determined using a poultry isolate of Salm. enteritidis carrying a hilA-lacZY transcriptional fusion from Salm. typhimurium. The media used were Luria Bertani (LB) broth and LB broth diluted 1:5 (DLB). The expression of hilA was 2.9-fold higher in DLB broth compared with LB broth which suggested that there is a nutritional component to the regulation of hilA. Addition of 0.2% glucose, fructose or mannose to LB and DLB reduced hilA expression 1.5 to twofold. Addition of 0.2% Casaminoacids, arabinose, fucose, or lactose had little effect on hilA expression. Lactate (25 and 50 mmmol 1-1) reduced hilA expression at pH 6, 5 and 4, with the lowest expression occurring at pH 4. Based on these results it appears that the composition of the crop lumen could potentially influence Salm. enteritidis virulence expression.

Tail-docking influences on behavioral, immunological, and endocrine responses in dairy heifers.

Behavioral and physiological changes were measured following tail-docking in primiparous heifers. One month before projected first parturition, 21 heifers were assigned to control (nondocked), docked, or docked with lidocaine groups. Heifers were banded to initiate tail-docking and the necrotic tail was removed after 144 h. Physiological, immunological, and behavioral measures were taken for 240 h following banding. Cortisol was not different for control and treated heifers. Haptoglobin increased for docked heifers by 168 h postbanding (24 h postdocking). Alpha1-acid glycoprotein decreased as haptoglobin increased, and alpha1-acid glycoprotein increased until 240 h postbanding. Tumor necrosis factor-alpha increased only with lidocaine and did not show an effect of docking by 240 h postbanding. Lymphocyte phenotyping demonstrated increased CD4+ and CD8+ peripheral blood mononuclear cells for docked plus lidocaine heifers and gammadelta+ cells of those heifers tended to be reduced compared with docked heifers. Eating was the only maintenance behavior affected by banding in both docked groups (increased with banding and decreased with docking). The initial banding procedure did not alter heifer physiology and altered only eating behavior, but the cutting of the tail (docking) increased haptoglobin in response to the tissue damage and returned eating behavior to baseline. The use of lidocaine to anesthetize the tail before banding affected lymphocyte phenotypes and TNF-alpha (banding alone did not alter these parameters).

Assessment of the long-term shedding pattern of Salmonella serovar choleraesuis following experimental infection of neonatal piglets.

In the United States, swine salmonellosis is most often attributed to infections by Salmonella serovar choleraesuis. As a host-adapted pathogen rarely found in nonswine sources, S. choleraesuis is thought to be spread primarily via horizontal transmission, with carrier animals playing an important role. Little has been reported regarding infection of neonatal piglets, particularly regarding their potential to become carriers. Evidence reported herein demonstrates that piglets experimentally infected by S. choleraesuis at 2 days of age were capable of shedding the pathogen for up to 85 days postinfection, at which time the study was concluded. This study also presents findings supporting the use of GN-Hajna as a preenrichment medium for the isolation of S. choleraesuis.

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.