Cynthia L. Sheffield

Differential nitric oxide production by chicken immune cells

Nitric oxide is a rapidly reacting free radical which has cytotoxic effects during inflammatory responses and regulatory effects as a component of signal transduction cascades. We quantified the production of nitrite, a stable metabolite of nitric oxide, in chicken heterophils, monocytes and macrophages after stimulation by IFNgamma, LPS and killed bacteria. Our results demonstrate a differential production of nitrite over 72 h by chicken peripheral blood heterophils, monocytes and the chicken macrophage cell line (HD11). HD11 cells produced an average of 10 fold more nitrite in comparison to monocytes and 30 fold more than heterophils upon stimulation. This production could be inhibited by S-methylisothiourea indicating that the inducible nitric oxide synthase enzyme was participating in the pathway leading to nitrite production.

The Acquisition and Internalization of Salmonella by the Lesser Mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)

In poultry broiler production facilities, it is important to understand the sources and contribution of reservoir populations of pathogens. The lesser mealworm beetle, Alphitobius diaperinus (Panzer), is a common pest in poultry litter that is reported to carry pathogens affecting both human and animal health. This study investigates whether the carriage of a bacterial pathogen occurs by the harboring of bacteria internally by these insects. Beetles were exposed to a marker bacterium, Salmonella enterica serovar Typhimurium-green fluorescent protein (ST-GFP), at concentrations up to 10(7) colony-forming units (cfu)/mL for 0.5 to 12 h, and then subsequently surface disinfected and dissected. The head, gastrointestinal tract and hemolymph were cultured for the presence of ST-GFP. This study definitively demonstrates the internal carriage of Salmonella by this insect and found that the beetles rapidly acquired bacteria from external sources and harbored the bacteria within their alimentary canal after exposure for 30 min at 10(4) cfu/mL and within the hemolymph after exposure for 2 h at 10(6) cfu/mL. Beetles internalized an average of 9.5 × 10(1) and 3.2 × 10(3) after a 2-h exposure to 2 × 10(4) and 2 × 10(6) cfu/mL, respectively. The lesser mealworm is a serious pest within the poultry brooder and laying industry and because of their mobility, voracious feeding habits, and prey potential may represent an active source facilitating the dissemination of Salmonella.

A continuous-flow high-yield process for preparation of lipid-free hemoglobin

Hypotonic hollow-fiber dialysis of bovine red blood cells followed by ultrafiltration through 0.1-micron pore hollow fibers provides a simple method for isolation of lipid-free hemoglobin. Hemoglobin (Hb) isolated by comparative techniques were all contaminated with membrane stroma. HPLC analysis of Hb revealed a protein peak of 99.6% purity and sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed a single band. The process requires hypoosmotic dialysis of bovine RBC to a final 160-180 mosmol/kg osmotic pressure. Additional reduction in osmotic pressure causes irreversible cell lysis which leads to lipid contamination of the Hb. Processing of 1/2 liter of packed red blood cells requires 4-5 h, resulting in an average of 90% hemoglobin recovery.

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.

Transient gut retention and persistence of Salmonella through metamorphosis in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)

Aims:  This study was undertaken to determine the retention of Salmonella through Alphitobius diaperinus metamorphosis and its contribution, through defecation, to external contamination.

Characterization of Salmonella enterica isolates from turkeys in commercial processing plants for resistance to antibiotics, disinfectants, and a growth promoter.

Salmonella enterica isolates from turkeys in two commercial processing plants (1 and 2) were characterized for susceptibility to antibiotics, disinfectants, and the organoarsenical growth promoter, 4-hydroxy-3-nitrophenylarsonic acid (3-NHPAA, roxarsone), and its metabolites, NaAsO(2) (As(III)) and Na(2)HAsO(4) • 7H(2)O (As(V)). The 130 Salmonella serovars tested demonstrated a low incidence of resistance to the antibiotics gentamicin (GEN), kanamycin (KAN), sulfamethoxazole (SMX), streptomycin (STR), and tetracycline (TET). Isolates resistant to antibiotics were most often multidrug resistant. Serovars Hadar and Typhimurium were resistant to KAN, STR, and TET and GEN, SMX, and STR, respectively. All isolated Salmonella serovars were resistant to the disinfectant chlorhexidine with minimum inhibitory concentrations (MICs; 1-8 μg/mL), and they were susceptible to triclosan and benzalkonium chloride. The didecyldimethylammonium chloride component was the most active ammonium chloride tested. No cross-resistance was observed between antibiotics and disinfectants. The MICs for 3-NHPAA (4096 μg/mL) were consistent between processing Plant 1 and Plant 2, but MICs for the 3-NHPAA metabolites (As(III) and As(V)) were higher in Plant 1 than in Plant 2. In Plant 1, 76% of the isolates had MICs >256 μg/mL for As(III) and 92% of the isolates had MICs >1024 μg/mL for As(V). In Plant 2, all of the isolates had MICs ≤256 μg/mL for As(III) and 90% of the isolates had MICs ≤1024 μg/mL for As(V). Only 4 Salmonella serovars were isolated from Plant 1, but 10 serovars were isolated from Plant 2. S. enterica serovar Derby from Plant 1 was highly resistant to As(III) and As(V) with MICs >1024 and >8192 μg/mL, respectively, suggesting previous exposure to high arsenic metabolite concentrations. These levels may have been high enough to kill other Salmonella serovars, thus possibly explaining the lack of serovar diversity observed in Plant 1. The application of a growth promoter may affect the serovar diversity in treated birds.

Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994-2003

To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants.

Evaluation of Salmonella Movement Through the Gut of the Lesser Mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)

AIMS The lesser mealworm, Alphitobius diaperinus is an important poultry pest prevalent during production that is capable of vectoring pathogens. This study was undertaken to determine the gut transit time of Salmonella for biosecurity risk analysis of pathogen dispersal into the environment. METHODS Adult and larval A. diaperinus were exposed to two concentrations of a fluorescently labeled Salmonella enterica for 15, 30, and 60 min time periods then externally disinfected to evaluate internal transfer of Salmonella. The insects were monitored every 30 min over 4 h and evacuated frass (feces) processed for the marker Salmonella. The minimum time monitored was 45 min (15 exposure+30 min time point), and the maximum was 5 h (60 exposure+4 h time point). RESULTS Adults treated with 10(6) or 10(8) colony-forming units (cfu)/mL, which produced Salmonella positive frass within the 5 h experimental time, displayed a mean gut transit time of 144.4 min (range 90-270 min) and 186.3 min (range 120-300 min), respectively. Larvae treated with 10(6) or 10(8) cfu/mL displayed a mean gut transit time of 172.5 min (range 120-300 min) and 131.7 min (range 60-300 min), respectively. SIGNIFICANCE AND IMPACT OF STUDY Understanding the sources and contribution of reservoir populations of pathogens in poultry production operations is important for development of biosecurity measures to mitigate their transfer. A. diaperinus are prevalent in production operations and difficult to suppress. Management standards accept the reutilization of litter in which insects survive between flock rotations. Removing litter and spreading it onto nearby fields results in the inadvertent dispersal of beetles. Few studies demonstrating the specific bacterial dispersal capacities of these insects have been performed. This study determined that Salmonella acquired internally, commonly transits the gut, allowed the insect to disperse viable pathogenic bacteria within 2-3 h.

Persistence of a Vancomycin-Resistant Enterococcus faecium in an Anaerobic Continuous-Flow Culture of Porcine Microflora in the Presence of Subtherapeutic Concentrations of Vancomycin

Recombined porcine continuous-flow culture (RPCF) maintained in a continuous-flow fermentation system is effective in protecting neonatal and weaned pigs against infection by enteropathogens. In the current study, we demonstrate the effect of RPCF on vancomycin-resistant enterococci (VRE) in the presence and absence of subtherapeutic levels of vancomycin. Also examined was the ability of VRE to transfer vancomycin resistance to endogenous Enterococcus faecalis 137.1. When RPCF was challenged with VRE, the rate of VRE clearance was dependent on the method of challenge. In the control experiment, RPCF was challenged with 7.0 log10/CFU/ml VRE. Clearance of VRE from the culture was observed within 7 days at a rate of 1.44 log10/day. RPCF containing 0.001 microg/ml vancomycin cleared VRE at a slightly lower rate of 0.94 log10/day. RPCF containing 0.01 microg/ml or 0.1 microg/ml vancomycin reduced the level of VRE from 7.0 log10/CFU/ml to 2.0 log10/CFU/ml within 9 days, but failed to clear the VRE after 24 days. During the period of decline, the VRE clearance rate for the 0.01 microg/ml and 0.1 microg/ml vancomycin-treated cultures was 0.52 log10/day, and 0.53 log10/day, respectively. E. faecalis 137.1 endogenous to RPCF did not acquire the vancomycin resistance genes throughout the experiment as evidenced by direct selection, ribotyping, and pulsed-field gel electrophoresis.

Disinfectant and antibiotic susceptibility profiles of Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States.

The disinfectant and antibiotic susceptibility profiles of 344 Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States were determined. A low prevalence of antibiotic resistance was observed (14%). The highest prevalences of resistance were to sulfisoxazole (10.5%), tetracycline (9.9%), streptomycin (7%), and chloramphenicol (4.9%). Four strains were resistant to eight antibiotics (two strains from ground beef and one strain each from hide and preevisceration carcass swabs of cull cattle at harvest). Pulsed-field gel electrophoresis analysis of the E. coli O157:H7 strains revealed two major groups (designated 1 and 2) composed of 17 and 20 clusters, respectively. Clusters 1A, 1B, 1C, and 1G.1 were associated with multidrug-resistant strains. There was no observed correlation between disinfectant resistance and antibiotic resistance. Sixty-nine (20%) of the 344 strains were resistant to chlorhexidine or benzalkonium chloride or the MICs of benzyldimethyldodecylammonium chloride were elevated. Inducible resistance was observed at elevated concentrations of antibiotics (1.4%) and disinfectants (6.1%). The highest rate of disinfectant inducible resistance was to OdoBan, quaternary ammonium chlorides, and the surface disinfectants F25, FS512, and MG, which are used in dairies, restaurants, and food processing plants. High MICs (1,024 to 4,096 m g/ml) of acetic, lactic, and citric acids were found. The decreasing order of acid potency based on molar MICs (MICs(molar)) was acetic, citric, and lactic acid. The correlation of the concentration of dissociated organic acids and MICs(molar) strongly suggests that the observed inhibition of E. coli O157:H7 was primarily due to dissociated forms of the acids.