The horizontal transfer of Salmonella between the lesser mealworm (Alphitobius diaperinus) and poultry manure.

There is need to determine the nature of enduring reservoirs of Salmonella contributing to perpetual contamination within poultry flocks. The dispersal of Salmonella between birds, litter and the lesser mealworm has been established, but the extent that these act as critical components in the epidemiology of Salmonella infection during broiler grow-out and flock rotation has not been delineated; in particular, the level of participation by the lesser mealworm beetles (LMB) as agents of retention and dispersal. This study defines this route of transmission and provides empirical data on bacterial loads that facilitate Salmonella transfer. Results showed differential Salmonella transfer dependent on bacterial concentration. At 103  cfu/ml, only a small, but not significant, amount of Salmonella was transferred, from the LMB to the manure and back to uninfected LMB; while from 105 to 107  cfu/ml, a significant acquisition and transfer occurred both internally and externally to the LMB over 4 and 24 hr exposures. These data will be used in correlation with facility management practices to develop intervention strategies to mitigate the establishment and spreading of reservoir Salmonella populations contributing to pre-harvest contamination of poultry flocks.

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

AIMS: To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants. METHODS AND RESULTS: Pseudomonas aeruginosa isolates collected from dogs (n = 155) and other animals (n = 20) from sixteen states during 1994-2003 were tested for susceptibility. Most isolates were resistant to twenty-one antimicrobials tested, and the highest prevalence of resistance was to ß-lactams (93.8%) and sulphonamides (93.5%). Fluoroquinolone resistance did not increase from 1994 to 2003. Ciprofloxacin and enrofloxacin had a 5 and 16% prevalence of resistance, respectively, while sarafloxacin and nalidixic acid had a prevalence of resistance of 97 and 98%, respectively. Strains were pan-resistant to triclosan and chlorhexidine, were highly resistant to benzalkonium chloride and demonstrated high susceptibility to other disinfectants. Didecyldimethylammonium chloride was the most active ammonium chloride. Inducible resistance was observed to cetyl ammonium halides, chlorhexidine and benzyl ammonium chlorides, which formulate disinfectants used in veterinary clinics and dairies. Organic acid inhibition was associated with the dissociated acid species. CONCLUSIONS: Dissociated organic acids appear able to inhibit Ps. aeruginosa, and rates of fluoroquinolone resistance merit sustained companion animal isolate surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of Ps. aeruginosa susceptibility to 24 disinfectants and illustrates the high resistance of Ps. aeruginosa to both antibiotics and disinfectants.

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. METHODS AND RESULTS: Insects were exposed to a tagged Salmonella enterica and evaluated for external elimination. (i) Each day for 3 weeks, a filter collected frass from a restrained insect for analysis. (ii) Exposed larvae in a closed container were followed through pupation, and newly emerged adults were examined for their retention of marker bacteria. CONCLUSIONS: Exposed adults and larvae produced Salmonella-positive frass for an average of 8 days, ranging from 6 to 11 days and 6 to 12 days, respectively. Nineteen per cent of the larvae carried Salmonella through metamorphosis and eclosion, with 5% of the pupal exuviae being positive as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Many sources of foodborne pathogens within the poultry production facilities, including reservoir populations, currently go unrecognized. This diminishes the ability of producers to mitigate the transfer of pathogens between animals, humans and the environment. Poultry management standards accept the reutilization of litter. Alphitobius diaperinus survive between flock rotations on the reutilized litter, and it was demonstrated in this study that the Salmonella they carry can survive with them.

Planktonic and biofilm communities from 7-day-old chicken cecal microflora cultures: characterization and resistance to Salmonella colonization.

Information implicating bacterial biofilms as contributory factors in the development of environmental bacterial resistance has been increasing. There is a lack of information regarding the role of biofilms within the microbial ecology of the gastrointestinal tract of food animals. This work used a continuous-flow chemostat model derived from the ceca of 7-day-old chicks to characterize these communities and their ability to neutralize invasion by Salmonella enterica serovar Typhimurium. We characterized and compared the biofilm and planktonic communities within these microcosms using automated ribotyping and the Analytical Profile Index biotyping system. Eleven species from eight different genera were identified from six culture systems. Klebsiella pneumoniae was isolated from all planktonic communities and four of the biofilm communities. Three of the communities resisted colonization by Salmonella enterica serovar Typhimurium, two communities suppressed growth, and one community succumbed to colonization. In cultures that resisted colonization, no Salmonella could be isolated from the biofilm; in cultures that succumbed to colonization, Salmonella was consistently found within the biofilms. This study was one of a series that provided a molecular-based characterization of both the biofilm and planktonic communities from continuous-flow culture systems derived from the cecal microflora of chicks, ranging in age from day-of-hatch to 14 days old. The one common factor relating to successful colonization of the culture was the presence of Salmonella within the biofilm. The capacity to sequester the introduced Salmonella into the biofilm appears to be a contributing factor to the inability of these cultures to withstand colonization by the Salmonella.

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.

Effect of oral sodium chlorate administration on Escherichia coli O157:H7 in the gut of experimentally infected pigs.

Strategies are sought to reduce pathogenic Escherichia coli concentrations in food animals. Because E. coli possess respiratory nitrate reductase activity, which also reduces chlorate to cytotoxic chlorite, we tested and found that oral sodium chlorate administration reduced gut concentrations of E. coli O157:H7 in experimentally infected pigs and wildtype E. coli concentrations in nonchallenged pigs. Mean +/- S.E. concentrations (log10 CFU/g) of E. coli O157:H7 in ileal, cecal, colonic and rectal contents from placebo-treated pigs were 4.03 +/- 0.66, 3.82 +/- 0.24, 4.42 +/- 0.25 and 4.03 +/- 0.16, respectively. In contrast, E. coli O157:H7 concentrations were reduced (P < 0.05) in ileal (1.56 +/- 0.22) cecal (2.65 +/- 0.38), colonic (3.05 +/- 0.38) and rectal (3.00 +/- 0.29) contents from pigs orally administered three successive (8 h apart) 10-ml doses of 100 mM chlorate. Wildtype E. coli concentrations in gut contents of non-E. coli O157:H7-challenged pigs likewise treated with chlorate were reduced by 1.1 to 4.5 log10 units compared to concentrations in placebo-treated pigs, which exceeded 6.0 log10 CFU/g. As before, the reductions were greater in anterior regions of the gut than regions more caudal. Similar treatment of E. coli O157:H7-challenged pigs with 200 mM chlorate caused reductions in gut concentrations of E. coli O157:H7; however, the reductions were not necessarily greater than those achieved with the 100 mM chlorate treatment.

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.

Nonencapsulated hemoglobin-based red blood cell substitutes: an updated view.

The characteristics and properties of the nonencapsulated hemoglobin (Hb)-based materials which have been used as substitutes for red blood cells are reviewed. The chemical and physical criteria such as oxygen dissociation, oxygen carrying capacity, antigenicity, viscosity, and circulatory retention time are described. The critical issue of Hb purity assurance testing is examined. The methods of the analyses which should be used to evaluate the lipid purity, endotoxin levels, and protein profile of Hb solutions are described. Additionally, the various approaches to Hb isolation, purification, and production are examined.

Preparation and in vivo evaluation of two bovine hemoglobin-based plasma expanders.

A hemoglobin (Hb)-based oxygen carrier was successfully transfused into rats. An ultrapure lipid-free bovine Hb was prepared by hypotonic dialysis and ultrafiltration. The Hb was polymerized with glutaraldehyde and the P50 was 24.3 mm Hg. On the basis of immunological analysis, immuno-dot blot, the Hb preparations were not antigenic. A second transfusion produced no adverse immunological side effects. A right shift in P50 was obtained by further treatment of polymerized Hb with inositol hexaphosphate; however, this Hb preparation was unsuitable for transfusion as all animals died within a few minutes. A 30% exchange transfusion in rats with the polymerized bovine Hb resulted in a 100% survival of all animals. P50 values of treated animals were reduced by about 2 mm Hg for 14 days. The Hb product circulated for 14 days as determined by 51Cr labeling. Ultrapure bovine Hb has the potential to circulate and carry oxygen in rats and causes no immunological side effects.

Encapsulation of interleukin-2 in murine erythrocytes and subsequent deposition in mice receiving a subcutaneous injection.

Radiolabeled recombinant human interleukin-2 (IL-2) was successfully encapsulated in both mouse and sheep erythrocytes. Of the added IL-2, 70% was recovered bound to or encapsulated within the carrier cells. Erythrocytes containing IL-2 were stable in vitro and most of the IL-2 remained associated with the cells following a 16-h incubation at 37 degrees C. When carrier erythrocytes containing IL-2 were injected subcutaneously into mice, intact [35S]IL-2 was detectable in a number of tissues 3 days after injection.

Hypoosmotic dialysis and ultrafiltration technique for preparation of mammalian hemoglobin: a comparison of three species.

Dialysis of human, bovine, and ovine red blood cells using a hypotonic solution and a commercial kidney dialysis unit, followed by ultrafiltration through 0.1 micron pore hollow-fibers provides an easily managed method for isolation of lipid-free hemoglobin (LFHB). High performance liquid chromatography (HPLC) analysis of hemoglobin indicated 99% protein purity. SDS polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. Processing one-half liter of packed red blood cells requires approximately 10 hours and resulting in an average of 93% hemoglobin recovery.

Preparation of lipid-free human hemoglobin by dialysis and ultrafiltration.

Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid-free hemoglobin. High pressure liquid chromatography analysis of lipid-free hemoglobin (LFHB) indicates 99-100% protein purity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119-139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one-half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.

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.