The impact of different housing systems on egg safety and quality.

A move from conventional cages to either an enriched cage or a noncage system may affect the safety or quality, or both, of the eggs laid by hens raised in this new environment. The safety of the eggs may be altered either microbiologically through contamination of internal contents with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. Quality may be affected through changes in the integrity of the shell, yolk, or albumen along with changes in function, composition, or nutrition. Season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account. An understanding of these different effects is prudent before any large-scale move to an alternative housing system is undertaken.

In vitro penetration of Salmonella Enteritidis through yolk membranes of eggs from 6 genetically distinct commercial lines of laying hens.

Although deposition of Salmonella Enteritidis inside yolks is less common than deposition in albumen or on the vitelline (yolk) membrane in naturally contaminated eggs laid by infected hens, bacterial migration into the yolk to reach its nutrient-rich contents could lead to extensive multiplication. The present study used an in vitro egg contamination model to assess the ability of small initial numbers of Salmonella Enteritidis to penetrate the vitelline membrane and multiply inside yolks of eggs laid by 6 genetically distinct commercial lines of hens during 24 h of storage at 30 degrees C. Eggs from each line were tested at 4 different hen ages by inoculation of approximately 100 cfu of Salmonella Enteritidis onto the outside of the vitelline membranes of intact yolks in plastic centrifuge tubes and then adding back the albumen into each tube before incubation. Overall, the frequency of penetration of Salmonella Enteritidis into the yolk contents of eggs from individual lines of hens ranged from 30 to 58% and the mean concentration of Salmonella Enteritidis in yolk contents after incubation ranged from 0.8 to 2.0 log(10) cfu/mL. For both of these parameters, values for one hen line were significantly higher than for 2 other lines, but no other differences were observed. Hen age did not have a significant effect on egg yolk penetration by Salmonella Enteritidis. These results indicate that opportunities for the migration and growth of small initial numbers of Salmonella Enteritidis to attain more dangerous levels inside contaminated eggs during storage at warm temperatures can sometimes vary between different lines of laying hens.

Crop immune response post-Salmonella enteritidis challenge in eight commercial egg-layer strains and specific-pathogen-free White Leghorn chickens.

The crop immune response against Salmonella Enteritidis (SE) challenge in eight commercial egg-layer strains (five white-egg layer and three brown-egg layer) and specific-pathogen-free (SPF) White Leghorn (WL) hens was investigated. Pre- and post-SE challenge mucosal immune responses within the crops were evaluated. Commercial layers and SPF WL hens were orally challenged with 10(8) CFU/ml SE PT13a and SE nalR PT13, respectively. Crop lavage samples were collected at weekly intervals from day 0 (pre-challenge) to day 25-27 postinfection (PI), and bacteriological examination was performed to monitor progression of SE infection. Crop lavage samples were analyzed for SE-lipopolysaccharide (LPS)-specific IgA using enzyme-linked immunosorbent assay (ELISA). H&E-stained slides of crop sections from day 34 PI and uninfected controls were assessed for lymphoid tissue via light microscopy. Lymphoid areas were graded based on morphology, size, and cellularity using a score 0 to 5 scale. The 0 to 5 (low to high) numerical values represented progressive increases in size and cellular density of lymphoid tissue. Bacterial culture results showed the highest percentage of SE-positive crop lavage samples from all hen groups at day 5-6 PI and day 11-12 PI. A progressive decline in percentage of SE-positive crop lavage samples did occur as time PI lengthened; however, at day 25-27 PI SE persisted in crop lavage samples from SPF WL hens and three commercial white-egg layer strains. A marked increase in SE-LPS-specific IgA was measured in crop lavage samples between day 0 and day 11-12 PI for all hen groups. Crop SE-LPS-specific IgA response remained elevated above day 0 baseline for the duration of the experiment. Well-defined score 3 to 5 lymphoid tissue aggregates were observed in crop tissue sections harvested at day 34 PI. Comparison of crop sections determined a 1.2-4.0 times increase in ratio of lymphoid tissue in day 34 PI SE-challenged hens vs. uninfected control hens.

Intestinal cytokine response of commercial source broiler chicks to Salmonella typhimurium infection.

Development of molecular-based immunotherapeutic strategies for controlling Salmonella Typhimurium (ST) infection in poultry requires a better understanding of intestinal and cecal cytokine responses. Accordingly, an experiment was conducted to measure changes in intestinal cytokine expression when commercial source broiler chickens were challenged with a nalidixic acid-resistant ST. Ross broiler chicks were nonchallenged with ST (control treatment) or challenged by orally giving 7.8 x 10(6) cfu at 4 d of age (STC treatment). Each treatment consisted of 4 replicate pens with 14 chicks per pen. Expression levels of proinflammatory cytokines, interferon-gamma, and antiinflammatory interleukin (IL)-10 were determined at 5 and 10 d postchallenge (PC). Intestinal flushes were also collected from each treatment at 7 d PC to estimate IgA and IgG. Results showed an upregulation in IL-1beta mRNA in STC chicks at 5 d PC. By 10 d PC, the expression of IL-1beta was further increased and accompanied by an upregulation of IL-6 and interferon-gamma mRNA, whereas IL-10 mRNA expression decreased. It was concluded that ST induced an intestinal mucosal inflammatory response in commercial source broiler chicks less than 2 wk of age.

In vitro penetration of egg yolks by Salmonella Enteritidis and Salmonella Heidelberg strains during thirty-six-hour ambient temperature storage.

Although Salmonella deposition inside yolks is uncommon in naturally contaminated eggs, migration through the vitelline membrane into the nutrient-rich yolk contents could enable rapid bacterial multiplication. Egg refrigeration restricts both penetration and growth, but a recently proposed national Salmonella Enteritidis control program would allow unrefrigerated ambient temperature storage of eggs on farms for up to 36 h. The present study used an in vitro egg contamination model to assess the ability of small numbers of 4 Salmonella Enteritidis strains and 4 Salmonella Heidelberg strains to penetrate the vitelline membrane and multiply inside yolks during 36 h of storage at either 20 or 30 degrees C. After inoculation onto the exterior surface of the vitelline membrane, all 8 Salmonella strains penetrated to the yolk contents (at a mean frequency of 45.1%), and most strains grew to significantly higher levels (with a mean (log)10 bacterial concentration of 2.2 cfu/mL) during incubation at 30 degrees C. Significant differences in penetration frequency and yolk multiplication were observed between individual strains and between serotypes (Salmonella Enteritidis > Salmonella Heidelberg for both parameters). Penetration and multiplication were significantly less frequent during incubation at 20 degrees C. These results demonstrate that controlling ambient temperatures during prerefrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness.

Optimization of ferrioxamine E concentration as effective supplementation for selective isolation of Salmonella enteritidis in egg white.

Utilization of ferrioxamine E (FE) as a sole source of iron distinguishes Salmonella from a number of related species, including Escherichia coli. FE is not able to serve as a source of iron for E. coli or the Proteus-Providencia-Morganella group. This confers a selective advantage on Salmonella Enteritidis in egg white supplemented with FE. The optimum concentration of FE that promoted a selective advantage for Salmonella in egg white was determined. Four supplementation concentrations were evaluated (25, 50, 200, and 500 microg/ml) in egg white artificially inoculated with proportionally mixed cultures of a rifampin-resistant strain of Salmonella Enteritidis (0.1 ml of 102 CFU/ml) and E. coli K-12 (0.1 ml of 10(1) through 10(8) CFU/ml). After a 24-h incubation at 37 degrees C, Salmonella and E. coli populations were enumerated. At higher concentrations of FE (>50 microg/ml), both Salmonella and E. coli were able to use the iron supplement (1 to 8.5 log CFU/ml and 1.8 to 8 log CFU/ml, respectively); however, lower FE concentrations (< or = 50 microg/ml) exclusively promoted Salmonella growth. Salmonella was unrecoverable without supplementation. This study indicates that optimum levels of FE supplementation in egg can improve the selective detection for Salmonella Enteritidis among other competitive organisms.

Enhanced gross visualization of chicken Peyer's patch: novel staining technique applied to fresh tissue specimens.

The ileal Peyer's patches (Pp), secondary gut-associated lymphoid tissue of the mucosal immune system, may serve as an important site for monitoring inflammatory and immunologic responses of the host against enteric pathogens. Chicken Pp are often difficult to observe grossly, and a simple technique to enhance visualization of the Pp is lacking. Therefore, we designed a novel staining method that is quick, easy, and accurate to aid in gross identification and recovery of the chicken Pp from fresh tissue specimens. Lower alimentary tracts were harvested from White Leghorn hens and commercial broilers. The ileocecocolic region was excised intact, flushed with deionized water to remove ingesta, and a dilute eosin-Y solution was infused. After 1 min, the eosin-Y was gently extruded. Modified-crystal violet (mCV) was then injected into the gastrointestinal segment, where on the lymphoid tissue area became apparent at the serosal surface. The distal ileal Pp was visible as a pale whitish pink ovoid-focalized area with surrounding gut tissue stained light purple. The exact Pp site could be delineated at the serosal and mucosal surface by gross assessment. Light microscopy evaluation of hematoxylin and eosin-stained tissue slides prepared from the excised Pp site revealed lymphoid tissue aggregations with multiple follicular units indicative of Pp. The novel eosin-Y + mCV staining technique promotes rapid identification and accurate recovery of chicken Pp lymphoid tissue from fresh tissue specimens.

The effect of feed deprivation on tissue invasion by Salmonella enteritidis.

A tissue culture procedure was utilized to compare tissue cell invasion by Salmonella enteritidis from molted and full feed hens. Three identical trials were performed in which 80-wk-old active laying hens were divided into 2 groups of 6 birds each. The molted hen group was subjected to a 14-d feed withdrawal, and the full-fed hen group was administered a standard layer ration. After feed treatment, crop, ileum, cecum, and ovary (small and large yellow follicles removed) were collected, rinsed in PBS, and placed into 50 mL of RPMI medium. The ends of intestine and crop tissues were tied to allow attachment of Salmonella only to the lumen surface. The RPMI medium containing 10(7) to 10(8) cfu of novobiocin and nalidixic acid-resistant phage type 13 Salmonella enteritidis was injected into the lumen of the intestine and crop tissues. Additionally, ovaries were incubated in 50 mL of RPMI medium containing 10(6) to 10(7) cfu of the Salmonella enteritidis. Tissues were incubated with Salmonella at 37 degrees C for 2 h, after which tissues were placed in 50 mL of fresh RPMI medium containing 500 microg/mL of gentamicin and incubated for 5 h at 37 degrees C to remove any Salmonella that had not penetrated tissues. Tissues were rinsed, stomached in 10 mL of PBS, serially diluted, and plated onto brilliant green agar containing novobiocin and nalidixic acid for Salmonella enumeration. Salmonella invasion of ovaries was reduced in tissues from molted hens in trials 1 and 2 as compared with full-fed controls (> 1.2 log reduction) but not in trial 3. Salmonella invasion of ceca from molted hens was numerically increased in trials 1 and 2 and significantly increased in trial 3 as compared with controls (> 0.8 log increase). No significant differences in Salmonella invasion were detected for crops and ileum. These data suggest that molting may affect invasion of tissues by Salmonella enteritidis.

Cellular effects of T-2 mycotoxin on two different cell lines.

The effects of T-2 toxin on protein synthesis, respiratory chain activity of the mitochondria, cell lysis and toxin-cell binding were compared in the toxin-sensitive bovine kidney cell line (MDBK) and in the toxin-resistant Chinese hamster ovary cell line (CHO). Protein synthesis and mitochondrial activity were 10-fold less sensitive in CHO cells as compared to MDBK (50% inhibition = 10-15 ng/ml vs. 1-1.5 ng/ml, respectively). Lytic activity, as determined by release of 51Cr from cells incubated at 4 degrees C, was not detected at 5 h or 24 h in either of the cell lines. However, at 24 h, CHO cells released less 51Cr than non-toxin-exposed controls, indicating that some membrane interaction does occur. Both cell lines equally bound [3H]T-2 toxin at 4 degrees C. At 37 degrees C, the MDBK cells take up twice as much [3H]T-2 toxin at 2 h and 6 h. These results indicate that T-2 toxin mediates a number of effects on the cell at the level of the membrane, protein synthesis and probably mitochondrial activity toward 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction.

Presence of Campylobacter jejuni in various organs one hour, one day, and one week following oral or intracloacal inoculations of broiler chicks.

Day-old broiler chicks (n=30) were obtained from a commercial hatchery and inoculated, either orally or intracloacally, with a characterized strain of Campylobacter jejuni. At 1 hr, 1 day, and 1 wk after inoculation, broilers (n = 5) from the orally and intracloacally inoculated groups along with control birds (n=4) were humanely killed by cervical dislocation. The broilers from the control and treatment groups were aseptically opened, and the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca were aseptically removed and individually analyzed for C. jejuni. Overall, C. jejuni was isolated after oral inoculation from 13% (10/ 75), 17% (13/75), and 28% (14/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 10% (4/ 40), 8% (3/40), 10% (4/40), 25% (10/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively. Following the intracloacal route of inoculation, C. jejuni was recovered from 32% (24/75), 8% (6/75), and 16% (8/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 5% (2/40), 5% (2/40), 5% (2/40), 45% (18/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively, for all sampling periods. Campylobacter spp. were not recovered from sample sites examined from the control broilers from trial one, trial two, or trial three samples examined after 1 hr and 1 day. However, one control sample was positive from the 1-wk sampling from repetition three; therefore, those data were omitted. The rapid movement of Campylobacter to internal organs following both oral and intracloacal inoculation may be significant, particularly if it persists in these organs as reservoirs throughout the 65-wk life cycle of breeding birds.

Penetration of Salmonella enteritidis and Salmonella heidelberg into egg yolks in an in vitro contamination model.

Eggs that harbor Salmonella in their edible contents pose a significant risk of transmitting disease to consumers. Although Salmonella deposition inside yolks does not usually occur at a high frequency in naturally contaminated eggs, bacterial penetration through the vitelline membrane could lead to rapid and extensive multiplication in the nutrient-rich yolk contents. The present study used an in vitro egg contamination model to assess the ability of Salmonella strains to penetrate the vitelline membrane and multiply inside yolks. An S. enteritidis strain and 2 Salmonella heidelberg strains, initially inoculated onto the outside of the vitelline membrane, were able to enter the yolk contents (at frequencies ranging from 10 to 25% of experimentally contaminated eggs) during 24 h of incubation at 30 degrees C. Variants of these parent strains, obtained by in vivo passage into eggs laid by infected hens, penetrated the yolk membrane at significantly higher frequencies. These results demonstrate that pathogens such as S. enteritidis and S. heidelberg can penetrate into and begin to multiply inside the yolks of contaminated eggs during the first day of storage at warm temperatures.

An improved chromatographic method for the separation of egg yolk IgG into subpopulations utilizing immobilized metal ion (Fe3+) affinity chromatography.

An improved methodology is described for the separation of yolk IgG into subpopulations using immobilized metal ion (Fe3+) affinity chromatography. The yolk IgG was first extracted using a prechilled, pre-acidified method. After extraction, the yolk IgG was then fractionated using an Fe3+ column. Using an ascending pH gradient, four IgG containing peaks were well resolved based upon the elution pH, specific activity and the relative avidity index.

Enhancement of chicken lymphocyte activation and lymphokine release by avian influenza virus.

We had previously found that inactivated avian influenza virus (AIV) could enhance the response of chicken lymphocytes to mitogen or antigen activation. An investigation into the possible mechanisms of this enhancement was undertaken. Peripheral blood lymphocytes (PBL) were incubated with AIV expressing different hemagglutinin (HA) types (H1-H13) along with doses of concanavalin A (Con A) which induce maximum (0.5 microgram) or submaximum (0.125 microgram) PBL activation. The lymphocyte activation was measured 72 h later. All of the HA types except H13 enhanced the Con A response. Diminished but significant enhancement could be observed when AIV administration was delayed by as much as 48 h of the 72-h incubation time. The AIV A/ck/Ala/75 (H4N8) was also examined for its effect on interleukin 2 (IL 2) synthesis by Con A-activated PBL and was found to modestly increase the synthesis of this lymphokine. All of the AIV hemagglutinin types agglutinated the PBL with titers slightly lower than that observed for the chicken erythrocyte agglutination. These results indicate that the AIV-induced enhancement of Con A responsiveness by chicken PBL is due, at least partly, to increased synthesis of IL 2 and that the effect may be due to some viral component other than the agglutinin.

Cytotoxic effect of T-2 mycotoxin on cells in culture as determined by a rapid colorimetric bioassay.

We developed a colorimetric assay for determining metabolic activity (viability) of cells exposed to toxic agents. This system is based on the ability of mitochondrial enzymes in viable cells to modify a tetrazolium salt into a blue formazan product that can be detected spectrophotometrically at 570 nm. The assay works equally well for mammalian and insect cell lines and at 48 hr color formation is linear over a cell input range of 1.56-50 X 10(4) cells/ml. The inhibitory effects of T-2 mycotoxin on tetrazolium cleavage in L929 cells is comparable to that observed for protein and DNA synthesis (50% inhibition = 6-8 ng/ml). Using this system to analyze the lethal effect of T-2 toxin on cells from various animal species, it was found that bovine cells were the most sensitive (50% inhibition at 2.2 ng/ml) while hamster cells were the most resistant (50% inhibition at 26.2 ng/ml). Murine cells exhibited intermediate sensitivity (50% inhibition at 10.9 ng/ml). Variable toxin susceptibility was also observed among different cell types. Lymphocytes were 3-fold more sensitive to the T-2 inhibitory effects than comparable tissue culture cell lines. These data indicate that the colorimetric assay system could have broad applications in toxicological studies. Further, the observed differences in species sensitivity may provide insight into the primary mechanism of the T-2 toxin-cell interaction that ultimately leads to cell death.

Detection of the lethal effects of T-2 mycotoxin on cells using a rapid colorimetric viability assay.

A colorimetric method of determining cell viabilities in cultured cells is described. The system is based on the ability of mitochondrial enzymes in live but not dead cells to chemically reduce a tetrazolium salt (MTT) into a colored formazan dye which can be detected at 570 nm using a multiwell scanning spectrophotometer. 48 h Chinese hamster ovary (CHO) cell cultures are used in the assay and the amount of colored product formed is directly proportional to cell number over a range of 0.39-12.5 X 10(4) cells/ml. The cytotoxic effects of T-2 mycotoxin can also be detected colorimetrically using this method. The toxin dose which inhibits formazan formation (50% endpoint = 14-16 ng/ml) is very comparable to that which inhibits cell viability (17 ng/ml), or protein and DNA synthesis (10 ng/ml). This system also works well with mitogen-stimulated primary lymphocyte cultures but these cells exhibit a much more sensitive response to T-2 effects having a 50% inhibition endpoint of 2 ng/ml. The assay is rapid to perform and gives a high degree of precision and could serve as a valid alternative to viability assays currently in use.

T-2 toxin effects on the serum amyloid P-component (SAP) response of Listeria monocytogenes- and Salmonella typhimurium-infected mice.

T-2 mycotoxin, given to mice 4 days prior to an intraperitoneal inoculation with Listeria monocytogenes EGD, increases the acute phase response as determined by measurements of serum amyloid protein-P (SAP), and decreases the severity of the infection. Conversely, when T-2 toxin is given simultaneously with L. monocytogenes the mice become more susceptible to the infection, and the SAP levels attained are diminished relative to the non-toxin-treated Listeria-infected controls. T-2 toxin given 4 days prior to intraperitoneal inoculation with Salmonella typhimurium had no effect on either the resultant infection or SAP levels. These results indicate that T-2 toxin modulates the acute phase response to infection, and are consistent with an in vivo role for SAP as a nonspecific host resistance factor.

Multiplication in egg yolk and survival in egg albumen of Salmonella enterica serotype Enteritidis strains of phage types 4, 8, 13a, and 14b.

Refrigeration of eggs is vital for restricting the multiplication of Salmonella enterica serotype Enteritidis contaminants, but differences between Salmonella Enteritidis strains or phage types in their survival and multiplication patterns in egg contents might influence the effectiveness of refrigeration standards. The present study compared the abilities of 12 Salmonella Enteritidis isolates of four phage types (4, 8, 13a, and 14b) to multiply rapidly in egg yolk and to survive for several days in egg albumen. The multiplication of very small numbers of Salmonella Enteritidis inoculated into yolk (approximately 10(1) CFU/ml) was monitored during 24 h of incubation at 25 degrees C, and the survival of much larger numbers of Salmonella Enteritidis inoculated into albumen (approximately 10(5) CFU/ml) was similarly evaluated during the first 3 days of incubation at the same temperature. In yolk, the inoculated Salmonella Enteritidis strains multiplied to mean levels of approximately 10(3) CFU/ml after 6 h of incubation and 10(8) CFU/ml after 24 h. In albumen, mean levels of approximately 10(4) CFU/ml or more of Salmonella Enteritidis were maintained through 72 h. Although a few differences in multiplication and survival were observed between individual isolates, the overall range of values was relatively narrow, and no significant differences (P < 0.05) were evident among phage types.

Supplementing pools of egg contents with broth culture media to improve rapid detection of Salmonella enteritidis.

Culturing egg contents to detect Salmonella enteritidis (SE) has become an important tool for identifying infected laying flocks and thereby reducing the transmission of SE to humans by contaminated eggs. The present study evaluated the efficacy of supplementing incubating egg pools with selective and nonselective enrichment broth media (prepared at higher than usual concentrations) for rapidly isolating SE by a direct plating culture method. When 100-ml pools of liquid whole egg from a mixture of 60 egg contents were contaminated with approximately 10 SE cells each, supplementation with ferrous sulfate or with concentrates of either tryptone soya broth or Rappaport-Vassiliadis broth significantly improved SE recovery. When 100-ml egg-contents pools were contaminated with approximately 2 SE cells each, the addition of concentrated tryptone soya broth to incubating egg pools resulted in significantly better SE recovery than did iron supplementation. Efficient presumptive detection of very low incidences and levels of SE contamination by direct plating was thus accomplished in a total of 48 h by adding concentrated tryptone soya broth to incubating egg pools.

Comparison of Salmonella Enteritidis infection in hens molted via long-term feed withdrawal versus full-fed wheat middling.

Molting is an important economic management tool for the layer industry as a means of maximizing the effective laying life of a flock. Previous work has shown that molting birds through feed removal (FM) increased the severity of a Salmonella Enteritidis (SE) infection. The current study was conducted to follow the progression of an SE infection in unmolted hens versus hens molted via 14-day FM or ad libitum feeding of wheat middlings (WM), in the presence or absence of 2.5% lactose administered in the drinking water. In two trials of the experiment, all hens were infected with approximately 1 x 10(7) SE at day 4 of molt and sampled for SE shedding on days 4, 10, 17, and 24 postinfection (PI). Organ levels of SE were determined on day 7 PI. All molt procedures caused cessation of egg lay within 3 to 7 days. In trials 1 and 2, birds subjected to total FM shed 3 to 5 logs more SE than either the control birds (unmolted) or the birds fed WM on days 4 and 10 PI. Liver and spleen, ovary, and cecum counts were also significantly (P < 0.05) higher in the fasted birds in one trial and liver and spleen and cecum counts in the second. No differences in any of the SE counts were observed in unmolted versus WM-fed birds. Lactose supplementation in drinking water did not provide any advantage in reducing SE infection in either trial. These results indicate that there are alternative methods to long-term FM that can be used to molt birds and not increase the risk for SE problems. How these alternative methods compare with FM with regard to second-cycle egg production and the mechanisms involved in the reduced SE shedding remain to be investigated.

Bactericidal effects of negative air ions on airborne and surface Salmonella enteritidis from an artificially generated aerosol.

The bactericidal effect of high levels of negative ions was studied using a custom-built electrostatic space charge device. To investigate whether the ion-enriched air exerted a bactericidal effect, an aerosol containing Salmonella Enteritidis (SE) was pumped into a sealed plastic chamber. Plates of XLT4 agar were attached to the walls, top, and bottom of the chamber and exposed to the aerosol for 3 h with and without the ionizer treatment. The plates were then removed from the chamber, incubated at 37 degrees C for 24 h, and colonies were counted. An average of greater than 10(3) CFU/plate were observed on plates exposed to the aerosol without the ionizer treatment (control) compared with an average of less than 53 CFU/plate on the ionizer-treated plates. In another series of experiments, the SE aerosol was pumped for 3 h into an empty chamber containing only the ionizer and allowed to collect on the internal surfaces. The inside surfaces of the chamber were then rinsed with 100 ml phosphate-buffered saline that was then plated onto XLT4 plates. While the rinse from the control chamber contained colony counts greater than 400 CFU/ml of wash, no colonies were found in the rinse from the ionizer-treatment chamber. These results indicate that high levels of negative air ions can have a significant impact on the airborne microbial load, and that most of this effect is through direct killing of the organisms. This technology, which also causes significant reduction in airborne dust, has already been successfully applied for poultry hatching cabinets and caged layer rooms. Other potential applications include any enclosed space such as food processing areas, medical institutions, the workplace, and the home, where reduction of airborne and surface pathogens is desired.