Introduction

Salmonella spp. may be found in the nest box of breeder chickens, cold egg-storage rooms at the farm, on the hatchery truck or in the hatchery environment. These bacteria may then be spread to fertilised hatching eggs on the shell or, in some cases, may penetrate the shell and reside just beneath the surface of the eggshell.

Research has demonstrated that contamination of raw poultry products with Salmonella spp. may be attributable to cross-contamination in the hatchery from Salmonella infected eggs or surfaces to uninfected baby chicks during the hatching process. Cox et al. (references 6 and 7 below) reported that broiler and breeder hatcheries were highly contaminated with Salmonella spp. Within the broiler hatchery, 71 per cent of eggshell fragments, 80 per cent of chick conveyor belts swabs, and 74 per cent of pad samples placed under newly hatched chicks contained Salmonella spp.

Cason et al. reported that, although fertile hatching eggs were contaminated with high levels of Salmonella typhimurium, they were still able to hatch. The authors stated that paratyphoid salmonellae do not cause adverse health affects to the developing and hatching chick. During the hatching process, Salmonella spp. are readily spread throughout the hatching cabinet due to rapid air movement by circulation fans. When eggs were inoculated with a marker strain of Salmonella during hatching, greater than 80 per cent of the chicks in the trays above and below the inoculated eggs were contaminated. In an earlier study, Cason et al. demonstrated that salmonellae on the exterior of eggs or in eggshell membranes could be transmitted to baby chicks during pipping.

Salmonella may persist in hatchery environments for long periods of time. When chick fluff contaminated with Salmonella was held for four years at room temperature, up to 1,000,000 Salmonella cells per gram could be recovered from these samples.

Researchers have demonstrated a link between cross-contamination in the hatchery and contaminated carcasses during processing. Goren et al. isolated salmonellae from three different commercial hatcheries in Europe and reported that the same serotypes found in the hatcheries could be found on processed broiler chicken carcass skin. Proper disinfection of the hatchery environment and fertile hatching eggs, therefore, is essential for reducing Salmonella on ready-to-cook carcasses.

Numerous studies have been conducted to evaluate sanitizing agents for disinfecting eggshell surfaces and membranes. Bailey et al. reported that 2.5 per cent hydrogen peroxide (H₂O₂), administered using 100 or 500mL per hour, reduced Salmonella typhimurium-positive eggshells by 55 per cent and the number of positive chicks by 53 per cent. Hydrogen peroxide reduced total aerobic bacterial counts (APC) in air in hatching egg incubators from 3.6 colony-forming units (CFU) per litre for water fogging to 0.35CFU per litre when the incubator was fogged with three per cent hydrogen peroxide. Sheldon and Brake (14) demonstrated that five per cent hydrogen peroxide reduced APC on hatching eggs from 3.98 CFU per egg for water sprayed eggs to 0.99 CFU per egg for treated eggs. Bailey et al. reported that ozone at 0.2 to 0.4ppm reduced Salmonella typhimurium-positive eggshells by 10 per cent and the number of positive chicks by 26.7 per cent. In another study, ozone at 3.03 per cent by weight was able to reduce APC on broiler hatching eggs by 2.57 log₁₀ cfu. Brake and Sheldon observed that a quaternary ammonium sanitiser at 3.0 per cent reduced APC on broiler hatching eggs by 99.9 per cent within 30 minutes of application.

The methods used to apply sanitisers in these studies varied from gaseous exposure to fogging and dipping. Method of application may have a dramatic impact on efficacy of the sanitisers used.

Law developed an electrostatic spray-charging system using air atomisation which achieved a seven-fold increase in spray deposition over conventional application methods.

In later studies, a 1.6- to 24-fold increase in deposition was reported. Herzog et al. observed that insect control on cotton plants was equal to or better than conventional spray application using only one-half the amount of insecticide. Thus, the electrostatic spraying method may be an appropriate means of applying sanitisers in the hatchery environment — by distributing the sanitiser more effectively over the surface of eggs and equipment and by reducing the amount of sanitiser needed to eliminate pathogenic bacteria.

If EO water applied using ESS can significantly reduce Salmonella on the surface of fertile eggs, it is believed that this method may result in significant reductions in Salmonella contamination of raw poultry products. Although these studies collectively point to the fact that reduction of Salmonella in the hatchery should result in reductions on final product, this relationship has never been proven scientifically. Moreover, if a safe, non-toxic sanitiser could be proven effective in the hatchery, its use would be a welcomed replacement for formaldehyde, since formaldehyde is objectionable to both chicks and workers.

As is demonstrated in the figures, the sanitiser is sprayed as a very fine fog and, in a short period of time, completely disappears. This fog completely covers every surface within the hatching cabinet, including eggs. Complete coverage has been demonstrated using fluorescent dye sprayed onto surfaces. After spraying, the area can be evaluated using a black light, and even the most difficult to reach spaces are completely covered.

Matching the Sanitiser to the Electrostatic Spraying System

A major consideration when using electrostatic spraying is the type of sanitiser being used. Applying an electrical charge or atomisation has the potential to completely eliminate the killing power of some sanitisers. When using electrostatic spraying, it is best to evaluate the sanitiser to be used in light of this limitation.

As mentioned previously, currently used sanitisers are objectionable for various reasons. Formaldehyde is difficult to work with and presents a worker safety hazard. Formaldehyde gas burns people's lungs and eyes when they are exposed to it, and many have compared the experience to that of being exposed to tear-gas. Glutaraldehyde is also unpleasant for workers. Hydrogen peroxide, while effective, is corrosive to equipment and is irritating to the lungs. It would seem that exposing baby chicks on day of hatch to these chemicals would be disadvantageous if other chemicals could be used that would be as effective at eliminating pathogenic bacteria.

Mixed oxidants

A method has been developed for splitting salt water into streams of mixed oxidants. One such system (EAU Limited) is displayed in Figure 5. By mixing a 20 per cent solution of salt water and placing the water in the container inside the machine, the water is then taken up by the instrument, passed over an electrode, and various oxidising chemicals are generated. The water comes out of the machine as two different mixed oxidant streams. One stream is acidic (pH 2.1) and the other is alkaline (pH 10.8). The acidic portion of this water has been shown to be effective for killing various pathogenic bacteria of concern to the poultry industry in an experimental setting (K.S. Venkitanarayanan et al., 1999a, 1999b).

Some of the possible mixed oxidants produced by electrolysing the salt water are hydrogen peroxide, chloride, HOCl, oxone (O₃) and chlorine dioxide (ClO₂). All of these compounds have been proven to be effective sanitisers, but the unique aspect of this methodology is that these compounds are produced in very low concentrations (three to 80ppm, depending on the system).

Collectively, these chemicals exhibit bacteriocidal activity while the water remains safe enough to drink and is not harmful to equipment. The advantage of using electrolysed water is that it costs almost nothing to produce, is safe enough to drink and breathe (when atomised), is effective against pathogenic bacteria, and will not corrode equipment.

The purpose of the studies described in this publication was to determine if electrostatic application (ESS) of a nontoxic, novel sanitiser would be effective in eliminating Salmonella spp. from fertile hatching eggs, and to evaluate if this reduction carries through to a significant reduction in colonisation of chickens during the grow-out process.

The Studies

Materials and methods

Study I

Pathogenic Bacterial Isolates
Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli were obtained from the United States Department of Agriculture, Agricultural Research Service’s (USDA-ARS) Poultry Microbiological Safety Unit laboratory. These isolates were originally collected from commercial broiler carcasses. Each isolate was assayed for Gram reaction, cytochrome oxidase activity and production of catalase, and was identified using either the Vitek, Biolog or Micro-ID rapid identification methods.

EO Water Preparation
A solution of EO water was prepared by electrolysis of a 20 per cent saline solution made with tap water. The final pH and oxidation-reduction potential of this solution were 2.1 and 1,150, respectively. Due to electrolysis of the saline solution, small concentrations of antimicrobial substances were produced including chlorine ions (8ppm free chlorine), chlorine dioxide, ozone and hydrogen peroxide (See Figure 5). It is believed that the combination of very low concentrations of these compounds in an acidic environment is the mechanism of action for EO water.

Egg Preparation
Eggs were collected from layer chickens housed at The University of Georgia Poultry Research Center. After collection, the eggs were washed using a commercially available chlorine based sanitiser and allowed to dry. Each egg was then rinsed thoroughly three times using sterile de-ionised water to remove any residual sanitiser that may have remained from the washing process.

Egg Inoculation
An inoculation solution was prepared by placing 0.1mL of an actively multiplying pure bacterial culture (incubated for 24 hours in brain heart infusion broth5 at 35°C) into 200mL of sterile one per cent peptone broth. The bacterial cultures used were Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli. Eggs were individually dipped into the inoculum and allowed to dry under a laminar flow hood for one hour. This procedure provided time for the bacteria to attach to the surface of the egg.

Electrostatic Spraying of Eggs
Each egg was placed into a clean egg flat and positioned in an electrostatic spraying chamber. Tap water (two repetitions) or EO water (four repetitions) was sprayed onto the eggs using two electrostatic spray nozzles for 15 seconds each hour for 24 hours (See Figures 6 and 7). After treatment, the eggs were allowed to dry under a laminar flow hood for one hour. In addition, two eggs were dipped in each bacterial isolate, allowed to dry and stored for 24 hours in an enclosed chamber with 96 per cent humidity as a control.

Neutralisation of sanitiser
Each control and treated egg was cracked using a sterile blade and the contents were removed. Egg-shells and membranes were placed into 25mL of sterile one per cent peptone broth containing three per cent Tween 80, 0.3 per cent lecithin and 0.1 per cent histidine to neutralise the sanitisers.

Microbiological Evaluation
One millilitre of this mixture was placed into 9mL of sterile BHI, which acts as a growth medium for conducting impedance or conductance assays, and vortexed. One mL of this mixture was placed into a Bactometer module well in duplicate. Samples were monitored using the Bactometer Microbial Monitoring System M128. All of the bacterial isolates tested were monitored at 35°C. All samples were monitored for 48 hours using impedance except for E.coli, which was monitored using conductance.

Statistical Analysis
The experimental design was a 4×4×2 of replication, bacterial type and treatment (EO water and controls). All microbiological analyses were conducted in duplicate. Data were analysed after averaging the duplicates. Results were analyzed using the General Linear Models (GLM) procedure of SAS software (SAS Institute, 1994). Treatment means were separated using Fisher's Least Significant Difference option of SAS software (SAS Institute, 1994). All values reported as significant were analysed at the P=0.05 level.

Study II

Pathogenic Bacterial Isolates
Salmonella typhimurium was obtained from the United States Department of Agriculture, Agricultural Research Service's (USDA-ARS) Poultry Microbiological Safety Unit laboratory. These isolates were originally collected from commercial broiler carcasses. Each isolate was assayed for Gram reaction and cytochrome oxidase activity, and identified using the Vitek rapid identification method.

EO Water Preparation
A solution of EO water was prepared by electrolysis of a 20 per cent saline solution made with tap water. The final pH and oxidation-reduction potential of this solution were 2.1 and 1,150, respectively. Due to electrolysis of the saline solution, small concentrations of antimicrobial substances were produced including chlorine ions (8ppm free chlorine), chlorine dioxide, ozone and hydrogen peroxide. It is believed that the combination of very low concentrations of these compounds in an acidic environment is the mechanism of action for EO water.

Industrial Hatchability Study
Thirty thousand eggs were placed into clean egg flats and positioned in two separate commercial hatchers (15,000 each) at a commercial primary broiler breeder facility. Tap water in one hatching cabinet and EO water in the other hatching cabinet were sprayed onto the eggs using two electrostatic spray nozzles. The timers were set to deliver EO water to the eggs for five minutes immediately upon placement and then two minutes every six hours for the first day in the hatching cabinet. On the second day, the nozzles delivered EO water for two minutes every four hours. On the final day of hatch, the nozzles produced EO water for two minutes every two hours. These nozzles were designed to spray a volume of 280mL of liquid per minute. After hatching, the percent hatchability was determined for each hatching cabinet.

Egg Preparation and Inoculation
Fertile hatching eggs were collected from broiler breeder chickens housed at The University of Georgia, Poultry Research Center and were incubated for 18 days in setters. An inoculation solution was prepared by placing 0.1mL of an actively multiplying culture of Salmonella typhimurium (incubated 24 hours in brain heart infusion broth at 35°C) into 200mL of sterile one per cent peptone broth. Eggs were individually dipped into the inoculum and allowed to dry for one hour. This procedure provided time for the bacteria to attach to the surface of the egg.

Electrostatic Spraying of Eggs
In the second portion of Study II conducted at the UGA Poultry Research Center, 40 eggs were placed into clean egg flats and positioned in two separate commercial hatchers in two separate repetitions. Tap water in one hatching cabinet and EO water in the other hatching cabinet were sprayed onto the eggs using two electrostatic spray nozzles. The timers were set to deliver EO water to the eggs for five minutes immediately upon placement and then two minutes every six hours for the first day in the hatching cabinet. On the second day, the nozzles delivered EO water for two minutes every four hours. On the final day of hatch, the nozzles produced EO water for two minutes every two hours. These nozzles were designed to spray a volume of 280mL of liquid per minute. After hatching, the percentage hatchability was determined for each hatching cabinet. After hatching, the percent hatchability was determined for each hatching cabinet.

Grow-out
After hatching, the chicks from each hatcher were transported separately to different research facilities that had been thoroughly disinfected. The chicks were reared to four weeks of age being fed and watered ad libitum. After four 4 weeks, the birds were euthanised using carbon dioxide gas. The lower digestive tract (including the ileal junction, caeca, rectum and cloacae) were removed from each bird, placed into a sterile plastic bag, encoded and transported to Woodsen-Tenant Laboratories for evaluation for the presence (colonisation) of Salmonella.

Microbiological Evaluation
The lower digestive tracts of each bird were evaluated using the following method:

1. Intestines and caeca were homogenised in 250mL of universal preenrichment broth and incubated for 24 hours at 35°C.
2. A 1-mL aliquot was transferred to 10mL selenite cysteine (SC) broth and a 1mL aliquot was transferred to 10mL tetrathionate (TT) broth. The SC broth tubes were incubated at 35°C for eight hours and the TT broth tubes were incubated at 42°C for eight hours in a water bath.
3. 1-mL aliquots from SC and TT broth tubes were placed separately into two tubes containing M- broth and incubated at 35°C for six hours in a water-bath.
4. Tecra ELISA visual immunoassays were used to evaluate the tubes for the presence of Salmonella
5. Presumptive positives were streaked onto xylose lysine desoxycholate (XLD), Hektoen enteric (HE), bismuth sulfate (BS), and xylose lysine tergitol (XLT4) agars and incubated at 35°C for 24 hours.
6. Colonies were streaked onto triple sugar iron (TSI) and lysine iron agar (LIA) slants and incubated at 35°C for 24 hours.
7. Slants exhibiting typical reactions for Salmonella were evaluated using Poly A-I and Vi and Poly a-z for "O" and "H" antigens.

Statistical Analysis
The experimental design for the industrial hatchability study was a 1×2×15,000 of replication, treatment (tap water or EO water), and egg. The experimental design for the hatchability study at the university was a 2×2×80 of replication, treatment (water or EO water), and egg. The experimental design for the Salmonella recovery portion of the study was a 2×2×40 of replication, treatment and egg. Results were analyzed using the logistic progression procedure of SAS software (SAS Institute, 1994). All values reported as significant were analyzed at the P=0.05 level.

Results and Discussion

Study I

Bacterial proliferation requires the availability of nutrients such as carbohydrates, proteins, or lipids. As bacteria break down and utilise these nutrients, they release charged byproducts such as lactic acid and acetic acid (Cady, 1974). As charged metabolites accumulate, the conductance and capacitance of the growth medium increases, and impedance decreases. A significant and dramatic shift in the electrical component of the medium occurs when bacterial populations reach a threshold of 10⁶ to 10⁷ cells per mL (Firstenberg-Eden, 1983). The time required for this shift to occur is called the detection time (DT).

DT is dependent on the initial concentration of bacteria, the rate at which bacteria in the sample reproduce, the temperature and the test medium used (Richards et al., 1978; Silley and Forsythe, 1996). Using electrical methods, highly contaminated samples would be detected first. For example, a sample that initially contains 10⁵ organisms would require fewer cell divisions to reach the 10⁶ detection threshold, than a sample that initially contains only 10¹ bacteria. Thus, DT is inversely proportional to the initial bacterial level in the sample. If impedance or conductance detection times are significantly increased when bacterial populations are exposed to a chemical sanitiser, then the sanitiser had an inhibitory effect on the proliferation of the bacterium or group of organisms. In addition, if no detection time is recorded in 48 hours, then it is assumed that the organism was deactivated or injured beyond repair by the sanitiser, as it was unable to multiply under optimal growth conditions.

In this study, significant differences in bacterial inhibition by EO water were observed between replicates. For each replicate, different concentrations of bacteria were used and the oxidation/reduction potential (ORP) of the EO water evolving from the electrostatic spray nozzle head varied within and between replicates. Thus, the differences observed between replicates may be attributed to application of high numbers of bacteria in some instances and fluctuation in ORP values.

Fluctuation in ORP at the nozzle head may be attributed to the charge of the liquid coming out of the nozzle, the air speed of compressed air carrying the sanitiser and the size of the liquid droplet coming from the nozzle. None of these variables are associated with the sanitiser but are able to be controlled by adjustments to the electrostatic spray nozzle system, especially if this system is to be used in an industrial setting.

Colony-forming units (cfu) of bacteria per millilitre of inoculum exposed to EO water are presented in Table 1. Please note that, in some cases, very high concentrations of bacteria were challenged in this study to determine the effect of the sanitiser on high numbers of actively growing pathogens and indicator populations of bacteria.

Impedance and conductance detection times (hours), and log₁₀ cfu estimations for pure cultures of Salmonella typhimurium (ST), Staphylococcus aureus (SA), Listeria monocytogenes (LM), and conductance detection times (hours) for Escherichia coli (EC) on eggs that have been treated with tap water (two replicates) or EO water (four replicates) using electrostatic spraying, and control eggs that were not treated are presented in Tables 2 and 3, respectively.

EO water completely eliminated all ST on three (20 per cent), seven (46.7 per cent), one (6.7 per cent) and eight (53.3 per cent) eggs of 15 tested in Reps 1, 2, 3 and 4, respectively. In all Reps, for the sanitiser to eliminate ST on an egg completely, a minimum of a 5-log₁₀ reduction would be required. In Rep 4, when 53.3 per cent of eggs were negative for ST, 6-log₁₀ ST were killed. In addition, for eggs that remained positive, the number of ST remaining were significantly reduced by a minimum of 4-log₁₀ when compared to control eggs.

EO water was able to eliminate SA on 12 (80 per cent), 11 (73.3 per cent), 12 (80 per cent) and 11 (73.3 per cent) eggs of 15 tested in Reps 1, 2, 3 and 4, respectively (Table 2). In Reps 3 and 4, for the sanitiser to eliminate SA on an egg completely, a minimum of a 6-log₁₀ and a 5-log₁₀ reduction would be required, respectively. In addition, for eggs that remained positive, the number of SA remaining were significantly reduced by a minimum of 3 log₁₀ when compared to control eggs.

For LM, EO water eliminated all bacteria on eight (53.3 per cent), 13 (86.7 per cent), 12 (80 per cent) and 14 (93.3 per cent) eggs of 15 tested in Reps 1, 2, 3 and 4, respectively (Table 2). In Reps 3 and 4, for the EO water to eliminate LM on an egg completely, a minimum of a 4-log₁₀ reduction would be required. In addition, for eggs that remained positive, the number of LM remaining were significantly reduced by a minimum of 1-log₁₀ (Rep 2) or 2.2-log₁₀ (Reps 3 and 4) when compared to control eggs, except in Rep 1.

EO water eliminated all EC on nine (60 per cent), 11 (73.3 per cent), 15 (100 per cent) and 11 (73.3 per cent) eggs of 15 tested in Reps 1, 2, 3 and 4, respectively (Table 2).

In all Reps, for the sanitiser to eliminate EC on an egg completely, a minimum of a 4-log₁₀ reduction would be required. In Rep 4, when 73.3 per cent of eggs were negative for EC, 6-log₁₀ EC were killed. In addition, for eggs that remained positive, the number of EC remaining were significantly (P<0.05) reduced by a minimum of 2-log₁₀ when compared to control eggs. In Rep 3, EO water performed especially well by eliminating all EC on all eggs, even when a concentration of 47,500 cfu/mL were used.

These data are promising in that EO water is non- toxic and can be consumed as produced. Moreover, this sanitiser is environmentally friendly and is not harmful to humans. Because Salmonella testing is part of the USDA Food Safety and Inspection Service (FSIS) Pathogen Reduction Final Rule (USDA- FSIS, 1996), and Salmonella is spread throughout the hatchery environment, leading to cross-contamination and eventual contamination of the product, this sanitiser should prove effective as a means of treating hatching eggs. Currently used hatchery sanitisers (formaldehyde gas and glutaraldehyde) are noxious to humans and chicks, and may pose a serious health risk. Thus, a sanitiser that does not harm chicks, is inexpensive to produce, and is effective would be a useful tool for the poultry industry.

Study II

Results for hatchability of commercial broiler breeder chicks from hatching eggs treated electrostatically with tap water or EO water during hatch are presented in Table 4. Although treatment with EO water seemed to lower hatchability slightly when compared to tap water treated fertile eggs, the hatchery manager indicated that this was expected because he used older fertile eggs for the EO water treated hatching cabinet. This effect was corroborated in the later hatchability study at the University of Georgia.

Results for hatchability of chicks from fertile hatching eggs obtained at the University of Georgia and treated electrostatically with tap water or EO water during hatch are presented in Table 5.

No differences were observed in hatchability between EO treated or tap water treated eggs at 93 per cent each. These data are very consistent with expected hatch percentages from the incubators at the UGA Poultry Research Center. Thus, hatchability does not seem to be a significant factor when considering the use of EO water for sanitising hatching eggs during the hatching process.

The researchers observed in these studies that electrostatic application of EO water completely removed the dust, fluff and dander from the air, upper surfaces of the hatching cabinet, and the eggs. It is believed that, by charging the EO water using electrostatic spraying, the dust and dander were also charged and fell to the floor, away from the eggs and chicks.

Results for Salmonella typhimurium prevalence in the lower intestines of broiler chickens from hatching eggs treated electrostatically with tap water or EO water during hatch are presented in Table 6. These results are extremely encouraging in that 65 to 95 per cent (Replicate 1 and 2, respectively) of the chickens were colonised when only tap water was used to treat the fertile hatching eggs, indicating that the method for inducing colonisation was appropriate; however, for electrostatically treated eggs using EO water, Salmonella was only able to colonise one chicken out of 40 tested over two repetitions under actual grow-out conditions.

This research has tremendous industrial application because many of the companies that are experiencing failures due to high Salmonella prevalence at the poultry plant are receiving flocks of birds that are 80 to 100 per cent positive for Salmonella as they enter the plant. It would seem logical to suppose that if the number of chickens in field that are colonised with Salmonella could be reduced to the levels observed in this study, the industry would be able to meet the Salmonella performance standard required by the USDA-FSIS.

This research describes a method that should have tremendous value to the poultry industry for reducing Salmonella in flocks arriving to the processing plant, which, according to our research, will translate directly into lower numbers of processed carcasses that are positive for Salmonella. Moreover, the electrostatic spraying system is not expensive to incorporate into a commercial hatchery.

Additionally, the EO water is very economical to produce and is so non-toxic as to be potable. This water does not degrade equipment and does not present an environmental hazard when discharged.

Summary

Electrolysed oxidative water applied using electrostatic spraying is an effective means of eliminating pathogenic and indicator populations of bacteria from hatching eggs. Using this method in a pilot scale hatchery, the percentage of chickens that were colonised with Salmonella was dramatically reduced.

These studies demonstrate that the use of EO water in combination with electrostatic spraying may provide a practical and inexpensive way for the industry to significantly lower the number of birds that arrive at the processing plant contaminated with Salmonella.

References

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March 2013