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Modeling disinfection of plastic poultry transport cages inoculated with Salmonella enteritids by slightly acidic electrolyzed water using response surface methodology
Modeling disinfection of plastic poultry transport cages inoculated with Salmonella enteritids by slightly acidic electrolyzed water using response surface methodology Y. T. Zang,∗ B. M. Li,∗ Sh. Bing,† and W. Cao∗,1 ∗
Key Laboratory of Structure and Environment in Agricultural Engineering, Ministry of Agriculture, China Agricultural University, Beijing 100083, China; and † Shandong Agricultural University, Shandong 271000, China ite design of the response surface methodology (RSM). The established RS model had a goodness of fit quantified by the parameter R2 (0.971), as well as a lack of fit test (P > 0.05). The maximum reduction of 3.12 log10 CFU/cm2 for S. Enteritidis was obtained for the cage treated with tap water cleaning for 15 s followed by SAEW treatment for 40 s at an ACC of 50 mg/l. Results indicate that the established RS model has shown the potential of SAEW in disinfection of bacteria on cages.
Key words: Slightly acidic electrolyzed water, poultry transport cages, disinfection, S. Enteritidis, modelling 2015 Poultry Science 00:1–7 http://dx.doi.org/10.3382/ps/pev188
INTRODUCTION
tants have been widely used as a preventative measure against bacterial infections in poultry (DeBenedictis et al., 2007), but potentially toxic, corrosive, or volatility problems may thereby arise (Gr¨ aslund and Bengtsson, 2001). Therefore it is crucial to develop a novel disinfectant with high efficacy and which leaves little residue. SAEW produced by electrolysis of a dilute hydrochloric acid and/or sodium chloride solution in a chamber without a membrane, and with a pH value of 5.0 to 6.5, is one of the emerging environment friendly antimicrobial disinfectants (Hricova et al., 2008; Huang et al., 2008). It has been proven to be safe, cheap, and exhibits high bactericidal and fungicidal efficacy (Abadias et al., 2008; Cao et al., 2009). Moreover, SAEW has the advantage of reducing corrosion of surfaces and minimizes of the potential for damage to human health (Abadias et al., 2008). Several studies have demonstrated that SAEW could be used as a disinfectant in poultry production (Huang et al., 2008; Loretz et al., 2010). Cao et al. (2009) indicated that a reduction of 6.5 log10 CFU/g of S. Enteritidis on shell eggs was obtained by SAEW at 15 mg/l available chlorine for 3 min. Hao et al. (2013a) reported that SAEW with an ACC of 300 mg/l resulted in a significant reduction in microbes on the wall, rail, and floor of swine barns after flushing disinfection (P < 0.05). However, little information is available on the disinfection of SAEW for poultry transport cages.
Salmonella Enteritids is an important pathogen of animals and humans and represents a serious public health concern worldwide (Baggesen et al., 1997). Over the years, S. Enteritids has proven to be a very difficult disease to consistently control over time and across farms (CDC, 2002). S. Enteritids can spread from infected farms through a number of routes and that of bacterial entry to the farm is therefore often difficult to ascertain. In addition, extensive efforts to identify the routes of S. Enteritids transmission between layer farms have been made. Many studies have been conducted on the routes of S. Enteritids transmission via infected animals, contaminated materials, insects, and soils (Totton et al., 2012), however, a potential route of S. Enteritids transmission between farms may be through the plastic poultry transport cages used for transport (Racicot et al., 2011, 2012). Thus, it is necessary to reduce the level of pathogenic microorganisms on the poultry cages that enter the farm to avoid chick infection. Disinfection is a generally employed method for preventing the introduction of both endemic and epidemic infections (B¨ ohm 1998; Totton et al., 2012). Several studies have shown that chemical disinfec C 2015 Poultry Science Association Inc. Received March 7, 2015. Accepted May 3, 2015. 1 Corresponding author: [email protected]
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ABSTRACT In order to reduce the risk of enteric pathogens transmission in animal farms, the disinfection effectiveness of slightly acidic electrolyzed water (SAEW, pH 5.85 to 6.53) for inactivating Salmonella Enteritidis on the surface of plastic poultry transport cages was evaluated. The coupled effects of the tap water cleaning time (5 to 15 s), SAEW treatment time (20 to 40 s), and available chlorine concentrations (ACCs) of 30 to 70 mg/l on the reductions of S. Enteritidis on chick cages were investigated using a central compos-
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The RSM has been recognized to be a powerful tool for determining the effects of different factors and the interactions among them (Bas and Boyaci, 2007). The central composite design (CCD) of RSM is the most common experimental design which has been successfully used to determine Salmonella inactivation on drycured ham (Bover-cid et al., 2012), and reduction of Bacillus subtilis (Aouadhi et al., 2013) and vibrio parahaemolyticus on cooked shrimp by acidic electrolyzed water (Wang et al., 2014). The purpose of this work was to evaluate the effectiveness of SAEW to inactive S. Enteritids on the surface of plastic cages under the chicken manure interference circumstance. RSW was used to determine the inactivation of S. Enteritidis by SAEW on plastics as a function of the tap water cleaning time, SAEW treatment time, and ACC.
Bacterial Cultures The strains of Salmonella Enteritidis (CVCC 2184) used were obtained from the China Veterinary Culture Collection (Beijing, China). The strain was transferred into tryptic soy broth (Beijing Land Bridge Technology Company Ltd., Beijing, China) and incubated at 35◦ C for 24 h. Following incubation, a 10 ml culture was pooled into a sterile centrifuge tube and centrifuged at 3000 × g and 4◦ C for 10 min. The supernatant was decanted, and the pellets were resuspended in 10 ml of sterilized 0.85% NaCl solution, washed 3 times, and resuspended in 10 ml of the same solution to obtain a final cell concentration of approximately 107 to 108 CFU/ml. The bacterial population in each culture was confirmed by plating 0.1 ml portions of appropriately diluted culture on tryptic soy agar (Beijing Land Bridge Technology Company Ltd., Beijing, China) plates and incubating the plates at 35◦ C for 24 h.
Inoculation A 6% solution of liquid manure was prepared by the addition of 30 g of chicken manure (obtained from poultry with no bedding) to 0.5 l of sterile distilled water, and then inactivated by an autoclave (YXQ-LS-18SI, Shanghai Boxun Industrial Co., Ltd., Shanghai China). Five ml of the 6% of sterilized liquid manure was shaken and then mixed with equal portions of the prepared culture mixtures to obtain the final populations of contaminated culture of approximately 108 to 109 CFU/ml and 3% concentration (Elassaad et al. 1993) of chicken liquid manure soiling interference in disinfection. The plastics were obtained from a plastic poultry transport cage (High Density Polyethylene materials, 7.35 × 5.45 × 2.60 cm, Shenzhen Lanhai Co., Ltd., Shenzhen, China). The plastic cage was washed with tap water to remove soil, and then trimmed to approximately 1.5 × 1.5 cm2 and packed in a polyethylene bag
Preparation of Slightly Acidic Electrolyzed Water Slightly acidic electrolyzed water was produced using a SAEW generator (Zhouji Biosafety Technology Co., Ltd., Beijing, China) which consists of a non-membrane electrolytic cell with anode and cathode electrodes. SAEW with a pH value of 6.15 to 6.53, an oxidationreduction potential (ORP) of 974 to 989 mV, and different ACCs were prepared for electrolysis of NaCl (10 g/l) solution in the SAEW generator. The physicochemical properties of SAEW were measured before use. The pH and ORP values were measured using a dual scale pH/ORP meter (CON60, Trans-Wiggens, Singapore) with a pH electrode (PE02; range, 0.00 to 14.00) and an ORP electrode (ORP06; range, −999 to +999 mV). The ACC was determined using a digital chlorine test kit (RC-2Z, Kasahara Chemical Instruments Co., Saitama, Japan). The detection range was 0 to 320 mg/l. The solutions were placed into an atmospheric pressure manual sprayer purchased from a supermarket which was used to treat the sample.
Treatment of Samples with SAEW and Microbiological Determination Inoculated or non-inoculated plastic pieces were first sprayed with tap water by an atmospheric pressure manual sprayer to clean them, and then sprayed with SAEW to disinfect them under different conditions. The non-inoculated plastic pieces were used as the control group. After treatment, moistened sterile swabs with a neutralizing agent (0.1% Na2 S2 O3 ) were used to collect surface samples from the plastic pieces. The sterilized cotton swabs, which had been wiped back and forth twenty times on the sample surfaces, were immediately transferred into 5 ml neutralizing agent tubes for microbiological analyses. The tubes were shaken on a platform shaker at 1800 rpm (MIR-S100, Sanyo Electric Biomedical Co., Ltd., Osaka, Japan). After 5 min of
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MATERIALS AND METHODS
for the experiment. Before inoculation, the plastics were inactivated by an autoclave (YXQ-LS-18SI, Shanghai Boxun Industrial Co., Ltd., Shanghai, China) and then air-dried under a biosafety hood (DH-920, Beijing East Union Hall Instrument Manufacturing Co., Ltd., Beijing, China) at a room temperature of 20 ± 2◦ C for 60 min to remove the water. Each plastic piece was inoculated by spreading 0.1 ml onto the front side region of the prepared contaminated culture inoculum. Subsequently, all inoculated plastic pieces were air-dried under a biosafety hood (DH-920, Beijing East Union Hall Instrument Manufacturing Co., Ltd., Beijing, China) for 30 min at room temperature to allow bacterial attachment. The final concentration of S. Enteritidis inoculated on the samples was 6.65 log10 CFU/cm2 on average. The samples for each treatment were prepared at least in duplicate.
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MODELING DISINFECTION OF PLASTIC POULTRY TRANSPORT CAGES Table 1. Codes and levels of factors for the trials. Factors
Symbols Coded
Uncoded
−1.68
Level1 −1
0
1
1.68
Cleaning time (s) Treatment time (s) ACC2
x1 x2 x3
X1 X2 X3
1.6 13.2 16.4
5 20 30
10 30 50
15 40 70
18.4 46.8 84.6
1 2
x1 = (X1 − 10)/5; x2 = (X2 − 30)/10; x3 = (X3– 50)/20. ACC: available chlorine concentration.
neutralization, the surviving bacteria was determined by serial dilutions in sterile 0.1% peptone water, and then 0.1 ml of each dilution was plated onto tryptic soy agar plates in triplicate. The plates were incubated at 37◦ C for 24 h before the colonies were counted. No viable cells in the blank control group were detected in each trial.
A central composite experimental design with 3 independent factors (α = 1.68) was used to study the inactivation of S. Enteritids on the surface of plastic cages. The factors were cleaning time, treatment time, and ACC. Each factor at 5 coded levels (Table 1) was determined using the software design expert (Version 8.0.5, Stat-Ease Inc., Minneapolis, MN). The experiments were designed according to the CCD, as shown in Table 2. The order of the experiments was fully randomized. All treatments were replicated 3 times and the results were reported as means. The response value was expressed as the log reductions between final load after the treatments, and the initial inoculate per inoculated surface area. An analysis of variance and an estimation of response surface were performed using the software
Model Validation In order to validate the adequacy of the inactivation model, 8 sets of additional random experiments were carried out under different conditions (Table 3). The line of correlation (y = x) was made to evaluate the performance of the RS model from the experimental values which were obtained from the 8 experiments.
RESULTS AND DISCUSSION Model Fitting The responses of inactivation of S. Enteritidis on the plastic pieces measured in terms of log reductions obtained from the experiments are shown in Table 2. Response surface analysis demonstrated log reductions ranged from 0.37 log10 CFU/cm2 to 3.12 log10 CFU/cm2 . The experimental data designed by CCD were used to calculate the coefficients of the quadratic equation, and the analysis of variance results for the significance of the coefficients of the models and
Table 2. Observed and predicted reduction of S. Enteritidis on the surface of cages by RS model according to the central composite design. Trials
Cleaning time x1 (s)
Treatment time x2 (s)
ACC1 x3 (mg/l)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
5.00 10.00 10.00 10.00 5.00 10.00 10.00 10.00 10.00 15.00 5.00 5.00 1.59 18.41 10.00 10.00 10.00 15.00 15.00 15.00
40.00 30.00 46.82 30.00 20.00 30.00 30.00 13.18 30.00 20.00 40.00 20.00 30.00 30.00 30.00 30.00 30.00 20.00 40.00 40.00
30.00 50.00 50.00 83.64 30.00 50.00 16.36 50.00 50.00 70.00 70.00 70.00 50.00 50.00 50.00 50.00 50.00 30.00 30.00 70.00
1 2
ACC: available chlorine concentration. Data reported as means ± SD.
Observed value2 (log10 CFU/cm2 )
Predicted value (log10 CFU/cm2 )
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.69 2.42 2.59 3.13 0.13 2.42 0.61 0.62 2.42 2.42 2.86 2.03 1.91 2.59 2.42 2.42 2.42 0.59 2.10 3.21
1.86 2.43 2.36 3.12 0.39 2.43 0.37 0.60 2.46 2.42 3.12 2.02 1.59 2.66 2.42 2.40 2.43 0.51 2.29 3.12
0.07 0.03 0.03 0.03 0.03 0.05 0.07 0.08 0.04 0.05 0.01 0.03 0.04 0.06 0.01 0.03 0.07 0.03 0.09 0.02
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Experimental Design
design expert (Version 8.0.5, Stat-Ease Inc., Minneapolis, MN).
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Table 3. Observed and predicted reduction values of S. Enteritidis on the surface of plastics by RSM under the additional 8 random experimental conditions. Cleaning time x1 (s)
Treatment time x2 (s)
ACC1 x3 (mg/l)
5 5 5 8 8 15 15 15
20 30 40 20 30 30 30 40
30 70 50 30 70 30 50 50
1 2
Observed Value2 (log10 CFU/cm2 )
Predicted value (log10 CFU/cm2 )
± ± ± ± ± ± ± ±
0.13 2.74 2.47 0.32 2.90 1.64 2.56 2.85
0.37 2.76 2.46 0.53 2.92 1.54 2.72 2.86
0.02 0.07 0.11 0.01 0.06 0.07 0.03 0.04
ACC: available chlorine concentration. Data reported as means ± SD.
Table 4. Analysis of variance for response surface quadratic model for reduction of S. Enteritidis on the surface of the cages. Sum of squares
Df1
Mean square
F-value
P value
Cleaning time (x1 ) Treatment time (x2 ) ACC2 (x3 ) Cleaning time × Treatment time (x1 x2 ) Cleaning time × ACC (x1 x3 ) Treatment time × ACC (x2 x3 ) Cleaning time × Cleaning time (x21 ) Treatment time × Treatment time (x22 ) ACC × ACC (x23 )
14.82 0.55 4.70 7.70 0.011 2.812 × 10−4 0.26 0.053 1.20 0.55
9 1 1 1 1 1 1 1 1 1
1.65 0.55 4.70 7.70 0.011 2.812 × 10−4 0.26 0.053 1.20 0.55
37.81 12.71 107.84 176.76 0.023 0.065 6.03 1.21 27.52 12.55
< 0.0001 0.0049 < 0.0001 < 0.0001 0.8819 0.8046 0.0339 0.2978 0.0004 0.0053
1 2
Df: degree of freedom. ACC: available chlorine concentration.
regression coefficient are illustrated in Table 4. The fitted quadratic response model is expressed by Eq. (1):
R = −7.193 + 0.105x1 + 0.279x2 + 0.115x3 − 2.25 −4
−4
Validation of the Model
−3
×10 x1 x2 − 1.875 × 10 x1 x3 − 9.062 × 10 x2 x3 −2.141 ×
10−3 x21
− 2.884 ×
10−3 x22
− 4.869 ×
x21 did not show any significant differences (P > 0.05) (Table 4).
10−4 x23 (1)
where R is the response value in log10 CFU/cm2 ; x1 is the cleaning time in s, x2 is the treatment time in s, and x3 is the ACC in mg/l. The coefficient of determination, R2 , and adjusted determination R2 were 0.971 and 0.946, respectively. The R2 is the proportion of variation in the response attributed to the model rather than to random error. It demonstrates the goodness of fit of the regression equation. Mendenhall (1975) reported that a large value of R2 does not always imply adequacy of the model because additional statistically significant variables may increase R2 . The adjusted R2 was used to evaluate model adequacy. An adjusted R2 higher than 0.90 indicated that no significant terms have been missed by the model (Myers, 1976). Moreover, the quality of fitness models were assessed by a lack of fit test (P > 0.05), which determines model accuracy. The linear coefficients (x1 , x2, and x3 ), the quadratic-term coefficients (x22 and x23 ) and the cross-term coefficients (x2 × 3 ) were significant (P < 0.05). The coefficients of x1 × 2 , x1 ×3 , and
In order to verify the adequacy of the polynomial equations, 8 additional random experimentals were carried out (Table 3). The experimental values were in good agreement with the predicted values with a correlation coefficient (R2 ) of 0.996 and a statistical significance level of P < 0.0001. Therefore, the model was proven to be applicable for predicting the disinfection of S. Enteritidis on the plastic pieces by SAEW with cleaning times in the range of 5 to 15 s, treatment times of 20 to 40 s, and ACC of 30 to 70 mg/l.
Response Surfaces Figures 1–3 illustrate the response surfaces and contour plots showing the effect of cleaning time, treatment time, and ACC of SAEW treatment on the inactivation of S. Enteritidis, respectively. Figure 1 shows the effect of cleaning time (x1 ) and treatment time (x2 ) of SAEW on the reduction of S. Enteritidis at an ACC of 50 mg/l. Treatment time had a positive linear effect and a negative quadratic effect on the reduction of S. Enteritidis (P < 0.0001; P < 0.05). Cleaning time showed a positive linear effect on the response (P < 0.05). The log10 reduction increased with increasing treatment time, which revealed that a longer
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Sources
MODELING DISINFECTION OF PLASTIC POULTRY TRANSPORT CAGES
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Figure 1. Response surface plots showing the effects of cleaning time, x1 , and treatment time, x2 , on the inactivation of S. Enteritidis by SAEW at an available chlorine concentration of 50 mg/l.
treatment time is favorable to obtain a high log10 reduction with a shorter cleaning time. With a larger cleaning time, the log10 reduction also increased when treatment time was increased. The maximum log10 reduction of S. Enteritidis (3.12 log10 CFU/cm2 ) was observed SAEW with an ACC of 50 mg/l, 15 s cleaning time, and 40 s treatment time. In addition, the interaction effect between cleaning time and treatment time was not significant (P > 0.05). Figure 2 gives the response contour plots showing the effect of cleaning time (x1 ) and ACC (x3 ) of SAEW on the inactivation of S. Enteritidis. The ACC showed a positive linear effect and a negative quadratic effect on the reduction of S. Enteritidis (P < 0.0001; P < 0.05). At a lower ACC, log10 reduction slowly increased with an increase in cleaning time. While at a higher ACC, log10 reduction increased more rapidly when cleaning time was increased. Treatment of SAEW at the highest ACC of 70 mg/l and lowest cleaning time of 5 s resulted in a reduction of 2.73 log10 CFU/cm2 for S. Enteritidis (initial population 3.12 log10 CFU/cm2 ). Increasing ACC led to a greater reduction of S. Enteritidis compared to cleaning time, which implies that the ACC may be the more important factor for the bacte-
ricidal activity of SAEW than cleaning time. Moreover, the log10 reductions were not significantly changed (P > 0.05) due to the interactions between ACC and cleaning time. The interaction of treatment time and ACC at a constant cleaning time (10 s) is shown in Figure 3. The ACC had a more significant effect on the bactericidal activity of SAEW than treatment time. This result is consistent with the report of Quan et al. (2010) which indicated that ACC may be the primary factor for the bactericidal activity of SAEW. At a lower ACC, the log10 reduction consistently increased with increasing treatment time, but at a higher ACC, the log10 reduction increased rapidly, and then more slowly with treatment time. In addition, the log10 reductions were changed significantly (P < 0.05) due to the interactions between treatment time and ACC. Allen et al. (2008) reported that soiled transport crates are not easy to clean and disinfect properly because the surfaces of the crates may become scratched and suffer other minor damage during long-term use, which easily traps organic debris and microbes. In this way, transport cages can become a potential risk for the buildup of high loads of pathogenic microorganisms
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Figure 2. Response surface plots showing the effects of cleaning time, x1 , and ACC, x3 , of SAEW on the inactivation of S. Enteritidis at a treatment time of 30 s.
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Figure 3. Response surface plots showing the effects of treatment time, x2 , and ACC, x3 , of SAEW on the inactivation of S. Enteritidis at a cleaning time of 10 s.
In conclusion, the established RS model exhibited a good statistical performance and was suitable for predicting the inhibition of S. Enteritidis on the plastic pieces as a function of cleaning time, treatment time, and ACC. SAEW can inactivate S. Enteritidis on the surface of plastic poultry transport cages. The disinfection activity of SAEW increased with increasing cleaning time, treatment time, and ACC, and the ACC attribute was the most importantfor the bactericidal activity of the SAEW. Moreover, the log10 reductions changed significantly (P < 0.05) with the interactions between treatment time and ACC. These results illustrate the potential of SAEW in the disinfection of bacterial cells on plastic cages, and thus in disinfection measures to control and reduce the transmission risk of the disease.
ACKNOWLEDGMENTS This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-41), the National Natural Science Foundation of China (grant number: 21106179), and Special Fund for Chinese Universities Scientific Fund (2013YJ009).
Competing interests The authors declare that they have no competing interests.
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MODELING DISINFECTION OF PLASTIC POULTRY TRANSPORT CAGES
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Key Laboratory of Structure and Environment in Agricultural Engineering, Ministry of Agriculture, China Agricultural University, Beijing 100083, China; and † Shandong Agricultural University, Shandong 271000, China ite design of the response surface methodology (RSM). The established RS model had a goodness of fit quantified by the parameter R2 (0.971), as well as a lack of fit test (P > 0.05). The maximum reduction of 3.12 log10 CFU/cm2 for S. Enteritidis was obtained for the cage treated with tap water cleaning for 15 s followed by SAEW treatment for 40 s at an ACC of 50 mg/l. Results indicate that the established RS model has shown the potential of SAEW in disinfection of bacteria on cages.
Key words: Slightly acidic electrolyzed water, poultry transport cages, disinfection, S. Enteritidis, modelling 2015 Poultry Science 00:1–7 http://dx.doi.org/10.3382/ps/pev188
INTRODUCTION
tants have been widely used as a preventative measure against bacterial infections in poultry (DeBenedictis et al., 2007), but potentially toxic, corrosive, or volatility problems may thereby arise (Gr¨ aslund and Bengtsson, 2001). Therefore it is crucial to develop a novel disinfectant with high efficacy and which leaves little residue. SAEW produced by electrolysis of a dilute hydrochloric acid and/or sodium chloride solution in a chamber without a membrane, and with a pH value of 5.0 to 6.5, is one of the emerging environment friendly antimicrobial disinfectants (Hricova et al., 2008; Huang et al., 2008). It has been proven to be safe, cheap, and exhibits high bactericidal and fungicidal efficacy (Abadias et al., 2008; Cao et al., 2009). Moreover, SAEW has the advantage of reducing corrosion of surfaces and minimizes of the potential for damage to human health (Abadias et al., 2008). Several studies have demonstrated that SAEW could be used as a disinfectant in poultry production (Huang et al., 2008; Loretz et al., 2010). Cao et al. (2009) indicated that a reduction of 6.5 log10 CFU/g of S. Enteritidis on shell eggs was obtained by SAEW at 15 mg/l available chlorine for 3 min. Hao et al. (2013a) reported that SAEW with an ACC of 300 mg/l resulted in a significant reduction in microbes on the wall, rail, and floor of swine barns after flushing disinfection (P < 0.05). However, little information is available on the disinfection of SAEW for poultry transport cages.
Salmonella Enteritids is an important pathogen of animals and humans and represents a serious public health concern worldwide (Baggesen et al., 1997). Over the years, S. Enteritids has proven to be a very difficult disease to consistently control over time and across farms (CDC, 2002). S. Enteritids can spread from infected farms through a number of routes and that of bacterial entry to the farm is therefore often difficult to ascertain. In addition, extensive efforts to identify the routes of S. Enteritids transmission between layer farms have been made. Many studies have been conducted on the routes of S. Enteritids transmission via infected animals, contaminated materials, insects, and soils (Totton et al., 2012), however, a potential route of S. Enteritids transmission between farms may be through the plastic poultry transport cages used for transport (Racicot et al., 2011, 2012). Thus, it is necessary to reduce the level of pathogenic microorganisms on the poultry cages that enter the farm to avoid chick infection. Disinfection is a generally employed method for preventing the introduction of both endemic and epidemic infections (B¨ ohm 1998; Totton et al., 2012). Several studies have shown that chemical disinfec C 2015 Poultry Science Association Inc. Received March 7, 2015. Accepted May 3, 2015. 1 Corresponding author: [email protected]
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ABSTRACT In order to reduce the risk of enteric pathogens transmission in animal farms, the disinfection effectiveness of slightly acidic electrolyzed water (SAEW, pH 5.85 to 6.53) for inactivating Salmonella Enteritidis on the surface of plastic poultry transport cages was evaluated. The coupled effects of the tap water cleaning time (5 to 15 s), SAEW treatment time (20 to 40 s), and available chlorine concentrations (ACCs) of 30 to 70 mg/l on the reductions of S. Enteritidis on chick cages were investigated using a central compos-
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The RSM has been recognized to be a powerful tool for determining the effects of different factors and the interactions among them (Bas and Boyaci, 2007). The central composite design (CCD) of RSM is the most common experimental design which has been successfully used to determine Salmonella inactivation on drycured ham (Bover-cid et al., 2012), and reduction of Bacillus subtilis (Aouadhi et al., 2013) and vibrio parahaemolyticus on cooked shrimp by acidic electrolyzed water (Wang et al., 2014). The purpose of this work was to evaluate the effectiveness of SAEW to inactive S. Enteritids on the surface of plastic cages under the chicken manure interference circumstance. RSW was used to determine the inactivation of S. Enteritidis by SAEW on plastics as a function of the tap water cleaning time, SAEW treatment time, and ACC.
Bacterial Cultures The strains of Salmonella Enteritidis (CVCC 2184) used were obtained from the China Veterinary Culture Collection (Beijing, China). The strain was transferred into tryptic soy broth (Beijing Land Bridge Technology Company Ltd., Beijing, China) and incubated at 35◦ C for 24 h. Following incubation, a 10 ml culture was pooled into a sterile centrifuge tube and centrifuged at 3000 × g and 4◦ C for 10 min. The supernatant was decanted, and the pellets were resuspended in 10 ml of sterilized 0.85% NaCl solution, washed 3 times, and resuspended in 10 ml of the same solution to obtain a final cell concentration of approximately 107 to 108 CFU/ml. The bacterial population in each culture was confirmed by plating 0.1 ml portions of appropriately diluted culture on tryptic soy agar (Beijing Land Bridge Technology Company Ltd., Beijing, China) plates and incubating the plates at 35◦ C for 24 h.
Inoculation A 6% solution of liquid manure was prepared by the addition of 30 g of chicken manure (obtained from poultry with no bedding) to 0.5 l of sterile distilled water, and then inactivated by an autoclave (YXQ-LS-18SI, Shanghai Boxun Industrial Co., Ltd., Shanghai China). Five ml of the 6% of sterilized liquid manure was shaken and then mixed with equal portions of the prepared culture mixtures to obtain the final populations of contaminated culture of approximately 108 to 109 CFU/ml and 3% concentration (Elassaad et al. 1993) of chicken liquid manure soiling interference in disinfection. The plastics were obtained from a plastic poultry transport cage (High Density Polyethylene materials, 7.35 × 5.45 × 2.60 cm, Shenzhen Lanhai Co., Ltd., Shenzhen, China). The plastic cage was washed with tap water to remove soil, and then trimmed to approximately 1.5 × 1.5 cm2 and packed in a polyethylene bag
Preparation of Slightly Acidic Electrolyzed Water Slightly acidic electrolyzed water was produced using a SAEW generator (Zhouji Biosafety Technology Co., Ltd., Beijing, China) which consists of a non-membrane electrolytic cell with anode and cathode electrodes. SAEW with a pH value of 6.15 to 6.53, an oxidationreduction potential (ORP) of 974 to 989 mV, and different ACCs were prepared for electrolysis of NaCl (10 g/l) solution in the SAEW generator. The physicochemical properties of SAEW were measured before use. The pH and ORP values were measured using a dual scale pH/ORP meter (CON60, Trans-Wiggens, Singapore) with a pH electrode (PE02; range, 0.00 to 14.00) and an ORP electrode (ORP06; range, −999 to +999 mV). The ACC was determined using a digital chlorine test kit (RC-2Z, Kasahara Chemical Instruments Co., Saitama, Japan). The detection range was 0 to 320 mg/l. The solutions were placed into an atmospheric pressure manual sprayer purchased from a supermarket which was used to treat the sample.
Treatment of Samples with SAEW and Microbiological Determination Inoculated or non-inoculated plastic pieces were first sprayed with tap water by an atmospheric pressure manual sprayer to clean them, and then sprayed with SAEW to disinfect them under different conditions. The non-inoculated plastic pieces were used as the control group. After treatment, moistened sterile swabs with a neutralizing agent (0.1% Na2 S2 O3 ) were used to collect surface samples from the plastic pieces. The sterilized cotton swabs, which had been wiped back and forth twenty times on the sample surfaces, were immediately transferred into 5 ml neutralizing agent tubes for microbiological analyses. The tubes were shaken on a platform shaker at 1800 rpm (MIR-S100, Sanyo Electric Biomedical Co., Ltd., Osaka, Japan). After 5 min of
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MATERIALS AND METHODS
for the experiment. Before inoculation, the plastics were inactivated by an autoclave (YXQ-LS-18SI, Shanghai Boxun Industrial Co., Ltd., Shanghai, China) and then air-dried under a biosafety hood (DH-920, Beijing East Union Hall Instrument Manufacturing Co., Ltd., Beijing, China) at a room temperature of 20 ± 2◦ C for 60 min to remove the water. Each plastic piece was inoculated by spreading 0.1 ml onto the front side region of the prepared contaminated culture inoculum. Subsequently, all inoculated plastic pieces were air-dried under a biosafety hood (DH-920, Beijing East Union Hall Instrument Manufacturing Co., Ltd., Beijing, China) for 30 min at room temperature to allow bacterial attachment. The final concentration of S. Enteritidis inoculated on the samples was 6.65 log10 CFU/cm2 on average. The samples for each treatment were prepared at least in duplicate.
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MODELING DISINFECTION OF PLASTIC POULTRY TRANSPORT CAGES Table 1. Codes and levels of factors for the trials. Factors
Symbols Coded
Uncoded
−1.68
Level1 −1
0
1
1.68
Cleaning time (s) Treatment time (s) ACC2
x1 x2 x3
X1 X2 X3
1.6 13.2 16.4
5 20 30
10 30 50
15 40 70
18.4 46.8 84.6
1 2
x1 = (X1 − 10)/5; x2 = (X2 − 30)/10; x3 = (X3– 50)/20. ACC: available chlorine concentration.
neutralization, the surviving bacteria was determined by serial dilutions in sterile 0.1% peptone water, and then 0.1 ml of each dilution was plated onto tryptic soy agar plates in triplicate. The plates were incubated at 37◦ C for 24 h before the colonies were counted. No viable cells in the blank control group were detected in each trial.
A central composite experimental design with 3 independent factors (α = 1.68) was used to study the inactivation of S. Enteritids on the surface of plastic cages. The factors were cleaning time, treatment time, and ACC. Each factor at 5 coded levels (Table 1) was determined using the software design expert (Version 8.0.5, Stat-Ease Inc., Minneapolis, MN). The experiments were designed according to the CCD, as shown in Table 2. The order of the experiments was fully randomized. All treatments were replicated 3 times and the results were reported as means. The response value was expressed as the log reductions between final load after the treatments, and the initial inoculate per inoculated surface area. An analysis of variance and an estimation of response surface were performed using the software
Model Validation In order to validate the adequacy of the inactivation model, 8 sets of additional random experiments were carried out under different conditions (Table 3). The line of correlation (y = x) was made to evaluate the performance of the RS model from the experimental values which were obtained from the 8 experiments.
RESULTS AND DISCUSSION Model Fitting The responses of inactivation of S. Enteritidis on the plastic pieces measured in terms of log reductions obtained from the experiments are shown in Table 2. Response surface analysis demonstrated log reductions ranged from 0.37 log10 CFU/cm2 to 3.12 log10 CFU/cm2 . The experimental data designed by CCD were used to calculate the coefficients of the quadratic equation, and the analysis of variance results for the significance of the coefficients of the models and
Table 2. Observed and predicted reduction of S. Enteritidis on the surface of cages by RS model according to the central composite design. Trials
Cleaning time x1 (s)
Treatment time x2 (s)
ACC1 x3 (mg/l)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
5.00 10.00 10.00 10.00 5.00 10.00 10.00 10.00 10.00 15.00 5.00 5.00 1.59 18.41 10.00 10.00 10.00 15.00 15.00 15.00
40.00 30.00 46.82 30.00 20.00 30.00 30.00 13.18 30.00 20.00 40.00 20.00 30.00 30.00 30.00 30.00 30.00 20.00 40.00 40.00
30.00 50.00 50.00 83.64 30.00 50.00 16.36 50.00 50.00 70.00 70.00 70.00 50.00 50.00 50.00 50.00 50.00 30.00 30.00 70.00
1 2
ACC: available chlorine concentration. Data reported as means ± SD.
Observed value2 (log10 CFU/cm2 )
Predicted value (log10 CFU/cm2 )
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.69 2.42 2.59 3.13 0.13 2.42 0.61 0.62 2.42 2.42 2.86 2.03 1.91 2.59 2.42 2.42 2.42 0.59 2.10 3.21
1.86 2.43 2.36 3.12 0.39 2.43 0.37 0.60 2.46 2.42 3.12 2.02 1.59 2.66 2.42 2.40 2.43 0.51 2.29 3.12
0.07 0.03 0.03 0.03 0.03 0.05 0.07 0.08 0.04 0.05 0.01 0.03 0.04 0.06 0.01 0.03 0.07 0.03 0.09 0.02
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Experimental Design
design expert (Version 8.0.5, Stat-Ease Inc., Minneapolis, MN).
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Table 3. Observed and predicted reduction values of S. Enteritidis on the surface of plastics by RSM under the additional 8 random experimental conditions. Cleaning time x1 (s)
Treatment time x2 (s)
ACC1 x3 (mg/l)
5 5 5 8 8 15 15 15
20 30 40 20 30 30 30 40
30 70 50 30 70 30 50 50
1 2
Observed Value2 (log10 CFU/cm2 )
Predicted value (log10 CFU/cm2 )
± ± ± ± ± ± ± ±
0.13 2.74 2.47 0.32 2.90 1.64 2.56 2.85
0.37 2.76 2.46 0.53 2.92 1.54 2.72 2.86
0.02 0.07 0.11 0.01 0.06 0.07 0.03 0.04
ACC: available chlorine concentration. Data reported as means ± SD.
Table 4. Analysis of variance for response surface quadratic model for reduction of S. Enteritidis on the surface of the cages. Sum of squares
Df1
Mean square
F-value
P value
Cleaning time (x1 ) Treatment time (x2 ) ACC2 (x3 ) Cleaning time × Treatment time (x1 x2 ) Cleaning time × ACC (x1 x3 ) Treatment time × ACC (x2 x3 ) Cleaning time × Cleaning time (x21 ) Treatment time × Treatment time (x22 ) ACC × ACC (x23 )
14.82 0.55 4.70 7.70 0.011 2.812 × 10−4 0.26 0.053 1.20 0.55
9 1 1 1 1 1 1 1 1 1
1.65 0.55 4.70 7.70 0.011 2.812 × 10−4 0.26 0.053 1.20 0.55
37.81 12.71 107.84 176.76 0.023 0.065 6.03 1.21 27.52 12.55
< 0.0001 0.0049 < 0.0001 < 0.0001 0.8819 0.8046 0.0339 0.2978 0.0004 0.0053
1 2
Df: degree of freedom. ACC: available chlorine concentration.
regression coefficient are illustrated in Table 4. The fitted quadratic response model is expressed by Eq. (1):
R = −7.193 + 0.105x1 + 0.279x2 + 0.115x3 − 2.25 −4
−4
Validation of the Model
−3
×10 x1 x2 − 1.875 × 10 x1 x3 − 9.062 × 10 x2 x3 −2.141 ×
10−3 x21
− 2.884 ×
10−3 x22
− 4.869 ×
x21 did not show any significant differences (P > 0.05) (Table 4).
10−4 x23 (1)
where R is the response value in log10 CFU/cm2 ; x1 is the cleaning time in s, x2 is the treatment time in s, and x3 is the ACC in mg/l. The coefficient of determination, R2 , and adjusted determination R2 were 0.971 and 0.946, respectively. The R2 is the proportion of variation in the response attributed to the model rather than to random error. It demonstrates the goodness of fit of the regression equation. Mendenhall (1975) reported that a large value of R2 does not always imply adequacy of the model because additional statistically significant variables may increase R2 . The adjusted R2 was used to evaluate model adequacy. An adjusted R2 higher than 0.90 indicated that no significant terms have been missed by the model (Myers, 1976). Moreover, the quality of fitness models were assessed by a lack of fit test (P > 0.05), which determines model accuracy. The linear coefficients (x1 , x2, and x3 ), the quadratic-term coefficients (x22 and x23 ) and the cross-term coefficients (x2 × 3 ) were significant (P < 0.05). The coefficients of x1 × 2 , x1 ×3 , and
In order to verify the adequacy of the polynomial equations, 8 additional random experimentals were carried out (Table 3). The experimental values were in good agreement with the predicted values with a correlation coefficient (R2 ) of 0.996 and a statistical significance level of P < 0.0001. Therefore, the model was proven to be applicable for predicting the disinfection of S. Enteritidis on the plastic pieces by SAEW with cleaning times in the range of 5 to 15 s, treatment times of 20 to 40 s, and ACC of 30 to 70 mg/l.
Response Surfaces Figures 1–3 illustrate the response surfaces and contour plots showing the effect of cleaning time, treatment time, and ACC of SAEW treatment on the inactivation of S. Enteritidis, respectively. Figure 1 shows the effect of cleaning time (x1 ) and treatment time (x2 ) of SAEW on the reduction of S. Enteritidis at an ACC of 50 mg/l. Treatment time had a positive linear effect and a negative quadratic effect on the reduction of S. Enteritidis (P < 0.0001; P < 0.05). Cleaning time showed a positive linear effect on the response (P < 0.05). The log10 reduction increased with increasing treatment time, which revealed that a longer
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Sources
MODELING DISINFECTION OF PLASTIC POULTRY TRANSPORT CAGES
5
Figure 1. Response surface plots showing the effects of cleaning time, x1 , and treatment time, x2 , on the inactivation of S. Enteritidis by SAEW at an available chlorine concentration of 50 mg/l.
treatment time is favorable to obtain a high log10 reduction with a shorter cleaning time. With a larger cleaning time, the log10 reduction also increased when treatment time was increased. The maximum log10 reduction of S. Enteritidis (3.12 log10 CFU/cm2 ) was observed SAEW with an ACC of 50 mg/l, 15 s cleaning time, and 40 s treatment time. In addition, the interaction effect between cleaning time and treatment time was not significant (P > 0.05). Figure 2 gives the response contour plots showing the effect of cleaning time (x1 ) and ACC (x3 ) of SAEW on the inactivation of S. Enteritidis. The ACC showed a positive linear effect and a negative quadratic effect on the reduction of S. Enteritidis (P < 0.0001; P < 0.05). At a lower ACC, log10 reduction slowly increased with an increase in cleaning time. While at a higher ACC, log10 reduction increased more rapidly when cleaning time was increased. Treatment of SAEW at the highest ACC of 70 mg/l and lowest cleaning time of 5 s resulted in a reduction of 2.73 log10 CFU/cm2 for S. Enteritidis (initial population 3.12 log10 CFU/cm2 ). Increasing ACC led to a greater reduction of S. Enteritidis compared to cleaning time, which implies that the ACC may be the more important factor for the bacte-
ricidal activity of SAEW than cleaning time. Moreover, the log10 reductions were not significantly changed (P > 0.05) due to the interactions between ACC and cleaning time. The interaction of treatment time and ACC at a constant cleaning time (10 s) is shown in Figure 3. The ACC had a more significant effect on the bactericidal activity of SAEW than treatment time. This result is consistent with the report of Quan et al. (2010) which indicated that ACC may be the primary factor for the bactericidal activity of SAEW. At a lower ACC, the log10 reduction consistently increased with increasing treatment time, but at a higher ACC, the log10 reduction increased rapidly, and then more slowly with treatment time. In addition, the log10 reductions were changed significantly (P < 0.05) due to the interactions between treatment time and ACC. Allen et al. (2008) reported that soiled transport crates are not easy to clean and disinfect properly because the surfaces of the crates may become scratched and suffer other minor damage during long-term use, which easily traps organic debris and microbes. In this way, transport cages can become a potential risk for the buildup of high loads of pathogenic microorganisms
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Figure 2. Response surface plots showing the effects of cleaning time, x1 , and ACC, x3 , of SAEW on the inactivation of S. Enteritidis at a treatment time of 30 s.
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ZANG ET AL.
Figure 3. Response surface plots showing the effects of treatment time, x2 , and ACC, x3 , of SAEW on the inactivation of S. Enteritidis at a cleaning time of 10 s.
In conclusion, the established RS model exhibited a good statistical performance and was suitable for predicting the inhibition of S. Enteritidis on the plastic pieces as a function of cleaning time, treatment time, and ACC. SAEW can inactivate S. Enteritidis on the surface of plastic poultry transport cages. The disinfection activity of SAEW increased with increasing cleaning time, treatment time, and ACC, and the ACC attribute was the most importantfor the bactericidal activity of the SAEW. Moreover, the log10 reductions changed significantly (P < 0.05) with the interactions between treatment time and ACC. These results illustrate the potential of SAEW in the disinfection of bacterial cells on plastic cages, and thus in disinfection measures to control and reduce the transmission risk of the disease.
ACKNOWLEDGMENTS This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-41), the National Natural Science Foundation of China (grant number: 21106179), and Special Fund for Chinese Universities Scientific Fund (2013YJ009).
Competing interests The authors declare that they have no competing interests.
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and their biofilms (Bailey et al., 2001). Some work has been done on the sanitizing of transport cages, including the use of disposable crate liners (Slader et al., 2002), and drying the crates as a means of lowering Campylobacter numbers (Berrang & Northcutt, 2005). The performance of different cleaning treatments in reducing microbial contamination of poultry transport crates was evaluated by Allen et al. (2008). The use of Virkon S disinfectant, or ultrasound, after a combination of soaking, brushing, and washing are likely to be the most effective treatments to against Campylobacter on the surfaces of poultry transport crates. Ramesh et al. (2003) also reported that transport cages with galvanised frames and fibreglass floors immersed for 2 min in 1000 mg/l of sodium hypochlorite at 70◦ C resulted in reductions in bacterial numbers of 4 to 8 log10 CFU per unit sampled. However, the high levels of chlorine have not been deemed acceptable because of the chemical residues and adverse environmental impacts. SAEW, with a pH of 5.0 to 6.5 and containing a high concentration of hypochlorous acid (approximately 95%), generated by electrolysis of dilute hydrochloric acid and/or NaCl solution in a non-membrane electrolytic chamber is effective, convenient, and less expensive. Due to a near-neutral pH, SAEW is more applicable to poultry farms as it results in less corrosion of facilities and irritation to the hands of farm workers. Additionally, the present work shows that SAEW is very effective for inhibiting S. Enteritidis on the surface of plastic cages. Other studies have also illustrated a significant reduction of bacteria on the poultry housing facilities by SAEW. Hao et al. (2013b) reported that SAEW with an ACC of 250 mg/l can significantly (P < 0.05) reduce the microbes on the walls, floors, feed troughs, and water pipes in layer house. Guentzel et al. (2008) found that at an equivalent ACC, SAEW kills microorganisms more quickly than other chemical products such as sodium hypochlorite. Therefore, SAEW can be consider to be a potential disinfectant for bacterial cells on plastic cages.
MODELING DISINFECTION OF PLASTIC POULTRY TRANSPORT CAGES
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