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An evaluation of the effect of sodium bisulfate as a feed additive on Salmonella enterica serotype Enteritidis in experimentally infected broilers
An evaluation of the effect of sodium bisulfate as a feed additive on Salmonella enterica serotype Enteritidis in experimentally infected broilers I. I. Kassem,* Y. M. Sanad,* R. Stonerock,† and G. Rajashekara*1 *Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster 44691; and †Kalmbach Feeds, 7148 St. Hwy. 199, Upper Sandusky, OH 43351 ABSTRACT The colonization of broiler chickens with Salmonella can pose serious health and economic risks for both consumers and the poultry industry. Because colonization with Salmonella can lead to subsequent contamination of chicken carcasses during processing, preemptive control measures should include the reduction of this pathogen in chickens before slaughter. In this study, we evaluated the effect of sodium bisulfate, a potential antimicrobial feed additive, on Salmonella colonization of experimentally infected broiler chickens. Two hundred and forty 1-d-old chickens were infected orally with Salmonella enterica serotype Enteritidis and divided into 4 groups (each comprised of 60 chickens). Three groups received different concentrations of sodium bisulfate integrated into their feed, while the feed of
the fourth group (positive control) was not treated. At time points before the broilers’ slaughter age, different organs/tissues (liver, spleen, cecum, and bone marrow) and feces were aseptically collected and tested for the occurrence and density of Salmonella Enteritidis. Our results show that at 3 d postinfection, high colonization with Salmonella Enteritidis was detected and affected all tested tissues and fecal samples. Although colonization decreased across time, Salmonella Enteritidis persisted in the cecum, feces, spleen, and bone marrow, but not in the liver, until slaughter age. Furthermore, the addition of sodium bisulfate to the feed did not significantly reduce Salmonella Enteritidis numbers in infected chickens or affect the shedding of the pathogen.
Key words: Salmonella, sodium bisulfate, broiler, internal organ, feed additive 2012 Poultry Science 91:1032–1037 http://dx.doi.org/10.3382/ps.2011-01935
INTRODUCTION Food-borne nontyphoidal salmonellosis affects approximately 1.0 million individuals, resulting in 378 deaths yearly in the United States (Scallan et al., 2011). Furthermore, Salmonella-associated illnesses are estimated to cost the United States around $2.4 billion annually, while Salmonella contamination of food causes additional financial burdens, such as expenditures associated with decontamination of slaughter plants and recalling of contaminated product (Mainali et al., 2009). Of interest is the contamination of broiler carcasses with Salmonella during slaughter and subsequent processing (Vandeplas et al., 2010; Hoelzer et al., 2011), which poses significant concerns for consumers and the poultry industry. For example, Salmonella in intestinal contents can be transmitted to poultry carcasses during evisceration and associated rupturing of ©2012 Poultry Science Association Inc. Received October 11, 2011. Accepted December 18, 2011. 1 Corresponding author: [email protected]
ceca (Rasschaert et al., 2008). Further, evisceration could also cross contaminate edible internal organs (offals) that might not be readily accessible for decontamination (Arroyo and Arroyo, 1995). Although offals are mostly believed to acquire Salmonella via cross contamination (Arroyo and Arroyo, 1995) during processing, it is conceivable that Salmonella colonizes these organs early in the life cycle of the broilers, possibly persisting to slaughter age (Gast and Holt, 1998). Therefore, there is a need to better understand the persistence and transmission dynamics of Salmonella in broiler chicken internal organs, including edible parts (offals), in order to evaluate additional approaches to control Salmonella contamination of chicken carcasses. For the latter, it is thought that reducing Salmonella colonization before slaughter might constitute a judicial practice. To achieve this, several strategies have been suggested, including the use of feed additives to reduce the pathogen before harvest and processing. Of particular interest is sodium bisulfate (NaHSO4), an acid salt, used as an agent for primarily lowering the pH of a variety of matrices. This compound is also added as a supplement to treat farm-animal litter, where it exhibits a consid-
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EFFECT OF SODIUM BISULFATE ON SALMONELLA IN BROILERS
erable antibacterial activity, resulting in 2 to 3 logs reduction in the litter’s bacterial populations (Pope and Cherry, 2000). Ruiz-Feria et al. (2011) observed that litter of broilers that were on a diet supplemented with sodium bisulfate contained lower levels of Salmonella, while the productive performance of the chickens was higher than those receiving untreated feed. Additionally, Ruiz-Feria et al. (2011) also reported that the effect of sodium bisulfate on Salmonella shedding was inconsistent, whereas the effect of the chemical on Salmonella colonization of the chicken’s gut was not investigated. This is important given that Line (2002) showed that amending the litter with sodium bisulfate did not result in favorable negative effects on Salmonella colonization of the chicken ceca. A marked difference between the aforementioned studies is that the sodium bisulfate was used by Ruiz-Feria et al. (2011) as a feed additive, whereas Line (2002) used the chemical to treat litter. To resolve these conflicting reports, we monitored the liver, spleen, cecum, feces, and bone marrow of experimentally infected broiler chickens and evaluated the effect of sodium bisulfate, as an antimicrobial feed additive, on Salmonella colonization of these chicken organs.
MATERIALS AND METHODS Experimental Design and Monitoring Before the onset of the experiment, twelve 1-d-old chickens were examined to further confirm that the specific pathogen-free flock was not precolonized with Salmonella. The 1-d-old chickens were euthanized and samples (liver, spleen, cecum, feces, and bone marrow) were aseptically harvested, suspended in buffered peptone water, and homogenized. The suspensions (100 μL) were then spread on XLT-4 agar (BD Diagnostics, Franklin Lakes, NJ), a medium that is selective for Salmonella, while 1 mL of the suspension was enriched in tetrathionate broth base with iodine-potassium iodide solution (TBB), as described by the manufacturer (BD Diagnostics), to detect if the bacterium occurred at lower densities. Inocula of TBB enrichments that exhibited positive growth were transferred to XLT-4 agar to determine the occurrence of Salmonella Enteritidis. The XLT-4 agar plates and TBB enrichments were incubated overnight at 37°C under aerobic conditions. To increase the selectivity for retrieving Salmonella Enteritidis from the infected chickens and further limit the growth of other bacterial contaminants, XLT-4 agar plates were supplemented with novobiocin and nalidixic acid, as described elsewhere (Gast and Holt, 1998). Subsequently, two hundred and forty 1-dold chickens were orally inoculated with 2 × 105 cfu of nalidixic acid-resistant Salmonella Enteritidis (Gast and Holt, 1998). The chickens were then divided into 4 groups of 60 birds each and the birds representing each group were evenly distributed to 5 cages (i.e., 12
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chickens per cage). Chickens from each group were also placed into different pens. To assess the effect of sodium bisulfate on Salmonella Enteritidis colonization, the chickens in group 1 received ad libitum feed that was supplemented with ~4.5 kg/t (low dose; treatment 1) of sodium bisulfate. Similarly, 2 experimental setups were used to test 7 and 9 kg/t of sodium bisulfate in feed, which corresponded to medium (treatment 2 for group 2) and high doses (treatment 3 for group 3), respectively. A control group with untreated feed (treatment 4 for group 4) was also setup to determine the progression of Salmonella Enteritidis colonization in the broiler chickens in the absence of sodium bisulfate. Three days postinfection, 10 chickens (2 from each cage) from each treatment were killed and samples (spleen, liver, bone marrow, cecum, feces) were analyzed for Salmonella Enteritidis. This was repeated on d 7 postinoculation and subsequently weekly for a period of 4 additional weeks, which spanned the life of the broiler flock from hatching to slaughter. Collectively, a total of 1,200 samples was monitored for Salmonella Enteritidis throughout the experiment. Animal experiments were conducted according to the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Throughout the experiment, chickens received the same type of feed, a commercially available crumble (Kalmbach Feeds, Upper Sandusky, OH) that contained approximately 22% CP, 2% crude fat, 5% crude fiber, 0.7 to 1.2% calcium, 0.65% phosphorus, 0.2 to 0.7% NaCl, 0.5% methionine, and 1.1% lysine. Other important parameters, such as average feed intake, average gain, feed conversion ratio, and mortality (Table 1), were collected, whereas the temperature of the pens was monitored daily to limit possible variations to the experimental design.
Statistical Analysis Counts of Salmonella cfu were presented as means ± SE. The means were determined by averaging the cfu counts from each organ samples (n = 10) per treatment. Data were transformed (log10) and analyzed using oneway ANOVA, followed by Tukey’s post hoc significance test. A P-value of <0.05 was used to indicate whether differences were statistically significant.
RESULTS AND DISCUSSION All of the organs/tissues retrieved from randomly selected 1-d-old chickens were negative for Salmonella, which confirmed that the specific pathogen-free flock used in this study was likely free of this pathogen. Subsequently, our postinfection analysis showed that as early as d 3 postinoculation, the chickens in all groups were colonized with high numbers of Salmonella Enteritidis, which infected every chicken and organ/ tissue tested, with the exception of only 2 bone marrow samples that belonged to chickens receiving the
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Table 1. Feed conversion and mortality of chickens treated with different doses of sodium bisulfate Time (wk)
Parameter
1
Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Mortality (%)
2 3 4 5 Overall
(g) (g) (g) (g) (g)
Treatment 1 (low dose)
Treatment 2 (medium dose)
Treatment 3 (high dose)
Treatment 4 (no dose)
13.4 29.1 2.1 31.8 45.2 1.4 47.7 78.2 1.6 68.6 115.7 1.6 72.7 138.3 1.9 5
13.3 29.3 2.2 37.4 49.2 1.3 49.6 85.1 1.7 70.4 123.8 1.7 83.3 147.0 1.7 3.34
13.6 29.1 2.1 34.5 50.5 1.4 54.3 87.2 1.6 67.9 116.6 1.7 48.6 121.3* 2.4* 0
13.1 20.6* 1.5* 31.2 45.3 1.4 47.9 82 1.7 71.3 120.3 1.6 49.8 104.2 2 0.6
*Indicates statistically different numbers (P < 0.05).
Figure 1. A. Enumeration of Salmonella Enteritidis cfu retrieved from liver samples across time. B. Enumeration of Salmonella Enteritidis cfu retrieved from spleen samples across time. Treatments 1, 2, and 3 correspond to low, medium, and high doses of sodium bisulfate, respectively. Treatment 4 is the control that did not receive any sodium bisulfate. Data were represented as means ± SE. n = 40 samples/time point. *Indicates significantly different numbers as compared with the control (P < 0.05).
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Figure 2. A. Enumeration of Salmonella Enteritidis cfu retrieved from feces across time. B. Enumeration of Salmonella Enteritidis cfu retrieved from ceca across time. Treatments 1, 2, and 3 correspond to low, medium, and high doses of sodium bisulfate, respectively. Treatment 4 is the control that did not receive any sodium bisulfate. Data were represented as means ± SE. n = 40 samples/time point. *Indicates significantly different numbers as compared with the control (P < 0.05).
sodium bisulfate regimens (Figures 1 and 2; Table 2). Overall, the addition of sodium bisulfate to the feed did not result in significant reductions (P < 0.05) in Salmonella Enteritidis colonization as compared with the control. Interestingly, some samples collected from treated chickens appeared to harbor either comparable or statistically higher numbers (P < 0.05) of Salmonella Enteritidis (Figures 1 and 2). For example, at wk 5 postinfection, (2.8 ± 1.5) × 106, (2.1 ± 1.5) × 105, (14.5 ± 6.5) × 105 cfu/g of ceca were detected in the groups that received low, medium, and high doses of sodium bisulfate, respectively, as opposed to (4.4 ± 2.5) × 105 cfu/g of ceca retrieved from the control group (Figure 2A). Similarly, (8 ± 3.3) × 105, (4 ± 3.9) × 105, (11 ± 6) × 103 cfu/g of feces was observed in treated samples as compared with (8.6 ± 6.2) × 103 cfu/g of feces collected from the control group (Figure 2B). Although at specific time points, the addition of sodium bisulfate to the feed did seemingly result in reductions in Salmonella Enteritidis numbers, these decreases were not consistent and did not follow a specific trend. For example, cecal and fecal samples collected at
d 20 postinfection from chickens receiving low and medium doses of sodium bisulfate harbored a significantly lower Salmonella Enteritidis number as compared with that from the control group (Figure 2). However, at d 27 and 34 postinfection, the Salmonella Enteritidis numbers retrieved from cecal and fecal samples subjected to the aforementioned treatments were either statistically not different or significantly higher than those from the control group (Figure 2). A similar observation was noted with Salmonella Enteritidis numbers recorded for liver and spleen samples, although the decrease in Salmonella Enteritidis numbers associated with low and medium doses of sodium bisulfate was observed at d 14 postinfection (Figure 1). This transient decrease in Salmonella Enteritidis numbers in infected organs might explain the aforementioned observations of Ruiz-Feria et al. (2011), who reported that the effect of sodium bisulfate on Salmonella shedding was inconsistent. However, the precise reasons behind the absence of a consistent effect on Salmonella colonization after treatment are not clear. Yet, because the effect of sodium bisulfate is not specific to Salmonella, it
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Table 2. The number of Salmonella Enteritidis-positive samples after enrichment in iodine-potassium iodide solution and subsequent inoculation onto XLT-4 agar1 Time postinfection (d) Sample Liver
Spleen
Cecum
Feces
Bone marrow
Total samples processed
Treatment2
3
7
13
20
27
34
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 All treatments
10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 9/10 10/10 10/10 200/00
10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 8/10 10/10 10/10 200/00
10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 6/10 7/10 7/10 7/10 200/00
7/10 7/10 9/10 8/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 2/10 2/10 6/10 4/10 200/00
9/10 4/10 5/10 3/10 8/10 9/10 7/10 9/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 6/10 3/10 2/10 5/10 200/00
5/10 3/10 5/10 0/10 5/10 8/10 9/10 6/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 9/10 0/10 4/10 0/10 2/10 200/00
1In
total, 1,200 samples were processed throughout the experiment. 1, 2, and 3 corresponded to low, medium, and high doses of sodium bisulfate, respectively. Treatment 4 was the control that did not receive any sodium bisulfate. 2Treatments
is conceivable that this compound might have affected normal flora in the chickens’ gut, perhaps reducing the organisms that might otherwise compete with Salmonella to occupy these niches. Alternatively, it is possible that Salmonella Enteritidis might have adapted to potential decreases in pH that were induced by sodium bisulfate. This is plausible becasue it has been shown that exposure of Salmonella cells to mild acidic conditions can lead to adaptation to acid and other stresses (Leyer and Johnson, 1992, 1993). Regardless of the reasons, it should be further noted that the occurrence of Salmonella in the samples after enrichment was also not affected by the addition of sodium bisulfate (Table 2). Analyzing samples collected from the control group lead to a better understanding of the occurrence and persistence of Salmonella Enteritidis in broiler chickens’ internal organs. As expected, fecal and cecal samples harbored the highest numbers of Salmonella Enteritidis at all time points. Furthermore, until d 20 postinfection, the numbers of Salmonella Enteritidis in feces and ceca of the chickens in the control group were significantly higher than that of the original oral inocula (Figure 2). As compared with the first 20 d, Salmonella Enteritidis densities in the feces and ceca from control samples decreased significantly by wk 5 but remained in considerable quantities that might pose a risk (Figure 2). It was notable that Salmonella Enteritidis could not be detected without pre-enrichment at d 20 and 27 postinfection in the liver (an offal) samples of the control group, whereas at d 34 it appears that the bacterium was absent in this organ (Table 2). Additionally, after enrichment, 20 and 60% of bone marrow (an offal) and the spleen samples, respectively, from the control
group were found to harbor Salmonella Enteritidis until d 34. Taken together, our data further confirm the importance of internal organs as potential sources for cross contamination during slaughter and processing. In conclusion, at the concentrations used in this study, feed supplemented with sodium bisulfate did not consistently reduce Salmonella Enteritidis colonization. Because Salmonella can colonize the broilers at any point in their life cycle and its spread to internal organs can occur rapidly, we suggest that dissemination of internal chicken organs consumed by humans or prepared as pet food should be accompanied by recommendations for appropriate handling and cooking.
ACKNOWLEDGMENTS This work was supported by the Ohio Agricultural Research and Development Center (OARDC), The Ohio State University (Wooster, OH), and Kalmbach Feeds Inc. (Upper Sandusky, Ohio).
REFERENCES Arroyo, G., and J. A. Arroyo. 1995. Detection of Salmonella serotypes in edible organ meats from markets in Madrid, Spain. Food Microbiol. 12:13–20. Gast, R. K., and P. S. Holt. 1998. Persistence of Salmonella Enteritidis from one day of age until maturity in experimentally infected layer chickens. Poult. Sci. 77:1759–1762. Hoelzer, K., A. I. Moreno Switt, and M. Wiedmann. 2011. Animal contact as a source of human non-typhoidal salmonellosis. Vet. Res. 42:34. Leyer, G. J., and E. A. Johnson. 1992. Acid adaptation promotes survival of Salmonella spp. in cheese. Appl. Environ. Microbiol. 58:2075–2080.
EFFECT OF SODIUM BISULFATE ON SALMONELLA IN BROILERS Leyer, G. J., and E. A. Johnson. 1993. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbiol. 59:1842–1847. Line, J. E. 2002. Campylobacter and Salmonella populations associated with chickens raised on acidified litter. Poult. Sci. 81:1473– 1477. Mainali, C., G. Gensler, M. McFall, R. King, R. Irwin, and A. Senthilselvan. 2009. Evaluation of associations between feed withdrawal and other management factors with Salmonella contamination of broiler chickens at slaughter in Alberta. J. Food Prot. 72:2202–2207. Pope, M. J., and T. E. Cherry. 2000. An evaluation of the presence of pathogens on broilers raised on Poultry Litter Treatmenttreated litter. Poult. Sci. 79:1351–1355. Rasschaert, G., K. Houf, C. Godard, C. Wildemauwe, M. Pastuszczak-Frak, and L. De Zutter. 2008. Contamination of carcasses
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with Salmonella during poultry slaughter. J. Food Prot. 71:146– 152. Ruiz-Feria, C. A., E. Larrison, M. Davis, M. Farnell, J. Carey, and J. Pitts. 2011. Productive performance and litter microbiology of broiler chickens fed sodium bisulfate. 2011 Int. Poult. Sci. For 2011:25 (Abstr.) Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M. A. Widdowson, S. L. Roy, J. L. Jones, and P. M. Griffin. 2011. Foodborne illness acquired in the United States—Major pathogens. Emerg. Infect. Dis. 7:7–15. Vandeplas, S., R. Dubois Dauphin, Y. Beckers, P. Thonart, and A. Théwis. 2010. Salmonella in chicken: Current and developing strategies to reduce contamination at farm level. J. Food Prot. 73:774–785.
the fourth group (positive control) was not treated. At time points before the broilers’ slaughter age, different organs/tissues (liver, spleen, cecum, and bone marrow) and feces were aseptically collected and tested for the occurrence and density of Salmonella Enteritidis. Our results show that at 3 d postinfection, high colonization with Salmonella Enteritidis was detected and affected all tested tissues and fecal samples. Although colonization decreased across time, Salmonella Enteritidis persisted in the cecum, feces, spleen, and bone marrow, but not in the liver, until slaughter age. Furthermore, the addition of sodium bisulfate to the feed did not significantly reduce Salmonella Enteritidis numbers in infected chickens or affect the shedding of the pathogen.
Key words: Salmonella, sodium bisulfate, broiler, internal organ, feed additive 2012 Poultry Science 91:1032–1037 http://dx.doi.org/10.3382/ps.2011-01935
INTRODUCTION Food-borne nontyphoidal salmonellosis affects approximately 1.0 million individuals, resulting in 378 deaths yearly in the United States (Scallan et al., 2011). Furthermore, Salmonella-associated illnesses are estimated to cost the United States around $2.4 billion annually, while Salmonella contamination of food causes additional financial burdens, such as expenditures associated with decontamination of slaughter plants and recalling of contaminated product (Mainali et al., 2009). Of interest is the contamination of broiler carcasses with Salmonella during slaughter and subsequent processing (Vandeplas et al., 2010; Hoelzer et al., 2011), which poses significant concerns for consumers and the poultry industry. For example, Salmonella in intestinal contents can be transmitted to poultry carcasses during evisceration and associated rupturing of ©2012 Poultry Science Association Inc. Received October 11, 2011. Accepted December 18, 2011. 1 Corresponding author: [email protected]
ceca (Rasschaert et al., 2008). Further, evisceration could also cross contaminate edible internal organs (offals) that might not be readily accessible for decontamination (Arroyo and Arroyo, 1995). Although offals are mostly believed to acquire Salmonella via cross contamination (Arroyo and Arroyo, 1995) during processing, it is conceivable that Salmonella colonizes these organs early in the life cycle of the broilers, possibly persisting to slaughter age (Gast and Holt, 1998). Therefore, there is a need to better understand the persistence and transmission dynamics of Salmonella in broiler chicken internal organs, including edible parts (offals), in order to evaluate additional approaches to control Salmonella contamination of chicken carcasses. For the latter, it is thought that reducing Salmonella colonization before slaughter might constitute a judicial practice. To achieve this, several strategies have been suggested, including the use of feed additives to reduce the pathogen before harvest and processing. Of particular interest is sodium bisulfate (NaHSO4), an acid salt, used as an agent for primarily lowering the pH of a variety of matrices. This compound is also added as a supplement to treat farm-animal litter, where it exhibits a consid-
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erable antibacterial activity, resulting in 2 to 3 logs reduction in the litter’s bacterial populations (Pope and Cherry, 2000). Ruiz-Feria et al. (2011) observed that litter of broilers that were on a diet supplemented with sodium bisulfate contained lower levels of Salmonella, while the productive performance of the chickens was higher than those receiving untreated feed. Additionally, Ruiz-Feria et al. (2011) also reported that the effect of sodium bisulfate on Salmonella shedding was inconsistent, whereas the effect of the chemical on Salmonella colonization of the chicken’s gut was not investigated. This is important given that Line (2002) showed that amending the litter with sodium bisulfate did not result in favorable negative effects on Salmonella colonization of the chicken ceca. A marked difference between the aforementioned studies is that the sodium bisulfate was used by Ruiz-Feria et al. (2011) as a feed additive, whereas Line (2002) used the chemical to treat litter. To resolve these conflicting reports, we monitored the liver, spleen, cecum, feces, and bone marrow of experimentally infected broiler chickens and evaluated the effect of sodium bisulfate, as an antimicrobial feed additive, on Salmonella colonization of these chicken organs.
MATERIALS AND METHODS Experimental Design and Monitoring Before the onset of the experiment, twelve 1-d-old chickens were examined to further confirm that the specific pathogen-free flock was not precolonized with Salmonella. The 1-d-old chickens were euthanized and samples (liver, spleen, cecum, feces, and bone marrow) were aseptically harvested, suspended in buffered peptone water, and homogenized. The suspensions (100 μL) were then spread on XLT-4 agar (BD Diagnostics, Franklin Lakes, NJ), a medium that is selective for Salmonella, while 1 mL of the suspension was enriched in tetrathionate broth base with iodine-potassium iodide solution (TBB), as described by the manufacturer (BD Diagnostics), to detect if the bacterium occurred at lower densities. Inocula of TBB enrichments that exhibited positive growth were transferred to XLT-4 agar to determine the occurrence of Salmonella Enteritidis. The XLT-4 agar plates and TBB enrichments were incubated overnight at 37°C under aerobic conditions. To increase the selectivity for retrieving Salmonella Enteritidis from the infected chickens and further limit the growth of other bacterial contaminants, XLT-4 agar plates were supplemented with novobiocin and nalidixic acid, as described elsewhere (Gast and Holt, 1998). Subsequently, two hundred and forty 1-dold chickens were orally inoculated with 2 × 105 cfu of nalidixic acid-resistant Salmonella Enteritidis (Gast and Holt, 1998). The chickens were then divided into 4 groups of 60 birds each and the birds representing each group were evenly distributed to 5 cages (i.e., 12
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chickens per cage). Chickens from each group were also placed into different pens. To assess the effect of sodium bisulfate on Salmonella Enteritidis colonization, the chickens in group 1 received ad libitum feed that was supplemented with ~4.5 kg/t (low dose; treatment 1) of sodium bisulfate. Similarly, 2 experimental setups were used to test 7 and 9 kg/t of sodium bisulfate in feed, which corresponded to medium (treatment 2 for group 2) and high doses (treatment 3 for group 3), respectively. A control group with untreated feed (treatment 4 for group 4) was also setup to determine the progression of Salmonella Enteritidis colonization in the broiler chickens in the absence of sodium bisulfate. Three days postinfection, 10 chickens (2 from each cage) from each treatment were killed and samples (spleen, liver, bone marrow, cecum, feces) were analyzed for Salmonella Enteritidis. This was repeated on d 7 postinoculation and subsequently weekly for a period of 4 additional weeks, which spanned the life of the broiler flock from hatching to slaughter. Collectively, a total of 1,200 samples was monitored for Salmonella Enteritidis throughout the experiment. Animal experiments were conducted according to the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Throughout the experiment, chickens received the same type of feed, a commercially available crumble (Kalmbach Feeds, Upper Sandusky, OH) that contained approximately 22% CP, 2% crude fat, 5% crude fiber, 0.7 to 1.2% calcium, 0.65% phosphorus, 0.2 to 0.7% NaCl, 0.5% methionine, and 1.1% lysine. Other important parameters, such as average feed intake, average gain, feed conversion ratio, and mortality (Table 1), were collected, whereas the temperature of the pens was monitored daily to limit possible variations to the experimental design.
Statistical Analysis Counts of Salmonella cfu were presented as means ± SE. The means were determined by averaging the cfu counts from each organ samples (n = 10) per treatment. Data were transformed (log10) and analyzed using oneway ANOVA, followed by Tukey’s post hoc significance test. A P-value of <0.05 was used to indicate whether differences were statistically significant.
RESULTS AND DISCUSSION All of the organs/tissues retrieved from randomly selected 1-d-old chickens were negative for Salmonella, which confirmed that the specific pathogen-free flock used in this study was likely free of this pathogen. Subsequently, our postinfection analysis showed that as early as d 3 postinoculation, the chickens in all groups were colonized with high numbers of Salmonella Enteritidis, which infected every chicken and organ/ tissue tested, with the exception of only 2 bone marrow samples that belonged to chickens receiving the
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Table 1. Feed conversion and mortality of chickens treated with different doses of sodium bisulfate Time (wk)
Parameter
1
Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Average daily gain (g) Average daily feed intake Feed conversion ratio Mortality (%)
2 3 4 5 Overall
(g) (g) (g) (g) (g)
Treatment 1 (low dose)
Treatment 2 (medium dose)
Treatment 3 (high dose)
Treatment 4 (no dose)
13.4 29.1 2.1 31.8 45.2 1.4 47.7 78.2 1.6 68.6 115.7 1.6 72.7 138.3 1.9 5
13.3 29.3 2.2 37.4 49.2 1.3 49.6 85.1 1.7 70.4 123.8 1.7 83.3 147.0 1.7 3.34
13.6 29.1 2.1 34.5 50.5 1.4 54.3 87.2 1.6 67.9 116.6 1.7 48.6 121.3* 2.4* 0
13.1 20.6* 1.5* 31.2 45.3 1.4 47.9 82 1.7 71.3 120.3 1.6 49.8 104.2 2 0.6
*Indicates statistically different numbers (P < 0.05).
Figure 1. A. Enumeration of Salmonella Enteritidis cfu retrieved from liver samples across time. B. Enumeration of Salmonella Enteritidis cfu retrieved from spleen samples across time. Treatments 1, 2, and 3 correspond to low, medium, and high doses of sodium bisulfate, respectively. Treatment 4 is the control that did not receive any sodium bisulfate. Data were represented as means ± SE. n = 40 samples/time point. *Indicates significantly different numbers as compared with the control (P < 0.05).
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Figure 2. A. Enumeration of Salmonella Enteritidis cfu retrieved from feces across time. B. Enumeration of Salmonella Enteritidis cfu retrieved from ceca across time. Treatments 1, 2, and 3 correspond to low, medium, and high doses of sodium bisulfate, respectively. Treatment 4 is the control that did not receive any sodium bisulfate. Data were represented as means ± SE. n = 40 samples/time point. *Indicates significantly different numbers as compared with the control (P < 0.05).
sodium bisulfate regimens (Figures 1 and 2; Table 2). Overall, the addition of sodium bisulfate to the feed did not result in significant reductions (P < 0.05) in Salmonella Enteritidis colonization as compared with the control. Interestingly, some samples collected from treated chickens appeared to harbor either comparable or statistically higher numbers (P < 0.05) of Salmonella Enteritidis (Figures 1 and 2). For example, at wk 5 postinfection, (2.8 ± 1.5) × 106, (2.1 ± 1.5) × 105, (14.5 ± 6.5) × 105 cfu/g of ceca were detected in the groups that received low, medium, and high doses of sodium bisulfate, respectively, as opposed to (4.4 ± 2.5) × 105 cfu/g of ceca retrieved from the control group (Figure 2A). Similarly, (8 ± 3.3) × 105, (4 ± 3.9) × 105, (11 ± 6) × 103 cfu/g of feces was observed in treated samples as compared with (8.6 ± 6.2) × 103 cfu/g of feces collected from the control group (Figure 2B). Although at specific time points, the addition of sodium bisulfate to the feed did seemingly result in reductions in Salmonella Enteritidis numbers, these decreases were not consistent and did not follow a specific trend. For example, cecal and fecal samples collected at
d 20 postinfection from chickens receiving low and medium doses of sodium bisulfate harbored a significantly lower Salmonella Enteritidis number as compared with that from the control group (Figure 2). However, at d 27 and 34 postinfection, the Salmonella Enteritidis numbers retrieved from cecal and fecal samples subjected to the aforementioned treatments were either statistically not different or significantly higher than those from the control group (Figure 2). A similar observation was noted with Salmonella Enteritidis numbers recorded for liver and spleen samples, although the decrease in Salmonella Enteritidis numbers associated with low and medium doses of sodium bisulfate was observed at d 14 postinfection (Figure 1). This transient decrease in Salmonella Enteritidis numbers in infected organs might explain the aforementioned observations of Ruiz-Feria et al. (2011), who reported that the effect of sodium bisulfate on Salmonella shedding was inconsistent. However, the precise reasons behind the absence of a consistent effect on Salmonella colonization after treatment are not clear. Yet, because the effect of sodium bisulfate is not specific to Salmonella, it
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Table 2. The number of Salmonella Enteritidis-positive samples after enrichment in iodine-potassium iodide solution and subsequent inoculation onto XLT-4 agar1 Time postinfection (d) Sample Liver
Spleen
Cecum
Feces
Bone marrow
Total samples processed
Treatment2
3
7
13
20
27
34
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 All treatments
10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 9/10 10/10 10/10 200/00
10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 8/10 10/10 10/10 200/00
10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 6/10 7/10 7/10 7/10 200/00
7/10 7/10 9/10 8/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 2/10 2/10 6/10 4/10 200/00
9/10 4/10 5/10 3/10 8/10 9/10 7/10 9/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 6/10 3/10 2/10 5/10 200/00
5/10 3/10 5/10 0/10 5/10 8/10 9/10 6/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 9/10 0/10 4/10 0/10 2/10 200/00
1In
total, 1,200 samples were processed throughout the experiment. 1, 2, and 3 corresponded to low, medium, and high doses of sodium bisulfate, respectively. Treatment 4 was the control that did not receive any sodium bisulfate. 2Treatments
is conceivable that this compound might have affected normal flora in the chickens’ gut, perhaps reducing the organisms that might otherwise compete with Salmonella to occupy these niches. Alternatively, it is possible that Salmonella Enteritidis might have adapted to potential decreases in pH that were induced by sodium bisulfate. This is plausible becasue it has been shown that exposure of Salmonella cells to mild acidic conditions can lead to adaptation to acid and other stresses (Leyer and Johnson, 1992, 1993). Regardless of the reasons, it should be further noted that the occurrence of Salmonella in the samples after enrichment was also not affected by the addition of sodium bisulfate (Table 2). Analyzing samples collected from the control group lead to a better understanding of the occurrence and persistence of Salmonella Enteritidis in broiler chickens’ internal organs. As expected, fecal and cecal samples harbored the highest numbers of Salmonella Enteritidis at all time points. Furthermore, until d 20 postinfection, the numbers of Salmonella Enteritidis in feces and ceca of the chickens in the control group were significantly higher than that of the original oral inocula (Figure 2). As compared with the first 20 d, Salmonella Enteritidis densities in the feces and ceca from control samples decreased significantly by wk 5 but remained in considerable quantities that might pose a risk (Figure 2). It was notable that Salmonella Enteritidis could not be detected without pre-enrichment at d 20 and 27 postinfection in the liver (an offal) samples of the control group, whereas at d 34 it appears that the bacterium was absent in this organ (Table 2). Additionally, after enrichment, 20 and 60% of bone marrow (an offal) and the spleen samples, respectively, from the control
group were found to harbor Salmonella Enteritidis until d 34. Taken together, our data further confirm the importance of internal organs as potential sources for cross contamination during slaughter and processing. In conclusion, at the concentrations used in this study, feed supplemented with sodium bisulfate did not consistently reduce Salmonella Enteritidis colonization. Because Salmonella can colonize the broilers at any point in their life cycle and its spread to internal organs can occur rapidly, we suggest that dissemination of internal chicken organs consumed by humans or prepared as pet food should be accompanied by recommendations for appropriate handling and cooking.
ACKNOWLEDGMENTS This work was supported by the Ohio Agricultural Research and Development Center (OARDC), The Ohio State University (Wooster, OH), and Kalmbach Feeds Inc. (Upper Sandusky, Ohio).
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