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Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers
Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers W. Q. Alali,*1 C. L. Hofacre,† G. F. Mathis,‡ and G. Faltys# *Center for Food Safety, University of Georgia, Griffin 30223; †Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens 30602; ‡Southern Poultry Research, Athens, GA 30607; and #Vytol BioSystems, Oakland, NE 68045 EO administered in drinking water significantly (P < 0.05) reduced Salmonella Heidelberg colonization in crops of challenged birds, significantly lowered the feed conversion ratio, and increased weight gain compared with controls. The 0.025% and 0.015% EO in drinking water significantly lowered the feed conversion ratio and increased weight gain compared with controls, but did not significantly reduce Salmonella Heidelberg colonization in the crops. The EO in drinking water did not significantly reduce Salmonella Heidelberg colonization in ceca or fecal shedding in broilers. The EO used in the study may control Salmonella Heidelberg contamination in crops of broilers when administered in drinking water and therefore may reduce the potential for cross-contamination of the carcass when the birds are processed.
Key words: broiler, Salmonella, essential oil, performance, feed withdrawal 2013 Poultry Science 92:836–841 http://dx.doi.org/10.3382/ps.2012-02783
INTRODUCTION
and bone or joint; Vugia et al., 2004). This Salmonella Heidelberg invasiveness percentage was higher than the 6% percentage for Salmonella Typhimurium and Enteritidis (Vugia et al., 2004). One of the preslaughter practices recommended by the USDA-FSIS to control pathogens in broilers is to use organic acids in drinking water during feed withdrawal (USDA-FSIS, 2008). This practice may lead to intestinal inflammation and reduction in water consumption in broiler birds. A well-timed feed withdrawal (8 to 12 h preslaughter) reduces the amount of ingesta and feces in the bird gut and subsequent carcasses contamination with pathogens during slaughter/processing (Rigby et al., 1980, 1981; Buhr et al., 1998; Lyon et al., 1991). However, feed withdrawal practice can lead to increase in crop and ceca Salmonella levels in broilers (Humphrey et al., 1993; Ramirez et al., 1997). There are very limited data on the use of plant-derived compounds during the feed withdrawal period to control Salmonella in poultry. The EO blends have in general antibacterial activity (in vitro and in vivo) and can enhance poultry bird performance (Brenes and Rou-
Reducing Salmonella colonization and fecal shedding in live chickens and subsequently chicken meat contamination can reduce the burden of salmonellosis in humans (FAO, 2002; USDA-FSIS, 2008). Salmonella enterica serovar Heidelberg is 1 of the top 6 Salmonella enterica serotypes associated with human salmonellosis and one of the most commonly isolated serotypes from poultry (Liljebjelke et al., 2005; Foley et al., 2008; FDA, 2010; CDC, 2011a). This Salmonella serotype was associated with 2 recent multistate outbreaks linked to consumption of chicken liver and ground turkey (CDC, 2011b, 2012). According to the data published by the CDC FoodNet, 11% of Salmonella Heidelberg infections (n = 551) were invasive salmonellosis cases (i.e., bacteria reached blood, cerebrospinal fluid, peritoneal fluid, ©2013 Poultry Science Association Inc. Received September 17, 2012. Accepted December 2, 2012. 1 Corresponding author: [email protected]
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ABSTRACT The objectives of this study were to determine the effect of an essential oil blend (EO; carvacrol, thymol, eucalyptol, lemon) administered in drinking water on the performance, mortality, water consumption, pH of crop and ceca, and Salmonella enterica serovar Heidelberg fecal shedding and colonization in broiler birds following Salmonella Heidelberg challenge and feed withdrawal. Chicks were randomly assigned to water treatments containing 0.05, 0.025, or 0.0125% EO or untreated controls. Treatments were administered in drinking water on 0 to 7 and 35 to 42 d. One-half of the chicks were challenged with Salmonella Heidelberg and placed in pens with unchallenged chicks on d 1. Performance, mortality, water consumption, and pH were determined during the 42-d study. Prevalence of Salmonella Heidelberg was determined on drag swabs (0, 14, and 42 d) and in the ceca and crops (42 d). The 0.05%
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MATERIALS AND METHODS Study Design One-day-old male broiler chicks (Cobb × Cobb; n = 1,000) were assigned randomly to 4 drinking water treatments on d 0 of the study [n = 250 birds per treatment, with 5 replicates per treatment, 50 birds per replication (i.e., pen)]. The treatments were 0.05, 0.025, and 0.015% EO (Mix-Oil) and nontreated water. The treatments were provided in the drinking water at 0 to 7 and 35 to 42 d. Broilers were housed in floor pens (n = 20 pens; 860 cm2∙bird−1) that contained approximately 10 cm of pine shavings as litter. Feed and water were available ad libitum. Broiler rations were formulated to meet or exceed animal nutrient requirements (NRC, 1994) and were fed starter (0 to 21 d of age), grower (21 to 35 d), and finisher (35 to 42 d) diets (Table 1). Rations were fed as crumbles (starter feed) or as pellets (grower and finisher). Rations did not contain any antibiotics or additional subtherapeutic antimicrobial agents. Pen weights of broilers were recorded on d 1 and 42. Weights of feed filled into feeders were recorded. Disappearance of feeds from feeders during starter, grower, and finisher phases was calculated by subtracting weight of feed remaining on d 21, 35, and 42 from total weight of feed issued during that phase. Approximately 8 h before broilers
Table 1. Composition and calculated analysis of the basal diets Item
Starter
Grower
Finisher
Ingredient (%) Corn Soybean meal Fat, animal Meat meal Dicalcium phosphate Calcium carbonate Salt dl-Methionine l-Lys HCl Broiler vitamin mix1 Trace mineral mix2 Choline 70 Calculate analysis ME (kcal/kg) CP (%) Lysine (%) Methionine (%) Calcium (%) Phosphorus, total (%)
59.62 30.83 2.82 4.00 1.08 0.70 0.44 0.23 0.03 0.09 0.10 0.06 3,096 21.86 1.22 0.58 0.95 0.69
63.91 26.51 3.07 4.00 1.03 0.57 0.41 0.21 0.03 0.09 0.10 0.07 3,140 20.09 1.10 0.53 0.91 0.67
68.86 21.94 2.97 4.00 0.79 0.62 0.41 0.15 0.01 0.09 0.10 0.06 3,191 18.22 0.96 0.46 0.82 0.60
1Vitamin mix provided the following (per kg of diet): thiamine mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; d-Ca pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 µg; pyridoxine HCl, 4.7 mg; d-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 µg; trans-retinyl acetate, 1,892 µg; all-rac α-tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4∙H2O), 60 mg; iron (FeSO4∙7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4∙5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg.
were weighed at 42 d; feed was removed from the feeders, weighed, and discarded. Feed conversion ratios (adjusted for mortality: total feed consumption/(final live weight + total mortality weight of the birds) for each treatment were calculated. Water consumption (liter per pen) was measured on d 0, 3, and 5, and then daily between 35 to 42 d.
Salmonella Challenge and Sample Collection A Salmonella Heidelberg isolate resistant to nalidixic acid (NA) was grown for 6 h in Tryptic Soy Broth (Difco Laboratories, Detroit, MI); thereafter, the number of cfu per milliliter was determined by plating 10-fold dilutions of the bacterial suspension on Xylose Lysine Tergitol-4 (Difco Laboratories) medium. A bacterial colony was picked randomly and diluted in PBS solution to obtain a challenge suspension (5 × 107 cfu∙mL−1). All challenged chicks were identified with individually numbered wing bands. One-half of the chicks (n = 25 per pen) received 0.1 mL of challenge solution per chick (5 × 106 cfu) via oral gavage on d 1 before being placed in a pen with unchallenged chicks (n = 25 per pen). In this animal model, challenged chicks represented infected birds that may shed Salmonella in the environment (seeders), whereas unchallenged chicks represented birds that may become infected with Salmonella through contact with a contaminated environ-
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ra, 2010). The variability in the antibacterial outcome and bird performance depends mainly on the EO blend composition. Kollanoor Johny et al. (2012) revealed that trans-cinnamaldehyde and eugenol added to feed significantly reduced Salmonella Enteritidis cecal colonization in 20-d-old broilers. The authors reported that eugenol significantly reduced broiler BW compared with control birds. A recent study by Amerah et al. (2012) found that a blend of essential oils (EO; cinnamaldehyde and thymol) used as a feed supplement for broilers over a 42-d period significantly reduced both the horizontal transmission of Salmonella Heidelberg in treated broilers and prevalence of Salmonella Heidelberg-positive ceca compared with controls. Furthermore, the EO used in that study significantly improved BW gain and feed efficiency. In a recent study, we revealed that a 0.025% EO blend (composed of carvacrol, thymol, eucalyptol, lemon) and 0.44% lactic acid administered in drinking water significantly reduced the percentage of Salmonella Heidelberg-positive crops of broilers, increased weight gain, and lowered mortality percentages compared with controls (Alali et al., 2013). The objectives of this study were to determine the effect of an EO blend (Mix-Oil, Vytol BioSystems Inc., Oakland, NE) at 3 concentrations (0.05, 0.025, and 0.0125%) in drinking water on 1) colonization and fecal shedding of Salmonella Heidelberg in broilers following Salmonella Heidelberg challenge and feed withdrawal, and 2) feed conversion ratio (FCR), weight gain, mortality, water consumption, and pH of crop and ceca of broilers.
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ment (contact; Figure 1). Paper pads (96 cm × 58 cm) lining chick transport boxes were collected at d 0 and stored in sterile sample bags. Sterile gauze drag swabs (7.6 cm2; one per pen), soaked in skim milk (Solar Biologicals Inc., Ogdensburg, NY), were used to collect bacterial samples from the litter at d 0 (before chick placement), 14, and 42. Paper pads and swabs were transported on ice within 24 h to the Poultry Diagnostic Research Center (PDRC; Athens, GA) for analysis of Salmonella Heidelberg. Swabbing litter with sterile gauze soaked in skim milk is considered to be the most sensitive method of sampling the environment for Salmonella spp. (USDA, 1996). Prevalence of Salmonella Heidelberg on the swabs represented the frequency of Salmonella Heidelberg shed by the challenged broilers. At d 42 (8 h after feed withdrawal, but not withdrawal of water), 10 broilers per pen (5 seeders and 5 contacts) were randomly selected, euthanized by cervical dislocation, and the ceca and crops were aseptically removed. The pH of 5 ceca and 5 crops per pen was measured using a pH probe (Sensorex, Garden Grove, CA) and a pH meter (Milwaukee Instruments, Rocky Mount, NC). The pH probe was inserted inside the ceca or crop to measure the pH. The probe was cleaned and disinfected with 80% ethanol between readings. The organs were placed in sterile plastic sample bags and transported to PDRC within 3 h of collection for Salmonella Heidelberg analysis. The Salmonella Heidelberg presence in the ceca and crops were indicative of colonization of this pathogen in these organs.
Salmonella Analysis Each paper pad was enriched in 400 mL of tetrathionate broth (TT; Difco) containing 0.001% aqueous brilliant green and 2 mL of iodine solution (25% iodine and 30% potassium iodide) and then the paper pad in broth was incubated at 42°C for 24 h. A loopful (~10 µL) of the broth was streaked on XLT4 agar supplemented with 25 µg of NA/L (XLT4-NA). The XLT4NA plates were then incubated at 37°C for 24 h. Each drag swab was enriched with 100 mL of TT as above and incubated (42°C, 24 h). A loopful of enrichment culture was streaked onto XLT4-NA. These plates were then incubated at 37°C for 24 h. One selected black colony per XLT4-NA plate was streaked on blood agar (Remel, Lenexa, KS). One colony per blood agar plate was tested against Salmonella O group B antiserum (Difco) and confirmed to be Salmonella Heidelberg if agglutination was noted. If black colonies were not present, the enriched drag swab was held at room temperature for 5 d and a delayed secondary enrichment was prepared by diluting the drag swab in fresh TT broth (1:10) and incubating at 37°C for 24 h. A loopful of the mixture was streaked onto XLT4-NA. The remaining isolation and confirmation of Salmonella Heidelberg was carried out according to the same protocol as the primary enrichment. Fifty and 30 mL of TT broth (containing brilliant green and iodine solution) were added to ceca and crops, respectively. The mixture was then macerated by
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Figure 1. A flowchart representation of the sample collection and Salmonella enterica serovar Heidelberg analysis over the study period. TT: tetrathionate broth (Difco Laboratories, Detroit, MI); XLT4-NA: xylose lysine tergitol-4 (Difco Laboratories) supplemented with 25 µg/L of nalidixic acid. (…) indicates that the secondary enrichment process was used once per sample when primary enrichment was Salmonella Heidelberg-negative.
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a stomacher (Technar Company, Cincinnati, OH) for 20 s. The homogenized ceca and crops were incubated overnight at 42°C for primary enrichment. If no black colonies were present on the XLT4-NA plates, then 1 loopful of enriched sample was streaked onto XLT4NA, which was incubated at 37°C for 24 h, and 1 black colony per plate was streaked on blood agar. One colony per blood agar plate was tested against Salmonella O group B antiserum and confirmed to be Salmonella Heidelberg if agglutination was observed. If no black colonies were present from a sample, the enriched ceca and crop samples were held at room temperature for 5 d, and a delayed secondary enrichment was performed as described above for the drag swabs.
The study design was a randomized complete block with 5 blocks and 4 treatments. The experimental unit was the pen. The study outcomes measured were 1) the percentage of Salmonella Heidelberg isolates from drag swabs, ceca, or crop, and 2) performance data (FCR, weight gain, and mortality), water consumption, and pH values. A sample was considered positive if Salmonella Heidelberg was recovered from direct plating, primary, or secondary enrichments. The percentages of Salmonella Heidelberg isolates from ceca or crops were compared between treatments within seeders or contacts using generalized estimating equation models (for binary data: presence or absence of Salmonella Heidelberg), with logit link function and binomial distribution using an independent correlation structure to account for the block effect in STATA statistical software (ver. 10.1, StataCorp, College Station, TX). The performance data, water consumption, and pH values were compared between treatment groups using generalized estimating equation models, with identity link function, in STATA software. Treatment effects were considered significant at P < 0.05.
RESULTS AND DISCUSSION All paper pads and drag swabs collected at d 0 were negative for Salmonella Heidelberg. All drag swabs collected at 14 d were positive for Salmonella Heidelberg; hence, administration of the EO at the 3 concentrations
Table 2. Effect of essential oil blend on percentages of Salmonella enterica serovar Heidelberg-positive ceca and crop in broilers on d 42 postchallenge1 % (no. of Salmonella-positives/total) Ceca Treatment 0.015% 0.025% 0.050% Control a,bPercentages
All 10 4 8 14
(5/50) (2/50) (4/50) (7/50)
Seeders 12 4 8 24
(3/25) (1/25) (2/25) (6/25)
Crop Contacts 8 4 4 4
(2/25) (1/25) (2/25) (1/25)
All
24a 22b 10b 38a
with different superscripts within a column differ significantly (P < 0.05).
(12/50) (11/50) (5/50) (19/50)
Seeders 32a 36a 12b 52a
(8/25) (9/25) (3/25) (13/25)
Contacts 16a 8a 8a 24a
(4/25) (2/25) (2/25) (6/25)
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Statistical Analysis
from 0 to 7 d did not eliminate shedding of Salmonella Heidelberg 2 wk posttreatment. On d 42, Salmonella Heidelberg was present on drag swabs from 40, 80, 80, and 100% of pens assigned to 0.05, 0.025, 0.015% EO, and controls, respectively. However, these percent differences were not significantly different (P > 0.05). This may indicate that EO treatments did not reduce the horizontal transmission of Salmonella Heidelberg between seeders and contacts. Overall, treatments with EO did not significantly (P > 0.05) decrease the percentage of Salmonella Heidelberg-positive ceca of contact and seeder broilers (Table 2). Treatment with EO significantly (P < 0.05) decreased the percentage of Salmonella Heidelberg-positive crops compared with controls (Table 2). Seeders receiving 0.05% EO had a significantly (P < 0.05) lowered percentage of Salmonella Heidelberg-positive crops compared with 0.025 and 0.015% EO and control. The percentage of Salmonella Heidelberg-positive crop in contacts receiving EO did not significantly (P > 0.05) decrease compared with controls. Based on the data from this study, it appears that EO effect on Salmonella Heidelberg in the crop and not in the ceca. This is in agreement with our previous study findings on EO (Alali et al., 2013). The principal components of the EO blend used in this study were carvacrol, thymol, eucalyptol, lemon, and citric acid. These components may have a synergistic antimicrobial effect against Salmonella Heidelberg. Lambert et al. (2001) reported an in vitro synergistic antimicrobial activity by carvacrol and thymol against Staphylococcus aureus and Pseudomonas aeruginosa. Borsoi et al. (2011) reported that a blend of both organic acids (butyric, propionic, lactic, or formic acids) and essential oils (thymol, carvacrol, citrol, cinnamon, or clove oils) reduced Salmonella cell populations in cloacal samples at 13 and 20 d postinoculation compared with a control group. Eugenol, transcinnamaldehyde, carvacrol, and thymol were tested in vitro against Salmonella Enteritidis in chicken cecal contents (Kollanoor Johny et al., 2010). The authors reported significant Salmonella Enteritidis reduction in cecal contents after 8 and 24 h of incubation at 40°C. Trans-cinnamaldehyde at 0.06% was reported to inactivate S. Enteritidis to undetectable levels in fecally contaminated water (Kollanoor Johny et al., 2008). The EO blend (cinnamaldehyde and thymol) in Amerah et
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Alali et al. Table 3. Effect of essential oil blend on performance results and mortality percentages in broilers following exposure to Salmonella enterica serovar Heidelberg1 Treatment 0.015% 0.025% 0.050% Control3
FCR2 (0 to 42 d)
Average weight gain (kg)
Mortality (%)
1.87a 1.83a 1.79a 1.93b
1.96a 1.97a 2.10a 1.83b
4.0a 4.4a 4.4a 6.0a
a,bMeans
or percentages with different superscripts within a column differ significantly (P < 0.05). or percentages represent 5 pens per treatment, with 50 birds per pen. 2FCR = feed conversion ratio. 3The control birds had no medications in their drinking water. 1Means
cantly different (P > 0.5) compared with controls. This suggests that the amount of water consumed by broilers that received EO did not decrease compared with nontreated controls. The average pH values for crop and ceca of EO treatments were not significantly different (P > 0.5) compared with controls. This suggests that EO treatments did not have a significant effect on the pH compared with nontreated controls. In our study, we used nalidixic acid-resistant Salmonella Heidelberg (1 serotype) as a surrogate for the following reasons: 1) the ability to differentiate the source of Salmonella (inoculated versus environmental), which could confound the results, and 2) inoculation with one serotype could reduce the possibility of multiple serotype interaction, which could confound the results (treatment effect versus interaction effect). The treatments were given to the birds during the first week and last week of the grow-out period. The first week treatment was to adapt the birds’ gastrointestinal system to the water treatments before a second application during the last week of life to reduce Salmonella Heidelberg colonization and shedding. In summary, the 0.05% EO blend in drinking water significantly reduced Salmonella Heidelberg colonization in crops of seeders, lowered the FCR, and increased weight gain compared with controls. The 0.025 and 0.015% EO blend in drinking water significantly lowered FCR and increased weight gain compared with controls.
ACKNOWLEDGMENTS This study was supported in part by a grant from the University of Georgia, Center for Food Safety (Griffin) and a grant from Animal Wellness Products, Reggio
Table 4. Effect of essential oil blend on water consumption and pH measures for ceca and crop in broilers following exposure to Salmonella enterica serovar Heidelberg1 Treatment 0.015% 0.025% 0.050% Control
Water 0 to 7 d
Water 35 to 42 d
Ceca pH2
Crop pH
16.48 16.02 16.00 16.31
112.55 107.64 107.49 103.06
6.6 6.7 6.5 6.4
5.0 4.8 4.8 4.7
1Means represent 5 pens per treatment, with 50 birds per pen. There were no significant differences (P > 0.05) detected among treatments within seeder and contact birds for water consumption and pH measures. 2Ceca and crop of 5 birds per pen were collected for pH measurements.
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al. (2012) significantly reduced Salmonella Heidelbergpositive ceca in broilers challenged with this organism compared with controls. In other studies, EO blends added to feed have resulted in a reduction in Escherichia coli population in the broiler gastrointestinal tract (Jang et al., 2007), whereas increased ceca Lactobacillus and E. coli proportions (Tiihonen et al., 2010). Performance data and mortality results of broilers that received Salmonella Heidelberg challenge are shown in Table 3. Broilers that received EO at the 3 concentrations had significantly (P < 0.05) lower FCR and greater average weight gain than controls. This is in agreement with Amerah et al. (2011, 2012) and Alali et al. (2013) studies. The mortality percentages did not differ significantly (P > 0.05) among EO-treated and control broilers. It was suggested by Bajpai et al. (2012) that EO compounds have a wide range of activities (additive, synergistic, or antagonistic), not only limited to the microflora in the bird, but also the metabolism of the bird. Essential oils from cloves and cinnamon reportedly improved the FCR and percentage of breast weight of broilers compared with those receiving calcium propionate or calcium formate (Isabel and Santos, 2009). Furthermore, carvacrol added to broiler bird feed at 200 ppm reduced weight gain and feed intake, but improved the feed:gain ratio after 4 wk of feeding this diet (Lee et al., 2003). Kollanoor-Johny et al. (2012) revealed that trans-cinnamaldehyde added to 20-d-old broilers feed had no effect on the BW; however, eugenol significantly reduced broiler BW compared with controls. The water consumption and pH results of broilers that received Salmonella Heidelberg challenge are shown in Table 4. The average water consumption over 0 to 7 and 35 to 42 d for EO treatments was not signifi-
ESSENTIAL OIL AND SALMONELLA IN BROILERS
Emilia, Italy. We thank Diezhang Wu, Abdel-Rahman Alsleh, Christine Lobsinger, and PDRC (Athens, GA) technicians for their assistance in processing samples and performing microbiological assays. We also thank Animal Wellness Products and Vytol BioSystems (Oakland, NE) for providing us with their essential oil product.
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Alali, W. Q., C. L. Hofacre, G. F. Mathis, G. Faltys, S. C. Ricke, and M. P. Doyle. 2013. Effect of non-pharmaceutical compounds on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers. Food Contr. 31:125–128. http://dx.doi. org/10.1016/j.foodcont.2012.10.001. In press. Amerah, A. M., G. Mathis, and C. L. Hofacre. 2012. Effect of xylanase and a blend of essential oils on performance and Salmonella colonization of broiler chickens challenged with Salmonella Heidelberg. Poult. Sci. 91:943–947. Amerah, A. M., A. Péron, F. Zaefarian, and V. Ravindran. 2011. Influence of whole wheat inclusion and a blend of essential oils on the performance, nutrient utilisation, digestive tract development and ileal microbiota profile of broiler chickens. Br. Poult. Sci. 52:124–132. Bajpai, V. K., K.-H. Baek, and S. C. Kang. 2012. Control of Salmonella in foods by using essential oils: A review. Food Res. Int. 45:722–734. Borsoi, A., L. R. Santos, G. S. Diniz, C. T. C. Salle, H. L. S. Moraes, and V. P. Nascimento. 2011. Salmonella fecal excretion control in broiler chickens by organic acids and essential oils blend feed added. Braz. J. Poult. Sci. 13:65–69. Brenes, A., and E. Roura. 2010. Essential oils in poultry nutrition: Main effects and modes of action. Anim. Feed Sci. Technol. 158:1–14. Buhr, R. J., J. K. Northcutt, C. E. Lyon, and G. N. Rowland. 1998. Influence of time off feed on broiler viscera weight, diameter, and shear. Poult. Sci. 77:758–764. CDC (Centers for Disease Control and Prevention). 2011a. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2010 (Final Report). US Department of Health and Human Services, Atlanta, GA. CDC. 2011b. Multistate Outbreak of Human Salmonella Heidelberg Infections Linked to Ground Turkey. Accessed Apr. 13, 2012. http://www.cdc.gov/salmonella/heidelberg/index.html. CDC. 2012. Multistate Outbreak of Human Salmonella Heidelberg Infections Linked to “Kosher Broiled Chicken Livers” from Schreiber Processing Corporation. Accessed Oct. 20, 2012. http:// www.cdc.gov/salmonella/heidelberg-chickenlivers/011112/index. html. FAO (Food and Agriculture Organization of the United Nations). 2002. Risk assessments of Salmonella in eggs and broiler chickens. Pages 277–296 in Risk Characterization of Salmonella in Broilers. Accessed Oct. 21, 2012. ftp://ftp.fao.org/docrep/fao/005/ y4392e/y4392e00.pdf. FDA. 2010. National Antimicrobial Resistance Monitoring System— Enteric bacteria (NARMS): 2007 Executive Report. US Department of Health and Human Services, FDA, Rockville, MD. Foley, S. L., A. M. Lynne, and R. Nayak. 2008. Salmonella challenges: Prevalence in swine and poultry and potential pathogenicity of such isolates. J. Anim. Sci. 86:E149–E162. Humphrey, T. J., A. Baskerville, A. Whitehead, B. Rowe, and A. Henley. 1993. Influence of feeding patterns on the artificial infection of laying hens with Salmonella enteritidis phage type 4. Vet. Rec. 132:407–409. Isabel, B., and Y. Santos. 2009. Effects of dietary organic acids and essential oils on growth performance and carcass characteristics of broiler chickens. J. Appl. Poult. Res. 18:472–476.
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Key words: broiler, Salmonella, essential oil, performance, feed withdrawal 2013 Poultry Science 92:836–841 http://dx.doi.org/10.3382/ps.2012-02783
INTRODUCTION
and bone or joint; Vugia et al., 2004). This Salmonella Heidelberg invasiveness percentage was higher than the 6% percentage for Salmonella Typhimurium and Enteritidis (Vugia et al., 2004). One of the preslaughter practices recommended by the USDA-FSIS to control pathogens in broilers is to use organic acids in drinking water during feed withdrawal (USDA-FSIS, 2008). This practice may lead to intestinal inflammation and reduction in water consumption in broiler birds. A well-timed feed withdrawal (8 to 12 h preslaughter) reduces the amount of ingesta and feces in the bird gut and subsequent carcasses contamination with pathogens during slaughter/processing (Rigby et al., 1980, 1981; Buhr et al., 1998; Lyon et al., 1991). However, feed withdrawal practice can lead to increase in crop and ceca Salmonella levels in broilers (Humphrey et al., 1993; Ramirez et al., 1997). There are very limited data on the use of plant-derived compounds during the feed withdrawal period to control Salmonella in poultry. The EO blends have in general antibacterial activity (in vitro and in vivo) and can enhance poultry bird performance (Brenes and Rou-
Reducing Salmonella colonization and fecal shedding in live chickens and subsequently chicken meat contamination can reduce the burden of salmonellosis in humans (FAO, 2002; USDA-FSIS, 2008). Salmonella enterica serovar Heidelberg is 1 of the top 6 Salmonella enterica serotypes associated with human salmonellosis and one of the most commonly isolated serotypes from poultry (Liljebjelke et al., 2005; Foley et al., 2008; FDA, 2010; CDC, 2011a). This Salmonella serotype was associated with 2 recent multistate outbreaks linked to consumption of chicken liver and ground turkey (CDC, 2011b, 2012). According to the data published by the CDC FoodNet, 11% of Salmonella Heidelberg infections (n = 551) were invasive salmonellosis cases (i.e., bacteria reached blood, cerebrospinal fluid, peritoneal fluid, ©2013 Poultry Science Association Inc. Received September 17, 2012. Accepted December 2, 2012. 1 Corresponding author: [email protected]
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ABSTRACT The objectives of this study were to determine the effect of an essential oil blend (EO; carvacrol, thymol, eucalyptol, lemon) administered in drinking water on the performance, mortality, water consumption, pH of crop and ceca, and Salmonella enterica serovar Heidelberg fecal shedding and colonization in broiler birds following Salmonella Heidelberg challenge and feed withdrawal. Chicks were randomly assigned to water treatments containing 0.05, 0.025, or 0.0125% EO or untreated controls. Treatments were administered in drinking water on 0 to 7 and 35 to 42 d. One-half of the chicks were challenged with Salmonella Heidelberg and placed in pens with unchallenged chicks on d 1. Performance, mortality, water consumption, and pH were determined during the 42-d study. Prevalence of Salmonella Heidelberg was determined on drag swabs (0, 14, and 42 d) and in the ceca and crops (42 d). The 0.05%
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MATERIALS AND METHODS Study Design One-day-old male broiler chicks (Cobb × Cobb; n = 1,000) were assigned randomly to 4 drinking water treatments on d 0 of the study [n = 250 birds per treatment, with 5 replicates per treatment, 50 birds per replication (i.e., pen)]. The treatments were 0.05, 0.025, and 0.015% EO (Mix-Oil) and nontreated water. The treatments were provided in the drinking water at 0 to 7 and 35 to 42 d. Broilers were housed in floor pens (n = 20 pens; 860 cm2∙bird−1) that contained approximately 10 cm of pine shavings as litter. Feed and water were available ad libitum. Broiler rations were formulated to meet or exceed animal nutrient requirements (NRC, 1994) and were fed starter (0 to 21 d of age), grower (21 to 35 d), and finisher (35 to 42 d) diets (Table 1). Rations were fed as crumbles (starter feed) or as pellets (grower and finisher). Rations did not contain any antibiotics or additional subtherapeutic antimicrobial agents. Pen weights of broilers were recorded on d 1 and 42. Weights of feed filled into feeders were recorded. Disappearance of feeds from feeders during starter, grower, and finisher phases was calculated by subtracting weight of feed remaining on d 21, 35, and 42 from total weight of feed issued during that phase. Approximately 8 h before broilers
Table 1. Composition and calculated analysis of the basal diets Item
Starter
Grower
Finisher
Ingredient (%) Corn Soybean meal Fat, animal Meat meal Dicalcium phosphate Calcium carbonate Salt dl-Methionine l-Lys HCl Broiler vitamin mix1 Trace mineral mix2 Choline 70 Calculate analysis ME (kcal/kg) CP (%) Lysine (%) Methionine (%) Calcium (%) Phosphorus, total (%)
59.62 30.83 2.82 4.00 1.08 0.70 0.44 0.23 0.03 0.09 0.10 0.06 3,096 21.86 1.22 0.58 0.95 0.69
63.91 26.51 3.07 4.00 1.03 0.57 0.41 0.21 0.03 0.09 0.10 0.07 3,140 20.09 1.10 0.53 0.91 0.67
68.86 21.94 2.97 4.00 0.79 0.62 0.41 0.15 0.01 0.09 0.10 0.06 3,191 18.22 0.96 0.46 0.82 0.60
1Vitamin mix provided the following (per kg of diet): thiamine mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; d-Ca pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 µg; pyridoxine HCl, 4.7 mg; d-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 µg; trans-retinyl acetate, 1,892 µg; all-rac α-tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4∙H2O), 60 mg; iron (FeSO4∙7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4∙5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg.
were weighed at 42 d; feed was removed from the feeders, weighed, and discarded. Feed conversion ratios (adjusted for mortality: total feed consumption/(final live weight + total mortality weight of the birds) for each treatment were calculated. Water consumption (liter per pen) was measured on d 0, 3, and 5, and then daily between 35 to 42 d.
Salmonella Challenge and Sample Collection A Salmonella Heidelberg isolate resistant to nalidixic acid (NA) was grown for 6 h in Tryptic Soy Broth (Difco Laboratories, Detroit, MI); thereafter, the number of cfu per milliliter was determined by plating 10-fold dilutions of the bacterial suspension on Xylose Lysine Tergitol-4 (Difco Laboratories) medium. A bacterial colony was picked randomly and diluted in PBS solution to obtain a challenge suspension (5 × 107 cfu∙mL−1). All challenged chicks were identified with individually numbered wing bands. One-half of the chicks (n = 25 per pen) received 0.1 mL of challenge solution per chick (5 × 106 cfu) via oral gavage on d 1 before being placed in a pen with unchallenged chicks (n = 25 per pen). In this animal model, challenged chicks represented infected birds that may shed Salmonella in the environment (seeders), whereas unchallenged chicks represented birds that may become infected with Salmonella through contact with a contaminated environ-
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ra, 2010). The variability in the antibacterial outcome and bird performance depends mainly on the EO blend composition. Kollanoor Johny et al. (2012) revealed that trans-cinnamaldehyde and eugenol added to feed significantly reduced Salmonella Enteritidis cecal colonization in 20-d-old broilers. The authors reported that eugenol significantly reduced broiler BW compared with control birds. A recent study by Amerah et al. (2012) found that a blend of essential oils (EO; cinnamaldehyde and thymol) used as a feed supplement for broilers over a 42-d period significantly reduced both the horizontal transmission of Salmonella Heidelberg in treated broilers and prevalence of Salmonella Heidelberg-positive ceca compared with controls. Furthermore, the EO used in that study significantly improved BW gain and feed efficiency. In a recent study, we revealed that a 0.025% EO blend (composed of carvacrol, thymol, eucalyptol, lemon) and 0.44% lactic acid administered in drinking water significantly reduced the percentage of Salmonella Heidelberg-positive crops of broilers, increased weight gain, and lowered mortality percentages compared with controls (Alali et al., 2013). The objectives of this study were to determine the effect of an EO blend (Mix-Oil, Vytol BioSystems Inc., Oakland, NE) at 3 concentrations (0.05, 0.025, and 0.0125%) in drinking water on 1) colonization and fecal shedding of Salmonella Heidelberg in broilers following Salmonella Heidelberg challenge and feed withdrawal, and 2) feed conversion ratio (FCR), weight gain, mortality, water consumption, and pH of crop and ceca of broilers.
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ment (contact; Figure 1). Paper pads (96 cm × 58 cm) lining chick transport boxes were collected at d 0 and stored in sterile sample bags. Sterile gauze drag swabs (7.6 cm2; one per pen), soaked in skim milk (Solar Biologicals Inc., Ogdensburg, NY), were used to collect bacterial samples from the litter at d 0 (before chick placement), 14, and 42. Paper pads and swabs were transported on ice within 24 h to the Poultry Diagnostic Research Center (PDRC; Athens, GA) for analysis of Salmonella Heidelberg. Swabbing litter with sterile gauze soaked in skim milk is considered to be the most sensitive method of sampling the environment for Salmonella spp. (USDA, 1996). Prevalence of Salmonella Heidelberg on the swabs represented the frequency of Salmonella Heidelberg shed by the challenged broilers. At d 42 (8 h after feed withdrawal, but not withdrawal of water), 10 broilers per pen (5 seeders and 5 contacts) were randomly selected, euthanized by cervical dislocation, and the ceca and crops were aseptically removed. The pH of 5 ceca and 5 crops per pen was measured using a pH probe (Sensorex, Garden Grove, CA) and a pH meter (Milwaukee Instruments, Rocky Mount, NC). The pH probe was inserted inside the ceca or crop to measure the pH. The probe was cleaned and disinfected with 80% ethanol between readings. The organs were placed in sterile plastic sample bags and transported to PDRC within 3 h of collection for Salmonella Heidelberg analysis. The Salmonella Heidelberg presence in the ceca and crops were indicative of colonization of this pathogen in these organs.
Salmonella Analysis Each paper pad was enriched in 400 mL of tetrathionate broth (TT; Difco) containing 0.001% aqueous brilliant green and 2 mL of iodine solution (25% iodine and 30% potassium iodide) and then the paper pad in broth was incubated at 42°C for 24 h. A loopful (~10 µL) of the broth was streaked on XLT4 agar supplemented with 25 µg of NA/L (XLT4-NA). The XLT4NA plates were then incubated at 37°C for 24 h. Each drag swab was enriched with 100 mL of TT as above and incubated (42°C, 24 h). A loopful of enrichment culture was streaked onto XLT4-NA. These plates were then incubated at 37°C for 24 h. One selected black colony per XLT4-NA plate was streaked on blood agar (Remel, Lenexa, KS). One colony per blood agar plate was tested against Salmonella O group B antiserum (Difco) and confirmed to be Salmonella Heidelberg if agglutination was noted. If black colonies were not present, the enriched drag swab was held at room temperature for 5 d and a delayed secondary enrichment was prepared by diluting the drag swab in fresh TT broth (1:10) and incubating at 37°C for 24 h. A loopful of the mixture was streaked onto XLT4-NA. The remaining isolation and confirmation of Salmonella Heidelberg was carried out according to the same protocol as the primary enrichment. Fifty and 30 mL of TT broth (containing brilliant green and iodine solution) were added to ceca and crops, respectively. The mixture was then macerated by
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Figure 1. A flowchart representation of the sample collection and Salmonella enterica serovar Heidelberg analysis over the study period. TT: tetrathionate broth (Difco Laboratories, Detroit, MI); XLT4-NA: xylose lysine tergitol-4 (Difco Laboratories) supplemented with 25 µg/L of nalidixic acid. (…) indicates that the secondary enrichment process was used once per sample when primary enrichment was Salmonella Heidelberg-negative.
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a stomacher (Technar Company, Cincinnati, OH) for 20 s. The homogenized ceca and crops were incubated overnight at 42°C for primary enrichment. If no black colonies were present on the XLT4-NA plates, then 1 loopful of enriched sample was streaked onto XLT4NA, which was incubated at 37°C for 24 h, and 1 black colony per plate was streaked on blood agar. One colony per blood agar plate was tested against Salmonella O group B antiserum and confirmed to be Salmonella Heidelberg if agglutination was observed. If no black colonies were present from a sample, the enriched ceca and crop samples were held at room temperature for 5 d, and a delayed secondary enrichment was performed as described above for the drag swabs.
The study design was a randomized complete block with 5 blocks and 4 treatments. The experimental unit was the pen. The study outcomes measured were 1) the percentage of Salmonella Heidelberg isolates from drag swabs, ceca, or crop, and 2) performance data (FCR, weight gain, and mortality), water consumption, and pH values. A sample was considered positive if Salmonella Heidelberg was recovered from direct plating, primary, or secondary enrichments. The percentages of Salmonella Heidelberg isolates from ceca or crops were compared between treatments within seeders or contacts using generalized estimating equation models (for binary data: presence or absence of Salmonella Heidelberg), with logit link function and binomial distribution using an independent correlation structure to account for the block effect in STATA statistical software (ver. 10.1, StataCorp, College Station, TX). The performance data, water consumption, and pH values were compared between treatment groups using generalized estimating equation models, with identity link function, in STATA software. Treatment effects were considered significant at P < 0.05.
RESULTS AND DISCUSSION All paper pads and drag swabs collected at d 0 were negative for Salmonella Heidelberg. All drag swabs collected at 14 d were positive for Salmonella Heidelberg; hence, administration of the EO at the 3 concentrations
Table 2. Effect of essential oil blend on percentages of Salmonella enterica serovar Heidelberg-positive ceca and crop in broilers on d 42 postchallenge1 % (no. of Salmonella-positives/total) Ceca Treatment 0.015% 0.025% 0.050% Control a,bPercentages
All 10 4 8 14
(5/50) (2/50) (4/50) (7/50)
Seeders 12 4 8 24
(3/25) (1/25) (2/25) (6/25)
Crop Contacts 8 4 4 4
(2/25) (1/25) (2/25) (1/25)
All
24a 22b 10b 38a
with different superscripts within a column differ significantly (P < 0.05).
(12/50) (11/50) (5/50) (19/50)
Seeders 32a 36a 12b 52a
(8/25) (9/25) (3/25) (13/25)
Contacts 16a 8a 8a 24a
(4/25) (2/25) (2/25) (6/25)
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Statistical Analysis
from 0 to 7 d did not eliminate shedding of Salmonella Heidelberg 2 wk posttreatment. On d 42, Salmonella Heidelberg was present on drag swabs from 40, 80, 80, and 100% of pens assigned to 0.05, 0.025, 0.015% EO, and controls, respectively. However, these percent differences were not significantly different (P > 0.05). This may indicate that EO treatments did not reduce the horizontal transmission of Salmonella Heidelberg between seeders and contacts. Overall, treatments with EO did not significantly (P > 0.05) decrease the percentage of Salmonella Heidelberg-positive ceca of contact and seeder broilers (Table 2). Treatment with EO significantly (P < 0.05) decreased the percentage of Salmonella Heidelberg-positive crops compared with controls (Table 2). Seeders receiving 0.05% EO had a significantly (P < 0.05) lowered percentage of Salmonella Heidelberg-positive crops compared with 0.025 and 0.015% EO and control. The percentage of Salmonella Heidelberg-positive crop in contacts receiving EO did not significantly (P > 0.05) decrease compared with controls. Based on the data from this study, it appears that EO effect on Salmonella Heidelberg in the crop and not in the ceca. This is in agreement with our previous study findings on EO (Alali et al., 2013). The principal components of the EO blend used in this study were carvacrol, thymol, eucalyptol, lemon, and citric acid. These components may have a synergistic antimicrobial effect against Salmonella Heidelberg. Lambert et al. (2001) reported an in vitro synergistic antimicrobial activity by carvacrol and thymol against Staphylococcus aureus and Pseudomonas aeruginosa. Borsoi et al. (2011) reported that a blend of both organic acids (butyric, propionic, lactic, or formic acids) and essential oils (thymol, carvacrol, citrol, cinnamon, or clove oils) reduced Salmonella cell populations in cloacal samples at 13 and 20 d postinoculation compared with a control group. Eugenol, transcinnamaldehyde, carvacrol, and thymol were tested in vitro against Salmonella Enteritidis in chicken cecal contents (Kollanoor Johny et al., 2010). The authors reported significant Salmonella Enteritidis reduction in cecal contents after 8 and 24 h of incubation at 40°C. Trans-cinnamaldehyde at 0.06% was reported to inactivate S. Enteritidis to undetectable levels in fecally contaminated water (Kollanoor Johny et al., 2008). The EO blend (cinnamaldehyde and thymol) in Amerah et
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Alali et al. Table 3. Effect of essential oil blend on performance results and mortality percentages in broilers following exposure to Salmonella enterica serovar Heidelberg1 Treatment 0.015% 0.025% 0.050% Control3
FCR2 (0 to 42 d)
Average weight gain (kg)
Mortality (%)
1.87a 1.83a 1.79a 1.93b
1.96a 1.97a 2.10a 1.83b
4.0a 4.4a 4.4a 6.0a
a,bMeans
or percentages with different superscripts within a column differ significantly (P < 0.05). or percentages represent 5 pens per treatment, with 50 birds per pen. 2FCR = feed conversion ratio. 3The control birds had no medications in their drinking water. 1Means
cantly different (P > 0.5) compared with controls. This suggests that the amount of water consumed by broilers that received EO did not decrease compared with nontreated controls. The average pH values for crop and ceca of EO treatments were not significantly different (P > 0.5) compared with controls. This suggests that EO treatments did not have a significant effect on the pH compared with nontreated controls. In our study, we used nalidixic acid-resistant Salmonella Heidelberg (1 serotype) as a surrogate for the following reasons: 1) the ability to differentiate the source of Salmonella (inoculated versus environmental), which could confound the results, and 2) inoculation with one serotype could reduce the possibility of multiple serotype interaction, which could confound the results (treatment effect versus interaction effect). The treatments were given to the birds during the first week and last week of the grow-out period. The first week treatment was to adapt the birds’ gastrointestinal system to the water treatments before a second application during the last week of life to reduce Salmonella Heidelberg colonization and shedding. In summary, the 0.05% EO blend in drinking water significantly reduced Salmonella Heidelberg colonization in crops of seeders, lowered the FCR, and increased weight gain compared with controls. The 0.025 and 0.015% EO blend in drinking water significantly lowered FCR and increased weight gain compared with controls.
ACKNOWLEDGMENTS This study was supported in part by a grant from the University of Georgia, Center for Food Safety (Griffin) and a grant from Animal Wellness Products, Reggio
Table 4. Effect of essential oil blend on water consumption and pH measures for ceca and crop in broilers following exposure to Salmonella enterica serovar Heidelberg1 Treatment 0.015% 0.025% 0.050% Control
Water 0 to 7 d
Water 35 to 42 d
Ceca pH2
Crop pH
16.48 16.02 16.00 16.31
112.55 107.64 107.49 103.06
6.6 6.7 6.5 6.4
5.0 4.8 4.8 4.7
1Means represent 5 pens per treatment, with 50 birds per pen. There were no significant differences (P > 0.05) detected among treatments within seeder and contact birds for water consumption and pH measures. 2Ceca and crop of 5 birds per pen were collected for pH measurements.
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al. (2012) significantly reduced Salmonella Heidelbergpositive ceca in broilers challenged with this organism compared with controls. In other studies, EO blends added to feed have resulted in a reduction in Escherichia coli population in the broiler gastrointestinal tract (Jang et al., 2007), whereas increased ceca Lactobacillus and E. coli proportions (Tiihonen et al., 2010). Performance data and mortality results of broilers that received Salmonella Heidelberg challenge are shown in Table 3. Broilers that received EO at the 3 concentrations had significantly (P < 0.05) lower FCR and greater average weight gain than controls. This is in agreement with Amerah et al. (2011, 2012) and Alali et al. (2013) studies. The mortality percentages did not differ significantly (P > 0.05) among EO-treated and control broilers. It was suggested by Bajpai et al. (2012) that EO compounds have a wide range of activities (additive, synergistic, or antagonistic), not only limited to the microflora in the bird, but also the metabolism of the bird. Essential oils from cloves and cinnamon reportedly improved the FCR and percentage of breast weight of broilers compared with those receiving calcium propionate or calcium formate (Isabel and Santos, 2009). Furthermore, carvacrol added to broiler bird feed at 200 ppm reduced weight gain and feed intake, but improved the feed:gain ratio after 4 wk of feeding this diet (Lee et al., 2003). Kollanoor-Johny et al. (2012) revealed that trans-cinnamaldehyde added to 20-d-old broilers feed had no effect on the BW; however, eugenol significantly reduced broiler BW compared with controls. The water consumption and pH results of broilers that received Salmonella Heidelberg challenge are shown in Table 4. The average water consumption over 0 to 7 and 35 to 42 d for EO treatments was not signifi-
ESSENTIAL OIL AND SALMONELLA IN BROILERS
Emilia, Italy. We thank Diezhang Wu, Abdel-Rahman Alsleh, Christine Lobsinger, and PDRC (Athens, GA) technicians for their assistance in processing samples and performing microbiological assays. We also thank Animal Wellness Products and Vytol BioSystems (Oakland, NE) for providing us with their essential oil product.
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