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Effect of xylanase and a blend of essential oils on performance and Salmonella colonization of broiler chickens challenged with Salmonella Heidelberg
Effect of xylanase and a blend of essential oils on performance and Salmonella colonization of broiler chickens challenged with Salmonella Heidelberg A. M. Amerah,*1 G. Mathis,† and C. L. Hofacre‡ *Danisco Animal Nutrition, Marlborough, SN8 1XN UK; †Southern Poultry Research Inc., Athens, Georgia 30607; and ‡Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602 ing GLM. The frequency of positive Salmonella in the untagged birds was compared between treatments by using a chi-squared test of homogeneity. Challenging the birds with Salmonella had no effect (P > 0.05) on any of the measured performance parameters. Xylanase and EO supplementation improved (P < 0.05) the 42-d BW gain and feed efficiency, with no effect (P > 0.05) on feed intake, compared with that of the control treatment. Xylanase supplementation improved (P < 0.05) BW gain and feed efficiency compared with the results of EO supplementation. The combination treatment of xylanase and EO numerically improved BW gain and feed efficiency compared with the xylanase treatment. Xylanase and EO supplementation reduced (P < 0.05) the incidence of horizontal transmission of Salmonella infection between birds by 61 and 77%, respectively, compared with the control. The results of the current study suggested that dietary addition of EO and xylanase could improve broiler performance and contribute to food safety by lowering the incidence of horizontal transmission of Salmonella infection.
Key words: xylanase, essential oil, wheat, broiler, Salmonella 2012 Poultry Science 91:943–947 http://dx.doi.org/10.3382/ps.2011-01922
INTRODUCTION
thermore, xylanase supplementation of birds fed wheatbased diets has been reported to influence gut microflora and reduce the coliforms, lactic acid bacteria, enterococci, and the total bacterial count in the small intestine (Vahjen et al., 1998; Bedford and Apajalathi, 2001). Sinlae and Choct (2000) reported a reduction in the number of Clostridium perfringens in birds fed diets supplemented with xylanase compared with the control birds in broilers fed a wheat-based diet. A recent study showed that xylanase supplementation to a wheat-based diet can reduce the number of coliform bacteria and Salmonellae and can increase the number of lactobacilli in the ileum (Nian et al., 2011). Essential oils (EO) are steam-volatile or organic-solvent extracts of plants, usually used for the pleasant odor of their essence, their flavor, their antiseptic or
Wheat is a major raw material in poultry feeds in many parts of the world. The nonstarch polysaccharide (NSP) fraction of the wheat (mainly arabinoxylans) is the major contributing factor for the viscous nature of intestinal digesta and the negative effects on broiler performance, nutrient digestibility, and gut microflora (Choct and Annison, 1992; Bedford and Schulze, 1998). Currently, exogenous xylanases are routinely used in broiler diets to mitigate the adverse effects of NSP. Fur©2012 Poultry Science Association Inc. Received October 7, 2011. Accepted December 26, 2011. 1 Corresponding author: [email protected]
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ABSTRACT The present experiment examined the influence of xylanase supplementation and a blend of essential oils (EO; cinnamaldehyde and thymol) on performance and Salmonella horizontal transmission in broiler chickens challenged with Salmonella. Two thousand 1-d-old broiler chicks were randomly assigned to 5 dietary treatments (8 pens/treatment of 50 male broilers each). Four dietary treatments were challenged with Salmonella: 1) control, 2) basal diets supplemented with EO, 3) basal diet supplemented with xylanase (2,000 U/kg of feed), and 4) basal diet supplemented with a combination of EO and xylanase (2,000 U/kg of feed). One treatment served as an unchallenged control and was not supplemented with either additive. Broiler starter and finisher diets, based on wheat and soybean meal, were formulated, pelleted, and fed ad libitum. At d 1, before placement, half of the birds from each pen were tagged and dosed with Salmonella enterica serovar Heidelberg (5 × 105 cfu/mL). On d 42, 5 random untagged birds from each pen were killed and their ceca removed and tested for Salmonella. Performance data were analyzed as a completely randomized design us-
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MATERIALS AND METHODS Diets and Bird Management One-day-old male broilers (Cobb × Cobb) were obtained from a commercial hatchery and randomly assigned to 5 dietary treatments (8 pens/treatment of 50 male broilers each). Treatment 1 (control) received feed containing no feed additives and was not challenged with Salmonella. Treatment 2 (challenge control) contained no feed additives but was challenged with Salmonella. Treatment 3 received feed supplemented with EO (100 g/tonne of diet; Enviva EO 101, Danisco Animal Nutrition, Marlborough, UK; active ingredients were cinnamaldehyde and thymol) and was challenged with Salmonella. Treatment 4 received feed supplemented with xylanase (2,000 U/kg of feed, Danisco Xylanase, Danisco Animal Nutrition) and was challenged with Salmonella. Treatment 5 received feed containing a combination of EO (100 g/tonne of diet; Enviva EO 101) and xylanase (2,000 U/kg of feed) and
Table 1. Composition and calculated analysis of the basal diets Item Ingredient (g/kg) Maize Wheat Soybean meal 48 Soybean oil dl-Methionine l-Threonine Salt Limestone Dicalcium phosphate Lysine HCl Vitamin and mineral premix1 Phytase2 Calculated analysis ME (kcal/kg) CP (%) Lysine (%) Methionine + cystine (%) Calcium3 (%) Available phosphorus3 (%)
Starter
150 512 289 9.3 2.9 1.2 3.3 134 11 3.6 3.0 + 2,900 21.5 1.35 0.97 1.00 0.45
Finisher
0.00 692 244 32 2.1 0.7 3.2 14 5.0 3.0 3.0 + 3,050 20.0 1.19 0.86 0.85 0.40
1The vitamin mix provided the following (per kilogram of diet): thiamine mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; d-calcium pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 μg; pyridoxine hydrochloride, 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; 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; and selenium (NaSeO3), 0.3 mg. 2Phyzyme XP 10000 TPT, Danisco Animal Nutrition, Marlborough, UK. The enzyme was included at a rate of 50 g/t to supply a guaranteed minimum of 500 FTU/kg of feed. 3Includes the contribution from phytase of 0.11% Ca and 0.12% available P.
was challenged with Salmonella. All birds were sprayvaccinated with Coccivac-B (Intervet/Schering-Plough Animal Health Corp., Millsboro, DE). Broiler starter and finisher diets, based on wheat and soybean meal (Table 1), were formulated, pelleted, and fed ad libitum. All diets were supplemented with 500 FTU/kg of diet phytase (Phyzyme XP, Danisco Animal Nutrition). For each pen, BW and feed intake were recorded at 21 and 42 d of age. Mortality was recorded daily. Any bird that died was weighed and the feed-to-gain values were calculated by dividing total feed intake by weight gain of live plus dead birds.
Salmonella Challenge and Testing Except for the unchallenged control (treatment 1), on d 1, before placement, 25 chicks per pen were tagged for identification and then orally dosed with 0.1 mL of nalidixic acid-resistant (32 μg/mL; Sigma, St. Louis, MO) Salmonella Heidelberg (5 × 105 cfu/mL). The Salmonella environmental sampling was by drag swabs (Kinde et al., 2004) from all pens on d 14 and 42 and analyzed for Salmonella as described by Alali et al. (2011). On d 42, 10 birds per pen (5 tagged and 5 nontagged) were killed by cervical dislocation and the ceca were aseptically removed and placed into sterile plastic sampling bags (Fisher Scientific Inc., Pittsburgh, PA) for Salmonella isolation. To these samples, 50 mL of
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preservative properties, as well as their antimicrobial effect (Burt, 2004; Lee et al., 2004; Wallace, 2004). The antimicrobial properties of EO have encouraged their use as a natural replacement for antibiotic growth promoters in animal feeds. In addition to the positive effects of EO against the colonization and proliferation of pathogenic bacteria (Lee et al., 2004), EO have been shown to improve nutrient digestibility and broiler performance (Cao et al., 2010; Amerah et al., 2011). Recent studies have highlighted the potential benefit of combining EO and carbohydrase enzymes on broiler performance and nutrient digestibility (Cao et al., 2010). Salmonella enterica serovars Enteritidis and Typhimurium are the common serovars causing salmonellosis (Van Immerseel et al., 2009). However, a recent outbreak of Salmonella enterica serovar Heidelberg infections in the United States has increased the interest in this serovar of Salmonella as a potential livestockborne pathogen for humans. Borsoi et al. (2011) reported similar changes in the intestinal mucosa caused by Salmonella Heidelberg compared with those caused by Salmonella Enteritidis in broiler chickens, suggesting that Salmonella Heidelberg may have importance as a pathogen to newly hatched chicks and is a potential broiler carcass contaminant. Using feed additives to beneficially affect both performance and Salmonella colonization in broilers would be of interest to the poultry industry and can be an important tool to control Salmonella in broilers (Eeckhaut et al., 2008). To our knowledge, combining the effects of EO and carbohydrase enzymes on Salmonella transmission control, with possible complementary effect, has not yet been investigated. Therefore, the objectives of this study were to investigate the effects of combining xylanase and EO on performance and Salmonella transmission in broiler chickens fed wheat-based diets.
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tetrathionate broth (Young et al., 2007; Difco, Sparks, MD) with brilliant green broth (Sigma) and iodine (Sigma) was added, and the samples were mechanically homogenized using a stomacher (Technar Company, Cincinnati, OH) for 20 s. These samples were then incubated at 41.5°C and cultured for Salmonella as described below.
Isolation and Identification of Salmonella
Statistical Analysis For performance parameters, pen means served as the experimental unit for statistical analysis. All data were subjected to one-way ANOVA using the GLM procedure of SAS (SAS Institute, 2002). The differences were considered significant at P < 0.05, and significant differences between means were separated by the least significant difference test. The frequency of positive Salmonella in the untagged birds was compared between treatments by using a chi-squared test of homogeneity.
RESULTS
Salmonella Challenge and Testing The influence of treatments on the incidence of horizontal transmission of Salmonella infection between birds and the Salmonella environmental sampling by drag swab is summarized in Table 3. Xylanase and EO supplementation reduced (P < 0.05) the incidence of horizontal transmission of Salmonella infection between birds compared with the challenged control. The combination treatment of EO and xylanase had no further reduction effect on the incidence of horizontal transmission of Salmonella infection between birds compared with the EO and xylanase treatments. On d 14, the combination treatment of EO and xylanase reduced (P < 0.05) the positive drag-swab samples compared with the control. On d 42, xylanase supplementation had no effect on the positive drag-swab samples compared with the challenged control. However, EO and the combination treatment of EO and xylanase reduced (P < 0.05) the positive drag-swab samples compared with the challenged control.
DISCUSSION
Bird Performance The influence of treatments on the performance of broilers is summarized in Table 2. During the start-
The reduction in human cases of salmonellosis caused by poultry meat consumption is of great importance for
Table 2. Weight gain, feed intake, and feed conversion ratio (FCR) of broilers as influenced by xylanase supplementation (2,000 U/ kg of feed) and essential oils (100 g/t)1 Item 1–21 d Weight gain (g) Feed intake (g) FCR 21–42 d Weight gain (g) Feed intake (g) FCR 1–42 d Weight gain (g) Feed intake (g) FCR a–cMeans
Challenged control
Control
422c 658 1.56a 1,360c 2,994 2.20a 1,781c 3,652 2.01a
419c 659 1.58a 1,381c 3,047 2.21a 1,800c 3,706 2.01a
Essential oils
442b 649 1.47b 1,481b 3,072 2.07b 1,924b 3,721 1.90b
in a row not sharing a common superscript are significantly different (P < 0.05). value represents the mean of 8 replicates (50 birds/replicate). 2Pooled SEM. 1Each
Essential oils + xylanase
Xylanase 469a 659 1.41c 1,615a 3,088 1.91c 2,084a 3,747 1.78c
461a 644 1.40c 1,681a 3,109 1.85c 2,142a 3,753 1.73c
SEM2
5.7 7.1 0.01 29 77 0.03 27 70 0.02
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A 10-μL loop of the enriched sample was streaked onto Xylose Lys Tergitol-4 (XLT4) agar plates containing nalidixic acid (25 μg/mL; Sigma) and incubated at 37°C overnight, and then they were held at room temperature for 24 h before reading. The H2S-positive isolated colonies were streaked onto blood agar and incubated at 37°C with 7% CO2 overnight. Suspect Salmonella colonies were confirmed using poly O group B Salmonella-specific antiserum. Negative enrichments were held for 5 d, enriched again, and streaked onto XLT4 to confirm results. Any Salmonella recovered on secondary enrichment were verified as described previously.
er phase (1–21 d), finisher phase (22–42 d), and over the whole trial period (1–42 d), challenging the birds with Salmonella had no effect (P > 0.05) on any of the measured performance parameters compared with the unchallenged control. Xylanase and EO supplementation improved (P < 0.05) the 42-d BW gain and feed efficiency with no effect (P > 0.05) on feed intake compared with those of the control treatment. Xylanase supplementation improved (P < 0.05) BW gain and feed efficiency compared with those of the EO treatment birds. The combination treatment of xylanase and EO numerically improved (P > 0.05) BW gain and feed efficiency compared with those of the xylanasesupplemented diet.
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Table 3. Effect of essential oils (100 g/t) and xylanase supplementation (2,000 U/kg of feed) on Salmonella prevalence Item Salmonella-positive cecal samples on d 42 (%)1 Positive drag swab samples at 14 d (%)2 Positive drag swab samples at 42 d (%)2
Unchallenged control
Challenged control
Essential oils
Xylanase
Essential oils + xylanase
0 0 0
32.5a 100a 100a
7.5b 87.5ab 62.5b
12.5b 87.5ab 100a
7.5b 62.5b 62.5b
a,bValues
in a row not sharing a common superscript are significantly different (P < 0.05). value represents the percentage of Salmonella-positive cecal samples from 40 replicates (5 unchallenged birds/replicate pen). 2Each value represents the percentage of positive drag swabs of 8 replicates. 1Each
spleen, liver, and ceca of broilers fed a maize-based diet in comparison to those given a wheat/rye-based diet. It has been suggested that xylanase improves nutrient digestibility and, therefore, reduces the nutrients available for pathogenic bacteria in the small intestine (Choct, 2006; Vandeplas et al., 2009). Another possible explanation is that xylanase hydrolysis of arabinoxylan into fragments of arabinoxylan-oligosaccharides acted as prebiotics and stimulated the intestinal immune responses or stimulated beneficial populations, which can compete in the intestine with Salmonella and restore the microbial balance (Patterson and Burkholder, 2003). Eeckhaut et al. (2008) concluded that dietary addition of arabinoxylooligosaccharides prepared from wheat bran by extraction with endoxylanase provides dose-dependent protection against oral infections with Salmonella Enteritidis in poultry. It should be noted, in the current study, that although xylanase supplementation reduced the number of Salmonella-positive cecal samples, it had no effect on positive drag-swab samples on d 42, suggesting no effect of xylanase on environmental Salmonella. Essential oils improve digestive physiology, stimulate blood circulation, exert antioxidant properties, reduce levels of pathogenic bacteria, and may enhance immune status (Brenes and Roura, 2010). Several in vitro studies showed antibacterial effects of some essential oils against Salmonella. Smith-Palmer et al. (1998) reported that the oils of bay, cinnamon, clove, and thyme had the most inhibitory effect on 5 important food-borne pathogens (Escherichia coli, Salmonella enteritidis, Campylobacter jejuni, Staphylococcus aureus, Listeria monocytogenes). Helander et al. (1998) reported that carvacrol, thymol, and trans-cinnamaldehyde showed an antibacterial inhibitory effect against Escherichia coli and Salmonella Typhimurium. In the current study, the EO combination of thymol and cinnamaldehyde reduced the percentage of Salmonella-positive samples of the uninfected birds and in the environment (drag-swab samples). Using a similar blend of thymol and cinnamaldehyde, Tiihonen et al. (2010) showed a reduction of Escherichia coli and Salmonella in cecal samples of broiler chickens fed a wheat-based diet. Cox et al. (1994) stated that reductions in the levels of Salmonella in the intestines mean less transmission of the bacteria in the growing house and during processing, and therefore, less contaminated final product. There-
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both producers and consumers alike. It is unlikely that the use of feed additives alone can eliminate Salmonella. However, reduction of Salmonella numbers in the intestine and the number of contaminated carcasses can help in improving food safety (Corry et al., 2002). Starting in July 2011, the USDA Food Safety and Inspection Service changed the performance standards, based on the percentgage of carcasses testing positive for Salmonella, to be no more than 9.8% positives, compared with 23% in the previous recommendations (USDA/FSIS, 2010). It is believed that with these changes, 26,000 cases of salmonellosis can be avoided each year (USDA/FSIS, 2010). In the last survey in the European Union, the prevalence of Salmonella-contaminated broiler carcasses was 15.6% in the abattoir (EFSA, 2008). Therefore, using certain feed additives to reduce the incidence of Salmonella-contaminated broiler carcasses would be of great interest to the industry. In the current study, challenging the birds with Salmonella had no effect on broiler performance. Similarly, Mountzouris et al. (2009) reported no effect on 42-d broiler performance when challenged with Salmonella Enteritidis. In contrast, other researchers reported that challenging broilers with Salmonella resulted in a significant deterioration of growth performance (Vandeplas et al., 2009; Marcq et al., 2011). Reduced growth performance has been attributed to the decreased feed intake by challenged birds due to mucosal damage (Vandeplas et al., 2009). These contradictory results may be explained by differences in the species and strains or dose of Salmonella used to challenge the birds, which results in different levels of intestinal damage (Malago et al., 2003) or because of differences in cereal type used in the study (Teirlynck et al., 2009). In the present study, xylanase supplementation improved broiler performance and reduced the number of Salmonella-positive cecal samples. This result is consistent with the findings of other researchers who have observed improved performance and a reduction of Salmonella in the cecum of broilers fed a wheat-based diet supplemented with xylanase (Nian et al., 2011). Similarly, Vandeplas et al. (2009) reported a significant reduction in Salmonella Typhimurium concentration in excreta of 30-d-old broilers infected with Salmonella Typhimurium when a wheat-based diet was supplemented with xylanase. Teirlynck et al. (2009) reported a significantly lower Salmonella colonization in the
XYLANASE, ESSENTIAL OILS, AND SALMONELLA IN BROILERS
fore, the current data suggest that EO supplementation based on thymol and cinnamaldehyde could be used as part of a Salmonella control program. In conclusion, feed additives, such as enzymes and EO, could be used as part of an integrated program to control Salmonella levels in poultry production. The results of the current study suggested that dietary addition of EO and xylanase could improve broiler performance and contribute to food safety by lowering the incidence of horizontal transmission of Salmonella infection between birds by 77 and 61%, respectively, compared with the control. Further trials are warranted to consolidate these findings at a commercial level.
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Helander, I. M., H. L. Alakom, K. Latva-Kala, T. Mattila-Sandholm, E. J. Smid, L. G. M. Gorris, and A. Von Wright. 1998. Characterization of the action of selected essential oil components on gram-negative bacteria. J. Agric. Food Chem. 46:3590–3595. Kinde, H., D. M. Castellan, P. H. Kass, A. Ardans, G. Cutler, R. E. Breitmeyer, D. D. Bell, R. A. Ernst, D. C. Kerr, H. E. Little, D. Willoughby, H. P. Riemann, J. A. Snowdon, and D. R. Kuney. 2004. The occurrence and distribution of Salmonella Enteritidis and other serovars on California egg laying premises: A comparison of two sampling methods and two culturing techniques. Avian Dis. 48:590–594. Lee, K. W., H. Everts, H. J. Kappert, H. Wouterse, M. Frehner, and A. C. Beynen. 2004. Cinnamanaldehyde, but not thymol, counteracts the carboxymethyl cellulose-induced growth depression in female broiler chickens. Int. J. Poult. Sci. 3:608–612. Malago, J. J., J. F. Koninkx, H. H. Ovelgönne, F. J. van Asten, J. F. Swennenhuis, and J. E. van Dijk. 2003. Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella Enteritidis. Cell Stress Chaperones 8:194–203. Marcq, C., E. Cox, I. M. Szalo, A. Théwis, and Y. Beckers. 2011. Salmonella Typhimurium oral challenge model in mature broilers: Bacteriological, immunological, and growth performance aspects. Poult. Sci. 90:59–67. Mountzouris, K. C., C. Balaskas, I. Xanthakos, A. Tzivinikou, and K. Fegeros. 2009. Effects of a multi-species probiotic on biomarkers of competitive exclusion efficacy in broilers challenged with Salmonella Enteritidis. Br. Poult. Sci. 50:467–478. Nian, F., Y. M. Guo, Y. J. Ru, F. D. Li, and A. Peron. 2011. Effect of exogenous xylanase supplementation on the performance, net energy and gut microflora of broiler chickens fed wheat-based diets. Asian-australas. J. Anim. Sci. 24:400–406. Patterson, J. A., and K. M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. Poult. Sci. 82:627– 631. SAS Institute. 2002. SAS/STAT User’s Guide: Release 9.03 ed. SAS Inst. Inc., Cary, NC. Sinlae, M., and M. Choct. 2000. Xylanase supplementation affects the gut microflora of broilers. Proc. Aust. Poult. Sci. Symp. University of Sydney, Sydney, NSW, Australia. 12:209. Smith-Palmer, A., J. Stewart, and L. Fyfe. 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26:118–122. Teirlynck, E., F. Haesebrouck, F. Pasmans, J. Dewulf, L. Bjerrum, R. Ducatelle, and F. Van Immerseel. 2009. The cereal type in feed influences Salmonella Enteritidis colonization in broilers. Poult. Sci. 88:2108–2112. Tiihonen, K., H. Kettunen, M. H. Bento, M. Saarinen, S. Lahtinen, A. C. Ouwehand, H. Schulze, and N. Rautonen. 2010. The effect of feeding essential oils on broiler performance and gut microbiota. Br. Poult. Sci. 51:381–392. USDA/FSIS. 2010. Potential public health impact of Salmonella and Campylobacter performance guidance for young chickens and turkeys. Accessed June 20, 2011. http://www.fsis.usda.gov/ PDF/Potential_Public_Health_Impact_Salmonella_Campylobacter%20Performance%20Guidance_Chickens_Turkeys.pdf. Vahjen, W., K. Glaser, K. Schafer, and O. Simon. 1998. Influence of xylanase-supplemented feed on the development of selected bacterial groups in the intestinal tract of broiler chicks. J. Agric. Sci. 130:489–500. Van Immerseel, F., L. De Zutter, K. Houf, F. Pasmans, F. Haesebrouck, and R. Ducatelle. 2009. Strategies to control Salmonella in the broiler production chain. World’s Poult. Sci. J. 65:367–391. Vandeplas, S., R. Dubois Dauphin, C. Thiry, Y. Beckers, G. W. Welling, P. Thonart, and A. Théwis. 2009. Efficiency of a Lactobacillus plantarum-xylanase combination on growth performances, microflora populations, and nutrient digestibilities of broilers infected with Salmonella Typhimurium. Poult. Sci. 88:1643–1654. Wallace, R. J. 2004. Antimicrobial properties of plant secondary metabolites. Proc. Nutr. Soc. 63:621–629. Young, S. D., O. Olusanya, K. H. Jones, T. Liu, K. A. Liljebjelke, and C. L. Hofacre. 2007. Salmonella incidence in broilers from breeders vaccinated with live and killed Salmonella. J. Appl. Poult. Res. 16:521–528.
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Alali, W. Q., C. L. Hofacre, G. F. Mathis, and A. B. Batal. 2011. Effect of plant-based protein meal use in poultry feed on colonization and shedding of Salmonella Heidelberg in broiler birds. Agric. Food Anal. Bacteriol. 1:45–53. Amerah, A. M., A. Peron, F. Zaefarian, and V. Ravindran. 2011. Influence of whole wheat inclusion and a blend of essential oils on the performance, nutrient utilization, digestive tract development and ileal microbiota profile of broiler chickens. Br. Poult. Sci. 52:124–132. Bedford, M. R., and J. Apajalathi. 2001. Microbial interactions in the response to exogenous enzyme utilization. Pages 299–314 in Enzymes in Farm Animal Nutrition. M. R. Bedford, and G. G. Partridge, ed. CABI Publishing, London, UK. Bedford, M. R., and H. Schulze. 1998. Exogenous enzymes for pigs and poultry. Nutr. Res. Rev. 11:91–114. Borsoi, A., L. R. Santos, L. B. Rodrigues, H. L. S. Moraes, C. T. P. Salle, and V. P. Nascimento. 2011. Behavior of Salmonella Heidelberg and Salmonella Enteritidis strains following broiler chick inoculation: Evaluation of cecal morphometry, liver and cecum bacterial counts and fecal excretion patterns. Braz. J. Microbiol. 42:266–273. Brenes, A., and E. Roura. 2010. Essential oils in poultry nutrition: Main effects and modes of action. Anim. Feed Sci. Technol. 158:1–14. Burt, S. 2004. Essential oils: Their antibacterial properties and potential applications in foods—A review. Int. J. Food Microbiol. 94:223–253. Cao, P. H., F. D. Li, Y. F. Li, Y. J. Ru, A. Peron, H. Schulze, and H. Bento. 2010. Effect of essential oils and feed enzymes on performance and nutrient utilization in broilers fed a corn/soy-based diet. Int. J. Poult. Sci. 9:749–755. Choct, M. 2006. Enzymes for the feed industry: Past, present and future. World’s Poult. Sci. J. 62:5–15. Choct, M., and G. Annison. 1992. Anti-nutritive effect of wheat pentosans in broiler: Roles of viscosity and gut microflora. Br. Poult. Sci. 33:821–834. Corry, J. E. L., V. M. Allen, W. R. Hudson, M. F. Breslin, and R. H. Davies. 2002. Sources of Salmonella on broiler carcasses during transportation and processing: Modes of contamination and methods of control. J. Appl. Microbiol. 92:424–432. Cox, N. A., F. McHan, and J. S. Bailey. 1994. Effect of butyric or lactic acid on the in vivo colonization of Salmonella typhimurium. J. Appl. Poult. Res. 3:314–318. Eeckhaut, V., F. Van Immerseel, J. Dewulf, F. Pasmans, F. Haesebrouck, R. Ducatelle, C. M. Courtin, J. A. Delcour, and W. F. Broeckaert. 2008. Arabinoxylooligosaccharides from wheat bran inhibit Salmonella colonization in broiler chickens. Poult. Sci. 87:2329–2334. EFSA. 2008. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008. Accessed June 20, 2011. http://www.efsa.europa.eu/fr/scdocs/doc/1503.pdf.
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Key words: xylanase, essential oil, wheat, broiler, Salmonella 2012 Poultry Science 91:943–947 http://dx.doi.org/10.3382/ps.2011-01922
INTRODUCTION
thermore, xylanase supplementation of birds fed wheatbased diets has been reported to influence gut microflora and reduce the coliforms, lactic acid bacteria, enterococci, and the total bacterial count in the small intestine (Vahjen et al., 1998; Bedford and Apajalathi, 2001). Sinlae and Choct (2000) reported a reduction in the number of Clostridium perfringens in birds fed diets supplemented with xylanase compared with the control birds in broilers fed a wheat-based diet. A recent study showed that xylanase supplementation to a wheat-based diet can reduce the number of coliform bacteria and Salmonellae and can increase the number of lactobacilli in the ileum (Nian et al., 2011). Essential oils (EO) are steam-volatile or organic-solvent extracts of plants, usually used for the pleasant odor of their essence, their flavor, their antiseptic or
Wheat is a major raw material in poultry feeds in many parts of the world. The nonstarch polysaccharide (NSP) fraction of the wheat (mainly arabinoxylans) is the major contributing factor for the viscous nature of intestinal digesta and the negative effects on broiler performance, nutrient digestibility, and gut microflora (Choct and Annison, 1992; Bedford and Schulze, 1998). Currently, exogenous xylanases are routinely used in broiler diets to mitigate the adverse effects of NSP. Fur©2012 Poultry Science Association Inc. Received October 7, 2011. Accepted December 26, 2011. 1 Corresponding author: [email protected]
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ABSTRACT The present experiment examined the influence of xylanase supplementation and a blend of essential oils (EO; cinnamaldehyde and thymol) on performance and Salmonella horizontal transmission in broiler chickens challenged with Salmonella. Two thousand 1-d-old broiler chicks were randomly assigned to 5 dietary treatments (8 pens/treatment of 50 male broilers each). Four dietary treatments were challenged with Salmonella: 1) control, 2) basal diets supplemented with EO, 3) basal diet supplemented with xylanase (2,000 U/kg of feed), and 4) basal diet supplemented with a combination of EO and xylanase (2,000 U/kg of feed). One treatment served as an unchallenged control and was not supplemented with either additive. Broiler starter and finisher diets, based on wheat and soybean meal, were formulated, pelleted, and fed ad libitum. At d 1, before placement, half of the birds from each pen were tagged and dosed with Salmonella enterica serovar Heidelberg (5 × 105 cfu/mL). On d 42, 5 random untagged birds from each pen were killed and their ceca removed and tested for Salmonella. Performance data were analyzed as a completely randomized design us-
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MATERIALS AND METHODS Diets and Bird Management One-day-old male broilers (Cobb × Cobb) were obtained from a commercial hatchery and randomly assigned to 5 dietary treatments (8 pens/treatment of 50 male broilers each). Treatment 1 (control) received feed containing no feed additives and was not challenged with Salmonella. Treatment 2 (challenge control) contained no feed additives but was challenged with Salmonella. Treatment 3 received feed supplemented with EO (100 g/tonne of diet; Enviva EO 101, Danisco Animal Nutrition, Marlborough, UK; active ingredients were cinnamaldehyde and thymol) and was challenged with Salmonella. Treatment 4 received feed supplemented with xylanase (2,000 U/kg of feed, Danisco Xylanase, Danisco Animal Nutrition) and was challenged with Salmonella. Treatment 5 received feed containing a combination of EO (100 g/tonne of diet; Enviva EO 101) and xylanase (2,000 U/kg of feed) and
Table 1. Composition and calculated analysis of the basal diets Item Ingredient (g/kg) Maize Wheat Soybean meal 48 Soybean oil dl-Methionine l-Threonine Salt Limestone Dicalcium phosphate Lysine HCl Vitamin and mineral premix1 Phytase2 Calculated analysis ME (kcal/kg) CP (%) Lysine (%) Methionine + cystine (%) Calcium3 (%) Available phosphorus3 (%)
Starter
150 512 289 9.3 2.9 1.2 3.3 134 11 3.6 3.0 + 2,900 21.5 1.35 0.97 1.00 0.45
Finisher
0.00 692 244 32 2.1 0.7 3.2 14 5.0 3.0 3.0 + 3,050 20.0 1.19 0.86 0.85 0.40
1The vitamin mix provided the following (per kilogram of diet): thiamine mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; d-calcium pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 μg; pyridoxine hydrochloride, 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; 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; and selenium (NaSeO3), 0.3 mg. 2Phyzyme XP 10000 TPT, Danisco Animal Nutrition, Marlborough, UK. The enzyme was included at a rate of 50 g/t to supply a guaranteed minimum of 500 FTU/kg of feed. 3Includes the contribution from phytase of 0.11% Ca and 0.12% available P.
was challenged with Salmonella. All birds were sprayvaccinated with Coccivac-B (Intervet/Schering-Plough Animal Health Corp., Millsboro, DE). Broiler starter and finisher diets, based on wheat and soybean meal (Table 1), were formulated, pelleted, and fed ad libitum. All diets were supplemented with 500 FTU/kg of diet phytase (Phyzyme XP, Danisco Animal Nutrition). For each pen, BW and feed intake were recorded at 21 and 42 d of age. Mortality was recorded daily. Any bird that died was weighed and the feed-to-gain values were calculated by dividing total feed intake by weight gain of live plus dead birds.
Salmonella Challenge and Testing Except for the unchallenged control (treatment 1), on d 1, before placement, 25 chicks per pen were tagged for identification and then orally dosed with 0.1 mL of nalidixic acid-resistant (32 μg/mL; Sigma, St. Louis, MO) Salmonella Heidelberg (5 × 105 cfu/mL). The Salmonella environmental sampling was by drag swabs (Kinde et al., 2004) from all pens on d 14 and 42 and analyzed for Salmonella as described by Alali et al. (2011). On d 42, 10 birds per pen (5 tagged and 5 nontagged) were killed by cervical dislocation and the ceca were aseptically removed and placed into sterile plastic sampling bags (Fisher Scientific Inc., Pittsburgh, PA) for Salmonella isolation. To these samples, 50 mL of
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preservative properties, as well as their antimicrobial effect (Burt, 2004; Lee et al., 2004; Wallace, 2004). The antimicrobial properties of EO have encouraged their use as a natural replacement for antibiotic growth promoters in animal feeds. In addition to the positive effects of EO against the colonization and proliferation of pathogenic bacteria (Lee et al., 2004), EO have been shown to improve nutrient digestibility and broiler performance (Cao et al., 2010; Amerah et al., 2011). Recent studies have highlighted the potential benefit of combining EO and carbohydrase enzymes on broiler performance and nutrient digestibility (Cao et al., 2010). Salmonella enterica serovars Enteritidis and Typhimurium are the common serovars causing salmonellosis (Van Immerseel et al., 2009). However, a recent outbreak of Salmonella enterica serovar Heidelberg infections in the United States has increased the interest in this serovar of Salmonella as a potential livestockborne pathogen for humans. Borsoi et al. (2011) reported similar changes in the intestinal mucosa caused by Salmonella Heidelberg compared with those caused by Salmonella Enteritidis in broiler chickens, suggesting that Salmonella Heidelberg may have importance as a pathogen to newly hatched chicks and is a potential broiler carcass contaminant. Using feed additives to beneficially affect both performance and Salmonella colonization in broilers would be of interest to the poultry industry and can be an important tool to control Salmonella in broilers (Eeckhaut et al., 2008). To our knowledge, combining the effects of EO and carbohydrase enzymes on Salmonella transmission control, with possible complementary effect, has not yet been investigated. Therefore, the objectives of this study were to investigate the effects of combining xylanase and EO on performance and Salmonella transmission in broiler chickens fed wheat-based diets.
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tetrathionate broth (Young et al., 2007; Difco, Sparks, MD) with brilliant green broth (Sigma) and iodine (Sigma) was added, and the samples were mechanically homogenized using a stomacher (Technar Company, Cincinnati, OH) for 20 s. These samples were then incubated at 41.5°C and cultured for Salmonella as described below.
Isolation and Identification of Salmonella
Statistical Analysis For performance parameters, pen means served as the experimental unit for statistical analysis. All data were subjected to one-way ANOVA using the GLM procedure of SAS (SAS Institute, 2002). The differences were considered significant at P < 0.05, and significant differences between means were separated by the least significant difference test. The frequency of positive Salmonella in the untagged birds was compared between treatments by using a chi-squared test of homogeneity.
RESULTS
Salmonella Challenge and Testing The influence of treatments on the incidence of horizontal transmission of Salmonella infection between birds and the Salmonella environmental sampling by drag swab is summarized in Table 3. Xylanase and EO supplementation reduced (P < 0.05) the incidence of horizontal transmission of Salmonella infection between birds compared with the challenged control. The combination treatment of EO and xylanase had no further reduction effect on the incidence of horizontal transmission of Salmonella infection between birds compared with the EO and xylanase treatments. On d 14, the combination treatment of EO and xylanase reduced (P < 0.05) the positive drag-swab samples compared with the control. On d 42, xylanase supplementation had no effect on the positive drag-swab samples compared with the challenged control. However, EO and the combination treatment of EO and xylanase reduced (P < 0.05) the positive drag-swab samples compared with the challenged control.
DISCUSSION
Bird Performance The influence of treatments on the performance of broilers is summarized in Table 2. During the start-
The reduction in human cases of salmonellosis caused by poultry meat consumption is of great importance for
Table 2. Weight gain, feed intake, and feed conversion ratio (FCR) of broilers as influenced by xylanase supplementation (2,000 U/ kg of feed) and essential oils (100 g/t)1 Item 1–21 d Weight gain (g) Feed intake (g) FCR 21–42 d Weight gain (g) Feed intake (g) FCR 1–42 d Weight gain (g) Feed intake (g) FCR a–cMeans
Challenged control
Control
422c 658 1.56a 1,360c 2,994 2.20a 1,781c 3,652 2.01a
419c 659 1.58a 1,381c 3,047 2.21a 1,800c 3,706 2.01a
Essential oils
442b 649 1.47b 1,481b 3,072 2.07b 1,924b 3,721 1.90b
in a row not sharing a common superscript are significantly different (P < 0.05). value represents the mean of 8 replicates (50 birds/replicate). 2Pooled SEM. 1Each
Essential oils + xylanase
Xylanase 469a 659 1.41c 1,615a 3,088 1.91c 2,084a 3,747 1.78c
461a 644 1.40c 1,681a 3,109 1.85c 2,142a 3,753 1.73c
SEM2
5.7 7.1 0.01 29 77 0.03 27 70 0.02
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A 10-μL loop of the enriched sample was streaked onto Xylose Lys Tergitol-4 (XLT4) agar plates containing nalidixic acid (25 μg/mL; Sigma) and incubated at 37°C overnight, and then they were held at room temperature for 24 h before reading. The H2S-positive isolated colonies were streaked onto blood agar and incubated at 37°C with 7% CO2 overnight. Suspect Salmonella colonies were confirmed using poly O group B Salmonella-specific antiserum. Negative enrichments were held for 5 d, enriched again, and streaked onto XLT4 to confirm results. Any Salmonella recovered on secondary enrichment were verified as described previously.
er phase (1–21 d), finisher phase (22–42 d), and over the whole trial period (1–42 d), challenging the birds with Salmonella had no effect (P > 0.05) on any of the measured performance parameters compared with the unchallenged control. Xylanase and EO supplementation improved (P < 0.05) the 42-d BW gain and feed efficiency with no effect (P > 0.05) on feed intake compared with those of the control treatment. Xylanase supplementation improved (P < 0.05) BW gain and feed efficiency compared with those of the EO treatment birds. The combination treatment of xylanase and EO numerically improved (P > 0.05) BW gain and feed efficiency compared with those of the xylanasesupplemented diet.
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Table 3. Effect of essential oils (100 g/t) and xylanase supplementation (2,000 U/kg of feed) on Salmonella prevalence Item Salmonella-positive cecal samples on d 42 (%)1 Positive drag swab samples at 14 d (%)2 Positive drag swab samples at 42 d (%)2
Unchallenged control
Challenged control
Essential oils
Xylanase
Essential oils + xylanase
0 0 0
32.5a 100a 100a
7.5b 87.5ab 62.5b
12.5b 87.5ab 100a
7.5b 62.5b 62.5b
a,bValues
in a row not sharing a common superscript are significantly different (P < 0.05). value represents the percentage of Salmonella-positive cecal samples from 40 replicates (5 unchallenged birds/replicate pen). 2Each value represents the percentage of positive drag swabs of 8 replicates. 1Each
spleen, liver, and ceca of broilers fed a maize-based diet in comparison to those given a wheat/rye-based diet. It has been suggested that xylanase improves nutrient digestibility and, therefore, reduces the nutrients available for pathogenic bacteria in the small intestine (Choct, 2006; Vandeplas et al., 2009). Another possible explanation is that xylanase hydrolysis of arabinoxylan into fragments of arabinoxylan-oligosaccharides acted as prebiotics and stimulated the intestinal immune responses or stimulated beneficial populations, which can compete in the intestine with Salmonella and restore the microbial balance (Patterson and Burkholder, 2003). Eeckhaut et al. (2008) concluded that dietary addition of arabinoxylooligosaccharides prepared from wheat bran by extraction with endoxylanase provides dose-dependent protection against oral infections with Salmonella Enteritidis in poultry. It should be noted, in the current study, that although xylanase supplementation reduced the number of Salmonella-positive cecal samples, it had no effect on positive drag-swab samples on d 42, suggesting no effect of xylanase on environmental Salmonella. Essential oils improve digestive physiology, stimulate blood circulation, exert antioxidant properties, reduce levels of pathogenic bacteria, and may enhance immune status (Brenes and Roura, 2010). Several in vitro studies showed antibacterial effects of some essential oils against Salmonella. Smith-Palmer et al. (1998) reported that the oils of bay, cinnamon, clove, and thyme had the most inhibitory effect on 5 important food-borne pathogens (Escherichia coli, Salmonella enteritidis, Campylobacter jejuni, Staphylococcus aureus, Listeria monocytogenes). Helander et al. (1998) reported that carvacrol, thymol, and trans-cinnamaldehyde showed an antibacterial inhibitory effect against Escherichia coli and Salmonella Typhimurium. In the current study, the EO combination of thymol and cinnamaldehyde reduced the percentage of Salmonella-positive samples of the uninfected birds and in the environment (drag-swab samples). Using a similar blend of thymol and cinnamaldehyde, Tiihonen et al. (2010) showed a reduction of Escherichia coli and Salmonella in cecal samples of broiler chickens fed a wheat-based diet. Cox et al. (1994) stated that reductions in the levels of Salmonella in the intestines mean less transmission of the bacteria in the growing house and during processing, and therefore, less contaminated final product. There-
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both producers and consumers alike. It is unlikely that the use of feed additives alone can eliminate Salmonella. However, reduction of Salmonella numbers in the intestine and the number of contaminated carcasses can help in improving food safety (Corry et al., 2002). Starting in July 2011, the USDA Food Safety and Inspection Service changed the performance standards, based on the percentgage of carcasses testing positive for Salmonella, to be no more than 9.8% positives, compared with 23% in the previous recommendations (USDA/FSIS, 2010). It is believed that with these changes, 26,000 cases of salmonellosis can be avoided each year (USDA/FSIS, 2010). In the last survey in the European Union, the prevalence of Salmonella-contaminated broiler carcasses was 15.6% in the abattoir (EFSA, 2008). Therefore, using certain feed additives to reduce the incidence of Salmonella-contaminated broiler carcasses would be of great interest to the industry. In the current study, challenging the birds with Salmonella had no effect on broiler performance. Similarly, Mountzouris et al. (2009) reported no effect on 42-d broiler performance when challenged with Salmonella Enteritidis. In contrast, other researchers reported that challenging broilers with Salmonella resulted in a significant deterioration of growth performance (Vandeplas et al., 2009; Marcq et al., 2011). Reduced growth performance has been attributed to the decreased feed intake by challenged birds due to mucosal damage (Vandeplas et al., 2009). These contradictory results may be explained by differences in the species and strains or dose of Salmonella used to challenge the birds, which results in different levels of intestinal damage (Malago et al., 2003) or because of differences in cereal type used in the study (Teirlynck et al., 2009). In the present study, xylanase supplementation improved broiler performance and reduced the number of Salmonella-positive cecal samples. This result is consistent with the findings of other researchers who have observed improved performance and a reduction of Salmonella in the cecum of broilers fed a wheat-based diet supplemented with xylanase (Nian et al., 2011). Similarly, Vandeplas et al. (2009) reported a significant reduction in Salmonella Typhimurium concentration in excreta of 30-d-old broilers infected with Salmonella Typhimurium when a wheat-based diet was supplemented with xylanase. Teirlynck et al. (2009) reported a significantly lower Salmonella colonization in the
XYLANASE, ESSENTIAL OILS, AND SALMONELLA IN BROILERS
fore, the current data suggest that EO supplementation based on thymol and cinnamaldehyde could be used as part of a Salmonella control program. In conclusion, feed additives, such as enzymes and EO, could be used as part of an integrated program to control Salmonella levels in poultry production. The results of the current study suggested that dietary addition of EO and xylanase could improve broiler performance and contribute to food safety by lowering the incidence of horizontal transmission of Salmonella infection between birds by 77 and 61%, respectively, compared with the control. Further trials are warranted to consolidate these findings at a commercial level.
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