- Email: [email protected]
Use of Bacitracin and Roxarsone to Reduce Salmonella Heidelberg Shedding Following a Necrotic Enteritis Challenge Model1
©2007 Poultry Science Association, Inc.
Use of Bacitracin and Roxarsone to Reduce Salmonella Heidelberg Shedding Following a Necrotic Enteritis Challenge Model1 C. L. Hofacre,2* G. F. Mathis,† S. H. Miller,‡ and M. W. LaVorgna§
Primary Audience: Veterinarians, Nutritionists, Production Managers SUMMARY This study was performed to determine if the enteric disease necrotic enteritis results in higher shedding of Salmonella. Fifty percent of the chicks per pen were challenged with a naladixic acidresistant Salmonella Heidelberg. A necrotic enteritis challenge model using a coccidia vaccine to induce intestinal epithelium damage was used to demonstrate that neither the disease necrotic enteritis nor the control of that disease with the antibiotic bacitracin methylene disalycilate had an effect on the incidence of Salmonella Heidelberg. However, the feed additive 3-nitro-4hydroxyphenylarsonic acid, which controls the cecal coccidia Eimeria tenella, significantly reduced the amount of Salmonella Heidelberg in the environment of the chick as measured by dragswabs and also reduced the number of ceca positive in the contact chicks. Key words: Salmonella, necrotic enteritis, roxarsone, bacitracin 2007 J. Appl. Poult. Res. 16:275–279
DESCRIPTION OF PROBLEM The reduction in the use of feed additives by the poultry industry may be resulting in potential increases in Salmonella shedding and also a decline in bird health. The use of antibiotic growth promoters has been dramatically decreased. In Europe, this has been by legislation, and in the United States, it has been by market pressures [1]. There has also been a reduction in the use of anticoccidal drugs in the United States to meet consumer demand for drug-free poultry. These factors have resulted in an increase in intestinal disease in commercial 1
poultry [2], with the primary disease condition observed being necrotic enteritis (NE) caused by Clostridium perfringens [3]. However, rarely is NE the result of only a proliferation of C. perfringens in the intestine. It most often occurs in conjunction with damage to the small intestinal mucosa that results from strains of coccidia that affect the small intestine (Eimeria acervulina, Eimeria brunette, Eimeria maxima) [3]. Thus the combined decrease in the usage of antibiotics and anticoccidial drugs may have resulted in the increased incidence of NE [4]. It has also been documented that a concurrent infection of Salmonella with Eimeria ten-
The use of trade names in this publication does not imply endorsement by the University of Georgia of the products mentioned nor criticism of similar products not mentioned. 2 Corresponding author: [email protected]
Downloaded from http://japr.oxfordjournals.org/ at UNIVERSIDAD DE SEVILLA on May 11, 2015
*Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens 30602; †Southern Poultry Research, Athens, Georgia 30607; ‡Alpharma Inc., Cumming, Georgia 30041; and §Alpharma Inc., Salisbury, Maryland 21804
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Table 1. Performance results, lesion scores, and mortality in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis (NE) challenge model
Treatment Control Challenged control Roxarsone1 BMD2 Roxarsone + BMD3
FCR (0 to 42 d)
Live weight
Cocci score (ceca)
1.765d 2.010a 1.930b 1.856c 1.821c
2.235a 2.037d 2.109c 2.169b 2.181b
0.2b 0.5a 0b 0.2b 0.1b
Mortality NE score
NE (%)
Overall (%)
0 0.4 0.3 0.5 0.3
0d 37.78a 21.39b 15.83c 11.67c
7.22d 40.56a 29.17b 25.00bc 21.11c
Means in a column with no common superscript letters are significantly different (P < 0.05). 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. 2 BMD = bacitracin methylene disalycilate, 50 g/ton. 3 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton. a–d
ella or Eimeria necatrix results in increased fecal shedding of Salmonella in young chickens [5, 6, 7] and increased Salmonella shedding by breeders or layers [8]. This study was undertaken to determine whether the disease NE induced with a model using a commercially available coccidia vaccine and C. perfringens inoculation would result in increased cecal colonization and environmental shedding of Salmonella in chickens given Salmonella Heidelberg at 1 d of age. This study also determined whether it was more important to control the Clostridium with an antibiotic or to control the coccidia with an anticoccidial drug that specifically targets the cecal coccidia, E. tenella, to reduce Salmonella shedding.
MATERIALS AND METHODS Study Design The 42-d study consisted of 5 treatment groups with 6 replicates per treatment. Treatment 1 (control) received feed containing no feed additives and was not challenged with C. perfringens. Treatment 2 (challenge control) contained no feed additives but was challenged with C. perfringens. Treatment 3 received feed containing 45.4 g/ton of 3-nitro-4-hydroxyphenylarsonic acid (roxarsone) [9] continuously for the 42 d and was challenged with C. perfringens. Treatment 4 received feed containing bacitracin methylene disalycilate (BMD) [10] at 50 g/ton for the 42 d and was challenged with C. perfringens. Treatment 5 (roxarsone and BMD) received feed containing both roxar-
sone (45.4 g/ton) and BMD (50 g/ton) for the 42 d and was challenged with C. perfringens. Each batch of feed was sampled and assayed to insure the proper level of each drug. The birds were 1-d-old male Cobb × Cobb broilers [11]. Sixty birds were housed in each floor pen (0.60 ft2/bird) that contained litter used from a previous nonsalmonella trial covered over with a layer of new softwood litter. Feed and water were available ad libitum. NE Model All birds were administered a 25× dose of a coccidia vaccine, Coccivac-B [12] on d 15 by mixing the product into the pans of the feeders of the birds. Coccivac-B contains oocysts of E. maxima, E. acervulina, E. tenella, and Eimeria mivati. To document coccidial challenge level, 2 birds/pen were euthanized on d 21, weighed, and coccidial lesions were scored in the upper, middle, and cecal region of the intestine. The lesion scoring was by the Johnson and Reid method [13]. On d 19, 20, and 21, all birds in treatments 2, 3, 4, and 5 were administered a 24-h broth culture of C. perfringens (1 to 3 × 108 cfu/mL each d). The culture was administered by mixing the broth dose (1 mL/ bird) onto the feed in feed pans for each of the treated pens. The NE lesions were scored in the ileum on d 23 in 5 randomly selected birds per pen. The scoring was as follows: 0 for normal intestines, 1 for slight mucus covering and loss of tone, 2 for severe necrotizing enteritis, and 3 for extreme necrotizing enteritis with presence of blood in the lumen [14]. All dead birds
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Table 2. Mean percentage of positive Salmonella Heidelberg ceca culture results at d 22 and 42 in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis challenge model All Treatment Control Challenged control Roxarsone1 BMD2 Roxarsone + BMD3
d 22 ab
56.67 45.00ab 26.67b 75.00a 30.00b
Seeders d 42 18.33 8.33 5.0 8.33 8.33
d 22
Contacts d 42
53.33 43.33 33.33 70.00 33.33
10 10 10 3.33 16.67
d 22 ab
60.00 46.67bc 20.00c 80.00a 26.67c
d 42 26.67a 6.67b 0b 13.33ab 0b
Means in a column with no common superscript letters are significantly different (P < 0.05). 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. BMD = bacitracin methylene disalycilate, 50 g/ton. 3 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton. a–c 1
dying after d 19 were weighed and necropsied for the presence of NE lesions. Salmonella Challenge and Testing At d 1, before placement, 30 of the 60 chicks per pen (all treatments) were orally administered by gavage 0.1 mL of a naladixic acidresistant (25 g/mL) Salmonella Heidelberg (5 × 107 cfu/mL). Each of these 30 chicks challenged with Salmonella Heidelberg were also wing-banded and were called seeders, whereas the remaining 30 chicks were called contacts. The Salmonella environmental sampling was by dragswabs [15] from all pens on d 0, 14, and 42. At 22 and 42 d, 10 birds per pen (5 seeders, 5 contacts) were randomly selected, euthanized by cervical dislocation, weighed, and the ceca were aseptically removed and then placed into sterile plastic bags. Ten milliliters of sterile PBS was added to each bag, the bags were homogenized for 1 min, and then 10-fold dilutions were performed and transferred onto xylose Lys tergitol-4 agar plates [16] containing 25 g of naladixic acid/mL. After 24 h of incubation at 37°C, the black Salmonella colonies were counted. One colony/plate was randomly selected and tested by a polymerase chain reaction to insure only the challenge Salmonella Heidelberg was present [17]. Ninety milliliters of brilliant green tetrathionate broth with I [18] was then added to the ceca and PBS mixture, and these were incubated at 41.5°C for 24 h, struck onto xylose Lys tergitol-4 plates, and incubated for 24 h at 37°C. Black colonies were identified, and polymerase chain reaction confirmed the challenge strain as described above.
If no black colonies were present from a sample, the enriched ceca-containing bag was held at room temperature for 7 d and delayed secondary enrichment was performed [19]. Therefore, the results for presence or absence of Salmonella will be a summation by pen of the positive ceca from the primary and secondary enrichments. Statistical Analysis This study was conducted using a randomized complete block design. There were 6 blocks, each with 5 treatments, giving a total of 30 pens (approximately 60 male broiler chickens per pen at placement). The pen of birds was the experimental unit. The model included block and treatment. Block was a random effect. Treatment was a fixed effect, so the model was mixed. The summary variables included Salmonella data (enumeration, percentage of positive birds, and dragswabs) and performance data [FCR, live weight, and mortality] for each pen. Coccidiosis and NE lesion scores were also summary variables for each pen. A mixed model analysis was performed on the data using the PROC MIXED procedure of SAS [20]. Percentage data were analyzed with and without transformation (arc sin square root) [21]. Treatment effects were considered significant P < 0.05. When treatment effects were determined to be significant, means were separated using t-test probabilities generated by the LSMEANS option of SAS.
RESULTS AND DISCUSSION The NE challenge was successful as evidenced by the challenge control group having
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Table 3. Environmental contamination level of Salmonella Heidelberg from dragswabs in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis challenge model Treatment
d0
d 14
d 42
Control Challenged control Roxarsone1 BMD2 Roxarsone + BMD3
0 0 0 0 0
100 100 100 100 100
66.77a 33.33ab 0b 33.33ab 0b
a,b
Means in a column with no common superscript letters are significantly different (P < 0.05). 1 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. 2 BMD = bacitracin methylene disalycilate, 50 g/ton. 3 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton.
ment kept the Salmonella level lower for the contact chicks (Table 2). The percentage of cecal positives for Salmonella Heidelberg was significantly lower at d 22 in the contact chicks receiving the roxarsone either alone or combined with BMD. The findings of Qin et al. [8] that E. tenella infection results in significant increases of Salmonella Enteritidis in the ceca supports our finding that the contact chicks treated with roxarsone either alone or combined with BMD would have significantly less Salmonella Heidelberg in their ceca at d 22 and numerically less (0%) at the end of the 42-d study. This work is the first to demonstrate that NE does not result in higher colonization and shedding of Salmonella Heidelberg and that control of the disease NE by the antibiotic BMD alone will not reduce the Salmonella Heidelberg in the ceca of contact chicks at 42 d. This Table 4. Mean Salmonella Heidelberg enumeration results per gram of cecal content in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis challenge model1 Treatment Control Challenged control Roxarsone2 BMD3 Roxarsone + BMD4 1
d 22
d 42
1.3 2.3 188.0 33.2 1,498.7
0.3 0.5 0 0 26.5
No significant differences were noted among any of the treatments at either age. 2 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. 3 BMD = bacitracin methylene disalycilate, 50 g/ton. 4 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton.
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37.78% mortality associated with NE and NE lesions in the ileum (Table 1). The antibiotic, BMD, either alone (15.83%) or in combination with roxarsone (11.67%), had significantly reduced NE mortality, as has been seen in previous studies [14]. Subclinical NE resulted in the reduced live weight and higher FCR seen in the challenge controls (Table 1). The BMD significantly lowered the FCR and prevented the reduction in live weight associated with subclinical NE, as has previously been demonstrated [14]. The roxarsone alone significantly reduced the NE mortality and FCR and significantly improved live weight over the challenge control but was not as effective as the BMD in any of these parameters. This improvement in performance may be due to reduction in NE and not solely in control of the coccidia, because all groups received the coccidia vaccine including the control, which did not have any NE mortality. Interestingly, the Salmonella Heidelberg culture results from the NE challenge control birds (6.67%) were either numerically or significantly lower than the negative control groups (26.67%) throughout the 42-d study (Table 2). This may be the result of the severe disruption of the gastrointestinal flora from the C. perfringens toxin that also disrupted the ability of Salmonella Heidelberg to colonize. There were no significant differences in the level of Salmonella in the seeder birds that were given Salmonella at d 1 among any of the treatment groups (Table 2). This was not unexpected, because Cox [22] found that the Salmonella a chick obtained early in life remains colonized and is often found when they are older. However, the level of Salmonella that was being shed into the environment by the roxarsonetreated groups was less than the control groups, as evidenced by the environmental sampling via the dragswabs at 42 d (Table 3). This result was not confirmed with the enumeration of the ceca, because there were no significant differences among any of the treated or control groups (Table 4). Because Salmonella is difficult to culture from an intestinal sample without enrichment, the failure to determine accurate levels on direct dilutions is not an uncommon finding. The dragswab results give a truer indication that the lower Salmonella in the environ-
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Salmonella Heidelberg-positive contact chicks at 22 and 42 d of age by lowering the amount of Salmonella Heidelberg in their environment.
CONCLUSIONS AND APPLICATIONS 1. The intestinal disease NE resulted in higher levels of Salmonella Heidelberg colonization of ceca. 2. The drug 3-nitro-4-hydroxyphenylarsonic acid, most likely via its control of E. tenella, resulted in reduced Salmonella Heidelberg colonization of the ceca.
1. Singer, R., and C. L. Hofacre. 2006. Potential impacts of antibiotic use in poultry production. Avian Dis. 50:161–172. 2. van der Sluis, W. 2005. Global impact of bacterial enteritis is worse than ever. World Poult. 21:46–48. 3. Wages, D. P., and K. Opengart. 2003. Necrotic enteritis. Pages 781–784 in Diseases of Poultry. 11th ed. Y. M. Saif, ed. Iowa State Univ. Press, Ames. 4. Cervantes, H. 2006. Learning from the European experience. Watt Poultry USA 2:34. 5. Baba, E., M. Yaono, T. Fukata, and A. Arakawa. 1985. Infection by Salmonella typhimurium, S. agona, S. enteritidis or S. infantis of chicks with caecal coccidiosis. Br. Poult. Sci. 26:505– 511. 6. Baba, E., K. Sawano, T. Fukata, and A. Arakawa. 1987. Paratyphoid infection induced by Eimeria tenella in the broilertype chicken. Avian Pathol. 16:31–42. 7. Stephens, J. F., and B. D. Barnett. 1964. Concurrent Salmonella Typhimurium and Eimeria necatrix infections in chicks. Poult. Sci. 43:352–356.
13. Johnson, J., and W. M. Reid. 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28:30–36. 14. Hofacre, C. L., R. Froyman, B. George, M. A. Goodwin, and J. Brown. 1998. Use of Aviguard, virginiamycin or bacitracin MD against Clostridium perfringens-associated necrotizing enteritis. J. Appl. Poult. Res. 7:412–418. 15. 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. 16. Xylose Lys tergitol-4, Difco Laboratories, Detroit, MI. 17. Liu, T., K. Liljebjelke, E. Barlett, C. Hofacre, S. Sanchez, and J. J. Maurer. 2002. Application of nested polymerase chain reaction to detection of Salmonella in poultry environment. J. Food Prot. 65:1227–1232. 18. Brilliant green tetrathionate broth, Difco Laboratories.
8. Qin, Z., A. Arakawa, E. Baba, T. Fukata, and K. Sasai. 1996. Effect of Eimeria tenella infection on the production of Salmonella enteritidis-contaminated eggs and susceptibility of laying hens to S. enteritidis infection. Avian Dis. 40:361–367.
19. Waltman, W. D., A. M. Horne, and C. Pirkle. 1993. Influence of enrichment incubation time on the isolation of Salmonella. Avian Dis. 37:884–887.
9. 3-Nitro-4-hydroxyphenylarsonic acid, Alpharma, Fort Lee, NJ.
21. Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of Statistics. McGraw-Hill Inc., New York, NY.
10. BMD, Alpharma. 11. Cobb × Cobb broilers, Cobb-Vantress, Siloam Springs, AR. 12. Coccivac B, Schering-Plough Animal Health, Union, NJ.
20. SAS, SAS Institute Inc. Cary, NC.
22. Cox, N. A. 1998. Incidence and impact of Salmonella in broiler hatcheries. Pages 88–89 in Proc. Am. Assoc. Avian Pathol.: Int. Symp. Food-Borne Salmonella Poult, Baltimore, MD. R. Gast and C. Hofacre, ed. Am. Assoc. Avian Pathol., Athens, GA.
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REFERENCES AND NOTES
Use of Bacitracin and Roxarsone to Reduce Salmonella Heidelberg Shedding Following a Necrotic Enteritis Challenge Model1 C. L. Hofacre,2* G. F. Mathis,† S. H. Miller,‡ and M. W. LaVorgna§
Primary Audience: Veterinarians, Nutritionists, Production Managers SUMMARY This study was performed to determine if the enteric disease necrotic enteritis results in higher shedding of Salmonella. Fifty percent of the chicks per pen were challenged with a naladixic acidresistant Salmonella Heidelberg. A necrotic enteritis challenge model using a coccidia vaccine to induce intestinal epithelium damage was used to demonstrate that neither the disease necrotic enteritis nor the control of that disease with the antibiotic bacitracin methylene disalycilate had an effect on the incidence of Salmonella Heidelberg. However, the feed additive 3-nitro-4hydroxyphenylarsonic acid, which controls the cecal coccidia Eimeria tenella, significantly reduced the amount of Salmonella Heidelberg in the environment of the chick as measured by dragswabs and also reduced the number of ceca positive in the contact chicks. Key words: Salmonella, necrotic enteritis, roxarsone, bacitracin 2007 J. Appl. Poult. Res. 16:275–279
DESCRIPTION OF PROBLEM The reduction in the use of feed additives by the poultry industry may be resulting in potential increases in Salmonella shedding and also a decline in bird health. The use of antibiotic growth promoters has been dramatically decreased. In Europe, this has been by legislation, and in the United States, it has been by market pressures [1]. There has also been a reduction in the use of anticoccidal drugs in the United States to meet consumer demand for drug-free poultry. These factors have resulted in an increase in intestinal disease in commercial 1
poultry [2], with the primary disease condition observed being necrotic enteritis (NE) caused by Clostridium perfringens [3]. However, rarely is NE the result of only a proliferation of C. perfringens in the intestine. It most often occurs in conjunction with damage to the small intestinal mucosa that results from strains of coccidia that affect the small intestine (Eimeria acervulina, Eimeria brunette, Eimeria maxima) [3]. Thus the combined decrease in the usage of antibiotics and anticoccidial drugs may have resulted in the increased incidence of NE [4]. It has also been documented that a concurrent infection of Salmonella with Eimeria ten-
The use of trade names in this publication does not imply endorsement by the University of Georgia of the products mentioned nor criticism of similar products not mentioned. 2 Corresponding author: [email protected]
Downloaded from http://japr.oxfordjournals.org/ at UNIVERSIDAD DE SEVILLA on May 11, 2015
*Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens 30602; †Southern Poultry Research, Athens, Georgia 30607; ‡Alpharma Inc., Cumming, Georgia 30041; and §Alpharma Inc., Salisbury, Maryland 21804
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Table 1. Performance results, lesion scores, and mortality in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis (NE) challenge model
Treatment Control Challenged control Roxarsone1 BMD2 Roxarsone + BMD3
FCR (0 to 42 d)
Live weight
Cocci score (ceca)
1.765d 2.010a 1.930b 1.856c 1.821c
2.235a 2.037d 2.109c 2.169b 2.181b
0.2b 0.5a 0b 0.2b 0.1b
Mortality NE score
NE (%)
Overall (%)
0 0.4 0.3 0.5 0.3
0d 37.78a 21.39b 15.83c 11.67c
7.22d 40.56a 29.17b 25.00bc 21.11c
Means in a column with no common superscript letters are significantly different (P < 0.05). 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. 2 BMD = bacitracin methylene disalycilate, 50 g/ton. 3 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton. a–d
ella or Eimeria necatrix results in increased fecal shedding of Salmonella in young chickens [5, 6, 7] and increased Salmonella shedding by breeders or layers [8]. This study was undertaken to determine whether the disease NE induced with a model using a commercially available coccidia vaccine and C. perfringens inoculation would result in increased cecal colonization and environmental shedding of Salmonella in chickens given Salmonella Heidelberg at 1 d of age. This study also determined whether it was more important to control the Clostridium with an antibiotic or to control the coccidia with an anticoccidial drug that specifically targets the cecal coccidia, E. tenella, to reduce Salmonella shedding.
MATERIALS AND METHODS Study Design The 42-d study consisted of 5 treatment groups with 6 replicates per treatment. Treatment 1 (control) received feed containing no feed additives and was not challenged with C. perfringens. Treatment 2 (challenge control) contained no feed additives but was challenged with C. perfringens. Treatment 3 received feed containing 45.4 g/ton of 3-nitro-4-hydroxyphenylarsonic acid (roxarsone) [9] continuously for the 42 d and was challenged with C. perfringens. Treatment 4 received feed containing bacitracin methylene disalycilate (BMD) [10] at 50 g/ton for the 42 d and was challenged with C. perfringens. Treatment 5 (roxarsone and BMD) received feed containing both roxar-
sone (45.4 g/ton) and BMD (50 g/ton) for the 42 d and was challenged with C. perfringens. Each batch of feed was sampled and assayed to insure the proper level of each drug. The birds were 1-d-old male Cobb × Cobb broilers [11]. Sixty birds were housed in each floor pen (0.60 ft2/bird) that contained litter used from a previous nonsalmonella trial covered over with a layer of new softwood litter. Feed and water were available ad libitum. NE Model All birds were administered a 25× dose of a coccidia vaccine, Coccivac-B [12] on d 15 by mixing the product into the pans of the feeders of the birds. Coccivac-B contains oocysts of E. maxima, E. acervulina, E. tenella, and Eimeria mivati. To document coccidial challenge level, 2 birds/pen were euthanized on d 21, weighed, and coccidial lesions were scored in the upper, middle, and cecal region of the intestine. The lesion scoring was by the Johnson and Reid method [13]. On d 19, 20, and 21, all birds in treatments 2, 3, 4, and 5 were administered a 24-h broth culture of C. perfringens (1 to 3 × 108 cfu/mL each d). The culture was administered by mixing the broth dose (1 mL/ bird) onto the feed in feed pans for each of the treated pens. The NE lesions were scored in the ileum on d 23 in 5 randomly selected birds per pen. The scoring was as follows: 0 for normal intestines, 1 for slight mucus covering and loss of tone, 2 for severe necrotizing enteritis, and 3 for extreme necrotizing enteritis with presence of blood in the lumen [14]. All dead birds
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Table 2. Mean percentage of positive Salmonella Heidelberg ceca culture results at d 22 and 42 in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis challenge model All Treatment Control Challenged control Roxarsone1 BMD2 Roxarsone + BMD3
d 22 ab
56.67 45.00ab 26.67b 75.00a 30.00b
Seeders d 42 18.33 8.33 5.0 8.33 8.33
d 22
Contacts d 42
53.33 43.33 33.33 70.00 33.33
10 10 10 3.33 16.67
d 22 ab
60.00 46.67bc 20.00c 80.00a 26.67c
d 42 26.67a 6.67b 0b 13.33ab 0b
Means in a column with no common superscript letters are significantly different (P < 0.05). 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. BMD = bacitracin methylene disalycilate, 50 g/ton. 3 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton. a–c 1
dying after d 19 were weighed and necropsied for the presence of NE lesions. Salmonella Challenge and Testing At d 1, before placement, 30 of the 60 chicks per pen (all treatments) were orally administered by gavage 0.1 mL of a naladixic acidresistant (25 g/mL) Salmonella Heidelberg (5 × 107 cfu/mL). Each of these 30 chicks challenged with Salmonella Heidelberg were also wing-banded and were called seeders, whereas the remaining 30 chicks were called contacts. The Salmonella environmental sampling was by dragswabs [15] from all pens on d 0, 14, and 42. At 22 and 42 d, 10 birds per pen (5 seeders, 5 contacts) were randomly selected, euthanized by cervical dislocation, weighed, and the ceca were aseptically removed and then placed into sterile plastic bags. Ten milliliters of sterile PBS was added to each bag, the bags were homogenized for 1 min, and then 10-fold dilutions were performed and transferred onto xylose Lys tergitol-4 agar plates [16] containing 25 g of naladixic acid/mL. After 24 h of incubation at 37°C, the black Salmonella colonies were counted. One colony/plate was randomly selected and tested by a polymerase chain reaction to insure only the challenge Salmonella Heidelberg was present [17]. Ninety milliliters of brilliant green tetrathionate broth with I [18] was then added to the ceca and PBS mixture, and these were incubated at 41.5°C for 24 h, struck onto xylose Lys tergitol-4 plates, and incubated for 24 h at 37°C. Black colonies were identified, and polymerase chain reaction confirmed the challenge strain as described above.
If no black colonies were present from a sample, the enriched ceca-containing bag was held at room temperature for 7 d and delayed secondary enrichment was performed [19]. Therefore, the results for presence or absence of Salmonella will be a summation by pen of the positive ceca from the primary and secondary enrichments. Statistical Analysis This study was conducted using a randomized complete block design. There were 6 blocks, each with 5 treatments, giving a total of 30 pens (approximately 60 male broiler chickens per pen at placement). The pen of birds was the experimental unit. The model included block and treatment. Block was a random effect. Treatment was a fixed effect, so the model was mixed. The summary variables included Salmonella data (enumeration, percentage of positive birds, and dragswabs) and performance data [FCR, live weight, and mortality] for each pen. Coccidiosis and NE lesion scores were also summary variables for each pen. A mixed model analysis was performed on the data using the PROC MIXED procedure of SAS [20]. Percentage data were analyzed with and without transformation (arc sin square root) [21]. Treatment effects were considered significant P < 0.05. When treatment effects were determined to be significant, means were separated using t-test probabilities generated by the LSMEANS option of SAS.
RESULTS AND DISCUSSION The NE challenge was successful as evidenced by the challenge control group having
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Table 3. Environmental contamination level of Salmonella Heidelberg from dragswabs in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis challenge model Treatment
d0
d 14
d 42
Control Challenged control Roxarsone1 BMD2 Roxarsone + BMD3
0 0 0 0 0
100 100 100 100 100
66.77a 33.33ab 0b 33.33ab 0b
a,b
Means in a column with no common superscript letters are significantly different (P < 0.05). 1 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. 2 BMD = bacitracin methylene disalycilate, 50 g/ton. 3 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton.
ment kept the Salmonella level lower for the contact chicks (Table 2). The percentage of cecal positives for Salmonella Heidelberg was significantly lower at d 22 in the contact chicks receiving the roxarsone either alone or combined with BMD. The findings of Qin et al. [8] that E. tenella infection results in significant increases of Salmonella Enteritidis in the ceca supports our finding that the contact chicks treated with roxarsone either alone or combined with BMD would have significantly less Salmonella Heidelberg in their ceca at d 22 and numerically less (0%) at the end of the 42-d study. This work is the first to demonstrate that NE does not result in higher colonization and shedding of Salmonella Heidelberg and that control of the disease NE by the antibiotic BMD alone will not reduce the Salmonella Heidelberg in the ceca of contact chicks at 42 d. This Table 4. Mean Salmonella Heidelberg enumeration results per gram of cecal content in broilers following exposure to Salmonella Heidelberg and a necrotic enteritis challenge model1 Treatment Control Challenged control Roxarsone2 BMD3 Roxarsone + BMD4 1
d 22
d 42
1.3 2.3 188.0 33.2 1,498.7
0.3 0.5 0 0 26.5
No significant differences were noted among any of the treatments at either age. 2 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton. 3 BMD = bacitracin methylene disalycilate, 50 g/ton. 4 3-Nitro-4-hydroxyphenylarsonic acid, 45.4 g/ton, and BMD, 50 g/ton.
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37.78% mortality associated with NE and NE lesions in the ileum (Table 1). The antibiotic, BMD, either alone (15.83%) or in combination with roxarsone (11.67%), had significantly reduced NE mortality, as has been seen in previous studies [14]. Subclinical NE resulted in the reduced live weight and higher FCR seen in the challenge controls (Table 1). The BMD significantly lowered the FCR and prevented the reduction in live weight associated with subclinical NE, as has previously been demonstrated [14]. The roxarsone alone significantly reduced the NE mortality and FCR and significantly improved live weight over the challenge control but was not as effective as the BMD in any of these parameters. This improvement in performance may be due to reduction in NE and not solely in control of the coccidia, because all groups received the coccidia vaccine including the control, which did not have any NE mortality. Interestingly, the Salmonella Heidelberg culture results from the NE challenge control birds (6.67%) were either numerically or significantly lower than the negative control groups (26.67%) throughout the 42-d study (Table 2). This may be the result of the severe disruption of the gastrointestinal flora from the C. perfringens toxin that also disrupted the ability of Salmonella Heidelberg to colonize. There were no significant differences in the level of Salmonella in the seeder birds that were given Salmonella at d 1 among any of the treatment groups (Table 2). This was not unexpected, because Cox [22] found that the Salmonella a chick obtained early in life remains colonized and is often found when they are older. However, the level of Salmonella that was being shed into the environment by the roxarsonetreated groups was less than the control groups, as evidenced by the environmental sampling via the dragswabs at 42 d (Table 3). This result was not confirmed with the enumeration of the ceca, because there were no significant differences among any of the treated or control groups (Table 4). Because Salmonella is difficult to culture from an intestinal sample without enrichment, the failure to determine accurate levels on direct dilutions is not an uncommon finding. The dragswab results give a truer indication that the lower Salmonella in the environ-
HOFACRE ET AL.: BACITRACIN AND ROXARSONE research also demonstrated that the use of the drug roxarsone to reduce E. tenella will significantly and numerically reduce the level of
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Salmonella Heidelberg-positive contact chicks at 22 and 42 d of age by lowering the amount of Salmonella Heidelberg in their environment.
CONCLUSIONS AND APPLICATIONS 1. The intestinal disease NE resulted in higher levels of Salmonella Heidelberg colonization of ceca. 2. The drug 3-nitro-4-hydroxyphenylarsonic acid, most likely via its control of E. tenella, resulted in reduced Salmonella Heidelberg colonization of the ceca.
1. Singer, R., and C. L. Hofacre. 2006. Potential impacts of antibiotic use in poultry production. Avian Dis. 50:161–172. 2. van der Sluis, W. 2005. Global impact of bacterial enteritis is worse than ever. World Poult. 21:46–48. 3. Wages, D. P., and K. Opengart. 2003. Necrotic enteritis. Pages 781–784 in Diseases of Poultry. 11th ed. Y. M. Saif, ed. Iowa State Univ. Press, Ames. 4. Cervantes, H. 2006. Learning from the European experience. Watt Poultry USA 2:34. 5. Baba, E., M. Yaono, T. Fukata, and A. Arakawa. 1985. Infection by Salmonella typhimurium, S. agona, S. enteritidis or S. infantis of chicks with caecal coccidiosis. Br. Poult. Sci. 26:505– 511. 6. Baba, E., K. Sawano, T. Fukata, and A. Arakawa. 1987. Paratyphoid infection induced by Eimeria tenella in the broilertype chicken. Avian Pathol. 16:31–42. 7. Stephens, J. F., and B. D. Barnett. 1964. Concurrent Salmonella Typhimurium and Eimeria necatrix infections in chicks. Poult. Sci. 43:352–356.
13. Johnson, J., and W. M. Reid. 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28:30–36. 14. Hofacre, C. L., R. Froyman, B. George, M. A. Goodwin, and J. Brown. 1998. Use of Aviguard, virginiamycin or bacitracin MD against Clostridium perfringens-associated necrotizing enteritis. J. Appl. Poult. Res. 7:412–418. 15. 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. 16. Xylose Lys tergitol-4, Difco Laboratories, Detroit, MI. 17. Liu, T., K. Liljebjelke, E. Barlett, C. Hofacre, S. Sanchez, and J. J. Maurer. 2002. Application of nested polymerase chain reaction to detection of Salmonella in poultry environment. J. Food Prot. 65:1227–1232. 18. Brilliant green tetrathionate broth, Difco Laboratories.
8. Qin, Z., A. Arakawa, E. Baba, T. Fukata, and K. Sasai. 1996. Effect of Eimeria tenella infection on the production of Salmonella enteritidis-contaminated eggs and susceptibility of laying hens to S. enteritidis infection. Avian Dis. 40:361–367.
19. Waltman, W. D., A. M. Horne, and C. Pirkle. 1993. Influence of enrichment incubation time on the isolation of Salmonella. Avian Dis. 37:884–887.
9. 3-Nitro-4-hydroxyphenylarsonic acid, Alpharma, Fort Lee, NJ.
21. Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of Statistics. McGraw-Hill Inc., New York, NY.
10. BMD, Alpharma. 11. Cobb × Cobb broilers, Cobb-Vantress, Siloam Springs, AR. 12. Coccivac B, Schering-Plough Animal Health, Union, NJ.
20. SAS, SAS Institute Inc. Cary, NC.
22. Cox, N. A. 1998. Incidence and impact of Salmonella in broiler hatcheries. Pages 88–89 in Proc. Am. Assoc. Avian Pathol.: Int. Symp. Food-Borne Salmonella Poult, Baltimore, MD. R. Gast and C. Hofacre, ed. Am. Assoc. Avian Pathol., Athens, GA.
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REFERENCES AND NOTES