- Email: [email protected]
Effects of litter amendments on broiler growth characteristics and Salmonella colonization in the crop and cecum
©2010 Poultry Science Association, Inc.
Effects of litter amendments on broiler growth characteristics and Salmonella colonization in the crop and cecum
*Department of Poultry Science, Texas A&M University, College Station 77843; and †USDA-ARS, Food and Feed Safety Research Unit, College Station, TX 77845 Primary Audience: Broiler Grow-Out Technicians, Supervisors SUMMARY Salmonella can be prevalent in poultry litter and can be a source of contamination for chicks newly arrived at the poultry house. The objective of this study was to determine the effects of litter amendments on broiler grow-out characteristics (BW and feed conversion), litter moisture, and Salmonella enterica serovar Typhimurium colonization in the litter, crop and cecum. Because Salmonella Typhimurium is a pathogen of concern to the poultry industry, 2 types of litter amendments and 2 types of feed additions were created and tested to determine the effects of broiler growth, litter moisture, and litter efficacy against Salmonella colonization. Litter amendments consisted of a combination of acidic calcium sulfate and either diatomaceous earth or hydrated sodium calcium aluminiosilicate. Feed additions consisted of differing amounts of sodium bisulfate. For the litter amendment experiments, chicks were placed into pens in isolation rooms. Each litter amendment was applied to 3 pens for replicates of experimental groups. Litter samples were taken weekly from 5 areas in each pen and were combined for the determination of Salmonella colony-forming units per gram of litter. At 3 and 6 wk after placement, birds from each pen were killed by CO2 asphyxiation. Efficacy of the litter amendments varied among experimental groups in broiler growth characteristics and efficacy against Salmonella. Key words: litter, Salmonella, broiler, poultry 2010 J. Appl. Poult. Res. 19:132–136 doi:10.3382/japr.2009-00083
DESCRIPTION OF PROBLEM Contamination of poultry products by pathogenic bacteria poses both health and financial risks to poultry integrators [1]. Consumer confidence in the food supply is a major driving force behind recommendations and regulations that deal with modern poultry production. The paratyphoid salmonellae, especially Salmonella
1
Corresponding author: [email protected]
enterica serovar Typhimurium, are pathogens of concern for the poultry industry because of their association with raw poultry products and because these organisms displayed a 4-fold increase in isolation from ill individuals from 1955 to the 1990s [2]. Many successful programs have been instituted to meet the federal requirements and consumer demands for pathogen control. Most of
Downloaded from http://japr.oxfordjournals.org/ at Frankfurt University Library, Section Stadt- und Universitaetsbibliothek on June 8, 2015
E. L. Larrison,* J. A. Byrd,† and M. A. Davis*1
Larrison et al.: LITTER AMENDMENTS ON SALMONELLA
MATERIALS AND METHODS Grow-Out Conditions Birds in this experiment were supervised under the USDA Animal Care and Use protocol. Day-of-hatch broiler chicks (180 total, 15 per pen, 45 per treatment, 4 treatments) were placed and provided free access to feed and water. Feed was weighed in grams to allow feed conversion to be calculated at the end of the study. Birds
were housed in 4 separate rooms in an isolation building with positive airflow and air filtration. Each room in the isolation unit was divided into 3 pens, and each pen was equipped with a nipple watering system and adequate feeders. The grow-out phase of the study consisted of 12 pens to allow for 3 replications of each experimental group. Birds were fed a starter broiler feed for the first 3 wk of the study and were switched to a grower ration for the remainder of the study. All birds were killed by CO2 asphyxiation at 5 wk and 3 d of age after placement. After the experiment was concluded, total BW (by pen) was determined to calculate feed conversion. Averages of individual bird BW in each pen were also calculated for comparison. Experimental Groups and Challenge There were 4 experimental groups with 3 replications per group. Group 1 (negative control) did not receive any litter amendment or challenge with Salmonella Typhimurium and thus was the negative control. Birds in the 3 replications for the negative control were placed in a separate isolation room from those challenged with Salmonella Typhimurium. Birds in the other group replications were dispersed evenly throughout the other 3 isolation rooms. Group 2 (positive control) did not receive the litter amendment, but birds were challenged with Salmonella Typhimurium and served as the positive control. Group 3 received the litter amendment mixture containing the DE, and birds were challenged with Salmonella Typhimurium. Group 4 received the litter amendment mixture containing the clay, and birds were challenged with Salmonella Typhimurium. Litter amendments were applied by sprinkling the amendment onto the litter in accordance with the directions of the manufacturer. The amendment was then raked into the litter throughout the pen to cover the floor space. For those groups that received the challenge with Salmonella Typhimurium, 150 mL of a solution containing 3.8 × 108 cfu/mL of novobiocin- and nalidixic acid-resistant Salmonella Typhimurium was applied to the litter in each pen with a spray bottle, making sure to cover the entire pen area with the spray. The Salmonella challenge inoculate was prepared from a primary poultry isolate of Salmonella
Downloaded from http://japr.oxfordjournals.org/ at Frankfurt University Library, Section Stadt- und Universitaetsbibliothek on June 8, 2015
these programs, such as the Hazard Analysis and Critical Control Point system, Food Safety Objectives, and the introduction of novel chemical antimicrobials, have centered on the processing plant as the main target for food safety control measures. Recently, there has been interest in implementing on-farm pathogen reduction strategies to ease the burden on the processing plant by decreasing the initial load of bacteria that enter the plant with live birds. Reducing pathogens before processing can be beneficial in that it may allow the processing plant to lower the cost of production because of decreased risk, and there may also be a decrease in the amount of crosscontamination in the processing environment. The use of litter amendments is one possible approach to achieve on-farm pathogen reduction. Many formulations of litter amendments have been developed and tested, with varying results. Most of the compounds that have been tested are acidic in nature. Because of this characteristic, it is reasonable to assume that proper application could significantly lower both the pH and water activity of the litter, conditions that directly affect the survivability of microorganisms that are typically present in poultry litter [3]. The litter amendments used must not interfere with the normal growth characteristics of the broilers subjected to the treatment and must not present any additional risks to the broilers or to employees of the grow-out facility. The objective of this study was to determine the effects of litter amendments on broiler grow-out characteristics (BW and feed conversion), litter moisture, and Salmonella Typhimurium colonization in the litter, crop, and cecum. Litter amendments consisted of acidic calcium sulfate combined with either diatomaceous earth (DE) or hydrated sodium calcium aluminosilicate (clay).
133
JAPR: Research Report
134 Table 1. Body weight comparison (g) for all broilers, initial placement to wk 3 Item
Week 0 49.08 48.08 48.85 49.24 0.29
Week 2
Week 3
b
329.57 328.00 341.78 350.62 4.54
552.38b 627.01a 617.73a 624.20a 10.15
130.94 132.86ab 136.05ab 141.92a 1.88
a,b
Means within a column with differing superscripts differ significantly (P < 0.05). Diatomaceous earth. 2 Hydrated sodium calcium aluminosilicate. 1
Typhimurium obtained from the National Veterinary Services Laboratory (Ames, Iowa). After application of the litter amendment and Salmonella Typhimurium challenge, 15 one-day-old chicks were wing-banded and randomly placed into each pen. Initial BW was recorded for each chick as it was placed into the pen. Litter Samples Litter samples from each pen were collected each week from 4 random areas in each pen and combined in a zipper bag. The bags were shaken to distribute the contents evenly so that a representative sample of the pen could be obtained. From each litter sample bag, a 25-g amount was weighed and placed into a separate Whirl-Pak [4] bag. Butterfield’s buffer [5] solution (50 mL) was added to each bag and mixed thoroughly. After mixing, 0.1 mL from each Whirl-Pak bag was plated, using the spread-plate method, onto a plate containing brilliant green sulfa agar (BGS) [6]. The BGS was prepared according to the instructions of the manufacturer, and nalidixic acid (20 µg/mL) and novobiocin (25 µg/ mL) were added to deter the growth of isolates other than the challenge isolate. Serial dilutions consisting of 1 mL of sample placed into 9 mL of Butterfield’s buffer solution were made up to
a 10−2 dilution. Each dilution was plated onto a separate BGS plate. These plates were used to quantify Salmonella Typhimurium in the sample, if possible. From each individual sample, 1 mL of solution was also removed and placed into a 9-mL tube of tetrathionate broth (TET) for enrichment. The BGS plates and TET tubes were incubated for 18 to 24 h at 35 to 37°C. Each step in this process was done in duplicate to ensure accuracy. After incubation, BGS plates that were presumptively positive for Salmonella Typhimurium were counted. For the enrichment tubes, 1 mL of the TET broth was plated onto the BGS plates and incubated for 18 to 24 h at 35 to 37°C. These plates were then examined for the presumptive presence of Salmonella Typhimurium. The minimum detectable limit for Salmonella in this experiment was 15 cfu/g of litter. Positive plates from the enrichment were assigned a log10 value of 1.5 [7]. Pink colonies on the BGS plates were presumed to be positive samples of Salmonella Typhimurium. Crop and Cecal Samples At 3 wk after placement and at the end of the trial, 6 birds from each pen were killed by CO2 asphyxiation and the crop and ceca were aseptically removed for analysis. The crop and
Table 2. Body weight comparison (g) for birds not killed at wk 3, initial placement to wk 6 Item Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE a,b
Week 0
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
49.56 47.22 48.96 48.95 0.43
132.33 132.82 138.89 140.16 2.48
329.54 327.53 343.58 348.79 5.53
551.17b 625.98a 640.91a 631.09a 11.56
1,087.22b 1,123.71ab 1,173.60a 1,154.55ab 18.10
1,754.88 1,709.80 1,821.14 1,760.90 26.99
2,044.33ab 1,975.58b 2,146.30a 2,074.67ab 30.25
Means within a column with differing superscripts differ significantly (P < 0.05). Diatomaceous earth. 2 Hydrated sodium calcium aluminosilicate. 1
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Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
Week 1
Larrison et al.: LITTER AMENDMENTS ON SALMONELLA Table 3. Litter moisture comparison and FCR for the entire trial Item
18.17 17.28 22.66 16.24 2.591
Cumulative FCR 1.80 1.71 1.72 1.70 0.034
1
Diatomaceous earth. Hydrated sodium calcium aluminosilicate.
Statistical tests used for the litter counts and BW comparisons included the GLM procedure, MEANS statements, and Duncan’s multiple range test. Statistical tests used for crop and cecal analysis included the GLM procedure, LSMEANS statement, and PDIFF option [9]. Outputs were deemed significant at P < 0.05.
RESULTS AND DISCUSSION
2
ceca from each bird were placed into separate Whirl-Pak bags and diluted with Butterfield’s buffer solution at a 1:2 ratio (wt/wt). Each bag was stomached [8] for 30 s. After stomaching, 0.1 mL was removed from the sample bag and dispensed into 9.9 mL of Butterfield’s buffer solution. Serial dilutions were than made up to a 10−4 dilution of the original sample. Samples from each of the dilutions were plated onto BGS plates using the spread-plate method. An additional 1 mL of the original solution was placed into 9 mL of TET broth for enrichment. The BGS plates and TET tubes were incubated for 18 to 24 h at 35 to 37°C. Each step in this process was done in duplicate to ensure accuracy. After incubation, BGS plates that were presumptively positive for Salmonella Typhimurium were counted. The minimum detectable limit for Salmonella in this experiment was 15 cfu/mL of rinse. For the enrichment tubes, 1 mL of the TET broth was plated onto BGS plates and incubated for 18 to 24 h at 35 to 37°C. These plates were then examined for the presumptive presence of Salmonella Typhimurium. Pink colonies on the BGS plates were presumed to be positive samples of Salmonella Typhimurium. Statistics All statistics for this experimental project were calculated using SAS version 9.1 [9].
Broiler Growth Characteristics Body weights of all birds placed in this study were not significantly different at the day of placement (Table 1). As a group, the negative control (group 1) birds exhibited a significantly lighter BW at wk 1 and 3 after placement. It is important to note that calculations for BW comparisons were for all birds placed in the study up to 3 wk after placement. At 3 wk after placement, 6 birds from each pen were killed to determine Salmonella Typhimurium isolation. The BW comparison for the entire trial was based on those birds that were not killed until the end of the experiment. For the complete grow-out period (Table 2), there were no significant BW differences at wk 1, 2, and 5. For the weeks that did exhibit significant differences in BW, the negative control (group 1) birds were significantly lighter at wk 3 and 4, whereas the positive control (group 2) birds were the lightest at the end of the experiment. Litter moisture percentages and FCR at the end of the experiment did not exhibit significant differences (Table 3). Bacterial Challenge No significant differences were found in the recovery of Salmonella Typhimurium from litter samples throughout the study (Table 4). It was expected that recovered amounts of this microbe
Table 4. Recovery of Salmonella from litter (average cfu/g of litter sampled), initial placement to wk 6 Item
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
10.00 7.12 5.98 60.25 0.274
3.16 7.94 40.45 3.24 0.218
0.00 9.33 13.71 7.24 0.233
59.84 14.89 11.56 13.27 0.256
325.83 1148.15 1164.12 558.47 0.149
3.06 2.10 13.27 4.17 0.145
1
Diatomaceous earth. Hydrated sodium calcium aluminosilicate.
2
Downloaded from http://japr.oxfordjournals.org/ at Frankfurt University Library, Section Stadt- und Universitaetsbibliothek on June 8, 2015
Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
Moisture, %
135
JAPR: Research Report
136
Table 5. Recovery of Salmonella from the crop and ceca of birds killed at wk 3 and 6 (average cfu/g of crop or ceca) Crop Item
Week 3
Week 6
Week 3
Week 6
0a 1.20a 0a 0a 0.028
0a 1.20a 1.20a 1.20a 0.047
0c 5.62a 1.78bc 3.24ab 0.681
1.20b 1.20b 2.51a 0b 0.270
a–c
Means within a column with differing superscripts differ significantly (P < 0.05). Diatomaceous earth. 2 Hydrated sodium calcium aluminosilicate. 1
would be high because of the high concentration of organisms placed onto the litter at the beginning of the trial. However, this was not the case. The investigators surmise that the low litter moisture in the pens was the overall determining factor in the low numbers found. Overall, 9% of the samples had recoverable Salmonella after enrichment. Recovery of Salmonella Typhimurium from the crops and ceca of the birds at wk 3 and 6 is shown in Table 5. There were no significant differences in recovery from the crop at either wk 3 or wk 6. The investigators suspect that the low numbers found in this portion of the experiment were because the birds were not orally gavaged with bacterium, but were expected to pick it up from the litter. Very small numbers of the challenge were recovered from the ceca at wk 3, but there were significant differences among groups, with the negative control having no recoverable Salmonella Typhimurium at that time. Recovery of the challenge from the ceca at wk 6 also exhibited very small numbers, with significant differences. In this case, the DE treatment (group 3) had the highest count of recoverable Salmonella. The treatment with the clay additive (group 4) had no recoverable Salmonella at wk 6. We suggest that, once again, the low recovery was due to the method of challenge.
CONCLUSIONS AND APPLICATIONS
1. Efficacy of the litter amendments against Salmonella Typhimurium varied widely during the experiment.
2. Litter amendments in this study did not result in any adverse effects on broiler growth rate. 3. Litter amendments in this study did not result in any adverse effects on litter moisture or feed conversion. 4. Further research should be done using a different challenge method to determine if the litter amendments tested in this study would be a viable intervention strategy for Salmonella in the grow-out phase of broiler production.
REFERENCES AND NOTES 1. Hayes, J. R., L. E. Carr, E. T. Mallinson, L. W. Douglass, and S. W. Joseph. 2000. Characterization of the contribution of water activity and moisture content to the population distribution of Salmonella spp. in commercial poultry houses. Poult. Sci. 79:1557–1561. 2. Centers for Disease Control. 1992. Summary of notifiable diseases, United States, 1991. Morb. Mortal. Wkly. Rep. 40:633–638. 3. Line, J. E. 2002. Campylobacter and Salmonella populations associated with chickens raised on acidified litter. Poult. Sci. 81:1473–1477. 4. Whirl-Pak bags, 7 oz., Nasco; purchased from VWR, West Chester, PA. 5. Butterfield’s buffer, BD Diagnostic Systems; purchased from VWR, West Chester, PA. 6. Brilliant green sulfa agar, BD Diagnostic Systems; purchased from VWR, West Chester, PA. 7. Corrier, D. E., D. J. Nisbet, A. G. Hollister, C. M. Scanlan, B. M. Hargis, and J. R. DeLoach. 1993. Development of defined cultures of indigenous cecal bacteria to control salmonellosis in broiler chicks. Poult. Sci. 72:1164– 1168. 8. Stomacher 80 Lab Blender, Seward Medical, London, UK. 9. SAS Institute. 2008. SAS User’s Guide. Version 9.1 ed. SAS Inst. Inc., Cary, NC.
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Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
Ceca
Effects of litter amendments on broiler growth characteristics and Salmonella colonization in the crop and cecum
*Department of Poultry Science, Texas A&M University, College Station 77843; and †USDA-ARS, Food and Feed Safety Research Unit, College Station, TX 77845 Primary Audience: Broiler Grow-Out Technicians, Supervisors SUMMARY Salmonella can be prevalent in poultry litter and can be a source of contamination for chicks newly arrived at the poultry house. The objective of this study was to determine the effects of litter amendments on broiler grow-out characteristics (BW and feed conversion), litter moisture, and Salmonella enterica serovar Typhimurium colonization in the litter, crop and cecum. Because Salmonella Typhimurium is a pathogen of concern to the poultry industry, 2 types of litter amendments and 2 types of feed additions were created and tested to determine the effects of broiler growth, litter moisture, and litter efficacy against Salmonella colonization. Litter amendments consisted of a combination of acidic calcium sulfate and either diatomaceous earth or hydrated sodium calcium aluminiosilicate. Feed additions consisted of differing amounts of sodium bisulfate. For the litter amendment experiments, chicks were placed into pens in isolation rooms. Each litter amendment was applied to 3 pens for replicates of experimental groups. Litter samples were taken weekly from 5 areas in each pen and were combined for the determination of Salmonella colony-forming units per gram of litter. At 3 and 6 wk after placement, birds from each pen were killed by CO2 asphyxiation. Efficacy of the litter amendments varied among experimental groups in broiler growth characteristics and efficacy against Salmonella. Key words: litter, Salmonella, broiler, poultry 2010 J. Appl. Poult. Res. 19:132–136 doi:10.3382/japr.2009-00083
DESCRIPTION OF PROBLEM Contamination of poultry products by pathogenic bacteria poses both health and financial risks to poultry integrators [1]. Consumer confidence in the food supply is a major driving force behind recommendations and regulations that deal with modern poultry production. The paratyphoid salmonellae, especially Salmonella
1
Corresponding author: [email protected]
enterica serovar Typhimurium, are pathogens of concern for the poultry industry because of their association with raw poultry products and because these organisms displayed a 4-fold increase in isolation from ill individuals from 1955 to the 1990s [2]. Many successful programs have been instituted to meet the federal requirements and consumer demands for pathogen control. Most of
Downloaded from http://japr.oxfordjournals.org/ at Frankfurt University Library, Section Stadt- und Universitaetsbibliothek on June 8, 2015
E. L. Larrison,* J. A. Byrd,† and M. A. Davis*1
Larrison et al.: LITTER AMENDMENTS ON SALMONELLA
MATERIALS AND METHODS Grow-Out Conditions Birds in this experiment were supervised under the USDA Animal Care and Use protocol. Day-of-hatch broiler chicks (180 total, 15 per pen, 45 per treatment, 4 treatments) were placed and provided free access to feed and water. Feed was weighed in grams to allow feed conversion to be calculated at the end of the study. Birds
were housed in 4 separate rooms in an isolation building with positive airflow and air filtration. Each room in the isolation unit was divided into 3 pens, and each pen was equipped with a nipple watering system and adequate feeders. The grow-out phase of the study consisted of 12 pens to allow for 3 replications of each experimental group. Birds were fed a starter broiler feed for the first 3 wk of the study and were switched to a grower ration for the remainder of the study. All birds were killed by CO2 asphyxiation at 5 wk and 3 d of age after placement. After the experiment was concluded, total BW (by pen) was determined to calculate feed conversion. Averages of individual bird BW in each pen were also calculated for comparison. Experimental Groups and Challenge There were 4 experimental groups with 3 replications per group. Group 1 (negative control) did not receive any litter amendment or challenge with Salmonella Typhimurium and thus was the negative control. Birds in the 3 replications for the negative control were placed in a separate isolation room from those challenged with Salmonella Typhimurium. Birds in the other group replications were dispersed evenly throughout the other 3 isolation rooms. Group 2 (positive control) did not receive the litter amendment, but birds were challenged with Salmonella Typhimurium and served as the positive control. Group 3 received the litter amendment mixture containing the DE, and birds were challenged with Salmonella Typhimurium. Group 4 received the litter amendment mixture containing the clay, and birds were challenged with Salmonella Typhimurium. Litter amendments were applied by sprinkling the amendment onto the litter in accordance with the directions of the manufacturer. The amendment was then raked into the litter throughout the pen to cover the floor space. For those groups that received the challenge with Salmonella Typhimurium, 150 mL of a solution containing 3.8 × 108 cfu/mL of novobiocin- and nalidixic acid-resistant Salmonella Typhimurium was applied to the litter in each pen with a spray bottle, making sure to cover the entire pen area with the spray. The Salmonella challenge inoculate was prepared from a primary poultry isolate of Salmonella
Downloaded from http://japr.oxfordjournals.org/ at Frankfurt University Library, Section Stadt- und Universitaetsbibliothek on June 8, 2015
these programs, such as the Hazard Analysis and Critical Control Point system, Food Safety Objectives, and the introduction of novel chemical antimicrobials, have centered on the processing plant as the main target for food safety control measures. Recently, there has been interest in implementing on-farm pathogen reduction strategies to ease the burden on the processing plant by decreasing the initial load of bacteria that enter the plant with live birds. Reducing pathogens before processing can be beneficial in that it may allow the processing plant to lower the cost of production because of decreased risk, and there may also be a decrease in the amount of crosscontamination in the processing environment. The use of litter amendments is one possible approach to achieve on-farm pathogen reduction. Many formulations of litter amendments have been developed and tested, with varying results. Most of the compounds that have been tested are acidic in nature. Because of this characteristic, it is reasonable to assume that proper application could significantly lower both the pH and water activity of the litter, conditions that directly affect the survivability of microorganisms that are typically present in poultry litter [3]. The litter amendments used must not interfere with the normal growth characteristics of the broilers subjected to the treatment and must not present any additional risks to the broilers or to employees of the grow-out facility. The objective of this study was to determine the effects of litter amendments on broiler grow-out characteristics (BW and feed conversion), litter moisture, and Salmonella Typhimurium colonization in the litter, crop, and cecum. Litter amendments consisted of acidic calcium sulfate combined with either diatomaceous earth (DE) or hydrated sodium calcium aluminosilicate (clay).
133
JAPR: Research Report
134 Table 1. Body weight comparison (g) for all broilers, initial placement to wk 3 Item
Week 0 49.08 48.08 48.85 49.24 0.29
Week 2
Week 3
b
329.57 328.00 341.78 350.62 4.54
552.38b 627.01a 617.73a 624.20a 10.15
130.94 132.86ab 136.05ab 141.92a 1.88
a,b
Means within a column with differing superscripts differ significantly (P < 0.05). Diatomaceous earth. 2 Hydrated sodium calcium aluminosilicate. 1
Typhimurium obtained from the National Veterinary Services Laboratory (Ames, Iowa). After application of the litter amendment and Salmonella Typhimurium challenge, 15 one-day-old chicks were wing-banded and randomly placed into each pen. Initial BW was recorded for each chick as it was placed into the pen. Litter Samples Litter samples from each pen were collected each week from 4 random areas in each pen and combined in a zipper bag. The bags were shaken to distribute the contents evenly so that a representative sample of the pen could be obtained. From each litter sample bag, a 25-g amount was weighed and placed into a separate Whirl-Pak [4] bag. Butterfield’s buffer [5] solution (50 mL) was added to each bag and mixed thoroughly. After mixing, 0.1 mL from each Whirl-Pak bag was plated, using the spread-plate method, onto a plate containing brilliant green sulfa agar (BGS) [6]. The BGS was prepared according to the instructions of the manufacturer, and nalidixic acid (20 µg/mL) and novobiocin (25 µg/ mL) were added to deter the growth of isolates other than the challenge isolate. Serial dilutions consisting of 1 mL of sample placed into 9 mL of Butterfield’s buffer solution were made up to
a 10−2 dilution. Each dilution was plated onto a separate BGS plate. These plates were used to quantify Salmonella Typhimurium in the sample, if possible. From each individual sample, 1 mL of solution was also removed and placed into a 9-mL tube of tetrathionate broth (TET) for enrichment. The BGS plates and TET tubes were incubated for 18 to 24 h at 35 to 37°C. Each step in this process was done in duplicate to ensure accuracy. After incubation, BGS plates that were presumptively positive for Salmonella Typhimurium were counted. For the enrichment tubes, 1 mL of the TET broth was plated onto the BGS plates and incubated for 18 to 24 h at 35 to 37°C. These plates were then examined for the presumptive presence of Salmonella Typhimurium. The minimum detectable limit for Salmonella in this experiment was 15 cfu/g of litter. Positive plates from the enrichment were assigned a log10 value of 1.5 [7]. Pink colonies on the BGS plates were presumed to be positive samples of Salmonella Typhimurium. Crop and Cecal Samples At 3 wk after placement and at the end of the trial, 6 birds from each pen were killed by CO2 asphyxiation and the crop and ceca were aseptically removed for analysis. The crop and
Table 2. Body weight comparison (g) for birds not killed at wk 3, initial placement to wk 6 Item Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE a,b
Week 0
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
49.56 47.22 48.96 48.95 0.43
132.33 132.82 138.89 140.16 2.48
329.54 327.53 343.58 348.79 5.53
551.17b 625.98a 640.91a 631.09a 11.56
1,087.22b 1,123.71ab 1,173.60a 1,154.55ab 18.10
1,754.88 1,709.80 1,821.14 1,760.90 26.99
2,044.33ab 1,975.58b 2,146.30a 2,074.67ab 30.25
Means within a column with differing superscripts differ significantly (P < 0.05). Diatomaceous earth. 2 Hydrated sodium calcium aluminosilicate. 1
Downloaded from http://japr.oxfordjournals.org/ at Frankfurt University Library, Section Stadt- und Universitaetsbibliothek on June 8, 2015
Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
Week 1
Larrison et al.: LITTER AMENDMENTS ON SALMONELLA Table 3. Litter moisture comparison and FCR for the entire trial Item
18.17 17.28 22.66 16.24 2.591
Cumulative FCR 1.80 1.71 1.72 1.70 0.034
1
Diatomaceous earth. Hydrated sodium calcium aluminosilicate.
Statistical tests used for the litter counts and BW comparisons included the GLM procedure, MEANS statements, and Duncan’s multiple range test. Statistical tests used for crop and cecal analysis included the GLM procedure, LSMEANS statement, and PDIFF option [9]. Outputs were deemed significant at P < 0.05.
RESULTS AND DISCUSSION
2
ceca from each bird were placed into separate Whirl-Pak bags and diluted with Butterfield’s buffer solution at a 1:2 ratio (wt/wt). Each bag was stomached [8] for 30 s. After stomaching, 0.1 mL was removed from the sample bag and dispensed into 9.9 mL of Butterfield’s buffer solution. Serial dilutions were than made up to a 10−4 dilution of the original sample. Samples from each of the dilutions were plated onto BGS plates using the spread-plate method. An additional 1 mL of the original solution was placed into 9 mL of TET broth for enrichment. The BGS plates and TET tubes were incubated for 18 to 24 h at 35 to 37°C. Each step in this process was done in duplicate to ensure accuracy. After incubation, BGS plates that were presumptively positive for Salmonella Typhimurium were counted. The minimum detectable limit for Salmonella in this experiment was 15 cfu/mL of rinse. For the enrichment tubes, 1 mL of the TET broth was plated onto BGS plates and incubated for 18 to 24 h at 35 to 37°C. These plates were then examined for the presumptive presence of Salmonella Typhimurium. Pink colonies on the BGS plates were presumed to be positive samples of Salmonella Typhimurium. Statistics All statistics for this experimental project were calculated using SAS version 9.1 [9].
Broiler Growth Characteristics Body weights of all birds placed in this study were not significantly different at the day of placement (Table 1). As a group, the negative control (group 1) birds exhibited a significantly lighter BW at wk 1 and 3 after placement. It is important to note that calculations for BW comparisons were for all birds placed in the study up to 3 wk after placement. At 3 wk after placement, 6 birds from each pen were killed to determine Salmonella Typhimurium isolation. The BW comparison for the entire trial was based on those birds that were not killed until the end of the experiment. For the complete grow-out period (Table 2), there were no significant BW differences at wk 1, 2, and 5. For the weeks that did exhibit significant differences in BW, the negative control (group 1) birds were significantly lighter at wk 3 and 4, whereas the positive control (group 2) birds were the lightest at the end of the experiment. Litter moisture percentages and FCR at the end of the experiment did not exhibit significant differences (Table 3). Bacterial Challenge No significant differences were found in the recovery of Salmonella Typhimurium from litter samples throughout the study (Table 4). It was expected that recovered amounts of this microbe
Table 4. Recovery of Salmonella from litter (average cfu/g of litter sampled), initial placement to wk 6 Item
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
10.00 7.12 5.98 60.25 0.274
3.16 7.94 40.45 3.24 0.218
0.00 9.33 13.71 7.24 0.233
59.84 14.89 11.56 13.27 0.256
325.83 1148.15 1164.12 558.47 0.149
3.06 2.10 13.27 4.17 0.145
1
Diatomaceous earth. Hydrated sodium calcium aluminosilicate.
2
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Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
Moisture, %
135
JAPR: Research Report
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Table 5. Recovery of Salmonella from the crop and ceca of birds killed at wk 3 and 6 (average cfu/g of crop or ceca) Crop Item
Week 3
Week 6
Week 3
Week 6
0a 1.20a 0a 0a 0.028
0a 1.20a 1.20a 1.20a 0.047
0c 5.62a 1.78bc 3.24ab 0.681
1.20b 1.20b 2.51a 0b 0.270
a–c
Means within a column with differing superscripts differ significantly (P < 0.05). Diatomaceous earth. 2 Hydrated sodium calcium aluminosilicate. 1
would be high because of the high concentration of organisms placed onto the litter at the beginning of the trial. However, this was not the case. The investigators surmise that the low litter moisture in the pens was the overall determining factor in the low numbers found. Overall, 9% of the samples had recoverable Salmonella after enrichment. Recovery of Salmonella Typhimurium from the crops and ceca of the birds at wk 3 and 6 is shown in Table 5. There were no significant differences in recovery from the crop at either wk 3 or wk 6. The investigators suspect that the low numbers found in this portion of the experiment were because the birds were not orally gavaged with bacterium, but were expected to pick it up from the litter. Very small numbers of the challenge were recovered from the ceca at wk 3, but there were significant differences among groups, with the negative control having no recoverable Salmonella Typhimurium at that time. Recovery of the challenge from the ceca at wk 6 also exhibited very small numbers, with significant differences. In this case, the DE treatment (group 3) had the highest count of recoverable Salmonella. The treatment with the clay additive (group 4) had no recoverable Salmonella at wk 6. We suggest that, once again, the low recovery was due to the method of challenge.
CONCLUSIONS AND APPLICATIONS
1. Efficacy of the litter amendments against Salmonella Typhimurium varied widely during the experiment.
2. Litter amendments in this study did not result in any adverse effects on broiler growth rate. 3. Litter amendments in this study did not result in any adverse effects on litter moisture or feed conversion. 4. Further research should be done using a different challenge method to determine if the litter amendments tested in this study would be a viable intervention strategy for Salmonella in the grow-out phase of broiler production.
REFERENCES AND NOTES 1. Hayes, J. R., L. E. Carr, E. T. Mallinson, L. W. Douglass, and S. W. Joseph. 2000. Characterization of the contribution of water activity and moisture content to the population distribution of Salmonella spp. in commercial poultry houses. Poult. Sci. 79:1557–1561. 2. Centers for Disease Control. 1992. Summary of notifiable diseases, United States, 1991. Morb. Mortal. Wkly. Rep. 40:633–638. 3. Line, J. E. 2002. Campylobacter and Salmonella populations associated with chickens raised on acidified litter. Poult. Sci. 81:1473–1477. 4. Whirl-Pak bags, 7 oz., Nasco; purchased from VWR, West Chester, PA. 5. Butterfield’s buffer, BD Diagnostic Systems; purchased from VWR, West Chester, PA. 6. Brilliant green sulfa agar, BD Diagnostic Systems; purchased from VWR, West Chester, PA. 7. Corrier, D. E., D. J. Nisbet, A. G. Hollister, C. M. Scanlan, B. M. Hargis, and J. R. DeLoach. 1993. Development of defined cultures of indigenous cecal bacteria to control salmonellosis in broiler chicks. Poult. Sci. 72:1164– 1168. 8. Stomacher 80 Lab Blender, Seward Medical, London, UK. 9. SAS Institute. 2008. SAS User’s Guide. Version 9.1 ed. SAS Inst. Inc., Cary, NC.
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Group 1 (negative control) Group 2 (positive control) Group 3 (DE1 + Salmonella) Group 4 (clay2 + Salmonella) SE
Ceca