DETECTION, QUANTIFICATION AND MODIFICATION OF BACTERIAL POPULATIONS ASSOCIATED WITH BROILER POULTRY LITTER

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0222163
• Project No.: ALA018-1-10027
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015

Project Director
Macklin, KE.

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Poultry Science

Non Technical Summary
Proper litter management is important for good bird health as well as their ability to resist colonization by foodborne pathogens. As the cost of bedding and the difficulty in disposal increased, the practice of keeping litter in the house longer has gone up. This has lead to an increase in ammonia exposure to newly placed chicks as well as an increased chance of exposure to pathogens from the preceding flock. These issues are controlled by the addition of litter amendments or by physically manipulating the litter and often times a combination of both. Litter amendments can be broken down into one of two categories, those being either chemical or biological. Among the chemical amendments there are two main classes, one being the acidifiers and the other the alkaline. The acidifiers work by lowering the litter's pH. This lowering of the pH causes a reduction in ammonia production as well as a decrease in microbial numbers. The alkaline amendments work by increasing litter pH, this results in a decrease in microbial numbers. However it doesn't decrease ammonia production. Biological litter amendments are more difficult to classify; essentially they are either bacterial, enzymatic, or plant based. There are several bacterial amendments and although they generally have different bacterial mixtures, they all have the same general mode of action. They, like the chemical amendments, work by either lowering or raising the pH of the litter and by acting as a competitive exclusion agent within the litter. The second category of biological amendment mentioned is the enzymes. These work by either preventing the nitrifying bacteria from forming ammonia or by causing an increase in ammonia production from this same category of bacteria. The third group of biological litter amendments are the plant based ones. These amendments work similarly to the enzyme based ones in that they inhibit the production of ammonia by means of enzymes present in the plant. These particular amendments can be either directly added to the litter or fed to the birds. The objective of physically manipulating the litter is to dry the litter. By drying the litter there will be a decrease in bacterial numbers which in turn leads to less potential pathogens as well as a decrease in nitrifying bacteria. There are several different methods for accomplishing this. Essentially all of them involve mixing the litter. This action allows moisture to be removed from the litter, thus drying the litter which in turn leads to lower bacterial and ammonia levels. Although the mentioned methodologies have been used for some time, relatively little is known as to how they affect the litter's microbial profile. Generally it is thought that they are manipulating this microbial profile in a positive way; however this has yet to be determined. Perhaps from the above mentioned, a combination of, or from as yet unknown methodology an ideal litter treatment program can be developed. This program would ideally minimize the exposure newly placed chicks have to ammonia as well as to pathogens.

Animal Health Component

40%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
306	3299	1100	40%
311	3299	1100	60%

Knowledge Area
311 - Animal Diseases; 306 - Environmental Stress in Animals;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

broiler chicken

litter

microbiology

Goals / Objectives
Objective 1 - Develop a novel quantitative PCR method to determine the amount and type of bacteria present in poultry litter. This PCR will be based on the highly conserved 16s rRNA regions of the bacteria's chromosome. It has been shown previously that bacterial numbers and types can be detected in complex microbial communities with little loss of resolution (Lu, et al., 2003). Using this technique it will be possible to measure the bacterial populations in untreated versus any one of the various litter treatment techniques described above. Objective 2 - The initial phase of the study will be performed at the Auburn University Poultry Research Farm (AUPRF). At the AUPRF, unsoiled bedding as well as litter that has been exposed to multiple flocks will be tested both before and after chemical, biological and physical treatment. From the results generated in these studies a baseline level of what to expect in the field will be obtained. The eventual goal of this project is to obtain samples and flock history records from commercial poultry farms. These data will be analyzed and any significant trend noted. It is expected that there will be variations from the AUPRF baseline. However there should be general trends as to what makes up good litter quality that minimizes disease and ammonia production. Objective 3 - Explore novel techniques in reducing antagonistic bacteria and ammonia production. From the results obtained in objective 2, it is believed that a better litter amendment can be developed. This will be performed by determining what microbes are present in litter associated with poultry flocks that had superior performance. Performance will be determined by where the flocks ranked when it went to market as well as to the health status of the flock. With this microbial profile it should be possible to develop a bacterial based litter amendment. Additionally, it will be possible to determine which, if any of the current litter amendments lead to this profile. Any developed litter amendment will be tested in grow out trials to determine the reproducibility of the results over an extended period of time.

Project Methods
Quantitative PCR should make identification of general as well as specific bacteria relatively simple. It is believed that this technique will lend itself to a fast, easily performed identification of litter microbes. Using the developed technique, the ability to sample various litter samples should proceed rather quickly. Using this technique to determine the affect of the various litter treatments on litter microflora will help answer questions as to how these treatments are manipulating the litter microflora. As more is learned about the effects of litter treatments on the litter's microflora it is anticipated that a novel litter treatment may be devised that can lend themselves to a better litter quality. Better litter quality will lead to less stressed birds, which in turn will lead to healthier birds. Healthier birds should lead to less pathogens entering the processing plant, hence into the food chain.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience: Poultry farmers, company representatives, researchers and government regulators. Changes/Problems:The most significant change from the initial proposal was the inability to test the developed litter amendments under commercial poultry conditions. This was due to concerns from the partnered poultry company about Avian Influenza. We had tested the products at the AUPRF and had encouraging results. It is our hope that in 2016 or 2017 we can test the developed products under commercial conditions. What opportunities for training and professional development has the project provided?Under this project I had trained one PhD, one MAg and three MS students. In addition I have three MS and one MAg in training. All of these students have been involved with this project. I have given several presentations both uninvited and invited on the subject of litter management and alternatives to commonly used litter amendments. In addition I have been consulted by several poultry and non-poultry companies as to litter management, both conventional methods as well as unconventional. How have the results been disseminated to communities of interest? The PI has an extension appointment. All of the results have been discussed in either scientific journals, popular press articles or directly with representatives from both commercial poultry as well as allied industry companies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1- Was completed in 2012. However due to advances in technology we now send samples off for sequencing using Illumina MiSeq. It is cheaper and the results better then that performed using standard 454 pyrosequencing. Objective 2- Has been completed. From these results we have identified good bacteria that under laboratory conditions reduces ammonia production as well as lowers Salmonella and Clostridium perfringens levels. Objective 3- From the results obtained from objective 2 we had tested the novel litter amendment at the AUPRF. The results were mixed in that there was a reduction in ammonia, though not as expected. In addition, Salmonella levels were unchanged between our "novel" litter amendment and a commercially available one. With some additional modifications there may be developed a product that can reduce ammonia and Salmonella in poultry litter. The product has not been tested at a commercial poultry house due to concerns of Avian Influenza.

Publications

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Poultry farmers, company representatives, researchers and governement regulators. Changes/Problems: Costs for sequencing have gone down, so we have been sending more samples out then before. In addition the technology has eclipsed that which was intially described in this project and we have adjusted to using the new technology/techniques. What opportunities for training and professional development has the project provided? I have graduated two graduate students (MS and PhD) off of this project and currenly have four graduate students (3x MS and 1 MAg) that are either working on this project full or partial time. In addition I have three undergraduates that participate in this project on a part time basis. How have the results been disseminated to communities of interest? Yes some of the results have been discussed with commercial and non- commercial poultry producers. As mentioned there is some interest in trying out the ammonia control interventions. What do you plan to do during the next reporting period to accomplish the goals? A commercial company has expressend interest in using the product to control ammonia. They are also interested in the effects the product has on the litter microbiome as well as the birds microbiome.

Impacts
What was accomplished under these goals? Objective 1- Was accomplised in 2012. However due to advances in technology we now send samples off for sequencing using Illumina MiSeq. It is overall cheaper and the results better then that performed using standard 454 pyrosequencing. Objective 2- Has been accomplished. Some of the results have been presented at meetings and the papers should be at the very least submitted in 2015. Objective 3- We have profiles of the "good" and "bad" bacteria. I have been in communication with a commercial company to try out our novel admendment under commercial conditions.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: English, K.G., M. Singh and K.S. Macklin. 2014. Survey of the biosecurity and management practices on broiler farms. Abstract 1. Poultry Science vol. 93 (E-Suppl. 1): 1.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Krehling, J.T., L.B. Olivia, K. English, A.N Frazier and K.S. Macklin. 2014. Evaluation of litter from three broiler complexes for coccidia resistance. Abstract 119. Poultry Science vol. 93 (E-Suppl. 1): 42
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hess, J.B., K.S. Macklin and J.P. Blake. 2014. House environmental impact of wood pellet burning stoves. Abstract M71(2). Poultry Science vol. 93 (E-Suppl. 1): 212.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fraizer, A.N., J.T. Krehling, L. Olivia, K. English and K.S. Macklin. 2014. Effect of two Bacillus subtilis strains on production and Salmonella colonization in male broilers. Abstract 94. Poultry Science vol. 93 (E-Suppl. 1): 33

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The target audience for this work is - researchers, farmers, company representatives and government regulators. Changes/Problems: We do not have easy access to the equipment for pyrosequencing and the cost to send them off for analysis is high. With that being said we will do what we can with the resources we have available. What opportunities for training and professional development has the project provided? To date two students have been trainied in the procedures need to accomplish this project. How have the results been disseminated to communities of interest? The PI - Macklin has an extension appointment. He regularly speeks to poultry farmers and company representatives about managing litter. To date I have freely shared what is approriate to those two groups my findings. That pretty much is that for controlling ammonia the acidifying litter amendmants are currently the best. What do you plan to do during the next reporting period to accomplish the goals? Perform more small scale studies at the AU poultry research farm. Collect samples from commercial poultry farms to get more data points as to what makes up a good microbiome in the litter.

Impacts
What was accomplished under these goals? Objective 1 - We have used pyrosequencing for characterizing litter bactrial populations. This work was performed and published by my former PhD student Zac Williams. We plan on continuing to use this technology to further our understainding of what bacteria are in the litter. Objective 2 - The first phase (working at the AU poultry farm) of this objecitve is well underway. We have a few more treatments to test before we take our findings into the field for real world testing. Objective 3 - This objective is tied closely to objective 1. As mentioned that objective is well underway. We plan on taking samples for sequencing this year with the goal of determining the "good" profile.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Williams, Z.T. and K.S. Macklin. 2013. Reduction of Salmonella and ammonia emissions in broiler litter using sulfuric acid and aluminum sulfate. International Journal of Poultry Science, 12 (6), 328-334.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Williams, Z.T. and K.S. Macklin. 2013. Stratification of bacterial concentrations from upper to lower, in broiler litter. Journal of Applied Poultry Research, 22 (3), 492-498.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Bailey, M.A., K.S. Macklin, *J.T. Krehling. 2013. Use of a multiplex PCR for the detection of toxin-encoding genes netB and tpeL in strains of Clostridium perfringens. ISRN Veterinary Science, http://www.hindawi.com/isrn/veterinary.science/2013/865702/
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Macklin, K.S., M.R. Liles, Z.T. Williams and J.P. Blake. 2013. The use of bacteria to control ammonia and pathogens in litter. Midwest Poultry Federation Convention, pg. 5. March 13-14, 2013. Saint Paul, MN
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Macklin, K.S., J.B. Hess and S.F. Bilgili. 2013. Poultry bedding sources and litter management. Second International Poultry Meat Congress, pg. 3. April 24-28, 2013. Antalya, Turkey
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Macklin, K.S. 2013. Managing Poultry Waste. 3rd Incoftech Meeting, p. 115-118. January 4-5, 2013. Thanjavur, Tamil Nadu, India
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Williams, Z.T., M.A. Bailey and K.S. Macklin. 2013. Using sulfuric acid and aluminum sulfate to control litter ammonia emissions and Salmonella concentration. Abstract 211. Poultry Science, vol 92 (E-Suppl. 1): 75
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Bailey, M.A., K.S. Macklin and J.T. Krehling. 2013. Use of PCR for the detection of the netB gene in strains of Clostridium perfringens isolated from cases of avian necrotic enteritis in Alabama. Abstract P274. Poultry Science, vol 92 (E-Suppl. 1): 98.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: My labs efforts have been focused on identifying a Bacillus spp bacterium that can effectively reduce ammonia production in litter. In addition we screened these bacteria for pathogen elimination, specifically Salmonella. To date only one of these bacterium have shown promise when tested under field like conditions; more research with this bacterium needs to be performed. In addition to using our biological litter amendments we have looked at commercial biological and chemical litter amendments to effect the litter bacterial populations. Again specifically targeting ammonia producing bacteria, as well as pathogens C. perfringens and Salmonella. Data produced has been disseminated at scientific meetings and scientific journals. In addition information has been shored at meetings aimed at the commercial poultry industry as well as in popular press/trade journals. The PI over this past year had graduated a PhD student and has one MS student still working in his lab. In addition to graduate students, the PI has had several undergraduates working on various poultry litter projects that contribute to this project. PARTICIPANTS: Kenneth S. Macklin - PI, Zachary T. Williams - PhD student, Matthew A. Bailey - MS student, John P. Blake - collaborator, Mark A. Liles - collaborator, ten undergraduates had received hands on training in the PI's lab. The PI has an extension appointment, as part of that appointment he is expected to give talks about various topics. The most noted of which is poultry litter management, this past year the PI spoke on this topic three times at trade meetings. TARGET AUDIENCES: Poultry industry personal, poultry farmers and environmental groups PROJECT MODIFICATIONS: None

Impacts
Although a lot of work has been performed in my lab. The outcome is similar to what has been reported. That being that acidifying litter is effective at lowering ammonia emissions from poultry litter; however by doing this enteric bacteria, like Salmonella, seem to be partially protected from being eliminated. Using Bacillus to control litter ammonia, seems to be possible. The commercial bacterial litter amendment tested seemed to work after the third flock (third application). Although not as effective as the chemical (acidifying) litter amendment it seemed to change the detected ammonia levels slightly. The Bacillus tested in the PIs lab seems to have some promise. Further work with it needs to be performed.

Publications

• Williams, Z.T. and K.S. Macklin. 2012. The effect of sodium bisulfate on Salmoenlla viability in broiler litter. Poultry Science 91(9): 2083-2088.
• Gholap, D.B., K.S. Macklin, J.P. Blake, and S.F. Bilgili. 2012. Efficacy of a chemical and a biological litter amendment in commercial broiler facilities. National Poultry Waste Management Symposium, pg. 5. October 23-25, 2012, Shreveport, LA.
• Gholap, D.B., K.S. Macklin, J.P. Blake, and S.F. Bilgili. 2012. Poultry Litter Treatment (PLT) usage in commercial broiler houses for three consecutive flocks. National Poultry Waste Management Symposium, pg. 5. October 23-25, 2012, Shreveport, LA.
• Williams, Z.T. and K.S. Macklin. 2012. Use of Microbial Litter Amendments to Control Pathogens in Poultry Litter. National Poultry Waste Management Symposium, pg. 3. October 23-25, 2012, Shreveport, LA.
• Williams, Z., M. Bailey, J. Blake, and K. Macklin. 2012. Controlling broiler litter ammonia emissions with chemical litter amendments. 61st Western Poultry Disease Conference, p.120. April 2-4, 2012, Scottsdale, AZ.
• Gholap, D., K. Macklin, J. Blake, and S. Bilgili. 2012. Estimation of the efficacy of a chemical and a biological litter amendment in reducing ammonia levels, bacterial load, and paw lesions in commercial broiler facilities. 61st Western Poultry Disease Conference, p.103-105. April 2-4, 2012, Scottsdale, AZ.
• Gholap, D.B., K.S. Macklin, J.P. Blake and S.F. Bilgili. 2012. Compartive study of poultry litter treatment (PLT) in maintaining air and litter quality with microbioogical fluctuations over three consecutive broiler flocks. Abstract 90. Poultry Science, suppl. 1 ed., vol 91: 32.
• Gholap, D.B., K.S. Macklin, J.P. Blake and S.F. Bilgili. 2012. Evaluation of poultry litter treatments in reducing ammonia emission rates, pH and microbial load in commercial broiler houses over three consecutive broiler flocks. Abstract 155. Poultry Science, suppl. 1 ed., vol 91: 55.
• Williams, Z., J. Stough, M. Liles and K.S. Macklin. 2012. Reducing Salmonella and broiler litter ammonia emissions using Bacillus subtilis. Abstract 156. Poultry Science, suppl. 1 ed., vol 91: 55.
• Gholap, D.B., K.S. Macklin, J.P. Blake and S.F. Bilgili. 2012. Comparison of ammonia emission rates, litter quality and microbiological fluctuations in commercial broiler facilities using a biological and a chemical litter treatment for two consecutive broiler flocks. Abstract M34. Poultry Science, suppl. 1 ed., vol 91: 192.
• Williams, Z. and K. Macklin. 2012. Concentration of total aerobic and anaerobic bacteria in broiler litter, at varying locations within the house, conducted over a one year time. Abstract M38. Poultry Science, suppl. 1 ed., vol 91: 193.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: This past year we have concentrated our efforts in better understanding the microbial profile in poultry litter, specifically in regards to Salmonella and Clostridium perfringens accumulation. We have also looked at the role of microbial litter amendments on mitigating test two bacteria as well as ammonia in poultry litter. The results generated have been shared in both at scientific meetings and at extension functions. The PI has an extension appointment and disseminates any information found in trade magazines (Alabama Poultry and Egg, Poultry Times and Poultry USA) as well as in meetings that are aimed at the broiler industry (both integrator and grower). In addition the PI has two graduate students that are working with him on trying to get a better understanding of the microbial dynamics of poultry litter. PARTICIPANTS: Kenneth S. Macklin - PI Zachary T. Williams - graduate student Matthew A. Bailey - graduate student John P. Blake - collaborator Joseph B. Hess - collaborator TARGET AUDIENCES: Poultry industry personal, poultry growers and environmental groups PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Some of the research which we have reported upon shows that acidifying litter amendments have no effect on litter Salmonella levels. Additionally we have shown that bacterial levels within poultry litter plateau rather quickly (2-3 weeks) during the poultry production cycle and are maintained at this level throughout the cycle. The use of Bacillus spp to mitigate ammonia in poultry litter has had mixed results; however further research by our group should improve these results.

Publications

• Shaw, A. L., F. van Ginkel, K. Macklin, J. Blake. 2011. Effects of phytase supplementation in broiler diets on a natural Eimeria challenge in naive and vaccinated birds. Poultry Science, 90: 781-790.
• Macklin, K.S. and J.T. Krehling. 2010. The use of metam-sodium to reduce bacteria in poultry litter. Journal of Applied Poultry Research, 19(3): 274-278.
• Williams, Z., A. Segrest, M. Bailey, J. Krehling and K. Macklin. 2010. Concentrations of different types of pathogenic bacteria in varying levels of used broiler litter: top, middle and bottom. In: Proceedings of the 2010 National Poultry Waste Symposium, p 4. Oct. 26-28, 2010. Greensboro, NC.
• Williams Z. and K. Macklin. 2011. Concentrations of different types of bacteria in poultry litter, at varying depths and locations within a commercial broiler house. Abstract M12, presented at International Poultry Science Forum, Atlanta, GA, 24-25 January 2011.
• Stough, J., N. Capps, K. Macklin, M. Singh and M.R. Liles. 2010. Activity of 25 Bacillus strains against Salmonella strains. Abstract CVM11, presented at the 96th Annual Meeting of the Southeastern Branch of the American Society of Microbiologists, Montgomery, AL, 4-6 November 2010.
• Blake, J.P., J.B. Hess and K.S. Macklin. 2010. Litter treatment usage and trace element accumulation in broiler litter. In: Proceedings of the 2010 National Poultry Waste Symposium, p 4. Oct. 26-28, 2010. Greensboro, NC.
• Williams, Z., K. Macklin. 2011. Concentrations of different types of bacteria in poultry litter, at varying depths and locations within commercial broiler houses. Poultry Science, suppl.1 ed., vol. 90: 209.
• Gholap, D., K. Macklin, J. Blake, S. Bilgili. 2011. Evaluation of air and litter quality with microbiological fluctuations in commercial broiler facilities using a biological and a chemical litter treatment. Poultry Science, suppl.1 ed., vol. 90: 54.
• Macklin, K.S., Z.T. Williams, M.A. Bailey, J.T. Krehling and J.P. Blake. 2011. The effect of sodium bisulfate on reducing Salmonella in poultry litter. Abstract M82, presented at International Poultry Science Forum, Atlanta, GA, 24-25 January 2011.