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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL INTERACTIONS AND INTERVENTIONS TO REDUCE TRANSMISSION OF FOODBORNE PATHOGENS THROUGH POULTRY Title: Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection

Authors
item Hume, Michael
item Barbosa, Nei -
item Dowd, Scot -
item Sakamura, Nilva -
item Nalian, Armen -
item Kley, Alexandra -
item Oviedo-Rondon, Edgar -

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 21, 2011
Publication Date: October 23, 2011
Citation: Hume, M.E., Barbosa, N.A., Dowd, S.E., Sakamura, N.K., Nalian, A.G., Kley, A.M., Oviedo-Rondon, E.O. 2011. Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection. Foodborne Pathogens and Disease. 8:1159-1167.

Interpretive Summary: Protective digestive bacteria help prevent and reduce infection and colonization in chickens by potential human pathogens. In the current experiment, chickens fed diets containing protective bacteria and plant oils were infected with a mixed chicken parasite to determine the effects of the protective bacteria and plant oils on other bacterial populations in the digestive tract. Digestive tract contents were collected, and DNA was isolated and subjected to two molecular techniques used to examine bacteria populations: Pyrosequencing was used to identify individual bacteria and fungi; denaturing gradient gel electrophoresis (DGGE) was used to detect overall changes in the populations. Pyrosequencing resulted in the detection of one hundred and forty-seven bacterial species and twenty-three fungal species. Additionally, pyrosequencing revealed very low levels of disease-causing bacteria that may have resulted from the mild infection caused by the chicken parasite. Expected changes were seen with DGGE that resulted from the parasite infection and with increased age of the chickens. Treatment with the protective bacteria and plant oils changed the digestive bacteria populations. Pyrosequencing was very sensitive at detecting shifts in individual identified bacteria and fungi, while DGGE was able to detect gross shifts in entire populations. These combined techniques offer added versatility towards identifying the effects of the growth-enhancing materials added to diets on chicken digestive bacteria.

Technical Abstract: A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed corn-soybean meal diets supplemented with probiotics and essential oil blends (EO) were infected with mixed Eimeria spp. to determine effects broiler performance enhancers have on ileal and cecal microbial populations. Eight treatment groups included four controls [Uninfected-Unmedicated (UU), Unmedicated-Infected (UI), BMD plus Coban as positive control (PC), and ionophore (Coban) as negative control (NC)] and four treatments [two probiotics, BC-30 and Calsporin, and two specific essential blends, Crina Poultry Plus (CPP) and Crina PoultryAF (CPF)]. Day-old male Ross broilers were raised to 14 d in floor pens on litter used by two previous flocks fed similar diets and the same feed additives. At 14 d, broilers were moved to Petersime battery cages and inoculated at 15 d with mixed Eimeria spp. Ileal and cecal samples were collected at 14 d and 9 d post-infection. Digesta DNA was subjected to two PCR-based molecular techniques to examine the effects of the treatment on microfloral communities: Pyrosequencing for sequence analysis of individual bacteria and fungi; denaturing gradient gel electrophoresis (DGGE) for determination of percentage similarity coefficients (%SC) on dendrogram band pattern. Pyrosequencing resulted in the detection of one hundred and forty-seven bacterial species sequences and twenty-three fungal species sequences. Additionally, pyrosequencing revealed in broilers at 24 d of age low levels of potential enteropathogens following mild infection with the mixed Eimeria spp. Expected changes in ileal and cecal DGGE profiles were seen resulting from the coccidia infection and with increased broiler aged with a 56%SC in ilea and a 78.5%SC in ceca for pre- and post-infection populations. Probiotics and EO changed microbial populations from those seen in UU ilea and ceca. Pyrosequencing was very sensitive at detecting shifts in individual identified bacterial and fungal sequences, while DGGE was able to detect gross shifts in entire populations. These combined techniques offer added versatility towards identifying the effects of the performance-enhancing feed additives on broiler digestive microflora.

Last Modified: 12/19/2014
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