Use of Pyrosequencing and Denaturing Gradient Gel Electrophoresis to Examine the Effects of Probiotics and Essential Oil Blends on Digestive Microflora in Broilers Under MixedEimeriaInfection

Abstract

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre-and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements.

Quantitative molecular assays for evaluating changes in broiler gut microbiota linked with diet and performance

“…Several studies have already used pyrotag sequencing to investigate the community structure of the chicken gut (Callaway et al 2009;Yin et al 2010;Hume et al 2011;Stanley et al 2012). Although this methodology gives a detailed phylogenetic description of the complexity of the microbial community, quantitative interpretation of results should be performed with some caution (Amend et al 2010;Zhou et al 2011).…”

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under MixedEimeriaInfection

“…A total of 384 1-day-old male Ross 708 chickens were randomly placed in 48 floor pens (8/pen) as previously described (Hume et al, 2011). Broilers were reared according to safe practices authorized by the Animal Use Protocol Committee of North Carolina State University.…”

“…Ileal and cecal digesta were collected aseptically by excision, and by voiding of contents into sterile sample tubes, from six broilers at pre-Eimeria infection per treatment group at 14 days of age and from six additional broilers post-Eimeria infection per group at 22 days of age. Samples were lyophilized and stored at -80°C until DNA extraction from 0.25 g each (Hume et al, 2003(Hume et al, , 2011.…”

“…Molecular techniques have been adapted for the use of a single community DNA preparation for differentiation of general and specific bacterial, protozoan, plant, and fungal genetic targets. Sample DNA assayed in the current study was collected during a related study in which we examined the impact of Eimeria infection and treatment with two commercial probiotics and two essential oil blends (EO) to minimize this impact on ileal and cecal bacterial populations (Hume et al, 2011). Mixed Eimeria infection was selected as a model to study intestinal modifications caused by a common pathogen that can cause profound changes in intestinal microbial communities (Hume et al, , 2011Oviedo-Rondó n et al, 2006).…”

“…Sample DNA assayed in the current study was collected during a related study in which we examined the impact of Eimeria infection and treatment with two commercial probiotics and two essential oil blends (EO) to minimize this impact on ileal and cecal bacterial populations (Hume et al, 2011). Mixed Eimeria infection was selected as a model to study intestinal modifications caused by a common pathogen that can cause profound changes in intestinal microbial communities (Hume et al, , 2011Oviedo-Rondó n et al, 2006). Coccidiosis is one of the most endemic enteric diseases in broiler production (Dalloul and Lillehoj, 2005).…”