Zootecnica International - World Poultry Journal

Charles HOFACRE
Margie LEE
Jingrang LU
Poultry Diagnostic and Research Center,
Department of Population Health,
The University of Georgia Athens, GA, U.S.A.

Introduction

Why do we even care about the bacteria flora in the intestines of chickens?
One answer is the healthy normal flora is the first line of defence for all animal’s bodies to invading pathogens. All birds have a normal flora of bacteria on their skin, respiratory tract and the intestines. It has been estimated that the normal flora of the chicken’s gastrointestinal tract is approximately 1 x 1011 colony forming units/gram of intestinal contents. The predominant bacteria present in the chicken ceca are primarily obligate anaerobes. There have been at least 38 different types of anaerobic bacteria isolated from the chicken ceca with more than 200 total bacterial strains isolated. However, we are limited in our knowledge of all of the bacteria in a bird’s ceca because many of the bacteria cannot be grown using cultural techniques.
A second reason to care about the healthy normal flora may be the economic benefit of improved bird growth and feed efficiency. We know that the use of antibiotics continuously in the feed of birds will improve their growth, is this due to the antibiotics effect on specific pathogenic bacteria, such as C. perfringens? It has been shown that antibiotic growth promotants (AGP) inhibit the negative effects of Clostridium perfringens to cause necrotic enteritis. However, the actual mode of action of the AGP’s has not been determined.
Microbial analysis of intestinal communities is increasing in interest because of the knowledge that the microflora is intimately involved in intestinal health. There are two major methods used to determine the composition of the bacterial community of the intestine. The culture method is recognized to have significant weaknesses due to the inability to culture many of the abundant organisms in some samples. A molecular method, analysis of 16S rDNA genes present among the community DNA, is currently used because of this gene’s discriminatory ability in identifying bacteria to the genus, and frequently species, level. The diverse bacterial community of many environments can be revealed by sequencing cloned PCR amplicons resulting from targeting the 16S rDNA genes present in the genomes isolated from microbial communities. The cloned amplicon method is currently considered the “gold standard” for community analysis but the method is very labour intensive and expensive. Several other techniques have been developed to detect changes in the diversity of amplified total community 16S rDNA including denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment length polymorphisms (T-RFLP). These methods are rapid and cost-effective but assigning bacterial identity to the bands or terminal restriction fragments (T-RFs) and quantitatively interpreting the bacterial community change is very complex.
T-RFLP analysis of amplified total community 16S rDNA can provide a rapid and reproducible means for observing bacterial population dynamics and comparing community structure. The method uses a similar approach as the clone library, in that community DNA is amplified using a universal bacterial 16S rDNA primer set. But instead of cloning the amplicons and sequencing individual 16S rDNA genes amplified from the community DNA, the diversity of amplicon sequences is evaluated using restriction digestion of amplicons that are terminally tagged. The terminal restriction fragments (T-RFs) can be identified using a database of 16S sequences of organisms that comprise the intestinal community. We characterized the bacterial community of the intestine of chickens fed a corn-soy diet over a standard commercial growout period of 49 days . In this current study, we developed a chicken ileal database in order to use T-RFLP analysis to detect changes in the bacterial community structure of the ileum of chickens and turkeys fed various feed additives. 

Discussion

This study suggests that the ileal community is very sensitive to feed additives. The bacterial community of the control group contained primarily lactobacilli while the monensin group had a community dominated by two species of Clostridium. Culture¬-based studies have also found that lactobacilli predominate in the small intestine of chickens fed a corn-based diet; Salanitro et al. showed that the predominant cultivable bacteria present in the ileum of young chicks (14 days old) were Lactobacillus (33.8-59%), while the other groups, such as Streptococcus, E. coli and eubacteria and clostridia were a small part. Abundant L. acidophilus are hypothesized to reduce colonization of transient enterobacteria by competitive exclusion. The changes in bacterial communities due to competitive exclusion have resulted in exclusion of pathogens, improved digestion and absorption of nutrients and decreased net ammonia production.
Previous culture-based studies also found that antibiotics caused alterations of the microbial community including significant decreases in cultivatable Micrococcaceae, lactobacilli, and Clostridium perfringens The ionophore monensin, has been shown to alter ruminal bacterial communities by inhibiting gram-positive bacteria. Stahl et al. reported that the relative numbers of Lachnospira multiparus-like organisms (clostridia), detected using 16S probe hybridization, were depressed about 2-fold during monensin treatment. However, of the abundant gram-positive bacteria detected in our chicken ileal communities, L. acidophilus populations appeared to be sensitive to monensin but Clostridia relative abundance was not reduced.
The abundance of Clostridiales in the ileum of these seemingly healthy chickens was surprising because an abundance of lactobacilli is commonly viewed as a significant indicator of intestinal health. Clostridium lituseburense and C. irregularis and their relatives were abundant components in the treatment group but the beneficial effects of these clostridia have not been explored.
No studies have been published that evaluate their correlations to the health and performance of chickens or other animals. These species may not contain the necessary array of clostridial virulence determinants and may be nonpathogenic. However, they may potentially be opportunist pathogens if the bacterial community exhibits major changes in composition. On the other hand, they may also participate in commensal host-bacterial relationships in the gut as proposed by Hooper and Gordon, who suggested that certain bacteria might directly influence the intestinal epithelium to limit immune activation and to help fortify the epithelial barrier. However, some seemingly innocuous bacteria may shift from commensalism to pathogenicity within certain bacterial community structure changes.
New paradigms, based on the discoveries of such shifts will aid in the elucidation of so called "microflora imbalances” that may lead to the development of necrotic enteritis both in the clinical and subclinical form which may ultimately influence the overall health of the bird. 


References are available on request.
From Proceedings of the “Midwest Poultry Federation Convention”, St. Paul, Minnesota, U.S.A.