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Microbial Dynamics of the Broiler

Intestinal Tract

Margie D. Lee*, Jingrang Lu, Umelaalim Idris, Barry Harmon,

Charles Hofacre, John. J. Maurer. Departments of

Medical Microbiology and Parasitology, Pathology, and Avian Medicine,

The University of Georgia, Athens, GA 30602 (*speaker).

The microbial ecology of the chicken small intestinal flora is relatively ill defined primarily because studies have focused on the cecum. In order  to better understand the ecology of this environment, we used 16S ribosomal DNA gene sequencing to identify the dominant members of the bacterial flora. More than 68.85% of sequences, at all the tested ages, were related to those of Lactobacillus. However, the sequences of bacterial populations varied significantly by age of the birds. At all ages, sequences were identified in the library showing homology to the genus Clostridium. There was a unique community structure at 3 days age with the sequences homologous to culturable bacteria such as L. delbrueckii, C. perfringens and Campylobacter coli. From 7 days of age to 21days, a similar community structure was maintained with dominant sequences related to L. acidophilus, Enterococcus and Streptococcus. To some extent the bacterial community at 49 days of age was similar to those

at age 28 with the abundant sequences homologous to L. crispatus, but it was significantly different from those sequence from the other ages. The role of those bacteria nutrient acquisition, intestinal heath and growth promotion remain to be defined.

Introduction

It has long been known that densely colonized intestinal bacteria play an important role in the health and performance through its effect on gut

morphology, nutrition, and pathogenesis of intestinal disease and immune response of animal. Intestinal bacteria are primarily responsible for degrading the copious amounts of mucus produced by goblet cells in the intestinal mucosa. The microbial flora is may also protect against colonization of the intestines by pathogens and to stimulate the immune response (Mead, 1989).

Studies based on the culturable bacteria flora of chickens have been extensively conducted. The predominant bacteria present in the chicken ceca

are obligate anaerobes (1011/g) (Barnes, 1972). There have been at least 38 different types of anaerobic bacteria isolated from the chicken ceca (Barnes et al., 1972) with more than 200 total bacterial strains isolated (Mead, 1989).

However, it is believed that only between 10 and 60% of the bacteria in the cecum grew in culture (Barnes et al. 1972; Barnes, 1972; Salanitro 1974). Netherwood et al. (1999) used hybridization methods to monitor the response of bacterial flora in chicken cecum to probiotics and diet related differences were analyzed by Apajalahti et al. (1998) based on a percent G+C profiling. These studies demonstrated showed that many of the 16S rDNA sequences found in the chicken cecum were not closely related to any previous known bacterial genus. Zhu et al. (2002) isolated 243 unique partial 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa.

Discussion and Conclusions

We used a molecular ecological approach to identify the bacterial composition and to determine community succession in the ileum of chickens

fed a corn-soy diet lacking coccidiostats and growth-promoting antibiotics. We isolated random clones of 16S ribosomal DNA gene sequences after multiple PCR amplification of bacterial genomic DNA isolated from the ileum of chickens at 3, 7, 14, 21, 28 and 49 days of age. From analysis of 614 clones isolated from the 16S rDNA libraries, we identified four major phyla. These phyla included low and high G+C gram-positives, proteobacteria and the CFB group (Table A-1 and Fig. A-1). Eleven families or groups and sixteen genera were identified among the 16S rDNA sequences analyzed. The bacterial microbiota consisted predominantly of low G+C gram-positive bacteria, whose representative distinct sequences were shown in Fig. A-1, with Lactobacillus accounting for 68.85% of the total 16S rDNA sequences in the libraries. The

low G+C gram-positives consisted of five families or groups represented by nine genera. Identification of members of dominant genera  lactobacillus, Enterococcus and Streptococcus were culturable and have been often isolated from normal ileum (Salanitro, 1978). However, we did not anticipate finding that Clostridium was a dominant group at age 3 and age 49 in the ileum according to previous studies (Barnes et al. 1972; Salanitro, 1978). We detected Clostridium spp. in the ileal flora at all ages. Stutz and Lawton (1984) reported detection of clostridia, including C. perfringens, by culture of the ileum of 2-day-old chicks. About 15% of our total sequences at 3 days of age had homology to C. perfringens, which is important cause of necrotic enteritis in broilers (George, 1982; Long, 1973). We also detected sequences of segmented,

filamentous Clostridium spp., commonly found in healthy animals, at 14d of age (Snel, 1995).

There are various formulations of antibiotics used as growth promotants. In the US, many companies use Virginiamycin in the grower and  finisher feed for broiler chickens. In order to determine its effect on the ileum microflora, we sequenced 16S rDNA genes isolated from libraries prepared from these birds at 28 and 49 days of age. Birds fed Virginiamycin contained significantly fewer Lactobacillus species in the ileum than controls at both ages. In addition, the ratios among the dominant Lactobacillus species and the dominant Clostridium species were different. Changes in the other bacterial populations appeared to be minor.

These findings may allow us to identify ways to achieve present day growth rate and feed efficiency without use antibiotics by manipulation of the intestinal flora. It may also identify methods to predict intestinal disease prior to the clinical manifestation of symptoms and methods to prevent colonization of pathogens, such as, C. perfringens, Salmonella spp. or Campylobacter spp.

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Appl. Environ. Microbiol. 68:124-137.

Fig. A-1. Percentage of bacterial sequences belonging to particular phylogenetic groups or subdivisions present

in the ileum of chickens at different ages.

The Elanco Global Enteritis Symposium July 9-11, 2002 Microbial Dynamics of the

Broiler Intestinal Tract, “Abstract”, A-3 to A-7