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United States Department of Agriculture

Agricultural Research Service

Title: Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: Bacterial population in the ileum of newly weaned Salmonella infected pigs.

Authors
item Dowd, Scot
item Sun, Yan - MED BIOFILM RES INSTIT
item Wolcott, Randy - MED BIOFILM RES INSTIT
item Carroll, Jeffery

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 22, 2008
Publication Date: August 1, 2008
Citation: Dowd, S.E., Sun, Y., Wolcott, R.D., Carroll, J.A. 2008. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: Bacterial population in the ileum of newly weaned Salmonella infected pigs. Foodborne Pathogens and Disease. 5(4):459-472.

Interpretive Summary: The bacteria populations in the gut of animals is diverse and vital to the health and well-being of the animal. Alterations in management practices such as diet can positively or negatively affect the gut bacterial flora, thereby promoting dramatic effects in the productivity of the animal. USDA-ARS scientists in Lubbock, TX, have developed a novel genetic approach using next generation genetic sequencing to enable high-throughput evaluation of bacterial diversity in the animal. A herd of newly weaned pigs, some of which were infected with Salmonella, were evaluated using this novel method. With traditional culture only 3 of the pigs were positive for Salmonella, while the novel sequencing method identified 8 pigs infected with Salmonella and 2 infected with Campylobacter jejuni. The diversity of the bacteria in the ileum of these pigs was also evaluated. This indicates a powerful potential of this technology in food safety, epidemiological applications, and other bacterial diversity applications. Using bTEFAP, we can expect to gain a much better understanding of how the microbiome of the animal contributes to their health and well-being.

Technical Abstract: The microbiota of an animal’s intestinal tract plays a vital role in the animal’s overall health. There is currently a surprising scarcity of information in the scientific literature on the microbial diversity in the gut of livestock species such as cattle and swine. The primary reason for the scarcity of data on livestock gastrointestinal microbiomes relates to high cost, terms of labor and price, and methods needed to generate such data. Here we describe a bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach that is able to perform deep and semi-quantitative diversity analysis of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. In 24 hours, we were able to generate and analyze a nominal 1,000 sequences per sample at a cost of only approximately $100 per sample. To test this new methodology we evaluated bacterial diversity from the ileum of 21 pigs. Three of these pigs had been shown to be positive for Salmonella spp using culture methods. Ubiquitous bacteria detected in the newly weaned pigs were Clostridium spp., Lactobacillus spp., and Helicobacter spp., yet many of the pigs had surprisingly low concentrations of primary beneficial bacteria such as Bifidobacterium spp. There was also a surprising lack of enterobacteriacea in the ileum of these newly weaned pigs. A total of 8 pigs were positive for Salmonella using bTEFAP, including all 3 of the pigs which had been culture positive. Two of the samples were also positive for another foodborne pathogen, Campylobacter jejuni. This indicates a powerful potential of this technology in food safety, epidemiological applications, and other bacterial diversity applications. Using bTEFAP, we can expect to gain a much better understanding of how the microbiome of the animal contributes to their health and well-being.

Last Modified: 8/22/2014
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