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

The microbiota of an animal's intestinal tract plays a vital role in the animal's overall health. There is a surprising scarcity of information on the microbial diversity in the gut of livestock species such as cattle and swine. Here we describe a bacterial 16S-based tag-encoded FLX amplicon pyrosequencing (bTEFAP) method that we have developed as a high-throughput universal tool for bacterial diversity, epidemiology, and pathogen detection studies. This method will allow hundreds of samples to be run simultaneously but analyzed individually or as groups. To test this new methodology, we individually evaluated the bacterial diversity in the ileum of 21 pigs. Ubiquitous bacteria detected in the newly weaned pigs were Clostridium spp., Lactobacillus spp., and Helicobacter spp. Many of the pigs had surprisingly low concentrations of beneficial bacteria such as Bifidobacterium spp. Only four of the pigs were shown to be positive for Salmonella spp. using traditional culture methods. A total of eight pigs were bTEFAP positive for Salmonella spp., including all four of the pigs that had been culture positive. Two of the pigs sampled were also positive for Campylobacter spp. tentative identified as jejuni. Using rarefaction curves modeled with the Richards equation, we estimated the maximum number of unique species level (3% dissimilarity) operational taxonomic units in the ileum of these pigs. These predictions indicated that there may be as many as 821 different species associated with the ileum in pigs. Together these data indicate a powerful potential of this technology in food safety and epidemiological and bacterial diversity applications. Using bTEFAP, we can expect to gain a better understanding of how the microbiome of an animal contributes to its health and well-being.