Characterization of the bacterial gut microbiota of piglets suffering from new neonatal porcine diarrhoea.

BACKGROUND: In recent years, new neonatal porcine diarrhoea (NNPD) of unknown aetiology has emerged in Denmark. NNPD affects piglets during the first week of life and results in impaired welfare, decreased weight gain, and in the worst-case scenario death. Commonly used preventative interventions such as vaccination or treatment with antibiotics, have a limited effect on NNPD. Previous studies have investigated the clinical manifestations, histopathology, and to some extent, microbiological findings; however, these studies were either inconclusive or suggested that Enterococci, possibly in interaction with Escherichia coli, contribute to the aetiology of NNPD. This study examined ileal and colonic luminal contents of 50 control piglets and 52 NNPD piglets by means of the qPCR-based Gut Microbiotassay and 16 samples by 454 sequencing to study the composition of the bacterial gut microbiota in relation to NNPD. RESULTS: NNPD was associated with a diminished quantity of bacteria from the phyla Actinobacteria and Firmicutes while genus Enterococcus was more than 24 times more abundant in diarrhoeic piglets. The number of bacteria from the phylum Fusobacteria was also doubled in piglets suffering from diarrhoea. With increasing age, the gut microbiota of NNPD affected piglet and control piglets became more diverse. Independent of diarrhoeic status, piglets from first parity sows (gilts) possessed significantly more bacteria from family Enterobacteriaceae and species E. coli, and fewer bacteria from phylum Firmicutes. Piglets born to gilts had 25 times higher odds of having NNPD compared with piglets born to multiparous sows. Finally, the co-occurrence of genus Enterococcus and species E. coli contributed to the risk of having NNPD. CONCLUSION: The results of this study support previous findings that points towards genus Enterococcus and species E. coli to be involved in the pathogenesis of NNPD. Moreover, the results indicate that NNPD is associated with a disturbed bacterial composition and larger variation between the diarrhoeic piglets.

The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.

BACKGROUND: The intestinal microbiota is a complex and diverse ecosystem that plays a significant role in maintaining the health and well-being of the mammalian host. During the last decade focus has increased on the importance of intestinal bacteria. Several molecular methods can be applied to describe the composition of the microbiota. This study used a new approach, the Gut Microbiotassay: an assembly of 24 primer sets targeting the main phyla and taxonomically related subgroups of the intestinal microbiota, to be used with the high-throughput qPCR chip 'Access Array 48.48', AA48.48, (Fluidigm®) followed by next generation sequencing. Primers were designed if necessary and all primer sets were screened against DNA extracted from pure cultures of 15 representative bacterial species. Subsequently the setup was tested on DNA extracted from small and large intestinal content from piglets with and without diarrhoea. The PCR amplicons from the 2304 reaction chambers were harvested from the AA48.48, purified, and sequenced using 454-technology. RESULTS: The Gut Microbiotassay was able to detect significant differences in the quantity and composition of the microbiota according to gut sections and diarrhoeic status. 454-sequencing confirmed the specificity of the primer sets. Diarrhoea was associated with a reduced number of members from the genus Streptococcus, and in particular S. alactolyticus. CONCLUSION: The Gut Microbiotassay provides fast and affordable high-throughput quantification of the bacterial composition in many samples and enables further descriptive taxonomic information if combined with 454-sequencing.

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype.

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.