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.

In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galacto-rhamnogalacturonan.

Postweaning diarrhea (PWD) in pigs is a leading cause of economic loss in pork production worldwide. The current practice of using antibiotics and zinc to treat PWD is unsustainable due to the potential of antibiotic resistance and ecological disturbance, and novel methods are required. In this study, an in vitro model was used to test the possibility of producing prebiotic fiber in situ in the gastrointestinal (GI) tract of the piglet and the prebiotic activity of the resulting fiber in the terminal ileum. Soluble fiber was successfully produced from potato pulp, an industrial waste product, with the minimal enzyme dose in a simulated upper GI tract model extracting 26.9% of the initial dry matter. The fiber was rich in galactose and galacturonic acid and was fermented at 2.5, 5, or 10 g/liter in a glucose-free medium inoculated with the gut contents of piglet terminal ileum. Fermentations of 5 g/liter inulin or 5 g/liter of a purified potato fiber were used as controls. The fibers showed high fermentability, evident by a dose-dependent drop in pH and an increase in the organic acid content, with lactate in particular being increased. Deep sequencing showed a significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fiber being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. For animal studies, a dosage corresponding to the 5-g/liter treatment is suggested.

Diet type dictates the gut microbiota and the immune response against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss).

This study investigated the influence of the rainbow trout (Oncorhynchus mykiss) commensal intestinal microbiota in connection to an experimental Yersina ruckeri infection, the causative agent of enteric redmouth disease. One marine and one plant diet was administered to two different groups of rainbow trout. The plant-based diet gave rise to an intestinal microbiota dominated by the genera Streptococcus, Leuconostoc and Weissella from phylum Firmicutes whereas phylum Proteobacteria/Bacteroidetes/Actinobacteria dominated the community in the marine fed fish. In connection to the Y. ruckeri bath challenge there was no effect of the diet type on the cumulative survival, but the number of Y. ruckeri positive fish as measured by plate count and the number of fish with a 'high' number of reads belonging to genus Yersinia as measured by 16S rRNA next-generation sequencing was higher for marine diet fed fish. Furthermore, the two experimental groups of fish showed a differential immune response, where Y. ruckeri challenged marine fed fish had a higher transcription of IL-1ß and MBL-2 relative to challenged plant diet fed fish. The data suggest that the plant diet gave rise to a prebiotic effect favouring the presence of bacterial taxons proving protective in connection to bath challenge by Y. ruckeri.

Discovery of bovine digital dermatitis-associated Treponema spp. in the dairy herd environment by a targeted deep-sequencing approach.

The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to the Treponema denticola/Treponema pedis-like and Treponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD.

Characterisation of gut microbiota in Ossabaw and Göttingen minipigs as models of obesity and metabolic syndrome.

BACKGROUND: Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs. METHODS AND FINDINGS: The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs' cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine. CONCLUSION: Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.

Fibroblasts express immune relevant genes and are important sentinel cells during tissue damage in rainbow trout (Oncorhynchus mykiss).

Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1beta, IL-8, IL-10, TLR-3 and TLR-9. IL-1beta and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1beta, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1beta. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1beta, IL-8 and TGF-beta already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells.

A highly phagocytic cell line TO from Atlantic salmon is CD83 positive and M-CSFR negative, indicating a dendritic-like cell type.

Leucocyte cell lines are valuable tools for immunological studies. In this study the TO cell line, originating from Atlantic salmon head kidney leucocytes, is described with respect to enzyme cytochemistry, functional studies, reactivity with leucocyte specific antibodies and immune gene expression. Pronounced characteristics of the TO cell line are the rapid adherence to the plastic growth surface, high phagocytic capacity and bactericidal functions. No respiratory burst activity, and little or no NO production were detected under the experimental conditions tested, and thus the TO cells appear to have other effective killing mechanisms. The cells are reactive with a leucocyte specific monoclonal antibody (MAb), but does not bind a neutrophil specific MAb or stain for myeloperoxidase. Real-time RT-PCR showed the expression in TO cells of several immune genes, some of which were significantly regulated following LPS stimulation. The expression of CD83 might indicate a dendritic cell (DC) origin of the TO cells, as this marker is considered a hallmark for DC. Expression of TCR-alpha or the macrophage marker M-CSFR was not detected. Based on the present analyses the TO cells display a mixture of known characteristics for macrophages and DCs. At the same time the TO cells lack some central functions of phagocytic/myeloid cells. As the TO cells are developed to a long-term culture one cannot exclude that some functions might have been lost in this process. Nevertheless, the features of the TO cells indicate their potential as a model system for immunological studies of salmon phagocytic cells.

Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.).

The expression levels of three commonly used housekeeping genes, EF1-alpha, RPS20 and Beta-Actin, were examined in seven different tissues and leucocytes from non-stimulated Atlantic salmon (Salmo salar L.). The tissues analysed by quantitative real-time PCR were gill, liver, intestine, muscle, spleen, head kidney leucocytes (HKL) and peripheral blood leucocytes (PBL). The experiments were performed to investigate the transcriptional stability within and between tissues and leucocytes and between individuals. For all tissues and leucocytes, an appropriate reference gene was identified except for muscle tissue. HKL were used as a calibrator and the expression of EF1-alpha varied maximally 2.5-fold in five out of the six tissues and leucocytes investigated relative to the expression of 18S rRNA. The RPS20 gene was more intermediate and varied at least by a factor of two and maximally by a 20-fold factor. Beta-Actin was generally the most regulated gene showing high variations for gill (5.8x) and spleen tissue (10.3x) relative to the calibrator. A suitable reference gene for muscle tissue was not found since the expression varied between 8.3- and 25-fold for the three genes compared to the calibrator. By comparing the expression results of the non-stimulated tissues and leucocytes using the Normfinder programme, it was further shown that EF1-alpha was the most stably expressed gene both between individuals and the different tissues/leucocytes. Stimulation with lipopolysaccharide (LPS) of TO cells and HKL from Atlantic salmon was additionally performed to reveal whether an immune stimulating agent would change the expression level of EF1-alpha, RPS20 and Beta-Actin. LPS stimulation of cells revealed that RPS20 and EF1-alpha were least regulated by the LPS treatment in the TO cells relative to 18S rRNA, but in HKL, Beta-Actin was the most appropriate gene. However, the variations were overall maximally two-fold in LPS-stimulated TO cells and HKL, which make all three genes suitable as reference genes in this case. A further experiment showed that no RT- and/or PCR inhibitors were present in the non-stimulated tissues and cells, indicating true transcriptional differences.