Determining Presence of Yolk Sac and Intestinal Bacterial Colonization in Pre-hatch Chicken Embryos

Chicks hatch with a sterile gastrointestinal tract and receive the first colonization of intestinal bacteria from the shell and hatching environment. Bacteria may translocate from the shell to remaining yolk and/or the developing embryo pre-hatch due to penetration of the protective bloom on the unwashed shell’s surface. The objective of this study was to determine the presence or absence of bacterial DNA via PCR in the gastrointestinal tract of pre-hatch and hatched chicken embryos. Pre-hatch chicken embryo digestive tracts and yolk sacs sampled in a biosafety cabinet on days 13, 20, and 21 of incubation showed no bacterial presence using sensitive 16s PCR. Further research needs to be done in this area, potentially utilizing sterile sampling and cultures to confirm the apparent sterility of the inner egg environment as well as impact of time post-hatch and diversity of bacterial colonization on future production

The Plant Mitochondrial Carrier Family: Functional and Evolutionary Aspects

Mitochondria play a key role in respiration and energy production and are involved in multiple eukaryotic but also in several plant specific metabolic pathways. Solute carriers in the inner mitochondrial membrane connect the internal metabolism with that of the surrounding cell. Because of their common basic structure, these transport proteins affiliate to the mitochondrial carrier family (MCF). Generally, MCF proteins consist of six membrane spanning helices, exhibit typical conserved domains and appear as homodimers in the native membrane. Although structurally related, MCF proteins catalyze the specific transport of various substrates, such as nucleotides, amino acids, dicarboxylates, cofactors, phosphate or H+. Recent investigations identified MCF proteins also in several other cellular compartments and therefore their localization and physiological function is not only restricted to mitochondria. MCF proteins are a characteristic feature of eukaryotes and bacterial genomes lack corresponding sequences. Therefore, the evolutionary origin of MCF proteins is most likely associated with the establishment of mitochondria. It is not clear whether the host cell, the symbiont, or the chimerical organism invented the ancient MCF sequence. Here, we try to explain the establishment of different MCF proteins and focus on the characteristics of members from plants, in particular from Arabidopsis thaliana

Fluoxetine-induced alteration of murine gut microbial community structure: evidence for a microbial endocrinology-based mechanism of action responsible for fluoxetine-induced side effects

Background Depression and major depressive disorder affect 25% of the population. First line treatment utilizing selective serotonin reuptake inhibitors (SSRIs) have met with limited success due to well-recognized negative side effects which include weight gain or loss. This inability to control unwanted side effects often result in patients stopping their antidepressant medications. The mechanisms underlying the failure of SSRIs are incompletely understood. Methods Male CF-1 mice (5 weeks of age, N = 10 per group) were per orally administered fluoxetine (20 mg per kg body weight) or diluent daily for 29 days. During this time fecal specimens were collected at three defined time points (0, 15 and 29 days). At the conclusion of the 29-day dosing regimen, animals were subjected to two behavioral assessments. For bacterial identification of the microbiota, 16S rRNA gene sequencing was performed on 60 fecal specimens (three specimens per mouse time course, N = 20 mice) using Illumina MiSeq. Analysis of community sequence data was done using mothur and LEfSe bioinformatic software packages. Results Daily per oral administration of fluoxetine for 29 days to male mice resulted in a significant, time dependent, alteration in microbial communities accompanying changes in body weight. The calculated species richness and diversity indicators of the murine fecal microbial communities were inconsistent and not significantly different between the groups. Among the phylotypes decreased in abundance due to fluoxetine administration were Lactobacillus johnsonii and Bacteroidales S24-7 which belong to phyla associated with regulation of body mass. The observed changes in body weight due to fluoxetine administration mimicked the dramatic shifts in weight gain/loss that has been observed in humans. Further, at the conclusion of the 29-day dosing regimen fluoxetine-dosed animals evidenced a mild anxiogenic-like behavior. Discussion We report that the most widely used antidepressant, fluoxetine, which is an SSRI-type drug, results in the selective depletion of gut microbiota, specifically the Lactobacilli which are involved in the regulation of body weight. Concomitantly, fluoxetine administration increases the abundance of phylotypes related to dysbiosis. Since Lactobacilli have been previously shown to possess a known biogenic amine transporter that regulates the uptake of fluoxetine, it is proposed that a microbial endocrinology-based mechanistic pathway is responsible for the ability of SSRIs to selectively negatively impact beneficial microbiota. The results of this study therefore suggest that the negative clinical side effects due to fluoxetine administration may be due to alterations in gut microbiota. Further, the data also suggests that supplementation of bacterial genera directly affected by fluoxetine administration may prove useful in ameliorating some of the well-known side effects of chronic fluoxetine administration such as weight alterations

Brevibacterium from Austrian hard cheese harbor a putative histamine catabolism pathway and a plasmid for adaptation to the cheese environment

The genus Brevibacterium harbors many members important for cheese ripening. We performed real-time quantitative PCR (qPCR) to determine the abundance of Brevibacterium on rinds of Vorarlberger Bergkäse, an Austrian artisanal washed-rind hard cheese, over 160 days of ripening. Our results show that Brevibacterium are abundant on Vorarlberger Bergkäse rinds throughout the ripening time. To elucidate the impact of Brevibacterium on cheese production, we analysed the genomes of three cheese rind isolates, L261, S111, and S22. L261 belongs to Brevibacterium aurantiacum, whereas S111 and S22 represent novel species within the genus Brevibacterium based on 16S rRNA gene similarity and average nucleotide identity. Our comparative genomic analysis showed that important cheese ripening enzymes are conserved among the genus Brevibacterium. Strain S22 harbors a 22 kb circular plasmid which encodes putative iron and hydroxymethylpyrimidine/thiamine transporters. Histamine formation in fermented foods can cause histamine intoxication. We revealed the presence of a putative metabolic pathway for histamine degradation. Growth experiments showed that the three Brevibacterium strains can utilize histamine as the sole carbon source. The capability to utilize histamine, possibly encoded by the putative histamine degradation pathway, highlights the importance of Brevibacterium as key cheese ripening cultures beyond their contribution to cheese flavor production

The Rumen Epithelial Microbiota: Possible Gatekeepers of the Rumen Epithelium and Its Potential Contributions to Epithelial Barrier Function and Animal Health and Performance

Ruminants are characterized by their unique mode of digesting cellulose-rich plant material in their forestomach, the rumen, which is densely populated by diverse microorganisms that are crucial for the breakdown of plant material. Among ruminal microbial communities, the microorganisms in the rumen fluid or attached to feed particles have attracted considerable research interest. However, comparatively less is known about the microorganisms attached to the rumen epithelium. Generally, the tissue lining the gastrointestinal tract serves the dual role of absorbing nutrients while preventing the infiltration of unwanted compounds and molecules as well as microorganisms. The rumen epithelium fulfills critical physiological functions for the ruminant host in energy absorption, metabolism, and nutrient transport. Essential host metabolites, such as short-chain fatty acids, ammonia, urea, and minerals, are exchanged across the rumen wall, thereby exposing the rumen epithelial microbiota to these nutrients. The integrity of the gastrointestinal barrier is central to animal health and productivity. The integrity of the rumen epithelium can be compromised by high ruminal microbial fermentation activity resulting in decreased rumen pH or by stress conditions such as heat stress or feed restriction. It is important to keep in mind that feeding strategies in cattle have changed over the last decades in favor of energy- and nutrient-rich concentrates instead of fiber-rich forages. These dietary shifts support high milk yields and growth rates but raised concerns regarding a possibly compromised rumen function. This paper will provide an overview of the composition of rumen epithelial microbial communities under physiological and disease conditions and will provide insights into the knowledge about the function and in situ activity of rumen epithelial microorganisms and their relevance for animal health and production. Given that an impaired intestinal barrier will negatively affect economically significant phenotypes, a better understanding of rumen wall microbiota is urgently needed

Autochthonous facility-specific microbiota dominates washed-rind Austrian hard cheese surfaces and its production environment

Cheese ripening involves the succession of complex microbial communities that are responsible for the organoleptic properties of the final products. The food processing environment can act as a source of natural microbial inoculation, especially in traditionally manufactured products. Austrian Vorarlberger Bergkäse (VB) is an artisanal washed-rind hard cheese produced in the western part of Austria without the addition of external ripening cultures. Here, the composition of the bacterial communities present on VB rinds and on different processing surfaces from two ripening cellars was assessed by near full length 16S rRNA gene amplification, cloning and sequencing. Non-inoculated aerobic bacteria dominated all surfaces in this study. VB production conditions (long ripening time, high salt concentration and low temperatures) favor the growth of psychro- and halotolerant bacteria. Several bacterial groups, such as coryneforms, Staphylococcus equorum and Halomonas dominated VB and were also found on most environmental surfaces. Analysis of OTUs shared between different surfaces suggests that VB rind bacteria are inoculated naturally during the ripening from the processing environment and that cheese surfaces exert selective pressure on these communities, as only those bacteria better adapted flourished on VB rinds. This study analyzed VB processing environment microbiota and its relationship with VB rinds for the first time, elucidating that the processing environment and the cheese microbiota should be considered as microbiologically linked ecosystems with the goal of better defining the events that take place during cheese maturation

Virulence Pattern Analysis of Three Listeria monocytogenes Lineage I Epidemic Strains with Distinct Outbreak Histories

Strains of the food-borne pathogen Listeria (L.) monocytogenes have diverse virulence potential. This study focused on the virulence of three outbreak strains: the CC1 strain PF49 (serovar 4b) from a cheese-associated outbreak in Switzerland, the clinical CC2 strain F80594 (serovar 4b), and strain G6006 (CC3, serovar 1/2a), responsible for a large gastroenteritis outbreak in the USA due to chocolate milk. We analysed the genomes and characterized the virulence in vitro and in vivo. Whole-genome sequencing revealed a high conservation of the major virulence genes. Minor deviations of the gene contents were found in the autolysins Ami, Auto, and IspC. Moreover, different ActA variants were present. Strain PF49 and F80594 showed prolonged survival in the liver of infected mice. Invasion and intracellular proliferation were similar for all strains, but the CC1 and CC2 strains showed increased spreading in intestinal epithelial Caco2 cells compared to strain G6006. Overall, this study revealed long-term survival of serovar 4b strains F80594 and PF49 in the liver of mice. Future work will be needed to determine the genes and molecular mechanism behind the long-term survival of L. monocytogenes strains in organs

Development of New Measurements and Tools to Mitigate Fescue Toxicosis in Beef Cattle

The objective of this study was to identify new ways to determine the severity of fescue toxicosis and identify genetic differences in fescue impacted traits as a basis in understanding how cattle could be selected for tolerance to fescue toxicosis. We identified across breed and within breed differences in heat stress related traits and growth rate in pregnant cows exposed to toxic fescue. In addition, new biomarkers were identified to differentiate susceptible and tolerant cattle in the form of specific fecal and vaginal microbes. Finally, we identified differentially expressed (DE) genes in high versus low tolerant cattle on toxic fescue. These findings may allow more accurate diagnosis of fescue toxicosis and provide a glimpse into the genes and microorganisms that may impact tolerance or susceptibility to toxic fescue

Abundance and potential contribution of Gram-negative cheese rind bacteria from Austrian artisanal hard cheeses

Many different Gram-negative bacteria have been shown to be present on cheese rinds. Their contribution to cheese ripening is however, only partially understood until now. Here, cheese rind samples were taken from Vorarlberger Bergkäse (VB), an artisanal hard washed-rind cheese from Austria. Ripening cellars of two cheese production facilities in Austria were sampled at the day of production and after 14, 30, 90 and 160 days of ripening. To obtain insights into the possible contribution of Advenella, Psychrobacter, and Psychroflexus to cheese ripening, we sequenced and analyzed the genomes of one strain of each genus isolated from VB cheese rinds. Additionally, quantitative PCRs (qPCRs) were performed to follow the abundance of Advenella, Psychrobacter, and Psychroflexus on VB rinds during ripening in both facilities. qPCR results showed that Psychrobacter was most abundant on cheese rinds and the abundance of Advenella decreased throughout the first month of ripening and increased significantly after 30 days of ripening (p\u3c0.01). Psychrobacter and Psychroflexus increased significantly during the first 30 ripening days (p\u3c0.01), and decreased to their initial abundance during the rest of the ripening time (p\u3c0.05). Genome sequencing resulted in 17 to 27 contigs with assembly sizes of 2.7 Mbp for Psychroflexus, 3 Mbp for Psychrobacter, and 4.3 Mbp for Advenella. Our results reveal that each genome harbors enzymes shown to be important for cheese ripening in other bacteria such as: Cystathionine/Methionine beta or gamma-Lyases, many proteases and peptidases (including proline iminopeptidases), aminotransferases, and lipases. Thus, all three isolates have the potential to contribute positively to cheese ripening. In conclusion, the three species quantified were stable community members throughout the ripening process and their abundance on cheese rinds together with the results from genome sequencing suggest an important contribution of these bacteria to cheese ripening

Exogenous carbohydrases added to a starter diet reduced markers of systemic immune activation and decreased Lactobacillus in weaned pigs

Although the impact of carbohydrases on performance and nutrient utilization has been well studied, their effects on immune status and intestinal microbiota are less known in pigs. This study aimed to evaluate the impact of xylanase (X) and a carbohydrase enzyme blend (EB; cellulase, ß-glucanase, and xylanase) on the immune profile of the intestine and peripheral system as well as intestinal microbes and microbial metabolites of weaned pigs fed higher fiber diets. Pigs (n = 460; 6.43 ± 0.06 kg BW; F25 × 6.0 Genetiporc) were blocked by initial BW. Pens (n = 48; 12 per treatment; 9 or 10 pigs per pen) were randomly assigned to 1 of 4 dietary treatments, including a higher fiber control diet (CON) and the CON supplemented with 0.01% X, 0.01% EB, or both enzymes (X + EB), arranged in a 2 × 2 factorial. The diets were based on corn, soybean meal, corn distillers dried grains with solubles, and wheat middlings. After 7-d adaptation to the environment, pigs were fed experimental diets ad libitum for 28 d. Blood samples were collected from the same pig within each pen on days 0, 7, 14, and 28. Intestinal tissues and digesta were collected on day 28. Bacteria 16S rRNA gene copy numbers were quantified using qPCR. The mRNA levels of colonic IL-17, occludin (OCLN), and claudin 3 (CLDN3) were greater in pigs fed diets with X + EB, but not X or EB, compared with those fed CON (P \u3c 0.05). The EB in the diet reduced plasma IL-8 over the 28-d trial compared with diets without EB (P \u3c 0.05). There was an X × EB interaction on plasma tumor necrosis factor α and IL-1ß (P \u3c 0.05); their levels were decreased when X and EB were added together, but not individually, compared with CON. The EB decreased cecal propionate, butyrate, and total volatile fatty acids (P \u3c 0.05). Pigs fed X had lower ileal Lactobacillus and greater ileal and cecal Enterobacteriaceae compared with those fed unsupplemented diets (P \u3c 0.05). The EB decreased Lactobacillus (P \u3c 0.05) and tended to decrease (P = 0.065) Enterobacteriaceae in the colon compared with diets without EB. In conclusion, the addition of X and EB together decreased systemic markers of immune activation, potentially diverting energy and nutrients towards growth. The EB reduced colonic Lactobacillus and cecal total volatile fatty acids, probably due to improved prececal fiber and starch degradation and thus reduced substrate availability in the large intestine. These data corroborated previously observed enhanced growth in pigs fed EB-supplemented diets