gThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ϳ1% and ϳ0.1% of the total non-rRNAs, respectively. The majority (ϳ98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (ϳ82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. In nature, the cow rumen represents a highly specialized bioreactor wherein plant cell wall polysaccharides (PCWPs) are efficiently deconstructed. The extraordinary efficiency results from the concerted action of various enzymes produced by rumen-resident bacteria, archaea, fungi, and protozoa. Three rumen bacteria, i.e., Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes, which can be isolated and cultivated in the laboratory, have been thought to serve a predominant role in the degradation of cellulosic PCWPs in this niche (1, 2). However, metagenomic quantitations based on 16S rRNA gene analysis indicate that these three species of bacteria account for only less than 5% of the total rumen microorganisms (3). In addition, Koike et al. estimated that ϳ77% of the rumen microorganisms attached to solid fibers are uncultured, as their 16S rRNA gene sequences share less than 97% similarity with those of known isolates (4).To bypass the cultivation step, metagenomic approaches involving the direct analysis of total DNA sequences have been extensively used to investigate the PCWP-degrading gastrointestinal microbes in a variety of herbivores, such as termite hindguts (5); cow (6-8), yak (9), and Svalbard reindeer (10) ...

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2", ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.

Diarrhea is a leading cause of increased mortality in neonatal and young piglets. Aberration of the gut microbiota is one important factor in the etiology of piglet diarrhea. However, information regarding the structure and function of the gut microbiome in diarrheic neonatal piglets is limited. To investigate the composition and functional potential of the fecal microbiota in neonatal piglets, we performed 16S rRNA gene sequencing on 20 fecal samples from diarrheic piglets and healthy controls, and metagenomics sequencing on a subset of six samples. We found striking compositional and functional differences in fecal microbiota between diarrheic and healthy piglets. Neonatal piglet diarrhea was associated with increases in the relative abundance of Prevotella, Sutterella, and Campylobacter, as well as Fusobacteriaceae. The increased relative abundance of Prevotella was correlated with the reduction in Escherichia coli and the majority of beneficial bacteria that belonging to the Firmicutes phylum (e.g., Enterococcus, Streptococcus, Lactobacillus, Clostridium, and Blautia) in diarrheic piglets. The differentially functional gene abundances in diarrheic piglets were an increase in bacterial ribosome, and contributed primarily by the genera Prevotella, this indicates a growth advantage of the Prevotella in diarrheic conditions. Additional functional gene sets were associated with the reduction of polyamine transport, monosaccharide and sugar-specific PTS transport, amino acid transport, and two-component regulatory system. These profiles likely impact the ability to transport and uptake nutrients, as well as the ability to fight microbial infections in the piglet gut ecosystem. This work identifies a potential role for Prevotella in the community-wide microbial aberration and dysfunction that underpins the pathogenesis of piglet diarrhea. Identification of these microbial and functional signatures may provide biomarkers of neonatal piglet diarrhea.

Simple SummaryHeat stress negatively impacts the health and milk production of dairy cows, and ruminal microbes play an important role in the animal’s milk production. Understanding the link between heat stress and the ruminal microbiome could help to develop strategies to relieve the influence of heat stress by manipulating the ruminal microbial composition. We found that heat-stressed cows had decreased ruminal pH and acetate concentration, whereas the ruminal lactate concentration increased. Heat-stressed cows also had a significantly higher relative abundance of lactate producing bacteria (e.g., Streptococcus and unclassified Enterobacteriaceae), Ruminobacter, Treponema, and unclassified Bacteroidaceae, all of which utilize soluble carbohydrate as an energy source. The relative abundance of the acetate-producing bacterium Acetobacter decreased with heat stress treatment. Therefore, heat stress is associated with changes in ruminal bacterial composition and metabolites, with more lactate and less acetate-producing species in the population, which potentially negatively affects milk production.AbstractHeat stress negatively impacts the health and milk production of dairy cows, and ruminal microbial populations play an important role in dairy cattle’s milk production. Currently there are no available studies that investigate heat stress-associated changes in the rumen microbiome of lactating dairy cattle. Improved understanding of the link between heat stress and the ruminal microbiome may be beneficial in developing strategies for relieving the influence of heat stress on ruminants by manipulating ruminal microbial composition. In this study, we investigated the ruminal bacterial composition and metabolites in heat stressed and non-heat stressed dairy cows. Eighteen lactating dairy cows were divided into two treatment groups, one with heat stress and one without heat stress. Dry matter intake was measured and rumen fluid from all cows in both groups was collected. The bacterial 16S rRNA genes in the ruminal fluid were sequenced, and the rumen pH and the lactate and acetate of the bacterial metabolites were quantified. Heat stress was associated with significantly decreased dry matter intake and milk production. Rumen pH and rumen acetate concentrations were significantly decreased in the heat stressed group, while ruminal lactate concentration increased. The influence of heat stress on the microbial bacterial community structure was minor. However, heat stress was associated with an increase in lactate producing bacteria (e.g., Streptococcus and unclassified Enterobacteriaceae), and with an increase in Ruminobacter, Treponema, and unclassified Bacteroidaceae, all of which utilize soluble carbohydrates as an energy source. The relative abundance of acetate-producing bacterium Acetobacter decreased during heat stress. We concluded that heat stress is associated with changes in ruminal bacterial composition and metabolites, with more lactate and less acetate-producing species in the population, which potentially ne...

Milk fat globule membrane (MFGM) proteins have exhibited a relatively larger diversity than other milk fractions, and implicated health beneficial effects. Proteomic analysis of MFGM protein was mainly focused on human, bovine and goat in previous studies. Recently, there is an increasing demand for natural milk from minor dairy animals. Differences in protein components were not yet elucidated that required the integration of this information across multiple species. Thus, iTRAQ analysis of the proteins in MFGM fractions from Holstein, Jersey, yak, buffalo, goat, camel, horse, and human was performed in this study. A total of 520 proteins were identified and quantified in the MFGM fractions. The results were contributed to a comprehensive overview and discriminative profiling of the MFGM proteome across species.

Fluorescence sensing of specific biological molecules by artificial chemosensors is a versatile technique. In the present work, a switch-on fluorescence sensor for rapid, sensitive, and selective sensing of glutathione (GSH) in food samples was developed. This method was based on the g-CNQDs-Hg(2+) system, in which the initial fluorescence from g-CNQDs was quenched by Hg(2+) with an electron transfer process. In the presence of GSH, the fluorescence sensor was switched to the "on" state, which was attributed to a competitive affinity of Hg(2+) to GSH and the functional groups on the surface of g-CNQDs. Under the optimal conditions, the limit of detection (LOD) of 37 nM for GSH was achieved with a wide range of 0.16-16 μM. The repeatability was better than 5.3% for GSH in both standard and food samples (n = 3). Finally, this fluorescence sensor was successfully employed for the determination of GSH in various kinds of food samples with excellent recoveries. Furthermore, this application may pave a new way for fluorescence sensing of other substances in food samples.

Ureolytic bacteria are key organisms in the rumen producing urease enzymes to catalyze the breakdown of urea to ammonia for the synthesis of microbial protein. However, little is known about the diversity and distribution of rumen ureolytic microorganisms. The urease gene (ureC) has been the target gene of choice for analysis of the urea-degrading microorganisms in various environments. In this study, we investigated the predominant ureC genes of the ureolytic bacteria in the rumen of dairy cows using high-throughput sequencing. Six dairy cows with rumen fistulas were assigned to a two-period cross-over trial. A control group (n = 3) were fed a total mixed ration without urea and the treatment group (n = 3) were fed rations plus 180 g urea per cow per day at three separate times. Rumen bacterial samples from liquid and solid digesta and rumen wall fractions were collected for ureC gene amplification and sequencing using Miseq. The wall-adherent bacteria (WAB) had a distinct ureolytic bacterial profile compared to the solid-adherent bacteria (SAB) and liquid-associated bacteria (LAB) but more than 55% of the ureC sequences did not affiliate with any known taxonomically assigned urease genes. Diversity analysis of the ureC genes showed that the Shannon and Chao1 indices for the rumen WAB was lower than those observed for the SAB and LAB (P < 0.01). The most abundant ureC genes were affiliated with Methylococcaceae, Clostridiaceae, Paenibacillaceae, Helicobacteraceae, and Methylophilaceae families. Compared with the rumen LAB and SAB, relative abundance of the OTUs affiliated with Methylophilus and Marinobacter genera were significantly higher (P < 0.05) in the WAB. Supplementation with urea did not alter the composition of the detected ureolytic bacteria. This study has identified significant populations of ureolytic WAB representing genera that have not been recognized or studied previously in the rumen. The taxonomic classification of rumen ureC genes in the dairy cow indicates that the majority of ureolytic bacteria are yet to be identified. This survey has expanded our knowledge of ureC gene information relating to the rumen ureolytic microbial community, and provides a basis for obtaining regulatory targets of ureolytic bacteria to moderate urea hydrolysis in the rumen.

Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are mycotoxins commonly found in milk; however, their effects on intestinal epithelial cells have not been reported. In the present study, we show that AFM1 (0.12 and 12 μM) and OTA (0.2 and 20 μM) individually or collectively increased the paracellular flux of lucifer yellow and fluorescein isothiocyanate (FITC)-dextrans (4 and 40 kDa) and decreased transepithelial electrical resistance values in differentiated Caco-2 cells after 48 h of exposure, indicating increased epithelial permeability. Immunoblotting and immunofluorescent analysis revealed that AFM1, OTA, and their combination decreased the expression levels of tight junction (TJ) proteins and disrupted their structures, namely, claudin-3, claudin-4, occludin, and zonula occludens-1 (ZO-1), and p44/42 mitogen-activated protein kinase (MAPK) partially involved in the mycotoxins-induced disruption of intestinal barrier. The effects of a combination of AFM1 and OTA on intestinal barrier function were more significant (p < 0.05) than those of AFM1 and OTA alone, yielding additive or synergistic effects. The additive or synergistic effects of AFM1 and OTA on intestinal barrier function might affect human health, especially in children, and toxin risks should be considered.