Isolation and characterization of tannin-degrading bacteria from the rumen of wild Hokkaido sika deer (Cervus nippon yezoensis).

We isolated tannin-degrading bacteria from the rumen of wild Hokkaido sika deer and characterized their phylogeny and tannase activity in relation to sample sources. The condensed tannin level was higher in all deer rumen samples (n = 20) than in forage-fed cattle rumen samples (n = 6), whereas no hydrolyzable tannins were detected in any of the rumen samples. Rumen bacteria were enumerated on nonselective brain heart infusion (BHI) agar medium and then transferred onto tannic acid-containing BHI agar plates to screen for bacteria only showing growth (tannin-resistant bacteria) and those showing both growth and a clear zone (tannin-degrading bacteria). Summer samples provided only tannin-resistant bacteria, none of which showed tannin-degrading activity. Although winter samples also provided tannin-resistant bacteria, most isolates exhibited tannin-degrading activity. A total of 70 isolates exhibiting tannin-degrading activity were classified as Streptococcus bovis group based on 16S rRNA gene sequencing and further classified into two groups, either group A or group B. Group A consisted of isolates showing weak tannase activity, whereas group B included a majority of the isolates exhibiting high tannase activity. These results suggest that wild Hokkaido sika deer develop tannin-degrading Streptococcus in the rumen during winter, which allows access to woody food materials rich in tannins.

Batch culture analysis to identify potent organic acids for suppressing ruminal methane production.

We performed an in vitro rumen batch culture study to screen 11 commercially available organic acids for methane-suppressing ability and analyzed the rumen microbiota to determine the mode of action of the acids that showed potent methane-suppressing activity. Nine of the 11 acids showed methane-suppressing activity. Maleic anhydride, itaconate, citrate, and fumarate, which showed the highest activity, were further examined. These four acids showed methane-suppressing activity irrespective of the hay-to-concentrate ratios of the substrate. Maleic anhydride and itaconate decreased total gas and short-chain fatty acid production. Maleic anhydride and fumarate increased propionate production, while itaconate increased butyrate production. Maleic anhydride, itaconate, and citrate increased lactate production. Fumarate increased the abundance of bacteria involved in propionate production. Maleic anhydride, itaconate, and citrate increased the abundance of bacteria involved in lactate production. Thus, the results indicate that maleic anhydride, itaconate, and citrate may decrease methane in part by stimulating the acrylate pathway.

In vitro Effects of Cellulose Acetate on Fermentation Profiles, the Microbiome, and Gamma-aminobutyric Acid Production in Human Stool Cultures.

Gamma-aminobutyric acid (GABA) is considered as a potential candidate substance that mediates the effects of intestinal bacteria on human mental health. In the present study, we evaluated the effect of water-soluble cellulose acetate (WSCA), a type of cellulose ester, on fermentation and microbial profiles, and GABA production in human stool cultures prepared from fresh feces from volunteers. In addition, the GABA-producing ability of Bacteroides uniformis, which can utilize WSCA, was evaluated in a pure-culture study. All incubations were conducted anaerobically. WSCA supplementation increased (P < 0.05) acetate and propionate production and decreased (P < 0.05) the pH in human fecal cultures. WSCA significantly altered the microbiota, selectively increasing the relative abundance of B. uniformis (P < 0.05). Pure-culture study results revealed that B. uniformis produces GABA, possibly via a glutamate-dependent acid resistance system under low pH conditions. In conclusion, WSCA could be a potential prebiotic material that is fermented by intestinal bacteria and increases short-chain fatty acid and GABA production in the human gut. Bacteroides uniformis might play an important role in both WSCA degradation and GABA production in the intestine.

Aristaeella hokkaidonensis gen. nov. sp. nov. and Aristaeella lactis sp. nov., two rumen bacterial species of a novel proposed family, Aristaeellaceae fam. nov.

Two strains of Gram-negative, anaerobic, rod-shaped bacteria, from an abundant but uncharacterized rumen bacterial group of the order 'Christensenellales', were phylogenetically and phenotypically characterized. These strains, designated R-7T and WTE2008T, shared 98.6-99.0 % sequence identity between their 16S rRNA gene sequences. R-7T and WTE2008T clustered together on a distinct branch from other Christensenellaceae strains and had <88.1 % sequence identity to the closest type-strain sequence from Luoshenia tenuis NSJ-44T. The genome sequences of R-7T and WTE2008T had 83.6 % average nucleotide identity to each other, and taxonomic assignment using the Genome Taxonomy Database indicates these are separate species within a novel family of the order 'Christensenellales'. Cells of R-7T and WTE2008T lacked any obvious appendages and their cell wall ultra-structures were characteristic of Gram-negative bacteria. The five most abundant cellular fatty acids of both strains were C16 : 0, C16 : 0 iso, C17 : 0 anteiso, C18 : 0 and C15 : 0 anteiso. The strains used a wide range of the 23 soluble carbon sources tested, and grew best on cellobiose, but not on sugar-alcohols. Xylan and pectin were fermented by both strains, but not cellulose. Acetate, hydrogen, ethanol and lactate were the major fermentation end products. R-7T produced considerably more hydrogen than WTE2008T, which produced more lactate. Based on these analyses, Aristaeellaceae fam. nov. and Aristaeella gen. nov., with type species Aristaeella hokkaidonensis sp. nov., are proposed. Strains R-7T (=DSM 112795T=JCM 34733T) and WTE2008T (=DSM 112788T=JCM 34734T) are the proposed type strains for Aristaeella hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively.

Expression of chemerin in intestinal mucosa of calves with comparable expression level with other antimicrobial proteins.

Neonatal calves are highly susceptible to infectious disorders including diarrhea. Therefore, epithelial innate immunity, including antimicrobial peptides/proteins (AMPs), is important during the early stage of their lives. Chemerin, a multifunctional protein that was originally identified as a chemokine, possesses a potent antimicrobial activity. The present study investigated the expression levels of chemerin in the gastrointestinal (GI) tract of growing calves. Chemerin and its coding gene, retinoic acid receptor responder protein 2 (RARRES2), were highly expressed in duodenum, jejunum, and ileum compared with other parts of the GI tract. Immunohistochemistry demonstrated that chemerin-producing cells were localized in the crypt of the intestinal mucosa. Finally, the expression level of RARRES2 was higher compared with those of other major AMPs in duodenum, although it was lower compared with that of enteric ß-defensin but mostly higher than those of other AMPs in jejunum and ileum at various ages in calves. The expression levels of RARRES2 were not influenced by the age of calves in duodenum and jejunum, whereas a higher expression level of RARRES2 in ileum was observed in younger calves. This study revealed that chemerin is produced in the small intestine of calves and has the potential to contribute to the gut epithelial barrier system.

Mouse Scarb2 Modulates EV-A71 Pathogenicity in Neonatal Mice.

Enterovirus A71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease, which can progress to severe neurological disease. EV-A71 infects humans via the human scavenger receptor B2 (hSCARB2). It can also infect neonatal mice experimentally. Wild-type (WT) EV-A71 strains replicate primarily in the muscle of neonatal mice; however, susceptibility lasts only for a week after birth. Mouse-adapted (MA) strains, which can be obtained by serial passages in neonatal mice, are capable of infecting both muscle and neurons of the central nervous system. It is not clear how the host range and tropism of EV-A71 are regulated and why neonatal mice lose their susceptibility during development. We hypothesized that EV-A71 infection in neonatal mice is mediated by mouse Scarb2 (mScarb2) protein. Rhabdomyosarcoma (RD) cells expressing mScarb2 were prepared. Both WT and MA strains infected mScarb2-expressing cells, but the infection efficiency of the WT strain was much lower than that of the MA strain. Infection by WT and MA strains in vivo was abolished completely in Scarb2-/- mice. Scarb2+/- mice, in which Scarb2 expression was approximately half of that in Scarb2+/+ mice, showed a milder pathology than Scarb2+/+ mice after infection with the WT strain. The Scarb2 expression level in muscle decreased with aging, which was consistent with the reduced susceptibility of aged mice to infection. These results indicated that EV-A71 infection is mediated by mScarb2 and that the severity of the disease, the spread of virus, and the susceptibility period are modulated by mScarb2 expression. IMPORTANCE EV-A71 infects humans naturally but can also infect neonatal mice. The tissue tropism and severity of EV-A71 disease are determined by several factors, among which the virus receptor is thought to be important. We show that EV-A71 can infect neonatal mice using mScarb2. However, the infection efficiency of WT strains via mScarb2 is so low that an elevated virus-receptor interaction associated with mouse adaptation mutation and decrease in mScarb2 expression level during development modulate the severity of the disease, the spread of virus, and the susceptibility period in the artificial neonatal mice model.

Alterations in rumen microbiota via oral fiber administration during early life in dairy cows.

Bacterial colonization in the rumen of pre-weaned ruminants is important for their growth and post-weaning productivity. This study evaluated the effects of oral fiber administration during the pre-weaning period on the development of rumen microbiota from pre-weaning to the first lactation period. Twenty female calves were assigned to control and treatment groups (n = 10 each). Animals in both groups were reared using a standard feeding program throughout the experiment, except for oral fiber administration (50-100 g/day/animal) from 3 days of age until weaning for the treatment group. Rumen content was collected during the pre-weaning period, growing period, and after parturition. Amplicon sequencing of the 16S rRNA gene revealed that oral fiber administration facilitated the early establishment of mature rumen microbiota, including a relatively higher abundance of Prevotella, Shuttleworthia, Mitsuokella, and Selenomonas. The difference in the rumen microbial composition between the dietary groups was observed even 21 days after parturition, with a significantly higher average milk yield in the first 30 days of lactation. Therefore, oral fiber administration to calves during the pre-weaning period altered rumen microbiota, and its effect might be long-lasting until the first parturition.

TAK - 021, an inactivated Enterovirus 71 vaccine candidate, provides cross-protection against heterologous sub-genogroups in human scavenger receptor B2 transgenic mice.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of outbreaks of hand, foot and mouth disease, most frequently in children, and is a public health concern in the Asia-Pacific region. Takeda is developing TAK-021, an inactivated EV71 vaccine candidate based on sub-genogroup B2 strain MS87. In a phase I clinical trial, TAK-021 was safe, well tolerated, and immunogenic in healthy adults and elicited cross-neutralizing antibodies against heterologous EV71 sub-genogroup viruses. TAK-021 confers protection from lethal challenge with a mouse-adapted homologous strain in AG129 mice. However, it has not been determined whether TAK-021 can provide cross-protection against heterologous EV71 sub-genogroups. METHODS: We examined the efficacy of TAK-021 against challenge with EV71 sub-genogroups B4, B5, C1, C2, and C4 on day 42 (short-term) and sub-genogroups B5 and C4 on day 120 (long-term) after immunization of human scavenger receptor B2 transgenic (hSCARB2-tg) mice with TAK-021 on days 0 and 28. Antibody titers were monitored over 120 days using plaque reduction neutralization test of the homologous vaccine virus. RESULTS: TAK-021 elicited neutralizing antibody (nAb) in greater than 90% of the mice and nAb persisted through day 120. Challenge of control animals led to weight loss and death, as well as virus detection in various organs and histopathological lesions in the brain. All mice that received two doses of TAK-021 developed nAb and survived a short-term challenge given on day 42, while more than 80% survived a long-term challenge given on day 120. EV71 was detected less frequently and at lower levels in organs of immunized mice compared to non-immunized control mice. CONCLUSIONS: The results show that TAK-021 can confer protection in mice against the EV71 sub-genogroups tested.

Application of MinION Amplicon Sequencing to Buccal Swab Samples for Improving Resolution and Throughput of Rumen Microbiota Analysis.

The Illumina MiSeq platform has been widely used as a standard method for studying the rumen microbiota. However, the low resolution of taxonomic identification is the only disadvantage of MiSeq amplicon sequencing, as it targets a part of the 16S rRNA gene. In the present study, we performed three experiments to establish a high-resolution and high-throughput rumen microbial profiling approach using a combination of MinION platform and buccal swab sample, which is a proxy for rumen contents. In experiment 1, rumen contents and buccal swab samples were collected simultaneously from cannulated cattle (n = 6) and used for microbiota analysis using three different analytical workflows: amplicon sequencing of the V3-V4 region of the 16S rRNA gene using MiSeq and amplicon sequencing of near full-length 16S rRNA gene using MinION or PacBio Sequel II. All reads derived from the MinION and PacBio platforms were classified at the species-level. In experiment 2, rumen fluid samples were collected from beef cattle (n = 28) and used for 16S rRNA gene amplicon sequencing using the MinION platform to evaluate this sequencing platform for rumen microbiota analysis. We confirmed that the MinION platform allowed species-level taxa assignment for the predominant bacterial groups, which were previously identified at the family- and genus-level using the MiSeq platform. In experiment 3, buccal swab samples were collected from beef cattle (n = 30) and used for 16S rRNA gene amplicon sequencing using the MinION platform to validate the applicability of a combination of the MinION platform and buccal swab samples for rumen microbiota analysis. The distribution of predominant bacterial taxa in the buccal swab samples was similar to that in the rumen samples observed in experiment 2. Based on these results, we concluded that the combination of the MinION platform and buccal swab samples may be potentially applied for rumen microbial analysis in large-scale studies.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Virulence of Enterovirus A71 Fluctuates Depending on the Phylogenetic Clade Formed in the Epidemic Year and Epidemic Region.

Although epidemics of hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) have occurred worldwide, the Asia-Pacific region has seen large sporadic outbreaks with many severe neurological cases. This suggests that the virulence of the circulating viruses fluctuates in each epidemic and that HFMD outbreaks with many severe cases occur when highly virulent viruses are circulating predominantly, which has not been experimentally verified. Here, we analyzed 32 clinically isolated strains obtained in Japan from 2002 to 2013, along with 27 Vietnamese strains obtained from 2015 to 2016 that we characterized previously using human SCARB2 transgenic mice. Phylogenetic analysis of the P1 region classified them into five clades belonging to subgenogroup B5 (B5-I to B5-V) and five clades belonging to subgenogroup C4 (C4-I to C4-V) according to the epidemic year and region. Interestingly, clades B5-I and B5-II were very virulent, while clades B5-III, B5-IV, and B5-V were less virulent. Clades C4-II, C4-III, C4-IV, and C4-V were virulent, while clade C4-I was not. The result experimentally showed for the first time that several clades with different virulence levels emerged one after another. The experimental virulence evaluation of circulating viruses using SCARB2 transgenic mice is helpful to assess potential risks of circulating viruses. These results also suggest that a minor nucleotide or amino acid substitution in the EV-A71 genome during circulation causes fluctuations in virulence. The data presented here may increase our understanding of the dynamics of viral virulence during epidemics. IMPORTANCE Outbreaks of hand, foot, and mouth disease (HFMD) with severe enterovirus A71 (EV-A71) cases have occurred repeatedly, mainly in Asia. In severe cases, central nervous system complications can lead to death, making it an infectious disease of importance to public health. An unanswered question about this disease is why outbreaks of HFMD with many severe cases sometimes occur. Here, we collected EV-A71 strains that were prevalent in Japan and Vietnam over the past 20 years and evaluated their virulence in a mouse model of EV-A71 infection. This method clearly revealed that viruses belonging to different clades have different virulence, indicating that the method is powerful to assess the potential risks of the circulating viruses. The results also suggested that factors in the virus genome cause an outbreak with many severe cases and that further studies facilitate the prediction of large epidemics of EV-A71 in the future.

Growth and morphologic response of rumen methanogenic archaea and bacteria to cashew nut shell liquid and its alkylphenol components.

The growth and morphology of rumen methanogenic archaea (15 strains of 10 species in 5 genera, including 7 strains newly isolated in the present study) and bacteria (14 species in 12 genera) were investigated using unsupplemented in vitro pure cultures and cultures supplemented with cashew nut shell liquid (CNSL) and its phenolic compound components, anti-methanogenic agents for ruminant animals. Growth of most of the methanogens tested was inhibited by CNSL and alkylphenols at different concentrations ranging from 1.56 to 12.5 µg/ml. Of the alkylphenols tested, anacardic acid exhibited the most potent growth inhibition. Three gram-negative bacterial species involved in propionate production were resistant to CNSL and alkylphenols (>50 µg/ml). All the methanogens and bacteria that were sensitive to CNSL and alkylphenols exhibited altered morphology; disruption of the cell surface was notable, possibly due to surfactant activity of the tested materials. Cells division was inhibited in some organisms, with cell elongation and unclear septum formation observed. These results indicate that CNSL and alkylphenols, particularly anacardic acid, inhibit both rumen bacteria and methanogens in a selective manner, which could help mitigate rumen methane generation.

Adaptation and Virulence of Enterovirus-A71.

Outbreaks of hand, foot, and mouth disease caused by enterovirus-A71 (EV-A71) can result in many deaths, due to central nervous system complications. Outbreaks with many fatalities have occurred sporadically in the Asia-Pacific region and have become a serious public health concern. It is hypothesized that virulent mutations in the EV-A71 genome cause these occasional outbreaks. Analysis of EV-A71 neurovirulence determinants is important, but there are no virulence determinants that are widely accepted among researchers. This is because most studies have been done in artificially infected mouse models and because EV-A71 mutates very quickly to adapt to the artificial host environment. Although EV-A71 uses multiple receptors for infection, it is clear that adaptation-related mutations alter the binding specificity of the receptors and allow the virus to adopt the best entry route for each environment. Such mutations have confused interpretations of virulence in animal models. This article will discuss how environment-adapted mutations in EV-A71 occur, how they affect virulence, and how such mutations can be avoided. We also discuss future perspectives for EV-A71 virulence research.

Addition of ginkgo fruit to cattle feces and slurry suppresses methane production by altering the microbial community structure.

The effect of ginkgo fruit addition on methane production potential of cattle feces and slurry was assessed in relation to other fermentation products and the microbial community. Holstein cattle fresh feces and slurry were left at 30°C for 0, 30, 60, 90, and 180 days with/without ginkgo fruit to monitor the effect on fermentation potential. With the addition of ginkgo fruit, methane production potential of feces was reduced on Day 30 and thereafter, and that of slurry was consistently reduced over the experimental period. As a general trend, ginkgo fruit addition resulted in decreased acetate and increased propionate in feces and acetate accumulation in slurry. With ginkgo fruit addition, MiSeq analyses indicated decreases in methanogen (in particular Methanocorpusculum), Ruminococcaceae, and Clostridiaceae populations and increases in Bacteroidaceae and Porphyromonadaceae populations, which essentially agreed with quantitative real-time polymerase chain reaction (qPCR) assay results. These data indicate that direct addition of ginkgo fruit to cattle excreta is useful for reducing methane emissions by altering the microbial community structure. The application of ginkgo fruit to lower methane emissions from cattle excreta is, therefore, useful in cases in which the excreta is left without special management for a long period of time.

Cashew nut shell liquid potentially mitigates methane emission from the feces of Thai native ruminant livestock by modifying fecal microbiota.

The methane-mitigating potency of cashew nutshell liquid (CNSL) was evaluated by investigating gas production from batch cultures using feces from Thai native ruminants that had been incubated for different periods. Feces was obtained from four Thai native cattle and four swamp buffaloes reared under practical feeding conditions at the Kasetsart University farm, Thailand. Fecal slurry from the same farm was also included in the analysis. CNSL addition successfully suppressed the methane production potential of feces from both ruminants by shifting short chain fatty acid profiles towards propionate production. Methane mitigation continued for almost 150 days, although the degree of mitigation was more apparent from Day 0 to Day 30. Bacterial and archaeal community shifts with CNSL addition were observed in feces from both ruminants; specifically, Bacteroides increased, whereas Lachnospiraceae and Ruminococcaceae decreased in feces to which CNSL was added. Fecal slurry did not show marked changes in gas production with CNSL addition. The findings showed that the addition of CNSL to the feces of ruminants native to the Southeast Asian region can suppress methane emission. Because CNSL can be easily obtained as a byproduct of the local cashew industry in this region, its on-site application might be ideal.

Effect of Bacillus subtilis C-3102 supplementation in milk replacer on growth and rumen microbiota in preweaned calves.

We aimed to assess the effect of feeding Bacillus subtilis C-3102 on the growth and rumen microbiota in the preweaned calves. Twelve newborn Japanese Black calves were randomly allocated to either the control (n = 6) or the treatment (n = 6) groups in the present study. Calves in the treatment group were offered B. subtilis C-3102 supplemented milk replacer throughout the preweaning period. Rumen fermentation during the first 21 days of life seemed to be slightly suppressed by feeding B. subtilis C-3102. This fermentation shift was probably attributed to the lower abundance of the core members of rumen microbiota until 21 days of age in the calves fed B. subtilis C-3102. However, feeding B. subtilis C-3102 did not influence the abundance of the core members of rumen microbiota at 90 days of age. Distribution of Sharpea spp. and Megasphaera spp., which potentially contribute to low methane production and are regarded as beneficial rumen bacteria, was higher in the rumen of calves fed B. subtilis C-3102 at 90 days of age. These results suggest that B. subtilis C-3102 supplementation in milk replacer could potentially contribute to the improvement of feed efficiency after weaning via the establishment of beneficial rumen bacteria.

Identification of the core rumen bacterial taxa and their population dynamics during the fattening period in Japanese Black cattle.

The rumen microbiota comprises a vast range of bacterial taxa, which may affect the production of high-quality meat in Japanese Black cattle. The aim of this study was to identify core rumen microbiota in rumen fluid samples collected from 74 Japanese Black cattle raised under different dietary conditions using 16S rRNA gene amplicon sequencing. In the rumen of fattening Japanese Black cattle, 10 bacterial taxa, showing >1% average relative abundance and >95% prevalence, irrespective of the dietary conditions and the fattening periods, were identified as the core rumen bacterial taxa, which accounted for approximately 80% of the rumen microbiota in Japanese Black cattle. Additionally, population dynamics of the core rumen bacterial taxa revealed two distinct patterns: Prevotella spp. and unclassified Bacteroidales decreased in the mid-fattening period, whereas unclassified Clostridiales, unclassified Ruminococcaceae, Ruminococcus spp., and unclassified Christensenellaceae increased during the same period. Therefore, the present study reports the wide distribution of the core rumen bacterial taxa in Japanese Black cattle, and the complementary nature of the population dynamics of these core taxa, which may ensure stable rumen fermentation during the fattening period.

Feeding cashew nut shell liquid decreases methane production from feces by altering fecal bacterial and archaeal communities in Thai local ruminants.

The effect of feeding cashew nut shell liquid (CNSL) on fecal fermentation products and microbiota was investigated in Thai native cattle and swamp buffaloes. Four of each animal were fed rice straw and concentrate diet with control pellets without CNSL for 4 weeks, followed by the same diet with pellets containing CNSL for another 4 weeks, so that CNSL was administered at a level of 4 g/100 kg body weight. Feces were collected the last 2 days in each feeding period. CNSL alkyl phenols were recovered from feces (16%-28%) in a similar proportion to those in the diet, indicating that most functional anacardic acid was not selectively removed throughout the digestive tract. In vitro production of gas from feces, particularly methane, decreased with CNSL feeding. The proportion of acetate in feces decreased with CNSL feeding, whereas that of propionate increased, without affecting total short-chain fatty acid concentration. CNSL feeding changed fecal microbial community, particularly in swamp buffaloes, which exhibited decreases in the frequencies of Treponema, unclassified Ruminococcaceae, and Methanomassiliicoccaceae. These results suggest that CNSL feeding alters not only rumen fermentation but also hindgut fermentation via modulation of the microbial community, thereby potentially attenuating methane emission from the feces of ruminant animals.

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves.

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.

Microbial community structure of the bovine rumen as affected by feeding cashew nut shell liquid, a methane-inhibiting and propionate-enhancing agent.

The effect of cashew nut shell liquid (CNSL) feeding on bacterial and archaeal community of the bovine rumen was investigated by analyzing clone libraries targeting 16S rRNA genes, methyl-coenzyme reductase A-encoding genes (mcrA), and their respective transcripts. Rumen samples were collected from three non-lactating cows fed on a hay and concentrate diet with or without CNSL supplementation. DNA and complementary DNA (cDNA) libraries were generated for investigating rumen microbial communities. MiSeq analysis also was performed to understand more comprehensively the changes in the microbial community structures. Following CNSL supplementation, the number of operational taxonomical unit (OTU) and diversity indices of bacterial and archaeal community were decreased. Bacterial OTUs belonging to Proteobacteria, including Succinivibrio, occurred at a higher frequency with CNSL feeding, especially in cDNA libraries. The methanogenic archaeal community became dominated by Methanomicrobium. A bacterial community shift also was observed in the MiSeq data, indicating that CNSL increased the proportion of Succinivibrio and other genera known to be involved in propionate production. Methanogenic archaeal community shifts to increase Methanoplanus and to decrease Methanobrevibacter also were observed. Together, these results imply the occurrence of significant changes in rumen communities, not only for bacteria but also for methanogens, following CNSL feeding.