Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid.

Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6| |mM) in 48 h, and 1,230.1| |mL L-1 of CO2 and 523.5| |mL L-1 of CH4 were produced. The major isozymes identified by zymograms during the first 12| |h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial ana-lysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid.

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community's structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.

Characteristics of various fibrolytic isozyme activities in the rumen microbial communities of Japanese Black and Holstein Friesian cattle under different conditions.

Rumen microorganisms produce various fibrolytic enzymes and degrade lignocellulosic materials into nutrient sources for ruminants; therefore, the characterization of fibrolytic enzymes contributing to the polysaccharide degradation in the rumen microbiota is important for efficient animal production. This study characterized the fibrolytic isozyme activities of a rumen microbiota from four groups of housed cattle (1, breeding Japanese Black; 2, feedlot Japanese Black; 3, lactating Holstein Friesian; 4, dry Holstein Friesian). Rumen fluids in all cattle groups showed similar concentrations of total volatile fatty acids and reducing sugars, whereas acetic acid contents and pH were different among them. Predominant genera were commonly detected in all cattle, although the bacterial compositions were different among cattle groups. Zymograms of whole proteins in rumen fluids showed endoglucanase activities at 55 and 57 kDa and xylanase activity at 44 kDa in all cattle. Meanwhile, several fibrolytic isozyme activities differed among cattle groups and individuals. Treponema, Succinivibrio, Anaeroplasma, Succiniclasticum, Ruminococcus, and Butyrivibrio showed positive correlations with fibrolytic isozyme activities. Further, endoglucanase activity at 68 kDa was positively correlated with pH. This study suggests the characteristics of fibrolytic isozyme activities and their correlations with the rumen microbiota.

Diversity of Sulfur-oxidizing Bacteria at the Surface of Cattle Manure Composting Assessed by an Analysis of the Sulfur Oxidation Gene soxB.

Sulfur-oxidizing bacterial diversity at the surface of cattle manure was characterized throughout the composting process using a sulfur oxidation gene (soxB) clone library approach. In the mesophilic phase, clones related to the genera Hydrogenophaga and Hydrogenophilus were characteristically detected. In the thermophilic phase, clones related to the genera Hydrogenophaga and Thiohalobacter were predominant. In the cooling phase, the predominant soxB sequences were related to the genus Pseudaminobacter and a new sulfur-oxidizing bacterium belonging to the class Alphaproteobacteria. The present study showed changes in the community composition of sulfur-oxidizing bacteria at the surface of compost throughout the composting process.

Identification of bacteria involved in the decomposition of lignocellulosic biomass treated with cow rumen fluid by metagenomic analysis.

We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platform was performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37°C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter, one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus, Clostridium, and Caldicellulosiruptor. Calicellulosiruptor, which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using Rumen Microbiota.

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.

Pretreatment of Lignocellulosic Biomass with Cattle Rumen Fluid for Methane Production: Fate of Added Rumen Microbes and Indigenous Microbes of Methane Seed Sludge.

The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH4 reactor-1 for a 6-h treatment) was markedly higher than that in fermenters fed untreated rapeseed (1,325.8 mL CH4 reactor-1). Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge (e.g., Cellulosilyticum lentocellum and Ruminococcus flavefaciens) and rumen fluid (e.g., Butyrivibrio fibrisolvens and Prevotella ruminicola) became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge (e.g., Ruminofilibacter xylanolyticum) and rumen fluid (e.g., R. albus) remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.

Preservation of rumen fluid for the pretreatment of waste paper to improve methane production.

It is necessary to preserve rumen fluid for transport from slaughterhouses to the pretreatment facilities for use in treating lignocellulosic biomass. In this study, we investigated how the preservation of rumen fluid at various temperatures affects its use in hydrolysis of waste paper. Rumen fluid was preserved anaerobically at 4, 20, and 35 °C for 7 days. The number of protozoa and fibrolytic enzyme activity after preservation at 4 °C were significantly higher than that after preservation at either 20 or 35 °C. Waste paper was subsequently treated with preserved rumen fluid at 37 °C for 48 h. Preservation at 20 °C remarkedly decreased the hydrolysis of waste paper. Xylanase activity in rumen fluid preserved at 35 °C increased during the treatment, which enhanced the solubilization of waste paper as comparable to the control and preservation at 4 °C. Pretreatment of waste paper with rumen fluid preserved at 4 °C showed that the fluid retained high fibrolytic activity, and reduced the loss of organic carbon as substrate for methanogens. Our results suggest that preservation of rumen fluid at 4 °C is most suitable for efficient pretreatment and methane fermentation of waste paper.

A solution for private assessment in indirect reciprocity using solitary observation.

Although indirect reciprocity is a fundamental mechanism in the evolution of human cooperation, most studies assume public assessment in which individuals are not permitted to obtain private assessments of others. Existing studies on private assessment have used individual-based simulations because of the analytical difficulty involved. Here, we develop an analytical method using solitary observation to solve private assessment in indirect reciprocity problem without any approximation. In this study, we formulate a model of solitary observation and calculate the replicator dynamics systems of five leading norms of indirect reciprocity. Indirect reciprocity in private assessment provides a different result to that in public assessment. According to the existence proofs of cooperative evolutionarily stable (CES) points in the system, strict norms (stern judging and shunning) have no CES point in private assessment, while they do in public assessment. Image scoring does not change the system regardless of the assessment types because it does not use second-order information. In tolerant norms (simple standing and staying), the CES points move to co-existence of norms and unconditional cooperators. Despite the fact that there is no central coercive assessment system in private assessment, the average cooperation rate at the CES points in private assessment is greater than that in public assessment. This is because private assessment gives unconditional cooperators a role. Our results also show the superiority of the staying norm. Compared with simple standing, staying has three advantages in private assessment: a higher cooperation rate, easiness of invasion into defectors, and robustness to maintain cooperative evolutionarily stable situations. Our results are applicable to general social dilemmas in relation to private information. Under some dilemmas, norms or assessment rules should be carefully chosen to enable cooperation to evolve.

Prevalence and etiology of mastitis in dairy cattle in El Oro Province, Ecuador.

This study described the occurrence of clinical and subclinical forms of mastitis in 250 cattle from 5 dairy farms around the cities of Santa Rosa and Machala, El Oro Province, Ecuador. Clinical mastitis (CM) was determined based on obvious changes in milk (mild), signs of inflammation in the udder (moderate), and/or generalized clinical symptoms (severe). Subclinical mastitis (SCM) was assessed using the California mastitis test. CM and SCM were detected in 30 (12.0%) and 150 (60%) of the 250 tested cattle, respectively. Prevalence at the udder quarter level was 57.7% (577/1,000), which was higher among forequarters (369/577; 63.9%) than hindquarters. Of the 577 mastitic milk samples subjected to microbiological analysis, 35 were excluded due to contamination and 20 tested negative. Identification of bacterial isolates revealed that 33.3% of the 93 CM samples contained coliforms, 25.8% coagulase-positive staphylococci, 20.4% coagulase-negative staphylococci (CNS), 9.7% streptococci, 7.5% Bacillus spp., and 3.2% Klebsiella spp. Bacterial profiling of the 429 SCM milk samples showed that 55.4% contained CNS, 22.1% Bacillus spp., 9.3% streptococci, and 6.1% coagulase-positive staphylococci. In vitro antibiotic susceptibility testing of the obtained isolates indicated that all were susceptible to amoxicillin, ampicillin, cefotaxime, enrofloxacin, sulfamethoxazole-trimethoprim, gentamicin, and neomycin. No multidrug-resistant strains were observed.

Pretreatment with rumen fluid improves methane production in the anaerobic digestion of paper sludge.

Because paper sludge discharged from the waste paper recycling process contains high levels of lignin and ash, it is not hydrolyzed effectively during anaerobic digestion. In this study, we investigated the effects of pretreatment with rumen fluid on paper sludge and on the methane fermentation process. Paper sludge was pretreated with rumen fluid at 37 °C for 6 h. Following pretreatment, 4.5% of the total solids in paper sludge were degraded and converted, and the dissolved chemical oxygen demand and volatile fatty acid concentration increased. Batch methane fermentation was conducted at 37 °C for 20 days. During methane fermentation, the degradation and hydrolysis of paper sludge were enhanced by pretreatment with rumen fluid. The amounts of total methane production from pretreated paper sludge (excluding methane generated from rumen fluid), rumen fluid and untreated paper sludge were 650.4, 819.9 and 190.8 ml, respectively. The volume of methane gas produced from pretreated paper sludge was 3.4 times larger than that from untreated paper sludge. These results indicate that pretreatment with rumen fluid enhances methane production from paper sludge.

Clinical, pathological, and molecular data concerning Coenurus cerebralis in Sheep in Egypt.

This article contains information related to a recent study "Prevalence and Identity of Taenia multiceps cysts "Coenurus cerebralis" in Sheep in Egypt" (Amer et al., 2017) [1]. Specifically, affected sheep showed neurological disorders manifested as depression, head shaking and circling, altered head position, incoordination and paralysis in some cases. Brain-derived cysts were molecularly identified by PCR-sequence analysis at mitochondrial 12S rRNA gene marker. Cyst-induced pathological changes included degenerative changes and demyelination in brain tissue, infiltration of lymphocytes and histiocytes. Cystic fluids were biochemically analyzed for protein, lipids and electrolytes. The data of this study provides more understanding on phylogeny, epidemiology and pathology of coenurosis in sheep.

Prevalence, genotyping and risk factors of Giardia duodenalis from dogs in Vietnam.

This study was carried out to determine the prevalence, genotypes/assemblages and possible risk factors associated with Giardia duodenalis infection in dogs in central Vietnam. A total of 209 dog fecal samples, randomly collected from private owned dogs (n=105) and dogs from stores (n=104), were examined for Giardia cysts by microscopy. Positive samples were genotyped by PCR-sequence analysis of ß-giardin and triosephosphate isomerase genes markers. Risk factors were studied using a structured questionnaire and collected data were analyzed by univariate and multivariate logistic regression analyses. Results indicated that the overall infection rate was 8.6% (18/209) with the detected parasites were belonging to the non-zoonotic assemblages C and D. Age, gender and origin of animals were the main risk factors associated with G. duodenalis infection in dogs under study. Occurrence of infection was more likely in young animals compared to old ones and in females compared to males. Dogs originated from stores were more prone to Giardia infection compared to private owned counterparts.

Prevalence and Identity of Taenia multiceps cysts "Coenurus cerebralis" in Sheep in Egypt.

Coenurosis is a parasitic disease caused by the larval stage (Coenurus cerebralis) of the canids cestode Taenia multiceps. C. cerebralis particularly infects sheep and goats, and pose a public health concerns. The present study aimed to determine the occurrence and molecular identity of C. cerebralis infecting sheep in Egypt. Infection rate was determined by postmortem inspection of heads of the cases that showed neurological manifestations. Species identification and genetic diversity were analyzed based on PCR-sequence analysis of nuclear ITS1 and mitochondrial cytochrome oxidase (COI) and nicotinamide adenine dinucleotide dehydrogenase (ND1) gene markers. Out of 3668 animals distributed in 50 herds at localities of Ashmoun and El Sadat cities, El Menoufia Province, Egypt, 420 (11.45%) sheep showed neurological disorders. Postmortem examination of these animals after slaughter at local abattoirs indicated to occurrence of C. cerebralis cysts in the brain of 111 out of 420 (26.4%), with overall infection rate 3.03% of the involved sheep population. Molecular analysis of representative samples of coenuri at ITS1 gene marker showed extensive intra- and inter-sequence diversity due to deletions/insertions in the microsatellite regions. On contrast to the nuclear gene marker, considerably low genetic diversity was seen in the analyzed mitochondrial gene markers. Phylogenetic analysis based on COI and ND1 gene sequences indicated that the generated sequences in the present study and the reference sequences in the database clustered in 4 haplogroups, with more or less similar topologies. Clustering pattern of the phylogenetic tree showed no effect for the geographic location or the host species.

Tolerant indirect reciprocity can boost social welfare through solidarity with unconditional cooperators in private monitoring.

Indirect reciprocity is an important mechanism for resolving social dilemmas. Previous studies explore several types of assessment rules that are evolutionarily stable for keeping cooperation regimes. However, little is known about the effects of private information on social systems. Most indirect reciprocity studies assume public monitoring in which individuals share a single assessment for each individual. Here, we consider a private monitoring system that loosens such an unnatural assumption. We explore the stable norms in the private system using an individual-based simulation. We have three main findings. First, narrow and unstable cooperation: cooperation in private monitoring becomes unstable and the restricted norms cannot maintain cooperative regimes while they can in public monitoring. Second, stable coexistence of discriminators and unconditional cooperators: under private monitoring, unconditional cooperation can play a role in keeping a high level of cooperation in tolerant norm situations. Finally, Pareto improvement: private monitoring can achieve a higher cooperation rate than does public monitoring.

Detection and molecular status of Isospora sp. from the domestic pigeon (Columba livia domestica).

The domestic pigeon, Columba livia domestica, is reared for meat production, as a pet, or for racing. Few reports have characterized the parasitic protists from the genus Isospora isolated from Columbiformes. We detected Isospora-like oocysts from C. livia reared for racing. The oocyst contained two sporocysts, and each sporocyst included four sporozoites. The sporulated oocysts (n=4) were spherical; their mean diameters were 25.6 (24.0-27.2)×24.7 (23.4-26.0) µm. Micropyles, polar granules, and oocyst residuum were absent. The mean length and width of the sporocysts (n=8) were 19.5 (18.5-20.5) and 11.2 (10.2-12.1) µm, respectively. Stieda and sub-Stieda bodies were observed. Single-oocyst PCR revealed two different 18S rRNA gene sequences and one 28S rRNA gene sequence in a single oocyst of Isospora sp. Based on a phylogenetic analysis of the 18S rRNA gene, the two sequences made a group which fell within a cluster of known avian Isospora species. A tree based on the 28S rRNA gene sequence indicated that sequences from the pigeon Isospora sp. fell within a cluster of avian Isospora species. Both trees failed to clarify the phylogenetic relationships among the avian Isospora species due to limited resolution. Because the morphological description of Isospora sp. is based on only four oocysts, Isospora sp. is not proposed as a novel species here. This is the first description of Isospora sp. isolated from the domestic pigeon C. livia.

Pretreatment of lignocellulosic biomass by cattle rumen fluid for methane production: Bacterial flora and enzyme activity analysis.

We attempted to develop a pretreatment method for methane fermentation of lignocellulosic biomass using cattle rumen fluid, treated as slaughterhouse waste. When rapeseed (Brassica napus L.) was added to the methane fermentation after being solubilized with rumen fluid, 1.5 times more methane was produced compared with untreated rapeseed. Analysis of the bacterial flora during rumen fluid treatment using the MiSeq next-generation sequencer showed that the predominant phylum shifted from Bacteroidetes, composed of amylolytic Prevotella spp., to Firmicutes, composed of cellulolytic and xylanolytic Ruminococcus spp., in only 6 h. In total, 7 cellulolytic, 25 cello-oligosaccharolytic, and 11 xylanolytic bacteria were detected after investigating the most abundant sequences of detected taxa. The relative abundance of two Ruminococcus species (Ruminococcus albus and R. flavefaciens), known as cellulolytic, cello-oligosaccharolytic, and xylanolytic bacteria, increased with increasing cellulose and hemicellulose degradation rates, and, finally, comprised 48% of all operational taxonomic units. The chronological observation of enzyme activities showed that cellulolytic and xylanolytic activities increased 6 h later, and that oligosaccharolytic activity increased 24 h later. This study detected six bacteria that participate in the degradation of aromatics derived from lignin, which have rarely been reported in rumen fluid. The constitution of the detected bacteria suggests that the aromatics were converted into acetate via benzoate. The list of microbes that cover all lignocellulose-degrading candidates will provide fundamental knowledge for future studies focusing on rumen microbes.

The evolution of conditional moral assessment in indirect reciprocity.

Indirect reciprocity is a major mechanism in the maintenance of cooperation among unrelated individuals. Indirect reciprocity leads to conditional cooperation according to social norms that discriminate the good (those who deserve to be rewarded with help) and the bad (those who should be punished by refusal of help). Despite intensive research, however, there is no definitive consensus on what social norms best promote cooperation through indirect reciprocity, and it remains unclear even how those who refuse to help the bad should be assessed. Here, we propose a new simple norm called "Staying" that prescribes abstaining from assessment. Under the Staying norm, the image of the person who makes the decision to give help stays the same as in the last assessment if the person on the receiving end has a bad image. In this case, the choice about whether or not to give help to the potential receiver does not affect the image of the potential giver. We analyze the Staying norm in terms of evolutionary game theory and demonstrate that Staying is most effective in establishing cooperation compared to the prevailing social norms, which rely on constant monitoring and unconditional assessment. The application of Staying suggests that the strict application of moral judgment is limited.

Indirect reciprocity can overcome free-rider problems on costly moral assessment.

Indirect reciprocity is one of the major mechanisms of the evolution of cooperation. Because constant monitoring and accurate evaluation in moral assessments tend to be costly, indirect reciprocity can be exploited by cost evaders. A recent study crucially showed that a cooperative state achieved by indirect reciprocators is easily destabilized by cost evaders in the case with no supportive mechanism. Here, we present a simple and widely applicable solution that considers pre-assessment of cost evaders. In the pre-assessment, those who fail to pay for costly assessment systems are assigned a nasty image that leads to them being rejected by discriminators. We demonstrate that considering the pre-assessment can crucially stabilize reciprocal cooperation for a broad range of indirect reciprocity models. In particular for the most leading social norms, we analyse the conditions under which a prosocial state becomes locally stable.