Gastrointestinal Tract Of Ruminants Research Articles

Unveiling novel antimicrobial peptides from the ruminant gastrointestinal microbiomes: A deep learning-driven approach yields an anti-MRSA candidate.

Antimicrobial peptides (AMPs) present a promising avenue to combat the growing threat of antibiotic resistance. The ruminant gastrointestinal microbiome serves as a unique ecosystem that offers untapped potential for AMP discovery. The aims of this study are to develop an effective methodology for the identification of novel AMPs from ruminant gastrointestinal microbiomes, followed by evaluating their antimicrobial efficacy and elucidating the mechanisms underlying their activity. We developed a deep learning-based model to identify AMP candidates from a dataset comprising 120 metagenomes and 10,373 metagenome-assembled genomes derived from the ruminant gastrointestinal tract. Both in vivo and in vitro experiments were performed to examine and validate the antimicrobial activities of the AMP candidates that were selected through bioinformatic analysis and subsequently synthesized chemically. Additionally, molecular dynamics simulations were conducted to explore the action mechanism of the most potent AMP candidate. The deep learning model identified 27,192 potential secretory AMP candidates. Following bioinformatic analysis, 39 candidates were synthesized and tested. Remarkably, all synthesized peptides demonstrated antimicrobial activity against Staphylococcus aureus, with 79.5% showing effectiveness against multiple pathogens. Notably, Peptide 4, which exhibited the highest antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), confirmed this effect in a mouse model with wound infection, exhibiting a low propensity for resistance development and minimal cytotoxicity and hemolysis towards mammalian cells. Molecular dynamics simulations provided insights into the mechanism of Peptide 4, primarily its ability to disrupt bacterial cell membranes, leading to cell death. This study highlights the power of combining deep learning with microbiome research to uncover novel therapeutic candidates, paving the way for the development of next-generation antimicrobials like Peptide 4 to combat the growing threat of MRSA would infections. It also underscores the value of utilizing ruminant microbial resources.

Probing the eukaryotic microbes of ruminants with a deep-learning classifier and comprehensive protein databases.

Metagenomics, particularly genome-resolved metagenomics, has significantly deepened our understanding of microbes, illuminating their taxonomic and functional diversity and roles in ecology, physiology, and evolution. However, eukaryotic populations within various microbiomes, including those in the mammalian gastrointestinal (GI) tract, remain relatively underexplored in metagenomic studies due to the lack of comprehensive reference genome databases and robust bioinformatics tools. The GI tract of ruminants, particularly the rumen, contains a high eukaryotic biomass although a relatively low diversity of ciliates and fungi, which significantly impacts feed digestion, methane emissions, and rumen microbial ecology. In the present study, we developed GutEuk, a bioinformatics tool that improves upon the currently available Tiara and EukRep in accurately identifying eukaryotic sequences from metagenomes. GutEuk is optimized for high precision across different sequence lengths. It can also distinguish fungal and protozoal sequences, further elucidating their unique ecological, physiological, and nutritional impacts. GutEuk was shown to facilitate comprehensive analyses of protozoa and fungi within more than one thousand rumen metagenomes, revealing a greater genomic diversity among protozoa than previously documented. We further curated several ruminant eukaryotic protein databases, significantly enhancing our ability to distinguish the functional roles of ruminant fungi and protozoa from those of prokaryotes. Overall, the newly developed package GutEuk and its associated databases create new opportunities for in-depth study of GI tract eukaryotes.

A metagenomic catalogue of the ruminant gut archaeome

While the ruminant gut archaeome regulates the gut microbiota and hydrogen balance, it is also a major producer of the greenhouse gas methane. However, ruminant gut archaeome diversity within the gastrointestinal tract (GIT) of ruminant animals worldwide remains largely underexplored. Here, we construct a catalogue of 998 unique archaeal genomes recovered from the GITs of ruminants, utilizing 2270 metagenomic samples across 10 different ruminant species. Most of the archaeal genomes (669/998 = 67.03%) belong to Methanobacteriaceae and Methanomethylophilaceae (198/998 = 19.84%). We recover 47/279 previously undescribed archaeal genomes at the strain level with completeness of >80% and contamination of <5%. We also investigate the archaeal gut biogeography across various ruminants and demonstrate that archaeal compositional similarities vary significantly by breed and gut location. The catalogue contains 42,691 protein clusters, and the clustering and methanogenic pathway analysis reveal strain- and host-specific dependencies among ruminant animals. We also find that archaea potentially carry antibiotic and metal resistance genes, mobile genetic elements, virulence factors, quorum sensors, and complex archaeal viromes. Overall, this catalogue is a substantial repository for ruminant archaeal recourses, providing potential for advancing our understanding of archaeal ecology and discovering strategies to regulate methane production in ruminants.

New insights into the enteric methane production based on the archaeal genome atlas of ruminant gastrointestinal tract

IntroductionAs one of the important components of ruminant gastrointestinal tract (GIT) microbiome, archaea are involved in many biological processes, especially methanogenesis. In spite of being a well-recognised member of the mammalian gut microbiome, it remains poorly characterized, partly due to the lack of a unified reference genome catalog. ObjectivesThis study aimed to construct a unified genome atlas that captures the wider diversity in archaea and is thus more appropriate for functional and taxonomic exploration of ruminant GIT archaea. MethodsWe collected archaeal genomes from public sources and new data of this study. We performed phylogenetic and functional genomics analysis, prophage identification based on the genomes. Using collected genomes as a reference, we conducted metagenomic and metatranscriptomic analysis on rumen fluid samples from 18 dairy cows, and investigated the correlation between rumen archaeal communities and methane (CH4) production profiles. ResultsWe constructed the ruminant GIT archaeal genomes (RGAG) by compiling 405 strain-level (160 species) non-redundant archaeal genomes from more than 10 ruminant species. Investigating the functional heterogeneity and methanogenic structure within RGAG revealed that it possessed 1,124 (99.5%) unknown microbial biosynthetic gene clusters. A survey of RGAG-borne prophages identified 63 prophages with 122 host-beneficial genes and 18 auxiliary metabolic genes. The pipeline for both metagenomics and metatranscriptomics generated in the study revealed the roles of archaeal genomes under-assessed in general multi-omics analysis. The highly expressed genus Methanosphaera was negatively correlated with CH4 production at the RNA level. ConclusionA unified genome atlas of ruminant GIT archaea is constructed in the study. Our analyses revealed the advantages of metatranscriptomics over metagenomics in studying rumen archaeal communities and further demonstrated that the multifaceted functions of ruminant archaea remain undiscovered. Differences in rumen archaeal community structure among cattle with different CH4 production profiles may reflect the balance between rumen hydrogen production and methanogenesis. Our work provides a new resource for interrogating archaeal functions in the ruminant GIT and potential targets for future CH4 reduction.

296 Effects of dietary antimicrobials on the ruminant gastrointestinal tract microbiome

Abstract Monensin, tylosin, and chlortetracycline (CTC) are antimicrobials frequently employed in high grain feedlot cattle diets to inhibit liver abscesses and enhance growth. However, increased public concerns regarding antibiotic resistance have created a pressing need to develop alternative strategies. A more mechanistic understanding of how antimicrobial drugs effect the microbiome in the ruminant gastrointestinal tract (GIT) may help develop effective alternatives. Therefore, the objective of this study was to determine changes in the GIT microbiome of grain-fed feedlot steers after antimicrobial drug administration. Thirty individually housed Angus x Simmental steers [body weight (BW) = 355 ± 16.8 kg] previously fed a 56% forage diet were immediately transitioned to a 93% concentrate diet and assigned to one of three treatments (10 steers/treatment): 1) control, without antibiotics, 2) 75 mg of tylosin and 200 mg of monensin per animal per day (TYLMON), and 3) 70 mg of CTC per animal per day. Fecal samples were collected on d 0, 7, 14, 28, 56, 112, and at slaughter and digesta were obtained from the rumen, jejunum, and cecum at slaughter. DNA was extracted from samples, amplified via 16S PCR, and sequenced. Jejunal samples had the least alpha diversity compared with ruminal, cecal, or fecal samples (P &amp;lt; 0.001). There were no differences in measures of alpha diversity in the ruminal, jejunal, or cecal digesta at slaughter (P ≥ 0.10). However, overall measures of alpha diversity (Shannon entropy, observed features) in feces were greater in CTC-fed steers (P ≤ 0.009) as well as in TYLMON-fed steers (P ≤ 0.04) compared with control steers. Measures of alpha diversity for all treatments decreased in feces from d 0 to 7, then increased from d 7 to slaughter (time effect, (P ≤ 0.001). There were no differences in fecal alpha diversity between treatment groups on d 0 (P ≥ 0.50), but by d 28 both TYLMON and CTC treated steers had greater fecal alpha diversity compared with the control steers (P ≤ 0.05). With regard to differential abundance, Phascolarctobacterium and Prevotellaceae UCG-003 were more abundant in feces of control steers relative to antimicrobial treated steers (P &amp;lt; 0.05). Bifidobacterium, Dorea, CAG-56, and Erysipelotrichaceae UCG-003, were the 4 genera found to be in the greatest abundance in the feces of TYLMON-fed relative to control steers (P &amp;lt; 0.05). Akkermansia, Succinivibrio, and Turicbacter were the 3 genera found to be in the greatest abundance in the feces of CTC-fed compared with control steers (P &amp;lt; 0.05). Taken together, these data suggest that differences in the microbiome associated with dietary antimicrobial treatment occur to a greater extent in the lower sections of the GIT, and that these changes in the microbiome occur after 28 d of feeding.

Ultralong CDR3 cattlebody use biased to switched immunoglobulin isotypes in bovine immune tissues

Abstract Alongside typical immunoglobulin (Ig), cattle express unique ultralong immunoglobulins (UL) that contain a CDR H3 comprised of a β-ribbon stalk and disulfide-linked knob that reaches over 70 amino acids in length. UL Ig exhibit potential "reach" to recessed pathogen epitopes and can provide broadly neutralizing antibodies to elusive antigens. This study explores the relative amounts of UL Ig in pre-switch isotypes (IgM and IgD) versus post-switch isotypes (IgG, IgA and IgE). Rearranged immunoglobulin heavy chain transcripts from primary and secondary lymphoid tissues of three steer were assessed with primers specific for the variable segments used in ultralong and canonical rearrangements as well as specific subisotype constant regions. Relative expression suggests biased selection for UL Ig in germinal centers and use in mucosal tissues of the ruminant gastrointestinal tract. Despite the explosion of studies exploring the immunogenetic diversification and binding possibilities of the UL Ig knob "picobody", our understanding of the use of the role of these antibodies in the Bovid immune system has been much slower. With a more complete understanding of tissue-specific expression patterns and isotype-specific immune responses using these exceptional paratopes, we'll be better poised to exploit them for improved bovine and human health.

A compendium of ruminant gastrointestinal phage genomes revealed a higher proportion of lytic phages than in any other environments

BackgroundRuminants are important livestock animals that have a unique digestive system comprising multiple stomach compartments. Despite significant progress in the study of microbiome in the gastrointestinal tract (GIT) sites of ruminants, we still lack an understanding of the viral community of ruminants. Here, we surveyed its viral ecology using 2333 samples from 10 sites along the GIT of 8 ruminant species.ResultsWe present the Unified Ruminant Phage Catalogue (URPC), a comprehensive survey of phages in the GITs of ruminants including 64,922 non-redundant phage genomes. We characterized the distributions of the phage genomes in different ruminants and GIT sites and found that most phages were organism-specific. We revealed that ~ 60% of the ruminant phages were lytic, which was the highest as compared with those in all other environments and certainly will facilitate their applications in microbial interventions. To further facilitate the future applications of the phages, we also constructed a comprehensive virus-bacteria/archaea interaction network and identified dozens of phages that may have lytic effects on methanogenic archaea.ConclusionsThe URPC dataset represents a useful resource for future microbial interventions to improve ruminant production and ecological environmental qualities. Phages have great potential for controlling pathogenic bacterial/archaeal species and reducing methane emissions. Our findings provide insights into the virome ecology research of the ruminant GIT and offer a starting point for future research on phage therapy in ruminants.CJAF8kS71Z3NBgTNJ_uZFeVideo

PSVIII-A-2 How Does the Duration of Low Feed Intake Affect the Ruminant Gastrointestinal tract?

Abstract The objective of this study was to determine the effect of 5 and 10 d low feed intake (LF) on the ruminant gastrointestinal tract (GIT). Twelve rams and nine wethers were used in a randomized complete block design where they were blocked by castration status and BW. After 5 d of ad libitum feeding, lambs were either exposed to 0 (CON; 100% DMI; n = 7), 5 (LF5; 30% DMI; n = 7), or 10 d (LF10; 30% DMI; n = 7) of LF. At the end of the study, lambs were euthanized, the GIT was removed to determine the digesta and tissue weights by region and the weights of the liver, spleen, and kidneys were recorded. There were no castration × treatment interactions (P ≥ 0.32). Initial (P = 0.97) and final (P = 0.19) BW did not differ by treatment. The weight of the reticulo-ruminal tissue tended to be less (P = 0.06) for LF10 than CON, with LF5 being intermediate but not different. Ruminal digesta weight was not affected (P = 0.98). Omasal tissue weight did not differ among treatments (P = 0.24), but digesta weight tended (P = 0.07) to be greater for CON compared with LF10 and LF5. Abomasal tissue weight was reduced for LF10 (P = 0.04), with LF5 being intermediate but not different. Abomasal digesta weight was less for LF5 and LF10 than CON (P = 0.04). The weight of the duodenum tended (P = 0.08) to be greater for CON than LF10 and LF5, but digesta weight was not affected (P = 0.16). For the jejunum, tissue and digesta weight tended (P = 0.07) to be greater for CON than LF10, with LF5 being intermediate. The weight of the ileum tended to be the greatest for CON, intermediate for LF5, and the least for LF10 (P = 0.10), but digesta weight was not affected (P = 0.81). Cecal tissue weight was not affected by treatment (P = 0.89); whereas digesta weight was the greatest (P = 0.03) for LF10, the least for CON, and intermediate but not different for LF5. In contrast, colonic tissue weight was less for LF10 relative to LF5 and CON (P = 0.01) and digesta weight tended (P = 0.05) to be greater for CON than LF5 and LF10. Liver weight was reduced (P &amp;lt; 0.01) for both LF5 and LF10 relative to CON, but kidney and spleen weights were not affected (P ≥ 0.33). This study indicates that exposing ruminants to LF rapidly reduces the weights for regions of the GIT and liver. These changes likely implicate a reduced absorptive capacity of the GIT and a lesser metabolic capacity of the liver.

Immunomodulation by cannabidiol in bovine primary ruminal epithelial cells

BackgroundRuminant livestock experience a number of challenges, including high concentrate diets, weaning and transport, which can increase their risk of disorders such as ruminal acidosis, and the associated inflammation of the ruminal epithelium. Cannabidiol (CBD), a phytochemical from hemp (Cannabis sativa), is a promising target as a therapy for gastrointestinal inflammation, and may be extremely valuable as either a treatment or prophylactic. However, the effects of CBD in the the ruminant gastrointestinal tract have not been explored, in part due to the restrictions on feeding hemp to livestock. Therefore, the objective of this study was to investigate the immunomodulatory properties of CBD using a model of inflammation in primary ruminal epithelial cells (REC). In addition, CBD dose was evaluated for possible cytotoxic effects.ResultsNegative effects on cell viability were not observed when REC were exposed to 10 μM CBD. However, when the dose was increased to 50 μM for 24 h, there was a significant cytotoxic effect. When 10 μM CBD was added to culture media as treatment for inflammation induced with lipopolysaccharide (LPS), expression of genes encoding for pro-inflammatory cytokine IL1B was less compared to LPS exposure alone, and CBD resulted in a down-regulation of IL6. As a pre-treatment, prior to LPS exposure, REC had decreased expression of IL6 and CXCL10 while CBD was present in the media, but not when it was removed prior to addition of LPS.ConclusionsResults suggest that CBD may reduce cytokine transcription both during LPS-induced inflammation and when used preventatively, although these effects were dependent on its continued presence in the culture media. Overall, these experiments provide evidence of an immunomodulatory effect by CBD during a pro-inflammatory response in primary REC in culture.

Abundance of Amino Acid Transporters and mTOR Pathway Components in the Gastrointestinal Tract of Lactating Holstein Cows.

Data from non-ruminants indicate that amino acid (AA) transport into cells can regulate mTOR pathway activity and protein synthesis. Whether mTOR is expressed in the ruminant gastrointestinal tract (GIT) and how it may be related to AA transporters and the AA concentrations in the tissue is unknown. Ruminal papillae and the epithelia of the duodenum, jejunum, and ileum collected at slaughter from eight clinically healthy Holstein in mid-lactation were used. Metabolites and RNA were extracted from tissue for liquid chromatography-mass spectrometry and RT-qPCR analysis. The glycine and asparagine concentrations in the rumen were greater than those in the intestine (p < 0.05), but the concentrations of other AAs were greater in the small intestine than those in the rumen. Among the 20 AAs identified, the concentrations of glutamate, alanine, and glycine were the greatest. The mRNA abundances of AKT1 and MTOR were greater in the small intestine than those in the rumen (p < 0.05). Similarly, the SLC1A1, SLC6A6, SLC7A8, SLC38A1, SLC38A7, and SLC43A2 mRNA abundances were greater (p < 0.05) in the small intestine than those in the rumen. The mRNA abundances of SLC1A5, SLC3A2, and SLC7A5 were greater in the rumen than those in the small intestine (p < 0.05). Overall, the present study provides fundamental data on the relationship between mTOR pathway components and the transport of AAs in different sections of the gastrointestinal tract.

Identification of nematodes of the genus Teladorsagia parasites of ruminants with the help of species-specific markers based on ITS2 rDNA

The present study delves into a methodological framework aimed at establishing species-specific markers via the utilization of sequences derived from the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA. This method, in conjunction with polymerase chain reaction (PCR) testing, serves as a diagnostic tool for discerning species belonging to the genus Teladorsagia Andreeva et Satubaldin, 1954. These species, constituents of the subfamily Ostertagiinae (Nematoda: Trichostrongylidae), exhibit wide distribution within the gastrointestinal tracts of ruminants across the geographic expanse of Uzbekistan. The heart of this endeavor is the development of species-specific primers, a pioneering creation in its own right. These primers are crafted using sequences emanating from the ITS2 region of the ribosomal DNA, an innovative approach that facilitates the precise identification of morphospecies within the Teladorsagia genus. Notably, the primers exhibit a nucleotide length of 153 base pairs, an attribute instrumental in their capacity to accurately distinguish and diagnose eggs and larvae of three distinct morphspecies: T. circumcincta, T. trifurcata, and T. davtiani. The potential implications of this method are significant, with ramifications reverberating across the field of veterinary diagnostics. Through the application of these primers, practitioners and researchers alike can effectively ascertain the presence of specific Teladorsagia morphospecies in ruminant animals. This holds the promise of not only enhancing diagnostic precision but also contributing to the broader comprehension of the prevalence and distribution of these nematode species within the local ruminant populations.

Cultivation-independent genomes greatly expand taxonomic-profiling capabilities of mOTUs across various environments

BackgroundTaxonomic profiling is a fundamental task in microbiome research that aims to detect and quantify the relative abundance of microorganisms in biological samples. Available methods using shotgun metagenomic data generally depend on the deposition of sequenced and taxonomically annotated genomes, usually from cultures of isolated strains, in reference databases (reference genomes). However, the majority of microorganisms have not been cultured yet. Thus, a substantial fraction of microbial community members remains unaccounted for during taxonomic profiling, particularly in samples from underexplored environments. To address this issue, we developed the mOTU profiler, a tool that enables reference genome-independent species-level profiling of metagenomes. As such, it supports the identification and quantification of both “known” and “unknown” species based on a set of select marker genes.ResultsWe present mOTUs3, a command line tool that enables the profiling of metagenomes for >33,000 species-level operational taxonomic units. To achieve this, we leveraged the reconstruction of >600,000 draft genomes, most of which are metagenome-assembled genomes (MAGs), from diverse microbiomes, including soil, freshwater systems, and the gastrointestinal tract of ruminants and other animals, which we found to be underrepresented by reference genomes. Overall, two thirds of all species-level taxa lacked a reference genome. The cumulative relative abundance of these newly included taxa was low in well-studied microbiomes, such as the human body sites (6–11%). By contrast, they accounted for substantial proportions (ocean, freshwater, soil: 43–63%) or even the majority (pig, fish, cattle: 60–80%) of the relative abundance across diverse non-human-associated microbiomes. Using community-developed benchmarks and datasets, we found mOTUs3 to be more accurate than other methods and to be more congruent with 16S rRNA gene-based methods for taxonomic profiling. Furthermore, we demonstrate that mOTUs3 increases the resolution of well-known microbial groups into species-level taxa and helps identify new differentially abundant taxa in comparative metagenomic studies.ConclusionsWe developed mOTUs3 to enable accurate species-level profiling of metagenomes. Compared to other methods, it provides a more comprehensive view of prokaryotic community diversity, in particular for currently underexplored microbiomes. To facilitate comparative analyses by the research community, it is released with >11,000 precomputed profiles for publicly available metagenomes and is freely available at: https://github.com/motu-tool/mOTUs.EtaL3gdjB5XhyYuYaq3JvNVideo

Isolation and characterization of bacteriophage Ib_pec2 against shigatoxigenic Escherichia coli.

This study was aimed to isolate and characterize bacteriophage against drug-resistant, shigatoxigenic Escherichia coli (STEC), one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n = 35) from neonatal calves, dairy workers, and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multidrug resistant to important antibiotics like ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin, and tetracycline. Bacteriophage namely Ib_pec2 was isolated against one of the STEC isolates and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophages belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. Proteomic analysis was able to identify five proteins identical to Tequatrovirus of myoviridae family. One-step growth curve experiment revealed a latency period of 40 min and a burst size of 893 pfu/bacteria. Temperature and pH ranging from 40°C to 50°C, pH6-8, respectively. Phage could able to lyse majority of the STEC isolates. STEC are commensal organisms in the gastrointestinal tract of ruminants but are pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control bacterial growth in ruminants and prevent its further spillage in the environment.

97 Immunology and the gut: The key to Animal Health

Abstract The gut immune system consists of three lines of defense systems: barriers, innate immunity and adaptive or acquired immunity that work together to give ruminant gastrointestinal tract (GIT) protection from disease. The gastrointestinal tract is the largest immune organ of the body. The mucosal barrier- the tight junctions and the “kill zone” along with the gut mucosa and maintaining an “anti-inflammatory” state are essential for “good gut health”. The microbiome- the microorganisms in the GIT, which has more cells than the entire animal’s body, is essential for immune development, immune response, and maximizing ruminant productivity. Homeostasis is an important function of the gut immune response to maintain health. Management of bovine GIT immune system is not a simple process. Stressors along with the intake of feed and hydration affect the microbiome and the intestinal epithelial cells resulting in important immune interactions. Nutraceuticals (i.e., probiotics, prebiotics, hen yolk IgY, essential oils, organic acids) aid in both microbiome stability “homeostasis” and immune function.

Isolation and characterization of bacteriophage Ib_pec2 against Shigatoxigenic E.coli

The current study aimed to isolate and characterize bacteriophage against drug resistant, Shiga toxigenic E.coli, one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n=35) from neonatal calves, dairy workers and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multi-drug resistant to important antibiotics like as ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin and tetracycline. Bacteriophage namely Ib_pec2 was isolated against NM—18-040 isolate and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophage belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. SDS-PAGE analysis of structural proteins followed by LC-MS/MS analysis could able to identify five proteins identical to Tequatrovirus of myoviridae family. One step growth curve experiments revealed a latency period of 40 mins and a burst size of 893 PFU/ bacteria. Temperature and pH ranging from 40-50°C, pH 7-8, respectively were ideal for phage survival and multiplication. Phage was able to lyse 22 out of 35 STEC isolates. Thus, the study could able to characterize Ib_pec2 which could be used in control of STEC in the near future. STEC is a commensal organism in the gastrointestinal tract of ruminants but pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control its growth in ruminants and prevent its further spillage in the environment.

Unraveling aerobic cultivable cellulolytic microorganisms within the gastrointestinal tract of sheep (Ovis aries) and their evaluation for cellulose biodegradation.

Anaerobic cellulolytic microbes in the gastrointestinal tract (GT) of ruminants have been well-documented; however, knowledge of aerobic microbes with cellulolytic activities in the ruminant GT is comparably limited. Here, we unraveled aerobic cultivable cellulolytic microbes in the GT of Ujimqin sheep (Ovis aries) and evaluated the cellulolytic potential of the promising isolates. Twenty-two strains were found to possess cellulose-degrading potential by Congo-red staining and phylogenetic analysis of the 16S rDNA/ITS sequence revealed that all strains belonged to nine genera, i.e., Bacillus, Streptomyces, Pseudomonas, Lactobacillus, Brachybacterium, Sanguibacter, Rhizobium, Fusarium, and Aspergillus. Strains with high cellulolytic activity were selected to further evaluate the activities of various enzymes in lignocellulosic alfalfa hay (Medicago sativa). Among them, isolate Bacillus subtilis RE2510 showed the highest potential for cellulose degradation, considering the high endoglucanase (0.1478 ± 0.0014 IU mL-1), exoglucanase (0.1735 ± 0.0012 IU mL-1), and β-glucosidase (0.3817 ± 0.0031 IU mL-1) after 10-day incubation with alfalfa hay. A significant destruction effect of the cellulose structure and the attachment of B. subtilis RE2510 to the hay were also revealed using a scanning electron microscope. This study expands our knowledge of aerobic cellulolytic isolates from the GT of sheep and highlights their potential application as a microbial additive in the aerobic process of cellulose bioconversion.

Biochemical and Structural Characterization of a Novel Bacterial Tannase From Lachnospiraceae bacterium in Ruminant Gastrointestinal Tract.

Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds present in hydrolyzable tannins to release gallic acid. Here, a novel tannase from Lachnospiraceae bacterium (TanALb) was characterized. The recombinant TanALb exhibited maximal activity at pH 7.0 and 50°C, and it maintained more than 70% relative activity from 30°C to 55°C. The activity of TanALb was enhanced by Mg2+ and Ca2+, and was dramatically reduced by Cu2+ and Mn2+. TanALb is capable of degrading esters of phenolic acids with long-chain alcohols, such as lauryl gallate as well as tannic acid. The Km value and catalytic efficiency (k cat /Km) of TanALb toward five substrates showed that tannic acid (TA) was the favorite substrate. Homology modeling and structural analysis indicated that TanALb contains an insertion loop (residues 341–450). Based on the moleculer docking and molecular dynamics (MD) simulation, this loop was observed as a flap-like lid to interact with bulk substrates such as tannic acid. TanALb is a novel bacterial tannase, and the characteristics of this enzyme make it potentially interesting for industrial use.

BioWorma as an Aid for Controlling Ruminant Nematode Parasites

Abstract Nematode-trapping fungi are biological control agents used against the larval stages of gastrointestinal nematodes in livestock feces. These fungi are normal soil inhabitants where they feed on a variety of non-parasitic soil nematodes. Of the various fungi tested, Duddingtonia flagrans spores (BioWorma, International Animal Health Products, Australia) have been shown to survive passage through the gastrointestinal tract of ruminants. After defecation, the spores germinate and grow in the feces to form sticky, sophisticated traps/loops which are able to trap the developing larval stages in the fecal environment. This form of control has been successfully applied under field conditions and is an environmentally safe biological approach for forage-based feeding systems. BioWorma has recently been approved for use in the United States. The primary delivery system is mixing BioWorma into supplement feedstuffs daily where each animal has the opportunity to consume an adequate amount of the mixture. To achieve optimum control of larvae during the transmission season (May–October), BioWorma needs to be fed for a period of no shorter than 60 days, starting at the beginning of the grazing season (especially young after weaning). Feeding BioWorma to dams during late pregnancy and lactation will also help to reduce pasture contamination, especially for growing young that will graze the same pasture. Another delivery system is mixing BioWorma into loose mineral supplement where animals will consume it free choice. The mineral needs to be kept covered and dry. The spores cannot be incorporated into pellets as the heat of the pelleting process will kill the spores. One drawback in using BioWorma is the relatively high cost. Research is being conducted to evaluate other delivery schemes that could be more cost effective. This product is the only control method that targets nematodes on pasture, where a majority of the total population reside.

Cellulolytic and xylanolytic faecal bacteria from tedong bonga, [Toraja buffalo, Bubalus bubalis carabanesis

The gastrointestinal tract of ruminants contains a considerable number of microbes that can utilize lignocellulosic biomass from plants. In this study, faecal samples of a local white spotted swamp buffalo or tedong bonga from North Toraja, South Sulawesi [Bubalus bubalis carabanesis] were investigated to aimed bacterial isolate that are capable of degrading both cellulose and xylan. Direct isolation and enrichment methods were performed by using selective medium containing 1% carboxymethyl cellulose [CMC] or xylan in the isolation process. Plate screening and enzyme quantification indicate that 11 out of 25 isolates have double actions of cellulase and xylanase with the range of 0.18-0.30 U/ml and 0.10-1.56 U/ml, respectively. Further, molecular identification using 16S rRNA gene for four selected isolates shows that three strains [KBX04, KBX07 and KBX08] were identical to Bacillus altitudinis 41KF2bT [100%] while another strain [KBX03] were identical to Cellulomonas flavigena DSM 20109T [99%]. We demonstrated that faeces from ruminants are a promising source for lignocellulose degrading bacteria that could be used for biomass conversion.

The effects of phytochemicals on methanogenesis: insights from ruminant digestion and implications for industrial biogas digesters management

The overarching objective of this review is to compare methanogenesis in ruminant gut systems and industrial digesters that employ plants, crops and their processing residues or by-products as feedstocks. An important consideration is the presence of phytochemicals in these feedstocks, with the understanding that depending on the type and quantities, these phytochemicals affect the activity of biomass degrading microorganisms to varying extents. This review aims to evaluate currently available knowledge on the effects of phytochemicals in ruminants’ methanogenesis as well as industrial anaerobic digestion processes. Technology and scale up in industrial fermentation have always benefited from our understanding of natural biological processes as such, knowledge from the process of methanogenesis in the gastrointestinal tract of ruminants can be used to make inferences about the performance of industrial digester systems for biogas production. This is particularly important when the same feedstock is used in both systems. Thus, in this review, comparisons of the two methane generating systems are discussed with an emphasis on phytochemical inhibitory effects on microorganisms that are involved in anaerobic digestion.