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Isolation and characterization of a new Methanoculleus bourgensis strain KOR-2 from the rumen of Holstein steers.

ABSTRACT: OBJECTIVE:To isolate and identify new methanogens from the rumen of Holstein steers in Korea. METHODS:Representative rumen contents were obtained from three ruminally cannulated Holstein steers (793±8 kg). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the isolated strain. Furthermore, the isolate was microscopically studied for its morphology. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification. RESULTS:One strain designated as KOR-2 was isolated and found to be a non-motile irregular coccus with a diameter of 0.2 to 0.5 ?m. KOR-2 utilized H2+CO2 and formate but was unable to metabolize acetate, methanol, trimethylamine, 2-propanol, and isobutanol for growth and methane production. The optimum temperature and pH for the growth of KOR-2 were 38°C and 6.8 to 7.0, respectively, while the optimum NaCl concentration essential for KOR-2 growth was 1.0% (w/v). KOR-2 tolerated ampicillin, penicillin G, kanamycin, spectromycin, and tetracycline. In contrast, the cell growth was inhibited by chloramphenicol. Phylogenetic analysis of 16S rRNA and mcrA genes revealed the relatedness between KOR-2 and Methanoculleus bourgensis. CONCLUSION:Based on the physiological and phylogenetic characteristics, KOR-2 was thought to be a new strain within the genus Methanoculleus and named Methanoculleus bourgensis KOR-2.

SUBMITTER: Battumur U

PROVIDER: S-EPMC6325391 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Isolation and characterization of a new Methanoculleus bourgensis strain KOR-2 from the rumen of Holstein steers.

Battumur Urantulkhuur U Lee Manhee M Bae Gui Sek GS Kim Chang-Hyun CH

Asian-Australasian journal of animal sciences 20180726 2

<h4>Objective</h4>To isolate and identify new methanogens from the rumen of Holstein steers in Korea.<h4>Methods</h4>Representative rumen contents were obtained from three ruminally cannulated Holstein steers (793±8 kg). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the iso ...[more]

PMID: 30056662

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Project description:Seasonal effects on rumen microbiome and enteric methane (CH4) emissions are poorly documented. In this study, 6 Holstein and 6 Jersey steers were fed the same total mixed ration diet during winter, spring, and summer seasons under a 2 × 3 factorial arrangement for 30 days per season. The dry matter intake (DMI), rumen fermentation characteristics, enteric CH4 emissions and rumen microbiota were analyzed. Holstein had higher total DMI than Jersey steers regardless of season. However, Holstein steers had the lowest metabolic DMI during summer, while Jersey steers had the lowest total DMI during winter. Jersey steers had higher CH4 yields and intensities than Holstein steers regardless of season. The pH was decreased, while ammonia nitrogen concentration was increased in summer regardless of breed. Total volatile fatty acids concentration and propionate proportions were the highest in winter, while acetate and butyrate proportion were the highest in spring and in summer, respectively, regardless of breed. Moreover, Holstein steers produced a higher proportion of propionate, while Jersey steers produced a higher proportion of butyrate regardless of season. Metataxonomic analysis of rumen microbiota showed that operational taxonomic units and Chao 1 estimates were lower and highly unstable during summer, while winter had the lowest Shannon diversity. Beta diversity analysis suggested that the overall rumen microbiota was shifted according to seasonal changes in both breeds. In winter, the rumen microbiota was dominated by Carnobacterium jeotgali and Ruminococcus bromii, while in summer, Paludibacter propionicigenes was predominant. In Jersey steers, Capnocytophaga cynodegmi, Barnesiella viscericola and Flintibacter butyricus were predominant, whereas in Holstein steers, Succinivibrio dextrinosolvens and Gilliamella bombicola were predominant. Overall results suggest that seasonal changes alter rumen microbiota and fermentation characteristics of both breeds; however, CH4 emissions from steers were significantly influenced by breeds, not by seasons.

Project description:Previous studies have focused on the rumen microbiome and enteric methane (CH4) emissions in dairy cows, yet little is known about steers, especially steers of dairy breeds. In the present study, we comparatively examined the rumen microbiota, fermentation characteristics, and CH4 emissions from six non-cannulated Holstein (710.33 ± 43.02 kg) and six Jersey (559.67 ± 32.72 kg) steers. The steers were fed the same total mixed ration (TMR) for 30 days. After 25 days of adaptation to the diet, CH4 emissions were measured using GreenFeed for three consecutive days, and rumen fluid samples were collected on last day using stomach tubing before feeding (0 h) and 6 h after feeding. CH4 production (g/d/animal), CH4 yield (g/kg DMI), and CH4 intensity (g/kg BW0.75) were higher in the Jersey steers than in the Holstein steers. The lowest pH value was recorded at 6 h after feeding. The Jersey steers had lower rumen pH and a higher concentration of ammonia-nitrogen (NH3-N). The Jersey steers had a numerically higher molar proportion of acetate than the Holstein steers, but the opposite was true for that of propionate. Metataxonomic analysis of the rumen microbiota showed that the two breeds had similar species richness, Shannon, and inverse Simpson diversity indexes. Principal coordinates analysis showed that the overall rumen microbiota was different between the two breeds. Both breeds were dominated by Prevotella ruminicola, and its highest relative abundance was observed 6 h after feeding. The genera Ethanoligenens, Succinivibrio, and the species Ethanoligenens harbinense, Succinivibrio dextrinosolvens, Prevotella micans, Prevotella copri, Prevotella oris, Prevotella baroniae, and Treponema succinifaciens were more abundant in Holstein steers while the genera Capnocytophaga, Lachnoclostridium, Barnesiella, Oscillibacter, Galbibacter, and the species Capnocytophaga cynodegmi, Galbibacter mesophilus, Barnesiella intestinihominis, Prevotella shahii, and Oscillibacter ruminantium in the Jersey steers. The Jersey steers were dominated by Methanobrevibacter millerae while the Holstein steers by Methanobrevibacter olleyae. The overall results suggest that sampling hour has little influence on the rumen microbiota; however, breeds of steers can affect the assemblage of the rumen microbiota and different mitigation strategies may be needed to effectively manipulate the rumen microbiota and mitigate enteric CH4 emissions from these steers.

Project description:BackgroundTrypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection.MethodsTrypanosomes isolated from the urethral tract of a stallion with suspected dourine, were directly cultivated using soft agarose media at 37 °C in 5 % CO2. For molecular characterization, 18S ribosomal RNA (rRNA) gene, the internal transcribed spacer (ITS) and 8 maxicircle DNA regions were amplified by a PCR and their sequences were determined. To analyze the ratio of the kinetoplastic/akinetoplastic population, the kinetoplasts and the nuclei of trypanosomes were subjected to Hoechst staining and observed by fluorescence microscopy.ResultsIn addition to the clinical symptoms and the molecular diagnosis, this stallion was definitively diagnosed with dourine by the detection of trypanosomes in the urethral mucosa. These results strongly suggested that the isolated trypanosome was true T. equiperdum. T. equiperdum isolated from the urethral tract was adapted in vitro using soft agarose media. Based on the results of a phylogenetic analysis of 18S rRNA and ITS, this T. equiperdum isolate was classified into the Trypanozoon clade. In a PCR of the maxicircle DNA region, only NADH-dehydrogenase subunits 4 and 5 was amplified. Clear kinetoplasts were observed in most of the T. equiperdum isolates. In contrast, most culture-adapted T. equiperdum were of the akinetoplastic form.ConclusionWe concluded that our isolated trypanosome was the first confirmed case of T. equiperdum in Mongolia and named it "T. equiperdum IVM-t1". T. equiperdum IVM-t1 was well adapted and propagated in soft agarose media, which indicates that this culture method is useful for isolation of T. equiperdum from horses with dourine.

Dataset Information

Isolation and characterization of a new Methanoculleus bourgensis strain KOR-2 from the rumen of Holstein steers.

Publications

Isolation and characterization of a new Methanoculleus bourgensis strain KOR-2 from the rumen of Holstein steers.

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