• Journal of The Korean Society of Grassland and Forage Science
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• v.37 no.1
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• pp.35-43
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• 2017

This study was to research the relationships between rice straw degradation and changes of fibrolytic bacteria population during the in vitro rumen fermentation. Dry matter(DM) digestion of rice straw and population of fibrolytic bacteria were measured at the 0. 4, 8, 12 and 48 hours during the incubation. The populations of F. succinogenes. R. albus and R. flavefaciens were defined as log copy number of 16S rDNA by technical method of Quantitative real-time PCR. Total population of F. succinogenes, R. flavefaciens and R. albus was sum of bactera attached on rice straw and suspended in medium. It's population was increased with incubation, reached top level of 29.0 Log copy No at the 24 hour and then decreased. In the meantime, DM digestion of rice straw showed the higher increasement from the 8 hour to the 24 hour than from the 0 hour to the 8 hour, and then a slowdown in increasing trend of digestibility. Attachments of F. succinogenes, R. flavefaciens and R. albus were detected immediately after start of in vitro rumen incubation. At the same time, the colonized bacterial share were respectively 34.5%, 84.4% and 67.9% in total population. All of them was reached the highest colonized bacterial share above 94.7% at the 4 hour incubation. However population of attached bacteria was shown the highest level at the 12 hour or the 24 hour incubation. Kinetics of colonization were formed area of top speed from the 12 hour to the 24 hour and respectively reached 10.33, 9.28 및 8.30 Log copy No/h/g DM at the 24 hour by F. succinogenes, R. flavefaciens and R. albus. The kinetics of rice straw degradation was formed top level of 0.95% DM/h at the 24 hour. The present results gave clear evidence that degradation of rice straw was increased with the development of total fibrolytic bacteria in process of rumen fermentation. Also, their attachment was largely occurred immediately after insertion of rice straw, the colonized bacteria was actively proliferated, and then degradation of rice straw was maximized.

• Journal of The Korean Society of Grassland and Forage Science
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• v.34 no.1
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• pp.60-67
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• 2014

The comparisons between cellulolytic bacteria adhesion on rice straw and fiber digestion in time course during rumen fermentation were studied in situ. The adhesions of cellulolytic bacteria, F. succinogenes. R. albus and R. flavefaciens, were measured by RT-PCR. When the rice straws were incubated at 0. 2, 4, 8, 12 and 24 hours of the in situ rumen, straw was degraded with increasing speed during the incubation and showed the highest disappearance increasing rate (DM g/h) from 8 to 12 hour. The adhesions of F. succinogenes, R. flavefaciens and R. albus were achieved above 80% in 1 hour of in situ rumen fermentation and then keep adhesive population up after the time of fermentation. When the in situ samples were collected at 0, 5, 10, 30 and 60 min to detect the early stages of adhesion on the rice straws ingested into rumen, the numberous adhesive colony of F. succinogenes, R. flavefaciens and R. albus were detected in 5 min. In case of rice straw treated with 0, 2, 4 and 8% NaOH, all of three cellulolytic bacteria showed the increasing trends of adhesion with increasing DM disappearance of rice straw by higher concentration of NaOH at 12 hour of in situ. However, there were showed respectively difference at 24 hour. The present results gave certain evidence that adhesion of cellulolytic bacteria is definitely achieved in early stage of roughage ingestion into rumen, their colony develop the stable communities on roughage in process of rumen fermentation and then fiber degradation is accelerated.

• IMMUNE NETWORK
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• v.4 no.1
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• pp.53-59
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• 2004

Background: Minor histocompatibility HY antigen, as a transplantation antigen, has been known to cause graft rejection in MHC (major histocompatibility complex) matched donor-recipient. The aim of our study is to investigate the role of male antigen (HY) disparity on MHC matched pancreatic islet transplantation and to examine the mechanism of the immune reaction. Methods: Pancreatic islets were isolated and purified by collagen digestion followed by Ficoll gradient. The isolated islets of male C57BL6/J were transplanted underneath the kidney capsule of syngeneic female mice rendered diabetic with streptozotocine. Blood glucose was monitored for the rejection of engrafted islets. After certain period of time, tail to flank skin transplantation was performed either on mouse transplanted with HY mismatched islets or on sham treated mouse. The rejection was monitored by scoring gross pathology of the engrafted skin. Results: HY mismatched islets survived more than 300 days in 14 out of 15 mice. The acceptance of second party graft (male B6 islets) and the rejection of third party graft (male BALB/c islets) in these mice suggested the tolerance to islets with HY disparity. B6 Skin with HY disparity was rejected on day $25{\pm}7$. However, HY mismatched skin transplanted on the mice tolerated to HY mismatched islets survived more than 240 days. Tetramer staining in these mice indicated the CTL recognizing MHC Db/Uty was not deleted or anergized. Conclusion: The islet transplantation across HY disparity induced tolerance to HY antigen in C57BL6 mouse, which in turn induced tolerance to HY mismatched skin, which otherwise would be rejected within 25 days. The MHC tetramer staining suggested the underlying mechanisms would not be clonal deletion or anergy.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.624-630
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• 2008

Terminal restriction fragment length polymorphism (T-RFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct T-RFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.1-8
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• 2016

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, $RIDA^{(R)}$ Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.603-615
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• 2008

Dietary fiber is an inevitable component in pig diets. In non-ruminants, it may influence many physiological processes in the gastrointestinal tract (GIT) such as transit time as well as nutrient digestion and absorption. Moreover, dietary fiber is also the main substrate of intestinal bacteria. The bacterial community structure is largely susceptible to changes in the fiber content of a pig's diet. Indeed, bacterial composition in the lower GIT will adapt to the supply of high levels of dietary fiber by increased growth of bacteria with cellulolytic, pectinolytic and hemicellulolytic activities such as Ruminococcus spp., Bacteroides spp. and Clostridium spp. Furthermore, there is growing evidence for growth promotion of beneficial bacteria, such as lactobacilli and bifidobacteria, by certain types of dietary fiber in the small intestine of pigs. Studies in rats have shown that both phosphorus (P) and calcium (Ca) play an important role in the fermentative activity and growth of the intestinal microbiota. This can be attributed to the significance of P for the bacterial cell metabolism and to the buffering functions of Ca-phosphate in intestinal digesta. Moreover, under P deficient conditions, ruminal NDF degradation as well as VFA and bacterial ATP production are reduced. Similar studies in pigs are scarce but there is some evidence that dietary fiber may influence the ileal and fecal P digestibility as well as P disappearance in the large intestine, probably due to microbial P requirement for fermentation. On the other hand, fermentation of dietary fiber may improve the availability of minerals such as P and Ca which can be subsequently absorbed and/or utilized by the microbiota of the pig's large intestine.

• Parasites, Hosts and Diseases
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• v.47 no.3
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• pp.307-310
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• 2009

Fishborne trematode (FBT) metacercariae were investigated in yellowfin goby, Acanthogobius flavimanus, collected from Shinan-gun and Muan-gun, Jeollanam-do (province), Korea. All collected fishes were examined using the artificial digestion method. In all of 15 gobies from Aphae-myeon in Shinan-gun, metacercariae of Stictodora spp. (334 metacercariae/fish), Heterophyes nocens (153/fish), and Heterophyopsis continua (20/fish) were detected. In 2 of 14 gobies from Jido-myeon in Shinan-gun, 8 Echinostoma hortense metacercariae in total were detected. In 15 gobies from Haeje-myeon in Muan-gun, the metacercariae of H. continua were found in 100%, Stictodora spp. in 86.7%, and H. nocens in 6.7% of fish examined. The average numbers of metacercariae per infected fish were 23.3 (H. continua), 416.0 (Stictodora spp.), and 2.0(H. nocens), respectively. The metacercariae of E. hortense found in gobies were elliptical, with 150 ${\times}$ 138 ${\mu}m$, in average size, and had 27 collar spines on the head crown. The above results suggest that yellowfin gobies from 2 localities may be the potential infection sources of FBT. Moreover, it is proved for the first time that the yellowfin goby, A. flavimanus, acts as a second intermediate host for E. hortense.

• YAKHAK HOEJI
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• v.51 no.4
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• pp.253-258
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• 2007

Bombycis corpus, a batryticated silkworm and white-stiff silkworm, is an oriental drug consisting of the dried larva of silkworm, dead and stiffened due to the infection of Beauveria. An peptidyl antimicrobial molecule was purified from B. corpus by reverse phase-column chromatography and HPLC. Its molecular weight was determined to be 2295.45 by using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Its antimicrobial activity was diminished by trypsin digestion. It exhibited a broad antimicrobial spectrum against not only Gram-negative, but also Gram- positive bacteria. Furthermore, it was found to have an antimicrobial activity against vancomycin-resistant enterococci (VRE), methicillin-resistant S. arureus (MRSA), and vancomycin-intermediate resistant S. arureus (VISA). It may be a useful molecule for a new antibiotic development, especially against antibiotic resistant microbe. This substance may play a role in the defense system of this animal against Beauveria bassiana. This is the first report of a peptidyl antimicrobial substance from B. corpus.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1397-1401
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• 2007

An in situ experiment was conducted to find out whether Tween 80 improves rice straw digestion through increased adhesion of major fibrolytic bacteria. Rice straw was sprayed with various levels of Tween 80 non-ionic surfactant or SDS ionic surfactant 24 h before incubation in the rumen of Holstein steers. Dry matter (DM) disappearance and adhesion of F. succinogenes, R. flavefaciens and R. albus on rice straw after in situ incubation were measured by real-time PCR. Application of Tween 80 increased DM disappearance, which was more noticeable at an application level of 1% compared to lower application levels. Application of SDS resulted in an opposite response in DM disappearance with highest reduction in DM disappearance at 1% level. In a subsequent in situ experiment, higher Tween 80 was applied to rice straw in an attempt to find the optimum application level. Tween 80 at 2.5% gave better DM disappearance than 1% with a similar result at 5%. Therefore, an adhesion study was carried out using rice straw treated with 2.5% Tween 80. Our results indicated that Tween 80 reduced adhesion of all three major rumen fibrolytic bacteria to rice straw. Present data clearly show that improved DM disappearance by Tween 80 is not due to increased bacterial adhesion onto substrates.

• BMB Reports
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• v.40 no.2
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• pp.163-171
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• 2007

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry $\delta$- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, $Trp^{243}$, $Phe^{246}$, $Tyr^{249}$ and $Phe^{264}$, in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at $Tyr^{249}$ and $Phe^{264}$ still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of $Tyr^{249}$ and $Phe^{264}$ within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.