Influence of repeated ochratoxin A ingestion on milk production and its carry-over into the milk, blood and tissues of lactating cows.

An experiment was conducted to investigate the influence of repeated ingestion of ochratoxin A (OTA) on milk production of lactating Holstein cows over 28 days, and the carry-over of OTA from the diets into the milk and tissues of the cows. Nine cows were divided into three groups, labeled OTA5, OTA50 and OTA100, and fed a diet containing 5, 50 and 100 µg OTA/kg of dry matter, respectively. Body weight, feed intake and daily milk yield in cows were not different among the three groups during the OTA-intake period. OTA residues were neither detected in the tissues, such as liver, kidney, muscles, fat and jejunoileum, nor in the milk of any cows in the OTA intake groups. In contrast, a small amount of OTA (0.1 µg/kg) was detected in the blood plasma of one sample in the OTA50 group and multiple samples in the OTA100 group. The results of this study show that the ingestion of diets containing up to 100 µg/kg of OTA over 28 days does not affect feed intake or milk production of cows, and the dietary OTA is not carried over into milk and edible tissues such as the liver, muscles and fat.

Isolation and identification of cellulose-binding proteins from sheep rumen contents.

To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.

Comparison of pulsed-field gel electrophoresis patterns of antimicrobial-resistant Escherichia coli and enterococci isolates from the feces of livestock and livestock farmers in Japan.

Seven hundred thirty-nine animal strains and 662 livestock-farmer strains, consisting of Escherichia coli and enterococci, were examined for their pulsed-field gel electrophoresis (PFGE) and antimicrobial-resistance patterns. Two hundred fifty-eight and 203 PFGE patterns were found among 739 animal strains isolated from animals comprising broilers, pigs and cattle, and 662 human strains isolated from livestock farmers, respectively, from 27 farms in Japan. These results demonstrated that the PFGE patterns found among E. coli and enterococci strains from animals and livestock-farmers were heterogeneous and considerably diverse. The strains having both the identical PFGE pattern and the same drug-resistance pattern were defined as a single clone in this study. Seven types of E. coli and enterococci clones were shared among animals within the same farms and between the different farms housing the same animal species. The 25 strains (3.4%) of 739 E. coli and enterococci animal strains belonged to these seven types of clones. Only three types of E. coli clones were shared among animals between the different farms housing different animal species, but no identical E. faecalis or E. faecium clones were found between different animal species farms. The 15 strains (2.0%) of 739 E. coli and enterococci animal strains belonged to these three types of clones. Additionally, the 11 strains (1.5%) of 739 E. coli and enterococci strains isolated from animals were identical clones to strains isolated from livestock farmers of the same farm. These results suggest that the transmission of animal clones to livestock farmers or vice versa is less common.

Biochemical characterization of cellulose-binding proteins (CBPA and CBPB) from the rumen cellulolytic bacterium Eubacterium cellulosolvens 5.

The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-beta-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch.

Cloning, nucleotide sequence and module structure of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5.

The nucleotide sequence of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3,429 nucleotides. The deduced amino acid sequence of CBPB contained one module highly similar to a catalytic module of glycosyl hydrolase family 9 (GHF9), one module partially similar to a family 3 carbohydrate-binding module (CBM3), two linkers, one module similar to a CBM of cellulose-binding protein A (CBPA) from E. cellulosolvens 5, and one module almost identical to a cell wall-binding module (CWBM) of CBPA. The module similar to GHF9 showed CMCase activity, and the modules similar to CBM3 and CBM of CBPA bound to cellulose. Moreover, the module highly similar to CWBM of CBPA bound to the cell walls prepared from E. cellulosolvens 5. The amino acid sequence of CBPB had a significant homology (64.15% sequence identity) with that of CBPA. These results suggest that cbpA and cbpB genes descended from the same ancestral cellulase gene.

Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A.

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.

A possible role of cellulose-binding protein A (CBPA) in the adhesion of Eubacterium cellulosolvens 5 to cellulose.

The cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 is a modular enzyme comprised of a catalytic domain, a cellulose-binding domain and a cell wall-binding domain. Cellobiose-grown cells changed their adhesion ability to cellulose depending on the growth phase. On the other hand, carboxymethyl cellulose (CMC)-grown cells bound to cellulose regardless of their growth phase. The distribution of CBPA in the culture supernatant and cell fractions changed depending on the carbon source contained in the medium and growth phase. The cellobiose-grown cells harvested from the culture of the late stationary growth phase did not bind to cellulose, but their adhesion ability was recovered by treatment with recombinant CBPA. Moreover, cellobiose-grown cells harvested from the culture of an early exponential growth phase bound to cellulose, but their adhesion ability was inhibited by treatment with anti-CBPA antiserum. CBPA rapidly decreased the viscosity of CMC, indicating that CBPA was endoglucanase. The results obtained in this study indicate that CBPA plays an important role in the adhesion of E. cellulosolvens 5 cells to cellulose.

Identification of the cellulose-binding and the cell wall-binding domains of Eubacterium cellulosolvens 5 cellulose-binding protein A (CBPA).

The cellulose-binding domain (CBD) and the cell wall-binding domain (CWBD) of Eubacterium cellulosolvens 5 cellulose-binding protein A (CBPA) have been determined. The gene (cbpA) encoding CBPA and its derivatives were expressed in Escherichia coli. We were able to obtain the eight recombinant proteins and examine for their cellulose-binding ability, cell wall-binding ability and carboxymethyl cellulase (CMCase) activity. Since five recombinant proteins, which contain the unknown domain (UD-2) located between two linker-like regions of CBPA, bound to cellulose, this region has been identified as the CBD. The CBD did not show a significant sequence similarity with any other CBDs. Moreover, the N-terminal region of CBPA showed a significant sequence similarity with a catalytic domain of glycosyl hydrolase family 9, and the recombinant proteins containing the region showed CMCase activity. Since the UD-3, which is located in the C-terminal region of CBPA, bound to the cell walls of E. cellulosolvens 5, the region has been identified as the CWBD. However, the CWBD did not show a significant sequence similarity with any other proteins previously reported.

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5.

The nucleotide sequence of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3453 nucleotides and encodes a protein of 1151 amino acids with a molecular mass of 126408 Da. The deduced amino acid sequence of CBPA contained one domain highly similar to a catalytic domain of glycosyl hydrolases belonging to family 9, two linker-like domains and four domains of unknown function. Among the four domains of unknown function, the domains 1 and 2 region had significant homology in amino acid sequence with the cellulose-binding domains in the family 9 glycosyl hydrolases. The cloned gene was inserted into an expression vector, pBAD-TOPO, and expressed in Escherichia coli as a fused protein. The fused protein was detected by immunoblotting using antiserum against CBPA.

Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5.

Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.

A comparison of features and the microbial constitution of the fresh feces of pigs fed diets supplemented with or without dietary microbes.

The features and the constitution of the microbial population of fresh feces were compared between pigs fed a diet supplemented with dietary microbes and pigs given nonsupplemented diets. The former were reared on farm C and the latter on farms A and B. The concentrations of ammonia-N, indole, and skatole of fresh feces were not significantly different between pigs reared on farm C and those raised on farms A and B, but the concentrations of ammonia-N and the skatole of fresh feces were significantly different between pigs reared on farms A and B. The total VFA (volatile fatty acids) concentration of fresh feces in pigs on farm C was slightly lower than in those on farms A and B. Moreover, the molar proportion of the acetic acid in feces in pigs on farm C was lower; inversely, that of propionic and butyric acids was higher in comparison with those on farms A and B. No differences were evident in the total viable counts of feces among pigs reared on the three different farms. Clostridium perfringens was abundant in feces of pigs raised on farms A and B, but it was not detected in pigs reared on farm C. Megasphaerae, bifidobacteria, and clostridia except for C. perfringens were more abundant in the feces of pigs fed a diet supplemented with dietary microbes on farm C, compared with pigs given the nonsupplemented diets on farms A and B.