One-pot synthesis of cycloamyloses from sucrose by dual enzyme treatment: combined reaction of amylosucrase and 4-α-glucanotransferase

Amylose-like α-(1,4)-glucan known as the most favorable substrate for the enzymatic production of cycloamyloses (CAs) using 4-α-glucanotransferase has a solubility issue, which requires an additional solubilization process. In our study, two glucosyltransferases, Synechocystis 4-α-glucanotransferase and Neisseria amylosucrase, were adopted to develop an efficient biocatalytic production process of CAs directly from sucrose. From one-pot synthesis, the maximum CA yield (9.6%, w/w) with 0.3 M sucrose was achieved with 10 units/mL of amylosucrase and 0.1 unit/mL of 4-α-glucanotransferase at 40 °C for a 3 h reaction in a simultaneous dual enzyme reaction mode. The size of linear α-(1,4)-glucan was positively related to the CA productivity by 4-α-glucanotransferase in a hyperbolic manner. Using our innovative bioprocess, there was no practical limitation on the initial sucrose concentration and no use of insoluble linear α-(1,4)-glucan substrate. Consequently, the concomitant dual enzyme reaction converted sucrose directly to CAs via in situ transient linear α-(1,4)-glucan as an soluble intermediate.     

Structural and biological characterization of sulfated-derivatized oat beta-glucan

The structural, physicochemical, and biological properties of sulfated oat beta-glucan were characterized. The degree of substitution of the sulfated oat-beta glucan was obtained by elemental analysis, which was 0.68. Compared to native oat beta-glucan, the FT-IR spectra of the derivative showed two new absorption bands at 1250 and 810 cm(-)(1), which would be attributed to (S=O) and (C-O-S) groups, respectively. The molecular weight of the sulfated beta-glucan was determined to be 68 kDa and its viscosity decreased by almost 2 orders of magnitude while its solubility increased by more than 100% compared to that of the native beta-glucan. In addition, the sulfation caused the reduction of in vitro bile acid binding capacity of oat beta-glucan due to the new anionic character and decreased molecular weight. The sulfated derivative exhibited, however, anticoagulant activity which showed a concentration-dependent increase.     

Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors

Full-length infectious cDNA clones have recently become available for both European and North American genotypes of porcine reproductive and respiratory syndrome virus (PRRSV), and it is now possible to alter the PRRSV genome and create genetically defined mutant viruses. Among many possible applications of the PRRSV infectious cDNA clones, development of genetically modified vaccines is of particular interest. Using infectious clones, the PRRSV genome has been manipulated by changing individual amino acids, deleting coding regions, inserting foreign sequences, and generating arterivirus chimeras. The limited available data suggest that all structural proteins of PRRSV are essential for replication of the virus, and that PRRSV infectivity is relatively intolerant of subtle changes within the structural proteins. The major tasks in PRRSV research are to identify virulence factors and pathogenic mechanisms, and to understand the structure-function relationships of individual viral proteins. Utilizing these infectious clones as tools, a new generation of safe and efficacious PRRS vaccines may be constructed.     

Induction of protective immune responses against challenge of Actinobacillus pleuropneumoniae by oral administration with Saccharomyces cerevisiae expressing Apx toxins in pigs

Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, a highly contagious endemic disease of pigs worldwide, inducing significant economic losses worldwide. Apx toxins, which are correlated with the virulence of A. pleuropneumoniae, were expressed in Saccharomyces cerevisiae and its possible use as an oral vaccine has been confirmed in our previous studies using a murine model. The present study was undertaken to test the hypothesis that oral immunization using S. cerevisiae expressing either ApxI or ApxII could protect pigs against A. pleuropneumoniae as an effective way of inducing both mucosal and systemic immune responses. The surface-displayed ApxIIA#5 expressing S. cerevisiae was selected as an oral vaccine candidate by finding on induction of higher immune responses in mice after oral vaccination. The surface-displayed ApxIIA#5 expressing S. cerevisiae and the ApxIA expressing S. cerevisiae were developed to serve as an oral vaccine in pigs. The vaccinated pigs showed higher specific IgG- and IgA-related antibody activities than the non-treated control and vector control pigs. Additionally, the induced immune responses were found to protect pigs infected with A. pleuropneumoniae according to the analysis of clinical signs and the gross and microscopic pulmonary lesions. These results suggested that the surface-displayed ApxIIA#5 and ApxIA in S. cerevisiae might be a potential oral vaccine to protect pigs against porcine pleuropneumonia. Thus the present study is expected to contribute to the development of a live oral vaccine against porcine pleuropneumonia as an alternative to current conventional vaccines.     

Profiling of antimicrobial resistance and plasmid replicon types in β-lactamase producing Escherichia coli isolated from Korean beef cattle

In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum β-lactamase (ESBL) and/or AmpC β-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC β-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type β-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type β-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on β-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of β-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established.     

Clinical haematology and biochemistry profiles of cattle naturally infected with Theileria orientalis Ikeda type in New Zealand

AIMS: To present the haematology and biochemistry profiles for cattle in New Zealand naturally infected with Theileria orientalis Ikeda type and investigate if the results differed between adult dairy cattle and calves aged <6 months.

METHODS: Haematology and biochemistry results were obtained from blood samples from cattle which tested positive for T. orientalis Ikeda type by PCR, that were submitted to veterinary laboratories in New Zealand between October 2012 and November 2014. Data sets for haematology and biochemistry results were prepared for adult dairy cattle (n=62 and 28, respectively) and calves aged <6 months (n=62 and 28, respectively), which were matched on the basis of individual haematocrit (HCT). Results were compared between age groups when categorised by HCT. Selected variables were plotted against individual HCT, and locally weighted scatterplot smoothing (Loess) curves were fitted to the data for adult dairy cattle and calves <6 months old.

RESULTS: When categorised by HCT, the proportion of samples with HCT <0.15 L/L (severe anaemia) was greater for adult dairy cattle than for beef or dairy calves, for both haematology (p<0.002) and biochemistry (p<0.001) submissions. There were differences (p<0.05) between adult dairy cattle and calves aged <6 months in the relationships between HCT and red blood cell counts, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentrations, lymphocyte and eosinophil counts, and activities of glutamate dehydrogenase and aspartate aminotransferase. In both age groups anisocytosis was frequently recorded. The proportion of blood smears showing mild and moderate macrocytosis was greater in adults than calves (p=0.01), and mild and moderate poikilocytosis was greater in calves than adults (p=0.005).

CONCLUSIONS AND CLINICAL RELEVANCE: The haematology and biochemistry changes observed in cattle infected with T. orientalis Ikeda type were consistent with extravascular haemolytic anaemia. Adult dairy cattle were more likely to be severely anaemic than calves. There were differences in haematology and biochemistry profiles between adult dairy cattle and calves, but most of these differences likely had a physiological rather than pathological basis. Overall, the haematological changes in calves aged <6 months appeared less severe than in adult dairy cattle.     

Subcutaneous granuloma caused by Mycobacterium avium complex infection in a cat

A localised subcutaneous swelling developed on the nasal bridge of a cat receiving chemotherapy for alimentary tract lymphosarcoma. Cytological and histological examination of representative samples of the lesion demonstrated pyogranulomatous inflammation and abundant acid-fast bacilli. A Mycobacterium sp was cultured from tissue excised from the lesion. Extensive testing at three reference laboratories indicated the strain was a member of the Mycobacterium avium complex. The infection was treated successfully by cytoreductive surgery and a 6 weeks course of orally administered clofazimine.     

Effects of chitooligosaccharide supplementation on growth performance, nutrient digestibility, blood characteristics and immune responses after lipopolysaccharide challenge in weanling pigs

The objective of this study was to determine the effects of dietary supplementation with chitooligosaccharide (COS) on growth performance, nutrient digestibility, blood characteristics and immune response in lipopolysaccharide-challenged weanling pigs. A total of 90 crossbred weanling pigs (5.44 ± 0.50 kg BW) were employed in Exp. 1. The three dietary treatments were basal diets supplemented with 0, 2.5, and 5 g COS/kg, and fed for 28 d. Each treatment had 6 replications with 5 pigs per pen. Increasing the level of supplemental COS tended to linearly (P < 0.10) improve ADG and ADFI during phase 2 and overall period, while there were no differences in G:F. The linear improvement in the apparent DM (P < 0.05) and N (P < 0.10) digestibility in pigs fed COS supplemented diets was noticed. The tested blood characteristics were not influenced under non-challenge conditions. In Exp. 2, a total of 20 pigs (5.22 ± 0.31 kg BW) were initially assigned to two dietary treatments and fed basal diets supplemented with 0 or 0.5 g COS/kg for 28 d. At the end of d 28, half of the pigs in each treatment (n = 5) were injected i.p. with Escherichia coli lipopolysaccharide at a concentration of 100 μg/kg of BW. The other half of the pigs in each treatment were injected with sterile saline solution at a concentration of 100 μg/kg of BW. This arrangement resulted in a 2 × 2 factorial design with diet and LPS challenge as the main effects. Blood sample and rectal temperature data were collected at 0, 2, 4 and 12 h post-challenge. Rectal temperatures increased as the result of LPS injection at 4 and 12 h post-challenge (P < 0.05). Serum cortisol, IGF-1, and TNF-α concentration were also increased as the result of LPS challenge (P < 0.05). The COS treatments resulted in lower cortisol concentrations at 2 h and higher IGF-1 concentrations at 4 h post-challenge (P < 0.05). COS and LPS interactions were also observed on cortisol and IGF-1 when the COS effects were presented (P < 0.05). Haptoglobin concentrations remained unaffected throughout the challenge period. White blood cell counts were increased in the LPS-treated pigs at 2 and 4 h post-challenge (P < 0.01). Lymphocyte count was elevated at 2 h and reduced at 12 h post-challenge as the result of LPS challenge (P < 0.05). However, there were no COS main effects observed on lymphocyte count throughout the challenge period. The comparison between two LPS challenged treatments also indicated that COS treatment has beneficial effects on rectal temperature, cortisol and IGF-1 concentrations. In conclusion, dietary supplementation with COS had little effect on nutrient digestibility and inflammatory stress markers in weanling pigs.