Single-nucleotide polymorphism-based epidemiological analysis of Korean Mycobacterium bovis isolates

BackgroundBovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea.ObjectivesTo survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing.MethodsA total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs.ResultsWe identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes.ConclusionsThrough SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.     

Full-length ORF2 sequence-based genetic and phylogenetic characterization of Korean feline caliciviruses

Feline calicivirus (FCV) is a highly infectious pathogen in cats and widely distributed worldwide with high genetic variation. Full-length open reading frame 2 of 5 from recently isolated Korean FCV isolates were sequenced and compared with those of global isolates. The results of phylogenetic analysis supported dividing global FCV isolates into two genogroups (type I and II) and demonstrated the presence of genogroup II in Korea, indicating their geographic spread in East Asia. High sequence variations in region E of the FCV isolates emphasizes that a novel vaccine needs to be developed to induce protective immunity against various FCV strains.     

Canter lead change kinematics of superior olympic dressage horses

Limb contact variables of the gaits of superior dressage horses were determined for competitors at the 1988 Seoul Summer Olympic Games in the finals of the Individual Dressage competition. Two 16-mm motion picture cameras were aimed perpendicular to the plane of motion along the HXF and KXM diagonals of the standard dressage arena, and filmed at 100 fps. Nineteen of the horses selected as finalists for individual dressage medals were filmed during the Grand Prix Special test executing one-stride canter lead changes, two-stride canter lead changes and the left lead extended canter. Velocities, stride lengths and stride durations were 7.03±.07 m/s, 4.15±.05 m and .592±.004 s for the extended canter. Across all stride frequencies, score was optimized with extended canter strides of the greatest length, with no upper limit detected to optimal stride length. Canter strides with lead changes had lower velocities, shorter strides and longer durations than strides without a lead change, with velocities, stride lengths and stride durations, respectively, of 3.36±.05 m/s, 2.08±.04 m and .617±.003 s for one-stride canter lead change strides; 3.65±.07 m/s, 2.21±.04 m and .605±.003 s for pre-change canter two-stride lead change strides; and 3.95±.07 m/s, 2.44±.04 m and .621±.004 s for post-change canter two-stride lead change strides. This study provided the first objective documentation of the canter limb contact patterns of world-class dressage horses.     

Identification of the DDAH2 Protein in Pig Reproductive Tract Mucus: A Putative Oestrus Detection Marker

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus‐specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous‐ and oestrous‐stage mucus proteins were purified and analysed with proteomic tools such as two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization–time‐of‐flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6‐fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti‐DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.     

Molecular epidemiology of bovine toroviruses circulating in South Korea

The prevalence of the bovine torovirus (BToV) and its genetic characterization have been reported in North America, Europe and Japan. Therefore, this study examined the prevalence and genetic diversity of the BToV in a total of 645 diarrheic fecal samples from 629 Korean native beef calf herds using RT-PCR and nested PCR with the primer pairs specific to a part of the BToV membrane (M) gene. Overall, 19 (2.9%) out of 645 diarrheic samples from 19 herds (6.9%) tested positive for BToVs by either RT-PCR or nested PCR. A comparison of the nucleotide (nt) and amino acid (aa) sequences of a part of the BToV M gene (409bp) among the BToVs showed the Korean BToVs to have comparatively higher sequence homology to the Japanese and Dutch BToVs than to the American and Italian BToVs. Generally, the Korean BToV strains clustered with the Japanese and Dutch BToV strains. However, the American and Italian BToV strains clustered on a separate major branch, suggesting that these are more distantly related to other known BToV strains. These results suggest that the BToV infections are sporadic in diarrheic calves in South Korea, and the Korean BToV strains are more closely related to the Japanese and Dutch BToVs than to the American and Italian BToVs.     

Sperm penetration assay as an indicator of bull fertility

To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.     

Molecular epidemiology of bovine noroviruses in South Korea

Since the prevalence of bovine norovirus (BNoV) and their genetic diversity have only been reported in the USA, England, Germany and The Netherlands, this study examined the prevalence and genetic diversity of BNoVs in diarrheic calves in South Korea using 645 diarrheic fecal specimens from calves by RT-PCR and nested PCR assays. Overall, 9.3% of the diarrheic fecal samples tested positive for BNoVs by either RT-PCR or nested PCR, of which 5.9% samples also tested positive for other enteric pathogens including the bovine coronavirus, bovine viral diarrhea virus, bovine torovirus, bovine groups A, B and C rotaviruses, bovine enteric Nebraska-like calicivirus and Escherichia coli. The genetic diversity was determined by direct sequencing of the partial RdRp region of 12 BNoVs detected from the fecal samples by nested PCR. Among the BNoVs examined, one Korean BNoV strain had the highest nucleotide (86.8%) and amino acid (99.1%) identity with the genotype 1 BNoV (GIII-1) strain, while the remaining 11 Korean BNoVs shared a higher nucleotide (88.0-90.5%) and amino acid (93.5-99.1%) identity with the genotype 2 BNoV (GIII-2) strains. The phylogenetic data for the nucleotide and amino acid sequences also demonstrated that one Korean BNoV strain clustered with GIII-1 but the remaining eleven strains clustered with GIII-2. In conclusion, BNoV infections are endemic and there are two distinct genotypes with GIII-2 being the main genotype circulating in the calf population in South Korea.     

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.     

Extracellular matrix of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> may have a role in host adhesion during fungal penetration and is digested by matrix metalloproteinases

Spores and infection structures such as germ tubes and appressoria of Magnaporthe oryzae, the fungus causing blast disease of wheat, produced an extracellular matrix (ECM) on the surfaces of host leaves during fungal differentiation. The chemical components and function of the ECM were studied to understand the pathological roles using two immunological techniques and ECM-digesting enzymes. The ECM was characterized by fibrous and amorphous materials, located in the spaces between fungal cell walls and plant cuticles. Immunohistochemical and immunoelectron microscopy suggested that ECM includes components positively reacted with antibodies of four animal cell adhesion factors (collagen VI, vitronectin, fibronectin and laminin) and an animal integrin α3. ECM, incubated on a cellulose membrane, was rapidly digested by matrix metalloproteinases (collagenase and gelatinase B), resulting in the detachment of most infection structures from membrane surfaces. Both ultrastructural observation and immunological responses showed that more ECM was located at the appressoria than at the spores and germ tubes. This result suggested that appressoria needed a powerful adhesion force for aggressive action of penetration pegs into plant cuticles. An erratum to this article can be found at     

Synthesis, structure analyses, and characterization of novel epigallocatechin gallate (EGCG) glycosides using the glucansucrase from Leuconostoc mesenteroides B-1299CB

In this study, three epigallocatechin gallate glycosides were synthesized by the acceptor reaction of a glucansucrase produced by Leuconostoc mesenteroides B-1299CB with epigallocatechin gallate (EGCG) and sucrose. Each of these glycosides was then purified, and the structures were assigned as follows: epigallocatechin gallate 7-O-alpha-D-glucopyranoside (EGCG-G1); epigallocatechin gallate 4'-O-alpha-D-glucopyranoside (EGCG-G1'); and epigallocatechin gallate 7,4'-O-alpha-D-glucopyranoside (EGCG-G2). One of these compounds (EGCG-G1) was a novel compound. The EGCG glycosides exhibited similar or slower antioxidant effects, depending on their structures (EGCG > or = EGCG-G1 > EGCG-G1' > EGCG-G2), and also manifested a higher degree of browning resistance than was previously noted in EGCG. Also, EGCG-G1, EGCG-G1', and EGCG-G2 were 49, 55, and 114 times as water soluble, respectively, as EGCG.