Prevalence and antimicrobial susceptibility of Brachyspira species in pigs in Korea / 대한수의학회지

The purpose of this study was to investigate the prevalence of Brachyspira species and antimicrobial susceptibility of Brachyspira (B.) hyodysenteriae isolates in Korea. A total of fifty-five Brachyspira species were isolated; five (1.0%) beta-hemolytic Brachyspira species and 50 (10.4%) weak hemolytic Brachyspira species from 116 different diarrheic pig samples and 367 apparently normal pig samples. In farm level, beta hemolytic and weak hemolytic Brachyspira species were detected in 7.4% (5/68) and 19.1% (13/68) of tested pig farms, respectively. By phenotypic and genotypic characterization, all beta hemolytic Brachyspira isolates was classified as group I (B. hyodysenteriae), whereas weak hemolytic Brachyspira species isolates were group III (B. innocens or B. murdochii). B. hyodysenteriae isolates showed high level of minimum inhibition concentrations to macrolide antimicrobials. This study shows that the prevalence of pathogenic B. hyodysenteriae in pigs is low but antimicrobial resistance of the pathogens is high in Korea. This is the first report of the prevalence of Brachyspira group III and antimicrobial susceptibility of B. hyodysenteriae in pigs in Korea. Our results could provide basic data for the management and treatment guidelines of Brachyspira infection.

Adenovirus Expressing Human Interferon Inhibits Replication of Foot and Mouth Disease Virus and Reduces Fatal Rate in Mice

Interferon is an important cytokine that plays a critical role in the initial host defense against viral infection. Recombinant human adenoviruses expressing human interferon-alpha (Ad-HIFNalpha) or pig interferon-beta fused with interleukin-18 (Ad-PIFNbeta-IL18) were constructed and used to induce an early protective response against foot and mouth disease (FMD). To analyze the antiviral effect, bovine thyroid and porcine kidney IBRS-2 cells and ICR mice were treated with Ad-HIFNalpha, Ad-PIFNbeta-IL18, and cocktail of Ad-HIFNalpha and Ad-PIFNbeta-IL18. The survival rate of suckling mice was monitored after foot and mouth disease virus (FMDV) challenge following intra-peritoneal (IP) administration of appropriate adenovirus. Indirect antigen ELISA was performed to evaluate inhibition of FMDV replication following challenge with the FMDV O, A, or Asia 1 serotypes in vitro. These recombinant adenoviruses reduced the replication of FMDV in susceptible cells, thereby decreasing the fatality in mice, suggesting that they can be a useful control method for the early protection against FMD infection in livestock after field trial.

Synthetic and Adenovirus Delivered Small Interference RNA Pools Targeting Conserved Regions of Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is an economically significant animal disease because of the speed of its transmission. Routine vaccination may not be effective for early protection in an outbreak situation. Small interfering RNA (siRNA) can be used as a rapid, effective, and an alternative antiviral approach. In this study, we screened 15 synthetic siRNAs to inhibit FMD virus replication in IBRS-2 cells and selected 10 siRNA sequences. Furthermore, we produced 7 adenoviruses expressing shRNA targeting conserved regions of FMDV, such as a leader sequence and nonstructural protein regions, and showed their antiviral effects. We compared the antiviral effects among them and compared between synthetic siRNAs and adenovirus-delivered siRNAs. In particular, the most efficient siRNA, 3C2, was the conserved sequence in the O, A, Asia 1, and C serotypes of FMDV and was located in the predicted loop structure. The pool of sequences including 3C2 and recombinant adenoviruses could be applied for multiple siRNAs and protection in a broad range of cells and animals.

An outbreak of fatal hemorrhagic pneumonia caused by Streptococcus equi subsp. zooepidemicus in shelter dogs

An outbreak of fatal hemorrhagic pneumonia with 70~90% morbidity and 50% mortality occurred in an animal shelter in Yangju, Gyeonggi Province, Korea. Clinically, the affected dogs showed severe respiratory distress within 48 h after arriving in the shelter. The dead were found mainly with nasal bleeding and hematemesis. At necropsy, hemothorax and hemorrhagic pneumonia along with severe pulmonary consolidation was observed, though histopathological analysis showed mainly hemorrhagic bronchopneumonia. Lymphoid depletion was inconsistently seen in the spleen, tonsil and bronchial lymph node. Gram-positive colonies were shown in blood vessels or parenchyma of cerebrum, lung, liver, spleen, and kidney. Also, Streptococcus (S.) equi subsp. zooepidemicus was isolated from the various organs in which the bacterium was microscopically and histologically detected. In addition, approximately 0.9 Kb specific amplicon, antiphagocytic factor H binding protein, was amplified in the bacterial isolates. In this study, we reported an outbreak of canine hemorrhagic bronchopneumonia caused by S. equi subsp. zooepidemicus in an animal shelter in Yangju, Korea.

Enhanced immune response with foot and mouth disease virus VP1 and interleukin-1 fusion genes

The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.

Identification and antigenic site analysis of foot-and-mouth disease virus from pigs and cattle in Korea

From May to June 2002, a total of 16 foot-and mouth disease (FMD) outbreaks due to the serotype O virus, Pan Asia strain, were recorded in Korea. The viruses were identified by antigen ELISA, RT-PCR and sequence analysis. The overall nucleotide sequence divergence of the VP1 region among the 4 isolates in 2002 was 0 to 1.4%, but between O/SKR/2002 and O/SKR/2000 isolates was 1.9-4.9%. Phylogenetic analysis with the some known strains from East Asian countries showed that the 4 Korean isolates in 2002 formed one distinct cluster, which different from clusters of Korean isolates in 2000, with in the same lineage of the ME-SA topotype strains. Deduced amino acid sequences around neutralizable antigenic site on VP1 site of O/SKR/2002 isolates were aligned and compared with other strains. At the antigenic site 1, the replacements of the critical amino acid residues at position 144 from V to L and at position 152 from A to T were observed in O/SKR/2002 viruses. For antigenic site 2 and 4, there were not significant variations in general. At the antigenic site 3, the substitutions of amino acid residues were present at positions 54 and 56 in O/SKR/2002 isolates and an alternative residue I at position 54 are observed only at the sequence of O/SKR/AS/2002 (cow) virus. And the substitution (L-->P) of significant residue at position 144 was detected at the amino acid sequence of the O/SKR/2002 (cow) virus.

Development of a Lightcycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus

One step TaqMan real-time reverse transcription polymerase chain reaction (R/T RT-PCR)using a set of primers/probes was developed for the detection of foot-and-mouth disease (FMD)virus. The gene-specific probes labeled fluorogen for the internal ribosomal entry site, Leader sequence and 2B regions were used to detect FMD virus (FMDV). This assay specifically detected FMDV both in cell culture preparations and clinical samples, and was capable of distinguishing FMD from other viral diseases similar to clinical signs (swine vesicular disease, vesicular stomatitis and bovine viral diarrhea). This assay was shown to be 1000-fold more sensitive than the conventional RT-PCR method. The detection limits of this assay was 1 TCID 50 /ml of the FMDV RNA concentration. Quantification was obtained by a standard curves plotting threshold cycle values versus known infectivity titer. The assay was sensitive, specific and rapid enough to detect FMDV RNA genome in probang samples. As such, the described method is reliable and provides faster disease diagnostics than the conventional RT-PCR procedure to detect FMDV.

Flow Cytometric Analysis of Lymphocyte Subpopulations of Cattle Infected with Bovine Leukemia Virus

We examined lymphocyte subpopulations of peripheral blood from BLV infected and noninfected Holstein-Friesian dairy cattle reared in Korea by flow cytometry using monoclonal antibodies specifically reactive with bovine leukocyte differentiation marker. Lymphocyte subpopulations expressing BoCD11b, B-B2, CD5, B, MHC II-DP, MHC II-DQ, and MHC II-DR antigens were significantly abundant in the BLV(+) group than the BLV(-) group (p<0.01). On double staining, subpopulation of B-1a(BoCD5+ BoCD11b+) lymphocytes was significantly increased in leukemic group. However, T-lymphocyte lineage expressing BoCD2, BoCD4, BoCD8, and WC1 antigens was significantly lower than in the BLV(+) group (p<0.01). However the absolute number of T-lymphocytes expressing BoCD2, BoCD4, BoCD8, and WC1 antigens in BLV(+) group remained with in the normal range. Furthermore mean ratio of BoCD4/BoCD8 in the BLV(+) groups was higher than that in the BLV(-) group. Taken together, cellular immune responses did not seem to significantly be decreased in the leukemic cattle.

Characterization of lymphocyte subpopulations and major histocompatibility complex haplotypes of mastitis-resistant and susceptible cows

Bovine mastitis is an infectious disease with a major economic influence on the dairy industry worldwide. Many factors such as environment, pathogen, and host affect susceptibility or resistance of an individual cow to bovine mastitis. Recently, there has been considerable interest in defining genetic and immunological markers that could be used to select for improved disease resistance. In this study we have analyzed the lymphocyte subpopulations of mastitis-resistant and susceptible cows using monoclonal antibodies specific for bovine leukocyte differentiation antigens and flow cytometry. We have also used a microarray typing technique to define the bovine leukocyte antigen (BoLA) class I and class II haplotypes associated with resistance or susceptibility to bovine mastitis. A striking finding of the present study is that susceptibility to mastitis was associated with major histocompatibility complex (MHC) haplotypes that have only a single set of DQ genes. The study also revealed that susceptible cows had CD4:CD8 ratios of less than one in both their mammary gland secretions and peripheral blood. These results raise the possibility that the number of DQ genes that a cow has and/or a cow's CD4:CD8 ratio could be used as indicators of susceptibility to bovine mastitis.

Characterization of Antigenic Sites on the Rinderpest Virus N Protein Uusing Monoclonal Antibodies

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.

Localization of Antigenic Sites at the Amino-terminus of Rinderpest Virus N Protein Using Deleted N Mutants and Monoclonal Antibody

The nucleocapsid (N) protein of rinderpest virus (RPV) is highly conserved, immunogenic, and abundantly expressed during infection. Six antigenic sites (sites A, B, C, D, E and F), defined previously by a competitive binding assay using corresponding monoclonal antibodies (Mabs), have been further localized by immunoassays using deleted N mutants. Five different forms of RPV N protein, containing residues aa 1-79, aa 1-149, aa 1-421, aa 414-525 and aa 1-525, were expressed as glutathione S transferase (GST) fusion proteins (designated as GST-N1-79, GST-N1-149, GST-N1-421, GST-N414-525, and GST-N1-525, respectively) in E.coli BL21 cells. In ELISA using deleted N mutants, Mabs recognizing sites A, B, C, D and E reacted with 3 GST fusion proteins (GST-N1-149, GST-N1-421 and GST-N1-525), indicating that they are located at aa 80-149. Mab recognizing site F reacted with 4 GST fusion proteins (GST-N1-79, GST-N1-149, GST-N1-421 and GST-N1-525), indicating that site F is located at aa 1-79. Identification of the amino-terminal antigenic sites of the N protein would provide antigen basis for developing sensitive and specific diagnostic reagents for RPV, although it remains to be further investigated antigenic sites at the carboxyl-terminus.