The aim of this study was to identify a new gene of Haemophilus parasuis that could be used to develop a polymerase chain reaction (PCR) test for this porcine pathogen. H. parasuis genomic DNA was cloned into a set of expression vectors, and transformants expressing His-tagged polypeptides were identified by colony blotting. An ABC transporter-like gene was isolated. The cloned DNA fragment is 1,105 base pair and shows 78% similarity at the nucleotide level with an ABC transporter gene of H. ducreyi. Based on this sequence, two PCR primers were designed to amplify the entire 1,105-bp fragment in the proposed diagnostic PCR test. PCR amplification was able to detect a minimum of 1 x 10(4) CFU/ml of H. parasuis organisms. Fifteen different H. parasuis serovars were positive using the PCR test. No amplification was observed when the test was done using DNA from 16 other bacterial species commonly isolated from swine.
Base Pairing ; Clone Cells ; Cloning, Organism ; DNA ; Haemophilus ; Haemophilus parasuis ; Peptides ; Polymerase Chain Reaction ; Swine

A comparative in vivo pharmacokinetic (PK) study of tilmicosin (TIL) was conducted in 6 crossbred healthy pigs and 6 crossbred pigs infected with Haemophilus (H.) parasuis following oral administration of a single 40 mg/kg dose. The infected model was established by intranasal inoculation and confirmed by clinical signs, blood biochemistry, and microscopic examinations. Plasma TIL concentrations were determined by a validated high-performance liquid chromatography method with ultraviolet detection at 285 nm. PK parameters were calculated by using WinNonlin software. After TIL administration, the main PK parameters of TIL in healthy and H. parasuis-infected pigs were as follows: Area under the concentration-time curve, maximal drug concentration, half-life of the absorption phase, half-life of the distribution phase, and half-life of the elimination phase were 34.86 ± 9.69 vs. 28.73 ± 6.18 µg · h/mL, 1.77 ± 0.33 vs. 1.67 ± 0.28 µg/mL, 2.27 ± 0.45 vs. 2.24 ± 0.44 h, 5.35 ± 1.40 vs. 4.61 ± 0.35 h, and 43.53 ± 8.17 vs. 42.05 ± 9.36 h, respectively. These results of this exploratory study suggest that there were no significant differences between the PK profiles of TIL in the healthy and H. parasuis-infected pigs.
Absorption ; Administration, Oral ; Biochemistry ; Chromatography, Liquid ; Haemophilus parasuis* ; Haemophilus* ; Half-Life ; Methods ; Pharmacokinetics* ; Plasma ; Swine*

Due to the serovar diversity in Haemophilus (H.) parasuis, it is difficult to develop a universal serological method for detection of this pathogen. Here, we report a universal plate-agglutination test for detecting H. parasuis. Diagnostic antisera were prepared by mixing antisera of serovars 4, 5, 12, 13 and 14 in the optimized ratio. The results of the plate-agglutination test showed that the diagnostic antisera could agglutinate with all 15 reference strains of H. parasuis and 74/75 clinical isolates. Further, the specificity of the method was validated with 22 bacterial strains from 12 related species.
Agglutination Tests/*methods ; Animals ; Cross Reactions ; Haemophilus parasuis/isolation & purification/*physiology ; Immune Sera/*metabolism ; Reproducibility of Results ; Sensitivity and Specificity

Oral fluid analysis for herd monitoring is of interest to the commercial pig production in Korea. The aim of this study was to investigate pathogen-positive rates and correlations among eight pathogens associated with porcine respiratory disease complex by analyzing oral fluid samples from 214 pig groups from 56 commercial farms. Samples collected by a rope-chewing method underwent reverse-transcriptase polymerase chain reaction (RT-PCR) or standard polymerase chain reaction (PCR) analysis, depending on the microorganism. Pathogens were divided into virus and bacteria groups. The former consisted of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 (PCV2), and the latter Pasteurella multocida, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae (MHP), Mycoplasma hyorhinis, and Streptococcus suis (SS). All pathogens were detected more than once by PCR. Age-based analysis showed the PCR-positive rate increased with increasing age for PCV2 and MHP, whereas SS showed the opposite. Correlations between pathogens were assessed among 36 different pair combinations; only seven pairs showed statistically significant correlations. In conclusion, the oral fluid method could be a feasible way to detect various swine respiratory disease pathogens and, therefore, could complement current monitoring systems for respiratory diseases in the swine industry.
Actinobacillus pleuropneumoniae ; Agriculture* ; Bacteria ; Circovirus ; Complement System Proteins ; Haemophilus parasuis ; Korea* ; Methods* ; Mycoplasma hyopneumoniae ; Mycoplasma hyorhinis ; Pasteurella multocida ; Polymerase Chain Reaction ; Porcine respiratory and reproductive syndrome virus ; Streptococcus suis ; Swine

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics