Investigation of morphological changes of HPS membrane caused by cecropin B through scanning electron microscopy and atomic force microscopy

Background@#Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. @*Objectives@#This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. @*Results@#Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 μg/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. @*Conclusions@#Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications.

Investigation of morphological changes of HPS membrane caused by cecropin B through scanning electron microscopy and atomic force microscopy

Background@#Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. @*Objectives@#This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. @*Results@#Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 μg/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. @*Conclusions@#Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications.

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (chi(2) test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.

The co-administrating of recombinant porcine IL-2 could enhance protective immune responses to PRV inactivated vaccine in pigs.

Three candidate cytokines: recombinant porcine interleukin-2 (rpIL-2), rpIL-6 and the fusion protein rpIL6-IL2 were used as adjuvants in this study to investigate the enhanced immune responses to PRV inactivated vaccine (IAV) in pigs. In this natural host trial, we demonstrated that rpIL-2 showed potential adjuvant effects on PRV IAV, which was characterized not only in antigen-specific immune responses, but also in protection against PRV infection. The use of rpIL-2 resulted in significantly higher virus neutralizing (VN) antibody levels and CTL activities on PRV IAV vaccination. The increased PRV-specific secretion of pIL-4 and pIFN-gamma from PBMC of pigs also demonstrated the adjuvant effects of rpIL-2. In addition, the co-administration of the rpIL-2 also produced an improved protection to the viral challenge, demonstrated by significant reduction of the ratios of fever and viral excretion in nasal swabs. However, there was no additional effect of adjuvant induced enhancement of immune responses and protection against challenge with the use of rpIL-6 and rpIL6-IL2 in this study.

The development and application of the latex agglutination test to detect serum antibodies against Japanese encephalitis virus.

The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20,000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1,613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis.