Dong Jin Yoo

Capillary and microchip gel electrophoresis for simultaneous detection of Salmonella pullorum and Salmonella gallinarum by rfbS allele-specific PCR.

We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54+/-1 degrees C and an MgCl(2) concentration of 2.8-5.6mM. Under an electric field of 333.3V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M(r) 600000), the amplified PCR products were analyzed within 6min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency.

Oral administration of Salmonella enterica serovar Typhimurium expressing swine interleukin-18 induces Th1-biased protective immunity against inactivated vaccine of pseudorabies virus.

Enhancing and/or modulating innate and adaptive immunity by cytokines appears to be greatly useful to provide effective protective immunity against infectious diseases. However, an effective delivery system for mass administration in livestock industry is needed because of limitations such as cost, labor, time, and protein stability. Here the immunomodulatory functions of swine interleukine-18 (swIL-18), known as IFN-γ-inducing factor (IGIF), were evaluated in a vaccination model of pseudorabies virus (PrV) using attenuated Salmonella enterica serovar Typhimurium as the oral delivery system. The oral administration of S. enterica serovar Typhimurium expressing swIL-18 prior to vaccination with inactivated PrV vaccine induced enhanced levels of serum PrV-specific IgG and its IgG2 isotype, compared to administration of S. enterica serovar Typhimurium harboring the empty vector. Furthermore, S. enterica serovar Typhimurium expressing swIL-18 mounted Th1-biased cellular immune responses against PrV antigen, as evaluated by the production of IFN-γ and IL-4 from peripheral blood mononuclear cells of piglets. Subsequently, Th1-biased immunity induced by S. enterica serovar Typhimurium expressing swIL-18 showed rapid response and rendered piglets displayed more alleviated clinical signs following the virulent PrV challenge. Also, this alleviation of clinical signs was further confirmed by the reduction of nasal excretion of PrV after challenge. The present study demonstrates the extended use of immunomodulatory functions of swIL-18 orally delivered by attenuated S. enterica serovar Typhimurium.

Portable capillary electrophoresis system for identification of cattle breeds based on DNA mobility

A portable CE system was developed for the identification of cattle breeds. The system had a width of 44u2009cm, depth of 27u2009cm, height of 13u2009cm, and a weight of only ∼8u2009kg and included an LIF detector, with everything integrated into a small box. The specific sizes of genes were quickly separated and detected with a high sensitivity based on the difference in the DNA mobility using a diode‐pumped solid‐state LIF detector. Using this system, the 100‐bp DNA ladder was analyzed under a 1.0% PVP (Mr=300u2009000) sieving gel matrix in a fused silica capillary with LODs of 4.4–13.0u2009pg/μL (S/N=3) for 100–3000u2009bp DNAs, which indicates ten times improved value than other commercialized portable CE system. The migration times and the peak areas showed good reproducibilities with relative standard deviations that were less than 0.49 and 1.3% (n=5), respectively. Based on the difference in the DNA mobility of the microsatellite and SNP markers, Korean cattle and Holstein were exactly identified as the model cattle breeds within 32 and 3.5u2009min, respectively.

Genetic co-transfer of CCR7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co‐transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV‐specific immunoglobulin (Ig) G by 2‐ to 2.5‐fold. In addition, the level of PrV‐specific IgG2a isotype was significantly enhanced by co‐injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1‐type pattern. The co‐injection of CCR7 ligand DNA consistently enhanced the level of Th1‐type cytokines (IL‐2 and IFN‐γ) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co‐transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co‐administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV‐immune CD4+ T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.

Oral administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α alleviates clinical signs caused by respiratory infection with avian influenza virus H9N2.

Low pathogenic avian influenza (LPAI) H9N2 has attracted considerable attention due to severe commercial losses in the poultry industry. Furthermore, avian influenza virus (AIV) H9N2-infected chickens can be a reservoir for viral transmission to mammals including pigs and humans, complicating control of viral mutants. Chicken interferon-alpha (chIFN-α) may be useful as an exogenous antiviral agent to control AIV H9N2 infection. However, a superior vehicle for administration of chIFN-α is needed because of challenges of protein stability, production cost, and labor associated with mass administration. Presently, oral administration of single dose of attenuated Salmonella enterica serovar Typhimurium expressing chIFN-α alleviated clinical signs and histopathological changes caused by respiratory infection with AIV H9N2 and reduced the excretion of virus in cloacal swab samples. Similarly, chickens administered S. enterica serovar Typhimurium expressing chIFN-α showed inhibited replication of AIV H9N2 in several different tissues including trachea, lung, cecal tonsil, and brain. Furthermore, immune responses specific for challenged AIV H9N2 were enhanced in chickens administered S. enterica serovar Typhimurium expressing chIFN-α, as determined by hemagglutination inhibition assay of sera, proliferation and IFN-γ and interleukin-4 expression by AIV H9N2 antigen-stimulated peripheral blood mononuclear cells and splenocytes. Therefore, oral administration of S. enterica serovar Typhimurium expressing chIFN-α can successfully control clinical signs caused by respiratory infection with AIV H9N2, which provides valuable insight into the use of attenuated Salmonella vaccine as an oral delivery system of chIFN-α to prevent the replication of AIV H9N2 in respiratory tract.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with β2-adrenergic agonist

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that β2-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since β2-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

Polarization of protective immunity induced by replication-incompetent adenovirus expressing glycoproteins of pseudorabies virus

Replication-incompetent adenoviruses expressing three major glycoproteins (gB, gC, and gD) of pseudorabies virus (PrV) were constructed and used to examine the ability of these glycoproteins to induce protective immunity against a lethal challenge. Among three constructs, recombinant adenovirus expressing gB (rAd-gB) was found to induce the most potent immunity biased to Th1-type, as determined by the IgG isotype ratio and the profile of the Th1/Th2 cytokine production. Conversely, the gC-expressing adenovirus (rAd-gC) revealed Th2-type immunity and the gD-expressing adenovirus (rAd-gD) induced lower levels of IFN-γ and IL-4 production than other constructs, except IL-2 production. Mucosal delivery of rAd-gB induced mucosal IgA and serum IgG responses and biased toward Th2-type immune responses. However, these effects were not observed in response to systemic delivery of rAd-gB. In addition, rAd-gB appeared to induce effective protective immunity against a virulent viral infection, regardless of whether it was administered via the muscular or systemic route. These results suggest that administration of replication-incompetent adenoviruses can induce different types of immunity depending on the expressed antigen and that recombinant adenoviruses expressing gB induced the most potent Th1-biased humoral and cellular immunity and provided effective protection against PrV infection.

Fast parallel detection of feline panleukopenia virus DNA by multi‐channel microchip electrophoresis with programmed step electric field strength

A multi-channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge-coupled device camera were used to simultaneously detect the separations in three parallel channels using laser-induced fluorescence detection. The parallel separations of a 100-bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M(r) = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single-run and one-step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi-channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease-related DNA fragments in parallel with high speed, throughput, and accuracy.