Evaluation of the adjuvant effect of Salmonella-based Escherichia coli heat-labile toxin B subunits on the efficacy of a live Salmonella-delivered avian pathogenic Escherichia coli vaccine.

Chaudhari, Atul A,Lee, John Hwa World Veterinary Poultry Association 2013 Avian pathology Vol.42 No.4

<P>The present study evaluated the adjuvant effect of live attenuated salmonella organisms expressing the heat-labile toxin of Escherichia coli B subunit (LTB) on the efficacy of an avian pathogenic Escherichia coli (APEC) vaccine. The Asd(+) (aspartate semialdehyde dehydrogenase) plasmid pMMP906 containing the LTB gene was introduced into a Salmonella enterica Typhimurium strain lacking the lon, cpxR and asd genes to generate the adjuvant strain. Live recombinant Salmonella-delivered APEC vaccine candidates were used for this study. The birds were divided into three groups: group A, non-vaccinated controls; group B, immunized with vaccine candidates only; and group C, immunized with vaccine candidates and the LTB strain. The immune responses were measured and the birds were challenged at 21 days of age with a virulent APEC strain. Group C showed a significant increase in plasma IgG and intestinal IgA levels and a significantly higher lymphocyte proliferation response compared with the other groups. Upon challenge with the virulent APEC strain, group C showed effective protection whereas group B did not. We also attempted to optimize the effective dose of the adjuvant. The birds were immunized with the vaccine candidates together with 110? or 110? colony-forming units of the LTB strain and were subsequently challenged at 3 weeks of age. The 110? colony-forming units of the LTB strain showed a greater adjuvant effect with increased levels of serum IgG, intestinal IgA and a potent lymphocyte proliferation response, and yielded higher protection against challenge. Overall, the LTB strain increased the efficacy of the Salmonella -delivered APEC vaccine, indicating that vaccination for APEC along with the LTB strain appears to increase the efficacy for protection against colibacillosis in broiler chickens.</P>

Construction of a <i>Salmonella</i> Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

Chaudhari, Atul A,Jawale, Chetan V,Kim, Sam Woong,Lee, John Hwa BioMed Central 2012 VETERINARY RESEARCH Vol.43 No.-

<P>In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a <I>Salmonella</I> Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene <I>E</I> was constructed using pMMP99 plasmid in this study. The formation of the <I>Salmonella</I> Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene <I>E</I> expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the <I>Salmonella</I> Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent <I>Salmonella</I> Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the <I>Salmonella</I> Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed <I>Salmonella</I> Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.</P>

Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis.

Chaudhari, Atul A,Matsuda, Kiku,Lee, John Hwa American Association of Avian Pathologists [etc.] 2013 Avian diseases Vol.57 No.1

<P>An attenuated Salmonella (deltalon, deltacpxR, and deltaasdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra-air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC.</P>

Electrospun Antimicrobial Polyurethane Nanofibers Containing Silver Nanoparticles for Biotechnological Applications

Sheikh, Faheem A.,Barakat, Nasser A.M.,Kanjwal, Muzafar A.,Chaudhari, Atul A.,Jung, In-Hee,Lee, John-Hwa,Kim, Hak-Yong The Polymer Society of Korea 2009 Macromolecular Research Vol.17 No.9

In this study, a new class of polyurethane (PU) nanofibers containing silver (Ag) nanoparticles (NPs) was synthesized by electrospinning. A simple method that did not depending on additional foreign chemicals was used to self synthesize the silver NPs in/on PU nanofibers. The synthesis of silver NPs was carried out by exploiting the reduction ability of N,N-dimethylformamide (DMF), which is used mainly to decompose silver nitrate to silver NPs. Typically, a sol-gel consisting of $AgNO_3$/PU was electrospun and aged for one week. Silver NPs were created in/on PU nanofibers. SEM confirmed the well oriented nanofibers and good dispersion of pure silver NPs. TEM indicated that the Ag NPs were 5 to 20 nm in diameter. XRD demonstrated the good crystalline features of silver metal. The mechanical properties of the nanofiber mats showed improvement with increasing silver NPs content. The fixedness of the silver NPs obtained on PU nanofibers was examined by harsh successive washing of the as-prepared mats using a large amount of water. The results confirmed the good stability of the synthesized nanofiber mats. Two model organisms, E. coli and S. typhimurium, were used to check the antimicrobial influence of these nanofiber mats. Subsequently, antimicrobial tests indicated that the prepared nanofibers have a high bactericidal effect. Accordingly, these results highlight the potential use of these nanofiber mats as antimicrobial agents.

Avian Colibacillosis Caused by Intestinal Pathogenic Escherichia coli Isolate from Calf Diarrhea

Atul A. Chaudhari,Jong Hyo Kim,In Hee Jung,Nandre Rahul,John Hwa Lee 대한수의학회 2009 대한수의학회 학술대회발표집 Vol.2009 No.-

Electrospun Antimicrobial Polyurethane Nanofibers Containing Silver Nanoparticles for Biotechnological Applications

Faheem A. Sheikh,Nasser A. M. Barakat,Muzafar A. Kanjwal,Atul A. Chaudhari,In Hee Jung,John Hwa Lee,Hak Yong Kim 한국고분자학회 2009 Macromolecular Research Vol.17 No.9

Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated <i>Salmonella</i> Gallinarum vaccine candidate

Won, Gayeon,Chaudhari, Atul A.,Lee, John Hwa 대한백신학회 2016 Clinical and Experimental Vaccine Research Vol.5 No.2

<P><B>Purpose</B></P><P><I>Salmonella enterica</I> serovar Gallinarum (SG) ghost vaccine candidate was recently constructed. In this study, we evaluated various prime-boost vaccination strategies using the candidate strain to optimize immunity and protection efficacy against fowl typhoid.</P><P><B>Materials and Methods</B></P><P>The chickens were divided into five groups designated as group A (non-immunized control), group B (orally primed and boosted), group C (primed orally and boosted intramuscularly), group D (primed and boosted intramuscularly), and group E (primed intramuscularly and boosted orally). The chickens were primed with the SG ghost at 7 days of age and were subsequently boosted at the fifth week of age. Post-immunization, the plasma IgG and intestinal secretory IgA (sIgA) levels, and the SG antigen-specific lymphocyte stimulation were monitored at weekly interval and the birds were subsequently challenged with a virulent SG strain at the third week post-second immunization.</P><P><B>Results</B></P><P>Chickens in group D showed an optimized protection with significantly increased plasma IgG, sIgA, and lymphocyte stimulation response compared to all groups. The presence of CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T cells and monocyte/macrophage (M/M) in the spleen, and splenic expression of cytokines such as interferon γ (IFN-γ) and interleukin 6 (IL-6) in the immunized chickens were investigated. The prime immunization induced significantly higher splenic M/M population and mRNA levels of IFN-γ whereas the booster showed increases of splenic CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T-cell population and IL-6 cytokine in mRNA levels.</P><P><B>Conclusion</B></P><P>Our results indicate that the prime immunization with the SG ghost vaccine induced Th1 type immune response and the booster elicited both Th1- and Th2-related immune responses.</P>

Immune responses to oral vaccination with Salmonella-delivered avian pathogenic Escherichia coli antigens and protective efficacy against colibacillosis.

Lee, John Hwa,Chaudhari, Atul A,Oh, In Gyoung,Eo, Seong Kug,Park, Sang-Youel,Jawale, Chetan V Canadian Veterinary Medical Association = Associat 2015 Canadian journal of veterinary research Vol.79 No.3

<P>In this study, the immune responses to and protective efficacy of a live attenuated Salmonella-delivered vaccine candidate secreting the papA, papG, iutA, and clpG antigens of Escherichia coli were evaluated against infection with avian pathogenic E. coli (APEC) in layer chickens. Primary vaccination was done at age 7 d and booster vaccination at age 5 wk. The levels of intestinal secretory immunoglobulin A specific to the 4 antigens were significantly higher in the vaccinated group than in the control group. A potent lymphocyte-proliferation response and increased levels of interferon-gamma, interleukin-2, and interleukin-6 in the plasma and in culture supernatants of antigen-stimulated lymphocytes from the vaccinated group suggested significant induction of the cell-mediated immune response in this group compared with the control group. Upon challenge with a virulent APEC strain at 8 wk of age, the vaccinated group had no deaths, whereas the control group had a 15% mortality rate. In addition, the morbidity rate was significantly higher in the control group (55%) than in the vaccinated group (15%). Thus, giving primary and booster vaccination with the Salmonella-delivered APEC vaccine candidate significantly elevated both mucosal and cellular immune responses, which protected the chickens against colibacillosis.</P>

Avian colibacillosis caused by an intestinal pathogenic <i>Escherichia coli</i> isolate from calf diarrhea

Matsuda, Kiku,Chaudhari, Atul A.,Lee, John Hwa Elsevier 2010 Research in veterinary science Vol.89 No.2

<P><B>Abstract</B></P><P>An intestinal pathogenic <I>Escherichia coli</I> isolate from calf diarrhea, containing the <I>iutA</I>, <I>f17A</I>, <I>afa</I>-<I>8D</I>, and <I>cnf2</I> genes, was able to cause avian colibacillosis after experimental infection in chickens. Intra-tracheal inoculation and spray of this strain caused 10% of mortality and gross lesions, including airsacculitis, pericarditis, and perihepatitis. These results suggest that some bovine pathogenic <I>E. coli</I> can cause extra-intestinal infections in other animal species.</P>

Physiology, pathogenicity and immunogenicity of <i>lon</i> and/or <i>cpxR</i> deleted mutants of <i>Salmonella</i> Gallinarum as vaccine candidates for fowl typhoid

Matsuda, Kiku,Chaudhari, Atul A.,Kim, Sam Woong,Lee, Kyeong Min,Lee, John Hwa EDP Sciences 2010 VETERINARY RESEARCH Vol.41 No.5

<P>To construct a novel live vaccine candidate for fowl typhoid (FT) caused by <I>Salmonella</I> Gallinarum (SG), the <I>lon</I> and <I>cpxR</I> genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Δ<I>lon</I>) and JOL916 (Δ<I>lon</I>Δ<I>cpxR</I>) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (Δ<I>cpxR</I>), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD<SUB>50</SUB> using 1-week-old chickens, were attenuated approximately at 10<SUP>1</SUP>-, 10<SUP>1</SUP>-, and > 10<SUP>3</SUP>-fold, respectively. The oral inoculations of 2 × 10<SUP>7</SUP> cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT.</P>