Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.

ABSTRACTThe efficacies oftrans-cinnamaldehyde (TC) and eugenol (EG) for reducingSalmonella entericaserovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks (n= 75/experiment) were randomly assigned to five treatment groups (n= 15/treatment group): negative control (-veS. Enteritidis, -ve TC, or EG), compound control (-veS. Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+veS. Enteritidis, -ve TC, or EG), low-dose treatment (+veS. Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+veS. Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherentSalmonella(n= 5/experiment). On day 8, birds were inoculated with ∼8.0 log10CFUS. Enteritidis, and cecal colonization byS. Enteritidis was ascertained (n= 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecalS. Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced (P< 0.05)S. Enteritidis in the cecum (≥3 log10CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments (P> 0.05); however, EG supplementation led to significantly lower (P< 0.05) body weights. Follow-upin vitroexperiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibitSalmonellagrowth) of TC and EG reduced the motility and invasive abilities ofS. Enteritidis and downregulated expression of the motility genesflhCandmotAand invasion geneshilA,hilD, andinvF. The results suggest that supplementation with TC and EG through feed can reduceS. Enteritidis colonization in chickens.

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β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

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