An OMV-Based Nanovaccine Confers Safety and Protection against Pathogenic Escherichia coli via Both Humoral and Predominantly Th1 Immune Responses in Poultry

Avian pathogenic Escherichia coli (APEC) infection in poultry causes enormous economic losses and public health risks. Bacterial outer membrane vesicles (OMVs) and nano-sized proteolipids enriched with various immunogenic molecules have gained extensive interest as novel nanovaccines against bacterial infections. In this study, after the preparation of APEC O2-derived OMVs (APEC_OMVs) using the ultracentrifugation method and characterization of them using electron microscopy and nanoparticle tracking analyses, we examined the safety and vaccination effect of APEC_OMVs in broiler chicks and investigated the underlying immunological mechanism of protection. The results showed that APEC_OMVs had membrane-enclosed structures with an average diameter of 89 nm. Vaccination with 50 μg of APEC_OMVs had no side effects and efficiently protected chicks against homologous infection. APEC_OMVs could be effectively taken up by chicken macrophages and activated innate immune responses in macrophages in vitro. APEC_OMV vaccination significantly improved activities of serum non-specific immune factors, enhanced the specific antibody response and promoted the proliferation of splenic and peripheral blood lymphocytes in response to mitogen. Furthermore, APEC_OMVs also elicited a predominantly IFN-γ-mediated Th1 response in splenic lymphocytes. Our data revealed the involvement of both non-specific immune responses and specific antibody and cytokine responses in the APEC_OMV-mediated protection, providing broader knowledge for the development of multivalent APEC_OMV-based nanovaccine with high safety and efficacy in the future

The ¹⁵N-leucine single-injection method allows for determining endogenous losses and true digestibility of amino acids in cecectomized roosters

<div><p>This study was conducted to assess the influence of dietary protein content in poultry when using the ¹⁵N-leucine single-injection method to determine endogenous amino acid losses (EAALs) in poultry. Forty-eight cecectomized roosters (2.39 ± 0.23 kg) were randomly allocated to eight dietary treatments containing protein levels of 0, 3%, 6%, 9%, 12%, 15%, 18% and 21%. Each bird was precisely fed an experimental diet of 25 g/kg of body weight. After feeding, all roosters were subcutaneously injected with a ¹⁵N-leucine solution at a dose of 20 mg/kg of body weight. Blood was sampled 23 h after the injection, and excreta samples were continuously collected during the course of the 48-h experiment. The ratio of ¹⁵N-enrichment of leucine in crude mucin to free leucine in plasma ranged from 0.664 to 0.763 and remained relatively consistent (<i>P</i> > 0.05) across all treatments. The amino acid (AA) profiles of total endogenous AAs, except isoleucine, alanine, aspartic acid, cysteine, proline and serine, were not influenced (<i>P</i> > 0.05) by dietary protein contents. The predominant endogenous AAs in the excreta were glutamic acid, aspartic acid, threonine, serine and proline. The order of the relative proportions of these predominant AAs also remained relatively constant (<i>P</i> > 0.05). The endogenous losses of total AAs determined with the ¹⁵N-leucine single-injection method increased curvilinearly with the dietary protein contents. The true digestibility of most AAs and total AAs was independent of their respective dietary protein levels. Collectively, the ¹⁵N-leucine single-injection method is appropriate for determining EAALs and the true digestibility of AAs in poultry fed varying levels of protein-containing ingredients.</p></div

Endogenous amino acid losses (mg/kg of DMI) determined with the ¹⁵N-leucine single-injection method in precision-fed cecectomized roosters fed the nitrogen-free diet and soybean meal diets at varying crude protein levels¹.

<p>Endogenous amino acid losses (mg/kg of DMI) determined with the ¹⁵N-leucine single-injection method in precision-fed cecectomized roosters fed the nitrogen-free diet and soybean meal diets at varying crude protein levels<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188525#t005fn002" target="_blank">¹</a>.</p

True amino acid digestibility calculated from the endogenous losses estimated by the ¹⁵N-leucine single-injection method in cecectomized roosters fed soybean meal diets at varying crude protein levels¹.

<p>True amino acid digestibility calculated from the endogenous losses estimated by the ¹⁵N-leucine single-injection method in cecectomized roosters fed soybean meal diets at varying crude protein levels<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188525#t006fn002" target="_blank">¹</a>.</p

Ingredient composition of the experimental diets (g/kg as-fed basis).

<p>Ingredient composition of the experimental diets (g/kg as-fed basis).</p

¹⁵N-enrichment of leucine in the deproteinized plasma, crude mucin and excreta sampled from precision-fed cecectomized roosters after a single-injection of ¹⁵N-leucine¹.

<p>¹⁵N-enrichment of leucine in the deproteinized plasma, crude mucin and excreta sampled from precision-fed cecectomized roosters after a single-injection of ¹⁵N-leucine<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188525#t002fn002" target="_blank">¹</a>.</p