Advances in Poultry Vaccines: Leveraging Biotechnology for Improving Vaccine Development, Stability, and Delivery.

With the rapidly increasing demand for poultry products and the current challenges facing the poultry industry, the application of biotechnology to enhance poultry production has gained growing significance. Biotechnology encompasses all forms of technology that can be harnessed to improve poultry health and production efficiency. Notably, biotechnology-based approaches have fueled rapid advances in biological research, including (a) genetic manipulation in poultry breeding to improve the growth and egg production traits and disease resistance, (b) rapid identification of infectious agents using DNA-based approaches, (c) inclusion of natural and synthetic feed additives to poultry diets to enhance their nutritional value and maximize feed utilization by birds, and (d) production of biological products such as vaccines and various types of immunostimulants to increase the defensive activity of the immune system against pathogenic infection. Indeed, managing both existing and newly emerging infectious diseases presents a challenge for poultry production. However, recent strides in vaccine technology are demonstrating significant promise for disease prevention and control. This review focuses on the evolving applications of biotechnology aimed at enhancing vaccine immunogenicity, efficacy, stability, and delivery.

The effects of timing of high immune response phenotyping in relation to weaning on immune responses of crossbred beef calves.

Genetic selection for immune response has the potential to increase the sustainability of the beef industry by breeding cattle that are productive yet with an increased capacity to resist disease. Determining the optimal time to immunophenotype beef cattle is crucial for the accurate prediction of an animal's immune response. The objective of this study was to determine the effect of time of immunophenotyping in relation to weaning on immune responses of beef calves. Antibody- (AMIR) and cell-mediated (CMIR) immune responses were measured on 97 calves on the day of weaning (WEANING, N = 56) or 2 mo post-weaning (POST-WEANING, N = 41). Within each period of immunophenotyping, on day 0, blood was collected, and calves received a 1.0 mL intramuscular injection of type 1 and 2 test antigens. On day 14, blood was collected, and baseline skinfold thickness (SFT) was measured. Calves received an intradermal injection of 0.1 mg of the type 1 antigen suspended in 0.1 mL phosphate buffered saline (PBS) in the right tail fold, and 0.1 mL of PBS in the left. Changes in SFT at 24 h was used to indicate CMIR. To assess AMIR, the titer of type 2 antigen-specific bovine immunoglobulin G in serum from blood collected on day 14 was determined by measuring optical density (OD) using an enzyme-linked immunosorbent assay (ELISA). Among heifers, AMIR was greater for the POST-WEANING group than for the WEANING group (P < 0.01). Among steers, AMIR was not different between the POST-WEANING group and the WEANING group (P = 1.0). Therefore, the AMIR of heifers may be more negatively affected by immunophenotyping at weaning than the AMIR of steers. For steers, CMIR was greater in the POST-WEANING group than the WEANING group (P < 0.001). For heifers, CMIR was not different between the POST-WEANING group and the WEANING group (P = 0.22). The CMIR of steers may be more negatively affected by immunophenotyping at weaning than the CMIR of heifers. Calf age was not associated with AMIR or CMIR for calves phenotyped at weaning or post-weaning. The effect of sire nested within dam age was significant for CMIR for calves in the POST-WEANING group (P < 0.01), but not for calves in the WEANING group (P = 0.67). The results suggest that measuring immunocompetence at weaning may not be representative of a calf's genetic ability to mount an effective immune response, and immunophenotyping should be performed outside the weaning period.

Understanding the optimal time to immunophenotype beef calves is important for the accurate estimation of their genetic ability to resist disease. The compound stressors experienced by a calf during weaning may have a similar impact on the immune system as chronic stress. Therefore, the immune response phenotype of a calf immunophenotyped during the weaning period may not truly reflect the animal's genuine capacity for immune response. To accurately identify cattle with a superior capacity for immune response, with the goal of genetically selecting cattle for immunocompetence, immunophenotypes must be measured accurately. In this study, the effect of time of immunophenotyping in relation to weaning on immune responses of beef calves was determined. Calves immunophenotyped at weaning had lesser antibody-mediated and cell-mediated immune responses than calves immunophenotyped 2 mo post-weaning, this effect was influenced by sex. Sire affected immune responses when calves were immunophenotyped 2 mo post-weaning, but not when calves were immunophenotyped at weaning, indicating that when immunophenotyped post-weaning, the genetic component of a calf's immune response is quantified without being obscured by other environmental factors.

Serum IgM antibody response to Clostridioides difficile polysaccharide PS-II vaccination in pony foals.

OBJECTIVES: Clostridioides difficile (formerly Clostridium difficile) is associated with colitis in foals and mature horses. C. difficile exposes specific phosphorylated polysaccharides (PSs), named PS-I, PS-II and PS-III. These cell-surface PSs are potential vaccine targets, especially the hexasaccharide phosphate PS-II, that has been found in all C. difficile ribotypes examined. Since we previously identified anti-PS-II circulating antibodies in horses, we postulated that vaccinating foals with PS-II may prevent colonization by C. difficile. In this study, we aim to evaluate the IgM antibody responses in foals to PS-II. METHODS: To evaluate the reactogenicity and immunogenicity of C. difficile PS-II in foals, three-to four-month-old foals were vaccinated intramuscularly three times at intervals of three weeks with 100 µg/dose (3 foals) or 500 µg/dose (3 foals) of purified PS-II antigen with aluminum hydroxide adjuvant, or with a placebo preparation (2 foals) containing adjuvant alone. RESULTS: No injection site swelling, pain or fever was observed after vaccination. Two of the three foals receiving 100 µg/dose, and three out of three foals receiving 500 µg/dose of PS-II responded with increases in serum IgM antibodies. No control foals that received the placebo had IgM responses to PS-II. There was a trend towards a higher response rate in foals receiving 500 µg PS-II one week after second vaccination when compared to control foals and towards higher concentrations of serum IgM antibodies in foals receiving 500 µg PS-II. CONCLUSIONS: No adverse reactions were observed following vaccination with PS-II in foals; Serum IgM immune responses were induced by vaccination. A polysaccharide-based vaccine for C. difficile in horses deserves further investigation.

Transmission of H9N2 Low Pathogenicity Avian Influenza Virus (LPAIV) in a Challenge-Transmission Model.

Migratory birds are major reservoirs for avian influenza viruses (AIV), which can be transmitted to poultry and mammals. The H9N2 subtype of AIV has become prevalent in poultry over the last two decades. Despite that, there is a scarcity of detailed information on how this virus can be transmitted. The current study aimed to establish a direct contact model using seeder chickens infected with H9N2 AIV as a source of the virus for transmission to recipient chickens. Seeder chickens were inoculated with two different inoculation routes either directly or via the aerosol route. The results indicate that inoculation via the aerosol route was more effective at establishing infection compared to the direct inoculation route. Shedding was observed to be higher in aerosol-inoculated seeder chickens, with a greater percentage of chickens being infected at each time point. In terms of transmission, the recipient chickens exposed to the aerosol-inoculated seeder chickens had higher oral and cloacal virus shedding compared to the recipient chickens of the directly inoculated group. Furthermore, the aerosol route of infection resulted in enhanced antibody responses in both seeder and recipient chickens compared to the directly inoculated group. Overall, the results confirmed that the aerosol route is a preferred inoculation route for infecting seeder chickens in a direct contact transmission model.

Immuno-phenotyping of Canadian beef cattle: adaptation of the high immune response methodology for utilization in beef cattle.

The high immune response (HIR) methodology measures the genetic performance of the adaptive immune system to identify and breed animals with balanced and robust immunity. The HIR methodology has previously been used in dairy and swine to reduce disease but has not been fully investigated in beef cattle. The first objective of the current study was to examine whether the HIR methodology as standardized for use in dairy cattle was appropriate for use in beef cattle. The second objective was to determine the earliest age for immune response phenotyping of beef calves. In this study, beef calves (n = 295) of various ages, as well as mature beef cows (n = 170) of mixed breeds, were immunized using test antigens to assess their antibody- (AMIR) and cell-mediated immune responses (CMIR). Heritability for AMIR and CMIR was estimated at 0.43 and 0.18, respectively. The HIR methodology was appropriate for use in beef cattle; beef calves as young as 2-3 wk of age were capable of mounting AMIR responses comparable with those seen historically in mature Holstein dairy cows. Three-week-old beef calves mounted CMIR responses comparable with those of Holstein cows, but 9-mo-old calves and mature beef cows had significantly higher CMIR responses than Holsteins. The HIR methodology can be used to measure both AMIR and CMIR in beef calves as young as 3 wk of age.

Concentration and heritability of immunoglobulin G and natural antibody immunoglobulin M in dairy and beef colostrum along with serum total protein in their calves.

Immunoglobulin (Ig) G and natural antibody (NAb) IgM are passively transferred to the neonatal calf through bovine colostrum. Maternal IgG provides pathogen- or vaccine-specific protection and comprises about 85% of colostral Ig. NAb-IgM is less abundant but provides broad and nonspecific reactivity, potentially contributing to protection against the dissemination of pathogens in the blood (septicemia) in a calf's first days of life. In the dairy and beef industries, failure of passive transfer (FPT) of colostral Ig (serum total protein [STP] <5.2 g/dL) is still a common concern. The objectives of this study were to: (1) compare colostral IgG concentrations and NAb-IgM titers between dairy and beef cows; (2) assess the effect of beef breed on colostral IgG; (3) compare passive transfer of colostral Ig in dairy and beef calves; and (4) estimate the heritability of colostral IgG and NAb-IgM. Colostrum was collected from Holstein dairy (n = 282) and crossbred beef (n = 168) cows at the University of Guelph dairy and beef research centers. Colostral IgG was quantified by radial immunodiffusion and NAb-IgM was quantified by an enzyme-linked immunosorbent assay. In dairy (n = 308) and beef (n = 169) calves, STP was estimated by digital refractometry. Beef cows had significantly greater colostral IgG (146.5 ± 9.5 standard error of the mean [SEM] g/L) than dairy cows (92.4 ± 5.2 g/L, P <0.01). Beef cows with a higher proportion of Angus ancestry had significantly lower colostral IgG (125.5 ± 5.8 g/L) than cows grouped as "Other" (142.5 ± 4.9 g/L, P = 0.02). Using the FPT cutoff, 13% of dairy and 16% of beef calves had FPT; still, beef calves had a significantly larger proportion with excellent passive transfer (STP ≥6.2 g/dL, P <0.01). The heritability of colostral IgG was 0.04 (±0.14) in dairy and 0.14 (±0.32) in beef. Colostral NAb-IgM titers in dairy (12.12 ± 0.22, log2 [reciprocal of titer]) and beef cows (12.03 ± 0.19) did not differ significantly (P = 0.71). The range of NAb-IgM titers was 9.18-14.60, equivalent to a 42-fold range in antibody concentration. The heritability of colostral NAb was 0.24 (±0.16) in dairy and 0.11 (±0.19) in beef cows. This study is the first to compare colostral NAb-IgM between dairy and beef cows. Based on the range in NAb-IgM titers and the heritability, selective breeding may improve colostrum quality and protection for neonatal calves in the early days of life.

Understanding how breed influences immunoglobulin (Ig) G and natural antibody (NAb) IgM concentrations in colostrum can improve bovine colostrum quality and calf health. Maternal colostral IgG is abundant, persistent, and pathogen specific. Natural antibody-IgM is less abundant but mediates broad, short-lived, nonspecific pathogen protection, and potentially important against septicemia. Colostral IgG and NAb-IgM concentrations were compared between dairy and beef cows and among cross-bred beef cows. Heritabilities were calculated to assess the practicality of selective breeding. Serum total protein (STP) in neonatal dairy and beef calves was estimated using refractometry. Colostrum from beef cows had higher concentrations of IgG than dairy cows. Beef cows with higher Angus ancestry produced colostrum with lower IgG concentrations than other mixed breeds. Heritability of colostral IgG was low (0.04­0.14). Failure of passive transfer was similar in dairy and beef calves, but a significantly larger proportion of beef calves had excellent STP (≥6.2 g/dL). There were no differences in NAb-IgM titers between dairy and beef cows or among beef breeds. Colostral NAb-IgM varied widely among individuals (42-fold) and was moderately heritable (0.11­0.24). These results suggest that selective breeding to improve colostrum quality is feasible and practical to improve calf health.

Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination.

Necrotic enteritis (NE), caused by Clostridium perfringens (CP), is one of the most common of poultry diseases, causing huge economic losses to the poultry industry. This review provides an overview of the pathogenesis of NE in chickens and of the interaction of CP with the host immune system. The roles of management, nutrition, probiotics, and vaccination in reducing the incidence and severity of NE in poultry flocks are also discussed.

Commensal gut microbiota can modulate adaptive immune responses in chickens vaccinated with whole inactivated avian influenza virus subtype H9N2.

Variations in the composition of commensal gut microbiota have been reported to be major contributors to differences in responses to vaccination among individuals. In chickens, there is limited information on the role of gut microbiota in responses to vaccination. The current study studied the role of gut microbiota in cell- and antibody-mediated immune responses to vaccination with a whole inactivated avian influenza virus, subtype H9N2. A total of 166 one-day-old specific pathogen free layer chickens (SPF) were randomly assigned to treatments, where a combination of antibiotic depletion, and probiotics (a combination of five Lactobacillus species) or fecal microbial transplant (FMT) reconstitution were used to study the dynamics of cell- and antibody-mediated immune responses to primary and secondary vaccinations at days 15 and 29 of age, respectively. Overall, at days 7 and 14 post primary vaccination (p.p.v.), administration of probiotics to non-depleted chickens resulted in significantly higher mean hemagglutination (HI) titre compared to antibiotic treated chickens. Furthermore, at day 21 p.p.v., chickens treated with probiotics or FMT post-antibiotic treatment showed a significantly higher mean HI titre compared to non-depleted chickens treated with probiotics. At day 7 p.p.v., a significantly higher virus specific IgM and IgG titres were observed in non-depleted chickens administered with probiotics compared to antibiotic depleted chickens, and a significantly higher IgG titre was observed in chickens treated with FMT following antibiotic treatment compared to only antibiotic treatment. Analysis of interferon gamma expression in splenocytes to assess cell-mediated immune responses showed a significantly lower expression in antibiotic-treated chickens compared to non-depleted chickens and FMT reconstituted chickens. Taken together, the current study suggests that shifts in the composition of gut microbiota of chickens may result in changes in cell- and antibody-mediated immune responses to vaccination against influenza viruses. Further studies will be needed to highlight the mechanisms involved in this modulation.

Development of innate immunity in chicken embryos and newly hatched chicks: a disease control perspective.

Newly hatched chickens are confronted by a wide array of pathogenic microbes because their adaptive immune defences have limited capabilities to control these pathogens. In such circumstances, and within this age group, innate responses provide a degree of protection. Moreover, as the adaptive immune system is relatively naïve to foreign antigens, synergy with innate defences is critical. This review presents knowledge on the ontogeny of innate immunity in chickens pre-hatch and early post-hatch and provides insights into possible interventions to modulate innate responses early in the life of the bird. As in other vertebrate species, the chicken innate immune system which include cellular mediators, cytokine and chemokine repertoires and molecules involved in antigen detection, develop early in life. Comparison of innate immune systems in newly hatched chickens and mature birds has revealed differences in magnitude and quality, but responses in younger chickens can be boosted using innate immune system modulators. Functional expression of pattern recognition receptors and several defence molecules by innate immune system cells of embryos and newly hatched chicks suggests that innate responses can be modulated at this stage of development to combat pathogens. Improved understanding of innate immune system ontogeny and functionality in chickens is critical for the implementation of sound and safe interventions to provide long-term protection against pathogens. Next-generation tools for studying genetic and epigenetic regulation of genes, functional metagenomics and gene knockouts can be used in the future to explore and dissect the contributions of signalling pathways of innate immunity and to devise more efficacious disease control strategies.

Characterization of innate responses induced by in ovo administration of encapsulated and free forms of ligands of Toll-like receptor 4 and 21 in chicken embryos.

Toll-like receptors (TLRs) are a family of innate receptors that recognize pathogen-associated molecular patterns, including double-stranded RNA, CpG DNA and lipopolysaccharide (LPS). After interaction with their ligands, TLRs initiate innate responses that are manifested by activating cells and inducing expression of cytokines that help mediate adaptive immune responses. TLR ligands (TLR-Ls) have the potential to be used prophylactically (alone) or as vaccine adjuvants to promote host immunity. Encapsulating TLR-Ls in nanoparticles, such as Poly (d,l-lactic-co-glycolic acid), may prolong responses through sustained release of the ligands. PLGA nanoparticles protect encapsulated TLR-Ls from degradation and extend the half-life of these ligands by reducing their rapid removal from the body. In this study, encapsulated and free forms of LPS and CpG ODN were administered to embryonation day 18 (ED18) chicken embryos. Spleen, lungs and bursa of Fabricius were collected at 6, 18 and 48hour post-stimulation (hps) and cytokine gene expressions were evaluated using quantitative real-time PCR. Results indicate that both the free and encapsulated forms of LPS and CpG ODN induced innate immune responses in ED18 chicken embryos. Innate responses induced in embryos seem similar to those reported in mature chickens. Significant upregulation of cytokine genes generally occurred by 48hps. Further studies are needed to evaluate long term immunomodulatory effects of encapsulated TLR-Ls and their ability to mediate protection against pathogens of young chicks.

Author Correction: Gut microbiota-mediated protection against influenza virus subtype H9N2 in chickens is associated with modulation of the innate responses.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Gut microbiota-mediated protection against influenza virus subtype H9N2 in chickens is associated with modulation of the innate responses.

Commensal gut microbiota plays an important role in health and disease. The current study was designed to assess the role of gut microbiota of chickens in the initiation of antiviral responses against avian influenza virus. Day-old layer chickens received a cocktail of antibiotics for 12 (ABX-D12) or 16 (ABX-D16) days to deplete their gut microbiota, followed by treatment of chickens from ABX-12 with five Lactobacillus species combination (PROB), fecal microbial transplant suspension (FMT) or sham treatment daily for four days. At day 17 of age, chickens were challenged with H9N2 virus. Cloacal virus shedding, and interferon (IFN)-α, IFN-ß and interleukin (IL)-22 expression in the trachea, lung, ileum and cecal tonsils was assessed. Higher virus shedding, and compromised type I IFNs and IL-22 expression was observed in ABX-D16 chickens compared to control, while PROB and FMT showed reduced virus shedding and restored IL-22 expression to levels comparable with undepleted chickens. In conclusion, commensal gut microbiota of chickens can modulate innate responses to influenza virus subtype H9N2 infection in chickens, and modulating the composition of the microbiome using probiotics- and/or FMT-based interventions might serve to promote a healthy community that confers protection against influenza virus infection in chickens.

PLGA-encapsulated CpG ODN and Campylobacter jejuni lysate modulate cecal microbiota composition in broiler chickens experimentally challenged with C. jejuni.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. Reducing Campylobacter numbers in the intestinal tract of chickens will minimize transmission to humans, thereby reducing the incidence of infection. We have previously shown that oral pre-treatment of chickens with C. jejuni lysate and Poly D, L-lactide-co-glycolide polymer nanoparticles (PLGA NPs) containing CpG oligodeoxynucleotide (ODN) can reduce the number of C. jejuni in infected chickens. In the current study, the effects of these pre-treatments on the composition and functional diversity of the cecal microbiota, in chickens experimentally infected with C. jejuni, were investigated using next-generation sequencing. The taxonomic composition analysis revealed a reduction in cecal microbial diversity and considerable changes in the taxonomic profiles of the microbial communities of C. jejuni-challenged chickens. On the other hand, irrespective of the dose, the microbiota of PLGA-encapsulated CpG ODN- and C. jejuni lysate-treated chickens exhibited higher microbial diversity associated with high abundance of members of Firmicutes and Bacteroidetes and lower numbers of Campylobacter than untreated-chickens. These findings suggest that oral administration of encapsulated CpG ODN and C. jejuni lysate can reduce colonization by C. jejuni by enhancing the proliferation of specific microbial groups. The mechanisms that mediate these changes remain, however, to be elucidated.

Effects of early feeding and dietary interventions on development of lymphoid organs and immune competence in neonatal chickens: A review.

With the ongoing intensification of the poultry industry and the continuous need to control pathogens, there is a critical need to extend our understanding of the avian immune system and the role of nutritional interventions on development of immune competence in neonatal chicks. In this review, we will focus on the ontogeny of the lymphoid organs during embryonic life and the first 2 weeks post-hatch, and how early feeding practices improve heath and modulate the development and function of the immune system in young chicks. The evidence for the positive impact of the nutrition of breeder hens on embryonic development and on the survival and immunity of their chicks will also be outlined. Additionally, we will discuss the vital role of supplemental feeding either in ovo or immediately post-hatch in chick health and immunity and the importance of these approaches in ameliorating immune system functions of heat-stressed chicks. To conclude, we provide some perspectives on a number of key issues, concerning the mechanisms of nutritional modulation of immunity, that need to be addressed. A thorough investigation of these mechanisms may assist in the formulation of diets to improve the immunity and general health status.

Oral administration of PLGA-encapsulated CpG ODN and Campylobacter jejuni lysate reduces cecal colonization by Campylobacter jejuni in chickens.

Campylobacter jejuni (C. jejuni) is a major cause of bacterial food-borne illness in humans. It is considered a commensal organism of the chicken gut and infected chickens serve as a reservoir and shed bacteria throughout their lifespan. Contaminated poultry products are considered the major source of infection in humans. Therefore, to reduce the risk of human campylobacteriosis, it is essential to reduce the bacterial load in poultry products. The present study aimed to evaluate the protective effects of soluble and PLGA-encapsulated oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (E-CpG ODN) as well as C. jejuni lysate as a multi-antigen vaccine against colonization with C. jejuni. The results revealed that oral administration of a low (5 µg) or high (50 µg) dose of CpG resulted in a significant reduction in cecal C. jejuni colonization by 1.23 and 1.32 log10 (P < .05) in layer chickens, respectively, whereas E-CpG significantly reduced cecal C. jejuni colonization by 1.89 and 1.46 log10 in layer and broiler chickens at day 22 post-infection (slaughter age in broilers), respectively. Similar patterns were observed for C. jejuni lysate; oral administration of C. jejuni lysate reduced the intestinal burden of C. jejuni in layer and broiler chickens by 2.24 and 2.14 log10 at day 22 post-infection, respectively. Moreover, the combination of E-CpG and C. jejuni lysate reduced bacterial counts in cecal contents by 2.42 log10 at day 22 post-infection in broiler chickens. Anti-C. jejuni IgG antibody (Ab) titers were significantly higher for broiler chickens receiving a low or high dose of E-CpG or a low dose of C. jejuni lysate than for chickens receiving the placebo. Furthermore, a positive correlation was observed between serum IgG Ab titers and cecal counts of C. jejuni in these groups. These findings suggest that PLGA-encapsulated CpG or C. jejuni lysate could be a promising strategy for control of C. jejuni in chickens.

Gene expression profiling of chicken cecal tonsils and ileum following oral exposure to soluble and PLGA-encapsulated CpG ODN, and lysate of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is a leading bacterial cause of food-borne illness in humans. Contaminated chicken meat is an important source of infection for humans. Chickens are not clinically affected by colonization, and immune responses following natural infection have limited effects on bacterial load in the gut. Induction of intestinal immune responses may possibly lead to a breakdown of the commensal relationship of chickens with Campylobacter. We have recently shown that soluble and poly D, L-lactic-co-glycolic acid (PLGA)-encapsulated CpG oligodeoxynucleotide (ODN) as well as C. jejuni lysate, are effective in reducing the intestinal burden of C. jejuni in chickens; however, the mechanisms behind this protection have yet to be determined. The present study was undertaken to investigate the mechanisms of host responses conferred by these treatments. Chickens were treated orally with soluble CpG ODN, or PLGA-encapsulated CpG ODN, or C. jejuni lysate, and expression of cytokines and antimicrobial peptides was evaluated in cecal tonsils and ileum using quantitative RT-PCR. Oral administration of soluble CpG ODN upregulated the expression of interferon (IFN)-γ, interleukin (IL)-1ß, CXCLi2, transforming growth factor (TGF)-ß4/1, IL-10 and IL-13, while treatment with PLGA-encapsulated CpG ODN upregulated the expression of IL-1ß, CXCLi2, TGF-ß4/1, IL-13, avian ß-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). C. jejuni lysate upregulated the expression of IFN-γ, IL-1ß, TGF-ß4/1, IL-13, AvBD1, and CATHL-3. In conclusion, induction of cytokine and antimicrobial peptides expression in intestinal microenvironments may provide a means of reducing C. jejuni colonization in broiler chickens, a key step in reducing the incidence of campylobacteriosis in humans.

Characterization of Innate Responses Induced by PLGA Encapsulated- and Soluble TLR Ligands In Vitro and In Vivo in Chickens.

Natural or synthetic Toll-like receptor (TLR) ligands trigger innate responses by interacting with distinct TLRs. TLR ligands can thus serve as vaccine adjuvants or stand-alone antimicrobial agents. One of the limitations of TLR ligands for clinical application is their short half-life and rapid clearance from the body. In the current study, encapsulation of selected TLR ligands in biodegradable poly(D,L-lactide-co-glycolide) polymer nanoparticles (PLGA NPs) was examined in vitro and in vivo as a means to prolong innate responses. MQ-NCSU cells (a chicken macrophage cell line) were treated with encapsulated or soluble forms of TLR ligands and the resulting innate responses were evaluated. In most cases, encapsulated forms of TLR ligands (CpG ODN 2007, lipopolysaccharide and Pam3CSK4) induced comparable or higher levels of nitric oxide and cytokine gene expression in macrophages, compared to the soluble forms. Encapsulated CpG ODN, in particular the higher dose, induced significantly higher expression of interferon (IFN)-γ and IFN-ß until at least 18 hr post-treatment. Cytokine expression by splenocytes was also examined in chickens receiving encapsulated or soluble forms of lipopolysaccharide (a potent inflammatory cytokine inducer in chickens) by intramuscular injection. Encapsulated LPS induced more sustained innate responses characterized by higher expression of IFN-γ and IL-1ß until up to 96 hr. The ability of TLR ligands encapsulated in polymeric nanoparticles to maintain prolonged innate responses indicates that this controlled-release system can extend the use of TLR ligands as vaccine adjuvants or as stand-alone prophylactic agents against pathogens.

Delivery of an inactivated avian influenza virus vaccine adjuvanted with poly(D,L-lactic-co-glycolic acid) encapsulated CpG ODN induces protective immune responses in chickens.

In poultry, systemic administration of commercial vaccines consisting of inactivated avian influenza virus (AIV) requires the simultaneous delivery of an adjuvant (water-in-oil emulsion). These vaccines are often limited in their ability to induce quantitatively better local (mucosal) antibody responses capable of curtailing virus shedding. Therefore, more efficacious adjuvants with the ability to provide enhanced immunogenicity and protective anti-AIV immunity in chickens are needed. While the Toll-like receptor (TLR) 21 agonist, CpG oligodeoxynucleotides (ODNs) has been recognized as a potential vaccine adjuvant in chickens, poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, successfully tested as vaccine delivery systems in other species, have not been extensively explored. The present study, therefore, assessed both systemic and mucosal antibody-mediated responses following intramuscular vaccination (administered at 7 and 21days post-hatch) of chickens with PLGA encapsulated H9N2 AIV plus encapsulated CpG ODN 2007 (CpG 2007), and nonencapsulated AIV plus PLGA encapsulated CpG 2007 vaccine formulations. Virus challenge was performed at 2weeks post-secondary vaccination using the oculo-nasal route. Our results showed that chickens vaccinated with the nonencapsulated AIV vaccine plus PLGA encapsulated CpG 2007 developed significantly higher systemic IgY and local (mucosal) IgY antibodies as well as haemagglutination inhibition antibody titres compared to PLGA encapsulated AIV plus encapsulated CpG 2007 vaccinated chickens. Furthermore, chickens that received CpG 2007 as an adjuvant in the vaccine formulation had antibodies exhibiting higher avidity indicating that the TLR21-mediated pathway may enhance antibody affinity maturation qualitatively. Collectively, our data indicate that vaccination of chickens with nonencapsulated AIV plus PLGA encapsulated CpG 2007 results in qualitatively and quantitatively augmented antibody responses leading to a reduction in virus shedding compared to the encapsulated AIV plus PLGA encapsulated CpG 2007 formulation.

Local Innate Responses to TLR Ligands in the Chicken Trachea.

The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV.

Characterization of host responses induced by Toll-like receptor ligands in chicken cecal tonsil cells.

The innate responses of cecal tonsils against invading microorganisms are mediated by conserved pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). TLRs expressed by mammalian and avian immune system cells have the capability to recognize pathogen-associated molecular patterns (PAMPs). Although, the role of TLR ligands in innate and adaptive responses in chickens has been characterized in spleen and bursa of Fabricius, considerably less is known about responses in cecal tonsils. The aim of the current study was to assess responses of mononuclear cells from cecal tonsils to treatment with the TLR2, TLR4 and TLR21 ligands, Pam3CSK4, lipopolysaccharide (LPS), and CpG oligodeoxynucleotide (ODN), respectively. All three ligands induced significant up-regulation of interferon (IFN)-γ, interleukin (IL)-1ß, IL-6 and CxCLi2/IL-8, whereas no significant changes were observed in expression of IL-13 or the antimicrobial peptides, avian ß-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). In general, CpG ODN elicited the highest cytokine responses by cecal tonsil mononuclear cells, inducing significantly higher expression compared to LPS and Pam3CSK4, for IFNγ, IL-1ß, IL-6 and CxCLi2 at various time points. These findings suggest the potential use of TLR21 ligands as mucosal vaccine adjuvants, especially in the context of pathogens of the intestinal tract.