The poultry industry has recently undergone transitions into antibiotic free production, and viable antibiotic alternatives, such as probiotics, are necessary. Through in ovo probiotic inoculation, beneficial microflora development in the gastrointestinal tract may occur prior to hatch without negatively impacting chick performance. Therefore, the objective of the present study was to observe the impacts of the injection of probiotic bacteria individually or combined into fertile broiler hatching eggs on hatch and live performance characteristics. A total of 2,080 fertile broiler hatching eggs were obtained from a commercial source. On day 18 of incubation, 4 in ovo injected treatments were applied: 1.) Marek's Disease (HVT) vaccination, 2.) L. animalis (∼106 cfu/50μl), 3.) E. faecium (∼106 cfu/50μl), and 4.) L. animalis + E. faecium (∼106 cfu & ∼106 cfu/50μl each). On day of hatch, hatchability and hatch residue data were recorded. A portion of male chicks from each treatment were placed in a grow-out facility for a 21 d grow-out (18 chicks/pen × 10 pens/treatment = 720 male chicks) with a corn and soy bean meal-based diet without antibiotics or antibiotic alternatives. Performance data and gastrointestinal samples were collected on days 0, 7, 14, and 21. Results indicated no differences in all hatch parameters between treatments (P > 0.05) except for % pipped, where the L. animalis treatment had lower % pipped eggs compared to the HVT control and E. faecium treatments (P = 0.04). No differences were observed in body weight gain or mortality (P > 0.05). Probiotic treatments altered gastrointestinal tissue length, weight, and pH. This resulted in all in ovo injected probiotic treatments increasing feed conversion ratio (FCR) from days 7 to 14 as compared to the control (P = 0.01). Differences in FCR were not observed in any other week of data collection (days 0 to 7, 14 to 21, or 0 to 21; P > 0.05). Although probiotics altered live performance from days 7 to 14, these data suggest that in ovo inoculations of L. animalis and E. faecium in combination are viable probiotic administration practices that potentially improve hatch characteristics and gastrointestinal tract development.

Vaccination strategies are used to manage Salmonella in chickens. Salmonella-killed vaccines are considered safer since they are inactivated. However, little is known regarding the cellular immune activities at the site of vaccine administration of Salmonella-killed vaccines. The growing feather (GF) cutaneous test has been shown to be an effective bioassay to monitor local tissue/cellular responses. We assessed local and systemic antibody responses initiated by intradermal injection of Salmonella-killed vaccines into GF-pulps of 14–15-week-old pullets. Treatments consisted of two autogenous Salmonella-killed vaccines (SV1 and SV2), S. Enteritidis (SE) lipopolysaccharide (SE-LPS), and the water-oil-water (WOW) emulsion vehicle. GF-pulps were collected before (0 h) and at 6, 24, 48, and 72 h post-GF-pulp injection for leukocyte population analysis, while heparinized blood samples were collected before (0 d) and at 3, 5, 7, 10, 14, 21, and 28 d after GF-pulp injections to assess plasma levels (a.u.) of SE-specific IgM, avian IgY (IgG), and IgA antibodies using an ELISA. Injection of GF-pulps with SV1, SV2, or SE-LPS, all in a WOW vehicle, initiated inflammatory responses characterized by the recruitment of heterophils, monocytes/macrophages, and a few lymphocytes. The WOW vehicle emulsion alone recruited more lymphocytes than vaccines or SE-LPS. The SV1 and SV2 vaccines stimulated Salmonella-specific IgM and IgA early, while IgG levels were greatly elevated later during the primary response. Overall, SV1 and SV2 stimulated a heterophil and macrophage-dominated local inflammatory- and SE-specific humoral response with an isotype switch from IgM to IgG, characteristic of a T-dependent primary antibody response. This study provides comprehensive information on innate and adaptive immune responses to autogenous Salmonella-killed vaccines and their components that will find application in the management of Salmonella in poultry.

To assess effects of environmental heat stress (HS) on the local and systemic inflammatory responses to lipopolysaccharide (LPS), broilers were reared under thermoneutral (TN) or cyclic HS conditions. Thermoneutral temperatures followed commercial production settings, with HS broilers exposed to 35 °C for 14 h/day from 4 days onward. At 37 days, HS- and TN-broilers were assigned to either LPS (100 μg/mL) or endotoxin-free phosphate-buffered saline (PBS; vehicle) treatments, eight each to HS- and TN-LPS, four each to HS- and TN-PBS. Treatments were administered by intradermal injection of growing feather (GF) pulps; 10 μL/GF; 12 GF/broiler. Blood and GF were collected before and at 6 and 24 h post-injection to assess leukocyte population changes in GF-pulps and blood, reactive oxygen species (ROS) generation and cytokine expression in GF-pulps, and plasma concentrations of alpha-1 acid glycoprotein (AGP-1). HS-LPS broilers had lower (p ≤ 0.05) infiltration of heterophils and macrophages, ROS generation, and inflammatory cytokine expression in GF-pulps, and lacked the increases in heterophil, monocyte, and plasma AGP-1 concentrations observed in TN-LPS broilers. HS-broilers had similar or greater drops in blood lymphocytes 6 h post-LPS or -PBS injection, respectively, and lower baseline levels (p ≤ 0.05) of circulating T- and B-lymphocytes than TN-broilers. Results indicated that cyclic HS reduced the local and systemic acute inflammatory responses to LPS in broilers, likely impairing their innate defense against microbial infection.

Selection for water consumption could impact broiler breeders’ immune capabilities. To assess these impacts of selection based on the water conversion ratio (WCR), three trials were conducted using broiler breeders from the modern random bred (MRB), low (L)WCR, and high (H)WCR lines. Ten- to 11-week-old male broilers received intradermal (i.d.) growing feather (GF)-pulp injections of LPS (Trial 1) or PGN (Trial 2), to assess local (GF-pulp) and systemic (blood) inflammatory responses over 24 h and 72 h p.i., respectively. Measurements included leukocyte profiles in GF-pulps and blood, GF cytokine mRNA expression and reactive oxygen species (ROS) generation, and plasma concentrations of α1-acid glycoprotein (AGP-1). In Trial 3, 14-week-old pullets were immunized by i.d. GF-pulp injection of SEV (10⁸ CFU/mL). Leukocyte profiles in the GF-pulp and blood were measured over 72 h and plasma levels of SEV-specific IgM, IgY(G), and IgA antibodies over 4 weeks p.i. Independent of the line, phagocytes infiltrated GF-pulps by 6 h post-LPS injection (p ≤ 0.05), while lymphocytes were the major leukocyte recruited in response to PGN (p ≤ 0.05). However, with both LPS and PGN, HWCR broilers were less effective in recruiting lymphocytes than MRB and LWCR broilers, which had similar lymphocyte infiltration levels. There were no line differences in GF-pulp cytokine mRNA expression and ROS generation, nor in blood leukocyte and AGP-1 concentrations, following LPS injections. Independent of the line, SEV immunization stimulated similar phagocyte recruitment profiles; however, the LWCR and MRB lines had a higher infiltration of lymphocytes (esp. B cells) than the HWCR line (p ≤ 0.05). Independent of the line, SEV immunization triggered a robust, high-quality, primary SE-specific antibody response (p ≤ 0.05). Collectively, selection for improved water efficiency in the LWCR broiler breeder lines did not negatively impact immune response capabilities to LPS, PGN, and a killed SEV.

Background/Objectives: Commercial poultry flocks undergo Salmonella vaccinations to manage salmonellosis outbreaks. Due to reports of severe injection site reactions to Salmonella bacterins, assessment of local inflammatory responses is necessary. The objective was to assess local inflammatory and systemic humoral immune responses to commercial autogenous Salmonella bacterin vaccines (SV1 or SV2) following primary or secondary intradermal (i.d.) vaccination in Light-Brown Leghorns (LBLs). Methods: LBL pullets received primary (14 wks) or secondary (19 wks) vaccination by i.d. growing feather (GF) pulp injection of SV1, SV2, Salmonella Enteritidis (SE) lipopolysaccharide (LPS), or water–oil–water emulsion (V). Local leukocyte levels and relative cytokine mRNA expression were monitored before (0 d) and at 6 h, 1 d, 2 d, 3 d, 5 d, and 7 d post-GF pulp injection (p.i.). Blood was collected through 28 d post-primary or -secondary vaccination, and SE-specific antibodies were quantified via ELISA. Results: Primary vaccine administration increased local heterophil and macrophage levels and increased IL-6 and IL-8 mRNA expressions at 6 h p.i., independent of treatment. Secondary administration extended these local immune activities through 3 d p.i. and included prolonged IL-17A mRNA expression. Primary and secondary GF-pulp injection with V resulted in rapid lymphocyte recruitment by 6 h p.i., comprised primarily of CD4⁺ and γδ T cells. SV1 and SV2 also produced a T-dependent systemic humoral immune response, as indicated by the IgM-to-IgG isotype switch, along with a memory phenotype in the secondary response. Conclusions: These commercial-killed Salmonella vaccines, when prepared in water–oil–water emulsions, stimulated prolonged innate and T helper (Th) 17-type inflammatory responses at the injection site and produced a classic systemic humoral immune response after a second vaccination. Further research is needed to determine if extended inflammation influences adaptive immune responses in eliminating Salmonella infection.

At the turn of the 21st century, the importance of maintaining a balanced microbiome was brought to the forefront of the microbiology, immunology, and physiology research fields. Exploring the complex interactions between vaccine administration, mucosal microbiome, oral tolerance, and enteric inflammation in health and disease is challenging since environmental factors (such as diet and sanitation) have major influences on gut microbiota composition. High enteric pathogen load has been shown to contribute to dampened cell-mediated and humoral immune responses to vaccines in human case studies, either through elevated enteric inflammation or increased tolerance to environmental microbes. Although antibiotic and probiotic interventions have been evaluated in human health as well as research animal models, effective measures to mediate vaccine hyporesponsiveness are still ill-defined. Research in this field is becoming increasingly important for managing flock health in commercial poultry production, especially as antibiotic-free production is more prevalent and vaccination programs remain extensive during the first weeks of a bird’s life. By understanding the cellular interactions between commensal microbiota, vaccine antigens, and the host immune system (particularly in avian models), advancements in bacterial and viral vaccine immunogenicity could lead to improved flock health in meat-type and egg-type chickens in the future.