The Effect of Necrotic Enteritis Challenge on Production Performance, Cecal Microbiome, and Cecal Tonsil Transcriptome in Broilers.

The objective of this study was to identify the effects of experimental necrotic enteritis (NE) infection on the production performance, gut microbiome, and cecal tonsil transcriptome in broiler birds. A total of 192 chicks were not-induced (control) or induced with NE. NE was induced by inoculating Eimeria maxima at 14 d of age and Clostridium perfringens at 19, 20, and 21 d of age. NE challenge increased (p < 0.01) NE lesion score at 7 days post-E.maxima infection (dpi), decreased (p < 0.01) average weight gain and increased (p < 0.01) mortality at 7 and 14 dpi. NE challenge increased (p < 0.05) gut permeability at 5, 6, and 7 dpi and increased ileal C. perfringens load at 5 dpi. NE challenge increased (p < 0.01) Eimeria oocyst shedding at 5, 6, 7, 8 and 14 dpi. NE challenge decreased (p < 0.05) the relative abundance of Lactobacillaceae and increased (p < 0.05) the relative abundance of Campylobacteriaceae, Comamonadaceae, and Ruminococcaceae at 6 dpi. NE challenge upregulated (p < 0.05) genes related to immune response and downregulated (p < 0.05) genes related to lipid metabolism at 6 dpi. It can be concluded that NE infection decreased beneficial bacteria and increased gut permeability.

Immunization of Broiler Chickens With a Killed Chitosan Nanoparticle Salmonella Vaccine Decreases Salmonella Enterica Serovar Enteritidis Load.

There is a critical need for an oral-killed Salmonella vaccine for broilers. Chitosan nanoparticle (CNP) vaccines can be used to deliver Salmonella antigens orally. We investigated the efficacy of a killed Salmonella CNP vaccine on broilers. CNP vaccine was synthesized using Salmonella enterica serovar Enteritidis (S. Enteritidis) outer membrane and flagella proteins. CNP was stable at acidic conditions by releasing 14% of proteins at pH 5.5. At 17 h post-incubation, the cumulative protein release for CNP was 75% at pH 7.4. Two hundred microliters of PBS with chicken red blood cells incubated with 20 µg/ml CNP released 0% hemoglobin. Three hundred chicks were allocated into 1) Control, 2) Challenge, 3) Vaccine + Challenge. At d1 of age, chicks were spray-vaccinated with PBS or 40 mg CNP. At d7 of age, chicks were orally-vaccinated with PBS or 20 µg CNP/bird. At d14 of age, birds were orally-challenged with PBS or 1 × 107 CFU/bird of S. Enteritidis. The CNP-vaccinated birds had higher antigen-specific IgY/IgA and lymphocyte-proliferation against flagellin (p < 0.05). At 14 days post-infection, CNP-vaccinated birds reversed the loss in gut permeability by 13% (p < 0.05). At 21 days post-infection, the CNP-vaccinated birds decreased S. Enteritidis in the ceca and spleen by 2 Log10 CFU/g, and in the small intestine by 0.6 Log10 CFU/g (p < 0.05). We conclude that the CNP vaccine is a viable alternative to conventional Salmonella poultry vaccines.

Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens.

Poultry infected with Salmonella mount an immune response initially, however the immune responses eventually disappear leading the bird to be a carrier of Salmonella. The hypothesis of this study is that Salmonella infection induces T regulatory cell numbers and cytokine production and suppress host T cells locally in the gut to escape the host immune responses. An experiment was conducted to comparatively analyze the effect of S. enterica ser. Enteritidis (S. Enteritidis) and S. enterica ser. Heidelberg (S. Heidelberg) infection on CD4+CD25+ T regulatory cell properties in chickens. A total of 144 broiler chicks were randomly distributed into three experimental groups of non-infected control, S. Enteritidis infected and S. Heidelberg infected groups. Chickens were orally inoculated with PBS (control) or 5x106 CFU/mL of either S. Enteritidis or S. Heidelberg at 3 d of age. Each group was replicated in six pens with eight chickens per pen. Chickens infected with S. Enteritidis had 6.2, 5.4, and 3.8 log10 CFU/g, and chickens infected with S. Heidelberg had 7.1, 4.8, and 4.1 log10 CFU/g Salmonella in the cecal contents at 4, 11, and 32 dpi, respectively. Both S. Enteritidis and S. Heidelberg were recovered from the liver and spleen 4 dpi. At 4, 11, and 32 dpi, chickens infected with S. Enteritidis and S. Heidelberg had increased CD4+CD25+ cell numbers as well as IL-10 mRNA transcription of CD4+CD25+ cells compared to that in the control group. CD4+CD25+ cells from S. Enteritidis- and S. Heidelberg-infected chickens and restimulated with 1 µg antigen in vitro, had higher (P < 0.05) IL-10 mRNA transcription than the CD4+CD25+ cells from the non-infected controls Though at 4dpi, chickens infected with S. Enteritidis and S. Heidelberg had a significant (P < 0.05) increase in CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, the CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, were comparable to that in the control group at 11 and 32dpi identifying that the host inflammatory response against Salmonella disappears at 11 dpi. It can be concluded that S. Enteritidis and S. Heidelberg infection at 3 d of age induces a persistent infection through inducing CD4+CD25+ cells and altering the IL-10 mRNA transcription of CD4+CD25+ cell numbers and cytokine production in chickens between 3 to 32 dpi allowing chickens to become asymptomatic carriers of Salmonella after 18 dpi.

Salmonella chitosan nanoparticle vaccine administration is protective against Salmonella Enteritidis in broiler birds.

Salmonella control strategies include vaccines that help reduce the spread of Salmonella in poultry flocks. In this study we evaluated the efficacy of administering a live Salmonella vaccine followed by a killed Salmonella chitosan nanoparticle (CNP) vaccine booster on the cellular and humoral immunity of broilers. The CNP vaccine was synthesized with Salmonella Enteritidis (S. Enteritidis) outer-membrane-proteins (OMPs) and flagellin-proteins. At d1-of-age, one-hundred-sixty-eight chicks were allocated into treatments: 1) No vaccine, 2) Live vaccine (Poulvac®ST), 3) CNP vaccine, 4) Live+CNP vaccine. At d1-of-age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7-of-age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14-of-age, birds were challenged with 1×109 CFU/bird S. Enteritidis. There were no significant differences in body-weight-gain (BWG) or feed-conversion-ratio (FCR). At 8h-post-challenge, CNP and Live+CNP-vaccinated birds had 17% and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d28-of-age, CNP, Live, and Live+CNP-vaccinated birds had 33%, 18%, and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d14-of-age, Live+CNP-vaccinated birds had 46% greater levels (P<0.05) of anti-Salmonella OMPs IgY in serum, compared to control. At d21-of-age, splenocytes from CNP and Live-vaccinated birds had increased (P<0.05) T-lymphocyte proliferation at 0.02 mg/mL OMPs stimulation compared to the control. At d28-of-age, CNP and Live+CNP-vaccinated birds had 0.9 Log10 CFU/g and 1 Log10 CFU/g decreased S. Enteritidis cecal loads (P<0.05), respectively, compared to control. The CNP vaccine does not have adverse effects on bird's BWG and FCR or IL-1ß, IL-10, IFN-γ, or iNOS mRNA expression levels. It can be concluded that the CNP vaccine, as a first dose or as a booster vaccination, is an alternative vaccine candidate against S. Enteritidis in broilers.

A Novel Approach against Salmonella: A Review of Polymeric Nanoparticle Vaccines for Broilers and Layers.

This work discusses the present-day limitations of current commercial Salmonella vaccines for broilers and layers and explores a novel approach towards poultry vaccination using biodegradable nanoparticle vaccines against Salmonella. With the increasing global population and poultry production and consumption, Salmonella is a potential health risk for humans. The oral administration of killed or inactivated vaccines would provide a better alternative to the currently commercially available Salmonella vaccines for poultry. However, there are currently no commercial oral killed-vaccines against Salmonella for use in broilers or layers. There is a need for novel and effective interventions in the poultry industry. Polymeric nanoparticles could give way to an effective mass-administered mucosal vaccination method for Salmonella. The scope of this work is limited to polymeric nanoparticles against Salmonella for use in broilers and layers. This review is based on the information available at the time of the investigation.

Effects of Lactobacillus reuteri and Streptomyces coelicolor on Growth Performance of Broiler Chickens.

There are well documented complications associated with the continuous use of antibiotics in the poultry industry. Over the past few decades, probiotics have emerged as viable alternatives to antibiotics; however, most of these candidate probiotic microorganisms have not been fully evaluated for their effectiveness as potential probiotics for poultry. Recent evaluation of a metagenome of broiler chickens in our laboratory revealed a prevalence of Lactobacillus reuteri (L. reuteri) and Actinobacteria class of bacteria in their gastrointestinal tract. In this study Lactobacillus reuteri and Streptomyces coelicolor (S. coelicolor) were selected as probiotic bacteria, encapsulated, and added into broiler feed at a concentration of 100 mg/kg of feed. In an 8-week study, 240 one day-old chicks were randomly assigned to four dietary treatments. Three dietary treatments contained two probiotic bacteria in three different proportions (L. reuteri and S. coelicolor individually at 100 ppm, and mixture of L. reuteri and S. coelicolor at 50 ppm each). The fourth treatment had no probiotic bacteria and it functioned as the control diet. L. reuteri and S. coelicolor were added to the feed by using wheat middlings as a carrier at a concentration of 100 ppm (100 mg/kg). Chickens fed diets containing L. reuteri and S. coelicolor mixture showed 2% improvement in body weight gain, 7% decrease in feed consumption, and 6-7% decrease in feed conversion ratios. This research suggests that L. reuteri and S. coelicolor have the potential to constitute probiotics in chickens combined or separately, depending on the desired selection of performance index.

Characterizing the immune response of chickens to Campylobacter jejuni (Strain A74C).

Campylobacter is one of the major foodborne pathogens causing bacterial gastroenteritis worldwide. The immune response of broiler chickens to C. jejuni is under-researched. This study aimed to characterize the immune response of chickens to Campylobacter jejuni colonization. Birds were challenged orally with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or with 0.5 mL of 0.85% saline. Campylobacter jejuni persisted in the ceca of challenged birds with cecal colonization reaching 4.9 log10 CFU/g on 21 dpi. Campylobacter was disseminated to the spleen and liver on 7 dpi and was cleared on 21 dpi from both internal organs. Challenged birds had a significant increase in anti-Campylobacter serum IgY (14&21 dpi) and bile IgA (14 dpi). At 3 dpi, there was a significant suppression in T-lymphocytes derived from the cecal tonsils of birds in the challenge treatment when compared to the control treatment after 72 h of ex vivo stimulation with Con A or C. jejuni. The T-cell suppression on 3 dpi was accompanied by a significant decrease in LITAF, K60, CLAU-2, IL-1ß, iNOS, and IL-6 mRNA levels in the ceca and an increase in nitric oxide production from adherent splenocytes of challenged birds. In addition, on 3 dpi, there was a significant increase in CD4+ and CD8+ T lymphocytes in the challenge treatment. On 14 dpi, both pro and anti-inflammatory cytokines were upregulated in the spleen, and a significant increase in CD8+ T lymphocytes in Campylobacter-challenged birds' ceca was observed. The persistence of C. jejuni in the ceca of challenged birds on 21 dpi was accompanied by an increase in IL-10 and LITAF mRNA levels, an increase in MNC proliferation when stimulated ex-vivo with the diluted C. jejuni, an increase in serum specific IgY antibodies, an increase in both CD4+ and CD8+ cells, and a decrease in CD4+:CD8+ cell ratio. The balanced Th1 and Th2 immune responses against C. jejuni might explain the ceca's bacterial colonization and the absence of pathology in Campylobacter-challenged birds. Future studies on T lymphocyte subpopulations should elucidate a pivotal role in the persistence of Campylobacter in the ceca.

In vitro characterization and immunogenicity of chitosan nanoparticles loaded with native and inactivated extracellular proteins from a field strain of Clostridium perfringens associated with necrotic enteritis.

There are currently no licensed vaccines against Clostridium perfringens which causes necrotic enteritis in poultry. Chitosan nanoparticles were formulated with native (CN) or toxoids (CT) of extracellular proteins (ECP) of C. perfringens, both surface-tagged with Salmonella flagellar proteins. In a pH stability assay, CN and CT nanoparticles released 6% and 0% of their protein at 8.0 pH. In a protein release assay, CN and CT nanoparticles released 16% and 10% of their protein respectively at 7.4 pH after 24 h. CN and CT nanoparticles incubated at 100 µg/mL PBS with Chicken RBCs released 1% and 0% hemoglobin respectively. Ninety broilers were randomly assigned to treatments; sham-vaccinated (Control), CN-vaccinated (CN), and CT-vaccinated (CT). Each bird was orally gavaged with 50 µg vaccine in 0.5 mL PBS or 0.5 mL PBS only on d 0, 3, 7 and 14 of age. At 21 d of age, the CN group had higher anti-ECP IgA than control (P < 0.05). At 21 d of age, the CN and CT group had higher anti-ECP IgA than control (P < 0.05). At 17 d of age, the CN group had higher anti-flagellar IgG than control (P < 0.05). At 10 d of age, the CN group had higher anti-flagellar IgA than control (P < 0.05). Splenic T cells from chickens in the CN and CT group ex-vivo stimulated with 0.05 mg/mL ECP, had higher proliferation control (P < 0.05, P < 0.01 respectively). Splenic T cells from chickens in the CN and CT groups ex-vivo stimulated with 0.1 mg/mL ECP had proliferation than control (P < 0.05). Pooled serum from 17 d of age CN and CT-vaccinated birds partially neutralized toxins in 50 µg of ECP (P < 0.05). Pooled serum from 28 d of age CN-vaccinated birds also partially neutralized toxins in 50 µg of ECP. The result from this study indicates the potential for chitosan loaded with Clostridium perfringens extracellular proteins to be applied to necrotic enteritis challenge studies.