Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks.

BACKGROUND: Infectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages. RESULTS: Although whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages. CONCLUSIONS: Overall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens.

Protection of Neonatal Broiler Chickens Following in ovo Delivery of Oligodeoxynucleotides Containing CpG Motifs (CpG-ODN) Formulated with Carbon Nanotubes or Liposomes.

Unformulated oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to stimulate the innate immune system against a variety of bacterial, viral, and protozoan infections in a variety of vertebrate species. We have previously shown that in ovo delivery of unformulated CpG-ODN was able to significantly protect neonatal broiler chickens against Escherichia coli or Salmonella Typhimurium infections. The objectives of this study were to examine the safety and immunoprotective effects of CpG-ODN formulated with 2 types of carbon nanotubes (CNTs) or 2 types of lipid-surfactant (LSC) delivery systems in neonatal broilers against E. coli septicemia. Embryonated eggs, which had been incubated for 18 days, received either 50 µg of CNT-CpG-ODN, 50 µg of LSC-CpG-ODN, 50 µg of unformulated CpG-ODN, or saline. Four days after exposure to CpG-ODN (day 1 posthatch), 1 x 10(4) or 1 x 10(5) colony-forming units of a virulent strain of E. coli isolated from a turkey with septicemia were inoculated subcutaneously in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 8 days following challenge with E. coli. Bacterial isolations and pathologic observations were conducted immediately after birds were dead or euthanatized. The survival rate of birds in groups receiving saline following E. coli infection was 20% to 30%. In contrast, birds receiving CpG-ODN formulations had a significantly higher survival rate of 60% to 80% (P < 0.01). Bacterial loads and clinical scores were significantly lower (P < 0.05) in groups treated with CNT- or LSC-CpG-ODN compared to the groups receiving CpG-ODN or saline. Moreover, there is no evidence of any adverse effects of these formulations in any organs or in growth rates of birds until 42 days of age. This is the first time that CpG-ODN formulated with CNT and LSC have been demonstrated to have an immunomodulatory effect against an E. coli infection in neonatal broiler chickens following in ovo delivery.

Development of a multivalent adjuvanted inactivated vaccine against variant arthrotropic avian reoviruses.

Variant avian reoviruses (ARVs) are economically important emerging pathogens of poultry, which mainly affect young broiler chickens and cause significant production losses. Currently, there are no effective commercial vaccines available for control and prevention of emerging variant ARVs. In this study, monovalent inactivated adjuvated (20% Emulsigen D) broiler breeder vaccines containing antigens from ARV genotype cluster (C) group -2, -4, -5, or -6, and a multivalent vaccine containing antigens from all the four indicated genotypic cluster groups were developed and evaluated for their efficacy in protecting broiler progenies against homologous or heterologous ARV challenge. The use of monovalent or multivalent inactivated vaccines in a prime-boost immunization strategy induced the production of ARV specific antibodies in broiler breeders. The maternal antibodies were effectively transferred to broiler progenies. Broiler progenies obtained from immunized breeders demonstrated milder clinical symptoms and reduced gross and histopathological lesions after homologous ARV challenge. More severe gross and histological lesions were observed in challenged progenies from unvaccinated broiler breeders. However, cross protection was not observed when either of the monovalent-vaccine groups were challenged with a heterologous virus. In addition, the progenies from the unvaccinated ARV challenged control or heterologous ARV challenged vaccinated groups had significantly reduced body weight gain (p < 0.01) than the unchallenged-control, challenged-multivalent, or homologous ARV-challenged monovalent vaccine groups. However, homologous ARV challenged progenies in the multivalent or monovalent vaccine groups had similar body weight gain as the control unchallenged group with significantly reduced viral load (p < 0.01) in the gastrocnemius tendon tissue. This study indicates that broad-spectrum protection of broiler progenies from variant ARV infections is feasible through the development of multivalent vaccines after proper characterization, selection and incorporation of multiple antigens based on circulating ARV genotypes in targeted regions.

Virulence of Emerging Arthrotropic Avian Reoviruses Correlates With Their Ability to Activate and Traffic Interferon-γ Producing Cytotoxic CD8+ T Cells Into Gastrocnemius Tendon.

Newly emerging arthrotropic avian reoviruses (ARVs) are genetically divergent, antigenically heterogeneous, and economically costly. Nevertheless, the mechanism of emerging ARV-induced disease pathogenesis and potential differences in virulence between virus genotypes have not been adequately addressed. In this study, the life cycle of ARV, including the formation of cytoplasmic ARV neo-organelles, paracrystalline structures, and virus release mechanisms, were characterized in the infected host cell by transmission electron microscopy (TEM). In addition, progressive changes in the structure of infected cells were investigated by time-lapse and field emission scanning electron (FE-SE) microscopy. ARVs from the four genotypic cluster groups included in the study caused gross and microscopic lesions in the infected birds. Marked infiltration of γδT cells, CD4+ and CD8+ T lymphocytes were observed in ARV infected tendon tissues starting day 3 post-infection. The ARV variant from genotype cluster-2 triggered significantly high trafficking of IFN-γ producing CD8+ T lymphocytes in tendon tissues and concomitantly showed high morbidity and severe disease manifestations. In contrast, the ARV variant from genotype cluster-4 was less virulent, caused milder disease, and accompanied less infiltration of IFN-γ producing CD8+ T cells. Interestingly, when we blunted antiviral immune responses using clodronate liposomes (which depletes antigen-presenting cells) or cyclosporin (which inhibits cytokine production that regulates T-cell proliferation), significantly lower IFN-γ producing CD8+ T cells infiltrated into tendon tissues, resulting in reduced tendon tissues apoptosis and milder disease manifestations. In summary, these data suggest that the degree of ARV virulence and tenosynovitis/arthritis are potentially directly associated with the ability of the virus to traffic massive infiltration of cytotoxic CD8+ T cells into the infected tissues. Moreover, the ability to traffic cytotoxic CD8+ T cells into infected tendon tissues and the severity of tenosynovitis differ between variants from different ARV genotype cluster groups. However, more than one virus isolate per genotype group needs to be tested to further confirm the association of pathogenicity with genotype. These findings can be used to further examine the interaction of viral and cellular pathways which are essential for the pathogenesis of the disease at the molecular level and to develop effective disease control strategies.

Comparison of Therapeutic Antibiotics, Probiotics, and Synthetic CpG-ODNs for Protective Efficacy Against Escherichia coli Lethal Infection and Impact on the Immune System in Neonatal Broiler Chickens.

The poultry industry needs alternatives to antibiotics, as there are growing public concerns about the emergence of antimicrobial resistance owing to antimicrobial use in animal production. We have reported that the administration of neonatal chicks with synthetic DNA oligodeoxynucleotides containing unmethylated cytosine guanine dinucleotide (CpG) motifs (CpG-ODN) can protect against bacterial pathogens in chickens. The objective of this study was to compare the immunoprotective effects of CpG-ODN and probiotics against Escherichia coli infection vs. commonly used therapeutic antibiotics. Day-old broiler chicks were divided into five groups (n = 35/group; 30 for the challenge experiment and 5 for the flow cytometry analysis). The chicks in Group 1 received a single dose of CpG-ODN by the intramuscular route on day 4 (D4) posthatch (PH), and Group 2 received drinking water (DW) with a probiotic product (D1-D15 PH, DW). The Group 3 chicks received tetracycline antibiotics during D9-D13 in DW; the Group 4 chicks got sodium sulfamethazine on D9, D10, and D15 PH in DW; and the Group 5 chicks were administered intramuscular (IM) saline D4 PH, DW. We challenged all the groups (n = 30/group) with E. coli (1 × 105 or 1 × 106 colony-forming units/bird) on D8 PH through the subcutaneous route. Our data demonstrated that the CpG-ODNs, but not the probiotics, could protect neonatal broiler chickens against lethal E. coli septicemia, as would the tetracycline or sodium sulfamethazine. The flow cytometry analysis (n = 5/group) revealed enrichment of immune cells in the CpG-ODN group and a marked decrease in macrophages and T-cell numbers in antibiotics-treated groups, indicating immunosuppressive effects. Our data showed that, like therapeutic antibiotics, CpG-ODNs reduced clinical signs, decreased bacterial loads, and induced protection in chicks against E. coli septicemia. Unlike therapeutic antibiotics-induced immunosuppressive effects, CpG-ODN caused immune enrichment by increasing chicken immune cells recruitment. Furthermore, this study highlights that, although therapeutic antibiotics can treat bacterial infections, the ensuing immunosuppressive effects may negatively impact the overall chicken health.

Comparación de antibióticos terapéuticos, probióticos y CpG-ODN sintéticos en su eficacia protectora contra la infección letal por Escherichia coli y el impacto en el sistema inmunológico en pollos de engorde recién eclosionados. La industria avícola necesita alternativas a los antibióticos ya que existe una creciente preocupación pública sobre la aparición de resistencia a los antimicrobianos debido a su uso en la producción animal. Se ha informado que la administración de oligodesoxinucleótidos de ADN sintético que contienen motivos de dinucleótidos de citosina guanina (CpG) no metilados (CpG-ODN) a pollitos recién eclosionados puede proteger contra patógenos bacterianos en pollos. El objetivo de este estudio fue comparar los efectos inmunoprotectores de CpG-ODN y de los probióticos contra la infección por Escherichia coli frente a los antibióticos terapéuticos de uso común. Los pollos de engorde de un día se dividieron en cinco grupos (n = 35/grupo; 30 para el experimento de desafío y 5 para análisis de citometría de flujo). Los pollitos del Grupo 1 recibieron una dosis única de CpG-ODN por vía intramuscular el día 4 (D4) después de la eclosión (PH), y el Grupo 2 recibió agua potable (DW) con un producto probiótico del día uno al quince después de la eclosion en agua de bebida. Los pollitos del Grupo 3 recibieron tetraciclina durante los días nueve a trece (D9­D13) en agua de bebida (DW9; los pollitos del Grupo 4 recibieron sulfametazina de sodio en los días nueve, diez y 15 (D9, D10 y D15) después de la eclosion en agua de bebida; ya los pollitos del Grupo 5 se les administró solución salina intramuscular (IM) al día cuatro después de la eclosión en agua de bebida. Se desafiaron todos los grupos (n = 30/grupo) con E. coli (1 × 105 o 1 × 106 unidades formadoras de colonias/ave) en el día ocho después de la eclosión por vía subcutánea. Nuestros datos demostraron que los CpG-ODN, pero no los probióticos, pudieron proteger a los pollos de engorde recién eclosionados contra la septicemia letal por E. coli, al igual que la tetraciclina o la sulfametazina sódica. El análisis de citometría de flujo (n = 5/grupo) reveló un enriquecimiento de células inmunes en el grupo CpG-ODN y una marcada disminución en el número de macrófagos y células T en los grupos tratados con antibióticos, lo que indica efectos inmunosupresores. Nuestros datos mostraron que, al igual que los antibióticos terapéuticos, los CpG-ODN redujeron los signos clínicos, disminuyeron las cargas bacterianas e indujeron protección en los pollitos contra la septicemia por E. coli. A diferencia de los efectos inmunosupresores inducidos por antibióticos terapéuticos, los CpG-ODN provocaron un enriquecimiento inmunitario al aumentar el reclutamiento de células inmunitarias de pollo. Además, este estudio destaca que, aunque los antibióticos terapéuticos pueden tratar las infecciones bacterianas, los efectos inmunosupresores resultantes pueden tener un impacto negativo en la salud general de los pollos.

Evaluation of five circulating strains of variant infectious bursal disease virus (varIBDV) for their immunogenicity as broiler breeder vaccines and protective efficacy in neonatal broiler chicks.

The majority of infectious bursal disease virus (IBDV) strains circulating in the broiler chicken industry in Canada are variant strains (varIBDV). Despite high levels of maternally derived antibodies (MtAb), the circulating varIBDVs can establish infection and cause severe immunosuppression in broiler chicks. The objective of this study was to evaluate circulating varIBDVs as broiler breeder vaccine candidates and investigate their protective efficacy against varIBDV challenge in their progeny chicks. Six groups of breeders (20 females/group) were vaccinated with varIBDV strains, SK09, SK10, SK11, SK12, and SK13 or saline at the age of 13 weeks and antibody response was determined by ELISA at 3-7-, and 20- weeks post-vaccination. We also included commercial chicks for the comparison. Results showed that SK-09 is the most antigenic strain, followed by SK-10, SK-12, and SK-13. In contrast, SK-11 showed the lowest antibody response, and over time, antibody titers steadily decreased. Eggs from breeders were collected at 21-week post-vaccination and incubated to produce their respective progenies. The serum antibody titer in day-old chicks showed a successful MtAb transfer. Progeny chicks (n = 40/group) were orally challenged with varIBDV-SK-09 strain at 6 days of age and serum antibody titer (19 d and 35 d of age), bursa to body weight ratio (19 d and 35 d of age), bursal viral load (9 d and 19 d of age) was examined to assess the protection against IBDV. Following the challenge, we found a significant increase in the antibody titers in MtAb-free and commercial vaccine groups than in the varIBDV groups, both at 19 d and 35 d of age. The BBW ratio and viral load data indicated a significant homologous and heterologous protection against varIBDV-SK-09 challenge by SK-09 and SK-10 MtAbs, respectively. Overall, this study demonstrated the feasibility of developing breeder vaccines using circulating varIBDV as candidate vaccine antigens.

Exposure of embryonating eggs to Enterococcus faecalis and Escherichia coli potentiates E. coli pathogenicity and increases mortality of neonatal chickens.

Enterococci and Escherichia coli are opportunistic pathogens of poultry and are associated with embryo and neonatal chick mortality. We have recently demonstrated that 56% of dead broiler chicken embryos in commercial hatcheries in western Canada were due to the coinfection of Enterococcus species and E. coli. The objective of this study was to investigate the host-pathogen interactions of Enterococcus faecalis and E. coli in developing chicken embryos. Embryonating eggs at 12 d of incubation were dipped in a solution of E. faecalis and/or E. coli for 30 s to expose the eggshell to study the migration and colonization of E. faecalis and E. coli in the internal organs of chicken embryos and subsequent neonatal chicken mortality following hatch. A multidrug-resistant E. faecalis isolate from a dead chicken embryo and an E. faecalis isolate from a case of yolk sac infection were able to colonize the internal organs of chicken embryos rapidly compared to an E. faecalis isolate from a healthy chicken without affecting viability or hatchability of embryos. Although E. faecalis colonized internal organs of chicken embryos, no evidence of inflammation of these organs nor the expression of virulence genes of E. faecalis was observed. Although E. faecalis and E. coli alone did not affect the viability of embryos, a significantly high neonatal chicken mortality (27%) was observed following exposure of embryos to both E. faecalis and E. coli. Upregulation of IL-1 and CXCR4 was evident 48 h before peak mortality of neonatal chickens; this could suggest a possible link of cytokine dysregulation to increased mortality in coinfected neonatal chickens. However, further studies are warranted to investigate this issue vis-à-vis coinfection with E. faecalis and E. coli in chicken embryos and neonatal chickens.

CpG-ODN induced antimicrobial immunity in neonatal chicks involves a substantial shift in serum metabolic profiles.

Synthetic CpG-ODNs can promote antimicrobial immunity in neonatal chicks by enriching immune compartments and activating immune cells. Activated immune cells undergo profound metabolic changes to meet cellular biosynthesis and energy demands and facilitate the signaling processes. We hypothesize that CpG-ODNs induced immune activation can change the host's metabolic demands in neonatal chicks. Here, we used NMR-based metabolomics to explore the potential of immuno-metabolic interactions in the orchestration of CpG-ODN-induced antimicrobial immunity. We administered CpG-ODNs to day-old broiler chicks via intrapulmonary (IPL) and intramuscular (IM) routes. A negative control group was administered IPL distilled water (DW). In each group (n = 60), chicks (n = 40) were challenged with a lethal dose of Escherichia coli, two days post-CpG-ODN administration. CpG-ODN administered chicks had significantly higher survival (P < 0.05), significantly lower cumulative clinical scores (P < 0.05), and lower bacterial loads (P < 0.05) compared to the DW control group. In parallel experiments, we compared NMR-based serum metabolomic profiles in neonatal chicks (n = 20/group, 24 h post-treatment) treated with IM versus IPL CpG-ODNs or distilled water (DW) control. Serum metabolomics revealed that IM administration of CpG-ODN resulted in a highly significant and consistent decrease in amino acids, purines, betaine, choline, acetate, and a slight decrease in glucose. IPL CpG-ODN treatment resulted in a similar decrease in purines and choline but less extensive decrease in amino acids, a stronger decrease in acetate, and a considerable increase in 2-hydroxybutyrate, 3-hydroxybutyrate, formic acid and a mild increase in TCA cycle intermediates (all P < 0.05 after FDR adjustment). These perturbations in pathways associated with energy production, amino acid metabolism and nucleotide synthesis, most probably reflect increased uptake of nutrients to the cells, to support cell proliferation triggered by the innate immune response. Our study revealed for the first time that CpG-ODNs change the metabolomic landscape to establish antimicrobial immunity in neonatal chicks. The metabolites highlighted in the present study can help future targeted studies to better understand immunometabolic interactions and pinpoint the key molecules or pathways contributing to immunity.

The dynamics of molecular evolution of emerging avian reoviruses through accumulation of point mutations and genetic re-assortment.

In the last decade, the emergence of variant strains of avian reovirus (ARV) has caused enormous economic impact in the poultry industry across Canada and USA. ARVs are non-enveloped viruses with ten segments of double-stranded RNA genome. So far, only six genotyping cluster groups are identified worldwide based on sequence analysis of the σC protein encoded by the S1 segment. In this study, we performed deep next generation whole-genome sequencing and analysis of twelve purified ARVs isolated from Saskatchewan, Canada. The viruses represent different genotyping cluster. A genome-wide sequence divergence of up to 25 per cent was observed between the virus isolates with a comparable and contrasting evolutionary history. The proportion of synonymous single-nucleotide variations (sSNVs) was higher than the non-synonymous (ns) SNVs across all the genomic segments. Genomic segment S1 was the most variable as compared with the other genes followed by segment M2. Evidence of positive episodic/diversifying selection was observed at different codon positions in the σC protein sequence, which is the genetic marker for the classification of ARV genotypes. In addition, the N-terminus of σC protein had a persuasive diversifying selection, which was not detected in other genomic segments. We identified only four ARV genotypes based on the most variable σC gene sequence. However, a different pattern of phylogenetic clustering was observed with concatenated whole-genome sequences. Together with the accumulation of point mutations, multiple re-assortment events appeared as mechanisms of ARV evolution. For the first time, we determined the mean rate of molecular evolution of ARVs, which was computed as 2.3 × 10-3 substitution/site/year. In addition, widespread geographic intermixing of ARVs was observed between Canada and USA, and between different countries of the world. In conclusion, the study provides a comprehensive analysis of the complete genome of different genotyping clusters of ARVs including their molecular rate of evolution and spatial distribution. The new findings in this study can be utilized for the development of effective vaccines and other control strategies against ARV-induced arthritis/tenosynovitis in the poultry industry worldwide.

Non-viable chicken embryos: an overlooked niche harbouring a significant source of multidrug resistant bacteria in the poultry production.

Antimicrobial resistance (AMR) is a global issue, posing a grave threat to the public, animal, and environmental health. The AMR surveillance at the level of the hatchery is crucial to develop an AMR control strategy in the poultry industry. The objective of this study was to investigate the AMR profiles of bacteria isolated from yolk material of non-viable broiler chicken embryos at hatch from commercial hatcheries in western Canada. Antimicrobial susceptibility testing was done using the Kirby-Bauer disk diffusion method focusing on Escherichia coli (n = 170) and Enterococcus (n = 256) species, which are commonly used as indicators of AMR evolution. E. coli isolates were resistant to tetracycline, ampicillin, amoxycillin-clavulanic acid, triple sulpha, ceftiofur, gentamycin, and spectinomycin at the rate of 52.9%, 50.6%, 40.0% 31.8%, 29.4%, 29.4%, 21.8% respectively. Among those, 37.1% of E. coli were multidrug resistant. The descending order of antimicrobial resistance of E. faecalis was; tetracycline (61.9%), ceftiofur (46.2%), bacitracin (43.9%), erythromycin (31.4%) and tylosin (27.4%). Multidrug resistance was detected in 40.4% of E. faecalis isolates, and 85.7% of E. faecium isolates. To the best of our knowledge, this is the first report on AMR surveillance of non-viable chicken embryos. Overall, the present study revealed that non-viable chicken embryos, an overlooked niche for AMR surveillance, harbour multidrug-resistant E. coli, and enterococci that can be a substantial source of superbugs in the environment. Our data also highlight the urgency of including non-viable chicken embryos in AMR surveillance programme to understand AMR dissemination and its control.

Mucosal delivery of CpG-ODN mimicking bacterial DNA via the intrapulmonary route induces systemic antimicrobial immune responses in neonatal chicks.

The transition to antibiotic-free poultry production in the face of pathogenic threats is a very challenging task. We recently demonstrated that mucosal delivery of CpG-ODN alone by the intrapulmonary route (IPL) has potential as an effective alternative to antibiotics in neonatal chicks against Escherichia coli septicemia. How exactly mucosal delivery of CpG-ODN elicits, protective antibacterial immunity remained poorly understood. In this study, CpG-ODN or saline was delivered via the intrapulmonary route to day-old chicks (n = 80/group) using a compressor nebulizer in an acrylic chamber (1 mg/mL CpG-ODN for 15 minutes). In the first part of the study, two days after mucosal CpG-ODN delivery, 40 chicks from each group were challenged subcutaneously with 1 × 105 cfu (n = 20) or 1 × 106 cfu (n = 20) of E. coli and the mortality pattern was monitored for seven days. We found significantly higher survival, better clinical conditions and lower bacterial loads in chicks that received mucosal CpG-ODN. To explore the mechanisms behind this protective immunity, we first looked at the kinetics of the cytokine gene expression (three birds/ group/ time for 10 time-points) in the lungs and spleens. Multiplex gene analysis demonstrated a significant elevation of pro-inflammatory cytokine genes mRNA in the CpG-ODN group. Interleukin (IL)-1ß robustly upregulated many folds in the lung after CpG-ODN delivery. Lipopolysaccharide-induced tumor necrosis factor (LITAF) and IL-18 showed expression for an extended period in the lungs. Anti-inflammatory cytokine IL-10 was upregulated in both lungs and spleen, whereas IL-4 showed upregulation in the lungs. To investigate the kinetics of immune enrichment in the lungs and spleens, we performed flow cytometry, histology, and immunohistochemistry at 24, 48 and 72 hrs after CpG-ODN delivery. CpG-ODN treated lungs showed a significant enrichment with monocytes/macrophages and CD4+ and CD8+ T-cell subsets. Macrophages in CpG-ODN treated group demonstrated mature phenotypes (higher CD40 and MHCII expression). Importantly, mucosal delivery of CpG-ODN via the intrapulmonary route significantly enriched immune compartment in the spleen as well, suggesting a systemic effect in neonatal chicks. Altogether, intrapulmonary delivery of aerosolized CpG-ODN orchestrates protective immunity against E. coli septicemia by not only enhancing mucosal immunity but also the systemic immune responses.

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 µg of CpG-ODN or saline (n = ~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n = 3-4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 µg and 50 µg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1 × 104 or 1 × 105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 µg and 50 µg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.

Synthetic CpG-ODN rapidly enriches immune compartments in neonatal chicks to induce protective immunity against bacterial infections.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3-4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4+ and CD8+ T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4+ and CD8+ T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.

Broad spectrum protection of broiler chickens against inclusion body hepatitis by immunizing their broiler breeder parents with a bivalent live fowl adenovirus vaccine.

Historically, fowl adenovirus (FAdV) associated inclusion body hepatitis (IBH) was considered a secondary disease in broiler chickens associated with immunosuppression. However, we previously reported the occurrence of IBH as a primary disease in the broiler chicken industry in Canada as a result of infections with various FAdV serotypes. Therefore, the objectives of this study were to develop an immunization strategy in broiler breeders using live FAdV 11-1047 and FAdV8a-TR59 to confer homologous and heterologous protection in broiler progeny against IBH and to study the efficacy of natural exposure of naïve broiler breeders to a vaccine virus from live FAdV vaccinated birds as an immunization technique. Broiler breeders vaccinated orally with FAdV8a-TR59 (1 × 104 TCID50/bird) and FAdV11-1047 (1 × 104 TCID50/bird), FAdV8a-TR59 (1 × 106 TCID50/bird) and FAdV11-1047 (1 × 106 TCID50/bird) or FAdV8b (1 × 106 TCID50/bird) transferred substantial levels of neutralizing antibodies to their progeny. The efficacy of maternal antibodies was studied by challenging 14-day old broiler chickens with 1 × 107 TCID50 of FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 which are the dominant serotypes causing IBH outbreaks in Canada. Broiler chickens from the low and high dose vaccinated breeders were significantly protected against all serotypes of FAdV (P < 0.05). Comingling of unvaccinated broiler breeders with FAdV-vaccinated broiler breeders was an effective immunization technique for in-contact naïve birds. This study confirms that IBH can be effectively controlled in Canada by vaccination of broiler breeder parents with a bivalent vaccine containing live FAdV8a-TR59 and FAdV11-1047.

Shell-Less Egg Syndrome (SES) Widespread in Western Canadian Layer Operations Is Linked to a Massachusetts (Mass) Type Infectious Bronchitis Virus (IBV) Isolate.

A disease with a sudden drop in egg production and shell-less eggs called, shell-less egg syndrome (SES) has been observed in Western Canada egg layer flocks since 2010. The etiology of this disease is not known. We hypothesize that SES is caused by an infectious bronchitis virus (IBV) strain since it is known that IBV replicates in the shell gland causing various eggshell abnormalities. In this study, we screened egg layer flocks, in the provinces of Alberta (AB) and Saskatchewan (SK), with and without a history of SES for the presence of IBV infection. During 2015⁻2016, a total of 27 egg layer flocks were screened in AB (n = 7) and SK (n = 20). Eighty-one percent of the screened flocks (n = 22) were positive for IBV infection. Thirty of these isolates were successfully characterized using molecular tools targeting the most variable spike (S) 1 gene. IBV isolates from this study clustered into three genotypes based on partial S1 gene variability. The majority of the IBV isolates (70%) were Massachusetts (Mass) type, and the rest were either Connecticut (Conn) type or an uncharacterized genotype with genetic characteristics of Mass and Conn types. Since the majority of the IBV isolates included within the Mass type, we used a Mass type IBV isolate to reproduce SES in specific pathogen free (SPF) white leghorn chickens in lay. Further studies are warranted to investigate whether other IBV isolates can cause SES, to clarify the pathogenesis of SES and to develop a vaccine in order to prevent SES as observed in Western Canadian layer flocks.

Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH).

Fowl adenovirus (FAdV) is comprised of five species (A to E) and 12 serotypes (1-7, 8a, 8b, 9-11). Inclusion body hepatitis (IBH) is caused by FAdV-7, 8a, 8b (species E) and FAdV-2 and 11 (species D). Commercial vaccines against IBH are not available in Canada. Autogenous FAdV broiler breeder vaccines are now used in some areas where outbreaks of IBH are occurring. The objective of this study was to evaluate the efficacy of a bivalent (species D and E) live and an inactivated FAdV broiler breeder vaccine in protecting broiler chicks against IBH through maternal antibody (MtAb) transfer. FAdV seronegative broiler breeders (n = 300/group) received either a live or inactivated bivalent (FAdV-8b-SK + FAdV-11-1047) vaccine. The live vaccine (1 × 104 TCID50 of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 106TCID50 of each virus + 20% Emulsigen D) was given intramuscularly at 16 and 19 weeks of age. Controls (n = 150) were given saline orally. The inactivated vaccine group was boosted 3 weeks later with the same vaccine. Neutralizing antibodies (NAb) in sera (n = 10) were detected at 19, 22, 30 and 48 weeks of age. NAb were able to neutralize various FAdV serotypes within species D and E. Mean NAb were similar in the both live and killed vaccine groups at 19, 30 and 48 weeks and ranged from 2.4 to 3.7 log10. Approximately 26 ±â€¯7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 107 TCID50/bird intramuscularly) of FAdV-8b-SK, FAdV-11-1047, or FAdV-2-685 (n = 90/group) at 14 days post-hatch (dph) showed 98-100% protection in broiler chicks to homologous or heterologous FAdV challenges. Our data suggests that a bivalent live and an inactivated FAdV vaccine are equally effective and have the potential for the control of IBH.

Intrapulmonary Delivery of CpG-ODN Microdroplets Provides Protection Against Escherichia coli Septicemia in Neonatal Broiler Chickens.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine phosphodiester guanine (CpG) motifs (CpG-ODN) are effective immunostimulatory agents against a variety of viral, bacterial, and protozoan diseases in different animals including poultry. We have recently demonstrated that in ovo injection of CpG-ODN confers protection in neonatal chickens against bacterial septicemias. The objective of this study was to investigate the effectiveness of needle-free intrapulmonary (IPL) delivery of CpG-ODN microdroplets against Escherichia coli infection in neonatal chicks. In the present study, we used 880 chicks in total keeping 40 chicks per group. Chicks were delivered CpG-ODN or saline by IPL at the day 1 of hatch. Three days later, chicks were challenged with two doses (1 × 104 CFU, n = 20 or 1 × 105 CFU, n = 20) of E. coli. Chicks treated with CpG-ODN by the IPL route had significantly lower clinical signs and bacterial load compared to the group treated with saline ( P < 0.05). CpG-ODN-treated groups were significantly protected against E. coli septicemia. We observed dose- and exposure time-dependent immunoprotective effects of IPL CpG-ODN in chicks. We found that IPL delivery of CpG-ODN can induce protective immunity as early as 6 hr that remains effective at least until day 5 post-treatment. Moreover, there were no adverse effects of IPL delivery of CpG-ODN on growth or mortality up to 42 days of age. Based on these findings, it can be suggested that CpG-ODN delivery by IPL route can be a promising alternative to antibiotics for inducing protective immunity in chicks during the critical first week of neonatal life.

Phenotypic, genotypic and antigenic characterization of emerging avian reoviruses isolated from clinical cases of arthritis in broilers in Saskatchewan, Canada.

In recent years, emerging strains of pathogenic arthrogenic avian reovirus (ARV) have become a challenge to the chicken industry across USA and Canada causing significant economic impact. In this study, we characterized emerging variant ARV strains and examined their genetic and antigenic relationship with reference strains. We isolated 37 emerging variant ARV strains from tendons of broiler chickens with clinical cases of arthritis/tenosynovitis at commercial farms in Saskatchewan, Canada. Viral characterization using immunocytochemistry, gold-immunolabeling and electron microscopy revealed distinct features characteristic of ARV. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the viral Sigma C gene revealed genetic heterogeneity between the field isolates. On phylogenetic analyses, the Sigma C amino acid sequences of the isolates were clustered into four distinct genotypic groups. These ARV field strains were genetically diverse and quite distant from the vaccine and vaccine related field strains. Antibodies produced against a commercial Reo 2177 ® vaccine did not neutralize these variants. Moreover, structure based analysis of the Sigma C protein revealed significant antigenic variability between the cluster groups and the vaccine strains. To the best of our knowledge, this is the first report on the genetic, phenotypic and antigenic characterization of emerging ARVs in Canada.

Modified live infectious bursal disease virus (IBDV) vaccine delays infection of neonatal broiler chickens with variant IBDV compared to turkey herpesvirus (HVT)-IBDV vectored vaccine.

Chickens are commonly processed around 35-45days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n=330), divided into 6 groups (n=50-60/group), namely Group 1 (saline), Group 2 (saline+varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV+varIBDV), Group 5 (MdLV) and Group 6 (MdLV+varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3×103 EID50) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8+ T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.