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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 P P P

Synthetic DNA containing CpG motifs (CpG-ODN) are potent innate immune stimulators in neonatal and adult broiler chickens against bacterial septicemia. We have recently demonstrated that intrapulmonary (IPL) delivery of CpG-ODN as microdroplets under laboratory conditions can protect neonatal chickens against lethal Escherichia coli septicemia. The objectives of this study were to develop a commercial-scale poultry nebulizer (CSPN) that can deliver CpG-ODN as microdroplets in neonatal broiler chicks in the hatcheries and study the efficacy of CSPN in inducing immune-protective effects under different environmental conditions in 2 geographical locations in Canada. Three field experiments were conducted in commercial poultry hatcheries during different seasons of the year in Saskatchewan and British Columbia, Canada. Neonatal broiler chicks (n = 8,000/experiment) received CpG-ODN by the IPL route in the CSPN chamber for 30 min, and control chicks received distilled water (DW) for 30 min. Broiler chicks (CpG-ODN—240 chicks/experiment and DW—40 chicks/experiment) were randomly sampled from all locations of the CSPN after nebulization and challenged with a lethal dose of E. coli to examine the CpG-ODN nebulization induced protection. We found a significant level (P < 0.05) of protection in broiler chicks against E. coli challenge, suggesting that the newly built CSPN successfully delivered CpG-ODN via the IPL route. We found that when the CSPN was maintained at humidex 28°C or below and relative humidity (RH) between 40 and 60%, neonatal birds were significantly (P < 0.05) protected against E. coli septicemia after IPL delivery of CpG-ODN. By contrast, protection in chicks was adversely affected when the CSPN was maintained at the humidex of 29°C or higher and RH of 70%. Overall, the present study successfully built a CSPN for CpG-ODN delivery in chicks at the hatchery and revealed that the temperature, humidity, and humidex were critical parameters in CSPN for efficient delivery of CpG-ODN.

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.

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 naive 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

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 ± 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.

The emergence of enterococcal infections in neonatal broiler chickens in the poultry industry has become common in many countries, including Canada. The objective of this study was to examine the bacterial infections in nonviable broiler chicken embryos in three western Canadian poultry hatcheries using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The pattern of embryo mortality that occurred during incubation and the breakout analysis results were similar in all three hatcheries. The majority of embryo mortality occurred during the late stage of incubation (35.08%), followed by the early stage of incubation (15.35%). The breakout analysis showed that 65.82% of swabs had at least one type of bacterial growth while 34.17% of swabs were negative for bacterial isolation. Of those 65.82% swabs with bacterial growth, 34.3% of swabs yielded a mixed bacterial population while 31.52% yielded one type of bacterial growth. The frequency of bacterial isolation from hatch debris (60%-75%) increased with the age of broiler breeders. MALDI-TOF MS was able to provide genus-level identification of 83.13% of isolates among all bacterial types isolated. MALDI-TOF MS identified Enterococcus and Escherichia coli isolates with 97.18% and 100% accuracy at species level, respectively, whereas Staphylococcus species were identified with 62.59% accuracy. The congruence between MALDI-TOF MS identification and 16S rRNA or cpn60 universal gene target sequencing was 100% or 90%, respectively. Of all bacteria isolated, Enterococcus species (29.71%) were the most prevalent, followed by E. coli (19.46%). About 56% of E. coli-infected samples were coinfected with Enterococcus species. Among all Enterococcus species isolated, Enterococcus faecalis (79.58%) was the most prevalent, followed by Enterococcus faecium (8.1%). Overall, our study showed that Enterococcus-associated embryo mortality was predominant in all three hatcheries investigated and suggests that MALDI-TOF MS technology can be applied to identify bacteria such as Enterococcus species isolated from poultry.

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.

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.

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% (P0.01). Bacterial loads and clinical scores were significantly lower (P0.05) 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.