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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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: We examined heat stress symptoms and urine markers of chronic kidney disease (CKDu) in Sri Lanka to assess differences between endemic vs. non-endemic regions and by occupation., Sample and Methods: We assessed a total of 475 villagers. In the endemic region, 293 were agricultural workers and 67 were not working primarily in agriculture. In the non-endemic region, 76 were agricultural workers. Of the residents, 218 were assessed for neutrophil gelatinase-associated lipocalin (NGAL), an early predictor of acute kidney injury, along with urine markers of chronic kidney disease., Results: The mean (sd) age of the sample was 45.2 (12.6), with males comprising 52.7%; 7.2% reported kidney disease ( n = 34), and 5.7% reported diabetes ( n = 27). The heat stress index (mean (sd)) was highest among agricultural workers in the endemic region (8.05 (5.9)), intermediate in non-agricultural workers in the endemic region (4.61 (4.5)), and lowest among agricultural workers in the non-endemic region (3.85 (3.3)); p < 0.0001. Correlations were higher between NGAL and serum microalbumin in the endemic agricultural worker sample than in the other two samples (Spearman's r = 0.34 vs. 0.15 and 0.20)., Conclusions: Both heat stress symptoms and NGAL values were higher among agricultural workers in endemic CKDu regions. Correlations between NGAL and microalbumin suggested a link between acute kidney injury and chronic kidney disease in the more-exposed sample.

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 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 × 10 5 cfu (n = 20) or 1 × 10 6 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)-1β 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.

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., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

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.

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 × 10 4 TCID 50 of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 10 6 TCID 50 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 log 10 . Approximately 26 ± 7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 10 7 TCID 50 /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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 × 10 4 CFU, n = 20 or 1 × 10 5 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.

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.

Inclusion body hepatitis (IBH) is an economically important diseases in broiler chicken industry. Several serotypes of fowl adenovirus (FAdV) can cause IBH, among them, serotype FAdV-8b is associated with the majority of the IBH cases in Canada. Here, we evaluated FAdV-8b virus-like particles (VLPs) and recombinant FAdV-8b fiber proteins (expressed in E. coli) as potential broiler-breeder vaccines against IBH. For assessing the immunogenicity of vaccines, we investigated both humoral and cellular immunity. The humoral immune response was evaluated by determining total IgY and virus-neutralizing antibody in serum at 14, 28, 35 and 60days post-immunization (dpi). We examined cellular immunity using flow cytometry by determining CD4:CD8 ratio change in peripheral blood after the booster vaccination. The protective effect of vaccines was tested by challenging 14day-old progeny (n=30/group) carrying maternal antibodies (MtAb) by challenging with virulent FAdV-8b virus (1×10 7 TCID 50 , FAdV-8b-SK). Although total IgY levels were comparable in all groups, the neutralizing antibody response in broiler-breeders at 35 and 60 dpi was significantly (p<0.05) higher those vaccinated with FAdV-8b VLPs followed by FAdV-8b fiber compared to fiber-knob. Moreover, vaccines comprised of FAdV-8b VLPs and FAdV-8b fiber rather than FAdV-8b fiber-knob efficiently elicited the cell-mediated immune response as evidenced by a statistically significant (p<0.05) CD8 + T-cell proliferative response in broiler-breeders four days after the booster vaccination. Unlike FAdV-8b fiber-knob, FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders were able to transfer a substantial amount (28.4±9%) of MtAb to their progeny. Challenge revealed that MtAb provided 100% and 82.7% protection in progeny hatched from FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders, respectively. Collectively, our data suggest that FAdV-8b subunit vaccine-induced MtAb efficiently protected progeny against clinical IBH and broiler-breeder vaccination with subunit vaccines is a potential approach to protect against IBH., (Copyright © 2017 Elsevier Ltd. All rights reserved.)