Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Students at all levels in preparation for employment in the poultry and allied industry. Effort: science-based laboratory instruction, experiential learning courses, undergraduate and graduate research opportunities; scientific meetings, outreach programs. Research scientists working on immune system development and function in poultry, vaccine developments, treatment testing in animals, agricultural and biomedical research. Effort: scheduled meetings, scientific meetings, publications, scientists at poultry breeding companies, commercial poultry growers, allied industries including animal health, vaccine, pharmaceutical, and nutrition companies. Effort: conversations, scheduled meetings, scientific meetings, workshops and outreach programs. Changes/Problems:Due to the Covid 19 pandemic-related restrictions, laboratory analyses of the samples collected for Objective 1b and 2 during this year were delayed and had to be switched from same-day analyses, such a flow cytometric analysis of fluorescently stained pulp cell supsensions prepared from injected GF, to the more labor intensive and technically difficult procedure of immunohistochemical staining of frozen tissue sections and imagine analysis. What opportunities for training and professional development has the project provided?One graduate student, one undergraduate, and a technician carried out the studies for Objective 1. One graduate student carried out the study described under Objective 2. All students and technician gained experience in carrying out the various methodologies reported. Data on local and systemic inflammatory responses were incorporated as case studies in immunology laboratory and lecture courses. Interns with Nanomatronix, LLC, the company that licensed the patent on the GF injection method in July 2018, have been taught the GF injection method and the procedures involved in the ex vivo analysis of collected GFs. How have the results been disseminated to communities of interest?Meetings with representatives of the Poultry and Allied Industry; graduate student seminar; presentation at USDA-NIFA Sustainable Agriculture Grant consortium; Presentation of our research program at departmental student recruitment workshops, and to international groups visiting the department. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will conduct an animal experiment to examine the "intra-assay" variation. This will be accomplished by injection of multiple GF with the same test-material and individually processing 3 GF per time point (like tissue cultures set up in triplicate wells) per chicken. Twelve GF of 10 (5 males and 5 females), 9-week-old chickens from the Light-brown Leghorn line maintained by the PI, will be injected with 1 μg LPS/GF. Three GF will be collected before (0 h), and at 6, 24, 48, and 72 h p.i. Blood samples (0.5-1 mL) will also be collected at each time-point for cell population analysis. The 3 GFs/bird at each time-point will be used to prepare individual pulp cell suspension for immunofluorescent staining and leukocyte population analysis by flow cytometry. Leukocyte infiltration data will be statistically examined for intra-bird variation at each time-point, as well as by repeated measures ANOVA. This study will be repeated using peptidoglycan (PGN), a cell wall product of Gram-positive bacteria, which we have shown to result in a qualitatively different leukocyte infiltration response than LPS from Gram-negative bacteria over the 72 h time course. The studies constitute validation and refinement of methods, needed preliminary data, and replication of previous studies on the inflammatory response initiated by LPS and PGN in Light-brown Leghorn chickens. Objective 2. Projects examining the vaccination response to different Salmonella vaccines will be carried out, examining local cellular responses to vaccine injected into GF as well as the antibody response in the peripheral blood circulation. Objective 3. As part of genetic selection and nutritional studies, we will use the GF in vivo test-tube system to examine the effects of treatments on the innate immune response in broilers.
Impacts
What was accomplished under these goals?
Objective 1. Objective 1a. To adapt the GF cutaneous-test (Erf and Ramachandran, 2016), which we established in egg-type chickens, to broilers, we first examined GF growth, size, and quality in commercial broilers. Ensheathed GF were already present on the breast tract at 2 weeks and reached an injectable size by 2.5- 3 weeks. To examine GF regeneration in broilers, a row of GF on each breast tract was plucked when the birds were two weeks old and allowed to regenerate for 19 days. The 19-day-old regenerated GF were uniform and similar to 19-day-old regenerating GF in 12-week-old egg-type chickens; i.e., broiler GF were comparable in size, pulp volume, and "stability" to same-age regenerating GF in layer chickens. Graduate student Chelsea Ellington French (M.S.) then examined the local (GF) and systemic (blood) inflammatory response by GF injection with lipopolysaccharide (LPS). Specifically, 19-d old GF of 12 male and 12 female, 5-wk-old broilers were injected with LPS (16 GF/chicken; 1 μg LPS/GF). Blood and GF were collected before (0 h), and at 6 and 24 h post-GF injection of LPS or PBS. GF-pulp was used to determine leukocyte-infiltration and gene-expression profiles. Additionally, new assays were developed to examine activities initiated in injected GF pulp, including assessment of reactive-oxygen-species (ROS) generation by pulp cell suspensions, and superoxide dismutase (SOD) activity in pulp homogenates. Blood was used to determine blood cell profiles and plasma SOD activity. A time effect (P ≤ 0.05) was observed for most aspects examined. In GF, LPS-injection resulted in heterophil and monocyte infiltration reaching maximal levels at 6 and 24 h, respectively. ROS generation, SOD activity, and mRNA levels of IL-1β, IL-6, IL-8, IL-10, and cathelicidin B1 were elevated, whereas those of TNF-α, LITAF, SOD1, and SOD2 decreased post-LPS injection. In blood, levels of heterophils and monocytes were elevated at 6 h, lymphocytes and RBC decreased at 6 h, and thrombocytes and SOD-activity increased at 24 h. Assessment of LPS-induced activities at the site of inflammation (GF) contributed novel insights into temporal, qualitative, and quantitative aspects of the LPS-induced inflammatory response. Knowledge generated from this two-window approach also resulted in many new questions regarding the LPS-induced inflammatory response in birds, that can be effectively addressed using this dual window approach (French et al., 2020). In addition to a first application of the GF dermal test technology in broilers, new assays were developed/adapted to examine local immune system related activities. Including ROS generation by pulp cell suspensions, SOD activity in pulp homogenates/plasma; as well as gene-expression data on the relatively newly define chicken tumor necrosis factor-alpha (TNF-α) in relation to the LPS-induced TNF-α factor involved in TNF-α gene-expression. Objective 1b. Animal experiments and sample collections weres accomplished for a study examining innate local and systemic responses to Salmonella bacterins intradermally injected into GF of Light-brown Leghorn chickens. The response to different Salmonella types, dosages, as well as, microbial cell wall product lipopolysaccharide (LPS) in vehicle emulsion, or vehicle emulsion control in injected GF was tested before and at 6, 24, 48 and 72 h post-GF-injection. Additionally, blood samples were collected before and at 3, 5, 7,10, 14, 21 and 28 days post-GF injection of test material to examine the antibody response to Salmonella bacterin by ELISA. Tissue and blood analyses are underway. Objective 2. Animal experiments and sample collections were accomplished for a study examining the local and systemic recall response to Salmonella bacterins intradermally injected into GF of Light-brown Leghorn chickens. For this, the chickens were immunized by subcutaneous (s.c.) injection of two types of Salmonella bacterins, LPS, or vehicle. Blood was sampled at 3, 5, 7, 10, 14, 21, and 28 days post-primary s.c. immunization. On day 28, local and systemic recall response was tested by intradermal injection of respective immunization treatment into the dermis of GF. Injected GF were tested before and at 6, 24, 48 and 72 h post-GF-injection. Additionally, blood samples were collected before and at 3 ,5, 7, 10, 14, 21, and 28 days post-GF injection of test material to examine the secondary antibody response to Salmonella bacterin by ELISA. Tissue and blood analyses are underway.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
French, C. E., M. A. Sales, S. J. Rochell, A. Rodriguez, and G. F. Erf. 2020. Local and systemic inflammatory responses to lipopolysaccharide in broilers: new insights using a two-window approach. Poult. Sci. 99:6593-6605. doi: 10.1016/j.psj.2020.09.078. Epub 2020 Oct 8. PMID: 33248575
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Students at all levels in preparation for employment in the poultry and allied industry. Effort: science-based laboratory instruction,experiential learning courses,undergraduate and graduate researchopportunities; scientific meetings, outreach programs Research scientists working on immune system development and function in poultry, vaccine developments, treatment testing in animals, agricultural and biomedical research.Effort:scheduled meetings, scientific meetings, publications Poultry scientists at poultry breeding companies, commercial poultry growers, allied industries including animal health, vaccine, pharmaceutical, and nutrition. Effort:scheduled meetings, scientific meetings, workshops and outreach programs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate student and a technician carried out the studies for Objective 1. One graduate student carried out the study described under Objective 3. All students and technician gained experience in carrying out the various methodologies reported. Data on local and systemic inflammatory responses were incorporated as case studies in immunologylaboratory and lecture courses. Interns with the company that licensed the patent on the GF injection method in July 2018, have been taught the GF injection method and the procedures involved in the ex vivo analysis of collected GFs. How have the results been disseminated to communities of interest?Presentations at the Poultry Science Association, as well as graduate student seminar, defense and thesis. Meetings with representatives of the Poultry and Allied Industry Presentation of our research program at High Schools, at departmental student recruitment workshops, and to international groups visiting the department. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will conduct an animal experiment to examine the "intra-assay" variation. This will be accomplished by injection of multiple GF with the same test-material and individually processing 3 GF per time point (like tissue cultures set up in triplicate wells) per chicken. Twelve GF of 10 (5males and 5females), 9-week-old chickens from the Light-brown Leghorn line maintained by the PI, will be injected with 1 μg LPS/GF. Three GF will be collected before (0 h), and at 6, 24, 48, and 72 h p.i. Blood samples (0.5-1 mL) will also be collected at each time-point for cell population analysis. The 3 GFs/bird at each time-point will be used to prepare individual pulp cell suspension for immunofluorescent staining and leukocyte population analysis by flow cytometry. Leukocyte infiltration data will be statistically examined for intra-bird variation at each time-point, as well as by repeated measures ANOVA. This study will be repeated using peptidoglycan (PGN), a cell wall product of Gram-positive bacteria, which we have shown to result in a qualitatively different leukocyte infiltration response than LPS from Gram-negative bacteria over the 72 h time course. The studies constitute validation and refinement of methods, needed preliminary data, and replication of previous studies on the inflammatory response initiated by LPS and PGN in Light-brown Leghorn chickens. Objective 2. Projects examining the vaccination response to different Salmonella vaccines will be carried out, examining local cellular responses to vaccine injected into GF as well as the antibody response in the peripheral blood circulation. Objective 3. As part of genetic selection and nutritional studies, we will use the GF in vivo test-tube system to examine the effects of treatments on the innate immune response in broilers.
Impacts
What was accomplished under these goals?
In this first year of this Animal Health project, our efforts focused primarily on Objective 1 and 3. Objective 1. To explore and define local innate responses of chickens to microbial products injected into a complex tissue, we focused on bacterial cell wall products that are known stimulants of inflammatory responses such as lipopolysaccharide (LPS) and peptidoglycan (PGN) of Gram-negative and Gram-positive bacteria, respectively. In chickens, like in mammals, LPS has been shown to activate heterophils and macrophages in culture, increase heterophil levels in the blood, and result in rapid heterophil accumulation at the site of injection. Heterophil infiltration is followed within hours by monocytes, whereas lymphocytes are a minor part of the LPS response. The response to LPS is transient, with heterophils and macrophages reaching peak levels by 6 h and 24 h, respectively, and returning to pre-injection levels by 48 h. Knowledge regarding leukocyte recruitment initiated by PGN is limited. To examine temporal, qualitative and quantitative aspects of the avian response to PGN in a complex tissue, we used our growing feather cutaneous test-system. Briefly, we developed the growing feather (GF), a skin derivative, as a minimally invasive platform to examine local cellular responses to test-materials injected into the dermis of the GF pulp. Simultaneous injection of multiple GFs of a chicken with test-material, and subsequent periodic collection of GFs for laboratory analysis, provides a window into local activities initiated by test-material, like the window into systemic activities provided by periodic blood sampling. Injection of LPS or PGN into the pulp of GFs resulted in leukocyte infiltration profiles in the dermis of PGN-injected GFs that differed (P < 0.05) from those observed in LPS-injected GFs. Specifically, GFs (20 per bird) were injected with 10 μL of LPS or PGN (50 μg/mL) or vehicle (endotoxin-free PBS) (n = 6 chickens per treatment) and GFs collected before (0 h), and at 0.25, 1, 2, 3, 5, and 7 d post GF-injection. At each time point, pulp cell suspensions prepared from collected GFs were immunofluorescently stained and subjected to leukocyte population analysis by flow cytometry. While the leukocyte infiltration profiles to LPS were as described above, the leukocytes infiltration response to PGN was dominated by mononuclear leukocyte infiltration consisting primarily of T and B lymphocytes. Lymphocyte levels (% pulp cells) in the dermis of PGN injected GFs increased by 6 h, continued to increase to maximal levels by 1 d, and then remained at this high level through 5 d. By 7 d post-injection, lymphocyte levels in the PGN injected pulps had dropped, but remained at above baseline levels. This immediate and sustained lymphocyte recruitment to the site of PGN injection was consistently observed independent of the type of chicken used, and when LPS and PGN were injected into different GFs of the same chickens. Current research is focused on gene-expression profiles that may explain the qualitative differences in the types of leukocytes recruited and stimulated by PGN compared with LPS. Objective 3. Acute phase inflammatory response to LPS in broiler chickens and effects of trace mineral supplementation Chelsea Ellington completed her MS project on the effects of micronutrients, specifically looking at the effect of source and amount of trace-mineral supplementation in the diet, on the early phase of the acute inflammatory response to LPS injected into the pulp of growing feathers. Trace minerals, particularly copper, zinc and manganese, play a role in ensuring optimal immune function. To examine the effects of diets containing different levels and sources of trace minerals on the inflammatory response, Cobb 500 broilers were fed four experimental diets containing 5 mg/kg Cu and 45 mg/kg of Zn and Mn or 10 mg/kg Cu and 90 mg/kg Zn and Mn in either the sulfate or hydroxychloride form. When the broilers were 5 weeks of age, 16 μg of lipopolysaccharide (LPS) was injected into the pulp of growing feathers (GF; 16 GF per chicken; 1 μg LPS per GF). Injected GF and blood were sampled before (0 hour) and at 6- and 24-hours post-GF-injection of LPS. Samples were used to determine leukocyte infiltration profiles in LPS-injected pulps, alterations in concentrations and proportions of blood leukocytes (WBC), concentrations of thrombocytes, red blood cells (RBC) and other RBC-related measurements, reactive oxygen species (ROS) generation, and superoxide dismutase (SOD) activity. A time effect (P ≤ 0.05) was observed for most of the aspects examined. In GF, LPS-injection into the pulp resulted in heterophil and monocyte/macrophage infiltration that reached maximal levels at 6- and 24-hours, respectively. Additionally, levels of ROS generation were greatly increased at 6 hours post-pulp injection of LPS. In the blood, GF injection of LPS increased heterophil and monocyte concentrations at 6 hours, thrombocyte concentration and plasma SOD activity at 24 hours, and decreased lymphocyte and RBC concentrations at 6 hours. While few dietary effects were observed, birds fed lower trace mineral levels had lower SOD activity in the GF pulp (but not in the plasma) than the chickens fed higher levels of trace minerals. Using the GF as a minimally invasive, cutaneous test-site, together with periodic sampling of injected GF and blood for laboratory analyses, the early phase of the LPS-induced inflammatory response could be observed both at the local (GF-pulp) and systemic (peripheral blood) level in the same broiler chickens. Furthermore, inclusion of the GF-injection approach revealed effects of dietary supplementation on the inflammatory activities in the inflamed tissue that were not detected in the blood. Through this study, we were able to apply new approaches, develop new assay systems, and generate new knowledge regarding the effect of diet on immune system activities in a living broiler chicken. The focus of this study was the LPS-induced inflammatory response. By definition, iflammation is a complex reaction of vascularized tissues to infection or cell injury that involves extravascular accumulation of plasma proteins and leukocytes. Considering the complex interactions and signaling processes at the site of inflammation, inflammatory processes are best evaluated by examination of affected tissue, rather than solely examining changes in the peripheral blood or responses of isolated WBC in cultures.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Erf, G. F., M. A. Sales, C. E. Ellington. 2019. Intradermal injection of lipopolysaccharide and peptidoglycan in chickens results in divergent leukocyte infiltration profiles. Poult. Sci. 98 (E-Suppl. 1):181-182.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2019
Citation:
Ellington, C. Effects of dietary copper, zinc and manganese source and level on the acute inflammatory response of broilers. M.S. Thesis. University of Arkansas, Fall 2019.
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