In vivo system to monitor tissue responses in birds

Methods of evaluating the tissue response to an agent in birds are provided as well as methods of monitoring exposure to agents in birds and of determining the efficacy of vaccines

Effects of a Low Crude Protein Diet With and Without Spirulina platensis Inclusion on White Blood Cell Profiles in Broilers

Spirulina microalgae is an alternative protein source under consideration for feed formulation in commercial broiler production. The purpose of this study was to determine the effects of a low crude protein (LCP) diet and a LCP diet formulated with 100 g/kg Spirulina (LCP-SP) on blood cell measurements in broilers. One-day-old Ross 708 male broilers were assigned to three dietary treatments: a standard crude protein (SCP), the LCP, or the LCP-SP diet, with five pens/treatment. When the chickens were 37-days old, blood samples were obtained from 2 birds/pen. Each blood sample was used to determine 1) the concentrations of white blood cells (WBC), thrombocytes, red blood cells (RBC), hemoglobin, and hematocrit by automated hematology; 2) the proportions among WBC populations by microscopic evaluation of \u3e300 WBC on Wright-stained blood smears; and 3) the proportions among lymphocyte-subsets by immunofluorescent staining and flow-cytometric cell population analysis. Except for monocytes, none of the blood cell measurements were affected (P \u3e 0.05) by diet. The LCP diet resulted in increased (P ≤ 0.05) monocyte concentration and proportion compared to the SCP diet, indicating heightened inflammatory activity with lower dietary protein content. The LCP-SP diet reversed the effect of the LCP diet, resulting in monocyte concentrations and proportions not different (P \u3e 0.05) from those of the SCP diet. The ability of Spirulina microalgae to maintain normal WBC profiles in broilers fed the LCP diet is a promising sign for its use as a proteinaceous feed component without compromising the health of the bird

Time Course Investigation of the Dermal Leukocyte Response to Lipoteichoic Acid in Chickens

Lipoteichoic acid (LTA) is a cell-wall polymer in Gram-positive bacteria that stimulates inflammation. Few studies have investigated in vivo immune response to LTA, and none of the in vivo studies have been performed in birds. For this project, the pulp (a skin derivative) of growing feathers (GF) of chickens was used to investigate the in vivo effects of intradermally injected LTA. In Study 1, GF of chickens were injected with 10 μL of differing concentrations of LTA (0.1, 1.0, 10, 100 or 250 μg LTA/mL; 3 chickens/dose). Growing feathers were plucked before injection (0 h) and at 6, 24, 48, and 72 h post-injection and frozen before staining using immunohistochemistry for visual inspection of leukocyte infiltration. Ten μg/mL LTA was found to be the optimal concentration to stimulate inflammation. In Study 2, GF were injected with 10 μL of either 10 μg/mL LTA (0.1 μg LTA/GF, 12 GF/bird, n = 8) or PBS (vehicle; n = 4). GF were collected at 0, 6, 24, 48, and 72 h. For each time point, pulp cell suspensions were prepared and immunofluorescently stained with a panel of chicken-leukocyte-specific monoclonal antibodies. Cell populations were analyzed via flow cytometry, revealing elevated levels (% pulp cells) in total leukocytes, monocytes/macrophages, and major histocompatibility complex (MHC) class-II expressing cells in GF injected with LTA when compared to the control. Infiltration of lymphocytes and heterophils was not different between treatment groups. This study suggests that the inflammatory response to LTA in chickens is characterized primarily by recruitment of monocytes/macrophages to the site of inflammation

Temporal, Phenotypic, and Quantitative Characterization of Thyroid Infiltrating Mononuclear Cells During Development of Spontaneous Autoimmune Thyroiditis in Obese Strain Chickens

The Obese strain (OS) of chickens spontaneously develops autoimmune thyroiditis (SAT) and is a well-established biomedical model for Hashimoto’s thyroiditis in humans. Both conditions are characterized by the infiltration of thyroid glands with mononuclear immune cells resulting in the destruction of thyroid tissue and impairment of the thyroid’s endocrinological functions. Past studies described immune cell infiltration in thyroids of the OS chickens, but the time-course, cell composition, and relative amounts of the various immune cells infiltrating the thyroids have not been well defined. In this project, frozen and stored thyroid glands that were previously collected at 1, 4, 7, 14, 21, 28, 35, and 42 days of age (n = 4 to 5 OS birds/age) were used. Frozen thyroid sections (8-μm thick) were prepared and used in an indirect immunohistochemical staining procedure to identify macrophages, B cells, T cells, T helper cells, cytotoxic T cells, γδ T cells, and MHC II-expressing cells. Stained sections were evaluated by microscopy, and the percentage of tissue area occupied by various cell types was determined. Thyroid infiltration was first observed at 7 days of age, and immune cells occupied the entire tissue in most samples from 3 weeks onwards. Macrophages were the first cells to infiltrate, but T cells dominated the response. MHC II expression reached very high levels by 14 days and remained at nearly 100% thereafter. This study provided new insights regarding the participating immune cells and the chronological order of their infiltration into thyroid glands during SAT development in OS chickens

Effects of virus infection on release of volatile organic compounds from insect-damaged bean, Phaseolus vulgaris

Insects can serve as important vectors of plant pathogens, especially viruses. Insect feeding on plants causes the systemic release of a wide range of plant volatile compounds that can serve as an indirect plant defense by attracting natural enemies of the herbivorous insect. Previous work suggests that the Mexican bean beetle (Epilachna varivestis) prefers to feed on plants infected by either of two viruses that it is known to transmit: Southern bean mosaic virus (SBMV) or Bean pod mottle virus (BPMV). A possible explanation for the preferred feeding on virus-infected tissues is that the beetles are attracted by volatile signals released from leaves. The purpose of this work was to determine whether volatile compounds from virus-infected plants are released differentially from those emitted by uninfected plants. To test the hypothesis, common bean plants (Phaseolus vulgaris cv. Black Valentine) were inoculated with either BPMV, SBMV, or a mixture of both viruses, and infected plants were compared to uninfected plants. An Ouchterlony assay was used with SBMVand BPMV-specific antisera to confirm the presence of virus in inoculated plants. RNA blot analysis was performed on tissue from each plant and indicated that a well-characterized defense gene, encoding phenylalanine ammonia-lyase (PAL), was not induced in systemic tissue following virus infection. Plant volatiles were collected—and analyzed via gas chromatography (GC)—from plants that were either undamaged or beetle-damaged. In undamaged plants, there were no measurable differences in profiles or quantities of compounds released by uninfected and virus-infected plants. After Mexican bean beetles were allowed to feed on plants for 48 h, injured plants released several compounds that were not released from undamaged plants. Lower quantities of volatile compounds were released from virus-infected plants suggesting that enhanced release of plant-derived volatile organic compounds is not the cause for attraction of Mexican bean beetles to virus-infected plants

Temporal, Phenotypic, and Quantitative Characterization of Thyroid Infiltrating Mononuclear Cells During Development of Spontaneous Autoimmune Thyroiditis in Obese Strain Chickens

The Obese strain (OS) of chickens spontaneously develops autoimmune thyroiditis (SAT) and is a well-established biomedical model for Hashimoto’s thyroiditis in humans. Both conditions are characterized by the infiltration of thyroid glands with mononuclear immune cells resulting in the destruction of thyroid tissue and impairment of the thyroid’s endocrinological functions. Past studies described immune cell infiltration in thyroids of the OS chickens, but the time-course, cell composition, and relative amounts of the various immune cells infiltrating the thyroids have not been well defined. In this project, frozen and stored thyroid glands that were previously collected at 1, 4, 7, 14, 21, 28, 35, and 42 days of age (n = 4 to 5 OS birds/age) were used. Frozen thyroid sections (8-μm thick) were prepared and used in an indirect immunohistochemical staining procedure to identify macrophages, B cells, T cells, T helper cells, cytotoxic T cells, γδ T cells, and MHC II-expressing cells. Stained sections were evaluated by microscopy, and the percentage of tissue area occupied by various cell types was determined. Thyroid infiltration was first observed at 7 days of age, and immune cells occupied the entire tissue in most samples from 3 weeks onwards. Macrophages were the first cells to infiltrate, but T cells dominated the response. MHC II expression reached very high levels by 14 days and remained at nearly 100% thereafter. This study provided new insights regarding the participating immune cells and the chronological order of their infiltration into thyroid glands during SAT development in OS chickens

The effects of classic and variant infectious bursal disease viruses on lymphocyte populations in specific-pathogen-free White Leghorn chickens

Infectious bursal disease virus (IBDV) is a pathogen that primarily infects B lymphocytes in domestic avian species. This viral infection has been associated with immunosuppression, clinical disease/mortality, and enteric malabsorption effects. The purpose of this experiment was to compare the effects of a classic (USDA-STC) and a new variant IBDV (RB-4, known to induce primarily the enteric disease) on immune cell populations in lymphoid organs. Seventeen-dayold specific-pathogen-free (SPF) White Leghorn chickens were either not infected (control) or inoculated with either USDA-STC or RB-4 IBD viral isolate. On days 3 and 5 post-inoculation (PI), lymphoid tissues were collected to prepare cell suspensions for immunofluorescent staining and cell population analysis by flow cytometry. Portions of the tissues were snap frozen for immunohistochemistry to localize various immune cells and IBD virus in the tissues. Tissue homogenates were prepared to test for IBDV by quantitative MTT assay. Both the USDA-STC and RB-4 viruses greatly altered lymphocyte populations in the spleen and bursa. At 5 d PI, bursal B cells were approximately 25% and 60% of lymphocytes in chicks infected with USDA-STC and RB-4, respectively, whereas in control birds, B cells constituted 99% of bursal lymphocytes. This reduction in the proportions of bursal B cells was associated with an infiltration of T cells. In the spleen, IBDV infection also reduced the percentage of B cells and increased the percentage of T cells. The differential effects of classic and variant IBDV infection on immune cell populations in lymphoid organs may explain the differences in clinical effects induced by these viruse

Effects of Cyclic Heat Stress on the Acute Inflammatory Response in Broilers

Heat stress (HS) is a growing concern in broiler production. Little is known regarding the effect of HS on immune function. To examine the effects of HS on innate immunity, the local- and systemic-inflammatory responses to lipopolysaccharide (LPS) were examined in Cobb 500 male broiler chicks reared under thermoneutral (TN) or cyclic HS conditions. Beginning at four days of age, HS birds were subjected to 35 °C from 8:00 a.m. to 10:00 p.m. and TN temperatures from 10:00 p.m. to 8:00 a.m. At 37 days of age, four groups of broilers were formed: LPS-TN (8 broilers), phosphate-buffered saline (PBS)-TN (4 broilers), LPS-HS (8 broilers), and PBS-HS (4 broilers), with each broiler receiving LPS- (100 μg/mL) or PBS-treatments by intradermal pulp-injection of 12 growing feathers (GF; 10 μL/GF). Blood and GF were collected before (0 h) and at 6 and 24 h post-injection to determine leukocyte population changes. Locally, LPS-HS broilers had lower (P ≤ 0.05) levels (% pulp cells) of infiltrating heterophils and macrophages in GF-pulps at 6 and 24 h, respectively, compared to LPS-TN birds. In the blood, TN and HS broilers had similar baseline (0 h) concentrations (cells/μL) of heterophils, monocytes, eosinophils, and basophils, but HS broilers had lower (P ≤ 0.05) T- and B-lymphocyte levels. Concentrations of heterophils and monocytes were greatly elevated (P ≤ 0.05) at 6 and 24 h, respectively, only in LPS-TN broilers. Overall, results indicated that cyclic HS reduced both the local and systemic acute inflammatory responses to LPS in broilers, likely impairing their innate defense against microbial infection

Effects of Cyclic Heat Stress on the Acute Inflammatory Response in Broilers

Heat stress (HS) is a growing concern in broiler production. Little is known regarding the effect of HS on immune function. To examine the effects of HS on innate immunity, the local- and systemic-inflammatory responses to lipopolysaccharide (LPS) were examined in Cobb 500 male broiler chicks reared under thermoneutral (TN) or cyclic HS conditions. Beginning at four days of age, HS birds were subjected to 35 °C from 8:00 a.m. to 10:00 p.m. and TN temperatures from 10:00 p.m. to 8:00 a.m. At 37 days of age, four groups of broilers were formed: LPS-TN (8 broilers), phosphate-buffered saline (PBS)-TN (4 broilers), LPS-HS (8 broilers), and PBS-HS (4 broilers), with each broiler receiving LPS- (100 μg/mL) or PBS-treatments by intradermal pulp-injection of 12 growing feathers (GF; 10 μL/GF). Blood and GF were collected before (0 h) and at 6 and 24 h post-injection to determine leukocyte population changes. Locally, LPS-HS broilers had lower (P ≤ 0.05) levels (% pulp cells) of infiltrating heterophils and macrophages in GF-pulps at 6 and 24 h, respectively, compared to LPS-TN birds. In the blood, TN and HS broilers had similar baseline (0 h) concentrations (cells/μL) of heterophils, monocytes, eosinophils, and basophils, but HS broilers had lower (P ≤ 0.05) T- and B-lymphocyte levels. Concentrations of heterophils and monocytes were greatly elevated (P ≤ 0.05) at 6 and 24 h, respectively, only in LPS-TN broilers. Overall, results indicated that cyclic HS reduced both the local and systemic acute inflammatory responses to LPS in broilers, likely impairing their innate defense against microbial infection

Intra-vascular administration of particles to induce pulmonary hypertension, pulmonary hypertension syndrome, and ascites in animals

The invention relates to a new process for inducing pulmonary hypertension (elevated blood pressure in the pulmonary arteries) in animals, with the objective of identifying and/or eliminating susceptible individuals or families to achieve genetic improvement in agriculturally important breeds of poultry (broiler chickens and turkeys), and cattle. The invention will also be useful for animal research directed toward understanding pulmonary hypertension and its sequelae in human patients as well as in animals. Sustained pulmonary hypertension leads to pulmonary hypertension syndrome, which adversely impacts poultry production throughout the world, as well as cattle production when cattle are kept at latitudes sufficiently high to challenge blood oxygenation. In poultry, the pathophysiological progression of pulmonary hypertension syndrome leads to terminal ascites (fluid accumulation in the abdominal cavity) followed by death of the animal