CD4+ T cells dominate the leukocyte infiltration response initiated by intra-dermal injection of phytohemagglutinin into growing feathers in chickens.

Phytohemagglutinin (PHA) is commonly used to evaluate cell-mediated immunocompetence. In chickens, PHA is typically injected intra-dermally (i.d.) into the skin (e.g., wing web, wattle, or footpad), and the tissue swelling response is monitored, whereby the extent of tissue swelling positively relates to the individual's cell-mediated immune system capabilities. Although i.d. injected PHA was shown to stimulate mononuclear cell and basophil infiltration to the site of injection, reports on temporal, qualitative, and quantitative aspects of the local cutaneous PHA response are limited. The objective of this study was to use the growing feather (GF) as a cutaneous test site to assess and monitor the type and relative amounts of leukocytes present in the pulp of PHA-injected GF. For this study, male, non-vaccinated Light-brown Leghorn chickens reared at the Arkansas Experiment Station Poultry Health Laboratory were used. At 9 wk of age, the dermis of 20 18-day-old regenerating GF was injected with 10 µL of either PBS diluent or 300 µg/mL PHA-P (5 chickens per treatment). GF were collected from each chicken before (zero) and at 0.25, 1, 2, 3, 4, 5, and 7 d post injection. At each time point, one GF was collected for immunofluorescent staining of pulp cell suspensions and leukocyte population analysis by flow cytometry, and another GF for histological analysis. Histological analysis confirmed participation of granulocytes and mononuclear cells, primarily lymphocytes, in the cutaneous PHA response. As revealed by flow cytometric cell population analysis, T cells, especially CD4+ T cells, constituted the major portion of the mononuclear cell infiltrate. Levels of CD4+ T cells were greatly elevated in PHA-injected GF within 6 h and remained elevated throughout the 7-day examination period. γδ T cells, CD8+ T cells, and B cells also infiltrated in response to PHA although at lower levels and with different time-course patterns from CD4+ T cells. The dominant presence of CD4+ T cells at the site of PHA injection further affirms this polyclonal response as an indicator of cell-mediated immunity.

T lymphocytes dominate local leukocyte infiltration in response to intradermal injection of functionalized graphene-based nanomaterial.

Graphene-based nanomaterials (GBN) have many potential biomedical applications. However, information regarding their biological properties and interactions with cells and/or soluble factors within a complex tissue is limited. The objective of this study was to use the growing feather (GF) of chickens as a minimally invasive cutaneous test-site to assess and monitor leukocyte recruitment in response to intradermal GBN injection. Specifically, the dermis of 20 GFs per chicken was injected with 10 µl of phosphate-buffered saline (PBS)-vehicle or 10 µl of 300 µg ml-1 oxygen-functionalized (f) GBN (6 chickens/treatment). GFs were collected before- (0) and at 0.25, 1, 2, 3, 4, 5, and 7 days post-injection and used for leukocyte-population analysis of immunofluorescently stained pulp cell suspensions or histological examination. Based on flow-cytometric cell population analysis, lymphocytes and macrophages were the major leukocyte-populations infiltrating GFs in response to f-GBN presence. Compared with PBS-controls, levels of T cells (γδ-, αß-, CD4- and CD8-T cells) were greatly elevated in f-GBN-injected GFs within 6 h and remained elevated throughout the 7-day examination period. f-GBN's effects on local tissue leukocyte recruitment were not reflected in the blood, except for a higher percentage of lymphocytes on 7 days. These observations together with a visual examination of f-GBN-injected GF tissue-sections suggest a delayed-type hypersensitivity-like, inflammatory cell-mediated response to the non-biodegradable f-GBN. The GF 'in vivo test-tube'system together with blood sampling provided unique insight into the time-course, qualitative, and quantitative aspects of immune system activities initiated by the presence of f-GBN in a complex tissue of a living animal. Copyright © 2017 John Wiley & Sons, Ltd.

The growing feather as a dermal test site: Comparison of leukocyte profiles during the response to Mycobacterium butyricum in growing feathers, wattles, and wing webs.

Using the response to Mycobacterium butyricum as the test-immune response, the main goal of this study was to demonstrate the suitability of the growing feather (GF) as a dermal test site and window into in vivo cellular/tissue responses (US-Patent 8,216,551). Using M. butyricum immunized chickens, the specific objectives were to: 1) compare the leukocyte infiltration response to intra-dermally injected M. butyricum in GF, wattles, and wing webs; 2) use GF as the test site to monitor leukocyte response profiles to recall antigen in the same individuals; and 3) gain new knowledge regarding the local response to M. butyricum in chickens. For objective 1, chickens were euthanized for tissue collection at 4 to 6, 24, 48, and 72 h after intra-dermal antigen injection. Leukocyte infiltration profiles were determined using immunochemical and conventional histology. Data from this study established the similarities between the cellular response in GF, wattles, and wing webs and uncovered many advantages of working with GF. For objective 2, antigen was injected into multiple GF per individual. GF were collected before and at 0.25, 1, 2, 3, and 7 d post injection and processed for cell population analysis by flow cytometry. Advantages of the approach used in objective 2 included a technically easier, more comprehensive, and more objective leukocyte profile analysis; same-day data acquisition; and, most importantly, easy, minimally invasive sample collection from the same individual throughout the study. Both studies contributed new knowledge regarding the local cutaneous response to M. butyricum in M. butyricum immunized chickens and confirmed the cell-mediated nature of the immune response to M. butyricum (e.g., elevated levels [P < 0.05] of T cells [CD4+ and CD8+], macrophages and MHC class II+-cells on days one to 3 post injection in M. butyricum- compared to PBS-injected tissues). The use of GF as an "in vivo test tube" to monitor local innate and adaptive immune activities will find direct application in vaccine development, as well as in the assessment and optimization of immune system development and function in poultry.

Acquisition of immunity to the protozoan parasite Eimeria adenoeides in turkey poults and cellular responses to infection.

Newly hatched turkey poults were infected with 10(2) oocysts of Eimeria adenoeides and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age, respectively. Three peaks in oocyst production were observed in the feces of poults following this series of infections. A second group of poults given the same dosing regimen was challenged with 5 × 10(4) oocysts/poult at different times to evaluate the acquisition of immunity. Judging by weight gain and mortality, no protection had been acquired at 6 d of age, but partial protection was observed by 12 and 18 d of age. A third group of poults were also infected with 10(2) oocysts and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age to evaluate cellular immune responses to infection. Sections of ceca from infected poults showed a significantly higher leukocyte infiltration on d 6, 10, 12, 16, and 18 after infection than uninfected controls. The percent area occupied by CD4+ and CD8+ lymphocytes in the ceca, as assessed by immunohistochemistry, was significantly elevated in infected poults on d 12, 16, and 18. The relative expression of chemokine CXCLi2, and cytokines IL1ß, IFNγ, IL10, IL13, IL2, IL12b, and IL18 was measured by real-time reverse-transcription PCR. The expression of CXCLi2 and IL10 was found to be elevated on d 12, and IFNγ on d 10, 12, and 16. Expression of IL13 and IL18 was increased on d 10 and IL2 on d 10 and 16, and that of IL12b on d 16 in infected poults. Increase in the infiltration of leukocytes, percent area occupied by CD4+ and CD8+ lymphocytes, and changes in the relative expression of cytokines in the ceca characterize the dynamics of immune responses in turkey poults infected with E. adenoeides early in life.

Pulmonary arterial hypertension (ascites syndrome) in broilers: a review.

Pulmonary arterial hypertension (PAH) syndrome in broilers (also known as ascites syndrome and pulmonary hypertension syndrome) can be attributed to imbalances between cardiac output and the anatomical capacity of the pulmonary vasculature to accommodate ever-increasing rates of blood flow, as well as to an inappropriately elevated tone (degree of constriction) maintained by the pulmonary arterioles. Comparisons of PAH-susceptible and PAH-resistant broilers do not consistently reveal differences in cardiac output, but PAH-susceptible broilers consistently have higher pulmonary arterial pressures and pulmonary vascular resistances compared with PAH-resistant broilers. Efforts clarify the causes of excessive pulmonary vascular resistance have focused on evaluating the roles of chemical mediators of vasoconstriction and vasodilation, as well as on pathological (structural) changes occurring within the pulmonary arterioles (e.g., vascular remodeling and pathology) during the pathogenesis of PAH. The objectives of this review are to (1) summarize the pathophysiological progression initiated by the onset of pulmonary hypertension and culminating in terminal ascites; (2) review recent information regarding the factors contributing to excessively elevated resistance to blood flow through the lungs; (3) assess the role of the immune system during the pathogenesis of PAH; and (4) present new insights into the genetic basis of PAH. The cumulative evidence attributes the elevated pulmonary vascular resistance in PAH-susceptible broilers to an anatomically inadequate pulmonary vascular capacity, to excessive vascular tone reflecting the dominance of pulmonary vasoconstrictors over vasodilators, and to vascular pathology elicited by excessive hemodynamic stress. Emerging evidence also demonstrates that the pathogenesis of PAH includes characteristics of an inflammatory/autoimmune disease involving multifactorial genetic, environmental, and immune system components. Pulmonary arterial hypertension susceptibility appears to be multigenic and may be manifested in aberrant stress sensitivity, function, and regulation of pulmonary vascular tissue components, as well as aberrant activities of innate and adaptive immune system components. Major genetic influences and high heritabilities for PAH susceptibility have been demonstrated by numerous investigators. Selection pressures rigorously focused to challenge the pulmonary vascular capacity readily expose the genetic basis for spontaneous PAH in broilers. Chromosomal mapping continues to identify regions associated with ascites susceptibility, and candidate genes have been identified. Ongoing immunological and genomic investigations are likely to continue generating important new knowledge regarding the fundamental biological bases for the PAH/ascites syndrome.

Consumer acceptance of visual appearance of broiler breast meat with varying degrees of white striping.

White striping is a condition associated with heavier broiler breast fillets and is observed grossly as white striations seen parallel to the direction of the muscle fibers. The present study was intended to assess the consumer acceptance of broiler fillets with different degrees of white striping condition. High resolution digital images of fillets, representative of varying degrees of white striping, were shown to 75 consumers in a blind study. Individual images were presented using a completely randomized design. There were 4 replicates of individual fillets within each white striping category (normal = NORM, moderate = MOD, and severe = SEV) and one picture of tray pack (3 fillets) for each category. The consumers were asked to express their overall liking for appearance with a 9-point hedonic scale (9 = like extremely; 1 = dislike extremely) and purchase intent using a 5-point scale (5 = definitely would buy; 1 = definitely would not buy). An open-ended comments section was also included. The results showed that NORM fillets had a significantly higher hedonic score (6.9) than the MOD fillets (6.1), which was also significantly higher than the SEV fillets (4.5), indicating that as severity of white striping increased, the consumer acceptance decreased. From the distribution of the responses, 10.7, 22.4, and 56.7% of the consumers disliked the NORM, MOD, and SEV fillets, respectively. Furthermore, the average purchase intent score for the NORM fillets (3.6) was significantly higher than those with 2 degrees of white striping (2.4 and 2.5, respectively), suggesting that the consumers were more likely to buy NORM fillets. Over 50% of the consumers indicated that they would probably not or definitely not buy MOD or SEV fillets. The correspondence analysis of open-ended comments revealed the major reasons for the dislike of the white-striped meat was that the fillets had a more fatty or marbled appearance. The results of the study suggest that the white striping does affect the consumer acceptance based on the appearance of the fillets.

Cellular immune responses, chemokine, and cytokine profiles in Turkey poults following infection with the intestinal parasite Eimeria adenoeides.

Cellular immune responses, chemokine, and cytokine profiles were investigated in 20-d-old turkey poults following an oral infection with 12.5 × 10(3) oocysts of Eimeria adenoeides, a protozoan parasite of the genus Eimeria that develops in the ceca. Large numbers of oocysts were produced in the feces of infected birds from d 5 after infection followed by a rapid decline by d 7. Local immune activities were characterized by observing the extent of leukocyte infiltration in the ceca by histology, measuring subsets of the lymphocyte population by immunohistochemistry, and determining the relative expression of cytokines by real-time, reverse-transcription PCR. Inflammation, assessed by scoring the extent of cellular infiltration of leukocytes in sections of ceca, was significantly higher in infected poults compared with uninfected poults on d 4, 7, 9, and 11 following infection. The percent area occupied by CD4+ and CD8+ cells in the ceca was significantly greater on d 9 and 11 (CD4+) and d 11 (CD8+) in infected poults compared with uninfected controls. The relative expression of the chemokine CXCLi2 and the cytokines interleukin (IL) 1ß, interferon (IFN) γ, IL13, and IL10 was investigated in tissue samples taken from the ceca. Increased expression of CXCLi2 occurred on d 4 and 7. Increased expression of IL10 and IFNγ occurred on d 4 and of IL1ß and IL13 occurred on d 7 postinfection. The increased leukocyte infiltration in the ceca, alterations in the lymphocyte subpopulations, and changes in expression of chemokines and cytokines are an indication of the cell-mediated immune mechanisms occurring in the host as a result of exposure to E. adenoeides.

Acquisition of immunity to the protozoan parasite Eimeria adenoeides in turkey poults and the peripheral blood leukocyte response to a primary infection.

A primary infection of 12.5 x 10(3) oocysts of Eimeria adenoeides , given to 20-d-old turkey poults, resulted in depression of weight gain, and the production of large numbers of oocysts in the feces, compared with uninfected controls. Poults were raised under conditions to prevent possible reinfection to determine the ability of the primary infection to confer protective immunity against a challenge infection of 5 x 10(4) oocysts given at 34 d of age. Using weight gain and oocyst production after challenge as criteria for protection, the results indicated that immunity had developed. The concentration and proportions among white blood cell (WBC) populations in peripheral blood were determined at different times after the primary infection. The WBC concentration of infected poults was elevated on d 7 and 11, primarily due to elevated levels of lymphocytes and monocytes on d 7 and eosinophils on d 11. There were no differences in heterophil and basophil concentrations between infected and uninfected poults at any of the time points examined. With the exception of increased percentages of eosinophils on d 11, infection was not associated with alterations in the proportions among WBC populations. Comparison of CD4+ and CD8+ defined lymphocyte subpopulations in the blood of infected versus uninfected poults revealed higher concentrations of CD4+ lymphocytes on d 11, lower concentrations of CD8+ cells on d 4, and higher concentrations of CD8+ cells on d 11 of infection, as well as elevated ratios of CD4+:CD8+ lymphocytes in infected birds on d 4 and 11. These alterations in WBC profiles after primary E. adenoeides infection in turkey poults suggest initiation of both innate and adaptive cellular immune activities designed to effectively cope with a parasitic, intracellular pathogen.

Serum ovotransferrin as a biomarker of inflammatory diseases in chickens.

Infectious and metabolic disorders are common in poultry and cause stress, poor performance, and mortality that results in considerable economic loss. Identifying the nature of stress in chickens will assist the development of appropriate measures to improve health and welfare. Acute phase proteins are hepatic proteins, the blood concentrations of which change significantly in the event of many health problems including inflammation and physical injuries. Thus, acute phase proteins are used as nonspecific diagnostic markers for various health disorders. Our previous studies showed that serum ovotransferrin (OVT) is an acute phase protein in chickens. Therefore, in the present study, we investigated whether OVT concentration can be a marker of physiological stress using sera from chickens with different infectious and metabolic disorders. A competitive enzyme immunoassay was developed to measure serum OVT concentrations. The results show that with experimentally induced pulmonary hypertension syndrome and tibial dyschondroplasia, there were no significant changes in OVT levels compared with matched controls. In contrast, when chickens were infected with microbes such as the bacterium Escherichia coli, or protozoan parasites such as Eimeria maxima and Eimeria tenella, there was a significant increase in the levels of OVT in the serum. Chickens with spontaneous autoimmune vitiligo also showed a significant increase in blood OVT levels. These studies suggest that blood OVT concentration is modulated under inflammatory and microbial stress and can therefore be used as a diagnostic marker of infection and inflammation in chickens.

Altered monocyte and macrophage numbers in blood and organs of chickens injected i.v. with lipopolysaccharide.

Lipopolysaccharide (LPS) is a Gram-negative bacteria cell wall component that activates monocytes and macrophages to produce nitric oxide (NO) from inducible nitric oxide synthase. Nitric oxide production in the plasma of chickens peaks 5-6-h post-i.v. LPS injection reflecting iNOS activation. To determine monocyte responsiveness after an i.v. LPS injection, a time course study was conducted examining the concentrations among peripheral blood leukocytes post-i.v. LPS injection in male and female chickens, the proportions among peripheral mononuclear leukocyte (PBMC; containing lymphocytes, thrombocytes, and monocytes) populations isolated from the blood samples collected at various times post-i.v. LPS treatment, and the ability of monocytes to produce NO with and without further LPS stimulation in vitro using the PBMC NO production assay. Additionally, monocyte extravasation activity was determined by analyzing macrophage proportions after the i.v. LPS injection in spleen, lung, and liver tissues. Blood was collected from male and female chickens at 0 h (pre-LPS injection control) and at 1, 3, 6, 24, and 48 h post-LPS injection, and additionally, at 72 h from female chickens. Tissues were collected 0, 1, 6, and 48 h post-i.v. LPS injection from male chickens. Monocyte concentrations dropped substantially by 1h in both males and females. In males, monocyte concentrations returned to control concentrations by 6h and increased at 24- and 48-h post-LPS injection, whereas in females, monocyte concentrations recovered more slowly, returning to near control concentrations by 24-48-h and increasing above control levels by 72 h. Lipopolysaccharide stimulated NO production by PBMC cultures established from blood samples obtained at various times post-LPS injection in vivo followed the same pattern as monocyte concentrations in the blood. Hence, NO concentrations within PBMC cultures were dependent upon the number of monocytes that were in the PBMC cultures isolated at different times post-i.v. LPS injection. Furthermore, macrophage proportions in spleen tissues responded similarly to monocyte concentrations in the blood, decreased in lung tissue, and varied widely in liver tissue throughout 48 h after an LPS injection. Monocytes and other leukocytes may attach to the endothelium post-i.v. LPS injection preventing the monocytes from entering the needle during blood collection resulting in what seems to be leukopenia in blood and in PBMC cultures attenuating NO production in PBMC cultures. Furthermore, monocyte differentiation and recruitment from the bone marrow is a likely contributor to the reconstitution and rise of monocyte concentrations in blood samples post-i.v. LPS injection.

Cellular component of lavage fluid from broilers with normal versus aerosol-primed airways.

Previously, we reported that intratracheal administration of lipopolysaccharide elicited pulmonary hypertension (PH) in broilers reared under commercial conditions and in broilers reared in environmental chambers and pretreated with aerosolized red food colorant # 3 and propylene glycol (Red#3+PG), but not in control broilers reared in environmental chambers. The objective of the present experiment was to determine possible changes in the number or proportion of airway leukocytes that could contribute to the magnitude of the PH responses elicited in broilers. Birds were aerosolized for 40 min with a saturated mixture of Red#3+PG. After 24 h, a blood sample was taken, the broilers were killed, and a pulmonary lavage process was conducted in each bird. Leukocyte concentration (white blood cells/microL) and differential leukocyte counts (%) were measured in blood and lavage fluid. Leukocyte concentration in blood did not differ between groups, but the percentage of blood lymphocytes was lower in broilers from the Red#3+PG group compared with birds from the control group (52.4+/-2.9 and 56.9+/-2.9%, respectively). Cells recovered from the lavage fluid from both groups were primarily heterophils. The concentration of leukocytes was greater in the lavage fluid of broilers from the Red#3+PG group compared with broilers from the control group (763.2+/-158.7 and 402.9+/-62.6 white blood cells/microL, respectively), but the proportions among leukocytes were not different between the 2 groups. We propose that the increased concentration of leukocytes present within the airways was one of the components that enabled broilers pre-treated with aerosolized Red#3+PG to exhibit PH responses to intratracheal lipopolysaccharide.

Broiler pulmonary hypertensive responses during lipopolysaccharide-induced tolerance and cyclooxygenase inhibition.

Bacterial lipopolysaccharide (LPS, endotoxin) triggers pulmonary hypertension (PH) characterized by an increase in pulmonary arterial pressure (PAP) that reaches a peak value within 20 to 25 min and then gradually subsides within 60 min. As the PAP subsides PH cannot be reinitiated, signifying the onset of a period of tolerance (refractoriness) to repeated LPS exposure. The present study was conducted to determine the duration of this tolerance, and to evaluate key mediators thought to contribute to LPS-mediated PH in broilers. Tolerance was shown to persist for 4 to 5 d after the initial exposure to LPS. In tolerant broilers supramaximal i.v. injections of LPS did not reinitiate PH, nor was a significant modulatory role for nitric oxide demonstrated. The pulmonary vasculature of tolerant broilers remains responsive to the thromboxane A(2) (TxA(2)) mimetic U44069, 5-hydroxytryptamine (5-HT, serotonin), and constitutive nitric oxide. Meclofenamate successfully blocked the conversion of arachidonic acid to vasoconstrictive eicosanoids such as TxA(2); nevertheless, meclofenamate failed to inhibit PH in response to LPS. Therefore, TxA(2) does not appear to be the primary vasoconstrictor involved in the PH response to LPS and neither does 5-HT. Broilers emerging from tolerance 5 d after the initial exposure to LPS exhibited interindividual variation in their PH responsiveness to a second LPS injection, ranging from zero response (individuals that remain fully tolerant) to large increases in PAP (post-tolerant individuals). Tolerance might be an important compensatory or protective mechanism for broilers whose pulmonary vascular capacity is marginally adequate under optimal conditions, and whose respiratory systems are chronically challenged with LPS in commercial production facilities. The key vasoconstrictors responsible for the PH elicited by LPS remain to be determined.

Expression of inducible nitric oxide synthase in lungs of broiler chickens following intravenous cellulose microparticle injection.

Intravenous microparticle (MP) injection is a patented method used to select broilers with a robust pulmonary capacity and improved resistance to pulmonary hypertension syndrome (PHS, ascites). Injected MP become entrapped in the terminal pulmonary arterioles where they elicit an increase in pulmonary arterial pressure attributable to vascular occlusion and focal thrombocyte aggregation. Within 2 to 48 h postinjection perivascular mononuclear cell aggregates begin to form around MP-occluded vessels. Nitric oxide (NO) has been shown to modulate the pulmonary arterial pressure response to MP entrapment, but a role of NO during the more chronic (2 to 48 h) focal inflammatory response has not been evaluated. In this study we determined the time-course of inducible nitric oxide synthase (iNOS) expression in the lungs of MP-injected broilers from PHS-resistant (RES) and PHS-susceptible (SUS) lines. Four-week-old broilers (10 broilers/line per time point) were injected i.v. with a minimally lethal dose of MP, and the right lung was collected at 0 h (no MP) and 2, 24, and 48 h postinjection. Immunohistochemistry revealed that macrophage infiltration increased over time in both lines and was higher in the RES line than the SUS line (P<0.0001) at all time points. Nicotinamide adenine dinucleotide phosphate diaphorase staining showed nitric oxide synthase activity around MP-occluded vessels and in the perivascular mononuclear cell aggregates. Relative iNOS expression in lung tissue was examined by 2-step reverse transcription PCR. Lines differed in relative iNOS mRNA expression only at 24 h (P<0.001; RES > SUS line). For the RES line iNOS mRNA expression increased consistently from 0 to 48 h, but for the SUS line iNOS mRNA expression increased at 2 h, decreased to baseline at 24 h, and increased again by 48 h. The decline in iNOS expression in the SUS line between 2 and 24 h coincides with the interval when most of the MP-induced mortality occurs, which suggests that NO synthesized by iNOS may contribute to lower MP-induced mortality in the RES line when compared with the SUS line.

Plasma nitric oxide concentrations in broilers after intravenous injections of lipopolysaccharide or microparticles.

Nitric oxide is a potent vasodilator synthesized from l-Arg by NO synthase (NOS). Constitutive NOS in endothelial cells (eNOS) produces transient bursts of NO in low but physiologically effective levels. Activated monocytes and macrophages express inducible NOS (iNOS), which produces copious quantities of NO. Previous studies showed that NO attenuates pulmonary hypertensive responses induced by i.v. injections of lipopolysaccharide (LPS) or cellulose microparticles (MP). The present study determined whether changes in plasma NO concentrations could be used to assess the time course of NO production in response to LPS or MP injections. Broilers were injected i.v. with 1 mL of PBS (control), 1 mL of LPS (1 mg/mL), or 0.4 mL of MP (0.02 g/mL). Plasma samples were collected from 10 different broilers per group at 15, 30, 45, and 60 min and at 2, 3, 4, 5, 6, 8, 10, and 12 h postinjection. Total plasma NO concentrations were analyzed by nitrate + nitrite assay. After PBS or MP injection, plasma NO levels did not change throughout the 12-h period. Nitric oxide measured in the plasma increased in LPS-injected broilers from 4.8 +/- 0.8 microM at 15 min to 46.6 +/- 5.7 microM by 4 h postinjection, reached peak levels of 85.1 +/- 10.6 microM at 5 h, and returned to baseline levels similar to PBS-injected broilers by 12 h postinjection. We conclude that LPS triggered widespread iNOS expression by circulating monocytes and macrophages, resulting in copious NO production as reflected by significant increases in total plasma NO. Proportionally few monocytes and macrophages responded to MP entrapped in pulmonary arterioles. Consequently, NO produced by iNOS in activated leukocytes or by eNOS in the pulmonary vasculature had a minimal impact on total plasma NO. Total plasma NO from broilers did reflect the time course of massive iNOS activation in response to LPS, but biologically relevant quantities of NO produced by iNOS and eNOS activated during the local inflammatory response to entrapped MPs were too low to affect total plasma NO concentrations.

Effect of probiotic treatment in broiler chicks on intestinal macrophage numbers and phagocytosis of Salmonella enteritidis by abdominal exudate cells.

Previous data have indicated that a Lactobacillus-based probiotic culture (FM-B11) is efficacious in reducing Salmonella Enteritidis colonization within 24 h when administered within 1 h of challenge. We hypothesized that the innate immune system, specifically macrophages, may play a role in the observed reduction of Salmonella Enteritidis colonization with probiotic treatment. Day-of-hatch chicks were challenged with Salmonella Enteritidis and then treated with the probiotic culture 1 h later. Three other treatment groups were not treated (negative control), challenged only, or treated with probiotic only. In all experiments, probiotic treatment on the day of hatch reduced (P < 0.05) cecal Salmonella Enteritidis recovery as compared with the control treatment. In experiments 1 and 2, immunohistochemistry was used to evaluate the presence of macrophages (KUL01+) in the ileum and cecum of 7 to 10 chicks per group at 24 h posttreatment. In experiment 1, the number of macrophages observed per 10,000 microm(2) in the ileum of Salmonella Enteritidis-challenged chicks was higher (P < 0.05) than that of nonchallenged chicks (4.87 +/- 0.31 vs. 3.05 +/- 0.19). In the cecum, there were more (P < 0.05) macrophages per 10,000 microm(2) in chicks receiving probiotic treatment without challenge than in negative control chicks (5.32 +/- 0.41 vs. 3.66 +/- 0.35). However, in experiment 2 we found no differences among treatments in the numbers of macrophages for both the ileum and cecum. Experiments 3 and 4 were performed to evaluate the ability of Sephadex-elicited abdominal exudate cells (AEC) from chicks to phagocytose Salmonella Enteritidis in vitro. Abdominal exudate cells were isolated from the abdominal cavity, maintained in tissue culture plates overnight, and then assayed for phagocytic activity by coincubating with Salmonella Enteritidis. In experiment 3, more (P < 0.05) Salmonella Enteritidis was recovered from AEC derived from probiotic-treated chicks than in any other treatment. However, in experiment 4, all treatments resulted in similar levels of elicited AEC, and phagocytosis of Salmonella Enteritidis was at low levels in all groups. Although not conclusive, the modest differences detected in experiments 1 and 3, and the fact that those differences were not repeatedly detectable, suggest that these macrophage-related changes were not solely responsible for the reductions of Salmonella Enteritidis following probiotic treatment.

An inadequate pulmonary vascular capacity and susceptibility to pulmonary arterial hypertension in broilers.

Broilers are susceptible to pulmonary hypertension syndrome (PHS; ascites syndrome) when their pulmonary vascular capacity is anatomically or functionally inadequate to accommodate the requisite cardiac output without an excessive elevation in pulmonary arterial pressure. The consequences of an inadequate pulmonary vascular capacity have been demonstrated experimentally and include elevated pulmonary vascular resistance (PVR) attributable to noncompliant, fully engorged vascular channels; sustained pulmonary arterial hypertension (PAH); systemic hypoxemia and hypercapnia; specific right ventricular hypertrophy, and right atrioventricular valve failure (regurgitation), leading to central venous hypertension and hepatic cirrhosis. Pulmonary vascular capacity is broadly defined to encompass anatomical constraints related to the compliance and effective volume of blood vessels, as well as functional limitations related to the tone (degree of constriction) maintained by the primary resistance vessels (arterioles) within the lungs. Surgical occlusion of 1 pulmonary artery halves the anatomical pulmonary vascular capacity, doubles the PVR, triggers PAH, eliminates PHS-susceptible broilers, and reveals PHS-resistant survivors whose lungs are innately capable of handling sustained increases in pulmonary arterial pressure and cardiac output. We currently are using i.v. microparticle injections to increase the PVR and trigger PAH sufficient in magnitude to eliminate PHS-susceptible individuals while allowing PHS-resistant individuals to survive as progenitors of robust broiler lines. The microparticles obstruct pulmonary arterioles and cause local tissues and responding leukocytes to release vasoactive substances, including the vasodilator NO and the highly effective vasoconstrictors thromboxane A(2) and serotonin [5-hydroxytryptamine (5-HT)]. Nitric oxide is the principal vasodilator responsible for modulating (attenuating) the PAH response and ensuing mortality triggered by i.v. microparticle injections, whereas microparticle-induced increases in PVR can be attributed principally to 5-HT. Our observations support the hypothesis that susceptibility to PHS is a consequence of anatomically inadequate pulmonary vascular capacity combined with the functional predominance of the vasoconstrictor 5-HT over the vasodilator NO. The contribution of TxA(2) remains to be determined. Selecting broiler lines for resistance to PHS depends upon improving both anatomical and functional components of pulmonary vascular capacity.

Variation in the pulmonary hypertensive responsiveness of broilers to lipopolysaccharide and innate variation in nitric oxide production by mononuclear cells.

Variability among broilers in their pulmonary hypertensive (PH) responsiveness to lipopolysaccharide (LPS) appears to reflect innate variation in the types or proportions of vasodilators and vasoconstrictors released by leukocytes and endothelial cells. Two experiments were designed to evaluate possible correlations between the PH responsiveness to LPS in vivo and the quantities of nitric oxide (NO; a potent pulmonary vasodilator) produced by mononuclear cells in vitro. In Experiment 1, blood samples were collected from male broilers from a base population (control group) and from survivors of a 60% lethal dose i.v. injection of cellulose microparticles (MP survivor group). In Experiment 2, blood samples were collected from male broilers from a relaxed line and from lines known to be susceptible or resistant to pulmonary hypertension syndrome. Peripheral mononuclear cells (PMNC) from each blood sample were cultured at 2 million cells per well, remained unstimulated, or were stimulated with LPS to elicit the expression of inducible NO synthase, and the 24-h production of NO was measured. In both experiments, unstimulated PMNC cultures did not produce consistently detectable levels of NO, whereas LPS-stimulated cultures produced quantities of NO that varied widely among individuals. Nitric oxide production by cultured PMNC also was evaluated by flow cytometry, demonstrating that LPS-stimulated PMNC produced substantially more NO than did unstimulated cells in all of the groups evaluated. Moreover, NO-producing PMNC were identified to be monocytes. The same broilers from which PMNC had been isolated were catheterized subsequently to record pulmonary arterial pressure, LPS was injected i.v. to assess the amplitudes of peak and postpeak PH responses, then N(omega)-nitro-L-arginine methyl ester was injected to inhibit ongoing NO production. In Experiment 1, the amplitude of the peak and postpeak PH responses to LPS were correlated with the quantity of NO produced by LPS-stimulated cultured PMNC from broilers in the control group but not for MP survivors. In Experiment 2, the postpeak PH response to LPS was correlated with the quantity of NO produced by LPS-stimulated PMNC from broilers in the relaxed line, but not in the susceptible or resistant lines. In all groups, N(omega)-nitro-L-arginine methyl ester injections triggered substantial increases in pulmonary arterial pressure (> or = 8 mm Hg), thereby revealing a significant ongoing modulation by NO of the PH response to LPS. We concluded that most of the modulatory NO generated in vivo during the acute PH response to LPS (within 60 min postinjection) likely is produced by constitutive NO synthase in the vascular endothelium. In addition, the NO produced by inducible NO synthase in PMNC appeared to have modulated the LPS-stimulated PH responses of unselected broilers having the broadest range of pulmonary vascular capacities (control broilers and relaxed line), but not in broilers whose pulmonary vascular capacities had been selected to represent the higher (MP survivors, resistant line) or lower (susceptible line) extremes of the population.

Maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens.

Maternal antibodies are transferred from hens to the chicks via the egg. To gain insight into maternal antibody transfer and endogenous production of antibodies in broiler chicks, total IgY, IgA, IgM, as well as anti-Newcastle disease virus (NDV) and anti-infectious bronchitis (IBV) antibody levels were examined in the dams' plasma, egg yolks, egg whites, and chicks' plasma on d 3, 7, 14, and 21. Blood was collected from 39-wk-old breeder hens (line 1, n = 17; line 2, n = 21). Fertile eggs were used for antibody extraction from the egg yolks and egg whites (4 to 5 eggs/dam) and for hatching. Unvaccinated chicks (4 to 5 chicks/dam) were reared in a HEPA-filtered room and were bled on d 3, 7, 14 and 21. Based on ELISA methods, plasma levels of IgY and IgM were higher (P < 0.0001), and those of IgA were similar (P = 0.31), in line 2 compared with line 1. Egg yolk IgY and IgA, as well as egg white IgY, IgA, and IgM levels were higher in line 2 compared with line 1 (P < 0.0001). Independent of line of chicken, the percentage dam-to-chick (3 d) plasma transfer of IgY was estimated to be approximately 30%, with that for IgM and IgA less than 1%. Chicks synthesized IgM first, followed by IgA and IgY. Anti-NDV and anti-IBV antibodies were detected in the dams' plasma, egg yolks, and in the chicks' plasma on d 3 and 7, with line 2 having higher anti-IBV and lower anti-NDV levels than line 1 in all samples (P < 0.0001). In summary, IgY levels, total or antigen-specific, in the dams' plasma or eggs were found to be a direct indicator of maternal antibody transfer to the chicks' circulation, with an expected percentage transfer of approximately 30%. This knowledge, together with the observed time course of endogenous antibody production in broiler chicks, may find direct application in formulating strategies for protecting chicks, especially during the first few weeks of age when their immune system is not yet fully functional.

The influence of different management systems and age on intestinal morphology, immune cell numbers and mucin production from goblet cells in post-weaning pigs.

At time of weaning, the immune system in piglets is not fully mature resulting in reduced growth and increased mortality. Early-weaned pigs transported to a segregated early weaning (SEW) facility have enhanced performance and gut development compared to conventional (CONV) pigs which may be due, in part, to decreased pathogen challenge. To gain further insight into SEW enhanced performance and gut development, gut samples from pigs weaned at 19+/-2 days were assessed during the post-weaning (PW) period. The numbers of cells expressing CD2, CD4, CD8, and CD172 (the 74-22-15 (SWC3) antibody is now known to be specific for CD172), MHC class II, and CD25 were quantified using immunohistochemistry. Additionally, samples of duodenum, jejunum, and ileum were evaluated for the production of neutral, acidic, and sulfuric mucins from goblet cells and morphological measurements were also made. No effects due to the management systems alone were observed for any of the parameters. However, there were interactive effects of age/time post-weaning and management system on the immune cells as well as on the mucin secreting goblet cells. There were no differences in gut morphology between SEW and CONV reared pigs.

Influence of aminoguanidine, an inhibitor of inducible nitric oxide synthase, on the pulmonary hypertensive response to microparticle injections in broilers.

The pulmonary hypertensive response to pulmonary vascular obstruction caused by intravenously injected microparticles is amplified by pretreatment with N(omega)nitro-L-arginine methyl ester (L-NAME). The L-NAME prevents the synthesis of the potent vasodilator nitric oxide (NO) by inhibiting both the constitutive [endothelial NO synthase (eNOS or NOS-3)] and inducible [inducible NO synthase (iNOS or NOS-2)] forms of NO synthase. In the present study we used the selective iNOS inhibitor aminoguanidine (AG) to evaluate the role of iNOS in modulating the pulmonary hypertension (PH) triggered by microparticle injections. Experiment 1 was conducted to confirm the ability of AG to inhibit NO synthesis by iNOS in broiler peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide (LPS, endotoxin). Mononuclear leukocytes treated with LPS produced 10-fold more NO than untreated (control) cells. The LPS-stimulated production of NO was partially inhibited by L-NAME and was fully inhibited by AG, thereby confirming that AG inhibits LPS-mediated iNOS activation in broilers. In Experiment 2 we evaluated the responses of male progeny from a base population (MP Base) and from a derivative line selected for one generation from the survivors of an LD50 microparticle injection (MP Select). The pulmonary arterial pressure (PAP) was lower in MP Select than in MP Base broilers. Both lines exhibited similar percentage increases in PAP after microparticles were injected, and AG modestly amplified the PH triggered by microparticles in both lines. In Experiment 3 we evaluated the responses of male progeny from a second base population (PAC Base) and from a derivative line selected for 3 generations using the unilateral pulmonary artery clamp technique (PAC Select). The PAP was lower in PAC Select than in PAC Base broilers, and both lines exhibited similar percentage increases in PAP in response to the microparticles. The PH triggered by microparticles was not amplified by AG but was doubled by L-NAME. These experiments demonstrate that during the 30 min following pulmonary vascular entrapment of microparticles, iNOS modulated the PH elicited in broilers derived from the MP pedigree line, but not in broilers from the PAC pedigree line. Different NOS-mediated responses among broiler populations may affect pulmonary hemodynamic characteristics of broiler lines selected using i.v. microparticle injections.