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.

Melanocyte-specific cell mediated immune response in vitiliginous Smyth line chickens.

Smyth line chickens (SL) are an animal model to study human autoimmune vitiligo. Vitiligo in SL chickens is characterized by a post-hatch loss of feather melanocytes (MC) resulting in feather depigmentation. The etiopathology of this disease remains unclear, however, a hypothesis that cell-mediated immunity (CMI) plays an important role in the destruction of melanocytes has been proposed based on previous studies. To support this hypothesis, the delayed wattle response (DWR) to MC lysates was examined in SL chickens and normally pigmented, MHC-matched controls (Brown line, BL; Light Brown Leghorn, LBL) to determine whether vitiliginous SL chickens have MC-specific CMI. Chickens were challenged in one wattle with a lysate of either embryo- or feather-derived cultured MC. Lysates of chick-embryo-fibroblasts (CEF) associated turkey herpesvirus (HVT) vaccine and syngeneic CEF were used as positive and negative control, respectively. Vitiliginous SL chickens exhibited a significantly greater DWR to feather-MC but not to embryo-MC and CEF than nonvitiliginous SL chickens and normally pigmented controls. The DWR to feather-MC peaked at 3 to 5 days post feather-MC lysate injection. Immunohistochemical staining of DWR-positive wattle tissue revealed extensive lymphocyte infiltration at the site of feather-MC injection consisting primarily of T cells (TCR2+, CD4+ or CD8+ T cells). These observations support the presence of MC-specific CMI in SL chickens with vitiligo. The anti-MC CMI activity appears to be specific to MC derived from feathers. SDS-PAGE analysis of different sources of MC lysates revealed a 43 kDa missing band in the lysate of feather-MC established from SL chickens. No other obvious protein pattern differences that could account for the feather MC-specific CMI response in vitiliginous SL chickens were observed among the MC lysates.

Divergent selection for delayed-type wattle reaction of domestic fowls to BCG antigen.

1. Two-way selection for delayed-type hypersensitivity wattle reaction (DWR) competence to BCG antigen was carried out over 4 generations, using a flock of White Leghorn chickens. DWR was measured by intradermal injection of BCG into the wattle of chickens. 2. Selection for DWR was effective. The generation means of average selection differential, selection response and realized heritability were 1.15 mm, 0.77 mm and 0.70 respectively. Correlated responses were found in body weight, egg weight and hatchability. In all of these traits, the L line (selected for low wattle reaction) showed higher values than the H line (high reaction). 3. Specific trends in gene frequencies were observed at the major histocompatibility B and alkaline phosphatase Akp loci. 4. Significant line differences were found in graft-versus-host reaction competence and Marek's disease resistance. In both characters, the L line was higher than the H line. No line differences were found in phytohaemagglutinin response, phagocytic activity or immune responses.

Rooster comb and wattle tissues contain an anti-keratan sulfate monoclonal antibody epitope.

Glycosaminoglycan fractions isolated from either comb or wattle tissue were examined using ELISA for their antigenicities to AH12, a monoclonal antibody recognizing keratan sulfate. The results showed a positive antibody binding to a sulfated glycosaminoglycan fraction from either tissue. The treatment of the same fraction with keratan sulfate degrading enzyme, keratanase, or endo-beta-galactosidase resulted in a decrease in its antigenicity. These findings indicated the presence of AH12 epitope (keratan sulfate disaccharide) in both tissues.

Kinetics of the wattle reaction to human plasma.

Human plasma produced cutaneous basophil hypersensitivity when injected into chicken wattles. The kinetics of the response development were affected by presensitization, whether or not complete Freund's adjuvant was used. Presensitized chickens developed their maximum response significantly sooner (12.6 hr earlier) than the controls.

Major histocompatibility (B) complex-associated differences in the delayed wattle reaction to staphylococcal antigen.

The influence of the major histocompatibility (B) complex on the delayed wattle reaction (DWR) to Staphylococcus aureus was studied in 109 segregants (B2/B2, B2/B5, and B5/B5) of a fourth generation cross between inbred Regional Poultry Research Laboratory lines 6(1) and 15(1). Chickens were sensitized at 6 weeks of age with S. aureus antigen. One week later, DWR was evaluated by injecting the right wattle with S. aureus antigen. Thickness measurements were taken 4, 24, 48, 72, and 96 hr after injection. A quadratic equation model was used for each bird to calculate maximum wattle thickness, hour of maximum response, and rate of response development and decline. In males, the maximum wattle thickness in response to S. aureus antigen was significantly greater in B2/B5 heterozygotes (1.77 +/- .07 mm) than in either homozygote, B2/B2 (1.36 +/- .13 mm) or B5/B5 (1.39 +/- .08 mm). Heterozygous males reached maximum response sooner and recovered more quickly than homozygous males but these differences were not statistically significant. In females, response developed later than in males but no B complex effect was detected in either rate of development or maximum response.

Monoamines suppress the phytohemagglutinin wattle response in chickens.

The influence of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE) on the phytohemagglutinin (PHA) wattle response was studied in three populations derived from line UNH 105 New Hampshire chickens. At 3 or 6 weeks of age, chicks were injected intravenously with either 5-HT, DA, NE, or a saline control. Thirty minutes after monoamine injection, 100 ug PHA-P was injected in the wattle and the thickness increase was calculated 24 hr later. Analysis of variance showed significant monoamine effects compared to controls but no difference in response among the three populations. 5-HT and NE each depressed significantly the wattle response in 3 and 6 week old chicks. DA reduced the response in both ages also but the significantly greater reduction in 6 than in 3 week old chicks indicated an age effect. These results suggest a regulatory role for monoamines in the PHA wattle response.

Major histocompatibility (B) complex and sex effects on the phytohaemagglutinin wattle response.

The influence of the major histocompatibility (B) complex and sex on the phytohaemagglutinin (PHA) wattle response was studied in 136 segregants (B2/B2, B2/B5 and B5/B5) of a fourth generation cross between inbred lines 6(1) and 15(1). At 6 weeks of age, chickens were injected with 100 micrograms purified PHA-P. Wattle thickness measurements were taken 4, 24, 48, 72 and 96 h after injection. Analysis of variance showed that 4 h after injection, males had a significantly higher response than females but the sex-genotype interaction was also significant. Females had higher responses than males 24 and 48 h after injection as a consequence of more rapid development and earlier resolution of the reaction in males. B2/B2 chickens had significantly lower responses than B5/B5 chickens 72 and 96 h after injection, signifying a faster late resolution phase in the B2/B2 genotype. The developmental and early resolution phases of the PHA wattle response were influenced by sex while the late resolution phase was influenced by B genotype.

Primary and secondary wattle swelling response to phytohemagglutinin as a measure of immunocompetence in chickens.

Wattle responses of 2-to-8-week-old chickens to phytohemagglutinins PHA-P and PHA-M were studied. Dilutions of PHA-M that did not induce wattle swelling after one injection did cause readily detectable swelling when injected a second time 1 week later. These responses were absent in birds subjected to thymectomy and gamma-irradiation at hatching and in birds treated with cyclosporin A during the week of sensitization, indicating that these responses are T-cell-dependent. Chickens bearing transplantable fibrosarcomas failed to show responses. It is suggested that reactivity to a second injection of PHA-M may be used as a measure of immunocompetence at the T-cell level in very young chickens.

The delayed and saline wattle reactions in broilers challenged with bovine serum albumin.

Bovine serum albumin was used to study the development of a wattle reaction in broiler-type chickens. A strong response developed in both nonsensitized and sensitized chickens and the frequency of such was shown to be dependent on the method used for the sensitization. A similar swelling developed in a proportion of the wattles injected with saline and this too seemed to be influenced by the sensitization method. Moreover, both types of responses were shown to be somewhat unstable during the first 24 hr postchallenge.

Adoptive transfer of delayed wattle reactivity in chickens with a dialyzable leukocyte extract containing transfer factor.

Delayed wattle reactions (DWR) to tuberculin, diphtheria toxoid (DT), and keyhole limpet hemocyanin (KLH) were transferred to chickens with dialyzable leukocyte extracts (DLE) prepared from splenic leukocytes of chickens sensitized and reactive by DWR to the tuberculin, DT, or KLH. A DLE prepared from chickens unsensitized and unreactive to tuberculin by DWR failed to transfer DWR to tuberculin in recipients. Only chickens injected with DLE from tuberculin sensitized and reactive chickens exhibited significant (P less than .01) DWR to tuberculin. Chickens that received DLE prepared from DT sensitized and reactive chickens exhibited significant (P less than .01) DWR to DT but not to KLH. Further, DWR to both DT and KLH was transferred with DLE prepared from chickens sensitized to both antigens. Adoptive transfer of DWR to KLH in comparison to DT was more successful. Finally, the serial transfer of DWR to KLH (P less than .05) but not to DT (P greater than .05) was accomplished using DLE prepared from chickens that previously were recipients of DLE prepared from chickens sensitized to KLH and DT. Results indicate that DLE prepared from chickens contain transfer factor (TF) responsible for adoptive transfer of DWR to tuberculin, DT, and KLH.

Effect of levamisole on the phytohemagglutinin induced basophil hypersensitivity reaction in the chicken wattle.

The potentiating effect of levamisole (L) on the phytohemagglutinin (PHA) induced hypersensitivity skin reaction in the chicken's wattle was studied. The L increased the number of heterophils and basophils 10 and 6 times, respectively. The number of mononuclear phagocytic cells in the PHA skin reaction was unaltered. These results contrast slightly with those of mammals.

Heat- and cold-stress suppresses in vivo and in vitro cellular immune responses of chickens.

The influence of hot and cold ambient temperatures on cellular immune responses of chickens was assessed, utilizing dinitrofluorobenzene-induced contact sensitivity, phytohemagglutinin (PHA) skin test, and PHA-stimulated, whole blood lymphocyte transformation assays. Dinitrofluorobenzene- and PHA-induced wattle swelling were reduced (P less than 0.05) in birds exposed to air temperatures of 36 degrees C for 1 C for 5 days when compared with wattle swelling in chickens maintained at 26 C. The PHA-induced lymphocyte transformation, as measured by tritiated thymidine uptake, was also suppressed (P less than 0.05) in chickens exposed to either heat or cold stress for 5 days. Some breed differences were observed. These data demonstrated that chronic heat and cold stress impaired expression of contact sensitivity in vivo and proliferation of T lymphocytes in vitro in the avian species. Synthesis of antibodies to sheep RBC on day 5 after antigen injection was not altered (P greater than 0.05) when birds were vaccinated 24 hours before the start of thermal stress. This indicated that B cell and T-helper cell functions were not compromised by chronic thermal stress. However, New Hampshire birds that were heat-stressed had higher (P less than 0.05) antibody titers than control birds 10 days after vaccination.

Kinetics of delayed-type hypersensitivity (DTH) reactions and Ig levels in FCA-induced dysgammaglobulinemic B 12 chickens.

Kinetic studies on delayed-type hypersensitivity (DTH) to Freund's complete adjuvant (FCA) have been done in spontaneous dysgammaglobulinemic chickens, which have drastically reduced serum IgG levels and highly elevated IgM and IgA levels - in comparison to chickens with normal immunoglobulin (Ig) levels. For a period of five weeks, serum Ig levels, leukocyte migration inhibition (LMIT), and wattle reaction were examined once a week. In normal chickens, FCA treatment resulted in stimulation of IgG, but did not affect IgM synthesis, whereas in spontaneous dysgammaglobulinemic chickens, FCA stimulated only IgM synthesis. Spontaneous dysgammaglobulinemic chickens could produce LMIT and wattle reactions as well as normal birds. Whereas in normal birds, both types of DTH reactions declined continuously about the third or fourth week, in immunodeficient chickens, further increments of LMIT and wattle reactions up to the fifth week persisted as evidenced by LMIT and wattle reactions even 15 weeks after sensitization. In contrast, only minimal signs of reactivity were seen in normal birds. Spontaneous dysgammaglobulinemic chickens, nearly unable to synthesize IgG even after FCA stimulation in vivo, lack suppressive mechanisms regulating the course of DTH reaction to FCA. The possible B-cell nature of the regulatory cell population is discussed.

Induction of delayed hypersensitivity to turkey herpesvirus in fowls.

Delayed hypersensitivity, as determined by the wattle skin reaction, to turkey herpesvirus developed in chickens sensitised with the infected cell antigen. A significant increase in skin thickness occurred in the sensitised wattles as compared with the controls.

Suppression of phytohemagglutinin skin response in thymectomized chickens.

Intradermal injection of phytohemagglutinin (PHA) in the wattles of chickens elicited marked swellings often accompanied by induration. Histologically, this reaction was characterized by a perivascular accumulation of lymphocytes and macrophage migration in the central layer of the wattles. Heterophilic infiltration was observed mostly at early hours and waned thereafter. Sometime basophils were prominently located around the vessels. These responses were significantly decreased in cases of neonatal thymectomy. Therefore, the PHA skin test was considered to be a thymus-dependent response. The use of the PHA skin test in chickens may provide useful information for the evaluation of thymus-dependent function.

Specific in vitro lymphocyte response of chickens injected with killed Mycobacterium tuberculosis.

An in vitro microassay for lymphocyte transformation, using 3H-thymidine incorporated into avian peripheral blood leukocytes, is described. The transformation responses of 1 X 10(6) leukocytes stimulated by the nonspecific mitogen, phytohemagglutinin-M (PHA-M), were equivalent with 68- and 92-hour incubation durations. The PHA-M in concentration of 50 micronl/ml produced greater stimulation than did that of 25 or 100 micronl/ml. The transformation response to PHA-M was significantly affected by the lot of bovine fetal serum used to supplement the RPMI 1640 culture medium. The specific antigen, purified protein derivative (PPD) of tuberculin, stimulated significant transformation in cultures of 1 X 10(6) leukocytes from Mycobacterium tuberculosis-sensitized fowl. The PPD at concentration of 50 microng/ml was superior to 100 or 150 microng/ml, and 68-hour incubation was significantly better than 92-hour incubation. The magnitude of the in vitro transformation response to PPD was greater at 6 weeks after sensitization than at 2 or 4 weeks, and it was not directly related to the magnitude of the in vivo wattle test response in sensitized chickens.

Delayed wattle reaction as a measure of cell-mediated immunity in the chicken.

The cell-mediated immune (CMI) response to diphtheria toxoid (DT) was studied in commercial broiler breeder lines. The chickens were immunized with DT in complete Freund's adjuvant 21 days before CMI assay by delayed wattle reactivity (DWR) to DT. The DWR was shown to be a valid measure of CMI and to correlate with spleen cell migration inhibition and blastogenesis assays of CMI in vitro. In addition, each criterion of CMI was met, except for the passive transfer of DWR. These criteria were delayed timing, gross morphology, histopathology of DWR, and effect of antilymyphocytic sera on DWR. The duration of DWR over a 1-year period differed in the two lines of chickens tested. A significantly greater DWR was found in one line with all birds remaining reactive for CMI whereas in the other line 16% did not have CMI by the end of the year. The differences may reflect genetic variability within and between lines in expression of CMI.

Age-dependent resistance of chickens to salmonella in vitro: phagocytic and bactericidal activities of splenic phagocytes.

The phagocytic and bactericidal activities to Salmonella pullorum (strain 9-25) or Salmonella senftenberg (strain 99D) were examined in chicken splenic phagocytes from 0-day-old to 2-month-old chickens. The phagocytic activity against S pullorum increased in splenic phagocytes from chickens older than 7 days, but significant changes in activity against S senftenberg were not observed during the experimental period. The bactericidal activity of splenic phagocytes against S senftenberg was higher than that of phagocytes against S pullorum during the same period. Increase of the bactericidal activity against S pullorum was observed with increasing age, but the activity of the splenic phagocytes from 0-day-old chickens against S senftenberg was similar to that of the phagocytes from 2-month-old chickens. Although delayed hypersensitivity was confirmed by delayed wattle reaction in 2-month-old chickens sensitized with living S pullorum, the sensitization did not markedly affect phagocytic and bactericidal activities.