Wing Web Research Articles

Social environment and immunity in male red jungle fowl

We examined the relationship between social dominance, immune response, and ornamentation in captive red jungle fowl by comparing these variables in males housed individually with a single female to those in the same males after they were placed in flocks with an unfamiliar male and three unfamiliar females. Males with larger combs before being placed in the flocks were more likely to become dominant, and dominant males’ combs grew after flock formation, whereas subordinate males’ combs shrank. Immune response as reflected in hematocrit, immunoglobulin levels, and wing web swelling (a measure of cell-mediated immunity) was stronger in males that later became dominant, both before and after flock formation, although the difference between dominant and subordinate birds was more pronounced after males were housed in the multi-male groups. Dominant and subordinate males also differed in the relationship between comb length and wing web swelling. Among dominant males, individuals with larger combs had significantly larger swellings after flock formation, whereas within the subordinate males, those with relatively larger combs had worse cell-mediated immunity than those with smaller combs. These results suggest that males of different quality pay different costs to maintain both ornamentation and immune defense.

Parasites and Tradeoffs in the Immune Response of Female red Jungle Fowl

Reproduction and survival are influenced by parasites, and recently, the ability of the immune system to fight infection has been included in life history theory. A condition dependent immune response predicts tradeoffs within the immune system and condition dependent tradeoffs with other life history traits. Female red jungle fowl (Gallus gallus) parasitized with an intestinal nematode (Ascaridia galli) had a higher concentration of eosinophils than controls, and parasitized and control females responded differently to injections of phytohemagglutinin (PHA). PHA injections stimulated an increase in granulocytes in parasitized females relative to controls. Although parasitized females had greater circulating levels of the white blood cells that are involved in the cutaneous hypersensitivity response, control females exhibited greater swelling of the wing web six hours after injection. A. galli seemed to slow the cellular immune response to injections of PHA. suggesting a tradeoff within the cellular component of the immune system. Parasitized and control females differed in the relationship between immunoglobulin G (IgG) and the strength of the cutancous hypersensitivity response. The decrease in cutaneous hypersensitivity with increasing IgG in parasitized vs control females suggests a condition dependent tradeoff between the cellular and humoral components of the immune system.

Simplifying the phytohaemagglutinin skin‐testing technique in studies of avian immunocompetence

1. Researchers involved in ecology and toxicology, as well as many other aspects of avian biology, use phytohaemagglutinin (PHA) skin testing as a means of evaluating the immune status of individuals. 2. Immune function, one measure of individual quality, can be used as a sensitive, non‐lethal variable that may be negatively affected in animals exposed to degraded, contaminated or otherwise disturbed ecological zones. 3. Typically this test has been applied by challenging one wing web with the immunostimulant PHA, while the other ‘control’ wing is injected with phosphate buffered saline (PBS). Injection sites on the wing web are measured before and 24 h after injection with PHA or PBS. The immune response is considered to be the difference between the two wings. 4. Results from PHA skin tests conducted on 608 birds in seven studies representing passerines, waterfowl, upland game birds and raptors are examined. 5. Numerous advantages to eliminating the PBS injection as the experimental control are: (i) decrease by half, the time required for testing; (ii) decrease handling‐related stress on the birds (proportional to handling time); (iii) reduce the probability of errors at injection time; (iv) spare the other wing for different tests or uses (e.g. tuberculin DTH testing); and (v) decrease the coefficient of variation that is due to measurement inaccuracies. 6. The only disadvantage identified is that hypersensitive individuals (outliers) could be missed, which in this case represents 2 of 608 individuals.

Introduction to poultry vaccines and immunity

The poultry industry constitutes a significant sector of world agriculture. In the United States, more than 8 billion birds are produced yearly with a value exceeding $20 billion. Broiler chickens are the largest segment of the industry. Birds raised under commercial conditions are vulnerable to environmental exposure to a number of pathogens. Therefore, disease prevention by vaccination is an integral part of flock health management protocols. Active immunization using live vaccines is the current industry standard. Routinely used vaccines in chickens include MDV, NDV, IBV, and IBDV, and in turkeys NDV and HEV. Newer vaccines, including molecular recombinants in which genes of immunogenic proteins from infectious agents are inserted into a live viral vector, are also being examined for commercial use. Efforts are under way to enhance vaccine efficacy by the use of adjuvants, particularly cytokines. The vaccine delivery systems include in ovo injection, aerosol, spray, drinking water, eye drop, and wing web injection. The in ovo vaccination procedure is relatively new and at the present time it is used primarily to vaccinate broiler chickens against MDV. Birds respond to vaccines by developing humoral and cellular immune responses. Bursa of Fabricius and the thymus serve as the primary lymphoid organs of the immune system. B cells use surface immunoglobulins as antigen receptors and differentiate into plasma cells to secrete antibodies. Three classes of antibodies are produced: IgM, IgG (also called IgY), and IgA. Successful vaccinal response in a flock is often monitored by demonstrating a rise in antibody titer within a few days of vaccination. ELISA is used most commonly for serologic monitoring. T cells are the principal effector cells of specific cellular immunity. T cells differentiate into alpha beta and gamma delta cells. In adult birds, gamma delta cells may constitute up to 50% of the circulating T cells. Functionally, CD4+ cells serve as helper cells and CD8+ cells as cytotoxic/suppressor cells.

Kinetic and immunohistochemical characteristics of mitogen-induced cutaneous hypersensitivity in chickens selected for antibody responsiveness

Mitogen-induced cutaneous hypersensitivity was evaluated in chickens selected for high and low antibody responses to SRBC, and in a random bred control line. Wing web swelling responses were found after subcutaneous administration of phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide (LPS), respectively, in all three lines. All mitogens induced significant acute 4 h wing web swelling responses, followed by a significant (classical) late 24 h wing web swelling response. The 4 h responses were significantly lower in the L line, whereas a tendency for lower responses at 24 h in the L line was found as well. Immunohistochemical evaluation of the early and late wing web swelling responses revealed extravascular localisation of leukocytes at 24 h after sensitization with mitogens, which consisted of CD4+ cells, CD8+ cells, TCR-1+ cells, and heterophils, but no B cells, whereas the 4 h swelling response was primarily characterized by oedema. Cutaneous hypersensitivity either initiated by T-cell mitogens as well as B-cell mitogens may depend for an important part on the rapid induction of local homing of lymphocytes towards the sensitizing agent, which may be mediated by an acute local expression of molecules with chemo-attractive capacities. Interpretation of cellular immunity responses in vivo such as delayed-type hypersensitivity should therefore incorporate oedema-initiating characteristics of sensitizing agents. The relationship between the magnitude of cutaneous hypersensitivity to mitogens and selection for antibody responsiveness is discussed.

Pasteurella Challenge and ELISA Serology Evaluation of Broiler Breeders Vaccinated with Live Cholera Vaccine

Broiler breeder pullets were vaccinated against fowl cholera at 10 and 20 wk of age using a live PM-1 Pasteurella multocida vaccine administered by wing web stick. Antibody production for P. multocida was measured at vaccination and at 1-4-wk intervals following vaccination by enzyme-linked immunosorbent assay. Groups of vaccinated birds were challenged at 23 and 32 wk of age. Two doses of a live PM-1 P. multocida vaccine protected broiler breeder hens against virulent challenge up to 32 wk of age when measured antibody levels had a range of 1951-4346 and a geometric titer of 3000.

Reduced immunocompetence of nestlings in replacement clutches of the European magpie (Pica pica)

Laying date is one of the most important determinants of reproductive success and recruitment probability in birds. Late breeders usually fledge fewer chicks than individuals with earlier breeding dates, and fledglings produced late in the season have high mortality rates. Food availability and nestling mass have been evoked as the principal mechanistic links between laying date and offspring survival. Here we suggest that another factor may actually account for the difference in survival rate between early and late offspring: immunocompetence. We predicted that nestlings produced later in the season or in replacement clutches should have lower immune responses when challenged with an antigen, than early nestlings or nestlings produced in first clutches. This hypothesis was tested in a population of magpies (Pica pica), in which we experimentally induced breeding failure in a group of nests and compared the immune response of nestlings in replacement clutches with the immune response of first clutch nestlings. Cellular immune response, as measured by wing web swelling (a correlate of T-lymphocyte production after injection of phytohaemagglutinin-P), significantly decreased with hatching date and was significantly lower in nestlings of replacement clutches. Furthermore, coefficients of intraclutch variation in immune response were higher in nestlings of replacement clutches. This experiment demonstrates an inverse relationship between immune responsiveness and breeding date, and reduced recruitment probability of late nestlings may be a direct consequence of their inability to cope with parasites.

Examination of the Dietary Methionine Requirements of Breeding Northern Bobwhite, Colinus virginianus

Adult Northern bobwhite were used to test the hypothesis that dietary methionine levels recommended by the NRC for breeding quail are excessive for wild bobwhite. We tested the hypothesis by comparing immunocompetence, reproductive performance, and chick viability of Northern bobwhite hens fed diets containing low (0.31%), moderate (0.39%), or high (0.47%) concentrations of methionine. Chick viability was determined by assessing immunocompetence, including evaluating the ability of hens to passively transfer immunity to their chicks. Hens were fed the experimental diets for 6 wk on an ad libitum basis. After 6 wk, methionine treatment had no measurable effect (P ≥ 0.20) on hen phytohemagglutinin wing web indices, organ weights, or serum anti-Pasteurella multocida titer indices. Mean egg weight, percentage egg production, total cumulative egg production, yolk weight, yolk volume, and percentage fertile and percentage hatch of fertile eggs did not differ (P ≥ 0.12) among diet treatments. Amount of albumen in eggs produced by hens fed the high methionine diet averaged 0.27 g more (P = 0.003) than eggs of hens fed the low methionine diet. Anti-P. multocida titer of yolks from eggs in Week 6 were not different (P = 0.36) between birds fed the high and the low methionine diets. The mortality rate of chicks after challenge with 23 cfu of P. multocida was not different (P ≥ 0.05) among diets. Chicks hatched from eggs laid by vaccinated hens during Weeks 2 and 3, however, had lower (P < 0.05) mortality than chicks of unvaccinated hens. It appears a dietary methionine concentration of 0.3% may be sufficient for wild Northern bobwhite to produce viable chicks.

Effect of Injection Site on Cutaneous Basophil Hypersensitivity Response to Phytohemagglutinin

Cutaneous basophil hypersensitivity response to phytohemagglutinin (PHA) is frequently used as a measure of cell-mediated immune response in chickens. Several anatomical sites have been used to measure this response. No correlation between response level at different sites, however, has been reported. This study compares responses to PHA by using two different injection sites (wing web and interdigital skin of the foot) in White Leghorn chickens. Response at each individual site was comparable whether the bird received a PHA injection at that site alone or at both sites. Correlations between responses at the two sites were not different from zero [–.1733 (P = .44)]. These data indicate that caution must be exercised in comparing cell-mediated immune responses when different PHA injection sites have been used to assess response.

Influence of dose and route of inoculation on responses of chickens to recombinant fowlpox virus vaccines

The influence of dose and route of inoculation on responses of chickens to vaccination with recombinant fowlpox viruses (rFPVs) expressing an influenza haemagglutinin (HA) (FPV-HA) and the infectious bursal disease virus (IBDV) VP2 antigen (FPV-VP2) has been evaluated. Antibody responses to influenza and fowlpox virus were generated following vaccination via the wing web by subcutaneous inoculation or skin scarification. Intranasal and conjunctival inoculation failed to induce antibodies to FPV or influenza. Following direct intratracheal inoculation antibodies developed to influenza but not FPV. Dose response studies with the FPV-HA and FPV-VP2 recombinants showed that good responses to FPV and the vaccine antigen could be generated over a wide ( 100000 fold) dose range following wing web inoculation. The responses generated by the FPV-VP2 recombinant over this vaccine dose range protected against IBDV infection of the bursae following challenge with the Australian IBDV 002/73 isolate. These data suggest that effective application of rFPVs for poultry vaccination may be restricted to wing web and parenteral routes of inoculation.

Protection against turkey rhinotracheitis pneumovirus (TRTV) induced by a fowlpox virus recombinant expressing the TRTV fusion glycoprotein (F).

A recombinant fowlpox virus was produced which expressed the fusion protein (F) of turkey rhinotracheitis virus (TRTV), a pneumovirus. Turkey poults were vaccinated twice, at an interval of 2 weeks, intramuscularly and by wing web on each occasion, with the recombinant or a control fowlpox virus. Two weeks after the second vaccination the poults were challenged superconjunctivally and intranasally with virulent TRTV. A partially protective immune response was achieved; turkeys vaccinated with the F recombinant showed milder clinical signs and 1000-fold less challenge virus was recovered from the nose and trachea compared with turkeys that had been vaccinated with control fowlpox virus. Expression of the F protein induced antibodies which were detectable both by an ELISA and a virus neutralization test. These results show that the immune responses to the F protein play a major role in protection against TRTV and indicate that recombinant viruses expressing the TRTV F protein have potential as vaccines against TRT.

Measurement of antibody titer to fowl pox virus by enzyme-linked immunosorbent assay.

The usefulness of the measurement of antibody titer to fowl pox virus (FPV) by enzyme-linked immunosorbent assay (ELISA) was evaluated in SPF chickens with or without inoculation with FPV. The optimum concentration of purified antigen was 10 micrograms/ml of protein. The absorbance at 492 nm was less than 0.10 in the chickens negative to FPV from 1 to 63 days old. By contrast, a higher titer was detected in SPF chickens with various FPVs inoculated into the wing web than in non-inoculated chickens. Moreover, there was no cross response to chicken sera immunized with Haemophilus paragallinarum, Marek's disease virus, Newcastle disease virus or infectious bronchitis virus. The titers increased after vaccination were not increased after subsequent challenge with virulent FPV. These findings suggested the usefulness of the measurement of the antibody response to FPV vaccine by ELISA.

Direct and Correlated Responses to Multitrait, Divergent Selection for Immunocompetence

Leghorn lines had been selected for an immunocompetence index based on four traits: antibody production to Mycoplasma gallisepticum (MG) and Pasteurella multocida (PM) vaccines, reticuloendothelial clearance of colloidal carbon (CCA), and cell-mediated, wing web response to phytohemagglutinin (PHA). The purpose of this study was to produce replicated lines of chickens with divergent levels of multitrait immunocompetence by index selection. The objectives of analyses of Generations 5 to 7 of this study was to characterize these lines with respect to immune-response traits, correlations among these traits, and correlated responses in other important production traits.Differences (P < .05) existed between the lines selected for high or low immune response and between the two replicates in mean breeding values and in individual immune-response traits. Averages of heritability estimates, weighted by number of offspring and pooled across three generations (two cycles of selection), estimated by using sire variance components and parent-offspring correlations were, respectively, .16 and .09 for the index, .31 and .08 for MG, .21 and −.02 for PM, .06 and .05 for CCA, and .08 and .12 for PHA. Realized heritabilities (response divided by effective selection differential) pooled across the two selection cycles, were .19 and .11 for the index, .06 and −.01 for MG, .44 and .32 for PM, 1.52 and −1.21 for CCA, and .48 and .15 for PHA, for Replicates 1 and 2, respectively. Phenotypic correlations among traits were generally small, and several estimates were negative. Estimates of genetic correlation varied widely. Juvenile and adult body weights, age of first egg, 32-wk egg weight, and rate of egg production were analyzed to evaluate effects of selection on these traits of direct economic importance. Very few differences were noted.

Cutaneous Hypersensitivity Responses in Chickens Divergently Selected for Antibody Responses to Sheep Red Blood Cells

Cellular immunity in vivo of chickens selected for high and low antibody responses to SRBC was evaluated by primary and secondary cutaneous hypersensitivity reactions to BSA and phytohemagglutinin (PHA)-induced delayed-type hypersensitivity (DTH).Similar to PHA-induced DTH, primary and secondary cutaneous hypersensitivity to BSA comprised acute (1- and 2-h), intermediate (4- and 6-h), and late (24-h) wing web swelling responses. Primary and secondary hypersensitivity to BSA revealed line differences between the High (H) line and a randombred Control (C) line on the one hand, and the Low (L) line on the other hand. Hypersensitivity to BSA was almost absent or decreased in the L line. The late phase of PHA-induced DTH was significantly elevated in the H line as compared with the L line. Naive recipients sensitized systematically with either serum from chickens sensitized with BSA, or with BSA-binding IgG, developed an antigen-specific early wing web swelling response following local challenge with BSA. It is suggested that the acute component of cutaneous hypersensitivity is mediated by Ig. The acute response may be a prerequisite for the development of classical (late) cutaneous DTH responses or may be initiated simultaneously with DTH. The current results indicate that the selection did not result in a higher hypersensitivity response to another T cell-dependent antigen and a mitogen in the H line, but in a lower response in the L line.

Infectious bursal disease virus structural protein VP2 expressed by a fowlpox virus recombinant confers protection against disease in chickens.

Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP4-VP3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP2 and VP3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP2 contained only the VP2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of the TK gene. The expression level of VP2 from FPV-VP2 was approximately 5 times higher than from FPV-VP2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.

Vaccination of chickens with live fowl pox (FP) vaccine in oil.

Live fowl pox (FP) vaccine was adjuvanted in oil just prior to the subcutaneous (SC) vaccination of one day old chicks and adult chickens. The birds were challenged by the wing web (WW) method and absence of "takes" were considered as protection. On 21 day post challenge, 90%-100% of the chicks or chickens were protected while on day 9 post challenge 60% were protected. Full protection of the live-in-oil adjuvanted vaccine is probably somewhat delayed as compared to protection endowed by the liquid vaccine. Incorporation of live FP vaccine in two different kinds of commercial Newcastle disease (ND) killed vaccine in oil, was shown to endow full protection following SC administration.

Tumor immunity generated in the course of regression of v-src-induced sarcomas.

Previous studies have indicated that the regression versus progression of v-src-DNA-induced sarcomas is dependent on chicken line. As a first step in analyzing the role of tumor immunity as a determinant of this line dependence, experiments were undertaken to ascertain whether an antisarcoma immune response is generated in the course of sarcoma growth in TK chickens, a regressor line. To assay for this response, test TK chickens in which v-src-induced wing web sarcomas had regressed, as well as control TK chickens that had not been exposed to v-src, were challenged in protocols known to yield v-src-dependent sarcoma formation and monitored for challenge sarcoma growth. Compared with the control chickens, the test chickens showed a significant resistance to the sarcomagenic challenge. These results raise the possibility that the antisarcoma response that is inducible in regressor lines, as demonstrated here in terms of a protective effect against a subsequent sarcomagenic challenge, may also underlie the regression of v-src-induced primary sarcomas.

Estimates of Quantitative Genetic Parameters of Immunological Traits in the Chicken

Three in vivo assays were used to measure the immunocompetence of chickens in two generations of a selection experiment. The obtained data were used to estimate the variance components for sire and dam for antibody production to Pasteurella multocida and Mycoplasma gallisepticum vaccines, for T-cell–mediated immunity evaluated by a phytohemagglutinin wing web assay, and for clearance of foreign particles from the circulatory system. Heritabilities of and genetic correlations among these immunological traits were calculated from the sire variance components. Heritability estimates of the immunological traits based on the sire component of variance ranged from .06 to .53, and genetic correlations among immunological traits were generally negative.

On the tumorigenicity of mitochondrial DNA-depleted avian cells

We have examined the tumorigenic potential of mitochondrial DNA-depleted (mtDNA −) cells derived from the tumorigenic chicken cell line DU24. The mtDNA − cells were unable to proliferate in the wing web of day-old chicks. Cytoplasmic hybrids resulting from crosses between the mtDNA − whole cells and cytoplasts from enucleated parental cells (mtDNA +) recover both mtDNA and tumorigenicity. These results are in accordance with those obtained in prior experiments [33] where mtDNA was shown to modulate the anchorage-independent phenotype of transformed avian cells.

V-src genes in two cell lines of so-called RSV-transformed human cells are defective and inactive.

Trial of Rous sarcoma virus (RSV) induction by cell fusion with chick embryo cells (CEC) and wing web test from so-called RSV-transformed human cells, KC and RSb cells, was unsuccessful. The loss of RSV inducibility was also confirmed by DNA transfection method. Southern blot and northern blot hybridization of DNA and RNA from those cells with the v-src probe revealed that the v-src genes in those cells were defective and not expressed. On the other hand, the v-src gene in RSV-transformed mouse and rat cells was complete and transforming virus was inducible from them.