Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 7
Issue 2
Veterinary Medicine
Available Online: http://www.ejpau.media.pl/volume7/issue2/veterinary/art-04.html


Stanisław Graczyk, Wojciech Zawadzki, Albert Czerski, Aleksandra Pliszczak-Król, Bogusław Kotoński



An experiment was carried out evaluating the course of cellular response in turkeys receiving propranolol and ACTH.

Key words: turkey, DTH, propranolol, ACTH.


Depriving animals of their natural environment has created many hazards, not only epizootic ones. The negative results of stress constitute a problem. In poultry-breeding it is particularly related to turkeys, whose breeding work has been directed at obtaining maximal muscle mass, while neglecting their inborn ability of adaptation to the changing living conditions. This favours not only falls and the development of pathological lesions of not fully explained etiopathogenesis but most of all, the deterioration of well-being [2, 6, 8, 9, 10, 19]. The well-being of animals exposed to stress (transport, concentration, extreme temperatures, etc.) constitutes currently one of the basic problems. This is caused by the fact that there is neither general consent to the determination methods of the quality of animal response nor the ways allowing the prevention of stress reaction and the type and intensity of stress stimulation in animals. The majority of research published so far concerning stress in birds concentrates on the relations within hypothalamic-pituitary-adrenal axis, playing a basic role in the adaptation processes [15, 29, 32]. The release of catecholamines during the initial stage of psychogenic stress causes the occurrence of not only of cardiovascular and metabolic effects. Also the influence of these substances on the function of cells and organs of lymphatic system is proved. The changes caused by the catecholamines released may be totally or partially eliminated by blocking, e.g. adrenergic receptors. A lot of attention is also devoted to finding means and methods allowing the prevention or attenuation of stress in animals. Many agents are used in order to decrease the negative results of stress – among others: xylasine, prazosin, acepromazine, diazepam, morphine or magnesium aspartate or ascorbic acid [1]. The selection of anti-stress agents is rather empirical or based on the established principles related mainly to the neurochemical aspects of stress. There is no universal means o r proceedings that would prevent stress [1].

Propranolol is one of the available and used drugs of adrenergic antagonists group. As the non-selective β-adrenergic receptor antagonist, propranolol is applied in medicine in the treatment of hypertension and cardiac diseases. It is also used in the prevention of stress (e.g. sudden cardiac death) in pigs [1].

Taking into consideration the data presented and particularly the role of adrenal hormones in the formation, course and development of stress, the authors decided to check the influence of propranolol as β-adrenergic blocker on the intensity of delayed type hypersensitivity reaction, after intracutaneous administration of PHA (phytohemagglutinin) in turkeys remaining under the influence of ACTH administered.


The material consisted of 24 turkeys of heavy BIG-6 type, females of 6 - 9 weeks of age. The birds were subject to 14-day acclimatisation after bringing them from the farm. Then, they were divided into four groups, with identical dietary and zoohygienic conditions. All the birds were weighed before the experiment in order to make dosage of the substances administered easier. Turkeys of groups 3 and 4 were given propranolol with drinking water during the whole period of the experiment (Uterotonic-Polfa in the dose of 1 mg/kg body weight/day). When administering propranolol, the procedure applied stayed in accordance with the description presented in the earlier research paper [13]. 24 hours after the beginning of propranolol administration, the birds of groups 2 and 4 received intramuscular injection of ACTH (Synacten – Ciba in the dose of 1 iu/100 g of body weight). The administration of ACTH was repeated after subsequent 24 hours.

The proposed system is presented in table 1.

Table 1. The schema of experimental procedure

Group 1

Group 2

Group 3

Group 4

Control turkeys

Turkeys which were administered ACTH

Turkeys which were administered propranolol

Turkeys which were administered propranolol and ACTH

Before the administration of the second dose of ACTH, 24 hours before the end of the experiment, all birds were subject to the wing test for the delayed type hypersensitivity (DTH) [11, 21, 33]. It consisted in intracutaneous injection of 0.1 cm3 PHA – (DIFCO LAB - 1000 µg/cm3) into the right wing. A similar procedure with the other wing, where PBS was administered, was assumed as the control. The thickness of the skin fold, exactly in the PBS or PHA place of injection, was measured with a micrometer before and 24 hours after injection. The response level was determined by calculating the wing index (WI) as the difference of the fold thickness before and 24 hours after the intracutaneous injection of one of the substances mentioned.


Cutaneous delayed-type hypersensitivity (DTH) with the use of mitogens is applied as an intravital test for the evaluation of T lymphocyte reactivity, and is successfully used in birds [12, 16, 19, 20, 24, 26, 31]. The wing web, intradigital skin of the foot and - in older individuals - wattle are convenient places of mitogen administration in case of birds. However, the testing place does not play a significant role in its evaluation and usefulness [4, 26]. In a classical DTH test in mice as well as in birds, the test reading takes place after 24 – 48 hours. A local oedema is formed as soon as after 1 – 2 hours as a result of response to antigen. It is caused by the release of vasoactive amines, such as histamine or serotonin, from mastocytes [4, 17, 26, 28]. An intermediate stage, during which the activation of immunoglobulins occurs, takes place 4-6 hours after the administration of antigen [26]. T lymphocytes are not included in the contents of inflammatory infiltration, neither during the early nor the intermediate stage, which is supposed to result from too short a time of exposure to the antigen. The mechanism of the early and intermediate stages of response to the administration of PHA (phytohemagglutinin) or another mitogen stimulating T and B lymphocyte response, has not been explained so far [25].

On the basis of the observations conducted in thymectomized birds one can state that DTH is a thymus-dependant reaction. It is confirmed by histological examinations of sections taken from PHA administration places, where cellular infiltrations and endothelial cell lesions similar to those present in thymus-dependant zones of lymphatic nodes of mammals were found [11, 21]. Many factors may influence the course and evaluation of DTH after the administration of antigens or mitogens, among others adrenal cortex hormones [12, 27].

It was demonstrated in the experiment discussed that 24 hours after the administration of the second dose of ACTH to the turkeys, the wing index value (WI) did not differ from that noted in control birds. Also no differences of this index were noted after the administration of PBS only. The wing index value in the control group of birds and in the group which was administered ACTH (groups 1 and 2) amounted to 0.993 ± 0,12 mm and 0.993 ± 0.16 mm respectively. On the other hand, the wing index in turkeys receiving propranolol increased by 12% (p≤0.20), and it amounted to 1.113 ± 0.48 mm. The administration of ACTH to the birds influenced by propranolol resulted in the wing index increase by 33.33% (p≤0.001), which amounted to 1.328 ± 0.39 mm (Fig 1).

Fig 1. Wing index value.
1 - control group of birds
2 – group of birds which were administered adrenocorticotrophic hormone (ACTH)
3 – group of birds which were administered propranolol
4 – group of birds which were administered propranolol and ACTH

Paramietier et al. [25], after the PHA injection (DTH test) into the wing of chicken, observed the wing index increase of 1.33 mm in the 24th hour. PBS injection, on the other hand, caused IS increase only by 0.7 mm (reading taken in 24th hour). The inflammatory infiltration included mainly mononuclear cells. A detailed cytological analysis demonstrated that TCR-1+, CD4+ and CD8+ lymphocytes dominate [25].

A weakened response of the immune system is often observed in animals exposed to stressors. This is related to the effects of glycorticoids released during stress. They are believed to be responsible for the suppression of humoral and cellular response [3, 7, 22]. It has been demonstrated that, apart from immunosuppression, glycocorticoids cause changes in the metabolism and behaviour of the animal [9]. Corticosterone is the main glycocorticoid released by the birds’ adrenal glands [5, 23, 30]. El-Lethey et al. [9] demonstrated that in chickens taking corticosterone with drinking water, the following occurs: the weakening of the response to the PHA (DTH test), neutrophil to lymphocyte ratio increase (H/L ratio), body weight as well as egg production decrease. Post et al. [27] suggest that DTH test is an easy and useful test for the evaluation of cellular response in chickens with the increased corticosterone level. The lack of the wing index value change - recorded in the experiment discuss ed - in the group of turkeys subject to ACTH activity, in comparison to the control group (Fig. 1), may be caused by the short period of ACTH administration (double injection of ACTH at 24 hour interval), as well as the dose applied of this hormone. It is possible that the double administration of ACTH results in short and not cumulating changes in the adrenal gland function. The reduction of adrenal glands sensitivity also seems possible [14].

On the other hand, the increase on the wing index observed in the groups of birds taking propranolol with drinking water (groups 3 and 4) proves the role of catecholamines in the control of the cellular type response. According to Konstandi et al. [18], the adrenergic system plays an important role in the formation of changes in the organism influenced by stress. The blockage of β-adrenergic receptors with propranolol results in the cancellation of negative results of stress for the organism, which is proved by the WI increase.


The changes demonstrated of the wing index after the administration of PHA indicate the contribution of catecholamines in the cellular response (increase of the wing index in the groups of birds taking propranolol) as well as their role in the interaction of the neuroendocrine and immune systems.


  1. Ali B.H., Al.-Qarawi A.A.: An evaluation of drugs used in the control of stressful stimuli in domestic animals: a review. Acta. Vet. Brno. 2002, 71, 205-216.

  2. Berezi I.: The stress concept and neuroimmunoregulation in modern biology. Ann. N. Y. Acad. Sci. 1998, 851, 3-12.

  3. Corrier D.E., DeLoach J.R.: Interdigital skin test for evaluation of delayed hypersensitivity and cutaneous basophil hypersensitivity in young chickens. Am. J. Vet. Res. 1990, 51, 950-954.

  4. Cotter P.F., Wing T., Swanson J.: The delayed and saline wattle reactions in broilers challenged with bovine serum albumin. Poult. 1985, 64, 1293-1295.

  5. Curtis M.J., Flack I.H., Harvey S.: The effect of Escherichia coli endotoxins on the concentrations of corticosterone and growth hormone in the plasma of the domestic fowl. Res. Vet. Sci. 1980, 28, 123-127.

  6. Dantzer R.: Stress and immunity: what have we learned from psychoneuroimmunology ?. Acta Physiol. Scand. Suppl. 1997, 640, 43-46.

  7. Davison T.F., Misson B.H.: Effect of corticosterone on antibody responses to a T-dependent and T-independent antigen in domestic fowl. Med. Sci. Res. 1987, 15, 967-968.

  8. Dohms J.E., Metz A.: Stress-mechanisms of immunosuppression. Vet. Immunol. Immunopathol. 1991, 30, 89-109.

  9. El-Lethey H., Huber-Eicher B., Jungi T.W.: Exploration of stress-induced immunosuppression in chickens reveals both stress-resistant and stress-susceptible antigen responses. Vet. Immunol. Immunopath. 2003, 95, 91-101.

  10. Glaser R., Kiecolt-Glaser J.K., Malarkey W.B., Sheridan J.F.: The influence of psychological stress on the immune response to vaccines. Ann. N. Y. Acad. Sci. 1998, 840, 649-655.

  11. Goto N., Kodama H., Okada K., Fujimoto Y.: Suppression of phytohemagglutinin skin response in thymectomized chickens. Poultry Sci. 1978, 57, 246-250.

  12. Graczyk S., Madej J., Pliszczak-Król A.: The influence of splenectomy on humoral and celullar immune response and morphology of central immune response and morphology of central lymphatic organs in the chickens immunized by different type of antigens. Sci. Lett. (Wrocław, Agric. University). 1998, 58, 7-15.

  13. Graczyk S., Pliszczak-Król A., Kotoński B., Wilczek J., Chmielak Z.: Examinations of hematological and metabolic changes mechanisms of acute stress in turkeys. Elec. J. Pol. Agric. Univ. s. Vet. Med. 2003,Vol. 6 Iss. 1.

  14. Graczyk S., Kuryszko J., Madej J.: The reactivity of spleen germinal centres in immunized and ACTH treated chickens. Acta Vet. Brno. 2003, 72, 523-531.

  15. Hashimoto M., Watanabe T., Fujioka T., Tan N., Yamashita H., Nakamura S.: Modulating effects of prenatal stress on hyperthermia induced in adult rat offspring by restraint or LPS-induced stress. Physiol. Behav. 2001, 73, 125-132.

  16. Kirby J.D., Froman D.P: Research note: evaluation of humoral and delayed hypersensitivity responses in cockerels reared under constant light or a 12 h light: 12 h dark photoperiod. Poult. Sci. 1991, 70, 2375-2378.

  17. Klesius P., Johnson W., Kramer T.: Delayed wattle reaction as a measure of cell mediated immunity in the chicken. Poult. Sci. 1977, 56, 249-256.

  18. Konstandi M., Johnson E.O., Marselos M., Kostakis D., Fotopoulos A., Lang M.A.: Stress-mediated modulation of B(α)P-induced hepatic CYP1A1: role of catecholamines. Chem. Biol. Interaction. 2004,147, 65-77.

  19. Magnusson U., Wattrang E., Tsuma V., Fossum C.: effects of stress resulting from short-term restraint on in vitro functional capacity of leukocytes obtained from pigs. Am. J. Vet. Res. 1998, 59, 421-425.

  20. McCorkle Jr. F.M., Stinson R., Glick B.: A biphasic graft vs. Host repsonse in aging chickens. Cell. Immunol. 1979, 46, 208-212.

  21. McCorkle Jr. F.M., Olah I., Glick B.: The morphology of the phytohemagglutinin-induced cell response in the chicken’s wattle. Poult. Sci. 1980, 59, 616-623.

  22. Murray D.L., Brake J., Thaxton J.P.: Effect of adrenocorticotropin and dietary ascorbic acid on cutaneous basophil hypersensitivity to phytohemaglutinin in chickens. Poult. Sci. 1987, 66, 1846-1852.

  23. Palladino M.A., Giimour D.G., Scafuri A.R., Stone H.A., Thorbecke G.J.: Immune response differences between two inbred chicken lines identical at the major histocompatibility complex. Immunogenetics. 1997, 5, 253-259.

  24. Palladino M.A., Grebenau M.D., Thorbecke G.J.: Requirements for induction of delayed hypersentivity in the chicken. Dev. Comp. Immunol. 1978, 2, 121-132.

  25. Parmentier H.K., De Vries Reilingh G., Nieuwland M.G.B.: Kinetic and immunohistochemical characteristics of mitogen-induced cutaneous hypersensitivity in chickens selected for antibody responsiveness. Vet. Immunol. Immunopath. 1998, 66, 367-376.

  26. Parmentier H.K., Schrama J.W., Meijer F., Nieuwland M.G.B.: Cutaneus hypersensitivity responses in chickens divergently selected for antibody responses to sheep red blood cells. Poult. Sci. 1993, 72, 1679-1692.

  27. Post J., Gielkens A.: Delayed type hypersensitivity reaction as indicator of cellular immune competence in broiler chickens exposed to dietary corticosterone. Acta Agric. Scan. 2004, 54, 30-35.

  28. Ptak W., Geba G.P., Askenase P.W.: Initiation of delayed-type hypersensitivity by low doses of monoclonal IgE antibody. Mediation by serotonin and inhibition by histamine. J. Immunol. 1991, 146, 3929-3936.

  29. Sheridan J.F., Dobbs C., Jung J., Chu X., Konstantinos A., Padgett D., Glaser R.: Stress-induced neuroendocrine modulation of viral pathogenesis and immunity. Ann. N. Y. Acad. Sci. 1998, 840, 803-808.

  30. Siegel H.S.: Physiological stress in birds. Bioscience. 1980, 30, 529-534.

  31. Smits J.E., Bortolotti G.R., Tella J.L.: Simplifying the phytohaemagglutinin skin-testing technique in studies of avian immunocompetence. Func. Ecol. 1999, 13, 567.

  32. Watanabe T., Fujioka T., Hashimoto M., Nakamura S.: Stress and brain angiotensin II receptors. Crit. Rev. Neurobiol. 1998, 12, 305-317.

  33. Zhu X.Y., Porter R.E., Hester P.Y.: Delayed-type hypersensitivity reaction induced in broilers by killed staphylococcus aureus1. Poul. Sci. 1999, 78, 1703-1710.

Stanisław Graczyk
Department of Pathological Anatomy
Pathophysiology, Microbiology and Forensic Veterinary Medicine
Division of Pathophysiology
Agricultural University
Norwida 31, 50-375 Wrocław, Poland
e-mail: graczyk@ozi.ar.wroc.pl

Wojciech Zawadzki,
Department of Animal Physiology
Agricultural University
Norwida 31, 50 – 375 Wrocław, Poland
e-mail: waza@ozi.ar.wroc.pl

Albert Czerski Department of Animal Physiology
Agricultural University
Norwida 31, 50 – 375 Wrocław, Poland
e-mail: Albert-Czerski@wp.pl

Aleksandra Pliszczak-Król
Department of Pathological Anatomy
Pathophysiology, Microbiology and Forensic Veterinary Medicine
Division of Pathophysiology Agricultural University
Norwida 31, 50-375 Wrocław, Poland
e-mail: graczyk@ozi.ar.wroc.pl

Bogusław Kotoński
Department of Biochemistry
Pharmacology and Toxicology, Faculty of Veterinary Medicine
Agricultural University
Norwida 31, 50-375 Wrocław, Poland

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed ‘Discussions’ in each series and hyperlinked to the article.