Aim. To determine the difference in immune responses of the harderian gland in clinically healthy chickens and the ones with infectious bronchitis based on the content, localization and morphometric estimation of the surface markers of Т- and В-lymphocytes and to determine the differentiation index as an indicator of assessing body defenses. Methods. Histological, immunohistochemical, optical, morphometric and statistical. Results. The histological study of the harderian gland of chickens with infectious bronchitis determined the swelling and proliferation of the connective tissue as well as infiltration of secretory lobules by lymphoid cells. It was found that the immunity of chickens with infectious bronchitis, in which the harderian gland plays a relevant role, depends considerably on the differentiation index of immunocompetent cells. There was a reliable 1.77- and 1.36-fold decrease in this indicator for 40- and 90-day-old chickens, respectively, in case of nephroso-nephritic form of infectious bronchitis which demonstrated a weaker function of the defense cells of this organ. According to the cytomorphometric analysis, the number of cells, expressing CD4+, CD8+, CD20+, CD45RA+ markers in the harderian gland of sick 20-, 40-, and 90-day-old chickens with respiratory and nephroso-nephritic forms of infectious bronchitis was reliably (P < 0.05) increasing compared to the clinically healthy chickens. For instance, the number of mature В-lymphocytes increased in sick 20-day-old chickens – 2.44 times, 40-day-old chickens – 1.88 times, and 90-day-old ones – 2.62 times compared to clinically healthy chickens. Conclusions. The data were obtained about the changes in quantitative and qualitative composition of lymphocytes with surface markers CD4+, CD8+, CD20+, CD45RA+ in the harderian gland of chickens with infectious bronchitis. Our results will supplement current knowledge about the feasibility of immunohistochemical methods in the diagnostics of avian infectious bronchitis.

Immune formations of birds' digestive organs, including the esophageal tonsil and Meckel’s diverticulum, protect the body from foreign antigens that enter the body with food and water and play an important role in maintaining the genetic constancy of its internal environment. This unique property of the immune system is formed during ontogenesis and is associated with maintaining the selection of lymphocyte clones that are able to respond to foreign antigens and carry out a specific immune response of two types: humoral and cellular. This article presents the results of a study of T- and B-lymphocyte subpopulations of the esophageal tonsil of Shever 579 cross chickens at the age of 25, 180 and 300 days, the Meckel diverticulum of the Blagovarsky cross ducks at the age of 30, 150 and 180 days. Immunohistochemical and statistical research methods were used to determine the localization and quantitative parameters of cell populations of lymphocytes (CD4+, CD8+, CD20+) using monoclonal antibodies and the DAKO EnVision FLEX+ imaging system (Dako Cytomation, Denmark). Separate subpopulations of T-lymphocytes (CD4+ - helpers, CD8+ -cytotoxic / T-suppressors) and mature B-lymphocytes (CD20+) were found in the esophageal tonsil and Meckel diverticulum of birds. Their presence confirms that antigen-independent proliferation and differentiation of lymphocytes into effector cells occur in the immune formations of the digestive system. The lymphoid tissue of these formations is represented mainly by a well-defined diffuse form and nodules with light centers (secondary). In the esophageal tonsil of chickens, these structures are located in the tunica mucosa and tela submucosa, and in the Meckel diverticulum of ducks – also in the tunica muscularis. The content of lymphocytes with these markers predominates in diffuse lymphoid tissue compared to that in secondary lymphoid nodules. In the diffuse lymphoid tissue of the esophageal tonsil, lymphocytes are located mainly near the adenomeres and excretory ducts of the esophageal glands, blood vessels, and under the surface epithelium, and in Meckel’s diverticulum – around the crypts, in their epithelium and in the epithelium of the villi. They are also found in the light centers of lymphoid nodules and on their periphery. The indices of the content of lymphocytes with the indicated markers in the esophageal tonsil and Meckel diverticulum which we determined were associated with age characteristics of the poultry in the postnatal period of ontogenesis. According to our observations, the content of CD20+ lymphocytes was the highest, while the populations of CD4+ and CD8+ lymphocytes were much smaller. This indicates an increase in the activity and predominance of the humoral immunity over the cellular one. The content of CD20+ lymphocytes was highest in birds at the age of 180 days, that is, during their sexual maturity. The data presented in the work can be used by morphologists researching the organs of the immune system, immunologists, poultry specialists involved in breeding, using and raising poultry and in educational work.

The article presents the results of histological studies of topography, microstructure and functional features of the stomach lymphoid tissue in birds. The research was carried out on 12 species of wild birds belonging to 6 orders. Classical methods of histological specimens staining with hematoxylin and eosin, according to Weigert, Van Gieson, and argentum nitrate impregnation according to Kelemen were used when performing the work. It has been confirmed that the birds' stomach has glandular (proventriculus), muscular (ventriculus, gizzard) and pyloric parts, which are not equally expressed in separate species. In all parts of the stomach, lymphoid tissue is represented by local clusters, which in birds of the orders Galliiformes (P. colchicus, B. bonasia, P. crictatus); Anseriformes (B. canadensis); Passeriformes (P. pica, C. cornix); Gruiformes (G. chloropus, F. atra); Ciconiiformes (C. ciconia) and Columbiiformes (C. livia) are located in the tunica mucosa and submucosa, and in B. canadensis, F. atra, G. chloropus also in the tunica muscularis and tunica serosa. In L. lagopus and G. glandarius, lymphoid tissue has been found only in the proventriculus and its intermediate zone. Structural levels of lymphoid tissue (diffuse form, prenodules, primary and secondary lymphoid nodules) are unequally expressed in the birds' stomach. In B. bonasia, B. canadensis, P. pica, C. cornix, C. ciconia, C. livia, only a diffuse form appears in all its parts. This form of lymphoid tissue is registered in the ventriculus of other birds species and proventriculus in L. lagopus and G. glandarius. All levels of the lymphoid tissue structural organization are detected in the proventriculus in P. crictatus, G. chloropus and in the proventriculus and pyloric part of the stomach in P. colchicus and F. atra. The content of lymphoid tissue is different in parts of the birds' stomach. Most of all it is contained in the intermediate zone of the proventriculus and the pyloric part of the stomach. Diffuse form is the most common among the levels of lymphoid tissue structural organization. The research results make it possible to more fully assess the morphofunctional status of wild birds and, accordingly, to find out the state of their immune system

Knowledge about the morphological features of the structure and functions of the digestive organs provide the basis for rational and effective use of feed, prevention and treatment of gastrointestinal diseases in poultry. In this regard, the study of the morphogenesis of the digestive system in birds and the mechanisms of their regulation is of great importance. The object of the study was the glandular part of the stomach (proventriculus) in chickens of Shaver 579 strain. The material for macro- and microscopic examinations was selected from birds at age of 1, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 days and 1, 2, and 3 years; it was fixed in a 10% neutral formalin solution and embedded into paraffin according to conventional methods. For submicroscopic examinations, the material was selected from hens of this strain at age of 180 days. The structure of epitheliocytes in the superficial epithelium and secretory cells of the deep glands was studied in ultrathin sections. Digital indicators of research results were statistically processed by a personal computer using Microsoft Excel program. The glandular stomach in chickens is a direct extension of the esophagus and has the form of a thick-walled tube, the wall of which is formed by mucous, muscular, and serous membranes. The superficial epithelium of the mucosa is represented by cylindrical epithelial cells that are located within the basal membrane. They are linked to each other by different types of contacts and have a well-defined polar differentiation. The lobules of the deep glands are formed by cells with well-developed synthesizing organelles and secretory granules. The morphogenesis of the glandular part of the stomach in chickens according to age aspect is manifested by changes in morphometric parameters of the thickness and the area of the membranes of its wall. The wall thickness increases (between the folds 4223.23 ± 189.25 and in the area of the folds 5561.32 ± 45.01 μm) unevenly in chickens up to age of 240 days. The most developed membrane of the wall of the glandular stomach is the mucosa. Its area increases (by 82.14 ± 0.56%) in chickens up to age of 180 days, and area of muscular and serous membranes decreases (by 15.54 ± 0.65 and 2.32 ± 0.33%, respectively). In older birds, the thickness of the wall and the area of the membranes of the glandular stomach do not change significantly.

Chronic diffuse liver diseases are characterized by continuous progression of fibrosis, ultimately leading to cirrhosis with the following loss of the normal functioning of this organ due to excessive accumulation of the components of extracellular matrix. To find new, more available diagnostic markers of detecting disorders in the liver, we used methods of antifungal cytofluorometry and quantitative real-time polymerase chain reaction. Intensity of exposure of fibronectin and plasmatic membrane of lymphocytes in the group of patients with chronic diffuse diseases compared with the control group of practically healthy donors decreased both inside and on the surface of the cells respectively by 45.3% and 16.2%. Similar tendency towards decrease was observed during the assays of the level of the exposure of fibronectin on the surface and inside the blood granulocytes: by 25.0% and 36.5%, respectively. In the blood of the patients suffering from chronic diffuse diseases, compared with the control group, there was determined reliable increase in percentage of lymphocytes and granulocytes which contain topical fibronectin, by 32.3% and 2.78 times, correspondingly. The level of monocytes (as a percentage) with cell-associated fibronectin and fibronectin localized inside, by contrast, reliably decreased in 2.07 and 4.50 times, respectively. Analysis of the expression of FN1 in lymphocytes of blood of the studied groups using quantitative real-time polymerase chain reaction revealed decrease in the level of FN1 mRNA expression by 34.0% in the group of ill patients compared with the control group. We determined excellent diagnostic informativeness of the parameters of the level of exposure of fibronectin inside and on the surface of granulocytes and prognostic accuracy of the classifier from these parameters at the level of 100% using the method of support vector machine, SVM. High levels of diagnostic informativeness were recorded for the tests of all types of analyzed leukocytes with cell-associated fibronectin, and the classifiers based on the pair combinations of the tests with cell-associated fibronectin and fibronectin localized within the cells provide high diagnostic accuracy of the prognosis. Because the mentioned indicators are highly-sensitive tests, they can be proposed for early diagnostics and evaluation of the effectiveness of the conducted therapy of chronic diffuse liver diseases, which would allow reducing the use of paracentetic trepanobiopsy, a painful and risky procedure, which still remains the main type of diagnostic.