Thymus cell pool of the common carp against the background of using Cellobacterin-T

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Thymus cell pool of the common carp against the background of using Cellobacterin-T

Viktoriya Vyacheslavovna Zhukova, Evgeniy Vladimirovich Mikhaylov,

Valeriya Sergeevna Bolotova, Yuliya Vladimirovna Shaposhnikova,

Boris Viktorovich Shabunin

Аннотация

Пул клеток тимуса карпа на фоне применения Целлобактерина-Т

Виктория Вячеславовна Жукова, Евгений Владимирович Михайлов, Валерия Сергеевна Болотова, Юлия Владимировна Шапошникова, Борис Викторович Шабунин

В статье описан клеточный состав тимуса сазана (Cyprinus carpio) в возрасте трех лет на фоне применения пробиотического препарата Целлобактерин-Т, содержащего Bacillus subtilitis. Показатели опытной группы свидетельствовали об уменьшении интенсивности воспалительного процесса, выявленного при фоновом исследовании, что выражалось снижением процентного содержания моноцитов, нейтрофилов и повышением лимфоцитов. В ходе эксперимента изготавливали мазки из тимуса, которые окрашивали по Паппенгейму. Цитоз микропрепаратов наблюдался от среднего до высокого, фон был базофильным, обнаруживались разрушенные эритроциты за счет гемодилюции. Большинство клеток при окраске по Паппенгейму идентичны клеткам высших позвоночных (лимфоциты, макрофаги, эпителиоциты и др.). Некоторые клетки (нейтрофилы, моноциты и др.) имеют свои цитоморфологические особенности. Преобладающей группой клеток были малые лимфоциты. Бластные формы лимфоцитов наблюдались в единичном количестве. При применении Целлобактерина-Т в опытной группе показатели моноцитов и нейтрофилов значительно ниже, чем в контрольной группе: D14 -- в 1,9 раза, D28 -- в 1,7 раза, D42 -- в 1,3 раза. Количество лимфоцитов близко к физиологической норме: D14 -- 68,6 %, D28 -- 80,7 %, D42 -- 79,4 %, что свидетельствует о снижении воспалительной реакции. Ключевые слова: тимус, цитология, сазан, лимфоциты, миелоциты, клетки Мотта.

Abstract

This article describes thymus cellular composition of the common carp (Cyprinus carpio) at the age of three years against the background of using the probiotic drug Cellobacterin-T that contains Bacillus subtilitis. The indicators of the experimental group showed a decrease in the intensity of the inflammatory process, revealed in the background study, which was expressed by a decrease in the percentage of monocytes, neutrophils and an increase in lymphocytes.

During the experiment, thymus smears were made, which were stained according to Pappenheim. Cytosis of microslides was observed from medium to high, the background was basophilic, destroyed erythrocytes were found due to hemodilution. Most of the cells, when stained by Pappenheim, are identical to those of higher vertebrates (lymphocytes, macrophages, epithelial cells, etc.). Some cells (neutrophils, monocytes, etc.) have their own cy- tomorphological features. The predominant group of cells were small lymphocytes. Blast forms of lymphocytes were observed in a single amount. When using Cellobacterin-T in the experimental group, the indicators of monocytes and neutrophils are much lower than in the control group: D14 -- by 1.9 times, D28 -- by 1.7 times, D42 -- by 1.3 times. The number of lymphocytes is close to the physiological norm: D14--68.6 %, D28--80.7 %, D42-- 79.4 %, which indicates a decrease in the inflammatory response.

Keywords: thymus, cytology, common carp, lymphocytes, myelocytes, Mott cells

Introduction

Fish diseases can cause great damage to fish farming, therefore, in order to successfully breed fish, in particular the common carp, to obtain high productivity of water bodies, it is important to know and be able to diagnose the most common fish diseases and effectively implement preventive measures. In some cases, the disease is caused by a pathogen that enters the body of the fish, in others -- the fish becomes sick with a lack or an excess of certain substances dissolved in water, sharp fluctuations in water temperature, mechanical damage, as well as insufficient nutrition or malnutrition. Therefore, it is important to study the vital systems of fish organs for their further correction under the conditions of anthropogenesis [1].

Organs or tissues of the immune system in teleosts (bony fish) include the thymus, head kidney, spleen and major mucosal-associated lymphoid tissues [2].

The immune system of the mucous membrane of teleosts is a lymphoid tissue of the skin, intestines, gills, nasopharynx. It is the main lymphoid structure in the body and plays a critical role in the response to infection [3]. The thymus in freshwater teleosts was the first organ to become lymphoid [4]. It is the primary lymphoid organ, in which bone marrow-derived lymphoid progenitor cells develop into functionally competent T cells with continued support from thymic stromal cells [5]. Thymus stromal cells in teleosts are represented by limiting epithelial cells located in the sub- capsular, perivascular and peritrabecular zones; reticular epithelial cells located in the medullary and cortical zone; cystic cells located in the medullary zone; thymic cells located in the corticomedullary border and Hassall's bodies [6]. Stromal cells also include various subtypes of fibroblasts and dendritic cells [7]. Other cellular components of the thymus are macrophages, myoid and mast cells [6].

Thymus tissue is densely filled with lymphoid cells, which contain typically deeply stained nuclei and sparse cytoplasm. Trabeculae may consist of strands of epithelial cells, capillaries or fibrocytes. Cortical and medullary epithelial cells form a network with their cytoplasmic processes, which increase the area of interaction with thymocytes and support the movement of lymphoid cells [6].

After the process of selection and differentiation, T lymphocytes leave the thymus and migrate to the periphery to fulfill their role in the body's immune response [9].

In mammals, the thymus is cytologically represented by numerous cells of the lymphoid series. In addition, epithelioreticular cells can be found in the cyto- gram, due to the hematopoietic factors of which lymphocyte differentiation occurs. These cells have a large oval or rounded basophilic nucleus with 2--3 nucleoli, the cytoplasm is elongated, with processes, the zone of enlightenment (Golgi complex) can be visualized. Thymus microslides can also contain single neutrophils, macrophages and mast cells (up to 3--5 %) [10].

In mammals, it has been demonstrated that various pathological conditions, such as infectious diseases, can affect the thymus, disrupting its architecture and function and negatively affecting the ongoing immune response [11].

Cellobacterin-T combines the functions of two feed additives -- a feed enzyme and a probiotic. The bacteria that make up Cellobacterin®-T (Bacillus sub- tilitis) have a pronounced probiotic effect, inhibit the development of pathogenic and opportunistic microorganisms, which ensures that the balance of the gastrointestinal microflora tract is maintained in the normal range. The enzyme complex of bacterial cellulases contributes to a more complete breakdown of structural fiber (cellulose, hemicellulose), providing a more complete release of feed nutrients. carp lymphocyte fish probiotic

The previous studies have demonstrated the beneficial effects of Bacillus subtilitis in broilers, in particular, high rates of livability, weight gain and a decrease in the number of intestine salmonella [18]. Pigs fed Bacillus ssubtilis had a higher weight gain to feed ratio and had an increased total peripheral blood leukocyte count [19].

Considering a great role of the thymus in immune processes, many studies have been carried out on this organ. There are publications on the thymus in fish that describe histological, immunohistochemical studies and expression analysis. Nevertheless, when analyzing the literature available to us, we have found rather scarce data on a cytological study that describes the morphology of cells.

In this regard, the study of the fish thymus cell pool will allow a more detailed study of the physiology and functioning of the immune system of hydrobionts.

Aims and objectives of the research. The aim of the study was to assess changes in the cellular composition of the Cyprinus carpio thymus parenchyma cells when using Cellobacterin-T.

Material and methods

The experiment was carried out on the common carps (Cyprinus carpio) on a specialized fish farm of Voronezh. Laboratory studies were carried out on the basis of the Research Center and the Department of Experimental Pharmacology of FSBSI «ARVRIPP&T».

For the experiment, one-year-old individuals weighing 250--300 grams were selected. In the baseline study (D0), the thymus was obtained from fish (n = 5) for cytological examination. Subsequently, hydrobionts were divided into two groups (experimental and control ponds).

The carps of the control pond (n = 8000) received the main diet, and the fish kept in the experimental pond (n = 8000) received the probiotic drug Cellobacterin-T at a dosage of 2 kg/t of feed for 30 days in addition to the main diet. Then during the test fishing, from the fish (n = 8) of the experimental and control ponds (n = 8), thymus for cytological examination was taken 14 days (D14), 28 days (D28) and 42 days (D42) after the beginning of using the drug. These drugs were stained according to Pappenheim: 3-minute fixation in a fixative-dye according to May-Grunwald, then for 40 minutes the glasses were immersed in a solution of azure-eosin according to Romanowsky, after which they were placed in polystyrene for further study.

Cytological examination was performed using the Hospitex Diagnostics microscopes. The built-in camera with a resolution of 5 megapixels was used to take photographs.

The percentage of cells was calculated based on 1000 counted cells. Statistical processing of the results was carried out using the Microsoft Excel software package. Reliability was assessed by paired comparisons using the Student's t-test.

Study results

Cytological examination of thymus imprint smears (Table 1) showed from moderate to high cytosis, basophilic background, destroyed erythrocytes due to hemodilution.

Table

Percentage of hematopoietic cells in the thymus of the common carp

p < 0.05 compared to the control group

Two weeks after the use of Cellobacterin-T in the control group, an increase in the percentage of neutrophils has been observed (Fig. 2). Throughout the experiment, these cells in the control group are by 2 times more than in the baseline study: D14 -- by 2.02 times, D28 -- by 2.7 times, D42 -- by 2.04 times. In the experimental group, the inflammatory reaction is less pronounced: on day 14, the ratio of neutrophils in the control and experimental groups does not have significant differences; on day 28, there are more of these cells in the control group by 57.3 %, on day 42 -- by 54.4 %.

During the experiment, a decrease in the number of monocytes was found (Fig. 3) in both groups, in the experimental group this decrease is more pronounced: on day 14 after the beginning of using the drug, it is lower than in the baseline study by 20.9 %, on day 28 -- by 2 .4 %, on day 42 -- by 3.2 %. In the control, on day 14, the ratio is lower by 15.6 %, on day 28 -- by 9.3 %, on day 42 -- by 5.7 % than in the baseline study. Reactive macrophages with cell phagocytosis, intercellular substance were extremely rare (Fig. 4).

The percentage of lymphocytes in the control group had no significant changes. Throughout the study it was within 10 % of the baseline study. In the experimental group, we observed an increase in lymphocytes by 2.8 % on day 14, by 21 % on day 28, and by 19 % on day 42. Mostly small lymphocytes were presented (the cell diameter is less than the diameter of an erythrocyte). The cells of the common carp had a structure similar to the lymphocytes of higher vertebrates: the shape of the cells was round, a high nuclear-cytoplasmic ratio, a meager amount of homogeneous light-basophilic cytoplasm, the nuclei were round, with coarse hyperchromic chromatin (Fig. 1). Reactive lymphocytes in most fish were not observed or were in a single amount.

When comparing the control (A) and experimental (B) groups, we see the difference in the number of neutrophils in one field of view: 2 in the control and 8 in the experimental group. Neutrophil granules do not perceive the dye according to Romanowsky, which is why their cytoplasm looks light and white [14, 15]. The shape of the cells themselves is round. The nuclei are eccentrically located, round or oval in shape with basophilic condensed chromatin.

Fig. 1. Small lymphocytes in the thymus of the Cyprinus carpio. Pappenheim staining, 1000x magnification

Fig. 2. Neutrophils in the specimen of fish thymus. Pappenheim staining, 1000x magnification

Fig. 3. Monocytes in the microslide of the common carp thymus. Pappenheim staining, 1000 x magnification

Fig. 4. Cytophagy in the specimen of the Cyprinus carpio thymus. Pappenheim staining, 1000x magnification

Fig. 5. Basophil against the background of lymphocytes in the microslide of the Cyprinus carpio thymus. Pappenheim staining, 1000x magnification

Fig. 6. Layer of epithelial cells in the Cyprinus carpio thymus. Pappenheim staining, 1000x magnification

Fig. 7. Reticuloepithelial cells in the Cyprinus carpio thymus. Pappenheim staining, 1000x magnification

The arrow indicates monocytes in a microslide of the Cyprinus carpio thymus. Monocytes were large mononuclear leukocytes with abundant granular cytoplasm from grey to bluish-grey. The nuclei are round or oval, less often bean-shaped or rod-shaped, with granular chromatin, displaced to the periphery.

The number of Mott cells, mast cells (Fig. 5) did not differ significantly. The stromal epithelium was represented by extensive layers (Fig. 6). The cell nuclei were round or oval with dusty chromatin, the cytoplasm was tender, weakly basophilic. The boundaries between cells were blurred.

Figure 7 shows putative reticuloepithelial cells. These cells have a rounded eccentrically located nucleus with granular chromatin, 2 nucleoli. The cytoplasm is processed, has a pronounced basophilic color, on the periphery there is a contour of the cytoplasm with a light basophilic tint. It is also worth noting the presence of cells with square vacuole-like inclusions resembling cholesterol crystals in all groups (Fig. 8A-D).

Similar inclusions are described in an iguana blood smear. There are two hypotheses about their origin.

The first of them states that these inclusions of the cytoplasm contain trace elements. The second assumption judges that these are viral or pyrhemocytonic inclusions. The exact cause of them is unknown, at the moment it is believed that they are not associated with hematological abnormalities or diseases.

These figures were also found in the extracellular space [14, 16]. Of particular interest were cells with light almond-shaped inclusions in the cytoplasm (15--20 % of all cells). Reliable data on the nature of these inclusions have not been found (Fig. 9).

Fig. 8. Square vacuole-like inclusions. Pappenheim staining, 1000x magnification

Fig. 9. Cells richly filled with almond-shaped inclusions. Pappenheim staining, 1000x magnification

Discussion

The cytological picture of the common carp thymus described by us is similar to the descriptions of this organ in fish earlier. The parenchyma of the organ is densely filled with lymphoid cells. Neutrophils, monocytes, mast cells and Mott cells are also present in a small amount. Among stromal cells, it was possible to describe epithelial and reticuloepithelial cells based on the previously obtained morphological characteristics of these cells [6, 7, 10].

Many cells, when stained by Pappenheim, are identical to those of higher vertebrates (e. g., lymphocytes, macrophages, epithelial cells). Some cells (neutrophils, monocytes) have their own characteristics, which are described in various literature sources [15, 16].

Judgments about Hassall's bodies have different data. In some studies, they were observed only in adults [17], in other studies, it is said that Hassall's bodies undergo degeneration during ontogenesis [8].

In our study, Hassall's bodies were not found. Probably, this may be due to age characteristics. It should also be taken into account that the judgments presented above are given on the basis of histological studies, and probably, the reliability of a cytological studyfor the detection of Hassal's bodies is low. In further studies, attention should be paid to objects that require additional research methods for their differentiation. In particular, these are cells with square vacuole-like inclusions and cells with light almond-shaped inclusions in the cytoplasm. These inclusions may contain trace elements and other components, be viral [14, 16].

Overall, the present results emphasize that the role of the thymus in pathological conditions in fish should not be overlooked, as this may contribute to a better understanding of host-antigen interactions in case of infectious and non-infectious pathologies.

The conducted experiment with a baseline study demonstrates the inflammatory response of the common carp thymus, as evidenced by the high values of such inflammatory cells as monocytes (20.5 %) and neutrophils (10.3 %). In the control group, high levels of neutrophil count were maintained throughout the study (20.8 % on day 14, 27.6 % on day 28, 21.0 % on day 42).

Most of the cells, when stained by Pappenheim, had the same cytological features as in higher vertebrates (lymphocytes, macrophages, etc.). Some of the cells (neutrophils, monocytes) had their own features typical of fish, described in various literature sources.

Two types of non-differentiable cells with eccentric nuclei and cytoplasm densely filled with rectangular unstained inclusions in one type of cells and almond-shaped weakly basophilic or unstained inclusions in another type of cells were observed in large numbers.

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