A STUDY OF THE PHAGOCYTIC ACTIVITY OF HOPLOBATRACHUS RUGULOSUS NUCLEAR ERYTHROCYTES

Во Ван Тхань¹, Чернявских С.Д.², Нгуен Тхань Тхуи³, До Хыу Кует⁴, Чан Тхи Монг Ко³

^{1, 4}Аспиранты, ² Кандидат биологических наук, доцент, Белгородский государственный национальный исследовательский университет, Россия; ¹ Преподаватель, ³ Студентки, Педагогический университет г. Хошимина, Вьетнам

ИЗУЧЕНИЕ ФАГОЦИТАРНОЙ АКТИВНОСТИ ЯДЕРНЫХ ЭРИТРОЦИТОВ HOPLOBATRACHUS RUGULOSUS

Аннотация

В опытах in vitro выявлена способность эритроцитов H. rugulosus к фагоцитозу микроорганизмов. Установлено, что фагоцитарная активность (ФА) красных клеток крови H. rugulosus к S. cerevisiae и Shigella sp. увеличивается как при повышенной (37^оС), так и при пониженной (20^оС) температурах инкубации, к S. aureus – повышается только при уменьшении температуры инкубации (до 20^оС) по сравнению с температурой 28^оС. Наиболее высокая ФА эритроцитов H. rugulosus при температурах инкубации 20^оС и 28^оС зарегистрирована к S. aureus, при 37^оС – к S. cerevisiae.

Ключевые слова: ядерные эритроциты; лягушки; фагоцитарная активность; микроорганизмы.

Vo Van Thanh¹, Chernyavskikh S.D.², Nguyen Thanh Thuy³, Do Huu Quyet⁴, Tran Thi Mong Co³

^{1, 4} Postgraduate students, ² PhD in Biology, Associate professor, Belgorod State National Research University, Russia; ¹ Teacher, ³ Students, Ho Chi Minh City University of Education, Vietnam

A STUDY OF THE PHAGOCYTIC ACTIVITY OF HOPLOBATRACHUS RUGULOSUS NUCLEAR ERYTHROCYTES

Abstract

Under in vitro conditions, erythrocytes were observed to have the ability to englobe microorganisms in H. rugulosus.  It was observed that phagocytic activity (PA) in H. rugulosus red blood cells for S. cerevisiae and Shigella sp. increase at elevated (37°C), and at low (20°C) incubation temperatures, whereas for S. aureus – PA increases only at a decreased incubation temperature (20°C) in comparison with incubation at a temperature of 28°C. The highest PA in H. rugulosus erythrocytes occurred at incubation temperatures of 20°C and 28°C for S. aureus; and at 37°C for S. cerevisiae.

Keywords: nuclear erythrocytes; frogs; phagocytic activity; microorganisms.

Introduction

In recent years, much attention has been paid to the study of phagocytic activity of vertebrate animal blood cells [2, 5, 6, 12, 9]. It’s known that the low vertebrate erythrocytes are capable of absorption of alien particles [11]. However, scientific research regarding the features of phagocytic reactions in nuclear erythrocytes remains little studied. Proceeding from the above, we conclude that the study of phagocytic activity in amphibious erythrocytes is critical.

Studying phagocytic activity in H. rugulosus nuclear erythrocytes is the purpose of our research.

Objects and methods

Our study utilized the peripheral blood of frogs Hoplobatrachus rugulosus (30 specimens). Nuclear erythrocytes served as objects of the study. The research was carried out at the Department of Anatomy and Physiology of Ho Chi Minh City University of Education (Vietnam).

Blood samples were taken from frog hearts after giving a light ether anesthesia. Heparin at 10 U/mL was used for prevention of blood clotting [9]. The obtained blood was centrifuged for 4 min. at 400g and the erythrocyte mass was collected. Suspension of erythrocytes was incubated with phagocytic objects at room (28°C), low (20°C) and elevated (37°C) temperatures within 30 min. Saccharomyces cerevisiae (S. cerevisiae), Staphylococcus aureus (S. aureus) and Shigella sp. were used as phagocytic objects. Then, smears were done, cells were fixed by ethanol and were stained with azure-eosin utilizing the Romanovsky technique. The phagocytic activity of erythrocytes was counted [5]. To avoid inaccuracies in the counting of absorbed particles because of difficulties in definition of their localization (inside or on the cell surface), an immersion magnification of 100× was used.

The obtained results were processed using statistical variation methods with the computer program Minitab 16. Changes were taken at the level of statistical significance at p ≤ 0.05.

Results and discussion

As a result of this research it is concluded that after incubation with S. cerevisiae, S. aureus and Shigella sp., nuclear erythrocytes in H. rugulosus frogs show phagocytic activity during reactions which correspond to reactions of leukocytes in the phagocytic process.

The figure shows that erythrocytes move close to phagocytic objects using pseudopodia (fig. A). This phenomenon is similar to the stage of attraction [6]. Contact of erythrocytes with phagocytic objects (fig. A), corresponds to the stage of adhesion (sticking) which is a result of their convergence. In the process of this reaction the surface charge, membrane receptor, as well as chemotactic attraction can play an important role [1, 7].

Fig. 1 - Phagocytosis of S. cerevisiae by H. rugolosus erythrocytes (×100)

1 – S. cerevisiae, 2 – erythrocytes, 3 – partially digested phagocytic objects

The ability of H. rugulosus erythrocytes for absorption and partial intracellular digestion of microorganisms is shown (fig. B). The plasmalemma of the englobing erythrocyte contacts the phagocytic object and the object plunges into the cell, the edges of the plasma membrane are then closed over the object. Thus the vacuole, which contains the phagocytosed particle, is formed. At the membrane is the wall of the newly formed vacuole [2].

Apparently, fine particles are revealed in the cytosol of some erythrocytes (fig. C), which can be partially digested phagocytosed objects, i.e. phagolysosomes [9].

The indicators of PA in H. rugulosus erythrocytes are shown in the table.

Table 1 - Phagocytic activity of H. rugulosus erythrocytes for different phagocytic objects at different incubation temperature, %

The table shows that the values of PA of H. rugulosus erythrocytes for S. cerevisiae, S. aureus and Shigella sp. are changed by different incubation temperatures. So, when incubation temperature reduces to 20°C, the studied indicator of H. rugulosus erythrocytes for S. cerevisiae, S. aureus, Shigella sp. increases by 43.16%, 6.97% and 15.55% respectively compared with incubation at 28°C.  When incubation temperature increases to 37°C, PA for S. cerevisiae and Shigella sp. also increases by 51.35% and 6.60%. This may be due to the intensive involvement of red blood cells in immunological processes at hit in unfavorable conditions [3, 9] (decrease or increase in the temperature). The PA of erythrocytes for S. aureus increases only at the low temperature, while at raised temperature – is changed insignificantly. Such multidirectional change of PA can be conditioned by the fact that S. aureus doesn’t develop at low temperatures [2, 10] and is more subject to phagocytosis, and at higher temperatures which are optimal for growth and development, these bacteria can excrete toxins hemolysins and leucocidin, causing hemolysis of hemocytes [2, 4, 10].

Comparative analysis of PA of erythrocytes for different phagocytic objects showed that at an incubation temperature of 20°C, the PA for S. aureus was higher than both S. cerevisiae and Shigella sp.; this could be due to the low viability of S. aureus at low temperatures [2].

At an incubation temperature of 28°C, the PA for S. aureus was 47.30% and 26.74%, higher, than for S. cerevisiae and Shigella sp., respectively. At this temperature the lowest PA was registered for S. cerevisiae. It could be due to the presence of a specific immunity in amphibians as the main habitats of S. cerevisiae are the soil, the surface of fruits and leaves [8, 10], which are the habitat of the frogs.

Comparative analysis at an incubation temperature of 37°C has shown that the PA of H. rugolosus erythrocytes for S. cerevisiae was the highest, and for Shigella sp – the lowest. The PA for S. cerevisiae was 6.93% and 27.60% higher than S. aureus and Shigella sp. respectively; while S. aureus was 22.21% higher than Shigella sp.

Thus, under conditions of both lowered and raised incubation temperatures the phagocytic activity of H. rugulosus erythrocytes for S. Cerevisiae, S. aureus and Shigella sp. are different. It is caused by a complex effect of temperature changes on the activity of the immune system. An increase or decrease in the temperature can influence the immunological function of amphibians in three ways: 1) to cause reactions to temperature stress; 2) to directly affect immune cells or the activity of functional proteins; 3) to allow pathogens to adapt to this more rapidly than their host organisms [12].

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