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HayKOBHH BicHHK .HbBiBCbKoro Ha^oHaibHoro ymBepcurery BeTepHHapHoi' MeguuHHH Ta 6ioTexHonoriH iMeHi C.3. I^H^Koro Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z. Gzhytskyj
doi: 10.15421/nvlvet7315
ISSN 2518-7554 print ISSN 2518-1327 online
http://nvlvet.com.ua/
UDC 612.62:612.017.11
The status of extracellular antimicrobial potential of phagocytes genitals of cats
M.M. Zhelavskyi, I.M. Shunin [email protected]
Podillya State Agrarian and Engineering University, Shevchenko str., 13, Kamyanets-Podilsky, 32300, Khmelnytsky region, Ukraine
This article shows the results of the experimental researches in the cellular link of local immune protection of cats' genitals. Due to the urgency of the problem the aim of our study was to investigate the functional state of the phagocytic cells and to explore and interpret their role in the formation of the antimicrobial potential of homeostasis in the system of local immunity of the cats' reproductive organs. Cytochemical research determined the antimicrobial reactive Oxygen-dependent mechanism for the protection of neutrophil granulocytes in reaction with NBT
It was found out, that the antimicrobial potential of phagocyte cells actively realizes via Oxygen dependent protection mechanisms. The total number of NBT + phagocytic cells in the investigated micropreparations was 21.35 ± 0.86%. Cytochemical reactivity of primary phagocytic cells was shown on the I and II level, which reflects the full manifestation of the phagocytic defense. The activated phagocytes have also shown the active adhesion and phagocytosis of epithelial cells and of apoptosial macrophages, which is also one of the signs of the maintenance of cellular homeostasis. The interpretation of the results was done according to the outcomes of the research and the conclusions and recommendations on the application of cytochemical studies in a comprehensive assessment of the antimicrobial potential ofphagocytic cells and for predicting clinical evaluation of reproductive diseases in small animals were developed.
It is recommended to perform cytochemical studies during complex testing of the local immune protection of the reproductive organs of animals, which will allow to objectively assess the state of cellular immunity, diagnose subclinical manifestation of reproductive pathology and predict the risk of complications.
Key words: cats, reproductive system, local immunity, cellular homeostasis, neutrophilic granulocytes, cytochemical reactivity, NBT-test.
Стан екстрацелюлярного потенщалу фагоципв статевих оргашв у кчшок
М.М. Желавський, 1.М. Шунш [email protected]
Подшъсъкий держаний аграрно-техтчний yHieepcumem, вул. Шевченка, 13, м. Кам 'янецъ-Подшъсъкий, 32300, Хмелъницъка обл., Украгна
У cmommi наведет резулътати експерименталъних до^дженъ вивчення стану клтинног ланки локалъног iMyHHoao за-хисту репродуктивних оргатв у тток. Метою натог роботи було до^дити функцюналъний стан фагоцитарних клтин, а також вивчити гх ролъ у формуванн iмyннoгo гомеостазу в cиcтемi локалъног захисту оргатв розмноження у кшок. Встановлено, що протимжробний потенщал фагоцитарних клтин активно реалiзyeтъcя через Оксигензалежн мехашзми захисту. Загалъна ктъюстъ НСТ + фагоцитарних клтин в мжропрепаратах становило 21,35 ± 0,86%. Цитoхiмiчний потенщал активованих фагоцитарних клтин проявляласъ на I та IIрiвнi реактивнocтi. До^дженнями встановлено також кореляцшний зв'язокмiж цитoлoгiчним тдексом та мжрофагами з активованим цитoхiмiчним (протимжробним) потенщ-алом, що вказуе про часткову праймерiзацiю клтинног ланки неcпецифiчнoгo iмyннoгo захисту. Активоваш фагоцити проявляли активну адгезiю епiтелioцитiв i апоптозних мiкрoфагiв. За резулътатами до^дженъ проведено ттерпретацт та
Citation:
Zhelavskyi, M.M., Shunin, I.M. (2017). The status of extracellular antimicrobial potential of phagocytes genitals of cats. Scientific Messenger LNUVMBT named after S.Z. Gzhytskyj, 19(73), 71-74.
зроблеш висновки про перспективи застосування qumoxrniumx до^джень в комплекснш оцтц протимжробног потенщалу фагоцитарних клтин для клШчног оцтки i прогнозування розвитку репродуктивноi патологп у dpi6mx домаштх тварин.
Ключовi слова: кшки, статева система, локальний iмунiтет, клтинний гомеостаз, нейтрофти, цитохiмiчна реакти-втсть, НСТ-тест.
Состояние экстрацеллюлярного противомикробного потенциала фагоцитов половых органов у кошек
Н.Н. Желавский, И.Н. Шунин [email protected]
Подольский государственный аграрно-технический университет, ул. Шевченка, 13, г. Каменец-Подольский, 32300, Хмельницкая область, Украина
В статье приведены результаты экспериментальных исследований изучения состояния клеточного звена локальной иммунной защиты репродуктивных органов у кошек. целью нашей работы было исследовать функциональное состояние фагоцитарных клеток, а также изучить их роль в формировании иммунного гомеостаза в системе локальной защиты органов размножения у кошек.
Определено, что противомикробный потенциал фагоцитарных клеток активно реализуется через кислородзависимые механизмы защиты. Цитохимическими исследованиями кислородзависимого механизма фагоцитарной защиты клеток слизистой оболочки в реакции нитросиним тетразолием определено, что фагоциты принимают активное участие в реализации противомикробной защиты. Общее количество НСТ+ фагоцитарных клеток в исследованных микропрепаратах составило 21,35 ± 0,86%. Цитохимическая реактивность праймированых фагоцитарных клеток проявлялась на I и IIуровне. Исследованиями определено корреляционную связь между цитологическим индексом и количеством микрофагов с активированным цитохимическим (противомикробным) потенциалом, что указывает о частичной праймеризации клеточного звена неспецифической иммунной защиты. Активированные фагоциты проявляли активную адгезию эпителиоцитов и апотозных микрофагов. По результатам исследований проведено интерпретацию и сделаны выводы о перспективах применения цитохимических исследований в комплексной оценке противомикробного потенциала фагоцитарных клеток для клинической оценки и прогнозирования развития репродуктивной патологии у мелких домашних животных.
Ключевые слова: кошки, половая система, локальный иммунитет, клеточный гомеостаз, нейтрофильные гранулоци-ты, цитохимическая реактивность, НСТ-тест.
Intoduction
Immune animal protection system was formed in the course of a long evolution. More than a century has already passed since the phenomenon of phagocytosis was discovered by Mechnikov I.I. Despite this, scientists around the world continue actively to carry out a comprehensive study of cellular factors protecting the immune defense and of the interaction of immunocompetent cells (Ignacio et al., 2005; Michael, 2012).
The system of local immune protection of animal breeding has complicated ontogenetic development, which is clearly subordinated to the genetic determination and neurohumoral mechanisms of regulation (Yablonsky et al., 2004; Kjelgaard-Hansen et al., 2007). The modern scientific publications show more data on the role of phagocytes in the induction of cytokines, peptides fusion, mediators and other biologically active substances, which take part in the formation of immune homeostasis as well as in triggering the inflammatory response cascade Jursza-Piotrowska et al., 2016). Today, the central object of research are mechanisms for the implementation of the antimicrobial protection of phagocytic cells (urinary antimicrobial compounds, the formation of the protective traps and so on). Scientists are increasingly interested in the study of regulatory factors of functional state of phagocytes (William et al., 2012, Zhelavskyi et al., 2016).
According to many researchers the origin and development of the reproductive pathology (vaginitis, endometritis, pyometra) often occur because of immunological
disorders (Batista-Arteaga et al., 2007; Payan-Carreira et al., 2015; Zhelavskyi et al., 2016).
Due to the urgency of the problem the aim of our study was to investigate the functional state of the phago-cytic cells and to explore and interpret their role in the formation of the antimicrobial potential of homeostasis in the system of local immunity of the cats' reproductive organs.
Material and methods
Clinical and experimental studies were carried out on 2 to 8 years old cats of various breeds. Laboratory studies were done in a specialized laboratory of animal reproduction in Podillya State Agrarian and Engineering University. The laboratory was founded by the doctor of biological sciences, professor, corresponding member of NAAS of Ukraine Yablonsky V. Immunological study determined cellular structure and functional state of the phagocytic cells. Cytochemical research determined the antimicrobial reactive Oxygen-dependent mechanism for the protection of neutrophil granulocytes in reaction with nitro blue tetrazolium (NBT +) using our own patented method (Zhelavskyi et al., 2016). At the same time we have determined the total percentage of reactive phagocytes (NBT +) index activation of phagocytic reactivity of neu-trophils (IAN), cytological index of reactivity of the antimicrobial potential of cells (CLI). During the processing of biometric data set we were using the statistical software Statistica v.10.
Results and discussion
Cytological studies have determined that the cellular composition of the vaginal mucosa in the metestrus stage was mainly represented by intermediaries (Pic. 1) and parabasal epithelial cells. In the microscope slide cyto-gramme the overall share of neutrophils was 14.70 ± 0.68%. Neutrophilic granulocytes were localized either singly or in groups and had a clear-cut segmented nucleus and cytoplasm (Pic. 2).
It is known that neutrophil granulocytes are able to activate the cellular part of the immune defense (Th1, Th2), as well as to coordinate the interaction of humoral and cellular immunity.
Neutrophils are located in the peripheral bloodstream only for 6 - 10 hours, and then get into the tissue where
Pic. 1. Micro photo.
Intermediate epithelial cell ^ 2500)
Furthermore, with the participation of superoxide dismutase other active forms of Oxygen are produced (H2O2, •OH, 102 et al.) that are released by phagocyte into extracellular space. All this is accompanied by the launch of a cascade of other immune-biological reactions: there is an active synthesis of chemotactic peptides, the IL is being induced and others (Michael, J., 2012, Jursza-Piotrowska, E. et al., 2016). It was determined by
they perform their effector function (Kjelgaard-Hansen et al., 2012; William et al., 2012). Priming phagocytic cells are capable of destroying pathogenic agents both in the immediate attack (killing) as well as by absorption and digestion. Phagocytes are also able to realize its function by activating metabolic reactivity, followed by the extracellular release of antimicrobial compounds. This phenomenon has been called in the scientific literature as a respiratory burst. In the phagocytes occurs biochemical activation of the hexose monophosphate shunt and NADPH oxidase of phagosome cell. This metabolic reaction occurs against the backdrop of increasing (in ten times) consumption of cell glucose and Oxygen. NADPH oxidase converts 02 - superoxide anion (02-).
Pic. 3. Micro photo. Metabolic neutrophilic granulocyte reactivity in the reaction NBT ^ 2500)
cytochemical studies of Oxygen-phagocytic mechanism of protection of mucosal cells in the reaction of NBT that phagocytes take an active part in the implementation of anti-microbial protection. The granules of diformasane were clearly visualized in the cytoplasm of reactive phagocytes. The total count of NBT + phagocytic cells in the investigated micropreparations was 21.35 ± 0.86% (Pic. 3).
Диаграмма рассеяния: NBT_% vs. IAN (Построч.удаление ПД) IAN = ,46835 + ,04617 * NBT_% Корреляция: r = ,52409
1,65 1,60 1,55 1,50 1,45 1,40 1,35 1,30
1,25
19,8 20,2 20,6 21,0 21,4 21,8 22,2 0 20,0 20,4 20,8 21,2 21,6 22,0
0,95 Дов.Инт.
Pic. 4. Correlational connection of IAN and NBT+
20
0
NBT %
К
Pic. 2. Micro photo. Neutrophil of vaginal mucosa ^ 2500)
It was also determined that the intensity of antimicrobial potential of priming neutrophils appears mainly on the of I and II reactivity levels, which can clearly be seen in the graph (Pic. 4).
Statistical studies also identified a positive correlation (r = 4.42, P < 0.001) between the cytological index and the number of macrophages with activated cytochemical (antimicrobial) potential. All this indicates a partial activation of phagocytic cells and their incomplete primarization.
Recently, researchers are increasingly interested in the role of phagocytic cells in the formation of microbiocenosis and regulation of cellular homeostasis (Michael, 2012, Jursza-Piotrowska et al., 2016; Zhelavskyi et al., 2016). Reactive phagocytic cells have been often identified in our experiments, which carried out adhesion of epithelial cells and apoptotic neutrophils. We assume that phagocytes are involved not only in antimicrobial defense, but also involved in the formation of cellular homeostasis of mucosal tissues. This phenomenon leads us to the hypothesis that the functional state of neutrophils and epithelial cells, as well as the regulation of their apoptosis can be regulated by immunocompetent cells (under the influence of antimicrobial compounds, peptides, neurotransmitters and other biologically active compounds), which is going to be a subsequent stage of our research.
Conclusions
1. Thus, it can be concluded that in the cytogram of cats' vaginal mucosa during metestrus period intermediate and parabasal epithelial cells are mainly dominated. Cellular link of local security of the nonspecific immunity is presented by phagocytes.
2. The total number of neutrophils in cytogramme is 14.70 ± 0.68%. The intensity of the antimicrobial potential of the Oxygen-dependent mechanism for the protection of neutrophils is found in the I and II levels of cyto-chemical reactivity.
3. It is recommended to perform cytochemical studies during complex testing of the local immune protection of the reproductive organs of animals, which will allow to objectively assess the state of cellular immunity, diagnose subclinical manifestation of reproductive pathology and predict the risk of complications.
Prospects for further research. Further research will be used to study the diagnostic criteria of reproductive pathology in cats.
References
Ignacio, G., Nordone, S., Howard, K. (2005). Toll-like receptor expression in feline lymphoid tissues. Veterinary Immunology and Immunopathology. 106, 229237.
Michael, J. (2012). Clinical Immunology of the Dog and Cat Revised And Updated J. Michael. Day Second Edition Copyright: Manson Publishing Ltd.
Yablonsky, V.A., Homin, S.P., Kalinovs'ky, G.M. (2006). Veterinarne akusherstvo, ginekologija ta biotehnologija vidtvorennja tvarin z osnovami an-drologiï. Vinnicja : Nova kniga (in Ukrainian).
Kjelgaard-Hansen, M., Luntang-Jensen, M., Willesen, J. (2007). Measurement of serum interleukin-10 in the dog. Veterinary Journal. 173, 361-365.
Jursza-Piotrowska, E., Marta, J. (2016). Siemieniuch Identifying diagnostic endocrine markers and changes in endometrial gene expressions during pyometra in cats. Reprod. Biol. 8, 16(2), 174-180.
Jursza-Piotrowska, E., Socha, P., Dariusz, J. (2016). Pros-taglandin release by cultured endometrial tissues after challenge with lipopolysaccharide and tumor necrosis factor a, in relation to the estrous cycle, treatment with medroxyprogesterone acetate, and pyometra. Theri-ogenology. 10, 85(6), 1177-1185.
Jursza-Piotrowska, E., Siemieniuch, E., Marta, J. (2016) Identifying diagnostic endocrine markers and changes in ndometrial gene expressions during pyometra in cats. Reprod. Biol. 8, 16(2), 174-180.
William, A., Rose, I.I., McGowin, C.L. (2012). Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures http://dx.doi.org/10.1371/journal.pone.0032728.
Zhelavskyi, M., Shunin, I. (2016). Cell factors' condition of local immunity of vaginas mucosa in cats. Scientific Messenger LNUVMBT named after S.Z. Gzhyt-skyj. 18, 32-36 (in Ukrainian).
Sposib ocinjuvannja lokal'nogo imunitetu statevih organiv u tvarin: pat. 109040 Ukraina. № u 201601190; vi-nahidniki ta vlasniki Zhelavs'kij Mykola Mykolajo-vich, Shunin Igor Mikitovich ; zajavl. 11.02.2016 ; opubl. 10.08.2016, Bjul. № 15. (in Ukrainian).
Zhelavs'kij, M.M. (2016). Osoblivosti sistemnogo ta loka-l'nogo imunitetu u period vagitnosti : metodichni vkazivki. Podil's'kij derzhavnij agrarno-tehnichnij universitet. Kam'janec'-Podil's'kij, 22 (in Ukrainian).
Batista-Arteaga, М., Alamo, D., Herraez, P. (2007). Segmental atresia of the uterus associated with hydromet-ra in a ferret. Vet. Rec. 161, 759-760.
Payan-Carreira, R., Pina, J., Costa, M. (2015). Oestrogen receptors in a case of hydrometra in a bitch. Vet. Rec. 158, 487-489.
Zhelavskyi, M.M., Shunin, I.M. (2016). Perspektivi doslidzhennja klitinnih faktoriv lokal'nogo imunitetu slizovih obolonok statevih organiv kishok. Zbirnik materialiv HV Mizhnarodnoï naukovo-praktichnoï konferenciï profesors'ko-vikladac'kogo skladu ta aspirantiv «Problemy veterinarnoy medicini ta jakosti i bezpeki produkciï tvarinnictva» fakul'tetu veterinarnoy medicini, 36-37 (in Ukrainian).
Стаття надiйшла до редакцИ' 26.02.2017
