Rezumatul tezei de doctorat în engleza

THE IMMUNE REACTIVITY OF DOMESTIC RUMINANTS IN SOMEBIOLOGICAL STRESS SITUATIONSABSTRACTThe PhD thesis is presented on 213 pages and includes 34 tables, 113 figures and 158bibliographic titles. The work is structured in two parts, one part devoted to bibliographicinformation and the other to personal research.The first part, on 50 pages, including 3 tables and 5 figures, presents the current stage ofknowledge in the approached field and it is based on the consultation of 158 bibliographic titles.54% of the works in the bibliography were published after 2000, and the first part of the paperrepresents 24% of the total.The second part of the thesis, on 163 pages, including 31 tables and 108 figures, presentsresults and conclusions of personal research.Proportion of the second part of the total work thesis is 76%.According to current legal regulations, the following are enclosed in the PhD thesis: one (a)summary in Romanian and the other translated into English and two annexes : a list of originalcontributions and a work which is the result of research related to the thesis, the work is recommendableand useful for specialized laboratories.In the PhD thesis, titles of tables and figures are presented in Romanian and English.I. THE AIM OF THE PAPERUnlike lifeless matter, living matter has a strong antientropic character, fundamental propertythat allows it to resist actively against the unfavourable or even disorganizing action ofenvironmental factors.Consequently, all beings react permanently through specific species mechanisms, to thefavourable or unfavourable challenges caused by a wide variety of external and internal factors.The characteristic mode of response (reactivity) of the organism to the action of thoseentropic factors was named in 1936 by Bruno Hans Selye with the term of stress.xvii

Later (1975), he also systematized this knowledge in the field and proposed the term ofdistress for the body's response to unfavourable (negative) actions on it (concretized through theproduction of morpho-functional imbalances that can evolve even to the stage of illness) and theterm of eustress for body reactions to the action of factors that determine favourable effects(positive) on its functions.In both types of stress, the body's response is based on reactions nervous, endocrine andimmunological.From the many biological stress factors acting in cattle and sheep, the present studyinvestigates the following two states of biological stress:- the state of distress caused by Mycobacterium avium paratuberculosis, usuallycharacterized by functional disorders with slow evolution, but with certainadverse consequences that lead to irreversible compromising of the organismhomeostasis and and passing of organism to the state of disease;- the state of eustress, caused by intense, but short-term stress on the body of thepregnant female, condition finished by increasing the body's functional capacitybased on intensification of certain functions, as it usually happens in the stressof parturitionAmong the many defense systems of the body against the action of biological stress factorsinvolved in both states of stress mentioned above, this paper sets out to research the immuneresponse of cattle and sheep in two stressful situation for the following reasons:- the immunological nature of characteristic stress reactions at these species ofgreat economical importance has been less studied compared with reactions ofthe neuro endocrine nature;- in Romanian veterinary medicine, this subject has not been yet approached in asystematic and holistic way;- Veterinary immunology now recognizes an intensely dynamic development, therefore itis estimated that many practical aspects of the animal pathology of farm will beresolved based on the solutions coming from this area of science- the logistics available base of the specialized institution (Institute forDiagnosis and Animal Health) in which the author of this thesis works is thexviii

most performant in the country and it is optimal for the selected and specifiedaspects mentioned in the contents of this paper.Essentially, in the research dedicated to this work, our aim is to investigate the reactivity ofT and B lymphocytes , as well as the lysozym from blood from bovine and sheep located in twodifferent states of stress (distress and eustress) studied in order to identify the involvement (orabstention) of those components of the immunological system, specific and nonspecific,in theadaptive reaction of organisms subject to the respective biological stressors studied.Whereas assessment of functional T and B lymphocytes as well as the evaluation of lysozymefrom blood of cattle and sheep in distress induced by Mycobacterium avium paratuberculosis and instress of parturition, were performed with high sensitivity methods, we consider that the results ofexperiments conducted in this work could benefit from confidence necessary for elaboration of validconclusions for each experimentII.STRUCTURE AND CONTENT OF THE THESISPart ISYNTHESIS OF ACTUAL DATA REGARDING THE REACTIVITY OFIMMUNE SYSTEM AND STRESS SITUATIONThis part is structured in two chapters:1. Functional organization of the immune system;2. Stress and stress typesData on the functional organization of the immune system and stress types are presented in thefirst 45 pages of the thesis and are developed based on literature data analysis specific to these twofields.xix

Part IIPERSONAL RESEARCHThis part expands on 163 pages and is divided into two main sections: Section A and Section B.SecŃion AREACTIVITY OF IMMUNE SYSTEM IN BOVINE AND SHEEP AFFECTEDBY BIOLOGICAL DISTRESS CAUSED BYMYCOBACTERIUM AVIUM PARATUBERCULOSISThis section extends on 120 pages, contains 24 tables, 97 figures and is divided into six chapters.A 1. Hemogram of ruminant in distress caused by Mycobacterium avium paratuberculosis(M.a.p.)Before carrying out detailed investigations over the body's immune reactivity of the organism,blood cell populations of the animals included in the study must be assessed in order to obtaingeneral information related to the functional status of blood, desideratum solved by performing ofthe hemogram and interpretation of the hematological parameters. The results of this experimentled to the following conclusions:- the chronic distress state, characteristic of subclinical infection induced and maintained byM.a.p. does not significantly change homogram parameters, neither in cattle nor in sheep;- the homogram is not an effective means of identifying the state of distress in its early stagesof paratuberculosis in cattle or sheep.A 2. Optimization of the technique for separation of leukocytes from the cattle and sheepbloodThis experiment aims to improve the traditional techniques of separating the cell in theseparating medium for optimization and validation of these techniques for ruminant specific bloodto allow the final evaluation of the lymphocytes reactivity in some state of distress and eustress incattle and sheep.Results from these sounding allowed us to elaborate the following conclusions:- our optimized method following a series of testing experiments described in this chapterallows proper separation of mononuclear blood cells of cattle and sheep blood;xx

- due to the lack of toxicity of Percoll solutions and their compatibility with the living cell itis possible to separate different subsets of leukocytes in the blood of ruminants and theircultivation, ,,in vitro,, to assess immune functions presented in the following chapters of thethesis.A 3. Reactivity of lymphocytes of cattle and sheep in distress determined by Mycobacteriumavium paratuberculosis (M.a. p.)This experiment aims to check the functional status of lymphocytes in the pathogenesis of theinitial phase (subclinical) of paratuberculosis and clarify whether, in the beginning of this state ofdistress, lymphocytes are, or are not reactive to contact with the specific antigen of disease.The results obtained in this experiment allowed us to draw the following conclusions:- lymphocytes of cattle and sheep naturally infected with M.a.p are operational ever since theearly stage of distress, they react to specific stimuli for this stressor;- the presence of large blast cells is an indicative of the presence of cell-mediated immunityand default of lymphocytes functionality of the animals infected.A 4. The reactivity of the T lymphocytes in distress determined by Mycobacterium aviumparatuberculosis (M.a.p.)This experiment aims to evaluate the cellular immune response in cattle and to obtain data onthe involvement of T cell reactivity in the immune defense cell type in the preclinical stage ofdevelopment of paratuberculosis.For this purpose the test, test the-TCS (simultaneously comparative allergic test) ,,in vivo,,and the – iELISA (immunoassay test for detection of γ-IFN) ,,in vitro,,, were used observing thefollowing:- at cattle in the early phase of the state of distress caused by M.a.p, T lymphocytes are functionaland their involvement in cellular type immune defenses can be highlighted both through the allergictesting and by determining ,,in vitro,, the IFN-γ- ului secreted by these;- the sensitivity test based on detection of γ -IFN, in vitro, is higher than that observed in allergictest compared simultaneously;- for optimal stimulation of T lymphocytes in blood culture better results have been obtained usingbovine PPD in concentrations exceeding (30000 ICU /ml) instead of commonly used by competentlaboratories (20000UTI/ml).xxi

A 5. The reactivity of the B lymphocytes in ruminants in distress determined byMycobacterium avium paratuberculosis (M.a.p)The objective of this chapter was to investigate the involvement of B lymphocytes in thespecific immune defense of humoral type in domestic ruminants (cattle and sheep) in a state ofdistress typical for the clinically unapparent debut of the paratuberculosis of these species.The purpose of this experiment was achieved by using two technical methods:- the qualitative highlighting (detection) of the presence of immunoglobulins (IgG) anti-Mycobacterium avium paratuberculosis in the blood of animals infected with M.a.p;- quantitative evaluation of of IgG in the blood of these animals (by IDSR)From the results obtained in this experiment we were able to indicate the following:- B cells are still active in the early stage of M.a.p infection, but anti-M.a.p antibodies can bedetected correctly only by very sensitive tests (indirect imunoenzimatic test);- participation of humoral immunity in these times of biological distress is weak (discrete) comparedwith the cell.- quantitative evaluation of of IgG by IDSR does not provide conclusive evidence in its earlystages of infection with M.a.pA 6. Non specific immune reactivity in ruminants distress determined by Mycobacteriumavium paratuberculosis (M.a.p.)This chapter aimed to investigate the possible participation of the lysozyme (componentfeature of nonspecific immune defense) to cattle in the state of distress caused by infection withM.a.p, state that precedes the clinical development stages of the disease.Performing this experiment is justified to highlight the possible reactivity of the immunesystem in all its three basic components: cellular, humoral and non specific immunity.The results obtained in this experiment have led to the following conclusions:-the lysozym concentration in the blood serum of cattle in the state of early distress caused byM.a.p (subclinical stage of evolution of the disease) is significantly higher compared with animalsfree of infection with M.a.p;- non specific immune defense mechanisms (illustrated in this experiment by assessing bloodlysozyme) are,'' primed early,, but are not effective enough for the aggression of this biologicalstressor.xxii

SecŃion BIMMUNE SYSTEM RECTIVITY IN BOVINE UNDER EUSTRESS CAUSEDBY PARTURITIONIn the second section of this thesis we intend to investigate the reactivity of T and Blymphocytes, and an element of nonspecific immunity (lysozyme) in cows under stress duringparturition. This condition is caused by the stress on the maternal body under the influence ofcomplex factors acting in late gestation and in the moments immediately preceding the puerperium.Experiments of this section aim to deepen knowledge in the complex and fascinating, but not fullyelucidated field of immunological reactivity of the maternal organism during parturition.B 1. Hemogram in cattle during the stress of parturitionBy making hemogram in cows in the last days of gestation, and then, in the momentsimmediately following parturition, we tried to obtain guiding data of the general status of thehematoformator system and mainly of lymphocytes and polymorphonuclear in the mentionedphysiological period.The results of this experiment allowed drawing the following conclusions:- hemogram parameters in cows in the last days of gestation and parameters of thehemogram of same animals available on the first day of parturition did not differ significantly.- in both physiological cases investigated (when the animal bodies are naturally under theinfluence of stress to parturition) the state of lymphopenia is found, accompanied by moderateneutrophilia;- determination of white blood cell counts, although it may highlight effective installation ofLymphocytopenia during the final stage of gestation, does not represent however a valid test forassessing the functionality of lymphocytes;- neutrophilia observed in late pregnancy and during parturition stress suggests increasednon-specific defense mechanisms, assertion also supported by increased blood concentrations oflysozyme (cap.B.4), substance secreted mainly by neutrophils.B 2. The reactivity of T lymphocytes from cattle during the stress of parturitionThis chapter aims to check the functional capacity of T lymphocytes and identify possiblefunctional correlations of these cells with other functional parameters of the immune system ofpregnant cows in the parturition stress.xxiii

One of the methods used to evaluate functional capacity of T lymphocytes during the stress ofparturition was the determination of gamma interferon (IFN-γ) secreted by T lymphocytesstimulated with specific antigen or mitogen (lectins).The results of this experiment revealed that during stress of parturition (last days of gestationand the first day of puerperium) in cows, T lymphocytes are functional and their ability to secretegamma interferon is significantly higher in the last days of gestation compared with the onset ofpuerperium.B 3. The reactivity of B lymphocytes from cattle during the stress of parturitionThis experiment aimed to investigate the functional capacity of B lymphocytes from cows inthe last days of gestation and the beginning of the puerperium to obtain additional data on currentknowledge related to reactivity of the immune system in these species under the stress of parturition.The way to highlight the functionality of B lymphocytes was to determine the bloodconcentration of immunoglobulins, support molecules of antibodies secreted exclusively by theselymphoid cells.The results obtained in this experiment show that at cows under stress during parturition inthe last days of gestation and the onset of puerperium, B lymphocytes have intense secretoryactivity, blood concentrations of G immunoglobulin being greater in the last days of gestationcompared to the onset puerperium.B 4. Non-specific immune reactivity in cattle during the stress of parturitionAddressing the lysozyme status during a particular physiological state of the pregnant body, asis the stress of parturition, fully justifies performing the present experiment because of itsomnipresent role in the nonspecific immune defenses of the body.Research during this experiment aimed to obtain data in addition to those mentioned inspecialized literature, and the results obtained showed that after parturition, the bloodconcentration of lysozyme is immediately reduced, which suggests reduced ability of the body'snonspecific defense calving which in such functional conjuncture, becomes vulnerable to theetiologic agents of certain postpartum diseases.xxiv

OVERALL CONCLUSIONSIn a synthetic and distinct manner, this chapter presents essential functional significancedeveloped on the results obtained from experiments included in this doctoral work, for each of thetwo functional states studied (distress and eustress).At domestic ruminants (cattle and sheep) under status of distress characteristic of early stage(subclinical) of infection determined by a given biological stressor (Mycobacterium aviumparatuberculosis) the following can be seen:1. Hemogram parameters do not change significantly and as a consequence, the hemogrammay not represent a valid means of laboratory to identify the state of distress in thisevolutionary stage of paratuberculosis.2. Lymphocytes of cattle and sheep infected with Mycobacterium avium paratuberculosishave the functional capacity to respond to this biological stressor, a fact highlighted bylymphoblastic transformation tests ,,in pure culture'' and respectively the, ,,the bloodculture '' test.3. Reactivity of T lymphocytes, as evidenced by evaluating the secretion of gammainterferon ,,in vitro'' is intense and is manifested from the early stage of infection withMycobacterium avium paratuberculosis.4. Reactivity of B lymphocytes are active since the early stage of infection but the presenceof antibodies anti-Mycobacterium avium paratuberculosis can be detected only by highlysensitive tests, as observed in our experiment based on iELISA test. Therefore, theparticipation of humoral immunity during this period of biologically distress is weak(discrete) compared with that of the cell (see Chapter 4).5. Concentration of the lysozym in blood serum of cattle under the status of distress causedby Mycobacterium avium paratuberculosis is significantly higher, compared with theanimals which are free from infection, which demonstrates that the non-specific immunedefense mechanisms (highlighted through the evaluation of the blood lysozym) areprimed early, but are also efficient enough for the aggression of this biological stressor.6. Our experimental results obtained in this first section of the work demonstrate that thereactivity of the immune system of cattle and sheep infected with Mycobacterium aviumparatuberculosis, although present from the onset of the biological stressor aggression,is not redoubtable enough to prevent the organism from passing from the discrete state ofdistress to the disease state.xxv

In cows under stress during parturition, our research revealed the following:1. The leukocytes formula changes significantly for the purpose of producing alymphocytopenia accompanied by neutrophilia, the last aspect being positivelycorrelated with an increase of blood lysozyme.2. The functional capacity of T lymphocytes evaluated by determining the secretion ofgamma interferon, is significantly higher in the last days of gestation compared with thefirst day of the puerperium.3. The functional capacity of B lymphocytes, evaluated based on blood concentrations ofIgG, is significantly higher in the last days of gestation compared with the onset ofpuerperium.4. Blood concentration of lysozyme decreases significantly after parturition, which suggestsnon-specific defense capacity is decreasing after parturition, the calving organism becomingmore vulnerable against the stress action of etiologic agents responsible for some postpartumdisorders.As in the field of fundamental research, progress in knowledge is done with 'small steps'', butwe hope that our modest contributions will be integrated in the recently acquired data in thisdomain and even gain further functional meanings, in addition to those we have already noticed.** *xxvi