Loranskaya I.D., Lavrentieva O.A.
The history of the study of the composition of the microflora of the gastrointestinal tract(GIT) began in 1681 when the Dutch explorer Anthony van Leeuwenhoek first reported his observations of bacteria and other microorganisms found in human feces and hypothesized that different types of bacteria coexisted in the gastrointestinal tract. tract. In 1850, Louis Pasteur developed the concept of functional the role of bacteria in the fermentation process. The German physician Robert Koch continued research in this direction and created a method for isolating pure cultures, which makes it possible to identify specific bacterial strains, which is necessary to distinguish between pathogens and beneficial microorganisms. In 1886, one of the founders of the doctrine of intestinal infections F. Escherich first described intestinal coli (Bacterium coli communae). Ilya Ilyich Mechnikov in 1888, working at the Louis Pasteur Institute, argued that in intestines a complex of microorganisms inhabits the human body, which have an "autointoxication effect" on the body, believing that the introduction of "healthy" bacteria into the gastrointestinal tract can modify the effect intestinal microflora and counteract intoxication. The practical implementation of Mechnikov's ideas was the use of acidophilic lactobacilli for therapeutic purposes, which began in the United States in 1920-1922. Domestic researchers began to study this issue only in the 50s of the XX century. In 1955 Peretz L.G. showed that intestinal coli of healthy people is one of the main representatives of the normal microflora and plays a positive role due to its strong antagonistic properties against pathogenic microbes. Started over 300 years ago, studies of the composition of the intestinal microbiocenosis, its normal and pathological physiology and the development of ways to positively influence the intestinal microflora continue to this day.
The main biotopes are: gastrointestinal tract(oral cavity, stomach, small intestine, large intestine), skin, respiratory tract, urogenital system.
The microflora of the gastrointestinal tract is the most representative, its weight in an adult is more than 2.5 kg, the number is 1014. It was previously believed that the composition microbiocenosis The gastrointestinal tract includes 17 families, 45 genera, more than 500 species of microorganisms. Taking into account the new data obtained in the study of the microflora of various biotopes of the gastrointestinal tract using molecular genetic methods and the method of gas-liquid chromatography-mass spectrometry, the total genome of bacteria in the gastrointestinal tract has 400 thousand genes, which is 12 times larger than the size of the human genome. exposed analysis on the homology of the sequenced 16S rRNA genes of the parietal (mucosal) microflora of 400 different sections of the gastrointestinal tract, obtained by endoscopic examination of various sections of the intestines of volunteers. As a result of the study, it was shown that the parietal and luminal microflora includes 395 phylogenetically isolated groups of microorganisms, of which 244 are absolutely new. At the same time, 80% of the new taxa identified in the molecular genetic study belong to non-cultivated microorganisms. Most of the proposed new phylotypes of microorganisms are representatives of the genera Firmicutes and Bacteroides. The total number of species is close to 1500 and requires further clarification.
The gastrointestinal tract through the system of sphincters communicates with the external environment of the world around us and at the same time through the intestinal wall - with the internal environment of the body. Due to this feature, the gastrointestinal tract has created its own environment, which can be divided into two separate niches: chyme and mucous membrane. The human digestive system interacts with various bacteria, which can be referred to as "endotrophic microflora of the human intestinal biotope". Human endotrophic microflora is divided into three main groups. The first group includes useful for humans eubiotic indigenous or eubiotic transient microflora; to the second - neutral microorganisms, constantly or periodically sown from the intestine, but not affecting human life; to the third - pathogenic or potentially pathogenic bacteria ("aggressive populations"). In microecological terms, the gastrointestinal biotope can be divided into tiers (oral cavity, stomach, intestines) and microbiotopes (cavitary, parietal and epithelial). The ability to apply in the parietal microbiotope, i.e. histadhesiveness (the ability to fix and colonize tissues) determines the essence of transient or indigenous bacteria. These signs, as well as belonging to a eubiotic or aggressive group, are the main criteria characterizing a microorganism interacting with the gastrointestinal tract. Eubiotic bacteria are involved in the creation of colonization resistance of the body, which is a unique mechanism of the system of anti-infective barriers. The cavity microbiotope throughout the gastrointestinal tract is heterogeneous, its properties are determined by the composition and quality of the contents of one or another tier. The tiers have their own anatomical and functional features, therefore their contents differ in the composition of substances, consistency, pH, speed of movement and other properties. These properties determine the qualitative and quantitative composition of cavity microbial populations adapted to them. The parietal microbiotope is the most important structure that limits the internal environment of the body from the external one. It is represented by mucous overlays (mucous gel, mucin gel), glycocalyx located above the apical membrane of enterocytes and the surface of the apical membrane itself. The parietal microbiotope is of the greatest interest from the point of view of bacteriology, since it is in it that interaction with bacteria, beneficial or harmful to humans, occurs - what we call symbiosis. To date, it is known that the microflora of the intestinal mucosa differs significantly from the microflora of the intestinal lumen and feces. Although every adult has a specific combination of predominant bacterial species in the gut, the composition of the microflora can change with lifestyle, diet, and age. A comparative study of the microflora in adults who are genetically related to one degree or another revealed that genetic factors influence the composition of the intestinal microflora more than nutrition.
Consider the composition of the normal microflora of different parts of the gastrointestinal tract. The oral cavity and pharynx carry out preliminary mechanical and chemical processing of food and assess the bacteriological hazard with respect to bacteria penetrating the human body. Saliva is the first digestive fluid that processes food substances and affects the penetrating microflora. The total content of bacteria in saliva is variable and averages 108 MK/ml. The composition of the normal microflora of the oral cavity includes streptococci, staphylococci, lactobacilli, corynebacteria, a large number of anaerobes. In total, the microflora of the mouth has more than 200 species of microorganisms. On the surface of the mucosa, depending on the hygiene products used by the individual, about 103-105 MK / mm2 are found. The colonization resistance of the mouth is carried out mainly by streptococci (S. salivarus, S. mitis, S. mutans, S. sangius, S. viridans), as well as representatives of the skin and intestinal biotopes. At the same time, S. salivarus, S. sangius, S. viridans adhere well to the mucous membrane and dental plaque. These alpha-hemolytic streptococci, which have a high degree of histadgesia, inhibit the colonization of the mouth by fungi of the genus Candida and staphylococci. The microflora transiently passing through the esophagus is unstable, does not show histadhesiveness to its walls and is characterized by an abundance of temporarily located species that enter from the oral cavity and pharynx. Relatively unfavorable conditions for bacteria are created in the stomach due to increased acidity, exposure to proteolytic enzymes, rapid motor-evacuation function of the stomach and other factors that limit their growth and reproduction. Here, microorganisms are contained in an amount not exceeding 102-104 per 1 ml of content. Eubiotics in the stomach master mainly the cavity biotope, the parietal microbiotope is less accessible to them. The main microorganisms active in the gastric environment are acid-resistant representatives of the genus Lactobacillus, with or without a histadhesive relationship to mucin, some types of soil bacteria and bifidobacteria. Lactobacilli, despite their short residence time in the stomach, are able, in addition to their antibiotic action in the stomach cavity, to temporarily colonize the parietal microbiotope. As a result of the joint action of protective components, the bulk of microorganisms that have entered the stomach die. However, in case of malfunction of the mucous and immunobiological components, some bacteria find their biotope in the stomach. So, due to pathogenicity factors, the population of Helicobacter pylori is fixed in the gastric cavity.
The main functions of the small intestine include cavitary and parietal hydrolysis of food, absorption, secretion, as well as barrier-protective. In the latter, in addition to chemical, enzymatic and mechanical factors, the indigenous microflora of the small intestine plays a significant role. She takes an active part in the cavity and parietal hydrolysis, as well as in the absorption of nutrients. The small intestine is one of the most important links that ensure the long-term preservation of the eubiotic parietal microflora. There is a difference in the colonization of cavitary and parietal microbiotopes with eubiotic microflora, as well as in the colonization of tiers along the length of the intestine. The cavity microbiotope is subject to fluctuations in the composition and concentration of microbial populations; the wall microbiotope has a relatively stable homeostasis. In the thickness of the mucous overlays, populations with histadhesive properties to mucin are preserved. The proximal small intestine normally contains a relatively small amount of gram-positive flora, consisting mainly of lactobacilli, streptococci and fungi. The concentration of microorganisms is 102-104 per 1 ml of intestinal contents. As we approach the distal parts of the small intestine, the total number of bacteria increases to 108 per 1 ml of content, while additional species appear, including enterobacteria, bacteroids, bifidobacteria.
The main functions of the large intestine are the reservation and evacuation of chyme, the residual digestion of food, the excretion and absorption of water, the absorption of some metabolites, the residual nutrient substrate, electrolytes and gases, the formation and detoxification of feces, the regulation of their excretion, and the maintenance of barrier-protective mechanisms. All of these functions are performed with the participation of intestinal eubiotic microorganisms. The number of microorganisms in the colon is 1010-1012 CFU per 1 ml of content. Bacteria account for up to 60% of stool. Throughout life, a healthy person is dominated by anaerobic species of bacteria (90-95% of the total composition): bifidobacteria, bacteroids, lactobacilli, fusobacteria, eubacteria, veillonella, peptostreptococci, clostridia. From 5 to 10% of the microflora of the colon are aerobic microorganisms: Escherichia, Enterococcus, Staphylococcus, various types of opportunistic enterobacteria (Proteus, Enterobacter, Citrobacter, Serrations, etc.), non-fermenting bacteria (pseudomonas, Acinetobacter), yeast-like fungi of the genus Candida and others Analyzing species composition of the colon microbiota, it should be emphasized that, in addition to the indicated anaerobic and aerobic microorganisms, its composition includes representatives of non-pathogenic protozoan genera and about 10 intestinal viruses. Two biotopes differing in anatomical, physiological and ecological characteristics - the small and large intestines are separated by an efficiently functioning barrier: a baugin valve that opens and closes, passing the contents of the intestine in only one direction, and keeps the contamination of the intestinal tube in the quantities necessary for a healthy organism. Thus, although the bacteria content in the mouth can be quite high - up to 106 CFU / ml, it decreases to 0-10 CFU / ml in the stomach, rising by 101-103 in the jejunum and 105-106 in the distal ileum, with followed by a sharp increase in the amount of microbiota in the colon, reaching a level of 1012 CFU/ml in its distal sections. As the contents move inside the intestinal tube, the partial pressure of oxygen decreases and the pH value of the medium rises, in connection with which there is a "number of storeys" of the settlement of various types of bacteria along the vertical: aerobes are located above all, facultative anaerobes are below, and even lower - strict anaerobes.
It has been proven that the microflora can affect the sensorimotor function of the intestine in three ways:
through the end products of bacterial fermentation and metabolism,
neuroendocrine factors
immune mediators.
Bacterial peptides stimulate the enteric nervous system and afferent innervation, and endotoxins (lipopolysaccharides) can affect intestinal motility. Metabolic products of saccharolytic bacteria - short chain fatty acids (SCFA), such as butyrate, acetate, propionate, play an important role in maintaining normal bowel function and may be involved in the pathogenesis of gastrointestinal diseases. SCFAs are important sources of energy required for colonocytes. Maintenance of anaerobic conditions in the colon is also carried out with the help of microbial metabolites.
SCFAs affect the production of serotonin, motilin and somatostatin contained in the enteroendocrine cells of the colon and ileum; they are key mediators of intestinal motility. The microflora is essential in the normal development of the intestinal immune system and lymphoid tissue. The importance of the immune system in the regulation of the sensorimotor function of the intestine cannot be underestimated either.
There are histochemical, morphological, molecular genetic methods for studying microorganisms, stress tests.
The most common method is bacteriological examination of feces. As a rule, the number of determined indicators ranges from 14 to 25. The advantage of the method is the accurate verification of pathogenic bacteria. The disadvantages of the method include the possibility of obtaining false positive and false negative results due to the heterogeneity of the isolation of microorganisms from different parts of the feces, the difficulty of cultivating anaerobic microorganisms. In addition, the cavitary and transit flora, which dominates the fecal flora, is determined, while the parietal flora is not assessed.
An alternative to bacteriological studies can be chromatographic methods of differentiation of microorganisms - gas-liquid, ion-exchange and, in particular, gas-liquid chromatography (GLC) in combination with mass spectrometry (MS) - GLC-MS. The GLC-MS method is based on the determination of the components of bacterial cells that appear as a result of their natural death or attack by components of the immune system. Minor lipid components of microbial membranes are used as markers. According to their content and quantity, up to 170 species of aerobic and anaerobic bacteria and fungi can be determined within a few hours in various biological media.
A method of GLC analysis based on the determination of SCFAs, which are metabolites mainly of anaerobic genera of microorganisms, has been developed and is being put into practice. Based on the data obtained, a metabolic passport was created for intestinal eubiosis. The method allows you to quickly and fairly accurately assess the state of the indigenous microflora.
Small Intestine Bacterial Overgrowth Syndrome (SIBO) involves an abnormal growth (greater than 105 CFU/mL) of endogenous bacteria in the small intestine, similar to those normally found in the large intestine. Direct and indirect methods are used to diagnose SIBO. Direct consists in sowing duodenal and jejunal contents obtained with a sterile probe. An indirect method includes the study of hydrogen released - a breath test. The rationale for creating a hydrogen breath test was the fact that in the process of metabolism of carbohydrates by the microflora of the colon, a large amount of gases, including hydrogen, is formed. The hydrogen test can be used to give an approximate idea of the degree of bacterial colonization of the small intestine. Recently, however, an opinion has appeared that the hydrogen breath test can only determine the orocecal transit of bacteria.
Currently, a method for determining the types of microorganisms using the polymerase chain reaction (PCR) has become widespread. The PCR method was developed back in 1983 by Kary Mullis, for which he was awarded the Nobel Prize in Chemistry in 1993. The PCR method is based on repeated copying (amplification) of the desired DNA fragment using the DNA polymerase enzyme. Compared with traditional diagnostic methods, PCR is characterized by high sensitivity and specificity. It allows the use of samples for diagnostics with less stringent requirements for the viability of the studied microorganisms than for microbiological research. Today, there is a more advanced PCR method than the "classical" one - with the detection of results in real time. This method is based on the automatic measurement of the level of the fluorescent signal, which increases with each cycle with a positive PCR reaction, which allows quantitative assessment of the DNA of the microorganism under study in a biological sample.
The evolution of man and animals took place with constant contact with the world of microbes, resulting in the formation of close relationships between macro- and micro-organisms. The influence of the microflora of the gastrointestinal tract on the maintenance of human health, its biochemical, metabolic and immune balance is undeniable and has been proven by a large number of experimental works and clinical observations. Its role in the genesis of many diseases continues to be actively studied (atherosclerosis, obesity, irritable bowel syndrome, nonspecific inflammatory bowel disease, celiac disease, colorectal cancer, etc.). Therefore, the problem of correcting microflora disorders, in fact, is the problem of maintaining human health, the formation of a healthy lifestyle.
It must be remembered that dysbiotic disorders are always secondary. Therefore, the elimination of the causes, the treatment of the underlying disease, against which violations develop microbiocenosis intestines, is one of the leading principles of its correction. Along with the impact on the underlying disease and an increase in the body's resistance, the correction of dysbiotic disorders shows: normalization of the motor function of the intestine, the use of enterosorbents, the appointment of antibacterial drugs, pre- and probiotics, synbiotics.
Bactistatin®, a complex preparation of natural origin, an innovative enterosorbent with a probiotic effect, used in modern clinical practice, has these properties.
The drug Bactistatin® consists of three components that mutually reinforce each other's action. The basis of the drug is a sterilized culture fluid of the natural microbe Bacillus Subtilis with high medical and biological properties, containing probiotic substances (lysozyme, bacteriocins, catalase), enzymes and amino acids. The bactericidal and bacteriostatic effect on pathogenic and opportunistic microbes is due to the content of probiotic substances synthesized during the vegetative growth of B. Subtilis strain bacteria and their concentration in the culture fluid during fermentation. Thus, the probiotic compounds in the composition of Bactistatin® ensure the restoration of the normal intestinal microflora, increase the nonspecific resistance of the body.
The second component is zeolite, a natural sorbent with ion-exchange properties. Zeolite is able to exhibit sorption properties mainly in relation to compounds with low molecular weight (methane, hydrogen sulfide, ammonia and other toxic substances), without directly interacting with vitamins, amino acids, proteins, leaving them in the gastrointestinal tract. tract. The ions contained in the body can be included in the crystal structure of the mineral, and, conversely, the body receives from the mineral those inorganic elements that it needs. There is a so-called selective ion exchange. Zeolites contribute to the normalization of fat, protein, carbohydrate metabolism; increase immunity; increase resistance to stress; improve reproductive function, liver cell function; normalizes intestinal motility, accelerating the movement of intestinal contents through the digestive tract.
Bactistatin® also contains soy flour hydrolyzate, which is a natural source of a complete protein of amino acids, oligosaccharides, provides the most favorable conditions for the uncompetitive growth of normal flora and restoration of the microbial landscape of the body.
Bactistatin® is especially effective for normalizing the intestinal microflora in acute and chronic diseases of the gastrointestinal tract with symptoms of dysbacteriosis, in dysbacteriosis that occurs with irritable bowel syndrome, as a result of antibiotic therapy, after intestinal infections.
Literature
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2. Bondarenko V.M., Matsulevich T.V. Intestinal dysbacteriosis as a clinical and laboratory syndrome: the current state of the problem. - M.: Publishing group "GEOTAR-Media". - 2007. - P.8-35
3. Grigoriev A.V. Gastrointestinal tract as a habitat for bacteria // Section 1. - M.: Publisher: CJSC "SILMA". - 2004.- P.5-7, P.16-32
4. Korovina N.A., Zakharova I.N., Kostadinova V.N. etc. Prebiotics and probiotics for intestinal disorders microbiocenosis in children. - M.: ID "Medpraktika-M". - 2004. - P.8-9
5. Tkachenko E.I., Uspensky Yu.P. Nutrition, microbiocenosis and human intelligence. - St. Petersburg: SpecLit. - 2006. - P.110-113
6. Ursova N.I. Modern technologies in the correction of dysbiosis in children. - Tutorial. - Moscow. - 2003. - P.4-6.
7. Eckburg P.B., Bik E.M., Bernstein C.N. et al. Diversity of the human intestinal microbial flora. // Science. 2005. - 308.- R.1635-1638.
8. Ghoshal U.C., Park H., Gwee K.-A. Bugs and Irritable Bowel Syndrome: The Good, the Bad and Ugly. // J Gastroenterology, Hepatology. - 2010. - 25 (2). - P.244-251.
9. O'Hara A.M., Shanahan F. Review. Intestinal microflora: analysis therapeutic potential. // Clinical gastroenterology and hepatology. Russian edition. - 2008.- Volume1, No. 4: 236-247.
10. Spiller R.C. Review Article: Probiotics and Prebiotics in Irritable Bowel Syndrome // Alimentary Pharmacology & Therapeutics. 2008; 28(4):385-396.
480 rub. | 150 UAH | $7.5 ", MOUSEOFF, FGCOLOR, "#FFFFCC",BGCOLOR, "#393939");" onMouseOut="return nd();"> Thesis - 480 rubles, shipping 10 minutes 24 hours a day, seven days a week and holidays
Mikhailova, Olesya Nikolaevna Theoretical and practical aspects of the prevention and treatment of gastrointestinal diseases of calves in the early postnatal period: dissertation ... candidate of veterinary sciences: 06.02.02 / Mikhailova Olesya Nikolaevna; [Place of protection: Kur. state s.-x. acad. them. I.I. Ivanov].- [Kursk], 2013.- 159 p.: ill. RSL OD, 61 14-16/47
Introduction
1.0 Literature review 8
1.1 Gastrointestinal diseases of calves in the early postnatal period: etiology, clinical and epizootic features 8
1.2 Prevention and treatment of gastrointestinal diseases in calves 20
1.3 The use of immunostimulants in gastrointestinal diseases of newborn calves 31
2.0 Own research 34
2.1 Material and methods of research 34
3.0 Results of own research 36
3.1 Theoretical substantiation of the dissertation research methodology 36
3.2 Finding out the causes and characteristics of the course and manifestations of gastrointestinal diseases in newborn calves 38
3.3. Theoretical and experimental substantiation of obtaining a new immunometabolic drug based on succinic acid and levamisole 44
3.3.1 Study of the effect of a complex preparation (levamisole amber) on hematological, immunological and biochemical parameters of newborn calves 48
3.4 Effect of levamisole succinic on diarrhea rates 55
3.5 Efficacy of levamisole amber for correction of metabolic and immune processes in deep-calving cows 56
3.6 Production test of the effectiveness of levamisole amber for the prevention of gastrointestinal diseases in newborn calves 61
3.7 The effectiveness of complex preparations of succinic acid for the prevention and treatment of diarrhea in newborn calves when administered orally 68
3.7.1 Theoretical and experimental substantiation of the possibility of combined use of succinic acid, ASD of the second fraction of iodoinol 68
3.7.2 The effect of succinic acid in combination with ASD of the second fraction, in combination with iodinol, on hematological, immunological and biochemical parameters of clinically healthy calves when administered orally 70
3.7.3 Efficacy of oral administration of a formulation based on succinic acid and ASD for prevention and in combination with iodinol in the clinic for the treatment of diarrhea in newborn calves 73
3.7.4 Efficacy of oral administration of a composition based on succinic acid and ASD for prevention and in combination with iodinol in the treatment of diarrhea in newborn calves with severe toxic infection syndrome 75
3.8 Results of field experience in evaluating the effectiveness of scientific developments for the prevention of diarrhea in newborn calves 77
4.0 Discussion of research results 81
5.0 Conclusions 104
6.0 Practical suggestions 106
7.0 References 107
Appendix 143
Introduction to work
Relevance of the topic. Gastrointestinal diseases, manifested by diarrhea syndrome, are widespread and cause great economic damage to industrial livestock. Despite the great attention paid by science and practice to the problem of prevention and treatment of gastrointestinal diseases in calves, there is no significant improvement in the situation. Calves that have been ill with diarrhea at an early age are later stunted and, as a rule, are prone to respiratory pathology.
The leading cause of massive gastrointestinal diseases in newborn calves are infectious pathogens, the virulence of which increases when passed through the body of susceptible animals. In diarrhea of newborn calves, it is very difficult to determine the leading role of one or another pathogen. In this regard, attempts to prevent these diseases using specific agents do not always have a positive result. At the same time, the widely practiced use of chemotherapy and antibiotic therapy often leads to the selection of drug-resistant strains of microorganisms.
The resistance of calves to diarrhea is completely determined by the activity of colostral immunity, which is directly dependent on the quality of colostrum (Mishchenko V.A. et al. 2004). The data of recent years indicate that under the conditions of industrial animal husbandry in the colostrum of cows there is a sharp decrease in immune defense factors, as a result of which newborn calves have a deficiency of the humoral immune system (Voronin E.S., Shakhov A.G., 1999). Taking into account that disorders of metabolic and immune processes underlie the clinical manifestations of all pathophysiological conditions, the modern concept of prevention and treatment of gastrointestinal diseases should include the mandatory use of immunometabolic agents.
All of the above determined the choice of the topic of the dissertation research on the search for effective means of preventing and treating gastrointestinal diseases with diarrheal syndrome.
Purpose of research. The main goal of the dissertation research was the theoretical and experimental substantiation of the preparation and use of succinic acid preparations in the system of measures for the prevention and treatment of gastrointestinal diseases in calves with diarrheal syndrome.
To achieve this goal, the following tasks were identified:
To study the features of the manifestation and course of gastrointestinal diseases of newborn calves;
To theoretically and experimentally substantiate methods for obtaining complex preparations with immunometabolic and anti-infectious activity;
To study the effectiveness of the use of succinic acid preparations for stimulating immunobiochemical processes, preventing and treating gastrointestinal diseases in calves.
Determination of the production efficiency of the author's approaches to the prevention and treatment of diarrhea in calves.
Scientific novelty. New compositions of complex immunometabolic preparations based on succinic acid have been scientifically substantiated, developed and patented, and the effectiveness of their use for stimulating metabolic and immune processes, preventing dysbacteriosis, and treating diarrhea in calves of the early postnatal period has been determined.
The practical significance of the work. As a result of scientific research in practical veterinary medicine, cost-effective, simple and effective means and practical proposals for the prevention and treatment of gastrointestinal diseases in newborn calves have been proposed. The results of the research were included as an integral part of the project of temporary guidance on the use of levamisole amber, approved by the director of the Kursk Research Institute of Agro-Industrial Production of the Russian Agricultural Academy and the Veterinary Administration of the Kursk Region.
The main provisions of the dissertation submitted for defense:
1. Causes, features of the manifestation and course of gastrointestinal diseases in newborn calves;
2. Theoretical and experimental substantiation of obtaining new compositions with immunometabolic, anti-infective and detoxification activity.
3. The results of studying the effectiveness of the use of new drugs in the system of measures to stimulate metabolic and immune processes, prevent and treat gastrointestinal diseases in calves with diarrheal syndrome.
Approbation and publication of the results of the study. The materials of the dissertation research were reported and discussed at the International Scientific and Practical Conference of the Belgorod State Agricultural Academy "Problems of agricultural production at the present stage and ways to solve it" - Belgorod, 2012; at the International Scientific and Practical Conference of the Kursk State Agricultural Academy "Agro-industrial complex: contours of the future" - Kursk, 2012; at the International Don agrarian scientific and practical conference "Actual problems of ensuring the veterinary welfare of the livestock industry" - Zernograd, 2012; at the International scientific and practical conference "Actual problems of veterinary medicine and intensive animal husbandry" of the Bryansk State Agricultural Academy - Bryansk - 2013.
Based on the materials submitted for the defense of the dissertation, 7 articles were published, including 3 in peer-reviewed journals.
The structure and scope of the dissertation.
The dissertation work is presented on 143 pages of a computer text, illustrated with 24 tables, consists of an introduction, a literature review, own research and a discussion of their results, conclusions, proposals for production, a list of references. The list of used literature includes 344 sources, including 122 foreign authors.
Prevention and treatment of gastrointestinal diseases in calves
According to Samokhin V.T. et al. (2002), Shakhova A.G. (2002) The biological complex "mother-fetus-newborn" should be considered as a single system in the development of rational methods for the prevention and control of neonatal diseases, because there is a direct relationship between the state of metabolism, the level of natural resistance of the body of cows, intrauterine development of the fetus, the state of health and safety of newborn calves. This point of view is the result of numerous scientific studies carried out at different times by E.S. Voronin. (1981) with co-authors (1989), Devrishev D.A. (2000), Zaroza V.G. (1983), Kasicha A.Yu. (1987), Nemchenko M.I. et al. (1986), Semenov V.G. (2002), Sidorov M.A. (1981, 1987), Suleimanov S.M. (1999), Urban V.P., Neimanov I.L. (1984), Fedorov Yu .Sch1988), Chekishev V.M. (1985), Sharabrin I.G. (1974), Shishkov V.P. et al. (1981,1985), Shkil N.A. (1997) and others.
Even normally developed calves have a number of physiological characteristics that make them especially vulnerable to gastrointestinal diseases. First of all, it is a physiological immunodeficiency.
In newborns, the immune system is underdeveloped, they are characterized by low functional activity of the cellular and inferiority of the humoral immunity. Compensation for age-related deficiencies in the immune system of newborns in the postnatal period of life occurs due to cellular and humoral factors of colostrum. With their lack in colostrum, immunodeficiency is aggravated (Voronin E.S., Shakhov A.G., 1999; Devrishev D.A., 2000; Terekhov V.I., 2002; Fedorov Yu.N. 1988, etc.).
According to the vast majority of researchers, the resistance of calves to diarrhea is determined by the activity of colostral immunity, which is directly dependent on the quality of colostrum, which is the only source of immunoglobulins (Mishchenko V.A. et al., 2005; Richou R 1981; Salt LJ, 1985; Selman JE, 1979).
Along with antibodies from the mother, immunologically active leukocyte cells are transmitted to the newborn with colostrum (Vieg A. ea, 1971 Concha C. ea, 1980; Selman J. 1979; Suling L. 1980; Smith Y. ea. , 1977; Tough DFea, 1996).
With timely, no later than 2 hours after birth, drinking colostrum, the proportion of gamma globulins in blood plasma proteins reaches 30-50%, which significantly reduces the risk of diarrhea. Conversely, calves in which by the age of one day the amount of gamma globulins in the blood does not exceed 10% fall ill and almost all die.
The importance of timely feeding of colostrum to newborns is most indicative of the comparison of clinical data on the registration of diarrheal disease in calves "night" and "day". The incidence and death of calves born at night significantly exceeds the indicators for "day" calves (Mishchenko V.A. et al., 2005). Feeding colostrum to calves "nightlights" is carried out in the morning, that is, after 5-6 or more hours.
Immediately after calving, colostrum should be checked for mastitis. The most important factor in the prevention of neonatal diarrhea is the timing of colostrum feeding. The best time for the first watering is when the calf develops a sucking reflex (most 30-40 minutes after birth). For mastitis in a mature cow, colostrum from other cows can be used. It is recommended to have a frozen colostrum bank.
An important factor influencing the level of colostral immunity is the concentration of immunoglobulins in colostrum (Weaver D.e.a. 2000). In high-yielding cows, the concentration of immunoglobulins in colostrum is lower than in animals with lower milk yield. In cows with impaired metabolic processes, calves are born with a similar metabolic symptom complex. Violations of the immunometabolic status in the mother has a direct impact on the embryonic development of the fetus, which can be one of the causes of the development of secondary immunodeficiencies, and, as a barely, a consequence of high morbidity.
The next feature that affects the propensity for the incidence of diarrhea is the sterility of the intestine at birth. The calf is born weakly protected and, getting into a new environment saturated with various pathogens, is easily infected.
The main route of infection of newborn calves is alimentary, as a result of contact with the so-called "shed" microflora, represented by an association of gram-negative and gram-positive microorganisms.
Conditionally pathogenic (“shed”) microflora decomposes milk in the stomach with the formation of a large amount of toxins that irritate the intestinal mucosa.
The phenomenon of dysbacteriosis reflects the conditions for the reproduction of lactic acid and opportunistic microorganisms that have changed in the intestine. The latter, penetrating into the abomasum, displace lactic acid microorganisms. The rapid reproduction of conditionally pathogenic microorganisms is accompanied by the formation of a large number of toxic products of their vital activity.
To compensate for physiological dysbacteriosis and earlier formation of intestinal colonization resistance after the first colostrum giving to newborn calves, it is recommended to prescribe probiotics (Antipov V.A., 1981; Bazhenov A.N. et al., 1986; Voronin E.S. et al., 1994; Grigoriev G. I. et al., 2000; Gryazneva T. N., 2005; Gudkov A. V. et al., 1986; Devrishev D. A., 1988; Intizarov M. M., 1989; Karpov V. N., 1987; Kvasnikov E. I. et al., 1975; Panin A. N. et al., 1988; Perdigon G. e. a. 2001; Shanahan F., 2001 and others).
Probiotics are biological preparations that are stabilized cultures of symbiotic microorganisms or their fermentation products that have antagonistic activity against putrefactive and pathogenic microorganisms, incl. and to Escherichia, in the intestines.
The multicomponent composition and versatile pharmacological action allow the use of probiotics with a high effect for the prevention and treatment of calf colibacillosis, dysbacteriosis, detoxification of individual endogenous and exogenous toxins, creation of nonspecific intestinal protection against pathogenic bacteria (intestinal colonization resistance). These are environmentally safe, physiological drugs in terms of pharmacokinetics and pharmacodynamics, technologically advanced for group use, do not have side effects, do not accumulate in organs and tissues of animals, do not cause addiction from pathogenic microflora (V.A. Antipov, 2001; A. Panin et al. ., 1993; Yu. N. Proskurin, 2000; S. Parnikova, 2002).
Early administration of probiotic preparations to newborn calves is also important because the normal intestinal microflora acts as the first stimulant of the immune system. It should be noted that the therapeutic efficacy of probiotics in gastrointestinal diseases of calves is not high enough. A contraindication to the use of probiotics is the inadmissibility of their combination with antibiotics or other antimicrobial drugs.
A starvation diet is prescribed when signs of frequent bowel movements appear. Exclude 1-3 (at the discretion of the veterinarian) feeding with colostrum (milk), replacing it with electrolyte-energy solutions (in the amount of 0.5-1.0 l) or decoctions (infusions) of medicinal plants (R.J. Bywater, 1983). To prevent dehydration and intoxication of sick calves, a large number of different electrolyte solutions have been proposed for both drinking and parenteral administration.
Means of chemo-antibiotic therapy remain the main group of drugs used in these pathologies.
Antibacterial drugs are etiotropic drugs that selectively inhibit the growth of microorganisms, which determines their most important property - specificity in relation to pathogens of infectious diseases in calves. The most important place among these drugs is occupied by antibiotics. Kovalev V.F. et al. (1988),. Vitebsky E.L. et al. (1998), Sokolov V.D. et al. (2000), Troshin A.N. et al. (2003):
Among the highly effective, antidiarrheal agents are drugs of the nitrofuran series. Possessing a wide range of bioactivity, nitrofurans, unlike antibiotics, are able to increase the overall resistance of the macroorganism (Shipitsyn A.G. et al., 1999).
As a result of the mass and often unsystematic use of this group of drugs, their effectiveness has noticeably decreased. The main reason for this is the accumulation of drug-resistant strains of opportunistic microorganisms in nature.
To overcome the drug resistance of microorganisms, a combination of two drugs and a combination of antibacterial therapy with drugs that enhance the body's defenses are most often practiced.
Study of the effect of a complex preparation (levamisole amber) on hematological, immunological and biochemical parameters of newborn calves
In this series of experiments, the composition of the drug was used, including: 1% succinic acid and 2% levamisole. We illustrate the method of obtaining a complex preparation with the following example.
To prepare a complex preparation, 950 ml of demineralized water was used, in which 10.0 g of succinic acid and 20 g of levamisole were successively dissolved when heated. The total volume was adjusted to 1000 ml. by adding demineralized water. The resulting solution had pH = 4.5-4.7. After packaging the drug in vials, sterilization was carried out. Sterilization by autoclaving in the mode of 1.0-1.1 atm. within 30 minutes did not change the physico-chemical properties of the drug. The precipitate did not fall out during storage for 12 months.
The research and production experience was carried out at the Kalininsky SGJ.
The objectives of the research included studying the effect of the drug (levamisole amber) on the hematological, immunological and biochemical parameters of normotrophic calves and hypotrophic calves.
Taking into account the clinical material at our disposal, 4 groups of calves were formed for the experiment. The first two groups were formed from physiologically developed calves. In two more groups, calves of the same age were selected, but from among hypotrophics.
Thus, by conducting the first series of experiments, we had the opportunity to study the effect of the drug on the body of physiologically developed calves and animals with obvious signs of immunodeficiency. The latter was confirmed by the data of background homeostasis, selected for the experiments of calves, displayed in tables 4,5,6,7.
As expected, even the first control results of the studies showed that hematological parameters, even in physiologically developed calves, were at the lower values of the physiological norm for this age group. The use of levamisole amber made it possible to activate metabolic processes, expressed in an increase and normalization of hemoglobin levels in normotrophic calves. In the hypotrophic calves of the experimental group, the level of hemoglobin content and the number of erythrocytes practically reached the background indicators of clinically healthy peers. When conducting subsequent studies (after 2 weeks), there were no significant changes in calves from the control groups over this period of time.
The study of the level of total protein and protein fractions in the blood serum of calves found that their content at the beginning fluctuated within the lower limits of the physiological norm. The use of levamisole amber already on the third day revealed a trend towards an increase in total protein and y - globulins, which reached their maximum value on the 7th day, after which there was a tendency for a gradual decrease. However, on the 14th day of control studies, the total protein content and gamma globulin fraction were significantly higher than those of their peers from the control groups (P 0.05). In the process of research, a slight increase in the level of albumins, fractions of a- and p-globulins was also noted.
Thus, the use of levamisole amber contributed to the improvement of hematological parameters and protein metabolism.
An increase in the level of gamma globulin fraction indicated an increase in the body's resistance.
The use of the drug allowed to significantly improve the biochemical parameters of blood (table 7). So, after 2 weeks in physiologically developed calves, the content of total calcium in the blood serum after was 3.02±0.11, against 2.41±0.19 in control animals (P 0.05); and in calves - hypotrophic, respectively 2.28±0.10 and 1.57±0.18 (Р 0.005). The content of inorganic phosphorus also had a pronounced growth trend. Thus, in normotrophic calves, the content of inorganic phosphorus increased on day 7 to 2.04±0.15, on day 14 to 2.09±0.16, which was 11.3% and 12.4% higher than in animals of the control group. A similar pattern was observed in terms of the content of inorganic phosphorus in normotrophic calves. Normalization of calcium-phosphorus metabolism significantly improved the alkaline reserve of the blood. Thus, in normotrophic calves, it increased from 29.4 ± 2.3 to 43.7 ± 3.1 vol% CO2 (on day 14), and in hypotrophic calves from 14.3 ± 2.1 vol% CO2 to 29.1±2.8 vol% CO2. The increase and normalization of the content of calcium, phosphorus and reserve alkalinity of the blood could not but indicate an improvement in the mineral metabolism in general, which is important in terms of increasing the overall resistance of the organism. Clinical observations indicated that the animals of the experimental groups grew better and were more calm than their peers from the control groups. The average increase in live weight of clinically healthy calves was 307 g, and in control calves - 250 g. In general, the absolute increase in live weight of clinically healthy experimental calves was 9.2 + 0.3 kg, against 7.5 + 0.4 kg in control calves, and in developmentally lagging 5.0 ± 0.2 kg and 2.4 kg, respectively. +0.2 kg (Table 8).
As you know, one of the factors of nonspecific defense of the body is phagocytosis. When analyzing the results of the phagocytic reaction, it was found that already on the 3rd day, the phagocytic activity of neutrophils in both clinically healthy and hypotrophic patients was 13% and 5.8% higher than before the administration of the drug, and 12% and 5.2% higher, according to compared with control animals.
The bactericidal and lysozyme activity of blood serum, reflecting, in general, the humoral mechanisms of natural resistance, already on the 3rd day after immunization was higher than in control animals (table 9).
The dynamics of immunological parameters indicates that levamisole amber provided a pronounced tendency to increase the phagocytic activity of neutrophils and the bactericidal activity of blood serum. On the 14th day, these indicators did not undergo pronounced changes.
The effect of succinic acid in combination with ASD of the second fraction, in combination with iodinol, on hematological, immunological and biochemical parameters of clinically healthy calves when administered orally
For experiments, we used a composition based on 1% succinic acid, 4% ASD of the second fraction in combination with iodinol in a ratio of 3:1. Three groups of calves were formed according to the principle of analogues in SPK Kalininsky. The first experimental group (n=5) was given an amber biostimulant, the second (n=5) an amber biostimulant in combination with iodinol. The third group (n=5) was the control group. The calves of this group were fed an aqueous solution of iodinol (3:1). The drugs were drunk in a volume of 100 ml, once a day for 5 days, 30 minutes before drinking milk.
Blood sampling for research was carried out before, on the third and fourteenth days after drinking the drugs. In tables - 19, 20,21 we provide information on the dynamics of hematological, immunological and biochemical studies.
The results of hematological studies indicated that the use of succinic acid preparations had a positive effect on the level of erythrocytes and their saturation with hemoglobin. The level of hemoglobin content on days 3 and 14 after the course of drug use was significantly higher in experimental calves in relation to their peers from the control group.
The index of reserve alkalinity in calves of the control groups in all periods of the study was below the physiological norm, while in animals of both experimental groups it was within the physiological parameters. This indicated that oral administration of succinic acid preparations ensured the elimination of metabolic acidosis. The elimination of metabolic acidosis had a positive effect on protein metabolism. The level of total protein in the blood of calves of the experimental groups was within the average physiological parameters, while in their peers it was slightly above the lower limit.
In the control periods of the studies after the course of using the drugs in calves of both experimental groups, the indicators of bactericidal and lysozyme activity were significantly higher than in individuals from the control group. This indicated a positive effect of succinic acid preparations on natural resistance factors, which is important in relation to the body's resistance to endogenous infection, including diarrhea.
Results of industrial experience in evaluating the effectiveness of scientific developments for the prevention of diarrhea in newborn calves
Based on the fact that the resistance of newborn calves to diarrhea is completely determined by the activity of colostral immunity, which directly depends on the quality of colostrum, an important role should be given to the health of deep-calving cows. However, the data of retrospective studies, including those conducted by us, indicate that deep pathobiochemical processes develop in most cows in the last months of pregnancy. Metabolic and immune processes are interconnected. This circumstance served as the basis for the development of a complex drug with immunometabolic activity. In the course of scientific and production experience, it was found that the use of levamisole amber provides an effective correction of metabolic and immune processes in deep-calving cows and calves. This had a positive impact on the incidence of diarrhea in newborn calves.
In turn, the oral administration of iodinol, an amber biostimulant, turned out to be an effective approach for the relief of diarrheal syndrome in newborn calves.
Research and production experience was carried out in the SPK "Kalininsky" in the period from January to May 2013. The procedure for conducting scientific and industrial experience included carrying out preventive measures both on deep-calving cows and on newborn calves.
Deep-calving cows and newborn calves up to 10 days of age were the object for conducting research and production experience.
During the course of preventive immunometabolic therapy, we were guided by the already approved scheme for the use of levamisole amber, which provides for three intramuscular injections of the drug to deep-calving cows in a volume of 10.0 ml with an interval of 10 days.
In the event of diarrhea in calves, drinking colostrum or milk was replaced by giving iodinol - amber biostimulant (100 ml) mixed with saline with simultaneous single administration of levamisole amber at a dose of 2.0 ml. The concentration of succinic acid in the preparation used for cows and calves was 1.5%.
From among the deep-calving cows, two experimental groups were formed. The cows of the experimental group (n=103) were treated with levamisole amber. The control group included 95 cows.
Based on the results of clinical observation of calves, the following data were obtained, which are presented in table 24.
During clinical observation, it was found that the disease of diarrhea in calves obtained from cows of the control group in March and April was recorded in almost all animals. At the same time, diarrhea often proceeded with symptoms of severe intoxication.
In calves born from cows of the experimental group, diarrhea proceeded mainly in mild and moderate severity.
It should be noted this feature. In calves born from cows in the control group, diarrheal syndrome usually developed on 2 less than 3 days. While in calves born from cows of the experimental group, diarrhea appeared on the 5-6th day.
For the treatment of diarrhea, we used iodinol-amber biostimulator. At the same time, it was found that a single feeding of iodinol, an amber biostimulant, to calves with symptoms of mild diarrhea, as a rule, was sufficient to stop it. With an average severity of the course of diarrhea, two, less often three, drinking of this composition was required with an interval of 5-6 hours.
In the clinic for the treatment of diarrhea with a pronounced toxic symptom complex, intravenous administration of 100 ml of Reamberin (1.5% succinic acid solution) with the addition of 50 ml of 40% glucose to its composition turned out to be very effective. The choice of Reamberin to relieve the symptoms of toxicosis is not accidental. The detoxification solution "Reamberin" contains 1.5% succinic acid in the form of its salt - sodium succinate. The practical use of "Reamberin" in medicine and veterinary medicine indicates that it has an exceptionally high therapeutic effect in toxicoinfectious diseases. However, the experience of its use indicates that it can also have side effects on the cardiac and respiratory systems. For infusions, it is used intravenously, drip. It is obvious that the drip administration of the drug to productive animals is difficult to perform under production conditions.
According to the results of our observations, the inclusion of glucose in the composition of the infusion solution made it possible to reduce the risk of side effects of Reamberin on the cardiovascular and respiratory systems with a slow (through a thin needle) jet method of administration. This composition was tested by us on 17 calves. No side effects were observed in any of the cases.
The results of the use of Reamberin in combination with glucose provided a favorable "break" of the intoxication syndrome. As a rule, after the first administration of the infusion composition, the clinical condition of the calves improved so much that it no longer raised concerns about an unfavorable outcome. It should be noted that the use of other infusion solutions, in particular glucose-salt solutions, in the clinic for the removal of toxic syndrome did not give such a pronounced positive effect.
Table of contents of the subject "Bacteriological Examination of the Gastrointestinal Tract. Examination of the Genitourinary System.":In the proximal parts of the esophagus can detect a small amount of bacteria living in the oropharynx, in the distal sections - staphylococci, diphtheroids, lactic acid bacteria, sarcins, Bacillus subtilis and candida. Bacteriological examination is carried out to identify pathogens of esophagitis. The main pathogens are HSV, CMV and fungi of the genus Candida.
for herpes infection indicate deep multiple small ulcers; with CMV infection, they are larger and tend to merge. Candida esophagitis is typical for patients with severe immunodeficiency. To isolate candida, biopsy specimens are taken during esophagoscopy, smears are microscopically stained by Gram, and the material is inoculated on nutrient media.
Bacteriological examination of the stomach
Bacteria in the stomach practically absent, or their number does not exceed 10 3 -10 4 ml of content, due to acidic pH. More bacteria are found in the pyloric part. With hypochlorhydria in the stomach, lactobacilli, sarcins, enterobacteria, aeruginosa, enterococci, spore-forming bacilli and various fungi are detected. Of particular importance is Helicobacter pylori, the causative agent of recurrent ulcerative gastroduodenitis.
For H. pylori detection The most optimal is the sampling of biopsy specimens during fibrogastroscopy. At food poisoning caused by S. aureus and B. cereus, study gastric lavage, which is examined immediately. The samples are centrifuged, the sediment is microscopically examined and cultured on nutrient media. If immediate analysis is not possible, the pH of the samples is adjusted to neutral values and frozen. The vomit is delivered to the laboratory on ice; they can be frozen if necessary.
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