CC BY
0VETERINARY SCIENCES
EFFECT OF ENROFLOXACIN ANTIBIOTIC ON THE MICROFLORA OF THE DIGESTIVE TRACT OF HONEYBEES OF CARPATHIAN BREED IN THE CONDITIONS OF KRASNODAR REGION
Serdyuchenko I. V.
candidate of veterinary sciences, associate professor, department of microbiology, virology epizootology faculty of veterinary medicine Federal state budgetary educational institution higher education "Kuban state agrarian University named after I. T. Trubilin»
Abstract
The relevance of the problem is due to the fact that to obtain high quality and useful products of beekeeping (honey, wax, pollen, perga, royal jelly, propolis, merva, zabrus, subpestilence, bee venom) is very important condition of the intestinal flora of bees, which subsequently contributes to healthy offspring. The article aims to study the effect of the antibiotic enrofloxacin on the quantitative and qualitative composition of the microflora of the digestive tract of bees. The leading method for the study of this problem is to conduct microbiological and viro-logical studies of the suspension from the bee intestine. In the process of research it was found that feeding bees carbohydrate feeding with enrofloxacin is accompanied by the development of their intestinal tract selective dysbacteriosis, manifested almost complete disappearance of gram-positive and gram-negative bacteria and an increase in the quantitative presence of yeast and fungi. With the purpose of correction of intestinal microflora of bees to improve their condition in the intestinal tract after wintering, increase in reproductive activity and medoproduktivnost recommend to use the incentive to carbohydrate feeding with the antibiotic enrofloxacin. Materials of the article can be useful for all beekeepers in the planning of seasonal work on apiaries, both industrial and private.
Keywords: apis mellifera, microorganisms, microflora, an intestine, enrofloxacin.
Introduction
One of the fundamental factors affecting the strength and productivity of the bee family is the health of its members (uterus, worker bee, drones). Bees, as well as other biological objects, are subject to various infectious and non-infectious diseases, and therefore are constantly under the threat of diseases. And under the threat of not only disease, but also under the threat of extinction as a species, because. The last decade was marked by the spread in Europe and the United States of the phenomenon of mass death of bees, called the collapse of bee colonies. It is assumed that the causes of mass death of bees can be a variety of biogenic and abiogenic factors, including microorganisms that make up the microbiocenosis of the body of bees [2, c. 234].
Microbiocenosis of bees is largely determined by the habitat of the insects themselves, and therefore its members are not only saprophytic, but are potentially pathogenic microscopic organisms that cause the development of diseases such as E. coli, Harris, Citrobacter, Salmonella, etc.
The presence of honeybees such biological features as the lack of emptying the colon during the winter period provides a risk of developing a particular infectious disease during or by the end of winter [3, c. 100].
Meanwhile, it should be assumed that the microbiocenosis of the intestinal tract of bees is formed during the entire active period of family life, and on what composition of the microflora will be formed in adults and young bees going into the winter, will depend on the health of the family as a whole, and, consequently, their reproductive and productive activity in adulthood.
To neutralize the negative effects of wintering, beekeeping widely use a variety of feeding, which include, in addition to sugar or honey, substances of an antibiotic nature, minerals, stimulants and vitamins that affect both the bees themselves and the microflora of their digestive tract. Therefore, when developing fertilizing, it should be taken into account how their use will affect the normal intestinal microflora.
Therefore, the study of intestinal microbiocenosis of honeybee, and the development of environmentally friendly methods of its regulation, is an urgent issue for modern beekeeping.
The main risk of intestinal diseases occurs at the end of wintering and early spring period, which is associated with the physiological characteristics of the intestine and the functional state of bees in this period, especially when omissions in conditions of maintenance and feeding. In this regard, all preventive measures of intestinal diseases of honeybees are reduced to compliance with veterinary and sanitary rules of their content, full feeding and the use of modern methods and tools to improve their productivity.
It should be noted that the normal state of its mi-crobial background plays an important role for the normal functioning of the digestive system. Consequently, the purposeful formation of the predominance of beneficial microflora with the help of various drugs, in particular antibiotics, seems relevant.
Antibiotics, even in small amounts, help to improve the General physiological state of the insect. This is manifested in the fact that increases the life expectancy of bees, increases the mass of larvae and bees, and dramatically increases the viability of the whole bee family. In addition, antibiotic drugs stimulate the
physiological activity of bees. This is characterized by the fact that the bees increases in duration and intensity of the summer period, increased nectar load, enhanced ovipositor of the uterus. This in turn leads to the fact that there is a more increased growth of individual's bee family, increases and increases the productivity of bees receiving antibiotics. At the same time, antibiotics also have a preventive effect on the functional state of the insect's body, strengthening its resistance to diseases.
Various antibiotics are widely used in beekeeping to prevent and treat bacterial infections. However, the fact that to many of them the pathogens of bacterioses of bees began to show resistance pushes beekeepers to find more effective antibacterial agents. Among these tools is enrofloxacin, which is widely used in the treatment of animals, and now began to be used in beekeeping. On apiaries of Krasnodar Krai enrofloxacin is used in the form of 10% solution, which is introduced into sugar syrup at the rate of 10 ml per 1 liter and fed to bees three courses at intervals of 7 days in late February early March.
Materials and Methods
To determine the level of the antibiotic - enrofloxacin effect on the microbial background of the digestive system of the bee's body, the following studies were conducted. Created 2 groups of bees-experienced and control, 50 individuals in each of them. The antibiotic was given to experimental insects together with sugar syrup according to the method mentioned above. Individuals of the control group were given syrup without antibiotic. After eating the last tab of feeding, after 24 hours, bees studied the qualitative and quantitative composition of the contents of the posterior intestine.
To do this, live bees were put to sleep and from 10 individuals of each studied family were prepared the intestine, which was placed in a sterile bux, weighed, then carefully homogenized in a sterile porcelain mortar in 1% peptonic water in a ratio of 1:10, and prepared a series of successive 10-fold dilutions on 1% peptonic water with 0.1% agar agar.
From the obtained dilutions, with the help of a graduated pipette on the surface of well-dried selective culture media, crops in the amount of 0.025 ml were made in the form of 3 isolated drops. As selective culture media were used: Endo agar for the isolation of en-terobacteria, agar Kvasnikova with 6% ethanol for isolation of lactic acid bacteria, yolk-salt agar - for isolation of staphylococci, nutrient agar with potassium tellurite for the isolation of enterococci, TSPKH-agar -for the isolation of Pseudomonas and other non-fermenting bacteria, the author's environment (patent RF №2407783) - for isolation of microscopic fungi, environment Wilson-Blair for the isolation of Clostridium, Wednesday Blaurock with neomycin for selection of bifidobacteria. In addition, used blood agar for isolation of streptococci and mastopathy agar for isolation of Bacillus [4, c. 145; 5, c. 113].
Identification of the isolated bacteria was carried out by cultural, morphological, tinctorial and biochemical properties. We used biochemical test system of the company Pliva-Lachema Diagnostika: ENTEROtest,
Deferment, EN-Accutest, Anartist, Statetest. The species of bacteria and fungi was determined with the use of special directories, keys and manuals.
After 24-72 hours of incubation at 370S carried out the accounting of the grown colonies and calculated the number of microorganisms in 1 g of intestinal contents of bees according to the formula [1, c. 45]:
where: A - number of living microbial cells;
a1-a3 - the number of colonies grown in the 3 counted drop;
n - is the number of breeding;
4 - conversion factor;
p - linkage of the intestine (g).
The state of the intestinal tract of bees was assessed by the ball method [7, c. 130]:
I score - the intestinal wall is a very thin shell, easily torn, filled with watery, flake-like content, the intestine is easily torn when extracting it from the abdomen of the bee;
II score - the wall of the intestine is loose, filled with liquid, homogeneous, easily spreading excrements, it is possible to extract from the abdomen of the bee only part of the intestine;
III point - the intestine is removed completely, its walls are not torn, filled with weakly spreading homogeneous excrements;
IV point - the intestine fully retains its structure, is extracted easily and entirely, its wall is elastic, does not break, well holds the excrement, on the outside clearly visible muscles with trachea in the form of white threads. Excrement is formed and dense.
Reproductive ability and honey productivity of bee colonies were determined by methods recommended by the research Institute of beekeeping.
Honey productivity was determined by weighing on the postal scales of the frames before and after pumping honey. The difference in the weight of the frames was used to calculate the amount of pumped honey.
The results of the research were subjected to mathematical and biometric processing using a computer program Microsoft Excel 2010. With the help of this program we calculated the average values, arithmetic mean errors, values of the degree of reliability and degree of correlation, as well as converted the data into graphs and charts.
Resultats
The results of these studies are shown in table 1 h table 2. The materials in this table indicate that in the control group of bees, after feeding, the number of different representatives of the microbiota in the intestinal contents of increased by 0.6-1 lg CFU/g. To a greater extent in the intestinal tract of insects has increased the number of enterobacteria (up to 6,9+0,4 lg CFU/g), enterococci (up to 6,6+0,6 lg CFU/g) and yeast (to 6.5+0,6 lg CFU/g).
Table 1.
Qualitative and quantitative composition of the microflora of the hind intestine of the control group of _bees receiving sugar syrup with enrofloxacin (February-March)_
Microorganisms Number of microorganisms, lg CFU / g
Control group (n = 50)
To experience After the experience
Enterobacteria 5,9+0,6 6,9+0,4
Lactobacillus 4,3+0,5 5,0+0,5
Staphylococcus 5,4+0,4 6,3+0,3
Enterococcus 6,0+0,3 6,6+0,6
Pseudomonas 5,9+0,5 6,0+0,5
Yeast 5,7+0,1 6,5+0,6
Moldy mushrooms 1,1+0,1 1,9+0,2
Note: * - P<0.05 relative to control group and initial data
Table 2.
Qualitative and quantitative composition of the microflora of the posterior intestine of bees of the _experimental group receiving sugar syrup with enrofloxacin (February-March)_
Microorganisms Number of microorganisms, lg CFU / g
Experimental group (n = 50)
To experience After the experience
Enterobacteria 5,8+0,6 0,6+0,1
Lactobacillus 4,1+0,4 1,2+0,2
Staphylococcus 5,6+0,2 0,2+0,1
Enterococcus 5,9+0,3 4,7+0,3
Pseudomonas 5,7+0,5 0,3+0,1
Yeast 5,9+0,4 6,3+0,6
Moldy mushrooms 1,3+0,2 2,0+0,3
Note: * - P<0.05 relative to control group and initial data
In the experimental group of bees, on the contrary, the number of representatives of bacterial flora has decreased significantly, and the fungus, on the contrary, has increased. However, the parameters of reducing the amount of bacterial microflora for different representatives were different. More decreased (and in some months and disappeared completely from the intestine of bees), the number of enterobacteria, staphylococci and Pseudomonas, significantly decreased the number of lactobacilli (from 4.1+0.4 to 1,2+0,2 lg CFU/g). Quite a bit decreased the number of enterococci (from 5.9+0.3 to 4.7+0.3 lg CFU/g).
The quantitative presence of fungi in the intestines of bees, unlike bacteria, on the contrary increased by 0.4-0.7 lg CFU / g and became almost the same as in the bees of the control group.
Thus, our studies have shown that giving bees sugar feeding in the form of syrup without antibiotic promotes increased development in their digestive system of all kinds of microorganisms, and to the greatest extent this applies to enterobacteria, staphylococci, en-terococci and fungi. When entering into the sugar syrup of the antibiotic, enrofloxacin, there is a significant decrease in the development of bacterial microflora in the intestinal tract of bees (with the exception of enterococci). But at the same time, the amount of fungal flora remains almost at the same level.
Consequently, giving bee individuals sugar syrup without any antibacterial drugs can contribute to the development of various diseases in insects, the causative agents of which can be enterobacteria, and staphylo-cocci, enterococci and fungi. But with the introduction
of enrofloxacin into the syrup, it immediately decreases, and even excludes the possibility of bacterial diseases in bees. Also, the percentage of fungal diseases in bees increases, as it leads to the development of selective dysbacteriosis in the intestinal tract of insects with a predominance of fungal (especially yeast) mi-crobiota.
Thus, studies have shown that the use of carbohydrate feeding bees with enrofloxacin accompanied by the development of their intestinal tract selective dysbacteriosis, manifested almost complete disappearance of gram-positive and gram-negative bacteria and an increase of 0.4-0.7 lg CFU / g quantitative presence of yeast and fungi.
References
1. Bochkov, I. A. Simplified technique of counting micro-organisms in the study of the autoflora of the man // A. I. Bochkov, O. D. Trofimova, O. S. Darbaeva etc. / / Laboratory work. - 1989. - №6. - P. 43-47.
2. Litvinova, A. R. Microbiocenosis of the adult tract of bees / A. R. Litvinova, I. V. serdyuchenko, V. I. Terekhov, N. N. Gugushvili / / in the collection: Scientific support of agro-industrial complex. Collection of articles on the materials of the X all-Russian conference of young scientists dedicated to the 120th anniversary of I. S. Kosenko. OTV. for vol. A. G. Kashchayev. - 2017. - Pp. 235-236.
3. Oskin S. V. Electrotechnological methods of treatment of bee families from diseases / S. V. OS'kin,
D. A. Ovsyannikov, A. A. Blyagoz // Emergency situations: industrial and environmental safety. - 2017. - № 31 (3). - P. 97-106.
4. Terekhov V. I. Nutrient medium for cultivation of lactobacilli / V. I. Terekhov, I. N. Nasonova, I. V. serdyuchenko, T. V. Malysheva // Proceedings of the Kuban state agrarian UNIVERSITY. - 2014. - №49. -P. 144-146.
5. Terekhov, V. I., Selective nutrient medium for isolation of lactic acid bacteria / V. I. Terekhov, A. Y. Arushanyan, I. V. serdyuchenko, S. G. Glushchenko, T. V. Malysheva // Proceedings of the SFU. - 2014. - №47.
- Pp. 112-114.
6. Increase of winter hardiness and productivity of bee families by use of mineral additives in winter forage: dis. ... kand. s-x Sciences / L. Shagun; fish, 1983.
- 136 p.
