Microbiological screening of bacterial infections in Russian duck breeding enterprises

One of the factors for the development and spread of antibacterial resistance of bacteria is untimely or incorrectly performed laboratory diagnostics, which results in randomly prescribed antibacterial therapy aimed at containing the infection and ensuring the safety of the livestock. This phenomenon is common in many industrial poultry and livestock enterprises, but at the same time, it is difficult to blame the veterinary service of these farms, since they must ensure safety which sometimes cannot be achieved without antibiotics. The use of advanced microbiological screening of all microbiological risks to which enterprises are exposed is proposed as means of solving the indicated problem. The results of such work done in a number of duck breeding enterprises in Russia, in which at least 192 species of cultivated bacteria are circulated, are presented as an example. Most of the isolated microbes are representatives of the normal or transient flora, but a certain part of the bacteria has an obvious pathogenic or conditionally pathogenic role. From the selected bacteria, we emphasize the relevance of the following pathogens for the duck breeding industry: Bordetella avium, Campylobacter coli, Campylobacter concisus, Campylobacter jejuni, Clostridium colinum, Clostridium perfringens, Myroides odoratimimus, Pasteurella multocida, Riemerella anatipestifer, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcus entericus, Streptococcus gallolyticus, Shigella sonnei, which are typical pathogens of poultry infections. Information from the literature about the remaining bacteria were analyzed and structured in the form of a table with the description of the possible clinical and morphological manifestations of the diseases that they cause.


Introduction
The control of bacterial infections is one of the most important tasks for the veterinary service of any livestock or poultry enterprise. Against the background of modern realities, this control should focus not only on ensuring the safety and productivity of the livestock, but also on careful monitoring of the safety of manufactured products. Of course, the mechanism of this work has already been carefully established with respect to some epidemiologically significant pathogens of human infectious pathologies and their toxic infections, in particular, Salmonella sp., Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, but this control is not always enough [1][2][3]. The widespread and often unreasonable use of antibiotics in industrial poultry and other livestock industries contributes to the formation and spread of bacteria multi-resistance to antibiotics [4][5][6]. The consequences of this phenomenon are often published in news portals in the form of the reports of The last way, in our opinion, is so far more realistic in execution in comparison with the first two. Since vaccines against the large number of bacterial species have not been developed or registered and alternative to antibiotics preparations often cannot demonstrate the desired effect (for example, with respiratory and septic infections). That is why more advanced laboratory diagnostic methods or approaches to diagnosing bacterial infections make it possible to rationalize the tactics of antibacterial therapy during outbreaks of infections. This way has its drawbacks, in particular, a number of regulatory documents governing the conduct of certain studies are missing or existing regulations require updating. In the fight against bacterial pathologies, it is worth taking into account such important factors as the intensity of reproduction, as well as the introduction of infections due to newly imported livestock or breeding material. In this case, the quarantine of animals is mainly aimed at identifying especially dangerous diseases or infected animals, which during the furlough period will show clinical signs of pathology. But a number of factor infections will not be detected in this way, since for them there is a dependence on the presence of certain trigger mechanisms. In order to effectively deal with these problems, it is necessary to broaden the horizons of veterinary and laboratory specialists regarding the possible microbiological risks to which their enterprises are exposed [12]. The use of only routine bacteriological methods for diagnosis is often not enough to obtain the correct result, since these methods are limited. For example, identification of microorganisms with commercial biochemical kits is possible only for a small number of species, which in turn are primarily of medical importance. In addition, biochemical identification will not be effective in differentiating closely related bacterial species. The purpose of this investigation was to study the current epizootological situation, using the industrial duck breeding enterprises of Russia as an example, through expanded microbiological screening using modern diagnostic methods and approaches.

Materials and methods
The scientific work was performed in the period of 2016-2019 on the basis of Federal State Budget Scientific Institution "Federal Scientific Centre VIEV" (FSC VIEV).
To achieve this goal, 120 heads of birds under the age of 4 weeks (10 birds from each enterprise) from 12 duck breeding enterprises of various regions of Russia, namely the Krasnodar Krai, Altai Krai, the Republics of Bashkortostan, Chuvashia and Tatarstan, Orenburg, Rostov, Ryazan, Tambov, Astrakhan, Nizhny Novgorod and Kurgan regions, was subjected to a comprehensive laboratory diagnosis. The enterprises from which the birds were obtained for this study will remain anonymous. The bird was delivered to the laboratory as a whole carcass, and the autopsy and selection of pathological material was carried out already in place. For the study, the following samples were taken from each bird: intestine, liver, lungs, oviducts, heart. Campylobacter coli / 5 80,00 20,00 0,00 0,00 0,00 32.
Chryseobacterium endophyticum / 9 0,00 0   According to the data shown in table 1, the maximum number of bacterial species was isolated from samples of the studied liver, the value of this indicator was 158 species. A slightly smaller number of microorganisms species was isolated from the digestive system of birds -142 species. 109 types of bacteria were isolated from oviducts and 97 species from lung tissue. The smallest species composition was determined in the heart muscle, namely 68 species. It is worth noting here that as a result of the work done, it is possible to analyze data only on cultivated types of bacterial agents, therefore, this bacterial composition of various organs and tissues of the bird's organism cannot be considered complete, and for the full disclosure of the microbiome it is necessary to use molecular biological methods, in particular metagenomic analysis. For clarity, data on the excretion of various genera of bacterial agents isolated from different organs and tissues of waterfowl were reflected in the form of graphic figure 1. Figure 1 indicates that the main bacterial flora isolated from the studied samples turned out to be coccal microorganisms of the genera Staphylococcus, Enterococcus, Streptococcus, as well as enterobacteria Escherichia, spore-forming bacteria of the genus Clostridium, etc. Despite the high incidence of the isolation of these bacteria, we cannot confirm their participation in the development of infectious pathologies of birds, since most of the isolated bacteria species are representatives of normal or transient flora.
The full structure of the microbiome of various organs and tissues of the bird (by family), defined in the framework of the study, is shown in figures 2-6.   According to the data reflected in graphic figures 2-6, the following conclusions can be made: In the structural composition of bacterial agents located in the intestines of waterfowl, the Enterobacteriaceae family prevails, the percentage of which was 34.22 %; then, by the incidence of excretion, bacteria of the Staphylococcaceae family were found to be 11.56 %; anaerobic microorganisms of the Clostridiaceae family were in third place by excretion -9.06 %; the following place in the overall structure was occupied by microorganisms of the Enterococcaceae family -7.  In the structural composition of bacterial agents located in the liver of waterfowl, the Enterobacteriaceae family prevails, the percentage of which was 27.60 %; then, by the incidence of excretion, bacteria of the Staphylococcaceae family were found to be 10.88 %; anaerobic microorganisms of the Clostridiaceae family were in third place by excretion -8.99 %; the following place in the overall structure was occupied by microorganisms of the Enterococcaceae family -7.73 %; Streptococcaceae - 6 To summarize the data obtained during bacteriological screening and analysis of literature, we compiled a table describing the possible forms and signs of the manifestation of an infectious disease in birds, manifested by various types of microorganisms (table 2). The summary table shows information on the most significant bacterial pathogens for poultry farming.
As can be seen from the data shown in table 2, bacterial isolates are able to provoke the development of many different clinical signs of diseases, both in the form of monoinfection and associated pathologies.

Conclusion
The obtained results indicate that the use of modern laboratory diagnostic tools such as MALDI-ToF mass spectrometric analysis allows microorganisms to be identified with high reliability, thereby increasing the effectiveness of subsequent treatment and prophylactic measures. The use of exclusively routine methods of bacteriology is not enough in the fight against the development and spread of antibiotic resistance. Thanks to a comprehensive laboratory diagnosis and the use of modern means of identification, we were able to discover wide range of microorganisms circulating in duck breeding enterprises of Russia. All types of bacteria that we have identified can be divided into various functional groups, such as: 1) the causative agents of the infectious pathologies of birds, including waterfowl: Bordetella avium, Campylobacter coli, Campylobacter jejuni, Clostridium colinum, Clostridium perfringens, Myroides odoratimimus, Pasteurella multocida, Riemerella anatipestifer, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcus entericus, Streptococcus gallolyticus, Shigella sonnei [13][14][15][16][17]. Some of these species are also causative agents of human toxic infections; 2) pathogens of nosocomial infections in humans, which include bacteria of the genera Acinetobacter, Pseudomonas, Klebsiella, Staphylococcus -which are most often multiresistant; 3) opportunistic and transient microorganismsthe remaining species. Of course, this gradation is not universal, and in each individual case it is necessary to deal with it individually, including through the use of the data shown in table 2, where it is clearly seen that the same clinical manifestations of infections can be provoked by many types of bacteria. Thus, we