CC BY
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Original article
UDK 639.3.09+ 579.832/.833 (631.463) https://doi.org/10.24143/2073-5529-2023-1-89-97 EDN DFMKGF
Bacteria of genus Bacillus as antagonists of pathogens in aquaculture
M. A. Morozova 1, A. V. Gorovtsov2^, E. V. Prazdnova3, V. M. Basankina4, V. A. Chistyakov5, А. Pepoyan6, Sh. Miralimova7, V. A. Grigoryev8
1Rostov Scientific Research Institute of Microbiology and Parasitology; Don State Technical University,
Rostov-on-Don, Russia
2,3,5Southern Federal University, Rostov-on-Don, Russia, gorovtsov@gmail.comM
4Krasnodar Interregional Veterinary Laboratory, Krasnodar, Russia
6National Agrarian University of Armenia, Yerevan, Armenia
7Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan,
Tashkent, Uzbekistan
8Astrakhan State Technical University, Astrakhan, Russia
Abstract. Due to the increasing resistance of aquaculture pathogens to antibiotics, the search for new approaches to the control of infectious diseases is of particular importance. A promising approach is the development of probi-otic preparations based on antagonist bacteria. The paper studied the antagonistic activity of Bacillus strains that are promising for developing probiotics for fish. The strains were isolated from the bottom sediments of the Don River in the area of the Donskoy Fish Reserve, as well as from the intestines of cyprinids of natural populations, namely, from silver carp (Cyprinus gibelio), roach (Rutilus heckelii), and bream (Abramis brama). The inhibitory effect of Bacillus strains against causative agents of aeromonosis, pseudomonosis and bacterial hemorrhagic septicemia was studied by the method of delayed antagonism. Aeromonas and Pseudomonas species pathogenic for fish were isolated from sturgeons, cyprinids and salmons grown in pond farms and recirculating aquaculture systems in the Krasnodar Region, Rostov Region and the Republic of Adygea. It has been shown that sensitivity to the action of bacilli is not only a species, but also a strain-specific trait among bacterial pathogens of fish. Five strains were identified that exhibited the maximum antagonistic activity against the studied pathogens, and their species identification was carried out by molecular biological and mass spectrometric methods. It has been established that the most pronounced antagonism is exhibited by strains of bacilli isolated from bottom sediments. The growth of all studied strains of fish pathogenic species of Pseudomonas was suppressed only the B. subtilis R4 strain. Thus, strains of aerobic spore-forming bacteria suitable for the creation of probiotic preparations for aquaculture objects have been isolated and characterized.
Keywords: aeromonosis, probiotics, fish diseases, antagonist bacilli, aeromonads, pseudomonads, aquaculture object
Acknowledgment: the study was supported by a grant of Russian Foundation of Basic Research №20-516-81004.
For citation: Morozova M. A., Gorovtsov A. V., Prazdnova E. V., Basankina V. M., Chistyakov V. A., Pepoyan А., Miralimova Sh., Grigoryev V. A. Bacteria of genus Bacillus as antagonists of pathogenes in aquaculture. Vestnik of Astrakhan State Technical University. Series: Fishing Industry. 2023;1:89-97. (In Russ.). https://doi.org/10.24143/2073-5529-2023-1-89-97. EDN DFMKGF.
© Morozova M. A., Gorovtsov A. V., Prazdnova E. V., Basankina V. M., Chistyakov V. A., Pepoyan A, Miralimova Sh., Grigoryev V. A., 2023
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Научная статья
Бактерии р. Bacillus - антагонисты патогенов аквакультуры
М. А. Морозова1, А. В. Горовцов2ш, Е. В. Празднова3, В. М. Басанкина4, В. А. Чистяков5, А. Пепоян6, Ш. Миралимова7, В. А. Григорьев8
1Ростовский НИИ микробиологии и паразитологии; Донской государственный технический университет
Ростов-на-Дону, Россия
2,3,5Южный федеральный университет, Ростов-на-Дону, Россия, gorovtsov@gmail.comB
4Краснодарская межобластная ветеринарная лаборатория, Краснодар, Россия
6Национальный аграрный университет Армении, Ереван, Армения
7Институт микробиологии Академии наук Республики Узбекистан, Ташкент, Узбекистан
8Астраханский государственный технический университет, Астрахань, Россия
Аннотация. На фоне возрастающей резистентности патогенов аквакультуры к антибиотикам особое значение приобретает поиск новых подходов к контролю инфекционных заболеваний. Перспективным подходом пред-Э ставляется разработка пробиотических препаратов на основе бактерий-антагонистов. Изучена антагонистиче-g ская активность бактерий р. Bacillus, перспективных для создания пробиотиков для рыб. Штаммы выделяли § из донных отложений р. Дон в районе Донского рыбного заповедника, а также из кишечника карповых рыб S природных популяций, а именно от серебряного карася (Cyprinus gibelio), тарани (Rutilus heckelii), леща ^ (Abramis brama). Ингибирующее действие бактерий р. Bacillus в отношении возбудителей аэромоноза, псев-^ домоноза и бактериальной геморрагической септицемии исследовали методом отсроченного антагонизма. Паи тогенные для рыб бактерии р. Aeromonas и р. Pseudomonas были выделены от осетровых, карповых и лососе-g вых рыб, выращиваемых в условиях прудовых хозяйств и установках замкнутого водоснабжения Краснодарец ского края, Ростовской области и Республики Адыгея. Показано, что чувствительность к действию бацилл яв-^ ляется не только видовым, но и штамм-специфичным признаком среди бактериальных патогенов рыб. Выде-щ лено 5 штаммов, проявляющих максимальную антагонистическую активность в отношении исследуемых па-g тогенов, проведена их видовая идентификация молекулярно-биологическим и масс-спектрометрическим ме-£ тодами. Установлено, что наиболее выраженный антагонизм проявляют штаммы бацилл, выделенные из дон-я ных отложений. Рост всех изученных штаммов патогенных для рыб видов р. Pseudomonas подавлял только . штамм B. subtilis R4. Таким образом, выделены и охарактеризованы штаммы аэробных спорообразующих бак-
^ терий, подходящие для создания на их основе пробиотических препаратов для объектов аквакультуры. и
| Ключевые слова: аэромоноз, пробиотики, заболевания рыб, бациллы-антагонисты, аэромонады, псевдомона-
g ды, объект аквакультуры
о
w Благодарности: исследование выполнено при поддержке гранта РФФИ № 20-516-81004.
® Для цитирования: Морозова М. А., Горовцов А. В., Празднова Е. В., Басанкина В. М., Чистяков В. А., Пепоян
g А., Миралимова Ш., Григорьев В. А. Бактерии р. Bacillus - антагонисты патогенов аквакультуры // Вестник
§ Астраханского государственного технического университета. Серия: Рыбное хозяйство. 2023. № 1. С. 89-97. https://doi.org/10.24143/2073-5529-2023-1-89-97. EDN DFMKGF.
Introduction of imported aquaculture objects. In addition, a high
In modern conditions, aquaculture is developing in risk of morbidity is caused by the environmental prob-
several directions, which have significant differences. lems due to the unsatisfactory quality of the aquatic
These are pasture, pond and industrial aquaculture (in environment, especially in cage farms concentrated in
pools, in recirculating aquaculture systems, in fish coastal zones and in freshwater reservoirs, which are
farms using cages), as well as artificial reproduction more often exposed to anthropogenic pollution. including sturgeon, salmon hatcheries and spawning As a result, the resistance of the fish organism
farms. For each of these directions the risk of diseases sharply decreases, which causes diseases from the op-
in breeding objects is quite high [1]. Diseases can be portunistic bacteria that constantly circulate in the
associated with a violation of biotechnological, sani- aquatic environment [2, 3]. tary standards for growing and quarantine measures
Bacterial diseases all over the world cause significant economic damage to fish farms, both due to fish death, the need for quarantine measures, and from a decrease in the consumer attractiveness of raw materials and, accordingly, the cost of fish. Bacterial diseases of fish in ponds and farms in the Russian Federation rank second after the parasitic ones among infectious diseases. The most common are aeromonosis of salmon and cyprinid fish species, pseudomonosis of carp, myxobacteriosis of sturgeon and trout fish species [4, 5]. For the period 2014-2018 80.55% of all detected outbreaks of diseases of bacterial etiology accounted for aeromonoses of cyprinids and salmon fish [4]. It should be noted that cyprinid and salmon aeromonoses are included in the list of especially dangerous quarantine diseases [6]. In addition, many fish diseases (vibriosis, aeromonosis, pseudomonosis, citrobacteriosis) are characterized by natural foci [7]. Certain environmental factors are favorable for the persistence of certain types of pathogens. The most common pathogens for the water bodies of the North Caucasus and the southern region as a whole are aer-omonads. It should be noted that bacteria of the genus Aeromonas are constantly present in the aquatic environment, and their epizootic significance is determined by abundance and virulence [8]. Mild climatic conditions contribute to the long-term persistence of virulent aeromonads in the aquatic environment. A major factor for the high incidence of aeromonosis in fish is an increase in water temperature above 14°C. At the same time, the percentage of death (10-100%) depends on the conditions of detention and the load per unit area of the reservoir of each particular farm. Outbreaks of aeromonosis can occur in the form of epizootics, affecting species of all age groups. However, in the acute form, the disease is more often recorded in fry and yearlings of sturgeon, salmon and some species of cyprinids (crucian carp, grass carp, mirror carp) [4]. For the Rostov region, cyprinid aeromonosis is the most dangerous because it spreads not only in pond farms, but also in natural reservoirs. Aeromonosis cases were recorded in the Don, Bolshaya Kalancha, Mius and Aksai rivers. A growing number of aeromonads in natural water bodies, especially strains with pathogenicity factors in the summer-autumn period, increases the risks for the development of infection or colonization of aquatic organisms by these bacteria [8]. Pseudomonas bacteria are the causative agents of pseudomonosis and this disease is rather rarely detected in aquaculture objects in the farms of the southern region. However, recently the incidence of diseases, caused by the associations of gram-negative microorganisms (aeromonads, pseudomonads, enterobacteria, etc.) has increased. In particular, a more severe course of the disease is observed and an acute form of the disease develops more often if the clinical signs of aeromonosis are accompanied by the presence of pseudomonads, enterobacteria, moraxella and other opportunistic bacteria [4]. In this
case, it is difficult to choose effective drugs to combat the disease.
The existing complex of veterinary-sanitary and fish-breeding and reclamation measures cannot fully provide protection against bacterial infections in aquaculture objects. This is largely due to the resistance of pathogens to drugs used in fish farming.
The use of probiotics based on spore-forming bacteria of the Bacillus genus is a modern trend in the prevention and treatment of infectious diseases in aquaculture. The presence of probiotic effects in spore-forming microorganisms has led to the development of preparations based on them, belonging to the group of "self-eliminating antagonists". Probiotic preparations offered on the market differ not only in price, but also in composition, quality, method and dose of application. Bacillus based probiotics are suitable candidates for the development of preparations for use in aquaculture. They exhibit antagonistic activity against pathogenic microorganisms and are also nontoxic to fish [9, 10]. At the same time, the ability of probiotic bacilli to manifest antagonism is a rather variable trait and depends predominantly not on the species, but on the spectrum of antimicrobial metabolites secreted by a particular strain [11]. Therefore, to combat fish diseases, it is necessary to find species-specific bacterial antagonists. In addition, it is important to consider their ability to multiply effectively and exhibit probi-otic properties under suboptimal temperature conditions. The effectiveness of the prevention and treatment of bacterial diseases in aquaculture objects is associated with the development of new probiotics, including those based on spore-forming bacteria. This approach will reduce the economic damage from the death of aquaculture objects and reduce the prevalence of antibiotic-resistant strains of pathogens.
In this regard, the aim of the work was to search for new natural probiotic Bacillus strains and evaluation of their antagonistic activity against pathogens of bacterial diseases relevant to aquaculture objects (pseudomonosis, aeromonosis, bacterial hemorrhagic septicemia) by the method of co-cultivation.
Materials and methods
The object of the study were Bacillus strains isolated from silver carp (Cyprinus gibelio), roach (Rutilus heckelii), bream (Abramis brama) as well as from bottom sediments of the lower reaches of the Don River in the area of the Donskoy Fish Reserve.
Inoculation of bacteria from bottom sediments and imprints of samples of fish gills and intestines was carried out on wort-agar. The species were identified by MALDI-TOF MS (matrix-associated laser desorp-tion/ionization time-of-flight mass spectrometry) on an Autoflex speed III device with Biotyper software (Bruker Daltoniss, Germany). For more precise species identification, DNA was isolated from bacterial cells and the 16S rRNA gene was sequenced. The conditions for DNA isolation and purification, as well as for
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sequencing, are similar to those described in our previous works [12].
The strains with no hemolytic and DNAse activity were included in the study.
7 species of bacteria were used as test cultures. Species of Aeromonas genus - Aeromonas salmonicida (7 strains), Aeromonas veronii (30), Aeromonas caviae (14), Aeromonas eucrenophila (4), Aeromonas ichthi-osmia (3), Aeromonas bestiarum (3), Aeromonas hy-
drophila (5), of which 14 are deposited in collections of microorganisms of Federal State Budgetary Institution "The Russian State Center for Animal Feed and Drug Standardization and Quality" (FGBU "VGNKI") (Table 1); 3 species of Pseudomonas genus - P. putida (2 strains), P. fuorescens and P. aureofaciens by 1 strain from each were also used to assess the antagonistic activity of bacilli.
Table 1
Deposited test strains of aeromonads from the collection of microorganisms of the FGBU "VGNKI"
Bacterial species of Aeromonas genus Registration number at depositing Fish species / sampling area
Aeromonas caviae VKSHM-B-297M Acipenseridae, Seversky district of the Krasnodar Region
VKSHM-B-300M Sterlet, Krasnodar
Aeromonas veronii VKSHM-B-296M Cyprinids, Dinskoy district of the Krasnodar Region
VKSHM-B-299M Cyprinids, Krasnodar
VKSHM-B-305M Cyprinids, Adygea
VKSHM-B-302M Cyprinids, Krasnodar
Aeromonas salmonicida VKSHM-B-295M Koi carp, Krasnodar
VKSHM-B-293M Russian sturgeon, village Starominskaya, the Krasnodar Region
VKSHM-B-307M Acipenseridae, village Bryukhovetskaya, the Krasnodar Region
Aeromonas eucrenophila VKSHM-B-294M Acipenseridae, village Starominskaya, the Krasnodar Region
VKSHM-B-298M Acipenseridae, Korenovsky district of the Krasnodar Region
Aeromonas ichthiosmia VKSHM-B-303M Cyprinids, Dinskoy district of the Krasnodar Region
VKSHM-B-304M Acipenseridae, village Starominskaya the Krasnodar Region
VKSHM-B-306M Cyprinids, Shcherbinovsky district of the Krasnodar Region
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Strains of Aeromonas and Pseudomonas were isolated from parenchymal organs and skin ulcers of fish with bacterial hemorrhagic septicemia and aeromonosis. The study included fish from 3 families: cyprinids (Cy-prinidae) - common carp (Cyprinus carpio), koi carp (Cyprinus carpio haematopterus), silver carp (Hy-pophthalmichthys molitrix), grass carp (Ctenopharyn-godon idella); sturgeons (Acipenseridae) - Russian sturgeon (Acipenser gueldenstaedtii), sterlet (Acipenser ruthenus), stellate sturgeon (Acipenser stellatus); salm-onids (Salmonidae) - rainbow trout (Salmo gairdneri irideus) and brook trout (Salmo trutta morpha fario) grown in pond farms and recirculating aquaculture system in the Krasnodar Region, Rostov Region, Republic of Adygea. Pathological anatomical autopsy, primary bacteriological inoculation of samples of fish organs and tissues, and isolation of bacteria were carried out in accordance with regulatory documents [13, 14].
To assess the antagonistic activity of Bacillus strains the method of delayed antagonism was used (method of perpendicular streaking) in accordance with MUK 4.2.2602-10.
Inoculation of antagonist strains on the rich nutrient medium (nutrient agar is used for cultivating microorganisms) was carried out using a loop with a diameter of (3.5 ± 0.5 mm) with a straight streaking along the diameter of a Petri dish. It was incubated for 3 days, after which a suspension of the overnight test culture (not less than 109 CFU/ml) was inoculated perpendicularly to the grown streak of the antagonist strain using a loop with a diameter of 2 mm. The cultures were incubated for 24 h at a temperature of (26 ± 1°C), followed by measurement of the growth inhibition zone of the test cultures.
Results and discussion
As a result of the screening of 28 strains, 5 promising strains of Bacillus were selected, of which 3 strains (R1, R4, R5) were isolated from cyprinids of natural populations and 2 strains (G5, G6) from bottom sediments.
The results of mass spectrometric analysis showed that strains R4, G5, G6 belong to Bacillus subtilis, R1 to Bacillus mojavensis, R5 to Bacillus sp. To clarify the species identification, an analysis of the 16S rRNA
gene was carried out, as a result of which strain R5 belonged to the species Bacillus velezensis.
It was shown that the level of antagonistic activity of Bacillus strains varies not only in relation to the
species of aeromonads, but also in relation to the strains of these species (Table 2).
Table 2
Species composition of aeromonad strains sensitive to antagonist bacilli
Strain Bacillus species Source of Bacillus strains isolation Antagonism towards aeromonad test strains Proportion of test strains sensitive to antagonist bacilli, %
R1 B. mojavensis Intestines of carp fish A. salmonicida 28
A. eucrenophila 62
R4 B. subtilis Intestines of carp fish A. salmonicida 28
A. caviae 100
A. eucrenophila 100
A. hydrophila 80
A. bestiarum 33
R5 B. velezensis Intestines of carp fish A. salmonicida 14
A. caviae 100
A. hydrophila 100
G5 B. subtilis Sediments A. salmonicida 86
A. eucrenophila 100
G6 B. subtilis Sediments A. salmonicida 86
A. caviae 100
A. eucrenophila 100
A. bestiarum 100
A. hydrophila 100
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Thus, the antagonistic activity of bacilli differed significantly against the strains of immobile aeromon-ads Aeromonas salmonicida. The most pronounced antagonistic activity was found in bacilli isolated from the bottom sediments (more than 35 mm). Antagonistic bacilli isolated from fish exhibited a strong inhibitory effect only against 2 deposited strains (growth inhibition zone > 35 mm), the remaining 5 test cultures of aeromonads had a weak response (growth inhibition zone from 1 to 3 mm).
During co-cultivation of bacilli with motile aer-omonads A. eucrenophila, A. bestiarum, and A. ichthi-osmia, their growth inhibition zones varied widely. All antagonist bacilli (except B. velezensis R5) showed a good inhibitory effect on A. eucrenophila test cultures (more than 35 mm). In relation to cultures of A. besti-arum, the effect of bacilli differed significantly (zones of growth inhibition from 1 to 35 mm). At the same time, strains of Bacillus subtilis R3 and G6 (more than 35 mm) showed the greatest antagonistic activity, while B. subtilis R4 and B. velezensis R5 inhibited the growth of the tested cultures to a lesser extent (growth inhibition zone 3-5 mm). The weakest antagonistic activity of bacilli was noted against A. ichtiosmia (from 1 to 3 mm).
It is known that mobile species A. caviae and A. hydrophila are most often found as causative agents
of aeromonosis and bacterial hemorrhagic septicemia. The inhibitory effect of bacilli to 5 cultures of A. hydrophila and 14 test cultures of A. caviae showed significant differences in the severity of antagonism (growth inhibition zones from 1 to 35 mm). However, the most effective antagonists were bacterial Bacillus strains R3, R4, R5, G6.
The inhibitory effect of 4 strains of antagonist bacilli against pathogenic for fish Pseudomonas species was studied. 3 species of Pseudomonas (P. putida, P. fluorescens, P. aureofaciens) isolated from fish with bacterial hemorrhagic septicemia were used as test cultures. It was found that most of the tested antagonist bacilli (B. mojavensis R1, B. velezensis R5) did not show a pronounced inhibitory effect on P. fluorescens and P. aureofaciens species. The growth of Pseudomonas test cultures was suppressed only by B. subtilis R4. Although P. putida strains differed in a number of biochemical properties and were isolated from different fish species (trout and Russian sturgeon), the antagonistic effect of bacilli against these strains was identical. B. subtilis R4 and Bacillus velezensis R5 strains suppressed the growth of P. putida test cultures. The results of the antagonistic activity of bacilli against Pseudomonas are presented in Table 3.
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Table 3
Evaluation of the antagonistic activity of Bacillus bacteria against strains of Pseudomonas species pathogenic for fish
——^Test strains of pseudomonads Bacteria Bacillus ——^^ Zone of Pseudomonas bacteria growth inhibition, mm
P. putida 100 P. putida 101 P fluorescens 75 P. aureofaciens 107
R1 B. mojavensis absent* absent not defined absent
R4 B. subtilis 10 10 more than 40 5
R5 Bacillus velezensis over 40 over 40 20 absent
Zone of growth inhibition is absent.
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The genus Bacillus is one of the most commonly used genera of probiotics in aquaculture due to its ability to produce bacteriocins, influence the growth rates, host immune system and resistance to pathogens [15].
It has been shown that in vivo conditions B. sub-tilis, B. velezensis, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus thuringiensis and Bacillus aeri-us increase the resistance of aquaculture objects to pathogenic bacteria, including Streptococcus, Aer-omonas, Vibrio, Enterococcus and Lactococcus [16]. Representatives of the genus Bacillus are a promising group for searching the new bacteria that inhibit quorum sensing in pathogens [17].
In addition, probiotics based on bacteria of the genus Bacillus, in particular B. subtilis and B. licheni-formis, can be used to purify water and bottom sediments in fish ponds by removing toxic substances, such as ammonia, nitrites, nitrates and carbon dioxide, as well as by competing with opportunistic bacterial species [15, 18].
Bacillus strains also have a beneficial effect on the microbiome of the gastrointestinal tract of aquaculture objects, which leads to improved feed conversion and accelerates the growth of aquatic organisms [16]. Their mechanisms of action, however, are still the subject of active discussion and study.
Probiotic strains of bacilli affect the process of intestinal colonization by other types of microorganisms, including pathogens, due to the mechanisms of both direct competition and influence on the adhesion process [15, 16].
Among the specific properties of bacillary probiot-ics, the immunostimulating properties are also worth mentioning. Thus, it was demonstrated that bacilli can modulate the mechanisms of the innate immune response of fish and molluscs, for example, phagocytic and lysozyme, antiprotease and peroxidase, superoxide dismutase and myeloperoxidase activities, and respiratory burst [15, 19]. In addition, probiotics based on Bacillus strains can cause changes in animal cell physiology, in particular, affect neutrophil migration, plasma bacte-
ricidal activity and increase the ability of neutrophils to attach, which ultimately can lead to an improvement in the immune response, for example, an increase in complement activity, immunoglobulin production in the gut-associated lymphoid fish tissues [16, 19-21].
Spore-forming bacteria have the ability to synthesize a wide range of compounds that inhibit the growth of other microorganisms. In particular, B. velezensis was shown to be capable of synthesizing bacilizin, which shows activity against opportunistic Gram-negative bacteria [22]. The ability to synthesize a wide range of biologically active compounds, including peptides, lipopeptides, siderophores, etc., makes it possible to effectively use probiotics based on Bacillus strains against bacteria pathogenic for aquaculture objects, such as representatives of Aeromonas, Vibrio, Pseudomonas, and others [23]. In particular, it has recently been shown that the B. coagulans strain had a pronounced antagonistic effect against P. aeruginosa isolated from diseased carps and also reduced the death of fish in the in vivo experiment [24]. Similar data were obtained for B. velezensis WLY23 which effectively suppressed 20 fish pathogenic bacteria, including A. schubertii, A. jandaei, A. hydrophila, A. veronii, A. aquariorum, P. shigelloides, N. seriolae, S. agalactiae and S. iniae [25]. It should be noted that in our study, the B. velezensis R5 strain was also characterized by the widest spectrum of antimicrobial activity.
Conclusion
Thus, the conducted studies make it possible to identify a group of natural strains of bacilli with antagonistic activity against aeromonads and pseudo-monads pathogenic for fish, which may be promising for developing the probiotic preparations for aquaculture. It has been found that sensitivity to the antagonistic action of Bacillus is not only a species, but also a strain trait among bacterial pathogens of fish.
The strains selected in the course of this work can be used to develop drugs that are active against pathogens which are of current importance for aquaculture.
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6. Ob utverzhdenii perechnia zaraznykh, v tom chisle osobo opasnykh, boleznei zhivotnykh, po kotorym mogut ustanavlivat'sia ogranichitel'nye meropriiatiia (karantin): prikaz Minselkhoza Rossii № 476 ot 19 dekabria 2011 g. [On approval of the list of contagious, including especially dangerous, animal diseases, for which restrictive measures (quarantine) can be established: order of the Ministry of Agriculture of Russia No. 476 dated December 19, 2011]. Available at: https://rsnadzor.ru/fperechen_476.pdf (accessed: 16.02.2022).
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9. Abrosimova N. A., Abrosimova K. S., Abrosimova E. B., Morozova M. A. Kormovoe syr'e i biologicheski aktivnye dobavki dlia rybnykh ob"ektov akvakul'tury: uchebnoe posobie [Feed raw materials and biologically active additives for aquaculture fish objects: textbook]. Saint-Petersburg, Lan' Publ., 2019. 151 p.
10. Golovko G. V., Chistiakov V. A., Sazykina M. A, Zipel't L. I., Kolenko M. A., Satarov V. V., Shipilo V. Iu. Ispol'zovanie probioticheskoi dobavki na osnove Bacillus subtilis «V-1895» v akvakul'ture [Use of probiotic supplement based on Bacillus subtilis "V-1895" in aquaculture]. Rybnoe khoziaistvo, 2009, no. 5, pp. 60-64.
11. Morozova M. A., D'iachenko, M. A., Chistiakov, V. A., Parkhomenko Iu. O., Stepanova Iu. V. Otsenka chu-vstvitel'nosti aeromonad k antibakterial'nym preparatam i sporovym probiotikam [Evaluation of sensitivity of aeromonads to antibacterial drugs and spore probiotics]. Ak-tual'nye voprosy rybolovstva, rybovodstva (akvakul'tury) i ekologicheskogo monitoringa vodnykh ekosistem: materialy Mezhdunarodnoi nauchno-prakticheskoi konferentsii, posvi-ashchennoi 90-letiiu Azovskogo nauchno-issledovatel'skogo instituta rybnogo khoziaistva (Rostov-na-Donu, 11-12 dekabria 2018 g.). Rostov-on-Don, Izd-vo Azov. nauch.-issledovat. in-ta ryb. khoz-va, 2018. Pp. 70-75.
12. Gorovtsov A. V., Vasilchenko N. G., Prazdnova E. V., Chistyakov V. A., Kukharenko L. E. The influence of soil type and preceding crop on the suppression of fusarium by indigenous spore-forming bacteria. Periodico Tche Quimica,
2019, vol. 16, no. 33, pp. 225-240.
13. Instruktsiia o meropriiatiiakh po bor'be s aeromo-nozom karpovykh ryb (utv. Minsel'khozprodom RF17 avgus-ta 1998 g. № 13-4-2/1366) [Instruction on measures to combat aeromonosis of cyprinids (approved by the Ministry of Agriculture and Food of the Russian Federation on Au-
gust 17, 1998 No. 13-4-2 / 1366)]. Available at: https://sv. yanao.ru/documents/active/22710/ (accessed: 24.01.2022).
14. Metodicheskie ukazaniia po laboratornoi diagnostike psevdomonoza ryb MU № 13-4-2/1403 ot 22 sentiabria 1998 g. (utv. Departamentom veterinarii Ministerstva sel'skogo khoziaistva Rossiiskoi Federatsii) [Guidelines for laboratory diagnosis of fish pseudomonosis MU No. 13-4-2 / 1403 of September 22, 1998 (approved by the Department of Veterinary Medicine of the Ministry of Agriculture of the Russian Federation)]. Available at: https://e-ecolog.ru/docs/zh49kSe 1HNrKqhT4CwedP (accessed: 24.01.2022).
15. Soltani M., Roy S., Lymbery A., Kumar V., Ringoe E. Genus Bacillus, promising probiotics in aquaculture: Aquatic animal origin, bio-active components, bioremedia-tion and efficacy in fish and shellfish. Reviews in Fisheries Science & Aquaculture, 2019, vol. 27 (3), pp. 331-379.
16. Ringoe E., Soon Ho Lee, Hoseinifar S. H., Harikrishnan R., Hien Van Doan, Soltani M., Seong Song. Probiotics, lactic acid bacteria and bacilli: interesting supplementation for aquaculture. Journal of Applied Microbiology, 2020, vol. 129, no. 1, pp. 116-136.
17. Chen B., Peng M., Tong W., Zhang Q., Songet Z. The quorum quenching bacterium Bacillus licheniformis T-1 protects zebrafish against Aeromonas hydrophila infection. Probiotics Antimicrob Proteins, 2019, vol. 12 (1), pp. 160-171.
18. Kewcharoen W., Srisapoome P. Probiotic effects of Bacillus spp. from Pacific white shrimp (Litopenaeus van-namei) on water quality and shrimp growth, immune responses, and resistance to Vibrio parahaemolyticus (AHPND strains). Fish Shellfish Immunol., 2019, vol. 94, pp. 175-189.
19. Yi Y., Zhang Z., Zhao F., Liu H., Yu L., Zha J., Wang G. Probiotic potential of Bacillus velezensis JW: Antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus. Fish Shellfish Immunol., 2018, vol. 78, pp. 322-330.
20. Zhou S., Song D., Zhou X., Mao X., Zhou X., Wang S., Wei J., Huang Y., Wang W., Xiao Su-M., Qin Q. Characterization of Bacillus subtilis from gastrointestinal tract of hybrid Hulong grouper (Epinephelus_ fuscoguttatus x E. lanceo-latus) and its effects as probiotic additives. Fish Shellfish Immunol., 2018, vol. 84, pp. 1115-1124.
21. Di J., Chu Z., Zhang S., Huang J. Evaluation of the potential probiotic Bacillus subtilis isolated from two ancient sturgeons on growth performance, serum immunity and disease resistance of Acipenser dabryanus. Fish Shellfish Immunol, 2019, vol. 93, pp. 711-719.
22. Nannan C., Vu H. Q., Gillis A., Caulier S., Nguyen Thuy T. T., Mahillon J. Bacilysin within the Bacillus subtilis group: Gene prevalence versus antagonistic activity against Gram-negative foodborne pathogens. Journal of Biotechnology, 2021, vol. 327, pp. 28-35.
23. Van Doan H., Soltani M., Ringoe E. In vitro antagonistic effect and in vivo protective efficacy of Gram-positive probiotics versus Gram-negative bacterial pathogens in fin-fish and shellfish. Aquaculture, 2021, vol. 540, pp. 736-581.
24. Ji T., Cao Y., Cao Q., Zhang Y., Yang H. The antagonistic effect and protective efficacy of gram-positive probiotics Bacillus coagulans to newly identified pathogens Pseudomonas aeruginosa in crucian carp Carassius auratus gibelio. Aquaculture Reports, 2022, vol. 24, pp. 101-126.
25. Zhang D. F., Xiong X. L., Wang Y. J., Gao Y. X., Ren Y., Wang Q., Shi C. B. Bacillus velezensis WLYS23 strain possesses antagonistic activity against hybrid snake-head bacterial pathogens. Journal of Applied Microbiology, 2021, vol. 131 (6), pp. 3056-3068.
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Список источников
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6. Об утверждении перечня заразных, в том числе особо опасных, болезней животных, по которым могут устанавливаться ограничительные мероприятия (карантин): приказ Минсельхоза России № 476 от 19 декабря 2011 г. URL: https://rsnadzor.rU/f/perechen_476.pdf (дата обращения: 16.02.2022).
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10. Головко Г. В., Чистяков В. А., Сазыкина М. А, Зи-пельт Л. И., Коленко М. А., Сатаров В. В., Шипило В. Ю. Использование пробиотической добавки на основе Bacillus subtilis «В-1895» в аквакультуре // Рыбное хозяйство. 2009. № 5. С. 60-64.
11. Морозова М. А., Дьяченко, М. А., Чистяков, В. А., Пархоменко Ю. О., Степанова Ю. В. Оценка чувствительности аэромонад к антибактериальным препаратам и споровым пробиотикам // Актуальные вопросы рыболовства, рыбоводства (аквакультуры) и экологического мониторинга водных экосистем: материалы Междунар. науч.-практич. конф., посвящ. 90-летию Азов. науч.-ис-следоват. ин-та рыб. хоз-ва (Ростов-на-Дону, 11-12 декабря 2018 г.). Ростов н/Д.: Изд-во Азов. науч.-иссле-доват. ин-та рыб. хоз-ва, 2018. С. 70-75.
12. Gorovtsov A. V., Vasilchenko N. G., Prazdnova E. V., Chistyakov V. A., Kukharenko L. E. The influence of soil type and preceding crop on the suppression of fusarium by indigenous spore-forming bacteria // Periodico Tche Quimica. 2019. V. 16. N. 33. Р. 225-240.
13. Инструкция о мероприятиях по борьбе с аэромо-нозом карповых рыб (утв. Минсельхозпродом РФ 17 августа 1998 г. № 13-4-2/1366). URL: https://sv.yanao.ru/ documents/active/22710/ (дата обращения: 24.01.2022).
14. Методические указания по лабораторной диагностике псевдомоноза рыб МУ № 13-4-2/1403 от 22 сентября 1998 г. (утв. Департаментом ветеринарии Министерства сельского хозяйства Российской Федерации). URL: https://e-ecolog.ru/docs/zh49kSe1HNrKqhT4CwedP (дата обращения: 24.01.2022).
15. Soltani M., Roy S., Lymbery A., Kumar V., Ringoe E. Genus Bacillus, promising probiotics in aquaculture: Aquatic animal origin, bio-active components, bioremediation and efficacy in fish and shellfish // Reviews in Fisheries Science & Aquaculture. 2019. V. 27 (3). Р. 331-379.
16. Ringoe E., Soon Ho Lee, Hoseinifar S. H., Harikrishnan R., Hien Van Doan, Soltani M., Seong Song. Probiotics, lactic acid bacteria and bacilli: interesting supplementation for aquaculture // Journal of Applied Microbiology. 2020. V. 129. N. 1. Р. 116-136.
17. Chen B., Peng M., Tong W., Zhang Q., Songet Z. The quorum quenching bacterium Bacillus licheniformis T-1 protects zebrafish against Aeromonas hydrophila infection // Probiotics Antimicrob Proteins. 2019. V. 12 (1). Р. 160-171.
18. Kewcharoen W., Srisapoome P. Probiotic effects of Bacillus spp. from Pacific white shrimp (Litopenaeus van-namei) on water quality and shrimp growth, immune responses, and resistance to Vibrio parahaemolyticus (AHPND strains) // Fish Shellfish Immunol. 2019. V. 94. Р. 175-189.
19. Yi Y., Zhang Z., Zhao F., Liu H., Yu L., Zha J., Wang G. Probiotic potential of Bacillus velezensis JW: Antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus // Fish Shellfish Immunol. 2018. V. 78. Р. 322-330.
20. Zhou S., Song D., Zhou X., Mao X., Zhou X., Wang S., Wei J., Huang Y., Wang W., Xiao Su-M., Qin Q. Characterization of Bacillus subtilis from gastrointestinal tract of hybrid Hulong grouper (Epinephelus_ fuscoguttatus x E. lanceo-latus) and its effects as probiotic additives // Fish Shellfish Immunol. 2018. V. 84. Р. 1115-1124.
21. Di J., Chu Z., Zhang S., Huang J. Evaluation of the potential probiotic Bacillus subtilis isolated from two ancient sturgeons on growth performance, serum immunity and disease resistance of Acipenser dabryanus // Fish Shellfish Immunol. 2019. V. 93. Р. 711-719.
22. Nannan C., Vu H. Q., Gillis A., Caulier S., Nguyen Thuy T. T., Mahillon J. Bacilysin within the Bacillus subtilis group: Gene prevalence versus antagonistic activity against Gram-negative foodborne pathogens // Journal of Biotechnology. 2021. V. 327. P. 28-35.
23. Van Doan H., Soltani M., Ringoe E. In vitro antagonistic effect and in vivo protective efficacy of Gram-positive probiotics versus Gram-negative bacterial pathogens in fin-fish and shellfish // Aquaculture. 2021. V. 540. P. 736-581.
24. Ji T., Cao Y., Cao Q., Zhang Y., Yang H. The antagonistic effect and protective efficacy of gram-positive probiotics Bacillus coagulans to newly identified pathogens Pseudomonas aeruginosa in crucian carp Carassius auratus gibelio // Aquaculture Reports. 2022. V. 24. P. 101-126.
25. Zhang D. F., Xiong X. L., Wang Y. J., Gao Y. X., Ren Y., Wang Q., Shi C. B. Bacillus velezensis WLYS23 strain possesses antagonistic activity against hybrid snake-head bacterial pathogens // Journal of Applied Microbiology. 2021. V. 131 (6). P. 3056-3068.
The article is submitted 31.08.2022; approved after reviewing 23.11.2022; accepted for publication 16.03.2023 Статья поступила в редакцию 31.08.2022; одобрена после рецензирования 23.11.2022; принята к публикации 16.03.2023
Information about the authors / Информация об авторах
Marina Ä. Morozova - Candidate of Sciences in Biology; Senior Researcher of the Laboratory of Sanitary Microbiology of Water Bodies and Human Microbial Ecology; Rostov Research Institute of Microbiology and Parasitology; Assistant Professor of the Department of Aquaculture Technical Means; Don State Technical University; morozova. q@mail. ru
Andrey V. Gorovtsov - Candidate of Sciences in Biology; Assistant Professor of the Department of Biochemistry and Microbiology; D. I. Ivanovsky Academy of Biology and Biotechnology, Southern Federal University; gorovtsov@gmail.com
Марина Александровна Морозова - кандидат биологических наук; старший научный сотрудник лаборатории санитарной микробиологии водных объектов и микробной экологии человека; Ростовский научно-исследовательский институт микробиологии и паразитологии; доцент кафедры «Технические средства аква-культуры»; Донской государственный технический университет; morozova.q@mail.ru
Андрей Владимирович Горовцов - кандидат биологических наук; доцент кафедры биохимии и микробиологии; Академия биологии и биотехнологии им. Д. И. Ивановского Южного федерального университета; gorovtsov@gmail.com
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Evgenia V. Prazdnova - Doctor of Sciences in Biology; Head of the Laboratory of Experimental Mutagenesis; D. I. Ivanovsky Academy of Biology and Biotechnology, Southern Federal University; prazdnova@sfedu.ru
Victoria M. Basankina - Candidate of Sciences in Veterinary; Veterinarian of the first category of the Department of Virology, Pathomorphology and PCR Research; Krasnodar Interregional Veterinary Laboratory; vbasankina@mail.ru
Vladimir A. Chistyakov - Doctor of Sciences in Biology; Chief Researcher of the Student Research Laboratory of New Biopreparations; D. I. Ivanovsky Academy of Biology and Biotechnology, Southern Federal University; vladimirchi@yandex.ru
Евгения Валерьевна Празднова - доктор биологических наук; заведующий лабораторией экспериментального мутагенеза; Академия биологии и биотехнологии им. Д. И. Ивановского Южного федерального университета; prazdnova@sfedu.ru
Виктория Михайловна Басанкина - кандидат ветеринарных наук; ветеринарный врач первой категории отдела вирусологии, патоморфологии и ПЦР исследований; Краснодарская межобластная ветеринарная лаборатория; vbasankina@mail.ru
Владимир Анатольевич Чистяков - доктор биологических наук; главный научный сотрудник студенческой научно-исследовательской лаборатории новых биопрепаратов; Академия биологии и биотехнологии им. Д. И. Ивановского Южного федерального университета; vladimirchi@yandex.ru
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Astghik Pepoyan - Doctor of Sciences in Biology, Professor; Head of the Food Safety and Biotechnology Department; National Agrarian University of Armenia; apepoyan@gmail.com
Shahlo Miralimova - Doctor of Sciences in Biology, Professor; Deputy Director for Science; Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan; mirshakhlo@yahoo.com
Vadim A. Grigoriyev - Candidate of Sciences in Biology; Researcher at the Research Laboratory of Biotechnologies for Preservation and Reproduction of Valuable Fish Species; Astrakhan State Technical University; aqua-group@yandex.ru
Астгик Пепоян - доктор биологических наук, профессор; заведующий отделом безопасности питания и биотехнологий; Национальный аграрный университет Армении, apepoyan@gmail.com
Шахло Миралимова - доктор биологических наук, профессор; заместитель директора по науке; Институт микробиологии Академии наук Республики Узбекистан; mirshakhlo@yahoo.com
Вадим Алексеевич Григорьев - кандидат биологических наук; научный сотрудник научно-исследовательской лаборатории биотехнологии сохранения и воспроизводства ценных видов рыб; Астраханский государственный технический университет; aqua-group@yandex.ru
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