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Project titleApproaches for application of D-lactate in early diagnostics of bacteria-associated pathologies for fish in aquaculture

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-209 - Biotechnology (including biological nanotechnology)

Annotation
Increased aquaculture production is an important prerequisite for food security of the Russian Federation and ensuring socio-economic development of the regions. The development of aquaculture is especially promising in the Northwest Russia and Siberia due to favorable environmental conditions for valuable salmon species. The Republic of Karelia is now the leader in cage trout farming; it contributes over 80% marked-size trout among all trout grown in Russia for the domestic market. To ensure economic efficiency of the farm, fish are grown in cages in at maximum density of fish, which leads to injuries, stress and, as a result, weakened immunity of fish. Therefore, the timely and effective prevention of epizootics in reservoirs is one of the key factors for the sustainable functioning of trout farms. Accordingly, an important task is the development of effective technologies to early identify and correctly determine the cause of the pathological conditions in fish, which contribute to informed decision-making on the use of drugs for the treatment and prevention of infectious diseases in fish. D-lactate is a specific metabolite secreted mainly by normal microbiota of the gastrointestinal tract of animals, as well as by some pathogenic bacteria, while vertebrates produce it in detectable amounts. In case the natural balance of microbiota is shifted, D-lactate of microbial origin accumulates, enters the bloodstream and then spreads to the internal environments of the body. This makes the content of D-lactate in the blood or intracellular medium a promising marker for the development of specific pathologies associated with bacterial infection of the internal environment of the body or for changes in the natural balance of skin and gastrointestinal tract microbiota. The use of D-lactate for these purposes has been well studied in humans and a number of other mammals, but the prospects for its use for fish remain completely unknown. At the same time, it is in the case of industrial aquaculture, when the procedure for analyzing the condition of each individual should be as simple and cheap as possible, D-lactate, penetrating into the body fluids in high concentration, may turn out to be an optimal marker of bacterial pathogenesis. The proposed interdisciplinary project aims to fill this gap and not only fully characterize the importance of D-lactate as a marker of pathological processes in fish, but also offer a number of cheap and convenient methods for determining its content in animal tissues, including those that do avoid the collection of biological samples thanks to the use of advanced implantable sensors. Parallel effort of both project teams will provide the greatest synergistic effect due to the combined efforts and experience of specialists from various fields. Within the framework of the project implemented at Petrozavodsk State University in the field Agricultural Sciences, a complex of analyzes and large-scale controlled experiments will be performed that will provide data on the relationship between the level of D-lactate and the health of the cultivated trout Oncorhynchus mykiss. The goal of this project is the theoretical justification for the use of D-lactate as an indicator of the state of microbiota and the creation of guidelines for the interpretation of the results of developed tests for D-lactate. The content of D-lactate and its correlation with immunity and nonspecific resistance of fish will be determined under the conditions of experimental microbial imbalance of fish under the influence of non-optimal biotic values(feed composition, pathogen infection) and abiotic (temperature) environmental factors typical for trout farms. An assessment of the level of D-lactate in fish with signs of infectious diseases at trout farms of the Republic of Karelia will also be made, which will determine the possibility of using D-lactate as one of the markers for the rapid differential diagnosis of diseases of a viral, fungal, and bacterial nature. At the same time, a number of tools based on different principles will be developed to ensure the possibility of regular monitoring of D-lactate in fish directly at the farm during the implementation of the project at the Irkutsk State University in the field of Biology and Life Sciences. These tools will include three types of implantable sensors and two types of test strips, sensitivity of which to D-lactate will be provided by a chiral selective enzymatic reaction or recognition by yeast. The use of implantable sensors will make it possible to constantly analyze the internal environment of the body without increasing the cost with an increase in the measurement frequency, while the practical implementation of test strips may be the fastest. A distinctive feature of one of the developed types of implantable sensors will also be unique opportunity to detect short-term maxima of the content of D-lactate that occur during periods when the operator does not record sensor readings or take blood samples. Thus, the proposed project directly corresponds to the direction of the Strategy for Scientific and Technological Development of the Russian Federation “#4 transition to highly productive and environmentally friendly agro- and aquaculture, the development and integration of the systems for the rational use of chemical and biological protection equipment for agricultural plants and animals, the storage and effective processing of agricultural products, the manufacture of safe and quality, as well as functional, food products.”

Expected results
Within the framework of the project at Irkutsk State University, it is planned to develop five different tools for determining the content of D-lactate in fish tissues (field "Biology and Life Sciences"). These tools will include two types of implantable sensors sensitive to the current metabolite content, one type of implantable sensors sensitive to its cumulative level over the entire period of use of the implanted sensor, as well as two types of test strips based on different principles. The sensitivity ranges of the developed tools to the content of D-lactate depending on the parameters of their preparation will be determined. In addition, the estimated operating time of the developed implantable sensors will be estimated, and the required parameters of a compact and mobile optical system for signal registration outside the laboratory (the power of the illuminator for exciting fluorescence, the sensitivity of the detector matrix, etc.), consisting of widespread electronic components, will be determined. In turn, the implementation of the project at Petrozavodsk State University (field "Agricultural Sciences") will allow for the first time to fully characterize the importance of D-lactate as a marker of various pathological conditions of cultivated trout, to assess the normal ranges of this metabolite content in fish’s internal environment and to study the effect of various biotic and abiotic factors on its concentration. Joint work within the framework of the interdisciplinary project will allow testing the developed tools for assessing the content of D-lactate in fish tissues, as well as formulating specific protocols and recommendations for their use. The widespread use of developed implantable sensors, cheap test strips and methods for their use will not only provide a fundamentally new research tool for studying fish pathologies, but will also allow the detection of bacterial infections in fish farms at the earliest stages, as well as more quickly determine the nature of the pathogen in outbreaks of unknown diseases nature. This, in turn, will make it possible to select targeted therapeutic measures in advance, which will reduce both the death of fish and the number of drugs used and ensure the growth of profitability and production efficiency. In addition, the developed tools tested on fish can subsequently be adapted for livestock farming, which can also have a significant economic effect.

Annotation of the results obtained in 2023
The global objective of the interdisciplinary project was to investigate the possibilities of using D-lactate as a marker of various pathologies in fish in aquaculture. The task of Petrozavodsk State University in the framework of the interdisciplinary project was to study the relationship between the level of D-lactate in blood and the development of various pathological processes in rainbow trout caused by changes in microbiota composition. In turn, the task of Irkutsk State University was to develop methods and approaches for rapid determination of D-lactate content in biological media under conditions of fish farms using a range of approaches including chemical and microbiological methods in vitro and in vivo. In 2023, the project participants from PetrSU completed the collection of a database on biochemical and physiological adaptations of trout during outbreaks of infectious diseases at trout farms. In particular, it was shown that fish with impaired growth and early development are more susceptible to various skin infections. At the same time, it was found that chronic skin infections have no effect on either the systemic immunity reactivity indicators or the level of D-lactate in the blood and intestines of trout. This year, the project team from PetrSU completed an analysis of the data obtained in field and aquarium experiments on the influence of inflammatory processes on the permeability of the intestinal barrier in rainbow trout. It was concluded that the influx of exogenous compounds from the intestine may depend on the degree of local inflammation in the hindgut, while systemic inflammation at the organism level does not affect the intestinal barrier functions, including the influx of D-lactate into fish blood. Project participants from ISU have completed work on genetic modification of microorganisms to produce recombinant proteins with chemical affinity to D-lactate or to produce microorganism strains sensitive to the presence of D-lactate in the medium. The chemical activity of recombinant protein SC-LOX (Dld3) from yeast S. cerevisiae was characterised, and it was shown that the enzyme is a functional D/L-lactate dehydrogenase and is not suitable for construction of a stereospecific sensor for D-lactate. Within the project, a number of model chemical lactate sensors based on lactatoxidase that recognise L- but not D-lactate, which can be implanted directly into body tissues and do not require biological samples for analysis, was also produced and tested in vitro. Characterisation of the yeast transcriptomic response to changes in D-lactate concentration in the medium has been completed, with the aim of potential molecular systems for the design of stereo-specific genetic constructs. It was shown that the regulation of yeast response to high concentrations of lactate and to high acidity of the medium is carried out by shared gene sets. The stability and functionality of implanted hydrogels of different compositions for subsequent introduction of microsensors sensitive to D-lactate in vivo and on test strips were evaluated by microscopy and histology. In vivo experiments on juvenile trout showed a fundamental possibility to register fluorescence from live genetically modified yeast immobilised in 13% polyacrylamide gel and implanted subcutaneously into the trout adipose fin.

Publications

1. A. Nazarova, A. Gurkov, Y. Rzhechitskiy, E. Shchapova, A. Mutin, A. Saranchina, A. Diagileva, N. Bolbat, P. Krivoshapkin, M. Timofeyev Turn a shrimp into a firefly: monitoring tissue pH in small crustaceans using an injectable hydrogel sensor with infrared excitation and visible luminescence Photonics, Photonics. 2023. V. 10, No 6. 697 (year - 2023) https://doi.org/10.3390/photonics10060697

2. E. Borvinskaya, S. Matrosova, I. Sukhovskaya, P. Drozdova, E. Titov, I. Anikienko, Y. Lubyaga, A. Gurkov, M. Timofeyev Tissue reaction to low-density polyacrylamide gel as a carrier for microimplants in the adipose fin of rainbow trout Gels, Gels. 2023. Vol. 9, No 8. Article number 629. (year - 2023) https://doi.org/10.3390/gels9080629

3. P. Drozdova, A. Gurkov, A. Saranchina, A. Vlasevskaya, E. Zolotovskaya, M. Timofeyev, E. Borvinskaya Transcriptional response of Saccharomyces cerevisiae to lactic acid enantiomers Applied Microbiology and Biotechnology, - (year - 2024)

4. A.N. Gurkov, E.P. Shchapova, Y.A. Rzhechitskiy, E.V. Borvinskaya, A.A. Nazarova, A.A. Diagileva, M.A. Timofeyev Возможности имплантируемых оптических сенсоров для мониторинга pH в различных органах водных животных Развитие жизни в процессе абиотических изменений на Земле: материалы IV Всероссийской научно-практической конференции, посвящённой 30-летнему юбилею Байкальского музея СО РАН, 25-29 сентября 2023 г., пос. Листвянка, Иркутская область /, Развитие жизни в процессе абиотических изменений на Земле: материалы IV Всероссийской научно-практической конференции, посвящённой 30-летнему юбилею Байкальского музея СО РАН, 25-29 сентября 2023 г., пос. Листвянка, Иркутская область – С. 188-190 (year - 2023) https://doi.org/10.24412/cl-34446-2023-4-188-190

5. E. Indosova, E. Zolotovskaya, A. Saranchina, P. Drozdova Exploring the perspectives of using the D-lldR protein from Pseudomonas fluorescens as a biosensor for D-lactic acid in vitro Molecular Biology of the Cell, Molecular Biology of the Cell. 2023. Vol. 33. Is.2. B28/P2427. (year - 2023) https://doi.org/10.1091/mbc.E22-12-0555

6. - Способ долговременного неинвазивного мониторинга различных физиологических параметров организма культивируемых рыб семейства лососевых -, 623011000032-0 (year - )

Annotation of the results obtained in 2020
During the first year of the project the team at PetrSU obtained the first data on D-lactate concentration in the blood of trout cultivated in water basins. A screening was conducted to find microorganisms suitable for a model experiment imitating infection in trout under controlled laboratory conditions. An E. coli strain potentially virulent for the trout was isolated, and its ability to cause inflammation and infection process in trout in vivo was tested. The equipment for trout cultivation in laboratory conditions and PCR analysis was installed and tested. The first results of molecular genetics testing of the samples were obtained. The data on variability of physiological, biochemical and molecular parameters of internal media of fish with signs of infection from trout farms of Northwestern Russia is accumulating. During the first year of the project, samples were obtained from two farms at Lake Ladoga and the White Sea. These samples were processed to analyze the correlation between markers of physiological state of the fish and signs of bacterial infection. The team at ISU designed, obtained and partially tested vector sequences essential for molecular genetics manipulations with microorganisms within the Project. A modified E. coli strain producing D-lactate oxidase (mostly accumulated in the insoluble fraction) was obtained. Several types of polymer films to immobilize the enzymes that metabolize D-lactate for further use in test strips were tested. The most promising approaches to design D-lactate test strips were identified with patent search. Techniques for immobilization of necessary fluorescent probes were verified. Methods for live yeast polymerization in polyacrylamide gel were tested, and preliminary work was conducted to estimate the immunogenicity of polyacrylamide gels and implantable microsensor carriers. The genetic modification of Saccharomyces cerevisiae to introduce the genetic system sensitive to D-lactate. The obtained data allow us to start the main part of the project, i.e. development of D-lactate sensors (including implantable vital sensors).

Publications

1. Borvinskaya E, Gurkov A, Shchapova E, Mutin A, Timofeyev M. Histopathological analysis of zebrafish after introduction of non-biodegradable polyelectrolyte microcapsules into the circulatory system PeerJ, 2021 May 5;9:e11337 (PMID: 33996284; PMCID: PMC8106396) (year - 2021) https://doi.org/10.7717/peerj.11337

2. Anna Nazarova, Ekaterina Shchapova, Anton Gurkov, Ekaterina Borvinskaya, Maxim Timofeyev Изучение иммуного ответа амфипод на полиакриламидный гель в первичной культуре гемоцитов Сборник тезисов VII Международной конференции молодых ученых: биофизиков, биотехнологов, молекулярных биологов и вирусологов — 2020, АНО «Иннов. центр Кольцово». — Новосибирск : ИПЦ НГУ, 2020. - С. 105-106 (year - 2020)

Annotation of the results obtained in 2021
In 2021, the ISU interdisciplinary project team continued to work on finding approaches and technical solutions for measuring D-lactate in test strips, in vitro and as part of implantable microsensors. Colorimetric response screening was performed to determine lactate in a concentration range corresponding to the physiological D-lactate concentrations. Based on the colorimetric reaction with guaiacol, a test strip design for the determination of D-lactate was proposed. To carry out this reaction using a genetically modified strain of E. coli, various variants of the recombinant D-oxidase enzyme from yeast were obtained. Evaluation of the enzyme's ability to metabolize D-lactate showed that this protein has lactate dehydrogenase, but not lactate oxidase activity. The sensor was chosen on the basis of Ru (ddp) and a silicone carrier, which is sensitive to the content of dissolved oxygen, and also makes it possible to take into account the photodegradation of the dye during repeated measurements. The proof-of-principle feasibility of the proposed oxygen sensor in combination with D-lactate oxidase for measuring D-lactate was confirmed in the glucose / glucose oxidase system. The work to obtain live biosensors for the detection of D-lactate based on GMO yeast was continued. For this, the transfer of the genetic system sensitive to D-lactate from Pseudomonas fluorescens to the yeast Saccharomyces cerevisiae was carried out. The immobilization of yeast in hydrogels based on alginate, polyhydroxyethyl methacrylate and polyacrylamide was tested. It has been shown that 10-15% polyacrylamide gels are a promising basis for the design of non-biodegradable implantable yeast biosensors. The adipose fin of trout has been shown to be a suitable site for the implantation of fluorescent microsensors. This organ transmits light in the 440-1000 nm range up to 4 times better than the skin in other parts of the fish body, has a sufficient volume for the introduction of a microsensor, is compact enough for quick imaging of the sensor, and is a promising place for assessing the internal physiological processes of salmonids. Work continued on assessing the immunogenicity of the introduced microsensors. The mechanism of elimination of microparticles from the body through the exfoliating epithelium of the intestine and skin has been described in fish tissues. The immunogenicity of the microcapsules with a fluorescent probe was significantly reduced by coating them with 2.5% polyacrylamide gel. As a result, in an in vivo experiment, the advantages of 2.5% PAGE as a carrier for microsensors were demonstrated, such as ease of administration; impermeability to immune cells; low cytotoxicity, decreased immune response to a foreign body; stability under physiological conditions for several weeks; tissue-like structure; and the ability to integrate into tissues without fibrous encapsulation. The team of an interdisciplinary project from PetrSU continued to study the level of D-lactate in the internal media of trout, the permeability of the intestines of fish, as well as the composition of the intestinal microflora under the influence of various factors in natural and artificial conditions. To assess the effect of the composition of the intestinal microbiota on the content of D-lactate in internal media, an aquarium experiment was carried out in which fry of trout were fed with food with different ratios of proteins and carbohydrates. An increase in intestinal permeability was shown when feeding trout diets with a high protein content. It was also shown that the major producers of D-lactate are the microflora of the posterior intestine of fish. Within the framework of the project, work was continued to study the composition of the microbiome, biochemical and physiological characteristics of diseased individuals of trout on trout farms in the North-West of Russia. The search for potential markers of the prodromal stage of bacterial infection and the general state of fish immunity was carried out on trout affected to various degrees by an infectious disease caused by the dangerous pathogen Vibrio anguillarum. It was concluded that extracellular histones, antiproteases, and activators of the blood coagulation cascade can participate in the elimination of the pathogen in the early stages of the disease in fish resistant to vibriosis. Also, proteins presumably associated with the progression of the disease have been identified. It was not possible to reveal changes in intestinal permeability in fish with vibriosis as the disease progressed; however, there was a trend towards increased expression of the gut intercellular contact protein, claudin, as the severity of the infection increased. To search for new markers that, along with D-lactate, can be used to assess the immunological status of trout and predict the course of bacterial infection, a search and description of molecules interacting with bacteria in the blood of healthy fish was carried out. For this, the effect of natural trout plasma on the growth and survival of the pathogenic bacterium Aeromonas hydrophila was studied. Using various microbiological methods, the antimicrobial properties of the high molecular weight protein fraction of trout plasma (> 10 kDa) were studied. As a result, material was selected for analyzing the composition of blood plasma proteins of trout interacting with the pathogen.

Publications

1. Nazarova A.A., Gavrilova M.A., Borvinskaya E.V. Testing polyacrylamide gel as the carrier for yeast biosensors FEBS Open Bio, T. 11 (S1.), стр. 275-276. (year - 2021) https://doi.org/10.1002/2211-5463.13205

Annotation of the results obtained in 2022
In 2022, the team of the interdisciplinary project from the ISU continued to work on finding approaches and technical solutions for measuring D-lactate in complex biological mixtures in the format of test strips, in vitro and in vivo. Molecular screening was performed for protein sequences capable of specifically recognizing the D-lactate isomer. To design an enzymatic system for measurement the concentration of D-lactate in fish plasma, recombinant GO-LOX protein from Gluconobacter oxydans was obtained using genetically modified E. coli. The obtained protein was able to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in presence of D-lactate, but did not have oxidase activity. Thus, it was shown that the enzymes of the Dld3 family are not suitable for creating colorimetric tests for D-lactate. In addition, transcriptional factor Dlld-R from Pseudomonas fluorescens has been successfully cloned in E. coli. The ability of Dlld-R to specifically bind to D-lactate was tested by the retardation in the gel. In 2022 development of tests for D-lactate measurement based on genetically modified organisms was continued. For this purpose, the D-lactate-sensitive genetic system from P. fluorescens was transferred to E. coli and Pseudomonas putida. The resulting GMOs produced fluorescent protein in the presence of D-lactate at concentrations from 12 mM. The resulting strains were immobilized in sodium alginate to create a prototype test strip for the determination of D-lactate. Participants of the project from ISU tested the stability and functionality of polyacrylamide hydrogels, as the scaffold for GMO-based microsensors of D-lactate in vivo. A microsensor based on a pH-sensitive 13% polyacrylamide gel showed its functionality for 9 days in live trout tissues. For multiple registration of optical signals from fish, a laboratory prototype of a hand-held device was developed and tested, providing the registration of a signal from a commercial-sized trout without anesthesia. The participants of the interdisciplinary project from PetrSU continued evaluation of D-lactate in the farmed trout, as well as fish intestinal permeability and the composition of the intestinal microflora of trout under various environmental factors. To evaluate the effect of systemic inflammation on the intestinal permeability and D-lactate plasma concentration, an experiment was conducted in which trout were treated with inactivated pathogenic bacteria Aeromonas hydrophila. It was revealed that intestinal permeability does not determine the level of D-lactate in the blood of trout. The collection of data on biochemical adaptation of trout to infectious disease was continued on the fish farms by participants of the project from PetrSU. In particular, it was found that a chronic infectious disease caused by F. psychrophilum does not affect the level of D-lactate in the blood of trout, while a fungal infection causes a significant increase in the level of this metabolite. To search for new markers, which can be used to assess the immunological status of trout and predict the course of a bacterial infection, the study of fish plasma proteins recognizing the bacterial antigen was continued. For this, mass spectrometry of trout plasma proteins capable of binding to the cell wall of A. hydrophila was performed. In the experiment, bacteria were incubated in the plasma of healthy and immunized trout from two fish farms on the river Angara and Onega Lake. In the bacteriostatic plasma of immunized fish, immunoglobulins together with unknown actin-like protein, lectin, lipocalin, and an isoform of apolipoprotein B-100 were able to bind to the bacterial cells. Thus, for the first time, a native trout plasma complex attacking a pathogen was investigated, and a group of presumably bacteriostatic proteins was described. Accordingly, the described proteins can be suggested as markers of trout resistance to bacterial diseases.

Publications

1. Rzhechitskiy Y, Gurkov A, Bolbat N, Shchapova E, Nazarova A, Timofeyev M, Borvinskaya E. Adipose Fin as a Natural "Optical Window" for Implantation of Fluorescent Sensors into Salmonid Fish Animals, Animals 2022, 12(21), 3042 (year - 2022) https://doi.org/10.3390/ani12213042

2. Shchapova E., Titov E., Gurkov A., Nazarova A., Borvinskaya E., Timofeyev M. Durability of Implanted Low-Density Polyacrylamide Hydrogel Used as a Scaffold for Microencapsulated Molecular Probes inside Small Fish Polymers, Polymers 2022, 14, 3956 (year - 2022) https://doi.org/10.3390/polym14193956

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