RU2603104C2 - Method of biochemiluminescent assessment of rumen fluid toxicity in vitro - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention relates to laboratory diagnostics and concerns method of biochemiluminescent assessment of rumen fluid toxicity in vitro. Disclosed method involves measuring luminescence intensity of bacteria strain E. coli K12 TG1 with luxCDABE cloned genes of Photobacterium leiognathi 54D10 “Ecolum-9" in test sample, containing rumen fluid, in comparison with control sample, containing physiologic saline, and accounting of values of rumen fluid toxicity (RFT) by formula $$RFT=100-\frac{I_{\text{experience}}^{\mathrm{0,5}}\times I_{\text{control}}^0}{I_{\text{experience}}^0\times I_{\text{control}}^{\mathrm{0,5}}}\times 100\%,$$

where $$I_{\text{control}}^0$$

is luminescence level of control sample at 0 minute; $$I_{\text{control}}^{\mathrm{0,5}}$$

is luminescence level of control sample at 0.5 minute; $$I_{\text{experience}}^0$$

is luminescence level of test sample at 0 minute; $$I_{\text{experience}}^{\mathrm{0,5}}$$

is luminescence level of test sample at 0.5 minute. If RFT is less than 5 %, sample is considered to be non-toxic, if it is from 5 to 19 %, sample is considered to be low-toxic, if it is from 20 to 49 %, sample is considered to be moderately toxic, if it is from 50 to 100 %, sample is considered to be highly toxic.

EFFECT: invention can be used in veterinary laboratories, solving issues of assessment of farm animals feeding efficiency in order to detect possible disorders and control effectiveness of corresponding correction measures.

1 cl, 3 dwg, 2 tbl

Description

The invention relates to the field of laboratory diagnostics and can be used to determine the toxicity of scar fluid in vitro.

The method is intended for use in laboratories with a veterinary profile that address issues of evaluating the effectiveness of feeding farm animals in order to identify possible violations and monitor the effectiveness of the relevant corrective measures.

The essence of the invention is to evaluate the toxicity of cicatricial fluid in vitro by the severity of the inhibitory effect of cicatricial fluid (CG) on the intensity of the luminescence of Escherichia coli K12 TG1 with the cloned luxCDABE genes of Photobacterium leiognathi 54D10 as compared to the control - the intensity of the luminescence of the same luminescent bacteria that did not have contact with the RG and then calculate the value of toxicity of cicatricial fluid according to the formula

where TRG is the toxicity of cicatricial fluid,%;

- уровень люминесценции контрольной пробы на 0 минуте;

- luminescence level of the control sample at 0 minute;

- уровень люминесценции контрольной пробы на 0,5 минуте;

- the luminescence level of the control sample at 0.5 minutes;

- уровень люминесценции опытной пробы на 0 минуте;

- the luminescence level of the experimental sample at 0 minute;

- уровень люминесценции опытной пробы на 0,5 минуте.

- the luminescence level of the experimental sample at 0.5 minutes.

The criterion of the toxic effect is the change in the bioluminescence intensity of the test object in the test sample compared to that for the sample with a solution that does not contain toxic substances. A decrease in the intensity of bioluminescence is proportional to the toxic effect.

Our proposed method eliminates the need for nutrient media, allows you to quickly and reliably evaluate the toxicity of diet components or liquids. The technical result achieved is to simplify, reduce the duration and complexity of the method of bio-chemiluminescent toxicity assessment of scar fluid in vitro.

A known bio-chemiluminescent method for determining the phagocytic activity of neutrophils, where recombinant luminescent bacteria are used as objects of phagocytosis, namely microorganisms Escherichia coli K12 TG1 with cloned luxCDABE genes Photobacterium leiognathi 54D10 [1].

A known method for determining the biotoxicity of nanocarbon by studying the effect on the luminosity of a recombinant luminescent strain of Escherichia coli K12 with genes of the luminescent system Photobacterium leiognathi [2].

A known method for determining the bactericidal activity of blood serum by the severity of its inhibitory effect on the intensity of the glow of serosensitive luminescent bacteria [3].

The known methods listed above are applicable in medicine and toxicology, are highly accurate and sensitive, but do not characterize approaches to assess the toxicity of scar fluid.

The composition of the feed has a direct effect on the species diversity and preservation of the rumen microbiota, but at the same time it can act as a toxicant, exerting a depressing effect on the rumen microflora and, as a consequence, on the animal organism.

Studying the effect of scar fluid toxicity when using various types of feed seems to be an urgent task, since it is the bacterial link of this biocenosis that first interacts with the feed received in the rumen. One of the possible ways to solve this problem is biotesting, which allows determining the degree of environmental toxicity by the degree of biosensor viability.

The studies used a recombinant strain of E. coli K12 TG1 with the cloned luxCDABE genes Photobacterium leiognathi 54D10 [5], produced in the form of a lyophilized preparation under the commercial name "Ekolyum", manufactured by NEY Immunotech (Moscow).

As the basic equipment for research, we used the “Artificial Scar” KPL-01 [5, 4], the LM-01T luminometer (Immunotech, Czech Republic), the Expert-001 pH meter ion meter (Eoniks-Expert, Russia), a centrifuge Laboratory SM-6M (Elmi, Russia).

The objects of study were:

- cicatricial fluid of beef cattle of Kazakh white-headed breed;

- model cicatricial fluid obtained on the basis of phosphate buffer, propionic - 17-21%, lactic - 5%, butyric - 14-34%, acetic acid - 49-69%, glucose and 10% ammonia solution;

- trophic substrates based on wheat bran extruded with the addition of Fe ²⁺ ;

- phosphate buffer (pH 7.0) by mixing 48.8 ml of a solution of 0.2 M KH ₂ PO ₄ and 51.2 ml of a solution of 0.2 M Na ₂ HPO ₄ .

128 ml of cicatricial and model fluid and 500 mg of trophic substrate were added to each of the containers of the “artificial scar” and incubated for 24 hours at 37 ° C. The sampling time interval was 3, 6, 12, 24 hours.

After 24 hours of incubation under "artificial scar" conditions, the scar fluid was separated from the substrate and from bacteria by centrifugation for 10 minutes at 3000 rpm and the supernatant was selected, in which the possible presence of the toxicant was determined.

The content of the first stage is the preparation of the luminescent bacterial biosensor E. coli K12 TG1 with the cloned luxCDABE genes Photobacterium leiognathi 54D10 with verification of its luminosity. At the same time, this problem is currently being solved by rather simple means, since existing commercial test systems usually contain them in a ready-to-use lyophilized form. As a result of this, there is no need to independently control the density of the bacterial population before each study, now carried out at the stage of biotest production, as well as the need to maintain the constancy of the characteristics of luminescent bacteria in museum cultures. The latter is especially relevant, since often the ability to luminescence is lost three years after the isolation of bacteria from natural ecosystems and a number of passages on artificial nutrient media.

To study the toxicity of cicatricial fluid, the procedures were performed according to the developed algorithm (Fig. 1). For this, the preparation “Ekolyum” was restored from the lyophilized state by adding 10 ml of distilled water, after which the bottle was kept for 30 minutes at 4 ° C. Then a mixture was formed in the wells of the tablet, consisting of 100 μl of the tested scar fluid (experimental sample) or 100 μl of a 0.9% NaCl solution (control sample), and 100 μl of bacteria. A sharp quenching of the luminescence of the strain E. coli K12 TG1 with the cloned luxCDABE genes Photobacterium leiognathi 54D10 was observed from the first seconds of contact, and the level of luminescence of the biosensor in the samples with a lower concentration of scar fluid was higher than in the samples with a higher concentration (Fig. 2), and the samples containing less than 3.125% of cicatricial fluid, provided luminescence induction.

Based on preliminary experimental results, it was suggested that the probable cause of this phenomenon may be the effect on the bacterial bioluminescence of any one or more of the components that make up the scar fluid. To confirm or refute this assumption, at the next stage of the work, a model mixture (model scar fluid) was created based on a phosphate buffer, which included propionic, lactic, butyric, acetic acid, glucose, and 10% aqueous ammonia solution taken in physiological concentrations in order to further clarify the effect of individual components of real scar fluid on the Ekolyum biosensor.

In general, the data obtained indicate that the real (native) cicatricial fluid and the model mixture gave similar effects of suppressing the level of bioluminescence at the beginning of the experiment (they had a similar character), but did not exhibit toxic effects. The results obtained allowed us to judge some identity of both liquids, and, therefore, to study the effect of individual components on the recombinant E. coli strain.

Analyzing data on the influence of individual components of the model scar fluid on the recombinant E. coli strain, we can conclude that none of the components had any pronounced toxic effect on this strain (Fig. 3).

An example of a specific implementation: cicatricial digestion helps to increase the efficiency of the use of feed nutrients due to efficient energy, nitrogen metabolism, while feeding diets with a high proportion of the grain part leads to disruption of the digestive processes. In this regard, studies have been conducted on the toxicity of scar fluid with the inclusion of various types of grain feed.

In the studies, a recombinant strain of E. coli K12 TG1 with the cloned luxCDABE genes Photobacterium leiognathi 54D10 was used. The objects of study were cicatricial fluid; trophic substrates - crushed grain of barley (Hordeum vulgare), wheat (Triticum aestivum), rye (Secale cereale). The experimental design involved the preparation of weights: barley (H) - 100 mg, rye (S) - 100 mg, wheat (T) - 100 mg). Next, weighed samples were placed in test tubes, in which 1 ml of scar fluid was added, one of which was a control. The resulting mixtures were incubated in a thermostat at 37 ° C with single shaking for 3 hours. To study the toxicity (activity) of cicatricial fluid, the Ekolyum luminescent strain was used, which was reconstituted from the lyophilized state by adding 10 ml of distilled water, after which the vial was kept for 30 minutes at 4 ° C. In the wells of the plate, incubated samples were serially diluted with physiological saline (wells 2-11, while in 11 wells the maximum concentration was 100%), well 1 contained only 0.9% NaCl solution. The fluid volume in each well was 100 μl. 100 μl of the restored Ekolyum biosensor was added to all wells; thus, the ratio of the analyzed liquid and the biosensor was 1: 1.

Glow was measured at 0.5 minutes on a luminometer. As the basic equipment for the research, the “Artificial scar” KPL-01, luminometer LM-01T (Immunotech, Czech Republic), pH meter ion meter Expert-001 (Eonix-Expert, Russia), and a laboratory centrifuge SM-6M (Elmi) were used , Russia).

According to the results of studies, it was found that all samples were highly toxic.

The final calculation is carried out according to the formula

where TRG is the toxicity of scar fluid,%;

- уровень люминесценции контрольной пробы на 0 минуте;

- luminescence level of the control sample at 0 minute;

- уровень люминесценции контрольной пробы на 0,5 минуте;

- the luminescence level of the control sample at 0.5 minutes;

- уровень люминесценции опытной пробы на 0 минуте;

- the luminescence level of the experimental sample at 0 minute;

- уровень люминесценции опытной пробы на 0,5 минуте,

- the luminescence level of the experimental sample at 0.5 minute,

and with an SFA value of less than 5%, the sample is considered non-toxic, from 5 to 19% low toxic, from 20 to 49% medium toxic, from 50 to 100% highly toxic.

Information sources

1. Patent for the invention of the Russian Federation No. 2366953. Biochemiluminescent method for determining the phagocytic activity of neutrophils // D.G. Deryabin, I.F. Karimov. Published on April 27th, 2009. Bull. Number 25.

2. Patent for the invention of the Russian Federation No. 2437938. A method for determining the biotoxicity of nanocarbon // D.G. Deryabin, E.G. Alyoshina. Published 08/20/2011. Bull. Number 36.

3. Patent for the invention of the Russian Federation No. 22797987. A method for determining the bactericidal activity of blood serum // D.G. Deryabin, V.A. Gritsenko, E.G. Poles. Published on January 22, 2003. Bull. Number 7.

4. Patent for utility model of the Russian Federation No. 106956. Device for in vitro research // K.G. Logachev, S.A. Miroshnikov A.G. Meshcheryakov. Published July 27, 2011

5. Popov VV, Rybina E.T. Method for determining the digestibility of dry matter "in vitro" // Livestock. - 1983. - No. 8. - S. 37-39.

Claims (1)

In vitro method for bio-chemiluminescent assessment of scar fluid toxicity based on the bioluminescent reaction of E. coli K12 TG1 strain with the cloned luxCDABE genes Photobacterium leiognathi 54D10 “Ecolyum-9” by measuring the intensity of bacterial luminescence in an experimental sample containing scar fluid, compared to a control sample containing saline solution, and taking into account the values of toxicity of cicatricial fluid (SCF) according to the formula

Where

- luminescence level of the control sample at 0 minute;

- the luminescence level of the control sample at 0.5 minutes;

- the luminescence level of the experimental sample at 0 minute;

- the luminescence level of the experimental sample at 0.5 minute,
with a value of TRI of less than 5%, the sample is considered non-toxic, from 5 to 19% low toxic, from 20 to 49% medium toxic, from 50 to 100% highly toxic.

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