Experimental evaluation of biological

risks of introduction of the gene-

engineering-modified microorganism
(GMM) B. subtilis VKPM V-7092 into
the environment
E.A. Stavskiy 1, A.I. Lelyak 2, N. M. Larina 3, O.N. Grishaeva 4, V.V.
Serebrov 5, Yu. A. Gorbunov 1,
L. G. Duben 6, L. A. Timukhina 3, L. V. Mitko 3,
R. S. Koval 3, V.P. Khodyrev 5
1
State Research Center of Virology and Biotechnology Vector, Koltsovo, Novosibirsk region;
2
JSC SPC Research Center, Koltsovo, Novosibirsk region;
3
SVDI Regional Veterinary Laboratory, Accredited Testing Center, Affiliated Agency
of Certification of Veterinary Preparations and Feeds FSA VSRCI, Novosibirsk;
4
JSC Vector-Best, Koltsovo, Novosibirsk region;
5
RI of Animal Systematic and Ecology SB RAS, RF, Novosibirsk;
6
Anti-plague Station, Medical and Sanitary Station 163 FSA Medbioextrem, Koltsovo,
Novosibirsk region

Abstract

Experimental evaluation of biological risks of introducing the genetically

modified microorganism (GMM) B. subtilis VKPM B7092, the active ingredient of the
probiotic VETOM 1.1, into an open system has been performed. The survival of the
above GMM in bovine gastroenteric tract, its influence on microbiocenosis, the species
composition of microflora of the gastroenteric tract, the possibility of transfer of the DNA
fragment cloned in B. subtilis bacterium and containing the gene of human leukocytic
alpha-2 interferon to the representatives of intestinal microflora with administration of
the probiotic VETOM 1.1, as well as its possible transfer to other microorganism species
in the environment (soil) were studied. There were no negative effects of the GMM on
the animal organism and the environment, including remote consequences.

Introduction

Probiotics are widely used in medicine and veterinary for prophylaxis of

dyspepsia, diarrhea, (dysbacteriosis) and gastroenteric diseases. They are often used to
correct intestinal bacteriocenosis after antibiotic- or chemotherapy, as well as, to increase
natural resistance and to stimulate growth in animals [9, 14, 16, 17, 23]. Probiotics are
live active antagonistic microorganism cultures producing a favorable effect on intestinal
bacteriocenosis. They are produced on the basis of one three species of lactate bacteria
and streptococci, Bifidobacteria, enterococci, nonpathogenic Escherichia, yeast and
certain representatives of Bacillus genus. A large number of officially registered and
approved probiotics is known [4, 9, 14, 16, 17, 23]. Recombinant probiotics with their
broad prospects of medical and veterinary applications take a special place among
probiotics [2, 5, 15, 22, 24]. However, the use of recombinant probiotics under the Law
of Russian Federation On the State Regulation in the Field of Gene Engineering (N
86_3 of 05.07.96), as well as international regulations, require deep and substantial
evidence of biological safety and demonstration of biological risks of using GMM (gene-
engineering modified microorganisms) that are the active ingredient of this group of
probiotics. This is especially urgent for open systems [6, 7, 28], where introduction of
GMM into the environment is inevitable and predictable.

An evaluation of the possibility of transfer of the cloned DNA fragment to other

microorganism species in the environment is required [8]. Available experience from
analyzing the viabilities of selective producer strains and GMM in the natural
environment suggests that the viabilities of these microorganisms in the environment are
considerably inferior to those of microorganisms inhabiting natural ecological niches [1,
5, 10, 13, 19, 25]. Under conditions of natural habitat and artificial introduction,
microorganisms including GMM can release chromosomal and plasmid DNA from lysed
cells into the environment. Depending on external conditions, plasmids may preserve
their transforming activity for a long time. This is one of the serious risk factors from the
introduction, which potentially promotes uncontrolled dissemination of recombinant
DNA with possible penetration into cells of new hosts [1, 6, 20, 25, 27]. Experimental
data on risk evaluation and consequences of GMM introduction into the environment is
urgent and practically important both from the points of view of obtaining and
accumulating data on different aspects of biological safety using recombinant
microorganisms and of mastering methods used to perform such studies.

The goal of the present work was the evaluation of survival of a GMM
(recombinant B. subtilis strain VKPM B7092) in the bovine gastroenteric tract,
determine its effect on microbiocenosis and species composition of the microflora of
bovine gastroenteric tracts, examine the possibility of transfer of the DNA fragment
cloned in B. subtilis bacterium, containing the gene of human leukocytic alpha-2
interferon, to the representatives of intestinal microflora when the probiotic VETOM 1.1
is given to animals, as well as its transfer to other microorganism species the environment
(soil).

Materials and methods

B. subtilis VKPM B7092 strain is a recombinant strain which cells contain a

plasmid with the structural gene of the mature form of human leukocytic alpha-2
interferon. The above plasmid provides for the synthesis and secretion of biologically
active interferon by B. subtilis VKPM B7092 cells [18, 25]. Bacteria of B. subtilis
VKPM B7092 strain are aerobes. When grown on LA medium, they form chamomile-
like colonies. The colonies are whitish. The bacteria are Gram positive baculiform. The
cells of a one-day agar culture are (2 4)(0.5 0.8) mm large. They form spores, but do
not form capsules. Bacteria of this strain can reproduce at 15 50C, optimal growth is
observed at the temperature of 36 37C. They hydrolyze starch and reduce nitrates.
They split glucose, sucrose, mannose, maltose and lactose. The strain is canamycin-
resistant; besides alpha-2 interferon, it synthesizes an antibiotic of broad spectrum of
action inhibiting the growth of fungi, staphylococci, streptococci and Pseudomonas
aeruginosa [18].

The probiotic VETOM 1.1 manufactured by the JSC Scientific and Production
Company Research Center (Koltsovo, Novosibirsk region) was used in the
experiments.

A solid medium with canamycin was used to isolate B. subtilis VKPM B7092
from bovine feces [18], and generally accepted methods and standard bacterial media
were used for intestinal bacteria, etc. [11, 18, 21].

Animals that were healthy by veterinary indices were used in the work. The
animals were given the probiotic VETOM 1.1 two times a day at the rate of 75 mg of the
preparation (not less than 7.5104 of bacterial spores) per 1.0 kg of bodyweight for 9
days. The survival of the recombinant strain was estimated by the period of isolation from
gastroenteric tract (till complete elimination) via seeding the contents of feces of
experimental animals on nutrient bacterial media. Fecal samples were selected before
giving the preparation, daily for the whole period of giving the preparation and up the
moment of complete excretion of B. subtilis VKPM B7092 from the animal
gastroenteric tract. Besides, the probiotic effect on the microbiocenosis structure in the
animal, the gastroenteric tract was evaluated by changes in the numbers of dominating
microbial populations making part of the microbiocenosis determined by microbiological
analyses of the above samples and those collected 30 days after the preparation was first
given [11, 21].

The study of the possibility of transfer of a DNA fragment cloned in B. subtilis

bacterium to the representatives of intestinal microflora was performed using the method
of two-round PCR with specific primers. Specific primers were developed and
synthesized for the detection of specific DNA fragments with the program Oligo-6 (for
artificial marker of the gene of leukocytic human interferon with the lengths of fragments
of 452 bp and 328 bp, respectively; for the marker of canamycin resistance gene 834 bp
and 314 bp, respectively). The specificity of primers was confirmed using the program
BLAST (/gi:396443/, / gi: 31415690/, /gi: 29568850/, /gi: 3953638/) of the National
Institute of Health, USA (http://www.ncbi.nlm.nih.gov/blast/). PCR conditions for these
primers were selected and perfected. As it was known that the sought genetic fragments
were in plasmid structures that had been used to transform the B. subtilis VKPM B7092
strain, plasmid DNAs were isolated from bacterial cultures [26] obtained from animal
feces and were analyzed in PCR. Standard reagents, solutions and equipment were used
to perform the two-round PCR.

Amplification was carried out in 30 ml of a mixture containing 10X PCR-buffer

(10 mM of Tris-HCl, pH 8.3, 50 mM of KCl ), 2 mM of MgCl , 0.2 mM of each dNTP,
20 pM of each primer, 2.5 U of Taq- DNA-polymerase and 5 ml of the tested sample.
Thirty amplification cycles were performed for all the used primers in the following
mode:

The first round of PCR

95_ 3 min 1 cycle.
94_ 30 sec
58_ 30 sec
72_ 30 sec 30 cycles.
72_ 3 min 1 cycle.

After the first round, amounts of 1.0 l of amplificate were transferred to test
tubes with the reactive mixture for the second round of PCR.

The second round of PCR

95_ - 3 min 1 cycle.
94_ 30 sec
68_ 30 sec
72_ 30 sec 30 cycles.
72_ 3 min 1 cycle.

The detection of the amplified DNA after PCR was performed with gel
electrophoresis method in 2% agarose gel. The obtained results were considered
satisfactory when there was a clearly seen DNA band of the calculated length in the gel.
Samples were considered positive when they had a DNA band in the gel corresponding to
the DNA fragment of the control sample by length as well as to an appropriate band in
the marker of the fragments lengths.

Soil samples were collected at agricultural enterprises that had used the probiotic
VETOM 1.1 for treatment and prophylaxis of bovine diseases, in particular, at the JSC
Kirzinskoye, Ordynsk district, Novosibirsk region, where the preparation had been
used for eight years. Control soils were from APC Rogalevskoye, Ordynsk district,
Novosibirsk region, that had not used the VETOM 1.1. Soil samples were collected from
plots where manure from the farms of the JSC Kirzinskoye (samples # 1, 2 and 3,
respectively) had been added eight, five and one year ago. Samples from the plot of APC
Rogalevskoye (sample 4) where manure from the farms had been added six years ago
served as samples for comparison. Bacterial cultures from soil samples were isolated on
liquid and solid bacterial media supplemented with canamycin according to generally
accepted technique [11, 25, 18, 21].

Initial identification of the GMM strain among bacterial cultures isolated from
soil samples was performed by its cultural and morphological characteristics [18]. Exact
identification of the GMM B. subtilis VKPM B7092 strain was carried out with the
method of two-round PCR using specific primers. Cultures of microorganisms isolated
from soil were grown on a solid medium supplemented with canamycin. Plasmid DNAs
were isolated from the cell culture obtained at the end of the exponential the beginning
of the stationary phase of development with a modified Birnboim - Doly method of
alkaline lysis [26]. The obtained DNA was analyzed with the two-round PCR method.
Aqueous extracts were obtained from soil samples # 1-3 to reveal the possible presence
of the plasmid DNA that entered the environment from lysed bacterial cells of the GMM
and was preserved in soil during the analyzed periods. Plasmid DNA was isolated from
aqueous extracts and analyzed with the two-round PCR method.

Standard statistical methods were employed for processing and analysis of

obtained results [12].

Results and discussion.

The results of evaluating the survival of recombinant B. subtilis VKPM B7092

strain in bovine gastroenteric tract when the probiotic VETOM 1.1 was given to animals,
its effect on microbiocenosis and species composition of microflora in gastroenteric tract
of these animals are presented in Tables 1 and 2. The number of bacterial cultures isolated
from the contents of feces of each of experimental animals, as well as the results of
evaluating the possible of transfer of the GMM plasmid genes into these bacterial
cultures, are presented in Tables 3 and 4.

Table 1

The dynamics of isolation of B. subtilis VKPM V-7092 strain from bovine

gastroenteric tract when the probiotic VETOM 1.1 is given to animals.

Time of sampling, Number of bacteria per 1.0 g of feces, 103

Cow Bull Calf
Before administration day 0 0 0
1 2.8 (1.14.5) 2.5 (0.44.6) 4.8 (2.57.1)
2 7.3 (3.411.2) 6.1 (3.58.7) 6.4 (3.89.0)
3 2.6 (0.94.3) 9.2 (7.011.4) 9.8 (7.512.1)
4 6.8 (4.29.4) 3.2 (1.84.6) 2.4 (1.23.6)
5 8.4 (5.411.4) 10.4 (7.113.7) 5.2 (3.47.0)
6 7.6 (4.810.4) 5.4 (3.07.8) 10.4 (6.414.4)
7 1.0 (0.71.3) 4.0 (1.96.1) 4.8 (2.37.3)
8 7.6 (4.710.5) 6.4 (3.89.0) 7.2 (4.69.8)
9 10.8 (7.414.2) 10.0 (6.713.3) 12.0 (8.315.7)
10 10.0 (7.212.8) 11.2 (8.114.3) 11.6 (8.315.0)
11 4.4 (2.86.0) 7.6 (4.810.4) 6.0 (3.88.2)
12 2.4 (1.23.6) 5.2 (3.47.0) 2.4 (1.23.6)
13 3.2 (1.83.6) 2.4 (1.23.6) 0.9 (0.41.4)
14 Not determined Not determined 0.8 (0.11.5)
15 As above As above 0.1 (00.4)
16 - // - - // - 0.1 (00.4)
17 - // - - // - Not determined
18 - // - - // - As above
30 - // - - // - - // -

The data of Table 1 show that the excretion of B. subtilis from gastroenteric tracts
of all experimental animals ceases 7 days after the cycle of administration of the probiotic
VETOM 1.1 is completed. The obtained results are close to the data of determining the
terms of elimination of bacteria of B. subtilis 2335 pBMB105 strain, the active ingredient
of another recombinant probiotic Subalin, from gastroenteric tracts of some animal
species [1, 2, 3, 5, 15, 22].

Table 2

The study of the effect of B. subtilis VKPM V-7092 on the microflora of bovine
gastroenteric tract when the probiotic VETOM 1.1 is given to animals

Before After
30 days after
Composition of microflora giving giving
preparation
of bovine gastroenteric tract VETOM VETOM
was first given
1.1 1.1
COW (Note: ND = Not
Determined)
Pathogenic enterobacteria ND ND ND
6 5
E.coli with normal 5_10 3_10 9_104
enzymatic activity (62.8%) (100%)
6
E.coli with low activity 3_10 ND ND
(lactosonegative) (37.2%)
Hemolytic E.coli ND ND ND
5
Protei ND 3_10 ND
Other conventionally ND ND ND
pathogenic enterobacteria
Plasmonegative staphylococci 3_105 1.2_105 ND
6 4
Hemolytic streptococci 1_10 10 104
Lactobacteria 104 104 104
Fungi ND ND ND
7 7
Bifidobacteria 10 10 108
Clostridia ND ND ND
CALF
Pathogenic enterobacteria ND ND ND
5 5
E.coli with normal 1_10 4_10 1_105
enzymatic activity (3,4%)
E.coli with low activity 29_105 ND ND
(lactosonegative) (96.6%)
Hemolytic E.coli ND ND ND
7
Protei ND 3_10 ND
Other conventionally ND ND ND
pathogenic enterobacteria
Plasmonegative staphylococci 2_105 1.3_104 ND
6 4
Hemolytic streptococci 1_10 10 104
Lactobacteria 104 104 104
Fungi ND ND ND
7 5
Bifidobacteria 10 10 108
Clostridia ND ND ND
BULL
Pathogenic enterobacteria ND ND ND
4 4
E.coli with normal 10 7_10 2_105
enzymatic
(3%) (100%) (100%)
activity
E.coli with low activity 5_107 ND ND
(lactosonegative) (97%)
Hemolytic E.coli ND ND ND
Protei 12_107 8_105 ND
Other conventionally ND ND ND
pathogenic enterobacteria
Plasmonegative staphylococci 8_105 ND ND
4 4
Hemolytic streptococci 1_10 10 104
Lactobacteria 105 104 105
Fungi ND ND ND
Bifidobacteria 108 106 108
Clostridia ND ND ND

Microbial maps presented in Table 2 showed that after the 9-day cycle of
introducing of B. subtilis VKPM V-7092 cells into gastroenteric tract, the number and the
ratio in the dominating taxonomic groups of intestinal microflora remained practically
unchanged as compared to the initial indices. However, 30 days after the probiotic
VETOM 1.1 was first given the data demonstrating the improvement of qualitative and
quantitative compositions of the microflora of bovine gastroenteric tract were obtained.

Table 3 presents the results of determining the markers of the genes of human
leukocytic -2 interferon and canamycin-resistance in 48 isolated bacterial cultures from
feces contents of a bull, a cow and a calf after giving them the preparation VETOM 1.1
and full elimination of B. subtilis VKPM V-7092 cells from gastroenteric tracts of these
animals.

Table 3

Determination of the markers of the gene of human leukocytic -2 interferon and

canamycin-resistance gene in bacterial cultures isolated from the contents of feces of
experimental animals with the two-round PCR method

Determination of marker
Determination of marker
ANIMAL Time of of canamyncin-resistance
of _-2 interferon gene
cultures gene
isolation, days Cultures Cultures
Cultures Cultures
after the obtained on obtained on
obtained on obtained on
preparation medium medium
medium with medium with
was first given without without
canamycin canamycin
canamycin canamycin
BULL 0 - - - -
- - - -
- - + +
- - + +

12 - - + +
- - + -
- - - -

23 - - + -
- +
- -

COW 0 - - + +
- - + +
- - + +
- - + -

12 - - + -
 - - + -
- - - -
- -

CALF 0 - - + +
- - + +
- - -

12 - - + -
- -
- -

23 - - + +
- - + +
- -
- -

Note: + presence of the sought markers;

- absence of the sought markers.

The data of Table 3 demonstrate that 48 studied bacterial cultures do not contain
the gene of human leukocytic _-2 interferon in the cell genomes. In a portion of the
analyzed bacterial cultures isolated from the contents of feces of experimental animals
the marker of canamycin-resistance gene was detected. However, in the control initial
samples of bacterial cultures isolated from the contents of feces of the above animals
before the cycle of VETOM 1.1 administration, the presence of the marker of canamycin-
resistance gene was revealed even in a greater number of cultures (in 2, 4 and 2 cultures,
respectively) than after the cycle had been completed. Most likely, the obtained results
and the available literature data on rather frequent occurrence of canamycin-resistance
both in soil populations and in the microflora of animal gastroenteric tract [1, 3, 25] are
indicative of the circulation of canamycin resistance genes in native populations.

Nineteen canamycin-resistant bacterial cultures were isolated from soil samples as

a result of the study of remote consequences of using the probiotic VETOM 1.1 in the
areas of potential getting of GMM into the environment. Table 4 presents some of their
cultural and morphological properties, the results of PCR analysis of DNA samples
obtained from the above cultures as well as aqueous extracts from soil samples # 1-3.

Table 4

Cultural and morphological properties of bacterial strains isolated from soil

samples, results of PCR analysis DNA samples obtained from the above cultures as well
as aqueous extracts from soil samples # 1- 3
 Determination of
Cultural and Determination of
Sample Bacterial marker of
morphological marker of -2
# culture # canamyncin-
properties of soilstrains interferon gene
resistance gene
Round, dull, grayish-
white with yellow-tint
1 - +
convex colonies.Gram
positive cocci.
Convex, large, slimy
(juicy), with smooth
2 edges, yellowish - +
colonies. Gram-negative
cocci-form rods.
1 Small-grayish-white,
slightly rising above
3 agar, with smooth edges - +
colonies. Gram-positve
rods.
Large (up to 16mm),
flat, with smooth edges,
4 grayish-white colonies. - +
Gram-negative rods and
spores.
Aqueuous extract from soil sample # 1 -
Convex, shining, round,
grayish-white colonies.
1 - +
Gram-negative small
rods.
Large, flat, with smooth
edges, grayish-white
2 - +
colonies.Gram-negative
polymorphous rods.
2
Very large, transparent,
3 grayish colonies. Gram- - +
positive rods.
Convex, grayish-white,
shining, smooth,
4 slimy(juicy), round - +
colonies. Gram-negative
rods.
Aqueuous extract from soil sample # 2 -
3 1 Convex, grayish-white, - +
 shining, smooth, slimy
(juicy), round colonies.
Gram-negative small
rods.
Large, convex, round,
slimy (juicy), grayish-
2 - +
white colonies. Gram-
negative cocci.
Rough (dry), convex,
yellowish colony, poorly
3 - +
taken from agar. Gram-
negative rods.
Large, round, convex,
juicy, yellowish
4 colonies. Gram-positive - +
and Gram-negative
cocci.*
Aqueuous extract from soil sample # 3 -
4 Round shining, convex,
smooth,grayish-white
1 with green tint and - +
greenish colonies.
Gram-negative rods.

Dull, grayish-white,
round, small colonies
2 - +
with round edges,
convex. Very small
Gram-negative rods.
Flat, large with rough
edges. Gram-positive
3 rods with and without - +
spores with central and
terminal arrangement.

Large, grayish-white,
4 dull colonies. Thin and - +
long Gram-positive
rods.
5 Small with smooth - +
edges, convex, shining,
grayish-white colonies.
Individual Gram-
 positive cocci.
Small, grayish-yellow,
convex, round colonies.
6 - +
Individual Gram-
positive cocci.
Small, convex, grayish-
white, transparent,
7 - +
shining, round colonies.
Gram-negative rods.
Large, whitish
camomile-like colonies.
VKPM _-7092
Gram-positive + +
(control)
sporiferous rods. Do not
form capsules.

Note: * - a mixture of cultures of two strains obtained by taking of a colony of one strain
grown on a colony of another strain or two confluent colonies using a bacterial loop.

The data of Table 4 demonstrate that 7 of 19 species of canamycin-resistant

bacterial cultures were isolated from soil samples from the plot of APC Rogalevskoye.
This farm had never used the probiotic VETOM 1.1 for prophylaxis and treatment of
animals. Four different bacterial cultures were isolated from the soils of the JSC
Kirzinskoye where the probiotic had been used for one - eight years. The study of 19
isolated bacterial cultures by the main cultural and morphological properties [18] did not
reveal the presence of B. subtilis culture VKPM V-7092 among them. The presence of the
marker of canamycin-resistance gene was revealed in the genomes of the cells of 19
isolated bacterial cultures. Most likely, the obtained results and the available literature
data are indicative of the circulation of canamycin-resistance genes in native microbial
populations of genes and frequent occurrence of canamycin resistance in soil populations
[1, 3, 20, 22]. The studies bacterial cultures do not contain the marker of the gene of
human leukocytic -2 interferon. The cultural and morphological data of Table 4 and the
results of two-round PCR demonstrated the absence of B. subtilis bacteria VKPM V-7092
among the 19 studied bacterial cultures. According to PCR data, the analyzed aqueous
extracts of soil samples from the JSC Kirzinskoye, did not contain the recombinant
plasmid DNA of the above GMM, which could be preserved in soil during the whole
observation period (from one to eight years).

Conclusions

1. Bacteria of the GMM B. subtilis strain VKPM V-7092 do not colonize bovine
gastroenteric tract when the probiotic VETOM 1.1 is given to the above animals and are
fully excreted from the intestines of experimental animals four-seven days after the
probiotic administration had been completed.
2. The introduction of the probiotic VETOM 1.1 does not produce a negative effect on
intestinal microbiocenosis of experimental animals and the number and ratio in the
dominating taxonomic groups of normoflora. It was shown that introducing of the
probiotic promoted the normalization of the microflora of bovine gastroenteric tract.

3. The absence of transfer of plasmid genes of GMM B. subtilis VKPM V-7092 strain to
bacteria of intestinal microflora at its introduction into the animal organism was shown in
experiments.

4. Experimental evaluation of remote consequences of risks of introducing the GMM B.

subtilis VKPM V-7092 strain into the environment at observation periods from 1 to 8
years showed that bacteria of the given GMM were not detected in soil samples of the
agricultural enterprise that had used the probiotic VETOM 1.1 for treatment and
prophylaxis of cattle diseases, with methods of initial and exact identification. The
introduction of B. subtilis VKPM _-7092 strain into the environment did not result in its
unlimited growth and spread in soil.

5. The absence of transfer of plasmid genes (the genes of human leukocytic -2

interferon) from the GMM B. subtilis VKPM V-7092 strain to other microorganism
species spread in the areas of the GMM getting into the environment (soil) was shown in
experiments. The presence of recombinant plasmid DNA from lysed cells of the above
GMM was not revealed in soil samples.

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ASA thanks Dr. Stavskiy and his Vector group for sharing this information
with the ASA family of professionals.