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UDC 619:578.828.11:636.2 HAC Code 06.02.01 DOI: 10.32417/1997-4868-2021-213-10-48-54
Nitric oxide as an indicator for assessing the resistance and susceptibility of cattle to leukemia
U. Zh. Kuzhebayeva1*, I. M. Donnik2, M. V. Petropavlovskiy2, S. G. Kanatbaev3, B. E. Nurgaliev1
1 Zhangir Khan West Kazakhstan Agrarian-Technical University, Uralsk, Kazakhstan
2 Ural Federal Agrarian Scientific Research Center of Ural Branch of the Russian Academy of Sciences, Ekaterinburg, Russia
3 West Kazakhstan Innovative and Technological University, Uralsk, Kazakhstan
*E-mail: usya_999@mail.ru
Abstract. The role of allelic variability of inducible nitric oxide synthase (iNOS) is significant in the study of the resistance and susceptibility of animals to leukemia infection. After analyzing the literature data, it can be stated that in the iNOS gene, allele A (with genotype AA) is responsible for resistance to the leukemia virus, and allele B (with genotype BB) is responsible for susceptibility. This is due to the frequency of occurrence of alleles and their genotypes of the polymorphic marker AN13-1 of the inducibeal nitric oxide synthase gene. The iNOS gene is capable of producing a large amount of nitric oxide, compared to other isoforms. In turn, nitric oxide causes death or can stop the growth of pathogenic microorganisms, including viruses. The purpose of this work is to further study o nitric oxide as an indicator for determining the resistance and susceptibility of animals to leukemia, as well as the selection of specific primers for PCR-PDRF used in genotyping. Methods. The iNOS gene sequence was analyzed and a pair of specific primers were selected and synthesized using the Vector NTI program. Scientific novelty of this work lies in the fact that we have selected specific primers that are important for the analysis of cattle genotyping by allelic variants of the polymorphic marker AH13-1 of the iNOS gene. Results. Based on this work, a pair of primers iNOSF_new and iNOSR_new, with a calculated annealing temperature of 52 °C, were selected and syn-s thesized, giving an amplicon with a length of 186 bp. The amplicon contains a polymorphic site that distinguishes n the A and B alleles. During PCR-RFLP, the following genotype-specific fragments are formed: AA-47/139 bp; O AB -186/139/47 bp; BB-186 bp.
^ Keywords: leukemia, bovine, interleukin, nitric oxide, oligonucleotides, resistance, susceptibility, polymorphic.
£ For citation: Kuzhebayeva U. Zh., Donnik I. M., Petropavlovskiy M. V., Kanatbaev S. G., Nurgaliev B. E. Nitric g oxide as an indicator for assessing the resistance and susceptibility of cattle to leukemia // Agrarian Bulletin of the ^ Urals. 2021. No. 10 (213). Pp. 48-54. DOI: 10.32417/1997-4868-2021-213-10-48-54.
Date ofpaper submission: 27.08.2021, date of review: 02.09.2021, date of acceptance: 06.09.2021.
'¿2, Introduction territory of 64 subjects, according to official data, the g Currently, one of the widespread viral diseases caus- number of disadvantaged leukemia sites in the Russian ]> ing significant economic damage to the cattle breeding Federation reaches 1789 [6, p. 8]. ¡^ industry is bovine leukemia [1, p. 1058], [2, p. 3688], Bovine leukemia virus is an RNA-containing onco-■g [3, p. 255]. In the Republic of Kazakhstan, this disease genic virus that causes malignant lymphoproliferative was first registered in early 1966 in two farms of the disease of animals. According to the classification of ^ Karaganda region and in one farm of the Almaty region the International Committee for Taxonomy of Viruses i-i among young cattle of brown Latvian and red Lithuanian (ICTV), it belongs to the family Retroviridae (2 subfami-^ breeds imported in 1960 from the Baltic republics. In lies, 7 genera), the subfamily Orthoretrovirinae, the geg 1969, the number of disadvantaged settlements increased nus Deltaretrovirus, which also includes T-lymphotropic
Q to 22, in 1970 - to 49 and in 1983 - to 186 in 14 regions primate viruses (PTLV) - humans and monkeys (HTLV
^ of the republic. To date, the disease has been registered and STLV).
N in 12 out of 14 regions on the territory of the Republic According to the authors involved in the study of
^ of Kazakhstan. According to the "National Reference leukemia, it is known that almost 70 % of infected ani-
> Center for Veterinary Medicine" and the "Republican mals have an asymptomatic disease, the aleikemic stage.
¡^ Veterinary Laboratory" of the Ministry of Agriculture However, after a few years of the asymptomatic period,
of the Republic of Kazakhstan for 2015-2018, 0.44 % of 25-30 % of animals infected with the leukemia virus
the total number of cattle were examined, the degree of develop persistent lymphocytosis characterized by poly-
^ infection was 5.8 % [4, p. 150], [5, p. 79]. In the Rus- clonal expression of the neoplastic population of CD5+B
sian Federation, the leukemia virus is established on the lymphocytes, and 1-5 % develop B-cell lymphoma [7]. 48
However, a mechanism capable of providing protection against enzootic leukemia for cows infected with leukemia is still unknown.
The leukemia virus can be transmitted vertically and horizontally, as well as during procedures related to the transfer of infected blood between animals, such as needle reuse, horn amputation, rectal palpation, tattooing, the so-called iatrogenic pathway [8].
It is worth noting the importance of the influence of the level of proviral load (PVL) on the course of the infectious process. The level of proviral load is the amount of retroviral genome embedded in the host genome and associated with the progression of the disease. There are two profiles or phenotypes of the level of proviral load - high and low, which causes the transmission of the virus from infected to healthy animals. Animals with high levels of PLV are considered the main source of transmission and a risk factor for leukemia. According to the results of foreign studies, it was found that the level of PVL does not always correlate with the development of the hematological stage of the disease. To date, most researchers assume the relationship of allelic polymorphism of genes of cattle resistance to leukemia with the level of PVL. However, the biological mechanisms controlling the level of PVL and the progression of infection in cattle in relation to the breed and origin of animals have not been fully elucidated. The determination of the fundamental foundations of the biological mechanisms leading to the progression of BLV infection in cows is of great importance for the development of effective programs to combat the bovine leukemia virus [9].
Of significant importance in the development of leukemia infection is the natural resistance of the body and immunological reactivity, when infected with the leukemia virus, violations of cellular and humoral links of immunity are observed, as well as violations of erythro-, leuko-, and hematopoiesis with a simultaneous significant decrease in all metabolic reactions of the body.
It was found out that bovine leukemia is a disease with a hereditary predisposition due to genetic polymorphism, i.e. the existence in the population of several forms of a gene occurring with a certain frequency, serving as a measure of genetic variability of the population [10, p. 1440], [11, p. 14], as well as weak immunity often developing as a result of violations of the technology of maintenance and feeding. The manifestation of hereditary predisposition is determined by a complex of hereditary and external factors [12, p. 22], [13, p. 38], [14, p. 999]. To date, single nucleotide polymorphism (SNP) is widely used as a genetic marker for assessing DNA polymorphism, the high density of which makes it easy to identify a sufficient number of them near or inside the required gene. They are highly conservative, and they allow haplotype analysis of relatively short PCR fragments, thereby increasing the accuracy of the study. It is worth noting the importance of the analysis of the PDRF (polymorphism of the lengths of restriction fragments), in which the fragment is used as the simplest genetic marker. This analysis makes it possible to search for al-
lelic variants of genes that determine the resistance and susceptibility of animals to infections [15, p. 107].
Due to the high economic costs caused by the disease, scientists are working to find an effective solution to the issue of selection and production of animals with low susceptibility to leukemia [16, p. 1063].
To date, studies on polymorphism in the main histocompatibility complex (MHC) of cattle by alleles of the BoLA gene are widely conducted. This highly polymorphic gene is registered in the Immuno-Polymorphism (IPD) - MHC database (https://www.ebi.ac.uk/ipd/mhc/ group/BoLA/) and serves as a marker for determining the disease, and also forms immunological signs in cattle. It is worth noting that along with MNS, the role of the influence of cytokine network genes, which in turn are involved in the pathogenesis of viral diseases, including bovine leukemia, has also been studied to date. Much attention is paid to the assessment of cytokine gene polymorphism and their role in pathology, since single nucle-otide gene substitutions can affect the functional activity of genes, which in turn affects the course of the inflammatory process [17, p. 163].
Cytokines are low-molecular-weight soluble proteins that ensure the transmission of signals between cells, they are important both for the realization of innate and adaptive immunity [18, p. 154]. Cytokine synthesis is an inducible process that is triggered in response to the penetration of pathogens into the body, they are mainly released from immune cells, including monocytes, macrophages and lymphocytes. Proinflammatory cytokines -chemokines play an important role in the study of the molecular mechanisms of the occurrence of pathological processes [19, p. 342], [20, p. 617]. They, in turn, interact with the mediator of apoptosis, with the regulator of the innate immunity system, short-lived nitric oxide (NO) molecules. It is NO that causes the cytotoxic effect of cells of the immune system or can stop the growth of pathogenic microorganisms, including viruses [21, p. 281].
In connection with the above, the purpose of this work is to study nitric oxide as an indicator for determining the resistance and susceptibility of animals to leukemia, as well as the selection of specific primers for PCR-RFLP used in genotyping.
Methods
We took the structure of locus-specific DNA fragments of the iNOS gene being analyzed from the DNA sequence database presented in the NCBI international database. Then, in the Primer-BLAST program, the selection of specific oligonucleotide primers was carried out, after which they were aligned in the BLAST program. The selection of primers for RFLP analysis was carried out by us in the Vector NTI program. We have selected primers suitable for the following characteristics: the length of the primers is 17-28 n; the percentage of G + C pairs is 40-60; to avoid sticking of the primers to itself; the formation of dimers; the melting point is in the range of 52-59 °C.
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Table 1
Characteristics isoforms of nitric oxide synthase
eNOS iNOS nNOS
Alias Endothelial Inducible Neuronal
Molecular weight 135 kD 125-135 kD 150-160 kD
Number of amino acid residues 1203 1153 1434
Activity (max value) About 15 nmol/mg/min Up to 1000 nmol/mg/min About 300 nmol/mg/min
Function Regulates vascular tissue, blood pressure, inhibits platelet aggregation and leukocyte adhesion Response to stress and inflammation, involved in host defense and tissue destruction Involved in neurotransmission, modulates response to pain and neuronal sensitization
Gene expression Constitutively expressed Induced in response to immunostimulatory cytokines or bacterial pathogens Constitutively expressed
Dependency on calcium Calcium - calmodulin dependent Calcium - calmodulin independent Calcium - calmodulin dependent
NO production Small amount (pico to nanomolar) Large sustained amount of NO (nano to micromolar) Small amount (pico to nanomolar)
Signal for discontinuation of NO production Self-limited NOS degradation Self-limited
Cellular expression Endothelial tissue, cardiac myocytes, platelets, some brain neurons, placental syncytiotrophoblast and kidney epithelial cells Chondrocytes, synoviocytes, hepatocytes, macrophages and microglia Certain neurons of the brain
Subcellular localization Golgi apparatus, cell membrane in the area of small invaginations that contain transmembrane caveolin, cell nucleus, mitochondria Phagosomes, peroxisomes, membrane, cell nucleus, mitochondria Cytoplasm, endoplasmic reticulum, sarcolemma
The forward and reverse sequences of oligonucle-otides modeled by us were synthesized on the Expedite 8909 oligonucleotide synthesizer, Applied Biosystems. According to the protocols attached to the device, DNA synthesis was performed.
Results
To date, three isoforms of NO synthase (NOS) have been studied: neuronal (nNOS/NOS 1), endothelial (ENOS/NOS3), inducible (iNOS/NOS2). These isoforms are distinguished depending on the structure and localization. It is worth noting that the formation of nitrogen monoxide occurs as a result of the oxidation reaction by the O2 atom of L-arginine in the presence of a specific enzyme NO-synthase. Table 1 shows the characteristics of isoforms of nitric oxide synthesis.
Under the influence of two isoforms (neuronal and endothelial), regulated, continuous, but low-intensity synthesis of nitric oxide occurs as a response to receptor and physiological stimulation. In turn, the isoform of nos that interests us consists of three types of NO synthases: NOS2A, NOS2B, NOS2C. This isoform appears in cells after their induction. Compared with other isoforms, this one shows the formation of a high concentration of nitric oxide, which has a cytotoxic effect. Inducible nitric oxide synthase is not present in most cells under normal conditions, however, in various malignant diseases, the intracellular mechanisms controlling iNOS are not regu-
lated, which leads to constant expression and activation of iNOS. According to research data, S.Widdison iNOS of cattle shows great similarity with ios of other species and has a common protein domain [22, pp. 302-309].
The gene we are investigating is located on the 19th chromosome of cattle (Fig. 1).
It is also worth noting that 3 polymorphic markers A H 13-1, AH 13-2, AH 13-3 in the third intron are contained in the enos gene. In the works of S. V. Chichinina (2005) and H. H. Gilmanov (2019), the allelic polymorphism of the AH13-1 polymorphic marker of the iNOS gene was investigated, and the relationship of resistance and susceptibility of animals with the leukemia virus was established, previously in the works of M. P. Heaton (2001), the diversity of DNA sequences in the genes of cattle cytokines was assessed. For the analysis of cattle genotyping, a search for nucleotide sequences was performed. As a result, several genome sequences were identified and analyzed using the NCBI database (Fig. 2).
A genome with GenBank ID: AF465168 was used as a reference sequence. The genomic sequence of Bos taurus BTANOS2DS1 contains highly polymorphic single nucleotide sites. This genome consists of 258 bp (Fig. 3).
As a result, we selected 6 pairs of synthetic oligonucleotides per iNOS gene (Table 2).
According to the main criteria for PCR, a preliminary selection of a pool of primers was carried out.
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LOCI 01904699 LGRLS9 LVRM9
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Fig. 1. Chromosome 19 - NC_037346.1. Source: https://www.ncbi.nlm.nih.gov/gene/?term=AF465168
Fig. 2. Genome sequence analysis
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Table 2
Combination of selected primer pairs
No. Forward primer Reverse primer
1 GGCGGGTTTAAGCTCCTAGA GATGAGGCACTAGGCTCACG
2 GAGCCTAGTGCCTCATCAGT AAGTGGGGACCCTAACCTCG
3 AGAGGCCAGAGAGGAAGAAAG GGACCCTAACCTCGAAGACTG
4 AGGCGGGTTTAAGCTCCTAGA GGACCCTAACCTCGAAGACTG
5 AAAGACCTTGGCTTCTTCAGGG AAAGTGGGGACCCTAACCTCG
6 AGAGGCCAGAGAGGAAGAAAGT GGGGACCCTAACCTCGAAGAC
The next step was to check the specificity of the selected primers using the BLAST program, primers with 100 % specificity were selected. After that, we selected primers for PDRF analysis using the Vector NTI program (Fig. 4). Primers with an estimated annealing temperature of 52 °C were selected, giving an amplicon with a length of 186 bp. The amplicon contains a polymorphic site that distinguishes the A and B alleles. Allele A contains a HinfI restriction site, the PCR product obtained from it is cut into fragments 47 and 139 bp long. Allele B does not contain a Hinfl restriction site.
Thus, during PCR-PDRF, the following genotype-specific fragments are formed: AA - 47/139 bp; AB -186/139/47 bp; BB - 186 bp (Table 3).
Automatic synthesis of oligonucleotides was carried out using special devices - DNA synthesizers. The synthesis of the primer was carried out by gradual completion of the nucleotide, according to its nucleotide sequence.
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Table 3
Primer sequence and PCR-RFLP genotype profiles of the iNOS gene
Gene Primer PCR-RFLP genotype profiles (bp)
iNOS iNOSF new: 5 AGAGGCCAGAGAGGAAGAAG-3 ' (21 b.p.) iNOSR new: 5'-GGACCCTAACCTCGAAGACTG-3' (21 b.p.) AA = 47/139 BB = 186 АВ = 186/139/47
Bos taurus BTANOS2DS1 genomic sequence containing highly polymorphic single nucleotide sites
Sequence ID: AF465168.1 Length: 258 Number of Matches: 1
Range l: 1 to 258 G en Bark Graphics
' Next Match A Previous Match
Score
462 bits(250)
Expect 4e-126
Identities 253/253(100%)
Gaps
0/253(0%)
Strand Plus/Plus
Query 1 AGGCGGGTTTAAGCTCCTAGAGAGGCCAGAGAGGAAGAAAGTAGAAAGACCTTGGCTTCT SO
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Sbjct 1 AGGCGGGTTTAAGCTCCTAGAGAGGCCAGAGAGGAAGAAAGTAGAAAGACCTTGGCTTCT SO Query 61 TCAGGGAGTYGTCACAGTGAGCCCGCCTCCGTGAGCCTAGTGCCTCATCAGTGRCCCATT 12 0
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Sbjct 61 TCAGGGAGTYGTCACAGTGAGCCCGCCTCCGTGAGCCTAGTGCCTCATCAGTGRCCCATT 12 0 Query 121 TCCTTGGTTTCTTTCT GAC T T GAAAC CAT T T RGAGGAGACAGGGGGGAT C T T TAAGAGGT ISO
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Sbjct 121 TCCTTGGTTTCTTTCT GAC T T GAAAC CAT T T RGAGGAGACAGGGGGGAT C T T TAAGAGGT ISO Query lai AAC TTCAGTCTTC GAGGT TAGGGTCCCCACTTT GTAGAGGGGAT GAGAART TGGTTTTGC 240
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Sbjct lai AAC TTCAGTCTTC GAGGT TAGGGTCCCCACTTT GTAGAGGGGAT GAGAART TGGTTTTGC 240 Query 2 41 AGCTGATGGGTCCAGTGT 25S
I I I I I I I I I I I I I I I I I I
Sbjct 2 41 AGCTGATGGGTCCAGTGT 258
Fig. 3. Bos taurus BTANOS2DS1 genomic sequence containing highly polymorphic single nucleotide sites
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1 iAGGCGGGTTT AAGCTCCTAG AGAGGCCAGA GAGGAAGAAA GTAGAAAGAC CTTGGCTTCT tcagggagtIy] GTCACAGTGA GCCCGCCTCC GTGAGCCTAG
ÏTCCGCCCAAA TTCGAGGАТС TCTCCGGTCT CTCCTTCTTT CATCTTTCTG GAACCGAAGA agtccctcjJr] CAGTGTCACT CGGGCGGAGG CACTCGGATC
101 TGCCTCATCA GTGRCCCATT TCCTTGGTTT CTTTCTGACT TGAAACCATT TRGAGGAGAC aggggggatc TTTAAGAGGT AACTTCAGTC TTCGAGGTTA
ACGGAGTAGT CACYGGGTAA AGGAACCAAA GAAAGACTGA ACTTTGGTAA AYCTCCTCTG TCCCCCCTAG AAATTCTCCA TTGAAGTCAG AAGCTCCAAT
R-52 new 100.0K
201 GGGTCCCCAC TTTGTAGAGG GGATGAGAAR TTGGTTTTGC AGCTGATGGG TCCAGTGT
CCCAGGGGTG АААСАТСТСС CCTACTCTTY AACCAAAACG TCGACTACСС AGGTCACA
R-52 new 100.05Í
Fig. 4. Selection of primers in the program "Vector NTI". The polymorphic site is highlighted with a red rectangle
Discussion and Conclusion
As mentioned above, bovine leukemia is a problem of the livestock industry all over the world, leading to significant economic losses due to premature death of animals from lymphomas, loss of carcasses during slaughter, reduced milk yield and reduced immunity of animals, as well as the impact on reproductive ability and longevity. In this regard, the search for genetically stable animal breeds is relevant today.
According to V. L. Petukhov (1992), animals that do not get sick for a long period, as well as breeding bulls with a low degree of daughter disease, are characterized by a high concentration of resistance genes and the duration of the body's resistance to pathogens, depends on the number of corresponding genes present in the genotype of animals.
In studies of animal resistance to leukemia, an active study of the allelic structure of the BoLA-DRB3 gene is currently underway, along with it, it is worth noting the role of the iNOS gene also responsible for resistance to infectious diseases.
Inducibeal nitric oxide synthase is an enzyme that catalyzes the formation of nitric oxide. In turn, as noted in the literature, NO is an important signaling molecule, which in turn is released by a large number of cells acting in many tissues. It has a regulating effect on a wide range of physiological and biological processes, such as neurotransmission, immune protection and apoptosis.
Earlier, in the works of S. V. Chichinina, H. H. Gil-manov, work was carried out to study the iNOS gene and its haplotypes. The allelic polymorphism of the iNOS gene is evaluated and the genotypic structure of the ana-
lyzed animal population is established in the context of carried out - iNOSF_new: 5'-AGAGGCCAGAGAG-genetic resistance and sensitivity to infection. The pos- GAAGAAG-3', iNOSR_new: 5'-GGACCCTAACCTC-sibility of using this gene as an SNP marker for cattle GAAGACTG-3', in the required amount on the oligoidentification was studied. An associative relationship nucleotide synthesizer, according to the manufacturer's was established between the genotypes of the polymor- protocols. Genotype-specific fragments were also iden-phic marker AH13-1 of the iNOS gene of cattle and their tified: AA - 47/139 bp; AB - 186/139/47 BP; BB - 186 bp breeding value in terms of milk productivity. formed during PCR-RFLP analysis.
Thus, we searched for complete nucleotide sequences Such studies, to determine the resistance and suscep-
of the iNOS gene for the analysis of cattle genotyping. tibility of animals, can be used to monitor the potential
A comparative analysis and multiple alignment of the of genetic resistance of a population to leukemia, which
complete nucleotide sequences of the gene were carried makes it possible to plan long-term breeding work in or-
out. And based on the analysis of gene sequences, us- der to combat the disease, as well as to develop animal
ing the Vector NTI program, primers for PDRF analysis breeding programs for individual farms that are disad-
were selected. And the synthesis of specific primers was vantaged by bovine leukemia.
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Authors' information:
Ulbolsyn Zh. Kuzhebayeva1, doctoral student, ORCID 0000-0002-7887-3376, AuthorlD 1106962;
+ 7 74 7 926-12-32, usya_999@mail.ru
Irina M. Donnik2, doctor of biological sciences, professor, honorary worker of Higher Professional Education of the
Russian Federation, Academician of the Russian Academy of Sciences, Vice President of the Russian Academy of
Sciences, ORCID 0000-0002-8593-7470, AuthorlD 313786; imdonnik@presidium.ras.ru
Maksim V. Petropavlovskiy2, candidate of veterinary sciences, senior researcher of the leukemia laboratory,
ORCID 0000-0002-9892-6092, AuthorlD 676746; +7 902 877-46-57, Petropavlovsky m@mail.ru
Serik G. Kanatbaev3, doctor of biological sciences, professor of the department of ecology and biotechnology,
ORCID 0000-0003-0640-4316, AuthorID 336795; +7 777 385-48-46, serik_kg@mail.ru
Birzhan E. Nurgaliev1, candidate of veterinary sciences, acting associate professor, head of the Higher School
of Veterinary Medicine and Biosafety, ORCID 0000-0001-5998-8250, AuthorID 1063767; +7 747 1 62-81-23,
nurgaliev. 79@mail.ru
1 Zhangir Khan West Kazakhstan Agrarian-Technical University, Uralsk, Kazakhstan
2 Ural Federal Agrarian Scientific Research Center of Ural Branch of the Russian Academy of Sciences, Ekaterinburg, Russia
3 West Kazakhstan innovative and technological University, Uralsk, Kazakhstan
