INTRODUCTION

Enzootic Bovine Leukosis (EBL) is a chronic infectious disease of a tumorous nature. It causes significant economic damage to the dairy and beef cattle industry due to poor production, low quality, cattle mortality, and expensive epidemic prevention measures [1, 2].

Foodstuffs of infected animals can be dangerous to humans due to the harmful metabolites it contains. The causative agent affects all kinds of raw material (milk, meat, by-products) and products, remaining a potential source of human infection [3–5].

Pasteurization of milk inactivates the virus but does not degrade its genome. The genetic material of the provirus maintains its integrity in canned meat [6]. Moreover, there are dairy products with partial pasteurization regime, e.g. classic cheeses, granulated cottage cheese, powdered milk with a low heating temperature, etc. The temperature processing parameters used in accordance with the regulatory and technical documentation cannot destroy harmful metabolites and, in some cases, do not kill the virus [7].

According to some researches, DNA of BLV provirus was found in epithelial cells of human mammary glands, including those of breast cancer patients. The hypothesis states that BLV may destabilize the host genome, thus leading to cancerous degeneration of cells [8–12].

Obtaining high-quality raw materials of animal origin is the most important challenge for meat and dairy industry. The challenge includes the development of functional and gerodietic foods [13–15].

According to the requirements of the Technical Regulations of the Customs Union “On safety of milk and dairy products” (TR CU 033/2013), BLV preventive measures and eradication activities are extremely important, given the significant prevalence of this incurable disease in the Russian Federation [16, 17].

An early genetic diagnosing of the pathogen is part of the system of anti-epizootic measures, followed by the removal of infected animals from the herd.  Molecular genetic research methods make it possible to assess the genetic diversity of BLV [18]. This is the most informative approach to the gene identification of the virus. Molecular genetic research methods work well for the phylogenetic analysis of nucleotide DNA sequences of the provirus, as well as for PCR-RFLP analysis according to the phylogenetic classification of the pathogen [19].

The current phylogenetic classification of BLV includes ten genotypes. The first seven genotypes were described by Argentinean scientists in 2009 [20], while genotype 8 was described by researchers from Russia [21–23], Croatia [24], and a European team of scientists [25] in 2011–2013. Genotype 9 was investigated by a team of Argentinean, Chilean, and Japanese scientists in 2016 [26]. Genotype 10 was described by a team of researchers from Thailand and South Korea [27] in 2016.

The objective of the current research was to study the scientific and methodological approaches to the genetic identification of BLV integrated into the molecular monitoring of infection of cattle herds with BLV genotypes. The following tasks were set:

• to establish the genotypes of BLV isolates in Tearstain cattle;
• to define the types of BLV isolates with deciphered nucleotide sequences of the env gene fragment, depending on the chosen gene identification strategy;
• to improve the strategy of PCR-RFLP-genotyping of BLV and make it consistent with the modern phylogenetic BLV classification.

STAGY OBYECTS AND METHODS

The research involved a total of 179 blood samples from AGID-positive cows. The samples were provided by agricultural enterprises from 21 districts of the Republic of Tatarstan. The samples were genetically examined for BLV. The examination included a phylogenetic analysis of sequenced env gene fragment of the pathogen and a PCR-RFLP-genotyping consistent with the phylogenetic classification of the infectious agent.

To extract DNA from the whole conserved blood, we used a commercial PCR diagnostic kit, ‘DNA-Sorb B’, produced by the Central Research Institute of Epidemiology of the Federal Supervisory Service for Consumer Rights and Human Welfare, Ministry of Health of the Russian Federation.

Nested PCR with extracted samples of BLV proviral DNA was performed with external (env5032 and env5608) and internal (env5099 and env5521) primers initiating the generation of env gene 444 bp fragment of causative agent at the final stage of reaction [28].

Restriction endonucleases used in PCR-RFLP-genotyping of BLV were consistent with its phylogenetic classification: BstYI (isoshizomer BstX2I), HphI (isoshizomer AsuHPI), HaeIII, PvuII, SspI. The NEBcutter v.2.0 web resource was used for PCR-RFLP modelling.

For the detection of the obtained results of PCR and PCR-RFLP analysis, 2.5% agarose gel horizontal electrophoresis was applied with a TBE buffer (pH 8.0) containing ethidium bromide. The electrophoregrammes were examined in a UV-transilluminator (λ = 310 nm). The sizes of the generated DNA fragments were compared with standard DNA molecular weight markers (SibEnzyme Ltd, Russia).

Sequencing of the PCR amplification products of the env gene fragment of detected BLV provirus isolates was performed on ABI PRISM 3100 Genetic Analyser (Applied Biosystems, USA) in the laboratory of Scientific and Technical Complex Sintol (Russia). Internal oligonucleotide primers env5099 and env5521 were used as sequencing. The sequenced fragments of the env gene of BLV provirus isolates were aligned with the corresponding nucleotide sequences of the reference BLV isolates from GenBank with the help of BLAST and MEGA-4 programmes. The last stage included a phylogenetic analysis.

RESULTS AND DISCUSSION

The study featured a PCR-RFLP-genotyping and a comparative phylogenetic analysis of the aligned sequences of the env gene fragment of BLV provirus isolates from 21 districts of the Republic of Tatarstan.

As a result, out of 179 identified isolates, ten isolates belonged to genotype 1; 106 isolates belonged to the cluster of genotype 4; 55 were characterized as genotype 7, and the remaining eight provirus isolates belonged to genotype 8 (Table 1).

According to the results obtained by PCR-RFLP-genotyping and phylogenetic analysis of sequenced env gene fragment, there are four out of ten currently known BLV genotypes in Tatarstan: 1, 4, 7, and 8.

Fig. 1 shows the genotypes of BLV isolated with the help of phylogenetic analysis of nucleotide sequences of env gene fragment.

An additional assessment of the heterogeneity of the reference BLV representatives for the env gene included an analysis of the intra- and intergenotypic heterogeneity of genotypes. The data in Table 2 indicate that it is impossible to use the ‘heterogeneous’ criterion for assessing the genetic diversity of BLV.

As part of the next task, BLV isolates with the decoded nucleotide sequences of the env gene fragment were identified according to the chosen genetic identification strategy. The degree of consistency of genotypic approaches was assessed by comparing the data of the in silico PCR-RFLP and the phylogenetic analyses.

A comparative analysis of 505 nucleotide sequences of the BLV env gene locus, including those deposited with GenBank NCBI, confirms the inconsistency of a number of earlier PCR-RFLP typing strategies [28–30] with the current approach in assessing the genotypic diversity by means of phylogenetic analysis.

Thus, the BLV isolates that belong to the Belgian subgroup according to D. Beier et al. (2001) [28], belong to genotype 4 according to the phylogenetic classification; Australian subgroup can be referred to genotypes 1, 3, 6, 8, or 9; Japanese subgroup – to genotypes 1, 6, or 7. In addition, the genotyping strategy [28] includes 11 additional unique combinations of PCR-RFLP profiles, conditionally identical to 11 unclassifiable BLV subgroups (Table 3).

Besides, BLV isolates that belong to genotype 1 and 7 according to the phylogenetic analysis, can be referred to Australian and Japanese subgroups, as well as to three unclassifiable subgroups, according to the strategy of D. Beier et al. (2001) [28]; genotypes 2, 5, and 10 belong to two unclassifiable subgroups; genotypes 3 and 8 – to Australian subgroup; genotype 4 – to the Belgian subgroup and four unclassifiable subgroups; the genotype 5 – to two unclassifiable subgroups; genotype 6 – to the Australian, Japanese and two unclassifiable subgroups; genotype 9 – to the Australian and one unclassifiable subset (Table 3).

BLV isolates that were genotyped according to
M. Licursi et al. (2002) [29] as genotype 1 may belong to genotypes 1, 4, 6, or 7 according to the phylogenetic classification; genotype 3 – to genotypes 1, 6, or 7; genotype 5 – to genotypes 1, 3, 6, 7, or 9; genotype 6 – to genotypes 2, 4, 5, or 7 (Table 4).

For the genotyping strategy described in [29], there are 19 unique combinations of PCR-RFLP profiles that are conditionally identical to 19 unclassifiable BLV genotypes (Table 4).

Besides, BLV isolates that are genotyped according to phylogenetic analysis as genotype 1 may refer to 1, 3, 5, and three unclassifiable BLV genotypes according to the strategy of M. Licursi et al. (2002) [29]; genotype 2 belongs to genotype 6 and two unclassifiable genotypes; the genotype 3 – to genotype 5 and one unclassifiable genotype; genotype 4 – to genotypes 1 and 6 and five unclassifiable genotypes; genotype 5 – to genotype 6 and two unclassifiable genotypes; genotype 6 – to genotypes 1, 3, and 5 and three unclassifiable genotypes; genotype 7 – to genotypes 1, 3, and 6 and four unclassifiable genotypes; genotype 8 – to one unclassifiable genotype; genotype 9 – to genotype 5; genotype 10 – to three unclassifiable genotypes (Table 4).

It should be mentioned that, when analyzing in silico PCR-RFLP data from 505 BLV representatives, we found not a single nucleotide sequence of the env gene fragment that would belong to genotypes 2 and 4 according to M. Licursi et al. (2002) (Table 4). This fact did not make it possible to prove the actual existence of PCR-RFLP profiles indicated for these two BLV genotypes.

Table 1. Distribution of 179 genotyped samples of BLV provirus DNA according to 21 districts of the Republic of Tatarstan, Russian Federation

Fig. 1. Dendrogramme of 99 isolates of 10 BLV genotypes, based on a phylogenetic analysis of the env gene fragment [MEGA-4: algorithm NJ, 400 nt, 99 seq.] Legend: black diamond marks GenBank NCBI nucleotide sequences of the env gene fragment of BLV provirus isolates in the Republic of Tatarstan.

Table 2. Intra- and intergenotypic heterogeneity of reference BLV representatives according to env gene (% ratio)

Table 3. Comparison of in silico data for PCR-RFLP (typification according to D. Beier et al., 2001) and the phylogenetic analysis of the BLV env gene fragment

Legend. N is the number of analysed BLV isolates with an established PCR-RFLP profile; “?” – an unclassifiable BLV taxon.

Table 4. Comparison of in silico data of PCR-RFLP (typification according to M. Licursi et al., 2002) and phylogenetic analysis of a fragment of the BLV env gene