1 Introduction

Anthrax is an especially dangerous infection that occurs from time to time as a potential hazard for human and animal health. Anthrax outbreaks are regularly reported in various countries, including relatively recent ones in China [1], India [2], Bangladesh [3], Bhutan [4], Georgia [5], Germany [6] and other countries. There were 43 confirmed outbreaks in the northern region of Ghana which resulted in 131 cattle, 44 sheep, 15 goat, 562 pig and 6 human deaths [7]. In 2011, an anthrax outbreak in the district of Chama, Zambia, caused 85 hippopotamus deaths and infection of 511 people including lethal ones [8].

The widespread use of live vaccines in veterinary and human medicine requires genotyping of vaccinal and pathogenic strains of a causative agent and isolates obtained from sick animals. Current bacteriological and serological methods do not provide quick and reliable strain characterization of isolated biological pathogens. This can be done by means of molecular and genetic methods, including those for field screening [9].

Scientific studies of the genetic apparatus of bacteria enable decoding of the genome nucleotide sequence, which underlies establishing the molecular mechanisms of their antigenicity, immunogenicity and pathogenicity. At the same time, not only B. anthracis genomics, but also its proteomics has been researched [10]. The B. anthracis virulence is associated with the pXO2 plasmid, which carries the genes essential for the antiphagocytic capsule biosynthesis and degradation [11]. The pXO1 plasmid contains the genes that encode for a lethal factor, an edema factor and a protective antigen [12] present in both pathogenic and vaccinal strains. Recent studies with the use of genome data for pXO1 and pXO2 identified virulent properties of the Brazilian vaccinal B. anthracis strain [13]. The pXO1 plasmid encodes the synthesis of the protein Hfq3, which interacts with RNAs involved in essential functions of cell formation and growth [14]. In addition to encoding for the protective antigen, the pag gene of pXO1 is also involved in modulating the B. anthracis capsule biosynthesis [15]. A positive correlation between the number of atxA copies and the expression level of the pagA gene, encoding for a virulence factor such as the B. anthracis protective antigen has been found [16].

The contamination reference in a laboratory by B. anthracis strain, which occurred in 1981, was unexpectedly detected and confirmed with methods of next-generation sequencing and subsequent phylogenetic analyses more than 30 years after [17]. The identification of marker genes plays an important role in the detection and genotyping of new strains. For example, two new Bacillus strains—B. cereus E41 and B. anthracis F34—have been recently identified in Algeria [18]. Whole genome genotyping of historical B. anthracis strains has been conducted in Georgia [19] and Austria [20]. Multi-primer PCR improved the identification of B. anthracis typical and atypical strains and differentiation between closely related bacilli [21] and for polymorphism analysis [22].

This research was aimed to determine the effectiveness of the use of multi-primer PCR for the identification of B. anthracis pathogen isolates and their epidemiological status through genotyping in comparison with the historical strains.

2 Materials and Methods

В. аnthracis 55, CH-7, 81, 71/12, F34 strains, collection isolates #22 and #36, as well as isolates extracted from soil samples and animal cadavers obtained in the Middle Volga region of Russia, were used in this research. Bacteria of heterologous species (such as В. cereus strain 8035) and other genera (such as Brucella abortus strain 19 and Listeria monocytogenes strain AUF) were used to control the reaction specificity. Vegetative forms of bacilli were obtained by 16-h culturing in beef-extract agar (BEA) or by 5- to 6-h culturing in 2% Hottinger’s agar at 37 °С. The specificity of used primers were analyzed by BLAST NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

RCP samples were prepared with thermolysis: the grown cultures were transferred with a loop into 2.0-ml centrifuge tubes with a thermal latch on the lid, followed by addition of a lysis buffer. The tubes were heated at 98 °С for 15 min in the heating/cooling dry block and then precipitated on a centrifuge at 12,000 rpm for 60 s. A DNA-containing supernatant was taken into other test tubes.

PCR was performed on a Tercyc amplifier (DNA-Technology, Russia) using a PCR kit (Syntol, Moscow, Russia) according to the manufacturer’s instruction. The final volume of a 30 μl PCR mixture contained 3 μl of a 25 mM MgCl2 solution, 1 μl of 10 pM of each primer solution, 3 μl of a 2.5 mM dNTP solution, 3 μl of a 10× buffer for PCR, 1 μl of Taq polymerase, 10 μl of a DNA extract and 8 μl of deionized water. The PCR program used was as follows: (I) denaturation at 95 °C for 2 min; (II) 42 cycles: 10 s at 95 °C, 10 s at 55 °C, 10 s at 72 °C; (III) final elongation at 72 °C for 1 min. The amplification results were assessed with 2% agarose gel electrophoresis.

For genotyping, the amplicon production was performed using PCR kit (Syntol, Moscow, Russia) according to the manufacturer’s instruction. The final volume of 50 μl PCR mixture contained the following: 2 μl of 25 mM MgCl2 solution; 1 μl of 10 pM of each primer solution; 5 μl of 2.5 mMdNTP solution; 5 μl of 10× buffer for PCR; 1 μl of Taq polymerase; 5 μl of DNA extract and 30 μl of deionized water. Loci amplification for genotyping had the following programs: (I) denaturation at 95 °C for 2 min; (II) 42 cycles: 10 s at 95 °C, 10 s at 65 °C–55 °C (65 °C for the loci vrrA and vrrB1; 62 °C for the loci vrrC1 and vrrC2; 60 °C for the locus vrrB2; 58 °C for the CG3 locus, 57 °C for the pX01 locus, 55 °C for the pX02 locus), 10 s at 72 °C; (III) final elongation 72 °C for 1 min. After amplification, the fragments were dispersed in 1% low melting point agarose with subsequent DNA extraction using a GeneJETPCR purification kit (ThermoScientific) according to the manufacturer’s instruction. Multilocus sequence typing (MLST) was performed on an automated Applied Biosystems 3500ХL genetic analyzer (HITACHI/ High-Technologies Corporation, Japan). Sequence Scanner V1.0 was applied for reading and comparison.

3 Results and Discussion

3.1 Condition Optimization and Primer Construction for PCR

Three pairs of primers conditionally designated as Bacillus, рХО1 and рХО2 (Table 1) were designed and synthesized for В. аnthracis indication experiments. The first pair of primers, Bacillus, flanked a DNA region common for the genus Bacillus, while рХО1 and рХ02 being complementary to separate В. аnthracis plasmid DNA loci. Eight paired primers such as vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, pX01 and рХ02 were designed and synthesized for В. anthracis DNA MLST.

Table 1 Nucleotide sequence of primers used for B. anthracis indication and genotyping in this research
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To determine whether the microorganisms studied belong to the genus Bacillus, the region of chromosomal DNA was amplified using forward and reverse primers BacillusF and BacillusR. More precise sample indication was carried out when amplifying the first and second B. anthracis plasmids, the sequence designated as pXO1 and pXO2, respectively.

In genotyping, the annealing temperature changed depending upon a primer structure. To separate DNA strands more completely, preliminary denaturation (initialization) was carried out at 95 °С for 2 min. For genotyping the abovementioned data, eight pairs of primers were applied for different loci of the bacillus genome.

3.2 Multi-primer PCR of Strains and Isolates

Lysates of various В. аnthracis strains and other species of the genus Bacillus as well as Brucella and Listeria were used to determine the reaction specificity with the primers selected.

When amplifying the DNA fragments, three specific fragments were synthesized on a matrix of В. аnthracis strains CH-7, 81 (Fig. 1, lanes 7 and 17), that indicates they belong to the genus Bacillus and to a species having two plasmids in its genome. At the same time, the synthesis of two fragments (Fig. 1, lanes 5 and 12) was initiated in non-encapsulated В. аnthracis strains 55 and F34, the primers neither bound with heterologous species or genus genomes nor initiated DNA fragment synthesis. Three amplicons with different molecular weights were synthesized on matrices of the derived isolates and collection strains and isolates; these amplicons coincided with those of known pathogenic strains upon this criterion. The results of these experiments demonstrate that the primers detected DNA loci of microorganisms which belong to the genus Bacillus, while the presence of рХО1 and рХО2 plasmids indicated the pathogenicity of В. аnthracis strains, i.e. the primers allowed to differentiate pathogenic and vaccinal strains as well as isolates.

Fig. 1
figure 1

Multi-primer PCR results for tested samples. Lanes: 1—negative control (ТЕ-buffer); 2—positive control of В. аnthracis, plasmids chromosome + рХО1; 3—positive control of B. anthracis, plasmid рХО2; 4—B. cereus strain 8035; 5—В. anthracis strain 55, vaccinal; 6—B. anthracis isolate Lai (derived from a cattle cadaver, Laishevo, Tatarstan, 2014); 7—B. anthracis strain CH-7, pathogenic; 8—B. abortus strain 19; 9—B. anthracis isolate #22, from collection (derived from a sheep cadaver, Tatarstan, 1972); 10—L. monocytogenes strain AUF; 11—soil sample collected on the territory of the meat packing plant, Zelenodolsk, Tatarstan, 2004; 12—B. anthracis strain F34; 13—isolate U2 (soil-derived, Ulyanovsk, 2004); 14—isolate U1 (derived from a pig cadaver, Ulyanovsk, 2004); 15—B. anthracis isolate ZEL (derived from a cattle cadaver, Zelenodolsk, 2004); 16—B. anthracis isolate #36, from collection (derived from a sheep cadaver, Chuvashia, 1971); 17—B. anthracis strain 81, pathogenic, master seed strain

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The lower amplicon is genus-specific, that is, this locus is found in all species of the genus Bacillus, for example, in B. cereus (Fig. 1, lane 4). However, these species have no В. anthracis-specific plasmids; therefore, two upper amplicons are not detectable in PCR. On the contrary, pathogenic В. аnthracis strains have both plasmids, рХ01 and рХ02; therefore, two more amplicons are synthesized on their matrices; they are larger in molecular weight than the Bacillus-specific amplicon.

We examined four isolates such as isolates ZEL and Lai from Tatarstan and isolates U1 and U2 from the Ulyanovsk region. The isolates Lai and ZEL were derived from cattle cadavers (Laishevo and Zelenodolsk, Tatarstan, respectively). Soil samples were collected on the territory of a meat packing plant (Zelenodolsk, Tatarstan). The isolates Lai and ZEL were found to be pathogenic В. аnthracis strains (Fig. 1, lanes 6 and 15), with no В. аnthracis detected in the soil samples (Fig. 1, lane 11).

The isolate U1 was obtained from a pig cadaver, while U2 derived from a soil sample collected on a farm in the Ulyanovsk region where the pig died. Pathogenic B. anthracis strains with a full set of plasmids were identified (Fig. 1, lanes 13 and 14). To validate the PCR results, the known pathogenic В. аnthracis isolates obtained in Tatarstan (#22) and Chuvashia (#36) in 1972 and 1971, respectively, were examined in parallel (Fig. 1, lanes 9 and 16).

3.3 Sequencing of Derived Isolates

To clarify the origin of derived isolates ZEL, Lai, U1 and U2, they were genetically typed using amplicon sequencing at eight marker loci such as vrrA (310 bp), vrrB1 (240 bp), vrrB2 (170 bp), vrrC1 (600 bp), vrrC2 (530-600 bp), CG3 (160 bp), pX01 (120 bp) and рХ02 (140 bp) (Table 2).

Table 2 Size of amplified B. anthracis strain and isolate loci, basepairs
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Six isolates and one strain of В. аnthracis were examined; they are isolates ZEL and Lai, both derived from cattle cadavers (Tatarstan), the collection isolate #22, derived from a sheep cadaver (Tatarstan), the collection isolate #36, derived from a sheep cadaver (Chuvashia); the isolate U1, derived from a pig cadaver (Ulyanovsk), the soil-derived isolate U2 (Ulyanovsk), a vaccine strain 55, which is non-encapsulated and has no рХ02 plasmid.

Using complementary primers to the marker sites studied oligonucleotides (amplicons) were synthesized on a DNA matrix which were later sequenced. The analysis performed demonstrated the genetic relationship of the strains and isolates studied (Fig. 2). The vaccine strain 55 cannot be included into a phylogenetic tree as it lacks one marker. A special software was used to analyze a structural composition of sequences (Table 3).

Fig. 2
figure 2

Distribution of B. anthracis isolates according to the degree of genetic homology. Group A: isolate ZEL (derived from a cattle cadaver, Zelenodolsk, 2004); isolate #22, from collection (derived from a sheep cadaver, Tatarstan, 1972); isolate Lai (derived from a cattle cadaver, Laishevo, 2014); isolate #36, from collection (derived from a sheep cadaver, Chuvashia, 1971); Group B: isolate U1 (derived from a pig cadaver, Ulyanovsk, 2004); isolate U2 (soil-derived, Ulyanovsk, 2004)

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Table 3 Nucleotide sequence of the resulting amplicons
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The sequencing results demonstrate that the derived isolate ZEL is the same strain as the collection isolate #22 which was obtained in Tatarstan in 1972 and is different from the collection strain #36 obtained in Chuvashia (Fig. 2). At the same time the derived isolate Lai is the same strain as the collection isolate #36. However, their similarity facilitated combining all four strains into conditional group A. The isolated strains U1 and U2 were different from the four abovementioned isolated strains by genotype, which indicates their separate grouping (group B).

Thus, the distribution of B. anthracis isolates according to the degree of genetic homology based on the sequencing results suggests that the derived isolates were local strains surviving for a long time in the environment rather than those brought from other regions of Russia.

4 Conclusion

Our research has shown that multi-primer PCR can in general be used to clarify the epidemic situation with anthrax. Multi-primer PCR has sufficient sensitivity and specificity that defines it as an effective method for B. anthracis detection in laboratory diagnostics. The used primers allow to differentiate isolates by their belonging to the genus Bacillus and as B. anthracis species by the presence of рХО1 plasmid. The detection of the рХ02 plasmid encoding for capsule formation indicates pathogenicity of the isolate studied. Using multi-primer PCR allowed to determine the epizootic situation of anthrax in the Middle Volga Region and to establish that two genotypic groups of the pathogen B. anthracis circulate in the region.