Borrelia DNA

Pukhovskaya, Natalia M., Morozova, Olga V., Vysochina, Nelya P., Belozerova, Nadejda B. & Ivanov, Leonid I., 2019, Prevalence of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in ixodid ticks in the Far East of Russia, International Journal for Parasitology: Parasites and Wildlife 8, pp. 192-202 : 197-198

3.3. Quantitative estimations of Borrelia DNA in ixodid ticks

Average threshold cycles (Ct) of real time PCR with the analyzed B. burgdorferi s.l. DNA significantly differed among four examined tick species with the maximal deduced bacterial loads for I. persulcatus and

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genome-equivalents

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miyamotoi tick B. (10 / 5.35) 3 2) 10 8.81 / () 3 10 (/ 1.40) 10 2 5.98 / (2.05 /) 10 5 (/ () 10 3 2.86) 3 / 10 (3.39) 10 4 / (1.79 / 10 () 3 7.95

Average a per, 3 × 03 8.07 × 1 01 ×, × 1.94 × 7.61 2.21 × 2, 03 × × 6.54 × 9.56. Russia of 1.63 1.16 7.60 7.33 1.18 6.34 East Far

. l s. miyamotoi / .. l ± / 13.59 ± / 12.20 / 21.62 ± ± 17.57 / / ± 11,85 / 16.37 ±

the collected in and B burgdorferi . Ct. B Average. s B burgdorferi ± 33.34 0.64 35.25 / 12.87 34.92 ± 1.17 30.04 / 37.31 6.51 ± 25.37 33.80 / 11.17 ± 33.92 1,26 91 10.30, 28 ± 31.70 5.72 ± ticks persulcatus miyamotoi ) min-max (/ miyamotoi . l. s burgdorferi / 33.79 14.74 12.87 36.02 / 13.84 30.04 30.80 / 28.85 11.17 35.00 / 25.97 / 36.19 12.68 / 36.19 30.04 Borrelia range – 32.89 – 12.44 / 35.25 – 33.66 – 10.84 37.31 / 17.53 – – 11.92 33.80 / 32.54 – 12.46 – 21.62 – – 11.01 – 21.62 – 10.84 Ixodes with Ct B. B. loads in infection Number of with samples Borrelia bacterial Mixed the DNA 2 1 5 1 5 1 3 2 20 and lato Ct sensu Average of quantity genome-equivalents tick per a (6.56 2 10 ×)) 2.55 4 (10 ×) (10 × 2.47 2 3.65 10) × 4 () 7 × (10 7.64 × 9.52 (10 3) burgdorferi Ct Average 1.54 35.55 ± 30.27 ± 1.73 36.96 ± 2.29 ± 6.03 29.75 5.36 31.69 ± Borrrelia miyamotoi and monoinfection min- Ct range () max 37.24 – 33.94 – 31.55 27.74 38.58 – 35.34 33.25 – 22.78 18.72 – 18.72 38.25 Borrelia miyamotoi Number of with samples Borrelia of Borrelia the DNA 0 0 0 4 5 2 3 1 15 analysis of tick site, 16 comparative Number collection region Autonomous 7 8 9 territory 10 11 12 14 16 – 12 7, 14 6 Table Quantitative District Jewish Oktyabrsky Leninsky Birobidzhansky Khabarovsk Lazo Khabarovsky Nanaisky Solnechny Komsomolsky In total

H. japonica whereas those for H. concinna and D. silvarum were essentially lower ( Table 5). Bacterial loads estimated on the base of quantitative real time PCR varied in a wide range from 10 2 to 10 9 genome-equivalents per a tick ( Table 5). Based on the calibration curve of Ct from quantities of B. burgdroferi B31 genome-equivalents ( Morozova et al., 2011) and the Lukyanov-Matz equation, one might estimate the average number of genome-equivalents per a tick 5.63 × 10 7 for I. persulcatus , 5.55 × 10 7 for H. japonica , 1.13 × 10 4 for H. concinna and 2.61 × 10 3 for D. silvarum ( Table 5). The highest bacterial loads for I. persulcatus along with infection rate with B. burgdorferi s.l. exceeded the corresponding values of other tick species in all the examined regions during the whole period of observations (1999–2014) despite different tick abundance and dominance structure revealed the leading role of the taiga ticks in transmission of Borrelia . Similar quantitations for B. miyamotoi in I. persulcatus revealed the average bacterial load near 10 4 genome-equivalents per a tick in mono- and mixed infections (Table 6) that were in 10–1000 times less compared to amounts of B. burgdroferi s.l. (p <0.001).

3.4. Phylogenetic analysis of Borrelia species diversity in the Far East of Russia

Genetic diversity of Borrelia in the Far East of Russia ( Fig. 2 ) was similar to surrounding area of Eurasia ( Kurtenbach et al., 2006) including sequence from a tick collected from migrating bird in Japan (AB015911). Sequences of 16S rRNA gene and 5S ‾ 23S rRNA intergeniс spacer (ITS) of B. garinii corresponding to Asian type with prototype NT29 and European type with reference strain 20047 were found in all the examined regions of the Far East. The majority of B. afzelii isolates from ixodid ticks was identical to B. afzelii HLJ 01 strain (CP003882) from China and clustered with strain VS461 (GenBank accession number NR164748) from Switzerland ( Fig. 2 ) with the single nucleotide polymorphism (SNP) in 5S ‾ 23S ITS of Borrelia isolate Vanino 2011-2 (KX685729) similar to Borrelia sp. isolate Tokachi-J-IP21f (EF160140) from Japan. One should note that the only B. garinii (9 isolates) were found on the Sakhalin Island despite the simultaneous circulation of both B. garinii (23 isolates) and B. afzelii (12 isolates) in the continental part of the Far East of Russia. Observed fluctuations of ratios between B garinii and B. afzelii in two areas of the Khabarovsk territory with prevalence of B. garinii over B. afzelii (18:8, respectively) and in two collection places of the Amur region with the corresponding ratio species between B. garinii and B. afzelii near 1 (5:4) were not significant due to small sampling sizes ( Fig. 2 ). Phylogenetic analysis of nucleotide sequences of both 16S rRNA gene and 5S ‾ 23S rRNA ITS does not permit to distinguish between B. garinii and Borrelia bavarensis ( Fig. 2 ). Both species belong to the same clade with good bootstrap indexes ( Fig. 2 ).

3.5. MLSA analysis of B. miyamotoi

Multiple locus sequence analysis (MLSA) of B. miyamotoi nucleotide sequences of three fragments of 16S rRNA, glpQ and p66 genes showed similar patterns ( Fig. 3). Isolates of B. miyamotoi from the Far East of Russia belong to the Asian group previously found in Japan, China, Siberia and Ural ( Fig. 3). All our nucleotide sequences of 16S rRNA and p66 genes were identical to each other and to the strain FR64b (CP004217) from Japan, the only glpQ gene sequence includes the SNP identical to Siberian strain 57Nsk (FJ940729).

Figures

Fig. 2. Phylogenetic analysis of the nucleotide sequences of Borrelia 16S rRNA gene fragment of 733 bp long (A) and 5S‾23S ITS of 211–218 bp long (B) using Mega 6.06 software, UPGMA algorithm and 1,000 replications. Phylogenetic trees constructed by means of 5 alternative algorithms (Maximum likelihood, Neighbor- Joining, Minimum-Evolution, UPGMA and Maximum Parsimony) show similar topologies and reasonable bootstrap support. Nucleotide sequences of Borrelia isolates determined in our study are shown in bold. The number of identical sequences of Borrelia isolates determined in these ixodid ticks is shown in parentheses. Branches corresponding to the reference strains are underlined.