Detection of Francisella tularensis in ticks and identification of their genotypes using multiple-locus variable-number tandem repeat analysis

Background Tularemia was reported in China over 50 years ago, however, many epidemical characteristics remain unclear. In the present study, the prevalence of Francisella tularensis in ticks was investigated during an epidemiological surveillance in China and then we measured their genetic diversity by conducting multiple-locus variable- number tandem repeat analysis (MLVA). Results 1670 ticks from 2 endemic areas (Inner Mongolia Autonomous Region and Heilongjiang Province) and 2 non-endemic areas (Jilin and Fujian Provinces) were collected and tested for evidence of tularemia by nested PCR. The prevalence of Francisella tularensis in ticks averaged 1.98%. The positive rates were significantly different among tick species, with Dermacentor silvarum and Ixodes persulatus responsible for all positive numbers. All F. tularensis that were detected in ticks belonged to F. tularensis subsp. holarctica and MLVA disclosed genetic diversity. One subtype was identified in 17 of 33 positive tick samples in three different study areas. Another subtype belonging to F. tularensis subsp. holarctica genotype was described for the first time in the current study. Conclusion The study showed two tick species, D. silvarum and I. persulatus harboring the pathogen of tularemia in natural environment, indicating these two tick species might have a role in tularemia existence in China. MLVA results disclosed the genetic diversity F. tularensis and identified one genotype as the most prevalent among the investigated ticks in China.


Background
Tularemia, also known as rabbit fever or deer-fly fever, is caused by the gram-negative intracellular pathogen Francisella tularensis (F. tularensis), which is highly infectious and considered as a potential bioweapon. Currently there are four recognized subspecies of F. tularensis: subsp. tularensis, holarctica, mediasiatica and novicida. The former two subspecies are clinically important. F. tularensis is widely distributed in the Northern hemisphere and is capable of infecting hundreds of different vertebrates and invertebrates [1][2][3]. In addition, a variety of arthropods, including multiple genera of ticks as well as deer flies, fleas, mites, and mice, have been found to be naturally infected or experimentally competent as vectors. In many endemic areas, ticks play an important role in transmission of the disease from animals to humans [3], with the genera Amblyomma, Dermacentor, Haemaphysalis, Ixodes and Ornithodoros acting as main vectors [4].
In China, F. tularensis was isolated in Citellus dauricus from Tongliao region, Inner Mongolia municipality as early as in 1957 [5,6]. The first human tularemia epidemic, including 14 reported cases, which was caused by contact with infected hares, was reported in Heilongjiang province in 1959 [7]. The natural foci of the disease were subsequently identified in Tibet and Xinjiang municipalities [8][9][10]. The agent was detected and isolated from patients, Ixodex liberelis, Dermacentor marginatus and wolly hares (Lepus oiostolos) from Qinghai Province and Tibet, Xinjiang Autonomous Regions during 1962-1986 [8][9][10][11]. An outbreak of tularemia occurred in Shandong province in 1986. All the 31 reported patients were workers in a foodprocessing factory, where hares were slaughtered and processed [12]. Serological investigations demonstrated that the seropositive reaction to F. tularensis was significantly associated with tick exposure [8,13]. However, little is known about the prevalence of F. tularensis in ticks in China, lack of such information has inhibited our fully understanding of the disease ecology and also under-estimated the threat of the pathogen to human health where ticks are abundant. In the present study, we try to investigate the presence of F. tularensis in ticks, and then to identify their genotypes using multiple-locus variable-number tandem repeat analysis (MLVA).

Prevalence of F. tularensis in ticks
Out of 1670 adult ticks examined, thirty three were positive for the fopA gene of F. tularensis. The overall positive rate was 1.98%. All positive samples amplified the 220-bp fragment for the ppI-helicase gene that identifies F. tularensis subsp.holarctica (GenBank accession no. EU526911). Thus, all ticks in the present study were infected with F. tularensis subsp.holarctica. The positive rates of F. tularensis in D. silvarum were 3.67% and 2.33% in I. persulatus (Table 1), while all of the 144 Haemaphysalis verticalis and 299 Boophilus microplus were PCR-negative. The difference in positive rates among different species was significant (χ 2 = 14.366, P = 0.002). We were able to detect F. tularensis in ticks from Jilin, Heilongjiang and Inner Mongolia, but not from Fujian during the present study. The positive rates varied according to their geographical origins (Table  1), with minor statistical significance (χ 2 = 7.865, P = 0.049).

MLVA
Two F. tularensis MLVA markers (Ft-M3 and Ft-M10) were successfully amplified from the 33 samples that tested positive for both the fopA gene and PPI-helicase PCRs. The sequences from Ft-M10 were invariant, 2 copies and 361 bp in size (GenBank accession no. EU544140), same as previously reported [14]. The Ft-M3 loci had two allelic variants (9A: AACAAAGAC and 9E: AATAAGGAT), exactly as defined by Johansson for F. tularensis subsp. holarctica [15,16]. The sequence size from Ft-M3 ranged greatly from 270 to 369 bp and the repeating units differed from 7 to 18 copies (GenBank accession no. EU544141 to EU544147). Based on the copy numbers of two loci, seven genotypes were identified ( Table 2). The Ft-M3 genotype containing 9 repeat copies was detected at 3 study sites in over half of the F. tularensis positive ticks (17/33, 51.5%). The significance of this genotype-host association remains unknown. Different genotypes could be detected from the same location (Table 2), among which, one Ft-M3 genotype containing 7 repeat copies from I. persulcatus was unique which had been found existence in F. tularensis subsp. tularesnsis in Goethert's study [17].

Discussion
Although F. tularensis can be cultured in the laboratory, it requires enriched growth media and BSL3 facilities for isolation, these problems, together with the difficulty of recognizing the bacterium make it lack rapid and safe methods for laboratory identification [18]. Nowadays, various PCR approaches amplifying different genomic segments have been applied to detecting F. tularensis [19][20][21][22]. The evaluations were performed during epidemics in Sweden in 1995 and 1998, respectively. It proved PCR

Conclusion
This study showed the role of two tick species in harboring the pathogen of tularemia in natural environment in China. In endemic foci where these genera ticks are abundant, public health officials and clinicians should be alert to the presence of tularemia, although it is not notifiable disease in China. In addition, MLVA results disclosed the genetic diversity of detected F. tularensis and identified one dominant genotype existing in ticks. Future work will involve more markers other than Ft-M3 and Ft-M10 detected in multiple-labs so to make comparisons with isolates of worldwide origin possible [29,30].

DNA extraction
Ticks were processed individually. DNA extraction was performed by a modification of a previously described method [31]. Briefly, each tick was soaked in 70% ethanol for 10 minutes, and then rinsed three times in sterile water. Then the tick was placed into microtube and mechanically disrupted with sterile scissors in 50 μl of DNA extraction buffer (10 mM Tris, pH 8.0, 2 mM EDTA, 0.1% SDS, 500 μg of proteinase K/ml). Each sample was incubated for 2 h at 56°C and then boiled at 100°C for 10 min to inactivate proteinase K. After centrifugation, the supernatant was transferred to a fresh sterile microtubule and purified by extracting twice with an equal volume of phenol-chloroform before use. Then TE (10 mM Tris, pH 8.0, 2 mM EDTA) was added to dissolve DNA for PCR use.

PCR amplification
Samples were screened for evidence of F. tularensis by nested PCR targeting the fopA gene, as described previously [17,23]. All positive specimens for the fopA gene were re-tested to obtain subspecies confirmation by amplifying the ppI-helicase region of F. tularensis gene structure using the primer pair of C6 (AGGCGGA-GATCTAGGAACCTTT, GenBank accession no. AF247642) and C8 (AGCCCAAGCTGACTAAAATCTTT, GenBank accession no. AF247685). This PCR yielded a variable-length amplicon due to a 30-bp deletion in F. tularensis subsp.holarctica: for F. tularensis subsp.holarctica, the amplicon was 220 bp (GenBank accession no. EU526911), and for F. tularensis subsp.tularensis, the amplicon was 250 bp in length [15]. All the PCR products were separated by 3% agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Negative controls and positive controls (Chinese first F. tularensis strain isolated from Citellus dauricus in 1957 from Inner Mongolia) were applied to ensure validation of the amplification. To minimize contamination, DNA extraction, the reagent setup, amplification, and agarose gel electrophoresis were performed in separate rooms. Amplicons from all PCR assays were sequenced to verify the validity of the PCR. For sequencing, amplicons were excised from the gels, purified through spin columns (Qiagen, Hilden, Germany) and sequenced on automated DNA sequencer (ABI PRISM 377, Perkin-Elmer, Foster City, USA). All sequencing primers were amplification primers which were read for both strands.

Multiple-locus variable number tandem repeat analysis (MLVA)
Using MLVA, two markers, Ft-M3 and Ft-M10 (previous designations SSTR9 and SSTR16, respectively) were used to identify the genetic diversity among positive ticks as previously described [14,15]. All amplicons obtained from two markers were sequenced on an automated DNA sequencer (ABI PRISM 377 Parkin-Elmer, Inc). The number of repeated units for each sample was counted. The genotype was defined simply as the number of repeats for Ft-M3 followed by that for Ft-M10.

Nucleotide sequence accession numbers
Nucleic acid sequences representative of each genotype and sequence from subspecies confirmation were deposited in GenBank under accession numbers EU526911, EU544140 to EU544147.